The Mechanistic Study Of MMSET Promoting Ferroptosis Sensitivity Via ACSL4 In T(4;14)-positive Multiple Myeloma

Objective: To investigate ferroptosis sensitivity in t(4;14)-positive multiple myeloma(MM)and its underlying mechanism.Methods: First,we analyzed enrichment pathways in multiple myeloma SET domain protein(MMSET)-related public datasets.The MMSET knockdown MM cell line was constructed by lentiviral transfection,and the effect of chromosome t(4;14)status or MMSET expression on MM ferroptosis sensitivity was detected by cell counting reagent-8(CCK-8),real-time reverse transcription polymerase chain reaction(RT-q PCR),flow cytometry,electron microscopy,etc.Then we performed the correlation analysis between MMSET and long-chain acyl-Co A synthetase 4(ACSL4)in MM cell lines and primary MM cells by bioinformatics,western blotting,and immunohistochemistry.The regulation of MMSET on ACSL4 expression was analyzed by RT-q PCR,western blotting,and dual luciferase reporter gene assay,etc.The role of MMSET/ACSL4 axis on MM ferroptosis sensitivity was investigated in vitro and in vivo by transient transfection,CCK-8,flow cytometry,etc.Next,the effect of ACSL4 inhibition or fatty acids supplementation on MM ferroptosis sensitivity was detected by CCK-8.Metabolomics was applied to study the impact of ACSL4 on fatty acid composition in MM.Additionally,the curative effect and cell death pathways of ferroptosis inducers combined with bortezomib on MM were evaluated in vitro and in vivo by CCK-8,flow cytometer,etc.Ferroptosis-associated proteins including cystine/glutamate transporter(x CT)and glutathione peroxidase 4(GPX4)were detected by western blotting in MM treated with bortezomib or combined with ferroptosis inducers.And intracellular reduced glutathione levels was analyzed by colorimetry in MM treated with bortezomib/ferroptosis inducers alone or together.Results: Both ferroptosis and polyunsaturated fatty acids(PUFAs)synthesis pathways are enriched in several MMSET-related public datasets.MM cell lines or primary MM cells with t(4;14)-positive are more vulnerable to class II ferroptosis inducers compared to t(4;14)-negative,while little damage is observed by ferroptosis inducers in normal cells.Knockdown of MMSET renders t(4;14)-positive MM remarked resistance to ferroptosis.Mechanistically,MMSET upregulates ACSL4 transcription by binding to its promoter,ACSL4 increases multiple PUFAs levels and promotes sensitivity of t(4;14)-positive MM to ferroptosis.Supplementation of PUFAs restores ferroptosis susceptibility of t(4;14)-positive MM with MMSET or ACSL4 knockdown or t(4;14)-negative MM.Moreover,bortezomib upregulates the expression of ferroptosis-associated protein x CT in MM,independent of t(4;14)status.Co-treatment with class II FINs abrogates the upregulation of x CT by bortezomib in t(4;14)-positive MM,accompanied by the decrease of cellular reduced glutathione levels,then triggers both apoptosis and ferroptosis in t(4;14)-positive MM,displaying a synergistic anti-tumor activity in vitro and in vivo.Conclusions: Class II ferroptosis inducers are a novel therapeutic approach for t(4;14)-positive MM,particularly in combination with bortezomib.