Introduction Although venetoclax (VEN) combined with hypomethylating agents exhibits significant therapeutic efficacy in acute myeloid leukemia (AML) patients, the treatment failure rate is still approximately 30% to 40%. This is particularly evident among patients with monocytic AML (Mono-AML). Studies have shown that Mono-AML cells frequently harbor RAS mutations, which confer VEN resistance through the adoption of the phenotype and metabolic profile of drug-resistant leukemia stem cells. Recent studies have demonstrated that BDA-366, a novel BCL-2 inhibitor, exhibits high antitumor efficacy across multiple solid tumors. Unlike VEN, which targets the BH3 domain of BCL-2, BDA-366 is specifically designed to bind the BH4 domain. This study focused on elucidating the effects and mechanisms of BDA-366 in RAS-mutated Mono-AML.

Methods We conducted IC50 assays to compare the sensitivity of Mono-AML to BDA-366 and VEN. Further validation was achieved through cell cycle analysis, energy metabolism assays, and tumor transplantation in mice. Mechanistically, we investigated the role of ferroptosis in FLT3-ITD Mono-AML. For RAS-mutated Mono-AML, we explored potential pathways targeted by BDA-366 through multiomics analysis and identified the molecular targets of BDA-366 using molecular docking and surface plasmon resonance. Finally, we detected the expression of cellular molecules and inflammatory factors and studied whether BDA-366 can induce cell death in RAS-mutated Mono-AML through pyroptosis.

Results BDA-366 demonstrated broad antitumor activity in various Mono-AML cell lines, particularly showing stronger cytotoxicity than VEN in RAS-mutated cell lines. This effect was validated in a patient's sample with multiple RAS mutations. In addition, BDA-366 significantly inhibited cell cycle progression and suppressed oxidative phosphorylation in Mono-AML cells.

In the FLT3-ITD cell line MOLM-13, BDA-366 induced cell death by promoting the expression of ferroptosis-related genes (such as CDKN1A and HMOX1) and increasing the levels of ferrous ions, ROS, and lipids. These results suggest that BDA-366 can effectively kill FLT3-ITD-mutated Mono-AML cells by activating the ferroptosis pathway. However, these ferroptosis-associated phenotypes were not significantly activated in RAS-mutated cell line.

In the RAS-mutated NOMO-1 cell line, transcriptomic and proteomic sequencing results indicated that BDA-366 significantly regulated the Toll-like receptor (TLR) signaling pathway. Notably, BDA-366 exhibits high-affinity binding to TLR4 with a dissociation constant of 3.99×10⁻⁷ M. When BDA-366 binds to TLR4, it markedly activates downstream signaling pathways, thereby promoting the differentiation of primitive monocytes into proinflammatory M1 macrophages (as evidenced by increased expression of CD80 and CD86). Moreover, BDA-366 can induce pyroptosis by activating the downstream JNK/c-Jun pathway, and the expression of inflammatory factors (such as IL1B and IL18) is significantly upregulated in NOMO-1. In the FLT3-ITD-mutated MOLM-13 cell line, these indicators only exhibited moderate changes. Notably, the levels of activated ERK1/2, key downstream effectors of the RAS pathway, did not significantly change. These results suggest that BDA-366 overcomes the drug resistance of RAS-mutated Mono-AML by activating the TLR4 pathway and inducing cell differentiation and pyroptosis.

Furthermore, BDA-366 also exhibited high antitumor efficacy in vivo. We found that BDA-366 treatment significantly suppressed tumor growth and the splenic infiltration of tumor cells in a NOMO-1 cell line-derived mouse model. Notably, it had no significant effect on routine blood parameters, underscoring its favorable safety and tolerability.

Conclusions Our results indicate that the novel BCL-2 inhibitor BDA-366 exerts antitumor effects on Mono-AML independent of the BCL-2 and RAS/ERK pathways and that targeting the TLR4 pathway may help overcome the drug resistance of RAS-mutated Mono-AML. This study further improves our understanding of the molecular abnormalities involved in the development of Mono-AML and provides new molecular therapeutic strategies to overcome drug resistance in Mono-AML.

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2025
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