Activity of Pan-Class I Isoform PI3K/mTOR Inhibitor PF-05212384 in Combination with Crizotinib in Ovarian Cancer Xenografts and PDX

Abstract

The Phosphatidyl inositol-3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and c-Met signaling pathways are often deregulated in cancer. The two pathways are interconnected and at least c-Met has been implicated in drug resistance. The aim of the study was to assess in ovarian cancer preclinical models, the efficacy and tolerability of a dual PI3K mTOR inhibitor (PF-05212384 or gedatolisib) and a c-Met inhibitor (crizotinib) either as single agents or in combination. In vitro, both PF-05212384 and crizotinib showed a concentration dependent activity in the two ovarian cancer cell lines. The combination of the two did not result in synergistic activity. A subline resistant to gedatolisib was obtained and showed an increased expression of MDR-1 gene. In vivo results show that crizotinib alone did not display any activity in all the tumors investigated, while PF-05212384 alone had some marginal activity. The combination of the two resulted in all the experiments superior to single agents with a good tolerability. Considering that crizotinib did not show activity in the models used, the results indicate that crizotinib is able to potentiate the activity of PF-05212384. Although the activity of the combination was not striking in these three models of ovarian cancer, due to the good tolerability of the combination, the results would suggest the possibility to combine the two drugs in settings in which gedatolisib or crizotinib alone have already some significant activity.

Figure 1

In vitro activity of PF-05212384 and crizotinib as single agents. Response of A2780 (A) and SKOV3 (B) cell lines treated with increasing concentrations of PF-05212384 (PF) and crizotinib (CZ) for 72 h, detected by MTS assay. The average of 3 independent experiments and SD are shown.

Figure 2

In vitro activity of the combination of PF-05212384 and crizotinib. Response of A2780 and SKOV3 cells treated with PF-05212384 as single agent or in combination with crizotinib, detected by MTS assay. Cells were treated for 72 h with non cytotoxic concentrations of the two drugs using different schedules: PF-05212384 was administered for 24 h and followed by crizotinib for 48 h (A and D), crizotinib was followed after 24 h by PF-05212384 (B and E) or the two drugs were given simultaneously (C and F). The average of 3 independent experiments and SD are shown.

Figure 3

Characterization of SKOV3 cell line resistant to PF-05212384. A. c-Met gene copy number in SKOV3 parental cell line and SKOV3 cell line resistant to PF-05212384 (SKPF), assessed using the TaqMan Copy Number Assay and using hTERT copy number as reference gene. B. Relative expression levels of MDR-1 in SKPF at two different culture passages. mRNA expression at basal conditions was assessed by Real-Time PCR and relative expression levels were calculated with ΔΔCt method, setting SKOV3 parental cell line as reference sample (=1). A-B. The histograms represent the average mean and SD of 3 technical replicates. C-D. In vitro activity of PF-05212384 (C) and crizotinib (D) in SKOV3 parental cell line and in SKPF. The average of 3 independent experiments and SD are shown.

Figure 4

Dose–response curves of SKPF treated with the combination of PF-05212384 and crizotinib. Response of SKPF treated with increasing concentrations of PF-05212384 as single agent or in combination with a non toxic concentration of crizotinib, detected by MTS assay. Cells were treated for 24 h with one of the two drugs and for 48 h with the other one or the two drugs were given simultaneously for 72 h. The average of 3 independent experiments and SD are shown.

Figure 5

Antitumor activity of PF-05212384, crizotinib or the combination in A2780 xenografts. A. Tumor growth inhibition activity of A2780 xenografts, treated with PF-05212384 10 mg/kg iv Q4dx4 (green arrows), crizotinib 50 mg/kg po every day (red line) or with the combination of the two drugs for 13 days. B. Percentage of tumor growth inhibition (T/C%) calculated for all the treatment groups. C. Body weight of A2780 bearing mice treated with PF-05212384, crizotinib or the combination. Means and SEM are shown. Statistical analysis was performed using two-way ANOVA test and Bonferroni post-test for multiple comparisons and no differences were detected.

Figure 6

Antitumor activity of PF-05212384, crizotinib or the combination in SKOV3 xenografts. A. Tumor growth inhibition activity of SKOV3 xenografts, treated with PF-05212384 10 mg/kg iv Q4dx4 (green arrows), crizotinib 50 mg/kg po every day (red line) or with the combination of the two drugs for 13 days. B. Percentage of tumor growth inhibition (T/C%) calculated for all the treatment groups. C. Body weight of SKOV3 bearing mice treated with PF-05212384, crizotinib or the combination. Means and SEM are shown. Statistical analysis was performed using two-way ANOVA test and Bonferroni post-test for multiple comparisons and no differences were detected.

Figure 7

Antitumor activity of PF-05212384, crizotinib or the combination in the PDX MNHOC218. A. Tumor growth inhibition activity of MNHOC218 PDX, treated with PF-05212384 10 mg/kg iv Q4dx4 (green arrows), crizotinib 50 mg/kg po every day (red line) or with the combination of the two drugs for 13 days. B. Percentage of tumor growth inhibition (T/C%) calculated for all the treatment groups. C. Body weight of MNHOC218 PDX bearing mice treated with PF-05212384, crizotinib or the combination. Means and SEM are shown.

Figure 8

Effects of treatment on PI3K and MAPK pathways molecular effectors. Representative Western blot analysis after 24 hours of treatment with PF-05212384, crizotinib or the combination on A2780 xenografts (A) and SKOV3 xenografts (B). The histograms below the Western blot represent protein band intensities of p-Akt (Ser⁴⁷³), p-Akt (Thr³⁰⁸), p-S6 (Ser^235/236) levels normalized with the amount of the relative total protein. Two independent experiments were performed.