γ-6-Phosphogluconolactone, a Byproduct of the Oxidative Pentose Phosphate Pathway, Contributes to AMPK Activation through Inhibition of PP2A.

The oxidative pentose phosphate pathway (oxiPPP) contributes to cell metabolism through not only the production of metabolic intermediates and reductive NADPH but also inhibition of LKB1-AMPK signaling by ribulose-5-phosphate (Ru-5-P), the product of the third oxiPPP enzyme 6-phosphogluconate dehydrogenase (6PGD). However, we found that knockdown of glucose-6-phosphate dehydrogenase (G6PD), the first oxiPPP enzyme, did not affect AMPK activation despite decreased Ru-5-P and subsequent LKB1 activation, due to enhanced activity of PP2A, the upstream phosphatase of AMPK. In contrast, knockdown of 6PGD or 6-phosphogluconolactonase (PGLS), the second oxiPPP enzyme, reduced PP2A activity. Mechanistically, knockdown of G6PD or PGLS decreased or increased 6-phosphogluconolactone level, respectively, which enhanced the inhibitory phosphorylation of PP2A by Src. Furthermore, γ-6-phosphogluconolactone, an oxiPPP byproduct with unknown function generated through intramolecular rearrangement of δ-6-phosphogluconolactone, the only substrate of PGLS, bound to Src and enhanced PP2A recruitment. Together, oxiPPP regulates AMPK homeostasis by balancing the opposing LKB1 and PP2A.

The Dietary Supplement Chondroitin-4-Sulfate Exhibits Oncogene-Specific Pro-tumor Effects on BRAF V600E Melanoma Cells.

Dietary supplements such as vitamins and minerals are widely used in the hope of improving health but may have unidentified risks and side effects. In particular, a pathogenic link between dietary supplements and specific oncogenes remains unknown. Here we report that chondroitin-4-sulfate (CHSA), a natural glycosaminoglycan approved as a dietary supplement used for osteoarthritis, selectively promotes the tumor growth potential of BRAF V600E-expressing human melanoma cells in patient- and cell line-derived xenograft mice and confers resistance to BRAF inhibitors. Mechanistically, chondroitin sulfate glucuronyltransferase (CSGlcA-T) signals through its product CHSA to enhance casein kinase 2 (CK2)-PTEN binding and consequent phosphorylation and inhibition of PTEN, which requires CHSA chains and is essential to sustain AKT activation in BRAF V600E-expressing melanoma cells. However, this CHSA-dependent PTEN inhibition is dispensable in cancer cells expressing mutant NRAS or PI3KCA, which directly activate the PI3K-AKT pathway. These results suggest that dietary supplements may exhibit oncogene-dependent pro-tumor effects.

The eEF1A protein in cancer: Clinical significance, oncogenic mechanisms, and targeted therapeutic strategies.

Eukaryotic elongation factor 1A (eEF1A) is among the most abundant proteins in eukaryotic cells. Evolutionarily conserved across species, eEF1A is in charge of translation elongation for protein biosynthesis as well as a plethora of non-translational moonlighting functions for cellular homeostasis. In malignant cells, however, eEF1A becomes a pleiotropic driver of cancer progression via a broad diversity of pathways, which are not limited to hyperactive translational output. In the past decades, mounting studies have demonstrated the causal link between eEF1A and carcinogenesis, gaining deeper insights into its multifaceted mechanisms and corroborating its value as a prognostic marker in various cancers. On the other hand, an increasing number of natural and synthetic compounds were discovered as anticancer eEF1A-targeting inhibitors. Among them, plitidepsin was approved for the treatment of multiple myeloma whereas metarrestin was currently under clinical development. Despite significant achievements in these two interrelated fields, hitherto there lacks a systematic examination of the eEF1A protein in the context of cancer research. Therefore, the present work aims to delineate its clinical implications, molecular oncogenic mechanisms, and targeted therapeutic strategies as reflected in the ever expanding body of literature, so as to deepen mechanistic understanding of eEF1A-involved tumorigenesis and inspire the development of eEF1A-targeted chemotherapeutics and biologics.

Discovery of a nitroaromatic nannocystin with potent in vivo anticancer activity against colorectal cancer by targeting AKT1.

The development of targeted chemotherapeutic agents against colorectal cancer (CRC), one of the most common cancers with a high mortality rate, is in a constant need. Nannocystins are a family of myxobacterial secondary metabolites featuring a 21-membered depsipeptide ring. The in vitro anti-CRC activity of natural and synthetic nannocystins was well documented, but little is known about their in vivo efficacy and if positive, the underlying mechanism of action. In this study we synthesized a nitroaromatic nannocystin through improved preparation of a key fragment, and characterized its in vitro activity and in vivo efficacy against CRC. We first described the total synthesis of compounds 2-4 featuring Heck macrocyclization to forge their 21-membered macrocycle. In a panel of 7 cancer cell lines from different tissues, compound 4 inhibited the cell viability with IC values of 1-6 nM. In particular, compound 4 (1, 2, 4 nM) inhibited the proliferation of CRC cell lines (HCT8, HCT116 and LoVo) in both concentration and time dependent manners. Furthermore, compound 4 concentration-dependently inhibited the colony formation and migration of CRC cell lines. Moreover, compound 4 induced cell cycle arrest at sub-G1 phase, apoptosis and cellular senescence in CRC cell lines. In three patient-derived CRC organoids, compound 4 inhibited the PDO with IC values of 3.68, 28.93 and 11.81 nM, respectively. In a patient-derived xenograft mouse model, injection of compound 4 (4, 8 mg/kg, i.p.) every other day for 12 times dose-dependently inhibited the tumor growth without significant change in body weight. We conducted RNA-sequencing, molecular docking and cellular thermal shift assay to elucidate the anti-CRC mechanisms of compound 4, and revealed that it exerted its anti-CRC effect at least in part by targeting AKT1.

Tetrameric Acetyl-CoA Acetyltransferase 1 Is Important for Tumor Growth.

Mitochondrial acetyl-CoA acetyltransferase 1 (ACAT1) regulates pyruvate dehydrogenase complex (PDC) by acetylating pyruvate dehydrogenase (PDH) and PDH phosphatase. How ACAT1 is "hijacked" to contribute to the Warburg effect in human cancer remains unclear. We found that active, tetrameric ACAT1 is commonly upregulated in cells stimulated by EGF and in diverse human cancer cells, where ACAT1 tetramers, but not monomers, are phosphorylated and stabilized by enhanced Y407 phosphorylation. Moreover, we identified arecoline hydrobromide (AH) as a covalent ACAT1 inhibitor that binds to and disrupts only ACAT1 tetramers. The resultant AH-bound ACAT1 monomers cannot reform tetramers. Inhibition of tetrameric ACAT1 by abolishing Y407 phosphorylation or AH treatment results in decreased ACAT1 activity, leading to increased PDC flux and oxidative phosphorylation with attenuated cancer cell proliferation and tumor growth. These findings provide a mechanistic understanding of how oncogenic events signal through distinct acetyltransferases to regulate cancer metabolism and suggest ACAT1 as an anti-cancer target.

Metabolic Rewiring by Oncogenic BRAF V600E Links Ketogenesis Pathway to BRAF-MEK1 Signaling.

Many human cancers share similar metabolic alterations, including the Warburg effect. However, it remains unclear whether oncogene-specific metabolic alterations are required for tumor development. Here we demonstrate a "synthetic lethal" interaction between oncogenic BRAF V600E and a ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCL). HMGCL expression is upregulated in BRAF V600E-expressing human primary melanoma and hairy cell leukemia cells. Suppression of HMGCL specifically attenuates proliferation and tumor growth potential of human melanoma cells expressing BRAF V600E. Mechanistically, active BRAF upregulates HMGCL through an octamer transcription factor Oct-1, leading to increased intracellular levels of HMGCL product, acetoacetate, which selectively enhances binding of BRAF V600E but not BRAF wild-type to MEK1 in V600E-positive cancer cells to promote activation of MEK-ERK signaling. These findings reveal a mutation-specific mechanism by which oncogenic BRAF V600E "rewires" metabolic and cell signaling networks and signals through the Oct-1-HMGCL-acetoacetate axis to selectively promote BRAF V600E-dependent tumor development.

Tyr phosphorylation of PDP1 toggles recruitment between ACAT1 and SIRT3 to regulate the pyruvate dehydrogenase complex.

Mitochondrial pyruvate dehydrogenase complex (PDC) is crucial for glucose homeostasis in mammalian cells. The current understanding of PDC regulation involves inhibitory serine phosphorylation of pyruvate dehydrogenase (PDH) by PDH kinase (PDK), whereas dephosphorylation of PDH by PDH phosphatase (PDP) activates PDC. Here, we report that lysine acetylation of PDHA1 and PDP1 is common in epidermal growth factor (EGF)-stimulated cells and diverse human cancer cells. K321 acetylation inhibits PDHA1 by recruiting PDK1, and K202 acetylation inhibits PDP1 by dissociating its substrate PDHA1, both of which are important in promoting glycolysis in cancer cells and consequent tumor growth. Moreover, we identified mitochondrial ACAT1 and SIRT3 as the upstream acetyltransferase and deacetylase, respectively, of PDHA1 and PDP1, while knockdown of ACAT1 attenuates tumor growth. Furthermore, Y381 phosphorylation of PDP1 dissociates SIRT3 and recruits ACAT1 to PDC. Together, hierarchical, distinct posttranslational modifications act in concert to control molecular composition of PDC and contribute to the Warburg effect.

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.

Identification of HGD and GSTZ1 as Biomarkers Involved Metabolic Reprogramming in Kidney Renal Clear Cell Carcinoma.

Kidney renal clear cell carcinoma (KIRC) with poor prognosis is the main histological subtype of renal cell carcinoma, accounting for more than 80% of patients. Most patients are diagnosed at an advanced stage due to being asymptomatic early on. Advanced KIRC has an extremely poor prognosis due to its inherent resistance to radiotherapy and chemotherapy. Therefore, a comprehensive understanding of the molecular mechanisms of KIRC and the development of effective early diagnostic and therapeutic strategies is urgently needed. In this study, we aimed to identify the prognosis-related biomarker and analyzed its relationship with tumor progression. Metabolic changes are an important feature of kidney cancer, where the reduction of fumarate allows us to target the tyrosine metabolic pathway. The homogentisate 1,2-dioxygenase (HGD) and glutathione S-transferase zeta 1 (GSTZ1) related with prognosis of KIRC was identified through bioinformatics analysis based on The Cancer Genome Atlas (TCGA) databases. Mechanistically, we found that decreased HGD and GSTZ1 promote aerobic glycolysis in KIRC, coordinate the balance of amino acid metabolism and energy metabolism in tumor cells, and ultimately activate the tumor cell cycle and tumor progression. In summary, we identified the tyrosine metabolizing enzymes HGD and GSTZ1 as biomarkers of KIRC, which will further the understanding of the tumor metabolism profile, provide novel strategies and theoretical support for diagnosing and treating KIRC and as referential for future clinical research.

PIKE-A Modulates Mitochondrial Metabolism through Increasing SDHA Expression Mediated by STAT3/FTO Axis.

Previous studies have shown that phosphoinositide 3-kinase enhancer-activating Akt (PIKE-A) is involved in the regulation of several biological processes in cancer. In our previous study, we demonstrated a crucial function of PIKE-A in cancer energy metabolism by regulating pentose phosphate pathway (PPP) flux. However, whether PIKE-A regulates energy metabolism through affecting mitochondrial changes are poorly understood. In the present study, we show that PIKE-A promotes mitochondrial membrane potential, leading to increasing proliferation of glioblastoma cell. Mechanistically, PIKE-A affects the expression of respiratory chain complex Ⅱ succinate dehydrogenase A (SDHA), mediated by regulating the axis of STAT3/FTO. Taken together, these results revealed that inhibition of PIKE-A reduced STAT3/FTO/SDHA expression, leading to the suppression of mitochondrial function. Thus, our findings suggest the PIKE-A/STAT3/FTO/SDHA axis as promising anti-cancer treatment targets.

Genome-wide identification and expression analysis of the bHLH gene family in cauliflower (Brassica oleracea L.).

Basic helix-loop-helix (bHLH) transcription factors (TFs) are one of the largest TF families in plant species, and they play important roles in plant growth, development and stress responses. The present study systematically identified members of the cauliflower (Brassica oleracea L.) bHLH gene family based on genomic data. Analysis of bHLH family gene numbers, evolution, collinearity, gene structures and motifs indicated that cauliflower contained 256 bHLH family genes distributed on 10 chromosomes. Most of these genes have been localized in the nucleus, and they were divided into 18 subgroups which have been relatively conserved during evolution. Promoter analysis showed that most cis-acting elements were related to MeJA and ABA. Expression analysis suggested that 14 bHLH genes may be involved in the transformation of cauliflower curd from white to purple. An expression analysis of these 14 genes in FQ136 material was performed using qRT-PCR, and 9 bHLH genes (BobHLH1, 14, 58, 61, 63, 84, 231, 239 and 243) showed significantly increased or decreased expression in cauliflower from white to purple, which suggests that these 9 genes play important roles in the accumulation of anthocyanins in cauliflower. The coexpression network of these 9 genes and anthocyanin synthesis-related key genes was analyzed using weighted gene coexpression network analysis (WGCNA). In conclusion, our observations suggested that the bHLH gene family plays an important role in the accumulation of anthocyanins in cauliflower and provide an important theoretical basis for further research on the functions of the bHLH gene family and the molecular mechanism of cauliflower coloration. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01238-9.

Octamer transcription factor-1 induces the Warburg effect via up-regulation of hexokinase 2 in non-small cell lung cancer.

Reprogramming of energy metabolism is a hallmark of cancer which is prevalent worldwide. Octamer transcription factor-1 (OCT1) is a well-known transcription factor. However, the role of OCT1 in metabolism remodeling has not been well defined. In the present study, we found that OCT1 was up-regulated in non-small cell lung cancer (NSCLC) and correlated with poor patient survival. Further data identified that OCT1 increased glycolysis flux, promoting proliferation in lung cancer cells. Mechanistically, OCT1 facilitated the aerobic glycolysis and cell proliferation via up-regulation of hexokinase 2 (HK2), a crucial enzyme of the Warburg effect. Hence, our findings indicate that, in NSCLC, high levels of OCT1 contribute to the Warburg effect through up-regulation of HK2, linking up the OCT1/HK2 axis and cancer progression, which provide a potential biomarker and therapeutic target for NSCLC treatment.

Discovery and characterization of a novel glucose-6-phosphate dehydrogenase (G6PD) inhibitor via high-throughput screening.

Altered glucose-6-phosphate dehydrogenase (G6PD) status is influential in many cellular pathophysiological processes and diseases, making G6PD a potential target for cancer therapy. However, the available G6PD inhibitors are very limited and restricted. Here we developed a reducing equivalent nicotinamide adenine dinucleotide phosphate (NADPH) absorption photometry assay based on enzyme kinetics to characterize G6PD activity. In this way, we performed a high-throughput screening (HTS) to an in house library. And then we identified compound named Wedelolactone inhibiting G6PD strongly in a non-competitive, reversible way. In addition, we did the surface Plasmon Resonance (SPR) assay and indicated the KD between Wedelolactone and G6PD protein was 3.64 µM. Furthermore, our basic colony formation assay showed the inhibitory effect of Wedelolactone on the proliferation of ovarian cancer cells (IC50 ~ 10 µM). Thus, we provided a high-throughput screening assay to quickly and efficiently discover G6PD inhibitors, and identified Wedelolactone as a G6PD inhibitor, implying that Wedelolactone suppresses ovarian cancer partly through targeting G6PD.

APEX2-based Proximity Labeling of Atox1 Identifies CRIP2 as a Nuclear Copper-binding Protein that Regulates Autophagy Activation.

Mammalian cell nuclei contain copper, and cancer cells are known to accumulate aberrantly high copper levels, yet the mechanisms underlying nuclear accumulation and copper's broader functional significance remain poorly understood. Here, by combining APEX2-based proximity labeling focused on the copper chaperone Atox1 with mass spectrometry we identified a previously unrecognized nuclear copper binding protein, Cysteine-rich protein 2 (CRIP2), that interacts with Atox1 in the nucleus. We show that Atox1 transfers copper to CRIP2, which induces a change in CRIP2's secondary structure that ultimately promotes its ubiquitin-mediated proteasomal degradation. Finally, we demonstrate that depletion of CRIP2-as well as copper-induced CRIP2 degradation-elevates ROS levels and activates autophagy in H1299 cells. Thus, our study establishes that CRIP2 as an autophagic suppressor protein and implicates CRIP2-mediated copper metabolism in the activation of autophagy in cancer cells.

YTH domain family 2 promotes lung cancer cell growth by facilitating 6-phosphogluconate dehydrogenase mRNA translation.

N6-methyladenosine (m6A) is one of widespread post-transcriptional mRNA modifications in eukaryotes and the m6A modification plays critical roles in various human cancers. However, the role of m6A-binding proteins in cancer metabolism remains elusive. Here, we report that YTH domain family 2 (YTHDF2) is upregulated in lung cancer tissues, promotes lung cancer cell growth and enhances the pentose phosphate pathway (PPP) flux, which is crucial for tumor growth. Mechanistically, YTHDF2 directly binds to the m6A modification site of 6-phosphogluconate dehydrogenase (6PGD) three prime untranslated region (3'-UTR) to promote 6PGD mRNA translation in lung cancer cells. Collectively, our data indicate that YTHDF2 acts as a tumor promoter to enhance tumor growth via facilitating 6PGD mRNA translation.

Metabolomics reveals the effect of valproic acid on MCF-7 and MDA-MB-231 cells.

1. Breast cancer is one of the most common malignancies in women worldwide. Metabolomics has been shown to be a promising strategy to elucidate the underlying pathogenesis of cancer and identify new targets for cancer diagnosis and therapy. Valproic acid (VPA), a histone deacetylase inhibitor, is a potential new drug in tumor therapy. This work used metabolomics to examine the effect of VPA on metabolism in breast cancer cells.2. Based on UPLC-MS/MS, we identified 3137 differential metabolites in human breast cancer MCF-7 cells and 2472 differential metabolites in human breast cancer MDA-MB-231 cells after VPA treatment.3. We selected 63 differential metabolites from MCF-7 samples and 61 differential metabolites from MDA-MB-231 cells with the more conspicuous changing trend. Furfural was up-regulated after VPA treatment in both cell lines. In both samples, VPA exerted an effect on the beta-alanine metabolism pathway and the taurine and hypotaurine metabolism pathway.4. This study identified the effect of VPA on metabolites and metabolic pathways in breast cancer cells, and these findings may contribute to the identification of new targets for breast cancer treatment.

Tyr-301 phosphorylation inhibits pyruvate dehydrogenase by blocking substrate binding and promotes the Warburg effect.

The mitochondrial pyruvate dehydrogenase complex (PDC) plays a crucial role in regulation of glucose homoeostasis in mammalian cells. PDC flux depends on catalytic activity of the most important enzyme component pyruvate dehydrogenase (PDH). PDH kinase inactivates PDC by phosphorylating PDH at specific serine residues, including Ser-293, whereas dephosphorylation of PDH by PDH phosphatase restores PDC activity. The current understanding suggests that Ser-293 phosphorylation of PDH impedes active site accessibility to its substrate pyruvate. Here, we report that phosphorylation of a tyrosine residue Tyr-301 also inhibits PDH α 1 (PDHA1) by blocking pyruvate binding through a novel mechanism in addition to Ser-293 phosphorylation. In addition, we found that multiple oncogenic tyrosine kinases directly phosphorylate PDHA1 at Tyr-301, and Tyr-301 phosphorylation of PDHA1 is common in EGF-stimulated cells as well as diverse human cancer cells and primary leukemia cells from human patients. Moreover, expression of a phosphorylation-deficient PDHA1 Y301F 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 distinct serine and tyrosine residues inhibits PDHA1 through distinct mechanisms to impact active site accessibility, which act in concert to regulate PDC activity and promote the Warburg effect.

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.

Transcriptome analysis identifies genetic risk markers and explores the pathogenesis for inflammatory bowel disease.

Inflammatory bowel disease (IBD) is an incurable and disabling bowel disease driven by multiple risk factors that severely limit patients' quality of life. We integrated the RNA-sequencing data of 1238 IBD patients, and investigated the pathogenesis of IBD by combining transcriptional element prediction analysis and immune-related analysis. Here, we first determined that KIAA1109 is inhibited in IBD patients. The expression of KIAA1109 and NOD2, the key receptor of NOD-like receptors, showed a negative correlation. The NOD-like receptor signaling pathway is activated and exerts transcriptional regulation on the chemokines CXCL1 and CXCL2 through the activation of the transcription factors NFκB and AP1. Analysis of immune infiltration revealed that the expression of chemokines CXCL1 and CXCL2 may regulate the inflammatory response induced by immune cells. These findings suggest that the KIAA1109-NOD2-NFκB/AP1-CXCL1/CXCL2 regulatory axis is the molecular mechanism of IBD pathogenesis, which will provide a new perspective for the diagnosis, treatment and management of IBD patients.

Malate, a natural inhibitor of 6PGD, improves the efficacy of chemotherapy in lung cancer.

OBJECTIVE: Metabolic reprogramming is an important coordinator of tumor development and resistance to therapy, such as the tendency of tumor cells to utilize glycolytic energy rather than oxidative phosphorylation, even under conditions of sufficient oxygen. Therefore, targeting metabolic enzymes is an effective strategy to overcome therapeutic resistance. MATERIALS AND METHODS: We explored the differential expression and growth-promoting function of MDH2 by immunohistochemistry and immunoblotting experiments in lung cancer patients and lung cancer cells. Pentose phosphate pathway-related phenotypes (including ROS levels, NADPH levels, and DNA synthesis) were detected intracellularly, and the interaction of malate and proteinase 6PGD was detected in vitro. In vivo experiments using implanted xenograft mouse models to explore the growth inhibitory effect and pro-chemotherapeutic function of dimethyl malate (DMM) on lung cancer. RESULTS: We found that the expression of malate dehydrogenase (MDH2) in the tricarboxylic acid cycle (TCA cycle) was increased in lung cancer. Biological function enrichment analysis revealed that MDH2 not only promoted oxidative phosphorylation, but also promoted the pentose phosphate pathway (PPP pathway). Mechanistically, it was found that malate, the substrate of MDH2, can bind to the PPP pathway metabolic enzyme 6PGD, inhibit its activity, reduce the generation of NADPH, and block DNA synthesis. More importantly, DMM can improve the sensitivity of lung cancer to the clinical drug cisplatin. CONCLUSION: We have identified malate as a natural inhibitor of 6PGD, which will provide new leads for the development of 6PGD inhibitors. In addition, the metabolic enzyme MDH2 and the metabolite malate may provide a backup option for cells to inhibit their own carcinogenesis, as the accumulated malate targets 6PGD to block the PPP pathway and inhibit cell cycle progression.