Pharmacological Inhibition of METTL3 Enhances the Acute Myeloid Leukemia Cell Death Induced By Venetoclax Via the METTL3-YTHDF2-FBXW7-MCL1 Axis

Overexpression of BCL2 leads to survival of acute myeloid leukemia (AML) cells and poor prognosis. Targeting BCL2 by Venetoclax, a BH3 mimetic and selective inhibitor of BCL2, is widely utilized in clinical practice. However, the clinical utility of venetoclax is constrained by the acquired drug resistance. STM2457, an inhibitor of METTL3, has demonstrated the ability to arrest the proliferation of AML cells in pre-clinical models. Nonetheless, the combined effects of STM2457 and venetoclax in AML have yet to be investigated.

To evaluate the potential synergistic interaction between venetoclax and STM2457 in AML cells in vitro, cell viability assays were conducted following treatment with STM2457, venetoclax, or their combination at varying concentrations. The results indicated that the combination of STM2457 and venetoclax exerts synergistic antileukemic effects in the MOLM-13 and THP-1 cell lines. Furthermore, our study validated that STM2457 potentiates the cytotoxic effect of Venetoclax, as evidenced by flow cytometry (PI/Annexin V), immunofluorescence (JC-1), and western blot analysis (cleaved caspase-3 and cleaved PARP).

Subsequently, we observed that STM2457 down-regulates the protein level of MCL1 and c-Myc in AML cells, in a time- and concentration-dependent manner. Notably, the mRNA levels in STM2457-treated AML cells did not decreased accompanied with the declined protein levels, which indicated STM2457 regulate MCL1 at post- transcriptional level. A similar phenomenon was noted upon knockdown or overexpression of METTL3. We further investigated the correlation between the GI₅₀ values and MCL1 protein expression across various AML cell lines, establishing a significant association between STM2457 and MCL1 protein levels.

To explore potential mechanisms by which STM2457 influences on MCL1 protein, we performed Gene Ontology (GO) enrichment analysis, revealing a correlation with protein stability[1]. Given that the ubiquitin-proteasome and autophagy-lysosome pathways are primary mechanisms of protein degradation, we employed cycloheximide (CHX) chase assays and pretreatment with MG132. These experiments indicated that MCL1 degradation predominantly occurs via the ubiquitin-proteasome pathway.

To explore whether MCL1 undergoes degradation via the ubiquitin-proteasome pathway by STM2457 treatment, Co-immunoprecipitation (Co-IP) assay was conducted, revealing an elevated level of immunocomplexes between ubiquitin and MCL1, precipitated by an MCL1 antibody, in AML cells following STM2457 treatment. Additionally, predictions from the UbiBrowser website (http://ubibrowser. bio-it.cn/ubibrowser_v3/) suggested that MCL1 might interact with FBXW7, an E3 ubiquitin ligase. Meanwhile, STM2457 enhanced the expression of FBXW7 protein in MOLM13 and THP-1 cells in a concentration-dependent manner.

To elucidate the role of FBXW7 in the STM2457-mediated downregulation of MCL1, siRNA was employed to knock down the expression of FBXW7. Notably, FBXW7 siRNA significantly mitigated STM2457-induced MCL1 degradation. To elucidate the potential mechanisms underlying the increase of FBXW7 in STM2457-treated AML cells, we demonstrated that STM2457 affects MCL1 protein levels through the METTL3-YTHDF2-FBXW7-MCL1 axis, as evidenced by western blot, RNA stability assays, and RIP-qPCR.

Finally, we have shown that STM2457 enhances the efficacy of venetoclax in vivo. Meanwhile, our study demonstrated that STM2457 effectively mitigated Venetoclax resistance through pharmacological intervention in MOLM13-VR cells(Venetoclax resistance), primary drug-resistant AML cells, and co-culture models.

Our findings suggest that STM2457 can synergistically enhance the antitumor efficacy of venetoclax and overcome Venetoclax-induced resistance, primarily via the METTL3-YTHDF2-FBXW7-MCL1 signaling pathway.

[1]Yankova E, Blackaby W, Albertella M, et al. Small-molecule inhibition of METTL3 as a strategy against myeloid leukaemia. Nature. 2021;593(7860):597-601. doi:10.1038/s41586-021-03536-w