The Gamma Secretase Inhibitor MRK-003 Attenuates Pancreatic Cancer Growth in Preclinical Models

MRK-003 treatment inhibits anchorage-independent growth of PDAC cell lines, which is rescued by enforced expression of N1ICD. A, downregulation of Hes-1 mRNA is seen in PDAC lines treated with MRK-003. B, colony formation in soft agar is inhibited in Pa03C cells with 5 μmol/L of MRK-003. C, normalized colony counts in PDAC cell lines, pretreated with 2 and 5 μmol/L of MRK-003 (**, P < 0.01). Three of the cells lines (Capan-1, Pa03C, and Pa14C) were sensitive based on significant inhibition of anchorage growth, whereas the other 2 (Pa16C and Pa29C) were resistant. D and E, qRT-PCR and Western blots of N1ICD stably transfected Pa03C, showing overexpression of transcripts corresponding to the ICD domain as compared with mock-transfected cells. F and G, treatment of MRK-003 significantly reduced the number of colonies in mock Pa03C cells, whereas no significant effects are seen in N1ICD expressing Pa03C cells. Notably, expression of N1ICD per se results in a significant expansion of colonies in soft agar in untreated Pa03C compared with the mock controls.

Ex vivo pretreatment with MRK-003 significantly inhibits engraftment in mice. A, representative photographs of mice injected with MRK-003 pretreated Capan-1 and Pa03C cells (tumor generated from vehicle and MRK-003 pretreatment-treated cell lines are on the right and left side of mice, respectively). B, tumor growth curves show that MRK-003 pretreatment significantly delayed engraftment in Capan-1 and Pa03C cells (**, P < 0.01) compared with the tumor generated from vehicle-treated cells. However, MRK-003 pretreatment did not impact the engraftment of Pa16C or Pa29C compared with the tumors generated from vehicle-treated cells.

MRK-003 treatment modulates the population of tumor-initiating cells in PDAC lines. PDAC cells were treated with MRK-003 (2 or 5 μmol/L) for 48 hours. The cells were harvested and stained for FACS analysis. A, representative data for CD44⁺CD24⁺ and ALDH⁺ population in MRK-003 treated in Capan-1 cells showing reduction of CD44⁺CD24⁺ and ALDH⁺ cells compared with the vehicle-treated cells. B and C, percentage of CD44⁺CD24⁺ and ALDH⁺ population, respectively, in 4 pancreatic cancer cell lines treated with MRK-003 for 48 hours. Dose-dependent reduction in the proportion of CD44⁺CD24⁺ and ALDH⁺ cells were noticed in 2 sensitive cell lines (Capan-1 and Pa03C). However, MRK-003 increased the proportion of CD44⁺CD24⁺ and ALDH⁺ cells in Pa16C and Pa29C. Experiments were conducted in triplicate, N = 3, error bars SD.

In vivo efficacy of MRK-003, gemcitabine, and combination of MRK-003 and gemcitabine in a panel of patient-derived PDAC xenografts. A, 9 individual cases for this study were selected from a pool of 30 PDAC xenografts based on expression of NOTCH1 transcripts at baseline. In the illustrated heatmap, red to green indicates decreasing transcript expression. The numbers on the top panel indicate each individual patient-derived PDAC xenograft. B, growth curves of 2 representative PDAC xenografts, Panc374 and Panc198, show that the combination of gemcitabine and MRK-003 can significantly inhibit tumor growth compared with the vehicle and/or gemcitabine-treated animals. Each treatment arm was comprised of 10 tumors implanted as bilateral subcutaneous xenografts in 5 mice. C, summary of tumor growth inhibition data. MRK-003 monotherapy significantly reduced tumor volume in 5 of 9 xenografts compared with the vehicle-treated xenografts, which were Panc420, Panc374, Panc219, Panc265, and JH033. There was a significant reduction in tumor growth of 4 of 9 xenografts in the combination therapy group compared with the gemcitabine alone, which were Panc198, Panc219, Panc291, and JH033. (*, P < 0.05, **, P < 0.01, ***, P < 0.001 versus vehicle-treated mice; #, P < 0.05, ##, P < 0.01 compared with the gemcitabine-treated mice). GEM, gemcitabine.

MRK-003 treatment downregulates nuclear N1ICD and Hes-1 protein expressions in PDAC xenografts as shown by immunohistochemical staining. A and B, representative photomicrographs of N1ICD and Hes-1 from Panc291 and Panc374 xenografts showing downregulation of nuclear N1ICD and Hes-1 expression in MRK-003 and combination of gemcitabine and MRK-003 treatment compared with the vehicle-treated and gemcitabine-treated tumor samples. GEM, gemcitabine.