Fig 2: PIGH is epigenetically downregulated in GPI-negative BLaER1 cells. (a) Alignment of the PIGH locus RefSeq with the Sanger sequencing result for genomic DNA from the GPI-negative clones. Exon 2 is depicted in blue. The two nucleotides that are deleted in the PIGHP1 sequence are shown in red. (b) Two GPI-negative BLaER1 monoclonal cell lines (1, circle and 2, square) were treated with 0.4 µM (light red) or 2.0 µM (dark red) 5-aza-2′-deoxycytidine (5-aza) or left untreated (black) for 2, 4, and 7 days. The percentage of CD48- or CD55-positive cells among GFP-expressing cells was determined by FACS analysis. Shown is the mean + SD of data from n = 3 independent experiments. (c) After treatment of GPI-negative BLaER1 monoclonal cell lines with 0.4 µM 5-aza or leaving them untreated for 4 days, cells were transdifferentiated for 5 days. The percentage of CD14-positive cells among GFP-expressing cells was determined by FACS analysis. Shown is the mean + SD of data from n = 3 independent experiments. 5-aza-treated cells were compared to untreated control cells using unpaired t tests (**p ≤ 0.01, ***p ≤ 0.001).

Fig 3: Loss of CD14 on the cell surface in BLaER1 monocytes is the result of a general GPI anchor defect. (a) CD14 mRNA expression of BLaER1 clones, quantified by qRT-PCR. Shown is the mean ± SD of n = 3 independent measurements, normalized to GAPDH expression. Dots represent individual values. (b) Western blot analysis of CD14 expression. One representative blot of two is shown. Full-length blots are presented in Supplementary Figure S9. (c) Quantification of the CD14 protein in (b). CD14 expression was normalized to the housekeeping protein β-actin. The mean ± SD of pooled data from three different monoclonal cell lines from n = 2 independent measurements is shown. Dots represent the individual values. (d–f) Surface expression of the GPI-anchored proteins CD48 and CD55 (d,f) and the transmembrane proteins CD45 and CD64 (e) was measured by flow cytometry in transdifferentiated (d,e) or undifferentiated (f) BLaER1 monoclonal cell lines. Shown is the percentage of cells expressing the protein of interest among all GFP-positive cells from n = 2 independent measurements (mean ± SD). Individual values are depicted as dots. (a,c–f) Monoclonal cell lines with higher responsiveness to LPS (dark blue) were compared to cell lines with lower responsiveness to LPS (light blue) by Mann–Whitney test (ns—not significant, **p ≤ 0.01).

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.

Fig 5: 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.