The Exportin-1 Inhibitor Selinexor Exerts Superior Antitumor Activity when Combined with T-Cell Checkpoint Inhibitors

Abstract

Selinexor, a selective inhibitor of nuclear export (SINE) compound targeting exportin-1, has previously been shown to inhibit melanoma cell growth in vivo We hypothesized that combining selinexor with antibodies that block or disrupt T-cell checkpoint molecule signaling would exert superior antimelanoma activity. In vitro, selinexor increased PDCD1 and CTLA4 gene expression in leukocytes and induced CD274 gene expression in human melanoma cell lines. Mice bearing syngeneic B16F10 melanoma tumors demonstrated a significant reduction in tumor growth rate in response to the combination of selinexor and anti-PD-1 or anti-PD-L1 antibodies (P < 0.05). Similar results were obtained in B16F10-bearing mice treated with selinexor combined with anti-CTLA4 antibody. Immunophenotypic analysis of splenocytes by flow cytometry revealed that selinexor alone or in combination with anti-PD-L1 antibody significantly increased the frequency of both natural killer cells (P ≤ 0.050) and CD4+ T cells with a Th1 phenotype (P ≤ 0.050). Further experiments indicated that the antitumor effect of selinexor in combination with anti-PD-1 therapy persisted under an alternative dosing schedule but was lost when selinexor was administered daily. These data indicate that the efficacy of selinexor against melanoma may be enhanced by disrupting immune checkpoint activity. Mol Cancer Ther; 16(3); 417-27. ©2017 AACRSee related article by Tyler et al., p. 428.

©2017 American Association for Cancer Research.

Figures

Figure 1. Expression of immune checkpoint molecules on leukocytes and tumor cells at baseline and in response to selinexor

(a and b) Selinexor alters the expression of PDCD1, CTLA4, and XPO1 on immune and tumor cells. (a) Peripheral blood leukocytes were cultured in selinexor (30–300 nM) for up to 24 hours and mRNA transcript levels of PDCD1, CTLA4, and XPO1 assessed via qPCR, relative to the level in untreated cells. n=6 donors. (b) Melanoma cells or (c) human breast cancer (MDA-MB-468), prostate cancer (PC3), and sarcoma cells (ASPS-KY) were cultured in vehicle or up to 1 µM selinexor for 24 hours and CD274 mRNA transcript levels assessed via qPCR, relative to the untreated cells. n=9 (b) or 3 (c) independent biological replicates. Mean ± S.D. (d) Melanoma cells express PD-L1 on the cell surface. Surface expression of PD-L1 on human and murine melanoma cells was assessed by flow cytometry. Grey histogram = isotype control, open histogram = anti-PD-L1. Representative of 3 independent experiments. *, p<0.05; **, p<0.01. Mean ± S.D.

Figure 2. Selinexor combines with immune checkpoint blockade to slow B16F10 melanoma tumor growth

C57BL/6 mice were injected subcutaneously with B16F10 cells on day 0 and were treated twice per week (Tuesdays and Fridays) with selinexor and immune checkpoint blockade (or appropriate vehicle/isotype control) beginning when tumors became palpable. (a) Selinexor + anti-PD-1. (b) Selinexor + anti-PD-L1. (c) Selinexor + anti-CTLA4. n=6 mice per group. Mean + S.D. *, p

Figure 3. Selinexor plus anti-PD-L1 antibody alters immune cell frequencies in melanoma bearing animals

The frequency of splenic immune cell subsets was determined by flow cytometry. (a) Gating strategy. Cells were initially gated for viable populations, on the basis of forward and side scatter. Subsequently, T cells were identified by gating on CD3+ cells and NK cells identified by gating on CD3− CD49b+ cells. DCs were identified by gating on CD11c+ cells. MDSCs were identified both by gating on CD11b+ Gr1+ double positive cells (white bars) or by first gating on CD11b+ cells and then gating on Ly6CHi Ly6GLow (monocytic MDSC, black bars) or Ly6GHi Ly6CLow (granulocytic MDSC, data not shown) cells. Representative FACS plots. (b) NK cell frequency, (c) T cell frequency, (d) MDSC frequency, (e) DC frequency. (b–e) n=5–6 mice per group; Mean ± S.D.; *, p<0.05 as compared to vehicle + isotype control.

Figure 4. Selinexor + anti-PD-L1 antibodies induce T cell activation and TH1 differentiation in melanoma bearing animals

(a–b) Helper T cells were quantified on the basis of their pattern of chemokine receptor expression. (a) gating strategy: CD4+ viable cells (based on forward/side scatter and anti-CD4) were gated based on CCR6 expression: CCR6+ cells were defined as TH17, while CC6− cells were further gated based on expression of CXCR3 (TH1 cells) and CCR4 (TH2 cells). (b) Proportion of TH1 cells among splenic CD4+ T cells. (c–e) T cell activation status was assessed on the basis of cell surface phenotype. (c) gating strategy: singly positive CD4+ or CD8+ viable cells (based on forward/side scatter and anti-CD4 and anti-CD8) were selected and identified as a naïve, early activated/central memory, or effector phenotype based on staining for CD44 and CD62L. (d & e) Proportion of CD4+ T cells (d) or CD8+ T cells (e) with an early activated/central memory phenotype (CD62L+ CD44+). n=5–6 mice per group; Mean ± S.D.; *, p<0.05 between indicated groups.

Figure 5. Evaluation of alternative dosing schedules for selinexor and anti-PD-1

Animals were injected subcutaneously with B16F10 on day 0 and were treated twice per week with selinexor and immune checkpoint blockade (or appropriate vehicle/isotype control) beginning when tumors became palpable using the alternative treatment schedules depicted in (a). (b) Growth curves of B16F10 tumors administered Selinexor (15 mg/kg) + anti-PD-1 on schedules 1 and 2. (c) Growth curves of B16F10 tumors administered Selinexor (10 mg/kg) + anti-PD-1 on schedules 3 and 4. (d) Growth curves of B16F10 tumors administered Selinexor (5 mg/kg) + anti-PD-1 on schedule 5. n=5 mice per group. Arrow indicates initiation of treatment. Note: The vehicle/isotype control group in 5.b. and 5.c. consisted of the same animals. This line is present on both graphs for ease of reading. *, p=0.0332 between Schedule 1 and Schedule 2; **, p