Acute myeloid leukemia (AML) is an aggressive hematologic malignancy marked by frequent relapse and therapeutic resistance. Although the BCL-2 inhibitor venetoclax (VEN) has improved patient outcomes, drug resistance limits its efficacy. Overexpression of AXL, a member of the TAM family of receptor tyrosine kinases, has been linked to poor outcomes in AML. A recent study from our lab demonstrated that AXL activity is upregulated in AML stem/progenitor cells, particularly in MLL-mutant cells. Combining a newly developed selective AXL inhibitor (SLC-391) with BCL-2 inhibition sensitized these cells to VEN, yielding strong synergistic effects in vitro and in PDX models. However, the underlying mechanism of this synergy remains unclear. This study investigated the molecular basis of synergistic cell death induced by combined inhibition of AXL and BCL-2 in MLL-rearranged AML models. Quantitative mass spectrometry-based proteomics identified a greater number of differentially expressed proteins following dual inhibition of AXL and BCL-2 than with either inhibitor alone in AML cells. Gene set enrichment analysis showed downregulation of mRNA processing and cytoskeletal pathways and upregulation of cellular transport and energy metabolism. Four promising candidate proteins, including STMN1, HCLS1, LCP1, and AHNAK, involved in the regulation of cytoskeletal remodeling and survival signaling, were selected for functional validation. Interestingly, shRNA knockdown demonstrated that STMN1 and HCLS1 depletion significantly reduced cell viability, increased apoptosis, and enhanced drug sensitivity, particularly in VEN-resistant cells, while LCP1 and AHNAK showed cell context-dependent effects. Western blot analyses further demonstrated alteration of key proteins in survival signaling pathways, including β-catenin, AKT, STAT3, and MCL-1, following protein knockdown, providing mechanistic evidence linking cytoskeletal regulation to AXL-BCL-2-mediated signaling networks in AML. In conclusion, this study uncovered several key proteins and pathways that were differentially expressed under combined inhibition of AXL and BCL-2 in AML cells. Among these, the cytoskeletal regulators STMN1 and HCLS1 were shown to mediate AML cell survival and drug sensitivity, suggesting that they may contribute to resistance to BCL-2 inhibition and could serve as therapeutic targets in MLL-mutated AML. These newly identified candidates may also serve as biomarkers to predict patients’ responses to AML therapies.
Hongyu Guo (Thu,) studied this question.