GPCR/MALT1 protease axis promotes production of a pro-tumor secretome in triple-negative breast cancer

Summary: Background: Patients with triple-negative breast cancer (TNBC) have the worst overall prognosis among the breast cancer subtypes due to their aggressive nature and lack of tractable targets. Treatment relies heavily on chemotherapeutics, highlighting the need for novel therapies to improve outcomes. Our lab ascertained that MALT1, the effector protein of the CARMA3-BCL10-MALT1 (CBM) signalosome, is activated in breast cancer downstream of several G protein-coupled receptors (GPCRs) including the receptors for thrombin (PAR1), angiotensin II (AGTR1), and lysophosphatidic acid (LPAR). MALT1 acts as a protease to cleave selected protein targets that are involved in regulating pro-inflammatory NF-kB signaling. Because CBM signalosome activity promotes growth of TNBC tumors in immuno-competent mouse models, we hypothesized that MALT1 protease activity drives the production of a pro-tumor secretome, directly influencing the TNBC microenvironment contexture and leading to enhanced tumor growth. Methods/Results: We first verified MALT1 protease activity in TNBC cell lines using western blot analysis. To identify secreted factors dependent on GPCR/MALT1 protease signaling, we performed a comprehensive transcriptomic and proteomic analysis of TNBC cell lines treated with or without MALT1 protease inhibition using RNA-seq and Olink®, respectively. We found numerous MALT1 protease-dependent secreted factors, including GM-CSF, CXCL1, and PTX3. We validated these results at the mRNA and protein level in TNBC cell lines. To enable specific detection of MALT1 protease activity in clinical specimens and query the unique features of the TNBC microenvironment, we developed a novel MALT1 activity gene signature by performing RNA-seq on MALT1-active cell lines. In conjunction with our MALT1 activity gene signature, we constructed a TNBC cell block microarray to validate a novel antibody that recognizes a MALT1 protease-specific cleavage product. Conclusions/Future Directions: Initial studies suggest that MALT1 protease increases the expression of numerous immuno-suppressive factors as part of a MALT1-dependent secretome. Next, we will integrate our findings with functional assays to investigate how MALT1-dependent secreted factors impact immune cell function via paracrine signaling. Additionally, we will utilize our MALT1 activity gene signature to test archived TNBC clinical specimens for an association between MALT1 activity and a dysfunctional tumor immune contexture. Dong Hu, Jeffrey Meridew, Cody Hoffmann, Akhilesh Pandey, Linda McAllister, and Peter Lucas