Genome-Wide CRISPR-Cas9 Screen Identifies KDM6A As a Modulator of Daratumumab Sensitivity in Multiple Myeloma

Daratumumab (Dara) is the first-in-class monoclonal antibody targeting CD38, which triggers both direct and immune-mediated cytotoxicity of multiple myeloma (MM) cells in the bone marrow (BM) milieu. Importantly, Dara combination therapies have a high frequency and extent of response in both relapsed/refractory and newly diagnosed MM. However, relapse is commonly observed due to the development of Dara resistance.

As an immune-targeting drug, the anti-tumor activity of Dara is closely associated with the expression of CD38, with nonresponders having a lower expression of CD38. To identify novel regulators mediating CD38 expression and Dara sensitivity in MM cells, we performed a genome-wide CRISPR-Cas9 knockout (KO) screen in vitro. We identified 46 different sgRNAs that were positively correlated with MM cytotoxicity triggered by Dara and primary NK cells. Using the cells with the lowest expression of CD38 (bottom 5%), we performed a second round of screening and found that KDM6A was the top-ranked gene out of the set of overlapping genes from these two screenings. We confirmed that CD38 expression was significantly decreased (>2 times, p<0.01) by KDM6A KO and that re-introducing KDM6A into KDM6A KO cells restored the expression level of CD38 and Dara-induced cytotoxicity.

KDM6A encodes lysine-specific demethylase 6A, which demethylates lysine 27 of histone H3 (H3K27) to promote gene expression. To confirm that CD38 expression was altered by the methylation status of H3K27, we compared the CD38 promoter area between KDM6A KO and control cells using ChIP-seq and ChIP qPCR and found that the H3K27me3 level was higher in KDM6A KO cells. H3K27 is methylated by EZH2, and we found that EZH2 inhibitor treatment significantly decreased the H3K27me3 level of the CD38 promoter area. Moreover, EZH2 inhibitor treatment upregulated both CD38 mRNA and protein expression in KDM6A KO cells. Importantly, EZH2 inhibitor also restored the sensitivity of KDM6A KO cells to Dara-mediated NK cell cytotoxicity in vitro and in xenograft mouse model.

We next re-introduced CD38 into KDM6A KO cells and found that CD38 overexpression only partially restored the sensitivity of KDM6A KO cells to Dara treatment. This suggested that besides CD38 downregulation, there are other mechanisms underlying Dara resistance induced by KDM6A KO. We performed RNA-seq in KDM6A KO cells and found that CD48, an NK-activating ligand, was downregulated. Overexpression of CD48 in KDM6A KO cells significantly increased secretion of Granzyme B and Perforin, restoring the activity of NK cells and re-sensitizing KDM6A KO cells to Dara treatment. These data suggest that KDM6A not only regulates CD38 expression, but also NK cell activity by CD48 regulation, thereby modulating the efficacy of Dara.

Taken together, our studies demonstrate that KDM6A regulates CD38 and CD48 expression in MM. Moreover, we validate that combination treatment with FDA-approved 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.