The gamma secretase inhibitor MRK-003 attenuates pancreatic cancer growth in preclinical models

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy, with most patients facing an adverse clinical outcome. Aberrant Notch pathway activation has been implicated in the initiation and progression of PDAC, specifically the aggressive phenotype of the disease. We used a panel of human PDAC cell lines as well as patient-derived PDAC xenografts to determine whether pharmacologic targeting of Notch pathway could inhibit PDAC growth and potentiate gemcitabine sensitivity. MRK-003, a potent and selective γ-secretase inhibitor, treatment resulted in the downregulation of nuclear Notch1 intracellular domain, inhibition of anchorage-independent growth, and reduction of tumor-initiating cells capable of extensive self-renewal. Pretreatment of PDAC cells with MRK-003 in cell culture significantly inhibited the subsequent engraftment in immunocompromised mice. MRK-003 monotherapy significantly blocked tumor growth in 5 of 9 (56%) PDAC xenografts. A combination of MRK-003 and gemcitabine showed enhanced antitumor effects compared with gemcitabine in 4 of 9 (44%) PDAC xenografts, reduced tumor cell proliferation, and induced both apoptosis and intratumoral necrosis. Gene expression analysis of untreated tumors indicated that upregulation of NF-κB pathway components was predictive of sensitivity to MRK-003, whereas upregulation in B-cell receptor signaling and nuclear factor erythroid-derived 2-like 2 pathway correlated with response to the combination of MRK-003 with gemcitabine. Our findings strengthen the rationale for small-molecule inhibition of Notch signaling as a therapeutic strategy in PDAC.

Figure 1. Chemical Structures of MRK-003 and Gemcitabine

A: MRK-003 (Ref.17) and B: Gemcitabine (Ref.18).

Figure 2. MRK-003 treatment inhibits anchorage-independent growth of PDAC cell lines, which is rescued by enforced expression of N1ICD

A: Down-regulation 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 5μM of MRK-003. C: Normalized colony counts in PDAC cell lines, pre-treated with 2 and 5μM 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, while the other two (Pa16C and Pa29C) were “resistant”. D and F: qRT-PCR and western blots of N1ICD stably transfected Pa03C showing fover-expression of transcripts corresponding to the ICD domain as compared to mock-trasfected cells. F and G: Treatment of MRK-003 significantly reduced the number of colonies in mock Pa03C cells, while 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 to mock controls.

Figure 3. Ex vivo pre-treatment 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 demonstrate that MRK-003 pretreatment significantly delayed engraftment in Capan-1 and Pa03C cells (**P<0.01) compared to the tumor generated from vehicle treated cells. However, MRK-003 pretreatment did not impact the engraftment of Pa16C or Pa29C compared to the tumors generated from vehicle treated cells.

Figure 4. MRK-003 treatment modulates the population of tumor initiating cells in PDAC lines

PDAC cells were treated with MRK-003 (2 μM or 5μM) 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 to vehicle treated cells. B and C: Percentage of CD44⁺CD24⁺ and ALDH⁺ population respectively in four 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 two sensitive cell lines (Capan-1 and Pa03C). How ever, MRK-003 increased the proportion of CD44⁺CD24⁺ and ALDH⁺ cells in Pa16C and Pa29C. Experiment were conducted in triplicate, N=3, error bars SD.

Figure 5. In vivo efficacy of MRK-003, GEM and combination of MRK-003 and GEM in a panel of patient-derived PDAC xenografts

A: Nine 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 two representative PDAC xenografts, Panc374 and Panc198 demonstrates that the combination of GEM and MRK-003 can significantly inhibit tumor growth compared to the vehicle and/or GEM treated animals. Each treatment arm was comprised of ten tumors, implanted as bilateral subcutaneous xenografts in five mice. C: Summary of tumor growth inhibition data. MRK-003 monotherapy significantly reduced tumor volume in 5 of 9 xenografts compared to 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 to GEM 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 to GEM treated mice).

Figure 6. MRK-003 treatment down-regulates 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 down-regulation of nuclear N1ICD and Hes-1 expression in MRK-003 and combination of GEM and MRK-003 treatment compared to vehicle treated and GEM treated tumor samples.

Figure 7. Combination of GEM and MRK-003 induces apoptosis and reduces cell proliferation in PDAC xenografts

A: Representative photomicrograph of TUNEL staining from Panc198 and Panc291. Histogram of TUNEL-positive nuclei per high power field (lower panel), showing that combination of GEM and MRK-003 significantly enhanced apoptotic cells per field as compared to GEM treatment. B: Representative photomicrograph of Ki-67 staining from Panc198 and Panc291. Histogram of Ki-67-positive nuclei per high power field (lower panel), showing that combination of GEM and MRK-003 significantly reduced proliferating cells as compared to GEM treatment. Histograms for TUNEL and Ki-67 were generated by evaluating five high power fields per xenograft section from two independent tumors per treatment arms; mean ± SEM (*P<0.001 compared to GEM).