Nuclear Export Inhibitor KPT-8602 Synergizes with PARP Inhibitors in Escalating Apoptosis in Castration Resistant Cancer Cells

Abstract

Aberrant nuclear protein transport, often observed in cancer, causes mislocalization-dependent inactivation of critical cellular proteins. Earlier we showed that overexpression of exportin 1 is linked to higher grade and Gleason score in metastatic castration resistant prostate cancer (mCRPC). We also showed that a selective inhibitor of nuclear export (SINE) selinexor and second generation eltanexor (KPT-8602) could suppress mCRPC growth, reduce androgen receptor (AR), and re-sensitize to androgen deprivation therapy. Here we evaluated the combination of KPT-8602 with PARP inhibitors (PARPi) olaparib, veliparib and rucaparib in 22rv1 mCRPC cells. KPT-8602 synergized with PARPi (CI < 1) at pharmacologically relevant concentrations. KPT-8602-PARPi showed superior induction of apoptosis compared to single agent treatment and caused up-regulation of pro-apoptotic genes BAX, TP53 and CASPASE 9. Mechanistically, KPT-8602-PARPi suppressed AR, ARv7, PSA and AR targets FOXA1 and UBE2C. Western blot analysis revealed significant down-regulation of AR, ARv7, UBE2C, SAM68, FOXA1 and upregulation of cleaved PARP and cleaved CASPASE 3. KPT-8602 with or without olaparib was shown to reduce homologous recombination-regulated DNA damage response targets including BRCA1, BRCA2, CHEK1, EXO1, BLM, RAD51, LIG1, XRCC3 and RMI2. Taken together, this study revealed the therapeutic potential of a novel combination of KPT-8602 and PARP inhibitors for the treatment of mCRPC.

Keywords: KPT-8602; PARP inhibitors; castration resistance; eltanexor; nuclear export; prostate cancer.

Figure 1

KPT-8602 with PARP inhibitors induced growth inhibition of 22rv1 prostate cancer cells. (A–C) 22rv1 cells were treated with 50–200 nM doses of KPT-8602 or 2–10 µM doses of olaparib, veliparib and rucaparib for 72 h. MTT assay was performed to determine the growth inhibition. Left panel is the growth inhibition bar diagram with single or combinations of KPT-8602 and PARP inhibitors. Middle and right panels are the corresponding combination indexes and isobolograms of A–C, respectively, which were generated using Calcusyn 2.1 software. (D–E) Colony formation with single or combinations of KPT-8602 and PARP inhibitors. D. Bar diagram showing the number of colonies with indicated doses of KPT-8602 or PARP inhibitors. E. Representative plates showing colonies with either alone or a combination of KPT-8602 and PARP inhibitors. All experiments have been done at least thrice. Error bars represent standard deviation. NS, not significant; * p < 0.05; ** p < 0.01.

Figure 2

Apoptotic cell death with KPT-8602 and PARP inhibitors in 22rv1 cells. Apoptosis was determined by the flow cytometric detection of annexin-V and propidium iodide (PI) and western blotting technique. (A) Apoptosis in KPT-8602 and olaparib treated cells. (B) Representative flow cytometric image of KPT-8602 and olaparib-treated cells. (C) Western blot analysis of PARP and CASPASE 3 expression in KPT-8602 and olaparib-treated cells and (D) Western blot analysis of PARP and CASPASE 3 expression in KPT-8602 and veliparib- or rucaparib-treated cells. ns = not significant; ** p < 0.01. Lower panel of C and D shows relative protein expression obtained from band densities. NIH ImageJ 1.5Oi software was used to determine band density.

Figure 3

Expression of apoptosis-associated genes in response to KPT-8602 and PARP inhibitors treatment in 22rv1 cells. Gene expression was determined after 24 h of treatment using real-time RT-qPCR. (A,B) mRNA expression of BAX; (C,D) mRNA expression of TP53 and (E,F) mRNA expression of CASPASE 9. All experiments have been done at least three times. Error bars represent standard deviation. * p < 0.05; ** p < 0.01.

Figure 4

Expression of androgen receptor (AR) and its downstream target proteins and genes in 22rv1 cells treated with KPT-8602 and PARP inhibitors. Protein expression was determined by western blot analysis after 72 h of treatment and gene expression was determined after 24 h of treatment using real-time RT-qPCR. (A) Expression of AR, ARv, FOXA1, SAM68 and PSA in KPT-8602 and olaparib treated cells. (B) Expression of AR, ARv, FOXA1, PSA and SAM68 in KPT-8602 and veliparib- or rucaparib-treated cells. Lower panel shows relative protein expression obtained from band densities. NIH ImageJ 1.5Oi software was used to determine band density. (C) The mRNA expression of AR; ARv7, PSA, FOXA1 and UBE2C under specified treatment conditions. The color codes of designated treatment are consistent among the groups. All experiments have been done in triplicate and error bars represent standard deviation. * p < 0.05; ** p < 0.01.

Figure 5

AR and ARv7 localization and DNA damage response of prostate cancer cells under KPT-8602 and PARP inhibitors treatment. (A,B) Immunofluorescence (IF) analysis of AR and ARv7 in 22rv1 cells. Cells were grown in cell culture-treated chamber slides and treated with 2 µM dose of KPT-8602 and 10 µM dose of rucaparib alone or in combination. After 24h cells were fixed, permeabilized and stained with 1:600 and 1:100 dilution of AR and ARv7 (green) antibodies, respectively. Nuclei were stained with DAPI (blue). (C) Gene expression was determined after 24 h of treatment using real-time RT-qPCR. mRNA expressions of BRCA1, BRCA2, CHEK1, EXO1, BLM, RMI1, RAD54L, RAD51, LIG1, XRCC3 and RMI2 in KPT-8602 and/or olaparib treated cells. Red dotted line indicates the expression level of the control. Doses for KPT-8602 and olaparib were 100 nM and 5 µM, respectively. Experiments have been done in triplicate. Error bars represent standard deviation. * p < 0.05; ** p < 0.01.

Figure 6

Schematic diagram illustrating the working hypothesis for the sensitization of mCRPC cells to XPO1 and PARP inhibitors. (A) Mechanism of drug resistance with traditional PARP inhibitor and AR-targeted agent treatment. (B) Sensitization of mCRPC cells using second generation SINE compound KPT-8602 and PARP inhibitors. BRCA1/2 deficient mCRPC growth cannot be sufficiently blocked by PARP inhibitors and AR blockers since the tumor suppressor proteins (TSPs) are functionally inactivated due to their excessive export to the cytoplasm. KPT-8602 can retain TSPs such as TP53, RB, FOXO, P27 and AR translation promoter eIF4e in the nucleus to facilitate the induction of cell death signals. Moreover, by keeping AR and ARv7 in the nucleus, KPT-8602 interrupts AR- and ARv7-mediated import of ARv1 and ARv6 in the nucleus.