GSK‐3ß INHIBITOR, 9‐ING‐41 REDUCES CELL VIABILITY AND HALTS PROLIFERATION OF B‐CELL LYMPHOMA

Abstract

Introduction: Glycogen synthase kinase-3 beta (GSK-3ß) is an important signaling molecule involved in cell proliferation and survival. The complexities of GSK-3ß interactions with the PI3K/AKT/mTOR signaling, cyclin-dependent kinases (CDK) and anti-apoptotic protein bcl-2 have been described but are poorly understood in the context of lymphomagenesis and cancer therapeutics. We explore the anti-tumor effects of GSK-3ß inhibitor, 9-ING-41, in lymphoma cell lines as a single agent and in combination with novel agents comprising Bcl-2 inhibitor (ABT-199), CDK-9 inhibitor (BAY-1143572) and p110δ-PI3K inhibitor (CAL-101). Methods: Burkitt (Daudi), GC DLBCL, (SUDHL-4, Karpas 422) double-hit DLBCL (KPUM-UH1), and ABC DLBCL (TMD8) cell lines were treated with 1 μM 9-ING-41. Cell viability on day 3 and proliferation over 7 days were measured using MTS assays. Apoptotic pathways recruited by 9-ING-41 treated cells were evaluated using Luminex assays and analyzed with unpaired t-test. For analyzing additive effects with other novel agents, cell lines were treated with dose-response series of 9-ING-41 (5 nM–5 μM) with either ABT-199 (0.5 nM–5 μM), BAY-1143572 (0.01–100 mM) or CAL-101(0.01–100 μM) and day 3 viability was measured using MTS assay. Results: Cell viability was reduced by 40–70% (p < 0.05; Fig. 1) and all cell lines underwent growth arrest (proliferation <30% relative to control; p < 0.05) upon 1 μM 9-ING-41 treatment. Luminex analysis of apoptotic pathways revealed a significant increase in active caspase 3 in all cell lines (p < 0.001) except TMD8. Co-treating SUDHL-4 and KPUM-UH1 cells with 0.5 μM 9-ING-41 showed eightfold and twofold reduction in IC50 values of ABT-199, respectively (Table 1). The remaining cell lines were insensitive to ABT-199. The combination of BAY-1143572 with 0.5 μM 9-ING-41 also showed eightfold reduction in IC50 value of the former in SUDHL-4 cells, but no significant benefit in other cell lines. For the combination of 9-ING-41 and CAL-101, no significant changes in IC50 values of CAL-101 were measured across cell lines. Conclusions: GSK-3ß inhibitor, 9-ING-41, reduced viability and proliferation in aggressive B-cell lymphoma cells, in an apoptosis-dependent manner. 9-ING-41 demonstrates promising in vitro activity, irrespective of cell of origin and adds to the effects of ABT-199 and BAY-1143572. Efforts to better characterize anti-tumor mechanisms of GSK-3ß inhibitors as single agents and in combination with novel agents are warranted. Table: IC-50 concentrations for novel agents ±0.5 mM 9-ING-41 Keywords: ABT-199; diffuse large B-cell lymphoma (DLBCL); non-Hodgkin lymphoma (NHL).