Oncogenic MALT1 Promotes Cell Survival and Mediates Ibrutinib Resistance and Ibrutinib-Venetoclax Resistance in Mantle Cell Lymphoma

Both as monotherapies and in combination, the Bruton's tyrosine kinase inhibitor ibrutinib and the BH3 mimetic BCL2 inhibitor venetoclax have proven to be efficacious and are now widely used treatment options for mantle cell lymphoma (MCL) patients. However, mono- and dual- resistance frequently develops, necessitating investigation into the mechanisms mediating resistance to these therapies.

To investigate the mechanism of ibrutinib resistance, we generated two ibrutinib-resistant cells due to marked BTK knockdown via CRISPR/CAS9 from JeKo-1, which is ibrutinib-sensitive and venetoclax-resistant. To understand the mechanism of venetoclax resistance, we generated three venetoclax-resistant cell lines with acquired resistance via chronic exposure to increasing doses of venetoclax from ibrutinib/venetoclax double sensitive Mino and Rec-1 cells, and ibrutinib-resistant but venetoclax-sensitive Granta519 cells. All these paired cell lines with various resistance to ibrutinib and venetoclax were subject to whole transcriptome sequencing of these paired MCL cell lines. We discovered that mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is significantly overexpressed in ibrutinib-resistant and ibrutinib-venetoclax dual-resistant MCL cells, especially in cells with BTK knockdown. This was further validated in primary MCL patient cells (n=24). Interestingly, MALT1 overexpression inversely correlates with CARD11 expression and enhances non-canonical NF-κB signaling, suggesting a switch from a highly-dependent BTK-CARD11 mechanism to an independent mechanism in both ibrutinib-resistant and ibrutinib-venetoclax dual-resistant MCL cells.

Chromosomal translocations of MALT1 are the hallmarks of MALT lymphoma, which result in oncogenic fusion of MALT1 products. MALT1 is constitutively active in driving aggressive ABC-type DLBCL. This indicates MALT1 can be oncogenic when its activity is dysregulated. Therefore, we hypothesized that constitutive MALT1 activity may be responsible for the resistance to ibrutinib and venetoclax in MCL cells. To demonstrate this relationship, MALT1 ablation using genetic manipulation resulted in significant growth defects both in vitro and in vivo. Pharmaceutical approaches using MALT1 inhibitor MI-2 resulted in similar effects on cell survival and using cell viability assays. Whole transcriptome sequencing analysis revealed that MYC, NF-kB, ROS, cell cycle and mTOR signaling are the most significantly downregulated pathways upon MI-2 treatment. Intriguingly, MYC, NF-kB, PI3K-AKT-mTOR and mTOR signaling pathways were reported to be upregulated in ibrutinib-resistant MCL cells compared to sensitive MCL cells. To address this further, proteomics analysis by reverse phase protein array (RPPA) using more than 400 antibodies confirmed that MI-2 significantly downregulated AKT-mTOR signaling. NF-kB modulation, ROS production, AKT-mTOR, and metabolism changes were further confirmed through multiple biochemical approaches. In addition, MI-2 treatment resulted in a dramatic reduction of MALT1 expression, suggesting that MI-2 treatment affected both its scaffold and paracaspase activities. Furthermore, MI-2 treatment resulted in significant inhibition of in vivo tumor growth of ibrutinib-venetoclax dual-resistant MCL subcutaneous xenografts and tumor homing to the spleen and bone marrow in an ibrutinib-venetoclax dual-resistant MCL patient-derived xenograft (PDX) mouse model.

In conclusion, we discovered that MALT1, an essential regulator of NF-κB signaling, is hyperactive in ibrutinib-resistant cells and ibrutinib-venetoclax dual-resistant MCL cells, which puts MALT1 forward as a potentially new therapeutic target in ibrutinib and venetoclax-resistant MCL tumors. Genetic depletion or pharmaceutical inhibition of MALT1 resulted in remarkable defects in cell survival and cell proliferation. The MALT1 inhibitor MI-2 proved its in vivo potency by its pro-apoptotic effect and its significant tumor growth inhibition. In conclusion, targeting a hyperactive MALT1 is a promising therapeutic strategy that could lead to clinical implementation of a new treatment strategy meant to overcome ibrutinib and ibrutinib-venetoclax dual resistance in MCL patients by reversing the NF-kB and ROS/mTOR- mediated resistance in these tumors.