Background Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Over the last decade, the overall survival of AML patients has improved due to steadfast progress in drug development, but it remains far from satisfactory. A considerable proportion of patients still relapse after achieving remission.MALT1(mucosa-associated lymphoid tissue lymphoma translocation protein 1), also known as paracaspase, transmits abnormal oncogenic signals in ABC-DLBCL (activated B-cell-like diffuse large B-cell lymphoma) and is a potentially important therapeutic target for ABC-DLBCL and MALT lymphoma. The protease activity of Malt1 can degrade MCPIP1, leading to the induction of apoptosis in breast cancer. Abnormal expression of the MALT1 protein is closely associated with various diseases, including lymphoma, solid tumors, and autoimmune diseases. Preclinical studies have demonstrated the efficacy of MALT1 inhibitors in multiple hematological malignancies and solid tumors. Database screening (TCGA, Beat-AML) revealed that MALT1 is highly expressed in AML cell lines. This study used MALT1 inhibitor alone and in combination with venetoclax to treat AML cell lines, demonstrating that MALT1 inhibitors can effectively suppress AML cell growth and exhibit synergistic activity with venetoclax.

Method To determine the effect of the MALT1 inhibitor on AML, we treated multiple AML cell lines (THP-1, MV4-11, Molm-13, and Kasumi-1) with MI-2（MALT1 inhibitor 2）, venetoclax, or their combination. We performed viability assays using CCK-8（Cell Counting Kit-8） to examine cell death and determine the IC50 and optimal exposure time of these drugs. After determining the IC50 values, we examined cell viability at various exposure times between 4 and 72 hours. AML cell lines were treated with MI-2 alone or in combination with venetoclax, and cell apoptosis and cell cycle were detected by flow cytometry. Protein expression levels of anti-apoptotic factors (MCL-1, BCL-XL, BCL-2), apoptotic markers (cleaved PARP, caspase-3), NF-κB signaling components (p65, C-rel), and MCPIP1 were quantified via Western blot.

Results Treatment of THP-1， MV4-11 for 48hours with MI-2 demonstrated a dose-dependent decrease in cell viability. Western blot analysis revealed upregulation of MCPIP1 and apoptotic markers (cleaved PARP, caspase-3), while anti-apoptosis（MCL-1, BCL-XL, BCL-2）and NF-KB（p65, C-rel） decreased after MI-2 treatment. Cell cycle analysis showed MI-2 treatment causes significant cell cycle arrest at the G1 phase. When MI-2 was combined with venetoclax, the CCK-8 assay showed that cell activity was significantly reduced compared with single-drug treatment, resulting in increased apoptosis by flow cytometry, and the expression of apoptotic proteins was significantly elevated.

Conclusions

This study shows that MALT1 inhibitors display potent anti-leukemic activity, whether used as monotherapy or in combination with venetoclax. These findings indicate that MALT1 is a promising therapeutic target for AML treatment. Next, we will conduct experiments testing this drug combination on normal human CD34+ cells and determine the optimal therapeutic concentration required for effective eradication of leukemia cells. This work will yield critical preclinical data to support future clinical translation.

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2025
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