GSK-3β inhibitor, 9-ING-41, reduces cell viability and halts proliferation of B-cell lymphoma cell lines as a single agent and in combination with novel agents

Abstract

The complexities of GSK-3β function and interactions with PI3K/AKT/mTOR signaling, cell cycling, and apoptotic pathways are poorly understood in the context of lymphomagenesis and cancer therapeutics. In this study, we explored the anti-tumor effects of the GSK-3β inhibitor, 9-ING-41, in lymphoma cell lines as a single agent and in combination with novel agents comprising BCL-2 inhibitor (Venetoclax), CDK-9 inhibitor (BAY-1143572) and p110δ-PI3K inhibitor (Idelalisib). Treatment of Daudi, SUDHL-4, Karpas 422, KPUM-UH1, and TMD8 lymphoma cell lines with 1 μM 9-ING-41 reduced cell viability by 40-70% (p<0.05) and halted proliferation. Luminex analysis of apoptotic pathways revealed a significant increase in active caspase 3 in all lymphoma cell lines (p<0.001) except TMD8 cells. Co-treating SUDHL-4 and KPUM-UH1 lymphoma cells with 0.5 μM 9-ING-41 showed 8-and 2-fold reduction in IC50 values of Venetoclax, respectively. No significant benefit for this combination was seen in other lymphoma cells tested. The combination of BAY-1143572 with 0.5 μM 9-ING-41 showed an 8-fold reduction in the IC50 value of the former in SUDHL-4 lymphoma cells alone. No significant changes in IC50 values of Idelalisib were measured across all cell lines for the combination of 9-ING-41 and Idelalisib. Further, signaling analysis via Western blot in the double-hit lymphoma cell line, KPUM-UH1, suggests that phospho-c-MYC is modified with 9-ING-41 treatment. Altogether, our data show that 9-ING-41 results in increased apoptosis and decreased proliferation in aggressive B-cell lymphoma cells and enhances the antitumor effects of BCL-2 and CDK-9 antagonists.

Keywords: Bcl-2 inhibitor; CDK9 inhibitor; DLBCL; GSK-3β inhibitor; Myc+ Lymphoma.

Conflict of interest statement

CONFLICTS OF INTEREST 9-ING-41 has been licensed to Actuate Therapeutics, Inc. Andrew Mazar and Andrey Ugolkov hold an equity interest in Actuate Therapeutics, Inc., and Francis Giles is an advisor to this company.

Figures

Figure 1. Viability and proliferation of lymphoma cells with 9-ING-41 treatment

(A) Schematic representing GSK-3 up-stream regulators and down-stream targets and their role in cellular processes in cells. It should be noted that several of the target proteins are misregulated in cancer cells. Only target proteins relevant for this paper are shown for simplicity. (B-F) 10,000 cells (SUDHL-4, KPUM-UH1, Karpas 422, TMD8) per 96-well plate well were left untreated or treated with 1μM 9-ING-41 in triplicate, and the number of cells on days 1, 3, 5 and 7 were calculated using the MTS assay. Briefly, 20μL of MTS reagent was added to cells and incubated for 2 hours and, the absorbance at 490 nm (A490) was read using a Biotek plate reader. Error bars represent std. deviation between replicates. Day 3 viability is shown in B.

Figure 2. Luminex signaling analysis of lymphoma cells treated with 9-ING-41 (Related to Table 1)

(A) 1 million cells were left untreated or treated with 1μM 9-ING-41 for 48 hrs and were then lysed in Millipore MAP lysis buffer. After protein concentration determination via BCA, an equal amount of protein around 10-15 μg (depending on the kit) of protein was used for luminex analysis. Samples were run in duplicates and according to manufacturer instructions. After setting the net MFI or absolute quantity of untreated control to 1 for each analyte, the fold change with 9-ING-41 treatment was calculated (>1 is an increase in levels, <1 is a decrease in levels). Analytes that show significant changes are shown as individual panels. (B) c-Myc, (C) pH2A.X, (D) Survivin, (E) Active Caspase 3, (F) Mcl-1/Bak Dimer, (G) Bcl-Xl/Bak dimer.

Figure 3. Viability of lymphoma cells with novel chemotherapy agents in combination with 9-ING-41 (Related to Table 2)

10,000 cells (A, F, K: Daudi; B, G, L: SUDHL-4; C, H, M: KPUM-UH1; D, I, N: Karpas 422; E, J, O: TMD8) were plated per well of a 96-well plate and treated with a dose response series of both 9-ING-41 (0-0.5 μM) and Venetoclax (0-5,000 nM) (A-E) or BAY-1143572 (0-50 μM) (F-J) or Idelalisib (0-50 μM) (K-O) in triplicate. Viability after 3 days was analyzed using the MTS assay. Briefly, 20μl of MTS reagent was added to cells and incubated for 2 hours and, the absorbance at 490 nm was read using a Biotek plate reader. Relative absorbance is calculated after setting the average absorbance of the no-treatment control as 1.