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. 2011 May 1;17(9):2799-806.
doi: 10.1158/1078-0432.CCR-10-2580. Epub 2011 Mar 9.

MK-1775, a potent Wee1 inhibitor, synergizes with gemcitabine to achieve tumor regressions, selectively in p53-deficient pancreatic cancer xenografts

N V Rajeshkumar  1 , Elizabeth De Oliveira, Niki Ottenhof, James Watters, David Brooks, Tim Demuth, Stuart D Shumway, Shinji Mizuarai, Hiroshi Hirai, Anirban Maitra, Manuel Hidalgo
Affiliations

MK-1775, a potent Wee1 inhibitor, synergizes with gemcitabine to achieve tumor regressions, selectively in p53-deficient pancreatic cancer xenografts

N V Rajeshkumar et al. Clin Cancer Res. .

Abstract

Purpose: Investigate the efficacy and pharmacodynamic effects of MK-1775, a potent Wee1 inhibitor, in both monotherapy and in combination with gemcitabine (GEM) using a panel of p53-deficient and p53 wild-type human pancreatic cancer xenografts.

Experimental design: Nine individual patient-derived pancreatic cancer xenografts (6 with p53-deficient and 3 with p53 wild-type status) from the PancXenoBank collection at Johns Hopkins were treated with MK-1775, GEM, or GEM followed 24 hour later by MK-1775, for 4 weeks. Tumor growth rate/regressions were calculated on day 28. Target modulation was assessed by Western blotting and immunohistochemistry.

Results: MK-1775 treatment led to the inhibition of Wee1 kinase and reduced inhibitory phosphorylation of its substrate Cdc2. MK-1775, when dosed with GEM, abrogated the checkpoint arrest to promote mitotic entry and facilitated tumor cell death as compared to control and GEM-treated tumors. MK-1775 monotherapy did not induce tumor regressions. However, the combination of GEM with MK-1775 produced robust antitumor activity and remarkably enhanced tumor regression response (4.01-fold) compared to GEM treatment in p53-deficient tumors. Tumor regrowth curves plotted after the drug treatment period suggest that the effect of the combination therapy is longer-lasting than that of GEM. None of the agents produced tumor regressions in p53 wild-type xenografts.

Conclusions: These results indicate that MK-1775 selectively synergizes with GEM to achieve tumor regressions, selectively in p53-deficient pancreatic cancer xenografts.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest: M. Hidalgo received funding from Merck Research Laboratories. J. Watters, D. Brooks, T. Demuth, SD. Shumway, S. Mizuarai, and H. Hirai are employees of Merck. The other authors declare no potential conflicts of interest.

Figures

Figure 1
Figure 1. Combination of MK-1775 and gemcitabine potentiates the efficacy of gemcitabine in established human pancreatic cancer xenografts
Nine individual patient-derived low passage pancreatic cancer xenografts (three with wild type-p53 (WT) and six with deficient-p53 (MUT) were implanted in athymic mice. Animals with established tumors were dosed with MK-1775, GEM or a combination of GEM with MK-1775 as mentioned in the materials and methods. Tumor size was evaluated twice per week by caliper measurements. Relative tumor growth index (TGI) was calculated by relative tumor growth of treated mice (T) divided by relative tumor growth of control mice (C) x 100. (A) Efficacy of MK-1775, GEM and MK-1775 and GEM combination on the growth inhibition of pancreatic cancer xenografts. MK-1775 treatment produced greater than 50% inhibition of tumor growth in two xenografts (286, 198) compared to control tumors. Five of nine xenografts treated with GEM and six of nine xenografts treated with combination of GEM and MK-1775 produced complete tumor growth inhibition resulting in tumor shrinkage. Combination treatment caused greater than 50% regression in tumor size in four of six xenografts with p53-deficient tumors. Error bars represent SE. *** dennotes significance (P < 0.0001), ** denotes significance (P < 0.005), compared with GEM treated tumors. (B) Combination therapy leads to tumor regression in p53-deficient tumors. Tumors that regressed greater than 50% of its size upon treatment on day 28 were calculated. Error bars represent SE
Figure 2
Figure 2. MK-1775 synergize with gemcitabine to inhibit tumor growth of human pancreatic cancer xenografts
Animals were dosed with MK-1775, GEM or a combination of GEM with MK-1775 for 4 weeks as mentioned in the materials and methods. Tumor growth curves of (A) PANC374, (B) PANC185 and (C) PANC215 suggest that the combination of MK-1775 and GEM lead to synergistic growth inhibition. Tumors in the vehicle and MK-1775 treated animals grew progressively and were sacrificed on day 28 due to tumor burden. Animals in the GEM and combination of GEM with MK-1775 were kept longer after the 4 week treatment. Combination of GEM with MK-1775 slowed the tumor growth progression compared to GEM alone treated animals. Points, mean (n = 8 to 10 tumors per group); bars, SE. Error bars represent SE. *** dennotes significance (P < 0.0001), * denotes significance (P < 0.01), compared with GEM treated tumors.
Figure 3
Figure 3. Combination of MK-1775 and gemcitabine inhibits Wee1 and attenuates Cdc2 phosphorylation to promote mitotic entry and apoptosis
Tumor lysates collected from vehicle, GEM, MK-1775 and combination of GEM with MK-1775 treated mice were resolved in SDS-PAGE and probed with specific antibodies against indicated proteins. MK-1775 as well as combination of MK-1775 and GEM treatment strongly inhibit Wee1 and phospho Cdc2. Combination of MK-1775 and GEM treatment up-regulate the expression of p-HH3, γ-H2AX and cleaved PARP. Upon combination with GEM, MK-1775 down regulate the expression of cIAP2 as compared to GEM. MK-1775 as well as combination of MK-1775 and GEM treatment strongly inhibit the Cyclin B1 expression as compared to control and GEM treatment of PANC198. Filled arrowheads indicate the molecular weight of corresponding proteins (KD). The empty arrowhead indicates the presence of a non-specific band in Cyclin B1. β-actin was used as a loading control.
Figure 4
Figure 4. Immunohistochemical staining of phospho-Cdc2 and phospho-histone H3
(A) Representative micrographs of p-Cdc2 from PANC215 showing high immunoreactivity for p-Cdc2 in the vehicle and GEM treated tumors. MK-1775 and combination treatment greatly inhibited the p-Cdc2 compared to vehicle and GEM treated tumors (left panel). There was a 45 and 31% of tumor cells were stained positive for p-Cdc2 in the control and GEM treated tumors, respectively, while only 11 (P = 0.0061 compared to GEM treated tumors) and 2% (P < 0.0001 compared to GEM treated tumors) of tumor cells were stained positive for p-Cdc2 in the MK-1775 and combination of MK-1775 and GEM treated tumors respectively. (B) Representative micrographs of p-histone H3, a marker of mitosis, from PANC215 were shown in the right panel. Number of p-histone H3 stained tumor cells were elevated in the MK-1775 and combination of MK-1775 and GEM treated tumors as compared to control and GEM treated tumors. There was a 7 and 2% of tumor cells were stained positive for p-histone H3 in the control and GEM treated tumors, respectively, while 12 (P = 0.0115 compared to GEM treated tumors) and 11% (P = 0.0201 compared to GEM treated tumors) of tumor cells were stained positive for p-histone H3 in the MK-1775 and combination of MK-1775 and GEM treated tumors respectively. (C) Histogram showing the number of positively stained p-Cdc2 tumor cells over total number of tumor cells. (D) Histogram showing the number of positively stained p-HH3 tumor cells over total number of tumor cells.
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