Serine Synthesis Pathway Research Articles

CSN6 mediates nucleotide metabolism to promote tumor development and chemoresistance in colorectal cancer.

Metabolic reprogramming can contribute to colorectal cancer (CRC) progression and therapy resistance. Identification of key regulators of CRC metabolism could provide new approaches to improve treatment and reduce recurrence. Here, we demonstrate a critical role for the COP9 signalosome subunit CSN6 in rewiring nucleotide metabolism in CRC. Transcriptomic analysis of CRC patient samples revealed a correlation between CSN6 expression and purine and pyrimidine metabolism. A colitis-associated colorectal cancer model established that Csn6 intestinal conditional deletion decreased tumor development and altered nucleotide metabolism. CSN6 knockdown increased the chemosensitivity of CRC cells in vitro and in vivo, which could be partially reversed with nucleoside supplementation. Isotope metabolite tracing showed that CSN6 loss reduced de novo nucleotide synthesis. Mechanistically, CSN6 upregulated purine and pyrimidine biosynthesis by increasing expression of PHGDH, a key enzyme in the serine synthesis pathway. CSN6 inhibited β-Trcp-mediated DDX5 polyubiquitination and degradation, which in turn promoted DDX5-mediated PHGDH mRNA stabilization, leading to metabolic reprogramming and CRC progression. Butyrate treatment decreased CSN6 expression and improved chemotherapy efficacy. These findings unravel the oncogenic role of CSN6 in regulating nucleotide metabolism and chemosensitivity in CRC.

PHGDH and cancer: new job for an old enzyme!

How do cancer cells bolster their energy metabolism under conditions of stress? Recent work by Shu etal (2022) unveils a novel, non-canonical function of the de novo serine synthesis pathway enzyme phosphoglycerate dehydrogenase (PHGDH) as a regulator of mitochondrial translation and tumor progression in liver cancer.

Target enzymes in serine-glycine-one-carbon metabolic pathway for cancer therapy.

Cancer cells selectively take up exogenous serine or synthesize serine via the serine synthesis pathway for conversion into intracellular glycine and one-carbon units for nucleotide biosynthesis. In this process, serine-glycine metabolism and the one-carbon cycle play vital roles, which is named serine-glycine-one-carbon metabolism (SGOC). The SGOC pathway is a metabolic network crucial for tumorigenesis with unexpected complexity and clinical importance. Accumulating evidence has demonstrated that metabolic enzymes in SGOC metabolism play key roles in tumorigenesis, metastasis and resistance to therapies. In this review, we focus on the involvement of serine and glycine in the folate-mediated one-carbon pathway during cancer progression and highlight the pathways through which cancer cells acquire and use one-carbon units. In addition, we discuss the recently elucidated effects of SGOC (folate cycle) metabolic enzymes in the occurrence and development of tumors and their links to drug resistance. Inhibitors of target enzymes in the SGOC pathway display promise as investigational new drug candidates for the treatment of tumors.

Fructose Treatment Targets Myeloid Leukemogenesis in IDH2 Mutants By Exhausting Alpha-Ketoglutarate Pools

Acute Myeloid Leukemia (AML) is a heterogeneous group of hematological malignancies and represents the most frequent cause of leukemia-related deaths. The uncontrolled growth of myeloblasts in the Bone Marrow (BM) leads to accelerated metabolic processes and high consumption of nutrients, especially glucose. Isocitrate dehydrogenase (IDH) 1 and 2 are frequently mutated in human cancer, affecting up to 15% of AML patients. The neo-morphic activity conferred by IDH 1 or 2 mutations (mIDH1/2) converts α-ketoglutarate (α-KG) to the R-enantiomer of 2-hydroxyglutarate (2-HG), which competitively inhibits α-KG-dependent dioxygenases (Figueroa et al., 2010, Su et al.,2018, Li et al.,2017) and contributes to a block in myeloid differentiation and leukemic transformation. mIDH1/2 are clinically validated therapeutic targets as specific inhibitors have been approved by the FDA for the treatment of AML(DiNardo et al., 2018; Rohle et al., 2013; Stein et al., 2019; Wang et al., 2013) However, only 40% or less of mIDH AML patients respond to IDH inhibitor therapy (DiNardo et al., 2018; Stein et al., 2017) and little is known of how IDH mutations can be exploited for new metabolic vulnerabilities. We recently reported a novel metabolic dependency in AML cells that express low levels of the fructose transporter GLUT5. In fructose conditions these AMLs rewired their metabolism away from glycolysis and toward the serine synthesis pathway (SSP), (Jeong and Savino, Cell Metabolism 2021). Inhibiting the SSP by blocking PHGDH under these metabolic conditions resulted in reduced tumor burden. Additionally, patients with FLT-ITD alterations were sensitive to SSP blockade (Bjelosevic et. al. 2021). Although we previously found that myeloid leukemia cells grow normally in fructose, AML cell lines engineered to express IDH2 R140Q (TF1 knock in, and transduced MOLM13, U937 and OCIAML2) had a 50% reduction in cellular growth and 1.6-fold increase in differentiation (CD15 marker). Remarkably, a similar effect was observed in mIDH1 or 2 chondrosarcoma cell lines, suggesting that this metabolic program is more generally applicable. Excitingly, this new fructose induced therapeutic vulnerability could be recapitulated in vivo. We found that the fructose treated mice could delay myeloid leukemogenesis by 9-10 days (MOLM13 IDH2 R140Q) or in a primary patient sample successfully engrafted with IDH2 R140Q (n=6 mice/group p=0.0012; 3 weeks, I.P. 4 g/kg). To understand the basic mechanism for this new metabolic vulnerability, we first focused on the flux though the SSP. We found an increase in the end product of the SSP, glycine, as measured by LC-MS in the fructose condition compared to glucose. This SSP addiction under fructose was further demonstrated by reduced growth upon PHGDH inhibition (mIDH2: 75% reduction vs 50% WT IDH2 p=.0004; NCT503 5uM). Utilization of the SSP results in the generation of cytosolic α-KG, the substrate of IDH mutant enzymes. Thus, we hypothesized that mIDH AML cells might display enhanced dependency due to the requirement of α-KG for SSP, mIDH2 activity and to sustain the otherwise defective TCA cycle. Consistent with α-KG's central role in fructose driven metabolic addiction, we rescued the cell growth reduction by treating with cell permeable a-KG. Furthermore, in fructose conditions, we observed significant accumulation of the TCA cycle intermediate malate as well as aspartate, suggesting an engagement of the malate-aspartate shuttle in maintaining the redox balance. Previously, we found that AML cell lines (i.e THP1 cells) with moderate to high abundance of surface GLUT5, were not dependent on SSP in fructose. Surprisingly, by introducing IDH2 R140Q or by treating the cells with exogenous D-2HG, we found a 2-fold reduction of surface GLUT5 protein. This reduction of GLUT5 rewired the metabolism toward the SSP-addiction and resulted in a 50% reduction in cell growth in fructose. Interestingly, GLUT5 was controlled through transcriptional regulation of the locus as early as 8hrs post D-2HG treatment. Importantly, this relationship was found to be associated in patients, where PHGDH expression was significantly increased in mIDH2 AML patients with low vs high GLUT5 (n=15, n=16 p<.038) compared to other mutational subtypes (n=155, n=155 ns). In summary, we find a surprising new metabolic dependency based on sugar conditions in mIDH2 AML that could pave the way for a new therapeutic strategy.

Abstract 12785: Psat1 Promotes Serine Synthesis Pathway and Cardiac Regeneration After Myocardial Infarction

Introduction: Permanent loss of cardiomyocytes (CM) after myocardial infarction (MI) and limited cardiac regenerative capacity lead to heart failure. The iPSC-derived extracellular vesicles (EV) have been shown to improve cardiac function and protection. However, the iPSC-EVs-mediated cardiac function improvement mechanism remains unclear and largely pertains to microRNAs and other RNAs, with a little elucidation of the role of iPSC-EV proteins. Hypothesis: The myocardial delivery of hiPSC-EV-specific protein PSAT1 (phosphoserine aminotransferase 1) induced cardiac regeneration and improved cardiac function post-MI by activating CM proliferation and anti-oxidative stress molecular pathways. Methods and Results: We show that hiPSC-EVs treatment augmented cardiac functions and induced a significant CM cell cycle in mice post-MI. Proteomic analysis identified PSAT1 as a protein expressed exclusively in hiPSC-EVs. The role of PSAT1 in CM proliferation and cardiac regeneration is not studied. The PSAT1 expression was significantly downregulated with heart development. Cardiac delivery of PSAT1 modified mRNA (modRNA) induced a significant CM proliferation post-MI. This increase in the CMs proliferation by the PSAT1 modRNA was associated with reduced scar size, improved cardiac function and CM survival, reduced oxidative stress, and CM apoptosis post-MI. Furthermore, the PSAT1 modRNA inhibited the CM apoptosis by reducing oxidative stress and DNA damage response post-MI. Moreover, we show that the YAP, a master regulator of CM proliferation and cardiac regeneration, binds to the promoter of PSAT1 and induces its expression. Finally, PSAT1 modRNA induced the serine biosynthesis pathway in CMs, resulting in increased nucleotide synthesis and reduced oxidative stress, thereby supporting CM proliferation. Conclusion: Our studies uncover a novel role of hiPSC-EV specific protein PSAT1, and serine synthesis pathway activation leads to post-MI CM proliferation, inhibition of oxidative stress, and improved cardiac function. Furthermore, this work emphasizes the therapeutic prospect of using PSAT1 modRNA as a gene delivery approach for ischemic heart diseases.

Metabolic remodeling of pyrimidine synthesis pathway and serine synthesis pathway in human glioblastoma

Glioblastoma is the most common brain tumor with dismal outcomes in adults. Metabolic remodeling is now widely acknowledged as a hallmark of cancer cells, but glioblastoma-specific metabolic pathways remain unclear. Here we show, using a large-scale targeted proteomics platform and integrated molecular pathway-level analysis tool, that the de novo pyrimidine synthesis pathway and serine synthesis pathway (SSP) are the major enriched pathways in vivo for patients with glioblastoma. Among the enzymes associated with nucleotide synthesis, RRM1 and NME1 are significantly upregulated in glioblastoma. In the SSP, SHMT2 and PSPH are upregulated but the upstream enzyme PSAT1 is downregulated in glioblastoma. Kaplan–Meier curves of overall survival for the GSE16011 and The Cancer Genome Atlas datasets revealed that high SSP activity correlated with poor outcome. Enzymes relating to the pyrimidine synthesis pathway and SSP might offer therapeutic targets for new glioblastoma treatments.

Circulating metabolites associated with tumor hypoxia and early response to treatment in bevacizumab-refractory glioblastoma after combined bevacizumab and evofosfamide.

Glioblastomas (GBM) are the most common and aggressive form of primary malignant brain tumor in the adult population, and, despite modern therapies, patients often develop recurrent disease, and the disease remains incurable with median survival below 2 years. Resistance to bevacizumab is driven by hypoxia in the tumor and evofosfamide is a hypoxia-activated prodrug, which we tested in a phase 2, dual center (University of Texas Health Science Center in San Antonio and Dana Farber Cancer Institute) clinical trial after bevacizumab failure. Tumor hypoxic volume was quantified by 18F-misonidazole PET. To identify circulating metabolic biomarkers of tumor hypoxia in patients, we used a high-resolution liquid chromatography-mass spectrometry-based approach to profile blood metabolites and their specific enantiomeric forms using untargeted approaches. Moreover, to evaluate early response to treatment, we characterized changes in circulating metabolite levels during treatment with combined bevacizumab and evofosfamide in recurrent GBM after bevacizumab failure. Gamma aminobutyric acid, and glutamic acid as well as its enantiomeric form D-glutamic acid all inversely correlated with tumor hypoxia. Intermediates of the serine synthesis pathway, which is known to be modulated by hypoxia, also correlated with tumor hypoxia (phosphoserine and serine). Moreover, following treatment, lactic acid was modulated by treatment, likely in response to a hypoxia mediated modulation of oxidative vs glycolytic metabolism. In summary, although our results require further validation in larger patients’ cohorts, we have identified candidate metabolic biomarkers that could evaluate the extent of tumor hypoxia and predict the benefit of combined bevacizumab and evofosfamide treatment in GBM following bevacizumab failure.

Identification of Novel Natural Inhibitors to Human 3-Phosphoglycerate Dehydrogenase (PHGDH) for Cancer Treatment.

Targeting the serine biosynthesis pathway enzymes has turned up as a novel strategy for anti-cancer therapeutics. 3- Phosphoglycerate dehydrogenase (PHGDH) is the rate-limiting enzyme that catalyzes the conversion of 3-Phosphoglyceric acid (3-PG) into 3-Phosphohydroxy pyruvate (3-PPyr) in the first step of serine synthesis pathway and perform a critical role in cancer progression. PHGDH has been reported to be overexpressed in different types of cancers and emerged as a novel target for cancer therapeutics. During this study, virtual screening tools were used for the identification of inhibitors of PHGDH. A library of phenolic compounds was docked against two binding sites of PHGDH using Molegro Virtual Docker (MVD) software. Out of 169 virtually tested compounds, Salvianolic acid C and Schizotenuin F possess good binding potential to co-factor binding site of PHGDH while Salvianolic acid I and Chicoric acid were identified as the best binding compounds toward the substrate binding site of PHGDH. The top selected compounds were evaluated for different physiochemical and ADMET properties, the obtained results showed that none of these hit compounds violated the Pfizer Rule and they possess acceptable ADMET profiles. Further, a commercially available hit compound, Chicoric acid, was evaluated for its anti-cancer potential against PHGDH-expressing gastric cancer cell lines (MGC-803 and SGC-7901) as well as cell lines with low expression of PHGDH (MCF-7 and MDA-MB2-31), which demonstrated that Chicoric acid possesses selective cytotoxicity toward PHGDH expressing cancer cell lines. Thus, this study has unveiled the potential of phenolic compounds, which could serve as novel candidates for the development of PHGDH inhibitors as anti-cancer agents.

Abstract P3051: Yap Regulates Serine Biosynthesis During Compensatory Cardiac Hypertrophy

Introduction: In response to increased workload, cardiomyocytes increase in size, and the heart undergoes hypertrophy. The initial hypertrophy is a compensatory process that adds contractile units but reduces wall stress to help maintain heart function. Yes-associated protein (YAP), a major effector of the Hippo pathway, is transiently activated in response to acute pressure overload (PO). Cardiac-specific heterozygous downregulation of YAP inhibits hypertrophy but promotes heart failure, suggesting that endogenous YAP is salutary and mediates compensatory hypertrophy during the acute phase of PO. However, the mechanism by which YAP mediates compensatory hypertrophy is not well understood. We have shown previously that 3-phosphoglyceric acid and serine accumulate in the heart during acute PO in a YAP-dependent manner. The serine biosynthetic pathway provides precursors for the TCA cycle and antioxidant and purine biosynthesis. Hypothesis: YAP induces serine synthesis, thereby promoting compensatory cardiac hypertrophy in the presence of acute PO. Methods and Results: We evaluated the levels of intermediate enzymes in the serine synthesis pathway in YAP-overexpressing neonatal rat ventricular myocytes (NRVMs). Furthermore, we investigated whether YAP-induced cardiomyocyte hypertrophy is inhibited by siRNA targeting serine biosynthesis enzymes. Immunofluorescent staining of NRVMs with cardiac troponin T was conducted to evaluate cardiomyocyte size, which was assessed using ImageJ software. YAP upregulated protein levels of phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), phosphoserine phosphatase (PSPH), and serine hydroxymethyltransferase 1 (SHMT1) in NRVMs. siRNA-mediated downregulation of PHGDH, PSAT1, PSPH, or SHMT1 significantly inhibited YAP-induced cardiomyocytes hypertrophy. Conclusions: YAP upregulates the expression of enzymes in the serine biosynthesis pathway, thereby inducing hypertrophy in NRVMs. Metabolites of the serine biosynthesis pathway may play an important role in mediating YAP-induced compensatory cardiac hypertrophy.

Abstract 6058: The serine synthesis pathway contributes to the radiation-induced metabolic plasticity in glioblastoma multiforme

Abstract Purpose: Glioblastoma multiforme (GBM) remains a fatal disease despite aggressive treatment approaches. Post-surgical radiation therapy (RT) is the only treatment that significantly improves survival of GBM patients but recurrence is inevitable. Elucidating the mechanisms of resistance to RT could transform GBM patient outcomes. The purpose of this study was to elucidate the effect of radiation on the serine synthesis pathway (SSP) and the role of SSP in boosting antioxidant defenses in irradiated GBM cells and promoting radioresistance. Methods: We compared copy number alterations (CNA) and amplification of SSP enzymes (PHGDH, PSAT1, PSPH, SHMT1/2) in low-grade gliomas (LGG, n = 511) and GBM (n = 575) via TCGA pan-cancer analysis. In vitro HPLC/MS-based metabolomics assays were used to study the changes in overall metabolite levels induced by radiation in patient-derived GBM specimens. Targeted metabolomics with 13C-labeled glucose was used for tracking the fate of glucose towards SSP intermediates and antioxidant species. Gene expression levels after radiation were measured via molecular biology approaches. De novo SSP was targeted via pharmacological inhibition of PHGDH. Modified clonogenic, sphere forming assays (SFA) on gliomasphere cultures were used to determine radiosensitivity. Dependence on extracellular serine/glycine was determined via serine/glycine depletion experiments. Results: The TCGA analysis revealed increased CNAs in all the de novo SSP enzymes in GBM tumors relative to the LGGs. 82% of GBM tumors had CNAs or amplification of the PSPH gene, which codes for the ultimate enzyme in serine synthesis. In vitro metabolomics assays revealed that radiation increases the levels of serine, glycine and cystine in gliomaspheres, and this is accompanied by elevated levels of reduced glutathione (GSH). Targeted glucose metabolomics revealed radiation-induced upregulation of the de novoSSP, with a concomitant increase in gene expression of SSP enzymes. Downstream of the SSP, metabolomics data show that irradiated GBM cells elevate nucleotide and precursor levels, likely to support DNA damage repair mechanisms after radiation. The inhibition of PHGDH, the first rate-limiting enzyme in de novo SSP, significantly reduced GBM cell survival in vitro. Extracellular serine/glycine appeared to also be important contributors to radiation survival. Gene expression of SLC1A4, an amino acid transporter, was upregulated by radiation, while depletion of extracellular serine/glycine led to significant decreased survival in in vitro SFAs. Conclusion: Here we provide evidence that the de novo serine synthesis pathway is an important contributor to the metabolic reprogramming of GBM cells after radiation that promotes radiation resistance. These data call for further investigations to evaluate the radiosensitizing potential of targeting de novo SSP in GBM. Citation Format: Justine Bailleul, Yangjingyi Ruan, Erina Vlashi. The serine synthesis pathway contributes to the radiation-induced metabolic plasticity in glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6058.

Abstract 3014: PHGDH- A therapeutic vulnerability in melanoma

Abstract Recurrently amplified oncogenes may represent melanoma vulnerabilities that can be exploited for therapy. The genomic locus containing the Phosphoglycerate Dehydrogenase (PHGDH) gene at chromosome 1p12 is frequently amplified in melanoma. PHGDH is the rate limiting enzyme that converts the glycolytic intermediate 3-Phospho Glycerate (3-PG) to serine through the serine synthesis pathway (SSP). Serine is utilized to make glycine, glutathione, one carbon metabolites, proteins and lipids. PHGDH inhibition suppresses proliferation of 1p12 amplified melanoma cells. However, the role of PHGDH in chromosome 1p12 non-amplified melanoma is poorly understood. We observed that 1p12 non-amplified melanoma cell lines have enhanced expression of the SSP enzymes as compared to melanocytes cultured in tetradecanoyl-phorbol acetate (TPA, a growth factor that stimulates MAPK pathway). Moreover, TPA starvation in melanocytes increased PHGDH expression. This led us to hypothesize that activation of oncogenic MAPK signaling leads to high expression of PHGDH which can serve as therapeutic vulnerability in melanomagenesis. Acute expression of oncogenic BRAFV600E in melanocytes resulted in upregulation of SSP enzymes, while treatment of melanoma cells with either BRAF or MEK inhibitor resulted in downregulation of SSP enzymes. We confirmed that PHGDH is regulated by BRAF-ERK-mTOR axis. Downregulation of PHGDH in 1p12 non-amplified melanoma cell lines decreased their cell proliferation and colony formation ability in vitro. We also observed that Phgdh knockdown in a BrafV600E/WT and Pten-/- (BPP) melanoma mouse model significantly increased survival. Furthermore, serine-glycine starvation (external source of Serine-glycine) in BPP chimeras did not affect the survival of these mice while on the other end, it suppressed the growth of the xenograft tumors injected in the NSG mice, which warrants further investigation. These results suggest the importance of PHGDH for melanoma cell proliferation and tumor growth irrespective of its amplification status. The exact mechanism of PHGDH regulation by BRAFV600E and the therapeutic effect of PHGDH inhibition in melanoma initiation and progression are the focus of our studies. Citation Format: Neel N. Jasani, Florian Karreth. PHGDH- A therapeutic vulnerability in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3014.

Purine nucleotide depletion prompts cell migration by stimulating the serine synthesis pathway

Purine nucleotides are necessary for various biological processes related to cell proliferation. Despite their importance in DNA and RNA synthesis, cellular signaling, and energy-dependent reactions, the impact of changes in cellular purine levels on cell physiology remains poorly understood. Here, we find that purine depletion stimulates cell migration, despite effective reduction in cell proliferation. Blocking purine synthesis triggers a shunt of glycolytic carbon into the serine synthesis pathway, which is required for the induction of cell migration upon purine depletion. The stimulation of cell migration upon a reduction in intracellular purines required one-carbon metabolism downstream of de novo serine synthesis. Decreased purine abundance and the subsequent increase in serine synthesis triggers an epithelial-mesenchymal transition (EMT) and, in cancer models, promotes metastatic colonization. Thus, reducing the available pool of intracellular purines re-routes metabolic flux from glycolysis into de novo serine synthesis, a metabolic change that stimulates a program of cell migration.

PHGDH is required for germinal center formation and is a therapeutic target in MYC-driven lymphoma.

The synthesis of serine from glucose is a key metabolic pathway supporting cellular proliferation in healthy and malignant cells. Despite this, the role that this aspect of metabolism plays in germinal center biology and pathology is not known. Here, we performed a comprehensive characterization of the role of the serine synthesis pathway in germinal center B cells and lymphomas derived from these cells. We demonstrate that upregulation of a functional serine synthesis pathway is a metabolic hallmark of B cell activation and the germinal center reaction. Inhibition of phosphoglycerate dehydrogenase (PHGDH), the first and rate-limiting enzyme in this pathway, led to defective germinal formation and impaired high-affinity antibody production. In addition, overexpression of enzymes involved in serine synthesis was a characteristic of germinal center B cell–derived lymphomas, with high levels of expression being predictive of reduced overall survival in diffuse large B cell lymphoma. Inhibition of PHGDH induced apoptosis in lymphoma cells, reducing disease progression. These findings establish PHGDH as a critical player in humoral immunity and a clinically relevant target in lymphoma.

Phosphoglycerate dehydrogenase positively regulates the proliferation of chicken muscle cells

Phosphoglycerate dehydrogenase (PHGDH) is the rate-limiting enzyme in the serine synthesis pathway. However, the regulatory role of PHGDH in muscle development is unclear. We report that the expression of PHGDH increased significantly during proliferation of chicken skeletal muscle satellite cells. Knockdown of PHGDH by an siRNA suppressed myoblast proliferation, whereas overexpression of PHGDH enhanced muscle cell proliferation. Furthermore, PHGDH promoted the expression of Forkhead box protein M1 (FoxM1). Knockdown of FoxM1 by an siRNA attenuated the proliferation of chicken muscle cells, whereas its overexpression significantly promoted proliferation. Additionally, siRNA-PHGDH inhibited pcDNA3.1-FoxM1-induced FoxM1 expression in chicken muscle cells. Moreover, PHGDH inhibition overcame the stimulation by pcDNA3.1-FoxM1 of cell cycle-related gene expression. We propose that PHGDH accelerates chicken muscle cell proliferation by increasing FoxM1 expression.

Pharmacological inhibition of serine synthesis enhances temozolomide efficacy by decreasing O6-methylguanine DNA methyltransferase (MGMT) expression and reactive oxygen species (ROS)-mediated DNA damage in glioblastoma

AbstractGlioblastoma (GBM) is the most malignant primary tumor in the central nervous system of adults. Temozolomide (TMZ), an alkylating agent, is the first-line chemotherapeutic agent for GBM patients. However, its efficacy is often limited by innate or acquired chemoresistance. Cancer cells can rewire their metabolic programming to support rapid growth and sustain cell survival against chemotherapies. An example is the de novo serine synthesis pathway (SSP), one of the main branches from glycolysis that is highly activated in multiple cancers in promoting cancer progression and inducing chemotherapy resistance. However, the roles of SSP in TMZ therapy for GBM patients remain unexplored. In this study, we employed NCT503, a highly selective inhibitor of phosphoglycerate dehydrogenase (PHGDH, the first rate-limiting enzyme of SSP), to study whether inhibition of SSP may enhance TMZ efficacy in MGMT-positive GBMs. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flowcytometry and colony formation assays demonstrated that NCT503 worked synergistically with TMZ in suppressing GBM cell growth and inducing apoptosis in T98G and U118 cells in vitro. U118 and patient-derived GBM subcutaneous xenograft models showed that combined NCT503 and TMZ treatment inhibited GBM growth and promoted apoptosis more significantly than would each treatment alone in vivo. Mechanistically, we found that NCT503 treatment decreased MGMT expression possibly by modulating the Wnt/β-catenin pathway. Moreover, intracellular levels of reactive oxygen species were elevated especially when NCT503 and TMZ treatments were combined, and the synergistic effects could be partially negated by NAC, a classic scavenger of reactive oxygen species. Taken together, these results suggest that NCT503 may be a promising agent for augmenting TMZ efficacy in the treatment of GBM, especially in TMZ-resistant GBMs with high expression of MGMT.Pharmacological inhibition of the serine synthesis pathway with a highly selective inhibitor NCT503 synergistically works with temozolomide in inhibiting O6-methylguanine DNA methyltransferase (MGMT)-positive glioblastoma cell growth and inducing apoptosis both in vitro and in vivo. Mechanistically, NCT503 treatment reduces MGMT expression possibly via Wnt/βcatenin pathway. Reactive oxygen species-mediated DNA damage is at least partially involved. Combinational administration of NCT503 and TMZ may represent a novel and promising treatment strategy to enhance TMZ efficacy in patients with MGMT-high glioblastoma.

Lineage-specific silencing of PSAT1 induces serine auxotrophy and sensitivity to dietary serine starvation in luminal breast tumors.

SUMMARYA major challenge of targeting metabolism for cancer therapy is pathway redundancy, in which multiple sources of critical nutrients can limit the effectiveness of some metabolism-targeted therapies. Here, we analyze lineage-dependent gene expression in human breast tumors to identify differences in metabolic gene expression that may limit pathway redundancy and create therapeutic vulnerabilities. We find that the serine synthesis pathway gene PSAT1 is the most depleted metabolic gene in luminal breast tumors relative to basal tumors. Low PSAT1 prevents de novo serine biosynthesis and sensitizes luminal breast cancer cells to serine and glycine starvation in vitro and in vivo. This PSAT1 expression disparity preexists in the putative cells of origin of basal and luminal tumors and is due to luminal-specific hypermethylation of the PSAT1 gene. Our data demonstrate that luminal breast tumors are auxotrophic for serine and may be uniquely sensitive to therapies targeting serine availability.

Phosphatases of regenerating liver are key regulators of metabolism in cancer cells - role of Serine/Glycine metabolism.

Purpose of reviewPhosphatases of regenerating liver (PRL) are dual-specificity phosphatases and comprise three members, PRL-1, -2 and -3. Despite the importance of PRLs as oncoproteins, there is no consensus function for this family of phosphatases. In the current review paper, we summarize recent findings on the role of PRLs in metabolic regulation.Recent findingsReprogramming of cellular metabolism is a cancer hallmark. Glucose is the major source of energy in cells. Glucose metabolism occurs through the glycolysis and can continue through the pathways such as serine synthesis pathway or the tricarboxylic acid cycle (TCA). Magnesium (Mg2+), the second most abundant cation in cells, plays an essential role in energy production by acting as a cofactor for most enzymes involved in glycolysis and in TCA. Recent findings have shown that the PRL family has a role in metabolic reprogramming mediated by (1) Mg2+ homeostasis, (2) shifting the energy source preference to glucose consumption and fueling serine/glycine pathway and (3) regulating PI3 kinase/Mammalian target of rapamycin complex. Both the phosphatase and nonphosphatase activity of PRLs appear to be important for its oncogenic role.SummaryThe PRL family contributes to the metabolic plasticity of cancer cells and, thereby, allows cancer cells to meet the high metabolic demands required for cell proliferation.

Metabolic Profiling Reveals Cellular Reprogramming of Acute Myeloid Leukemia By Omipalisib through Serine Synthesis Pathway

Background: More than 50% of AML patients had hyperactivation of PI3K-AKT-mTOR signaling. Those patients are supposed to be associated with poor prognosis and chemotherapy resistance. The PI3K-AKT-mTOR signaling involves many cellular processes, including mRNA translation, cellular metabolism, and protein turnover. Omipalisib is a dual PI3K/mTOR inhibitor that exhibits anti-tumor activity in several cancers. However, the precise metabolic consequences in response to PI3K/mTOR dual inhibitor are still not fully studied in AML.Aims: To evaluate the efficacy and to elucidate the metabolic alteration of the anti-cancer effects of omipalisib on leukemic cells from both in vitro and in vivo aspects.Materials and Methods: HL60, THP1, and OCI-AML3 myeloid leukemia cell lines were used in this study. Omipalisib (GSK2126458) was used for in vitro and in vivo experiments. Cell proliferation was measured by Cell Titer 96 AQueous One Solution Cell Proliferation Assay. The protein expression and phosphorylation status were analyzed by immunoblotting. Flow cytometry was used for cell cycle and mitochondrial analysis. The metabolomics profiles were analyzed by Agilent 1290 UHPLC system coupled with 6540-QTOF. RNA-seq was performed using an Illumina NovaSeq 6000 platform. Differentially expressed genes (DEGs) between control and omipalisib groups were identified by EBseq. A threshold of fold change ≥2 (or ≤0.5) and p ≤ 0.05 was used to select the DEGs. The mRNA quantification was measured by QuantStudio 3 Real-Time PCR Systems. The oxygen consumption rate (OCR) was analyzed by the XFe 24 extracellular flux analyzer. The CAnN.Cg-Foxn1 nu/CrlNarl mice were used for evaluating in vivo efficacy of omipalisib in murine model.Results: We demonstrated the anti-proliferation effect of omipalisib on AML cell lines with different genetic background. The IC 50 of OCI-AML3, THP1, and HL60 were 16.97nM, 9.35 nM, and 18.69 nM, respectively. Omipalisib could significantly induce G 0/G 1 cell cycle arrest in all three cell lines. As expected, omipalisib could significantly down-regulate the phosphorylation of AKT, mTOR, S6K and 4E-BP1. Metabolomics profiling analysis revealed that 24 of the 137 tested metabolites were significantly different between the control group and the omipalisib-treated groups in OCI-AML3 cells. Further metabolic pathway enrichment analysis demonstrated that metabolites related to amino acid metabolisms were significantly reduced following omipalisib treatment. In addition, we identified 300 DEGs between control and omipalisib-treated OCI-AML3 cells; of these, 251 were upregulated and 49 were downregulated. Further gene set enrichment analysis (GSEA) of hallmark gene sets indicated omipalisib treatment was significantly negatively associated with E2F targets, Myc targets, G2M checkpoint, mTORC1 signaling pathway, and oxidative phosphorylation. Joint-Pathway analysis (MetaboAnalyst 5.0) revealed that ‘glycine, serine and threonine metabolism’ was the most downregulated pathway in the omipalisib-treated group with p-value of 1.0076E-5 and impact value of 0.86567. qRT-PCR confirmed that several important genes, PHGDH, PSAT1, PSPH, SHMT1/2 and MTHFD1/2 in the serine and glycine synthesis pathway were significantly decrease in the OCI-AML3 cells following treated with omipalisib. OCR analysis indicated that the capacity of the mitochondria to produce energy was reduced after omipalisib treatment. Mitochondrial analysis showed that mitochondria mass and membrane potential decreased after omipalisib treatment, indicating the biosynthesis and functions of mitochondrial may be affected by omipalisib. In vivo studies showed that oral administration of 0.2 or 1 mg/kg omipalisib in mice could significantly retard tumor growth without obvious changes in body weight.Summary: We found that nanomolar levels of omipalisib could significantly inhibit cell growth and induce G 0/G 1 cell cycle arrest in myeloid leukemia cells. Joint-Pathway analysis of RNA-seq and metabolomics data revealed that omipalisib mainly altered serine and glycine metabolism. Further experiments indicated that serine synthesis pathway could be suppressed by omipalisib at least in part through disrupting PI3K-AKT-mTOR signaling. In vivo xenograft model, omipalisib could retard tumor growth at as low as 0.2 mg/kg. This information may be potentially suitable for future clinical application. [Display omitted] DisclosuresChou: Kirin: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Other: Advisory Board; Pfizer: Honoraria, Other: Advisory Board; IQVIA: Honoraria, Other: Advisory Board; Abbvie: Honoraria, Other: Advisory Board, Research Funding; Celgene: Honoraria, Other: Advisory Board, Research Funding. Tien: AbbVie: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria.

Phosphoglycerate Dehydrogenase Is Required for Germinal Center Formation and Is a Therapeutic Target in MYC-driven Lymphoma

The fields of cancer- and immuno-metabolism have re-emerged as areas of significant translational potential. Even though the upregulation of glycolysis by proliferating lymphocytes is the basis for widely used clinical tests such as FDG-PET, little is known about which metabolic pathways are involved in the utilization of glucose to support B-cell proliferation. The synthesis of serine from glucose has been demonstrated to be a key metabolic pathway supporting cellular proliferation in some healthy and malignant cell types. Importantly, this pathway is regulated by MYC, which is known to be essential for germinal centre formation and is commonly dysregulated in lymphoma. Despite this, the role that the serine synthesis pathway (SSP) plays in germinal center biology and pathology has not been previously investigated. We performed a comprehensive characterization of the role of the SSP in germinal center B cells and lymphomas derived from these cells.We demonstrate that upregulation of a functional SSP is a metabolic hallmark of B-cell activation and the germinal center reaction. We show that both human and murine resting naïve B cells lack expression of the first two enzymes in this pathway, phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT1) enzymes. However, B-cell activation, predominantly through the B-cell receptor, robustly induces the expression of these enzymes in vitro, resulting in an acquired ability to synthesize serine, glycine and the purine nucleotides adenosine and guanosine from glucose. This is reflected in striking expression of PHGDH and PSAT1 within germinal centers but not in marginal zones confirming that this upregulation is occurring in germinal B cells activated in vivo. We then proceeded to investigate the impact of inhibiting PHGDH on germinal center formation and high-affinity antibody production in vivo. This was done both genetically, using a conditional B-cell knockout mouse model, and pharmacologically using a specific inhibitor of PHGDH, PH-755. Importantly, we show that PHGDH inhibition impairs germinal center formation with a resultant reduction in high-affinity antibody production. Mechanistic experiments demonstrate that PHGDH inhibition effectively blocks cells from synthesising serine and glycine from glucose, making them unable to proliferate in environments that lack these amino acids.We then investigated role of PHDGH and PSAT1 in Burkitt Lymphoma (BL), Diffuse Large B Cell Lymphoma (DLBCL) and Chronic Lymphocytic Leukemia (CLL). Notably, very high expression of these two proteins was observed in BL, with intermediate-to-high expression in DLBCL and relatively low expression in CLL, where expression was restricted to proliferation centers. Given the heterogeneity of expression in DLBCL patients, we next interrogated a published GSE database (Lenz et al. NEJM 2008) to investigate the impact on outcome. Notably high expression of PSAT1 was significantly associated with a poorer overall survival rate in DLBCL.We then investigated whether the SSP could be a therapeutic target in lymphoma. We demonstrate that PHGDH inhibition effectively inhibits de novo serine, glycine and purine nucleotide synthesis from glucose, resulting in impaired proliferation and increased apoptosis in a panel of human BL cell lines in vitro. We then analyzed the impact of PHGDH inhibition on lymphoma development in vivo using Eµ-myc mice, which harbour Myc coupled to the IgH enhancer characteristic of BL. Importantly we confirm the role of MYC by demonstrating that Eµ-Myc B-cells show significantly higher expression of PHGDH and PSAT1 expression resulting in increased serine and glycine synthesis when compared to control cells. We demonstrate that pharmacological inhibition of PHGDH using PH-755 impairs lymphoma progression in this model. We confirm the importance of PHGDH by showing that genetic ablation of Phgdh in Eµ-myc cells in a tamoxifen inducible system (using Eµ-myc/+;Rosa26-CreER T2/+;Phgdh fl/fl mice) also results in a significant reduction in lymphoma progression.Taken together, this work represents the first report of the role of the SSP in the biology of the germinal centre response and lymphomas derived from these cells. These findings establish PHGDH as a critical player in humoral immunity and a clinically relevant target in MYC-driven lymphoma, which is an area of significant unmet need. DisclosuresGribben: Abbvie: Honoraria; AZ: Honoraria, Research Funding; BMS: Honoraria; Gilead/Kite: Honoraria; Janssen: Honoraria, Research Funding; Morphosys: Honoraria; Novartis: Honoraria; Takeda: Honoraria; TG Therapeutis: Honoraria. Calado: Myricx Pharma: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Patents & Royalties: Cancer Treatments. WO patent WO 2020/128475 A1 (2020).

Proteomics analysis reveals the correlation of programmed ROS-autophagy loop and dysregulated G1/S checkpoint with imatinib resistance in chronic myeloid leukemia cells.

Although tyrosine kinase inhibitors (TKIs), including imatinib, have greatly improved clinical treatment of patients with chronic myeloid leukemia (CML), drug resistance remains a major obstacle. Studies on the mechanisms underlying imatinib resistance and other alternative drugs are urgently needed. Liquid chromatography tandem mass spectrometry was applied to investigate the differences in proteomics and phosphoproteomics between K562 and K562/G (imatinib resistant K562). Multiple bioinformatics analyses were performed to unveil the differential signal pathways. CCK-8 was used to detect cell proliferation. Flow cytometry was performed to analyze reactive oxygen species (ROS), cell cycle, and cell apoptosis. Western blotting and quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) were used to observe the changes of ROS and autophagy associated with imatinib resistance in CML. Our results indicated that ROS-autophagy formed one negative feedback loop and was associated with imatinib resistance. Additionally, the limited-rate enzymes of serine synthesis pathway were escalated in K562/G, which could contribute to the increased cyclin-dependent kinases and cell proliferation index. According to phosphoproteomics data, K562/G cells exhibited abnormal phosphorylation of splicing signals. These results revealed that it could be one useful strategy to correct metabolism shift and oxidative stress, or moderately regulate autophagy. Future research should focus on the discovery of potential targets in ROS-autophagy loop.