Clinical Response to Azacitidine in Myelodysplastic Neoplasms Is Associated with Distinct DNA Methylation Changes in Haematopoietic Stem and Progenitor Cells <i>In Vivo</i>

Hypomethylating agents are used as frontline therapy for myelodysplastic neoplasms (MDS), but clinical response is unpredictable. In particular, the association between HMA incorporation and DNA hypomethylation, and the clinical efficacy of these drugs is unclear. To address these relationships, we prospectively collected bone marrow (BM) and peripheral blood (PB) as part of a phase II clinical trial (NCT03493646) designed to evaluate in vivo AZA incorporation in mononuclear cells (MNCs) throughout the course of Vidaza (injection AZA; 6 cycles) or CC-486 (oral AZA; 6 cycles) therapy. The primary objective of the trial was to assess longitudinal drug incorporation into DNA and global changes in DNA methylation using an LC-MS/MS based method, while secondary objectives included observation of cell cycle changes in haematopoietic stem and progenitor cells (HSPCs), tracking of clonal variants, and assessment of any relationship between these measures and clinical response. Forty patients commenced treatment and clinical response was assessed following cycle (C) 6 and C12. Of the 24 who completed 6 cycles of injection AZA, there were 16 responders and 8 non-responders. 11/22 patients who commenced oral AZA reached the second response assessment point comprising 7 responders and 4 non-responders. DNA incorporation of AZA derivatives is cell cycle dependent. The fraction of HSPCs progressing through the cell cycle was correlated with the amount of drug incorporated in DNA but not with the degree of global DNA demethylation. Although baseline cell cycle state in HSPCs did not predict patient response, a significantly greater proportion of cells exited quiescence (C7 vs C1) during treatment and continued to cycle at C12 in AZA responders versus non-responders underlining the continued need for S phase dependent drug incorporation for response. Global DNA demethylation in PB and BM MNCs was not proportionate to in vivo DAC incorporation in these cells and was not a reliable measure of clinical response although all patients showed some degree of demethylation. By contrast, HSPCs in responders had lower baseline CpG methylation compared to non-responders and hypomethylated CpGs mapped to pathways involved in tissue patterning and cell migration. Baseline differences in CpG methylation were not accompanied by substantial differences in gene expression, a feature consistent with overlapping clinical phenotypes at presentation. CpG hypomethylation in MDS HSPCs during the first AZA cycle was skewed to non-coding regions, and in responders, mapped to pathways involved in activating myeloid programs, a feature that may have facilitated early myeloid differentiation of these cells. Differentially expressed genes (DEGs) at this time point were dominated by cell cycle and DNA repair pathways in both responders and non-responders, although the degree of enrichment was greater in non-responders suggesting enhanced early drug-induced toxicity in this group. Following multiple cycles of AZA when response was clinically apparent and HSPC populations have likely changed from those at baseline, only a small set of CpGs were hypomethylated, with top enriched pathways including cell-cell adhesion and positive regulation of hemopoiesis and leukocyte differentiation that pointed to altered relationships with other cells in the bone marrow microenvironment. DEGs at this timepoint mapped to inflammatory pathways, which were more highly expressed in responders. Although differences in CpG methylation or gene expression were minimal following a cycle of low-dose oral AZA in patients who had been pre-exposed to 6 cycles of injection AZA, families of transposable elements were re-expressed in responder HSPCs suggesting a possible role for these elements in the inflammatory response. In alignment with previous reports (Unnikrishnan et al Cell Reports 2017, Schnegg-Kaufmann et al Blood 2023), most patients in this cohort also had stable clonal composition over treatment phases, regardless of response status. Changes in variant allele frequencies (VAF) had no relationship to drug incorporation or global DNA demethylation. Overall, this study revealed that clinical response to AZA was related less to VAFs or how much drug incorporation or global demethylation occurred during treatment but more to where specifically demethylation occurred and in what cell types these changes occurred.