Abstract 5352: Genome-wide CRISPR-cas9 screening identifies KDM6A as a modulator of CD38 expression in multiple myeloma: Therapeutic implications

Abstract

Abstract Daratumumab (Dara) is the first-in-class monoclonal antibody targeting CD38 which triggers both direct and immune mediated cytotoxicity in preclinical in vitro and in vivo models of multiple myeloma (MM) in the bone marrow (BM) milieu. Importantly, Dara combination therapies can achieve high frequency and extent of response in both relapsed/refractory and newly diagnosed MM. However, relapse of MM is commonly observed due, at least in part, to downregulation of CD38 on MM cells. Here we utilize genome-wide CRISPR-cas9 knockout (KO) screening to identify novel regulators mediating CD38 expression and Dara sensitivity in MM cells. We identified 46 different sgRNAs to be positively correlated with MM cytotoxicity triggered by Dara and NK cells treatment. We further performed second round screening using the cell population with lowest 5% CD38 expression after CRISPR lentiviral library transduction. Importantly, KDM6A was the top ranked of the overlapping genes from these two screenings. Next, we individually KO of KDM6A in MM cells and found that both mRNA and protein levels of CD38 were significantly decreased, as well as the cell surface CD38 expression detected by flow cytometry. Re-introduction of KDM6A into KDM6A KO cells restored the expression level of CD38 and Dara-induced cytotoxicity, indicating that KDM6A positively regulates CD38 expression in MM cells. Since KDM6A regulates demethylation of lysine 27 at histone H3 (H3K27) to activate gene expression, we next examined whether CD38 expression was altered by methylation status of H3K27. From our ChIP-seq and ChIP Q-PCR data, we identified that H3K27me3 level was highly enriched on the promotor area of CD38 in KDM6A KO cells compared to control cells. These data indicate that KDM6A modulates CD38 expression by regulating H3K27me3 level on the CD38 promotor, thereby mediating sensitivity of MM cells to Dara-induced cytotoxicity. H3K27me3 is regulated by the balance of demethylase (KDM6A) and methyltransferase (EZH2). However, downregulating H3K27me3 on the CD38 promoter by enhancing KDM6A activity is challenging. Therefore, we next explored whether EZH2 inhibitor can restore CD38 expression and overcome Dara resistance in KDM6A KO cells. Indeed, H3K27me3 level on the CD38 promotor area was significantly decreased by EZH2 inhibitor. Moreover, EZH2 inhibitor treatment upregulated both CD38 mRNA and protein levels in KDM6A KO cells. Importantly, EZH2 inhibitor also restored sensitivity of KDM6A KO cells to Dara-mediated NK cell cytotoxicity. Taken together, our studies demonstrate that KDM6A regulates H3K27me3 level on CD38 promotor area and CD38 expression in MM. Moreover, we validate that combination treatment with EZH2 inhibitor and Dara can overcome Dara resistance in preclinical MM models, providing the rationale for combination clinical trials to overcome Dara resistance and improve patient outcome. Citation Format: Jiye Liu, Lijie Xing, Teru Hideshima, Kenneth Carl Anderson. Genome-wide CRISPR-cas9 screening identifies KDM6A as a modulator of CD38 expression in multiple myeloma: Therapeutic implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5352.