Kevin Marks

Cancer-associated IDH1 mutations produce 2-hydroxyglutarate

Mutations in the enzyme cytosolic isocitrate dehydrogenase 1 (IDH1) are a common feature of a major subset of primary human brain cancers. These mutations occur at a single amino acid residue of the IDH1 active site, resulting in loss of the enzyme’s ability to catalyse conversion of isocitrate to α-ketoglutarate. However, only a single copy of the gene is mutated in tumours, raising the possibility that the mutations do not result in a simple loss of function. Here we show that cancer-associated IDH1 mutations result in a new ability of the enzyme to catalyse the NADPH-dependent reduction of α-ketoglutarate to R(-)-2-hydroxyglutarate (2HG). Structural studies demonstrate that when arginine 132 is mutated to histidine, residues in the active site are shifted to produce structural changes consistent with reduced oxidative decarboxylation of isocitrate and acquisition of the ability to convert α-ketoglutarate to 2HG. Excess accumulation of 2HG has been shown to lead to an elevated risk of malignant brain tumours in patients with inborn errors of 2HG metabolism. Similarly, in human malignant gliomas harbouring IDH1 mutations, we find markedly elevated levels of 2HG. These data demonstrate that the IDH1 mutations result in production of the onco-metabolite 2HG, and indicate that the excess 2HG which accumulates in vivo contributes to the formation and malignant progression of gliomas.

Functional genomics reveal that the serine synthesis pathway is essential in breast cancer

Cancer cells adapt their metabolic processes to drive macromolecular biosynthesis for rapid cell growth and proliferation (1,2). RNAi-based loss of function screening has proven powerful for the identification of novel and interesting cancer targets, and recent studies have used this technology in vivo to identify novel tumor suppressor genes (3). Here, we developed a method for identifying novel cancer targets via negative selection RNAi screening in solid tumours. Using this method, we screened a set of metabolic genes associated with aggressive breast cancer and stemness to identify those required for in vivo tumourigenesis. Among the genes identified, phosphoglycerate dehydrogenase (PHGDH) is in a genomic region of recurrent copy number gain in breast cancer and PHGDH protein levels are elevated in 70% of ER-negative breast cancers. PHGDH catalyzes the first step in the serine biosynthesis pathway, and breast cancer cells with high PHGDH expression have elevations in serine synthesis flux. Suppression of PHGDH in cell lines with elevated PHGDH expression, but not those without, causes a strong decrease in cell proliferation and a reduction in serine synthesis. We find that PHGDH suppression does not affect intracellular serine levels, but causes a drop in the levels of alpha-ketoglutarate, another output of the pathway and a TCA cycle intermediate. In cells with high PHGDH expression, the serine synthesis pathway contributes approximately 50% of the total anaplerotic flux of glutamine into the TCA cycle. These results reveal that certain breast cancers are dependent upon increased serine pathway flux caused by PHGDH over-expression and demonstrate the utility of in vivo negative selection RNAi screens for finding potential anticancer targets.

Erratum: Cancer-associated IDH1 mutations produce 2-hydroxyglutarate

This corrects the article DOI: 10.1038/nature08617

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.

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.

mTORC1-dependent AMD1 regulation sustains polyamine metabolism in prostate cancer

Activation of the PTEN–PI3K–mTORC1 pathway consolidates metabolic programs that sustain cancer cell growth and proliferation. Here we show that mechanistic target of rapamycin complex 1 (mTORC1) regulates polyamine dynamics, a metabolic route that is essential for oncogenicity. By using integrative metabolomics in a mouse model and human biopsies of prostate cancer, we identify alterations in tumours affecting the production of decarboxylated S-adenosylmethionine (dcSAM) and polyamine synthesis. Mechanistically, this metabolic rewiring stems from mTORC1-dependent regulation of S-adenosylmethionine decarboxylase 1 (AMD1) stability. This novel molecular regulation is validated in mouse and human cancer specimens. AMD1 is upregulated in human prostate cancer with activated mTORC1. Conversely, samples from a clinical trial with the mTORC1 inhibitor everolimus exhibit a predominant decrease in AMD1 immunoreactivity that is associated with a decrease in proliferation, in line with the requirement of dcSAM production for oncogenicity. These findings provide fundamental information about the complex regulatory landscape controlled by mTORC1 to integrate and translate growth signals into an oncogenic metabolic program.

Abstract 33: Cancer-associated IDH1 mutations produce 2-hydroxyglutarate

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Mutations in the enzyme isocitrate dehydrogenase 1 (IDH1) are a common feature of most gliomas and secondary glioblastomas, as well as approx 10% acute myeloid leukemias. This event results in loss of the enzymes ability to catalyze conversion of isocitrate to α -ketoglutarate. However, these mutations are all heterozygous and occur at a single amino acid residue of the IDH1 active site consistent with an enzymatic gain of function rather than a simple loss of function. To test this hypothesis we characterized mutant IDH1 (IDH1m) biochemically. We have shown that cancer-associated IDH1 mutations result in a new ability of the enzyme to catalyze the NADPH-dependent reduction of α-ketoglutarate to R(-)-2-hydroxyglutarate (2-HG). Patients with an inherited, neurometabolic disorders called 2-hydroxyglutaric aciduria exhibit an accumulation of 2-HG in their CNS, and an increased risk of developing malignant brain tumors. Similarly, in human malignant gliomas harboring IDH1 mutations, we find elevated levels of 2-HG. Altogether our data demonstrate that the IDH1 mutations result in production of 2-HG, and suggest that the excess 2HG which accumulates in vivo contributes to the formation and malignant progression of gliomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 33.

Corrigendum: mTORC1-dependent AMD1 regulation sustains polyamine metabolism in prostate cancer

This corrects the article DOI: 10.1038/nature22964

Abstract LB-307: MTAP deletions in cancer create vulnerability to a MAT2A/PRMT5/RIOK1 axis

Homozygous deletions of the CDKN2A tumor suppressor locus occur in approximately 15% of all cancer and represent a substantial therapeutic need(1). Targeted therapies selective for loss of CDKN2A have proven elusive, so we sought to evaluate whether the frequent co-deletion of the adjacent metabolic gene methylthioadenosine phosphorylase (MTAP) leads to collateral vulnerabilities. We conducted an shRNA screen in isogenic cells that vary only in their MTAP-deletion status, and identified an axis of targets that become vulnerable upon MTAP loss. Central in this axis is the arginine methyltransferase, PRMT5. Targeting PRMT5 with doxycycline-inducible shRNA impaired the growth of MTAP-deleted HCT116 colon carcinoma cells without impact on the growth of MTAP-wt HCT116 cells. Having identified this synthetic lethality between PRMT5 and MTAP, we sought next to elucidate the mechanistic basis for this relationship. Untargeted metabolomics revealed that MTA, the substrate of the MTAP enzyme reaction, accumulates dramatically in MTAP-deleted cancers. Biochemical profiling across the methyltransferase enzyme family revealed that MTA is a potent and selective inhibitor of PRMT5. Consistent with this in vitro finding, MTA accumulation in MTAP-deleted cancers led to reduced basal PRMT5 methylation. This selective vulnerability extends beyond PRMT5 to methionine adenosyltransferase 2, a (MAT2A), the metabolic enzyme upstream of PRMT5. MAT2A was the top hit in the shRNA screen, and targeting of MAT2A with inducible shRNA led to selective inhibition of growth in MTAP-deleted HCT116 cells in vitro and in vivo. Targeting of MAT2A led to substantial reduction of PRMT5 methylation activity in MTAP-deleted cells, indicating that PRMT5 activity is controlled by levels of substrate SAM and inhibitory metabolite MTA. To assess the therapeutic relevance of this axis in settings with endogenous deletion of the CDKN2A/MTAP locus, we depleted MAT2A in a panel of cancer cell lines and observed selective growth inhibition in the lines with endogenous deletion of CDKN2A/MTAP. Lastly, we noted that RIO Kinase 1 (RIOK1), a PRMT5-interacting protein(2), also scored in the shRNA screen. Doxycycline-inducible shRNA studies validated RIOK1 as an MTAP-selective target, and additional siRNA studies indicated that multiple PRMT5 co-complex members are selectively essential in MTAP-deleted cells. Thus, PRMT5 is the central node in an axis of targets that could be exploited therapeutically in cancers with deletion of CDKN2A/MTAP. References: 1 Beroukhim, R. et al. The landscape of somatic copy-number alteration across human cancers. Nature 463, 899-905, doi:10.1038/nature08822 (2010). 2 Guderian, G. et al. RioK1, a new interactor of protein arginine methyltransferase 5 (PRMT5), competes with pICln for binding and modulates PRMT5 complex composition and substrate specificity. J. Biol. Chem. 286, 1976-1986, doi:10.1074/jbc.M110.148486 (2011). Citation Format: Kevin Marks. MTAP deletions in cancer create vulnerability to a MAT2A/PRMT5/RIOK1 axis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-307.