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3223 Clinical Response to Azacitidine in Myelodysplastic Neoplasms Is Associated with Distinct DNA Methylation Changes in Haematopoietic Stem and Progenitor Cells In Vivo

Program: Oral and Poster Abstracts
Session: 637. Myelodysplastic Syndromes: Clinical and Epidemiological: Poster II
Hematology Disease Topics & Pathways:
Research, Clinical trials, Acquired Marrow Failure Syndromes, Translational Research, Bone Marrow Failure Syndromes, Clinical Research, Diseases, Myeloid Malignancies
Sunday, December 8, 2024, 6:00 PM-8:00 PM

Julie A.I. Thoms, PhD1*, Feng Yan, PhD2*, Henry R. Hampton, PhD1*, Sarah Davidson, RN3*, Swapna Joshi, MSc4*, Jesslyn Saw, BSc5*, Chowdhury H. Sarowar, PhD4*, Xin Ying Lim, PhD4*, Andrea C Nunez, PhD4*, Purvi M. Kakadia, PhD6*, Golam Sarower Bhuyan, PhD4*, Xiaoheng Zou, MSc1*, Mary Nguyen, BSc1*, Elaheh S Ghodousi, MSc1*, Forrest C Koch, BSc7*, Fatemeh Vafaee, PhD7*, I. Richard Thompson8*, Mohammad M. Karimi, PhD8*, Russell Pickford, PhD9*, Mark J Raftery, PhD9*, Sally Hough10*, Griselda Buckland3*, Michelle Bailey10*, Yuvaraj Ghodke3*, Noorul Absar10*, Lachlin Vaughan, MBBS, PhD11*, Leonardo Pasalic, MBBS, PhD12*, Chun Yew Fong, MBBS, FRACP, FRCPA, PhD13*, Melita Kenealy14*, Devendra Hiwase, MD, MBBS, PhD, FRACP, FRCPA15, Rohanna Stoddart, MD16*, Soma Mohammed17*, Linda Lee18*, Freda H Passam, MD, PhD19, Stephen Robert Larsen, MBBS PhD FRACP FRCPA20, Kevin J Spring21*, Kristen K Skarratt22*, Patricia Rebeiro23*, Peter Presgrave, MBBS24, William S Stevenson, MBBS, FRACP, FRCPA, PhD25*, Silvia Ling26*, Campbell Tiley27*, Stephen Fuller22*, Fernando Roncolato28*, Anoop K Enjeti, MBBS, FRCPA, MD, MRCP29, Dirk Hoenemann, MD30*, Charlotte Lemech31*, Christopher J Jolly, PhD1*, Stefan K. Bohlander, MD6*, David J. Curtis, MBBS, PhD5, Jason Wong, PhD32*, Ashwin Unnikrishnan, PhD4, Mark S. Hertzberg, MD33, Jake Olivier, PhD34*, Mark N Polizzotto3* and John E Pimanda, MBBS, PhD, FRACP, FRCPA35

1School of Biomedical Sciences, University of New South Wales, Randwick, Australia
2Bioinformatics Division, Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
3ANU Clinical Hub for Interventional Research (CHOIR), John Curtin School of Medical Research, Canberra, Australia
4School of Clinical Medicine, University of New South Wales, Randwick, Australia
5Australian Centre for Blood Diseases, Monash University, Melbourne, VIC, Australia
6Leukaemia and Blood Cancer Research Unit, Department of Molecular Medicine and Pathology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand
7School of Biotechnology and Biomolecular Sciences, University of New South Wales, Randwick, Australia
8Comprehensive Cancer Centre, School of Cancer and Pharmaceutical Sciences, King’s College London, London, United Kingdom
9Bioanalytical Mass Spectrometry Facility, University of New South Wales, Randwick, Australia
10Kirby Institute, University of New South Wales, Randwick, Australia
11Westmead Hospital, Sydney, Australia
12Westmead Hospital, Westmead, Australia
13Department of Hematology, Austin Health, Heidelberg, VIC, Australia
14Cabrini Hospital, Melbourne, Australia
15Royal Adelaide Hospital, Adelaide, SA, Australia
16University of New South Wales, Randwick, Australia
17ICPMR, Department of Haematology, Westmead Hospital, Westmead, Australia
18Royal North Shore Hospital, Sydney, Australia
19Haematology Research Group, Heart Research Institute, Sydney, NSW, Australia
20Royal Prince Alfred Hospital, Camperdown, Australia
21Medical Oncology Group, Liverpool Clinical School, Western Sydney University, Liverpool, Australia
22Sydney Medical School, Nepean Clinical School, University of Sydney, Kingswood, Australia
23Blacktown Hospital, Blacktown, Australia
24Wollongong Hospital, Wollongong, Australia
25Royal North Shore Hospital, Sydney, AUS
26Liverpool Hospital, Liverpool, Australia
27Central Coast Health, Gosford Hospital, Gosford, Australia
28St George Hospital, Kogarah, Australia
29Department of Haematology, Calvary Mater Hospital, Waratah, NSW, Australia
30Otway Pharmaceutical Development and Consulting Pty Ltd, Forrest, Australia
31Scientia Clinical Research Limited, Sydney, Australia
32School of Biomedical Sciences, The University of Hong Kong, Hong Kong, SAR, China
33Prince of Wales Hospital and University of New South Wales, Sydney, Australia
34School of Mathematics and Statistics, University of New South Wales, Randwick, AUS
35School of Biomedical Sciences, University of New South Wales, Sydney, NSW, Australia

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.

Disclosures: Vafaee: OmniOmics.AI Pty Ltd: Consultancy, Current equity holder in private company. Fong: Servier: Speakers Bureau; Novotech: Consultancy; Jazz: Membership on an entity's Board of Directors or advisory committees; Otsuka: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BeiGene: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotech: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; BMS: Speakers Bureau; Astella: Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hiwase: Abbvie: Honoraria; Astella Pharma: Honoraria; Otsuka: Honoraria. Enjeti: Jazz: Honoraria; Servier: Honoraria; RACE Oncology: Consultancy, Honoraria; Otsuka: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astellas: Honoraria, Speakers Bureau; Amgen: Honoraria; AbbVie: Speakers Bureau; Amgen: Honoraria. Hoenemann: GlaxoSmithKline: Consultancy; Pharming Corp: Consultancy. Lemech: Sanofi: Consultancy; Amgen: Other: TRAVEL, ACCOMODATIONS, EXPENSES. Hertzberg: Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Otsuka: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Beigene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Yakeda: Consultancy, Membership on an entity's Board of Directors or advisory committees. Polizzotto: Celgene/BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; ViiV pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Research Funding; BRII Biosciences: Research Funding; CSL Behring: Research Funding; Eli Lilly: Research Funding; Emergent Biosciences: Research Funding; GlaxoSmithKline: Research Funding; Grifols: Research Funding; Janssen/Johnson and Johnson: Research Funding; Takeda: Research Funding. Pimanda: Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene/BMS: Research Funding; Astex: Research Funding; Verastem Oncology: Research Funding.

*signifies non-member of ASH