Fig 2: KDM6A regulates CD48 expression in MM cells.a Normalized lysis percentage of KDM6A WT or KO H929 cells after co-culture with primary NK cells at different E:T ratios with IL-2 for 6 hours (mean ± SEM, n = 3 biologically independent experiments). *p < 0.05; **p < 0.01; ***p < 0.001 (two-sided student’s t test). b Intracellular IFN-γ staining of primary NK cells co-cultured with KDM6A WT or KO H929 cells with IL-2 for 6 hours (mean ± SEM, n = 3 biologically independent experiments). **p < 0.01 (two-sided student’s t test). c Volcano plot of differentially expressed genes in KDM6A KO H929 cells compared with WT cells assessed by RNA-seq. d Venn diagram showing the overlapped enriched genes between the CRISPR screen and RNA-seq. e, Representative flow cytometry analysis of CD48 expression in H929 transfected with indicated sgRNAs. f q-RT-PCR for CD48 mRNA in H929 cells transfected with indicated sgRNAs. Data were normalized against GAPDH (mean ± SEM, n = 3 biologically independent experiments). ****p < 0.0001 (two-sided student’s t test). g, ChIP-seq density profiles for H3K27me3 on the CD48 gene in KDM6A WT and KO H929 cells. h, H3K27me3 ChIP-qPCR analysis at the CD48 gene in KDM6A WT and KO H929 cells (mean ± SEM, n = 3 biologically independent experiments). ***p < 0.001 (two-sided student’s t test). i, ATAC-seq density profiles at the CD48 promoter region in KDM6A WT and KO H929 cells. j q-RT-PCR for CD48 mRNA in newly diagnosed (N/D) (n = 7) and Dara-resistant (R/R) (n = 12) patient samples. Data were normalized against GAPDH (mean ± SEM). *p < 0.05 (two-sided student’s t test). Source data are provided as a Source Data file.

Fig 3: Schema of epigenetic regulation of NK cell-mediated immune response in multiple myeloma monoclonal antibody therapy.The loss or inactivation of KDM6A increased the level of H3K27me3, resulting in the downregulation of both CD38 and CD48 expression, which led to reduced ADCC. Lowering the H3K27me3 with an EZH2 inhibitor restored sensitivity to Dara through CD38 and CD48 upregulation. This Figure was created with BioRender.com.

Fig 4: EZH2 inhibitors increase CD38 and CD48 expression and enhance Dara-mediated ADCC.a Schematic utilizing EZH2 inhibitor to balance the expression of CD38 and CD48 in KDM6A-KO MM cells. b, Western blot of CD38 and CD48 protein levels in KDM6A-KO and control H929 cells treated with Taze (5 μM) for 4 days. q-RT-PCR for CD38 (c) and CD48 (d) mRNA in KDM6A-KO and control H929 cells treated with Taze (5 μM) for 4 days. Data were normalized against GAPDH (mean ± SEM, n = 3 biologically independent experiments). ns, not significant; ***p < 0.001 (two-sided student’s t test). e Representative flow cytometry analysis of CD38 and CD48 expression in KDM6A WT and KO H929 cells treated with Taze (5 μM) for 4 days. H3K27me3 ChIP-qPCR analysis at the CD38 (f) and CD48 (g) genes in KDM6A WT and KO H929 cells (mean ± SEM, n = 3 biologically independent experiments). ***p < 0.001 (two-sided student’s t test). KDM6A WT and KO H929 cells were treated with Taze (5 μM) for 4 days, then co-cultured with primary human NK cells and Dara (h) or Isa (i) and subjected to ADCC assay (mean ± SEM, n = 3 biologically independent experiments). ns, not significant; **p < 0.01 (two-sided student’s t test). j KDM6A WT and KO H929 cells were treated with Taze (5 μM) for 4 days, and then co-cultured with Dara and primary NK cells for 6 hours. The supernatant was collected for granzyme B ELISA assay (mean ± SEM, n = 3 biologically independent experiments). **p < 0.01; ***p < 0.001 (two-sided student’s t test). k The patients’ MM cells were treated with DMSO or Taze (5 μM) for 4 days, followed by the addition of Dara and incubation for 12 hours. Cell cytotoxicity was assessed by flow cytometry. (n = 10 for each group). Box plots represent the median, 25th, and 75th percentiles, and whiskers represent the values of min and max. *p < 0.05 (two-sided paired student’s t test). Source data are provided as a Source Data file.