Vitamins B9 (folate) and B12 are essential water-soluble vitamins that play a crucial role in the maintenance of one-carbon metabolism: a set of interconnected biochemical pathways driven by folate ...and methionine to generate methyl groups for use in DNA synthesis, amino acid homeostasis, antioxidant generation, and epigenetic regulation. Dietary deficiencies in B9 and B12, or genetic polymorphisms that influence the activity of enzymes involved in the folate or methionine cycles, are known to cause developmental defects, impair cognitive function, or block normal blood production. Nutritional deficiencies have historically been treated with dietary supplementation or high-dose parenteral administration that can reverse symptoms in the majority of cases. Elevated levels of these vitamins have more recently been shown to correlate with immune dysfunction, cancer, and increased mortality. Therapies that specifically target one-carbon metabolism are therefore currently being explored for the treatment of immune disorders and cancer. In this review, we will highlight recent studies aimed at elucidating the role of folate, B12, and methionine in one-carbon metabolism during normal cellular processes and in the context of disease progression.
Therapy induced senescence (TIS) in tumors and TIS cancer cells secrete proinflammatory senescence-associated secretory phenotype (SASP) factors. SASP factors promote TIS cancer cells to re-enter the ...growth cycle with stemness characteristics, resulting in chemo-resistance and disease relapse. Herein, we show that the immunotherapeutic HCW9218, comprising transforming growth factor-β (TGF-β) receptor II and interleukin (IL)-15/IL-15 receptor α domains, enhances metabolic and cytotoxic activities of immune cells and reduces TIS tumor cells in vivo to improve the efficacy of docetaxel and gemcitabine plus nab-paclitaxel against B16F10 melanoma and SW1990 pancreatic tumors, respectively. Mechanistically, HCW9218 treatment reduces the immunosuppressive tumor microenvironment and enhances immune cell infiltration and cytotoxicity in the tumors to eliminate TIS cancer cells. Immuno-depletion analysis suggests that HCW9218-activated natural killer cells play a pivotal role in TIS cancer cell removal. HCW9218 treatment following docetaxel chemotherapy further enhances efficacy of tumor antigen-specific and anti-programmed death-ligand 1 (PD-L1) antibodies in B16F10 tumor-bearing mice. We also show that HCW9218 treatment decreases TIS cells and lowers SASP factors in off-target tissues caused by chemotherapy of tumor-bearing mice. Collectively, HCW9218 has the potential to significantly enhance anti-tumor efficacy of chemotherapy, therapeutic antibodies, and checkpoint blockade by eliminating TIS cancer cells while reducing TIS-mediated proinflammatory side effects in normal tissues.
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An immunotherapeutic molecule, HCW9218, demonstrated anti-tumor efficacy by eliminating chemotherapy-induced senescent cells and reducing the off-target effects mediated by chemotherapy. HCW9218 represents a potent anti-cancer immunotherapeutic for use in combination with approved therapies to improve the health span and life span of treated patients.
Abstract
TET2 loss-of-function mutations induce a pre-malignant state known as clonal hematopoiesis of indeterminate potential (CHIP). CHIP occurs in approximately 10% of people over 65 years of age ...and confers a 10-fold greater risk of developing hematological malignancy. Several environmental factors, including radiation, sleep deprivation, atherosclerosis, and diet, have been associated with the expansion of pre-malignant clones in CHIP patients. Tet2-deficiency in mice has also been shown to trigger a pro-inflammatory state with increased intestinal permeability and accelerated myeloid expansion. Gut microbes exert an influence on host disease progression through the synthesis of many compounds including short-chain fatty acids (SCFAs), which promote gut barrier integrity. Dietary levels of one-carbon metabolites and cofactors, including methionine and folate, have been found to alter gut microbial composition in disease-free adults and influence SCFA production in the gut in murine models. Given the connection between diet, SCFAs and gut permeability, we sought to determine the impact of dietary one-carbon metabolites on gut microbial composition and function in a murine model of pre-malignant hematopoiesis. We performed competitive bone marrow transplantation assays in mice fed diets with altered one-carbon metabolite supplementation, such as high and low folate or methionine. We found that differential supplementation with these one-carbon metabolites did not influence the competitiveness of Tet2-deficient hematopoietic cells, however, increased dietary methionine promoted a myeloid lineage differentiation bias and an elevation in circulating pro-inflammatory cytokines. After 8 months of dietary treatment, fecal samples were collected, and shotgun sequencing was performed to examine the role of one-carbon metabolite levels on gut microbial diversity. Alterations in dietary methionine and folate caused significant changes to gut microbial composition in Tet2-deficient mice. High folate or methionine supplementation led to a decrease in the ratio of Firmicutes to Bacteroidetes and high folate supplementation was also associated with decreased alpha diversity and a decrease in SCFA-producing bacteria in the gut. These studies highlight the potential influence of dietary one-carbon metabolites on the microbiome and inflammatory microenvironment of pre-malignant hematopoiesis.
Citation Format: Peter Lyon, Praveen Singh, Byron Fang, Victoria Strippoli, Sabita Roy, Luisa Cimmino. The influence of dietary one-carbon metabolites on gut dysbiosis during pre-malignant hematopoiesis abstract. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A44.
TET2 haploinsufficiency is a driving event in myeloid cancers and associated with a worse prognosis in patients with AML. Enhancing residual TET2 activity using vitamin C increases oxidized ...5-methylcytosine (oxi-mC) formation and promotes active DNA demethylation via base-excision repair (BER) that slows leukemia progression. We utilized genetic and compound library screening approaches to identify rational combination treatment strategies to improve the use of vitamin C as an adjuvant therapy for AML. In addition to increasing the efficacy of multiple FDA approved drugs, vitamin C treatment with poly-ADP-ribose polymerase inhibitors (PARPi) elicited a strong synergistic effect at blocking AML self-renewal in murine and human models. AML cells treated with a combination of vitamin C and PARPi in vitro led to reduced replating capacity in colony-forming assays, reduced viability in liquid culture, and increased survival upon treatment in vivo. These phenotypes were associated with increased p21 expression, cell-cycle stalling in S-phase and differentiation of AML cells toward a more mature myeloid phenotype. Furthermore, we show that vitamin C-mediated TET activation combined with PARPi causes an enrichment of chromatin-bound PARP1 protein specifically at 5-formylcytosine (5fC) oxi-mCs, and an enrichment for yH2AX at these sites in addition to its accumulation during mid-S phase. This work provides the first proof of PARP1 as a direct reader of 5fC, and enrichment of 5fC at sites marked by yH2AX. The generation of BER-inducing oxi-mCs imparts a novel therapeutic potential of PARPi, which are currently in clinical trial to determine their therapeutic efficacy for AML. Given the majority of AML subtypes maintain residual TET2 expression, vitamin C could elicit broad efficacy as a PARPi therapeutic adjuvant to improve treatment outcome.
Accumulation of senescent cells (SNCs) with a senescence‐associated secretory phenotype (SASP) has been implicated as a major source of chronic sterile inflammation leading to many age‐related ...pathologies. Herein, we provide evidence that a bifunctional immunotherapeutic, HCW9218, with capabilities of neutralizing TGF‐β and stimulating immune cells, can be safely administered systemically to reduce SNCs and alleviate SASP in mice. In the diabetic db/db mouse model, subcutaneous administration of HCW9218 reduced senescent islet β cells and SASP resulting in improved glucose tolerance, insulin resistance, and aging index. In naturally aged mice, subcutaneous administration of HCW9218 durably reduced the level of SNCs and SASP, leading to lower expression of pro‐inflammatory genes in peripheral organs. HCW9218 treatment also reverted the pattern of key regulatory circadian gene expression in aged mice to levels observed in young mice and impacted genes associated with metabolism and fibrosis in the liver. Single‐nucleus RNA Sequencing analysis further revealed that HCW9218 treatment differentially changed the transcriptomic landscape of hepatocyte subtypes involving metabolic, signaling, cell‐cycle, and senescence‐associated pathways in naturally aged mice. Long‐term survival studies also showed that HCW9218 treatment improved physical performance without compromising the health span of naturally aged mice. Thus, HCW9218 represents a novel immunotherapeutic approach and a clinically promising new class of senotherapeutic agents targeting cellular senescence‐associated diseases.
Bifunctional immunotherapeutic HCW9218 functions as a novel SNC‐reducing and senomorphic agent in mice. Subcutaneous administration of HCW9218 activates NK, innate lymphoid group‐1, and CD8+ T cells, and neutralizes TGF‐ß to reduce senescent cells (SNC‐reducing) and SASP (senomorphic) leading to lower chronic inflammation and restored tissue homeostasis.
Exotoxins, including the hemolysins known as the alpha (alpha) and beta (beta) toxins, play an important role in the pathogenesis of Staphylococcus aureus infections. A random transposon library was ...screened for S. aureus mutants exhibiting altered hemolysin expression compared to wild type. Transposon insertions in 72 genes resulting in increased or decreased hemolysin expression were identified. Mutations inactivating a putative cyclic di-GMP synthetase and a serine/threonine phosphatase (Stp1) were found to reduce hemolysin expression, and mutations in genes encoding a two component regulator PhoR, LysR family transcriptional regulator, purine biosynthetic enzymes and a serine/threonine kinase (Stk1) increased expression. Transcription of the hla gene encoding alpha toxin was decreased in a Deltastp1 mutant strain and increased in a Deltastk1 strain. Microarray analysis of a Deltastk1 mutant revealed increased transcription of additional exotoxins. A Deltastp1 strain is severely attenuated for virulence in mice and elicits less inflammation and IL-6 production than the Deltastk1 strain. In vivo phosphopeptide enrichment and mass spectrometric analysis revealed that threonine phosphorylated peptides corresponding to Stk1, DNA binding histone like protein (HU), serine-aspartate rich fibrinogen/bone sialoprotein binding protein (SdrE) and a hypothetical protein (NWMN_1123) were present in the wild type and not in the Deltastk1 mutant. Collectively, these studies suggest that Stk1 mediated phosphorylation of HU, SrdE and NWMN_1123 affects S. aureus gene expression and virulence.
Somatic gain-of-function mutations in isocitrate dehydrogenases (
) 1 and 2 are found in multiple hematologic and solid tumors, leading to accumulation of the oncometabolite (
)-2-hydroxyglutarate ...(2HG). 2HG competitively inhibits α-ketoglutarate-dependent dioxygenases, including histone demethylases and methylcytosine dioxygenases of the TET family, causing epigenetic dysregulation and a block in cellular differentiation.
studies have provided proof of concept for mutant IDH inhibition as a therapeutic approach. We report the discovery and characterization of AG-221, an orally available, selective, potent inhibitor of the mutant IDH2 enzyme. AG-221 suppressed 2HG production and induced cellular differentiation in primary human
mutation-positive acute myeloid leukemia (AML) cells
and in xenograft mouse models. AG-221 also provided a statistically significant survival benefit in an aggressive IDH2
-mutant AML xenograft mouse model. These findings supported initiation of the ongoing clinical trials of AG-221 in patients with
mutation-positive advanced hematologic malignancies.
Mutations in
are identified in approximately 20% of patients with AML and contribute to leukemia via a block in hematopoietic cell differentiation. We have shown that the targeted inhibitor AG-221 suppresses the mutant IDH2 enzyme in multiple preclinical models and induces differentiation of malignant blasts, supporting its clinical development.
.
A number of human cancers harbor somatic point mutations in the genes encoding isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2). These mutations alter residues in the enzyme active sites and confer ...a gain-of-function in cancer cells, resulting in the accumulation and secretion of the oncometabolite (R)-2-hydroxyglutarate (2HG). We developed a small molecule, AGI-6780, that potently and selectively inhibits the tumor-associated mutant IDH2/R140Q. A crystal structure of AGI-6780 complexed with IDH2/R140Q revealed that the inhibitor binds in an allosteric manner at the dimer interface. The results of steady-state enzymology analysis were consistent with allostery and slow-tight binding by AGI-6780. Treatment with AGI-6780 induced differentiation of TF-1 erythroleukemia and primary human acute myelogenous leukemia cells in vitro. These data provide proof-of-concept that inhibitors targeting mutant IDH2/R140Q could have potential applications as a differentiation therapy for cancer.
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