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Targeting Metabolic Enzymes in Cancer – Clinical Trials Update

Julie O’Neal and Jason Chesney##

Division of Medical Oncology and Hematology, Department of Medicine, University of Louisville,

Molecular Targets Program, James Graham Brown Cancer Center, Louisville, Kentucky

## Communicating Author, 505 South Hancock Street, Clinical and Translational Research Building

Room 424, University of Louisville, Louisville, Kentucky, 40202

Key Words: Glycolysis, Chemotherapy, Glucose

Financial Support: DOD CDMRP BC112204 (JO) and NCI R01-CA149438 (JC)

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Abstract

The uptake and utilization of glucose and glutamine by cancer cells is markedly higher than by

most non-transformed, normal epithelial and mesenchymal cells. This metabolic shift enables the

production of ATP and anabolic precursors necessary for the synthesis of proteins, lipids and nucleotides

required for survival, proliferation and invasiveness. The observations that certain oncogenic proteins

(Ras, c-Myc and HIF-1α) and tumor suppressors (P53, PTEN, Rb and VHL) regulate the expression and

activity of several metabolic enzymes has supported their potential as molecular targets for the

development of anti-neoplastic agents. Indeed, recent pre-clinical studies have shown that several

established and novel inhibitors of metabolic enzymes exhibit reasonable therapeutic indices when tested

in xenograft models of tumorigenesis. In this review, we will discuss the rationale of targeting metabolic

enzymes for the treatment of cancer and then will describe published pre-clinical and clinical data for

several inhibitors of metabolism in cancer.

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Introduction

Death due to cancer remains the second highest cause of mortality in the United States behind

heart disease (www.cdc.gov) indicating that new approaches to identifying and implementing effective

anti-cancer treatments may improve the overall survival and quality of life of our population. The

relatively recent shift from generalized cytotoxic chemotherapies to treatments targeted at genetic

alterations of cancer has dramatically improved clinical outcomes in certain types of cancer. For example,

Imatinib (Gleevec), which inhibits the fusion BCR/ABL protein caused by a t(9;22)(q34;q11)

translocation in patients with chronic myelogenous leukemia (CML), was developed in the 1990s and

now has essentially eradicated CML as a cause of serious morbidity and mortality in the world [1].

Today, approximately 37 targeted agents in the form of small molecule antagonists or blocking antibodies

are FDA-approved for the treatment of cancer. Such therapies are designed to inhibit cancer-specific

signaling pathways (e.g. downstream of oncogenic Ras or the EGF receptor) or processes (e.g. tumor

angiogenesis induced by VEGF) with the goal of preferentially killing tumor cells without affecting the

viability of normal cells. Although an old concept, metabolic reprogramming recently has been

recognized as an additional essential characteristic of human cancers [2] and several agents targeted

against signaling pathways have been found to modulate the metabolism of cancer cells (Table I). These

observations have encouraged the development of small molecules designed to take advantage of the

metabolic differences of normal versus tumor cells with the goal of generating improved anti-cancer

drugs with unique mechanisms of action. In this review, we will discuss the rationale for metabolic

reprogramming in cancer cells and describe several drugs that target metabolism and show promise in

preclinical studies and/or clinical trials.

Rationale for Increased Metabolism In Cancer

Tumor cells consume a relative excess of glucose via passive glucose transporters and the

quantitation of 18F-2-fluoro-2-deoxy-glucose (FDG) uptake by tumors using positron emission

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tomography (PET) is a commonly used diagnostic measure that correlates directly with tumor

aggressiveness and patient prognosis [3, 4]. The increased metabolism of glucose to lactate by cancer

cells occurs even in the presence of oxygen (i.e. aerobic glycolysis or the “Warburg Effect”) [5].

Although currently an area of active research, it is generally accepted that rapidly dividing cancer cells

increase glycolytic flux for anabolic and energetic purposes enabling them to thrive in a low nutrient,

oxygen poor environment. Paradoxically, glycolysis only yields two ATP per glucose as opposed to 38

ATP via oxidative phosphorylation. However, the rate of ATP production via glycolysis is faster than via

oxidative phosphorylation with less free radical formation [6]. In addition to producing ATP, multiple

enzymes in glycolysis generate products that can be shunted to alternative pathways allowing for nucleic

acid, amino acid, and lipid synthesis essential for cell division. For example, glucose-6-phosphate,

fructose-6-phosphate and glyceraldehyde-3-phosphate can all be metabolized via the oxidative or non-

oxidative pentose phosphate pathways allowing for ribose-5-phosphate production and ultimately

nucleotide biosynthesis. Additionally, 3-phosphoglycerate (3PG) and pyruvate feed into pathways that

generate amino acids and 3PG and dihydroxyacetone are precursors for lipid biogenesis. High glycolytic

flux also lowers the extracellular pH causing p53 dependent apoptosis of normal cells facilitating

invasiveness [7] and the glycolytic end-product lactate also provides an essential carbon source for

supporting fibroblasts [8]. Increased glycolytic flux thus permits a ready supply of ATP and anabolic

precursors essential for cancer cell proliferation while simultaneously promoting invasiveness and support

from host cells.

Regulation of Metabolism by Oncogenes and Tumor Suppressors

Although the altered metabolism of cancer cells was discovered almost a century ago by Otto

Warburg, more recent research has demonstrated that changes in tumor metabolism may be causally

related to the selection of oncogenic/tumor suppressor mutations that directly induce and/or activate

glycolytic and mitochondrial proteins which, in turn, are required for survival, proliferation and

invasiveness. Expression of mutated Ras oncogenes causes increased glucose uptake [9, 10] and

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glycolytic flux [11] likely as a result of the ability of Ras to increase transcription and translation of

hypoxia-inducible factor 1-α (HIF1α) which promotes the transcription of multiple metabolic genes

involved in glycolysis including the glucose transporter-1 (GLUT1), hexokinase-2 (HK2), 6-

phosphofructo-1-kinase (PFK1) and lactate dehydrogenase-A (LDHA) [12]. Additionally, ectopic

expression of oncogenic Ras or HIF-1α increases protein levels of 6-phosphofructo-2-kinase/fructose-2,6-

biphosphatase-3 (PFKFB3), which, by producing fructose-2,6-bisphosphate (F26BP), activates 6-

phosphofructo-1-kinase (PFK1), a key regulated, irreversible and committed step of glycolysis [9, 13].

Like Ras, c-Myc alters tumor cell metabolism specifically through regulation of glycolytic genes

including hexokinase-2, PFK-1, LDH-A, enolase and pyruvate kinase M2 [14]. Congruently, the loss of

tumor suppressor function also has been found to activate glycolysis. For example, loss of functional p53

stimulates the expression of glucose transporters (GLUT1 and GLUT4) [15] and the glycolytic enzyme

phosphoglycerate mutase [16]. Additionally, the loss of PTEN has recently been found to reduce

APC/Cdh1-mediated degradation of PFKFB3 which activates glycolytic flux through PFK-1 [17]. Tumor

oxidative phosphorylation is also linked to oncogenes as expression of oncogenic Ras (H-RASV12) in

immortalized cells increases flux through the tricarboxylic acid cycle (TCA), oxygen consumption, and

sensitivity to electron transport (Complex I) inhibition [9] and cytochrome c oxidase Vb is a translational

target of H-RASV12 [18]. Additionally, multiple mitochondrial genes are c-Myc targets including

cytochrome c oxidase 5b and cytochrome c and c-Myc-deficient cells display reduced oxygen

consumption relative to Myc expressing cells [19]. The relatively recent observation that some tumor

cells die if glutamine is withdrawn led to the idea that some tumors are “glutamine addicted” [20]. The

glutamine requirement of cancer cells is at least in part due to c-Myc, which leads to mitochondrial

glutaminolysis [21]. As with glycolysis, the loss of a tumor suppressor, in this case Rb, also has been

found to increase glutamine metabolism [22, 23].

Basis for Targeting Metabolism as a Therapeutic Strategy in Cancer

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Efforts to directly target the proteins that result from genetic changes that activate oncogenes (e.g.

Ras, c-Myc, etc.) have largely proven ineffective. Two notable exceptions are the previously mentioned

BCR/ABL inhibitor Imatinib which has had a great impact on the lives of CML patients and a new B-

RafV600E inhibitor, Vemurafenib, that is yielding dramatic albeit relatively transient partial responses in

stage IV melanoma patients [1, 24]. Unfortunately, the vast majority of these novel signaling inhibitors

have had only limited success in the clinic as a result of the acquisition of resistance mutations and hyper-

activation of alternative signaling pathways [25]. Small molecule antagonists of metabolic proteins have

a high potential for being effective and druggable targets since: (i) many metabolic regulators are

differentially expressed in normal versus tumor cells; (ii) cancer cells are frequently addicted to certain

metabolites, for example glutamine; (iii) tracing metabolites provides a feasible biomarker for clinical

responses (e.g. FDG-PET imaging); (iv) testing for genetic deletion of upstream oncogenes or tumor

suppressor genes should be predictive of clinical effectiveness (e.g. PTEN status and PFKFB3 inhibitors);

and (v) there is prior success targeting metabolism (i.e. inhibitors of nucleotide synthesis such as anti-

folates in acute lymphoblastic leukemia and 5-fluorouracil in gastrointestinal cancers). Below, we will

discuss a subset of promising small molecules that target tumor-specific metabolic proteins and that are

currently in preclinical and/or clinical development for the treatment of cancer.

Targeting LDA-A: AT-101 and FX11

The lactate dehydrogenase enzyme is a tetrameric complex comprised of LDH-A (LDH-M,

muscle) and/or LDH-B (LDH-H, heart) subunits encoded by two separate genes (LDHA and LDHB,

respectively) that combine to generate five separate isozymes (LDH1-5) which contain differing ratios of A

and B subunits. A separate gene, LDH-C encodes LDH-C that is expressed in sperm and testes only. The

LDH enzyme complex catalyzes the reversible conversion of pyruvate to lactate. Generation of lactate

from pyruvate replenishes NAD+ required for enhanced flux through the glyceraldehyde-3-phosphate

dehydrogenase step of glycolysis and can provide a carbon source to adjacent cells. Increased expression

of LDH-A has been observed in several tumor types [26]. Additionally, reduction of LDH-A expression

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in tumor cells with siRNA molecules causes a dramatic decrease in tumor size and increase life span in a

murine model of breast cancer [27] as well as reduced tumor growth in a separate lung tumor xenograft

model [26] suggesting that inhibition of LDH-A activity in tumor cells may be an effective anti-tumor

therapy.

Gossypol is naturally found in the cotton plant and was originally tested in China as a male

contraceptive. AT-101 (Ascenta Therapeutics) is an orally bioavailable form of the R-(-) enantiomer of

gossypol that inhibits LDH activity. It also is reported to antagonize the anti-apoptotic BCL2 family of

proteins by acting as a BH3 domain mimetic [28]. Clinical efficacy results of AT-101 were mixed.

Reduced PSA levels were observed in some patients when AT-101 was given as a single agent to

castrate-resistant prostate cancer (CRPC) patients [29]. Preliminary results in a phase II study

demonstrated that 26% of newly diagnosed, metastatic, androgen dependent prostate cancer patients

treated with AT-101 and androgen deprivation therapy had undetectable PSA at seven months [30].

However, in a phase II, placebo controlled trial in metastatic CRPC patients, AT-101 given with

docetaxel and prednisone resulted in no statistical improvements in survival or progression free survival

[31]. Treatment of advanced small cell lung cancer (SCLC) patients with AT-101 and topotecan enabled

limited partial responses (PR) and stable disease (SD); however the study did not meet criteria for further

enrollment into the expansion Phase II of this Phase I/II study [32]. No improvement in PFS or response

rate (RR) were seen in patients with advanced non-small cell lung cancer (NSCLC) treated with AT-101

plus docetaxel, although an increase of 1.9 months in median survival was observed [33].

Disappointingly, no responses were seen in a trial of chemotherapy sensitive recurrent NSCLC patients

treated with AT-101 as a single agent [34]. A phase I study conducted in 24 patients with a variety of

refractory solid tumors treated with AT-101, paclitaxel, and carboplatin resulted in four CRPC patients

with SD and multiple PRs (two CRPC, one NSCLC, and one esophageal adenocarcinoma) [35].

Preliminary results from a Phase I trial conducted in previously untreated CLL patients with high risk

features treated with AT-101 resulted in multiple patients achieving decreases in lymphocyte counts,

lymphadenopathy and spleen size [36]. There are currently two ongoing Phase II clinical trials combining

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AT-101 with chemotherapy. In the first, AT-101 and docetaxel are being studied in patients with

squamous cell carcinoma of the head and neck (NCT01285635) and the second will assess AT-101 in

combination with chemotherapy (docetaxol and cisplatin or carboplatin) in laryngeal cancer patients

(NCT01633541).

FX-11 was originally identified in a study that sought to synthesize a specific inhibitor of malarial

LDH [37, 38]. It is a small molecule derivative of the LDH inhibitor, gossypol and is competitive to the

LDH substrate NADH. It has a Ki for LDH-A that is twenty times that of LDH-B (LDH-A: 0.05µM;

LDH-B: 1µM) [37, 38] and almost 40 times better than gossypol for LDH-A (1.9µM) [38]. Treatment of

cells with FX11 (or siRNA mediated knockdown of LDH-A) increases oxygen consumption and

generation of reactive oxygen species through elevated mitochondrial glucose oxidation [39] [27]. FX11

has been found to be effective in reducing tumor burden in three mouse models: (i) a P4398 B cell

xenograft model where treatment was initiated just after tumors were palpable; (ii) a human lymphoma

xenograft model; and (iii) a P198 pancreatic adenocarcinoma model where treatment was initiated after

tumors were well established (200 mm3). Mice treated with FX11 displayed no weight loss, normal

hematology and blood chemistry that included multiple markers of kidney (blood urea nitrogen, creatine)

and liver (aspartate aminotransferase, alanine aminotransferase or alkaline phosphatase) function,

suggesting that FX11 is not toxic [39]. Humans deficient for LDHA develop normally although they

suffer from exertional myopathy [40], suggesting that inhibition of LDH-A will be well-tolerated under

non-exertional circumstances. Although there are no current clinical trials involving FX11, a recent study

reports the synthesis of new gossypol derivatives that show toxicity to tumor cell lines suggesting that

these agents will continue to be developed for possible clinical trial testing [41].

Targeting PDK: CPI-613 and Dichloroacetate (DCA)

The pyruvate dehydrogenase complex (PDH) is composed of three enzymes: pyruvate

dehydrogenase (E1; comprised of two α and two β subunits with the active site in the α subunit),

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dihydrolipoamine acetyltransferase (E2) and lipoamide dehydrogenase (E3). Together, they catalyze the

conversion of pyruvate to Acetyl CoA for further oxidation in the TCA cycle. PDH is positively regulated

in situations of low energy by pyruvate dehydrogenase phosphatase (PDP) and negatively regulated by

phosphorylation of the E1α subunit by the serine/threonine pyruvate dehydrogenase kinase (PDK). When

PDH is active, it becomes saturated with cofactors, including covalently bound lipoate (lipoamide) that,

when sensed by PDK, results in phosphorylation of the E1α subunit of the PDH complex leading to its

inactivation and reduced conversion of pyruvate into Acetyl CoA. Glycolysis and the TCA cycle are

linked by PDH which, when activated, directs carbons away from lactate production and into the TCA

cycle. Since the TCA cycle is important for the production of anabolic precursors, inhibition of PDH is

potentially an effective anti-tumor therapy. There are two promising drugs that target PDH: CPI-613 that

inhibits PDH and DCA that activates PDH (discussed below). At first glance it seems counterintuitive

that both inactivating and activating PDH would be toxic to tumor cells. However, since tumor cells

require an activated glycolytic pathway, TCA cycle and electron transport chain, maintaining a controlled

integration between these pathways via PDH is thought to be essential for cancer cell survival [42].

A liopamide mimic, CPI-613 activates PDK, leading to phosphorylation and inactivation of PDH

[43]. Preclinical studies have revealed that CPI-613 reduced PDH activity and effectively killed tumor

cells but was less toxic to primary normal cell counterparts. Knockdown of all four PDK isoforms

resulted in resistance to CPI-613 treatment, indicating a requirement of PDK expression for CPI-613-

mediated cell death. Significantly, reduced tumor volumes were seen in pancreatic and lung xenograft

models with median survival in the pancreatic model extended to 192.5 days with CPI-613 treatment

versus 48 days with vehicle [43]. CPI-613, the lead clinical candidate in Cornerstone Pharmaceuticals

Altered Energy Metabolism Directed Platform, was granted orphan drug status for pancreatic cancer by

the FDA, and is currently being tested in clinical trials. A phase I trial to determine the safety,

tolerability, MTD, efficacy and pharmacokinetic profile of CPI-613 given IV twice a week for three

weeks in patients with advanced hematologic malignancies (NCT01034475) and a phase I/II trial

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(NCT00741403) to assess the same criteria in patients with advanced solid malignancies and lymphoma

are currently ongoing. Promising results from the hematologic study showed that CPI-613 was well

tolerated in the thirteen patients (dose ranged from 420-1386mg/M2) with no bone marrow suppression or

dose-limiting-toxicities (DLT). Seven of the thirteen patients had SD or better with an overall response

rate of 54%, suggesting CPI-613 may be an effective treatment for hematologic malignancies [44]. A

Phase I/II study (NCT00907166) will assess the safety of CPI-613 and gemcitabine in patients with solid

tumors (phase I) with the phase II part of the trial comparing CPI-613 and gemcitabine treatment to

gemcitabine alone in pancreatic cancer patients. Results from the phase I part of the trial revealed that

CPI-613 was well tolerated (no DLTs), and four of the eight patients with breast and colon cancer treated

had SD (4-16 weeks) with reductions in glucose uptake (4-42%) by FDG-PET imaging [45]. Initial

results from the phase II study showed that administration of CPI-613 and gemcitabine was well tolerated

and that prolonged survival correlated with increased CPI-613 dose [46]. The lack of toxicity and initial

positive patient outcomes suggests that targeting PDH with CPI-613 may hold promise as an anti-cancer

therapeutic agent in patients with solid tumors including pancreatic cancer.

As opposed to CPI-613, which activates PDK, and therefore inhibits PDH, DCA inhibits PDK,

leading to activation of PDH [47, 48]. This results in a decrease in glycolytic flux to lactate and increase

in oxidative metabolism which may starve adjacent cancer cells and supporting host cells of oxygen.

Additionally, as mentioned above, since both activating and inhibiting PDH activity can be toxic to

cancer cells, cancer cells may be especially sensitive to disruption of the normal metabolic integration of

the glycolytic pathway and the TCA cycle. The validation of DCA as an anti-cancer agent stems from an

initial report that demonstrated dramatic reductions in tumor size after DCA treatment in a nude rat A549

xenograft tumor model [48]. DCA is appealing as a therapeutic agent for widespread testing as a

monotherapy and in combination with standard agents since it is orally bioavailable and has little if any

patent restrictions. However, DCA was not found to be effective in reducing tumor size or improving

survival in a xenograft mouse model of breast cancer [49] suggesting that DCA may be effective in only

certain types of tumors. Since glioblastoma multiforme (GBM) tumors are very glycolytic, and DCA can

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cross the blood brain barrier, it was tested in a small clinical trial that included five GBM patients. Three

patients had failed prior standard therapy (debulking surgery, radiation therapy (RT), temozolomide

(TMZ)) and subsequent chemotherapies. These three patients were treated with DCA as a single agent.

One patient had a large tumor with brain edema at the start of treatment and died three months later from

complications related to the edema. However, after fifteen months of DCA treatment, the remaining four

patients had stable disease by CT imaging and all were still alive at eighteen months [50]. Although a

small trial, the results of this study provide hope that DCA may inhibit tumor growth and prolong survival

in GBM patients. There now are three ongoing cancer clinical trials involving DCA. The first is a Phase I

safety and efficacy study in patients with recurrent brain tumors (NCT0111097). The second is a Phase I

trial that is examining the safety of DCA in patients with recurrent or metastatic solid tumors

(NCT00566410) and the third is a placebo controlled Phase II study that will determine safety and

efficacy of DCA treatment in combination with cisplatin and radiation in patients with head and neck

carcinomas (NCT01386632).

Targeting Glucose Transporter 1 (GLUT1): STF-31

Von Hippel Lindau (VHL) is a tumor suppressor inactivated in most (~80%) spontaneous renal

cell carcinomas (RCC). A synthetic lethal screen designed to find agents that specifically kill VHL null

cells identified STF-31, a compound that is toxic to VHL negative RCCs in a HIF-1α−dependent manner

[51]. Since HIF-1α regulates expression of multiple glycolytic enzyme genes including the glucose

transporter GLUT1, STF-31 was tested for its ability to inhibit GLUT1 functions. STF-31 blocked

glucose uptake and reduced tumor size in renal cell carcinoma xenograft models with no reported

toxicities. STF-31 is thought to inhibit GLUT1 and the prediction thus is that it will be effective against

multiple tumor types that express GLUT1, and not just VHL negative RCCs, although this has yet to be

tested. STF-31 is currently licensed to Ruga Incorporated and is in preclinical testing with Phase I trials

predicted for 2013/2014 (Rugacorp.com).

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Targeting 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB3): 3PO/PFK158

The PFKFB3 kinase domain phosphorylates fructose 6-phosphate (F6P) to generate fructose 2,6-

bisphosphate (F26BP), a potent allosteric activator of PFK-1. Since activation of PFK-1 is a key

regulatory step in glycolysis, modulation of PFKFB3 activity directly affects flux through the entire

glycolytic pathway [52]. PFKFB3 is a member of a family of dual kinase:bisphosphatases (PFKFB1-4)

that phosphorylate and dephosphorylate F6P. Unlike the other family members, PFKFB3 functions

essentially solely as a kinase with a kinase:bisphosphatase ratio of ~740:1 [53]. PFKFB3 is expressed at

low levels in most normal human tissues, and is not expressed in neurons [54], but is highly expressed in

many human cancers including lung, breast, prostate and colon tumors when compared to matched

normal samples [55]. Importantly, PFKFB3 is upregulated by multiple oncoproteins including HIF-1α

and Ras [9, 56]. Additionally, recent data suggests that PFKFB3 is negatively regulated by the PTEN

tumor suppressor gene [57] which promotes the APC/CDH1 degradation complex that post-

translationally negatively regulates PFKFB3 [58]. PTEN null cells therefore are predicted to have higher

PFKFB3 expression and potentially higher reliance on the activity PFKFB3. Accordingly, PTEN status in

human tumors may be a predictive biomarker for sensitivity to PFKFB3 inhibitors. Last, the requirement

of PFKFB3 for neoplastic transformation was recently demonstrated by the observations that

heterozygous genomic deletion of the Pfkfb3 gene reduced the concentration of F2,6BP, glucose uptake,

glycolytic flux to lactate and anchorage-independent growth of the LT/H-RasV12-transformed fibroblasts

as xenograft tumors in syngeneic mice [52, 59].

In silico screening identified an inhibitor of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-

one (3PO), that inhibits glucose uptake, cellular F26BP production, glycolytic flux to lactate, growth of

cancer cell lines, and importantly, glucose uptake and tumor growth in multiple mouse tumor models

[60]. Development of 3PO derivatives by the biotechnology company, Advanced Cancer Therapeutics,

has resulted in the identification of an initial lead compound, PFK15 with improved (~100X) potency for

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inhibition of recombinant enzyme activity over 3PO   (2011 American Association for Cancer Research

Annual Meeting, Abstract #2825). PFK15 provided a synthetic platform for the synthesis of third

generation derivatives, which led to the pharmaceutical-grade agent, PFK158, that exhibits increased

potency and improved pharmacokinetic properties. PFK158 has undergone IND-enabling rat and beagle

toxicity testing with Phase I clinical trials slated to begin in 2013

(www.advancedcancertherapeutics.com). Kancera AB (Sweden) is also testing PFKFB3 inhibitors that

are in preclinical lead optimization phase with the plan to identify a candidate for clinical trial testing by

the end of 2012 (www.kancera.com).

Targeting Complex I: Metformin

For  millennia,  the  herb  Galega  officinalis  (French  Lilac,  Italian  Fitch  or  Goat’s  Rue)  has  been  

used  to  produce  tea  to  relieve  frequent  urination  and  sweet-­‐smelling  breath.    This  herbal  remedy  

for  what  was   eventually   found   to   be   caused   by   the   hyperglycemia   of  Diabetes  Mellitus   (DM)   led  

several   investigators   during   the   20th   century   to   purify   the   active   components   of   the   herb,  

biguanides,   including   Phenformin,   Buformin   and   Metformin   (i.e.   N’,N’-­‐dimethylbiguanide).    

Although  Phenformin  and  Buformin  were  limited  by  toxicity  related  to  lactic  acidosis,  Metformin  is  

currently  FDA-­‐approved  and  widely  used  for  the  treatment  of  DM.    The  precise  mechanism  of  action  

of   metformin   is   not   well   defined   but   the   agent   does   inhibit   complex   I   of   the   electron   transport  

chain,  oxygen  consumption  and  ATP   in  hepatocytes   [61,  62].    Such  a  decrease   in   the   intracellular  

concentration  of  ATP  will  cause  an  allosteric  activation  of  6-­‐phosphofructo-­‐1-­‐kinase  and  resultant  

elevation   in   glucose   uptake   and   glycolytic   flux   [63]   as  well   as   activation   of   AMP   kinase   (AMPK)  

which  increases  glucose  transporter  (GLUT4)  expression  and  translocation  in  myocytes  [64]  which  

may   in   part   explain   the   anti-­‐diabetic   effects   of  Metformin.     Several   retrospective   epidemiological  

studies  of  diabetic  patients  who  were  treated  with  Metformin  have  found  that  these  patients  had  a  

lower  risk  of  developing  all  types  of  cancer  and  of  cancer-­‐related  deaths  relative  to  diabetic  patients  

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who   received   other   oral   glucose-­‐lowering   agents   [65-­‐68].     Additionally,   diabetic   breast   cancer  

patients   on   Metformin   were   found   after   resection   to   experience   a   higher   rate   of   microscopic  

complete  responses  after  neoadjuvant  chemotherapy  than  diabetic  patients  not  being  treated  with  

Metformin  or  non-­‐diabetic  patients  [69].      The  mechanism  for  these  statistically  significant  effects  is  

an  area  of  active  investigation  and  several  pre-­‐clinical  and  clinical  investigators  are  now  attempting  

to  improve  outcomes  of  standard  anti-­‐neoplastic  agents  with  Metformin.    One  potential  hypothesis  

is   that   inhibition   of   electron   transport   chain   activity   will   limit   the   availability   of   NAD+   that   is  

required  for  TCA  cycling  which,  in  turn,  is  required  for  the  production  of  anabolic  precursors.      

  Importantly,  preclinical   studies   in  multiple  non-­‐diabetic  mouse   tumor  models   in  vivo  have  

demonstrated   that   metformin   may   be   effective   in   patients   with   normal   metabolism   (i.e.   non-­‐

diabetics).  For  example,  treatment  of  mice  with  metformin  prior  to  development  of  breast  tumors  

(MMTV-­‐HER-­‐2/neu),   resulted   in   delayed   tumor   onset,   reduced   number   of   tumors,   and   improved  

overall  survival  [70].  Metformin  also  has  been  found  to  reduce  tumorigenicity  in  mouse  models  of  

liver  cancer  [71],  lung  cancer  [72]  and  intestinal  cancer  [73]. Several clinical trials are now underway

to assess whether metformin as a single agent or in combination with other chemotherapies can improve

patient outcomes specifically in non-diabetic patients. A completed Phase I trial combining metformin

and temsirolimus conducted in patients with solid tumors evaluated eight patients for disease outcomes.

Two patients had disease progression, five had SD, and one had a PR. One of the patients with SD was a

melanoma patient that had radiologic progression on chemotherapy prior to the study who had SD for 22

months. Although a small study, these clinical results demonstrated that some solid cancers may be

sensitive to combined temsirolimus and metformin treatment [74].  

Final Comments and Future Directions

There is and always will be concern that targeting metabolic pathways required for both normal

and tumor cells will yield unacceptable therapeutic indices in cancer patients. However, given the lack of

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effective therapies against the majority of human cancers that have metastasized and the abundance of

pre-clinical data demonstrating efficacy without overt clinical toxicity, the clinical testing of metabolic

inhibitors seems warranted. Importantly, AT-101 has thus far been well tolerated in humans, as has been

the BCR/ABL inhibitor Imatinib, which is a potent inhibitor of glycolysis in CML cells [75].

Additionally, unanticipated mechanisms of action may be appreciated in clinical trials of metabolic

inhibitors which may in turn explain positive pre-clinical in vivo data. For example, rapidly dividing T

cells utilize glycolysis [76] and the PFKFB3 inhibitor, 3PO, has been found to suppress T cell activation

in vitro and in vivo [77]. Given the immunosuppressive effects of regulatory T cells in cancer patients

[78], PFKFB3 inhibitors may have positive anti-tumor effects on the immune system which may

contribute to their anti-tumor properties. Last, since metabolic pathways are increased in tumor versus

normal cells, a therapeutic window for metabolic modulators may be reached whereby treatment is below

the threshold for causing toxicity, but sufficient to kill tumor cells. Since metabolic reprogramming is

universal to tumor cells, modulating this phenotype with drugs has the potential to be useful for the

treatment of a wide range of tumor types. This does not imply however that the same metabolic inhibitors

will be effective in all tumor types. For example, as discussed above, PTEN status may predict tumor

sensitivity to inhibition of PFKFB3 but perhaps not to inhibition of other metabolic proteins. Given the

distinct mechanisms of action of the aforementioned metabolic inhibitors, we expect the development of

multiple phase I/II trials in which these inhibitors are combined with FDA-approved agents that are the

standard of care for distinct cancer types (e.g. vemurafenib in melanoma) as well as the rational

combination of glycolytic inhibitors such as PFK158 with agents that suppress angiogenesis, electron

transport chain activity, glutamine metabolism or alternative pathways for energy and anabolic precursor

production such as autophagy.

16    

Figure Legend

Figure 1. Metabolic Inhibitors In or Entering Clinical Trials. Ras, HIF-1α and c-Myc increase the

expression of metabolic transporters and enzymes which in turn cause a reprogramming of metabolic

utilization that supports the enhanced energetic and anabolic requirements of cancer cells. Several

metabolic inhibitors are under study in clinical trials, including the following inhibitors (targets in

parantheses): (i) STF-31 (Glut1); (ii) 3PO/PFK158 (PFKFB3); (iii) FX11/AT-101 (LDH-A); (iv)

DCA/CPI-613 (PDK); and (v) Metformin (Met; Complex I). Black lines indicate suppression and red

lines indicate stimulation of activity and/or expression. Key regulators, transporters and enzymes are

highlighted in red. GLUT1: Glucose transporter 1; HK2: Hexokinase 2; PFK1: 6-phosphofructo-1-kinase;

PK-M2: Pyruvate kinase M2; PDH: Pyruvate dehydrogenase; LDH-A: Lactate dehydrogenase A;

PFKFB3: 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3.

17    

Acknowledgements

We would like to thank Brian Clem, Yoannis Imbert-Fernandez, Sucheta Telang, Alden Klarer and John

Eaton for their critical reviews of this article. We also acknowledge the essential support of the National

Cancer Institute (R01-CA149438; JC) and the Congressionally Directed Medical Research Program

(BC112204; JO) for the completion of this review article.

18    

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28    

Table I. Effects of FDA-Approved Targeted Cancer Agents on Glycolysis*

*The PubMed Database was searched for abstracts that contained the generic name of each agent (n = 37) and one or both of the following medical subject headings: glucose and/or glycolysis. The following are targeted agents in which no published articles were found that demonstrated direct effects on glycolysis: Dasatinib, Nilotinib, Bosutinib, Pertuzumab, Lapatinib, Gefitinib, Erlotinib, Panitumumab, Crizotinib, Vorinostat, Romidepsin, Bexarotene, Aliretinoin, Tretinoin, Bortezomib, Sunitinib, Pazopanib, Regorafenib, Cabozantinib, Rituximab, Alemtuzumab, Ofatumumab, Ipilimumab, Tositumomab, Ibritumomab, Denileukin Diftitox, Brentuximab and Carfilzomib.

Generic Name Cancer Indications Target(s) Metabolic Effects Imatinib Chronic Myelogenous Leukemia,

Gastrointestinal Stromal Tumors Receptor Tyrosine Kinases (RTKs), Bcr-Abl, c-Kit

↓ Glucose Uptake [79] ↓ Glycolytic Flux to Lactate [79] ↑ TCA Cycling [79]

Tamoxifen Breast Cancer Estrogen Receptor ↓ Glycolytic Flux to Lactate [80]

Trastuzumab Breast Cancer ErbB2/HER-2 ↓ LDH-A [81]

Cetuximab Head and Neck, Colorectal Cancer ErbB1/EGFR ↓ Glucose Uptake [82]

Temsirolimus Renal Cell Carcinoma (RCC) mTOR ↓ Glucose Transporter Glut1[83] ↓ Glucose Uptake [84]

Everolimus RCC, Astrocytoma, Breast Cancer, Pancreatic Neuroendocrine Tumors

Immunophilin FKp12/mTOR

↓ LDH-A and HK-2 [85] ↓ Lactate Production [85]

Vandetanib Medullary Thyroid Cancer RTKs ↓ Glucose Uptake [86] ↓ Glycolytic mRNAs [86]

Bevacizumab Glioblastoma, Colorectal Cancer, Non-Small Cell Lung Cancer, RCC

VEGF/ Angiogenesis

↑ Lactate Secretion [87] ↓ Mitochondria [87]

Sorafenib RCC, Hepatocellular Carcinoma RTKs/ Angiogenesis

↑ Glycolysis [88] ↓ Oxygen Consumption [88]

Figure 1

Acetyl CoA

PDH

NADH

NAD+

2e-+2H+

H+

I

IIIII

IV

+½O2→H2O

TCA Cycle

Glucose

G6P

Glucose

HK2

F16P

F6P

PFK1

G3PDHA

Pyruvate

Ribose-5P

LDH-A

Lactate

X5P

PFKFB3

F26BP

PEP

PK-M2

GLUT1

+ + +

HIF-1a

Glutamine

Ras

Glutamine

PDKs

c-Myc

Glutamate

ATP-SMitochondria

STF-31

3PO

PFK158

FX11/AT-101

DCA

CPI-613+

Met