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Halting Multiple Myeloma with MALT1 Inhibition: Suppressing BCMA-Induced NF-κB and Inducing Immunogenic Cell Death

ABSTRACT: As multiple myeloma (MM) poses a formidable therapeutic challenge despite recent progress, exploring novel targets is crucial. Mucosa-associated lymphoid tissue lymphoma translocation protein-1 (MALT1) emerges as a promising paracaspase with druggable potential, especially unexplored in MM. Our study provided compelling evidence demonstrating a statistically significant elevation of MALT1 expression in human primary MM cells. Moreover, elevated MALT1 expression was associated with a poorer prognosis in MM. Genetic deletion of MALT1 reduced cell growth, colony formation, and tumor growth in vivo. Pharmacological inhibition with 1 μM Mi-2 effectively inhibited cell growth, inducing mitochondria-dependent apoptotic cell death. Mechanistically, MALT1 inhibition disrupted diverse signal transduction pathways, notably impeding nuclear factor κB (NF-κB). Significantly, the inhibition of MALT1 demonstrated a substantial suppression of NF-κB activation by elevating IκB, disrupting the nuclear localization of p65 and C-Rel. This effect was observed in both the basal state and when stimulated by BCMA, highlighting the pivotal role of MALT1 inhibition in influencing MM cell survival. It was noteworthy that Mi-2 induces properties associated with immunogenic cell death (ICD), as evidenced by increased calreticulin (CRT), ATP release, and high-mobility group protein B1 (HMGB1) upregulation, consequently triggering ICD-associated immune activation and enhancing CD8+ T-cell cytotoxicity in vitro. In conclusion, our research highlights MALT1 as a promising druggable target for therapeutic interventions in MM, providing insights into its molecular mechanisms in MM progression.

ORGANISM(S): Homo sapiens

PROVIDER: GSE266548 | GEO | 2024/08/28

REPOSITORIES: GEO

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Project description:Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a para-caspase required for B-cell receptor (BCR)-mediated NF-κB activation. We sought to examine the role of MALT1 in B-ALL and determine the biological consequences of inhibiting its activity. First, we tested MALT1 expression by immunoblot in B-cell acute lymphoblastic leukemia (B-ALL) using 16 cell lines representing the disease spectrum and found that MALT1 was expressed in all cell lines at different levels. To determine sensitivity to MALT1 inhibition we used two molecules: Z-VRPR-fmk, a highly selective MALT1 blocking peptide, and MI2, a small molecule MALT1 inhibitor. Z-VRPR-fmk resulted in a dramatic cell growth inhibition in most of our B-ALL cell lines, with appropriate positive (JEKO) and negative (JURKAT) controls. We did not observe a clear correlation between MALT1 level and degree of sensitivity to Z-VRPR-fmk. Interestingly, the two ibrutinib-resistant cell lines RS4;11 and 697, were amongst the top sensitive cell lines to MALT1 inhibition. A similar pattern of cell sensitivity was observed when these cell lines were treated with MI2, with a positive correlation between the IC50 of MI2 and the amount of cell growth inhibition with Z-VRPR-fmk (r=0.74 95%CI (0.47-0.88) p=0.0001). Interestingly, freshly collected PBMCs from patients with B-ALL showed a high sensitivity to MI2 following incubation at serial dilutions for 48h. Furthermore, apoptotic and cell cycle analyses showed a dose-dependent apoptotic induction and cell cycle arrest in the studied ell lines. Surprisingly, the proteolytic activity of MALT1, studied by measuring its ability to cleave its targets such as CYLD, BCL10, and RelB, was not detected at baseline, nor after proteasomal inhibition with MG-132 or following crosslinking of pre-BCR with anti-IgM in pre-B ALL, the latter successfully increased AKT phosphorylation. Collectively, these data are suggestive of distinct role for MALT1 in B-ALL. To explore this possibility, we used RNA sequencing to determine the changes in gene expression profiling following an 8h MI2 treatment in 12 sensitive B-ALL cell lines. Out of 39,514 tested genes, there were 160 genes whose expression changed ≥ 2-fold at P < 0.05 (84 down- and 76 up-regulated). Gene Set Enrichment Analysis (GSEA) identified 34 Hallmark and Oncogenic Signature gene sets relevant to B-ALL that were all downregulated by MI2 (FDR < 10%, and normalized enrichment score (NES) ≥ 1.50). Among those gene sets the c-MYC, gene signatures stood out as the most affected, further confirmed with downregulation of the MYC protein by immunoblot analysis in MI2-sensitive cell lines. In conclusion, MALT1 plays a critical role in B-ALL survival likely through a novel mechanism that involves MYC regulation, independently from pre-BCR/BCR signaling.

Project description:Manuscript Title: Co-targeting of BTK and MALT1 overcomes resistance to BTK inhibitors in mantle cell lymphoma Journal: Journal of Clinical Investigation Authors Vivian Changying Jiang1, Yang Liu1, Junwei Lian1, Shengjian Huang1, Alexa Jordan1, Qingsong Cai1, Fangfang Yan3, Joseph Mitchell McIntosh1, Yijing Li1, Yuxuan Che1, Zhihong Chen1, Jovanny Vargas1, Maria Badillo1, JohnNelson Bigcal1, Heng-Huan Lee1, Wei Wang1, Yixin Yao1, Lei Nie1, Christopher Flowers1, and Michael Wang1, 2* Abstract BrutonÃ¢Â€Â™s tyrosine kinase (BTK) is a proven target in mantle cell lymphoma (MCL), an aggressive subtype of non-Hodgkin lymphoma. However, resistance to BTK inhibitors is a major clinical challenge. We here report that MALT1 is one of the top overexpressed genes in ibrutinib-resistant MCL cells, while expression of CARD11, which is upstream of MALT1, is decreased. MALT1 genetic knockout or inhibition produced dramatic defects in MCL cell growth regardless of ibrutinib sensitivity. Conversely, CARD11 knockout cells showed anti-tumor effects only in ibrutinib-sensitive cells, suggesting that MALT1 overexpression could drive ibrutinib resistance via bypassing BTK-CARD11 signaling. Additionally, BTK knockdown and MALT1 knockout markedly impaired MCL tumor migration and dissemination, and MALT1 pharmacological inhibition decreased MCL cell viability, adhesion, and migration by suppressing NF-ÃŽÂºB, PI3K-ATK-mTOR, and integrin signaling. Importantly, co-targeting MALT1 with safimaltib and BTK with pirtobrutinib induced potent anti-MCL activity in ibrutinib-resistant MCL cell lines and patient-derived xenografts. Therefore, we conclude that MALT1 overexpression associates with resistance to BTK inhibitors in MCL, targeting abnormal MALT1 activity could be a promising therapeutic strategy to overcome BTK inhibitor resistance, and co-targeting of MALT1 and BTK should improve MCL treatment efficacy and durability as well as patient outcomes. Dataset description: The bulk RNA-seq dataset was generated for the cell lines below and used for two major purposes: 1. DEG analysis and GSEA analysis comparing IBN-R and IBN-S cells 2. DEG analysis and GSEA analysis comparing MCL cells with/without MI-2 treatment. sample Cell MI-2 Ibrutinib (IBN) Venetoclax (VEN) Used for IBN-R vs IBN-S comparison Used for MI-2 vs untreated (DMSO) H9 Granta519 - R S yes H21 Granta519 - R S yes H33 Granta519 - R S yes H10 Granta519-VEN-R - R R yes H22 Granta519-VEN-R - R R yes H34 Granta519-VEN-R - R R yes H3 JeKo BTK KD_1 - R R yes yes H15 JeKo BTK KD_1 - R R yes yes H27 JeKo BTK KD_1 - R R yes yes H5 JeKo BTK KD_2 - R R yes yes H17 JeKo BTK KD_2 - R R yes yes H29 JeKo BTK KD_2 - R R yes yes H1 JeKo-1 - S R yes yes H13 JeKo-1 - S R yes yes H25 JeKo-1 - S R yes yes H7 Mino - S S yes H19 Mino - S S yes H31 Mino - S S yes H8 Mino-VEN-R - S R yes H20 Mino-VEN-R - S R yes H32 Mino-VEN-R - S R yes H11 Rec-1 - S S yes H23 Rec-1 - S S yes H12 Rec-VEN-R - S S yes H24 Rec-VEN-R - S R yes H36 Rec-VEN-R - S R yes H35 Rec-1 -- S R yes H4 JeKo BTK KD_1 + MI-2 + yes H16 JeKo BTK KD_1 + MI-2 + yes H28 JeKo BTK KD_1 + MI-2 + yes H6 JeKo BTK KD_2 + MI-2 + yes H18 JeKo BTK KD_2 + MI-2 + yes H30 JeKo BTK KD_2 + MI-2 + yes H2 JeKo-1 + MI-2 + yes H14 JeKo-1 + MI-2 + yes H26 JeKo-1 + MI-2 + yes

Project description:Immunogenic cell death (ICD) in cancer represents a functionally unique therapeutic response that can induce tumor-targeting immune responses. ICD is characterized by the exposure and release of numerous damage-associated molecular patterns (DAMPs), which confer adjuvanticity to dying cancer cells. The spatiotemporally defined emission of DAMPs during ICD has been well described; whereas the epigenetic mechanisms that regulate ICD hallmarks have not yet been deeply elucidated. Here, we aimed to examine the involvement of miRNAs and their putative targets taking advantage from well-established in vitro models of ICD. To this end, B cell lymphoma (Mino) and breast cancer (MDA-MB-231) cell lines were exposed to two different ICD inducers: the combination of retinoic acid (RA) and interferon-alpha (IFN-a) and doxorubicin, and to non ICD inducers like gamma irradiation. Then, miRNA and mRNA profiles were studied by next generation sequencing. Co-expression analysis identified 16 miRNAs differentially modulated in cells subjected to ICD. Integrated miRNA-mRNA functional analysis revealed candidate miRNAs, mRNAs, and modulated pathways associated with Immune System Process (GO Term). In this sense, ICD induced a distinctive transcriptional signature hallmarked by regulation of antigen presentation, a crucial step for a proper immune system antitumor response activation. Interestingly, the major histocompatibility complex class I (MHC-I) pathway was upregulated whereas class II (MHC-II) was downregulated. Analysis of MHC-II associated transcripts and HLA-DR surface expression validated the inhibition of this pathway by ICD on lymphoma cells. miR-4284 and miR-212-3p were the strongest miRNAs upregulated by ICD associated with this event. It is well known that MHC-II expression on tumor cells facilitates the recruitment of CD4+ T cells. However, the interaction between tumor MHC-II and the inhibitory co-receptor LAG-3 on tumor-associated lymphocytes could provide an immunosuppressive signal that directly represses effector cytotoxic activity. In this context, MHC-II downregulation by ICD could enhance antitumor immunity. Overall, we found that the miRNA profile was significantly altered during ICD. Several miRNAs are predicted to be involved in the regulation of Class I and II MHC pathway, whose implication in ICD was demonstrated herein for the first time, which could eventually modulate tumor recognition and attack by the immune system.