XPO1 Inhibition using Selinexor Synergizes with Chemotherapy in Acute Myeloid Leukemia by Targeting DNA Repair and Restoring Topoisomerase IIα to the Nucleus

Abstract

Purpose: Selinexor, a selective inhibitor of XPO1, is currently being tested as single agent in clinical trials in acute myeloid leukemia (AML). However, considering the molecular complexity of AML, it is unlikely that AML can be cured with monotherapy. Therefore, we asked whether adding already established effective drugs such as topoisomerase (Topo) II inhibitors to selinexor will enhance its anti-leukemic effects in AML.

Experimental design: The efficacy of combinatorial drug treatment using Topo II inhibitors (idarubicin, daunorubicin, mitoxantrone, etoposide) and selinexor was evaluated in established cellular and animal models of AML.

Results: Concomitant treatment with selinexor and Topo II inhibitors resulted in therapeutic synergy in AML cell lines and patient samples. Using a xenograft MV4-11 AML mouse model, we show that treatment with selinexor and idarubicin significantly prolongs survival of leukemic mice compared with each single therapy.

Conclusions: Aberrant nuclear export and cytoplasmic localization of Topo IIα has been identified as one of the mechanisms leading to drug resistance in cancer. Here, we show that in a subset of patients with AML that express cytoplasmic Topo IIα, selinexor treatment results in nuclear retention of Topo IIα protein, resulting in increased sensitivity to idarubicin. Selinexor treatment of AML cells resulted in a c-MYC-dependent reduction of DNA damage repair genes (Rad51 and Chk1) mRNA and protein expression and subsequent inhibition of homologous recombination repair and increased sensitivity to Topo II inhibitors. The preclinical data reported here support further clinical studies using selinexor and Topo II inhibitors in combination to treat AML. Clin Cancer Res; 22(24); 6142-52. ©2016 AACR.

Figure 1. Combination of selinexor and idarubicin or Topo IIα inhibitors results in synergistic inhibition of proliferation and induction of apoptosis in AML cells in vitro

(A) Combination index (CI) plots of selinexor with idarubicin (IDA) and daunorubicin (DAUNO) concomitant treatment in AML cell lines MV4-11 and MOLM-13 and patient blasts (B). The effect of the combinations was assessed by WST-1 assay after 48 hrs of concomitant drug treatment. The doses for both drugs were chosen according to their individual IC₅₀ (2 fold dilutions) that were determined by using WST-1 assay (Supplemental Table 1). (C) CI plots of selinexor with Topo IIα inhibitors, etoposide and mitoxantrone in MV4-11 and MOLM-13 AML cell lines. The effects of the combinations were calculated using CalcuSyn software, where CI< 1 indicates synergy, CI=1 is additive and CI>1 is antagonistic. The results of the WST-1 assays are representative of at least two independent experiments performed in quadruplicate. (D) Apoptosis in MV4-11, MOLM-13 and AML primary patient blast was measured by Annexin-V/PI staining 48 hrs after drug treatment at indicated concentrations.

Figure 2. Selinexor restores nuclear localization of Topo IIα

(A) Topo IIα cellular localization assessed by confocal microscopy in MV4-11 and MOLM-13 cells after treatment with DMSO (control) or selinexor for 24 hrs. At least 500 cells were counted and one representative experiment of three is shown. Arrows pointing to cytoplasmic distribution of Topo IIα (B) Topo IIα cellular localization and protein expression (C) measured by confocal microscopy and western blotting of whole cell lysate in MV4-11 cells resistant to idarubicin (MV4-11 R). (D) Confocal microscopy of Topo IIα in two primary refractory and one relapsed AML patient samples after treatment with DMSO (control) or selinexor for 24 hrs and in a pretreatment sample from patient 1 (E). The left panel shows the DAPI staining (cell nucleus). The center panel is Topo IIα staining and the right panel is the merged image of DAPI and Topo IIα staining. (F) Topo IIα expression measured by western blotting in a pretreatment and relapsed AML samples from patient 1 and from a primary refractory AML samples (patient 2).

Figure 3. Selinexor reduces expression of DNA Damage Repair genes

(A) Expression levels of Chk1, MSH2, Rad51, MLH1, PMS2 and MSH6 were measured by quantitative PCR from total mRNA extracted from AML cell lines 6 hrs after selinexor treatment. The average relative expression and standard deviation of two independent experiments is shown. Selinexor treated versus untreated, * p<0.05. (B) Immunoblots of whole proteins from MOLM-13 and MV4-11 cell lines after treatment with DMSO or selinexor at the indicated doses and time points. Increased γH2A.X concurrently with increased caspase 3 cleavage are apoptosis indicators. One representative experiment of two is shown. Total mRNA (C) and protein expression (D) of Chk1 and Rad51 measured by real-time PCR and Western Blotting after DMSO or selinexor treatment in primary AML blasts. Quantification of RNA expression was done by quantitative PCR from whole RNA patient samples treated with selinexor for 10 and 24 hrs and protein expression was analyzed by immunoblots of whole protein extracts treated with selinexor for 24 and 48 hrs. Selinexor treated versus untreated, * p<0.05.

Figure 4. Selinexor blocks homologous recombination after DNA damage and prevents recovery from DNA damage caused by idarubicin treatment

(A) Percentage of GFP positivity in HeLa DR cells after ISCE1 cleavage and DMSO or selinexor treatment. HeLa DR cells carry two copies of inactive GFP genes integrated into the genome. The cells were treated with the ICSE1 enzyme that cuts within the specific DNA sequence of the GFP gene. If HR occurs, there is repair of the double strand breaks and GFP fluorescence is observed. (B) Percentage of viable cells after DMSO and selinexor treatment showing no difference, evidence that lack of GFP repair was due to inhibition of HR and not toxicity or cell death from drug treatment. (C) Immunofluorescence staining of γH2A.X, a marker of DNA damage in MV-4-11 cells treated with 10nM idarubicin for 2 hours. Idarubicin was washed out and cells were either allowed to recover or treated with 100nM selinexor for 48 hrs.

Figure 5. Selinexor downregulates c-Myc expression and binding to DNA Damage Repair gene promoters in AML

(A) c-Myc protein expression in AML cell lines MV4-11 and MOLM-13 treated with selinexor for 24 hrs. One representative western blot of three experiments is shown. (B) Chromatin immunoprecipitation (ChIP) assays of c-Myc on the Rad51 and Chk1 promoter regions in MV4-11 cells after treatment with DMSO or selinexor for 24 hrs.

Figure 6. Idarubicin enhances selinexor anti-leukemic activity in vivo

(A) Survival curve of NSG injected with MV4-11 xenografts and treated with indicated drugs. Survival comparison was made with log-rank test. (B) White blood cell count on day 25 (n=5 per group), p-values obtained using t-test. (C) Spleen weights (mg) on day 25 (n=5 per group), p-values obtained using t-test.