Hypomethylating agents are used as frontline therapy for myelodysplastic neoplasms (MDS), but clinical response is unpredictable. To determine whether response was associated with in vivo dynamics of DNA hypomethylation, we conducted a phase 2 trial for MDS using both injection and oral azacitidine (AZA). We established that global DNA methylation levels in peripheral blood and bone marrow mononuclear cells were comparable in AZA responders and non-responders during their course of treatment. However, there were distinct baseline and early drug induced differences in CpG methylation in haematopoietic stem and progenitor cells (HSPCs) in responders compared to non-responders that overlapped with regulatory regions of genes associated with tissue patterning, cell migration and myeloid differentiation. Following six cycles of therapy when clinical response typically manifests, differential hypomethylation in responder HSPCs pointed to marrow adaptation as a driver of enhanced haematopoiesis. Taken together, CpG methylation differences in HSPCs may explain variable response to AZA.

F.V. is affiliated with OmniOmics.AI Pty Ltd. C.F. is an advisory board member at Amgen, AbbVie, Adaptive Biotech, BeiGene, Pfizer, Otsuka, and Jazz, a consultant at Novotech, and received speaker fees from Amgen, Pfizer, Servier, BMS, and Astella. D.H. has consultancy agreements with GlaxoSmithKline and Pharming Corp. M.H. is a consultant/advisory board member at Roche, Gilead, Otsuka, Janssen, Beigene, and Takeda. M.N.P. received research funding and/or provision of drug for clinical trials (to institution) from AstraZeneca, BRII Biosciences, Celgene/BMS, CSL Behring, Eli Lilly, Emergent Biosciences, Gilead Pharmaceuticals, GlaxoSmithKline, Grifols, Janssen/Johnson and Johnson, Takeda, ViiV Pharmaceuticals and has advisory roles with Celgene/BMS, Gilead Pharmaceuticals, and ViiV Pharmaceuticals. J.E.P. received research funding and/or provision of drug for clinical trials (to institution) from Celgene/BMS, Astex, Verastem Oncology and received honoraria from Abbvie as an advisory board member. The remaining authors declare no competing financial interests.

NCT03493646

The investigator initiated clinical trial was funded in part by Celgene/BMS (RG172029) with research support from the National Health and Medical Research Council (RG170246, RG211412), Anthony Rothe Memorial Trust (RG182042, RG202657, RG213236), Leukaemia Foundation (RG231257).

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The trial protocol received ethical approval from the South Eastern Sydney Local Health District Human Research Ethics Committee, and participating sites received Institution approval to conduct the trial prior to commencing recruitment.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

All data produced in the present study are available upon reasonable request to the authors