Synergistic Killing Effect and Molecular Mechanism of Dual Targeting of Lymphoma By GSK-3 Inhibitor (9-ING-41) and BCL2 Inhibitor (ABT-199)

Background:

Double-hit lymphoma (DHL) is an aggressive hematological malignance with distinct genetic features and poor clinical prognosis, characterized by concurrent rearrangements of the MYC and BCL2 or BCL6 genes.Current standard chemoimmunotherapy fails to confer satisfying outcomes and few targeted therapeutics are available for the treatment against DHL. Despite promising results with BCL2 inhibitors like ABT-199 (Venetoclax), the therapeutic efficacy remains limited. The GSK-3 inhibitor 9-ING-41 may enhance the treatment effect by indirectly affecting MYC gene expression.

Objectives:

This study aims to investigate the combined therapeutic effects of 9-ING-41 and ABT-199 in DHL cell lines and to elucidate the underlying mechanisms of their synergistic action.

Methods:

The effects of ABT-199 and 9-ING-41 on DHL cell lines (Karpas-422 and SuDHL2) were assessed using the CCK-8 assay. Cells were treated with varying concentrations of 9-ING-41 and ABT-199, both individually and in combination, for 24 and 48 hours. IC50 values were calculated for each treatment condition.Apoptosis rates and cell cycle changes were analyzed by flow cytometry. Protein expression levels of GSK-3, phosphorylated GSK-3, and WNT/β-Catenin pathway-related proteins were evaluated using Western blotting.

Results:

Treatment with 9-ING-41 and ABT-199 alone effectively inhibited cell growth in a dose- and time-dependent manner. Notably, co-administration of 9-ING-41 and ABT-199 led to markedly enhanced growth inhibition in both Karpas-422 and SuDHL2 cell lines.The combination index (CI) values confirmed strong synergy between the two drugs. Flow cytometry revealed that the combined treatment significantly increased the apoptosis rate compared to single-drug treatments, as indicated by a higher proportion of Annexin V-positive cells.Combined treatment induced significant cell cycle arrest at the G0/G1 phase, reducing the percentage of cells in the S phase, which suggests an inhibition of cell cycle progression.Western blot analysis showed that the antitumour effect of 9-ING-41 and ABT-199 alone or in combination was further verified in vitro. In both DHL cell lines,compared with the 9-ING-41 or ABT-199 alone, the combination group further inhibited the expression of anti-apoptotic BCL-2 and promoted the expression of pro-apoptotic BAX. Similarly, the expression of key proteins in the WNT/β-Catenin pathway were significantly inhibited. Western blot revealed that the combination treatment modulated the expression of key proteins in the GSK-3 and WNT/β-Catenin pathways, indicating a potential mechanism for the observed synergistic effects.

Conclusion:

The combination of 9-ING-41 and ABT-199 demonstrates significant synergistic effects in inhibiting cell proliferation and inducing apoptosis in DHL cell lines. This dual targeting strategy presents a promising approach for improving treatment outcomes in DHL by modulating the GSK-3 and WNT/β-Catenin pathways.

Keywords:

DHL;BCL2 inhibitor; GSK-3 inhibitor;GSK-3

No relevant conflicts of interest to declare.