Serine Synthesis Pathway Research Articles

NAT10-mediated mRNA N4-acetylcytidine reprograms serine metabolism to drive leukaemogenesis and stemness in acute myeloid leukaemia.

RNA modification has emerged as an important epigenetic mechanism that controls abnormal metabolism and growth in acute myeloid leukaemia (AML). However, the roles of RNA N4-acetylcytidine (ac4C) modification in AML remain elusive. Here, we report that ac4C and its catalytic enzyme NAT10 drive leukaemogenesis and sustain self-renewal of leukaemic stem cells/leukaemia-initiating cells through reprogramming serine metabolism. Mechanistically, NAT10 facilitates exogenous serine uptake and de novo biosynthesis through ac4C-mediated translation enhancement of the serine transporter SLC1A4 and the transcription regulators HOXA9 and MENIN that activate transcription of serine synthesis pathway genes. We further characterize fludarabine as an inhibitor of NAT10 and demonstrate that pharmacological inhibition of NAT10 targets serine metabolic vulnerability, triggering substantial anti-leukaemia effects both in vitro and in vivo. Collectively, our study demonstrates the functional importance of ac4C and NAT10 in metabolism control and leukaemogenesis, providing insights into the potential of targeting NAT10 for AML therapy.

PHGDH Induction by MAPK is Essential for Melanoma Formation and Creates an Actionable Metabolic Vulnerability.

Overexpression of PHGDH, the rate-limiting enzyme in the serine synthesis pathway, promotes melanomagenesis, melanoma cell proliferation, and survival of metastases in serine-low environments such as the brain. Here, we found that PHGDH is universally increased in melanoma cells and required for melanomagenesis. While PHGDH amplification explained PHGDH overexpression in a subset of melanomas, oncogenic BRAFV600E also promoted PHGDH transcription through mTORC1-mediated translation of ATF4. Importantly, depletion of PHGDH in genetic mouse melanoma models blocked tumor formation. In addition to BRAFV600E-mediated upregulation, PHGDH was further induced by exogenous serine restriction. Surprisingly, BRAFV600E inhibition diminished serine restriction-mediated PHGDH expression by preventing ATF4 induction. Consequently, melanoma cells could be specifically starved of serine by combining BRAFV600E inhibition with exogenous serine restriction, which promoted cell death in vitro and attenuated melanoma growth in vivo. In summary, this study identified that PHGDH is essential for melanomagenesis and regulated by BRAFV600E, revealing a targetable vulnerability in BRAFV600E-mutant melanoma.

MYC-dependent upregulation of the de novo serine and glycine synthesis pathway is a targetable metabolic vulnerability in group 3 medulloblastoma.

Group 3 medulloblastoma (MBGRP3) represents around 25% of medulloblastomas and is strongly associated with c-MYC (MYC) amplification, which confers significantly worse patient survival. Although elevated MYC expression is a significant molecular feature in MBGRP3, direct targeting of MYC remains elusive, and alternative strategies are needed. The metabolic landscape of MYC-driven MBGRP3 is largely unexplored and may offer novel opportunities for therapies. To study MYC-induced metabolic alterations in MBGRP3, we depleted MYC in isogenic cell-based model systems, followed by 1H high-resolution magic-angle spectroscopy (HRMAS) and stable isotope-resolved metabolomics, to assess changes in intracellular metabolites and pathway dynamics. Steady-state metabolic profiling revealed consistent MYC-dependent alterations in metabolites involved in one-carbon metabolism such as glycine. 13C-glucose tracing further revealed a reduction in glucose-derived serine and glycine (de novo synthesis) following MYC knockdown, which coincided with lower expression and activity of phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in this pathway. Furthermore, MYC-overexpressing MBGRP3 cells were more vulnerable to pharmacological inhibition of PHGDH compared to those with low expression. Using in vivo tumor-bearing genetically engineered and xenograft mouse models, pharmacological inhibition of PHGDH increased survival, implicating the de novo serine/glycine synthesis pathway as a pro-survival mechanism sustaining tumor progression. Critically, in primary human medulloblastomas, increased PHGDH expression correlated strongly with both MYC amplification and poorer clinical outcomes. Our findings support a MYC-induced dependency on the serine/glycine pathway in MBGRP3 that represents a novel therapeutic treatment strategy for this poor prognosis disease group.

N6-Methyladenosine modification activates the serine synthesis pathway to mediate therapeutic resistance in liver cancer

Metabolic adaptation serves as a significant driving force for cancer growth and poses a substantial obstacle for cancer therapies. Herein, we unravelled the role of m6A -mediated serine synthesis pathway regulation in both hepatocellular carcinoma (HCC) development and therapeutic resistance. We demonstrated that the highly specific m6A inhibitor (STM2457) treatment effectively inhibited HCC cell line growth and suppressed spontaneous HCC formation in mice driven by liver specific Tp53 knockout and Myc overexpression. Using GLORI-seq, we delineated a single-base resolution m6A landscape in human HCC cell lines. Interestingly, we identified three core enzymes in the serine synthesis pathway (PHGDH, PSAT1, and PSPH) as novel targets of METTL3-mediated m6A modification. In these SSP genes, m6A modification recruited IGF2BP3 reader to stabilize their mRNA transcripts, thereby enhancing their mRNA and protein expression in HCC cells. Most importantly, our GLORI-seq data revealed that sorafenib-resistant HCC cells elevated m6A modification in SSP genes to promote protein expression and antioxidant production. STM2457 treatment attenuated the serine synthesis pathway, induced oxidative stress, and sensitized HCC cells to sorafenib and lenvatinib treatments. In conclusion, our findings suggest that targeting m6A could be a potential therapeutic strategy for HCC treatment.

Loss of USP10 promotes hepatocellular carcinoma proliferation by regulating the serine synthesis pathway through inhibition of LKB1 activity.

Metabolic dysregulation is emerging as a critical factor in tumorigenesis, and reprogramming of serine metabolism has been identified as an essential factor in the progression of hepatocellular carcinoma (HCC). Studies have shown that LKB1 deficiency can activate mTOR to upregulate the serine synthesis pathway (SSP) and promote tumor progression. Our team discovered that ubiquitin-specific protease 10 (USP10) can inhibit HCC proliferation through mTOR, but its relationship with SSP needs further investigation. The metabolite assays revealed a significant increase in serine content in HCC tissues. Through the LKB1/mTOR/activating transcription factor 4 (ATF4) axis, loss of USP10 may increase serine biosynthesis and promote the proliferation of HCC invitro and invivo. Furthermore, it was found that USP10 could activate LKB1 through deubiquitination. Analyzing clinical HCC tissues revealed a positive correlation between USP10 and LKB1. Additionally, those with high expression of USP10 in HCC tissues showed a better degree of tumor differentiation and longer overall survival time. Moreover, we found increased expression of both serine and its synthase in liver tumor tissues of USP10 liver-specific KO mice. Loss of USP10 inhibits the activity of LKB1, contributing to the stimulation of the mTOR/ATF4 axis and SSP and then promoting the proliferation of HCC. This work presents a novel approach for serine-targeted treatment in HCC.

PHGDH/SYK: a hub integrating anti-fungal immunity and serine metabolism.

Immune cells modify their metabolic pathways in response to fungal infections. Nevertheless, the biochemical underpinnings need to be better understood. This study reports that fungal infection drives a switch from glycolysis to the serine synthesis pathway (SSP) and one-carbon metabolism by inducing the interaction of spleen tyrosine kinase (SYK) and phosphoglycerate dehydrogenase (PHGDH). As a result, PHGDH promotes SYK phosphorylation, leading to the recruitment of SYK to C-type lectin receptors (CLRs). The CLR/SYK complex initiates signaling cascades that lead to transcription factor activation and pro-inflammatory cytokine production. SYK activates SSP and one-carbon metabolism by inducing PHGDH activity. Then, one-carbon metabolism supports S-adenosylmethionine and histone H3 lysine 36 trimethylation to drive the production of pro-inflammatory cytokines and chemokines. These findings reveal the crosstalk between amino acid metabolism, epigenetic modification, and CLR signaling during fungal infection.

Identification of novel PHGDH inhibitors based on computational investigation: an all-in-one combination strategy to develop potential anti-cancer candidates.

Biological studies have elucidated that phosphoglycerate dehydrogenase (PHGDH) is the rate-limiting enzyme in the serine synthesis pathway in humans that is abnormally expressed in numerous cancers. Inhibition of the PHGDH activity is thought to be an attractive approach for novel anti-cancer therapy. The development of structurally diverse novel PHGDH inhibitors with high efficiency and low toxicity is a promising drug discovery strategy. A ligand-based 3D-QSAR pharmacophore model was developed using the HypoGen algorithm methodology of Discovery Studio. The selected pharmacophore model was further validated by test set validation, cost analysis, and Fischer randomization validation and was then used as a 3D query to screen compound libraries with various chemical scaffolds. The estimated activity, drug-likeness, molecular docking, growing scaffold, and molecular dynamics simulation processes were applied in combination to reduce the number of virtual hits. The potential candidates against PHGDH were screened based on estimated activity, docking scores, predictive absorption, distribution, metabolism, excretion, and toxicity (ADME/T) properties, and molecular dynamics simulation. Finally, an all-in-one combination was employed successfully to design and develop three potential anti-cancer candidates.

The unique catalytic properties of PSAT1 mediate metabolic adaptation to glutamine blockade.

Cultured cancer cells frequently rely on the consumption of glutamine and its subsequent hydrolysis by glutaminase (GLS). However, this metabolic addiction can be lost in the tumour microenvironment, rendering GLS inhibitors ineffective in the clinic. Here we show that glutamine-addicted breast cancer cells adapt to chronic glutamine starvation, or GLS inhibition, via AMPK-mediated upregulation of the serine synthesis pathway (SSP). In this context, the key product of the SSP is not serine, but α-ketoglutarate (α-KG). Mechanistically, we find that phosphoserine aminotransferase 1 (PSAT1) has a unique capacity for sustained α-KG production when glutamate is depleted. Breast cancer cells with resistance to glutamine starvation or GLS inhibition are highly dependent on SSP-supplied α-KG. Accordingly, inhibition of the SSP prevents adaptation to glutamine blockade, resulting in a potent drug synergism that suppresses breast tumour growth. These findings highlight how metabolic redundancy can be context dependent, with the catalytic properties of different metabolic enzymes that act on the same substrate determining which pathways can support tumour growth in a particular nutrient environment. This, in turn, has practical consequences for therapies targeting cancer metabolism.

E-Cadherin Induces Serine Synthesis to Support Progression and Metastasis of Breast Cancer.

The loss of E-cadherin, an epithelial cell adhesion molecule, has been implicated in metastasis by mediating the epithelial-mesenchymal transition, which promotes invasion and migration of cancer cells. However, recent studies have demonstrated that E-cadherin supports the survival and proliferation of metastatic cancer cells. Here, we identified a metabolic role for E-cadherin in breast cancer by upregulating the de novo serine synthesis pathway (SSP). The upregulated SSP provided metabolic precursors for biosynthesis and resistance to oxidative stress, enabling E-cadherin+ breast cancer cells to achieve faster tumor growth and enhanced metastases. Inhibition of phosphoglycerate dehydrogenase, a rate-limiting enzyme in the SSP, significantly and specifically hampered proliferation of E-cadherin+ breast cancer cells and rendered them vulnerable to oxidative stress, inhibiting their metastatic potential. These findings reveal that E-cadherin reprograms cellular metabolism, promoting tumor growth and metastasis of breast cancers. Significance: E-Cadherin promotes the progression and metastasis of breast cancer by upregulating the de novo serine synthesis pathway, offering promising targets for inhibiting tumor growth and metastasis in E-cadherin-expressing tumors.

Skeletal muscle hypertrophy rewires glucose metabolism: An experimental investigation and systematic review.

Proliferating cancer cells shift their metabolism towards glycolysis, even in the presence of oxygen, to especially generate glycolytic intermediates as substrates for anabolic reactions. We hypothesize that a similar metabolic remodelling occurs during skeletal muscle hypertrophy. We used mass spectrometry in hypertrophying C2C12 myotubes in vitro and plantaris mouse muscle in vivo and assessed metabolomic changes and the incorporation of the [U-13C6]glucose tracer. We performed enzyme inhibition of the key serine synthesis pathway enzyme phosphoglycerate dehydrogenase (Phgdh) for further mechanistic analysis and conducted a systematic review to align any changes in metabolomics during muscle growth with published findings. Finally, the UK Biobank was used to link the findings to population level. The metabolomics analysis in myotubes revealed insulin-like growth factor-1 (IGF-1)-induced altered metabolite concentrations in anabolic pathways such as pentose phosphate (ribose-5-phosphate/ribulose-5-phosphate: +40%; P=0.01) and serine synthesis pathway (serine: -36.8%; P=0.009). Like the hypertrophy stimulation with IGF-1 in myotubes in vitro, the concentration of the dipeptide l-carnosine was decreased by 26.6% (P=0.001) during skeletal muscle growth in vivo. However, phosphorylated sugar (glucose-6-phosphate, fructose-6-phosphate or glucose-1-phosphate) decreased by 32.2% (P=0.004) in the overloaded muscle in vivo while increasing in the IGF-1-stimulated myotubes in vitro. The systematic review revealed that 10 metabolites linked to muscle hypertrophy were directly associated with glycolysis and its interconnected anabolic pathways. We demonstrated that labelled carbon from [U-13C6]glucose is increasingly incorporated by ~13% (P=0.001) into the non-essential amino acids in hypertrophying myotubes, which is accompanied by an increased depletion of media serine (P=0.006). The inhibition of Phgdh suppressed muscle protein synthesis in growing myotubes by 58.1% (P<0.001), highlighting the importance of the serine synthesis pathway for maintaining muscle size. Utilizing data from the UK Biobank (n=450243), we then discerned genetic variations linked to the serine synthesis pathway (PHGDH and PSPH) and to its downstream enzyme (SHMT1), revealing their association with appendicular lean mass in humans (P<5.0e-8). Understanding the mechanisms that regulate skeletal muscle mass will help in developing effective treatments for muscle weakness. Our results provide evidence for the metabolic rewiring of glycolytic intermediates into anabolic pathways during muscle growth, such as in serine synthesis.

Preclinical efficacy of CBR-5884 against epithelial ovarian cancer cells by targeting the serine synthesis pathway

Reprogramming of the serine synthesis pathway (SSP) is intricately linked to the progression of epithelial ovarian cancer (EOC). CBR-5884, a selective small-molecule inhibitor targeting phosphoglycerate dehydrogenase (PHGDH), effectively impedes the de novo synthesis of serine within cancer cells. This study aimed to evaluate the inhibitory effect of CBR-5884 on EOC cells and delineate its specific mechanism, thereby proposing a novel therapeutic approach for treating EOC. The suppression of serine biosynthesis after CBR-5884 treatment was evaluated using RNA sequencing and a serine assay kit, and the results showed that CBR-5884 effectively downregulated serine biosynthesis in EOC cells, particularly those expressing high levels of PHGDH. In vitro studies revealed that CBR-5884 demonstrated significant antitumor effects and suppressed migration and invasion of EOC cells through down-regulation of the integrin subunit beta 4 (ITGB4)/extracellular signal-regulated kinase (ERK)/epithelial–mesenchymal transition signal axis. Additionally, CBR-5884 mitigated the stemness of EOC cells and heightened their sensitivity to chemotherapy. Moreover, in vivo studies revealed that CBR-5884 significantly delayed tumor growth, with histological analysis indicating the safety profile of CBR-5884. Finally, the patient-derived organoid (PDO) models were utilized to explore the preclinical efficacy of CBR-5884 against EOC cells, and the results unveiled that CBR-5884 impeded proliferation and downregulated the expression of ITGB4 in EOC PDO models. Our findings supports the anticancer properties of CBR-5884 in EOC cells exhibiting high PHGDH expression, manifesting through the suppression of proliferation, migration, and invasion, while enhancing chemotherapy sensitivity, suggesting that CBR-5884 holds promise as an efficacious strategy for the treatment of EOC.

ASS1 inhibits triple-negative breast cancer by regulating PHGDH stability and de novo serine synthesis

Argininosuccinate synthase (ASS1), a critical enzyme in the urea cycle, acts as a tumor suppressor in many cancers. To date, the anticancer mechanism of ASS1 has not been fully elucidated. Here, we found that phosphoglycerate dehydrogenase (PHGDH), a key rate-limiting enzyme in serine synthesis, is a pivotal protein that interacts with ASS1. Our results showed that ASS1 directly binds to PHGDH and promotes its ubiquitination-mediated degradation to inhibit serine synthesis, consequently suppressing tumorigenesis. Importantly, the tumor suppressive effects of ASS1 were strongly abrogated by PHGDH knockout. In addition, ASS1 knockout and knockdown partially rescued cell proliferation when serine and glycine were depleted, while the inhibitory effect of ASS1 overexpression on cell proliferation was restored by the addition of serine and glycine. These findings unveil a novel role of ASS1 and suggest that the ASS1/PHGDH serine synthesis pathway is a promising target for cancer therapy.

Serine Metabolism Regulates the Replicative Senescence of Human Dental Pulp Cells through Histone Methylation.

Tissue regeneration therapy based on human dental pulp cells (hDPCs) faces the distinct challenge of cellular senescence during massive expansion in vitro. To further explore the regulatory mechanism of cellular senescence in hDPCs, we conduct experiments on young cells (Passage 5, P5) and replicative senescent (Passage 12, P12) hDPCs. The results confirm that hDPCs undergo replicative senescence with passaging, during which their ability to proliferate and osteogenic differentiation decreases. Notably, during replicative senescence, phosphoglycerate dehydrogenase (PHGDH), the key enzyme of the serine synthesis pathway (SSP), is significantly downregulated, as well as S-adenosylmethionine (SAM) levels, resulting in reduced H3K36me3 modification on Sirtuin 1 (SIRT1)and Runt-related transcription factor 2 (RUNX2) promoters. Inhibition of PHGDH leads to the same phenotype as replicative senescence. Serine supplementation fails to rescue the senescence phenotype caused by replicative senescence and inhibitors, in which folate metabolism-related genes, including serine hydroxymethyl transferase 2 (SHMT2), methylenetetrahydrofolate dehydrogenase 1(MTHFD1), methylenetetrahydrofolate dehydrogenase 2(MTHFD2), are notably decreased. Our research raised a possibility that PHGDH may be involved in cellular senescence by affecting folate metabolism and histone methylation in addition to serine biosynthesis, providing potential targets to prevent senescence.

Abstract 3079: AMPK-HIF-1α signaling enhances glucose-derived de novoserine biosynthesis to promote glioblastoma growth

Abstract Cancer cells undergo cellular adaptation through metabolic reprogrammin to sustain survival and rapid growth under various stress conditions. However, it remains unknown how brain tumors modulate their metabolic flexibility in the naturally serine/glycine (S/G)-deficient brain microenvironment. We used a range of primary/stem-like and established glioblastoma (GBM) cell models in vitro and in vivo. To identify regulatory mechanisms of S/G deprivation induced metabolic flexibility, we employed high throughput RNA-sequencing, transcriptomic analysis, metabolic flux analysis, metabolites analysis, chromatin immunoprecipitation (ChIP), luciferase reporter, nuclear fractionation, cycloheximide-chase, and glucose consumption. The clinical significances were analyzed in the genomic database (GSE4290) and in human GBM specimens. The high throughput RNA-sequencing and transcriptomic analysis demonstrate that the de novo serine synthesis pathway (SSP) and glycolysis are highly activated in GBM cells under S/G deprivation conditions. Mechanistically, S/G deprivation rapidly induces reactive oxygen species (ROS)-mediated AMP-activated protein kinase (AMPK) activation and AMPK dependent hypoxia-inducible factor (HIF)-1α stabilization and transactivation. Activated HIF-1α in turn promotes expression of SSP enzymes phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH). In addition, HIF-1α-induced expression of glycolytic genes (GLUT1, GLUT3, HK2, and PFKFB2) promotes glucose uptake, glycolysis, and glycolytic flux to fuel SSP, leading to elevated de novo serine and glycine biosynthesis, NADPH/NADP+ ratio, and proliferation and survival of GBM cells. Analyses of human GBM specimens reveal that levels of overexpressed PHGDH, PSAT1, and PSPH are positively correlated with levels of AMPK T172 phosphorylation and HIF-1α expression and poor prognosis of GBM patients. Our findings reveal that metabolic stress-enhanced glucose-derived de novo serine biosynthesis is a critical metabolic feature of GBM cells and highlight the potential to target SSP for treating human GBM. Citation Format: Hye Jin Yun, Jong-Ho Lee. AMPK-HIF-1α signaling enhances glucose-derived de novoserine biosynthesis to promote glioblastoma growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3079.

Abstract 7091: Significance of the tryptophan catabolism pathway in triple-negative breast cancer shows potential targets in certain subsets relating to the serine pathway

Abstract Background: Breast cancer (BC) is the most prevalent form of cancer worldwide, encompassing 12.5% of all forms of cancers with the fifth highest mortality rate. Triple negative cancers (TNBC), which account for 15~20% of BCs, are one of the most aggressive and heterogeneous forms of BCs, characterized by the loss of ER, PR, and low HER2 expression. This heterogeneity and plasticity are partly due to the adaptable nature of their metabolism, a dynamic phenotype which is commonly observed in many aggressive forms of cancer. One of the key metabolic pathways that can confer this aggressive proliferative characteristic is the tryptophan catabolism pathway. Tryptophan is an essential amino acid required for the proper function of all mammalian cells, with IDO1 being a key enzyme that converts tryptophan to kynurenine, marking the start of the catabolism pathway. IDO1 expression is observed to be elevated in various types of cancer including BCs and has been associated with immune suppression/evasion via resource competition and tumor progression by supplying resources required for proliferation. Despite this, tryptophan catabolism is one of the lesser studied and understood pathways in cancer metabolism, especially in the context of TNBCs. Methods: Previous analysis had shown the tryptophan catabolism pathway is downregulated in certain subsets of TNBCs. To understand the effective function of the pathway with regards to proliferation, we targeted the upper-most step of the catabolism pathway by inhibiting IDO1 with epacadostat and indoximod in different types of TNBC cell-lines and observed for changes in their growth rates via proliferation assays. We also performed parallel stable-isotope tracer-based flux analysis with [U-13C11]-Tryptophan and [U-13C3]-Serine with mass spectrometry to characterize flux changes with the goal of discovering potentially lethal metabolic pathways. Results: We observed higher proliferative sensitivity to IDO1 inhibition in TNBC cell-lines with a genetic dysfunction in the de novo serine synthesis pathway. To understand these differences at the metabolic level, we quantified the differential contributions of tryptophan and serine to nucleotide and NAD+ synthesis and the TCA cycle with the parallel tracer experiments. Comparison of the lines with dysfunctional de novo serine synthesis to those with normal or amplified flux towards the pathway showed a higher interdependence with the kynurenine pathway in the dysfunctional group. These results indicate a differential potential for targeting the kynurenine pathway in relation with the serine synthesis pathway. Conclusion: Targeting the IDO1 enzyme in the kynurenine pathway shows significant correlations between the de novo serine pathway and the tryptophan catabolism pathway in TNBCs that may lead to metabolic targets conferring potential therapeutic benefits. Citation Format: Jin Heon Jeon, Abhinav Achreja, Mark Slayton, Olamide Animasahun, Minal Nenwani, Miya Paserba, Zackariah Farah, Sofia D. Merajver, Deepak Nagrath. Significance of the tryptophan catabolism pathway in triple-negative breast cancer shows potential targets in certain subsets relating to the serine pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7091.

Abstract 6608: Therapeutic enzyme depletion of L-serine for the treatment of serine auxotrophic tumors

Abstract The non-essential amino acid L-serine (serine) plays an important role in cancer cell growth due to its diverse biosynthetic functions. In many cancer cells, serine can be taken up from the circulation or can be synthesized through the serine synthesis pathway (SSP). We have recently discovered that luminal breast tumors are auxotrophic for serine due to lineage-specific silencing of the SSP gene PSAT1, rendering luminal breast cancer cells sensitive to serine deprivation both in vitro and in vivo. At present the only way to reduce serine availability for tumors in vivo is through dietary serine starvation, which in mice reduces circulating serine levels by ~50% and has been shown to inhibit the growth of numerous mouse models of cancer. Importantly, dietary serine is achieved in mice using a synthetic protein-free diet that may be difficult for humans to sustain. Nevertheless, dietary serine starvation is currently being explored in a clinical trial for pancreatic cancer. To circumvent some of the challenges associated with dietary serine starvation, we have developed a therapeutic serine degrading enzyme that we are exploring as a potential treatment for serine auxotrophic tumors. Our enzyme, eSDH, is based on the human serine dehydratase enzyme that facilitates the pyridoxal phosphate-dependent irreversible deamination of serine to pyruvate and ammonia. Relative to wild-type serine dehydratase, eSDH has significantly improved stability in serum as well increased activity towards serine. Injection of eSDH into mice is well-tolerated and leads to long-term, near-complete (&amp;gt;95%) reduction in circulating serine levels without the need for dietary changes. Importantly, eSDH treatment inhibits the growth of serine auxotrophic cancer cells in vitro and in vivo. We are currently assessing the efficacy of eSDH treatment in advanced models of luminal/ER+ breast cancer and when utilized in combination with endocrine therapy and other standard-of-care treatments for luminal/ER+ cancer patients. In addition to the degradation of serine, eSDH also depletes circulating L-threonine (threonine), which could contribute to the in vivo activity of eSDH. However, we have recently developed new eSDH variants that are highly selective for serine over threonine and show enhanced specificity for serine auxotrophic cancer cells. In conclusion, we have developed a prototypical therapeutic serine degrading enzyme that may provide an opportunity to clinically achieve more complete serine starvation in patients without the need for restrictive dietary changes. Citation Format: Vipin Rawat, Huiping Zhao, Ladan Mashouri, Prarthana Prasanth, Kelly Conger, Sarita Bhetawal, Alessandra Araujo, Everett Stone, Jonathan Coloff. Therapeutic enzyme depletion of L-serine for the treatment of serine auxotrophic tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6608.

O-GlcNAc signaling increases neuron regeneration through one-carbon metabolism in Caenorhabditis elegans.

Cellular metabolism plays an essential role in the regrowth and regeneration of a neuron following physical injury. Yet, our knowledge of the specific metabolic pathways that are beneficial to neuron regeneration remains sparse. Previously, we have shown that modulation of O-linked β-N-acetylglucosamine (O-GlcNAc) signaling, a ubiquitous post-translational modification that acts as a cellular nutrient sensor, can significantly enhance in vivo neuron regeneration. Here, we define the specific metabolic pathway by which O-GlcNAc transferase (ogt-1) loss of function mediates increased regenerative outgrowth. Performing in vivo laser axotomy and measuring subsequent regeneration of individual neurons in C. elegans, we find that glycolysis, serine synthesis pathway (SSP), one-carbon metabolism (OCM), and the downstream transsulfuration metabolic pathway (TSP) are all essential in this process. The regenerative effects of ogt-1 mutation are abrogated by genetic and/or pharmacological disruption of OCM and the SSP linking OCM to glycolysis. Testing downstream branches of this pathway, we find that enhanced regeneration is dependent only on the vitamin B12 independent shunt pathway. These results are further supported by RNA sequencing that reveals dramatic transcriptional changes by the ogt-1 mutation, in the genes involved in glycolysis, OCM, TSP, and ATP metabolism. Strikingly, the beneficial effects of the ogt-1 mutation can be recapitulated by simple metabolic supplementation of the OCM metabolite methionine in wild-type animals. Taken together, these data unearth the metabolic pathways involved in the increased regenerative capacity of a damaged neuron in ogt-1 animals and highlight the therapeutic possibilities of OCM and its related pathways in the treatment of neuronal injury.

Increased Expression of PHGDH Under High-Selenium Stress In Vivo.

The purpose of this study is to explore the glycolytic remodeling under high-selenium (Se) stress. Three groups of male C57BL/6J mice were fed on diets with different Se contents (0.03, 0.15, and 0.30mg Se/kg). Glucose tolerance test (GTT) and insulin tolerance test (ITT) were measured at the third month. Mice were killed at the fourth month. Plasma, liver, and muscle tissues were fetched for biochemistry and Se analysis. The expressions of insulin signaling pathway (PI3K-AKT-mTOR), glutathione peroxidase 1 (GPX1), selenoprotein N (SELENON), 3-phosphoglycerate dehydrogenase (PHGDH), serine hydroxymethyltransferases 1 (SHMT1), 5,10-methylenetetrahydrofolate reductase (MTHFR), and methionine synthase (MS) were analyzed by western blotting (WB) in liver and muscle tissues. The results of GTT and ITT showed that glucose tolerance and insulin tolerance were both abnormal in the 0.03mg Se/kg and 0.3mg Se/kg groups. Se concentrations in plasma, liver, and muscle of 0.03mg Se/kg group were significantly lower than that of 0.15mg Se/kg and 0.30mg Se/kg groups (p < 0.05 or p < 0.01). The expressions of P-Akt (Thr-308) in muscle (p < 0.05) and PI3K and mTOR in liver (p < 0.001) of 0.30mg Se/kg group were downregulated. The expressions of GPX1 in liver and muscle (p < 0.05 and p < 0.001), SELENON in muscle (p < 0.05), PHGDH in liver and muscle (p < 0.05), and SHMT1 (p < 0.05), MTHFR (p < 0.001), and MS (p < 0.001) in muscle of 0.3mg Se/kg group were upregulated. The de novo serine synthesis pathway (SSP) was found to be activated in liver and muscle tissues of mice with a high-Se diet for the first time.

AMPK-HIF-1α signaling enhances glucose-derived de novo serine biosynthesis to promote glioblastoma growth

BackgroundCancer cells undergo cellular adaptation through metabolic reprogramming to sustain survival and rapid growth under various stress conditions. However, how brain tumors modulate their metabolic flexibility in the naturally serine/glycine (S/G)-deficient brain microenvironment remain unknown.MethodsWe used a range of primary/stem-like and established glioblastoma (GBM) cell models in vitro and in vivo. To identify the regulatory mechanisms of S/G deprivation-induced metabolic flexibility, we employed high-throughput RNA-sequencing, transcriptomic analysis, metabolic flux analysis, metabolites analysis, chromatin immunoprecipitation (ChIP), luciferase reporter, nuclear fractionation, cycloheximide-chase, and glucose consumption. The clinical significances were analyzed in the genomic database (GSE4290) and in human GBM specimens.ResultsThe high-throughput RNA-sequencing and transcriptomic analysis demonstrate that the de novo serine synthesis pathway (SSP) and glycolysis are highly activated in GBM cells under S/G deprivation conditions. Mechanistically, S/G deprivation rapidly induces reactive oxygen species (ROS)-mediated AMP-activated protein kinase (AMPK) activation and AMPK-dependent hypoxia-inducible factor (HIF)-1α stabilization and transactivation. Activated HIF-1α in turn promotes the expression of SSP enzymes phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH). In addition, the HIF-1α-induced expression of glycolytic genes (GLUT1, GLUT3, HK2, and PFKFB2) promotes glucose uptake, glycolysis, and glycolytic flux to fuel SSP, leading to elevated de novo serine and glycine biosynthesis, NADPH/NADP+ ratio, and the proliferation and survival of GBM cells. Analyses of human GBM specimens reveal that the levels of overexpressed PHGDH, PSAT1, and PSPH are positively correlated with levels of AMPK T172 phosphorylation and HIF-1α expression and the poor prognosis of GBM patients.ConclusionOur findings reveal that metabolic stress-enhanced glucose-derived de novo serine biosynthesis is a critical metabolic feature of GBM cells, and highlight the potential to target SSP for treating human GBM.

Modulating Metabolic Reprogramming By Phosphoglycerate Dehydrogenase (PHGDH) Inhibitors in Omipalisib-Refractory AML

Background: Numerous reports indicated the significance of the PI3K/AKT/mTOR signaling pathway in AML. Up to now, there is no FDA-approved PI3K/AKT/mTOR inhibitors available for patients with acute myeloid leukemia (AML). Recently, we demonstrated that PI3K/mTOR dual inhibitor omipalisib had a potent anti-leukemic effect, but it could not completely eliminate leukemia cells in a subcutaneous murine model. To explore the refractory features of omipalisib, we established several OCI-AML3 sublines refractory to omipalisib by an in vivo selection procedure. The OCI-AML3-R cell line was the most refractory one and showed cross-resistance to another PI3K/mTOR inhibitor dactolisib and multi-tyrosine kinase inhibitor dasatinib, in addition to omipalisib. Several genes associated with multidrug-resistance, ABCC1, ABCC2, ABCB1, ABCG2, MDR1, and MRP1, did not express significant difference between OCI-AML3 and OCI-AML3-R. Aims: To investigate the molecular mechanisms of OCI-AML3-R using transcriptomics and metabolomics analysis, and to explore potential therapeutic strategies from both in vitro and in vivo aspects. Methods: OCI-AML3 and OCI-AML3-R leukemia cell lines were used in this study. MTS assay was performed to assess cell viability and proliferation. The differential expression genes (DEGs) were examined using RNA-seq (Illumina NovaSeq 6000). Metabolomic analysis was conducted by the C-SCOPE service using capillary electrophoresis mass spectrometry CE-TOFMS and CE-QqQMS with Agilent 6460 TripleQuad LC/MS (HMT, Yamagata, Japan). Q-RT-PCR was used for mRNA quantification. Immunoblotting were used to clarify the variations of total- and phosphor-proteins. Flow cytometry was used for apoptosis and mitochondrial ROS content analysis. OCI-AML3-R xenograft models in nude mice were used for scrutinizing efficacy of PHGDH inhibitor in vivo. The protocol for the xenograft experiments was approved by the IACUC, National Taiwan University. Results: A total of 1946 DEGs between OCI-AML3 and OCI-AML3-R cells were identified with the selection criteria of p&amp;lt; 0.05 and [log 2 (fold change)]&amp;gt; 1 or &amp;lt;1, of which 477 and 1469 DEGs were up- and down-regulated, respectively. g:Profiler was used for functional enrichment analysis and revealed that NFkB signaling-related pathways ( p&amp;lt; 0.001), cell cycle-related pathways ( p&amp;lt; 0.001), and nucleotide metabolic pathways ( p&amp;lt; 0.001) were enriched in OCI-AML3-R. Q-RT-PCR confirmed that a series of genes related to pentose phosphate pathway (PPP; TKT and 6PGD), serine synthesis pathway (SSP; PHGDH and PSAT1), purine synthesis and folate/methionine cycle ( PAICS, SHMT2, MTHFD2, and MTHFD1L) were increased in OCI-AML3-R cells. Among them, PHGDH and PSAT1 increased the most, 4.75x and 11.38x, respectively. A total of 103 metabolites were screened out by metabolomic analysis, 58 of which showed significant differences between OCI-AML3 and OCI-AML3-R cells ( p&amp;lt; 0.05). Metabolite enrichment analysis also revealed that the purine metabolism, urea cycle, glycolysis/gluconeogenesis, and several amino acid metabolism pathways were enriched in OCI-AML3-R cells. A comprehensive overview of metabolites in glycolysis and TCA cycle in OCI-AML3-R cells revealed significantly increased levels of PRPP and serine, as well as steady-state levels of glycine, folate, and several purine metabolites. These results indicated that a reprogramming of glucose metabolism towards the PPP and SSP in OCI-AML3-R cells was noted. Based on these results, we proposed targeting the PPP and SSP as a therapeutic approach for omipalisib-refractory AML. The PHGDH inhibitors, WQ-2101 and NCT-503, showed strong anti-leukemic effects in OCI-AML3-R with an IC 50 of 26.17 and 16.81 μM, respectively, while the G6PD inhibitor 6-AN had limited effect. The PHGDH inhibitors increased mitochondrial ROS levels and induced apoptosis in OCI-AML3-R. Additionally, in vivo experiment showed that treatment with NCT-503 significantly prolonged mouse survival and inhibited tumor growth in a dose-dependent manner without adverse effects on body weight. Summary/Conclusion: Transcriptomic and metabolomic analyses revealed that OCI-AML3-R upregulated PPP and SSP, leading to increased nucleotide synthesis, which contribute cell growth and survival. Targeting PHGDH could significantly suppress omipalisib-refractory in AML and may have implications for future clinical trials.