Vitamin A Directly Enhances TET2 Activity and Suppresses Clonal Hematopoiesis

Introduction: TET2 haploinsufficiency is a driving event in up to 30% of individuals with clonal hematopoiesis (CH), a pre-malignant blood condition that can increase the risk of developing a hematological malignancy. Loss of TET2 function imparts aberrant self-renewal to hematopoietic stem and progenitor cells (HSPCs) and a pro-inflammatory myeloid lineage expansion. Studies have shown that restoring Tet2 activity using genetic models, or treatment with the TET catalytic cofactor ascorbate, can reverse the clonal advantage and myeloid bias of Tet2-deficient pre-malignant HSPCs, and slow the development of myeloid leukemia. Preliminary clinical studies have shown that increased supplementation with ascorbate can improve survival in patients with clonal cytopenia or low risk myeloid malignancy. These data suggest that enhancing TET2 activity represents a viable therapeutic strategy for the prevention of CH progression. To identify additional pharmacological interventions that could enhance and restore TET2 activity to suppress CH progression, we tested the efficacy of retinoic acid as a TET2 activator, given it had previously been shown in ESCs to upregulate Tet2 transcription and 5hmC formation, and is a known positive regulator of hematopoietic stem cell quiescence and myeloid differentiation.

Results: We show that all-trans retinoic acid (ATRA) induces TET2 transcription in myeloid leukemia cells and primary murine and human HSPCs. In combination with ascorbate, ATRA synergistically enhances TET2-mediated DNA hydroxymethylation when assayed by base-resolution 5hmC-sequencing in myeloid leukemia cells, with enrichment at enhancers and CEBP, ETS and FOS/JUN transcription factor binding motifs. RNA- and ATAC-sequencing showed that genes with increased 5hmC levels upon ATRA treatment alone or in combination with ascorbate overlap with gene expression changes and altered chromatin accessibility linked to terminal myeloid differentiation and reduced inflammatory signaling. Furthermore, using Tet2^+/+, Tet2^+/- and Tet2^-/- murine models, we show that ATRA treatment can block aberrant HSPC self-renewal in colony formation assays in a Tet2 allelic dose-dependent manner, and suppress the clonal advantage of Tet2 haploinsufficient cells in competitive bone marrow reconstitution assays when combined with ascorbate. Moreover, we find that increased dietary supplementation with vitamin A alone (in the form of retinyl palmitate), can reduce the levels of circulating inflammatory cytokines and suppress the myeloid expansion of Tet2-deficient HSPCs in an aging cohort of primary mice (treated for 12 months), and block their competitive advantage in bone marrow reconstitution assays (treated for 6 months post-transplant). Tet2-deficient hematopoiesis is also known to cause gut dysbiosis and bacteremia that triggers chronic inflammation in a positive feedback loop. Shotgun sequencing of the fecal microbiome revealed that increased vitamin A dietary supplementation promotes a healthy anti-inflammatory gut microbiome compared to normal or low vitamin A supplementation.

Conclusion: Our data implicate vitamin A and retinoic acid signaling as a regulator of Tet2 activity in HSPCs that could be exploited to suppresses the clonal expansion of Tet2 haploinsufficient cells to reduce the risk of developing a hematological malignancy in individuals with TET2 mutant CH.