DUAL INHIBITION OF BCL-2 AND GSK3Β TO IMPROVE RESPONSE IN VENETOCLAX-RESISTANT ACUTE MYELOID LEUKEMIA CELL LINES

Acute myeloid leukemia (AML) is a hematological malignancy with poor prognosis, particularly in patients who are ineligible or refractory to chemotherapy with anthracycline and cytarabine. Venetoclax (VEN), a selective BCL-2 inhibitor, has improved outcomes in AML, but many patients develop resistance, limiting their efficacy. Glycogen synthase kinase 3β (GSK3β), a multifunctional protein with a pro-oncogenic role in AML, represents a potential therapeutic target. Although not yet clinically approved, its inhibitors have shown promising in vitro results in AML and other cancers. This study aimed to evaluate the therapeutic potential of combining VEN with the GSK3β inhibitor BIO in AML cell lines. We used AML cell lines with distinct sensitivity to VEN, including sensitive (S: HL-60 and MOLM-13) and resistant (R: HEL and U937) models, to perform proliferation (cell counting - Neubauer chamber), apoptosis (Annexin V -FITC/ FACS), and gene expression (qPCR) assays. To define the inhibitor concentrations for each cell line, we performed IC50 assays. Proliferation assays showed that the combined treatment (BIO + VEN) nearly eliminated the cell population in sensitive cells line, with an average reduction of 94% compared to isolated treatments. In the resistant cell lines, VEN + BIO also reduced cell populations, but to a lesser extent: 84% reduction in R-HEL and only 17% in R-U937 relative to isolated treatments. Despite the sensitivity profile, the combined treatment showed a greater effect than isolated treatments in all AML cell lines, suggesting an additive effect. In cell death assays, BIO + VEN increased cell death by 55% in S-MOLM-13, 64% in S-HL-60, and 16% in R-U937 compared with single treatments. In contrast, R-HEL showed no additive effect, as cell death was primarily driven by VEN, with no significant difference between VEN alone and the combination. In gene expression assays, no changes were observed in pro-apoptotic genes. However, we detected an increase in GSK3β expression in sensitive cell lines (1.27- and 1.56-fold change, respectively), while in R-HEL, BCL-2 expression was upregulated 2.43-fold change. These findings suggest that the additive effect is driven by the complementary actions of the inhibitors: BIO primarily blocking cell cycle progression and VEN inducing cell death. Our results are partially consistent with the literature, which reports greater sensitivity of U937 to BIO compared to HL-60, though other cell lines have not been previously described. Sensitivity to VEN was consistent with prior studies. No data exists on the VEN + BIO combination, highlighting the novelty of this analysis. In summary, our findings demonstrate that combining VEN with the GSK3β inhibitor BIO enhances antileukemic activity not only in VEN-sensitive AML cell lines but also in resistant models. These results indicate that this strategy holds promise for future therapeutic approaches in AML and highlight the need for further analyses to investigate the underlying pathways.