MMSET enhances ferroptosis susceptibility by regulating ACSL4. (A-B) MMSET and ACSL4 protein levels were evaluated in primary plasma cells from patients with t(4;14)-positive and -negative MM using immunohistochemistry (scale bar: 25 μm) (A) and western blotting (B). (C) MMSET and ACSL4 expression levels were analyzed using western blotting (C) in the groups of NCI-H929 and OPM-2 cells with MMSET knockdown (shM) or not (shNC). (D) The relative dual-luciferase activity of NCI-H929 and OPM-2 cells with MMSET knockdown (shM) or without (shNC) with subsequent transfection with wild-type ACSL4 promotor or blank control for 48 hours. (E) Schematic diagram of the primer pair location in the ACSL4 promoter is shown in the top of the figure. OPM-2 cell lysates were used for ChIP assays with MMSET antibodies. ChIPs and input DNA were analyzed using qPCR for both the MMSET knockdown (shM) and negative control (shNC) groups. P1 to P3, primer pairs. (F-G) Relative cell viability (F) and lipid ROS levels (G) of NCI-H929 and OPM-2 cells with or without co-transfection with MMSET shRNA (shM) and ACSL4 overexpression (oeACSL4) constructs after 48 hours with subsequent treatment with RSL3 at the indicated concentrations for 24 hours. (H) Relative levels of PUFAs in OPM-2 cells with MMSET/ACSL4 co-transfection determined using Gas Chromatography-Mass Spectrometry (n = 6). (I) Relative cell viability of NCI-H929 and OPM-2 cells with or without ACSL4 knockdown treated with RSL3 for 24 hours. The IC₅₀ analysis was conducted using GraphPad software 8.0. (J) Metabolomics analysis of NCI-H929 and OPM-2 cells with or without ACSL4 knockdown (n = 5). ns, not significant; TSS, transcription start site. P > .05, ∗ P <0 .05, ∗∗ P < .01, and ∗∗∗ P < .001.