Nucleotide Biosynthesis Research Articles

Pilot Study of High-Dose Pemetrexed in Patients with Progressive Chordoma.

Chordomas are ultrarare tumors of the axial spine and skull-base without approved systemic therapy. Most chordomas have negative expression of thymidylate synthase (TS), suggesting a potential for responding to the antifolate agent pemetrexed, which inhibits TS and other enzymes involved in nucleotide biosynthesis. We evaluated the therapeutic activity and safety of high-dose pemetrexed in progressive chordoma. Adult patients with previously treated, progressive chordoma participated in an open-label, single-institution, single-arm, pilot clinical trial of intravenous pemetrexed 900 mg/m2 every 3 weeks and supportive medications of folic acid, vitamin B12, and dexamethasone. The primary endpoint was objective response rate according to RECIST v1.1. Secondary endpoints included adverse events, progression-free survival (PFS), tumor molecular profiles, and alterations in tissue and blood-based biomarkers. Fifteen patients were enrolled and the median number of doses administered was 15 (range, 4-31). One patient discontinued treatment due to psychosocial issues after four cycles and one contracted COVID-19 after 13 cycles. Of the 14 response-evaluable patients, 2 (14%) achieved a partial response and 10 (71%) demonstrated stable disease. Median PFS was 10.5 months (95% confidence interval: 9 months-undetermined) and 6-month PFS was 67%. Adverse events were expected and relatively mild, with one grade 3 creatinine increased, and one each of grade 3 and 4 lymphopenia. No grade 5 adverse events, unexpected toxicities, or dose-limiting toxicities were observed. Several patients reported clinical improvement in disease-related symptoms. High-dose pemetrexed appears tolerable and shows objective antitumor activity in patients with chordoma. Phase II studies of high-dose pemetrexed are warranted.

Glutamine Metabolism Is Altered in Myeloproliferative Neoplasms and Represents a Potential Novel Therapeutic Target

Cancer cells exhibit metabolic reprogramming to ensure sufficient production of macromolecules used to promote cell growth and proliferation. Previous studies have shown that proliferating cancer cells rely significantly on glutamine metabolism as a source of nitrogen and carbon to facilitate nucleotide and lipid biosynthesis, respectively, and to generate metabolites and/or maintain redox homeostasis. Despite this phenomenon being well characterized in many different solid and blood cancer types, very few studies have investigated the metabolic landscape of myeloproliferative neoplasms (MPNs). Glutamine catabolism begins via the uptake of glutamine through sodium-dependent transporters such as SLC1A5, and its transport to the mitochondria where it then undergoes deamination by glutaminase (GLS1) to produce glutamate. This conversion by GLS1 is the rate limiting step in the central glutaminolysis axis, making it a preferential point of study and therapeutic targeting. Indeed, many studies have explored the glutaminolysis pathway in a variety of cancers to decipher potential targets for treatment, but these studies have been extremely limited in MPNs and the potential mechanisms with respect to MPN driver mutations (CALR, JAK2, MPL) and their differential utilization of glutamine have yet to be fully explored. One previous study demonstrated that in JAK2V617F mutant-Ba/F3 cells, GLS1 expression was significantly increased and that GLS1 inhibition served as a promising intervention to suppress colony formation in patient samples but not healthy donors. To more comprehensively decipher the role of glutamine metabolism in MPNs driven by all three driver mutations ( CALR, JAK2, and MPL), we performed RNA-sequencing analysis on peripheral blood mononuclear cells (PMBCs) from MPN patients and found that genes involved in glutamine metabolism are significantly altered across the board. In contrast to the previous study mentioned above, our data demonstrate that GLS1 is not significantly up-regulated, and that MPN PBMCs are not sensitive to GLS1 inhibition. Rather, we characterized another key enzyme in glutamine metabolism, glutamine synthetase (GS) as a molecular and metabolic dependency in MPNs. GS catalyzes the reversion of glutamate into glutamine and serves to regulate nitrogen metabolism, support nucleotide biosynthesis, and most importantly synthesize glutamine to satiate the demand caused by the cancer cells' affinity for high glutamine levels. We found that GS is significantly up-regulated at the mRNA, protein, and activity level in MPNs driven by all three driver mutations, and that genetic and pharmacological inactivation of GS abrogates MPN cell proliferation in vitro. In summary, we have demonstrated that GLS1 may not be an effective therapeutic target for MPNs, and instead characterize GS as a novel dependency and potential point of therapeutic intervention.

Disruption of the Malate-Aspartate Shuttle Leads to Anemia

Erythropoiesis, the process of differentiating multipotent hematopoietic stem cells (HSCs) into mature enucleated red blood cells (RBCs), is a metabolically intensive process, resulting in the generation of over 2 million new RBCs per second. Although anemia due to impaired erythropoiesis is a common condition, therapeutic options are often limited to RBC transfusions. However, the emergence of effective metabolite therapies, such as glutamine supplementation for sickle cell disease, underscores the potential of understanding metabolism for developing additional treatments. Two key enzymes in the malate-aspartate shuttle (MAS), glutamic-oxaloacetic transaminase 1 (GOT1) and 2 (GOT2), play crucial roles in transferring energy in the form of NADH from the cytosol into the mitochondria for oxidative phosphorylation. NADH cannot directly cross the mitochondrial membrane, so its electrons are transported on malate, which enters the mitochondria through a specific transporter. Within this shuttle, mitochondrial GOT2 utilizes glutamine to generate aspartate, which is then transported back into the cytoplasm to be metabolized by GOT1. Previous research has shown that aspartate-dependent nucleotide biosynthesis is essential for HSC regeneration. To investigate the roles of GOT1 and GOT2 in erythropoiesis, we crossed mice expressing tamoxifen-inducible erythroid-specific Cre recombinase ( Gata1-Cre ERT2 BAC transgenic mice) to Got1 or Got2 floxed lines, generating Got1 flox/flox;Gata1-Cre ERT2( Got1 CKO) or Got2 flox/flox;Gata1-Cre ERT2( Got2 CKO) mice, respectively. Upon tamoxifen administration to these mice, conditional Got1 or Got2 deletion occurs in megakaryocytic-erythrocytic progenitor (MEP) cells, which give rise to both RBCs and platelets. Complete blood count analysis revealed decreased hemoglobin levels and RBC counts, along with a reciprocal increase in platelet count in both Got1 and Got2 CKO mice. These results were unexpected, as previous studies showed that hematopoietic Got1 loss led to increased aspartate-driven nucleotide biosynthesis and HSC regeneration, while the opposite effects were observed in mice with hematopoietic Got2 loss. Although these enzymes are expected to have opposite effects on aspartate levels, the deletion of either enzyme disrupts the transfer of reducing equivalents between the cytosol and mitochondria. This results in NADH accumulation and reductive stress, which in turn leads to metabolic dysfunction, affecting glycolysis and oxidative phosphorylation. Therefore, the occurrence of anemia in mice with either Got1 or Got2 deletion in the erythroid compartment suggests that impaired redox balance, rather than aspartate-driven nucleotide biosynthesis, is likely the cause of the anemia. To further understand the basis of anemia in Got1 and Got2 CKO mice, we harvested bone marrow cells and used conventional cell surface markers to quantify MEPs, burst-forming unit erythroid cells (BFU-Es), colony-forming unit erythroid cells (CFU-Es), and terminally differentiated erythroblast populations. Compared to littermate controls with no deletion of Got1 and Got2, the absolute numbers of pre-CFU-Es and CFU-Es were higher in both Got1 and Got2CKO mice, while the absolute numbers of differentiated erythroblasts were lower in both mutant mice. These findings indicate a block in erythroid differentiation, which is currently under further investigation. Collectively, these preliminary data suggest a novel role for MAS in regulating erythropoiesis. Ongoing studies are actively exploring the mechanisms by which dysfunctional MAS leads to anemia.

Novel Role of Mitochondrial Folate Metabolism in Cell Fate Decisions and Leukaemogenesis

Folate-mediated one carbon (1C) metabolism sustains proliferation of rapidly dividing cells through its direct contribution to nucleotide biosynthesis. In brief, new 1C units are donated from the amino acid serine (the primary source of 1C units), while folate molecules function as 1C unit carriers. Accordingly, antifolates have been used in the treatment of several malignancies, particularly in the field of haemato-oncology. Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disorder that arises in a single haematopoietic stem cell with the introduction of the BCR::ABL1 oncoprotein. Although it is generally accepted that CML is driven by leukaemic stem cells (LSCs) that are insensitive to standard therapy due to their quiescent/slow-cycling status, the role of folate metabolism in LSCs remains undescribed. Transcriptomic analysis of patient-derived CML LSCs (CD34 +38 -) revealed a significant upregulation of folate metabolism genes, including the mitochondrial hydroxymethyltransferase (SHMT2; p≤0.05), when compared with normal counterparts. Furthermore, CML stem/progenitor (CD34 +) cells displayed a significant increase in the exchange rate of formate (folate intermediate necessary for nucleotide synthesis) compared to the secretome of normal CD34 + cells (p<0.05), suggesting an upregulation of 1C metabolism in therapy resistant CML cells. Following SHMT2 knockout (KO), we observed a strong antiproliferative effect and significantly impaired tumour formation in CML cell line xenograft models (p<0.01), verifying that mitochondrial 1C metabolism is not dispensable for CML cells. Metabolic characterisation using liquid chromatography-mass spectrometry revealed that both genetic and pharmacological inhibition (using the SHMT1/2 inhibitor, SHIN1) of 1C metabolism results in a significant decrease in de novo purine synthesis. Furthermore, we uncovered activation of AMPK signalling and suppression of mTORC1 activity following mitochondrial 1C metabolism inhibition. Notably, formate supplementation or reconstitution of purine levels was sufficient to reverse the effect on AMPK and mTORC1. Phenotypically, SHIN1 treatment induced the expression of erythropoiesis markers CD71 and Glycophorin A in CML cells, including patient derived CD34 + cells. AMPKα1/α2 KO revealed that the increased expression of these markers was independent of AMPK activity. Mechanistically, we discovered that reconstitution of purine levels (adenine) and mTORC1 activity prevented erythroid differentiation following SHMT1/2 inhibition, highlighting that depletion of 1C units drives differentiation of leukaemic cells through mTORC1-mediated purine sensing. Of clinical relevance, combination of SHIN1 with imatinib, a frontline treatment for CML patients, decreased the number of therapy-resistant CML LSCs in a patient-derived xenograft model (p<0.05). Lastly, stable isotope-assisted metabolomics using 13C 315N 1-serine revealed that imatinib reduced labelling into purine nucleotides, but it maintained labelling into glutathione (GSH), the most abundant antioxidant in the cell, and its oxidised derivate (GSSG). Through generation of glycine, folate metabolism directly contributes to the GSH pool. These data suggest that standard therapy does not affect the incorporation of serine into GSH, implying that the synergistic effect with folate metabolism inhibition might be related to impeded antioxidant defence. Overall, our findings highlight a novel role for mitochondrial folate metabolism in stem cell fate decisions and leukaemogenesis. This work is available as a preprint: https://www.biorxiv.org/content/10.1101/2022.12.21.521404v1

Liquid chromatography-mass spectrometry-based metabolomics and fluxomics reveals the metabolic alterations in glioma U87MG multicellular tumor spheroids versus two-dimensional cell cultures.

Multicellular tumor spheroids (MCTSs) that reconstitute the metabolic characteristics of in vivo tumor tissue may facilitate the discovery of molecular biomarkers and effective anticancer therapies. However, little is known about how cancer cells adapt their metabolic changes in complex three-dimensional (3D) microenvironments. Here, using the two-dimensional (2D) cell model as control, the metabolic phenotypes of glioma U87MG multicellular tumor spheroids were systematically investigated based on static metabolomics and dynamic fluxomics analysis. A liquid chromatography-mass spectrometry-based global metabolomics and lipidomics approach was adopted to survey the cellular samples from 2D and 3D culture systems, revealing marked molecular differences between them. Then, by means of metabolomic pathway analysis, the metabolic pathways altered in glioma MCTSs were found using 13 C6 -glucose as a tracer to map the metabolic flux of glycolysis, the tricarboxylic acid (TCA) cycle, de novo nucleotide synthesis, and de novo lipid biosynthesis in the MCTS model. We found nine metabolic pathways as well as glycerolipid, glycerophospholipid and sphingolipid metabolism to be predominantly altered in glioma MCTSs. The reduced nucleotide metabolism, amino acid metabolism and glutathione metabolism indicated an overall lower cellular activity in MCTSs. Through dynamic fluxomics analysis in the MCTS model, we found that cells cultured in MCTSs exhibited increased glycolysis activity and de novo lipid biosynthesis activity, and decreased the TCA cycle and de novo purine nucleotide biosynthesis activity. Our study highlights specific, altered biochemical pathways in MCTSs, emphasizing dysregulation of energy metabolism and lipid metabolism, and offering novel insight into metabolic events in glioma MCTSs.

CTNI-15. PILOT STUDY OF HIGH-DOSE PEMETREXED FOR PROGRESSIVE CHORDOMA

Abstract INTRODUCTION Chordomas are ultra-rare tumors of the spine, and no systemic therapy is approved for refractory disease. Pemetrexed is a multitargeted antifolate that inhibits thymidylate synthase (TS) and other enzymes involved in nucleotide biosynthesis. Considering the majority of chordomas do not express TS, we sought to evaluate the therapeutic activity and safety of high-dose pemetrexed. METHODS Adults with previously-treated chordoma were enrolled on an open-label, single-institution, single-arm, pilot clinical trial. Pemetrexed 900 mg/m2 was administered intravenously every 3 weeks, with supportive medications of folic acid, vitamin B12, and dexamethasone. The primary endpoint was objective response rate according to RECIST v1.1. Secondary and exploratory endpoints included adverse events, progression-free survival (PFS), tumor molecular profiles, and alterations in potential tissue and blood-based biomarkers before and after treatment. RESULTS Fifteen patients with chordoma received a median of 15 (range 4-31) doses of pemetrexed. Median PFS was 10.5 months (95% CI 9 months-undetermined) and 6-month PFS was 67%. Out of 14 response-evaluable patients, two (14%) achieved a partial response and ten (71%) demonstrated stable disease. Adverse events were generally mild at grades 1-2, the most common being fatigue, rash, nausea, constipation, diarrhea, mucositis, vomiting, alopecia, pruritus, lower extremity edema, and alanine transaminase increased. One Grade 3 creatinine increase, one Grade 3 lymphopenia, and one Grade 4 lymphopenia occurred and there were no Grade 5 adverse events, unexpected toxicities, or dose-limiting toxicities. Clinical improvement in disease-related symptoms occurred in several patients. Tumor molecular profiling revealed low mutational burden and though not statistically significant, positive TS expression trended negatively with disease progression. Cell-free microRNAs (cfmiRs) were concordantly detected in plasma and tissues and a blood cfmiR signature was identified that distinguished chordoma from health donors. CONCLUSION High-dose pemetrexed is safe and well-tolerated in chordoma patients. A phase II study is in development. (NCT03955042)

Transcriptomic and metabolomic analysis of the mechanism by which Bacillus tequilensis inhibits Alternaria alternata to control pear black spot

Pear black spot caused by Alternaria alternata is widespread in Chinese orchards, causing serious losses. Screening of effective biocontrol microorganisms is important for controlling this disease. Bacillus tequilensis 2_2a has biocontrol potential. This study aimed to verify the antifungal activity of B. tequilensis against A. alternata and biocontrol effect on pear black spot, to elucidate by transcriptomic and metabolomic approach, the molecular pathway underlying to effect of strain against A. alternata, to validate the effect on genes and metabolic components related to the molecular action mechanisms, and to analyse the mechanisms and molecular targets of the biocontroller bacteria. The results showed that B. tequilensis inhibited mycelial growth. B. tequilensis caused A. alternata mycelial aggregation, twisting, and entanglement. B. tequilensis controlled pear black spot by over 90%. Six pathways were related to the key differentially expressed genes and metabolites of B. tequilensis-treated A. alternata: glycolysis, citric acid cycle, oxidative phosphorylation, pentose phosphate pathway, pyrimidine metabolism and cell cycle. Validated changes in the expression of key genes were consistent with the transcriptomic data, and the interactions among these pathways mainly affected glucose conversion, citric acid synthesis, intracellular redox-related biosynthesis of pyridine nucleotides, and adenine nucleotides that provide energy. This research helps to promote the biocontrol agent B. tequilensis, as it provides the mechanism behind its biocontrol efficacy.

IL-6/JAK2-dependent G6PD phosphorylation promotes nucleotide synthesis and supports tumor growth

ObjectiveTumor cells hijack inflammatory mechanisms to promote their own growth. IL-6 is one of the major cytokines, and is frequently upregulated in tumors. The pentose phosphate pathway (PPP) generates the indispensable building blocks to produce various nucleotides. Here we aimed to determine whether and how PPP is timely tuned in response to IL-6 to support tumor growth. MethodsProtein expression was examined by immunoblot. Protein interaction was examined by immunoprecipitation. Tumor cell proliferation in in vitro culture was examined by BrdU assay and colony formation assay. Tumor cell proliferation in mouse xenograft model was examined by Ki-67 staining. ResultsHere we show that the metabolic flux of PPP and enzymatic activity of glucose-6-phosphate dehydrogenase (G6PD) is rapidly induced under IL-6 treatment, without obvious changes in G6PD expression level. Mechanistically, Janus kinase 2 (JAK2) phosphorylates G6PD Y437 under IL-6 treatment, which accentuates G6PD enzymatic activity by promoting G6PD binding with its substrate G6P. Further, JAK2-dependent G6PD Y437 phosphorylation is required for IL-6-induced nucleotide biosynthesis and tumor cell proliferation, and is associated with the progression of oral squamous cell carcinoma. ConclusionsOur findings report a new mechanism implicated in the crosstalk between tumor cells and inflammatory microenvironment, by which JAK2-dependent activation of G6PD governs nucleotide synthesis to support tumor cell proliferation, thereby highlighting its value as a potential anti-tumor target.

PSII-8 Comparison of Different Fattening Stages on Rumen Microbiota and Physiological Characteristics in Japanese Black Cattle

Abstract This study aimed to investigate the relationship between rumen microbiota and physiological characteristics in Japanese Black cattle during the fattening period, which are known for their distinctive physiological characteristics due to prominent intramuscular fat deposition. The experimental period was categorized into three stages: early (12 to 14 months of age; T1), middle (15 to 22 months; T2), and late fattening phases (23 to 30 months; T3). We analyzed the rumen microbiota of 21 Japanese Black cattle in each phase using 16S rRNA gene amplicon sequencing. In addition, we examined the correlation between the rumen microbiota and various physiological parameters, such as blood metabolites, hormones, and rumen fermentation characteristics. Our results showed that rumen microbial diversity was greater in T1, and the inter-group distribution of rumen microbiota in T1 was differentiated from T2 and T3. At the genus level, Flexilinea, Streptococcus, Butyrivibrio, Selenomonas and Kandleria were the most abundant in T1. Bifidobacterium, Shuttleworthia and Sharpea were the most abundant in T2. Acetitomaculum, Mycoplasma, Atopobium and Howardella were the most abundant in T3. In correlation analysis, Monoglobus and Olsenella negatively correlated with IGF-1 and positively correlated with cortisol in T1. Firmicutes/Bacteroidota ratio positively correlated with daily gain, blood total cholesterol, and lactic acid dehydrogenase in T3. Planctomycetota correlated with volatile fatty acid components during the entire period, while Verrucomicrobiota positively correlated with carcass traits in T1. Additionally, functional genetic profiles indicated that rumen microbiota related to carbohydrate metabolism and nucleotide biosynthesis was significantly enriched in T1 and T3, respectively. The composition of rumen microbial in Japanese Black cattle varied depending on fattening stages, and this variation is likely attributable to changes in feed composition. Furthermore, specific microorganisms were significantly associated with physiological parameters. Our findings could help in the development of strategies to enhance gut health and productivity by manipulating the rumen microbiota through the changes in diet compositions considering the fattening stages of Japanese Black cattle.

In situ profiling reveals metabolic alterations in the tumor microenvironment of ovarian cancer after chemotherapy

In this study, we investigated the metabolic alterations associated with clinical response to chemotherapy in patients with ovarian cancer. Pre- and post-neoadjuvant chemotherapy (NACT) tissues from patients with high-grade serous ovarian cancer (HGSC) who had poor response (PR) or excellent response (ER) to NACT were examined. Desorption electrospray ionization mass spectrometry (DESI-MS) was performed on sections of HGSC tissues collected according to a rigorous laparoscopic triage algorithm. Quantitative MS-based proteomics and phosphoproteomics were performed on a subgroup of pre-NACT samples. Highly abundant metabolites in the pre-NACT PR tumors were related to pyrimidine metabolism in the epithelial regions and oxygen-dependent proline hydroxylation of hypoxia-inducible factor alpha in the stromal regions. Metabolites more abundant in the epithelial regions of post-NACT PR tumors were involved in the metabolism of nucleotides, and metabolites more abundant in the stromal regions of post-NACT PR tumors were related to aspartate and asparagine metabolism, phenylalanine and tyrosine metabolism, nucleotide biosynthesis, and the urea cycle. A predictive model built on ions with differential abundances allowed the classification of patients’ tumor responses as ER or PR with 75% accuracy (10-fold cross-validation ridge regression model). These findings offer new insights related to differential responses to chemotherapy and could lead to novel actionable targets.

Targeting Nucleotide Biosynthesis Ameliorates Neutropenia in a Zebrafish Model of Dnajc21-Mutant Shwachman-Diamond Syndrome

Germline mutations in the ribosome maturation factors, SBDS, DNAJC21 and EFL1, lead to Shwachman-Diamond syndrome (SDS), a type of inherited bone marrow failure syndrome characterized by neutropenia, exocrine pancreatic insufficiency and growth restriction. Further, SDS patients have a 36% increased cumulative risk of developing myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML) by 30 years of age. Given the rarity of SDS, large numbers of primary human samples are not readily available for mechanistic studies, warranting the use of animal models. Mouse and zebrafish models of SBDS mutations exhibit early lethality. In contrast, no in vivo models of DNAJC21 mutations have been published to date. We generated the first in vivo model of dnajc21 loss using zebrafish ( Danio rerio), given their highly conserved hematopoietic program and ease of genetic manipulation. Introduction of a homozygous deletion mutation in the zebrafish dnajc21 gene using CRISPR-Cas9 genomic editing resulted in stunted body growth and neutropenia, recapitulating hallmark SDS patient phenotypes. Whole-mount in situ hybridization for key hematopoietic lineage markers revealed reduced l-plastin +mature leukocytes and hbbe3 + mature erythrocytes in dnajc21 mutant embryos at 48 hours post-fertilization (hpf). A concomitant accumulation of myb + hematopoietic stem and progenitor cells (HSPCs) both in the dorsal aorta (equivalent to aorta-gonad-mesonephros) and the caudal hematopoietic tissue (fetal liver equivalent) was observed. Treatment of embryos with zebrafish granulocyte colony stimulating factor (gcsf) mRNA resulted in a modest expansion of neutrophils in dnajc21 mutant zebrafish compared to wildtype. Further, we observed reduced cell proliferation marked by phosphohistone-H3 (pH3) as well as low ATP:AMP levels measured by liquid chromatography/mass-spectrometry (LC/MS) in the dnajc21 mutants, suggesting a poor bioenergetic profile. At 4 months of age, flow cytometry and histopathological analysis identified reduced mature myelomonocytes and mild erythroid dysplasia in the whole kidney marrow (human bone marrow equivalent) of mutant fish. The link between TP53 somatic mutations and MDS/AML has been established in SBDS-mutant SDS, but has not been explored for DNAJC21-mutant SDS. We hence crossed our dnajc21 mutant zebrafish with a zebrafish line carrying a gain-of-function mutation (R217H) in the tp53 gene. Compound mutant fish had improved neutrophil counts but importantly, this was accompanied by an expansion of myeloid progenitors and significant erythroid dysplasia. To understand the disease mechanisms underlying the neutropenia and hypoproliferation in SDS, we performed bulk RNA sequencing of whole zebrafish larvae at 48 hpf. Biological processes such as oxidative stress response, drug catabolism and pre-mRNA spliceosome assembly were downregulated; whereas, acyl-CoA and nucleotide metabolism were upregulated in the mutants. In parallel, we performed untargeted metabolomics by LC/MS to determine the metabolic changes induced by dnajc21 loss. Upregulated pathways included urea cycle, amino acid and phenylacetate metabolism. Pyrimidine biosynthesis, including both the de novo and salvage pathways were downregulated in the mutants. Remarkably, exogenous supplementation with uridine or thymidine significantly improved neutrophil counts in the mutants. Our findings suggest that in dnajc21-mutant SDS1) neutropenia is caused by a combination of impaired granulocyte differentiation and cell proliferation; 2) the kidney marrow features myelodysplasia-like changes and 3) nucleotide biosynthesis may be a previously unrecognized therapeutic strategy in SDS.

Metabolic Programming of Hematopoietic Stem Cell Function By Prenatal Folate

Hematopoietic stem cells (HSCs) are programmed by metabolic changes resulting in alterations to HSC function. Here, we discovered that varying prenatal folate status metabolically programs HSCs during development and affects adult HSC self-renewal and engraftment potential into adulthood. Folate status varies greatly in the global population based on nutritional intake, common genetic polymorphisms, and widespread supplementation. Folate-derived one-carbon metabolism (OCM) regulates cellular methylation, de novo nucleotide biosynthesis and mitochondrial metabolism, processes critical to HSC function and establishment. Despite immense therapeutic potential, there is limited understanding of mechanisms driving HSC developmental programming. We hypothesize that varying prenatal folate status affects risk for hematologic dysfunction by developmentally programming HSCs. To test how prenatal folate modulated HSC function, female mice were assigned to one of three experimental diets to model population-wide folate consumption: 0mg/kg (deficient, FD), 2mg/kg (control, FC) and 8mg/kg (supplemented, FS). Profiling of fetal liver (FL) hematopoietic cells revealed that FD diet caused expansion of long term (LT)-HSCs that was propagated across the hematopoietic hierarchy, including myeloid and lymphoid progenitors and mature myeloid cells. In contrast, FS decreased FL cell output. We next determined if these changes were driven by metabolic activity. FD fetal HSPCs exhibited higher mitochondrial membrane potential and lower glycolytic activity, whereas FS fetal HSPCs displayed both higher glycolytic activity and oxygen consumption despite impaired production of downstream hematopoietic cells. Together, these data suggest that HSPC metabolic programming initiates during fetal development and is folate-dose dependent. To determine if prenatal folate programs hematopoiesis into adulthood, we quantified bone marrow (BM) hematopoietic cells in adult offspring after weaning onto standard chow. Surprisingly, FD expanded lymphoid progenitors in adult offspring with no impact on mature lymphoid cell output. In contrast, FS diet expanded myeloid and lymphoid progenitors in the BM as well as downstream mature cells, including monocytes and B-cells. To determine if these effects were HSC cell-intrinsic, we performed competitive transplantation assays of LT-HSCs isolated from adult offspring. FD offspring HSCs showed worse engraftment by chimerism across all peripheral blood (PB) populations compared to FC. In comparison, FS offspring HSCs showed enhanced total cellular output by donor cells/uL across all PB mature lineages except platelets. Secondary whole BM transplantation revealed that FS HSCs retained enhanced total cellular output and superior engraftment compared to FC, whereas FD HSCs demonstrated complete exhaustion. These data indicate that adult HSC function is programmed by prenatal folate status. To investigate molecular alterations driving metabolic programming of adult hematopoiesis by prenatal folate, we performed single-cell RNA sequencing (Scseq) and metabolomics on adult offspring HSPCs. Scseq analysis revealed most differential gene expression (DE) in HSC clusters, with less DE in progenitors. Re-clustering of all HSCs revealed distinct heterogeneity within this population, where both FD and FS HSCs were identified as distinct clusters compared to FC. Metabolomic profiling further suggested that FD HSCs exhibited signs of mitochondrial dysfunction through, 1) accumulating glucose and lactate suggesting reliance on glycolysis and, 2) accumulating succinate and glutamine suggesting complex II impairment and inadequate utilization of mitochondrial metabolism. Surprisingly, the same changes associated with mitochondrial dysfunction in FD HSCs persisted downstream into lymphoid-biased multipotent progenitors. In contrast, limited metabolic changes were observed in FS HSCs, suggesting alternative pathways for sustained programming. These changes indicate that FD and FS offspring have distinct metabolic programming. Together, our data suggest that prenatal folate status programs hematopoietic stem cell function into adulthood and is driven by alterations to metabolic activity. Ongoing studies examine how prenatal folate status affects the adult hematological and immune response to challenge.

CTPS1 is a novel therapeutic target in multiple myeloma which synergizes with inhibition of CHEK1, ATR or WEE1.

Targeting nucleotide biosynthesis is a proven strategy for the treatment of cancer but is limited by toxicity, reflecting the fundamental nucleotide requirement of dividing cells. The rate limiting step in de novo pyrimidine synthesis is of interest, being catalyzed by two homologous enzymes, CTP synthase 1 (CTPS1) and CTPS2, that could be differentially targeted. Herein, analyses of publicly available datasets identified an essential role for CTPS1 in multiple myeloma (MM), linking high expression of CTPS1 (but not CTPS2) with advanced disease and poor outcomes. In cellular experiments, CTPS1 knockout induced apoptosis of MM cell lines. Exposure of MM cells to STP-B, a novel and highly selective pharmacological inhibitor of CTPS1, inhibited proliferation, induced S phase arrest and led to cell death by apoptosis. Mechanistically, CTPS1 inhibition by STP-B activated DNA damage response (DDR) pathways and induced double-strand DNA breaks which accumulated in early S phase. Combination of STP-B with pharmacological inhibitors of key components of the DDR pathway (ATR, CHEK1 or WEE1) resulted in synergistic growth inhibition and early apoptosis. Taken together, these findings identify CTPS1 as a promising new target in MM, either alone or in combination with DDR pathway inhibition.

Combining multi-omics analysis to identify host-targeted targets for the control of Brucella infection.

Human infections caused by Brucella (called brucellosis) are among the most common zoonoses worldwide with an estimated 500,000 cases each year. Since chronic Brucella infections are extremely difficult to treat, there is an urgent need for more effective therapeutics. As a facultative intracellular bacterium, Brucella is strictly parasitic in the host cell. Here, we performed proteomic and transcriptomic and metabolomic analyses on Brucella infected patients, mice and cells that provided an extensive "map" of physiological changes in brucellosis patients and characterized the metabolic pathways essential to the response to infection, as well as the associated cellular response and molecular mechanisms. This is the first report utilizing multi-omics analysis to investigate the global response of proteins and metabolites associated with Brucella infection, and the data can provide a comprehensive insight to understand the mechanism of Brucella infection. We demonstrated that Brucella increased nucleotide synthesis in the host, consistent with increased biomass requirement. We also identified IMPDH2, a key regulatory complex that controls nucleotide synthesis during Brucella infection. Pharmacological targeting of IMPDH2, the rate-limiting enzyme in guanine nucleotide biosynthesis, efficiently inhibits B. abortus growth both in vitro and in vivo. Through screening a library of natural products, we identified oxymatrine, an alkaloid obtained primarily from Sophora roots, is a novel and selective IMPDH2 inhibitor. In further in vitro bacterial inhibition assays, oxymatrine effectively inhibited the growth of B. abortus, which was impaired by exogenous supplementation of guanosine, a salvage pathway of purine nucleotides. This moderately potent, structurally novel compound may provide clues for further design and development of efficient IMPDH2 inhibitors and also demonstrates the potential of natural compounds from plants against Brucella.

Overexpression of PSAT1 is Correlated with Poor Prognosis and Immune Infiltration in Non-Small Cell Lung Cancer.

Current evidence suggests that phosphoserine aminotransferase 1 (PSAT1) is overexpressed in various tumors. Herein, we investigate the significance of PSAT1 in non-small cell lung cancer (NSCLC) and its correlation with immune infiltration. The expression profile of PSAT1 in NSCLC patients and related clinical information was obtained from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA-NSCLC) databases. In silico and experimental validation were conducted to assess the role of PSAT1 in NSCLC. Gene set enrichment analysis (GSEA) was performed to investigate the disparities in biological functions between groups with high and low PSAT1 expression. Additionally, the biological characteristics and immune cell infiltration were compared between these two groups. We also assessed whether PSAT1 expression could predict the sensitivity of NSCLC patients to immunotherapy using the immunophenotype score (IPS) and an anti-PD-L1 immunotherapy cohort (IMvig-or210). Furthermore, the difference in drug sensitivity between PSAT1-high and PSAT1-low expression cell lines was investigated. Analysis of transcriptional expression profiles using TCGA data revealed overexpression of PSAT1 in NSCLC tissues correlated with poor overall survival (OS). GSEA results showed enrichment of DNA recombination and repair, nucleotide biosynthesis, and the P53 signaling pathway in the PSAT1-high group. Experimental validation demonstrated that the knockdown of PSAT1 suppressed cell proliferation, migration, and invasion of NSCLC. Immune cell infiltration analysis revealed an immune-activated tumor microenvironment in the PSAT1-low group. It was also observed that PSAT1-low cell lines were more likely to benefit from immunotherapy and several chemotherapy drugs. PSAT1 has enormous potential for applications in the prediction of NSCLC patient outcomes and provides the foothold for more precise individualized treatment of this patient population.

A Sedentary Lifestyle Changes the Composition and Predicted Functions of the Gut Bacterial and Fungal Microbiota of Subjects from the Same Company

A sedentary lifestyle affects the diversity and composition of the gut microbiota, but previous studies have mainly focused on bacteria instead of fungi. Here, we compared both the fecal bacterial and fungal microbiota compositions and functions in sedentary persons and controls. Subjects from the China Railway Corporation, including 99 inspectors and 88 officials, were enrolled in our study. Fecal microbiota communities were analyzed using 16S rRNA gene sequencing for bacteria and ITS sequencing for fungi. We found that the diversity of the gut microbiota of the sedentary group was significantly lower than that of the control group (P < 0.05). The sedentary group had a higher abundance of Firmicutes, a lower abundance of Actinobacteria and Proteobacteria and a higher abundance of Ascomycota, and a lower abundance of Basidiomycota. Furthermore, functional prediction analysis of the fungal microbiota revealed more L-tryptophan degradation to 2-amino-3-carboxymuconate semialdehyde, more phospholipid remodeling (phosphatidylethanolamine, yeast), and more L-tyrosine degradation I, as well as less pentose phosphate pathway (non-oxidative branch), less adenosine nucleotide biosynthesis and less L-valine biosynthesis in the sedentary group (P < 0.05). Thus, a sedentary lifestyle changes the composition and function of the gut microbiota. It may change the pentose phosphate pathway (non-oxidative branch), nucleic acid and amino acid biosynthesis and phospholipid metabolism in fungi.

Metabolic Hallmarks for Purine Nucleotide Biosynthesis in Small Cell Lung Carcinoma.

SCLC tumors preferentially utilize either the de novo or salvage pathway in purine nucleotide biosynthesis, and HPRT1 has therapeutic implications in SCLC.

The Strange Case of Orotic Acid: The Different Expression of Pyrimidines Biosynthesis in Healthy Males and Females.

Orotic acid (OA) is an intermediate metabolite of pyrimidine nucleotide biosynthesis and represents a minor diet constituent. The measurement of urinary orotic acid is useful in confirming the diagnosis of hereditary metabolic diseases. Moreover, it could be of interest to know how the physiological concentration of this metabolite changes in relation to different conditions of clinical normality. The purpose of this study was to determine the orotic acid concentration in the urine of healthy patients, to observe normal oroticuria and to evaluate if the expression of pyrimidine intermediate biosynthesis differs between healthy males and females. The orotic acid concentration in urine was performed via the ICH M10-validated analytical method. Unexpectedly, females showed a greater oroticuria than males in pediatric age (0-10); conversely, we did not find significant differences until 70 years of age. The LC-MS/MS method was suitable for use in the differential diagnosis of hereditary metabolic disease and metabolic monitoring of anticancer drug-induced toxicity. The analytical protocol was found to be rapid and ideal, and was used in the routine analysis of a clinical chemistry laboratory. The biochemical aspects related to the expression of pyrimidine biosynthesis should be further investigated in light of the obtained results.

Weight Loss Promotion in Individuals with Obesity through Gut Microbiota Alterations with a Multiphase Modified Ketogenic Diet.

The occurrence of obesity and related metabolic disorders is rising, necessitating effective long-term weight management strategies. With growing interest in the potential role of gut microbes due to their association with responses to different weight loss diets, understanding the mechanisms underlying the interactions between diet, gut microbiota, and weight loss remains a challenge. This study aimed to investigate the potential impact of a multiphase dietary protocol, incorporating an improved ketogenic diet (MDP-i-KD), on weight loss and the gut microbiota. Using metagenomic sequencing, we comprehensively analyzed the taxonomic and functional composition of the gut microbiota in 13 participants before and after a 12-week MDP-i-KD intervention. The results revealed a significant reduction in BMI (9.2% weight loss) among obese participants following the MDP-i-KD intervention. Machine learning analysis identified seven key microbial species highly correlated with MDP-i-KD, with Parabacteroides distasonis exhibiting the highest response. Additionally, the co-occurrence network of the gut microbiota in post-weight-loss participants demonstrated a healthier state. Notably, metabolic pathways related to nucleotide biosynthesis, aromatic amino acid synthesis, and starch degradation were enriched in pre-intervention participants and positively correlated with BMI. Furthermore, species associated with obesity, such as Blautia obeum and Ruminococcus torques, played pivotal roles in regulating these metabolic activities. In conclusion, the MDP-i-KD intervention may assist in weight management by modulating the composition and metabolic functions of the gut microbiota. Parabacteroides distasonis, Blautia obeum, and Ruminococcus torques could be key targets for gut microbiota-based obesity interventions.

Incorporation of inosine into DNA by human polymerase eta (Polη): kinetics of nucleotide misincorporation and structural basis for the mutagenicity.

Inosine, a purine nucleoside containing the hypoxanthine (HX) nucleobase, can form in DNA via hydrolytic deamination of adenine. Due to its structural similarity to guanine and the geometry of Watson-Crick base pairs, inosine can mispair with cytosine upon catalysis by DNA polymerases, leading to AT → GC mutations. Additionally, inosine plays an essential role in purine nucleotide biosynthesis, and inosine triphosphate is present in living cells. In a recent publication, Averill and Jung examined the possibility of polη catalyzed incorporation of deoxyinosine triphosphate (dITP) across dC and dT in a DNA template. They found that dITP can be incorporated across C or T, with the ratio of 13.7. X ray crystallography studies revealed that the mutagenic incorporation of dITP by human polη was affected by several factors including base pair geometry in the active site of the polymerase, tautomerization of nucleobases, and the interaction of the incoming dITP nucleotide with active site residues of polη. This study demonstrates that TLS incorporation of inosine monophosphate (IMP) into growing DNA chains contributes to its mutagenic potential in cells.