Multi- Omics Reveals Azacitidine Partially Restores Hematopoiesis-Supportive Functions in Mesenchymal Stromal Cells from Patients with Chronic Myelomonocytic Leukemia

Abstract Hypomethylating agents (HMAs) including azacitidine (AZA) represent the only FDA-approved first-line treatments for patients with chronic myelomonocytic leukemia (CMML). However, the mechanism by which HMAs produce therapeutic responses (e.g., hematological improvement) remains unclear. Bone marrow mesenchymal stromal cells (MSCs) play a crucial role in regulating the self-renewal, survival and differentiation of hematopoietic stem and progenitor cells (HSPCs). Recently, the identified sensitivity of patient-derived MSCs to HMAs underlines a critical yet unexplored role of MSCs in regulating post-HMA efficacies. By utilizing high-throughput approaches including targeted exon sequencing, DNA methylation profiling, and RNA sequencing, our present study aims to delineate the modifications and consequences of AZA on CMML-MSCs. Demonstrated by integrated multi-omics analysis, our results reveal that cytogenetically independent CMML-MSCs exhibit strikingly high amenability to AZA. Through selectively de-methylating/methylating CpGs of 1395 sensitive genes, AZA re-activates HSPC-supportive signatures and enhances the protective effect of CMML-MSCs on healthy HSPCs. Along with the reconstitution of the dysregulated methylome/transcriptome, AZA partially restored the impaired functions of CMML-MSCs to support healthy hematopoiesis in long-term co-culture conditions. Our findings suggested a niche-dependent mechanism of post-HMA recovery of normal hematopoiesis. Identifying AZA-sensitive genes and functions may be of particular value in developing niche-targeting strategies in treating myeloid malignancies.