MALT1 Protease Research Articles

MALT1 protease inhibition restrains glioblastoma progression by reversing tumor-associated macrophage-dependent immunosuppression.

MALT1 protease is an intracellular signaling molecule that promotes tumor progression via cancer cell-intrinsic and cancer cell-extrinsic mechanisms. MALT1 has been mostly studied in lymphocytes, and little is known about its role in tumor-associated macrophages. Here, we show that MALT1 plays a key role in glioblastoma (GBM)-associated macrophages. Mechanistically, GBM tumor cells induce a MALT1-NF-κB signaling axis within macrophages, leading to macrophage migration and polarization toward an immunosuppressive phenotype. Inactivation of MALT1 protease promotes transcriptional reprogramming that reduces migration and restores a macrophage "M1-like" phenotype. Preclinical in vivo analysis shows that MALT1 inhibitor treatment results in increased immuno-reactivity of GBM-associated macrophages and reduced GBM tumor growth. Further, the addition of MALT1 inhibitor to temozolomide reduces immunosuppression in the tumor microenvironment, which may enhance the efficacy of this standard-of-care chemotherapeutic. Together, our findings suggest that MALT1 protease inhibition represents a promising macrophage-targeted immunotherapeutic strategy for the treatment of GBM.

Abstract PO-036: Targeting the CBM signalosome with a MALT1 scaffolding inhibitor for treatment of non-Hodgkin lymphomas

Abstract 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.

DIPG-85. MALT1 IN H3K27-ALTERED DIFFUSE MIDLINE GLIOMA

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.

MALT1 substrate cleavage: what is it good for?

CARD-BCL10-MALT1 (CBM) signalosomes connect distal signaling of innate and adaptive immune receptors to proximal signaling pathways and immune activation. Four CARD scaffold proteins (CARD9, 10, 11, 14) can form seeds that nucleate the assembly of BCL10-MALT1 filaments in a cell- and stimulus-specific manner. MALT1 (also known as PCASP1) serves a dual function within the assembled CBM complexes. By recruiting TRAF6, MALT1 acts as a molecular scaffold that initiates IκB kinase (IKK)/NF-κB and c-Jun N-terminal kinase (JNK)/AP-1 signaling. In parallel, proximity-induced dimerization of the paracaspase domain activates the MALT1 protease which exerts its function by cleaving a set of specific substrates. While complete MALT1 ablation leads to immune deficiency, selective destruction of either scaffolding or protease function provokes autoimmune inflammation. Thus, balanced MALT1-TRAF6 recruitment and MALT1 substrate cleavage are critical to maintain immune homeostasis and to promote optimal immune activation. Further, MALT1 protease activity drives the survival of aggressive lymphomas and other non-hematologic solid cancers. However, little is known about the relevance of the cleavage of individual substrates for the pathophysiological functions of MALT1. Unbiased serendipity, screening and computational predictions have identified and validated ~20 substrates, indicating that MALT1 targets a quite distinct set of proteins. Known substrates are involved in CBM auto-regulation (MALT1, BCL10 and CARD10), regulation of signaling and adhesion (A20, CYLD, HOIL-1 and Tensin-3), or transcription (RelB) and mRNA stability/translation (Regnase-1, Roquin-1/2 and N4BP1), indicating that MALT1 often targets multiple proteins involved in similar cellular processes. Here, we will summarize what is known about the fate and functions of individual MALT1 substrates and how their cleavage contributes to the biological functions of the MALT1 protease. We will outline what is needed to better connect critical pathophysiological roles of the MALT1 protease with the cleavage of distinct substrates.

Abstract A002: MALT1 as a regulator of tumor progression in H3K27-altered diffuse midline glioma

Abstract A002: MALT1 as a regulator of tumor progression in H3K27-altered diffuse midline glioma

Abstract C127: Discovery of AUR-112, a novel MALT1 protease inhibitor for the treatement of B cell lymphomas

Abstract MALT1 is a key regulator of antigen receptor signaling in B and T cells. MALT1 partners with BCL10 and CARMA1 to form the CBM complex in which protease activity of MALT1 cleaves the negative regulators (A20, CYLD, RelB) leading to activation of canonical NF-kB pathway. Constitutive activation of NF-kB signaling is a key driver in B-cell lymphomas including diffuse large B-cell lymphomas (DLBCL). Therapeutic targeting of MALT1 protease activity can therefore be a useful approach for the treatment of B-cell lymphomas with dysregulated NF-kB signaling. We sought to discover and develop MALT1 inhibitors with the “best-in-class” profile with an emphasis on lowering plasma protein binding (PPB) to achieve improved pharmacological outcome. After extensive SAR efforts utilizing various computational methods, AUR-112 has been identified as a potential development candidate. The allosteric inhibitor AUR-112 exhibited potent and selective inhibition of MALT1 protease activity in both biochemical and cellular assays. Inhibition of MALT1 correlated with potent antiproliferative activity in an ABC-DLBCL cell line. The mechanistic activity of AUR-112 is supported by the inhibition of IL-10 in a cell line and IL-2 in human whole blood. Consistent with our objective, AUR112 showed low plasma protein binding across multiple species, which translated to greater potency in whole blood compared to published MALT1 inhibitors, positioning it as a best-in-class contender. AUR-112 has excellent oral bioavailability across species and exhibited desirable selectivity profile in kinase, protease and CEREP safety screen panels. Oral administration of AUR-112 led to dose dependent anti-tumor activity including durable tumor regression in cell line and patient derived xenograft models. Treatment with AUR-112 led to potent pharmacodynamic modulation in xenograft models as measured by significant and sustained reduction in serum IL-10 levels that correlated with the compelling efficacy. AUR-112 also demonstrated dose dependent inhibition of T-reg cells in vitro and in vivo supporting the immune modulatory role of MALT1 inhibition. AUR-112 is identified as a development candidate with potentially best-in-class physico-chemical properties resulting in a high free fraction for treatment of ABC-DLBCL patients. IND enabling studies are currently ongoing to support the clinical development of AUR-112. Citation Format: Wesley Roy Balasubramanian, Sammeta Srinivasa Raju, Vasantha K.Y, Archana Bhumi Reddy, Janith Mary Maben, Sandeep Sadashiv Patil, Raghavendra N.R, Kiran Aithal B, Charamanna K.B, Dinesh Chikkanna, Kavitha Nellore, Samiulla D.S, Girish Daginakatte, Shekar Chelur, Murali Ramachandra, Susanta Samajdar. Discovery of AUR-112, a novel MALT1 protease inhibitor for the treatement of B cell lymphomas [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C127.

Discovery of Novel, First-in-Class Allosteric Modulators of MALT1 Scaffolding Function with Differentiated Pharmacology for NFκB-Driven Malignancies

Constitutive activation of NFκB is an oncogenic driver for many lymphoid and selected solid tumor malignancies. BTK inhibitors, which affect the upstream NFκB pathway, have been successfully used in some diseases, but their utility is limited. The majority of DLBCL patients, for instance, are insensitive to BTK inhibition, supporting the need for alternative mechanisms to block NFκB-driven tumor progression. Knockdown of MALT1, a key component of the CBMT complex (CARD-BCL10-MALT1-TRAF6) involved in direct signal transduction of NFκB, is able to block NFκB-dependent DLBCL tumor growth in BTKi sensitive or resistant tumors, but pharmacologic modulation has been challenged by the dual-functionality of MALT1 in a complexed signalosome. MALT1 functions as the scaffolding component of the CBMT complex. Activated MALT1 recruits multiple proteins and directly activates NFκB canonical signaling. MALT1 also has protease activity that fine-tunes various signaling pathways including NFκB via the cleavage of many substrates. The scaffolding activity of MALT1 is critical for cancer cell survival. Most MALT1 inhibitors discovered so far only block protease activity, which results in incomplete NFκB inhibition, suboptimal anti-tumor activity, and the induction of severe autoimmunity due to protease inhibition. Using our Smart Allostery platform, we elucidated and then drugged a natural hotspot on MALT1 that selectively impacts scaffolding but not protease function. The series of scaffolding modulators demonstrate potent inhibition of MALT1 scaffolding activity with cellular IC 50 values ranging from 15 to 250 nM, while exerting minimal influence on MALT1 protease function. Unlike protease inhibitors, which only partially affect NFκB activity, scaffolding modulators completely inhibit the NFκB downstream signaling manifested by the suppression of NFκB activity with cellular IC 50 values ranging from 2 to 90 nM. Consequently, scaffolding modulators exhibit a superior and broader anti-proliferation profile compared to MALT1 protease inhibitors and BTK inhibitors in a panel of lymphoma cell lines. Pathway and genetic analyses indicate that B-cell lymphoma lines driven by NFκB are especially sensitive to scaffolding modulators. In vivo studies demonstrate a robust, dose-dependent tumor growth inhibition (TGI) by one representative scaffolding modulator in several B-cell lymphoma xenograft models. In a protease inhibitor sensitive ABC-DLBCL model, OCI-LY10, our scaffolding modulator achieved a maximal TGI of 94%, while a clinical MALT1 protease inhibitor showed 62% maximal TGI (p<0.001). In protease resistant NFκB driven DLBCL models, MALT1 scaffolding modulation resulted in tumor regression or stasis. These findings highlight the differentiated pharmacology of our scaffolding modulators. Distinct from protease inhibitors that suppress T cell activation and Treg population in vivo, scaffolding modulators also did not deplete Treg cells nor suppress T cell activation in a 14-day study. This potentially mitigates the risk of development of autoimmune disease caused by chronic MALT1 protease inhibition. In summary, we have discovered novel, first-in-class allosteric modulators of MALT1 scaffolding activities. These scaffolding modulators drive enhanced and broader anti-tumor effect in models resistant to MALT1 protease or BTK inhibitors and do so without immune dysregulation. Scaffolding modulation represents a potential best-in-class therapeutic opportunity to modulate MALT1 in patients with NFκB-driven malignancies.

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 inhibition alleviates intestinal impairment induced by chronic heat stress in finisher broilers

Heat stress (HS) in poultry has deleterious effects on intestinal development and barrier function, along with inflammatory outbursts. In the present study, chronic HS reduced body weight of broilers and activated mucosa-associated lymphoid tissue lymphoma translocation protein 1 (Malt1) /nuclear factor kappa B (NF-κB) signaling pathways to elicit the inflammatory cytokine response in jejunum. Subsequently, this study investigated the protective effects of the Malt1 inhibitor on the intestine of broilers under HS conditions. The 21-day-old male broilers were allocated to 8 pens housed in HS room (34°C for 7 h/d) until 28 d of age. During this period, 4 birds were selected from each heat-stressed pen and received intraperitoneal injection of 20 mg/kg body weight Mepazine (a Malt1 inhibitor) or the equivalent volume of phosphate buffer saline (PBS) every other day. When compared to PBS broilers, birds received Mepazine injection exhibited increased relative weight and higher villus height in jejunum (both P < 0.05). Mepazine treatment also increased (P < 0.05) the mRNA of zonula occludens-1 (ZO-1), claudin-1, and cadherin 1 of jejunum, which was companied by the reduced caspase-3 transcription under HS condition. Meanwhile, the gene expression levels of toll-like receptor 4 (TLR4), Malt1, NF-κB, interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) in the jejunum were significantly downregulated by Mepazine administration (P < 0.05). Although there were no significant differences in the relative weight of the thymus and bursa, the transcription levels of T helper 1 (Th1)- and Th17-related cytokines were lower in thymus of birds injected with Mepazine. The cytokines of Treg cytokine transforming growth factor beta (TGF-β) and forkhead box protein P3 (Foxp3) in both the thymus and bursa were not influenced. These results suggest that inhibition of Malt1 protease activity can protect intestinal integrity by promoting the production of tight junction proteins and attenuating NF-κB-mediated intestinal inflammation response under HS conditions.

Chemical Probes for Profiling of MALT1 Protease Activity.

The paracaspase MALT1 is a key regulator of the human immune response. It is implicated in a variety of human diseases. For example, deregulated protease activity drives the survival of malignant lymphomas and is involved in the pathophysiology of autoimmune/inflammatory diseases. Thus, MALT1 has attracted attention as promising drug target. While many MALT1 inhibitors have been identified, molecular tools to study MALT1 activity, target engagement and inhibition in complex biological samples, such as living cells and patient material, are still scarce. Such tools are valuable to validate MALT1 as a drug target in vivo and to assess yet unknown biological roles of MALT1. In this review we discuss the recent literature on the development and biological application of molecular tools to study MALT1 activity and inhibition.

CARD11 gain of function upregulates BCL2A1 expression and promotes resistance to targeted therapies combination in B-cell lymphoma

AbstractA strategy combining targeted therapies is effective in B-cell lymphomas (BCL), such as mantle cell lymphoma (MCL), but acquired resistances remain a recurrent issue. In this study, we performed integrative longitudinal genomic and single-cell RNA-sequencing analyses of patients with MCL who were treated with targeted therapies against CD20, BCL2, and Bruton tyrosine kinase (OAsIs trial). We revealed the emergence of subclones with a selective advantage against OAsIs combination in vivo and showed that resistant cells were characterized by B-cell receptor (BCR)–independent overexpression of NF-κB1 target genes, especially owing to CARD11 mutations. Functional studies demonstrated that CARD11 gain of function not only resulted in BCR independence but also directly increased the transcription of the antiapoptotic BCL2A1, leading to resistance against venetoclax and OAsIs combination. Based on the transcriptional profile of OAsIs-resistant subclones, we designed a 16-gene resistance signature that was also predictive for patients with MCL who were treated with conventional chemotherapy, underlying a common escape mechanism. Among druggable strategies to inhibit CARD11-dependent NF-κB1 transduction, we evaluated the selective inhibition of its essential partner MALT1. We demonstrated that MALT1 protease inhibition led to a reduction in the expression of genes involved in OAsIs resistance, including BCL2A1. Consequently, MALT1 inhibition induced synergistic cell death in combination with BCL2 inhibition, irrespective of CARD11 mutational status, both in vitro and in vivo. Taken together, our study identified mechanisms of resistance to targeted therapies and provided a novel strategy to overcome resistance in aggressive BCL. The OAsIs trial was registered at www.clinicaltrials.gov #NCT02558816.

MALT1 protease inhibition overcomes BTK inhibitor resistance and shows synergistic activity with venetoclax in models of B cell lymphoma and leukemia

MALT1 protease inhibition overcomes BTK inhibitor resistance and shows synergistic activity with venetoclax in models of B cell lymphoma and leukemia

Proteolytic Activity of the Paracaspase MALT1 Is Involved in Epithelial Restitution and Mucosal Healing.

The paracaspase MALT1 is a crucial regulator of immune responses in various cellular contexts. Recently, there is increasing evidence suggesting that MALT1 might represent a novel key player in mucosal inflammation. However, the molecular mechanisms underlying this process and the targeted cell population remain unclear. In this study, we investigate the role of MALT1 proteolytic activity in the context of mucosal inflammation. We demonstrate a significant enrichment of MALT1 gene and protein expression in colonic epithelial cells of UC patients, as well as in the context of experimental colitis. Mechanistically we demonstrate that MALT1 protease function inhibits ferroptosis, a form of iron-dependent cell death, upstream of NF-κB signaling, which can promote inflammation and tissue damage in IBD. We further show that MALT1 activity contributes to STAT3 signaling, which is essential for the regeneration of the intestinal epithelium after injury. In summary, our data strongly suggests that the protease function of MALT1 plays a critical role in the regulation of immune and inflammatory responses, as well as mucosal healing. Understanding the mechanisms by which MALT1 protease function regulates these processes may offer novel therapeutic targets for the treatment of IBD and other inflammatory diseases.

Abstract 4442: Dual benefit of MALT1 blockade in glioblastoma

Abstract Background: Glioblastoma multiforme (GBM) is the most common and aggressive malignant primary brain tumor. Despite advances in treatment modalities, GBM remains largely incurable and new treatment approaches are desperately needed. An innovative strategy for cancer therapy is to combine the inhibition of cancer cell-intrinsic oncogenic signaling with cancer cell-extrinsic immunological activation of the tumor microenvironment (TME). MALT1 represents a candidate target whose inhibition may provide this dual benefit. Originally identified as a proto-oncoprotein in lymphoma, MALT1 has recently also been implicated in multiple solid tumors, including GBM, and significant effort has been made to develop MALT1 protease inhibitors as anti-cancer agents. Interestingly, blockade of MALT1 protease has also been shown to selectively reprogram tumor infiltrating Treg cells into antitumor effector cells. Based on these observations, we hypothesized that in GBM, MALT1 acts both within tumor cells and within cells of the TME to promote tumorigenesis and MALT1 inhibitor treatment could thus have dual benefit. Methods/Results: We assembled a panel of mouse and human GBM cell lines and performed western-blot analysis to show that these cells demonstrate constitutive MALT1 expression and proteolytic activity. MALT1 blockade in GBM cells, using siRNA or MALT1 protease inhibitors, reduces viability and clonogenic potential, suggesting that inhibition of MALT1 could impair tumor cell survival. Single-cell RNAseq data from public datasets demonstrated that myeloid cells have the highest MALT1 expression in the GBM TME. Our data indicate that co-culture of macrophages with GBM cells induces MALT1-NF-κB activation within the macrophages and this is associated with polarization towards an M2-like immunosuppressive phenotype. Additionally, treating primary human or mouse macrophages with MALT1-protease inhibitor prevents GBM-induced M2-polarization, increases M1-polarization and enhances GBM cell killing. In a syngeneic orthotopic mouse model, GBM tumor growth is impaired, and survival is extended when tumor cells are implanted into MALT1-protease dead (PD) host mice (mice which harbor a point mutation in the MALT1-protease catalytic cysteine) as compared to when implanted into wild-type host mice. MALT1-PD mice demonstrate a less immunosuppressive GBM TME with increased M1-macrophages and decreased M2-macrophages, MDSCs and Tregs. Taken together, our findings suggest that inhibiting MALT1 protease in TME cells promotes anti-tumor immunity against brain tumors. Conclusions: Our studies suggest MALT1 protease as a new therapeutic target in GBM whose inhibition could have dual benefit via both direct effect on tumor cells and by reversing the immunosuppressive TME. Citation Format: Juliana Hofstatter Azambuja, Saigopalakrishna S. Yerneni, Gabriela N. Debom, Lisa M. Maurer, Hannah E. Butterfield, Linda R. Klei, Gary Kohanbash, Peter C. Lucas, Linda M. McAllister-Lucas. Dual benefit of MALT1 blockade in glioblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4442.

Abstract 1762: MALT1 is activated by doxorubicin and mediates therapy resistance in triple-negative breast cancer

Abstract Background: Breast cancer is the most commonly diagnosed malignancy in American women. The triple-negative breast cancer (TNBC) subtype has among the worst prognosis due to high rates of recurrence and metastasis. Since TNBC lacks targetable receptor proteins, treatment relies upon non-specific chemotherapy, which can become ineffective upon onset of resistance. One potential driver of TNBC treatment resistance is MALT1, the effector component of the CARMA-BCL10-MALT1 signaling complex, which activates NF-κB in multiple cancer cell types, including TNBC. Notably, breast cancer cells demonstrate increased sensitivity to chemotherapies such as doxorubicin and cisplatin when MALT1 is depleted. NF-κB activation has been suggested to promote DNA repair and the MALT1-NF-κB inducible factor, IL-6 has been shown to repress the immunogenic effects of doxorubicin. Hence, we hypothesize that MALT1 is a pharmaceutically targetable driver of TNBC treatment resistance. Mechanistically, we hypothesize that MALT1 promotes chemotherapy resistance by enhancing DNA repair and suppressing immunogenic cell death. Methods/Results: We analyzed RNAseq and proteomic data from TCGA and CPTAC, respectively, and found that MALT1 is highly expressed in basal breast cancer (a subtype largely composed of TNBC) and its expression level in this context is associated with reduced pathological complete response and survival. With the purpose of evaluating the effect of MALT1 protease inhibition on chemotherapy sensitivity, we first sought to identify TNBC cell lines that were most resistant to doxorubicin using GDSC and CTRP databases. Results indicated that MDA-MB-231, BT20 and HCC1143 were highly resistant. We then performed western blots to assess MALT1 expression and CellTiter-Glo assays to determine the doxorubicin IC50s for these cell lines. Results indicate that MALT1 expression correlates with the degree of doxorubicin resistance among these cell lines. To determine if MALT1 blockade, via siRNA-knockdown or MALT1 protease inhibitor treatment (JNJ-67856633 or MLT-748), increased doxorubicin sensitivity, CellTiter-Glo and Incucyte Caspase-3/7 assays were performed. Findings indicated that MALT1 blockade results in decreased cell viability and increased apoptosis in response to doxorubicin. Mechanistically, MALT1 protease is activated by doxorubicin and MALT1 inhibition increases expression of the immunogenic cell death protein, calreticulin (CRT) in response to doxorubicin. Conclusions and Future Directions: Initial studies suggest that targeting MALT1 enhances TNBC sensitivity to doxorubicin. We will next assess the effect of MALT1 blockade on DNA repair mechanisms in doxorubicin-treated TNBC cells. Additionally, we will test the role of MALT1 in suppressing immunogenic cell death. Through these studies, we hope to inform new approaches for improving treatment response in TNBC. Citation Format: Maria Beecher, Dong Hu, Linda Klei, Jack Little, Linda M. McAllister-Lucas, Peter C. Lucas. MALT1 is activated by doxorubicin and mediates therapy resistance in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1762.

Abstract 6156: MALT1 protease inhibition overcomes BTK inhibitor resistance and shows synergistic activity with venetoclax in models of B cell lymphoma and leukemia

Abstract Introduction: Specific B cell malignancies, including CLL and the aggressive non-GCB subtype of DLBCL, are driven by constitutive activation of the B cell receptor (BCR) pathway and the transcription factor NF-κB. Pharmacological inhibition of MALT1 protease, a key mediator of the BCR/NF-κB signal transduction pathway, may therefore provide an attractive treatment option for patients with these cancers. Further, as combination therapy is often required for the treatment of aggressive B cell malignancies, the identification of therapies that synergistically combine with MALT1 inhibitors could afford additional and promising treatment options. Experimental Procedures: A highly potent and orally bioavailable MALT1 protease inhibitor (ABBV-MALT1) was used to test the hypothesis that MALT1 inhibition will abrogate the proliferation of preclinical models of B cell malignancies in vitro and in vivo. Tumors treated with ABBV-MALT1 were subjected to transcriptomic and functional proteomic assays to elucidate molecular mechanisms of action and rational combination partners. Results: Mechanistic studies reveal that ABBV-MALT1 effectively inhibits signal transduction of the BCR pathway and reduces NF-κB gene activation in non-GCB DLBCL cell lines resulting in cell cycle arrest and diminished viability. In vivo, oral administration of this compound demonstrates robust tumor growth inhibition in several models of B cell tumors, including non-GCB DLBCL models that are resistant to Bruton’s tyrosine kinase (BTK) inhibitors. NF-κB target genes include the pro-survival family members BCL-XL and BCL2-A1, which aid in regulation of the intrinsic apoptosis pathway. As ABBV-MALT1-induced inhibition of the NF-κB pathway resulted in downregulation of these genes, we hypothesized that the associated tumor models would become increasingly dependent on the pro-survival family member BCL-2. To test this hypothesis, combination studies of ABBV-MALT1 and the selective BCL-2 inhibitor venetoclax were performed in both cell line and patient-derived xenograft models of DLBCL. Herein we show that concomitant administration of ABBV-MALT1 and venetoclax results in dramatic antitumor activity in all models tested in vivo. This efficacy also translates to primary patient CLL cells in vitro where the combination confers greater levels of apoptosis compared to either agent alone. Conclusion: ABBV-MALT1 demonstrates robust single agent anti-tumor activity in malignant B cell models that are resistant to BTK inhibitors. Moreover, combination of ABBV-MALT1 with the BCL-2 inhibitor venetoclax shows synergistic cell killing of B cell tumors in vitro and dramatic tumor regression in vivo. Together, these data indicate that MALT1 inhibition may overcome BTK inhibitor resistance and combine with venetoclax to effectively treat patients with B cell malignancies. Citation Format: Joshua P. Plotnik, Raghuveer S. Mali, Velitchka Bontcheva, Colleen Dowell, Jun Chen, Adam E. Richardson, Ryan A. McClure, Paul Jung, Lan Pham, Andrew J. Souers, Jonathan A. Meulbroek, William N. Pappano. MALT1 protease inhibition overcomes BTK inhibitor resistance and shows synergistic activity with venetoclax in models of B cell lymphoma and leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6156.

TRAF6 controls T cell homeostasis by maintaining the equilibrium of MALT1 scaffolding and protease functions.

MALT1 is a core component of the CARD11-BCL10-MALT1 (CBM) signalosome, in which it acts as a scaffold and a protease to bridge T cell receptor (TCR) ligation to immune activation. As a scaffold, MALT1 binds to TRAF6, and T cell-specific TRAF6 ablation or destruction of MALT1-TRAF6 interaction provokes activation of conventional T (Tconv) effector cells. In contrast, MALT1 protease activity controls the development and suppressive function of regulatory T (Treg) cells in a T cell-intrinsic manner. Thus, complete loss of TRAF6 or selective inactivation of MALT1 catalytic function in mice skews the immune system towards autoimmune inflammation, but distinct mechanisms are responsible for these immune disorders. Here we demonstrate that TRAF6 deletion or MALT1 paracaspase inactivation are highly interdependent in causing the distinct immune pathologies. We crossed mice with T cell-specific TRAF6 ablation (Traf6-ΔT) and mice with a mutation rendering the MALT1 paracaspase dead in T cells (Malt1 PD-T) to yield Traf6-ΔT;Malt1 PD-T double mutant mice. These mice reveal that the autoimmune inflammation caused by TRAF6-ablation relies strictly on the function of the MALT1 protease to drive the activation of Tconv cells. Vice versa, despite the complete loss of Treg cells in Traf6-ΔT;Malt1 PD-T double mutant mice, inactivation of the MALT1 protease is unable to cause autoinflammation, because the Tconv effector cells are not activated in the absence of TRAF6. Consequentially, combined MALT1 paracaspase inactivation and TRAF6 deficiency in T cells mirrors the immunodeficiency seen upon T cell-specific MALT1 ablation.

Normal lymphocyte homeostasis and function in MALT1 protease-resistant HOIL-1 knock-in mice.

The uniqueness of MALT1 protease activity in controlling several aspects of immunity in humans has made it a very attractive therapeutic target for multiple autoimmune diseases and lymphoid malignancies. Despite several encouraging preclinical studies with MALT1 inhibitors, severe reduction in regulatory T cells and immune-mediated pathology seen in MALT1 protease-dead (MALT1-PD) mice and some, but not all, studies analysing the effect of prolonged pharmacological MALT1 protease inhibition, indicates the need to further unravel the mechanism of MALT1 protease function. Notably, the contribution of individual MALT1 substrates to the immune defects seen in MALT1-PD mice is still unclear. Previous in vitro studies indicated a role for MALT1-mediated cleavage of the E3 ubiquitin ligase HOIL-1 in the modulation of nuclear factor-κB (NF-κB) signalling and inflammatory gene expression in lymphocytes. Here, we addressed the immunological consequences of inhibition of HOIL-1 cleavage by generating and immunophenotyping MALT1 cleavage-resistant HOIL-1 knock-in (KI) mice. HOIL-1 KI mice appear healthy and have no overt phenotype. NF-κB activation in T or B cells, as well as IL-2 production and in vitro T-cell proliferation, is comparable between control and HOIL-1 KI cells. Inhibition of HOIL-1 cleavage in mice has no effect on thymic T-cell development and conventional T-cell homeostasis. Likewise, B-cell development and humoral immune responses are not affected. Together, these data exclude an important role of MALT1-mediated HOIL-1 cleavage in T- and B-cell development and function in mice.

3D-QSAR, drug-likeness, ADMET prediction, and molecular docking studies in silico of novel 5-oxo-1-thioxo-4,5-dihydro-1H-thiazolo[3,4-a]quinazoline derivatives as MALT1 protease inhibitors for the treatment of B cell lymphoma

3D-QSAR, drug-likeness, ADMET prediction, and molecular docking studies in silico of novel 5-oxo-1-thioxo-4,5-dihydro-1H-thiazolo[3,4-a]quinazoline derivatives as MALT1 protease inhibitors for the treatment of B cell lymphoma

CARD11 and BCL2A1 Join Forces to Promote Resistance to Ibrutinib/Venetoclax Combination in Lymphoma Patients

Background Mantle cell lymphoma (MCL) is a hard to cure malignancy with conventional chemotherapy. We previously reported that combinations targeting CD20 (obinutuzumab), BTK (ibrutinib) and BCL2 (venetoclax) counteracted both tumor intrinsic anomalies and dialogs within corrupted MCL ecosystems. Importantly, this chemo-free combination provided durable complete responses in patients both at relapse or naïve of treatments (OAsIs trial, NCT02558816). Despite high initial response rates, nearly 1/3 of patients rapidly relapsed. To understand this in vivo resistance, we performed targeted sequencing and single-cell RNA-seq to identify key actors involved. Methods Tumor DNA from patients enrolled in the OAsIs trial were sequenced at baseline (n=12) or relapse (n=5) (Capture xGen technology, IDT). Forty-nine genes and the 9p21 region, described for their impact in lymphoma resistance, were targeted for recurrent SNVs/CNVs. Single cell RNA-seq (3' gene expression, 10X genomics) was performed before treatment (n=4) or after relapse (n=3). Integrated omics analysis was performed to define resistance signatures, at the single cell resolution and associated to gain of function (GOF) mutations. Functional analysis (i.e., reporter assay, BH3-profiling, drug testing) using primary MCL cells and cell lines were performed. Results Targeted sequencing of tumor cells at relapse highlighted an enrichment of CARD11 GOF mutations (p=0.01). The selective advantage of CARD11MUT cells under OAsIs pressure was further documented with a patient who achieved a complete response before relapsing 12 months after inclusion. Whereas CARD11MUT tumor cells were in a minority (0.0005 %) at the onset of treatment, all the cells carried a heterozygous mutation at relapse (VAF: 50.56%). scRNA-seq of the same longitudinal samples identified a minor cluster (187/4860 sequenced cells) enriched at relapse that was characterized by expression of NFKB1 target genes consistent with constitutive CARD11 activity (Enrichr p<5x10-4). By combining scRNA-seq and mutations, we identified a signature associated with CARD11 GOF, which we named "OAsIs-R" signature. Applied to additional patients' samples, this novel signature allowed the segregation of OAsIs sensitive from resistant cells (scRNA-seq). Of major interest, OAsIs-R signature was also predictive for OS/PFS in MCL patients treated with conventional chemotherapy (GSE93291 and in-house cohorts). Among the top genes of OAsIs-R signature, we further focused on BCL2A1. We first highlighted its frequent expression in MCL primary cells (n=63) and cell lines (n=7). BCL2A1 overexpression, BH3-profiling (FS2 peptide) and ex vivo drug testing (AnnexinV) further validated a key role of CARD11-related BCL2A1 dysregulation in OAsIs resistance. Ibrutinib deeply reduced BCL2A1 level in CARD11WT cell lines but not in CARD11MUT cells (RNA expression inhibition: 82%, n=4 vs. 0%, n=4), leading to synergistic apoptosis when associated to venetoclax in BCL2A1+ but not in BCL2A1- cells (mean synergy score: 17 vs. 1, n=6). Using reporter assay experiments we showed that CARD11 mutations directly led to an increase activity of BCL2A1 promoter (2 fold more activity, Mut vs. wt), reinforcing the need to target its activity to counteract resistance. Among druggable strategies to inhibit CARD11, selective inhibition of its partner MALT1, essential for CARD11-BCL10-MALT1 (CBM) activity, is currently tested in early phase clinical trials. We demonstrated that MALT1 protease inhibition led to the reduction of genes involved in OAsIs resistance, including BCL2A1 (RNA inhibition: 40%, n=6), and induced synergistic cell death in combination with venetoclax (mean synergy score: 20, n=4), irrespective of CARD11 mutational status. Conclusions In summary, our data uncovered control of the mitochondrial apoptosis by CARD11 through BCL2A1 expression. This network has critical implication in the resistance to BTK/BCL2 targeted inhibition. Indeed, CARD11 GOF mutations led not only to BCR-independence and consequently ibrutinib resistance but also to BCL2A1-mediated venetoclax resistance. Nevertheless, our data show that targeting the CBM through MALT1 inhibition reverts this resistance and that MALT1 targeting appears as a promising therapeutic option for counteracting MCL resistance, especially in the context of acquired CARD11 mutation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract A28: BCL10 mutations define distinct dependencies guiding precision therapy for DLBCL

Abstract Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy and the activated B cell-like subtype (ABC-DLBCL) is the most aggressive form and harbors frequent mutations of immune signaling pathways that culminate in constitutive activation of CARD11-MALT1-BCL10 (CBM) complex and downstream NF-κB pathway. CBM complexes form large macromolecular structures due to signal-induced polymerization of the BCL10 subunit, which is affected by recurrent somatic mutations in ABC-DLBCLs. Through biochemical, structural and functional dissection of these mutations, we find that BCL10 mutations fall into two functionally distinct classes: missense mutations of the BCL10 CARD domain (hotspot R58Q) and truncation of its C-terminal tail (hotspot E140X). To explore the functional consequences of BCL10 mutations, we established reporter systems to evaluate their impact on MALT1 and NF-𝜅B activities which are BCL10 downstream signaling cascades. We found that almost all mutants induced aberrantly strong NF-𝜅B and MALT1 activities in lymphoma cells as compared to WT BCL10, indicating the gain-of-function effect of BCL10 mutations. By performing immunohistochemistry staining of p65 in a set of tumor tissue microarray from DLBCL patients (n=298), we revealed that BCL10 mutant tumors have significantly (Mann-Whitney p&amp;lt;0.0001) increased p65 nuclear staining score compared to BCL10 WT tumors, suggesting enhanced NF-𝜅B activity. To investigate the biochemical impact of BCL10 mutants on CBM complex formation, we performed fluorescence polarization and filamentation formation assays with purified WT and mutant BCL10 proteins and found that both BCL10R58Q and BCL10E140X manifested faster and more spontaneous polarization compared to BCL10WT. Surprisingly, through mapping the BCL10-MALT1 interaction, we found that truncating mutation (E140X) abrogated a novel protein interaction motif through which MALT1 inhibits BCL10 polymerization, thus unleashing spontaneous CBM filament formation and inducing addiction to MALT1 activity. In marked contrast, the CARD missense mutation (R58Q) on BCL10 filament interface not only does not disrupt but enhances filament formation and it also alters CBM complex kinetics forming glutamine network structures that stabilize BCL10 filaments, but this still may require the upstream signal to activate MALT1. Importantly, we found that BCL10 mutant cells were less dependent on upstream CARD11 activation in MALT1 activation, NF-𝜅B signaling and cell growth assays performed in ABC-DLBCL lines. Furthermore, in vitro and in vivo xenograft studies revealed that BCL10 mutant lymphomas are resistant to BTK inhibitors, whereas BCL10 truncating (E140X) but not missense CARD (R58Q) mutants were hypersensitive to MALT1 protease inhibitors. Therefore, BCL10 mutations are potential biomarkers for BTK inhibitor resistance in ABC-DLBCL and further precision can be achieved by tailoring therapy (e.g. MALT1 inhibitors that are currently being tested in clinical trials) according to specific biochemical effects of distinct mutation classes. Citation Format: Min Xia, Liron David, Matthew R Teater, Johana Gutierrez, Xiang Wang, Cem Meydan, Andrew Lytle, Graham Slack, David Scott, Ozlem Onder, Kojo Elenitoba-Johnson, Nahuel Zamponi, Leandro Cerchietti, Tianbao Lu, Ulrike Philippar, Lorena Fontan, Hao Wu, Ari Melnick. BCL10 mutations define distinct dependencies guiding precision therapy for DLBCL [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A28.