Supporting Aspartate Biosynthesis Is an Essential Function of Respiration in Proliferating Cells

SummaryMitochondrial respiration is important for cell proliferation; however, the specific metabolic requirements fulfilled by respiration to support proliferation have not been defined. Here, we show that a major role of respiration in proliferating cells is to provide electron acceptors for aspartate synthesis. This finding is consistent with the observation that cells lacking a functional respiratory chain are auxotrophic for pyruvate, which serves as an exogenous electron acceptor. Further, the pyruvate requirement can be fulfilled with an alternative electron acceptor, alpha-ketobutyrate, which provides cells neither carbon nor ATP. Alpha-ketobutyrate restores proliferation when respiration is inhibited, suggesting that an alternative electron acceptor can substitute for respiration to support proliferation. We find that electron acceptors are limiting for producing aspartate, and supplying aspartate enables proliferation of respiration deficient cells in the absence of exogenous electron acceptors. Together, these data argue a major function of respiration in proliferating cells is to support aspartate synthesis

Circadian Rhythm Disruption Promotes Lung Tumorigenesis

Circadian rhythms are 24-hr oscillations that control a variety of biological processes in living systems, including two hallmarks of cancer, cell division and metabolism. Circadian rhythm disruption by shift work is associated with greater risk for cancer development and poor prognosis, suggesting a putative tumor-suppressive role for circadian rhythm homeostasis. Using a genetically engineered mouse model of lung adenocarcinoma, we have characterized the effects of circadian rhythm disruption on lung tumorigenesis. We demonstrate that both physiologic perturbation (jet lag) and genetic mutation of the central circadian clock components decreased survival and promoted lung tumor growth and progression. The core circadian genes Per2 and Bmal1 were shown to have cell-autonomous tumor-suppressive roles in transformation and lung tumor progression. Loss of the central clock components led to increased c-Myc expression, enhanced proliferation, and metabolic dysregulation. Our findings demonstrate that both systemic and somatic disruption of circadian rhythms contribute to cancer progression

Environment Dictates Dependence on Mitochondrial Complex I for NAD+ and Aspartate Production and Determines Cancer Cell Sensitivity to Metformin

Metformin use is associated with reduced cancer mortality, but how metformin impacts cancer outcomes is controversial. Although metformin can act on cells autonomously to inhibit tumor growth, the doses of metformin that inhibit proliferation in tissue culture are much higher than what has been described in vivo. Here, we show that the environment drastically alters sensitivity to metformin and other complex I inhibitors. We find that complex I supports proliferation by regenerating nicotinamide adenine dinucleotide (NAD)+, and metformin's anti-proliferative effect is due to loss of NAD+/NADH homeostasis and inhibition of aspartate biosynthesis. However, complex I is only one of many inputs that determines the cellular NAD+/NADH ratio, and dependency on complex I is dictated by the activity of other pathways that affect NAD+ regeneration and aspartate levels. This suggests that cancer drug sensitivity and resistance are not intrinsic properties of cancer cells, and demonstrates that the environment can dictate sensitivity to therapies that impact cell metabolism. Keywords: cancer metabolism; metformin; biguanide; NAD+/NADH ratio; drug sensitivity; complex I; mitochondria; aspartateNational Institutes of Health (U.S.) (Grant P30CA1405141)National Institutes of Health (U.S.) (Grant GG006413)National Institutes of Health (U.S.) (Grant R01 CA168653)National Institutes of Health (U.S.) (Grant R01 CA201276

B cells populating the multiple sclerosis brain mature in the draining cervical lymph nodes

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterized by autoimmune-mediated demyelination and neurodegeneration. The CNS of patients with MS harbors expanded clones of antigen-experienced B cells that reside in distinct compartments including the meninges, cerebrospinal fluid (CSF), and parenchyma. It is not understood whether this immune infiltrate initiates its development in the CNS or in peripheral tissues. B cells in the CSF can exchange with those in peripheral blood, implying that CNS B cells may have access to lymphoid tissue that may be the specific compartment(s) in which CNS-resident B cells encounter antigen and experience affinity maturation. Paired tissues were used to determine whether the B cells that populate the CNS mature in the draining cervical lymph nodes (CLNs). High-throughput sequencing of the antibody repertoire demonstrated that clonally expanded B cells were present in both compartments. Founding members of clones were more often found in the draining CLNs. More mature clonal members derived from these founders were observed in the draining CLNs and also in the CNS, including lesions. These data provide new evidence that B cells traffic freely across the tissue barrier, with the majority of B cell maturation occurring outside of the CNS in the secondary lymphoid tissue. Our study may aid in further defining the mechanisms of immunomodulatory therapies that either deplete circulating B cells or affect the intrathecal B cell compartment by inhibiting lymphocyte transmigration into the CNS

Treatment Response Assessment in IDH-Mutant Glioma Patients by Noninvasive 3D Functional Spectroscopic Mapping of 2-Hydroxyglutarate

Purpose: Measurements of objective response rates are critical to evaluate new glioma therapies. The hallmark metabolic alteration in gliomas with mutant isocitrate dehydrogenase (IDH) is the overproduction of oncometabolite 2-hydroxyglutarate (2HG), which plays a key role in malignant transformation. 2HG represents an ideal biomarker to probe treatment response in IDH-mutant glioma patients, and we hypothesized a decrease in 2HG levels would be measureable by in vivo magnetic resonance spectroscopy (MRS) as a result of antitumor therapy. Experimental Design: We report a prospective longitudinal imaging study performed in 25 IDH-mutant glioma patients receiving adjuvant radiation and chemotherapy. A newly developed 3D MRS imaging was used to noninvasively image 2HG. Paired Student t test was used to compare pre- and posttreatment tumor 2HG values. Test-retest measurements were performed to determine the threshold for 2HG functional spectroscopic maps (fSM). Univariate and multivariate regression were performed to correlate 2HG changes with Karnofsky performance score (KPS). Results: We found that mean 2HG (2HG/Cre) levels decreased significantly (median=48.1%; 95% confidence interval=27.3%-56.5%; P=0.007) in the posttreatment scan. The volume of decreased 2HG correlates (R2=0.88, P=0.002) with clinical status evaluated by KPS. Conclusions: We demonstrate that dynamic measurements of 2HG are feasible by 3D fSM, and the decrease of 2HG levels can monitor treatment response in patients with IDH-mutant gliomas. Our results indicate that quantitative in vivo 2HG imaging maybe used for precision medicine and early response assessment in clinical trials of therapies targeting IDH-mutant gliomas

Targeting serine hydroxymethyltransferases 1 and 2 for T-cell acute lymphoblastic leukemia therapy

Despite progress in the treatment of acute lymphoblastic leukemia (ALL), T-cell ALL (T-ALL) has limited treatment options, particularly in the setting of relapsed/refractory disease. Using an unbiased genome-scale CRISPR-Cas9 screen we sought to identify pathway dependencies for T-ALL which could be harnessed for therapy development. Disruption of the one-carbon folate, purine and pyrimidine pathways scored as the top metabolic pathways required for T-ALL proliferation. We used a recently developed inhibitor of SHMT1 and SHMT2, RZ-2994, to characterize the effect of inhibiting these enzymes of the one-carbon folate pathway in T-ALL and found that T-ALL cell lines were differentially sensitive to RZ-2994, with the drug inducing a S/G2 cell cycle arrest. The effects of SHMT1/2 inhibition were rescued by formate supplementation. Loss of both SHMT1 and SHMT2 was necessary for impaired growth and cell cycle arrest, with suppression of both SHMT1 and SHMT2 inhibiting leukemia progression in vivo. RZ-2994 also decreased leukemia burden in vivo and remained effective in the setting of methotrexate resistance in vitro. This study highlights the significance of the one-carbon folate pathway in T-ALL and supports further development of SHMT inhibitors for treatment of T-ALL and other cancers

Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis

Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. The interaction of PKM2 with phosphotyrosine-containing proteins inhibits enzyme activity and increases the availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small-molecule PKM2 activators inhibits the growth of xenograft tumors. Structural studies reveal that small-molecule activators bind PKM2 at the subunit interaction interface, a site that is distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small-molecule activation of PKM2 can interfere with anabolic metabolism.National Institutes of Health (U.S.) (NIH grant R01 GM56203)National Institutes of Health (U.S.) (grant NIH 5P01CA120964)Dana-Farber/Harvard Cancer Center (NIH 5P30CA006516)National Institutes of Health (U.S.) (NIH grant R03MH085679)National Human Genome Research Institute (U.S.) (Intramural Research Program)National Institutes of Health (U.S.) (Molecular Libraries Initiative of the NIH Roadmap for Medical Research

2-Oxo-N-aryl-1,2,3,4-tetrahydroquinoline-6-sulfonamides as activators of the tumor cell specific M2 isoform of pyruvate kinase

Compared to normal differentiated cells, cancer cells have altered metabolic regulation to support biosynthesis and the expression of the M2 isozyme of pyruvate kinase (PKM2) plays an important role in this anabolic metabolism. While the M1 isoform is a highly active enzyme, the alternatively spliced M2 variant is considerably less active and expressed in tumors. While the exact mechanism by which decreased pyruvate kinase activity contributes to anabolic metabolism remains unclear, it is hypothesized that activation of PKM2 to levels seen with PKM1 may promote a metabolic program that is not conducive to cell proliferation. Here we report the third chemotype in a series of PKM2 activators based on the 2-oxo-N-aryl-1,2,3,4-tetrahydroquinoline-6-sulfonamide scaffold. The synthesis, structure activity relationships, selectivity and notable physiochemical properties are described.National Human Genome Research Institute (U.S.) (Molecular Libraries Initiative of the NIH Roadmap for Medical Research

EGLN1 Inhibition and Rerouting of α-Ketoglutarate Suffice for Remote Ischemic Protection

Ischemic preconditioning is the phenomenon whereby brief periods of sublethal ischemia protect against a subsequent, more prolonged, ischemic insult. In remote ischemic preconditioning (RIPC), ischemia to one organ protects others organs at a distance. We created mouse models to ask if inhibition of the alpha-ketoglutarate (αKG)-dependent dioxygenase Egln1, which senses oxygen and regulates the hypoxia-inducible factor (HIF) transcription factor, could suffice to mediate local and remote ischemic preconditioning. Using somatic gene deletion and a pharmacological inhibitor, we found that inhibiting Egln1 systemically or in skeletal muscles protects mice against myocardial ischemia-reperfusion (I/R) injury. Parabiosis experiments confirmed that RIPC in this latter model was mediated by a secreted factor. Egln1 loss causes accumulation of circulating αKG, which drives hepatic production and secretion of kynurenic acid (KYNA) that is necessary and sufficient to mediate cardiac ischemic protection in this setting.Broad Institute of MIT and Harvard. SPARC ProgramBurroughs Wellcome Fun