Preclinical Assessment with Clinical Validation of Selinexor with Gemcitabine and Nab-Paclitaxel for the Treatment of Pancreatic Ductal Adenocarcinoma

Abstract

Purpose: Pancreatic ductal adenocarcinoma (PDAC) remains a deadly disease urgently requiring new treatments. Overexpression of the protein transporter exportin-1 (XPO1) leads to mislocalization of tumor-suppressor proteins (TSP) and their inactivation. Earlier, we showed that blocking XPO1 by CRISPR/Cas9 validated Selective Inhibitor of Nuclear Export (SINE) compounds (selinexor and analogs) restores the antitumor activity of multiple TSPs leading to suppression of PDAC in vitro and in orthotopic models.

Experimental design: We evaluate the synergy between SINE compounds and standard-of-care treatments in preclinical models and in a PDAC Phase Ib trial.

Results: SINE compounds synergize with gemcitabine (GEM) and nanoparticle albumin-bound (nab)-paclitaxel leading to suppression of PDAC cellular growth and cancer stem cell (CSC) spheroids disintegration. Label-free quantitative proteome profiling with nuclear and cytoplasmic enrichment showed superior enhancement in nuclear protein fraction in combination treatment. Selinexor inhibited the growth of PDAC CSC and two patient-derived (PDX) subcutaneous xenografts. Selinexor-GEM-nab-paclitaxel blocked PDX and orthotopic tumor growth. In a phase 1b study (NCT02178436), 9 patients were exposed to selinexor (60 mg oral) with GEM (1,000 mg/m2 i.v.) and nab-paclitaxel (125 mg/m2 i.v.) on days 1, 8, and 15 of 28-day cycle. Two patients showed partial response, and 2 had stable disease. An outstanding, durable objective response was observed in one of the responders with progression-free survival of 16 months and overall survival of 22 months.

Conclusions: Our preclinical and ongoing clinical study lends support to the use of selinexor-GEM-nab-paclitaxel as an effective therapy for metastatic PDAC.

Conflict of interest statement

Conflict of interest statement: None to declare.

©2019 American Association for Cancer Research.

Figures

Figure 1.. Selinexor synergizes with GEM and nab-paclitaxel in PDAC cellular models.

MiaPaCa-2 [A] or L3.6pl [B] cells growing in 96 well plates (5,000 cells/well) were exposed to vehicle, selinexor, GEM-nab-paclitaxel or selinexor-GEM-nab-paclitaxel for 72 hrs and standard MTT assay was performed. Isobologram analysis was performed and combination index (CI) was calculated using GraphPad Prism software. Data is representative of 3 independent experiments. MiaPaCa-2 [C] or L3.6pl [D] cells growing in 6 well plates (50,000 cells/well) for 24 hrs and exposed to selinexor (300 nM), GEM (90 nM), nab-paclitaxel (1.5 nM), selinexor+GEM, selinexor+nab-paclitaxel, GEM+nab-paclitaxel or a triple combination of selinexor, GEM and nab-paclitexal for 72 hrs. The cells were trypsinized and collected. 1000 cells from each treatment group were reseeded in 100 mm petri dishes. Clonogenic assay was performed. The plates were photographed under a light microscope (Nikon). L3.6pl [E] or MiaPaCa-2 [F] cells were grown in 6 well plates in duplicate and exposed to different drugs (at doses mimicking clonogenic assay) for 72 hrs. At the end of the treatment period, cells were trypsinized and 10,000 cells were subjected to Annexin V FITC analysis (Biovision USA).

Figure 2.. Selinexor synergizes with GEM-nab-paclitaxel in PDAC CSCs.

[A and B] Equal number of Panc-1 or MiaPaCa-2 CSC spheroids were seeded in ultra-low attachment plates for 1 week. Spheroids were exposed to KPT-330, GEM, nab-paclitaxel (100 nM) each or their combination for 1 week. Spheroids were counted and photographed under an inverted light microscope fitted with camera (at 100X magnification). [C] MiaPaCa-2 cells growing in 6 well plate (50,000 cells/well) in duplicate were exposed to different SINE analogs (100 nM each) for 72 hrs. RNA was isolated using standard methods. The RNA was subjected to RT-PCR using standard methods. The expression of CSC markers CD24, vimentin and CD44 was normalized to GAPDH (*p<0.05 and **p<0.001). Spheroid data is representative of two independent experiments.

Figure 3.. Selinexor-GEM-nab-paclitaxel retains TSPs in the nuclei of PDAC cellular models.

L3.6pl or MiaPaCa-2 cells grown on slides (3000 cells/well) and were exposed to vehicle or a combination of selinexor (300 nM), GEM (150 nM) and nab-paclitaxel (1.5 nM) for 24 hrs followed by IF analysis for nuclear TSPs (FOXO3a, Hsp90 and PAR-4).

Figure 4.. Proteomic profiling for nuclear retention of proteins.

MiaPaCa-2 cells were exposed to 1 μM selinexor, 1 μM selinexor plus 300 nM GEM and 3 nM nab-paclitaxel for 24 hours. The cell pellets were collected and analyzed by Label-Free Quantitative (LFQ) Proteome Profiling with nuclear and cytoplasmic enrichment using Thermo Q-Exactive HF-X Orbitrap Mass Spectrometer at Bioproximity. The intensity value of total 27528 proteins within 3617 protein sets in each samples was read. [A] Protein intensity value changes across samples. [B] Percentage of up-regulated and down regulated genes in nuclei of MiaPaCa-2 cells post selinexor single agent treatment. [C] Annotation of molecular function of proteins up-regulated in nucleus after single agent selinexor treatment. [D] Annotation of the protein class up-regulated in the nucleus post selinexor single agent treatment. [E] Percentage of nuclear proteins up-regulated post selinexor-GEM-nab-paclitaxel treatment.

Figure 5.. XPO1 CRISPRCas9 genome editing abrogates the activity of selinexor-GEM-nab-paclitaxel.

[A] Schema of CRISPR/Cas9-induced homologous recombination of XPO1 (adopted from our previous publication (Chem and Biol 22 (1) 107–116, 2015). [B] HEK-293 wild-type or HEK-XPO1C528S mutant cells were grown at a density of 5,000 cells/well in chambered slides. The next day, cells were exposed to indicated concentrations of either vehicle (DMSO) or selinexor for 2 hrs. After the treatment was over, IF analysis was performed using RanBP1 antibody (Cell Signaling USA). [C] HEK-293 wild-type or [D] HEK-XPO1C528S mutant cells were grown at a density of 5,000 cells/well in 96 well plates. Cells were treated with selinexor or GEM-nab-paclitaxel or their combination for additional 72 hrs at indicated doses. MTT assay was performed using standard methods. [E] HEK-293 wild-type or [F] HEK-XPO1C528S mutant cells were grown at a density of 50,000 cells/well in six well plates and exposed to selinexor (150 nM), GEM (75 nM) and nab-paclitaxel (0.75 nM) for 72 hrs. Apoptosis analysis was performed using Annexin V FITC analysis. [G] HEK-293 wild-type or HEK-XPO1C528S mutant cells were grown in chambered slides (5,000 cells per well) in duplicate and were exposed to selinexor (150 nM), GEM (75 nM) and nab-paclitaxel (0.75 nM) for 24 hrs. IF assay was performed using Hsp90 antibody.

Figure 6.. Selinexor-GEM-nab-paclitaxel anti-tumor activity in PDAC CSC and patient derived xenograft.

MiaPaCa-2 cells were flow sorted for CSC markers (CD44, CD133, EpCAM). These CSCs form tumor from 20,000 cells [A] Twelve mice were injected with CSCs. When palpable tumors were formed, mice were divided into two groups of five mice each and exposed to selinexor as single agent (used at 15 mg/kg every other day x3 weeks). Tumor weight was recorded every three days. [B] 10 mice bearing CSCs were divided into two groups. Group I vehicle and group II selinexor at sub-MTD (5 mg/kg twice a weekx3weeks) in combination with GEM (50 mg/kg i.v.) and nab-paclitaxel (30 mg/kg i.v.) (**p<0.01). [C] Anti-tumor activity of selinexor or KPT-8602 (Eltanexor) in PDX sub-cutaneous xenograft. [D] Photograph of residual tumors post treatment. [E] Anti-tumor activity of selinexor in combination with GEM-nab-paclitaxel (triple combination) compared to selinexor (single agent) or vehicle control in PDAC Pdx. Selinexor was administered orally (5 mg/kg), while gemcitabine (GEM) and nab-paclitaxel (PAC) were given i.v. 30 mg/kg and 20 mg/kg respectively. [F] Photographs showing reduction in tumor size in the combination treatment on day 53 post MiaPACa-2 orthotopic implantation. [G] Dot Graph representing gross tumor weights 72 hours post last treatment. Evaluable response to selinexor-GEM-nab-paclitaxel based treatment in patient with metastatic PDAC. [H] Before and after treatment CT-Scan showing the changes in the liver metastatic disease. [I] Serial measurements of the tumor marker CA19-9 in the patient with PDAC treated with a selinexor based regimen. Serum CA19-9 showed a precipitous drop after the first few treatments and a lower, steady concentration thereafter.