McAllister‐Lucas, Linda M.

Abstract Background: Antigen receptor stimulation induces assembly of the CARMA1-BCL10-MALT1 (CBM) signaling complex, which leads to activation of downstream transcriptional regulators including NF-κB and subsequent lymphocyte activation. Gain-of-function mutations in B-cell receptor subunits, CARMA1, or BCL10 which lead to constitutive activation of MALT1 underly the pathogenesis of multiple lymphoid malignancies, including activated B-cell–type diffuse large B-cell lymphoma (ABC-DLBCL). During previous efforts to identify regulators of MALT1 activity in lymphocytes, we discovered that G-protein-coupled receptor kinase 2 (GRK2) binds to MALT1, inhibits MALT1 scaffolding and proteolytic activities, and functions as a tumor suppressor in MALT1-dependent lymphomas. Notably, GRK2 mRNA expression is markedly lower in a subset of DLBCL tumors in comparison to normal B cells and lower GRK2 is associated with decreased patient survival in ABC-DLBCL. In light of these findings, we investigated the molecular mechanisms regulating GRK2 expression and then characterized the roles of specific GRK2-targeting miRNAs in ABC-DLBCL. Methods and Results: After finding that global impairment of miRNA processing through DICER1 depletion leads to increased GRK2 protein, we conducted bioinformatic analyses of patient tumor samples and microRNA Data Integration Portal screening to identify candidate microRNAs that target GRK2 in ABC-DLBCL. Direct targeting of GRK2 by candidate miRNAs was then confirmed by GRK2 3’UTR reporter assay. We tested for expression of three candidate microRNAs in ABC-DLBCL cells and observed elevated expression in both OCI-LY3 and TMD8 cells, which exhibited reduced GRK2 mRNA expression compared with primary B cells. Inhibition of candidate miRNAs in ABC-DLBCL cells, using anti-miRNA Locked Nucleic Acids (LNA) inhibitors, led to enhanced GRK2 expression, reduced MALT1 activity and decreased cell proliferation. Conversely, stable overexpression of candidate microRNAs led to reduced GRK2 expression, enhanced MALT1 activity and increased cell proliferation. In vivo, treatment of mice with inhibitors of candidate microRNAs abrogates the growth of ABC-DLBCL xenograft tumors. Conclusion: We have identified three miRNAs that negatively regulate GRK2 expression in ABC-DLBCL. By targeting GRK2 for downregulation, these miRNAs promote MALT1 oncoprotein scaffold and protease activities. Furthermore, inhibitors of these miRNAs enhance GRK2 expression and thereby suppress MALT1 activity and MALT1-dependent tumor cell proliferation in vitro and inhibit MALT1-dependent tumor growth in vivo. MicroRNA inhibitors that enhance GRK2 expression could represent a new approach for restraining MALT1-dependent lymphoma. Citation Format: Jing Cheng, Mei Smyers, Matt Trotta, Neil M. Carleton, Lisa M. Maurer, Uma R. Chandran, Min Xia, Ari M. Melnick, Peter C. Lucas, Linda M. McAllister-Lucas. Inhibitors of GRK2-targeting-microRNAs: New therapeutic strategies for MALT1-dependent lymphomas [abstract]. In: Proceedings of the Fifth AACR International Meeting on Advances in Malignant Lymphoma: From Discovery to Clinical Impact; 2026 Jun 24-27; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2026;7(3_Suppl):Abstract nr A033.

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: Pediatric and AYA sarcomas are rare, biologically heterogeneous tumors with limited noninvasive biomarkers to guide diagnosis, personalized treatment, and disease monitoring. Standard-of-care evaluation relies on invasive biopsies that carry procedural risks and imaging modalities with limited sensitivity for detecting minimal residual disease and distinguishing tumor types. Cell-free DNA (cfDNA) methylation profiling offers a minimally invasive alternative capable of capturing tumor-derived epigenomic alterations and has consistently shown strong clinical utility in adult malignancies. However, genome-wide cfDNA methylation patterns across diverse pediatric and AYA sarcoma subtypes remain poorly defined. Methods: Plasma was collected from 93 pediatric and AYA patients (pediatric <18 years; AYA 15-39 years) representing 27 Ewing sarcoma, 17 rhabdomyosarcoma, 11 liposarcoma, 10 chordoma, 8 synovial sarcoma, 7 chondrosarcoma, 6 osteosarcoma, and 6 fibrosarcoma cases, plus 19 matched healthy donors. cfDNA was isolated from 1 mL plasma, quantified by Qubit, and assessed by Bioanalyzer. Libraries were prepared using the NEBNext EM-seq v2 workflow with PCR cycles scaled to cfDNA input amount. Genome-wide sequencing (15× target depth) was followed by alignment, deduplication, and methylation calling. MultiQC was used to assess depth, duplication rate, conversion efficiency, and CpG coverage. Global methylation summaries were computed, and methylKit was used to generate per-CpG and 1-kb tiling matrices and initiate differential methylation analyses. Results: Ewing sarcoma patients had significantly higher plasma cfDNA concentrations than age- and sex-matched healthy donors (24.4 vs 4.6 ng/mL, p < 0.0001), whereas cfDNA levels for other sarcoma subtypes overlapped with the healthy cohort. Low or undetectable cfDNA concentrations by Qubit were far more common among non-Ewing sarcoma samples (23 of 66) than Ewing sarcoma (1 of 27). All samples achieved sufficient CpG coverage for genome-wide methylation modeling. Global methylation summaries revealed no significant differences in mean β-values across sarcoma subtypes or versus healthy donors. An independent analysis of the Ewing subset identified 299 significant differentially methylated regions (DMRs), supporting the detectability of disease-specific methylation changes. Conclusions: These results demonstrate the feasibility of high-quality cfDNA methylome profiling across a large and diverse pediatric and AYA sarcoma cohort, including in plasma samples with cfDNA concentrations below the limit of detection. While global methylation levels appear broadly similar across subtypes, proof-of-concept DMR analysis in Ewing sarcoma suggests that subtype-specific epigenomic signatures could enable future noninvasive diagnostic tools for pediatric and AYA sarcomas. Citation Format: Lilli J. Greiner, Roman O. Kowalchuk, Jeff Szymanski, Pradeep S. Chauhan, Adam Amundson, Peter J. Schoettler, Wendy Allen-Rhoades, Stephanie F. Polites, Peter S. Rose, Kelly Bailey, Linda McAllister-Lucas, Anita Mahajan, Nadia Laack, Aadel Chaudhuri. Defining genome-wide cfDNA methylation landscapes in pediatric and adolescent and young adult (AYA) bone and soft-tissue sarcomas [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 7885.

Abstract Introduction/Objectives: The CARMA1-BCL10-MALT1 (CBM) signalosome plays crucial role in mediating antigen receptor-induced activation of NF-κB transcription factor and subsequent lymphocyte activation. The effector protein of the signalosome, MALT1, functions both as a scaffold to recruit downstream NF-κB signaling machinery, and as a protease to cleave and inactivate multiple substrates including negative regulators of NF-κB. Constitutive MALT1 activation, which can occur as a result of gain-of-function mutations of the B cell receptor, CARMA1, BCL10 or chromosomal translocation involving the MALT1 gene, underlies the pathogenesis of multiple lymphoid malignancies including activated B-cell type-diffuse large B-cell lymphoma (ABC-DLBCL) and MALT lymphoma. We previously identified G-protein-coupled receptor kinase 2 (GRK2) as a tumor suppressor in MALT1-dependent lymphomas which binds MALT1 and inhibits MALT1 scaffolding and proteolytic activities. Accordingly, GRK2 mRNA levels are markedly lower in a subset of DLBCL tumors compared to normal B cells and lower GRK2 level in ABC-DLBCL is associated with reduced patient survival. In current study, we sought to investigate the mechanism of how GRK2 levels are regulated in ABC-DLBCL. Methods/Results: Using DICER1 depleted cells, we showed that globally impaired miRNA processing leads to increased GRK2 protein levels. We then conducted bioinformatic analyses of patient tumor samples and performed screening via microRNA Data Integration Portal to identify microRNAs that target GRK2 in ABC-DLBCL. Further GRK2 3’UTR reporter assay confirmed direct targeting of GRK2 by candidate miRNAs. ABC-DLBCL cell lines, OCI-LY3 and TMD8, which demonstrate reduced GRK2 mRNA expression compared to primary B cells, show elevated expression of candidate GRK2-targeting microRNAs. Stable overexpression of miR-125a, 125b, or 148b in OCI-LY3 or TMD8 cells led to reduced GRK2 expression, enhanced MALT1 activity and increased cell proliferation. Conversely, in vitro inhibition of candidate miRNAs using anti-miRNA Locked Nucleic Acids (LNA) inhibitors led to enhanced GRK2 expression, reduced MALT1 activity and decreased cell proliferation. Importantly, treatment of mice with inhibitors of miR-125b and miR-148b abrogates the growth of ABC-DLBCL xenograft tumors in vivo. Conclusion: We have identified miRNAs that negatively regulate GRK2 expression in ABC-DLBCL. These GRK2-targeting miRNAs serve a pro-tumorigenic role by promoting MALT1 oncoprotein’s scaffold and protease activity. Furthermore, inhibitors of these miRNAs can enhance GRK2 expression and thereby suppress MALT1 activity and MALT1-dependent tumor cell proliferation, both in vitro and in vivo. These studies suggest that microRNA inhibitors which enhance GRK2 expression could represent a novel approach for restraining MALT1-dependent lymphomagenesis. Citation Format: Jing Cheng, Mei Smyers, Matt Trotta, Neil M. Carleton, Lisa M. Maurer, Uma R. Chandran, Min Xia, Ari M. Melnick, Peter C. Lucas, Linda M. McAllister-Lucas. MicroRNAs targeting tumor suppressor GRK2: Novel potential therapeutic strategies for restraining MALT1-dependent DLBCL [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 2061.

Abstract Introduction: Detectable circulating tumor DNA (ctDNA) is associated with inferior outcomes in Ewing sarcoma (ES); however, ctDNA clears in nearly all patients within 2 of the 6 chemotherapy cycles before radiotherapy (RT) or surgery. Given a 3-year EFS of 37% on AEWS 1221 (metastatic) and 5-year EFS of 78% on AEWS 1031 (localized), current ctDNA testing fails to predict most relapses. Extracellular vesicles (EVs) are microscopic particles studied as a biomarker in multiple cancer types. We hope to improve the sensitivity of ctDNA in ES and provide the first application of EVs in ES. Methods: cfDNA was isolated from 1mL plasma, quantified by Qubit, and sequenced as NEBNext Enzymatic Methyl-seq v2 libraries. We performed a systematic unsupervised clustering analysis of 27 ES plasma cfDNA profiles using 4 differentially methylated regions (DMR) subsets (63 strong DMRs, top 30, top 15, and 10 promoter-proximal loci), 3 clustering algorithms (k-means, Ward hierarchical, PAM), and k=2-5 clusters, testing associations with tumor burden and progression. EV analysis involved antibody detection against ES surface markers, including LINGO1, ENPP1, CDH11, and CD99. Concentrations of EVs were determined by standardized and calibrated high-resolution flow cytometry then analyzed with custom-built software to automatically determine and apply gates. Samples were de-identified, randomly coded, and run in triplicates. The senior research technologist responsible for EV analysis was blinded to patient information and clinical data. Results: EV analysis included 36 pre-RT samples from 16 metastatic and 20 localized ES patients from a prospectively maintained biobank, with 27 pre-RT samples in the cfDNA analysis and 19 healthy donor (HD) samples. 35 of 36 patients had chemotherapy before sample collection. cfDNA concentrations were markedly elevated in ES patients compared with HD (24.4 vs 4.6 ng/mL, p<0.0001), and 74% of ES samples exceeded the HD 95th-percentile detectability threshold (7.0 ng/mL). Across clustering configurations, ES cfDNA profiles consistently exhibited reproducible methylation substructure. One configuration (PAM clustering, k = 5, 63 strong DMRs) showed a modest but permutation-supported association with primary tumor size (nominal p ≈ 0.04; empirical p ≈ 0.03-0.04). ES samples had increased EV concentration compared to HD (p<0.001), with AUC 0.988 for distinguishing between ES and HD ENPP1 (p<0.0001). The highest ENPP1 EV values were in patients with large primary tumors (>8 cm vs. ≤ 8cm) and metastatic disease (p=0.066). Conclusions: cfDNA- and EV-based liquid biopsy provide encouraging signals for high ES detectability pre-RT. We also show the first study of EVs as a novel biomarker in ES, which may provide complementary information to ctDNA. Multimodal liquid biopsy in ES may identify candidate patients for systemic or local therapy escalation and facilitate personalized care. Citation Format: Roman O. Kowalchuk, Lilli J. Greiner, Yohan Kim, Rubia Noori, Jeffrey Szymanski, William S. Harmsen, Pradeep S. Chauhan, Adam C. Amundson, Andrea M. McMahon, Peter J. Schoettler, Wendy A. Allen-Rhoades, Stephanie F. Polites, Peter S. Rose, Kelly M. Bailey, Linda M. McAllister-Lucas, Anita Mahajan, Fabrice Lucien-Matteoni, Nadia N. Laack, Aadel A. Chaudhuri. Multimodal liquid biopsy of Ewing sarcoma reveals high sensitivity prior to radiotherapy [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 1149.

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.

Diffuse large B cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma, and the activated B cell–like subtype (ABC-DLBCL) is associated with particularly poor outcome. Many ABC-DLBCLs harbor gain-of-function mutations that cause inappropriate assembly of the CARMA1-BCL10-MALT1 (CBM) signalosome, a cytoplasmic complex that drives downstream NF-κB signaling. MALT1 is the effector protein of the CBM signalosome such that its recruitment to the signalosome via interaction with BCL10 allows it to exert both protease and scaffolding activities that together synergize in driving NF-κB. Here, we demonstrate that a molecular groove located between two adjacent immunoglobulin-like domains within MALT1 represents a binding pocket for BCL10. Leveraging this discovery, we performed an in silico screen to identify small molecules that dock within this MALT1 groove and act as BCL10-MALT1 protein-protein interaction (PPI) inhibitors. We report the identification of M1i-124 as a first-in-class compound that blocks BCL10-MALT1 interaction, abrogates MALT1 scaffolding and protease activities, promotes degradation of BCL10 and MALT1 proteins, and specifically targets ABC-DLBCLs characterized by dysregulated MALT1. Our findings demonstrate that small-molecule inhibitors of BCL10-MALT1 interaction can function as potent agents to block MALT1 signaling in selected lymphomas, and provide a road map for clinical development of a new class of precision-medicine therapeutics.

Abstract Safe delivery of mRNA to the brain will revolutionize the treatment of brain tumors. While lipid nanoparticles (LNPs) are clinically most advanced non-viral delivery vehicles for therapeutic mRNA, LNP-mediated mRNA delivery to the brain remains challenging. We hypothesized that rationally designed LNPs based on extracellular vesicle mimicry would enable efficient delivery of RNA therapeutics to brain cells without undue toxicity. We engineered LNPs consisting of four components similar to the formulation used in the mRNA COVID-19 vaccines (Moderna and Pfizer-BioNTech): ionizable lipid, cholesterol, helper lipid and polyethylene glycol (PEG)-lipid. We screened ten classes of helper lipids based on lipids enriched in extracellular vesicles to engineer biomimetic LNPs and tested their GFP mRNA delivery efficacy in SIM-A9 mouse microglia cell line. Several unique LNP formulations with potent delivery efficacy (>90% cells transfected) and stable GFP expression kinetics (5 days) were identified. LNP formulations with high transfection efficacy were then tested in vivo for luciferase mRNA delivery via intrathecal injection in C57BL/6 mice. Luciferase expression in vivo confirmed widespread mRNA delivery in the brain. We then tested Cre recombinase mRNA delivery in Ai9 mouse to identify LNP-targeted cells via flow cytometry and histology. Flow cytometry and expansion microscopy confirmed Cre recombinase mRNA delivery to a variety of brain cells, including microglia (75-90%), neurons (31-40%), neural stem cells (39-62%), oligodendrocytes (70-90%), and astrocytes (44-76%). LNPs were further evaluated for Cas9 mRNA and CD81 sgRNA delivery in C57BL/6 mouse brains to assess brain-targeted gene editing. Sanger sequencing showed that CRISPR-Cas9 editing was successful in ~40% of cells in the mouse brain. In summary, we engineered extracellular vesicle-based LNP library that can deliver RNA therapeutics to a variety of brain cells in vivo. With further development, this technology could potentially enable genetic and epigenetic therapies targeting drivers of brain tumors.

Abstract BACKGROUND Glioblastoma (GBM), one of the deadliest primary tumors of the central nervous system, is characterized by a highly immunosuppressive tumor microenvironment (TME). Tumor-associated macrophages and microglia (TAMs) are a dominant population of immune cells in the GBM TME and substantially contribute to immunosuppression, tumor progression, and treatment resistance. Elucidating the molecular mechanisms underlying TAM behavior is crucial to the development of effective immuno-therapeutic strategies for treating GBM. The adaptor protein, Caspase Recruitment Domain-containing protein 9 (CARD9) is primarily expressed in myeloid cells and its activation triggers assembly of the CARD9-BCL10-MALT1 (CBM) complex, which then promotes activation of NF-κB and/or MAPK pathways. The aim of this study was to investigate the role of the CBM complex in the GBM TME and assess its potential as a therapeutic target. METHODS/RESULTS In silico analyses revealed that GBM tumors demonstrate increased CARD9 and MALT1 expression in comparison to non-tumor brain tissue, with CARD9 expressed only within the myeloid compartment. Using co-culture, scratch wound, and transwell assays, we found that GBM tumor cells induce macrophage M2 polarization and migration via a mechanism that is dependent on CARD9/MALT1 signaling within macrophages. scRNA-Seq and flow-cytometric analysis of orthotopic syngeneic immunocompetent GBM tumor models revealed that the GBM TME is dominated by macrophages. Disruption of MALT1 protease activity within the TME leads to reduced tumor infiltration by immunosuppressive TAMs, remodeling of TAMs toward an immunoreactive anti-tumor phenotype, and improved survival of GBM-bearing mice. Additionally, we found that tumor growth was abrogated when GBM-bearing mice were treated with either a MALT1-protease inhibitor or a CARD9 inhibitor. CONCLUSIONS Our study suggests that blockade of the CARD9-MALT1 signaling axis impairs GBM-induced macrophage recruitment and M2 polarization and inhibits tumor progression. These findings nominate the CARD9-MALT1 signaling axis as a new target for TAM-directed immunotherapy for the treatment of GBM.