Sung Choe

Small Molecule Activation of PKM2 in Cancer Cells Induces Serine Auxotrophy

Proliferating tumor cells use aerobic glycolysis to support their high metabolic demands. Paradoxically, increased glycolysis is often accompanied by expression of the lower activity PKM2 isoform, effectively constraining lower glycolysis. Here, we report the discovery of PKM2 activators with a unique allosteric binding mode. Characterization of how these compounds impact cancer cells revealed an unanticipated link between glucose and amino acid metabolism. PKM2 activation resulted in a metabolic rewiring of cancer cells manifested by a profound dependency on the nonessential amino acid serine for continued cell proliferation. Induction of serine auxotrophy by PKM2 activation was accompanied by reduced carbon flow into the serine biosynthetic pathway and increased expression of high affinity serine transporters. These data support the hypothesis that PKM2 expression confers metabolic flexibility to cancer cells that allows adaptation to nutrient stress.

Metabolic and Functional Genomic Studies Identify Deoxythymidylate Kinase as a target in LKB1 Mutant Lung Cancer

The LKB1/STK11 tumor suppressor encodes a serine/threonine kinase, which coordinates cell growth, polarity, motility, and metabolism. In non-small cell lung carcinoma, LKB1 is somatically inactivated in 25% to 30% of cases, often concurrently with activating KRAS mutations. Here, we used an integrative approach to define novel therapeutic targets in KRAS-driven LKB1-mutant lung cancers. High-throughput RNA interference screens in lung cancer cell lines from genetically engineered mouse models driven by activated KRAS with or without coincident Lkb1 deletion led to the identification of Dtymk, encoding deoxythymidylate kinase (DTYMK), which catalyzes dTTP biosynthesis, as synthetically lethal with Lkb1 deficiency in mouse and human lung cancer lines. Global metabolite profiling showed that Lkb1-null cells had a striking decrease in multiple nucleotide metabolites as compared with the Lkb1-wild-type cells. Thus, LKB1-mutant lung cancers have deficits in nucleotide metabolism that confer hypersensitivity to DTYMK inhibition, suggesting that DTYMK is a potential therapeutic target in this aggressive subset of tumors.

AG-221, a First-in-Class Therapy Targeting Acute Myeloid Leukemia Harboring Oncogenic IDH2 Mutations.

Somatic gain-of-function mutations in isocitrate dehydrogenases (IDH) 1 and 2 are found in multiple hematologic and solid tumors, leading to accumulation of the oncometabolite (R)-2-hydroxyglutarate (2HG). 2HG competitively inhibits α-ketoglutarate-dependent dioxygenases, including histone demethylases and methylcytosine dioxygenases of the TET family, causing epigenetic dysregulation and a block in cellular differentiation. In vitro studies have provided proof of concept for mutant IDH inhibition as a therapeutic approach. We report the discovery and characterization of AG-221, an orally available, selective, potent inhibitor of the mutant IDH2 enzyme. AG-221 suppressed 2HG production and induced cellular differentiation in primary human IDH2 mutation-positive acute myeloid leukemia (AML) cells ex vivo and in xenograft mouse models. AG-221 also provided a statistically significant survival benefit in an aggressive IDH2R140Q-mutant AML xenograft mouse model. These findings supported initiation of the ongoing clinical trials of AG-221 in patients with IDH2 mutation-positive advanced hematologic malignancies.Significance: Mutations in IDH1/2 are identified in approximately 20% of patients with AML and contribute to leukemia via a block in hematopoietic cell differentiation. We have shown that the targeted inhibitor AG-221 suppresses the mutant IDH2 enzyme in multiple preclinical models and induces differentiation of malignant blasts, supporting its clinical development. Cancer Discov; 7(5); 478-93. ©2017 AACR.See related commentary by Thomas and Majeti, p. 459See related article by Shih et al., p. 494This article is highlighted in the In This Issue feature, p. 443.

MTAP Deletions in Cancer Create Vulnerability to Targeting of the MAT2A/PRMT5/RIOK1 Axis

Homozygous deletions of p16/CDKN2A are prevalent in cancer, and these mutations commonly involve co-deletion of adjacent genes, including methylthioadenosine phosphorylase (MTAP). Here, we used shRNA screening and identified the metabolic enzyme, methionine adenosyltransferase II alpha (MAT2A), and the arginine methyltransferase, PRMT5, as vulnerable enzymes in cells with MTAP deletion. Metabolomic and biochemical studies revealed a mechanistic basis for this synthetic lethality. The MTAP substrate methylthioadenosine (MTA) accumulates upon MTAP loss. Biochemical profiling of a methyltransferase enzyme panel revealed that MTA is a potent and selective inhibitor of PRMT5. MTAP-deleted cells have reduced PRMT5 methylation activity and increased sensitivity to PRMT5 depletion. MAT2A produces the PRMT5 substrate S-adenosylmethionine (SAM), and MAT2A depletion reduces growth and PRMT5 methylation activity selectively in MTAP-deleted cells. Furthermore, this vulnerability extends to PRMT5 co-complex proteins such as RIOK1. Thus, the unique biochemical features of PRMT5 create an axis of targets vulnerable in CDKN2A/MTAP-deleted cancers.

D-2-hydroxyglutarate produced by mutant IDH2 causes cardiomyopathy and neurodegeneration in mice

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) have been discovered in several cancer types and cause the neurometabolic syndrome D2-hydroxyglutaric aciduria (D2HGA). The mutant enzymes exhibit neomorphic activity resulting in production of D2-hydroxyglutaric acid (D-2HG). To study the pathophysiological consequences of the accumulation of D-2HG, we generated transgenic mice with conditionally activated IDH2(R140Q) and IDH2(R172K) alleles. Global induction of mutant IDH2 expression in adults resulted in dilated cardiomyopathy, white matter abnormalities throughout the central nervous system (CNS), and muscular dystrophy. Embryonic activation of mutant IDH2 resulted in more pronounced phenotypes, including runting, hydrocephalus, and shortened life span, recapitulating the abnormalities observed in D2HGA patients. The diseased hearts exhibited mitochondrial damage and glycogen accumulation with a concordant up-regulation of genes involved in glycogen biosynthesis. Notably, mild cardiac hypertrophy was also observed in nude mice implanted with IDH2(R140Q)-expressing xenografts, suggesting that 2HG may potentially act in a paracrine fashion. Finally, we show that silencing of IDH2(R140Q) in mice with an inducible transgene restores heart function by lowering 2HG levels. Together, these findings indicate that inhibitors of mutant IDH2 may be beneficial in the treatment of D2HGA and suggest that 2HG produced by IDH mutant tumors has the potential to provoke a paraneoplastic condition.

Durable Remissions with Ivosidenib in IDH1-Mutated Relapsed or Refractory AML

Background Mutations in the gene encoding isocitrate dehydrogenase 1 (IDH1) occur in 6 to 10% of patients with acute myeloid leukemia (AML). Ivosidenib (AG‐120) is an oral, targeted, small‐molecule inhibitor of mutant IDH1. Methods We conducted a phase 1 dose‐escalation and dose‐expansion study of ivosidenib monotherapy in IDH1‐mutated AML. Safety and efficacy were assessed in all treated patients. The primary efficacy population included patients with relapsed or refractory AML receiving 500 mg of ivosidenib daily with at least 6 months of follow‐up. Results Overall, 258 patients received ivosidenib and had safety outcomes assessed. Among patients with relapsed or refractory AML (179 patients), treatment‐related adverse events of grade 3 or higher that occurred in at least 3 patients were prolongation of the QT interval (in 7.8% of the patients), the IDH differentiation syndrome (in 3.9%), anemia (in 2.2%), thrombocytopenia or a decrease in the platelet count (in 3.4%), and leukocytosis (in 1.7%). In the primary efficacy population (125 patients), the rate of complete remission or complete remission with partial hematologic recovery was 30.4% (95% confidence interval [CI], 22.5 to 39.3), the rate of complete remission was 21.6% (95% CI, 14.7 to 29.8), and the overall response rate was 41.6% (95% CI, 32.9 to 50.8). The median durations of these responses were 8.2 months (95% CI, 5.5 to 12.0), 9.3 months (95% CI, 5.6 to 18.3), and 6.5 months (95% CI, 4.6 to 9.3), respectively. Transfusion independence was attained in 29 of 84 patients (35%), and patients who had a response had fewer infections and febrile neutropenia episodes than those who did not have a response. Among 34 patients who had a complete remission or complete remission with partial hematologic recovery, 7 (21%) had no residual detectable IDH1 mutations on digital polymerase‐chain‐reaction assay. No preexisting co‐occurring single gene mutation predicted clinical response or resistance to treatment. Conclusions In patients with advanced IDH1‐mutated relapsed or refractory AML, ivosidenib at a dose of 500 mg daily was associated with a low frequency of grade 3 or higher treatment‐related adverse events and with transfusion independence, durable remissions, and molecular remissions in some patients with complete remission. (Funded by Agios Pharmaceuticals; ClinicalTrials.gov number, NCT02074839.)

Differential Aspartate Usage Identifies a Subset of Cancer Cells Particularly Dependent on OGDH

Although aberrant metabolism in tumors has been well described, the identification of cancer subsets with particular metabolic vulnerabilities has remained challenging. Here, we conducted an siRNA screen focusing on enzymes involved in the tricarboxylic acid (TCA) cycle and uncovered a striking range of cancer cell dependencies on OGDH, the E1 subunit of the alpha-ketoglutarate dehydrogenase complex. Using an integrative metabolomics approach, we identified differential aspartate utilization, via the malate-aspartate shuttle, as a predictor of whether OGDH is required for proliferation in 3D culture assays and for the growth of xenograft tumors. These findings highlight an anaplerotic role of aspartate and, more broadly, suggest that differential nutrient utilization patterns can identify subsets of cancers with distinct metabolic dependencies for potential pharmacological intervention.

Abstract 1194: IDH1 mutant inhibitor induces cellular differentiation and offers a combination benefit with Ara-C in a primary human Idh1 mutant AML xenograft model

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Somatic point mutations in isocitrate dehydrogenase 1/2 have a gain-of-function neomorphic activity that converts alpha-ketoglutarate to the oncometabolite, R (-)-2-hydroxyglutarate (2HG). Prospective studies of AML patients carrying IDH mutations have shown that intracellular concentrations of 2HG can range from 3-10 mM. This abnormal level of 2HG results in dysregulation of alpha-ketoglutarate dependent enzymes leading to alterations in the epigenetic state of hematopoietic progenitor/stem cells and functionally blocks their ability to fully differentiate. We have developed a potent and selective, orally available IDH1 mutant inhibitor AGI-14100, that is able to reduce intracellular 2HG concentrations to baseline levels found in wildtype cells. We next treated a primary human IDH1 (R132H)/FLT3-ITD mutant xenograft model with AGI-14100 either alone or in combination with Ara-c. In these studies, AGI-14100 alone significantly decreased tumor burden in the peripheral blood after 1 month of continuous BID treatment. In combination with a short-term, low-dose course of Ara-C, we also observed a decrease in the bone marrow tumor burden that was better than either treatment alone. Furthermore, this response was sustainable for >3 weeks even after dosing of both drugs had been terminated. Taken together, these data suggest that inhibition of mutant IDH1with AGI-14100 and low dose Ara-c could provide a combination benefit for patients with AML. Citation Format: Kate Ellwood-Yen, Yue Chen, Fang Wang, Rene Lemieux, Janeta Popovici-Muller, Hua Yang, Kimberly Straley, Sung Choe, Marion Dorsch, Sam Agresta, David Schenkein, Scott Biller, Michael Su. IDH1 mutant inhibitor induces cellular differentiation and offers a combination benefit with Ara-C in a primary human Idh1 mutant AML xenograft model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1194. doi:10.1158/1538-7445.AM2014-1194

Abstract 3116: AG-221 offers a survival advantage in a primary human IDH2 mutant AML xenograft model

Somatic point mutations in isocitrate dehydrogenase 1/2 (IDH1/2) confer a gain-of-function in cancer cells resulting in the accumulation and secretion of an onco-metabolite, R (-)-2-hydroxyglutarate (2HG). High levels of 2HG have been shown to inhibit α-KG-dependent dioxygenases including histone and DNA demethylases, which play a key role in regulating the epigenetic state of cells. Recently, ex vivo treatment with AGI-6780, a potent IDH2 R140Q inhibitor induced cellular differentiation of leukemic blast cells isolated from primary human AML patient samples harboring an IDH2 R140Q mutation. These data provided the first evidence that inhibition of mutant IDH2 can reverse the block in cellular differentiation conferred by high levels of 2HG and could provide a therapeutic benefit to patients. AG-221 is a potent and selective inhibitor of the IDH2 mutant enzyme and is currently being evaluated in a first-in-human study entitled: A Phase 1, Multicenter, Open-Label, Dose-Escalation, Safety, Pharmacokinetic, Pharmacodynamic, and Clinical Activity Study of Orally Administered AG-221 in Subjects with Advanced Hematologic Malignancies with an IDH2 Mutation. The compound has been demonstrated to reduce 2-HG levels by >90% and reverse histone and deoxyribonucleic acid (DNA) hypermethylation in vitro, and to induce differentiation in leukemia cell models. We evaluated the efficacy of AG-221 in a primary human AML xenograft model carrying the IDH2 R140Q mutation. This is an aggressive model with mortality from AML consistently occurring by day 80, following tail vein engraftment. Results show that AG-221 is able to potently reduce 2HG found in the bone marrow, plasma and urine of engrafted mice. Treatment also induced a dose dependent, statistically significant, survival benefit where all mice in the high dose treatment group survived to the end of study. We also saw a dose dependent proliferative burst of the human specific CD45+ blast cells followed by cellular differentiation as measured by the expression of CD11b, CD14 and CD15 and cell morphology. Furthermore, the onset of differentiation correlated with survival, whereas mice that died in the low dose groups failed to show signs of cellular differentiation. These data provide strong preclinical in vivo evidence that AG-221 may have clinical benefit for IDH2 mutant patients through the reduction of 2HG and the induction of blast differentiation. Citation Format: Kate Ellwood-Yen, Fang Wang, Jeremy Travins, Yue Chen, Hua Yang, Kim Straley, Sung Choe, Marion Dorsch, Sam Agresta, David Schenkein, Scott Biller, Michael Su. AG-221 offers a survival advantage in a primary human IDH2 mutant AML xenograft model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3116. doi:10.1158/1538-7445.AM2014-3116

Abstract 1002: Induced dependency on amino acid metabolism in proliferating cancer cells upon PKM2 activation

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL It is well recognized that proliferating tumor cells invariably utilize aerobic glycolysis to support their high metabolic demands. Recent studies have further elucidated the paradoxical observation that this increase in glycolysis in proliferating tumor cells is often coupled with a molecular switch from the constitutively high activity PKM1 isoform to the modulated lower activity PKM2 isoform1,2, effectively constraining lower glycolysis at the last pyruvate kinase step. Here, we report an unanticipated link between glucose metabolism and amino-acid metabolic pathways in cancer cells. We characterized the biochemical and cellular effects of a small-molecule allosteric activator of the M2 isoform of pyruvate kinase (AGX-257) in various cancer cell types. We demonstrate that activation of PKM2 results in a metabolic rewiring of specific cancer cells that results in a profound dependency on the presence of the normally non-essential amino acid serine for continued cell proliferation. The induced serine auxotrophy by PKM2 activation is concomitant with reduced flux through the endogenous serine biosynthetic pathway and increased levels of high affinity serine transporters. PKM2 activators, possibly in combination with agents that modulate the cellular and extracellular serine pools, could form the basis for a new approach toward developing therapy for treating cancer. References: 1 Christofk, H. R. et al. The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature 452, 230-233 (2008). 2 Clower, C. V. et al. The alternative splicing repressors hnRNP A1/A2 and PTB influence pyruvate kinase isoform expression and cell metabolism. Proc Natl Acad Sci U S A 107, 1894-1899 (2010). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1002. doi:1538-7445.AM2012-1002