Smac mimetic increases chemotherapy response and improves survival in mice with pancreatic cancer

Abstract

Failure of chemotherapy in the treatment of pancreatic cancer is often due to resistance to therapy-induced apoptosis. A major mechanism for such resistance is the expression and activity of inhibitors of apoptosis proteins (IAP). Smac (second mitochondria-derived activator of caspase) is a mitochondrial protein that inhibits IAPs. We show that JP1201, a Smac mimetic, is a potent enhancer of chemotherapy in robust mouse models of pancreatic cancer. Combination of JP1201 with gemcitabine reduced primary and metastatic tumor burden in orthotopic xenograft and syngenic tumor models, induced regression of established tumors, and prolonged survival in xenograft and transgenic models of pancreatic cancer. The effect of JP1201 was phenocopied by XIAP small interfering RNA in vitro and correlated with elevated levels of tumor necrosis factor alpha protein in vivo. The continued development of JP1201 and other strategies designed to enhance therapy-induced apoptosis in pancreatic cancer is warranted.

Figure 1. JP1201 in combination with TRAIL is effective in controlling pancreatic tumor xenografts

A, Early intervention treatment algorithm. 1×10⁶ MIA PaCa-2 cells were injected into mice on Day 0. Therapy was initiated on Day 7 post tumor cell injection (TCI). Therapy was given by iv or ip injection every other day (MWF) for two weeks on the days indicated. Mice received saline, TRAIL (20 mg/kg), or TRAIL + JP1201 (JP, at 0.2, 0.6, 2.0, or 6.0 mg/kg). Animals were sacrificed 2.5 weeks after the last dose of therapy (D36). B, A scatter plot of tumor weights at the time of sacrifice showing the mean +/− SEM as well as the weight of each tumor from individual mice. *, p < 0.05 by one-way ANOVA with Dunn’s posttest. C, Representative H&E staining of formalin-fixed, paraffin embedded sections of tumor tissue from mice treated with Saline or JP/TRAIL. The upper row are images at 100× total magnification (scale bar, 100 µm) while the lower row is at 400× total magnification (scale bar, 50 µm). The white box in the upper row is the area magnified. D, The mean +/− SEM number of metastases in each treatment group as well as the number of metastatic events in each animal is shown in the scatter plot. Metastases were evaluated by careful visual inspection at the time of necropsy. Note the animals treated with JP/TRAIL at the highest dose did not have any visible metastases. The data represent a single animal experiment with 7–9 animals in each treatment group.

Figure 2. Inhibition of XIAP improves sensitivity to gemcitabine in pancreatic cancer cell lines

A, Pancreatic cancer cells, MIA PaCa-2 and PANC-1, were treated with increasing concentrations of gemcitabine alone, or in combination with siRNA (100 nM) specific for the indicated target or JP1201 (100 nM). Cell viability was estimated 4 days after initiation of treatment by a MTS assay in which each condition was replicated 8 times. The data shown is representative of three independent experiments. Transfection with siRNA specific for luciferase (si-Luciferase) was used as a control for cell toxicity mediated by transfection alone. B, Western blot analysis of XIAP, cIAP1, cIAP2 demonstrating specificity of siRNA-mediated knockdown. Controls included no transfection (Cntl 1), transfection reagent alone (Cntl 2), and transfection with a siRNA specific for luciferase (Luc).

Figure 3. JP1201 combined with gemcitabine has potent anti-tumor activity against established pancreatic tumors

A–C, MIA PaCa-2 (1×10⁶) were injected into the pancreas of nude mice on day 0. On day 28 post tumor cell injection (TCI) therapy was initiated and consisted of saline, JP1201 (JP, 6.0 mg/kg), JP + TRAIL (TR, 20 mg/kg) 3x/week via iv injection on the days indicated. Gemcitabine (GEM) alone (n=5) or in combination with JP (A & B, n=4) was given ip at a dose of 175 mg/kg on day 28, and at 100 mg/kg on days 30, 37, and 40 (A, B) and 25 mg/kg 3x/week for two weeks (C, n=12/group). Scatter plots showing mean +/− SEM tumor weight as well as the weights individual tumors for each group are shown (A, C). *, p<0.05; **, p<0.01; ***, p<0.005 by one-way ANOVA. B, Three tumors from each treatment group shown in panel B (n=15 total) were harvested on day 29 post TCI. Final tumor size of each group (day 42) was compared to the mean tumor size on day 29. The percent change in tumor size is shown. *, p<0.05; ***, p<0.0001 vs. saline by one-way ANOVA. D, Pan02 murine pancreatic cancer cells (1×10⁶) were injected orthotopically into C57Bl/6J mice on day 0. Therapy with saline, GEM (25 mg/kg), JP (6.0 mg/kg) or the combination was initiated on day 21 post TCI and given 3x/week for two weeks. The mice were sacrificed on day 35 post TCI and resulting tumor weights are displayed as % of body weight in a scatter plot showing mean +/− SEM (n= 9–10/group). *, p<0.05; **, p<0.01 vs. saline by one-way ANOVA.

Figure 4. Induction of apoptosis using combination therapy

A–D, Tumors from animals sacrificed one day after initiation of therapy (EARLY (D29)) or after the cessation of therapy (LATE (D42)) were formalin fixed and paraffin embedded. A–C, Data from a minimum of 4 tumors from each group are normalized to the saline group and are representative of at least two independent assays on the same tumor tissue. A, TUNEL analysis was performed and analyzed using immunofluorescence. *, p<0.05; **, p<0.01; ***, p<0.001 vs. saline by one-way ANOVA. B, Cell proliferation was measured by immunofluorescence for PCNA. **, p<0.01; ***, p<0.001 by one way ANOVA. C & D, The expression level of TNFα was determined by immunofluorescence. *p<0.05; ***, p<0.001 by one way ANOVA. D, Representative images of TNFα (green) immunofluorescence, scale bar, 50 µm.

Figure 5. Combination of JP1201 with gemcitabine enhances survival of mice with pancreatic cancer

A, Mice bearing established MIA PaCa-2 tumors were treated using the late intervention protocol described in Fig. 3A. Mice were treated with saline (black line), gemctiabine (GEM, 25 mg/kg, green line), JP1201 (6.0 mg/kg, red line), or the combination of JP/GEM (blue line) 3x/week for two weeks. Animals were monitored and sacrificed when they demonstrated objective signs of tumor burden or when they appeared moribund (as determined by a blinded observer). B, p48-Cre:KRas^G12D:Ink4a/Arf^lox/lox PDAC animals were treated with saline, GEM or combination of JP/GEM starting at 4 weeks of age. Therapy was delivered ip continuously 3x/week until sacrifice. C, A second cohort of PDAC animals underwent 4 weeks of therapy (Saline, n=5; GEM, n=4; JP/GEM, n=6) and were sacrificed at 8 weeks of age. Tumors were excised and weighed and a scatter plot showing mean +/− SEM tumor weight is displayed. Compared to control (1.124 ± 0.1135 g), JP/GEM significantly reduced tumor growth (0.3833 ± 0.1492 g) **, p<0.01. Each panel represents a single animal experiment.