6 research outputs found
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Epigenetic Regulation of Genomic Stability by Vitamin C
DNA methylation plays an important role in the maintenance of genomic stability. Ten-eleven translocation proteins (TETs) are a family of iron (Fe
2+
) and α-KG -dependent dioxygenases that regulate DNA methylation levels by oxidizing 5-methylcystosine (5mC) to generate 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). These oxidized methylcytosines promote passive demethylation upon DNA replication, or active DNA demethylation, by triggering base excision repair and replacement of 5fC and 5caC with an unmethylated cytosine. Several studies over the last decade have shown that loss of TET function leads to DNA hypermethylation and increased genomic instability. Vitamin C, a cofactor of TET enzymes, increases 5hmC formation and promotes DNA demethylation, suggesting that this essential vitamin, in addition to its antioxidant properties, can also directly influence genomic stability. This review will highlight the functional role of DNA methylation, TET activity and vitamin C, in the crosstalk between DNA methylation and DNA repair
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3112 – RETINOIC ACID AND ASCORBATE REMODEL THE EPIGENOME OF LEUKEMIA CELLS TO IMPROVE THERAPEUTIC EFFICACY BY ENHANCING TET ACTIVITY
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Retinoic Acid and Ascorbate Remodel the Epigenome of Leukemia Cells to Improve Therapeutic Efficacy By Enhancing TET Activity
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
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Vitamin C Enhances PARPi Efficacy for the Treatment of AML
Abstract
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.
Disclosures
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
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3039 – OXIDIZED 5-METHYLCYTOSINE MODULATES SYNTHETIC LETHALITY TO POLY-ADP-RIBOSE POLYMERASE INHIBITORS IN ACUTE MYELOID LEUKEMIA
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
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Oxidized mC modulates synthetic lethality to PARP inhibitors for the treatment of leukemia
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