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.
TET2 haploinsufficiency is a driving event in myeloid cancers and is associated with a worse prognosis in patients with acute myeloid leukemia (AML). Enhancing residual TET2 activity using vitamin C ...increases oxidized 5-methylcytosine (mC) formation and promotes active DNA demethylation via base excision repair (BER), which slows leukemia progression. We utilize genetic and compound library screening approaches to identify rational combination treatment strategies to improve use of vitamin C as an adjuvant therapy for AML. In addition to increasing the efficacy of several US Food and Drug Administration (FDA)-approved drugs, vitamin C treatment with poly-ADP-ribosyl polymerase inhibitors (PARPis) elicits a strong synergistic effect to block AML self-renewal in murine and human AML models. Vitamin-C-mediated TET activation combined with PARPis causes enrichment of chromatin-bound PARP1 at oxidized mCs and γH2AX accumulation during mid-S phase, leading to cell cycle stalling and differentiation. Given that most AML subtypes maintain residual TET2 expression, vitamin C could elicit broad efficacy as a PARPi therapeutic adjuvant.
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•Vitamin C synergizes with PARPis, causing S phase stalling and AML differentiation•Combination treatment efficacy is dependent on TET2 expression in AML•PARPi treatment with vitamin C causes 5fC accumulation in the genome of AML cells•PARPi treatment with vitamin C enriches for PARP1 binding and γH2AX at 5fC sites
Vitamin C treatment can slow the progression of AML by enhancing TET2 activity but is not curative as a single agent therapy. Using genetic and compound screening approaches, Brabson et al. identify a rational combination treatment strategy where vitamin C enhances the therapeutic efficacy of PARPis for AML treatment.
TET methylcytosine dioxygenase 2 ( 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. It has previously been shown that hematopoietic Tet2 deficiency in mice triggers a pro-inflammatory state with increased intestinal permeability, gut bacterial translocation, and accelerated clonal expansion. Gut microbes themselves can exert an influence on myeloid leukemia progression through synthesis of compounds including short-chain fatty acids (SCFAs), which promote intestinal barrier integrity. Dietary levels of one-carbon metabolites and cofactors have been found to alter gut microbial composition, affecting SCFA production and intestinal permeability, in disease-free adults. 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 CHIP progression.
We performed competitive bone marrow transplantation assays with Tet2+/- cells in mice supplemented with altered one-carbon metabolites: a control diet, high or low methionine diets, high or low folate diets, and high or low vitamin B12 supplementation. Mice were treated for a total of 7 months before timed sacrifice. Altered supplementation of the one carbon metabolites tested did not influence the competitiveness of Tet2-deficient cells in peripheral blood, spleen or bone marrow, however, high vitamin B12 levels promoted a myeloid differentiation bias in the spleen and bone marrow. Plasma cytokine analysis also showed that high vitamin B12 supplementation caused increased circulating inflammatory cytokines (IL-1b, IL-23, IL-27, CCL3/4, CXCL2). Based on this strong inflammatory phenotype, we performed scRNA-sequencing of splenic Tet2+/- CD11b+ cells. These data confirmed a neutrophilic cell expansion in the spleens of vitamin B12-treated mice along with increased expression of innate inflammatory genes S100a8/9 and Lyz2 as well as IL-1 b and Cxcl2 in the neutrophil clusters.
Gut microbial-dependent inflammation is known to drive TET2-dependent myeloproliferation, thus we collected fecal samples before timed sacrifice and performed shotgun sequencing. Vitamin B12 supplementation decreased alpha diversity and the composition of genera that produce butyrate, an SCFA known to promote intestinal barrier integrity. Metagenomic analysis revealed increased amino acid metabolism and fatty acid biosynthesis in the gut microbiome of vitamin B12-supplemented mice, along with decreased butanoate metabolism, suggesting an alteration in microbial function that could influence SCFA content. To test whether these alterations in SCFA-producers led to increased gut permeability, we performed fluorescence in situ hybridization (FISH) against bacterial ribosomal 16S in livers from our mouse cohorts and observed increased levels of bacterial dissemination in mice with vitamin B12 supplementation. Our findings suggest that vitamin B12 supplementation in mice exacerbates the myeloid lineage differentiation bias of Tet2+/- hematopoiesis, possibly due to decreased gut barrier integrity which contributes to a heightened innate inflammatory response. This study highlights the potential for micronutrients of one-carbon metabolism to influence CHIP progression through maintenance of proper gut microbial homeostasis.
The formation, inheritance, and removal of DNA methylation in the genome of mammalian cells is directly regulated by two families of enzymes–DNA methyltransferases (DNMTs) and Ten-Eleven ...Translocation proteins (TETs). DNMTs generate and maintain the inheritance of 5-methylcytosine (5mC), which is the substrate targeted by the TET enzymes for conversion to 5-hydroxymethylcytosine (5hmC) and its downstream oxidized derivatives. The activity of DNMT and TET is dependent on the availability of micronutrients and metabolite co-factors, including essential vitamins, amino acids, and trace metals, highlighting how DNA methylation levels can be directly enhanced, suppressed, or remodeled via metabolic and nutritional perturbations. Dynamic changes in DNA methylation are required during embryonic development, lineage specification, and maintenance of somatic cell function that can be fine-tuned based on the influence of essential micronutrients. As we age, DNA methylation and hydroxymethylation levels drift in patterning, leading to epigenetic dysregulation and genomic instability that underlies the formation and progression of multiple diseases including cancer. Understanding how DNA methylation can be regulated by micronutrients will have important implications for the maintenance of normal tissue function upon aging, and in the prevention and treatment of diseases for improved health and lifespan.
A cost‐effective way of undertaking comprehensive, continental‐scale, assessments of ecological condition is needed to support large‐scale conservation planning, monitoring, reporting, and ...decision‐making. Currently, cross‐jurisdictional inconsistency in assessment methods limits the capacity to scale‐up monitoring. Here we present a novel way to build a coherent continent‐wide site‐level ecological condition dataset, using cross‐calibration methods to integrate assessments from many observers. We focus on the use of condition assessments from individual expert observers, a currently untapped resource. Our approach has two components: (1) a simple online tool that captures expert assessments at specific locations; (2) a process of calibrating and rescaling disparate expert evaluations that can be applied to the data to provide a consistent dataset for use in conservation assessments. We describe a pilot study, involving 28 experts, who contributed 314 individual site condition assessments across a wide range of ecosystems and regions throughout continental Australia. A correction factor for each expert was used to rescale the contributed site condition assessment scores, based on a set of 77 photographic images, each scored for their condition by multiple experts, using a linear mixed model. Our approach shows strong promise for delivering the volumes of data required to develop continental‐scale reference libraries of site condition assessments. Although developed from expert elicitation, the approach could also be used to harmonize the collation of existing condition datasets. The process we demonstrate can also facilitate online citizen scientists to make site condition assessments that can be cross‐calibrated using contributed images.
Ten-eleven translocation (TET) enzymes are commonly mutated in leukemia, leading to aberrant DNA methylation and impaired hydroxymethylation. All-trans retinoic acid (ATRA) is a vitamin A derivative ...and cofactor of retinoic acid receptors (RARs) which, in combination with arsenic trioxide, can curatively treat acute promyelocytic leukemia. ATRA has also been shown to upregulate expression of Tet2 and Tet3 in murine embryonic stem cells, and retinol and ascorbic acid (vitamin C) have been shown to enhance pluripotent stem cell reprogramming through increasing hydroxymethylcytosine and promoting DNA demethylation. Our previous work has demonstrated ascorbate can slow disease progression in vivo and, combined with the PARP inhibitor Olaparib, promote cell cycle dysregulation and induce cellular differentiation in acute myeloid leukemia (AML) models both in vitro and in vivo.
Here, we show ATRA can induce TET2 and/or TET3 expression in AML cells, and, combined with ascorbate, increase genome-wide oxidized methylcytosine in a TET-dependent manner. Combination treatment with ATRA and ascorbate also induces transcriptional reprogramming, leading to upregulation of DNA repair genes and pathways leading to increased expression of myeloid differentiation markers. Additionally, we observe chromatin remodeling associated with an enrichment for ETS motifs including genes and regions regulated by the transcription factor PU.1, an essential modulator of hematopoietic cell fate. We also show enrichment of retinoic acid receptor A (RARA) at the TET2 locus and at several RAR elements in response to ATRA in AML cells. Using very low dose ascorbate and ATRA, we show that the combination treatment can enhance the efficacy of Olaparib and Venetoclax to alter the cell cycle, promote differentiation, and induce apoptosis of AML cells. These data demonstrate the potential for ATRA and ascorbate to increase TET activity and drive myeloid differentiation and death of AML cells that can be exploited as an adjuvant therapy for the treatment leukemia.
To assess the feasibility of embolization of aortic side branches and its impact on the incidence of type II endoleak after endovascular aneurysm repair.
Endovascular aneurysm repair was performed in ...74 patients. Aortic side branch vessels were evaluated on the preoperative angiogram and computed tomography (CT) and, where embolization of lumbar and inferior mesenteric vessels was considered technically possible, this was attempted prior to endovascular repair. Follow-up CT was used to assess the presence of type II endoleak.
Seventy-two patients were followed up for longer than 1 month. Embolization was attempted in 25 cases, successfully in 10, with partial success in 11, and failure in four. Twenty patients with successful or partly successful preoperative embolization were discharged and followed-up. Four (20%) had demonstrable type II endoleak during follow-up, with two of these persisting at latest follow-up. Of 43 patients without previous embolization, there were 10 (23.3%) type II endoleaks during the follow-up period, four of these persisting. In cases with type II endoleak, mean sac diameter change was -0.5 mm in the cases with previous embolization and +3.1 mm without. The mean period to onset of type II endoleak was 6.9 months without, and 15.3 months with, previous embolization.
Although the cohort size is below a level that would confer significance, the trend of these findings is such as to suggest a lack of influence of aortic side branch embolization on the incidence of type II endoleak during the follow-up period.
Poly-ADP-ribose polymerase inhibitors (PARPi) are currently in clinical trial to determine their therapeutic efficacy for the treatment of acute myeloid leukemia (AML). We have shown that vitamin C ...(VitC), an essential micronutrient and co-factor of Ten-Eleven translocation (TET) proteins, enhances AML sensitivity to PARPi, potentially due to an increased dependency on base-excision repair (BER) enzymes needed to remove TET-catalyzed oxidized methylcytosine bases via active DNA demethylation. TET2 is the most frequently mutated TET gene in patients with AML, and vitamin C treatment can mimic genetic restoration of TET2 function, leading to DNA demethylation, differentiation, and leukemia cell death. Whether vitamin C efficacy in combination with PARPi depends on the level of TET2 functional alleles is not yet known and may stratify whether TET2 wild-type or mutant patients should be targeted by vitamin C adjuvant therapy.
We have generated primary murine AML-ETO9a+ and MLL-AF9+ leukemia models with Tet2 +/+, Tet2 +/- and Tet2 -/- alleles to determine the Tet2-dependent efficacy of PARPi treatment when combined with vitamin C. Furthermore, we have performed CRISPR gene knockout and drug library screening in human AML cell lines in combination with vitamin C treatment, and tested a panel of 10 AML cell lines with titrating concentrations of PARPi (Olaparib, Talazoparib, Veliparib and Rucaparib) alone or in combination with vitamin C (L-ascorbic acid) mimicking physiological to pharmacological in vivo doses. Primary murine AML cells and human cell lines were assayed for colony-forming capacity, differentiation, cell cycling, viability and effects on DNA methylation, levels of oxidized 5-mC and gene expression upon combination treatment in vitro and in vivo. TET2 mutant PDX and primary murine AMLs treated in vivo with L-ascorbate (4g/kg) and Olaparib (50mg/kg) by daily IP injection were also monitored for disease burden, cellular differentiation and survival.
Vitamin C is known to drive the TET-catalyzed iterative oxidation of 5-methylcytosine (5-mC) leading to the formation of 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). We show that VitC-PARPi combination treatment causes an accumulation of oxidized 5-mC intermediates in the AML genome that correlates with increased yH2AX formation in mid-S phase and cell cycle stalling. Vitamin C reduces the IC 50 of Olaparib and Talazoparib by greater than 10-fold in human AML cells lines and primary murine leukemia cells, and treatment in combination promotes myeloid differentiation and blocks colony-forming capacity greater than either alone. In both our in vitro and in vivo studies, Tet2 +/- AML cells exhibit increased sensitivity to vitamin C treatment alone or in combination with PARPi compared to either Tet2 +/+ or Tet2 -/- cells, suggesting that patients with TET2 haploinsufficiency, which represents the majority of TET2 mutant cases, could benefit the most from combined treatment.
Our findings confirm that vitamin C can act synergistically with PARPi to block AML cell viability, reduce colony-forming capacity, and decrease leukemia burden in PDX and primary murine leukemia models in a TET2 allelic dose-dependent manner. The combinatorial effect works at clinically relevant concentrations of PARPi, and low-pharmacological doses of vitamin C. These studies suggest that vitamin C can be used as a non-toxic therapeutic adjuvant to PARPi therapy for the treatment of AML.
Neel: Northern Biologics, LTD: Current equity holder in publicly-traded company, Other: Co- Founder; SAB: Other: Co-Founder; Navire Pharma: Consultancy, Current equity holder in publicly-traded company; Jengu Therapeutics: Consultancy, Current equity holder in publicly-traded company, Other: Co-Founder; Arvinas, Inc: Consultancy, Current equity holder in publicly-traded company; Recursion Pharma: Current equity holder in publicly-traded company.
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.
Transfection of transgenes into Drosophila cultured cells is a standard approach for studying gene function. However, the number of transgenes present in the cell following transient transfection or ...stable random integration varies, and the resulting differences in expression level affect interpretation. Here we developed a system for Drosophila cell lines that allows selection of cells with a single-copy transgene inserted at a specific genomic site using recombination-mediated cassette exchange (RMCE). We used the φC31 integrase and its target sites attP and attB for RMCE. Cell lines with an attP-flanked genomic cassette were transfected with donor plasmids containing a transgene of interest (UAS-x), a dihydrofolate reductase (UAS-DHFR) gene flanked by attB sequences, and a thymidine kinase (UAS-TK) gene in the plasmid backbone outside the attB sequences. In cells undergoing RMCE, UAS-x and UAS-DHFR were exchanged for the attP-flanked genomic cassette, and UAS-TK was excluded. These cells were selected using methotrexate, which requires DHFR expression, and ganciclovir, which causes death in cells expressing TK. Pure populations of cells with one copy of a stably integrated transgene were efficiently selected by cloning or mass culture in ∼6 weeks. Our results show that RMCE avoids the problems associated with current methods, where transgene number is not controlled, and facilitates the rapid generation of Drosophila cell lines in which expression from a single transgene can be studied.
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