HDAC inhibition overcomes acute resistance to MEK inhibition in BRAF mutant colorectal cancer by...

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HDAC inhibition overcomes acute resistance to MEK inhibition in BRAF mutant

colorectal cancer by down-regulation of c-FLIPL.

Robbie Carson1,†, Basak Celtikci1, †, Cathy Fenning1, Arman Javadi1, Nyree Crawford1, Lucia

Perez Carbonell1, Mark Lawler1, Daniel B. Longley1, Patrick G. Johnston1, †, Sandra Van

Schaeybroeck1,2, †

1. Centre for Cancer Research and Cell Biology, School of Medicine, Dentistry and

Biomedical Science, Queen’s University Belfast, 97 Lisburn Road, Belfast, BT9 7AE, UK

† equal contribution

Running title: Regulation of STAT3 and c-FLIPL in BRAF mutant colon cancer.

Keywords: BRAF, colorectal cancer, MEK, c-MET-JAK-STAT3, c-FLIP

Financial support from Cancer Research UK (C212/A7402); Cancer Research UK fellowship

(C13749/A7261).

2. Corresponding author: Dr. Sandra Van Schaeybroeck, Centre for Cancer Research and

Cell Biology, Queen’s University Belfast, 97 Lisburn Road, Belfast, Northern Ireland, BT9

7AE. [email protected]; Telephone: +44 2890 972954; Fax: +44 2890972776.

Conflicts of interest: P.G. Johnston has an ownership interest in both Almac Diagnostics and

Fusion Antibodies. He is a consultant/advisor for, and has received honoraria from, Chugai

pharmaceuticals, Sanofi-Aventis and Pfizer. All other authors have no conflicts of interest to

declare.

Research. on March 31, 2015. © 2015 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 26, 2015; DOI: 10.1158/1078-0432.CCR-14-2701

http://clincancerres.aacrjournals.org/

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TRANSLATIONAL RELEVANCE

Oncogenic V600E BRAF mutations are associated with poor clinical prognosis in colorectal

cancer (CRC). Inhibitors of the BRAF-MAPK pathway are effective therapies for BRAF

mutant (MT) melanoma, but are ineffective in the majority of BRAFMT CRCs. Understanding

MAPK inhibitor resistance is therefore an important unmet clinical need in BRAFMT CRC. In

this study, we have identified a novel resistance mechanism to MEK1/2 inhibition that is

mediated via STAT3 and the anti-apoptotic protein c-FLIPL and which is dependent on c-

MET activity. Furthermore, inhibiting c-MET or down-regulating c-FLIP using RNAi or HDAC

inhibitors significantly increased MEK inhibitor-induced apoptosis in BRAFMT CRC in vitro

and in xenograft models. Taken together, our results indicate that combining MEK1/2

inhibitors with c-MET or HDAC inhibitors may be promising therapeutic strategies for this

poor prognostic CRC subgroup and should be explored in future clinical trials.




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ABSTRACT

Purpose: Activating mutations in the BRAF oncogene are found in 8-15% of colorectal

cancer (CRC) patients and have been associated with poor survival. In contrast to BRAF

mutant (MT) melanoma, inhibition of the MAPK pathway is ineffective in the majority of

BRAFMT CRC patients. Therefore, identification of novel therapies for BRAFMT CRC is

urgently needed.

Experimental Design: BRAFMT and WT CRC models were assessed in vitro and in vivo.

Small molecule inhibitors of MEK1/2, MET and HDAC were employed, over-expression and

siRNA approaches were applied, and cell death was assessed by flow cytometry, Western

blotting, cell viability and caspase activity assays.

Results: Increased c-MET-STAT3 signalling was identified as a novel adaptive resistance

mechanism to MEK inhibitors (MEKi) in BRAFMT CRC models in vitro and in vivo. Moreover,

MEKi treatment resulted in acute increases in transcription of the endogenous caspase-8

inhibitor c-FLIPL in BRAFMT cells, but not in BRAFWT cells, and inhibition of STAT3 activity

abrogated MEKi-induced c-FLIPL expression. In addition, treatment with c-FLIP-specific

siRNA or HDAC inhibitors abrogated MEKi-induced upregulation of c-FLIPL expression and

resulted in significant increases in MEKi-induced cell death in BRAFMT CRC cells. Notably,

combined HDAC inhibitor/MEKi treatment resulted in dramatically attenuated tumor growth

in BRAFMT xenografts.

Conclusions: Our findings indicate that c-MET/STAT3-dependent upregulation of c-FLIPL

expression is an important escape mechanism following MEKi treatment in BRAFMT CRC.

Thus, combinations of MEKi with inhibitors of c-MET or c-FLIP (eg. HDAC inhibitors) could

be potential novel treatment strategies for BRAFMT CRC.




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INTRODUCTION

BRAF is mutated in ∼8% of all cancers, and approximately 10% of colorectal cancer tumors

harbour a BRAFT1799A transversion, encoding the constitutively active V600E-BRAF

oncoprotein (1, 2). BRAF belongs to the RAF protein-serine/threonine kinase family, which

also comprises RAF-1/c-RAF and A-RAF (3). In normal signalling conditions, RAF proteins

phosphorylate and activate the mitogen-activated protein kinase (MAPK) signalling axis in

response to active GTP-bound RAS. BRAFV600E mutations lead to constitutively active

BRAF, resulting in sustained MAPK signalling, independent of upstream kinase activity (4).

Recent phase III studies have shown that patients with BRAF mutant (MT) tumors have the

worst overall survival compared to patients with RASMT (KRAS or NRAS) or KRAS/BRAF

wild type (WT) CRC tumors (5-7). Furthermore, some studies have suggested that the

BRAFV600E mutation should be included as marker of resistance to epidermal growth factor

receptor (EGFR)-targeted therapies, such as cetuximab and panitumumab (8-10). Therefore,

novel therapeutic strategies are urgently needed for BRAFMT CRC patients.

Tumors carrying a mutationally activated oncogene frequently show “addiction” to the

oncoprotein or activated signalling pathway upon which the cancer cells became dependent

(11). Recently, the Food and Drug Administration (FDA) approved the selective BRAF

inhibitor Vemurafenib for the treatment of patients with unresectable or metastatic melanoma

with the BRAFV600E mutation. Although the BRAF inhibitors Vemurafenib or Dabrafenib,

MEK1/2 inhibition (Trametinib) or a BRAF/MEKi combination resulted in dramatic responses

between 60%-80% in BRAFMT melanoma (12-14), disappointing responses of 5% and 12%

were seen for Vemurafenib alone or Dabrafenib/Trametinib combination respectively in

BRAFMT CRC (15, 16). Understanding mechanisms of resistance to MAPK inhibition in

BRAFMT CRC may lead to new therapeutic strategies for this poor prognostic CRC

subgroup.




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Herein, we report a novel resistance mechanism to MEK1/2 inhibition in BRAFMT CRC,

which is mediated via STAT3 and the anti-apoptotic protein c-FLIPL. This survival response

is dependent on c-MET and JAK1/2 activity and is abrogated using c-MET and JAK1/2-

STAT3 inhibitors. We also show that concomitant treatment with inhibitors of c-MET or c-

FLIP (using HDAC inhibitors) and MEK1/2 inhibition leads to marked increases in

therapeutic efficacy in BRAFMT in vitro and xenograft models. Taken together, our results

indicate that combining MEK1/2i with inhibitors of c-MET or c-FLIP (eg. HDAC inhibitors)

could be potential novel treatment strategies for BRAFMT CRC.

MATERIALS AND METHODS

Materials

AZD6244, AZD1480 and AZD9150 were obtained from AstraZeneca (Macclesfield, UK),

Crizotinib from Pfizer (Peapack, New Jersey, USA), TG101348 from Axon Medchem

(Groningen, The Netherlands), Trametinib, AZD8931, Vorinostat and Entinostat (MS-275)

from Selleck Chemicals LLC (Suffolk, UK), Stattic from Sigma-Aldrich (Dorset, UK), UO126

from Promega (Southampton, UK) and PD98059 from Cell Signaling (Beverly, MA, USA).

siRNAs targeting STAT3 (STAT3_7), JAK1 (JAK1_1), JAK2 (JAK2_7) and caspase 9

(CASP9_5) were purchased from Qiagen (Crawley, UK); the c-FLIP siRNA sequence used

was 5’ AAG CAG TCT GTT CAA GGA GCA; the caspase 8 sequence used was 5’ GAG

UCU GUG CCC AAA UCA ATT (17).

Cell culture.

Authentication and culture of LIM2405, LIM1215, HT-29, VACO432/VT1, RKO F6-8 and

CACO-2 CRC cells have previously been described (18-20). All cells were passaged for a

maximum of 2 months, after which new seed stocks were thawed for experimental use.

LIM2405 and LIM1215 cell lines, established in 1992, were a gift from Dr. Whitehead

(Ludwig Institute of Melbourne and Vanderbilt University, Nashville, TN) in 2010. These cell




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lines were tested for morphology/growth rate/response to mitogens/xenograft

growth/expression of brush-border and mucin-related antigens/mutational analysis (21, 22).

VACO432, VT1 and RKO F6-8 were kindly provided by Prof B. Vogelstein (Johns Hopkins

University School of Medicine, Baltimore) in 2012. HT-29 (2001), CACO2 (2005), COLO205

(2012) and COLO320 (2012) cells were obtained from the American Type Culture Collection

(Authentication by short tandem repeat (STR) profiling/karyotyping/isoenzyme analysis).

Generation of stable c-FLIPL-overexpressing cells.

c-FLIPL containing pBABE puro vector was transfected into Phoenix 293T cells along with a

VSVG (vesicular stomatitis virus (vsv) glycoprotein) coat protein plasmid. Following 24h

transfection, filtered medium with polybrene (1:1000) was added to HT-29 cells for 48 hours,

and thereafter 1μg/mL puromycin (Life Technologies, Inc.) added for selection of c-FLIPL

overexpressing HT-29 cells. Overexpression of c-FLIPL was confirmed by Western blotting.

Cell Viability assays

Cell viability assays were done as previously described (20). Representative results of at

least 3 independent experiments are shown.

Flow cytometric analysis and cell death measurement.

Apoptosis was determined using propidium iodide (PI) staining to evaluate the percentage of

cells with DNA content <2N as previously described (18). For annexin/PI analysis, cells were

harvested and analysed according to the manufacturer’s instructions (BD Biosciences).

Representative results of at least 3 independent experiments are shown.




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Western blotting.

Western blot analysis was carried out as previously described (20). Anti-FLIP (NF6; Alexis,

San Diego, CA, USA), anti-caspase 8 (12F5; Alexis) and anti-poly(ADP-ribose) polymerase

(PARP; eBioscience; San Diego, CA, USA) mouse monoclonal antibodies were used in

conjunction with a horseradish peroxidase–conjugated anti-mouse secondary antibody

(Amersham). Anti-cleaved caspase 3 (Cell Signaling Technology, Beverly, MA, USA), anti-

hyperacetylated Hystone H4 (Millipore, Watford, UK) were used in conjunction with a HRP-

conjugated anti-rabbit secondary antibody (Amersham, Buckinghamshire, UK). Equal

loading was assessed using β-actin (Sigma) mouse monoclonal primary antibody.

Caspase-activity assays

Caspase-8 or caspase-3/7-GLO reagents (25μl) (Promega, Southampton, UK) were

incubated with 5μg of protein lysate diluted in cell culture medium in a total volume of 50μl

for 45 minutes at room temperature. Luciferase activity was measured using a luminometer.

STAT3 reporter assay

The STAT3 reporter, negative and positive control constructs were obtained from Qiagen

(Crawley, UK). Luminescence was measured following addition of 50μl of luciferase assay

reagent into each well.

siRNA transfections

siRNA transfections were carried out using Hiperfect (Qiagen) as previously described (20).

ELISA

Soluble c-MET and HGF ELISA assays were carried out as previously described (20).




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Real-time reverse transcription-PCR analysis

RNA was isolated using the GeneJET RNA purification kit (Thermo Scientific, Leicestershire,

UK) and reverse transcribed using the Moloney murine leukemia virus-based reverse

transcriptase kit (Invitrogen). Q-PCR analysis was performed using the LightCycler® 480

probes master mix or the Roche SYBR Green method (LightCycler® 480II, Roche).

In vivo study. In vivo studies were conducted as previously described using BALB/c nude

mice (18). AZD6244 10mg/kg/bid p.o, Entinostat (MS-275) 10mg/kg/qd p.o and Crizotinib

25mg/kg/qd were administered by oral gavage. The control groups received vehicle

(methocel/polysorbate buffer). In the combination groups, AZD6244 was administered

together with MS-275 or Crizotinib. Each treatment group contained 8 animals. Two hours

following final dosing, mice were sacrificed and tumor tissue was excised and snap-frozen in

liquid nitrogen for immunoblotting (PPL2704). All animal experiments were carried out

according to UKCCCR guidelines.

Statistical analysis

Student’s t-tests and 2-way ANOVA were calculated using the GraphPad software (Prism4).

2-way ANOVA test was used to determine the significance of change in levels of apoptosis

between different treatment groups. All changes in levels of apoptosis that are described as

significant had p values that were <0.05 (* denotes p<0.05; ** denotes p<0.01; *** denotes

p<0.001; ns denotes not significant compared with control).




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RESULTS

MEK1/2 inhibition decreases cell viability but does not induce apoptosis in BRAFV600E

CRC. To explore the sensitivity of BRAFMT CRC cells to MEK1/2 inhibition, we evaluated

the effects of the clinically employed MEK1/2 inhibitors AZD6244 and Trametinib

(GSK1120212) on viability of the parental VACO432 CRC cell line, which carries the V600E

mutation in BRAF (BRAFV600E/+), its isogenic VT1 clone with a disrupted BRAFV600E

allele (23), and a panel of BRAFMT (LIM2405, HT-29, COLO205, RKO F6-8) and WT

(CACO-2, LIM1215 and COLO320) CRC cells (Supplementary Fig. S1A, upper panel).

BRAFMT CRC cells showed a higher sensitivity to MEK1/2 inhibition with IC50 values

between 20nM-3.7μM and 0.1nM-9.4nM for AZD6244 and Trametinib respectively,

compared with IC50 values of >6.8μM and >13nM for BRAFWT cells (p<0.001 for both

AZD6244 and Trametinib). Although BRAFMT CRC cells were more sensitive to MEKi than

BRAFWT cells, apoptosis induction 24h after treatment with MEKi was relatively inefficient

as assessed by activation of caspase-3 (Supplementary Fig. S1A, lower panel) and flow

cytometry (Supplementary Fig. S1B).

STAT3 pathway activation and JAK1/JAK2 are key mediators of resistance to MEK

inhibitors in BRAFMT colorectal cancer cells. We have recently shown that KRAS mutant

cells respond to MEK1/2 inhibitors with increased STAT3Y705 phosphorylation (20). Hence,

we investigated the effect of AZD6244, PD98059, UO126 and Trametinib on STAT3

activation in our panel of BRAFMT and WT CRC cell lines and found that STAT3

phosphorylation (Y705) was acutely increased in the BRAFMT cell lines in response to each

agent, but not in the BRAFWT/KRASWT cells (Fig. 1A). In addition, we also found acute

increases in STAT3 transcriptional activity and expression of the STAT3 target genes IRF-7,

RPS18 and SOCS3 following AZD6244 treatment in the BRAFMT CRC cells

(Supplementary Fig. S1C). Constitutive pSTAT3 levels were higher in BRAFMT VACO432

cells compared to its isogenic WT clone; however there was no clear pattern of pSTAT3




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expression across a panel of non-matched BRAFMT and WT CRC cells (Supplementary Fig.

S1D). STAT3 is activated in the context of MEKi in BRAFMT but not BRAFWT cells, a

phenomenon known as an adaptive, acutely induced resistance mechanism (24). We also

assessed the effect of STAT3 silencing on apoptosis induction by flow cytometry and

Western blotting for PARP cleavage and activation of caspase 3. A significant increase in

apoptosis was observed when BRAFMT cells were co-treated with STAT3 siRNA and

AZD6244 (Supplementary Fig. S1B, E). Collectively, these data indicate that STAT3 is an

important survival pathway following MEK1/2 inhibition in BRAFMT CRC.

Next, we investigated the involvement of the upstream kinases JAK1 and JAK2 in regulating

STAT3 activity and survival of BRAFMT and WT CRC cells. MEK inhibitor-induced STAT3

phosphorylation was associated with increased JAK1 and JAK2 phosphorylation

(Supplementary Fig. S2A). In addition, RNAi against JAK1 decreased basal and MEKi-

induced pJAK2 levels and resulted in more potent inhibition of both basal and MEKi-induced

STAT3 activity compared to the effects of JAK2 silencing (Fig. 1B). Moreover, treatment with

the JAK1/2 inhibitor AZD1480 (25) resulted in potent inhibition of MEKi-induced STAT3

transcriptional activity, as indicated by decreased STAT3 reporter activity and mRNA

expression of IRF-7 (Fig. 1C, upper and lower panels). These results indicate that JAK1 and

JAK2 cooperate to regulate basal and MEKi-induced STAT3 survival response in BRAFMT

CRC, which is consistent with our previous data in KRASMT CRC cells (20).

We next investigated the involvement of JAK1/2 in regulating survival of BRAFMT and WT

CRC cells. Combined treatment of AZD1480 or TG101348 (20, 26) with AZD6244 resulted in

potent increases in apoptosis as indicated by flow cytometry and increased PARP cleavage

and caspase 3 processing in the BRAFMT VACO432 cell line but not the WT VT1 clone (Fig.

1D, upper and lower panels). Calculation of combination index (CI) values confirmed strong

synergy between AZD1480 or TG101348 and AZD6244 in the BRAFMT VACO432 cell line

(Supplementary Fig. S2B). Importantly, similar results were obtained in a wider panel of




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BRAFMT (HT-29, LIM2405, RKO F6-8, COLO205), but not BRAFWT CRC cell line models

(Fig. 1D; Supplementary Fig. S2B-D).

c-MET regulates MEKi-induced STAT3 activation and survival in BRAFMT CRC cells.

Previous studies have shown the role of the epidermal growth factor receptor family in

resistance to BRAF inhibitors in BRAFMT cancers, including CRC (27, 28). Secretion of the

hepatocyte growth factor (HGF), the ligand for c-MET, has also been shown to confer

resistance to BRAF inhibition in BRAFMT melanoma (29). Based on these studies and our

previous data in KRASMT CRC (20), we assessed the effect of MEK inhibition on

EGFRY1068, HER2Y1248 and c-METY1234/1235 phosphorylation and found acute increases in

phosphorylation of these receptors, as early as 3 hours following treatment with AZD6244 in

BRAFMT cells (Supplementary Fig. S3A). Basal and AZD6244-induced STAT3

phosphorylation were inhibited following treatment with the c-MET/ALK inhibitor Crizotinib,

but unaffected by treatment with the dual EGFR/HER2 inhibitor AZD8931 in a panel of

BRAFMT CRC cells, indicating that activation of STAT3 following MEK inhibition is not

driven by EGFR or HER2 in BRAFMT CRC (Fig. 2A; Supplementary Fig. S3B).

We next assessed the effect of c-MET inhibition on MEK-induced apoptosis in BRAFMT

CRC cells. Co-treatment with Crizotinib and AZD6244 resulted in significant increases in

apoptosis in the BRAFMT VACO432 cell line, but not in the BRAFWT daughter cell line (Fig.

2B). Similar results were obtained when apoptosis was assessed by Western blotting for

PARP and caspase 3 cleavage, flow cytometry and caspase-8 and -3/7 activity assays

following combined AZD6244/Crizotinib treatment in BRAFMT LIM2405, COLO205, HT-29

and RKO cells, but not in BRAFWT cells (Fig. 2B, C; Supplementary Fig. S3C-E).

Importantly, although both AZD8931/AZD6244 and Crizotinib/AZD6244 combinations

resulted in significant decreases in cell viability in BRAFMT cells, only Crizotinib enhanced

apoptotic cell death when combined with AZD6244 in the panel of BRAFMT cells (Fig. 2B, C;

Supplementary Fig. S3D and F). In order to define the relative importance of the extrinsic




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and intrinsic apoptotic pathways in mediating AZD6244/Crizotinib-induced apoptosis, we

used siRNA specifically directed against caspase 8 (extrinsic pathway) or caspase 9

(intrinsic pathway) (Supplementary Fig. S3G). Notably, silencing of either caspase

significantly decreased apoptosis following AZD6244/Crizotinib treatment in BRAFMT

LIM2405 cells. This suggests that the cell death induced by this combination proceeds via a

caspase-8-mediated activation of the caspase-9-dependent intrinsic apoptosis pathway.

Taken together, these data would indicate that the c-MET/STAT3 signalling is an important

survival response induced by MEK1/2 inhibition in BRAFMT CRC.

Based on the compelling in vitro evidence, we next assessed the therapeutic efficacy of

combined c-MET and MEK inhibition in a BRAFMT HT-29 xenograft model using AZD6244

and Crizotinib (Fig. 2D). We detected significantly increased pSTAT3 levels in xenografts

treated with AZD6244; moreover, Crizotinib resulted in decreased basal and AZD6244-

induced pSTAT3 levels, consistent with our in vitro observation (Supplementary Fig. S3H).

Importantly, the METi/MEKi combination led to a marked reduction in growth of BRAFMT

HT-29 xenografts compared with treatment with each agent individually (Fig. 2D, left panel).

Furthermore, caspase 3 cleavage was observed in the HT-29 tumors following treatment

with AZD6244 in combination with Crizotinib, indicative of apoptosis induction (Fig. 2D, right

panel). These results indicate that agents directed against c-MET may be highly effective

combination partners for MEK1/2-targeted therapies in BRAFMT CRC.

Inhibitors of MEK1/2 upregulate c-FLIPL expression in BRAFMT CRC. To investigate the

mechanism of apoptosis further, we assessed expression levels of pro- and anti-apoptotic

proteins following AZD6244/Crizotinib treatment in BRAFMT HT-29, COLO205 and LIM2405

CRC cells (Fig. 3A). Notably, expression of c-FLIPL, but not c-FLIPS, was acutely increased

following AZD6244 treatment in all three BRAFMT cell lines (Fig. 3A). In contrast, AZD6244

treatment had little impact on c-FLIPL levels in BRAFWT CRC models (Supplementary Fig.

S4A). We also found that the increases in c-FLIPL levels in BRAFMT CRC cells was a




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transcriptional event, as AZD6244 increased c-FLIPL mRNA levels as early as 6h post-

treatment (Fig. 3B; Supplementary Fig. S4B). Importantly, co-treatment with AZD6244 and

Crizotinib, but not AZD8931, abrogated AZD6244-induced c-FLIPL expression, which was

associated with processing of pro-caspase 8 and potent caspase-3, -9 and PARP cleavage

(Fig. 3A).

c-FLIPL is important in regulating sensitivity to MEKi in BRAFMT CRC cells

To assess further the importance of c-FLIPL in regulating response to MEK1/2 inhibition, we

developed a BRAFMT HT-29 cell line model that stably overexpresses c-FLIPL (Fig. 3C). c-

FLIPL expression in HT-29-FL cell line was approximately 2-2.5-fold higher compared with EV

(Empty vector) (Fig. 3C, upper panel). Overexpression of c-FLIPL in this cell line correlated

with reduced levels of AZD6244-induced apoptosis compared with the EV cell line (Fig. 3C,

lower panel). These results suggested a role for c-FLIPL in mediating resistance to MEK-

targeted agents in BRAFMT CRC cells. To investigate this, we assessed the effect of c-FLIP

silencing on apoptosis induction using Western blotting for PARP cleavage and activation of

caspase-3. Co-treatment with c-FLIP siRNA and AZD6244 resulted in significant increases

in apoptosis in the BRAFMT LIM2405, RKO F6-8, VACO432 and HT-29 cell lines, but not in

the BRAFWT CACO-2, LIM1215, COLO320 and VT1 cells (Fig. 3D; Supplementary Fig.

S4C). These results are the first to demonstrate the importance of c-FLIPL in regulating

sensitivity to MEK inhibition in BRAFMT CRC.

Co-inhibition of HDAC 1-3 and MEK1/2 results in cell death and inhibits growth of

BRAFMT xenograft models. Our previous studies and those of others have shown that

HDAC inhibitors with anti-HDAC1-3 activity are efficient post-transcriptional suppressors of

FLIP expression (30, 31). Treatment with the pan-HDAC inhibitor Vorinostat or the HDAC 1-

3 inhibitor Entinostat inhibited both basal and AZD6244-induced c-FLIPL expression, and this

was associated with marked increases in apoptosis in the BRAFMT cell line panel (Fig. 4A;

Supplementary Fig. S5A and B). We also determined that the apoptosis induced by




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Entinostat/AZD6244 was highly dependent on caspase 8 (Fig. 4B), consistent with a FLIP-

dependent mechanism of cell death.

To extend these in vitro findings, we next assessed the therapeutic efficacy of combined

Entinostat and MEK inhibition in the BRAFMT HT-29 xenograft model. Although both

Entinostat and AZD6244 slowed tumor growth, the Entinostat/MEKi combination led to a

supra-additive reduction in growth in HT-29 BRAFMT xenograft model (Fig. 5). Treatment

with Entinostat resulted in increased acetylation of H4, a marker of HDAC inhibition and

decreased basal and AZD6244-induced STAT3 activation (Fig. 5; Supplementary Fig. S5C).

In addition, combined Entinostat/AZD6244 treatment resulted in high levels of caspase 3

activation in HT-29 tumors (Fig. 5). These findings indicate that HDAC1-3-targeted agents

may be highly effective when used in conjunction with MEK1/2 targeted therapies to treat

BRAFMT CRC.

STAT3 regulates MEK1/2 inhibitor induced increases in c-FLIP expression. To

investigate the relationship between MEK1/2 inhibitor-induced increases in STAT3 activity

and c-FLIPL expression levels, we used the STAT3 small molecule inhibitor Stattic, which

targets the STAT3 SH2 domain and prevents dimerization (32), RNAi and antisense against

STAT3. Treatment with Stattic resulted in decreases in basal and AZD6244-induced

STAT3Y705 phosphorylation levels, and this was associated with decreased AZD6244-

induced c-FLIPL mRNA and protein levels (Fig. 6A; Supplementary Fig. S6A). To exclude

any off-target effects from Stattic that may affect c-FLIPL expression, we analysed the effect

of STAT3 siRNA and AZD9150, a 16 oligonucleotide antisense molecule targeting the 3’

untranslated region of the STAT3 mRNA which is in clinical development (33). Both

AZD9150 and STAT3 silencing decreased basal and AZD6244-induced STAT3Y705

phosphorylation levels and c-FLIPL expression levels (Fig. 6B; Supplementary Fig. S6B). In

addition, co-treatment with AZD6244 and Stattic or AZD9150 resulted in significant

increases in apoptosis (Fig. 6), similar to those observed with HDACi/MEKi combination (Fig.




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4). Taken together, these results would suggest that STAT3 directly regulates c-FLIPL

survival response in response to MEK inhibition in BRAFMT CRC.

DISCUSSION

Activating mutations in the BRAF oncogene occur in 8-15% of CRC and are associated with

the poor clinical outcome (34). The studies showing a lack of clinical benefit from EGFR-

targeted agents in BRAFMT CRC, remain controversial (9, 35). Although selective BRAF

and MEK1/2 inhibitors such as Vemurafenib and Trametinib have resulted in dramatic

responses in BRAFMTV600E melanoma, BRAFMT CRC with the identical missense mutation

has shown very poor responses to these treatments (15, 16). In both our in vitro and in vivo

data, we corroborate the findings seen in human patients. Understanding of the intrinsic

resistance mechanisms to MAPK inhibition in BRAFMT CRC has the potential to identify

novel therapeutic strategies for this poor prognostic genetic subgroup. Recently, there have

been a number of studies that have investigated resistance mechanisms to the BRAF

inhibitor Vemurafenib in BRAFMT CRC. Indeed, two studies have observed increased

activity of EGFR following treatment with Vemurafenib in BRAFMT CRC cells (27, 36).

Moreover, BRAFMT melanoma cells were found to have low levels of EGFR, which might

explain the favorable response of BRAFMT melanoma to BRAFi. Other studies reported the

role of the PI3K/AKT and mTOR pathways in mediating resistance to Vemurafenib in

BRAFMT CRC cells (37-39). Our study provides evidence that STAT3-dependent c-FLIPL

upregulation is a major resistance mechanism to MEK inhibitors in BRAFMT CRC models.

Constitutive activation of STAT3 has been reported in a broad range of tumors and tumor-

derived cell lines, including melanoma and colon cancer (40). A number of studies have

shown that deregulated STAT3 not only enhances tumor cell proliferation and survival, but

also can promote oncogenesis by bridging chronic inflammation to tumor formation (41-43).

Furthermore, recent data from our lab and other studies have shown a role for STAT3 in

regulating resistance to EGFR and MEK1/2 targeted therapies in lung and colon cancer,




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driven by activated EGFR, HER2, ALK, MET and mutant KRAS (20, 44). In this study, we

have found that treatment with different MEK inhibitors resulted in acute increases in STAT3

phosphorylation in BRAFMT CRC cells.

Although recent studies have identified mutations within the SH2 dimerization and activation

domain of STAT3 in large granular lymphocytic leukaemia (45) and inflammatory

hepatocellular adenomas (IHCAs) (46), STAT3 activation is usually dependent on growth

factor receptors signalling and their associated janus kinases. Here, we found that basal and

MEKi-induced STAT3 activation in BRAFMT CRC cells was mediated through JAK1 and

JAK2. Using selective inhibitors of JAK1/2, we further demonstrated the differential

dependency of BRAFMT and BRAFWT cells on STAT3 for survival, particularly in the

context of co-treatment with MEK inhibitors. Collectively, these results indicate that JAK

activation in response to inhibition of MEK1/2 in BRAFMT CRC cells contributes to

resistance to MEK inhibitors in these cancer cells.

In addition, we found that STAT3 was activated by upstream activation of c-MET. Soluble

HGF, the ligand for c-MET, was not detected in the culture medium of BRAFMT cell line

models; however, consistent with our data in KRASMT cell lines (20), we found that MEK

regulated the levels of soluble decoy MET, the natural antagonist of c-MET, in BRAFMT

CRC models in vitro and in vivo (Supplementary Fig. S7). Based on their findings of a

Vemurafenib-induced feedback activation of EGFR and their in vivo data, previous studies

have suggested that the addition of an EGFR inhibitor to Vemurafenib could be a novel

treatment approach for BRAFMT CRC (27). Interestingly, our studies showed that the c-

MET-STAT3 signalling axis appears to have a more significant role in survival and MEKi-

induced resistance in BRAFMT CRC cells, as the Crizotinib/AZD6244 combination was a

more effective inducer of apoptosis compared to AZD6244 combined with gefitinib (EGFR

inhibitor) or AZD8931 (EGFR/HER2 inhibitor). The importance of c-MET as mediator of

acute resistance to MEK1/2 inhibitors was demonstrated in vivo, where co-treatment of

BRAFMT CRC xenografts with Crizotinib blocked AZD6244-induced STAT3 activation and




17

resulted in supra-additive reductions in tumor growth and marked induction of apoptosis

(24). Collectively, these results indicate that inhibitors of the c-MET pathway in conjunction

with MEK inhibitors could be a novel treatment strategy for BRAFMT CRC tumors.

Mechanistically, we found that STAT3 activation protects BRAFMT cancer cells from

apoptosis following MEK inhibition, by acutely upregulating expression of the anti-apoptotic

protein c-FLIPL. Notably, treatment with AZD6244 resulted in acute increases in c-FLIPL

mRNA and protein levels only in BRAFMT cells, and inhibition of STAT3 using stattic or

AZD9150 attenuated MEKi-induced upregulation of c-FLIPL expression and resulted in

significant increases in MEKi-induced apoptosis. Moreover, we also demonstrate for the first

time that FLIPL overexpression abrogates the effect of MEKi in BRAFMT CRC cells. We

previously have shown that c-FLIP is an important regulator of apoptosis and drug

resistance and a poor prognostic marker in CRC (17, 47, 48). Using siRNA against c-FLIP

and multiple cell line models, we now show that c-FLIP is also a critical mediator of

resistance to MEKi in BRAFMT CRC. Our previous studies and those of others have shown

that pan-HDAC inhibitors and more specific HDAC1-3 inhibitors act as efficient post-

transcriptional suppressors of FLIP expression (30, 49). Combined treatment of BRAFMT

CRC models with the HDAC1-3 inhibitor Entinostat and AZD6244 blocked AZD6244-induced

c-FLIPL upregulation and resulted in enhanced levels of apoptosis induction. In addition, we

also demonstrated that MEK inhibition in conjunction with Entinostat was highly effective at

blocking the growth of BRAFMT CRC xenografts. This is the first study showing that

combined HDAC/MEK inhibition could be a promising treatment strategy for BRAFMT CRC

patients. Before carrying forward into a clinical trial, this combination study needs to be

confirmed in additional BRAFMT models.

In conclusion, we have identified STAT3-mediated c-FLIP upregulation as an important

resistance mechanism to MEKi in BRAFMT CRC that is acutely induced as a consequence

of increases in c-MET and JAK1/2 activation (Fig. 6C). From a cancer therapeutics




18

perspective, the substantial tumor growth inhibition observed in our xenograft studies

support the evaluation of c-MET/MEK co-inhibition or HDAC1-3/MEK co-inhibition in clinical

trials for patients with metastatic BRAFMT colorectal cancer.




19

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FIGURE LEGENDS

Figure 1. JAK1/2 regulate MEK inhibitor-induced STAT3 survival response in BRAFMT

CRC cells. A. Upper panel: BRAFMT and WT CRC cells were treated with AZD6244 for

24h and STAT3 and ERK1/2 expression/activity levels determined by Western blotting.

Lower panel: HT-29 and LIM2405 cells were treated with indicated doses of PD98059 (PD),

UO126 (UO), Trametinib (TRA) or AZD6244 (AZD) for 24h and STAT3 and ERK1/2

expression and activity levels determined by Western blotting. B. Left panel: LIM2405 cells

were transfected with 10nM JAK1 siRNA, JAK2 siRNA or co-transfected with 10nM JAK1

and JAK2 siRNA for 24h. Right panel: LIM2405 cells were transfected with 10nM JAK1

siRNA (J1), JAK2 siRNA (J2) or co-transfected with 10nM JAK1 and JAK2 siRNA (J1/2) for

24 hours, followed by treatment with AZD6244 for 6h. JAK1, pJAK1Y1022/1023, JAK2,

pJAK2Y1007/1008, pSTAT3Y705, STAT3, pERK1/2 and ERK1/2 levels were determined by

Western blotting. C. Upper panel: HT-29 cells were pre-incubated with AZD1480 for 3 hours

and thereafter treated with AZD6244 for 24 hours and IRF7 mRNA levels determined by

real-time PCR. Relative mRNA expression was calculated using the DDCt method with

normalisation to β-actin and GAPDH. Lower panel: HT-29 cells were co-treated with

AZD1480 and AZD6244 for 24 hours and a luciferase construct STAT3 reporter assay was

used to measure STAT3 activity. D. BRAFMT and WT cells were pre-incubated with

TG101348 (TG) or AZD1480 (AZ) for 4 hours, followed by treatment with AZD6244 for 24h.

Upper panel: PARP, cleaved caspase 3, pERK1/2 and ERK1/2 levels were determined by

Western blotting; Lower panel: apoptosis was assessed by flow cytometric analysis with

Propidium Iodide (PI).

Figure 2. Combined c-MET and MEK inhibition results in apoptosis in BRAFMT in vitro

and in vivo models. A. COLO205 and HT-29 cells were pre-treated with Crizotinib or

AZD8931 for 3h, and thereafter treated with AZD6244 for 24 hours and pHER2Y1248, HER2,

pEGFRY1068, EGFR, pcMETY1234/1235, MET, pSTAT3Y705, STAT3, pERK1/2 and ERK1/2 levels




23

determined by Western blotting. B. Upper panel: VACO432, VT1, LIM2405, COLO205 and

HT-29 cells were pre-treated with Crizotinib or AZD8931 for 3h, and thereafter treated with

AZD6244 for 48 hours and apoptosis was assessed by flow cytometric analysis with PI.

Lower panel: Caspase 3/7 activity levels were measured in HT-29, LIM2405 and COLO205

cells, following pre-incubation with Crizotinib or AZD8931 for 3h, and treatment with

AZD6244 for 24 hours. C. Expression levels of PARP and cleaved C3 in COLO205 and HT-

29 cells following treatment with Crizotinib or AZD8931 and AZD6244 for 24h. D. Left panel:

Growth rate of HT-29 xenografts in BALB/c Nude mice treated with vehicle, AZD6244,

Crizotinib or combined AZD6244/Crizotinib treatment. Differences in growth were

determined using Student’s t test and by calculating subsequent P values. ***, P < 0.001.

Right panel: WB analysis of cleaved C3 in HT-29 vehicle- (Vh), AZD6244-, Crizotinib-

(CRIZ) and AZD6244/Crizotinib (COMB)-treated xenografts.

Figure 3. c-FLIPL regulates sensitivity of BRAFMT CRC cells to MEK inhibition. A. HT-

29, COLO205, and LIM2405 cells were pre-treated with Crizotinib or AZD8931 for 3h, and

thereafter treated with AZD6244 for 24 hours and PARP, pro-caspase 8, caspase 8 p41/43,

caspase 8 p18, cleaved caspase 3, cleaved caspase 9, FLIPL, FLIPS, XIAP, BCL-2 and BAX

levels determined by Western blotting. B. HT-29 and COLO205 cells were treated with

AZD6244 for the indicated time. Upper panel: FLIPL, FLIPS, pSTAT3Y705, STAT3, pERK1/2

and ERK1/2 levels were determined by Western blotting. Lower panel: c-FLIP mRNA levels

were determined by real-time PCR. Relative mRNA expression was calculated using the

DDCt method with normalisation to β-actin and GAPDH. C. Upper panel: expression levels

of FLIPL and FLIPS in HT-29 empty vector (EV) and FLIPL overexpressing cells, determined

by Western blotting and Real-time PCR. Lower panel: HT-29 empty vector (EV) and FLIPL

overexpressing cells were treated with AZD6244 for 48 hours and apoptosis assessed by

flow cytometric analysis. D. BRAFMT LIM2405, RKO F6-8, HT-29 and VACO432 cells and

BRAFWT CACO-2, LIM-1215 and COLO320 cells were transfected with c-FLIP siRNA




24

(10nM) for 24 hours and thereafter treated with AZD6244 for 24h. PARP, cleaved caspase 3,

FLIPL, FLIPS, pERK1/2 and ERK1/2 levels were determined by Western blotting.

Figure 4. Combined HDAC1-3-MEK1/2-inhibition results in marked apoptosis in

BRAFMT CRC cells. A. HT-29, LIM2405, VACO432, RKO F6-8 and COLO205 cells were

pre-incubated with Entinostat for 12 hours (2.5μM in HT-29, LIM2405 and VACO432; 5μM in

RKO and 1μM in COLO205), followed by treatment with AZD6244 for 24h. Upper panel:

PARP, FLIPL, pSTAT3Y705, STAT3, pERK1/2 and ERK1/2 levels were determined by

Western blotting. Lower panel: Apoptosis was assessed by flow cytometric analysis. B.

LIM2405 cells were transfected with caspase 8 siRNA (10nM) for 24 hours and thereafter

treated with Entinostat and AZD6244 for 24h. PARP, cleaved caspase 3, pERK1/2, ERK1/2,

pro-caspase 8, cleaved C8 p41/43, cleaved C8 p18 levels were determined by Western

blotting.

Figure 5. Combined HDAC and MEK inhibition results in synergistic reduction in

growth of BRAFMT CRC in vivo. Upper panel: Growth rate of HT-29 xenografts in BALB/c

Nude mice treated with vehicle, AZD6244, Entinostat or combined AZD6244/Entinostat

treatment. Differences in growth were determined using Student’s t test and by calculating

subsequent P values. ***, P < 0.001. Lower panel: WB analysis of cleaved C3 and Ac-

Histone H4 in HT-29 vehicle- (Vh), AZD6244-, Entinostat- (ENT) and AZD6244/Entinostat

(COMB)-treated xenografts.

Figure 6. STAT3 regulates MEKi-induced FLIPL expression levels. A. Upper panel: HT-

29 cells were co-treated with Stattic and AZD6244 for 24 hours and pSTAT3Y705, STAT3,

FLIPL, FLIPS, pERK1/2 and ERK1/2 levels determined by Western blotting. Lower panel:

Flow cytometric analysis of HT-29 following treatment with 5μM Stattic and 1μM AZD6244

for 48 hours. B. Upper panel: pSTAT3Y705, STAT3, FLIPL, FLIPS, cleaved C3, pERK1/2 and




25

ERK1/2 levels in LIM2405 cells following transfection with 10nM AZD9150 for 48 hours and

treatment with AZD6244 for 24 hours. Lower panel: Flow cytometric analysis of LIM2405

following transfection 10nM AZD9150 for 48 hours and treatment with 1μM AZD6244 for 24

hours. C. Schematic overview of mechanism of STAT3 mediated c-FLIPL expression

following MEKi treatment in BRAFMT CRC.




Published OnlineFirst March 26, 2015.Clin Cancer Res Robbie Carson, Basak Celtikci, Catherine S. Fenning, et al. c-FLIPL.in BRAF mutant colorectal cancer by down-regulation of HDAC inhibition overcomes acute resistance to MEK inhibition

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