Structural Basis for Substrate Binding, Catalysis and Inhibition of Breast Cancer Target Mitochondrial Creatine Kinase by Covalent Inhibitor via Cryo-EM.

Mitochondrial creatine kinases are key players in maintaining energy homeostasis in cells by working in conjunction with cytosolic creatine kinases for energy transport from mitochondria to cytoplasm. High levels of MtCK observed in Her2+ breast cancer and inhibition of breast cancer cell growth by substrate analog, cyclocreatine, indicate dependence of cancer cells on the 'energy shuttle' for cell growth and survival. Hence, understanding the key mechanistic features of creatine kinases and their inhibition plays an important role in the development of cancer therapeutics. Herein, we present the mutational and structural investigation on understudied ubiquitous mitochondrial creatine kinase (uMtCK). Our cryo-EM structures and biochemical data on uMtCK showed closure of the loop comprising residue His61 is specific to and relies on creatine binding and the reaction mechanism of phosphoryl transfer depends on electrostatics in the active site. In addition, the previously identified covalent inhibitor CKi showed inhibition in breast cancer BT474 cells, however our biochemical and structural data indicated that CKi is not a potent inhibitor for breast cancer due to strong dependency on the covalent link formation and inability to induce conformational changes upon binding.

Lactic acid induces transcriptional repression of macrophage inflammatory response via histone acetylation.

Lactic acid has emerged as an important modulator of immune cell function. It can be produced by both gut microbiota and the host metabolism at homeostasis and during disease states. The production of lactic acid in the gut microenvironment is vital for tissue homeostasis. In the present study, we examined how lactic acid integrates cellular metabolism to shape the epigenome of macrophages during pro-inflammatory response. We found that lactic acid serves as a primary fuel source to promote histone H3K27 acetylation, which allows the expression of immunosuppressive gene program including Nr4a1. Consequently, macrophage pro-inflammatory function was transcriptionally repressed. Furthermore, the histone acetylation induced by lactic acid promotes a form of long-term immunosuppression ("trained immunosuppression"). Pre-exposure to lactic acid induces lipopolysaccharide tolerance. These findings thus indicate that lactic acid sensing and its effect on chromatin remodeling in macrophages represent a key homeostatic mechanism that can provide a tolerogenic tissue microenvironment.

Salvage therapy expands highly cytotoxic and metabolically fit resilient CD8+ T cells via ME1 up-regulation.

Patients with advanced cancers who either do not experience initial response to or progress while on immune checkpoint inhibitors (ICIs) receive salvage radiotherapy to reduce tumor burden and tumor-related symptoms. Occasionally, some patients experience substantial global tumor regression with a rebound of cytotoxic CD8+ T cells. We have termed the rebound of cytotoxic CD8+ T cells in response to salvage therapy as T cell resilience and examined the underlying mechanisms of resilience. Resilient T cells are enriched for CX3CR1+ CD8+ T cells with low mitochondrial membrane potential, accumulate less reactive oxygen species (ROS), and express more malic enzyme 1 (ME1). ME1 overexpression enhanced the cytotoxicity and expansion of effector CD8+ T cells partially via the type I interferon pathway. ME1 also increased mitochondrial respiration while maintaining the redox state balance. ME1 increased the cytotoxicity of peripheral lymphocytes from patients with advanced cancers. Thus, preserved resilient T cells in patients rebound after salvage therapy and ME1 enhances their resiliency.

Succinate based polymers drive immunometabolism in dendritic cells to generate cancer immunotherapy.

Boosting the metabolism of immune cells while restricting cancer cell metabolism is challenging. Herein, we report that using biomaterials for the controlled delivery of succinate metabolite to phagocytic immune cells activates them and modulates their metabolism in the presence of metabolic inhibitors. In young immunocompetent mice, polymeric microparticles, with succinate incorporated in the backbone, induced strong pro-inflammatory anti-melanoma responses. Administration of poly(ethylene succinate) (PES MP)-based vaccines and glutaminase inhibitor to young immunocompetent mice with aggressive and large, established B16F10 melanoma tumors increased their survival three-fold, a result of increased cytotoxic T cells expressing RORγT (Tc17). Mechanistically, PES MPs directly modulate glutamine and glutamate metabolism, upregulate succinate receptor SUCNR1, activate antigen presenting cells through and HIF-1alpha, TNFa and TSLP-signaling pathways, and are dependent on alpha-ketoglutarate dehydrogenase for their activity, which demonstrates correlation of succinate delivery and these pathways. Overall, our findings suggest that immunometabolism-modifying PES MP strategies provide an approach for developing robust cancer immunotherapies.

Overexpression of the energy metabolism transcriptome within clonal plasma cells is associated with the pathogenesis and outcomes of patients with multiple myeloma.

Altered energy metabolism and changes in glycolytic and oxidative phosphorylation pathways are hallmarks of all cancer cells. The expression of select genes associated with the production of various enzymes and proteins involved in glycolysis and oxidative phosphorylation were assessed in the clonal plasma cells derived from patients with newly diagnosed multiple myeloma (NDMM) enrolled in the Multiple Myeloma Research Foundation (MMRF) CoMMpass data set. A scoring system consisting of assigning a point for every gene where their fragments per kilobase of transcript per million (FPKM) was above the median yielded a minimum of 0 and a maximum of 12 for the set of genes in the glycolytic and oxidative phosphorylation pathways to create a total energy metabolism molecular signature (EMMS) score. This EMMS score was independently associated with worse progression free survival (PFS) and overall survival (OS) outcomes of patients with NDMM. A higher EMMS score was more likely to be present in clonal plasma cells derived from Multiple myeloma (MM) patients than those from patients with monoclonal gammopathy of undetermined significance (MGUS). This was functionally confirmed by the clonal plasma cells from MM patients having a higher rate of mitochondrial and glycolysis-derived ATP formation than clonal plasma cells from MGUS patients. Thus, this study provides evidence for the effect of energy metabolism within clonal plasma cells on pathogenesis and outcomes of patients with MM. Exploiting the energy-producing metabolic pathways within clonal plasma cells for diagnostic and therapeutic purposes in MM should be explored in the future.

Enzymatic activation of pyruvate kinase increases cytosolic oxaloacetate to inhibit the Warburg effect.

Pharmacological activation of the glycolytic enzyme PKM2 or expression of the constitutively active PKM1 isoform in cancer cells results in decreased lactate production, a phenomenon known as the PKM2 paradox in the Warburg effect. Here we show that oxaloacetate (OAA) is a competitive inhibitor of human lactate dehydrogenase A (LDHA) and that elevated PKM2 activity increases de novo synthesis of OAA through glutaminolysis, thereby inhibiting LDHA in cancer cells. We also show that replacement of human LDHA with rabbit LDHA, which is relatively resistant to OAA inhibition, eliminated the paradoxical correlation between the elevated PKM2 activity and the decreased lactate concentration in cancer cells treated with a PKM2 activator. Furthermore, rabbit LDHA-expressing tumours, compared to human LDHA-expressing tumours in mice, displayed resistance to the PKM2 activator. These findings describe a mechanistic explanation for the PKM2 paradox by showing that OAA accumulates and inhibits LDHA following PKM2 activation.

TFEB links MYC signaling to epigenetic control of myeloid differentiation and acute myeloid leukemia.

MYC oncoproteins regulate transcription of genes directing cell proliferation, metabolism and tumorigenesis. A variety of alterations drive MYC expression in acute myeloid leukemia (AML) and enforced MYC expression in hematopoietic progenitors is sufficient to induce AML. Here we report that AML and myeloid progenitor cell growth and survival rely on MYC-directed suppression of Transcription Factor EB (TFEB), a master regulator of the autophagy-lysosome pathway. Notably, although originally identified as an oncogene, TFEB functions as a tumor suppressor in AML, where it provokes AML cell differentiation and death. These responses reflect TFEB control of myeloid epigenetic programs, by inducing expression of isocitrate dehydrogenase-1 (IDH1) and IDH2, resulting in global hydroxylation of 5-methycytosine. Finally, activating the TFEB-IDH1/IDH2-TET2 axis is revealed as a targetable vulnerability in AML. Thus, epigenetic control by a MYC-TFEB circuit dictates myeloid cell fate and is essential for maintenance of AML.

In vivo assessment of glutamine anaplerosis into the TCA cycle in human pre-malignant and malignant clonal plasma cells.

BACKGROUND: Overexpression of c-Myc is required for the progression of pre-malignant plasma cells in monoclonal gammopathy of undetermined significance (MGUS) to malignant plasma cells in multiple myeloma (MM). c-Myc also increases glutamine anaplerosis into the tricarboxylic acid (TCA) cycle within cancer cells. Whether increased glutamine anaplerosis is associated with the progression of pre-malignant to malignant plasma cells is unknown. METHODS: Human volunteers (N = 7) and patients with MGUS (N = 11) and MM (N = 12) were prospectively recruited to undergo an intravenous infusion of 13C-labeled glutamine followed by a bone marrow aspiration to obtain bone marrow cells and plasma. RESULTS: Despite notable heterogeneity, stable isotope-resolved metabolomics (SIRM) revealed that the mean 13C-labeled glutamine anaplerosis into the TCA cycle was higher in malignant compared to pre-malignant bone marrow plasma cells relative to the remainder of their paired bone marrow mononuclear cells. RNA sequencing demonstrated a higher relative mRNA expression of c-Myc and glutamine transporters such as ASCT2 and SN2 in malignant compared to pre-malignant bone marrow plasma cells. Finally, higher quantitative levels of TCA cycle intermediates in the bone marrow plasma differentiated MM from MGUS patients. CONCLUSION: Measurement of the in vivo activity of glutamine anaplerosis into the TCA cycle provides novel insight into the metabolic changes associated with the transformation of pre-malignant plasma cells in MGUS to malignant plasma cells in MM. TRIAL REGISTRATION: NCT03384108 and NCT03119883.

Metabolomic and Lipidomic Profiling of Bone Marrow Plasma Differentiates Patients with Monoclonal Gammopathy of Undetermined Significance from Multiple Myeloma.

Oncogenic drivers of progression of monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) such as c-MYC have downstream effects on intracellular metabolic pathways of clonal plasma cells (PCs). Thus, extracellular environments such as the bone marrow (BM) plasma likely have unique metabolite profiles that differ from patients with MGUS compared to MM. This study utilized an untargeted metabolite and targeted complex lipid profiling of BM plasma to identify significant differences in the relative metabolite levels between patients with MGUS and MM from an exploratory cohort. This was followed by verification of some of the metabolite differences of interest by targeted quantification of the metabolites using isotopic internal standards in the exploratory cohort as well as an independent validation cohort. Significant differences were noted in the amino acid profiles such as decreased branch chain amino acids (BCAAs) and increased catabolism of tryptophan to the active kynurenine metabolite 3-hydroxy-kynurenine between patients with MGUS and MM. A decrease in the total levels of complex lipids such as phosphatidylethanolamines (PE), lactosylceramides (LCER) and phosphatidylinositols (PI) were also detected in the BM plasma samples from MM compared to MGUS patients. Thus, metabolite and complex lipid profiling of the BM plasma identifies differences in levels of metabolites and lipids between patients with MGUS and MM. This may provide insight into the possible differences of the intracellular metabolic pathways of their clonal PCs.

Enzymatic and metabolic regulation of lysine succinylation.

Lysine succinylation (Ksucc), defined as a transfer of a succinyl group to a lysine residue of a protein, is a newly identified protein post-translational modification1-3. This chemical modification is reversible, dynamic, and evolutionarily conserved 4 where it has been comprehensively studied in both bacterial and mammalian cells5-7. Numerous proteins involved in the regulation of various cellular and biological processes have been shown to be heavily succinylated5-7. Emerging clinical data provides evidence that dysregulation of Ksucc is correlated with the development of several diseases, including cardiovascular diseases and cancer7-9. Therefore, an in-depth understanding of Ksucc and its regulation is important not only for understanding its physiological function but also for developing drug therapies and targeted agents for these diseases. In this review, we highlight some of the recent advances in understanding the role of Ksucc and desuccinylation under physiological and pathological conditions.

BRCA1 Deficiency Upregulates NNMT, Which Reprograms Metabolism and Sensitizes Ovarian Cancer Cells to Mitochondrial Metabolic Targeting Agents.

BRCA1 plays a key role in homologous recombination (HR) DNA repair. Accordingly, changes that downregulate BRCA1, including BRCA1 mutations and reduced BRCA1 transcription, due to promoter hypermethylation or loss of the BRCA1 transcriptional regulator CDK12, disrupt HR in multiple cancers. In addition, BRCA1 has also been implicated in the regulation of metabolism. Here, we show that reducing BRCA1 expression, either by CDK12 or BRCA1 depletion, led to metabolic reprogramming of ovarian cancer cells, causing decreased mitochondrial respiration and reduced ATP levels. BRCA1 depletion drove this reprogramming by upregulating nicotinamide N-methyltransferase (NNMT). Notably, the metabolic alterations caused by BRCA1 depletion and NNMT upregulation sensitized ovarian cancer cells to agents that inhibit mitochondrial metabolism (VLX600 and tigecycline) and to agents that inhibit glucose import (WZB117). These observations suggest that inhibition of energy metabolism may be a potential strategy to selectively target BRCA1-deficient high-grade serous ovarian cancer, which is characterized by frequent BRCA1 loss and NNMT overexpression. SIGNIFICANCE: Loss of BRCA1 reprograms metabolism, creating a therapeutically targetable vulnerability in ovarian cancer.

The Transcription Factor Bhlhe40 Programs Mitochondrial Regulation of Resident CD8+ T Cell Fitness and Functionality.

Tissue-resident memory CD8+ T (Trm) cells share core residency gene programs with tumor-infiltrating lymphocytes (TILs). However, the transcriptional, metabolic, and epigenetic regulation of Trm cell and TIL development and function is largely undefined. Here, we found that the transcription factor Bhlhe40 was specifically required for Trm cell and TIL development and polyfunctionality. Local PD-1 signaling inhibited TIL Bhlhe40 expression, and Bhlhe40 was critical for TIL reinvigoration following anti-PD-L1 blockade. Mechanistically, Bhlhe40 sustained Trm cell and TIL mitochondrial fitness and a functional epigenetic state. Building on these findings, we identified an epigenetic and metabolic regimen that promoted Trm cell and TIL gene signatures associated with tissue residency and polyfunctionality. This regimen empowered the anti-tumor activity of CD8+ T cells and possessed therapeutic potential even at an advanced tumor stage in mouse models. Our results provide mechanistic insights into the local regulation of Trm cell and TIL function.

Tyrosine Phosphorylation of Mitochondrial Creatine Kinase 1 Enhances a Druggable Tumor Energy Shuttle Pathway.

How mitochondrial metabolism is altered by oncogenic tyrosine kinases to promote tumor growth is incompletely understood. Here, we show that oncogenic HER2 tyrosine kinase signaling induces phosphorylation of mitochondrial creatine kinase 1 (MtCK1) on tyrosine 153 (Y153) in an ABL-dependent manner in breast cancer cells. Y153 phosphorylation, which is commonly upregulated in HER2+ breast cancers, stabilizes MtCK1 to increase the phosphocreatine energy shuttle and promote proliferation. Inhibition of the phosphocreatine energy shuttle by MtCK1 knockdown or with the creatine analog cyclocreatine decreases proliferation of trastuzumab-sensitive and -resistant HER2+ cell lines in culture and in xenografts. Finally, we show that cyclocreatine in combination with the HER2 kinase inhibitor lapatinib reduces the growth of a trastuzumab-resistant HER2+ patient-derived xenograft. These findings suggest that activation of the phosphocreatine energy shuttle by MtCK1 Y153 phosphorylation creates a druggable metabolic vulnerability in cancer.

Tyrosine Kinase Signaling in Cancer Metabolism: PKM2 Paradox in the Warburg Effect.

The Warburg Effect, or aerobic glycolysis, is one of the major metabolic alterations observed in cancer. Hypothesized to increase a cell's proliferative capacity via regenerating NAD+, increasing the pool of glycolytic biosynthetic intermediates, and increasing lactate production that affects the tumor microenvironment, the Warburg Effect is important for the growth and proliferation of tumor cells. The mechanisms by which a cell acquires the Warburg Effect phenotype are regulated by the expression of numerous oncogenes, including oncogenic tyrosine kinases. Oncogenic tyrosine kinases play a significant role in phosphorylating and regulating the activity of numerous metabolic enzymes. Tyrosine phosphorylation of glycolytic enzymes increases the activities of a majority of glycolytic enzymes, thus promoting increased glycolytic rate and tumor cell proliferation. Paradoxically however, tyrosine phosphorylation of pyruvate kinase M2 isoform (PKM2) results in decreased PKM2 activity, and this decrease in PKM2 activity promotes the Warburg Effect. Furthermore, recent studies have shown that PKM2 is also able to act as a protein kinase using phosphoenolpyruvate (PEP) as a substrate to promote tumorigenesis. Therefore, numerous recent studies have investigated both the role of the classical and non-canonical activity of PKM2 in promoting the Warburg Effect and tumor growth, which raise further interesting questions. In this review, we will summarize these recent advances revealing the importance of tyrosine kinases in the regulation of the Warburg Effect as well as the role of PKM2 in the promotion of tumor growth.

Impaired ß-cell glucokinase as an underlying mechanism in diet-induced diabetes.

High-fat diet (HFD)-fed mouse models have been widely used to study early type 2 diabetes. Decreased ß-cell glucokinase (GCK) expression has been observed in HFD-induced diabetes. However, owing to its crucial roles in glucose metabolism in the liver and in islet ß-cells, the contribution of decreased GCK expression to the development of HFD-induced diabetes is unclear. Here, we employed a ß-cell-targeted gene transfer vector and determined the impact of ß-cell-specific increase in GCK expression on ß-cell function and glucose handling in vitro and in vivo Overexpression of GCK enhanced glycolytic flux, ATP-sensitive potassium channel activation and membrane depolarization, and increased proliferation in Min6 cells. ß-cell-targeted GCK transduction did not change glucose handling in chow-fed C57BL/6 mice. Although adult mice fed a HFD showed reduced islet GCK expression, impaired glucose tolerance and decreased glucose-stimulated insulin secretion (GSIS), ß-cell-targeted GCK transduction improved glucose tolerance and restored GSIS. Islet perifusion experiments verified restored GSIS in isolated HFD islets by GCK transduction. Thus, our data identify impaired ß-cell GCK expression as an underlying mechanism for dysregulated ß-cell function and glycemic control in HFD-induced diabetes. Our data also imply an etiological role of GCK in diet-induced diabetes.This article has an associated First Person interview with the first author of the paper.

Glutamine-derived 2-hydroxyglutarate is associated with disease progression in plasma cell malignancies.

The production of the oncometabolite 2-hydroxyglutarate (2-HG) has been associated with c-MYC overexpression. c-MYC also regulates glutamine metabolism and drives progression of asymptomatic precursor plasma cell (PC) malignancies to symptomatic multiple myeloma (MM). However, the presence of 2-HG and its clinical significance in PC malignancies is unknown. By performing 13C stable isotope resolved metabolomics (SIRM) using U[13C6]Glucose and U[13C5]Glutamine in human myeloma cell lines (HMCLs), we show that 2-HG is produced in clonal PCs and is derived predominantly from glutamine anaplerosis into the TCA cycle. Furthermore, the 13C SIRM studies in HMCLs also demonstrate that glutamine is preferentially utilized by the TCA cycle compared with glucose. Finally, measuring the levels of 2-HG in the BM supernatant and peripheral blood plasma from patients with precursor PC malignancies such as smoldering MM (SMM) demonstrates that relatively elevated levels of 2-HG are associated with higher levels of c-MYC expression in the BM clonal PCs and with a subsequent shorter time to progression (TTP) to MM. Thus, measuring 2-HG levels in BM supernatant or peripheral blood plasma of SMM patients offers potential early identification of those patients at high risk of progression to MM, who could benefit from early therapeutic intervention.

Inhibition of intracellular lipolysis promotes human cancer cell adaptation to hypoxia.

Tumor tissues are chronically exposed to hypoxia owing to aberrant vascularity. Lipid droplet (LD) accumulation is a hallmark of hypoxic cancer cells, yet how LDs form and function during hypoxia remains poorly understood. Herein, we report that in various cancer cells upon oxygen deprivation, HIF-1 activation down-modulates LD catabolism mediated by adipose triglyceride lipase (ATGL), the key enzyme for intracellular lipolysis. Proteomics and functional analyses identified hypoxia-inducible gene 2 (HIG2), a HIF-1 target, as a new inhibitor of ATGL. Knockout of HIG2 enhanced LD breakdown and fatty acid (FA) oxidation, leading to increased ROS production and apoptosis in hypoxic cancer cells as well as impaired growth of tumor xenografts. All of these effects were reversed by co-ablation of ATGL. Thus, by inhibiting ATGL, HIG2 acts downstream of HIF-1 to sequester FAs in LDs away from the mitochondrial pathways for oxidation and ROS generation, thereby sustaining cancer cell survival in hypoxia.

6-Phosphogluconate dehydrogenase links oxidative PPP, lipogenesis and tumour growth by inhibiting LKB1-AMPK signalling.

The oxidative pentose phosphate pathway (PPP) contributes to tumour growth, but the precise contribution of 6-phosphogluconate dehydrogenase (6PGD), the third enzyme in this pathway, to tumorigenesis remains unclear. We found that suppression of 6PGD decreased lipogenesis and RNA biosynthesis and elevated ROS levels in cancer cells, attenuating cell proliferation and tumour growth. 6PGD-mediated production of ribulose-5-phosphate (Ru-5-P) inhibits AMPK activation by disrupting the active LKB1 complex, thereby activating acetyl-CoA carboxylase 1 and lipogenesis. Ru-5-P and NADPH are thought to be precursors in RNA biosynthesis and lipogenesis, respectively; thus, our findings provide an additional link between the oxidative PPP and lipogenesis through Ru-5-P-dependent inhibition of LKB1-AMPK signalling. Moreover, we identified and developed 6PGD inhibitors, physcion and its derivative S3, that effectively inhibited 6PGD, cancer cell proliferation and tumour growth in nude mice xenografts without obvious toxicity, suggesting that 6PGD could be an anticancer target.

Lysine acetylation activates 6-phosphogluconate dehydrogenase to promote tumor growth.

Although the oxidative pentose phosphate pathway is important for tumor growth, how 6-phosphogluconate dehydrogenase (6PGD) in this pathway is upregulated in human cancers is unknown. We found that 6PGD is commonly activated in EGF-stimulated cells and human cancer cells by lysine acetylation. Acetylation at K76 and K294 of 6PGD promotes NADP(+) binding to 6PGD and formation of active 6PGD dimers, respectively. Moreover, we identified DLAT and ACAT2 as upstream acetyltransferases of K76 and K294, respectively, and HDAC4 as the deacetylase of both sites. Expressing acetyl-deficient mutants of 6PGD in cancer cells significantly attenuated cell proliferation and tumor growth. This is due in part to reduced levels of 6PGD products ribulose-5-phosphate and NADPH, which led to reduced RNA and lipid biosynthesis as well as elevated ROS. Furthermore, 6PGD activity is upregulated with increased lysine acetylation in primary leukemia cells from human patients, providing mechanistic insights into 6PGD upregulation in cancer cells.

Tyr-94 phosphorylation inhibits pyruvate dehydrogenase phosphatase 1 and promotes tumor growth.

Many cancer cells rely more on aerobic glycolysis (the Warburg effect) than mitochondrial oxidative phosphorylation and catabolize glucose at a high rate. Such a metabolic switch is suggested to be due in part to functional attenuation of mitochondria in cancer cells. However, how oncogenic signals attenuate mitochondrial function and promote the switch to glycolysis remains unclear. We previously reported that tyrosine phosphorylation activates and inhibits mitochondrial pyruvate dehydrogenase kinase (PDK) and phosphatase (PDP), respectively, leading to enhanced inhibitory serine phosphorylation of pyruvate dehydrogenase (PDH) and consequently inhibition of pyruvate dehydrogenase complex (PDC) in cancer cells. In particular, Tyr-381 phosphorylation of PDP1 dissociates deacetylase SIRT3 and recruits acetyltransferase ACAT1 to PDC, resulting in increased inhibitory lysine acetylation of PDHA1 and PDP1. Here we report that phosphorylation at another tyrosine residue, Tyr-94, inhibits PDP1 by reducing the binding ability of PDP1 to lipoic acid, which is covalently attached to the L2 domain of dihydrolipoyl acetyltransferase (E2) to recruit PDP1 to PDC. We found that multiple oncogenic tyrosine kinases directly phosphorylated PDP1 at Tyr-94, and Tyr-94 phosphorylation of PDP1 was common in diverse human cancer cells and primary leukemia cells from patients. Moreover, expression of a phosphorylation-deficient PDP1 Y94F mutant in cancer cells resulted in increased oxidative phosphorylation, decreased cell proliferation under hypoxia, and reduced tumor growth in mice. Together, our findings suggest that phosphorylation at different tyrosine residues inhibits PDP1 through independent mechanisms, which act in concert to regulate PDC activity and promote the Warburg effect.