Deletion of TRAF3 and CYLD and NIK amplification activates NF-kb signalling and drives resistance to T cell based therapies in multiple myeloma by suppressing T cell activity

Abstract Patients with Multiple Myeloma (MM) receiving adaptive T cell therapies such as chimeric antigen receptor (CAR) T cells and T cell engagers (TCEs) targeting BCMA have shown encouraging responses. However, despite initial response, most patients experience relapse. We have recently observed an enrichment of NF-kB activation mutations, including deletion of TRAF3 and CYLD or amplification of MAP3K14, also known as NF-kB Inducing Kinase (NIK), in patients with BCMA acquired resistance. Furthermore, in a genome-wide CRISPR screen performed using the YUSA library containing 90,709 unique sgRNA targeting 18,010 genes in OPM2 cells, sgRNAs targeting TRAF3 were the top enriched gene in MM cells surviving multiple exposures to an anti-BCMA TCE. Additional enriched hits include sgRNAs targeting genes involved in apoptotic regulation, such as CASP8, and key regulators of the NF-kB pathway, including BIRC3, NFKBIA and NFKBIB. Therefore, we aimed to determine the impact of NF-kB alterations (TRAF3, CYLD and NIK) on the development of resistance to anti-BCMA therapies in MM. Through CRISPR Cas9 gene editing, we have generated stable TRAF3 and CYLD knockout (KO) clones in OPM2 and ARP1 MM cells. Moreover, we have cloned NIK in a pLEX-307 vector to overexpress (OE) the NIK protein in both OPM2 and ARP1 cells. As controls, we generated a non-targeting Cas9 and pLEX-307 empty vector cells. The protein changes were confirmed by western blot and the activation of the non-canonical NF-kB pathway was validated by ELISA assay. By using flow cytometry-based cytotoxicity assays, we have next demonstrated that the KO and OE cells co-cultured with healthy donors' PBMCs exhibited resistance to anti-BCMA therapies, including CAR-T cells and TCEs. Of interest, similar resistance was observed in the presence of anti-GPRC5D targeting CAR-T cells and TCE, suggesting that their resistance is rather intrinsic and target antigen agnostic. To elucidate the molecular signature associated with the development of this acquired intrinsic resistance, we have next performed transcriptome profiling (scRNA-seq) on the OPM2 KO and OE clones and their isogenic controls. Several differentially activating pathways, including upregulation of anti-apoptotic signaling and an enrichment of the interferon alpha (IFN) and TNF-a signaling, were transcriptionally enriched in these TCE/CAR T resistance cells, with several targets confirmed at the protein level. Of note, this transcriptionally enriched IFN response was also functionally validated in vitro by demonstrating the lack of infection and replication of the VSV-ΔM51-GFP virus in the TRAF3KO and CYLDKO MM cells relative to their isogenic controls. In addition, multiplex cytokine assay performed on the supernatant of these TRAF3/CYLD KO and NIK OE TCE/CAR T resistant cells revealed elevated levels of multiple factors and cytokines with known inhibitory effect on T cell function, such as sFas, sFasL, TGF-b3, TNF-a, IL-10 and IP-10 when compared to the control isogenic cells. Importantly, we demonstrated that IP-10 (encoded by CXCL10) addition to healthy donor PBMCs co-cultures with MM cell lines was able to attenuate T-cell mediated killing in the presence of anti-BCMA CAR-T cells or TCEs. Lastly, by flow cytometry, we have observed upregulation of Fas expression in the KO cells as the likely source for the increased levels of soluble Fas (sFas). We suggest that sFas may interfere with T cell function by acting as a decoy receptor to immune cells FAS ligand (FASL) blocking its binding to FAS on MM cells.In summary, we have identified a novel NF-kB dependent tumor intrinsic mechanism of resistance to T cell-based therapies mediated by TRAF3/CYLD deletion and NIK amplification that promotes an inflammatory and immunosuppressive microenvironment suppressing T cell activity.