Citations

Abstract Background: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and remains difficult to treat due to the absence of targeted therapies. While recent advances in immunotherapy, particularly with immune checkpoint inhibitors (ICIs), have transformed the treatment landscape for many cancers, response rates to ICIs remain low for TNBC, largely due to tumor-intrinsic immune evasion mechanisms. Objective: We identify and characterize a novel immune evasion pathway in TNBC driven by deregulation of cancer cell-intrinsic protease-activated receptor 1 (PAR1), a thrombin receptor frequently upregulated in aggressive tumors. Methods and Results: Through integrative bioinformatic analyses, genetic perturbations, xenograft models (immunodeficient and immunocompetent), flow cytometry-based tumor immune profiling, and RNA-seq, we demonstrate that PAR1 activation enhances immune evasion in TNBC. Mechanistically, we show that the paracaspase MALT1 acts as a critical downstream effector of PAR1 signaling and promotes immune escape by driving PD-L1 expression in TNBC cells. Functional assays reveal that genetic depletion of MALT1 or PD-L1 increases TNBC cell susceptibility to T cell-mediated cytotoxicity in vitro and significantly suppresses tumor growth in vivo. Notably, through knock-in and rescue experiments using a catalytically inactive MALT1 mutant, we reveal that MALT1’s scaffolding - rather than protease - function is essential for PD-L1 regulation. Immune cell depletion assays further identify CD4+ T cells, CD8+ T cells, and natural killer (NK) cells as key mediators of the anti-tumor immune response suppressed by the PAR1-MALT1 axis. These findings are supported by human TNBC specimen analyses, where MALT1 expression inversely correlates with T cell activation and positively associates with PD-L1 in PAR1-high, but not PAR1-low, tumors. Conclusion: Our study reveals immune evasion as a novel mechanism of PAR1-driven breast cancer pathogenesis, mediated by a PAR1/MALT1/PD-L1 signaling cascade. These findings bridge a critical gap by positioning MALT1 at the interface between tumor-intrinsic signaling and the immune microenvironment - two areas of CARMA-BCL10-MALT1 (CBM) complex research that have largely evolved in parallel over the years. Importantly, targeting MALT1 may enhance immunotherapy efficacy in TNBC. Given the availability of MALT1 inhibitors in clinical development, our work supports combining MALT1 inhibition with ICIs or other immune-oncology agents as a promising strategy to overcome immune resistance in TNBC. Citation Format: Dong Hu, Prasanna Ekambaram, Zheqi Li, Linda Klei, Maria L. Beecher, Yi Liu, Zongyou Cai, John Little, Jeffrey A. Meridew, Jia-Ying Lee, E. Aubrey Thompson, Tullia C. Bruno, Lidija Covic, Seung-Oe Lim, Anushka Dongre, Heide L. Ford, Mien-Chie Hung, Adrian V. Lee, Steffi Oesterreich, Linda McAllister-Lucas, Peter C. Lucas. A cancer cell-intrinsic PAR1/MALT1/PD-L1 signaling pathway drives immune evasion in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7392.

Abstract Background: Treatment for patients with triple-negative breast cancer (TNBC) relies on non-specific chemotherapy, underscoring the need for precision-based approaches. Our lab discovered that MALT1, the effector protein of the CARMA3-BCL10-MALT1 (CBM) signaling complex, is activated downstream of several oncogenic G protein-coupled receptors (GPCRs) implicated in breast cancer, including the thrombin (PAR1), angiotensin II (AGTR1), and lysophosphatidic acid (LPAR) receptors. When activated, MALT1 functions as a protease to cleave select protein substrates. Based on our finding that CBM activity in TNBC cells promotes tumor growth in immunocompetent but not in immunodeficient mouse models, we hypothesized that MALT1 protease activity induces a pro-tumor secretome that creates an immune-suppressed tumor immune microenvironment (TIME). Methods/Results: We confirmed by western that MALT1 protease is active in multiple human and mouse TNBC cell lines. Next, to identify MALT1-dependent secreted factors, we performed transcriptomic and proteomic analyses comparing TNBC cells treated with or without MALT1 protease inhibitors. We found that several secreted factors, including GM-CSF, CXCL1, and Pentraxin 3, were reduced by MALT1 protease inhibition. To assess the functional impact of the MALT1 protease-dependent secretome, we performed media transfer experiments comparing the effects of conditioned media (CM) from TNBC cells treated with or without MALT1 protease inhibitors. To account for species differences, experiments were performed evaluating human TNBC cell CM added to human PBMCs and mouse TNBC cell CM added to murine bone marrow cells. We found that the MALT1 protease-dependent secretome reduces the expression of iNOS in both human and murine macrophages. For use in validating our findings in human TNBC clinical specimens, we developed a “MALT1 protease active gene signature” using RNA-seq analysis. Conclusions/Future Directions: Our studies indicate that MALT1 protease activity in TNBC cells promotes expression of numerous immune-suppressive factors. In both human and mouse systems, blockade of MALT1 protease alters the tumor cell secretome and promotes an increase in expression of the anti-tumor M1-like marker iNOS in macrophages. Future studies will evaluate how this change in iNOS expression impacts functional properties of TNBC-associated macrophages. We will also assess the influence of the TNBC MALT1-dependent secretome on the TIME in vivo by comparing the effects of intratumoral injection of CM from MALT1 wild-type or MALT1 protease-deficient TNBC cells. Finally, we will utilize our MALT1 activity gene signature to evaluate TNBC clinical specimens for an association between MALT1 activity and tumor immune contexture, with an emphasis on tumor-associated macrophages. Our studies could nominate MALT1 protease as a potential therapeutic target for improving anti-tumor immunity in TNBC. Since multiple MALT1 protease inhibitors are in clinical trial, our findings could be rapidly translated to improve outcomes for patients with TNBC. Citation Format: John W. Little IV, Jeffrey A. Meridew, Dong Hu, Juliana Hofstätter Azambuja, Linda M. McAllister-Lucas, Peter C. Lucas. The GPCR-MALT1 protease signaling axis drives an immunosuppressive secretome in triple-negative breast cancer [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr B046.

Abstract BACKGROUND H3K27-altered diffuse midline glioma (DMG) is a devastating pediatric brainstem tumor that affects 200-300 individuals in the US per year. Median survival is 9-11 months, and there are virtually no long-term survivors. Despite decades of clinical trials, radiation therapy remains standard of care, extending survival by 2-3 months. Elucidating the mechanisms that drive H3K27-altered DMG pathogenesis in order to uncover therapeutic vulnerabilities is of critical importance. Recent studies implicate MALT1 as a potential therapeutic target in gliomas. MALT1 is the effector molecule of the CARMA-BCL10-MALT1 (CBM) signalosome, a cytoplasmic protein complex that drives downstream pro-survival transcriptional activity. MALT1, which possesses scaffolding and protease functions, promotes cell viability, proliferation, and migration/invasion in multiple cancer types. We aim to evaluate the hypotheses that MALT1 promotes DMG cancer cell proliferation and migration and that MALT1 inhibition will abrogate tumor progression. METHODS/RESULTS We screened a panel of cells including astrocytes, neural stem cells, and DMG cell lines by Western blot and found that CBM complex members BCL10 and MALT1 are present in all cells tested. We next demonstrated that MALT1 is proteolytically active in DMG cell lines SF8628 and HSJD-DIPG-007. Specifically, we showed cleavage of the MALT1 protease substrate HOIL, which was abrogated by treatment with the MALT1 protease inhibitor MLT-748. We next generated multiple dox-inducible MALT1 shRNA DMG cell lines to assess the impact of MALT1 deficiency on DMG biology. Thus far, we find that shRNA knockdown of MALT1 does not impact SF8628 cell proliferation. CONCLUSIONS MALT1 is expressed and proteolytically active in DMG cells. Initial studies show that MALT1 knockdown does not affect proliferation of SF8628 cells. We will next evaluate the effect of MALT1 knockdown on proliferation of additional DMG cell lines, as well as the impact on other malignant features of DMG cells including migration/invasion.

Abstract Purpose: Tumor-infiltrating B lymphocytes (TIL-B) have demonstrated prognostic and predictive significance in solid cancers. In this study, we aimed to distinguish TIL-Bs from malignant B-cells in diffuse large B-cell lymphoma (DLBCL) and determine the clinical and biological significance. Experimental Design: A total of 269 patients with de novo DLBCL from the International DLBCL R-CHOP Consortium Program were studied. Ultra-deep sequencing of the immunoglobulin genes was performed to determine B-cell clonotypes. The frequencies and numbers of TIL-B clonotypes in individual repertoires were correlated with patient survival, gene expression profiling (GEP) data, and frequencies of DLBCL-infiltrating immune cells quantified by fluorescent multiplex IHC at single-cell resolution. Results: TIL-B abundance, evaluated by frequencies of normal B-cell clonotypes in the immunoglobulin repertoires, remarkably showed positive associations with significantly better survival of patients in our sequenced cohorts. DLBCLs with high versus low TIL-B abundance displayed distinct GEP signatures, increased pre-memory B-cell state and naïve CD4 T-cell state fractions, and higher CD4+ T-cell infiltration. TIL-B frequency, as a new biomarker in DLBCL, outperformed the germinal center (GC) B-cell–like/activated B-cell–like classification and TIL-T frequency. The identified TIL-B–high GEP signature, including genes upregulated during T-dependent B-cell activation and those highly expressed in normal GC B cells and T cells, showed significant favorable prognostic effects in several external validation cohorts. Conclusions: TIL-B frequency is a significant prognostic factor in DLBCL and plays a crucial role in antitumor immune responses. This study provides novel insights into the prognostic determinants in DLBCL and TIL-B functions with important therapeutic implications.

The fine structure of the microvasculature in areas of focal cerebral ischemia was studied in squirrel monkeys and the changes in areas of impaired and unimpaired microvascular filling, as defined by carbon perfusion, were compared. Microcirculatory obstruction became evident three hours following middle cerebral artery (MCA) occlusion and appeared to be partly the result of compression of capillaries by perivascular glial swelling and developing cerebral edema. Slight endothelial swelling was a common finding. Intraluminal membrane-bound bodies were occasionally identified but they did not appear to be producing significant obstruction. The tight endothelial junctions remained intact and there was no evidence of accelerated micropinocytosis. Severe neuronal injury frequently preceded the development of the microvascular obstruction and was more widespread than the zones of impaired perfusion.

The sequential synaptic alterations which occur during the early phase of developing cortical infarction in the squirrel monkey were studied with the electron microscope. Mild swelling of a few dendritic terminals and clumping of synaptic vesicles were the earliest detectable changes, being present at 45 minutes. The two basic patterns of degeneration of the terminal boutons were shrinkage and swelling, with shrinkage being the predominant response. Many of the shrunken boutons became electron-dense, resembling the changes present in anterograde axonal degeneration. The alterations which developed in the dendritic terminals were relatively unimpressive. Disruption of axosomatic synapses occurred at an earlier stage and was more severe than that of axodendritic synapses. Reduction in the number of synaptic vesicles was first observed at six hours and appeared to be progressive. The edema which developed was morphologically distinct from both "vasogenic" and "cytotoxic" edema in that there was early and progressive enlargement of the extracellular space.