Mebendazole induces ZBP-1 mediated PANoptosis of acute myeloid leukemia cells by targeting TUBA1A and exerts antileukemia effect

Abstract

Background: Despite notable advancements in AML therapy in recent years, a substantial proportion of patients remain refractory or at high risk of recurrence with limited efficacy. Therefore, it's urgent to develop novel drugs for treating AML.

Methods: The small molecule drug library was utilized to screen for drugs that elicit the inflammatory death of AML cells. Cell viability, cell morphological analysis, western blotting, and RNA-seq were used to determine the pathway of Mebendazole (MBD)-induced AML cell death. Cell cycle analysis, protein expression profiling, molecular docking, western blotting and lentivirus overexpression were used to analyze the target protein of MBD in AML cells. The anti-AML activity of MBD in vivo was evaluated using tumor xenograft models constructed by AML cell lines and patient-derived primary AML cells.

Results: In this study, we have identified Mebendazole (MBD), a conventional anthelmintic drug known for its low toxicity and cost, as a potent agent that exerts significant anti-AML effects in vitro. Furthermore, we have observed its inhibitory effects on the invasion of AML cell lines and primary AML cells in xenograft mouse models, while noting its negligible toxic side effects in normal mice in vivo. Mechanically, MBD inhibits the cell cycle in G2/M phase by inhibiting tubulin α1A (TUBA1A) and promotes ZBP-1 mediated PANoptosis in AML cells. Our results confirm that MBD exerts anti-AML activity in preclinical models.

Conclusion: These results highlight the remarkable clinical translational potential of MBD, providing new potential medicine for AML patients. In addition, TUBA1A can be used potential novel therapeutic target in tumors with abnormal TUBA1A expression.

Keywords: Acute myeloid leukemia; Mebendazole; PANoptosis; TUBA1A; ZBP-1.

Graphical abstract

Fig. 1

MBD inhibits AML progression in vivo. 1 × 10⁶ THP-1 cells were injected into B-NDG mice via the tail vein to generate AML mice model. A, Screening experimental procedures for MBD. B, Tumor burden in mice were detected by bioluminescence value. C, Representative images (n = 3) of hematoxylin-eosin staining (HE staining) of the bone marrow, liver and spleen of mice from the MBD-treated and control groups. Red arrow indicates tumor cell mass. D, Kaplan-Meier survival analysis was performed through log-rank test (n = 6, P = 0.0006). E, Changes in body weight of mice during MBD treatment. F, Effect of MBD on normal BALB/c mice. ALT, alanine aminotransferase (I U/L); AST, aspartate aminotransferase (I U/L); CREA, creatinine (m g/mdL); UREA, urea (m mol/L). Data shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar, 50 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

MBD induces PANoptosis in AML cells. A, Chemical structure of MBD. B, Cell viability was measured based on ATP level. C, Cell morphology was examined by microscopy. D, RIP1, p-RIP1, RIP3, p-RIP3, MLKL, p-MLKL, cleaved caspase-1, NLRP3 and cleaved caspase-3 were detected by western blotting. ACTB and GAPDH as internal control. E, The RIP1 inhibitor Nec-1 and pan-caspase inhibitor Z-VAD partially abolished MBD-induced AML cell death. MBD (6 μM), Nec-1 (10 μM), Z-VAD (20 μM), and NSA (1 μM). F, AIM2 and ZBP-1 expression in AML patients, from GEPIA 2 (http://gepia2.cancer-pku.cn/) based on TCGA and GTEx data. G, ZBP-1 in AML cells was detected by western blotting. ACTB as an internal control. H, The integrated effect of MBD on AML cells. Differentially expressed genes were measured by RNAseq, and the top 20 genetic changes were listed as log fold change rankings. I, KEGG pathway analysis of AML cells with MBD stimulation.J, MBD induced IL1β secretion by myeloid cells but failed on lymphocytes. K, pan-caspase inhibitor Z-VAD abrogated most of the MBD-induced IL1β secretion in THP-1 cells. Data shown as mean ± SEM. *P < 0.05, #P < 0.05 between the indicated group. Full-length gels are presented in Supplementary Fig. S13.

Fig. 3

MBD targets TUBA1A inhibition to induce AML cell death. A, THP-1 and U937 cells treated with MBD were analyzed for cell cycle by flow cytometry. B, Proteome data are presented as heat maps. C, TUBA1A expression is significantly up-regulated in AML patients from GEPIA 2 (http://gepia2.cancer-pku.cn/) based on TCGA and GTEx data. D, Binding mode of MBD to TUBA1A by molecular docking. Cartoon representation, overlay of the crystal structures of MBD and TUBA1A was illustrated. The Blue solid line represents Hydrogen bonds, grey dashed line represents hydrophobic interaction, and green dashed line represents π-stacking (paralell). E, TUBA1A mRNA levels in THP-1 and U937 cells treated with MBD were analyzed by QPCR. F, Western blot analysis of TUBA1A levels in MBD-treated AML cells. GAPDH as an internal control. Full-length gels are presented in Supplementary Fig. S14. G, Verification of the overexpression efficiency of TUBA1A in AML cells. Full-length gels are presented in Supplementary Fig. S14. H, AML cells overexpressing TUBA1A were treated with the indicated concentrations of MBD, and then cell viability was determined by ATP level. ATP, adenosine triphosphate. OE, overexpression. Data shown as mean ± SEM. *P < 0.05 between the indicated group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

MBD exerts significant anti-AML effect in primary AML cells and patient-derived xenograft tumor model mice. A, Cell viability and IL1β secretion of primary AML cells treated with MBD. B, The construction process of PDX model. C, Photographs of spleens were shown in MBD-treated and control mice. D, Weight of spleen. E-H, The proportion of Human CD45 in spleen (E), liver (F), bone marrow (G), and peripheral blood (H) was analyzed by flow cytometry in model mice. I, HE staining was used to show the tumor cell in spleen, liver and bone marrow of mice. Red arrow indicates tumor cell mass.J, Human CD45 was detected by IHC in spleen, liver and bone marrow of mice. Data shown as mean ± SEM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)