Selinexor reduces the expression of DNA damage repair proteins and sensitizes cancer cells to DNA damaging agents

Abstract

Introduction: The goal of this study was to examine the effects of selinexor, an inhibitor of exportin-1 mediated nuclear export, on DNA damage repair and to evaluate the cytotoxic effects of selinexor in combination with DNA damaging agents (DDAs) in cancer cells.

Results: Selinexor reduced the expression of DNA damage repair (DDR) proteins. This did not induce significant DNA damage in tested cell lines. Inhibition of DDR protein expression resulted in enhanced cancer cell death when cells were pretreated with DDAs. In contrast, enhanced cell death was not detected in cells that were pretreated with selinexor then with DDAs. In vivo, single-agent selinexor, docetaxel, or cisplatin treatment resulted in 66.7%, 51.5%, and 26.6% tumor growth inhibition (TGI), respectively, in an MDA-MB-231 xenograft model. Consequently, combination treatment with docetaxel or cisplatin followed by selinexor in vivo resulted in 93.9% and 103.4% TGI, respectively. Immunohistochemical staining and immunoblot analysis of tumor sections confirmed reduced expression of DDR proteins.

Conclusion: Selinexor treatment inhibited DDR mechanisms in cancer cell lines and therefore potentiated DNA damage-based therapy. The sequential combination of DDAs followed by selinexor increased cancer cell death. This combination is superior to each individual therapy and has a mechanistic rationale as a novel anticancer strategy.

Methods: Cancer cells treated with selinexor ± DDAs were analyzed using reverse phase protein arrays, immunoblots, quantitative PCR and immunofluorescence. Mice bearing MDA-MB-231 tumors were treated with subtherapeutic doses of selinexor, cisplatin, docetaxel and selinexor in combination with either cisplatin or docetaxel. Tumor growth was evaluated for 25 days.

Keywords: DNA damage repair; chemotherapy; nuclear export; selinexor.

Conflict of interest statement

CONFLICTS OF INTEREST The authors from Karyopharm Therapeutics are all full time Karyopharm employees and have no conflicts of interest to disclose. All authors have read and approved the manuscript for publication in “Oncotarget”.

Figures

Figure 1. Selinexor reduces the expression of DDR proteins

(A) Ingenuity pathway analysis (IPA) of 150 proteins from cell lysates of sarcoma cell lines ASPS-KY and HT1080 treated with 10 μM and 1 μM, respectively, of selinexor for 24 hours tested by reverse phase protein array (RPPA) revealed reduction in the expression of 8 proteins (red stars) with a role in DDR. Node shapes represent functional classes of protein products; rectangles with solid lines for cytokines, rectangles with dotted lines for growth factors, triangles for phosphatases, concentric circles for groups or complexes, diamonds for enzymes, and ovals for transcriptional regulators or modulators. (B) Western blot of proteins from whole cell lysates of HT1080 cells treated with 0, 0.1, or 1 μM selinexor and ASPS-KY cells treated with 0, 1, or 10 μM selinexor confirmed the RPPA results suggesting down-regulation of CHEK1, MLH1, MSH2, PMS2 and Rad51 protein levels from selinexor treatment in both cell lines.

Figure 2. Selinexor suppresses expression of DNA damage gene products at the transcriptional and post-transcriptional levels in both solid and hematological cancer cells

(A) MM.1S cells were treated with 0 (control), 0.05, or 0.5 μM selinexor and MDA-MB-231 cells were treated with 0, 0.1, or 1 μM selinexor for 24 hours. Real-time PCR indicated that selinexor reduced the transcript levels of MSH6, MSH2, CHEK1, MLH1 and Rad51 in a dose-dependent manner. (B) Immunoblots of whole cell lysates from MM.1S cells treated with 0, 0.05, or 0.5 μM selinexor and MDA-MB-231 treated with 0, 0.1, or 1 μM selinexor for 24 hours showed a reduction of the DNA damage repair proteins CHEK1, MLH1, MSH2, PMS2 and Rad51 in a dose-dependent manner. (C) Immunoblots of whole cell lysates from A549 cells treated with 0, 0.1, or 1 μM selinexor and Toledo, MOLM-13, and THP-1 cells treated with 0, 0.05, or 0.5 μM selinexor for 24 hours also showed a reduction in the expression of CHEK1, MLH1, MSH2, PMS2 and Rad51 (D) The reduction in the levels of DDR proteins is compared for MM.1S, MOLM13 and Toledo. The reduced levels of DDR proteins were found to be directly proportional to the sensitivity of the cells to selinexor.

Figure 3. Reduced expression of selinexor dependent expression of DNA damage repair proteins detected prior to cell death

Immunoblots of whole cell lysates from (A) MV-4-11 and (B) MDA-MB-231 cells treated with 200 nM or 1 μM selinexor for 2, 4, 6, 12 and 24 hours. Reduction in the levels of the DDR proteins CHEK1, MLH1, MSH2, PMS2 and Rad51 after selinexor treatment is seen before cell death.

Figure 4. Selinexor blocks recovery from damage caused by DDA

Immunofluorescent detection of γH2A.X in U2-0S cells either untreated or treated for 2 hours with 0.5 μg/mL doxorubicin. The cells were then washed and treated with 100 nM selinexor or vehicle for 48 hrs. DNA damage was detected by staining with γH2A.X. DNA damage due to doxorubicin persisted longer in the presence of selinexor. Selinexor alone did not induce DNA damage.

Figure 5. Selinexor exhibits synergistic cytotoxic effects in combination with chemotherapeutic agents

Immunoblots of whole cell lysates from MDA-MB-231 cells that were treated with (A) 1 μM docetaxel or (C) 100 nM cisplatin with or without 1 μM selinexor for 24 hours or (B) MiaPaCa-2 cells that were treated with either 5 μM gemcitabine, 1 μM selinexor or the combination for 24 hours. Selinexor inhibited the expression of the DDR proteins CHEK1, MLH1, MSH2 and Rad51 upon exposure to the DDAs and resulted in synergistic cell killing.

Figure 6. Pre-treatment with DDA followed by selinexor treatment is more cytotoxic than concomitant dosing or pretreatment with selinexor

(A) Immunoblot analysis of MOLM-13 cells that were either untreated or treated with idarubicin and (B) H929 cells that were either untreated or treated with doxorubicin for 24 hours (indicated by “+”), followed by a washout and subsequent treatment with selinexor. The sequence of treatment was then reversed such that cells were first treated with selinexor for 24 hours, washed and then treated with idarubicin or doxorubicin for additional 24 hours. The cells were also treated with each combination together for 48 hours as a control. Pre-treatment of MOLM-13 cells with idarubicin (green box) or H929 (blue box) cells with doxorubicin followed by exposure to selinexor induced more DNA damage and cell death than pre-treatment with selinexor followed by idarubicin or dexorubicin.

Figure 7. Selinexor demonstrates synergistic anti-tumor effects in combination with cisplatin or docetaxel and inhibits the expression of DDR proteins in an in vivo breast cancer model

Nu/nu mice were allocated to six groups of 4 mice and treated with vehicle (1), 2.5 mg/kg selinexor (2), 4 mg/kg cisplatin (3), 4 mg/kg docetaxel (4), selinexor in combination with cisplatin (5) or docetaxel (6) for 25 days. For groups V and Vi, selinexor was administered 6 hours after treatment with cisplatin and docetaxel respectively. Selinexor was administered orally, whereas cisplatin and docetaxel were administered by intraperitoneal injection. (A) Mean tumor volumes were calculated from the length and width measurements. Group means were calculated and are shown with error bars representing standard error of the mean (SEM) for each group. Combinatory treatments inhibited tumor growth better than each single agent. (B) The percent daily weight changes for each animal and the means for each treatment group were calculated. Error bars represent the SEM. There was no significant weight change among the groups at the end of the study. (C) At the end of the in vivo xenograft study (day 25), excised tumors from the vehicle, selinexor, cisplatin and docetaxel treated groups were assayed either by immunoblots for the expression of DDR proteins. Selinexor, but not cisplatin or docetaxel, reduced the levels of DDR proteins: CHEK1, MLH1, MSH2, PMS2, Rad51.