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4380 ASXL1 Mutations Are Associated with a Response to the Combination of Alvocidib and 5-Azacytidine in Higher-Risk Myelodysplastic Syndromes

Program: Oral and Poster Abstracts
Session: 636. Myelodysplastic Syndromes—Basic and Translational: Poster III
Hematology Disease Topics & Pathways:
Research, MDS, Translational Research, Chronic Myeloid Malignancies, Diseases, Myeloid Malignancies
Monday, December 12, 2022, 6:00 PM-8:00 PM

Vladimir Riabov, PhD1*, Qingyu Xu1*, Nanni Schmitt, MSc1*, Alexander Streuer, MD1*, Guo Ge2*, Johann-Christoph Jann, MD1*, Alina Wein1*, Eva Altrock, PhD1*, Felicitas Rapp, PhD1*, Verena Nowak1*, Nadine Weimer1*, Julia Obländer1*, Iris Palme1*, Melda Göl1*, Mark Wunderlich, MS3*, Ahmed Jawhar, MD4*, Ali Darwich, MD4*, Patrick Wuchter, MD5, Christel Weiss, PhD6*, Jason M Foulks7*, Daniel T. Starczynowski, PhD3, Feng-Chun Yang, MD, PhD2, Georgia Metzgeroth, MD1*, Laurenz Steiner, MD1*, Wolf-Karsten Hofmann, MD1, Daniel Nowak, MD1* and Mohamad Jawhar, MD1*

1Department of Hematology and Oncology, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
2Department of Cell Systems & Anatomy, University of Texas Health Science Center at San Antonio, San Antonio
3Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH
4Department of Orthopedic Surgery, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
5Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
6Department of Medical Statistics and Biomathematics, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
7Sumitomo Pharma Oncology, Inc, Lehi

Introduction: Inhibitors of anti-apoptotic BCL-2 family proteins in combination with chemotherapy and hypomethylating agents (HMAs) are perspective therapeutic approaches in acute myeloid leukemia (AML) and higher-risk myelodysplastic syndromes (MDS). Inhibitors of BCL-2 family proteins previously showed potent activity against AML and higher-risk MDS. Alvocidib (Alv), a cyclin-dependent kinase 9 inhibitor and indirect transcriptional repressor of the anti-apoptotic BCL-2 family member MCL-1, has previously shown clinical activity in AML (Lee DJ et al, Expert Opin Investig Drugs 2019; Zeidner JF et al, Leuk Res 2015). A phase 1b/2 study with Alv and HMAs 5-Azacytidine (5-AZA) or decitabine in higher-risk MDS patients was recently completed (NCT03593915). However, biomarkers of response to 5-AZA + Alv combination are insufficiently characterized. In order to possibly identify biomarkers of response, we performed a comprehensive in vitro assessment of Alv and 5-AZA effects in a cohort of n=45 higher-risk MDS patients.

Methods: CD34+ hematopoietic stem and progenitor cells (HSPCs) were isolated from bone marrow (BM) aspirates using positive selection with MACS microbeads. CD34+ cells of aged-matched healthy controls (HC, n=11) were obtained from bone specimen after femur endoprosthesis surgery. HSPCs were expanded for four days in StemSpan SFEM II medium containing StemSpan Myeloid Expansion Supplement (Stem Cell Technologies) and treated with 5-AZA for 48h, Alv for 24h or their sequential combination (5-AZA for 48h followed by Alv for 24h). Cell viability was determined using CellTiter-Glo (CTG) and Annexin-V apoptosis assays. MCL-1 dependency of primary MDS HSPCs was assessed using NOXA-derived T-MS1 peptide. MDS recurrent mutations in BM mononuclear cells were assessed using myeloid NGS panel deep sequencing containing 67 genes. For in vivo tumor transfer experiments, splenic cells from CD45.2+ Asxl1Y588X transgenic (Tg) mice were injected intravenously into sublethally irradiated CD45.1+ recipients through tail veins. Engrafted mice were treated with 1 mg/kg 5-AZA intraperitoneally (i.p.) on days 1-3 (qdx) every other week. Alv was administered i.p. at a dose of 2.5 mg/kg on day 5 (qw) in the weeks when 5-AZA was applied. Mice were sacrificed 11 weeks after the transplantation and the percentage of CD45.2+ cells in the BM was assessed using flow cytometric analysis.

Results: The combination of 5-AZA+Alv showed an additive cytotoxic effect on MDS cells in CTG cell viability assays (median cell viability was 74%, 74% and 55% for 5-AZA, Alv and combination, respectively, p<0.0001). In annexin-V apoptotic assays, MDS cells were more sensitive to the cytotoxic effect of the combination treatment compared to HC (median percent of apoptotic and dead cells was 37% for MDS vs 26% for HC, p=0.0256). In analogy to in vitro studies, accelerated clearance of xenografted MDS-L cells from the peripheral blood of NSGS mice was observed after 5-AZA+Alv treatment compared to single agents. MCL-1 dependency of primary MDS samples did not significantly correlate with cytotoxic effects of Alv and combination treatment. However, the presence of ASXL1 mutations was significantly associated with higher cytotoxic activity of 5-AZA+Alv combination in univariable (p=0.0044) and multivariable (p=0.042) analyses. Increased sensitivity of BM cells to the cytotoxic effects of 5-AZA+Alv was also observed in Asxl1Y588XTg mice. The combination treatment facilitated clearance of CD45.2+ Asxl1Y588X mutant leukemic cells from CD45.1+ recipients compared to 5-AZA monotherapy (p=0.0156). Histological analysis of BM cells after 5-AZA+Alv treatment in CD45.1+ recipients showed decrease in the percentage of immature blasts and improved development of mature BM cells when compared to 5-AZA monotherapy (p=0.0391). Improved cytotoxic effects of Alv and 5-AZA+Alv in ASXL1 mutant compared to ASXL1 wild type MDS samples were associated with higher expression of pro-apoptotic MCL-1 antagonist NOXA (p=0.0017).

Conclusions: We provided a pre-clinical rationale for the use of the 5-AZA+Alv combination for higher risks MDS and proposed ASXL1 mutations as potential genetic biomarkers of response in prospective clinical trials.

Disclosures: Foulks: Sumitomo Pharma Oncology, Inc: Current Employment. Starczynowski: Kymera Therapeutics: Consultancy; Treeline Biosciences: Research Funding; Sumitomo Pharma Oncology, Inc: Research Funding; Captor Therapeutics: Consultancy; Kurome Therapeutics: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Tolero Therapeutics: Research Funding. Yang: Sumitomo Pharma Oncology, Inc: Research Funding. Nowak: AbbVie: Other: Investigator funded clinical trial; Apogenix: Research Funding; Celgene: Honoraria; Takeda: Honoraria; Sumitomo Dainippon Pharma Co. Ltd: Research Funding; Affimed GmbH: Research Funding; Pharmaxis Ltd.: Current equity holder in private company, Research Funding.

*signifies non-member of ASH