Background: The combination therapy of hypomethylating agents and venetoclax (HMA+VEN) has emerged as a standard of care for acute myeloid leukemia (AML) patients unfit for intensive chemotherapy. Nonetheless, significant clinical challenges persist, including relapse and treatment resistance. Previous studies have revealed genetic ( RAS/RTK pathway mutations and TP53 mutations) and phenotypic (monocytic differentiation) resistance mechanisms in the context of HMA+VEN therapy. Despite these advancements, our comprehensive understanding of the HMA+VEN resistance mechanisms remains incomplete. To gain further insights into the resistance mechanism in HMA+VEN therapy, we implemented single-cell proteogenomics analysis on longitudinally collected samples from AML patients treated with decitabine+venetoclax (DAC+VEN) trial.

Methods: We performed a high-throughput, single-cell targeted DNA-antibody sequencing (DAb-seq) using Tapestri platform from Mission Bio Inc. Sixty-three AML samples sequentially collected from 33 unique patients who participated in DAC+VEN clinical trial (NCT03404193) were analyzed. Our custom DNA panel covered 37 recurrently mutated genes in AML, whereas the custom antibody panel included either 15 cell surface proteins (N = 20 patients) or 48 proteins (N = 13 patients). Cellular phenotypes were inferred based on the patterns of cell surface protein expression. Median 2,767 cells were sequenced per sample, with total 196,719 cells sequenced for this study.

Results: The median age of the cohort was 70 years (range 21-84). Twenty-four (73%) patients had primary AML and 9 (27%) had secondary or therapy-related AML. Based on the FAB classification, 13 patients had M0/M1/M2, 6 had M4/M5, one had M7, and the rest had unknown subtypes. The most frequent gene mutation at baseline was DNMT3A (45%), followed by NPM1 (39%), TET2 (30%), and TP53 (24%). Among the 33 patients, 32 had evaluable treatment response; 27 (82%) were defined as responders by achieving CR or CRi, whereas 5 (15%) were non-responders. Six patients experienced relapse after responding to the therapy, and their baseline-relapse pairs were analyzed by the single-cell platform. Using the composition of cellular phenotype data, AML samples were classified into either stem-like (N = 5), progenitor-like (N = 1), predominantly monocytic (N = 4), predominantly erythroid (N = 2) and mixed population AML (mixed population of stem, progenitor, monocyte or erythroid-like population, N = 21). Genotype-phenotype analysis showed a significant correlation between the KRAS mutation and the mixed phenotype (p= 0.0403), as well as between the U2AF1 mutation and the mixed phenotype (p= 0.0471). There was no significant association between treatment response and AML phenotype. Analysis of six baseline-relapse pairs showed that relapse was associated with phenotypic shift of AML cells. Stem-like cells were largely eradicated at relapse and replaced by more differentiated cells. Among the six baseline-relapse pairs, three showed monocytic shift, one showed erythroid shift and two showed mixed monocytic/erythroid shift. Phenotypic shifts at relapse were not always associated with expansion of new genetic mutations, except for in two cases: one had monocytic shift accompanied with expansion of KRAS mutation, and another had erythroid shift accompanied with expansion of NRAS mutation.

Conclusions: Phenotypic transition from stem-like cells to more differentiated cells (either monocytic or erythroid) was frequently observed in AML relapse following DAC+VEN therapy. These findings are consistent with the known dependency of these cells on non-BCL-2 anti-apoptotic proteins such as MCL-1 and BCL-xL. Close monitoring of these phenotypic alterations throughout the course of HMA+VEN treatment may potentially facilitate the precise identification of cellular populations predisposed to relapse.

Maiti:Celgene: Research Funding; Lin BioScience: Research Funding. Daver:Celgene: Consultancy; Astellas: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Trillium: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; Servier: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Agios: Consultancy; Novimmune: Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Hanmi: Research Funding; FATE: Research Funding; Trovagene: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Novartis: Consultancy; AROG: Consultancy; Jazz: Consultancy; Syndax: Consultancy; Shattuck Labs: Consultancy; Glycomimetics: Research Funding; Kite, a Gilead company: Consultancy, Research Funding; Kronos Bio: Research Funding. Pemmaraju:ClearView Healthcare Partners: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; ASCO Cancer.Net Editorial Board: Other: Leadership; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Karger Publishers: Other: Licenses; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; United States Department of Defense (DOD): Research Funding; Magdalen Medical Publishing: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Medscape: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CareDx: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CancerNet: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Curio Science: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Intellisphere: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; EUSA Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Neopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; PeerView Institute for Medical Education: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Cimeio Therapeutics AG: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Physician Education Resource (PER): Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; OncLive: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Patient Power: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Menarini Group: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Harborside Press: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Imedex: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; ASH Committee on Communications: Other: Leadership; Dan's House of Hope: Membership on an entity's Board of Directors or advisory committees; Aplastic Anemia & MDS International Foundation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Dava Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; PharmaEssentia: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Protagonist Therapeutics, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Stemline: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pacylex: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; ImmunoGen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CTI BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Blueprint: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Aptitude Health: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; National Institute of Health/National Cancer Institute (NIH/NCI): Research Funding; HemOnc Times/Oncology Times: Other: Uncompensated; Bristol Myers Squibb Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Kadia:Pulmotect, Inc.: Consultancy, Research Funding; Iterion: Research Funding; Genentech: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Sanofi-Aventis: Consultancy; Glycomimetics: Research Funding; Pinotb-Bio: Consultancy; Liberum: Consultancy; Regeneron Pharmaceuticals: Research Funding; Novartis: Consultancy; Astellas Pharma Global Development: Research Funding; Jazz Pharmaceuticals, Pfizer, Pulmotect, Inc, Regeneron Pharmaceuticals, SELLAS Life Sciences Group: Research Funding; GenFleet Therapeutics: Research Funding; Delta-Fly Pharma, Inc.: Research Funding; Cyclacel: Research Funding; AstraZeneca: Research Funding; Janssen Research and Development: Research Funding; Ascentage Pharma Group: Research Funding; Amgen, Inc.: Research Funding; Daiichi Sankyo, Genentech, Inc., Genzyme, Jazz Pharmaceuticals, Liberum, Novartis, Pfizer, PinotBio, Inc, Pulmotect, Inc, Sanofi-Aventis, Servier: Consultancy; Agios: Consultancy; Servier: Consultancy; Hikma Pharmaceuticals: Speakers Bureau; AbbVie, Amgen, Inc, Ascentage Pharma Group, Astellas Pharma Global Development, Astex, AstraZeneca, BMS, Celgene, Cellenkos Inc, Cyclacel, Delta-Fly Pharma, Inc, Genentech, Inc., Genfleet, Glycomimetics, Iterion, Janssen Research and Development: Research Funding; Cellenkos Inc.: Research Funding; Biologix, Cure, Hikma Pharmaceuticals: Speakers Bureau; Genzyme: Honoraria; Cure: Speakers Bureau; Celgene: Research Funding; BMS: Consultancy, Research Funding; Astex: Honoraria; SELLAS Life Sciences Group: Research Funding. Ravandi:Astex/taiho: Membership on an entity's Board of Directors or advisory committees, Research Funding; Xencor: Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Prelude: Research Funding; Amgen: Honoraria, Research Funding; Celgene/BMS: Consultancy, Honoraria, Research Funding; Biomea fusion: Honoraria, Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Syros: Consultancy, Honoraria, Research Funding. Short:Stemline therapeutics: Research Funding; Astellas: Research Funding; Novartis: Consultancy; AstraZeneca: Consultancy; Takeda: Consultancy, Research Funding; Amgen: Honoraria; Pfizer: Consultancy. Borthakur:Catamaran Bio, Abbvie, PPD Development, Protagonist Therapeutics, Janssen: Consultancy; Astex Pharmaceuticals, Ryvu, PTC Therapeutics: Research Funding; Pacylex, Novartis, Cytomx, Bio Ascend:: Membership on an entity's Board of Directors or advisory committees. Jain:Aprea Therapeutics: Research Funding; Adaptive Biotechnologies: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; ADC Therapeutics: Research Funding; Ipsen: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Takeda: Research Funding; Cellectis: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; BMS: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; TransThera Sciences: Research Funding; MEI Pharma: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Fate Therapeutics: Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Genentech: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Mingsight: Research Funding; AstraZeneca: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Novalgen: Research Funding; Incyte: Research Funding; Servier: Research Funding; Pfizer: Research Funding; Loxo Oncology: Research Funding; AbbVie: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; TG Therapeutics: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses; Dialectic Therapeutics: Research Funding; Kite/Gilead: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Precision Biosciences: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Newave: Research Funding; Beigene: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses; Medisix: Research Funding; CareDX: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses; Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses. Issa:Celgene: Research Funding; Merck: Research Funding; Syndax: Research Funding; Novartis: Consultancy, Research Funding; NuProbe: Consultancy; Kura Oncology: Consultancy, Research Funding. Masarova:MorphoSys US: Membership on an entity's Board of Directors or advisory committees. Wierda:Miragen: Research Funding; KITE Pharma: Research Funding; AbbVie: Consultancy, Research Funding; AstraZeneca/Acerta Pharma: Consultancy, Research Funding; GlaxoSmithKline: Research Funding; Loxo Oncology, Inc./Lilly: Research Funding; GSK/Novartis: Research Funding; Gilead Sciences: Research Funding; Accutar Biotechnology: Research Funding; Genentech: Research Funding; Pharmacyclics LLC: Research Funding; Janssens Biotech Inc: Research Funding; Janssens Biotech: Research Funding; Nurix THerapeutics: Research Funding; Juno Therapeutics: Research Funding; Cyclacel: Consultancy, Research Funding; Numab THerapeutics: Research Funding; Oncternal Therapeutics, Inc.: Research Funding; Bristol Myers Squibb (Juno & Celgene): Consultancy, Research Funding; NIH P30 CA016672/MDACC Cancer Center Support Grant: Research Funding; Sunesis: Research Funding; National Comprehensive Cancer Network: Other: Nonrelevant Financial Relationship/Chair, CLL). Supported by the NIH/NCI under award number P30 CA016672 and used MDACC Cancer Center Support Grant (CCSG) shared resources. Loghavi:QualWorld: Consultancy; Abbvie: Consultancy; Caris Diagnostics: Consultancy; Blueprint Medicine: Consultancy; Abbvie: Current equity holder in publicly-traded company; Astellas: Research Funding; Amgen: Research Funding; Recordati/ EUSA Pharma: Consultancy; Daiichi Sankyo: Consultancy; Guidepoint: Consultancy; Gerson Lehrman Group: Consultancy. Garcia-Manero:Bristol Myers Squibb: Other: Medical writing support, Research Funding; Genentech: Research Funding; AbbVie: Research Funding. Kantarjian:Ipsen: Honoraria; KAHR Medical: Honoraria; Daiichih-Sankyo (Inst): Honoraria, Research Funding; AstraZeneca/MedImmune: Honoraria; Astellas Pharma: Honoraria; Novartis (Inst): Research Funding; Taiho Pharmaceutical: Honoraria; Ascentage Pharma (Inst): Research Funding; Jazz Pharmaceuticals (Inst): Honoraria, Research Funding; Precision Biosciences: Honoraria; Immunogen (Inst): Honoraria, Research Funding; Pfizer: Honoraria; Abbvie (Inst): Research Funding; Novartis: Honoraria; Shenzhen Target Rx: Honoraria; Amgen (Inst): Research Funding; Ascentage Pharma Group: Honoraria; Abbvie: Consultancy, Honoraria; Amgen: Honoraria; Bristol-Myers Squibb (Inst): Research Funding. DiNardo:Astellas: Honoraria; AbbVie/Genentech: Honoraria; Takeda: Honoraria; Servier: Honoraria; BMS: Honoraria; Fogham: Honoraria; ImmuniOnc: Honoraria; Schrödinger: Consultancy; Notable Labs: Honoraria; Novartis: Honoraria. Konopleva:Abbvie, Allogene Therapeutics, Cellectis, Forty Seven, Gilead Sciences, Genentech, Sanofi, MEI Pharma, Rafael Pharmaceuticals, Daiichi Sankyo Pharmaceutical, AstraZeneca Co., Menarini, Precision BioSciences.: Research Funding; Reata Pharmaceuticals.: Current holder of stock options in a privately-held company, Patents & Royalties; AbbVie, Forty Seven, Precision Biosciences, Gilead Sciences, Genentech, Janssen, Sanofi, MEI Pharma, Daiichi Sankyo Pharmaceutical, AstraZeneca Co., Menarini.: Consultancy.

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2023
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