GSK3β Inhibition Synergizes with PARP Inhibitors through the Induction of Homologous Recombination Deficiency in Colorectal Cancer

Abstract

Background: Monotherapy with poly ADP-ribose polymerase (PARP) inhibitors results in limited objective response rate (≤ 60% in most cases) in patients with homologous recombination repair (HRR)-deficient cancer, which suggests a high rate of resistance in this subset of patients to PARP inhibitors (PARPi). To overcome resistance to PARPi and to broaden their clinical use, we performed high-throughput screening of 99 anticancer drugs in combination with PARPi to identify potential therapeutic combinations. Methods: The effects of PARPi combined with glycogen synthase kinase 3 (GSK3) inhibitors were investigated in vitro with respect to cell viability, cell cycle and apoptosis. The synergy was assessed by calculation of the combination index (CI). GSK3α null and GSK3β null cells were generated using CRISPR/Cas9 technique. The underlying mechanism was examined by western blotting, flow cytometry, qRT-PCR and fluorescence microscopy. This combination was also evaluated in the mouse xenograft model; tumor growth and tumor lysates were analyzed, and the immunohistochemistry assay was performed. All data are presented as mean ± SD. Comparison between two groups was performed with the Student’s t-test.Result: The data showed that ~25% of oncological drugs and kinase inhibitors that were evaluated displayed synergy with PARPi in HCT-15 cells. Among the tested agents, GSK3 inhibitors (GSK3i) exhibited the strongest synergistic effect with PARPi. Moreover, the synergistic antitumor effect of GSK3 and PARP inhibition was confirmed in a panel of colorectal cancer (CRC) cell lines with diverse genetic backgrounds. Additionally, inhibition or genetic depletion of GSK3β was found to impair HRR of DNA and reduce the mRNA and protein level of BRCA1. Finally, we demonstrated that inhibition or depletion of GSK3β could enhance the in vivo sensitivity to simmiparib without toxicity.Conclusion: Our results provide a mechanistic understanding of combination of PARP and GSK3 inhibition, and support the clinical development of this combination therapy for CRC patients.

Thioparib inhibits homologous recombination repair, activates the type I IFN response, and overcomes olaparib resistance

“…To this end, we performed a two-step screening strategy, comprising a histone-based ELISA assay for the primary hit finding, followed by further assessment of the hit compounds of interest using the DSB FA model. Our screen led to the identification of thioparib as a potential PARP inhibitor, with an IC 50 of 0.13 nM; the structure is shown in Fig 1A (Zhang et al, 2015). We observed an approximately 2-fold difference in potency between thioparib and its enantiomer, entthioparib (Cpd-391; PARP1 IC 50 = 0.26 nM) (Appendix Table S1).…”

“…The compound 4- 6,7,2,4]triazolo [4,3-a]pyrazine-7-carbonyl)benzyl)phthalazin-1(2H)-one (Cpd-391) and (R)-4- (4-fluoro-3-(5-methyl-3-(thiazol-2yl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)ben zyl)phthalazin-1(2H)-one (thioparib) were synthesized at the Shanghai Institute of Material Medica at the Chinese Academy of Sciences following a similar procedure as described in the Chinese patent, CN201510818057.7 (Zhang et al, 2015). High-performance liquid chromatography (HPLC) analysis confirmed that the purity of both thioparib and Cpd-391 was ≥ 98%.…”