Vitamin C deficiency is found in patients with cancer and might complicate various therapy paradigms. Here we show how this deficiency may influence the use of DNA methyltransferase inhibitors (DNMTis) for treatment of hematological neoplasias. In vitro, when vitamin C is added at physiological levels to low doses of the DNMTi 5-aza-2′-deoxycytidine (5-aza-CdR), there is a synergistic inhibition of cancer-cell proliferation and increased apoptosis. These effects are associated with enhanced immune signals including increased expression of bidirectionally transcribed endogenous retrovirus (ERV) transcripts, increased cytosolic dsRNA, and activation of an IFNinducing cellular response. This synergistic effect is likely the result of both passive DNA demethylation by DNMTi and active conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) by ten-eleven translocation (TET) enzymes at LTR regions of ERVs, because vitamin C acts as a cofactor for TET proteins. In addition, TET2 knockout reduces the synergy between the two compounds. Furthermore, we show that many patients with hematological neoplasia are markedly vitamin C deficient. Thus, our data suggest that correction of vitamin C deficiency in patients with hematological and other cancers may improve responses to epigenetic therapy with DNMTis.

The hypomethylating agents (HMAs) are standard therapy for patients with higher-risk myelodysplastic syndrome (MDS); however, the majority of the patients will lose their response to HMAs over time due to unknown mechanisms. It has recently been shown that T cell expression of the immunoinhibitory receptor PD-1 is regulated by DNA methylation. In 12 of 27 patients (44%) PD-1 promoter demethylation was observed in sorted peripheral blood T cells isolated over consecutive cycles of treatment with 5-azacytidine (5-aza). The PD-1 promoter demethylation correlated with an increase in PD-1 expression. Moreover, demethylation of the PD-1 promoter correlated with a significantly worse overall response rate (8% vs. 60%, p = 0.014), and a trend towards a shorter overall survival (p = 0.11) was observed. A significantly higher baseline methylation level of the PD-1 promoter was observed in T cells of non-responding patients compared to healthy controls (p = 0.023).Accordingly, in addition to their beneficial function, HMAs induce PD-1 expression on T cells in the MDS microenvironment, thereby likely hampering the immune response against the MDS blasts. Thus, we suggest that activation of the PD-1 checkpoint during HMA treatment can be a possible resistance mechanism, which may be overcome by combination therapy with a PD-1 pathway inhibitor.

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