Abstract 444: ASXL1 mutations in AML are associated with a distinct epigenetic state that results in vulnerabilities to epigenetic-targeted agents
Authors: Christopher P. Mill, Warren C. Fiskus, Christine E. Birdwell, John A. Davis, Kaberia Das, Hanxi Hou, Surbhi Sharma, Koji Sasaki, Sanam Loghavi, Tapan M. Kadia, Naval Daver, Courtney D. DiNardo, Lu Wang, Kapil N. Bhalla
Journal: Cancer Research (2025)

Abstract ASXL1 is frequently mutated in myeloid malignancies, often causing a stabilization of ASXL1 and the polycomb-repressive deubiquitinase (PR-DUB) complex, that results in a global reduction in H2AK119Ub and increased mRNA transcription. Poorer prognosis of AML with mutant (mt) ASXL1 creates an unmet need to identify and test novel therapies for this AML sub-type. We generated two knock-in (KI) models of the clinically relevant mtASXL1 Y591* into the AML cell lines OCI-AML3 and UCSD-AML1. Notably, ASXL1/BAP1 stabilization caused an increased expression of c-Myc at both the RNA and protein level. We observed via ChIP-Seq reduced H2AK119Ub at H3K27Ac-marked chromatin on Wnt/β-catenin stem cell pathway genes that resulted in increased mRNA expression of FZD5, TCF7L2, and WNT5A. KI of mtASXL1 in OCI-AML3 cells also increased G0/G1 with a concomitant reduction in S and G2/M phases of the cell-cycle. RNA-Seq analysis showed reduced expression of the REACTOME cell-cycle mitotic gene-set, associated with depletion of E2F1, AURKA/B, and PLK1 mRNA. A CRISPR screen in parental and KI cells revealed SPI1, CDK9, and EP300 among the top dependencies specific to mtASXL1 AML. Notably, compared to parental cells, OCIAML3 ASXL1 Y591* cells exhibited reduced sensitivity to standard anti-AML, chemotherapeutic agents, including cytarabine, etoposide and daunorubicin. Our findings confirm the previously published discovery that the presence of mtASXL1 confers an increased sensitivity to BETi inhibitors, e.g., pelabresib or birabresib. These cells were also more sensitive to other epigenetically targeted agents, including HATi (GNE-781 or CBP-IN-1), CDK8/19 mediator kinase inhibitor (SEL120-34A/RVU120) and the dual HAT/BETi (NEO2734/EP31670). Treatment with NEO2734 reduced the permissive chromatin marks especially on c-Myc and SPI1 super enhancers. There was also a significant reduction in mRNA and protein expressions of c-Myc, c-Myb, and PU.1. RNA-seq and mass-spectrometric analyses revealed that treatment with NEO2734 negatively enriched gene sets of c-Myc targets, rRNA processing, mRNA splicing, and translation. Treatment with SEL120 globally reduced active chromatin marks in the mtASXL1 expressing AML cells. This was associated with reduced protein expression of BAP1, pSTAT1 (S727), pSTAT5 (S726), MEIS1 and HOXA9, exclusively in mtASXL1 expressing cells. Co-treatment with pelabresib and NEO2734 or SEL120 and NEO2734 synergistically induced apoptosis in mtASXL1-expressing cells. Importantly, in the NSG mice engrafted with OCIAML3 ASXL1 Y591*, monotherapy with NEO2734, pelabresib or SEL120 significantly reduced AML burden. Collectively these findings highlight previously uncharacterized biologic effects of the presence of mtASXL1 and support the rationale for further evaluating AML therapies incorporating BETi, HAT-BETi or mediator-kinase inhibitor. Citation Format: Christopher P. Mill, Warren C. Fiskus, Christine E. Birdwell, John A. Davis, Kaberia Das, Hanxi Hou, Surbhi Sharma, Koji Sasaki, Sanam Loghavi, Tapan M. Kadia, Naval Daver, Courtney D. DiNardo, Lu Wang, Kapil N. Bhalla. ASXL1 mutations in AML are associated with a distinct epigenetic state that results in vulnerabilities to epigenetic-targeted agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 444.