Anti-tumor activity of selective inhibitor of nuclear export (SINE) compounds, is enhanced in non-Hodgkin lymphoma through combination with mTOR inhibitor and dexamethasone

Abstract

In previous studies we demonstrated that targeting the nuclear exporter protein exportin-1 (CRM1/XPO1) by a selective inhibitor of nuclear export (SINE) compound is a viable therapeutic strategy against Non-Hodgkin Lymphoma (NHL). Our studies along with pre-clinical work from others led to the evaluation of the lead SINE compound, selinexor, in a phase 1 trial in patients with CLL or NHL (NCT02303392). Continuing our previous work, we studied combinations of selinexor-dexamethasone (DEX) and selinexor-everolimus (EVER) in NHL. Combination of selinexor with DEX or EVER resulted in enhanced cytotoxicity in WSU-DLCL2 and WSU-FSCCL cells which was consistent with enhanced apoptosis. Molecular analysis showed enhancement in the activation of apoptotic signaling and down-regulation of XPO1. This enhancement is consistent with the mechanism of action of these drugs in that both selinexor and DEX antagonize NF-κB (p65) and mTOR (EVER target) is an XPO1 cargo protein. SINE compounds, KPT-251 and KPT-276, showed activities similar to CHOP (cyclophosphamide-hydroxydaunorubicin-oncovin-prednisone) regimen in subcutaneous and disseminated NHL xenograft models in vivo. In both animal models the anti-lymphoma activity of selinexor is enhanced through combination with DEX or EVER. The in vivo activity of selinexor and related SINE compounds relative to 'standard of care' treatment is consistent with the objective responses observed in Phase I NHL patients treated with selinexor. Our pre-clinical data provide a rational basis for testing these combinations in Phase II NHL trials.

Keywords: CRM1; Exportin-1; NHL; SINE; Selective inhibitors of nuclear export; XPO1.

Fig. 1

Structures of the Specific Inhibitors of Nuclear Export (SINE) used in this study.

Fig. 2

Selinexor synergizes with dexamethasone and everolimus leading to superior cytotoxicity in NHL. 1 × 10⁶ WSU-FSCCL or WSU-DLCL2 cells were seeded in triplicate in 24 well plates and incubated with 100 nM selinexor (SEL) or 100 nM dexamethasone (DEX) or 1.25 µM everolimus (EVER), each drug alone, SEL + DEX or SEL + EVER for 72 h. Resulting cell viability was determined using trypan blue staining [trypan blue (0.4%), Sigma Chemical Co. St. Louis, MO, USA] and cell counting. Data representative of three independent experiments with three replicates per concentration. *p < 0.05 and **p < 0.01.

Fig. 3

Apoptosis analysis. [A and B] 1 × 10⁶ cells were seeded per well in triplicate in 24 well plates and exposed to 100 nM selinexor (SEL) or 100 nM DEX or 1.25 µM everolimus (EVER), each drug alone, SEL + DEX or SEL + EVER for 72 h. After the treatment period was over, the cells were centrifuged at 3000 rpm and the media was removed. The cell pellet was dissolved in 500 µL of Annexin binding buffer and mixed with 5 µL of Annexin and 5 µL of propidium iodide reagent (Biovision, USA). [C and D] Apoptosis analysis using Histone DNA ELISA under similar treatment conditions (Annexin V FITC assay was performed according method supplied by the manufacturer Roche Death assay kit (Roche Cat #11774425001). Results are representative of three independent experiments. *p < 0.05 and **p < 0.01 when compared to single agent treatment.

Fig. 4

Molecular analysis of SEL-DEX and SEL-EVER combination. WSU-DLCL2 or WSU-FSCCL were grown at a density of 1 × 10⁶ per well in six well plates and exposed to indicated concentrations of drugs for 72 h followed by protein isolation and western blotting. [A and B] Results showing enhanced PARP cleavage, full length caspase 3 reduction and decrease in XPO1 expression for combination treatments compared to single agents. β-actin was used as loading control. Blots are representative of three independent experiments. [C] WSU-DLCL2 cells were grown in 24 well plates at a density of 200,000 cells per well overnight. The next day cells were exposed to either DMSO, selinexor, DEX or their combination for additional 72 h in quadruplets. RNA was isolated and RT-PCR was performed according to procedure described in methods section. Relative expression of mRNAs was analyzed by utilizing the Ct method and was normalized to GAPDH. p Values were calculated using Graph Pad Prism software. [D] WSU-DLCL2 cells were grown in 24 well plates at a density of 200,000 cells per well overnight. The next day cells were exposed to either DMSO, selinexor, DEX or their combination for additional 72 h in quadruplets. Protein isolation and western blotting was performed according to procedure described in methods section. Membranes were probed for Akt (Cell signaling Danvers, MA USA). The blots were re-probed for β-actin as loading control. [E] WSU-DLCL2 cells were grown at a density of 2 × 10³ per well in duplicate in 24 well plates and exposed to vehicle or selinexor (100 nM) + DEX (100 nM) or selinexor (100 nM) + Ever (1.25 µM) for 24 h. At the end of the treatment period, cells were spun down on a glass slide using cytospin (3000 rpm). The slides were subjected to immunofluorescence assay with p65 antibody (Santa Cruz). Briefly, the slides were fixed with 10% paraformaldehyde for 15 min. Followed by 3 washes in TBST (5 min each) and permeabilization in 0.5% Triton for 10 min. After additional 3 washes (in PBST 5 min each), the slides were blocked in 0.2% BSA and probed with primary and secondary antibodies according to our previously published methods [31]. The slides were dried and mounting medium was added to it and covered with a coverslip and were analyzed under an inverted fluorescent microscope. A total of two independent experiments were performed.

Fig. 5

Equivalent in vivo efficacy of single agent selinexor vs rituximab or CHOP. [A and B] Xenograft model of DLBCL. SINE Compounds KPT-251 and KPT-276 were administered sc and po, respectively, in cycles of once daily for ten consecutive days with a one day break prior to start of a new cycle. Cyclophosphamide, doxorubicin and vincristine (CHO) was administered once IV at MTD and prednisolone (P) was administered po QDX5. [C] 10 × 10⁶ WSU-FSCCL follicular lymphoma cells were injected IV in the tail veins of ICR-SCID mice. After 1 week of inoculation, the mice were randomly divided into different group (n = 6) and vehicle or drug treatments were started one week later as indicated.

Fig. 6

Selinexor at sub-optimal doses enhances the activity of DEX or EVER in subcutaneous DLBCL xenografts. WSU-DLCL2 xenograft were established as described above. Drugs were administered at indicated doses 5 days a week for three weeks. [A] SEL + EVER and [B] SEL + DEX combination (study continuing beyond 25 days). selinexor (orally 10 mg/kg every other day for three weeks); EVER (2.5 mg/kg orally); combination of selinexor (orally 10 mg/kg every other day for three weeks); EVER (2.5 mg/kg orally); DEX (7.5 mg/kg ip once a week for three weeks) and selinexor (orally 10 mg/kg every other day for three weeks) + DEX (7.5 mg/kg ip once a week for three weeks). **p < 0.01 when compared to single agent treatments.

Fig. 7

Evaluation of selinexor-DEX and selinexor-EVER combination in systemic (disseminated) model of NHL. 10 × 10⁶ WSU-FSCCL cells were injected via tail vain. One week after cell injection, mice were randomly divided in different treatment groups (n = 6) and treated with selinexor (orally 10 mg/kg every other day for three weeks); EVER (2.5 mg/kg orally); combination of selinexor (orally 10 mg/kg every other day for three weeks); EVER (2.5 mg/kg orally); DEX (7.5 mg/kg ip once a week for three weeks) and selinexor (orally 10 mg/kg every other day for three weeks) + DEX (7.5 mg/ kg ip once a week for three weeks). The mice were followed for 90 days (till death occurs). Color coded lines to differentiate the differences between each treatment groups. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).