First-in-class dual BTK-MALT1 fusion inhibitors for treating resistant Mantle Cell Lymphoma

Abstract Background Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with poor prognosis in relapsed or refractory (R/R) cases, driven by constitutive activation of the B-cell receptor (BCR) signaling pathway. Bruton's tyrosine kinase (BTK) is a validated therapeutic target within this pathway, and BTK inhibitors (BTKi) have shown clinical benefit. However, acquired resistance to BTKi remains a major challenge. MALT1, a downstream effector of BCR-BTK signaling, plays a pivotal role in BCR-NF-κB activation and tumor progression, making it an attractive target for combination therapy. Methods To overcome resistance and enhance therapeutic efficacy, we developed a novel class of bifunctional molecules that simultaneously target BTK and MALT1. Initial compounds were generated using fusion strategies and evaluated across a range of preclinical models, including established MCL cell lines, patient-derived samples and organoid models. Functional assays included cell viability, cell cycle analysis, apoptosis induction, and transcriptomic profiling. EMT and metastasis-related gene expression were assessed to determine effects on tumor invasiveness. Lead compounds were identified through iterative chemical optimization and efficacy/toxicity assessments. Results The initial lead compound, MZ0150-1, demonstrated potent anti-tumor activity across all tested models. Compared to reference agents—pirtobrutinib (BTKi) and safimaltib (MALT1i)—MZ0150-1 showed significantly enhanced cytotoxicity (IC50 = 1.6-2.4 µM in MCL cell lines, 3-4 fold lower than that of pirtobrutinib-safimaltib combination), both as a monotherapy and relative to the BTKi-MALT1i combination. Importantly, MZ0150-1 was well tolerated in peripheral blood mononuclear cells (PBMCs) from healthy donors, indicating a favorable safety profile. Mechanistically, MZ0150-1 induced G0/G1 cell cycle arrest and apoptosis, accompanied by downregulation of G1/S-specific transcriptional programs. Furthermore, MZ0150-1 significantly inhibited EMT and reduced expression of metastasis-associated genes, resulting in impaired tumor cell migration and invasiveness. To further enhance potency and pharmacological properties, we synthesized one optimized fusion derivative MZ0481. These compounds retained the bifunctional targeting mechanism and demonstrated superior efficacy in vitro and in vivo. In primary patient samples collected from R/R MCL patients, MZ0481 exhibited enhanced cytotoxicity, with lower IC50 values (IC50 = 0.8-1.8 µM) compared to MZ0150-1, This is also the case in multiple patient-derived organoid models from R/R patients. They are highly tolerant in healthy PBMCs (IC50 = 8.1-11.0 µM), confirming a favorable therapeutic window and supporting their potential for safe clinical application. In Z138-derived xenograft mouse models, treatment with MZ0481 resulted in marked tumor growth suppression, significantly outperforming the pirtobrutinib-safimaltib combination. Notably, no apparent toxicity or adverse effects were observed in treated mice, as assessed by body weight monitoring and serum chemistry panels. Conclusion This study introduces a promising therapeutic strategy for R/R MCL through dual targeting of BTK and MALT1 using single-molecule BTK-MALT1 fusion inhibitors. These first-in-class agents demonstrate superior efficacy, overcome resistance mechanisms, and hold strong potential to improve clinical outcomes in aggressive B-cell lymphomas.