Serine Biosynthesis Pathway Research Articles

YY2/PHGDH axis suppresses tumorigenesis by inhibiting tumor cell de novo serine biosynthesis

Metabolic reprogramming is one of the key features of tumors facilitating their rapid proliferation and adaptation to harsh microenvironments. Yin Yang 2 (YY2) has recently been reported as a tumor suppressor downregulated in various types of tumors; however, the molecular mechanisms underlying its tumor-suppressive activity remain poorly understood. Furthermore, the involvement of YY2 in tumor cell metabolic reprogramming remains unclear. Herein, we aimed to elucidate the novel regulatory mechanism of YY2 in the suppression of tumorigenesis. Using transcriptomic analysis, we uncovered an unprecedented link between YY2 and tumor cell serine metabolism. YY2 alteration could negatively regulate the expression level of phosphoglycerate dehydrogenase (PHGDH), the first enzyme in the serine biosynthesis pathway, and consequently, tumor cell de novo serine biosynthesis. Mechanistically, we revealed that YY2 binds to the PHGDH promoter and suppresses its transcriptional activity. This, in turn, leads to decreased production of serine, nucleotides, and cellular reductants NADH and NADPH, which subsequently suppresses tumorigenic potential. These findings reveal a novel function of YY2 as a regulator of the serine metabolic pathway in tumor cells and provide new insights into its tumor suppressor activity. Furthermore, our findings suggest the potential of YY2 as a target for metabolic-based antitumor therapeutic strategies.

Metabonomic analysis of the anti-hepatic fibrosis effect of Ganlong capsules.

Context: Hepatic fibrosis is a progressive condition, often attributed to metabolic disorders, which may promote cirrhosis and liver cancer. Ganlong capsules derived from Periplaneta Americana have been shown to have a therapeutic effect on liver fibrosis but little is known about the molecular mechanisms involved. Objective: To investigate the metabolic modulations produced by Ganlong capsules in liver fibrosis. Methods: A carbon tetrachloride- (CCl4) treated rat model of liver fibrosis was constructed and Ganlong capsules administered. Levels of serum liver enzymes and pathological changes to the liver were evaluated. Non-targeted metabolomics of liver, serum and urine were used to investigate metabolic regulatory mechanisms. Results: Ganlong capsules reduced serum levels of liver enzymes and improved pathological changes in the rat model of fibrosis. Non-targeted metabolomics showed that Ganlong capsules ameliorated pathways of glycerophospholipid, linoleic acid, pyrimidine, glycine, butyric acid, valine, serine, threonine and arachidonic acid metabolism and biosynthesis of leucine and isoleucine. Such pathways influence the development of CCl4-induced liver fibrosis. Conclusion: Ganlong capsules had an anti-fibrotic hepatoprotective effect and regulated lipid, butyric acid, amino acid and arachidonic acid metabolism.

Molecular Structure of Phosphoserine Aminotransferase from Saccharomyces cerevisiae

Phosphoserine aminotransferase (PSAT) is a pyridoxal 5'-phosphate-dependent enzyme involved in the second step of the phosphorylated pathway of serine biosynthesis. PSAT catalyzes the transamination of 3-phosphohydroxypyruvate to 3-phosphoserine using L-glutamate as the amino donor. Although structural studies of PSAT have been performed from archaea and humans, no structural information is available from fungi. Therefore, to elucidate the structural features of fungal PSAT, we determined the crystal structure of Saccharomyces cerevisiae PSAT (ScPSAT) at a resolution of 2.8 Å. The results demonstrated that the ScPSAT protein was dimeric in its crystal structure. Moreover, the gate-keeping loop of ScPSAT exhibited a conformation similar to that of other species. Several distinct structural features in the halide-binding and active sites of ScPSAT were compared with its homologs. Overall, this study contributes to our current understanding of PSAT by identifying the structural features of fungal PSAT for the first time.

PHGDH arginine methylation by PRMT1 promotes serine synthesis and represents a therapeutic vulnerability in hepatocellular carcinoma

Serine synthesis is crucial for tumor growth and survival, but its regulatory mechanism in cancer remains elusive. Here, using integrative metabolomics and transcriptomics analyses, we show a heterogeneity between metabolite and transcript profiles. Specifically, the level of serine in hepatocellular carcinoma (HCC) tissues is increased, whereas the expression of phosphoglycerate dehydrogenase (PHGDH), the first rate-limiting enzyme in serine biosynthesis pathway, is markedly downregulated. Interestingly, the increased serine level is obtained by enhanced PHGDH catalytic activity due to protein arginine methyltransferase 1 (PRMT1)-mediated methylation of PHGDH at arginine 236. PRMT1-mediated PHGDH methylation and activation potentiates serine synthesis, ameliorates oxidative stress, and promotes HCC growth in vitro and in vivo. Furthermore, PRMT1-mediated PHGDH methylation correlates with PHGDH hyperactivation and serine accumulation in human HCC tissues, and is predictive of poor prognosis of HCC patients. Notably, blocking PHGDH methylation with a TAT-tagged nonmethylated peptide inhibits serine synthesis and restrains HCC growth in an HCC patient-derived xenograft (PDX) model and subcutaneous HCC cell-derived xenograft model. Overall, our findings reveal a regulatory mechanism of PHGDH activity and serine synthesis, and suggest PHGDH methylation as a potential therapeutic vulnerability in HCC.

Overexpression of the Phosphoserine Phosphatase-Encoding Gene (AtPSP1) Promotes Starch Accumulation in Lemna turionifera 5511 under Sulfur Deficiency

Duckweeds are well known for their high accumulation of starch under stress conditions, along with inhibited growth. The phosphorylation pathway of serine biosynthesis (PPSB) was reported as playing a vital role in linking the carbon, nitrogen, and sulfur metabolism in this plant. The overexpression of AtPSP1, the last key enzyme of the PPSB pathway in duckweed, was found to stimulate the accumulation of starch under sulfur-deficient conditions. The growth- and photosynthesis-related parameters were higher in the AtPSP1 transgenic plants than in the WT. The transcriptional analysis showed that the expression of several genes in starch synthesis, TCA, and sulfur absorption, transportation, and assimilation was significantly up- or downregulated. The study suggests that PSP engineering could improve starch accumulation in Lemna turionifera 5511 by coordinating the carbon metabolism and sulfur assimilation under sulfur-deficient conditions.

Discovery of Novel Drug-like PHGDH Inhibitors to Disrupt Serine Biosynthesis for Cancer Therapy.

Being the rate-limiting enzyme within the serine biosynthesis pathway, phosphoglycerate dehydrogenase (PHGDH) is abnormally overexpressed in numerous malignant tumor cells and is a promising target for cancer treatment. Here, we report a series of novel PHGDH inhibitors using a focused compound screening and structural optimization approach. The lead compound D8 displayed good enzymatic inhibitory activity (IC50 = 2.8 ± 0.1 μM), high binding affinity (Kd = 2.33 μM), and sensitivity to the cell lines with the PHGDH gene amplification or overexpression. Furthermore, D8 was proven to restrict the de novo serine synthesis from glucose within MDA-MB-468 cells. X-ray crystallographic analysis, molecular dynamics simulations, and mutagenesis experiments on PHGDH revealed the binding site at D175 inside the NAD+-binding pocket. Finally, D8 exhibited excellent in vivo pharmacokinetic properties (F = 82.0%) and exerted evident antitumor efficacy in the PC9 xenograft mouse model.

Deubiquitinating enzyme Josephin-2 stabilizes PHGDH to promote a cancer stem cell phenotype in hepatocellular carcinoma.

Deubiquitinating enzymes (DUBs) have been shown to be possible targets for hepatocellular carcinoma (HCC) treatment. This study was designed to reveal the effect and underlying mechanism of Josephin-2, a relatively newly defined DUB, in HCC progression. SNU-387 and PLC/PRF/5 cells were used for in vitro functional assays. The levels of Josephin-2 and phosphoglycerate dehydrogenase (PHGDH) were determined using RT-qPCR and western blotting. Cell proliferation, migration and invasion were assessed by CCK-8, colony formation and Transwell. Spheroid-forming assay was performed to assess the cancer stem cell (CSC)-phenotype of HCC cells. A xenograft mice model was applied to verify the effect of Josephin-2 on HCC cell growth in vivo. Herein, we showed that Josephin-2 expression was negatively correlated with HCC patient survival in data from the online database. Cell experiments indicated that knockdown of Josephin-2 attenuated HCC cell malignant biological behaviors. Besides, Josephin-2 silencing also decreased the spheroid-formation while inhibited the expression of CSC biomarkers (CD133, OCT4, SOX2 and EpCAM) in HCC cells. Mechanistically, Josephin-2 had a deubiquitinating activity towards the regulation of PHGDH protein, the rate-limiting enzyme in the first step of serine biosynthesis pathway. Depletion of Josephin-2 enhanced the ubiquitination degradation of PHGDH and ultimately inhibited the proliferation and CSC-phenotype of HCC in vitro and in vivo. Our work uncovered the regulatory effects of Josephin-2 on PHGDH protein stability and profiled its contribution in HCC malignant progression, which might provide a potential therapeutic target for HCC.

Activin-like Kinase 2 (ALK2/ACVR1) Is a Resistance Factor and Therapeutic Vulnerability to FLT3 Inhibition in Acute Myeloid Leukemia

Background: FMS-like tyrosine kinase-3 (FLT3), a transmembrane receptor, is frequently mutated in acute myeloid leukemia (AML), and this is associated with poor prognosis. Although FLT3 inhibitors (FLT3i) have significantly improved overall survival, their clinical effectiveness is limited due to treatment resistance, and the molecular mechanism remains unknown. We previously reported that the bone morphogenetic protein (BMP) signaling pathway supports AML cell growth via osteogenic differentiation in mesenchymal stromal cells. However, the role of BMP in FLT3i resistance has not been studied. We hypothesized that BMP type-1 receptor (ALK2/ACVR1) is a resistance factor for FLT3i, and therefore targeting FLT3 and ACVR1 using a novel dual inhibitor, TP-0184, would overcome FLT3i resistance and sensitize AML cells to standard therapy. Methods: We assessed the association of ACVR1 expression with molecular mutations in AML by analyzing gene expression datasets (OHSU and TCGA). We also measured ACVR1 expression using qRT-PCR in FLT3-mutant AML cell lines (molm-13, molm-14, and MV4-11) treated with different doses of FLT3i (gilteritinib or midostaurin), as well as in peripheral blood mononuclear cells isolated from patients with FLT3-mutant AML treated with FLT3i. To determine the role of ACVR1 in FLT3i resistance, we knocked down ACVR1 in FLT3-mutant cells, treated them with various doses of FLT3i, and measured apoptosis using IncuCyte. We also measured the effect of TP-0184 on cell proliferation and the cell cycle in FLT3-wild-type (WT) and FLT3-mutant cells. To determine the mechanism of TP-0184-mediated cell cycle arrest, we measured the effect of TP-0184 on cell signaling pathways using Western blot analysis and RNA sequencing. We also performed human RTK kinase binding assay to understand the binding specificity of TP-0184 with 11 different FLT3 mutants. MTT assay was used to determine the synergistic effect of TP-0184 with cytarabine or venetoclax (BCL2 inhibitor). We investigated the effect of TP-0184 on AML growth in vivo using FLT3-mutant and FLT3-WT AML xenograft and patient-derived xenograft models. Results: Analysis of AML datasets showed that ACVR1 expression is upregulated in FLT3-mutant compared with FLT3-WT AML patient samples (p<0.01) and predicts poor overall survival (p=0.05). ACVR1 expression was also higher in FLT3-mutant than in FLT3-WT cell lines (p=0.039). Interestingly, ACVR1 expression was upregulated in patient samples of FLT3-mutant AML treated with gilteritinib (p=0.02) and FLT3-mutant cell lines treated with FLT3i in a dose- and time-dependent manner. Moreover, ACVR1 knockdown in FLT3-mutant cell lines sensitized AML cells to FLT3i 2- to 3-fold compared with scrambled control cells, suggesting that ACVR1 is a resistance factor in AML cells treated with FLT3i, and that targeting ACVR1 could sensitize AML cells to FLT3i. In FLT3-WT and FLT3-mutant AML cell lines treated with TP-0184, cell growth was significantly reduced in FLT3-mutant cells (IC50<25nM) but not in FLT3-WT cells. TP-0184 induced G1/G0 arrest in FLT3-mutant cells but had minimal or no effect in FLT3-WT cells, suggesting that TP-0184 overcomes ACVR1-mediated resistance in FLT3-mutant cells. Treatment with TP-0184 significantly inhibited multiple proteins downstream of FLT3, including STAT-5, ERK, PI3K, and mTOR pathways, and decreased in pSmad1/5 proteins downstream of ACVR1 in AML cells. These findings confirmed that TP-0184 inhibits both mutant FLT3 and ACVR1 in AML cells. Gene expression analysis revealed that TP-0184 significantly downregulated the serine biosynthesis pathway in FLT3-mutant cell lines. Moreover, molecular docking and kinase-binding studies confirmed that TP-0184 binds to FLT3 mutants with high affinity (KD=<5nM). Interestingly,TP-0184 with venetoclax or cytarabine showed a synergistic effect in FLT3-mutant cells (combination index 0.01; p<0.01). In vivo data also showed that TP-0184 inhibited AML growth and significantly prolonged survival in FLT3-mutant AML xenograft and patient-derived xenograft models (p<0.01). Conclusion: Our data indicate that ACVR1 causes resistance to FLT3 inhibitors. TP-0184, a dual inhibitor, targets mutant FLT3 and ACVR1 in AML cell lines. TP-0184 sensitizes AML cells with chemotherapy and venetoclax and inhibits AML growth in vivo.

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.

Eisosome disruption by noncoding RNA deletion increases protein secretion in yeast.

Noncoding RNAs (ncRNAs) regulate many aspects of gene expression. We investigated how ncRNAs affected protein secretion in yeast by large-scale screening for improved endogenous invertase secretion in ncRNA deletion strains with deletion of stable unannotated transcripts (SUTs), cryptic unstable transcripts (CUTs), tRNAs, or snRNAs. We identified three candidate ncRNAs, SUT418, SUT390, and SUT125, that improved endogenous invertase secretion when deleted. As SUTs can affect expression of nearby genes, we quantified adjacent gene transcription and found that the PIL1 gene was down-regulated in the SUT125 deletion strain. Pil1 is a core component of eisosomes, nonmobile invaginations found throughout the plasma membrane. PIL1 knockout alone, or in combination with eisosome components LSP1 or SUR7, resulted in further increased secretion of invertase. Secretion of heterologous GFP was also increased upon PIL1 deletion, but this increase was signal sequence dependent. To reveal the potential for increased biopharmaceutical production, secretion of monoclonal antibody Pexelizumab scFv peptide was increased by PIL1 deletion. Global analysis of secreted proteins revealed that approximately 20% of secreted proteins, especially serine-enriched secreted proteins, including invertase, were increased upon eisosome disruption. Eisosomes are enriched with APC transporters and sphingolipids, which are essential components for secretory vesicle formation and protein sorting. Sphingolipid and serine biosynthesis pathways were up-regulated upon PIL1 deletion. We propose that increased secretion of endogenous and heterologous proteins upon PIL1 deletion resulted from sphingolipid redistribution in the plasma membrane and up-regulated sphingolipid biosynthesis. Overall, a new pathway to improve protein secretion in yeast via eisosome disruption has been identified.

AML-326 Novel FLT3-ALK2 Dual Inhibitor TP-0184 Inhibits Leukemia Growth by Targeting Serine Biosynthesis in FLT3-ITD-Positive AML Cells

FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) inhibitors have been studied; however, molecular factors contributing to this resistance are unknown. i) To investigate the clinical significance of BMP type 1 receptor ALK2 in AML patients with FLT3-ITD mutations. ii) To elucidate the therapeutic effect of FLT3-ALK2 dual inhibitor TP-0184 on leukemia growth. To determine whether ALK2 is a potential target in FLT3-mutated AML patients, we analyzed OHSU and TCGA datasets. We treated AML cell lines with FLT3-WT or -ITD mutations with ALK2 inhibitors and measured their effect on cell cycle and proliferation. To determine the mechanism of TP-0184, we measured the activation of FLT3 downstream signaling by using western blotting and RNA sequencing. Further, we performed kinase profiling to understand the binding specificity of TP-0184 with 11 different FLT3 mutants. Finally, we investigated the effect of TP-0184 on AML growth in vivo using FLT3-mutated PDX and xenograft models. Analysis of AML datasets showed that ALK2 is significantly upregulated in AML patients with FLT3 mutations compared to those with WT-FLT3 (P<0.00001) and predicts poor overall survival (P=0.05). Treatment of FLT3-WT and -mutated AML cell lines with the ALK2 inhibitors LDN-212854 and TP-0184 resulted in significant inhibition of FLT3-ITD-mutant cell growth at low concentrations (IC50<25 nM), while WT-FLT3 cells were affected only at high concentrations (IC50>100 nM). Interestingly, TP-0184 induced G1/G0 arrest in AML cell lines with FLT-ITD mutations but had minimal to no effect in FLT3-WT AML cells. Further, we observed that treatment with TP-0184 in AML cell lines significantly inhibited multiple signaling proteins downstream of FLT3, such as p-STAT5 and p-ERK, as well as p-PI3K, p-mTOR, and p-S6K. Gene expression analysis revealed that treatment with TP-0184 in FLT3-ITD cell lines significantly downregulated the serine biosynthesis pathway. Lastly, treatment with TP-0184 significantly prolonged the survival of FLT3-ITD-mutated AML-bearing mice (P<0.0001). Our data indicate that ALK2 is a prognostic marker in FLT3-mutated AML. TP-0184 inhibits cell proliferation by inhibiting FLT3 downstream signaling pathways in AML cells. Kinase assays confirmed that TP-0184 is a highly specific FLT3-ALK2 dual inhibitor. TP-0184 inhibits AML growth in FLT3-mutated PDX and xenograft models.

Serine metabolism orchestrates macrophage polarization by regulating the IGF1-p38 axis.

Serine metabolism is reportedly involved in immune cell functions, but whether and how serine metabolism regulates macrophage polarization remain largely unknown. Here, we show that suppressing serine metabolism, either by inhibiting the activity of the key enzyme phosphoglycerate dehydrogenase in the serine biosynthesis pathway or by exogenous serine and glycine restriction, robustly enhances the polarization of interferon-γ-activated macrophages (M(IFN-γ)) but suppresses that of interleukin-4-activated macrophages (M(IL-4)) both in vitro and in vivo. Mechanistically, serine metabolism deficiency increases the expression of IGF1 by reducing the promoter abundance of S-adenosyl methionine-dependent histone H3 lysine 27 trimethylation. IGF1 then activates the p38-dependent JAK-STAT1 axis to promote M(IFN-γ) polarization and suppress STAT6-mediated M(IL-4) activation. This study reveals a new mechanism by which serine metabolism orchestrates macrophage polarization and suggests the manipulation of serine metabolism as a therapeutic strategy for macrophage-mediated immune diseases.

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.

PERK promotes immunosuppressive M2 macrophage phenotype by metabolic reprogramming and epigenetic modifications through the PERK-ATF4-PSAT1 axis.

The endoplasmic reticulum (ER) is a specialized organelle that participates in multiple cellular functions including protein folding, maturation, trafficking, and degradation to maintain homeostasis. However, hostile conditions in the tumor microenvironment (TME) disturb ER homeostasis. To overcome these conditions, cells activate ER stress response pathways, which are shown to augment the suppressive phenotypes of immune cells; however, the molecular mechanisms underpinning this process remain elusive. Here, we discuss a recent study by Raines et al, that suggests the role of the helper T-cell 2 (TH2) cytokine interleukin-4 (IL-4), and the TME in facilitating a protein kinase RNA-like ER kinase (PERK)-signaling cascade in macrophages, which promotes immunosuppressive M2 macrophage activation and proliferation. Further, the authors showed that PERK signaling promotes both mitochondrial respirations to fulfill cellular energy requirements and signaling through ATF4, which regulate phosphoserine aminotransferase 1 (PSAT1) activity to mediate the serine biosynthesis pathway. These results highlight a previously uncharacterized role for PERK in cellular metabolism and epigenetic modification in M2 macrophages, and thus offers a new therapeutic strategy for overcoming the immunosuppressive effects in the TME.

Serine biosynthesis as a novel therapeutic target for dilated cardiomyopathy.

Genetic dilated cardiomyopathy (DCM) is a leading cause of heart failure. Despite significant progress in understanding the genetic aetiologies of DCM, the molecular mechanisms underlying the pathogenesis of familial DCM remain unknown, translating to a lack of disease-specific therapies. The discovery of novel targets for the treatment of DCM was sought using phenotypic sceening assays in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) that recapitulate the disease phenotypes in vitro. Using patient-specific iPSCs carrying a pathogenic TNNT2 gene mutation (p.R183W) and CRISPR-based genome editing, a faithful DCM model in vitro was developed. An unbiased phenotypic screening in TNNT2 mutant iPSC-derived cardiomyocytes (iPSC-CMs) with small molecule kinase inhibitors (SMKIs) was performed to identify novel therapeutic targets. Two SMKIs, Gö 6976 and SB 203580, were discovered whose combinatorial treatment rescued contractile dysfunction in DCM iPSC-CMs carrying gene mutations of various ontologies (TNNT2, TTN, LMNA, PLN, TPM1, LAMA2). The combinatorial SMKI treatment upregulated the expression of genes that encode serine, glycine, and one-carbon metabolism enzymes and significantly increased the intracellular levels of glucose-derived serine and glycine in DCM iPSC-CMs. Furthermore, the treatment rescued the mitochondrial respiration defects and increased the levels of the tricarboxylic acid cycle metabolites and ATP in DCM iPSC-CMs. Finally, the rescue of the DCM phenotypes was mediated by the activating transcription factor 4 (ATF4) and its downstream effector genes, phosphoglycerate dehydrogenase (PHGDH), which encodes a critical enzyme of the serine biosynthesis pathway, and Tribbles 3 (TRIB3), a pseudokinase with pleiotropic cellular functions. A phenotypic screening platform using DCM iPSC-CMs was established for therapeutic target discovery. A combination of SMKIs ameliorated contractile and metabolic dysfunction in DCM iPSC-CMs mediated via the ATF4-dependent serine biosynthesis pathway. Together, these findings suggest that modulation of serine biosynthesis signalling may represent a novel genotype-agnostic therapeutic strategy for genetic DCM.

IL-35 suppresses pathogenic T cell response in atopic dermatitis by inhibiting PSAT1 activation

Abstract Interleukin 35 (IL-35) is a member of the IL-12 family and immune-suppressive cytokine and a critical regulator of immunity during autoimmune and infectious diseases. However, it is unclear whether IL-35 can control skin inflammation by regulating the biosynthesis of biomolecules. Here, we demonstrated that IL-35 ameliorated atopic dermatitis (AD), a Th2-mediated inflammatory disease, by inhibiting activated phosphoserine aminotransferase 1 (PSAT1). We DNCB/DFE-induced AD mouse model, monitored progression by ear thickness and histology, and investigated whether IL-35 treatment would suppress AD. Mice treated with IL-35 developed mild or low skin inflammation and disease protection correlated with expansion of Foxp3+ Treg cells with concomitant suppression of Th2 and Th1/2 responses. We further show that IL-35 regulates alterations in glycolytic metabolism in AD skin inflammation. Importantly, the IL-35 mediated suppression of Th2 and Th1/2 cells and its amelioration of AD correlated with inhibition of serine biosynthesis pathways such as PSAT1 in the skin. These results suggest that IL-35 inhibits Th2 and Th1/2 cell differentiation and proliferation by directly regulating PSAT1 immune metabolites in atopic dermatitis. Therefore, the development of targeted therapy for serine-dependent metabolic pathways via IL-35 would be an effective therapeutic strategy for AD. Supported by 21st Century Medical Science Creative Human Resource Development Center

Abstract P5-05-05: Unraveling the mechanism of Lipid-induced malignant transformation in non-transformed breast epithelial cells

Abstract Introduction. With the goal of defining the in-breast environment that facilitates the development of estrogen receptor negative (ER-) breast cancer (BC), we have previously identified a set of genes involved in lipid metabolism with increased expression in the contralateral unaffected breast of women with ER- BC (PMID: 28263391). MCF-10A cells exposed to lipids in vitro display profound changes in gene expression, chromatin packing density, chromatin accessibility and histone posttranslational modifications (PTMs). In addition, flux through thirty-eight metabolic reactions was significantly increased by this exposure, including those of the electron transport chain (ETC). However, the mechanisms by which lipids induce molecular changes that result in epigenomic reprogramming, and potentially promote malignant transformation remain to be elucidated. We hypothesized that dysfunction of the ETC and increased serine, glycine, one carbon (SGOC) metabolism pathway activity play a role in the modulation of epigenetics. Methods. To interrogate potential mechanisms that link lipids to ER- BC genesis, estrogen and progesterone receptor negative MCF-10A cells were cultured in MEBM/MEGM and exposed to vehicle, octanoic acid (C8) or linoleic acid (C18) for 24 hours. Histone PTMs were assayed by liquid chromatography-mass spectrometry. Gene expression was measured by RNA-seq and differentially expressed genes between vehicle and lipid treated groups were identified using the DESeq2 R package. ATAC-seq was performed to study chromatin accessibility. MCF-10A cells were exposed to C8 in presence or absence of inhibitors of Complex I (Metformin) and Complex III (Antimycin A) of the ETC. Real-time qPCR was performed to measure epigenetic modulators, Unfolded Protein Response (UPR) pathway and SGOC metabolism genes. Results. Histone PTMs changed significantly following 24 hours exposure to C8 or C18. Exposure to C8 resulted in the modulation of the expression of genes involved in the UPR/recognition of mitochondrial dysfunction (ATF4), de novo serine biosynthesis (PHGDH, PSAT1, SPH) and one-carbon metabolism (SHMT2, MTHFD2) pathways as well as numerous genes coding for epigenetic modulators. Upon C18 exposure, the binding motif for ATF4 was among the most significantly enriched motifs in the open chromatin regions and p53 was among the motifs with significantly decreased accessibility. Interestingly ATF4 actives PHGDH expression while p53 suppresses it. Exposure to C8 in presence of ETC inhibitors affected gene expression in the same direction as lipid exposure and co-treatment had an additive effect. Fatty acid oxidation and inhibition of the ETC increased the expression of PHGDH: C8, 2.6-fold (p-value = 0.005); Metformin, 2.3-fold (p-value = 0.0052); Antimycin A, 5.1-fold (p-value &amp;lt; 0.0001); C8 plus Metformin, 5.3-fold (p-value &amp;lt; 0.0001), and C8 plus Antimycin A, 5.2-fold (p-value &amp;lt; 0.0001). Conclusions. The modulation of epigenetic effectors does not appear to be a consequence of increased flux through the ETC but rather a consequence of increased SGOC metabolism, and the methionine cycle. Our in vitro model shows that lipid-induced stress increases ATF4 expression. ATF4 controls the expression of PHGDH, PSAT1, SPH, SHMT2 and MTHFD2 which influences epigenetics through S-adenosyl methionine production, the major methyl donor. PHGDH overexpression appears to be the key in our model as it has been associated with ER- BC and its engineered overexpression in non-transformed breast cells causes alterations associated with malignant transformation. The increase in PDGDH expression is hypothesized to compensate for mitochondrial dysfunction and to restore redox homeostasis by producing precursors that can be used for the production of antioxidants, such as glutathione. Citation Format: Mariana Bustamante Eduardo, Shivangi Yadav, Seema Khan, Susan Clare. Unraveling the mechanism of Lipid-induced malignant transformation in non-transformed breast epithelial cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-05-05.

Nitric Oxide Turnover Under Hypoxia Results in the Rapid Increased Expression of the Plastid-Localized Phosphorylated Pathway of Serine Biosynthesis.

The plant mitochondrial electron transport chain influences carbon and nitrogen metabolism under near anoxic conditions through its involvement in the phytoglobin-nitric oxide cycle, where the respiratory chain reduces nitrite to nitric oxide (NO), followed by NO conversion to nitrate by class 1 phytoglobin. Wild type (WT) and transgenic tobacco (Nicotiana tabacum L.) with differing amounts of alternative oxidase (AOX) were used to manipulate NO generation under hypoxia, and to examine whether this in turn influenced the gene expression of two stress-related amino acid biosynthetic pathways, the plastid-localized phosphorylated pathway of serine biosynthesis (PPSB), and the γ-aminobutyric acid (GABA) shunt. Under hypoxia, leaf NO emission rate was highest in AOX overexpressors and lowest in AOX knockdowns, with WT showing an intermediate rate. In turn, the rate of NO emission correlated with the degree to which amino acids accumulated. This amino acid accumulation was associated with the increased expression of the enzymes of the stress-related amino acid biosynthetic pathways. However, induction of the PPSB occurred much earlier than the GABA shunt. This work shows that high rates of NO turnover associate with rapid gene induction of the PPSB, establishing a clear link between this pathway and the maintenance of carbon, nitrogen and energy metabolism under hypoxia.

Metabolic shift to serine biosynthesis through 3-PG accumulation and PHGDH induction promotes tumor growth in pancreatic cancer.

Cancer cells craftily adapt their energy metabolism to their microenvironment. Nutrient deprivation due to hypovascularity and fibrosis is a major characteristic of pancreatic ductal adenocarcinoma (PDAC); thus, PDAC cells must produce energy intrinsically. However, the enhancement of energy production via activating Kras mutations is insufficient to explain the metabolic rewiring of PDAC cells. Here, we investigated the molecular mechanism underlying the metabolic shift in PDAC cells under serine starvation. Amino acid analysis revealed that the concentrations of all essential amino acids and most nonessential amino acids were decreased in the blood of PDAC patients. In addition, the plasma serine concentration was significantly higher in PDAC patients with PHGDH-high tumors than in those with PHGDH-low tumors. Although the growth and tumorigenesis of PK-59cells with PHGDH promoter hypermethylation were significantly decreased by serine starvation, these activities were maintained in PDAC cell lines with PHGDH promoter hypomethylation by serine biosynthesis through PHGDH induction. In fact, DNA methylation analysis by pyrosequencing revealed that the methylation status of the PHGDH promoter was inversely correlated with the PHGDH expression level in human PDAC tissues. In addition to PHGDH induction by serine starvation, PDAC cells showed enhanced serine biosynthesis under serine starvation through 3-PG accumulation via PGAM1 knockdown, resulting in enhanced PDAC cell growth and tumor growth. However, PHGDH knockdown efficiently suppressed PDAC cell growth and tumor growth under serine starvation. These findings provide evidence that targeting the serine biosynthesis pathway by inhibiting PHGDH is a potent therapeutic approach to eliminate PDAC cells in nutrient-deprived microenvironments.