NF-Kb Pathway Activation Driven By TRAF3 Loss Mediates Resistance to Anti-BCMA T-Cell Based Therapies in Multiple Myeloma
Authors: Jung David , Lee Holly , Chikhale Sejal , Poorebrahim Mansour , Ahn Sungwoo , Benaoudia Sacha , Leblay Noemie , Maura Francesco , Bahlis Nizar J. , Neri Paola 
Published: December 2024

Background: T-cell based therapies such as chimeric antigen receptor (CAR) T cells and bispecific T cell engagers (TCEs) targeting the B cell maturation antigen (BCMA) have shown promising activities in multiple myeloma (MM) patients with advanced disease. However, responses to these therapies are not universal, and acquired resistance does invariably occur. Antigen escape has been reported as a major mechanism of resistance following anti-BCMA CAR-T and TCEs. We have recently reported that nearly 50% of anti-BCMA TEC resistant patients acquired biallelic BCMA deletion or mutation in the BCMA extracellular domain. Notably, an enrichment of NF-kB activating mutations, including deletion of its negative regulators TRAF3 or CYLD or amplifications of MAP3K14 (encoding NIK), was observed in patients with acquired BCMA function loss and/or resistance. TRAF3 acts mainly as an inhibitor of the non-classical NF-kB pathway through NIK degradation and can positively regulate type I interferon (IFN) production while negatively modulating MAPK activation. Based on these data, we have postulated that NF-kB activating mutations may facilitate BCMA antigenic loss and acquired resistance to anti-BCMA TCE. To this end, we aimed to determine the impact of NF-kB alterations on the development of resistance to anti-BCMA therapies in MM.
Methods and Results: Through CRIPR Cas9 mediated genomic editing we have knocked out (KO) TRAF3 in OPM2 MM cells generating a stable OPM2TRAF3ko clone along with a lentiviral non-targeting Cas9 transduced OPM2 cells as negative control (OPM2TRAF3wt). Targeted TRAF3 KO protein expression was quantified by western blotting. In addition, an ELISA assay was performed to assess the activation of the NF-kB canonical (p65) or non-canonical (p52) pathways in TRAF3KO cells relative to their control. In vitro sensitivity to anti-BCMA immunotherapies was evaluated in flow cytometry based cytotoxicity assays of OPM2TRAF3wt or OPM2TRAF3ko co-cultures with anti-BCMA CAR-T cells (idecabtagene vicleucel, ide-cel) or anti-BCMA TCEs (elranatamab) together with healthy donors PBMCs.
Significant activation of the non-canonical NF-kB pathway was detected in OPM2TRAF3ko cells compared to OPM2TRAF3wt. Increased protein expression of several anti-apoptotic genes, including MCL-1 and BCL-2, known to be regulated by the NF-kB pathway, was also observed in OPM2TRAF3ko cells by western blot analysis. However, OPM2TRAF3ko proliferation rate and BCMA surface expression, was not significantly different to that of OPM2TRAF3wt cells as measured by colorimetric MTT assay and flow cytometry, respectively. Importantly, in vitro PBMCs co-cultures with OPM2TRAF3ko or OPM2TRAF3wt cells with ide-cel or elranatamab (concentrations ranging from 0.1 to 10 nM), at variable E:T ratios (2:1 or 1:4) for 48 hours revealed a significantly decreased sensitivity of OPM2TRAF3ko to both anti-BCMA targeting therapies when compared to OPM2TRAF3wt controls. Furthermore, we examined whether these anti-BCMA TCE and CART resistant cells (OPM2TRAF3ko) acquired any dependencies, cross-resistance or vulnerability to established anti-MM therapies. In particular we examined their sensitivity to immunomodulatory drugs such as lenalidomide and pomalidomide, the novel CELMoD mezigdomide and the proteasome inhibitor bortezomib. Notably, these OPM2TRAF3ko cells demonstrated a significantly reduced sensitivity to IMiDs and CELMoD when compared to OPM2TRAF3wt cells. Since TRAF3 is known to positively regulate interferon response genes, this resistance to IMiDs/CELMoD is likely mediated by the attenuation of the interferon response genes induced by Aiolos/Ikaros degradation. Further validation of the role of NF-kB pathway activation in acquired anti-BCMA TCE/CART resistance in MM is being validated in H929 TRAF3 KO cells as well as in CYLD mutants cells. These data will be updated at the meeting along with scRNA transcriptome and regulome profiling of these resistant cells.
In summary, we have demonstrated that TRAF3 deletion with the ensuing activation of the non-canonical NF-kB mediates resistance to anti-BCMA targeting TCE and CART cells therapies through tumor intrinsic and targeted antigen-independent mechanisms.