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.

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) and 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 (MTX), which requires DHFR expression, and ganciclovir (GCV), 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 approximately six weeks. Our results show 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.

Background: Social adversity from neighborhood disadvantage (ND) is associated with shorter breast cancer survival. Although studies have identified associations between ND and DNA methylation (DNAme) or gene expression (mRNA), a study integrating DNAme and mRNA to understand how the epigenome regulates key biological pathways in ND merits further inquiry. Methods: DNAme-, mRNA-, miRNA-, and tRNA-derived fragment data were analyzed from 80 estrogen receptor+/HER2− breast cancer samples. We analyzed the association among ND, DNAme, coding, and noncoding data to understand how the epigenome regulates biological pathways. Results: Twenty-five patients lived in ND and 55 in neighborhood advantage. In patients from ND, calcium signaling and cell adhesion pathways were hypermethylated, immune-related pathways hypomethylated, immune response genes upregulated, and estrogen response genes downregulated. Small RNA analysis showed differential expression of miRNA isoforms and tRNA-derived fragments related to ND. Subjective ND further correlated with epigenetic changes in calcium signaling, cell adhesion, and metabolic pathways, along with upregulation of proliferative and stemness pathways. Conclusions: We discovered novel associations between ND and epigenomic regulation of clinically relevant oncogenesis pathways associated with aggressive biology, such as estrogen response pathways. These findings lay the foundation for multi-institutional studies to validate our findings. Impact: This study demonstrates that ND, which is associated with shorter breast cancer survival, is also associated with epigenetic reprogramming of key oncogenesis pathways. These findings highlight pathways through which ND may affect tumor biology and lay the foundation for cancer control policy and behavioral interventions that may reverse the negative effects of ND on cancer biology.

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.

Supplementary Figure S3: Signaling and small non-coding RNAs rewiring in tumors from individuals with higher disadvantage scores