Introduction: The CBM signalosome (CARDs/BCL10/MALT1) is an evolutionarily conserved key regulator of canonical NF-κB signaling. Activating mutations of the CBM signalosome constitutively activate NF-κB, driving tumorigenesis in B-cell Non-Hodgkin Lymphoma (B-NHL). Depletion of individual components of the CBM complex including MALT1 was shown preclinically to suppress NF-κB driven Activated B-Cell Diffuse Large B-Cell Lymphoma (ABC-DLBCL), inspiring many efforts to develop MALT1 inhibitors for therapeutic use. MALT1 is a dual function protein with two independent roles. Primarily, MALT1 is a scaffold protein organizing the CBM complex and directly transducing canonical NF-κB signaling. Secondarily, MALT1 is a paracaspase, fine-tuning multiple signaling pathways, including NF-κB, via substrate cleavage. Research indicates that targeting MALT1 scaffolding activity is likely the preferred approach for effectively suppressing NF-κB activity while minimizing impact on the immune system. Using our Smart AllosteryTM platform, we have developed potentially the first-in-class MALT1 scaffolding inhibitors targeting the CBM signalosome as a more directed approach to specifically block NF-κB signaling. Here, we disclose the distinctive mechanism of action of scaffolding inhibitors.

Experiments: The mechanistic studies of MALT1 scaffolding inhibition were explored through comprehensive cellular assays and in vivo analyses. Engineered cell models were utilized to elucidate novel mechanisms of action.

Results: We have developed a series of potent MALT1 scaffolding selective inhibitors with cellular IC50 values approximating 250nM for scaffolding activity, 80nM for NF-κB activity, and 590nM for AP-1 activity. Using TR-FRET, NanoBRET, and TurboID assays, we observed scaffolding inhibitors did not interrupt the interaction of MALT1-TRAF6. Rather, they induced a non-productive signalosome blocking NF-κB activity. Scaffolding inhibitors did not inhibit MALT1 protease activity; in fact, inhibition of MALT1 scaffolding activity modestly increased its protease activity, which is consistent with observations in MALT1 scaffolding mutant mice. This distinctive consequence of MALT1 scaffolding inhibition translated to no detrimental effects on T-cell activation or Treg differentiation, both of which are dependent on MALT1 protease activity and cause associated safety concerns with chronic MALT1 protease inhibitor treatment. The distinct and superior mode of action of MALT1 scaffolding inhibitors manifested in a more potent and broad anti-proliferative activity against a panel of B-NHL cell lines dependent on intrinsic BCR or NF-κB activity for growth.

Conclusions: The potential first-in-class MALT1 scaffolding inhibitor offers what we believe is a best-in-class approach to targeting CBM-complex-driven NF-κB signaling. Scaffolding inhibitors effectively blocked both NF-κB and AP-1 signaling without impairing protease activity and avoided negative effects on T-cell function. Scaffolding inhibitors offer a promising therapeutic approach for NF-kB driven cancers.

Citation Format: Yi Yao, Kevin Ling, Huili Xia, Huadong Sun, Yingzhi Bi, Jun Kuai, Joe Wahle, Wei Liu, Ken Carson, Gerry Harriman, Fang Wang. Targeting the CBM signalosome with a MALT1 scaffolding inhibitor for treatment of non-Hodgkin lymphomas [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-036.