MASARYK UNIVERSITY

Faculty of Science

erRORs in cellular crosstalk- Importance in development and

disease

Zankruti Dave

Dissertation Thesis

Brno 2020

Supervisor: Prof. Mgr. Vítězslav Bryja, PhD

BIBLIOGRAPHIC ENTRY

Author: Zankruti Dave

Faculty of Science, Masaryk University, Department of Experimental Biology, Section of Animal Physiology and Immunology

Thesis title: erRORs in cellular crosstalk – importance in development and disease

Degree Program: Animal Physiology, Immunology and Developmental Biology

Supervisor: Prof. Mgr. Vítězslav Bryja, PhD.

Academic Year: 2019/2020

Number of Pages: 77

Key words: ROR1, ROR2, crosstalk, Wnt, chronic lymphocytic leukemia, Lyn, BMP signaling

BIBLIOGRAFICKÝ ZÁZNAM

Autor: Zankruti Dave, M.Sc

Přírodovědecká fakulta, Masarykova univerzita, Ústav experimentální biologie, Oddělení fyziologie a imunologie živočichů

Název dizertační práce:

erRORs in cellular crosstalk – importance in development and disease

Studijní program: Fyziologie, imunologie a vývojová biologie živočichů

Vedoucí práce: Prof. Mgr. Vítězslav Bryja, Ph.D.

Akademický rok: 2019/2020

Počet stran: 77

Klíčová slova: ROR1, ROR2, funkční interakce, Wnt, chronická lymfocytární leukémie, Lyn, BMP signalizace

PERMISSIONS Published articles have been reproduced with permission from the publisher.

ABSTRACT (Czech)

Buněčná signalizace je fascinující svou mírou komplexity.

Většina proteinů integruje vstupy z několika různých zdrojů a vytváří

výstup, který je pro buňku výhodný. Ve své práci se zaměřuji na dva

proteiny z rodiny tyrozinových kináz, receptor tyrosine kinase-orphan

receptor-1 a 2 (ROR1 a ROR2). Tyto proteiny slouží jako receptory

v rámci Wnt signální dráhy, která je jednou z nejdůležitějších

signalizací pro udržení buněčné homeostázy a během embryonálního

vývoje. Hlavním, spojujícím tématem této studie je otázka, jak proteiny

z ROR rodiny umožňují rychlou komunikaci v buňkách – ROR

receptory jsou křižovatkou několika buněčných signálních drah, což

buňkám přináší výhodu modulování odpovědí na externí a interní

signály. V prvním manuskriptu popsaném v této práci jsem se zaměřila

na popsání role ROR1 v kontextu chronické lymfocytární leukémie

(CLL) a ve druhém manuskriptu na roli ROR2 v kontextu vývoje

končetin.

CLL maligní buňky nesou zvýšenou hladinu ROR1 proteinu,

který patří mezi embryonální proteiny. Ve své práci jsem popisovala

interakci ROR1 s kinázou Lyn z rodiny Src kináz, nezávislou na jeho

primárním ligandu Wnt5a. Analyzovali jsme efekt kinázy Lyn na post-

translační modifikace ROR1 a s využitím CRISPR metodiky pro vyřazení

exprese Lyn kinázy jsme odhalili možné mechanismy propojení Wnt-

ROR a B-buněčné signalizace v CLL buňkách.

Správný vývoj končetin závisí na koordinaci několika signálních

drah, mezi které patří BMP, Wnt, Notch a TGF- Ve své práci jsme

poskytli důkazy propojující ligand Noggin, antagonistu BMP

signalizace, a Wnt-ROR2 dráhu.

ABSTRACT

Cellular signaling is a fascinating phenomenon because of the scale of

complexity involved in it. Most proteins have to integrate inputs from

multiple sources and generate an output beneficial to the cell. In my

thesis, I have focused on two proteins of the receptor tyrosine kinase

family, receptor tyrosine kinase-orphan receptor-1 and 2 (ROR1 &

ROR2). These proteins serve as receptors in the Wnt signaling pathway,

which is one of the most important pathways during embryonic

development and for cellular homeostasis. The overarching theme of my

thesis is how the ROR family of proteins facilitates rapid intracellular

communication by engaging in crosstalks with other pathways, offering

an advantage to the cell to modulate its external as well as internal

response. In manuscript 1, I have described the role of ROR1 in the

context of chronic lymphocytic leukemia (CLL) and in manuscript 2 that

of ROR2 in the context of limb development.

In CLL, malignant cells upregulate ROR1, an embryonic protein.

I have characterized the interaction of ROR1 and Src family kinase-Lyn,

independent of its primary ligand WNT5a. By analyzing the interaction

of these proteins when over-expressed and by using a CRISPR knock out

of LYN in a B-cell line, we have uncovered a possible crosstalk

mechanism between WNT-ROR and B-cell receptor (BCR) signaling in

CLL cells.

Normal limb development hinges on the co-ordination of

multiple signaling pathways such as bone morphogenetic factor (BMP),

WNT, Notch and transforming growth factor-beta (TGF-). In my work

related to ROR2 we provide evidence for crosstalk between the

antagonist of the BMP pathway - Noggin and WNT-ROR2 signaling.

LIST OF PUBLICATIONS INCLUDED IN THESIS 1) Zankruti Dave, Olga Vondálová Blanářová, Štěpán Čada, Pavlína

Janovská, Nikodém Zezula, Martin Běhal, Kateřina Hanáková, Sri

Ranjani Ganji, Pavel Krejci, Kristína Gömöryová, Helena Peschelová,

Michael Šmída, Zbyněk Zdráhal, Šárka Pavlová, Jana Kotašková, Šárka

Pospíšilová, and Vítězslav Bryja. Lyn controls chemotaxis and motility

of CLL cells via phosphorylation of ROR1.

bioRxiv 2020.05.29.124156; doi: https://doi.org/10.1101/2020.05.29.124156

I designed and carried out most of the experiments, analyzed the results

and wrote the manuscript.

2) Bernatik, O., Radaszkiewicz, T., Behal, M., Dave, Z., Witte, F., Mahl,

A., Cernohorsky, N. H., Krejci, P., Stricker, S., & Bryja, V. (2017). A Novel

Role for the BMP Antagonist Noggin in Sensitizing Cells to Non-

canonical Wnt-5a/Ror2/Disheveled Pathway Activation. Frontiers in

Cell and Developmental Biology, 5, 47.

https://doi.org/10.3389/fcell.2017.00047

I carried out the supplementary experiments to confirm the main

findings.

LIST OF OTHER PUBLICATIONS 1) Pospichalova, V., Svoboda, J., Dave, Z., Kotrbova, A., Kaiser, K.,

Klemova, D., Ilkovics, L., Hampl, A., Crha, I., Jandakova, E., Minar, L.,

Weinberger, V., & Bryja, V. (2015). Simplified protocol for flow

cytometry analysis of fluorescently labeled exosomes and microvesicles

using dedicated flow cytometer. Journal of Extracellular Vesicles, 4,

25530. https://doi.org/10.3402/jev.v4.25530

2) Harnoš, J., Cañizal, M., Jurásek, M., Kumar, J., Holler, C., Schambony,

A., Hanáková, K., Bernatík, O., Zdráhal, Z., Gömöryová, K., Gybeľ, T.,

Radaszkiewicz, T. W., Kravec, M., Trantírek, L., Ryneš, J., Dave, Z.,

Fernández-Llamazares, A. I., Vácha, R., Tripsianes, K., Hoffmann, C., …

Bryja, V. (2019). Dishevelled-3 conformation dynamics analyzed by

FRET-based biosensors reveals a key role of casein kinase 1. Nature

Communications, 10(1), 1804. https://doi.org/10.1038/s41467-019-

09651-7

3) Kotrbová, A., Štěpka, K., Maška, M., Pálenik, J. J., Ilkovics, L.,

Klemová, D., Kravec, M., Hubatka, F., Dave, Z., Hampl, A., Bryja, V.,

Matula, P., & Pospíchalová, V. (2019). TEM ExosomeAnalyzer: a

computer-assisted software tool for quantitative evaluation of

extracellular vesicles in transmission electron microscopy

images. Journal of Extracellular Vesicles, 8(1), 1560808.

https://doi.org/10.1080/20013078.2018.1560808

Table of Contents

1) Introduction 1

2) Receptor Tyrosine Kinases 4

2.1) ROR1 and ROR2 6

2.1.1) Structure 8

2.2) Receptors of Wnts 11

2.2.1) Canonical Wnt pathway 13

2.2.2) Non-Canonical Wnt pathway 14

2.2.3) ROR signaling in limb development 19

3) Chronic Lymphocytic Leukemia 22

3.1) IGHV status 25

3.2) Cytogenetic aberrations 26

3.3) B-cell receptor signaling in CLL 27

3.3.1) BCR pathway 28

3.3.2) Lyn kinase 30

3.3.3) Regulation of Lyn 33

4) Current therapeutic strategies in CLL 35

4.1) Targeting BCR 35

4.2) Targeting ROR1 37

5) Aims 40 6) Results and Discussion 41 7) Conclusions 50 8) Acknowledgements 52 9) References 58

LIST OF ABBREVIATIONS

Acute lymphoblastic leukemia ALL

Adenomatous polyposis coli APC Adenosine Triphosphate ATP Ataxia-telangiectasia mutated ATM B-cell lymphoma 2 BCL2 B-cell receptor BCR

Beta-transducin repeats containing protein -TrCP Bone morphogenetic protein BMP Brachydactyly B1 BDB1

Bruton’s tyrosine kinase BTK C-C chemokine receptor type 7 CCR7 C-C motif chemokine ligand-19 CCL19 c-casitas B lineage lymphoma c-CBL c-Jun N-terminal kinases JNK C-terminal Src kinase CSK Canal associated neuron abnormal migration – 1 CAM-1

Casein kinase CK Chimeric antigen receptor T cells CART Chronic Lymphocytic Leukemia CLL

Cluster of differentiation CD Colon carcinoma kinase-4 CCK4 Convergent extensions CE Csk homologous kinase CHK Cysteine rich domain CRD

Dedicator of cytokinesis-2 DOCK-2 Digit crescent DG Dishevelled Dvl Fibroblast growth factor FGF Frizzled domain Fzd Glycogen synthase kinase GSK Hematopoietic lineage specific protein-1 HS-1 Hematopoietic Stem Cell HSC

Immunoglobulin Ig

Immunoglobulin heavy chain variable region IGHV

Immunoreceptor tyrosine-based activation motifs ITAM Immunoreceptor tyrosine-based inhibitory motifs ITIM

Low density lipoprotein receptor related protein LRP Lymphoid enhancer-binding factor LEF Lyn knock out LKO Mantle cell lymphoma MCL

microRNA miRNA

Minimal residual disease MRD Monoclonal antibody mAb Muscle specific receptor kinase MuSK

Mutated CLL mCLL Myeloid differentiation primary response MYD Neurotrophic tropomyosin receptor kinase related NTRKR

Nuclear factor kappa-light-chain-enhancer of activated B cells NFB Phalyx-forming region PFR phosphatidyl inositol 3 kinase PI3K Phospho-tyrosine pY phospholipase C-2 PLC-2 Planar cell polarity. PCP Post-translational modification PTM

Proline rich domain PRD

Protein kinase C PKC Protein tyrosine phosphatase non-receptor PTPN Receptor Tyrosine Kinase RTK

Receptor tyrosine kinase orphan receptor ROR

Recessive robinow syndrome RRS

Serine/Threonine S/T

Short nucleotide polymorphisms SNP Src family kinase SFK Src homology SH Transforming growth factor TGF Tropomyosine receptor kinase TRK

Tyrosine kinase TK Tyrosine kinase domain TK Tyrosine-protein like kinase-7 PTK-7 Unmutated CLL uCLL Zinc finger protein ZNF −associated protein, molecular weight 70kDa ZAP-70

1

1. Introduction

he life of a cell is defined by 3 tightly regulated processes:

proliferation, differentiation, apoptosis. It is mindboggling to

even imagine that all the information needed by the cell to

carry out these processes is encoded and stored in its DNA— a

repository of information. To carry out each or all of these in a regulated

manner, cells rely on proteins - the work horses of a cell. Evolution has

fine-tuned these processes so fantastically that a cell can co-ordinate

multiple events occurring simultaneously; one would therefore think

that almost half of the DNA might be utilized in encoding proteins.

Instead, a germ line cell in humans uses up to only 2% of its 3 billion

base pairs to encode roughly 20,000 proteins.

Cancer is the end result of deregulated cellular signaling, when

a cell forgets to stop proliferating or has found means to overcome

apoptosis allowing cells to divide at a frantic pace. This is generally due

to the perturbation of dedicated signal transduction pathways.

Cancerous cells have an advantage over normal cells because they have

bypassed the regulatory mechanisms controlling the multiple activities

of a cell: cell cycle, division, migration or apoptosis. These rogue cells

either overexpress proteins which confer them with properties to

enhance proliferation capacity or repress proteins which instruct the

cell to self-abort in case of gross mistakes. In cancer, genetic insults

causing point mutations or large-scale chromosomal translocations

generally tend to strike a certain class of proteins and affect pathways

that play an important role in growth, development and homeostasis.

T

2

A normal adult, on average, has about 400 different types of cells

(Vickaryous and Hall 2006) and cancer is broadly categorized into 4

types based upon the cell type of origin: carcinomas in epithelial tissues,

sarcomas in mesenchymal tissues, neuroblastomas or glioblastomas in

the nervous tissues, and leukemias and lymphomas in hematopoietic

tissues (Sever and Brugge 2015). Among leukemias, chronic lymphocytic

leukemia (CLL) is the most common, prevalent in the western

countries, affecting every 4,1 out of 100,000 individuals diagnosed at a

median age of 72 years (Hallek 2019). Among the many abnormal

features of CLL, one of the most striking is the expression of an

embryonic protein receptor tyrosine kinase-orphan receptor (ROR1), a

receptor tyrosine kinase (RTK) on CLL cells (S. Baskar et al. 2008;

Fukuda et al. 2008; Klein et al. 2001; Rosenwald et al. 2001). For this very

reason, understanding ROR1 biology and using ROR1 as a lucrative

therapeutic target has been an area of intense research in the past

decade (Choi et al. 2015; Hudecek et al. 2010). Extant therapies already

exploit the dependence of CLL on the B-cell receptor (BCR) pathway for

survival and proliferation; key kinases of this pathway are targeted in

CLL patients (Ferrer and Montserrat 2018; ten Hacken et al. 2019).

However, there are a host of problems accompanying the present forms

of therapy: side effects, financial burden and resistance to drugs. Thus,

there is an urgent need for alternative targets in CLL, to alleviate the

problems associated with current therapies. Recent evidence suggesting

a communication between ROR1 and BCR pathway components thus

warrants further research (Bicocca et al. 2012; Karvonen, Chiron, et al.

2017; Q. Zhang et al. 2019).

3

With regards to cancer, ROR1 and its paralog ROR2 are unique

cancer markers in their own right (Borcherding et al. 2014; Rebagay et

al. 2012); however, these proteins also have very important roles to play

during embryonic development. Understanding signaling through

RORs has been the focus of my study while the overarching theme of

my thesis is cellular crosstalk. Since my primary project focuses on a

study of ROR1 and Lyn in the context of CLL and my secondary project

relates to Wnt5a-ROR2 signaling in the context of limb development,

in the following chapters I have tried to introduce each of these topics

individually and also attempted to provide a more general background

about them.

4

2. Receptor Tyrosine Kinases Proteins converse with each other by means of various post-

translational modifications (PTMs), namely, phosphorylation,

ubiquitination, acylation, glycosylation or methylation (Fabbro, Cowan-

Jacob, and Moebitz 2015). Among these, phosphorylation is the most

common form of modification found in a cell and almost 2% of the

human genome is dedicated to protein kinases, a specialized group of

enzymes that catalyse this process. Kinases are a specialized group of

enzymes which work by transferring the -phosphate group of the

adenosine triphosphate (ATP) molecule to amino acids having a free

hydroxyl group, namely serine, threonine or tyrosine. Protein kinases

can be divided into two major classes: Serine/Threonine (S/T) kinases

and Tyrosine kinases (TKs). The human genome encodes 58 types of

receptor tyrosine kinases (RTKs), which are broadly classified into 20

subfamilies (Fig 1), and 32 non-receptor tyrosine kinases which are

classified into 10 subfamilies (Blume-Jensen and Hunter 2001). It was

previously believed that tyrosine kinases were unique to metazoans and

that the emergence of these enzymes aided the process of

multicellularity; however, this theory was abandoned when it was

discovered that a unicellular organism like the choanoflagellate,

Monosiga brevicollis, also has a complex tyrosine kinase system

comprising of 128 tyrosine kinases (Manning et al. 2008). Nonetheless,

a general consensus in the field is that the ability to phosphorylate

tyrosine residues gives the cells additional signaling bandwidth (Mayer

2008) enabling it to forge new networks without disrupting the existing

signaling networks which rely on Ser/Thr kinases.

5

Fig 1: Scheme of Receptor tyrosine kinase families and their general structural features of, borrowed from the review (Lemmon and Schlessinger 2010)

The discovery and meticulous study of every new member of the RTK

family showed that members of this family are key regulators of the cell-

cycle, proliferation, differentiation and migration. Further, any changes

in the distribution, expression, or regulation of RTKs leads to disease

(Blume-Jensen and Hunter 2001; Lemmon and Schlessinger 2010). Thus,

it is not at all surprising that a significant number of cancers result from

mutations that impair the function of RTKs. In the case of CLL,

malignant cells rely on RTKs like ROR1, VEGF, insulin-like growth

factor-1 (IGF-1), and AXL for survival and evasion of apoptosis (Ghosh

6

and Kay 2013). Among these, ROR1 is of particular interest as it is

primarily an embryonic protein expressed by CLL cells (S. Baskar et al.

2008; Fukuda et al. 2008). It is highly expressed during embryonic

development with greatly reduced to no expression in tissues after birth

(Al-Shawi et al. 2001; Masiakowski and Carroll 1992). However, though

ROR1 expression was not detected on adult brain, lung, heart tissues, it

was detected in several parts of the gut, pancreas and parathyroid gland

(Balakrishnan et al. 2017).

2.1 ROR1 and ROR2

The ROR family comprises of ROR1 and its paralog ROR2, both type I

transmembrane RTKs. They were discovered in a neuroblastoma cell

line SH-SY5Y, using degenerate oligonucleotides as probes during a PCR

screen of the kinase domain during a search for additional RTKs, which

could be close relatives of the tropomyosin receptor kinase (Trk) family,

that play a role in the development of the nervous system. Owing to the

manner of their discovery, they were also initially known as

neurotrophic tropomyosin receptor kinase related (NTRKR) 1 and 2,

respectively (Masiakowski and Carroll 1992). The ROR1 gene, located on

chromosome 1, encodes a protein that is 937aa long and ROR2 on

chromosome 9 encodes a 943aa protein. Overall, ROR proteins share

about 58% amino acid identity with predicted molecular weights of

about 102 kDa, but their observed molecular weight is close to 130kDa

due to N-glycosylation, a PTM. It has been shown that ROR1 undergoes

multiple N-glycosylations (as well as mono-ubiquitination) which

influence the trafficking of ROR1 to the cell membrane and that these

modifications may play a role in ROR1 signaling (Kaucká et al. 2011).

7

Orthologs of ROR1 and ROR2 have been found in rat and mouse (mRor1

and mRor2) (Masiakowski and Carroll 1992; Oishi et al. 1999), in

D.melanogaster ( Dror and Dnrk ) (Oishi et al. 1997; Wilson, Goberdhan,

and Steller 1993) although, Dnrk may actually be the true ortholog of

drosophila MuSK receptor (Sossin 2006). In C.elegans, only a single

ortholog has been found known as CAM-1 (canal associated neuron

abnormal migration) (Forrester et al. 1999). RORs are highly expressed

at all embryonic stages, in cells belonging to all the 3 germ layers, but

the most prominent role they play is in neurogenesis and skeletal system

development. Their expression is however repressed to a large extent in

adult tissues (Balakrishnan et al. 2017; Rebagay et al. 2012). Mutations in

ROR2 have been known to cause heritable skeletal development

disorders: the autosomal recessive Robinow syndrome (RRS) — a

skeletal dysplasia and the autosomal dominant brachydactyly B1 (BDB1),

which causes developmental deformities in the fingers and toes (Afzal

et al. 2000; Afzal and Jeffery 2003). The mutations that cause RRS can

be found scattered all over the ROR2 sequence and they usually include

frame-shift, nonsense or missense mutations. BDB1 results from

mutations limited to two hotspots that give rise to a truncated protein,

almost always lacking the S/T and PRD (Stricker, Rauschenberger, and

Schambony 2017). I will delve deeper into the role of RORs in the context

of limb development in a later section. An autosomal recessive mutation

in ROR1 (pR736T) has been found to cause deafness due to inner ear

malformation and auditory neuropathy (Diaz-Horta et al. 2016). Both

the ROR proteins are implicated in cancer; initially it was thought that

ROR1 is seen to be upregulated in hematological malignancies such as

CLL, acute lymphoblastic leukemia (ALL) and mantle cell lymphoma

(MCL) while ROR2 plays a more prominent role in solid tumors such as

8

osteosarcoma or renal cell carcinoma (Rebagay et al. 2012). It is now

understood that both RORs are expressed in a wide variety of tumors

and their expression is generally related to worse overall survival (Saleh

et al. 2019).

RORs owe their classification as orphan receptors to the

considerable gap between their discovery and identification of their

ligands; this same gap also made it difficult to assess the possible roles

of ROR’s. Owing to their similarities to the Trk neurotropin receptors

and muscle-specific receptor kinase (MuSK) family, it was speculated

that RORs may play a role in synapse development(Forrester et al. 1999).

It is now known that ROR1 and ROR2 are receptors of WNT ligands,

specifically WNT5A, which constitutes a major pathway in embryonic

development (Ho et al. 2012; Oishi et al. 2003). This relationship is also

relevant to CLL since WNT signaling pathway has an important role to

play in the progression of the disease (Janovská and Bryja 2017).

2.1.1 Structure

As is the case with members of the RTK family, the RORs have a very

generic molecular architecture: an extracellular domain that can

respond to ligands, a transmembrane domain and an intracellular

tyrosine kinase domain (Fig 2). In case of human ROR1 and ROR2, the

extracellular domain has 3 subdomains: an immunoglobulin (Ig) like

domain, a cysteine-rich domain (CRD) like the one found in members

of the Frizzled family, and a kringle domain. The presence of a kringle

domain distinguishes the RORs from the rest of the RTK family; these

domains are highly folded structures, rich in cysteine residues found

predominantly in blood coagulation factors where they help in protein-

9

protein interactions. A short transmembrane domain is followed by an

intracellular domain which again is divided into 4 subdomains: a

tyrosine kinase (TK) domain, a proline rich domain (PRD) flanked by

serine/threonine (S/T) domains on each side.

Fig 2: Comparison of the domains of ROR, its C.elegans homolog CAM-1 and receptor

tyrosine kinase muscle specific kinase (MuSK). Figure borrowed from review on the

evolutionary divergence of tyrosine kinase domains(Bainbridge et al. 2014).

The phosphorylation of tyrosine residues in the TK domain opens up 2

possibilities: either it facilitates the stimulation of the inherent catalytic

activity of the TK or it serves to recruit adaptor proteins possessing the

phosphotyrosine recognizing domains e.g Src-homology 2 (SH2)

domain (Hubbard, Mohammadi, and Schlessinger 1998).

10

Interestingly, TK domains of ROR1 and ROR2 lack key amino acid

residues required for kinase activity in its catalytic loop (Bainbridge et

al. 2014; Masiakowski and Carroll 1992). Among RTKs, the kinase

domain is the most useful domain to trace the evolutionary history of a

receptor. Thus, a thorough comparison of protein sequences of the

catalytic or kinase domain in 65 kinases revealed a very tight

conservation in certain stretches of amino acids (Hanks and Hunter

1995). Accordingly, there are about 40 residues which are conserved

across all tyrosine kinases, except in the case of ROR1 and ROR2 which

have differences in 7 and 5 amino acids, respectively. The kinase domain

itself has about 11 sub-domains and it folds itself to give rise to 2 lobes –

the N-terminal lobe and the C-terminal lobe. Generally, the N-terminal

lobe comprises of subdomain I-IV and the residues here are involved in

anchoring the ATP molecule and stabilizing it while the C-terminal lobe

is involved in binding to the peptide substrate and carrying out the

transfer. Subdomain I forms the glycine rich loop and has the ‘GxGxxG’

sequence which is highly conserved and present in all tyrosine kinases,

including S/T kinases. This stretch of amino acids is involved in

stabilizing the ATP molecule and orienting it correctly for the phospho-

transfer to occur with the middle glycine residue (GxGxxG) playing an

important role in doing this. Crucially, in ROR1, this residue at position

482 changes to a cysteine and in ROR2 to aspartate. Two other

significant changes are in the C-terminal lobe containing the ‘HRD’ and

the ‘DFG’ motifs; in ROR1/2 where this changes to ‘HKD’ and ‘DLG’.

Even so, ROR2 has been shown to have some kinase activity in vitro but

ROR1 lacks any (Masiakowski and Carroll 1992; A. Mikels, Minami, and

Nusse 2009). Dror and CAM-1, on the other hand, retain the consensus

sequence as well as the kinase activity (Bainbridge et al. 2014).

11

2.2 Receptors of WNTs

The pathways on which the cells rely to undergo the regular cell-cycle,

maintain homeostasis, growth, division, or apoptosis are made up of

many individual components working together in a controlled fashion.

Most genetic insults to a cell are well tolerated and might not do much

long-term damage; cells do have very stringent modes of control for

such scenarios and can trigger apoptosis to deal with the problem. Even

so, there are some genes, called proto-oncogenes which are pivotal to a

cell and any mutation in these genes would turn them into oncogenes,

which results into cancer. Under normal circumstances though, these

proto-oncogenes are involved in key pathways, especially important for

the normal development of mammalian embryos. These proto-

oncogenes could be receptors, growth factors, cytoplasmic components

or nuclear factors and are a part of some important pathways such as

transforming growth factor – beta / bone morphogenetic protein (TGF-

/BMP), Hippo, Notch-Delta and WNT signaling pathways (Nusse and

Clevers 2017; Nusse and Varmus 1992).

Of these, the Wnt pathway genes were instrumental in establishing the

connection between key role players in development and oncogenesis.

Historically, tumorigenic viruses played a key role in aiding the

discovery of cellular oncogenes (Rijsewijk et al. 1987). By means of

transduction, these viruses lead to the expression of the proto-oncogene

in a modified form which helps the cell turn into tumorigenic form. A

study employing tumorigenic viruses led to the discovery of the int-1

gene, which upon transduction, led to the formation of tumors in the

mammary glands of mice (Nusse and Varmus 1982). It was later

12

discovered to be the homolog of the drosophila segment polarity gene

wingless, which if mutated was lethal zygotically (Nüsslein-volhard and

Wieschaus 1980; Sharma and Chopra 1976). Thus, Wnt is actually a

portmanteau of wingless and int-1. Wnts are small (42-48kDa), cysteine

rich, lipid modified secreted glycoproteins. In higher vertebrates, Wnts

form a large family comprising 19 members orchestrating different

functions in a cell such as, differentiation, polarity, migration and

proliferation (Kestler and Kühl 2008).

Broadly speaking, Wnts can be divided into 2 groups: one set of Wnts

(Wnt-1/3a/8/8b) can induce a secondary body axis formation in Xenopus

embryos and has the ability to transform cells; and the other set (Wnt-

4/5a/11) controls movements of cells and cell adhesion. There is also

evidence to suggest that these 2 sets of Wnts can antagonize each other

(Kestler and Kühl 2008; Kühl et al. 2000). Wnt pathways are broadly

classified as the canonical pathway, which culminates into the

stabilization of the -catenin protein, and the non-canonical pathway

that is -catenin independent. The complexity increases further with

regards to their receptors. The foremost receptor of Wnt, identified in

drosophila, was the Frizzled (Fzd) protein, (Bhanot et al. 1996), which

itself is a family of 10 members (Huang and Klein 2004). In addition,

there are co-receptors involved lending specificity with regards to the

function of the individual Wnts or Fzds in the canonical or the non-

canonical pathway. Lastly, the non-canonical pathways employ Wnts as

ligands but have receptors other than Fzd. This initial classification of

Wnts as canonical or non-canonical seems like an over-simplification of

a very complex event, since the same Wnt can have very different

outcomes based on its spatio-temporal distribution. Thus, it has been

13

suggested that the specificity of a signal is determined by (or in relation

to) the receptors / co-receptors and not the Wnt ligand per se

(Amerongen 2012; A. J. Mikels and Nusse 2006).

2.2.1 Canonical Wnt Pathway

A major goal of the canonical pathway is the cytoplasmic stabilization

of -catenin. In the absence of Wnt initiation, a group of 3 proteins –

adenomatous polyposis coli (APC), Axin, casein kinase – 1 epsilon (CK-

1) and glycogen synthase kinase 3b (GSK-3), come together to form

the ‘destruction complex’ and bind to -catenin. Ck-1 & Gsk-3 then

sequentially phosphorylate -catenin close to its N-terminal. The

phosphorylation now primes -catenin for ubiquitination by beta-

transducin repeats-containing protein (-TrCP), a subunit of an E3

ligase which results into the subsequent proteasomal degradation of it

(Fig 3). The binding of a Wnt ligand to its receptor Fzd, in the presence

of a co-receptor low density lipoprotein receptor related protein (LRP-

5/6), leads to the recruitment of disheveled (DVL) protein. The ensuing

cascade of events culminates into the disbanding of the destruction

complex, allowing the cytoplasmic accumulation of -catenin, which

then moves into the nucleus and binds to transcription factors of the T-

cell transcription factor/Lymphoid enhancer binding factor (TCF/LEF)

family and activates transcription (Kestler and Kühl 2008).

14

Fig 3: Figured borrowed from review on Wnt/-Catenin (MacDonald, Tamai, and He

2009). Panel A describes the scenario in the absence of Wnt ligands when the

destruction complex, made of Apc, Axin, Gsk3, Ck1, destroys -catenin and curbs

further downstream signaling. Panel B describes the scenario when in the presence of

the Wnt ligand, Fzd and co-receptor Lrp5/6, recruit Dvl to the membrane. The events

which follow disrupt the destruction complex. This allows the accumulation of -

catenin which is free to traverse into the nucleus and initiate the transcription of Wnt

responsive genes.

2.2.2 Non-canonical Wnt pathway(s)

To give rise to a 3-dimensional shape in an organism, cells have to be

directed to migrate in a certain way or have to be rearranged within the

tissue. Movements of cells which extend the body axis are generally

termed as convergent extensions (CE) and this morphogenetic event is

essential for the closure of the neural tube and extension of the body

axis during development. If it were to be absolutely simplified, one can

say that the canonical Wnt pathway specifies the fate of a cell and

whether or not it should proliferate, while the non-canonical pathway

dictates its orientation and migration capabilities (Amerongen 2012).

15

The non-canonical Wnt pathways encompass a set of pathways which

are usually triggered into action by Wnt or Fzd but downstream of that

they encompass a diverse range of receptors, cytoplasmic effectors and

transcription factors, each with a varying outcome, as can be seen in Fig

4. However, a unifying factor for all of these non-canonical Wnt

pathway sub-types is the lack of -catenin dependent transcription and

antagonism of the canonical pathway (Veeman, Axelrod, and Moon

2003).

The first clue regarding Wnts playing a role in gastrulation movements

came from studies in Xenopus embryos where overexpression of

XWnt5a, caused defects in the convergent extension movements, but

not the fates of cells (Moon et al. 1993). This observation suggested the

possibility of the existence of a pathway in vertebrates that was very

similar to Drosophila planar cell polarity (PCP). PCP, in flies, dictates

the polarity of a cell within a plane of tissue e.g: the arrangement of the

ommatidium or individual optical units which make up the compound

eye or the arrangement of the hair cells on the wing, in a fly. It was well

established that Fzd and Dvl played important roles in the PCP pathway

(Axelrod et al. 1998) but the ligand of this pathway remained obscure

until it was discovered in Drosophila that it was wingless which was the

ligand (Bhanot et al. 1996; Deardorff et al. 1998). In time, the list of non-

canonical Wnt pathways was extended to include the Wnt/PCP,

Wnt/Calcium pathway, Wnt/ c-Jun N-terminal kinases (JNK) pathway

and many others, though the latter ones are less well characterized. For

the purpose of this thesis, I will only discuss the Wnt5a-ROR axis.

16

Fig 4: Cartoon summary of the different branches of non-canonical Wnt pathways

borrowed from (Semenov et al. 2007)

It was first suggested that Wnts might be the elusive Ror receptor

ligands when it was discovered that Ror proteins also had a CRD on the

extracellular side (Rehn et al. 1998; Saldanha, Singh, and Mahadevan

1998; Y. K. Xu and Nusse 1998). By the late 1990’s, there was ample proof

from various sources that Ror1 and Ror2 played an important role during

mouse development especially in the neuronal tissues, cartilaginous

tissues, development of facial structure, heart and lungs (DeChiara et al.

2000; Matsuda et al. 2001; Oishi et al. 1997; Takeuchi et al. 2000). In most

instances their expression patterns were partly overlapping but on the

whole, it seemed that they were functionally redundant (Nomi et al.

2001). Around the same time, it was becoming increasing clear that

Wnt5a controlled the morphogenetic movement of cells and Wnt5a

signaling was essential in the vertebrate embryo for the proper

development of the outgrowing limbs and the proximal-distal axis of

various body structures (Moon et al. 1993; T. P. Yamaguchi et al. 1999).

Wnt5a-/- mutants recapitulated the developmental abnormalities

observed in ROR2-/- mutants (Takeuchi et al. 2000; T. P. Yamaguchi et

al. 1999). It all came together in when it was finally shown in Xenopus

17

that XRor2 was a receptor of the non-canonical Wnt5a and that it played

a role in CE movements (Hikasa et al. 2002; Schambony and Wedlich

2007) and that mROR2 and Wnt5a worked synergistically in activating

the JNK pathway. Ror2 was recognized as a bona fide receptor of Wnt5a

and not simply a co-receptor of Fzd (A. J. Mikels and Nusse 2006; Oishi

et al. 2003).

Over the years, various attempts were made to understand how Wnt5a

signaled through the Ror receptors. It was shown that Wnt5a induced

homodimerization and activation of Ror2 (Liu et al. 2008) and that upon

stimulation by Wnt5a, Ror2 underwent phosphorylation by CK1e and

GSK-3 on Ser/Thr residues (and not tyrosine residues) (Kani et al. 2004;

H. Yamamoto et al. 2007); Gsk-3 could specifically phosphorylate

serine 834 in Ror2 (Grumolato et al. 2010). The ability of Rors to function

as typical tyrosine kinases has always been disputed (as explained in the

‘structure’ section ) however it has been shown that Ror2 kinase activity

is required for signaling through Wnt5a (A. Mikels, Minami, and Nusse

2009). It was not long before Ror1 was also found to be a receptor of

Wnt5a. In a span of 5 years it was shown that Ror1 was a receptor of

Wnt5a (Fukuda et al. 2008), its interaction with Ror1/2 heterodimers

played an important role in synaptogenesis in hippocampal neurons

(Paganoni, Bernstein, and Ferreira 2010), and that Wnt5a-Ror signaling

axis was important for proper tissue morphogenesis (Ho et al. 2012).

Interestingly, while the first study showed that Wnt5a-Ror1 interaction

induced the activation of nuclear factor kappa-light-chain-enhancer of

activated B cells (NFkB), the last study found that among all the

downstream effectors of Wnt-Ror signaling which were reported

previously such as phosphorylation(s) of c-Jun, protein kinase C (PKC),

18

vang-like protein 2 (Vangl2) or Dvl (Gao et al. 2011; Oishi et al. 2003; X.

Zhang et al. 2007) or antagonism of canonical Wnt signaling (A. J.

Mikels and Nusse 2006) it was only phosphorylation of Dvl which was

affected. These studies highlight that the Wnt5a-Ror axis forms an

independent branch among the non-canonical pathways, though it

frequently can cooperate with the Wnt/PCP branch (Gao et al. 2011), and

that Wnt5a-Ror downstream signaling will differ among cell types. I

believe the initial intense scrutiny regarding the role of ROR2 in non-

canonical signaling was prompted by the fact that mutations in Ror2

were known to be responsible for the severe skeletal defects in RRS and

BDB1. Once it was discovered the ROR1 is a unique marker on CLL cells,

the focus shifted to also understanding this signaling branch better,

especially in CLL cells. First, it was shown that autocrine WNT5a by

regulating ROR1 activity conferred CLL cells with higher basal motility

and rendered them unable to respond to chemokines, and that

inhibiting the Wnt/PCP pathway in these cells restored migratory

defects (Janovska et al. 2016). It has also been shown that Wnt5a

promotes the interaction of ROR1 to intracellular proteins such as

dedicator of cytokinesis 2 (DOCK2), 14-3-3, hematopoietic-lineage-

specific protein 1 (HS1) and cortactin to activate Rho-GTPases,

ultimately leading to an enhanced rate of proliferation and migration in

CLL cells (M. Hasan et al. 2017; M. K. Hasan et al. 2018, 2019; J Yu et al.

2017). There have been reports suggesting that Wnt5a enhanced the

migration and proliferation in CLL cells by promoting the hetero-

oligomerization of ROR1 and ROR2 through the interaction of their

respective kringle domains (Jian Yu et al. 2016), however this has been

disputed since NMR studies show that these domains are not involved

in the interaction (Ma et al. 2019).

19

In general, a considerable body of work has helped us to understand

signaling through RORs via its ligand Wnt5a; however, significant gaps

of knowledge remain in our understanding of the importance of their

intracellular domains or any alternative modes of signaling since these

receptors do have other domains on the extracellular side which can

possibly interact with a wide repertoire of ligands.

2.2.3 ROR signaling in limb development

The skeletal system is composed of bone and cartilage made of dense

and semi-rigid connective tissues, respectively. It can be divided into

the axial and appendicular skeleton wherein the head and body trunk

are a part of the axial skeleton; forelimbs and hindlimbs part of the

appendicular skeleton. One of the first observable phenotypes in Ror-/-

or Wnt5a-/- mice were the skeletal defects (DeChiara et al. 2000; T. P.

Yamaguchi et al. 1999). While genetically Ror2 deficient mice exhibited

skeletal abnormalities (Takeuchi et al. 2000), Ror1 deficient mice did not

display any obvious skeletal defects but they died at birth due to

respiratory failure (Nomi et al. 2001). In a different study, though Ror1

mice were observed to have skeletal defects, they were limited to the

axial skeleton and the Ror1 deficient pups were severely growth

compromised (Lyashenko et al. 2010).

Between the RORs, ROR2 was firmly implicated to have a more

prominent role in skeletal development when it was discovered to

harbor mutations which caused RRS and BDB1 (Afzal et al. 2000; Afzal

and Jeffery 2003; Oldridge et al. 2000; Schwabe et al. 2000). RRS is

characterized by severe craniofacial malformations, overall skeletal

20

defects which affect the axial and appendicular system, heart defects,

and genital hypoplasia. On the other hand, brachydactylies are a group

of disorders characterized by the shortening of the limbs. Of the 5 types

(A-E), BDB1 subtype is the most severe which results from mutations in

ROR2 (Stricker and Mundlos 2011). For the purpose of this thesis, I will

discuss signaling events during limb development which can help us

appreciate the molecular milieu of ROR2 signaling.

During limb development, mesenchymal cells from the lateral plate

mesoderm initiate the formation of a limb bud. The limb bud then has

to coordinate development along 3 axes: proximo-distal(P-D), anterior-

posterior (A-P), and dorso-ventral (D-V). Each of these is under the

control of different signaling center: the apical ectodermal ridge (AER)

controls the P-D axis, the zone of polarizing activity (ZPA) controls the

A-P axis and the D-V axis is under the control of Wnts from the

overlying ectoderm (Petit, Sears, and Ahituv 2017). While the three axes

have independent signaling mechanisms, they have to be coordinated

spatio-temporally for correct limb formation (Spielmann and Stricker

2016). The limb can be divided into 3 regions from proximal to distal

end- stylopod, zeugopod and autopod. In the developing mouse

embryo, Ror1 expression is restricted to the proximal regions of the limb

while Ror2 expression can be detected throughout the limb, especially

in the distal regions (Matsuda et al. 2001). It is the autopod that is most

severely affected in BDB1 (Stricker, Rauschenberger, and Schambony

2017).

Bone formation is called ossification that can occur via

intramembranous ossification and endochondral ossification (Kamizaki

21

et al. 2020). Intramembranous ossification involves bone formation

directly from the connective tissue and is commonly seen in flat bones

while in endochondral ossification, commonly observed in long bones,

eg. Limb bones, the cartilage is laid first as a template. Chondrogenesis

is the process by which the cartilage is formed. It starts with the

condensation of undifferentiated mesenchymal stem cells into

aggregates which reflect the pattern of the future limb. These cells,

called chondrocytes, undergo progressive differentiation giving rise to

pre-hypertrophic and then hypertrophic chondrocytes; this entire

process is controlled by BMP signaling. It has been shown that Ror2

plays a role in chondrocyte differentiation but not so much in

proliferation (Schwabe et al. 2004). Mesenchymal cells which surround

the cartilage form the perichondrium and these cells express Wnt5a;

overexpression of Wnt5a prolongs the differentiation of

prehypertrophic to hypertrophic chondrocytes (Hartmann 2002;

Hartmann and Tabin 2000). The formation of the individual digits at

limb extremities is dependent on a signaling center called the phalynx-

forming region or digit crescent (PFR/DC) and it is characterized by a

highly active BMP signaling pathway (Witte et al. 2010). The AER

maintains an active fibroblast growth factor (FGF) signal to maintain

the cells in an undifferentiated state but AER signals are important for

the digit outgrowth driven by BMP signals. Ror2 and Wnt5a co-operate

to inhibit Wnt/-catenin signals from the ectoderm, a failure of which

leads to a break-down of the PFR/DC ultimately affecting the phalanges

as evinced by the BDB1 phenotype. However, the exact molecular and

biochemical details of these signaling crosstalks are yet to be elucidated.

22

3. Chronic Lymphocytic Leukemia CLL was first described in 1960’s by Dr William Dameshek, from Boston

and Dr D. Galton, from London (Dameshek 1967; G Galton 1966), almost

simultaneously, as an immunoproliferative disorder in which the B-cells

are immune-incompetent. The characteristic of CLL is accumulation in

peripheral blood, of small, mature looking, CD5+ B cells, which have

undergone clonal proliferation. The presence of CD5 on B-cells is an

obvious anomaly since it is actually a T-cell antigen (Burgess et al. 1992).

These cells have faulty apoptotic mechanisms and tend to accumulate

within the blood, spleen, bone marrow and lymph nodes and cause

lymphocytosis, splenomegaly and lymphadenopathy. For a very long

time, since the course of the disease is slow, it was believed the CLL

results due to the accumulation of faulty cells rather than being a

proliferative disorder. However it was shown that in fact CLL cells do

proliferate at an astounding rate (Messmer et al. 2005). In fact, the

prognosis is usually worse in patients in whom this rate is greater than

0.35%. These cells also express CD19; a biomarker of B-cells, and CD23;

a low affinity receptor of IgE. CD23 is used to distinguish CLL from other

lymphoproliferative disorders, mainly mantle cell lymphoma (MCL)

(DiRaimondo et al. 2002; Kilo and Dorfman 1996). CLL cells also express

lower levels of IgM and IgD in comparison to normal B-cells.

A perplexing aspect of CLL is that in some patients, the disease may

remain indolent for years and the patient may actually succumb due to

natural causes or some other ailment. These individuals may not even

know that they have CLL, if it were not for some routine blood check.

In other cases, the course of CLL may turn aggressive which leads to a

greatly decreased life expectancy, in some cases despite the therapy.

23

This heterogenous nature of CLL prompted Rai and colleagues in 1975

to devise a staging system of CLL based on the symptoms of the patients

(Rai et al. 1975). This staging system was revised and improved further

by Binet and colleagues (Binet et al. 1977) and has been used ever since

to stratify CLL patients into risk groups (as shown in Table 1) thereby

helping clinicians in identifying those who need treatment versus (vs)

those who just need to be under observation.

Table 1: Borrowed from the review – Chronic Lymphocytic Leukemia, A clinical review.

(Nabhan and Rosen 2014). It summarizes the criteria suggested by Rai and Binet to

stratify CLL patients into risk groups.

a-Note that the overall survival has improved over the years due to improved therapies

b-nodal areas such as cervical, axillary, inguinal, spleen and liver.

CLL is of unknown etiology and there are many theories about its cell of

origin. The identification of the cell of origin is imperative to understand

the pathobiology of the disease. Many groups have tried to hypothesize

about or identify the cellular origins of CLL. One view which has

prevailed in the field is that the disease develops from a self-renewing

hematopoietic stem cell (HSC) which may turn rogue and as a result be

the CLL cell of origin (Kikushige et al. 2011). Historically, it was also

believed that CLL arose from naïve, antigen inexperienced B-cells;

24

however, work by multiple groups has shown that since B-CLL cells are

functionally similar to the ones from splenic marginal zone, so they

must have arisen from antigen- experienced marginal zone, CD27+

memory B cells (Chiorazzi, Rai, and Ferrarini 2005; Damle et al. 2002;

Klein et al. 2001).

According to the Armitage and Doll ‘multistep model of carcinogenesis’,

it is not a single mutational event but a series of critical mutational

events which cause the transformation of a normal cell into a cancerous

one (Armitage and Doll 1954). Likewise, many factors contribute not just

to the development of the disease but also its course. In the case of CLL,

it is now understood that though the disease may be initiated by

changes in the genetic material it is the burden of additional factors /

mutations that makes it more aggressive. That genetics and hereditary

also play a role is evinced by the fact that approximately 9% of CLL

patients had a relative who also suffered from the disease. In addition,

in cases of familial CLL, about 30 genetic loci were found to carry short

nucleotide polymorphisms (SNPs)(Kipps et al. 2017). Curiously, gender

seems to play a role in CLL, with women having a much slower course

of the disease and a better chance at overall survival (D. Catovsky,

Fooks, and Richards 1989). Important hallmarks of CLL include: the

mutational status of the immunoglobulin heavy chain variable region

gene (IGHV); chromosomal changes in untreated patients such as

deletion of the long arm of chromosome 13 (del13q), trisomy 12, deletion

of the long arm of chromosome 11(del11q) or deletion of the short arm of

chromosome 17(del17p); expression of somatically mutated genes

especially NOTCH1, myeloid differentiation primary response

(MYD88), TP53, ZNF292, or PTPN11 (Hallek 2019). All together these

25

observations suggest that the central pathways on which a cell depends,

such as DNA damage and repair, RNA processing, MAPK signaling are

disrupted.

3.1 IGHV status

The course that CLL might take can be gauged by the mutational status

of the immunoglobulin heavy chain variable region gene. If a sequence

differs from the germline sequence more than 2%, it is considered

mutated. Many CLL patients, grouped as mCLL, harbor more than this

2% of mutations in their VH genes, and they have been observed to have

the less aggressive form of the disease and thus prolonged survival

(Damle et al. 1999). The other group of patients with < 2% or no

mutations in VH genes are referred to as uCLL, u referring to the

unmutated status. They usually suffer the more aggressive form of the

disease and can have decreased life expectancy (Hamblin et al. 1999).

Since there are 2 subtypes it was also believed that uCLL arose from

naïve or pre-germinal center B cells while mCLL arose from post-

germinal center B cells. uCLL cells have a greater capacity to proliferate

and can respond to immune-stimulation but are also prone to apoptosis

(Longo et al. 2007). Contrarily, mCLL do not respond to external

stimulus as much, but they do have a very active intracellular signaling

(Chiorazzi, Rai, and Ferrarini 2005). Interestingly, it was a study of the

mutational status of the IGHV that gave clues to researchers about the

difference in genders as well; the occurrence of uCLL is higher in males

(Daniel Catovsky, Wade, and Else 2014). Irrespective of the subtype, CLL

cells do have a very limited repertoire of antibodies, so much so that

26

about every 1 in 75 CLL patients will have nearly identical antibodies, a

phenomenon referred to as stereotypy (Widhopf et al. 2004).

3.2 Cytogenetic aberrations

In terms of genetic aberrations or alterations, CLL patients have some

classic cytogenetic lesions, namely: deletion of 13q14, trisomy 12,

deletion 11q22-23 and deletion of 17p13; however, these may not be the

primary triggers of CLL. Nonetheless, the presence of these cytogenetic

abnormalities does influence how patients will respond to treatments

hence it is important that CLL patients are tested for these lesions before

embarking upon treatments (Hallek et al. 2008).

The most common genetic insult found in CLL patients is the del13q,

specifically involving band 14. The frequency of this abnormality is close

to 50%, which means that the disruption of genes in this cluster, must

offer some advantage to the cell. Rightly so, this cluster has been found

to encode genes controlling cell cycle and apoptosis in B-cells. This

particular region has been found to encode a non-transcribed gene and

two micro RNAs (miRNA) 15-a and 16-1 (Calin et al. 2002, 2005; Veronese

et al. 2015),which serve to repress the expression of B-cell lymphoma 2

(BCL2) protein and -associated protein, molecular weight 70kDa (ZAP-

70), respectively. Lack of these miRNA controls allows the cells to

express BCL2 and ZAP70 which are anti-apoptotic (Klein et al. 2010).The

worst are the del17p and del11q22-23 lesions, since these stretches contain

p53 and ataxia-telangiectasia mutated (ATM) genes, respectively

(Döhner et al. 2000; Stilgenbauer et al. 2002). p53 protein is a well

27

characterized tumor suppressor and ATM kinase is involved in the DNA

damage repair pathway.

In spite of all the above mentioned molecular and genetic abnormalities

found in CLL, none of them are the direct causes nor are they specific to

CLL. The correct method of CLL diagnosis, as recommended by the

World Health Organization (WHO), international workshop on chronic

lymphocytic leukemia (iwCLL) and National Family Caregivers

Association (NCCN), relies on immunophenotyping to distinguish it

from other B-cell lymphoproliferative disorders (Rawstron et al. 2018).

Thus, in addition to the minimum set of markers such as CD19, CD5,

CD23, CD20, kappa & lambda, the assessment of the patient sample

should include CD200, CD10 and ROR1. The inclusion of ROR1 makes

sense since it was shown that higher levels of cell surface ROR1 were

associated with a rapid disease progression (Cui et al. 2016).

3.3 B-Cell Receptor signaling in CLL

There is no doubt that CLL is a BCR-dependent malignancy. Within

lymphoid tissues, CLL cells actively proliferate within areas termed as

psuedofollicles and BCR signaling is the most prominent pathway active

in these cells (Burger and Chiorazzi 2013). B-cells rely on this pathway

for their survival and a functional BCR is imperative for the survival of

B-cells (Lam, Kühn, and Rajewsky 1997). In a normal scenario, BCR

signaling allows for the expansion of a foreign antigen activated B-cell

and removal of B-cells which react to self-antigens. With an increase in

the knowledge about this pathway, it became clearer that in CLL a lot of

the BCR pathway components signal aberrantly which is advantageous

28

for the malignant cells; CLL-BCRs can be active even in the absence of

an external antigen, known as tonic stimulation (Burger and Chiorazzi

2013).

3.3.1 BCR Pathway

For about 500 years, vertebrates have relied on the adaptive immune

branch to protect themselves from an increasingly complex

environment. One of the key players in this branch of immune system

is the immunoglobulin (Ig) molecule, commonly referred to as an

antibody. Higher classes of vertebrates usually have 5 different isotypes

of Igs: IgM, IgD, IgG, IgA, IgE. Not only do these Ig molecules serve as

circulating effectors to stimulate other components of the immune

system, but also, they serve as antigen receptors on the surfaces of B-

cells (Schroeder 2015). The default antibody that a B-cell is equipped

with, when it is ‘born’ is the IgM; it is present even before the B-cell ever

encounters an external antigen. In a normal cell, this surface Ig molecule

is usually paired with a heterodimer comprising of cluster of

differentiation (CD) 79A-79B, also known as Ig/Ig, wherein the IgM

binds externally to the antigen while the Ig/Ig heterodimer handles

the intracellular signaling. The cytoplasmic domains of both these Ig

associated proteins contain multiple phosphorylatable sites known as

immunotyrosine based activation motifs (ITAMs). Upon antigen

stimulation, the BCR components undergo rearrangements in the

plasma membrane such that the tyrosines in the ITAMs are positioned

to get phosphorylated. These phosphorylations are carried out by Src-

family kinase (SFK) family members which are found to be pre-

associated with the BCRs, primarily Lyn (Burkhardt et al. 1991; T.

29

Yamamoto, Yamanashi, and Toyoshima 1993), and others like Fyn and

Blk. The tyrosine kinase Syk, which is a target of Lyn (Kurosaki et al.

1994), has 2 SH2 domains but lacks the N-terminal acylation to help it

anchor itself to the membrane. The SFK phosphorylated ITAM motifs

provide a binding site for Syk via its SH2 domain, such that gets apposed

to its activator Lyn.

Fig 5: Cartoon summary of the BCR pathway. Stimulation of the BCR by an antigen,

leads to the activation of various kinases downstream of the primary kinases Lyn and

Syk. Figure borrowed from review on B-cell receptor signaling by ten Hacken and Burger

(2016).

These SFKs, which fall under the class non-RTKs, are also responsible

for phosphorylating and activating further downstream kinases of the

BCR pathway which ensures that the incoming signal gets amplified and

30

is propagated via 3 main routes: phospholipase C-2 (PLC-2),

phosphatidyl inositol 3 kinase (PI3K) and Bruton’s tyrosine kinase

(BTK). The ensuing cascade of events leads ultimately to signals which

promote the survival and proliferation of B-cells (Burger and Chiorazzi

2013; Woyach, Johnson, and Byrd 2012). The importance of this pathway

is underscored by the current number of drugs which target the

individual pathway components. I will discuss more on that in the next

section but first I would like to highlight the importance of Lyn, a crucial

regulator of the BCR pathway.

3.3.2 Lyn kinase

Lyn is a member of a family of non-receptor tyrosine kinases, composed

of 9 structurally related members, which include Src, Yes, Fyn and Fgr

(which form the SrcA subfamily), Blk, Lck, Lyn and Hck (which form

the SrcB subfamily) and Yrk (Parsons and Parsons 2004). These

proteins, collectively referred to as the SFKs play an important role in

modulating the signals propagated by multiple RTKs on the membrane.

Lyn, short for Lck/Yes-related novel tyrosine kinase, is a SFK which was

discovered using v-yes DNA as a probe (Y Yamanashi et al. 1987) and its

gene is localized on human chromosome 8 (mouse chromosome 4). In

a mature B-cell, BCR signaling requires Lyn in its capacity as a positive

facilitator of the pathway through phosphorylations of ITAMs in Ig,

Ig and CD19; however, its role as a negative regulator is far more

important, whereby it phosphorylates immunoreceptor tyrosine-based

inhibitory motifs (ITIMs) in cell surface receptors which helps to end

the BCR signaling. Due to this dual role Lyn has been described as a

cellular signaling rheostat (Lowell 2004; Y. Xu et al. 2005).

31

It was known that the signal transducing entity in BCR, the Ig and Ig

heterodimer, lacked catalytic domains and therefore an alternate

protein was required to interpret the incoming signal and pass it

forward. By drawing parallels with the role of Lck in T cells and its

association with T cell receptors, it was discovered that Lyn physically

associates with IgM and mediates IgM signaling (Yuji Yamanashi et al.

1991). Subsequently it was found to associate with Syk, HS1, Vav, PI3K

and BTK. Lyn also co-operates heavily with the co-receptor CD19 to

form a signal amplification loop, wherein Lyn phosphorylates tyrosine

residues on CD19, creating an additional site for Lyn to bind and

undergo further activation in proximity of the BCR (Fujimoto et al. 2000;

Gauld and Cambier 2004). While its positive role in BCR signal

modulation can be shared to some extent by the other two SFKs, its role

as a negative modulator of this pathway is irreplaceable as was

highlighted by the work which shed light on the interaction of Lyn and

CD22 (Smith et al. 1998). As mentioned above, Lyn and CD19 enter into

a kind of activation loop. Among the many targets of Lyn, which it can

phosphorylate, one is CD22. The phosphorylation of CD22 initiates the

recruitment of SHP-1, a phosphatase of CD19 which ultimately leads to

the levels of activation signals going down. This finding was also

supported further by studies in Lyn-/- mice in which the B-cell

development was not affected but they were found to be

hyperproliferative, leading to autoimmune disorders (DeFranco, Chan,

and Lowell 1998; Hibbs et al. 1995). B-cell development was more or less

normal in these mice because at during this stage Lyn plays a minor role,

albeit a positive one, along with Fyn and Blk as a redundant SFKs (Y. Xu

et al. 2005). Surprisingly enough, the phenotype of mice overexpressing

32

Lyn (Lyn up/up ) recapitulated the effects of the Lyn-/- mice (Hibbs et al.

2002).

Owing to splice variants of its transcripts on exon 2 Lyn protein

can have 2 isoforms, p53 and p56 (also known as LynB and LynA,

respectively).The 2 isoforms were believed to be functionally redundant,

until it was shown in mast cells that the 2 isoforms have different roles

in signaling and associate with different effectors (Alvarez-Errico et al.

2010). Lyn is highly expressed in all hematopoietic cells of myeloid and

lymphoid lineage, except T cells, although it does play an important role

as a negative regulator of Th2 immune responses as seen in Lyn-/- mice

(Beavitt et al. 2005).

All in all, these studies highlight the importance of Lyn in B-cells

and suggest that Lyn fine-tunes BCR signaling by forming and

coordinating an extremely complex network of targets, activators and

regulators as shown in Fig 6.

Fig 6: Graphical summary of the positive, through CD19, and negative regulation,

through CD22, of BCR pathway by Lyn. Figure borrowed from review on Src family

kinases in B-cells.(Gauld and Cambier 2004)

33

3.3.3 Regulation of Lyn (and other SFKs)

In my work I have utilized pharmacological inhibition or Lyn deletion

mutants to control the kinase activity of Lyn and thereby influence its

interaction with ROR1. Hence, I would like to elaborate on how SFKs are

physiologically regulated.

Among the SFK family members there exists a considerable

amount of structural homology within their domains and thus, all SFKs

are regulated the same way. Each member has the following functional

domains: an N-terminal SH4 domain, unique for each kinase and which

hosts one or two acylation sites for both myristoyl and palmitoyl groups

to help it remain anchored to the membrane ( with the exception of Blk

which only has myristoyl groups), SH3 domains, SH2 domain, a linker

region and a kinase domain ( also referred to as the catalytic domain).

SH2 and SH3 domains are protein-protein interaction domains wherein

SH2 domains binds to phosphor-tyrosine (pY) residues (Moran et al.

1990) and SH3 binds to proline rich domains (PRD) (Ren et al. 1993).

The catalytic activity of SFKs has to be very tightly regulated and this is

done by a series of phosphorylations and dephosphorylations to keep it

active or inactive. To suppress the kinase activity, the catalytic domain

is maintained in a closed conformation (shown in Fig 7). This is

achieved by means of phosphorylating a Y residue at position 527, close

to the C-terminal. By convention, this position refers to the one

observed in c-Src but it differs slightly in each SFK. In the case of Lyn, it

is Y508. This phosphorylated Y527 can now bind to the SH2 domain and

keep the catalytic domain closed and the two PTKs responsible for this

34

phosphorylation are C-terminal Src kinase (CSK) and Csk homologous

kinase (CHK).

Fig 7: Regulation of Src family kinases. Figure on the left side shows SFK in an inactive

state kept phosphorylation of Y(508 in Lyn) residue in the C-terminal. Figure on the

right side shows an activated kinase in which the C-term phosphate is removed by a

phosphatase first, which allows the Y (397 in Lyn) in the kinase domain to become

accessible. Figure borrowed from a review on Src family kinases (Salter and Kalia 2004)

To activate the SFK, this Y527 has to first be dephosphorylated, which is

carried out by different phosphatases. Alternatively, even the binding of

external ligands to SH3/SH2 domains can interfere with this inactivating

35

intramolecular interaction, thereby opening up the catalytic domain

which exposes Y416 (Y396 in Lyn). Phosphorylation of this residue not

only dislodges it from the site to which SFK substrates can bind but also

completely activates the SFK (Salter and Kalia 2004).

36

4. Current therapeutic strategies in CLL

The objective of any treatment related to cancer is to achieve a minimal

residual disease (MRD) negative state. Since CLL is such a complex

disease, clinicians have found it beneficial to target it from multiple

directions using a wide array of available drugs. Thus most current

treatment options include combinations of cytostatic agents eg:

chlorambucil, fludarabine; monoclonal antibodies (mAb) eg: rituximab,

alemtuzumab; agents targeting BCR or BCL2 signaling eg: ibrutinib,

idelalisib, venetoclax; chimeric antigen receptor T cells (CART) therapy

(Hallek 2019). Overall it was observed that the addition of agents

targeting kinases in BCR pathway, to the combination treatments, was

more beneficial.

4.1 Targeting BCR as a therapeutic strategy in CLL

Given the importance of this pathway it was natural for it to be the focus

of therapies in CLL and hence this field has exploded in the recent years.

As can be seen in Fig 8, a host of small molecule kinase inhibitors have

been developed to target SYK , BTK and PI3K known as fostamatinib,

ibrutinib and idelalisib, respectively (Byrd et al. 2013; Friedberg et al.

2010; Furman et al. 2014). However, notwithstanding the evidence of

how efficient they are resistance to these drugs has been observed in

clinic. Point mutations in BTK (C481S) or activating mutations in PLC

have been observed (ten Hacken et al. 2019). BTK is a member of the Tec

family comprising of 5 other members, all of which are highly expressed

in cells of the hematopoietic system and play important roles in growth

and differentiation (Mano 1999). Members of this family are known to

37

mediate signals emerging from phosphotyrosine and phospholipid-

based systems and thus additional problems arise when drugs like

ibrutinib cross-react with other members of the family or other kinases

e.g: epidermal growth factor receptor (EGFR) (Byrd et al. 2016).

Moreover, these drugs are associated with expensive treatments, very

nasty side effects such a bleeding diathesis and arrythmias, and disease

relapse post discontinuation, hence there is a dire need of alternate

targets.

Fig 8: Cartoon summary of BCR kinases targeted for therapy in CLL. Figure borrowed

from review on the importance of BCR in CLL (ten Hacken et al. 2019)

Given the importance of BCR to CLL cells, it was not long before

the role of Lyn in CLL came under scrutiny. Even though BCR

stimulation in B-CLL cells failed to stimulate Lyn (Kawauchi,

Ogasawara, and Yasuyama 2002), Lyn was found to be highly expressed

in B-CLL cells as compared to normal B-cells (Contri et al. 2005; Hussein

38

et al. 2009). CLL cells thrive in areas of the bone marrow called

pseudofollicles and the microenvironment these CLL cells is made up of

mesenchymal stromal cells, nurse-like cells derived from monocytes

and T cells (ten Hacken and Burger 2016). CLL cells need input from all

of these to survive and this was evident from the observation that

culturing of CLL cells in vitro was a near impossible task, due to the

tendency of CLL cells to undergo apoptosis (Collins et al. 1989), unless

the culture was supported with factors normally found in the tumor

microenvironment (Nguyen et al. 2016). The importance of the

microenvironment on CLL cells was also gleaned from the observation

that CLL cells can have distinct biologies based on their location,

peripheral blood vs lymph nodes or bone marrow microenvironments

(Hayden et al. 2012). Given the importance of Lyn to BCR and cells of

the myeloid lineage, Nguyen et al attempted to uncover the role of Lyn

in influencing the interactions which occur in these

microenvironments. Indeed, its importance was underscored when it

was found that macrophages without Lyn failed to support the growth

of CLL cells. Thus targeting Lyn in CLL would be an option worth

exploring (Wiestner 2012).

4.2 Targeting ROR1 as a therapeutic strategy in CLL

The first line of treatment in CLL patients includes a combination of

cytostatic reagents, such as fludarabine and cyclophosphamide, and

mAbs, such as rituximab. The disadvantage of current mAbs which are

approved as first or second line of treatment in CLL, such as rituximab

(mAb against CD20) and alemtuzumab (mAb against CD52) is that

these target the normal B-cells leading to an overall

39

immunosuppression in patients (Yang et al. 2011). This need for a CLL

specific target paved the way for research into developing mAbs to

target other CLL-cell specific targets.

Two independent gene expression profiling studies had already

categorized ROR1 as a marker which was upregulated in CLL cells (Klein

et al. 2001; Rosenwald et al. 2001) but it was few more years before it was

proposed by different groups that ROR1 would be an ideal target to treat

this disease (S. Baskar et al. 2008; Amir H. Daneshmanesh et al. 2008;

Fukuda et al. 2008). ROR1 CLL cells did indeed undergo apoptosis when

treated with ROR1 siRNA which gave further impetus to this idea

(Choudhury et al. 2010) and within time different groups had developed

mAbs (Sivasubramanian Baskar et al. 2012; Choi et al. 2015; A. H.

Daneshmanesh et al. 2012) or CAR-T (Hudecek et al. 2010) against ROR1

which showed an apoptotic effect specifically towards CLL cells. One of

these anti-ROR1 mAbs, cirmtuzumab ( also known as UC-961), is

currently in clinical trials and has shown promising results (Choi et al.

2018).

It must be mentioned that B-cells have been observed to express

ROR1 normally during an intermediate stage of development and these

B-cells are termed hematogones (Broome et al. 2011; Hudecek et al.

2010). In light of this knowledge, a very interesting connection was made

between ROR1 and the pre-BCR in a subset of acute lymphoblastic

leukemia (ALL) patients, specifically ones which carried the genetic

abnormality t(1;19) (Bicocca et al. 2012). ALL is childhood malignancy

and about 5% of ALL patients carry this translocation t(1;19) and a study

of cells which carry this genetic abnormality showed that these were

cells which were arrested at a later stage of B-cell development,

compared to the other 95%. In this elegant study, the authors observed

40

that crosstalk between ROR1 and pre-BCR promoted the survival of CLL

cells by activating the Akt pathway via ROR1/MEK/ERK when only the

pre-BCR was inhibited, but a synergistic effect leading to cell death

when ROR1 and pre-BCR, both were inhibited. This line of thought was

further supported by a study which showed that downregulation of

ROR1 had a synergistic effect with BCR inhibition in BCR sensitive cells

(Karvonen, Chiron, et al. 2017). It was shown that NFB signaling was

downstream of ROR1 and this was affected when ROR1 was targeted.

This observation falls in line with the previous one implicating Akt lying

downstream of ROR1 since it is known that Akt lies upstream of NFkB

and regulates it (Scheid and Woodgett 2000).

In light of these discoveries, there has been an increasing

interest in pursuing combinatorial treatments targeting ROR1 and BCR

(Karvonen, Niininen, et al. 2017). However, how these crosstalks are

molecularly orchestrated is yet to be understood. In our work, we have

tried to decipher the crosstalk between Lyn and ROR1. We prioritized

our focus on Lyn owing to its importance in BCR pathway and prior

evidence which pointed to the relationship between ROR1 / ROR2 with

Src (Akbarzadeh et al. 2008; Gentile et al. 2014).

41

5. AIMS

1. Given the importance of ROR1 and Lyn in CLL and the problems of

drug resistance in disease relapse patients there is a need for additional

therapeutic targets. We wanted to ascertain if and how these tyrosine

kinases interacted intracellularly and what is the consequence of such

interaction.

Our main aims were

- To confirm interaction of ROR1 and Lyn

- To identify phosphorylations, if any, on ROR1 since Lyn is a

tyrosine kinase

- To identify any molecular and functional consequences of this

interaction/phosphorylation

2. Given the importance of ROR2 and Noggin mutations in causing BDB1

and BDB2, respectively, we wanted to ascertain if these proteins

interacted.

Our main aims were

- To confirm a genetic interaction of Noggin and Ror2

- To characterize a functional interaction of Noggin and Ror2

42

6. RESULTS & DISCUSSION

Article 1

(BIORXIV/2020/124156) Lyn controls chemotaxis and motility of CLL cells via phosphorylation of

ROR1.

Our study is the first to show that Lyn can interact with ROR1 and

phosphorylate it. By utilizing ROR1 intracellular domain-deletion

mutants, we were able to show that ROR1 could interact with Lyn in the

absence of its PRD and that Lyn could phosphorylate wild type (WT)

ROR1 on tyrosine residues. We further corroborated this result by

testing mutant forms of Lyn which either lacked the kinase domain,

possessed a kinase dead domain, or a constitutively active Lyn kinase;

indeed, the tyrosine (Y) phosphorylation of ROR1 depended on an active

kinase. This result was also supported by our experiments using

pharmacological inhibition of Lyn using Dasatinib, a pan-Src kinase

inhibitor. Two other SFKs are also found in B-cells, Fyn and Blk and

hence we wondered if ROR1 could also interact them. Fyn interacted

with ROR1 but could not phosphorylate it on tyrosine residues, while

Blk did not interact with ROR1 (data not shown in manuscript), proving

that ROR1- Lyn interaction was very specific.

Previously, ROR1 and ROR2 have been reported to interact with

Src (Akbarzadeh et al. 2008; Gentile et al. 2014; T. Yamaguchi et al. 2012).

In work shown by Akbarzadeh et al, it was shown that Src recruitment

and activation was incumbent upon Wnt5a stimulation of ROR2. In our

study we observed that Wnt5a was dispensable as far as ROR1 and Lyn

43

interaction was concerned. By means of immunocytochemistry we

could observe that this interaction occurred at or close to the plasma

membrane. While ROR1 is a RTK which spans the membrane; Lyn, as

well as other members of the SFK, usually stay anchored to the

membrane via their N-terminal dual fatty-acylation (Kovarova et al.

2001). In the work by Yamaguchi et al, the authors showed that in lung

adenocarcinoma cells, ROR1 was required to sustain the EGFR-ERBB3-

PI3K survival signaling. In the study, they also showed that Src and ROR1

could interact, but the interaction was dispensable for the role of ROR1

to cooperate with EGFR. Importantly, the authors claim that ROR1

kinase activity was required for c-Src activation, but this can be debated

on two accounts. One, ROR1 has been shown to lack in vitro kinase

activity (Bicocca et al. 2012; Masiakowski and Carroll 1992), hence has

been classified as a pseudokinase (Gentile et al. 2014). ROR1 can possibly

bind to ATP, since it retains the conserved lysine (K) in the b3-strand

(Rajakulendran and Sicheri 2010) but it lacks the conserved residues

needed to phosphorylate targets. Secondly, in the experiments carried

out by the authors, they do not address the issue of SFKs being capable

of undergoing autophosphorylation. Hence to ascertain if ROR1 indeed

is required for Src phosphorylation, additional experiments and

rigorous controls are warranted.

Using immunoprecipitation-mass spectrometry (IP-MS) we were able to

identify the residues phosphorylated by Lyn, namely Y645 and Y646.

These tyrosines are a part of a triad of tyrosines is commonly seen in

RTK family members with the consensus sequence ‘YxxxYY’ and they

are crucial for the autoregulation of the RTK (Hubbard, Mohammadi,

and Schlessinger 1998). In enzymatically active TKs, phosphorylation of

44

all 3 residues is required for the activation loop to undergo a

conformational change and make space to accommodate an ATP

molecule and a substrate. We have identified that Lyn can

phosphorylate at least 2 residues in this triad. Phosphorylation of the

second Y residue in the triad, in our case Y645, is essential for the RTK

to get out of its auto-inhibitory state. This observation becomes relevant

in light of the evidence that of ROR1 in CLL patients has been found to

be heavily phosphorylated (Hojjat-Farsangi et al. 2013). The same group

reported phosphorylation on residues Y641 Y646 and S652 but these

results were only presented in a conference perhaps as preliminary

results (Hojjat-Farsangi et al. 2012). On the other hand, this very triad

‘YxxxYY’, was found to be phosphorylated also by Src (Gentile et al.

2014). In that study, the RTK MET was shown to utilize ROR1, in its

capacity as a pseudokinase, to help drive tumorigenesis. Their work

demonstrated that phosphorylation of ROR1 in different domains

influenced different outcomes in a cell. The influence of MET on cell

proliferation was incumbent on phosphorylations in the PRD while the

capacity of a malignant cell to invade tissues was influenced by the

phosphorylation of the ‘pseudo’kinase domain by Src. Importantly, Src

needed the PRD of ROR1 to be present in order for it to phosphorylate

the kinase domain Y residues; Lyn somehow surpasses this need. This is

possible since individual SFK members do have different preferences of

sequences to bind to (Alexandropoulos, Cheng, and Baltimore 1995;

Zhou et al. 1993).

8 out of 58 human RTKs are most likely pseudokinases. Coincidentally,

4 of these happen to be receptors in the Wnt signaling pathway: ROR1,

ROR2, tyrosine-protein like kinase-7 (PTK7)/colon carcinoma kinase

45

(CCK4), and muscle specific kinase (MuSK). There is some evidence to

support the idea that these pseudokinases may function to allosterically

activate other kinases or may serve as scaffolding proteins (Mendrola et

al. 2013). ROR1 has been shown to play a scaffolding role in lung

adenocarcinoma cells (T. Yamaguchi et al. 2016) and hence the

possibility that ROR1 might serve such a function in CLL did not seem

too far-fetched. Once again, we relied on IP-MS to reveal binding

partners of ROR1 phosphorylated by Lyn. Among the many binding

partners which came up, one was Dvl2 which has been shown to work

downstream of ROR1, but another interesting candidate was c-casitas B

lineage lymphoma (c-Cbl) protein. c-Cbl is a proto-oncogene with E3

ligase activity known to negatively regulate RTKs by targeting them for

degradation (Kaabeche et al. 2004) or endocytosis (Petrelli et al. 2002).

It shares a very interesting relationship with Lyn, wherein not only does

it serve as its substrate in B-cells post BCR engagement (Tohru Tezuka

et al. 1996) but it can also target Lyn for degradation (Kaabeche et al.

2004; Shao et al. 2004). c-CBL is highly expressed in hematopoietic cells

and cells of the testis (Thien and Langdon 2001). Backed by our MS

results we sought to confirm this interaction in vitro and found that

indeed ROR1 could interact with c-Cbl but only after undergoing

phosphorylation by Lyn. Further study is definitely needed to

understand the downstream consequences of this interaction. In CLL c-

Cbl plays a role as an adaptor protein more prominently than that of an

E3 ligase (Martini et al. 2018). Structurally c-Cbl is a multi-domain

protein consisting of a N-terminal tyrosine-kinase binding domain

(TKB), a RING finger motif, a PRD and a ubiquitin associated domain

(UBA) close to the C-term. The TKB and PRD can engage in protein-

protein interactions while a number of phosphorylatable tyrosine and

46

serine residues provide an opportunity for c-Cbl to be differentially

regulated (Schmidt and Dikic 2005). A lot of work has been done to

show that Wnt5a exerts its effects in CLL through the promotion of

ROR1 interaction with various partners that play a role in the

modulation of cytoskeleton and migration (M. Hasan et al. 2017; M. K.

Hasan et al. 2019; J Yu et al. 2017). Naturally, it would be critical to

understand how this new interaction influences ROR1 response to

Wnt5a.

To further our understanding of how Lyn controls ROR1, we sought to

create Lyn deficient cells, using CRISPR-Cas technology. For this we

used a commercial cell line, derived from a patient suffering from MCL,

called Maver-1. Our Lyn knock-out (LKO) cells pointed to a defect in

ROR1 protein trafficking since ROR1 mRNA levels were lower in LKO

cells, but protein levels were more or less equal, or even higher in some

cases. Moreover, flow cytometric analysis showed that the LKO

expressed higher levels of surface level ROR1. A similar observation was

made by Bicocca et al when they treated cells with Dasatinib to inhibit

Lyn activity; they observed an increase in ROR1 levels. If c-Cbl plays a

role in ROR1 endocytosis and trafficking, this phenotype of ROR1 in the

absence of Lyn also makes sense, since ROR1-c-Cbl interaction is very

much dependent on Lyn. An increase in ROR1 surface levels would also

increase the chances of ROR1 becoming available for Wnt5a to exert its

effects. Our lab had previously shown that autocrine Wnt5a signaling

deregulated chemotaxis of leukemic cells in a way that CLL cells

displaying high levels of Wnt5a had greater levels of basal migration and

displayed defective response to chemokine stimulation (Janovska et al.

2016). Surprisingly, our LKOs displayed lowered surface levels of C-C

47

chemokine receptor type 7 (CCR7), a receptor for chemokine CCL19,

compared to the wild type (WT) cells. In trans-well migration assays our

LKOs showed reduced chemotaxis to CCL19 stimulation. These results

suggest that Lyn plays a pivotal role in balancing pathways mediating

basal migration (Wnt5a-ROR1) vs chemotaxis (CCL19-CCR7). Our

experiments with CLL patient samples support this model since CLL

cells which displayed higher levels of ROR1, were least chemotactic.

Conversely, CLL cells which displayed higher levels of pLyn, which

indicates the kinase active Lyn phosphorylated at Y397, were the most

chemotactic.

In conclusion, our work provides evidence that ROR1 is indeed engaged

in a crosstalk with BCR. Do cancer cells use such crosstalk mechanisms

as a backup to rewire pro-survival signaling? It may be the case, as seen

in t(1;19) ALL cells where ROR1 is the backup to activate the PI3K/Akt

pathway when input from pre-BCR is blocked (Bicocca et al. 2012). A

similar phenomenon has been observed in MCL cells in which ROR1

complexes with CD19 to activate PI3k/Akt independent of BCR/BTK axis

(Q. Zhang et al. 2019). Further understanding of how this crosstalk

benefits CLL, as well as MCL, cells will help us understand the best way

to target this interaction. Phosphorylation of ROR1 in the PRD has been

reported extensively (Gentile et al. 2014; M. Hasan et al. 2017; M. K.

Hasan et al. 2019; Karvonen et al. 2018) in comparison to

phosphorylation in TKD of ROR1. Clearly, they do represent two

different modes of ROR1 regulation but is one necessary for the other or

do they represent independent modes of regulation is yet to be defined.

48

Article II

(published in 2017)

A Novel Role for the BMP Antagonist Noggin in Sensitizing Cells to Non-

canonical Wnt-5a/Ror2/Disheveled Pathway Activation

During development different signaling pathways need to coordinate

for proper development, more so since a lot of signaling pathways end

up sharing common effector molecules. Hence crosstalk among

pathway components is common. Cell communication is facilitated by

the interaction of a host of growth factors such as BMP, WNT,

Hedgehog, Notch, FGF and their receptors during mammalian limb

development. In this paper we provided evidence of a crosstalk, between

an antagonist of the BMP pathway, Noggin and the non-canonical

Wnt5a pathway (Bernatik et al. 2017). Brachydactylies are classified as a

molecular disease family owing to overlapping phenotypes among the

subtypes. Thus, it is reasonable to hypothesize that mutations within

closely interacting pathway components would give rise to overlapping

phenotypes. Phenotypic resemblances in BDB1 and BDB2, which are

caused by mutations in ROR2 and NOGGIN, respectively, prompted us

to ask if they genetically interacted. We were able to confirm their

interaction genetically in mice, by analyzing the phenotypes of Ror2 /

Noggin compound heterozygotes and Noggin heterozygote in a Ror2-/-

background. In the ROR2-/- background, the Noggin heterozygosity was

not well tolerated and exacerbated the shortening or absence of the

phalanges phenotype. Our results indicated that Ror2 and Noggin not

only interacted genetically, but also functionally.

49

We were unable to observe a direct interaction of Noggin-Ror2 but

instead Noggin could potentiate the Wnt5a-Ror2-Dvl axis of signaling.

In the presence of Noggin, the threshold of Wnt5a required to activate

Dvl in mouse embryonic fibroblasts (MEF) was lowered. We were able

to demonstrate using the Ror double knock out (DKO) MEFs that this

Noggin-Wnt5a synergism was dependent on Ror1/Ror2. We even used

pharmacological inhibition of BMP signaling, to recapitulate the Noggin

effect on activation of Dvl2 by Wnt5a-ROR2 (data not shown). Finally,

using a rat chondrosarcoma (RCS) cell line, we were able to show that

pre-treatment of these RCS cells with FGF2 was a requirement to

observe the Noggin potentiation of Wnt5a-Ror2 signaling.

In conclusion, our work demonstrates that Noggin exerts an indirect

control over the sensitization of cells towards Wnt5a-ROR2 signaling

and that this control by Noggin is dependent on FGF. However, at the

molecular level how exactly does this interaction work, needs to be

elucidated. Ror2 has been shown to interact with the Ser/Thr kinase

Bmp receptor type 1 b(Bmpr1b) and get phosphorylated by it,

independent of its ligand growth differentiation factor 5 (GDF5) and this

interaction inhibits downstream signaling through Bmpr1b (Sammar et

al. 2004). At this point, we can only speculate that treatment with

Noggin, may interfere with some such intracellular ROR2 interaction,

thereby freeing up Ror2 to interact with Wnt5a and signal through it. In

the limb, the PCP pathway has been shown to operate downstream of

Wnt5a-Ror2 axis and mutations in Vangl2 have also shown BDB1 like

effects (Wang et al. 2011). Just like the BMP pathway, the developing

limb relies on the PCP pathway for proper digit shaping and outgrowth.

Clearly, the pathways have to complement one another. Interestingly,

50

halving the dose of Bmp4 can alleviate the severe symptoms due to loss

of Vangl2 (Wang et al. 2011). In future, it would be interesting to probe

for how this Noggin synergism can affect Wnt5a-Ror2 signaling to the

PCP pathway. The positive effect of FGF2 pretreatment suggests that in

vivo it is possible that the FGF2 induced growth arrest in cells makes

them responsive to differentiation signals promoted by ROR2, since

ROR2 has been shown to have a positive effect on chondrocyte

differentiation (Schwabe et al. 2004). In a complex process like limb

development which is under the influence of multiple pathways which

often work in opposition to one another, it is not too far-fetched to

hypothesize that crosstalk between individual pathway components

could potentially modulate signaling.

51

7. Conclusions

We provide evidence that both the ROR receptors by means of engaging

in crosstalks with other pathways have profound effects on cellular

signaling. ROR1-BCR communication is more direct through the action

of Lyn and has important consequences for CLL/MCL while Noggin-

ROR2 relationship seems to be more indirect but could play an

important role in the developing limb.

Based on the evidence so far, it seems that ROR1 functions largely as a

pseudokinase and serves as scaffold protein role while ROR2 can have

kinase independent as well as dependent roles. In cancer, RORs play an

important role in regulating cell migration and they are heavily

implicated in tumorigenesis and metastasis and owing to their

uniqueness as oncofetal antigens, they are prime candidates for drug

development. However, signaling through RORs is yet to be well

understood. So far, only Wnt ligands have been shown to signal through

ROR-CRD domains. The presence of Ig and kringle domains on the

extracellular side raise the possibility of additional ligands of RORs.

Intracellularly, RORs can be regulated by phosphorylations on TKD,

PRD as well as the S/T domains. Do the different domain

phosphorylations represent a means to interact with different classes of

effector proteins or protein regulatory mechanisms? There is evidence

to suggest that RORs can engage in intracellular interactions even in the

absence of their cognate ligands.

Our results with ROR1-Lyn are backed by research which has

shown that, in the event of inhibition of BCR signaling, cancer cells use

52

ROR1 as a back-up mechanism to transmit pro-survival signals. It has

been shown that Wnt5a promotes the interaction of its receptor ROR1

with proteins which affect cellular cytoskeleton, such as cortactin and

HS1, or which can function as molecular adaptors, such as 14-3-3. Is Lyn

the missing link in this equation since cortactin and HS-1 are substrates

of Lyn? Surely these new molecular connections facilitate us with

greater insight into how convoluted signaling in cancer cells can be.

Thus, targeting just one axis may not be enough. Moreover, evidence for

such an intracellular interaction provides targets for the development of

small molecule inhibitors in instances where other forms of treatments

can be prohibitively expensive. Our results with Noggin-Wnt5a-Ror2

signaling highlight that the strength of cell signaling is not just

dependent on the individual ligand - receptor pair but can be influenced

by many other factors which can potentiate or hamper signaling.

53

8. Acknowledgements

It would be foolish of me to believe, even for a second, that I have gotten

this far solely on the basis of my own endeavors. I have had the fortune

of being in the company of some of the best that humankind has to offer

and though words will not do enough justice, this is all I have to thank

them with.

None of this would have been possible without Vita. The sanskrit word

for a teacher is ‘guru’, the remover of darkness (meaning ignorance) and

in these past few years, Vita really has been a guru for me. Vita, you have

been incredibly understanding and generous in your guidance and

scientifically given me ample amount of space to come into my own.

Over the past few years, I have learnt so much from you and for that I

shall be indebted to you forever. Really, thank you for everything.

Bryjalab is not just Vita though. I am also forever indebted to my friends

and colleagues in Bryjalab; a group of unique and amazing individuals.

My interaction(s) with each member has definitely impacted my

scientific and personal growth. They welcomed me so readily and

helped me blend into the group with so much ease, it was unbelievable.

My first thankyou vote goes to Karol because he was my savior when I

first stepped into Brno. A walking-talking thesaurus in his own right, I

grew accustomed to that loud voice in the corridor or office and I have

been missing it ever since I left. Thank you for the Frodo-Sam moment

in Krknose . Bara was so kind and patient during the time of my

settling in and help with the visa office or student dorm or anything

related to official documents needed from me. Most importantly, with

54

her around, I did not have to worry about vegetarian food for me during

get togethers. I am glad you came back. The people I first started to work

with, Vendy and Mates, both fantastic teachers. If my IP and WB results

are any good, its thanks to them! Vendy thank you for trusting me with

the exosomes work and a lot many other things over the years. Tomek,

my fellow Wntsapper. I have been in awe of your multi-tasking and DIY

skills since the time I met you. Thank you introducing me to beer. I have

not yet learnt to appreciate it as well as you would like but getting there!

My ‘deer’ James, a fellow BSB and F.R.I.E.N.D.S lover, thank you for

introducing me to some great music and appreciating my crappy sense

of humor. You have been so helpful with so many things, especially the

humongous task of helping us move! Last, but by no means the least,

Paja. A walking talking repository of practically everything related to

CLL (and Brno). I could not have done a lot of things without your help

and guidance. Olga thank you for always being so kind and patient with

me. Anicka, Petra and Marek the masters students who evolved into

Phd students and a bunch of fantastic office mates. Petra thanks to you

I was introduced to the wonders of Policka ;)! You have always been so

kind with your generous compliments and smiles across the desk.

Thank you also for periodically bringing in the perfect tonic - Tonique.

Marek, I should actually thank you for tolerating me as your desk mate.

I took the liberty to spread my mess and you never complained. Thanks

for also making me appreciate the importance of adventure in life.

Anicka, you have introduced me to the BEST thing ever – Maximus. I

have to still take you up on the offer of going skiing but thank you for

patiently teaching me the joys of ice-skating. You made it look so

effortless and elegant. Lucka, what can I say, are you an angel? You

effuse warmth and joy, and your organization skills have always put me

55

to shame. Thank you for your help with EVERYTHING. You are the

BEST lab manager. Kristina thanks for always being so kind and

pleasant to work with and Tomas, thanks for being the CRISPR nerd

and the perfectionist that you are . My dear Labradors, you guys were

single handedly responsible for one of the best things I got to do while

in Czech- visit Krknose. Thank you for organizing that trip and all the

other random get-togethers or bbqs. Stepan, thank you for putting up

with me during the difficulties of generating the Lyn knock-outs. Petra

K, miss your mischief and smiles, Misa G, who ALWAYS had a smile on

her face when you walked into the lunchroom and the only other person

who understood the pains of being short, Nikodem, a.k.a Mr Helpful

and always eager to do something new. Mirek, thank you for trying to

encourage my sense of humor with your smiles and laughter, but more

importantly for visiting me in Uppsala! Pavlina M and Lenka thanks for

being awesome lab techs and making sure that we kept a clean lab.

Everything that you did in the background made our life so much easier.

Alka, your entry almost overlapped my exit but it was certainly nice

getting to know you. A big thank you to all the lab alumni as well -

Katka S, Ondra, Simca and Igor. Katka S, thank you for being so

appreciative, gentle and joyful always and Ondra for always having a

useful suggestion every time I ran into a roadblock. Kasia, thank you

for all the scrumptious baked products. You have a gift! Honza K, my

favorite stranger and the best guitar player in the department. Katka

and Mina, thanks for all the short weekend trips and ESPECIALLY for

helping me get to the top in Krknose. I would not have been able to

ascend that monster without your help! Pavel D, I always looked

forward to bumping into you in the corridor and carrying out our daily

ritual of saying ‘Hi’ to one another . Nadia and I endured an obvious

56

language barrier, but I have to thank her since she always found a way

to help. Lenka, Stasia, Jipro, Jipa, and all my other co-workers,

temporary bachelors/masters/high school students, thank you all for

making the department such a lively place to work in.

I landed in Brno thanks to WntsApp so how could I forget them? First

thanks to all the amazing supervisors of Wntsapp with their helpful and

critical comments during the meetings and organizing some fantastic

courses/workshops. Ingrid, Lena, Luca and Anna-Iris, thank you for

all the help during my secondment in Utrecht and Lelystad. My

Wntsapp colleagues Jana, Chelo, Alessandra, Lena, Gianmarco,

Nicola, Luca, Davide, Tomasso, Michael, Tomica and Shane with all

the usual shenanigans made all the meetings so memorable. I will

cherish the memories of all our times together. Brno also introduced me

to some very interesting people outside of my usual lab arena. Amrita

drove me crazy with her madness, but I am glad I had her around.

Sonali, thank you for all the chai, ‘charchas’ and introducing me to the

badminton group: Nandan, Anna, Neha and JD. Saturdays had turned

into so much fun thanks to you guys. Thanks for all the coffee(s) and

conversations, especially Neha and JD. Carlos & Kira, I am so glad I

found you guys. Jiri, Helena, Lida and Hinek, you guys were more than

just landlords and provided me with a comfortable home. For the past

one and half year, I have been working in IGP – Neuro-Oncology,

Uppsala (Sweden) all thanks to Michael. Thank you for being so

understanding and incredibly supportive. Tack for allt! My friends and

colleagues at IGP have been so helpful these past few months especially

Milena, Joaquin, Varsha, Anders and Grzegorz, to name a few.

57

3 important people have supplemented me with unconditional love and

always put a lot of faith in me which has propelled me this far. I am

blessed to have a lovely family and THE MOST amazing parents one

could ask for. Maa and Papa, thank you (a thousand times over) for

being my support, guidance and everything-under-the-sun system. Ishu

thank you for being such an understanding and loving brother. Your

numerous ‘Its ok, shit happens’ assurances have helped me quite a few

times in these past few years. Again, family is not just Maa-Papa-Ishan!!

Lipi, thanks for being the adorable person you are. You are indeed a

welcome addition to our crazy family . A big thankyou to my dear

Pune family: Amma, Appa, Subhashbhaiya, Kalpana and Vrushabh.

I would also like to thank my extended family, my mashi-masa, mama-

mami, fois-fuas, members of saptarshi gang, the Santacruz Trivedis,

Nikaka, Abhikaka-Kalyanikaki and especially Pka. Your blessings

and good-wishes have always been a source of encouragement and have

brought me this far. A big thank you for that. My dearest Pallavi, what

would I do without you! I am incredibly lucky to have a friend like you.

Thank you for everything over the years. The friendship, calls, chats,

laughter, advise, complaints. My Pittsburgh ashrama gang and

‘chronicles’ gang, thank you for being there for me during the roughest

moments of my life. Hari-Chandan, मम प्रिय ममत्र: अहम ्न जानामम क िं

रणीयिं तव दयािं ितत रोमम! Uppsala friends Tati & Mats thank you for

everything.

Last, but by no means the least, I would like to thank Sujikoo,

my life partner and best friend. We made Brno-Uppsala work

for 5 years and make it look so easy and doable, when in fact it was not.

58

All of this would not have been possible if it were not for your

accommodating, encouraging and supportive nature. You have played a

huge role in all my decisions in the past few years and have always given

me advise which has been in my best interest. Thank you not just for

putting up with me, always, but also for bringing out the best in me.

59

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I

1

Lyn controls chemotaxis and motility of CLL cells via phosphorylation of ROR1. 1

Zankruti Dave1, Olga Vondálová Blanářová1, Štěpán Čada1, Pavlína Janovská1, Nikodém 2

Zezula1, Martin Běhal1, Kateřina Hanáková2, Sri Ranjani Ganji2, Pavel Krejci3,4, Kristína 3

Gömöryová1, Helena Peschelová2, Michael Šmída2, Zbyněk Zdráhal2,5, Šárka Pavlová2,6, 4

Jana Kotašková2,6, Šárka Pospíšilová2,6, and Vítězslav Bryja1,7* 5

6

1) Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech 7

Republic 8

2) Central European Institute of Technology, Masaryk University (CEITEC), Brno, Czech Republic 9

3) Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic 10

4) International Clinical Research Center, St. Anne's University Hospital, Brno, Czech Republic 11

5) National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Brno, Czech 12

Republic 13

6) Department of Internal Medicine – Hematology and Oncology, University Hospital Brno and Faculty 14

of Medicine, Masaryk University, Brno, Czech Republic 15

7) Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic 16

v.v.i., Brno, Czech Republic 17

Corresponding author (*): 18

Prof. Vítězslav Bryja, PhD. 19

Department of Experimental Biology, Faculty of Science, Masaryk University 20

Kotlářská 2, 611 37 21

Brno Czech Republic 22

Tel:+420-549493291 23

e-mail: bryja@sci.muni.cz 24

25

Abstract word count: 168 words 26

Main text word count: 4183 words 27

Number of Figures: 6 28

Number of tables: 2 29

Running title: Lyn controls chemotaxis and motility of CLL cells via phosphorylation of ROR1. 30

Key words: Signaling, Chronic Lymphocytic Leukemia, Cross-talk, 31

32

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2

Abstract 33

Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) are malignancies 34

characterized by the dependence on B-cell receptor (BCR) signaling and by the high 35

expression of the cell surface receptor ROR1. Both, BCR and ROR1 are therapeutic targets 36

in these diseases and the understanding of their mutual cross talk is thus of direct 37

therapeutic relevance. In this study we analyzed the role of Lyn, a kinase from the Src 38

family, as a mediator of the BCR-ROR1 crosstalk. We confirm the functional interaction 39

between Lyn and ROR1 and demonstrate that Lyn kinase efficiently phosphorylates ROR1 40

in its kinase domain and aids the recruitment of an E3 ligase c-CBL. The absence of Lyn in 41

Lyn KO Maver-1 cells produced by CRISPR-Cas9 resulted in the increased ROR1 cell 42

surface levels and deregulated migratory properties. Similar correlations between ROR1 43

surface dynamics, levels of active Lyn and chemotactic properties were confirmed in primary 44

CLL samples. Our data establish Lyn-mediated phosphorylation of ROR1 as a point of 45

crosstalk between BCR and ROR1 signaling pathways. 46

47

48

49

50

51

52

53

54

55

56

57

58

59

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3

Introduction 60

The ROR protein family comprises of ROR1 and ROR2, which are both type 1 61

transmembrane receptors. Upon discovery, ROR proteins were referred to as orphan 62

receptors on account of the lack of identity of their ligands. However, subsequent studies 63

identified their ligands to be the Wnt proteins, mostly Wnt-5a protein1,2. Wnt-5a/ROR 64

pathway is an essential signaling pathway that controls cell polarity and migration during 65

embryonic development and tissue homeostasis 3,4. During embryonic development, RORs 66

are highly and uniformly expressed, most prominently in the skeletal and neural tissues, but 67

postnatally their expression becomes highly restricted 5. 68

Interestingly, ROR1 or ROR2 upregulation has been observed in many cancers: 69

ROR1 is upregulated in solid tumors or hematologic malignancies while ROR2 is 70

overexpressed in osteosarcomas or renal cell carcinomas 6. High expression of ROR1 is 71

typical for some B-cell lymphomas such as mantle cell lymphoma (MCL)7 and chronic 72

lymphocytic leukemia(CLL)8,9. CLL is a form of hematologic cancer which is manifested as a 73

steady accumulation of mature CD5+ B-cells in the bone marrow, lymphoid tissues and 74

peripheral blood. It is the most common form of adult leukemia in the western hemisphere, 75

with an incidence of 5 per 100 000 each year and an average median age of on-set around 76

70 years. Most of the CLL cases remain asymptomatic for a long time, in which case 77

therapeutic intervention is not necessary. However, part of the CLL cases progress rapidly, 78

require treatment and their overall life expectancy is decreased 10. 79

CLL cells are in most cases highly ROR1 positive 1,8 and there are currently several 80

therapies in development that target ROR1 11,12. Another typical feature of CLL is the 81

dependency on the B-cell receptor signaling (BCR) pathway that promotes survival and 82

proliferation of CLL cells 13,14. Modern treatments in CLL are thus designed to target the BCR 83

pathway components of which some examples include: Bruton tyrosine kinase (BTK) 84

inhibitor ibrutinib, and PI3K targeted by idelalisib 10. Importantly, there are several pieces of 85

evidence that propose a mutual crosstalk between ROR1 and BCR signaling 15–17. Given the 86

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4

importance of both pathways in the novel therapeutic strategies for CLL/MCL, identification 87

of the molecular basis of such crosstalk would be of utmost importance. 88

In this study we have analyzed the role of Lyn, a kinase from the Src family, as a 89

candidate for such a function. It is the predominant Src family kinase in lymphoid cells and it 90

plays a dual role as the positive as well as negative regulator of the BCR pathway 18. We 91

focused on Lyn because of prior studies which identified the important role of Src in the 92

regulation of ROR1 or ROR2 in other experimental systems 19,20. Further, Lyn as an 93

important component of BCR pathway, has been found to be over expressed in CLL patients 94

21. We were able to confirm the mechanistic and functional interaction between Lyn and 95

ROR1 in several cell types including MCL cell line Maver-1 and primary CLL. Our study 96

establishes Lyn-mediated phosphorylation of ROR1 as another point of crosstalk between 97

BCR and ROR1 signaling pathways. 98

99

Materials and Methods 100

Cell culture 101

All cell lines used in the experiments were grown at 37°C and 5% CO2. HEK-293T wild type 102

cells (ATCC-CRL-11268, LGC Standards, Manassas, VA) were cultured in DMEM medium 103

(Thermo Fischer Scientific, USA) supplemented with 10% FBS (#10270, Gibco, Thermo 104

Fisher Scientific) and 1% Penicillin/Streptomycin (#sv30010, HyClone, GE Healthcare, 105

Chicago, IL). The mantle cell lymphoma cell line Maver-1 (#ACC 717, DSMZ Gmbh, 106

Braunschweig, Germany) was cultured in HyCloneTM RPMI 1640 medium (GE Healthcare) 107

supplemented with 10% heat-inactivated FBS and 1% Penicillin/Streptomycin. 108

109

Primary CLL samples 110

Patient samples were obtained from Dept. of Internal Medicine – Hematology and Oncology, 111

University Hospital Brno. B cells were isolated from the peripheral blood of CLL patients 112

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undergoing monitoring and treatment at the hospital, as described here 22. CLL samples 113

were obtained after written informed consent in accordance to the Declaration of Helsinki 114

and by following protocols approved by the ethical committee of the University Hospital, 115

Brno. Primary CLL cells were grown in HyCloneTM RPMI 1640 medium (GE Healthcare) 116

supplemented with 10% heat-inactivated FBS and 1% Penicillin/Streptomycin at 37°C and 117

5% CO2. 118

119

Transfections, treatments and plasmids 120

Transient transfections of HEK-293T cells were carried out using polyethyleneimine (PEI 1 121

mg/ ml); for transfections in a 10 cm plate, a total of 6 µg of DNA and for the 24 well plate a 122

total of 0.2 µg of DNA per well was used. The DNA:PEI ratios were kept at 1:3 (w/v) in all 123

cases. All the ROR1 plasmids, apart from the ROR1-v5-his, were provided by Prof. Paolo 124

Comoglio and have been described previously 20. All the Lyn expression plasmids were a 125

kind gift from Dr. Naoto Yamaguchi 23. Dasatinib (#sc-218081, Santa Cruz Biotechnology, 126

Santa Cruz, CA) was used to inhibit the kinase activity of Lyn. Further details are provided in 127

the online Supplementary Materials and Methods. 128

129

Immunoprecipitation, western blotting and immunocytochemistry 130

An extensive description is provided in the online Supplementary Materials and Methods but, 131

in brief, for the immunoprecipitation experiments, transfected cells were first washed in cold 132

PBS and then lysed in cold 0.5% NP-40 Lysis buffer and kept at 4°C. Prior to lysis, the buffer 133

was supplemented with 1mM Na3VO4, 1mM DTT, 1mM NaF and with cOmpleteTM protease 134

inhibitor cocktail and phosphatase inhibitor cocktail set II (Merck, Kenilworth, NJ). For the 135

western blotting, samples were loaded on 8% gels and separated by SDS-PAGE followed by 136

the transfer done on to Immobilon-P® (Merck) PVDF membranes at 106 V for 75 minutes. 137

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6

For immunocytochemistry, HEK-293T cells were grown on glass coverslips, transfected, and 138

immunostained. The images were taken using Leica SP8 confocal microscope. 139

140

Generation Lyn KO Maver-1 cell line 141

The knockout of Lyn gen in Maver-1 was performed using Crispr/Cas9 system. Selection of 142

the Lyn KO was based on the results of western blot and confirmed using next generation 143

sequencing of PCR product as described here 24. A detailed description can be found in the 144

online supplementary methods. 145

146

Quantitative real-time PCR 147

Quantitative real-time qPCR was used to assess the relative gene expression of ROR1 in 148

WT and Lyn KO Maver-1 cells. Data were analyzed by Delta-Delta CT method and are 149

shown as 2-ΔΔCT 25. A detailed description can be found in the online supplementary methods. 150

151

Transwell migration assay 152

Cell migration assays were carried out using HTS Transwell 24-well plates with a 5 µm pore 153

size polycarbonate membranes (Corning, New York, NY). 0.2 x 106 WT or Lyn KO Maver-1 154

cells or 1 x 106 primary CLL cells were seeded into the transwell upper inserts while media 155

were supplemented with chemokine CCL19 (#361-MI-025, R&D Systems, Minneapolis, MN) 156

at 200 ng/mL or 0.1% BSA in PBS (control) in the lower chamber. After 3 h (Maver-1 cells) 157

or 6 h (primary CLL cells) at 37°C with 5% CO2, the cells in the lower chamber were 158

collected and counted using the Accuri C6 Flow Cytometer (Becton Dickinson, Franklin 159

Lakes, NJ). 160

161

BCR Stimulation assay 162

Maver-1 cells (1x 106) were subjected to BCR stimulation for 4 min at 37°C, with 10 μg/mI 163

F(ab’)2 Anti-human anti-IgM-UNLB (#2022-01, Southern Biotech, Birmingham, AL) 164

resuspended in PBS. The control samples were treated with 0.1% BSA in PBS. Post 165

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stimulation the cells were immediately spun at 200 g for 5’ and washed with PBS. The cells 166

were finally pelleted and resuspended in 1x Laemmli buffer. The samples were sonicated for 167

15 s and further subjected to Western blotting. 168

Flow cytometric analysis of surface expression of CCR7 and ROR1 169

Cells either from culture or from transwell assay were washed in PBS and incubated in 2% 170

FBS in PBS with anti-CCR7-FITC (1:25, #561271, BD Biosciences) and anti-ROR1-APC 171

(1:25, #130-119-860, Miltenyi Biotec, Bergish Gladbach, Germany) antibodies on ice for 20 172

minutes. The cells were washed and resuspended in PBS and analyzed using Accuri C6 173

Flow Cytometer (BD Biosciences). Data were analyzed using NovoExpress (ACEA 174

Biosciences, Inc., San Diego, CA) and presented as a median fluorescence intensity (MFI 175

index) or as a fold of MFI of wt cells (Maver Lyn KO cells) or as a ratio of MFI of cells from 176

lower and upper compartment. 177

178

Mass spectrometry 179

Unbiased identification of ROR1 phosphorylation sites and ROR1 interaction partners was 180

performed by mass spectrometry. A detailed description can be found in the online 181

supplementary methods. 182

183

Statistics 184

All statistical tests were performed using GraphPad Prism software 6.0 (GraphPad Prism 185

Software, Inc., San Diego, CA). Number of replicates, format of data visualization and 186

statistical tests used for comparison are indicated in the individual figure legends. 187

188

Results 189

Lyn interacts with the Wnt-5a receptor ROR1 190

ROR1 has been reported earlier to interact with the members of the Src kinase family 20,26. In 191

order to test if this holds true for ROR1 and Lyn, we overexpressed both proteins in HEK-192

293T cells and performed immunoprecipitation experiments. We observed a strong and 193

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specific pulldown of ROR1 by anti-Lyn antibody and vice versa (Fig. 1a and 1b). In order to 194

visualize this interaction intracellularly we used immunocytochemistry. Overexpressed 195

ROR1 and Lyn co-localized in the cell membrane and filopodia of HEK-293T cells (Fig. 1c). 196

Next, we attempted to identify the domains of ROR1 involved in its interaction with Lyn. For 197

this purpose, we used a set of ROR1 deletion mutants 20 (Fig. 1d). Lyn was able to interact 198

with the WT ROR1 as well as with the ROR1 lacking the C-terminal tail formed by two 199

Ser/Thr-rich and one Pro-rich (PRD) domains. Further deletion of the complete intracellular 200

domain of ROR1 abolished the binding to Lyn (Fig. 1e). These results indicated that the 201

ROR1 kinase domain, and/or nearly adjacent regions, represent a crucial interaction 202

interface for Lyn. 203

204

Lyn phosphorylates ROR1 205

Lyn is a tyrosine kinase and as such we wanted to test whether ROR1 represents its 206

substrate. ROR1, a member of the receptor tyrosine kinase family, has a considerable 207

number of tyrosine (Y) residues, which can be phosphorylated. Thus, we utilized a set of Lyn 208

plasmids [23] (schematized in Fig. 2a) to test this hypothesis. Lyn activity is regulated by 209

phosphorylation: Phosphorylation of the Y508 at its C-terminus keeps Lyn inactive and 210

dephosphorylation of this site is necessary for the activation of Lyn. On the other hand, 211

(auto)phosphorylation at Y396 turns it into an active kinase 27,28. We used the following Lyn 212

variants: WT Lyn; kinase domain deleted (Δ, aa 1-298) mutant lacking a significant portion of 213

the kinase domain; kinase active Lyn (KA, aa 1-506) lacking the C-terminal inhibitory Y508 214

and allowing the constant activation of Lyn; and kinase dead Lyn (KD, aa 1-506, K275A) 215

Lys->Ala mutation in the ATP binding pocket rendering Lyn kinase dead. 216

WT Lyn and kinase-active (KA) Lyn triggered a strong phosphorylation of ROR1 that could 217

be detected by phospho-tyrosine (pY) specific antibody of immunoprecipitated ROR1 (Fig 218

2b). In contrast, Lyn Δ and Lyn KD failed to phosphorylate ROR1 (Fig. 2b), which suggests 219

that the phosphorylation depends on Lyn kinase activity. Interestingly, all 4 different forms of 220

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Lyn efficiently interacted with ROR1 (see Fig. 2b; IP ROR1, WB: Lyn). To further 221

corroborate the analysis, we pharmacologically inhibited the kinase activity of Lyn by 222

Dasatinib, a pan-Src family inhibitor 29. Dasatinib did not interfere with the interaction 223

between ROR1 and Lyn WT or Lyn KA, however, it did interfere in the phosphorylation of 224

ROR1 as seen by the substantial decrease of tyrosine phosphorylation on ROR1 (Fig. 2c). 225

226

Identification and validation of ROR1 tyrosine residues phosphorylated by Lyn 227

In order to identify ROR1 residues that are phosphorylated by Lyn, we immunoprecipitated 228

ROR1 in presence and absence of Lyn from HEK-293T cells and subjected them to mass-229

spectrometry analysis of phosphorylation(s). The experimental design is schematized in Fig. 230

3a. Proteomic analysis detected phosphorylated tyrosines only when Lyn was co-expressed. 231

In total 6 tyrosine residues - Y459, Y645, Y646, Y666, Y828, and Y836 - were found 232

phosphorylated exclusively in the presence of Lyn (Fig. 3b). Three residues are part of 233

ROR1 tyrosine kinase domain (TKD) and two of those – Y645 and Y646 overlap with the 234

residues reported to be phosphorylated by Src 20. In order to decipher which of those 235

residues are functionally important, we generated ROR1 individual point mutants as well as 236

Y645/Y646 double mutant (schematized in Fig. 3c). Even though all tested mutants showed 237

decreased levels of phosphorylation, the double Y645/Y646 was clearly the most deficient 238

(Fig 3d). This suggests that Y645 and Y646 are the most critical residues phosphorylated by 239

Lyn. 240

241

Lyn-induced phosphorylation of ROR1 induces recruitment of E3 ligase c-CBL 242

Phosphorylation at tyrosines is a well described signaling event with various functional 243

consequences 30. Often, phosphorylated tyrosines serve as molecular motifs recognized by 244

downstream proteins containing SH2 domain. We thus hypothesized that ROR1 245

phosphorylation by Lyn will lead to the recruitment of further signal regulators. In order to 246

address this question, we decided to identify ROR1 interaction partners induced by Lyn 247

phosphorylation using unbiased immunoprecipitation coupled to mass spectrometry (IP/MS). 248

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10

We overexpressed ROR1 either alone or with WT or Lyn KD; pcDNA and Lyn WT-only 249

transfected cells served as a control. Design of the experiment is schematized in Fig. 4a. 250

IP/MS analysis identified 13 proteins that were uniquely detected as binding partners of 251

ROR1 phosphorylated by Lyn (Fig. 4b). Among the hits (Fig. 4c), c-Casitas B lineage 252

lymphoma (c-CBL) protein attracted our attention. c-CBL is an E3 ligase that recognizes pY 253

motifs31 and often downregulates its targets by triggering them for degradation 32 or 254

endocytosis 33. Of note, it is a known binding partner of Lyn 32, as well as its substrate 34. 255

We overexpressed the combinations of c-CBL, ROR1 and Lyn plasmids in HEK-293T cells 256

and performed a set of immunoprecipitation assays. In line with the mass spectrometry data, 257

c-CBL efficiently interacted with ROR1 only when WT Lyn was present. The interaction 258

between ROR1 and c-CBL was dependent on the Lyn-mediated phosphorylation of ROR1 259

since in the presence of the Lyn KD the binding to ROR1 was reduced (Fig. 4d, IP ROR1, 260

WB V5, lanes 6 vs. 8). Lyn was a part of the complex since it was pulled down both by 261

ROR1 and c-CBL (Fig. 4d, IP ROR1 & IP V5). Of note, co-expression of c-CBL clearly 262

attenuated the phosphorylation of ROR1 by Lyn and level of active Lyn itself (Fig. 4d, IP 263

ROR1 and IP pY, WB ROR1, Lyn and pY). Altogether, this data opens the possibility that the 264

consequence of phosphorylation-induced recruitment of c-CBL is the inactivation of the 265

phosphorylated ROR1, similar to a described c-CBL function in other RTKs targeted by c-266

CBL 31. 267

268

Lyn KO cells display increased surface levels of ROR1 269

Our findings reported in Figs. 1 – 4 showed that Lyn can efficiently phosphorylate ROR1 that 270

can be subsequently recognized by c-CBL. ROR1 and Lyn are important regulators of 271

signaling pathways driving chronic lymphocytic leukemia (CLL) 1,8,21,35 and several 272

lymphomas, namely mantle cell lymphoma (MCL) 15. Both, Lyn and ROR1, have been 273

evaluated as therapeutic targets in these malignancies and the understanding of their mutual 274

cross talk is thus of direct therapeutic relevance. In order to find out if Lyn controls ROR1 275

biology in CLL/MCL we decided to generate Lyn-deficient Maver-1 cells. Maver-136 are of 276

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11

MCL origin, express high levels of ROR1 and Lyn, and respond well to BCR activation 37. 277

We produced Maver-1 Lyn knockout (KO) cells using the Crispr-Cas9 system. We validated 278

four independent clones of Maver-1 Lyn KO cells by western blotting (Fig. 5a) and 279

sequencing (Supplementary Table 1). Functionally, Lyn KO Maver-1 cells are deficient in the 280

activation of BCR signaling triggered by IgM. As shown in Fig. 5b, activation of several BCR 281

components downstream of Lyn, namely Syk, PLCγ, PI3K, as well as phosphorylation of Lyn 282

substrate HS1 was dramatically reduced in Lyn KO cells. These observations are in line with 283

what is already known about the role of Lyn in the BCR signaling cascade 18. 284

After this initial characterization of the BCR signaling deficiency in Lyn KO cells we focused 285

on their ROR1 status. As shown in Fig. 5c, global ROR1 protein levels determined by 286

Western blotting were comparable in WT and Lyn KO cells, despite the fact that ROR1 287

mRNA expression, determined by qPCR, was slightly lower in the Lyn KO cells (Fig. 5d). 288

However, when we assessed cell surface ROR1 by flow cytometry we could observe a 289

consistent increase in surface ROR1 in all four clones (Fig. 5e). This suggested that 290

endogenous Lyn controls ROR1 trafficking or endocytosis to reduce ROR1 availability on the 291

surface. 292

ROR1 and its ligand Wnt-5a were shown to control CLL cell migration and chemotaxis 38,39. 293

More specifically, Wnt-5a-ROR1 axis increases basal migration and reduces chemotaxis 38. 294

Surprisingly, Lyn KO Maver-1 cells showed not only increased cell surface ROR1 (Fig. 5e) 295

but also much lower levels of CCR7 (Fig. 5f), a receptor for chemokine CCL19 and essential 296

component of CCL19-induced chemotaxis 40. Despite some variability in the individual Lyn 297

KO clones, the observed trends showing higher ROR1 and lower CCR7 were always the 298

same (Fig. 5 e/f, histograms in the bottom part of the panel). 299

To address whether the changes in ROR1 and CCR7 levels translate into the changes in the 300

migratory behavior of WT and Lyn KO Maver-1 cells we used transwell assays without 301

(basal motility) or with CCL19 in the bottom chamber (chemotaxis). Lyn KO cells had 302

significantly higher basal motility (Fig. 5g, i). This feature was preserved across individual 303

Lyn KO clones and reflected variability of the cell surface ROR1 (Fig. 5g, ii) and the cell 304

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surface CCR7 (Fig. 5g, iii). On the other hand, Lyn KO cells showed reduced chemotaxis to 305

CCL19 (Fig. 5h, i) that correlated negatively with cell surface ROR1 (Fig. 5h, ii) and 306

positively with the cell surface CCR7 (Fig. 5h, iii). Altogether, the analysis of the migratory 307

properties of Lyn KO Maver-1 cells suggested that Lyn, via control of cell surface levels of 308

ROR1 and CCR7, controls the migratory modes of lymphoid cells. Specifically, it suggests 309

that Lyn controls a balance between two migration modes – Wnt-5a/ROR1-mediated basal 310

migration (attenuated by Lyn) and CCL19/CCR7-mediated chemotaxis (promoted by Lyn). 311

312

Cell surface ROR1 is upregulated during CLL migration 313

Functional experiments with Lyn KO cells indicate that cell surface ROR1 can be under a 314

dynamic control during cell migration. In order to test whether this applies also to primary 315

CLL samples we performed a set of experiments in the patient cohort specified in Fig. 6a. 316

Cells from these patients were analyzed by Western blotting by probing for phospho-Y396-317

Lyn, which is auto-phosphorylated and as such represents a good hallmark for Lyn 318

activation (Fig. 6b). p-Lyn levels positively correlated with the phosphorylation of HS1 on 319

tyrosine 397, which is a well described target of Lyn 41 (Fig. 6c). On the other hand, ROR1 320

levels assessed by Western blotting did not correlate with active Lyn (Fig. 6d). 321

Chemotactic properties of CLL cells were analyzed in transwell assays as the migratory 322

response to the chemokine CCL19. In parallel, we analyzed the surface levels of ROR1 and 323

CCR7 in the non-migratory (upper chamber) and migratory (lower chamber) CLL cells (for 324

schematics see Fig. 6e). In some cases, because of low number of migrating cells we were 325

unable to assess in parallel all the functional parameters (migration, CCR7 and ROR1 levels 326

in lower and upper chamber, pLyn levels; see Supplementary Table 2 for details) and as 327

such the number of samples in individual analyses presented below slightly varies. 328

Surprisingly, ROR1 surface expression was clearly increased in the cells that passed 329

through the transwell membrane (Fig. 6f). No such increase was observed for CCR7, which 330

is a CCL19 receptor (Fig. 6g). Interestingly, the CLL samples that upregulated ROR1 most 331

efficiently, were the least chemotactic (Fig. 6h). CCR7 showed an opposite behavior to 332

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ROR1 and CCR7 levels correlated positively with the chemotaxis (Fig. 6i). This data shows 333

that even in primary CLL cells, similar to Maver-1 cells, ROR1 surface levels are dynamically 334

regulated during cell migration and that ROR1 and CCR7 show opposite behavior, in this 335

respect. 336

It remained to be analyzed whether the balance between ROR1-driven and CCR7-controlled 337

migration can be correlated with the activity of Lyn in primary CLL cells. pLyn-high CLL 338

samples (based on Fig. 6b), were in general more chemotactic (Fig. 6j), which is in line with 339

the positive role of Lyn in chemotaxis identified in Maver-1 cells. On the other hand, pLyn 340

levels negatively correlated with the capacity of CLL cells to upregulate ROR1 during 341

migration (Fig. 6k). Altogether, this suggests that the function of Lyn that switches between 342

ROR1- and CCR7-mediated migratory modes, uncovered in Maver-1 cells, is conserved 343

also in primary CLL cells. 344

345

Discussion 346

A significant amount of research and preclinical development is being conducted on 347

developing monoclonal antibodies to target surface ROR1 in CLL and other malignancies 348

11,42,43. Also, a considerable body of work has helped us to understand signaling on the 349

extracellular side of ROR1 via Wnt5a in CLL cells 39,44. However, significant gaps of 350

knowledge remain in our understanding of the importance of the intracellular domains of 351

ROR1, especially the TKD, as well as of the regulation of ROR1 levels on the cell surface. 352

Our study is the first to show that the Src family kinase Lyn, an important component of BCR 353

signaling, phosphorylates ROR1 intracellularly and controls its surface levels. 354

The phosphorylation of ROR1 by Lyn identifies a novel crosstalk between ROR1 and 355

BCR signaling. This crosstalk can be of particular importance in CLL and MCL where both, 356

ROR1 and BCR pathways, represent therapeutic targets. It has been shown earlier that in 357

these malignancies the non-canonical Wnt pathway and BCR signaling can be targeted in a 358

combinatorial manner. Namely, it has been observed in vivo in the mouse models of CLL 359

where BTK inhibitor ibrutinib and anti-ROR1 antibody45 or casein kinase 1 (CK1) inhibitors 46 360

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showed synergistic effects. Similar behavior has been observed by Karvonen and 361

colleagues in the in vitro model of MCL 15. Our data suggest that active BCR (correlating 362

with high Lyn activity) negatively controls the surface ROR1. This is an interesting 363

observation in the context of the recent report showing that in MCL ROR1/CD19 membrane 364

complex can functionally compensate for BCR/BTK activity and activate pro-survival and 365

pro-proliferative PI3K-Akt and MEK-Erk cascades 17. Lyn action towards ROR1 can explain 366

how BCR inhibited cells with low Lyn activation “switch” to the survival mode dependent on 367

ROR1. In addition, study by Zhang et al. opens the possibility that ROR1 and BCR-centered 368

complexes in MCL and CLL share even more components than Lyn described in this study. 369

Our analysis of Lyn KO Maver-1 cells has identified different migratory properties in 370

comparison to WT cells. Lyn KO cells had higher motility in the absence of external stimuli, a 371

feature correlating with the increased cell surface ROR1, but failed to respond to 372

physiological chemotactic stimulus CCL19, a feature correlating with the decrease in CCR7, 373

a CCL19 receptor. Of interest, similar behavior – i.e. deregulated motility and decreased 374

chemotaxis - was described for aggressive CLL characterized by high Wnt5a expression 38. 375

This opens the possibility that basal motility, promoted by Wnt5a/ROR1, and chemotaxis 376

represent distinct migratory modes that are coordinated by Lyn activity. In vivo phenotype of 377

Lyn KO leukemic lymphocytes in the TCL1 mouse model, which were more efficient in the 378

spleen infiltration than wt CLL cells 35 supports this view. 379

We demonstrate that at least one consequence of Lyn-induced ROR1 380

phosphorylation is the recruitment of c-Cbl. c-Cbl, a member of a family of RING finger E3 381

ligases, has been shown to be upregulated in CLL 47. Out of three 3 different family 382

members - Cbl (a.k.a c-Cbl or RNF55), Cbl-b (RNF56) and Cbl-c (RNF57), Cbl and Cbl-b 383

are known to be highly expressed in B and T lymphocytes. It is tempting to speculate that 384

Lyn activity towards ROR1-induced migration is not limited to the regulation of the interaction 385

with c-CBL but includes also action towards other cytoskeletal modulators of migration such 386

as HS1 and cortactin. Both these proteins serve in CLL cells as substrates of Lyn 41,48 and at 387

the same time were found to dynamically interact with ROR1 and control ROR1-induced 388

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migration 44,49. HS1-deficient leukemic cells in the mouse model of CLL are more aggressive 389

compared to Lyn wt mainly due to preferential homing to bone marrow 50 – the molecular 390

mechanism is not known but loss of Lyn capacity to control Wnt5a-driven migration is one 391

possible explanation. 392

In addition to Lyn, ROR1 has been shown to get phosphorylated also by other TK’s, 393

namely two receptor TK (RTK)s - Met and Src20, and MuSK51. In the study by Gentile et al 20, 394

it was shown that ROR1 is first phosphorylated by Met kinase in its PRD and this helps 395

recruit Src which then leads to the phosphorylation of ROR1 in the kinase domain. It remains 396

to be tested whether some membrane associated TKs, such as Axl 52 or ZAP70 53 can 397

synergize with Lyn in the regulation of ROR1. 398

In summary, our study is the first to show the interaction between ROR1, important 399

BCR kinase Lyn and c-Cbl. Our work also provides a molecular mechanism of the crosstalk 400

for two signaling pathways essential for CLL: BCR signaling and the non-canonical Wnt 401

pathway. This crosstalk mechanism provides a basis for the rational combinational therapies 402

targeting BCR and non-canonical Wnt in CLL and MCL. 403

404

Acknowledgement 405

VB, PK and ZZ gratefully acknowledge the support of the Czech Science Foundation 406

(the projects GA17-09525S, 17-16680S, GA19-20123S). ZD was supported by the 407

European Union Grant FP7 Marie Curie ITN 608180 ‘Wntsapp’. CIISB (LM2018127) and 408

NCMG (LM2015091) research infrastructures funded by MEYS CR are acknowledged for 409

the financial support of the measurements at the Proteomics and Genomics Core Facilities. 410

Further supported by Ministry of Education, Youth and Sports of the Czech Republic 411

(National Program of Sustainability II projects LQ1605 and LQ1601), by the Ministry of 412

Health of the Czech Republic (FNBr 65269705) and by European Structural and Investment 413

Funds, Operational Programme Research, Development and Education – "Preclinical 414

Progression of New Organic Compounds with Targeted Biological Activity” (PreclinProgress; 415

CZ.02.1.01/0.0/0.0/16_025/0007381). 416

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417

Conflict of interest 418

Authors declare that they have no conflict of interest. 419

420

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53. Dürig J, Nückel H, Cremer M, et al. ZAP-70 expression is a prognostic factor in 583

chronic lymphocytic leukemia. Leukemia. 2003;17(12):2426-2434. 584

doi:10.1038/sj.leu.2403147 585

586

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23

587

Figure legends 588

Figure 1: Lyn interacts with ROR1. a, b) Lyn and ROR1 were overexpressed in HEK-293T 589

cells. co-IP and Western blot analysis showing a pull-down of ROR1 when the lysates were 590

immunoprecipitated with Lyn (a) and a pull-down of Lyn when the lysates were 591

immunoprecipitated with ROR1 (b). c) Representative images of immuno-cytochemistry 592

analysis of HEK-293T cells overexpressing ROR1 (green) and Lyn (red) in the indicated 593

combinations. Co-localization of ROR1 and Lyn is observed at the membrane. Scale bar 7.5 594

μm. d) Scheme of ROR1 mutants used for domain mapping. e) Lyn was co-expressed with 595

the ROR1 intracellular deletion mutants in WT HEK-293T cells. Immunoprecipitation was 596

done using ROR1 as the bait. WB – Western blotting, IP – immunoprecipitation, TCL – total 597

cell lysate. Results in all panels are representative of at least 3 biological replicates. 598

599

Figure 2: Lyn phosphorylates ROR1. Indicated combinations of Lyn and ROR1 plasmids 600

were overexpressed in HEK-293T cells. ROR1 was immunoprecipitated and the binding of 601

Lyn and Y phosphorylation was assessed by Western blotting. a) General scheme of the Lyn 602

mutants used in b/c. b) Only the Lyn mutants with the intact kinase activity were able to 603

phosphorylate ROR1 on its tyrosine residues. c) Small molecule inhibitor of Lyn, Dasatinib 604

(Das, 0.2 µM), did not affect the interaction of ROR1 and Lyn, however it did block the ability 605

of Lyn to phosphorylate ROR1. WB – Western blotting, IP – immunoprecipitation, TCL – total 606

cell lysate. Results in all panels are representative of at least 3 biological replicates. 607

608

Figure 3: Mapping of the ROR1 residues phosphorylated by Lyn 609

a) Scheme of the experimental set-up for mass-spec analysis of ROR1 phosphorylation. 610

Indicated combinations were transfected in HEK-293T cells. ROR1 was immunoprecipitated, 611

separated on SDS-PAGE and bands corresponding to ROR1 were analyzed by MS/MS. b) 612

ROR1 tyrosine (Y) residues that were found phosphorylated only when Lyn was 613

coexpressed as identified by MS/MS analysis. The phospho-peptide signal intensity (up) and 614

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24

the position (bottom) of each detected phospho-tyrosine is presented. c) Schematics of the 615

point mutants of ROR1 made for the validation experiments. d) Phosphorylation analysis of 616

the point mutants showed that Y645/646F ROR1 mutation almost completely eliminated the 617

phosphorylation by Lyn. WB – Western blotting, IP – immunoprecipitation, TCL – total cell 618

lysate. Results in d are representative of at least 3 biological replicates. 619

620

Figure 4: Lyn induced phosphorylation of ROR1 triggers interaction with the E3 ligase 621

c-CBL 622

a) Scheme of the experimental set-up for the analysis of ROR1 interacting partners by 623

MS/MS. ROR1 and Lyn WT and Lyn KO were overexpressed in HEK-293T. ROR1 was 624

immunoprecipitated and the protein composition of the pulldown was analyzed by MS/MS. 625

b) Upset plot demonstrating the numbers of proteins identified as ROR1 interactors in 626

ROR1, ROR1+WT Lyn and ROR1+Lyn kinase dead (KD) conditions. Only proteins absent in 627

the control pulldowns (pcDNA and Lyn expression) were considered. c) List of ROR1 628

interactors identified only when phosphorylated by Lyn. d) Analysis of the interactions and 629

phosphorylation status of ROR1 and c-CBL. Indicated combinations were overexpressed in 630

HEK-293T, immunoprecipitated (IP) as indicated and subsequently analysed by WB. Lyn 631

promotes the interaction of ROR1 with c-Cbl. WB – Western blotting, IP – 632

immunoprecipitation, TCL – total cell lysate. Results in d are representative of at least 3 633

biological replicates. 634

635

Figure 5: Lyn KO cells display increased surface levels of ROR1 636

Lyn gene in Maver-1 cells was targeted by Crispr/Cas9. Parental WT and four clones of Lyn 637

KO cells coded #1E3, #1C2, #1E10, and #2F5 were analyzed functionally. a) Western blot 638

analysis of expression of Lyn in wt and KO cells. b) Activation of BCR signaling after 639

incubation with IgM antibody in WT and Lyn KO (#1E3) cells was assessed by Western 640

blotting of pPLCγ, pSYK, pLyn, pHS1, pPI3K and Lyn. GAPDH was used as a loading 641

control. c) Expression of ROR1 protein in WT and Lyn KO clones was analyzed by Western 642

.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted May 31, 2020. . https://doi.org/10.1101/2020.05.29.124156doi: bioRxiv preprint

25

blotting. β-actin was used as a loading control. Representative blots from 3 independent 643

experiments. d) Expression of ROR1 mRNA in WT and Lyn KO cells. Graphs show mean ± 644

SD of 3 independent experiments. Differences were analyzed by Kruskal-Wallis test (Dunn’s 645

multiple comparison test). e, f) Basal surface expression of ROR1 (e) and CCR7 (f) in 646

Maver-1 WT and individual clones of Lyn KO cells was analyzed by flow cytometry. Mean ± 647

SD from 3 independent experiments and Kruskal-Wallis test (Dunn’s multiple comparison 648

test). Representative histograms for all analyzed clones are shown. g, h) Basal motility (g) 649

or chemotaxis towards CCL19 (h) of Maver wt and Lyn KO cells was analyzed in a transwell 650

assay. Number of cells that migrated to lower chamber of transwell plate after 3 hours of 651

incubation is indicated. Panel i shows mean ± SD from 6 independent experiments for WT 652

and KO clone #1E3, Mann-Whitney nonparametric test. Other panels show variability among 653

4 different clones; migratory properties are plotted with the surface expression of ROR1 654

(panel ii) and CCR7 (panel iii) in Maver-1 WT and Lyn KO cells. 655

656

Figure 6: Correlation of Lyn activity with the chemotactic properties of primary CLL 657

cells 658

a) Table with basic clinical characteristics of the patient cohort used for functional analysis of 659

the primary CLL cells. Rai stage at the time of sampling, IGHV status and cytogenetic 660

analysis are indicated (n=13). b) Protein levels of ROR1, pLyn, Lyn and pHS1 expression in 661

the panel of CLL primary cells was analyzed by Western blotting. c, d) Correlation of pLyn 662

expression with pHS1 (c) and ROR1 (d). e) Scheme of the transwell assay indicating the 663

upper and lower compartment that was used for the separate analysis of the surface 664

markers. f,g) Surface expression of ROR1 (f) and CCR7 (g) in the upper (●) and lower (●) 665

compartments of transwell chamber in the panel of primary CLL cells was analyzed by flow-666

cytometry. Wilcoxon matched pairs signed test (n=9). h, i) Correlation of the change in the 667

surface expression of ROR1 (h) and CCR7 (i) during migration represented as the ratio of 668

receptor levels in the lower:upper compartment with the chemotactic properties of cells 669

(expressed as the number of cells in the lower chamber) in the panel of primary CLL cells. j) 670

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26

Correlation of the chemotaxis towards CCL19 with pLyn levels (WB). k) Correlation of ROR1 671

surface levels dynamics (expressed as the ratio of ROR1 surface levels in the lower:upper 672

well of transwell) with pLyn levels (WB). h-k) Pearson correlation coefficient. 673

674

.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted May 31, 2020. . https://doi.org/10.1101/2020.05.29.124156doi: bioRxiv preprint

ROR1

Figure 1

ROR1 + - + + - +Lyn - + + - + +

TCLIP Lyn

WB: ROR1

WB: Lyn

a. b.

c.

d.

ROR1 WT

Extra cellular domain Intra cellular domain

TM domain Kinase domain

ROR1 ΔCyto

ROR1 ΔTail

e.

TCL IP ROR1

ROR1 + - + + - +Lyn - + + - + +

WB: ROR1

WB: Lyn

130

55

IP ROR1

WB: ROR1

WB: Lyn

TCL

ROR1 - + Lyn + - - - + + + + - - - + + +

kDa kDa

aa 1-937

aa 1-750

aa 1-443

Δ Cy

to

Δ Ta

il

+ Δ Cy

to

Δ Ta

il

- + Δ Cy

to

Δ Ta

il

+ Δ Cy

to

Δ Ta

il

130

55

50

13080

55

ROR1

ROR1 Lyn

Lyn

Lyn

DRAQ5

DRAQ5

DRAQ5

ROR1 + Lyn

ROR1 + Lyn

ROR1 + Lyn

kDa

ROR1

Lyn

ROR1

+ Ly

n.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under a

The copyright holder for this preprint (whichthis version posted May 31, 2020. . https://doi.org/10.1101/2020.05.29.124156doi: bioRxiv preprint

SH4

a.

Figure 2

Wild type (aa 1-512)

Kinase active (aa 1-506; KA)

Kinase deleted (aa 1-298; Δ)

Kinase dead (aa 1-506; K275A; KD)

HA tag

WB: pY

WB: ROR1

Das

IP V

5

WB: Lyn

WB: ROR1

TCL

WB: Lyn

TKD

K275M

Phosphorylation

WB: Lyn

WB: ROR1

WB: pY

IP R

OR1

TCL

b.

WB: ROR1

130

55

55

130

55

130

130

130

c.

WB: Lyn

ROR1 + - - - - + + + +

Lyn - Wt Δ KA KD + Δ KA KD

55

35

35

ROR1-v5 Lyn

+ - - + + + +

- + KA + KA + KA

SH3 SH2

P

SH4 SH3 SH2

SH4 SH3 SH2

SH4 SH3 SH2

kDa

130

kDa

P

P

P

Lyn

.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted May 31, 2020. . https://doi.org/10.1101/2020.05.29.124156doi: bioRxiv preprint

a.

Figure 3

b.

d.pcDNA ROR1 ROR1 + Lyn

Transfection

Immunoprecipitation

Coommassie staining Of gel

WB: pY

WB: ROR1IP R

OR1

Mass-spec analysis

TCL

WB: ROR1

pcDNA ROR1 ROR1+Lyn

WB: pY

WB: pLyn

WB: ROR1

WB: Lyn

WB: pY

WB: pLyn

WB: ROR1

WB: Lyn

IP R

OR1

IP Ly

nWB: ROR1

WB: Lyn

TCL

55

55

130

130

130

130

55

55

ROR1Lyn - - - - - + + + + + +

130

55

+ Y641

F

Y645

F

Y646

F

Y645

/646

F

- + Y641

F

Y645

F

Y646

F

Y645

/646

F

kDa

WT ROR1 SREIYSADYYRVQSK

ROR1 Y641F SREIFSADYYRVQSK

ROR1 Y645F SREIYSADFYRVQSK

ROR1 Y646F SREIYSADYFRVQSK

ROR1 Y645/646F SREIYSADFFRVQSK

641

645

646

c.

ROR1 WT

.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted May 31, 2020. . https://doi.org/10.1101/2020.05.29.124156doi: bioRxiv preprint

a.

pcDNA ROR1 Lyn

ROR1+

Lyn

Mass-spec analysis

IP ROR1

Figure 4

b.

WB: pY

WB: V5

WB: Lyn

WB: pLyn

WB: ROR1

d.

IP V

5 WB: ROR1

WB: Lyn

WB: v5

IP p

Y

WB: c-Cbl

WB: Lyn

WB: ROR1

ROR1 + - - + + + + +

Lyn - + - + - + KD KD

c-Cbl-v5 - - + - + + - +

IP R

OR1

TCL

WB: ROR1

WB: Lyn

WB: c-Cbl

130kDa

55

120

55

130

130

55

120

130

55

120

130

55

120

ID Name Full name

Q16850 CYP51A1 Lanosterol 14-alpha demethylase

O14641 DVL2 Segment polarity protein dishevelled homolog DVL-2

Q8N4V1 MMGT1 Membrane magnesium transporter 1

Q8WVC6 DCAKD Dephospho-CoA kinase domain-containing protein

P01860 IGHG3 Immunoglobulin heavy constant gamma 3

Q9ULX6 AKAP8L A-kinase anchor protein 8-like

H3BRN7 CLN6 Ceroid-lipofuscinosisneuronal protein 6

Q9NR12 PDLIM7 PDZ and LIM domain protein 7

Q8TC12 RDH11 Retinol dehydrogenase 11

Q15392 DHCR24 Delta(24)-sterol reductase

Q9BSJ8 ESYT1 Extended synaptotagmin-1

P22681 CBL E3 ubiquitin-protein ligase CBL

P30876 POLR2B DNA-directed RNA polymerase II subunit RPB2

c.

WB: pY

WB: ROR1

WB: Lyn

WB: ROR1

WB: Lyn

IP R

OR1

TCL

ROR1+

Lyn KD

.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted May 31, 2020. . https://doi.org/10.1101/2020.05.29.124156doi: bioRxiv preprint

ROR1

wt#1

E3#1

C2#1

E10#2

F50.0

0.2

0.4

0.6

0.8

1.0

1.2 **

LYN KO

Figure 5a.

e. f.

WT

#1C2

#1E3

#1E1

0

#2F5

WB: Lyn

WB: β-ACTIN

55

c.

WB: ROR1

WB: Lyn55

130

WT

#1C2

#1E3

#1E1

0

#2F5

b.

WB: pLyn

WB: Lyn

WT Lyn KO #1E3

WB: GAPDH

WB: PPLCγ

WB: pSYK

WB: pHS1

WB: pPI3K55

kDa

155

72

80

56

56

37

g.

h.

d.

WB: β-ACTIN

kDa kDa

ii.

surfa

ce C

CR7

(fold

of w

t)

iii.i.

ii. iii.i.

cell

coun

t

wt

LYN KO #1E3

Lyn KO Lyn KO .CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted May 31, 2020. . https://doi.org/10.1101/2020.05.29.124156doi: bioRxiv preprint

Figure 6

a

e.

# SexRai

stage IGHV Cytogenetics1 M III mut ∆11q2 M IV unmut ∆13q3 M II unmut normal karyotype4 F I unmut ∆13q5 M I unmut ∆13q6 M II unmut ∆13q7 M IV unmut ∆13q8 M IV unmut ∆11q9 F III unmut tris12

10 F III mut tris12

11 F IV mut ∆11q

12 F III unmut tris1213 M II mut normal karyotype

f.

i.h.

RO

R1

low

er/u

pper

(MFI

)

g.

CCR

7 lo

wer

(MFI

)

c.

k.

d.

j.

pLyn (WB)0 5 10 15

0

50000

100000

2

3

5

6

9

10

1112 13

r=0.86p=0.0029

RO

R1

low

er/u

pper

(MFI

)

12987654321 1110 13

WB: ROR1

WB: pHS1

WB: pLyn

WB: Lyn

WB: GAPDH

kDa

130

70

55

55

35

uppercompartment

lowercompartment

uppercompartment

lowercompartment

CCL19

6 h

Chemotaxis (CCL19)

MFI

ROR1 upper

ROR1 lower

0

1000

2000

30000.0078

MFI

CCR7 upper

CCR7 lower

0.9375

b. .CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted May 31, 2020. . https://doi.org/10.1101/2020.05.29.124156doi: bioRxiv preprint

II

ORIGINAL RESEARCHpublished: 04 May 2017

doi: 10.3389/fcell.2017.00047

Frontiers in Cell and Developmental Biology | www.frontiersin.org 1 May 2017 | Volume 5 | Article 47

Edited by:

Gregory Kelly,

University of Western Ontario, Canada

Reviewed by:

Terry Van Raay,

University of Guelph, Canada

Ole-Morten Seternes,

University of Tromsø, Norway

*Correspondence:

Sigmar Stricker

sigmar.stricker@fu-berlin.de

Vitezslav Bryja

bryja@sci.muni.cz

Specialty section:

This article was submitted to

Signaling,

a section of the journal

Frontiers in Cell and Developmental

Biology

Received: 30 January 2017

Accepted: 13 April 2017

Published: 04 May 2017

Citation:

Bernatik O, Radaszkiewicz T, Behal M,

Dave Z, Witte F, Mahl A,

Cernohorsky NH, Krejci P, Stricker S

and Bryja V (2017) A Novel Role for

the BMP Antagonist Noggin in

Sensitizing Cells to

Non-canonical Wnt-5a/Ror2/

Disheveled Pathway Activation.

Front. Cell Dev. Biol. 5:47.

doi: 10.3389/fcell.2017.00047

A Novel Role for the BMP AntagonistNoggin in Sensitizing Cells toNon-canonical Wnt-5a/Ror2/Disheveled Pathway ActivationOndrej Bernatik 1, Tomasz Radaszkiewicz 1, Martin Behal 1, Zankruti Dave 1, Florian Witte 2,

Annika Mahl 2, Nicole H. Cernohorsky 3, Pavel Krejci 1, 3, Sigmar Stricker 2* and

Vitezslav Bryja 1, 4*

1 Faculty of Sciences, Institute of Experimental Biology, Masaryk University, Brno, Czechia, 2 Institute for Chemistry and

Biochemistry, Freie Universität Berlin, Berlin, Germany, 3Department of Biology, Faculty of Medicine, Masaryk University,

Brno, Czechia, 4Department of Cytokinetics, Institute of Biophysics AS CR, v.v.i., Brno, Czechia

Mammalian limb development is driven by the integrative input from several signaling

pathways; a failure to receive or a misinterpretation of these signals results in skeletal

defects. The brachydactylies, a group of overlapping inherited human hand malformation

syndromes, are mainly caused by mutations in BMP signaling pathway components.

Two closely related forms, Brachydactyly type B2 (BDB2) and BDB1 are caused by

mutations in the BMP antagonist Noggin (NOG) and the atypical receptor tyrosine kinase

ROR2 that acts as a receptor in the non-canonical Wnt pathway. Genetic analysis

of Nog and Ror2 functional interaction via crossing Noggin and Ror2 mutant mice

revealed a widening of skeletal elements in compound but not in any of the single

mutants, thus indicating genetic interaction. Since ROR2 is a non-canonical Wnt co-

receptor specific for Wnt-5a we speculated that this phenotype might be a result of

deregulated Wnt-5a signaling activation, which is known to be essential for limb skeletal

elements growth and patterning. We show that Noggin potentiates activation of the

Wnt-5a-Ror2-Disheveled (Dvl) pathway in mouse embryonic fibroblast (MEF) cells in a

Ror2-dependent fashion. Rat chondrosarcoma chondrocytes (RCS), however, are not

able to respond to Noggin in this fashion unless growth arrest is induced by FGF2. In

summary, our data demonstrate genetic interaction between Noggin and Ror2 and show

that Noggin can sensitize cells to Wnt-5a/Ror2-mediated non-canonical Wnt signaling,

a feature that in cartilage may depend on the presence of active FGF signaling. These

findings indicate an unappreciated function of Noggin that will help to understand BMP

and Wnt/PCP signaling pathway interactions.

Keywords: noggin, Wnt5a, non-canonical Wnt pathways, BMP signaling, brachydactyly, Ror2

Bernatik et al. Crosstalk of Noggin and Non-canonical Wnt Pathway

INTRODUCTION

Limb bud development and the concomitant formation oflimb skeletal structures are regulated by the intricate interplayand integration of various signaling pathways, with majorroles played by the Shh, BMP, FGF, and Wnt/β-cateninpathways (reviewed for example in, Robert, 2007; Zuniga,2015). The BMP signaling pathway is of pivotal importanceespecially for skeletal development. The analysis of inheritablehuman hand malformation syndromes has been instrumentalin understanding the contribution of BMP signaling andother pathways for skeletal development. One example arethe brachydactylies, a group of inheritable syndromes thatare characterized by shortening or absence of phalanges.Most brachydactyly subtypes are caused by mutations inBMP signaling components or factors that, at different levels,intersect with BMP signaling. Therefore brachydactylies havebeen interpreted in terms of a molecular disease family (Strickerand Mundlos, 2011). This hypothesis predicts that overlappingphenotypes are likely caused by mutations affecting componentsthat show a close functional interaction within a commonsignaling network.

Intriguingly, two closely related brachydactyly subtypes,BDB1 and BDB2, are caused by mutations in ROR2 or NOGGIN,respectively (Oldridge et al., 2000; Lehmann et al., 2007). WhileNOG is well known as a secreted BMP antagonist, ROR2 isan atypical receptor tyrosine kinase that is involved in theinhibition of Wnt/β-catenin signaling (Mikels and Nusse, 2006).In developing digits, Ror2-mediated Wnt/β-catenin inhibitionallows BMP-mediated digit outgrowth (Witte et al., 2010).In addition, Ror2 is a Wnt (co)receptor, mainly for Wnt-5a, acting in non-canonical Wnt signaling (Oishi et al., 2003;Schambony and Wedlich, 2007). Recently, activation of thenon-canonical Wnt/planar cell polarity (PCP) pathway by Wnt-5a and ROR2 was shown to be critically involved in theregulation of limb skeleton development (Gao et al., 2011;Wang et al., 2011; Ho et al., 2012; Kuss et al., 2014).Moreover, a separate set of mutations in ROR2 causes autosomalrecessive Robinow syndrome (RS), which is characterized bydiverse malformations including the axial and limb skeleton(Afzal et al., 2000; van Bokhoven et al., 2000). A dominantform of RS is caused by mutations in Wnt/PCP componentsDVL1, DVL3, and WNT-5A, it is therefore believed that thedevelopmental defects seen in Robinow syndrome are causedby a deregulation of Wnt-5a/Ror2/PCP signaling (Stricker et al.,2017).

The skeletal elements of the limbs are formed byendochondral ossification. In this process a cartilage templateis formed that mediates growth of the skeletal element andbecomes later replaced by bone. This process is dependent onthe formation of stacked columns of proliferating chondrocytesoriented perpendicular to the longitudinal axis of the growingskeletal element (Romereim and Dudley, 2011). Deregulation ofPCP signaling in proliferating chondrocytes leads to perturbationof column formation, and to arbitrary chondrocyte orientationthat ultimately leads to skeletal malformations typically resultingin a shortening and widening of the skeletal elements (Ahrens

et al., 2009; Li and Dudley, 2009; Kuss et al., 2014; Romereimet al., 2014).

Based on the close phenotypic overlap of humanbrachydactyly-causing mutations in ROR2 and NOG, wehypothesized that NOG may directly interact with the Wnt-5a/Ror2 pathway. We show here a subtle genetic interactionof Noggin with Ror2 during mouse limb development.Mechanistically, we provide evidence that Noggin can sensitizecells to Wnt/PCP pathway activation mediated by ROR2,providing first evidence for a yet uncharacterized level ofcross-talk between BMP and Wnt/PCP signaling.

MATERIALS AND METHODS

Mouse Lines and Phenotypical AnalysisRor2+/− (Takeuchi et al., 2000) and Nog+/− (McMahon et al.,1998) were maintained as heterozygous lines and intercrossedto yield compound mutants. Timed matings were set up andembryos were collected at E18.5. Skeletal preparations wereperformed as described previously (Mundlos, 2000). All animalprocedures were carried out in accordance with European Unionand German law. Animals were maintained in the SPF mousefacility of the Max Planck Institute for Molecular Genetics, Berlinunder license from the Landesamt für Gesundheit und Soziales(LAGeSo) under license numbers ZH120 and G0346/13.

Cell Culture and TreatmentsRor1−/− Ror2−/− mouse embryonic fibroblasts (MEF) werederived from Ror1 flox/flox Ror2 flox/flox MEF cells as describedpreviously (Ho et al., 2012). MEF and RCS cells were propagatedin DMEM, 10% FCS, 2 mM L-glutamine, 50 units/ml penicillin,and 50 units/ml streptomycin. RCS cells were seeded in 24-well plates, grown for 24 h and treated as indicated. Followingreagents: Wnt-5a (R&D systems, 645-WN-010), Noggin (R&DSystems, 1967-NG-025), FGF2 (5 ng/ml, R&D Systems) andWnt-C59 5 µM (Tocris Bioscience, 5148) were used fortreatment. Wnt-5a conditioned media was produced from LWnt-5a cells (ATCCCRL-2814) according to ATCC instructions.RCS cells intended for WB analysis were treated by FGF2 for48 h, then were treated by the porcupine inhibitor Wnt-C59 (toreduce background autocrine Wnt activity), Noggin and Wnt-5a in indicated doses for additional 24 h. Total time of FGF2treatment was 72 h.

Western BlottingLysates for western blotting were prepared as follows: Growthmedium was removed and cells were directly lysed in 100mMTris/HCl (pH 6.8), 20% glycerol, 1% SDS, 0.01% bromophenolblue and 1% 2-mercaptoethanol.Western blotting was performedaccording to manufacturer’s instructions with minor adjustments[SDS-PAGE run on 150 V, transfer onto PVDF membrane1 h on 100 V, both steps on ice (BIO-RAD)]. Antibodieswere from Santa Cruz Biotechnologies: anti-Dvl2 (dephospho-Dvl2)–sc8026, anti-beta-Actin–sc1615-R, anti-Dvl3 sc8027 andfrom Cell Signaling Technologies: anti-Dvl2–CS3224. Anti-Ror2antibody was a gift from Henry Ho (UC Davis) (Ho et al.,2012). Phosphorylation status of Dvl2 and Dvl3 was quantified

Frontiers in Cell and Developmental Biology | www.frontiersin.org 2 May 2017 | Volume 5 | Article 47

Bernatik et al. Crosstalk of Noggin and Non-canonical Wnt Pathway

by densitometric analysis of Western Blot in three independentreplicates using Fiji distribution of ImageJ software as described(Bernatik et al., 2014). For pDvl/Dvl rations the peak area forthe upper band representing P-Dvl was divided by the peak areaof the lower band (Dvl). Data was analyzed by paired t-test(GraphPad Prism).

Dual Luciferase AssayRCS cells were transfected using pRLtkLuc and Super8XTopFlash plasmid. 9µg Super8X TopFlash and 3 µg pRLtkLucplasmid were mixed with 38.4 µl of Fugene6 (E2691, Promega)in 1200 µl of DMEM. Cells were treated by 0.3% collagenasetype II (GIBCO, cat.no.17101015) before transfection, 50 µl oftransfection mixture and 500 µl of collagenase treated RCS cellsin DMEM were used per 1 well of 24 well plate. Transfectionwas carried out overnight, cells were treated according to theexperimental scheme for 20 h, and samples were processedby Dual-Luciferase R© Reporter Assay System according to themanufacturer instructions (Promega, E1960).

RESULTS

Noggin Genetically Interacts with Ror2To get a first indication whether Ror2 and Noggin mightfunctionally interact we generated compound mutants forRor2 and Noggin. Ror2+/− mice (Takeuchi et al., 2000)were crossed to Noggin+/− mice (Brunet et al., 1998;McMahon et al., 1998). Heterozygous inactivation of eitherRor2 or Noggin does not result in any skeletal alteration(Figure 1A). In Ror2+/−;Nog+/−compound heterozygotes theoverall appearance of the limb skeleton was normal; howeverthe skeletal elements of the stylopod (the humerus) and thezeugopod (radius and ulna) showed a consistent small lateralexpansion (Figure 1A, width of skeletal elements in wild type andsingle mutants indicated in yellow, width in compound mutantindicated in orange). All skeletal elements showed a tendencytoward widening at both metaphyseal sides, however statisticalsignificance was only reached for the distal humerus and radius,respectively. This feature was not seen in single heterozygotes,indicating genetic interaction between Nog and Ror2.

Ror2−/− mice are a model for RRS, recapitulating severalof its features including mesomelic limb shortening as well asmild brachydactyly (Schwabe et al., 2004). Ror2−/− mice haveshortened digits, however all phalanges (two in the thumb/digit 1,three in digits 2–5) as well as the interphalangeal joints separatingthe phalanges are present (Takeuchi et al., 2000; Schwabe et al.,2004; Schwarzer et al., 2009) (Figure 1B). Noggin heterozygousmice have phenotypically normal digits. When one allele ofNoggin was removed on the Ror2−/− background, shorteningof phalanges was further increased. In digit 3 the appearanceof 3 individual phalanges, which were smaller than those inthe Ror2−/−, was preserved. In digits 2 and 5 loss of oneNoggin allele on the Ror2−/− background led to loss of anindividual phalanx 2, concomitant with a longer phalanx 3,indicating failure of distal joint formation. Distal joint fusion isalso a feature seen sometimes in BDB1 (ROR2 mutation) andfrequently in BDB2 (NOG mutation). In addition, joint fusions

are the hallmark of proximal symphalangism 1A (SYM1A) andmultiple synostosis syndrome (SYSN1), two conditions causedby a different set of NOG mutations (Stricker and Mundlos,2011). Altogether the compound mutants support the notion ofa genetic and functional interaction of Ror2 and Nog in skeletaldevelopment.

Noggin Potentiates Wnt/PCP Signaling in aRor2-Dependent MannerIn digit formation, Ror2 acts in part via inhibition of β-cateninsignaling leading to derepression of BMP/SMAD signaling ina structure called phalanx-forming region (Witte et al., 2010).Evidence however has accumulated that in addition or in parallelto this function Ror2 and its paralog Ror1 are both required forWnt-5a/PCP signaling activation during digit development (Gaoet al., 2011; Ho et al., 2012). Our genetic interaction experimentscannot distinguish the origin of the interaction seen, i.e., whetherit originated from Nog function in the BMP pathway, or a yetuncharacterized role in the Wnt-5a/PCP pathway. Noggin thusmight not only influence activity of BMP, but also of Wnt-5a-Ror2 pathway. To test if Noggin is able to activate Ror2we treated mouse embryonal fibroblasts (MEF) with increasingdoses of Noggin. The activation of endogenous Ror2 can bemonitored as a phosphorylation-dependent mobility shift onWestern blotting (Oishi et al., 2003). As we show in Figure 2A,even in the highest concentrations used (1,500 ng/ml) Noggin didnot induce phosphorylation of Ror2 and was unable to promotephosphorylation of Ror2 induced by its cognate ligand Wnt-5a.This suggests that at the receptor level Noggin is unable to acteither directly as a ligand for Ror2, or indirectly.

In the next step we tested if Noggin can promote anyof the Ror2-downstream events. A robust readout of non-canonical Wnt pathways activation is the Wnt-5a-inducedphosphorylation of Disheveled (Dvl) 2, an event dependenton the Ror1 and Ror2 receptors (Ho et al., 2012). We tookadvantage of an anti-Dvl2 antibody that recognizes only theinactive, dephosphorylated form of Dvl2 in MEF cells (Gonzalez-Sancho et al., 2013). Disappearance of non-phosphorylatedDvl2 currently represents one of the most sensitive tools forvisualization of Dvl2 phosphorylation and hence Wnt/PCPpathway activation. When we treated MEF cells with increasingdoses of Wnt-5a, the non-phospho Dvl2 signal disappeared(Figure 2Bi), indicative of activated Wnt-5a-Ror-Dvl signaling.No such phenotype was observed when cells were treatedby Noggin, confirming our previous observation that Nogginitself is not able to activate signaling via Ror2 (Figure 2Bii).However, when cells were treated with 100 ng/ml of Noggin,we could clearly observe stronger effects of Wnt-5a on Dvl2activation (compare Figure 2Bi vs. Figure 2Biii). This indicatesthat Noggin can sensitizeMEF cells toWnt-5a/Ror2 signaling. Toconfirm this observation, we treated cells with 25 ng/ml of Wnt-5a, which is a suboptimal dose unable to trigger Dvl2 activation(Figure 2Bi). When cells pre-treated by 25 ng/ml ofWnt-5a weresupplemented with increasing doses of Noggin, activation ofDvl2 was observed in a dose dependent manner (Figure 2Biv),

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FIGURE 1 | Noggin genetically interacts with Ror2. Skeletal preparations of E18.5 embryos of the indicated allelic combinations are shown. Cartilage stains blue,

bone stains red. (A) Top panel: Limbs of compound Ror2 and Noggin heterozygous mutants have a normal appearance. Ror2−/− skeletal elements are visibly

shortened and enlarged. Bottom panel: magnifications of humerus and radius/ulna. The width of the wild type or single heterozygous skeletal elements is indicated by

a yellow line on either side of the ossification center. Width of the double heterozygous or Ror2−/− skeletal elements is indicated by orange line for comparison. A

quantification of skeletal element width is shown right; significant effects were observed for the distal humerus and distal radius (p < 0,05; student’s t-test). (B) Digit

development in compound mutants. Ror2−/− digits are shortened, but individual phalanges (p1, p2, and p3) are present, separated by synovial joints. In

Ror2−/−;Nog+/− animals, the medial phalange (p2) shows additional shortening, which in digits 2 and 5 leads to distal symphalangism of p2 and p3.

indicating that presence of Noggin can reveal biological activityof previously sub-threshold Wnt-5a concentrations.

All these data suggest that Noggin, despite its inability toactivate Ror2 on its own, can efficiently potentiate the Wnt-5a-Ror2 signaling axis and sensitize cells to low amounts ofWnt-5a. Ror2 can have redundant function with closely relatedRor1 (Ho et al., 2012) that can also bind Wnt-5a. To confirmthat the effects of Noggin are indeed dependent on Ror1/Ror2,Ror1−/− Ror2−/− double knockout MEF cells were isolatedfrom conditional Ror1/Ror2 knockout mice (as described in Hoet al., 2012). Individual clones were tested by Western blotting(Figure 2C) and one of the Ror1/Ror2 double negative clones(#13) was further used for functional analysis. When Ror1/Ror2-deficient MEF cells were treated with 30 ng/ml of Wnt-5a and500 ng/ml of Noggin simultaneously, no shift of Dvl2 mobility(upper panels) or effects on non-phospho Dvl2 (middle panel)was observed, in contrast to wt MEF where Dvl2 was activated by

the combination of Wnt-5a (30 ng/ml) and Noggin (500 ng/ml)(Figure 2D). This data show that Noggin is able to potentiate theactivation of the Wnt-5a-Ror2 signaling circuit and demonstratethat the observed Noggin/Wnt-5a synergism toward Dvl2 isdependent on Ror1/Ror2.

FGF2-Induced Chondrocyte Growth ArrestEnables Noggin-Mediated Wnt/PCPPotentiation in RCS CellsThe genetic interaction between Ror2 and Noggin observed inmice as well as the skeletal involvement in human syndromescharacterized by NOG and ROR2 mutations pointed towardthe importance of a functional Noggin-Ror2 interaction forskeletal development. To test the Noggin-Ror2 synergy in amodel system that is more relevant to skeletal developmentwe decided to use the rat chondrosarcoma (RCS) cell line.

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FIGURE 2 | Noggin potentiates Wnt/PCP signaling. (A) MEF cells were treated by Wnt-5a conditioned medium (CM) and stimulated by increasing doses of

Noggin protein. Activation of Ror2 was analyzed as a phosphorylation-dependent shift by Western blotting. Noggin alone, in contrast to Wnt-5a CM, is not able to

trigger activation of Ror2. (B) MEF cells were treated with increasing doses of Wnt-5a (0, 25, 50, 100, 150, and 200 ng/ml) and Noggin (0, 25, 50, 100, 150, and 200

ng/ml) for 2 h. The activation of Wnt signaling was assessed by Western blotting as a decrease in the signal of dephospho-Dvl2. Wnt-5a could cause phosphorylation

of Dvl2 visible as a disappearance of dephospho-Dvl2 signal (i), whereas Noggin is inactive in the same assay (ii). Interestingly, pre-treatment of MEF cells by Noggin

(100 ng/ml) enhanced the effect of Wnt-5a (iii). On the other hand, Noggin, in a dose-dependent manner, potentiated the response to suboptimal doses of Wnt-5a

(25 ng/ml), which are otherwise ineffective—see lane 2 in panel “i” (iv). Actin is used as a loading control. (C) Ror1flox/flox; Ror2flox/flox MEF cells were treated by

tamoxifen and Ror1−/−; Ror2 −/− isogenic MEF line was isolated by serial dilutions method. The presence of Ror2 was tested by Western blotting and the clone no.

13 used for further studies is indicated. (D) MEF wt and MEF Ror1−/−; Ror2−/− (Ror1/2 dKO) cells were treated by combinations of Noggin and Wnt-5a as

indicated. Noggin itself cannot stimulate activation of Dvl2—visible as a phosphorylation-dependent shift of Dvl2 (upper blots) or decrease in dephospho-Dvl2 (middle

blots) signal. Noggin, however, increases activity of suboptimal dose of Wnt-5a (30 ng/ml), an effect that is lost in Ror1−/− Ror2−/− MEF cells.

RCS chondrocytes maintain a fully differentiated proliferatingchondrocyte phenotype in culture, manifested by abundantproduction of cartilaginous extracellular matrix rich in sulfatedproteoglycans and collagen type 2, but not collagen type 10characteristic for hypertrophic chondrocytes (Mukhopadhyayet al., 1995). Moreover, RCS chondrocytes faithfully recapitulateFGF-receptor 3 (FGFR3) signaling in the growth plate cartilage.Many essential features of FGFR3 signaling in the growth platecartilage, such as the FGF-mediated chondrocyte growth-arresthave been unraveled using the RCS chondrocyte model system(Aikawa et al., 2001; Dailey et al., 2003; Krejci et al., 2005).

To define this experimental system, we first investigatedwhether the FGF-induced growth arrest in RCS cells is influencedby addition of Noggin andWnt-5a. Noggin, Wnt-5a and/or theircombination did not induce a growth arrest by themselves, andalso did not modulate the FGF-induced growth arrest of RCScells (Figure 3A). We also wanted to exclude that any possibleobservations in RCS cells are caused by modulation of canonicalWnt pathway that was shown to oppose Wnt/PCP pathway inchondrogenesis. Since it was shown that RCS cells are responsiveto canonical Wnt ligands, e.g., Wnt3a (Krejci et al., 2012), we

tested whether Noggin andWnt-5a treatment alters the canonicalWnt pathway in RCS cells using TopFlash reporter assay. Theseresults (Figure 3B) showed that Noggin and Wnt-5a could notactivate or inhibit the canonical Wnt pathway even though RCScells responded well to canonical Wnt ligands such as Wnt-3a(Figure 3B). We conclude that combined treatment of RCS cellswith Noggin/Wnt-5a does not influence FGF2 induced growtharrest or the canonical Wnt signaling pathway in RCS cells.

Finally, we analyzed whether RCS cells respond to combinedNoggin/Wnt-5a treatment similarly to MEF cells. As we couldnot detect any signal by using the dephospho-Dvl2 antibodyused in MEF cells (not shown), we have used an alternativereadout—electrophoretic mobility shift of Dvl induced by Wnt-5a. Such mobility shift indeed represents a phosphorylationand can be effectively abrogated by alkaline phosphatase (AP)treatment (Figure 3C). Using this readout we next tested whetherNoggin could potentiate the response to Wnt-5a in RCS cellssimilarly as was observed in MEF cells. When RCS cells weretreated by combination of Wnt-5a and Noggin, no potentiationof Wnt-5a-Ror2 signaling was observed (Figure 3D, quantifiedin Figure 3E), and only the highest dose of Wnt-5a triggered

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FIGURE 3 | Positive effects of Noggin on Wnt/PCP activation in chondrocytes is induced by co-stimulation with FGF2. RCS cells were treated by FGF2 to

induce growth arrest, and then treated by the porcupine inhibitor Wnt-C59 (5 µM) to reduce the autocrine Wnt activity and by Noggin and Wnt-5a as indicated.

Timepoints are specified in Materials and Methods section. (A) Wnt-5a, Noggin and their combination does not alleviate growth arrest of RCS cells induced by FGF2

(72 h), graph shows average and SD from two independent experiments, ***p < 0.001 [One-way ANOVA (ANalysis Of VAriance) with post-hoc Tukey test]. (B)

Treatment of RCS cells by FGF2, Noggin and Wnt-5a does not activate canonical Wnt pathway analyzed by TopFlash reporter system. Treatment with Wnt3a was

used as a positive control. RLU—relative light units, graph shows average and SD from two independent experiments, ***p < 0.001 [One-way ANOVA (ANalysis Of

VAriance) with post-hoc Tukey test]. (C) Alkaline phosphatase (AP) treatment can remove the electrophoretic mobility shift of Dvl3 induced by FGF2/Noggin/Wnt-5a

treatment, which suggests that the mobility changes (used in D–G) are caused by phosphorylation. (D) Wnt-5a can activate downstream signaling—visible as

phosphorylation-dependent shift (p-Dvl) of Dvl2 and Dvl3—at 50 ng/ml and this effect is not positively modulated by the addition of Noggin (125 ng/ml). (E)

Quantification of p-Dvl/Dvl ratios for Dvl2 and Dvl3 from three independent experiments. (F) Similar experiment as in (D) but RCS cells were pre-treated also by FGF2

(5 ng/ml) for total 72 h to induce FGFR3-mediated growth arrest. Under these conditions 24 h treatment by C59, Noggin (125 ng/ml) and Wnt-5a can dramatically

induce the Wnt-5a-induced activation of Dvl2 and Dvl3. (G) Quantification of three independent experiments. *p < 0.01 (paired t-test).

phosphorylation of Dvl2 and Dvl3. However, when RCS cellswere pre-treated with FGF2 for 2 days in order to inducegrowth arrest (Krejci et al., 2010), Noggin dramatically improvedthe response of RCS cells to low doses of Wnt-5a (Figure 3F,quantified in Figure 3G). Importantly, acute treatment of RCScells with FGF2, Noggin and Wnt-5a was unable to induce

such “sensitization” (data not shown). These data thus arguethat the synergism between Noggin and Wnt-5a-Ror2 is not aproximal effect of FGF2-induced signaling or an inhibition ofthe canonical branch of Wnt signaling but is rather induced bycell changes caused by prolonged FGF2 treatment and cell cyclearrest.

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Bernatik et al. Crosstalk of Noggin and Non-canonical Wnt Pathway

DISCUSSION

Signaling pathways do not operate as standalone units butfunctionally cooperate and interact. Inspired by the phenotypicresemblance of BDB1 and BDB2, inheritable syndromes causedby mutations in ROR2 or NOGGIN, respectively, we decidedto study how Noggin, an inhibitor of BMP pathway, and non-canonical Wnt signaling, driven by Ror2 receptor, can interact.We could show that Noggin increased biological activity ofWnt-5a and rendered cells sensitive to Wnt-5a concentrationsotherwise not causing cellular responses. This function wasdependent on the presence of Ror2, but Noggin did not elicit asignal on its own via Ror2.

Our study does not elucidate the molecular mechanismbehind this interaction. One mechanism may involve BMPreceptor type 1 b (Bmpr1b), which ismutated in BDA2 (Lehmannet al., 2003). In vitro, Ror2 and Bmpr1b were shown to interactand Ror2 is phosphorylated by Bmpr1b (Sammar et al., 2004,2009). The functional consequence of this phosphorylationremains unclear but one can speculate that the effects ofBmpr1b on Ror2 are controlled by BMP ligands, whose activeconcentration is controlled by Noggin. Another possibility,which we were, however, not able to prove (data not shown)can be formation of Noggin-Wnt-5a-Ror2 ternary complex withthe increased signaling capacity in comparison to Wnt-5a-Ror2only. As another alternative, Noggin can, via regulation of BMPpathway, control signaling competence or cell surface amountof Ror2—here a possible point of crosstalk can be representedby Smurf family E3-ligases, which were reported to controlboth BMP pathway (negatively) as well as Wnt/PCP pathway(positively) (Narimatsu et al., 2009).

The importance of the BMP pathway and its tight regulationby antagonists for digit development is underscored by thefact that the majority of human brachydactylies are causedby mutations in different members of this signaling network(reviewed in Stricker and Mundlos, 2011). A necessity forintegration of BMP and Wnt/β-catenin pathways has beenreported for numerous developmental processes (Itasaki andHoppler, 2010). For example, in digit outgrowth, BMP/SMADsignaling is fine-tuned by inhibition from the Wnt/β-cateninpathway, which itself is kept in check by Ror2 (Witte et al.,2010). Non-canonical (or alternative) Wnt pathways regulateentirely different aspects of tissue development compared tothe Wnt/β-catenin pathway, but are connected with the BMPpathway as well, albeit the connection has not been studied tothe same depth (Narimatsu et al., 2009; Schille et al., 2016).In developing limbs, Wnt/PCP signaling was involved in bothdigit shaping and outgrowth (Gao et al., 2011; Wang et al.,2011; Ho et al., 2012). Altogether this substantiates that bothBMP and non-canonical Wnt pathways are required and actin concert during the establishment of the limb skeleton. Ror2appears to be a pivotal intersection point between these twopathways.

Our work on RCS chondrocytes, a cell model for chondrocyte

growth and differentiation that to some extent recapitulatethe behavior of developing limb growth plate cartilage (Krejciet al., 2012) showed that Noggin could potentiate Wnt-5a-Ror2

pathway activity much more effectively when growth arrest

was induced by FGF2 stimulation. It was previously shownin RCS chondrocytes that the FGF pathway can stimulatephosphorylation of LRP6, a co-receptor of the Wnt/β-cateninpathway (Krejci et al., 2012; Buchtova et al., 2015). We speculated

that FGF signaling might be involved in activation of Wnt-5a-Ror2 in RCS cells, as it is known that Wnt/β-catenin and non-

canonical Wnt pathways receptors can be activated by common

mechanisms (Bryja et al., 2009; Grumolato et al., 2010). However,Wnt/β-catenin is likely not involved in the Noggin/Wnt-5a/Ror2 crosstalk in RCS cells because no differences inthe activity analyzed by the TopFlash reporter system wereobserved.

Where can such FGF-dependent Noggin-induced activationof Wnt-5a-Ror2 signaling pathway in chondrocytes take place invivo? In limb cartilage development, Wnt/PCP signaling appearsto be involved at two steps: during condensation of cartilageelements, especially the digits (Gao et al., 2011; Wang et al.,2011; Ho et al., 2012), and for establishing cartilage growthplate morphology (Ahrens et al., 2009; Li and Dudley, 2009;Kuss et al., 2014; Romereim et al., 2014). In the first scenario,Wnt-5a is required for digit formation, and mice deficient forWnt-5a form rudimentary digits (Yamaguchi et al., 1999). TheWnt-5a null phenotype is recapitulated by either Ror1/Ror2double null mutants (Ho et al., 2012) or Ror2/Vangl2 doublenull mutants (Gao et al., 2011), clearly establishing that a Wnt-5a/Ror2/PCP pathway is necessary for digit formation. Noggin isexpressed in forming cartilage condensations (Brunet et al., 1998)and could hence facilitate this process. During digit outgrowth,FGFs are expressed in the apical ectodermal ridge (AER). FGFsignaling from the AER is thought to keep distal mesenchymalcells proliferating and undifferentiated (ten Berge et al., 2008). Invitro, FGFs inhibit chondrogenesis (Buchtova et al., 2015), buton the other hand application of FGF beads can induce ectopicdigit formation in vivo (Montero et al., 2001). One possibilityis that FGF signaling that acts at a distance from the AER onprechondrogenic cells provides competence for Noggin activitytoward the Wnt-5a/Ror2/PCP pathway, and is thus enforcingPCP signaling in cells undergoing chondrogenic differentiation.In the growth plate, both Wnt-5a and Ror2 are essential forcellular polarity (Yang et al., 2003; Schwabe et al., 2004), andWnt-5a acts via a PCP pathway (Gao et al., 2011; Kuss et al., 2014).Noggin is expressed throughout the growth plate (Brunet et al.,1998), and FGF signaling, which is a major regulator of growthplate chondrocyte proliferation, is active here as well (Hortonet al., 2007).

In summary our data pinpoint a novel, yet unappreciated rolefor Noggin in sensitizing cells toWnt-5a. The cellular mechanismby which Noggin accomplishes this effect on the Wnt-5a-Ror2pathway remains to be elucidated.

AUTHOR CONTRIBUTIONS

PK, SS, and VB designed research; MB, TR, OB, ZD, FW, AM,NC, and PK performed research; all authors analyzed data; andOB, SS, and VB wrote the paper.

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ACKNOWLEDGMENTS

This work was supported by the Czech Science Foundation(15-21789S, 17-16680S, 17-09525S). TR and ZD are supportedby the Marie Curie ITN WntsApp. PK was supported by

Ministry of Education, Youth and Sports of the CzechRepublic (KONTAKT II LH15231) and Ministry of Healthof the Czech Republic (15-33232A, 15-34405A). This workwas supported by the Deutsche Forschungsgemeinschaft(DFG SFB 577).

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Conflict of Interest Statement: The authors declare that the research was

conducted in the absence of any commercial or financial relationships that could

be construed as a potential conflict of interest.

Copyright © 2017 Bernatik, Radaszkiewicz, Behal, Dave, Witte, Mahl, Cernohorsky,

Krejci, Stricker and Bryja. This is an open-access article distributed under the terms

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