Citrate is a critical metabolite required to support both mitochondrial bioenergetics and cytosolic macromolecular synthesis. When cells proliferate under normoxic conditions, glucose provides the acetyl-CoA that condenses with oxaloacetate to support citrate production. Tricarboxylic acid (TCA) cycle anaplerosis is maintained primarily by glutamine. Here we report that some hypoxic cells are able to maintain cell proliferation despite a profound reduction in glucose-dependent citrate production. In these hypoxic cells, glutamine becomes a major source of citrate. Glutamine-derived α-ketoglutarate is reductively carboxylated by the NADPH-linked mitochondrial isocitrate dehydrogenase (IDH2) to form isocitrate, which can then be isomerized to citrate. The increased IDH2-dependent carboxylation of glutamine-derived α-ketoglutarate in hypoxia is associated with a concomitant increased synthesis of 2-hydroxyglutarate (2HG) in cells with wild-type IDH1 and IDH2. When either starved of glutamine or rendered IDH2-deficient by RNAi, hypoxic cells are unable to proliferate. The reductive carboxylation of glutamine is part of the metabolic reprogramming associated with hypoxia-inducible factor 1 (HIF1), as constitutive activation of HIF1 recapitulates the preferential reductive metabolism of glutaminederived α-ketoglutarate even in normoxic conditions. These data support a role for glutamine carboxylation in maintaining citrate synthesis and cell growth under hypoxic conditions. C itrate plays a critical role at the center of cancer cell metabolism. It provides the cell with a source of carbon for fatty acid and cholesterol synthesis (1). The breakdown of citrate by ATP-citrate lyase is a primary source of acetyl-CoA for protein acetylation (2). Metabolism of cytosolic citrate by aconitase and IDH1 can also provide the cell with a source of NADPH for redox regulation and anabolic synthesis. Mammalian cells depend on the catabolism of glucose and glutamine to fuel proliferation (3). In cancer cells cultured at atmospheric oxygen tension (21% O 2 ), glucose and glutamine have both been shown to contribute to the cellular citrate pool, with glutamine providing the major source of the four-carbon molecule oxaloacetate and glucose providing the major source of the two-carbon molecule acetyl-CoA (4, 5). The condensation of oxaloacetate and acetyl-CoA via citrate synthase generates the 6 carbon citrate molecule. However, both the conversion of glucose-derived pyruvate to acetyl-CoA by pyruvate dehydrogenase (PDH) and the conversion of glutamine to oxaloacetate through the TCA cycle depend on NAD + , which can be compromised under hypoxic conditions. This raises the question of how cells that can proliferate in hypoxia continue to synthesize the citrate required for macromolecular synthesis.This question is particularly important given that many cancers and stem/progenitor cells can continue proliferating in the setting of limited oxygen availability (6, 7). Louis Pasteur first highlighted the impact of hypoxia on nutrient metabol...

SUMMARY Somatic mutations in isocitrate dehydrogenase 1 or 2 (IDH1/2) contribute to the pathogenesis of cancer via production of the ‘oncometabolite’ D-2-hydroxyglutarate (D-2HG). Elevated D-2HG can block differentiation of malignant cells by functioning as a competitive inhibitor of alpha-ketoglutarate (α-KG)-dependent enzymes, including Jumonji family histone lysine demethylases. 2HG is a chiral molecule that can exist in either the D- or L- enantiomer. Although cancer-associated IDH1/2 mutations produce D-2HG, biochemical studies have demonstrated that L-2HG also functions as a potent inhibitor of α-KG-dependent enzymes. Here we report that under conditions of oxygen limitation, mammalian cells selectively produce L-2HG via enzymatic reduction of α-KG. Hypoxia-induced L-2HG is not mediated by IDH1 or IDH2, but instead results from promiscuous substrate usage primarily by lactate dehydrogenase A (LDHA). During hypoxia, the resulting increase in L-2HG is necessary and sufficient for the induction of increased methylation of histone repressive marks, including histone 3 lysine 9 (H3K9me3).

MicroRNAs repress mRNA translation by guiding Argonaute proteins to partially complementary binding sites, primarily within the 3′ untranslated region (UTR) of target mRNAs. In cell lines, Argonaute-bound microRNAs exist mainly in high molecular weight RNA-induced silencing complexes (HMW-RISC) associated with target mRNA. Here we demonstrate that most adult tissues contain reservoirs of microRNAs in low molecular weight RISC (LMW-RISC) not bound to mRNA, suggesting that these microRNAs are not actively engaged in target repression. Consistent with this observation, the majority of individual microRNAs in primary T cells were enriched in LMW-RISC. During T-cell activation, signal transduction through the phosphoinositide-3 kinase–RAC-alpha serine/threonine-protein kinase–mechanistic target of rapamycin pathway increased the assembly of microRNAs into HMW-RISC, enhanced expression of the glycine-tryptophan protein of 182 kDa, an essential component of HMW-RISC, and improved the ability of microRNAs to repress partially complementary reporters, even when expression of targeting microRNAs did not increase. Overall, data presented here demonstrate that microRNA-mediated target repression in nontransformed cells depends not only on abundance of specific microRNAs, but also on regulation of RISC assembly by intracellular signaling.

More than 50% of patients with chondrosarcomas exhibit gain-of-function mutations in either isocitrate dehydrogenase 1 (IDH1) or IDH2. In this study, we performed genome-wide CpG methylation sequencing of chondrosarcoma biopsies and found that IDH mutations were associated with DNA hypermethylation at CpG islands but not other genomic regions. Regions of CpG island hypermethylation were enriched for genes implicated in stem cell maintenance/differentiation and lineage specification. In murine 10T1/2 mesenchymal progenitor cells, expression of mutant IDH2 led to DNA hypermethylation and an impairment in differentiation that could be reversed by treatment with DNA-hypomethylating agents. Introduction of mutant IDH2 also induced loss of contact inhibition and generated undifferentiated sarcomas in vivo. The oncogenic potential of mutant IDH2 correlated with the ability to produce 2-hydroxyglutarate. Together, these data demonstrate that neomorphic IDH2 mutations can be oncogenic in mesenchymal cells.

Ras-transformed cells can grow in amino acid-poor environments by recovering amino acids through macropinocytosis and lysosomal catabolism of extracellular proteins. However, when studying nontransformed fibroblasts, we found that Ras GTPases are dispensable for growth-factor-stimulated macropinocytosis and lysosomal catabolism of extracellular proteins. Instead, we establish a critical role for phosphatidylinositol 3-kinase (PI3-kinase) signaling in cell proliferation that is supported by protein macropinocytosis. Downstream of PI3-kinase, distinct effectors have opposing roles in regulating uptake and catabolism of extracellular proteins. Rac1 and PLC are required for nutritional use of extracellular proteins. In contrast, Akt suppresses lysosomal catabolism of ingested proteins when free amino acids are abundant. The interplay between these pathways allows cells with oncogenic PIK3CA mutations or PTEN deletion to grow using diverse amino acid sources. Thus, the prevalence of PI3-kinase and PTEN mutations in cancer may result in part because they allow cells to cope with fluctuating nutrient availability.

Somatic mutations in isocitrate dehydrogenase 1 or 2 (IDH1/2) contribute to the pathogenesis of cancer via production of the ‘oncometabolite’ D-2-hydroxyglutarate (D-2HG). Elevated D-2HG can block differentiation of malignant cells by functioning as a competitive inhibitor of alpha-ketoglutarate (alpha-KG) dependent enzymes, including Jumonji family histone lysine demethylases. 2HG is a chiral molecule that can exist in either the D- or L- enantiomer. Although cancer-associated IDH1/2 mutations exclusively produce D-2HG, biochemical studies have demonstrated that L-2HG functions as a more potent inhibitor of alpha-KG-dependent enzymes. Here we report that under conditions of oxygen limitation, mammalian cells selectively produce L-2HG via enzymatic reduction of alpha-KG. Hypoxia-induced L-2HG is not mediated by IDH1 or IDH2, but instead results from promiscuous substrate usage by lactate dehydrogenase A (LDHA). During hypoxia, the resulting increase in L-2HG is necessary and sufficient for the induction of increased methylation of histone repressive marks, including histone 3 lysine 9 (H3K9me3). Thus, L-2HG appears to function as a metabolic signaling intermediate, translating information about oxygen availability into epigenetic modifications that can influence gene expression and cellular differentiation. Citation Format: Craig B. Thompson, Andrew M. Intlekofer, Raymond G. Dematteo, Sriram Venneti, Lydia W. S. Finley, Chao Lu, Ariën S. Rustenburg, Patrick B. Grinaway, John D. Chodera, Justin R. Cross, Alexander R. Judkins. Stereochemistry matters: L-2HG as a tumor response to hypoxia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr SY43-01. doi:10.1158/1538-7445.AM2015-SY43-01

Purpose Phosphoinositide 3-kinase (PI3K) inhibition is used for the treatment of certain cancers, but can cause profound hyperglycemia and insulin resistance, for which sodium-glucose cotransporter-2 (SGLT2) inhibitors have been proposed as a preferred therapy. The objective of this research is to assess the effectiveness and safety of SGLT2 inhibitors for hyperglycemia in PI3K inhibition. Methods We conducted a single-center retrospective review of adults initiating the PI3k inhibitor alpelisib. Exposure to different antidiabetic drugs and adverse events including diabetic ketoacidosis (DKA) were assessed through chart review. Plasma and point-of-care blood glucoses were extracted from the electronic medical record. Change in serum glucose and the rate of DKA on SGLT2 inhibitor versus other antidiabetic drugs were examined as co-primary outcomes. Results We identified 103 patients meeting eligibility criteria with median follow-up of 85 days after starting alpelisib. When SGLT2 inhibitors were used to treat hyperglycemia, they were associated with a decrease in mean random glucose by -54 mg/dL (95% CI -99 to -8) in adjusted linear modeling. Five cases of DKA were identified, two occurring in patients on alpelisib plus SGLT2 inhibitor. Estimated incidence of DKA was: alpelisib plus SGLT2 inhibitor, 24 DKA cases per 100 patient-years (95% CI 6, 80); alpelisib with non-SGLT2 inhibitor antidiabetic drugs, 7 (95% CI 0.1, 34); alpelisib only, 4 (95% CI 0.1, 21). Conclusions SGLT2 inhibitors are effective treatments for hyperglycemia in the setting of PI3K inhibition, but given possible adverse events, SGLT2 inhibitors should be used with caution.

Introduction Risk of central nervous system (CNS) relapse and its management in patients with newly diagnosed diffuse large B-cell lymphoma (DLBCL) have been extensively studied. However, similar data in patients with relapsed or refractory (R/R) disease are lacking. Specifically, it is unknown whether choice of treatment in this setting may influence the CNS risk. A number of commonly used platinum-based regimens do not contain agents that consistently penetrate the CNS at therapeutic concentrations. High-dose cytarabine, used in standard salvage therapy alongside rituximab, dexamethasone, and either cisplatin (R-DHAP) or oxaliplatin (R-DHAX), is known to cross the blood-brain barrier. Therefore, we asked whether the use of R-DHAP/R-DHAX may be associated with lower risk of CNS relapse than other platinum-based regimens in patients with R/R DLBCL. Methods We reviewed consecutive adult patients who initiated a standard platinum regimen (R-ICE, R-GDP, R-GemOx, R-DHAP, or R-DHAX) for the treatment of DLBCL in first relapse at MSKCC. Patients with evidence of CNS involvement prior to, or at initiation of salvage, history of human immunodeficiency virus, or whose front-line treatment included a platinum agent were excluded. The primary objective of the analysis was to compare the cumulative incidence of CNS relapse in patients receiving R-DHAP/R-DHAX versus other salvage regimens (OSR). Death without relapse was treated as a competing risk. Results Between April 1996 and September 2020, we identified 302 eligible patients (Table 1), of whom 51 received R-DHAP/R-DHAX and 251 received OSR. Median age at the time of R/R disease was 61 years (range, 19 to 92 years). Patients who received R-DHAP/R-DHAX were significantly more likely to have germinal center B-cell-like phenotype by Hans algorithm (61% vs 24%, p < 0.001) and to have double- or triple-hit lymphoma (DHL/THL; 22% vs 2%, p < 0.001). A higher proportion of patients who received R-DHAP/R-DHAX received CNS prophylaxis during front-line treatment (37% vs 21%, p = 0.01), possibly owing to the number of patients with DHL/THL. Intrathecal methotrexate was used in 68% of patients receiving R-DHAP/R-DHAX and 17% of patients receiving OSR; high-dose methotrexate was used in 5% of the R-DHAP/R-DHAX group and 2% of OSR. A higher proportion of patients who received R-DHAP/R-DHAX underwent consolidative chimeric antigen receptor T-cell therapy (CAR-T) for residual disease (33% vs 5%, p < 0.001), but fewer underwent consolidative autologous hematopoietic cell transplantation (HCT; 33% vs 54%; p = 0.008). After median overall follow-up of 68 months (34 months for R-DHAP/R-DHAX vs 79 months for OSR, p < 0.001), CNS relapse was observed in 20 patients: 2 in the R-DHAP/R-DHAX group and 18 in the OSR group (Table 2). CNS relapse occurred as leptomeningeal disease only in both patients who received R-DHAP/R-DHAX; all brain parenchyma relapses occurred in patients who received OSR. This numerical difference did not translate into a statistically significant difference in cumulative incidence of CNS relapse (p = 0.52, Figure 1). No independent predictors of CNS relapse were identified in multivariate analysis, and cumulative incidence of CNS relapse was similar when the OSR group was restricted to patients who received R-ICE (n = 198). Median progression-free survival (PFS) was shorter in patients who received R-DHAP/R-DHAX compared to those who received OSR (5.4 months vs 24 months; p = 0.008). However, in Cox regression analysis controlling for effects of salvage regimen (R-DHAP/R-DHAX vs OSR), international prognostic index, DHL/THL biology, and consolidative use of either CAR-T or autologous HCT, only autologous HCT appeared to be an independent predictor of longer PFS (p < 0.001). There was no significant difference in median overall survival (38 months vs 58 months; p = 0.5). Conclusion In patients with R/R DLBCL receiving platinum-based salvage therapy, the cumulative incidence of CNS relapse after salvage initiation was not significantly different between patients who received R-DHAP/R-DHAX and those who received other salvage regimens. The lower number of CNS relapses and absence of brain parenchyma relapses in the R-DHAP/R-DHAX group, despite a greater proportion of patients with DHL/THL, suggests that parenchymal penetration by cytarabine may influence the pattern of relapse, warranting study in a larger cohort. Figure 1 Figure 1. Disclosures Batlevi: Seattle Genetics: Consultancy; TG Therapeutics: Consultancy; Regeneron: Current holder of individual stocks in a privately-held company; Moderna: Current holder of individual stocks in a privately-held company; Bayer: Research Funding; Dava Oncology: Honoraria; Life Sciences: Consultancy; GLG Pharma: Consultancy; Juno/Celgene: Consultancy; Kite Pharma: Consultancy; TouchIME: Honoraria; BMS: Current holder of individual stocks in a privately-held company; Pfizer: Current holder of individual stocks in a privately-held company; ADC Therapeutics: Consultancy; Viatris: Current holder of individual stocks in a privately-held company; Memorial Sloan Kettering Cancer Center: Current Employment; Medscape: Honoraria; Karyopharm: Consultancy; Xynomic: Research Funding; Roche/Genentech: Research Funding; Novartis: Research Funding; Epizyme: Research Funding; Janssen: Research Funding; Autolus: Research Funding. Hamlin: Incyte, Janssen, Molecular Templates: Research Funding; Alexion, AstraZeneca Rare Disease (formerly Portola Pharmaceuticals): Other: Study investigator, Research Funding; Kite, Karyopharm, Celgene: Membership on an entity's Board of Directors or advisory committees. Horwitz: ADC Therapeutics, Affimed, Aileron, Celgene, Daiichi Sankyo, Forty Seven, Inc., Kyowa Hakko Kirin, Millennium /Takeda, Seattle Genetics, Trillium Therapeutics, and Verastem/SecuraBio.: Consultancy, Research Funding; Affimed: Research Funding; Aileron: Research Funding; Acrotech Biopharma, Affimed, ADC Therapeutics, Astex, Merck, Portola Pharma, C4 Therapeutics, Celgene, Janssen, Kura Oncology, Kyowa Hakko Kirin, Myeloid Therapeutics, ONO Pharmaceuticals, Seattle Genetics, Shoreline Biosciences, Inc, Takeda, Trillium Th: Consultancy; Celgene: Research Funding; C4 Therapeutics: Consultancy; Crispr Therapeutics: Research Funding; Daiichi Sankyo: Research Funding; Forty Seven, Inc.: Research Funding; Kura Oncology: Consultancy; Kyowa Hakko Kirin: Consultancy, Research Funding; Millennium/Takeda: Research Funding; Myeloid Therapeutics: Consultancy; ONO Pharmaceuticals: Consultancy; Seattle Genetics: Consultancy, Research Funding; Secura Bio: Consultancy; Shoreline Biosciences, Inc.: Consultancy; Takeda: Consultancy; Trillium Therapeutics: Consultancy, Research Funding; Tubulis: Consultancy; Verastem/Securabio: Research Funding. Joffe: AstraZeneca: Consultancy; Epizyme: Consultancy. Khan: Seattle Genetics: Research Funding. Kumar: Adaptive Biotechnologies, Celgene, Abbvie Pharmaceticals, Pharmacyclics, Seattle Genetics: Research Funding; Kite Pharmaceuticals: Other: advisory board , Research Funding; Abbvie Pharmaceuticals: Research Funding; Celgene: Honoraria, Other: advisory board, Research Funding; Pharmacyclics: Research Funding; Seattle Genetics: Research Funding; Astra Zeneca: Honoraria, Other: Advisory Board, Research Funding. Lee: Intellisphere, LLC: Consultancy. Matasar: Takeda: Consultancy, Honoraria; GlaxoSmithKline: Honoraria, Research Funding; Teva: Consultancy; Juno Therapeutics: Consultancy; Genentech, Inc.: Consultancy, Honoraria, Research Funding; IGM Biosciences: Research Funding; Memorial Sloan Kettering Cancer Center: Current Employment; ImmunoVaccine Technologies: Consultancy, Honoraria, Research Funding; Rocket Medical: Consultancy, Research Funding; Pharmacyclics: Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Merck Sharp & Dohme: Current holder of individual stocks in a privately-held company; TG Therapeutics: Consultancy, Honoraria; Janssen: Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Consultancy; Merck: Consultancy. Moskowitz: ADC Therapeutics: Research Funding; Imbrium Therapeutics L.P./Purdue: Consultancy; Seattle Genetics: Consultancy, Research Funding; Miragen: Research Funding; Janpix Ltd.: Consultancy; Takeda: Consultancy; Incyte: Research Funding; Beigene: Research Funding; Merck: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding. Noy: Epizyme: Consultancy; Rafael Parhma: Research Funding; Morphosys: Consultancy; Targeted Oncology: Consultancy; Medscape: Consultancy; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy, Honoraria. Palomba: Notch: Honoraria, Other: Stock; PCYC: Consultancy; Pluto: Honoraria; Nektar: Honoraria; WindMIL: Honoraria; Lygenesis: Honoraria; Priothera: Honoraria; Ceramedix: Honoraria; Wolters Kluwer: Patents & Royalties; BeiGene: Consultancy; Seres: Honoraria, Other: Stock, Patents & Royalties, Research Funding; Rheos: Honoraria; Juno: Patents & Royalties; Magenta: Honoraria; Kite: Consultancy; Novartis: Consultancy. von Keudell: Incyte: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria; Merck: Consultancy, Honoraria; AbbVie: Research Funding; Merck: Research Funding; BMS: Research Funding; Janssen: Research Funding. Zelenetz: Abbvie: Honoraria, Research Funding; MethylGene: Research Funding; Beigene: Honoraria, Other, Research Funding; MorphoSys: Honoraria; LFR: Other; Gilead: Honoraria, Research Funding; Janssen: Honoraria; NCCN: Other; AstraZeneca: Honoraria; Pharmacyclics: Honoraria; BMS/Celgene/JUNO: Honoraria, Other; MEI Pharma: Honoraria, Research Funding; Genentech/Roche: Honoraria, Research Funding; Gilead: Honoraria; Amgen: Honoraria; Verastem: Honoraria; SecuraBio: Honoraria; Novartis: Honoraria. Sauter: Novartis: Consultancy; Celgene: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; GSK: Consultancy; Genmab: Consultancy; Kite/Gilead: Consultancy; Precision Biosciences: Consultancy; Gamida Cell: Consultancy; Spectrum Pharmaceuticals: Consultancy; Juno Therapeutics: Consultancy, Research Funding; Sanofi-Genzyme: Consultancy, Research Funding. Salles: Novartis: Consultancy; Kite/Gilead: Consultancy; Morphosys: Consultancy, Honoraria; Miltneiy: Consultancy; Regeneron: Consultancy, Honoraria; Incyte: Consultancy; Rapt: Consultancy; Takeda: Consultancy; Ipsen: Consultancy; Loxo: Consultancy; Genmab: Consultancy; Epizyme: Consultancy, Honoraria; Genentech/Roche: Consultancy; Janssen: Consultancy; Velosbio: Consultancy; Allogene: Consultancy; Debiopharm: Consultancy; BMS/Celgene: Consultancy; Beigene: Consultancy; Abbvie: Consultancy, Honoraria; Bayer: Honoraria. Falchi: Roche: Research Funding; Abbvie: Consultancy, Research Funding; Genmab: Consultancy, Research Funding; Genetech: Research Funding.