Targeting Metabolic Enzymes in Cancer – Clinical Trials Update

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) 1 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. 2 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 18 F-2-fluoro-2-deoxy-glucose (FDG) uptake by tumors using positron emission 3 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 nonoxidative 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 4 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), 6phosphofructo-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-2,6biphosphatase-3 (PFKFB3), which, by producing fructose-2,6-bisphosphate (F26BP), activates 6phosphofructo-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 5 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 BRafV600E 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 hyperactivation 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 antifolates 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 6 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 7 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), 8 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-613mediated 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 9 (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 10 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). 11 Targeting 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB3): 3PO/PFK158 The PFKFB3 kinase domain phosphorylates fructose 6-phosphate (F6P) to generate fructose 2,6bisphosphate (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 posttranslationally 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-1one (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 12 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 13 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 14 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. 15 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. 16 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. 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Effects of FDA-Approved Targeted Cancer Agents on Glycolysis* Generic Name Cancer Indications Target(s) Imatinib Chronic Myelogenous Leukemia, Gastrointestinal Stromal Tumors Receptor Tyrosine ↓ Glucose Uptake [79] Kinases (RTKs), ↓ Glycolytic Flux to Lactate [79] Bcr-Abl, c-Kit ↑ 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- VEGF/ Small Cell Lung Cancer, RCC Angiogenesis ↑ Lactate Secretion [87] ↓ Mitochondria [87] Sorafenib RCC, Hepatocellular Carcinoma ↑ Glycolysis [88] ↓ Oxygen Consumption [88] RTKs/ Angiogenesis Metabolic Effects *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. 28 STF-31 Glutamine Glucose GLUT1 Glutamine Glucose Ribose-5P HK2 Ras G6P +++ HIF-1a Glutamate X5P F6P F26BP PFK1 F16P PFKFB3 DHA H+ Mitochondria G3P 3PO PFK158 c-Myc ATP-S IV III Met II 2e-+2H+ NAD+ +½O2→H2O I NADH TCA Cycle Acetyl CoA PDH PEP PK-M2 FX11/AT-101 Pyruvate LDH-A Lactate DCA PDKs + CPI-613 Figure 1