Identification and Validation of FOXA3 as a Candidate Gene Involved in Carfilzomib Resistance in Multiple Myeloma

Multiple myeloma (MM) is an incurable haematological cancer originating from malignant plasma cells in the bone marrow, characterized by excessive immunoglobulin production. Although treatment regimens incorporating proteasome inhibitors (PIs) have significantly improved patient outcomes, most patients eventually develop drug resistance and relapse, highlighting the urgent need for novel strategies to better manage both early-stage and relapsed/refractory MM. To investigate the molecular drivers of PIs resistance, we conducted a genome-wide CRISPR activation (CRISPRa) screen in a MM cell line under the selective pressure of PI carfilzomib (CFZ), using the Synergistic Activation Mediator (SAM) system. Next-Generation Sequencing analysis identified several candidate genes enriched in CFZ-resistant cells, with FOXA3, PPAT, and IRF1 emerging among the most significantly enriched genes and therefore selected for validation. Based on preliminary validation results, the transcription factor FOXA3 was prioritised for detailed functional characterization. We demonstrated that sgRNA-mediated transactivation of FOXA3, using two independent sgRNAs, enhanced MM cells recovery following CFZ-induced cytotoxicity and conferred a selective growth advantage under PIs treatment (CFZ and bortezomib - BTZ), while no enrichment was observed with non-PI agents such as pomalidomide, methotrexate, or doxorubicin. Consistently, ectopic overexpression of FOXA3 similarly promoted recovery from CFZ treatment, while shRNA-mediated silencing of FOXA3 sensitized resistant cells to CFZ and BTZ respectively, supporting a functional role for FOXA3 in mediating PIs resistance. To elucidate the underlying molecular mechanisms, RNA-sequencing was performed and identified XBP1 and ABCB1 as potential downstream effectors of FOXA3-mediated CFZ resistance, suggesting that FOXA3 may promote PIs resistance through modulation of stress response and drug efflux pathways. Notably, pharmacological inhibition of either XBP1 or ABCB1 in combination with CFZ, significantly delayed the recovery of FOXA3- transactivating cells, supporting their contribution to FOXA3-mediated resistance. In conclusion, this study identifies FOXA3 as a novel transcriptional regulator of PIs resistance in MM and provides mechanistic insight into adaptive stress and drug efflux pathways, underlying therapeutic failure. These findings suggest that targeting FOXA3- associated signalling networks may represent a rational strategy to enhance PIs efficacy and overcome drug resistance in MM