Dual Pharmacological Targeting of the MAP Kinase and PI3K/mTOR Pathway in Preclinical Models of Colorectal Cancer

<div><p>Background</p><p>The activation of the MAPK and PI3K/AKT/mTOR pathways is implicated in the majority of cancers. Activating mutations in both of these pathways has been described in colorectal cancer (CRC), thus indicating their potential as therapeutic targets. This study evaluated the combination of a PI3K/mTOR inhibitor (PF-04691502/PF-502) in combination with a MEK inhibitor (PD-0325901/PD-901) in CRC cell lines and patient-derived CRC tumor xenograft models (PDTX).</p><p>Materials and Methods</p><p>The anti-proliferative effects of PF-502 and PD-901 were assessed as single agents and in combination against a panel of CRC cell lines with various molecular backgrounds. Synergy was evaluated using the Bliss Additivity method. In selected cell lines, we investigated the combination effects on downstream effectors by immunoblotting. The combination was then evaluated in several fully genetically annotated CRC PDTX models.</p><p>Results</p><p>The <i>in vitro</i> experiments demonstrated a wide range of IC₅₀ values for both agents against a cell line panel. The combination of PF-502 and PD-901 demonstrated synergistic anti-proliferative activity with Bliss values in the additive range. As expected, p-AKT and p-ERK were downregulated by PF-502 and PD-901, respectively. In PDTX models, following a 30-day exposure to PF-502, PD-901 or the combination, the combination demonstrated enhanced reduction in tumor growth as compared to either single agent regardless of KRAS or PI3K mutational status.</p><p>Conclusions</p><p>The combination of a PI3K/mTOR and a MEK inhibitor demonstrated enhanced anti-proliferative effects against CRC cell lines and PDTX models.</p></div

Effect of single agent the PI3K/mTORi, PF-04691502, the MEKi, PD-0325901 or the combination on downstream effector proteins assessed by immunoblotting.

<p>Effect of single agent the PI3K/mTORi, PF-04691502, the MEKi, PD-0325901 or the combination on downstream effector proteins assessed by immunoblotting.</p

Growth inhibition of the PI3K/mTORi, PF-04691502 (PF-502) combined with the MEKi, PD-0325901 (PD-901) on four colorectal cancer cell lines.

<p>LOVO (KRAS^G13D), HCT116 (KRAS^G13D/PIK3CA^H1047R), WIDR (BRAF^V600E/PIK3CA^P449T), GEO (KRAS^G13D). Cells were exposed to various combinations of PF-502 and PD-901 for 72 hours. (A) Fraction inhibited was graphed following exposure to single agent and combination. (B) Bliss additivity was calculated and to assess combinatorial effects. Average bliss scores were graphed.</p

Tumor growth rate analysis on patient-derived tumor xenograft models (PDTX). Tumor growth rates were determined for each individual tumor by fitting tumor volume data over the course of the treatment period to an exponential growth rate equation.

<p>Each point represents a single tumor. Mean tumor growth rate ± standard deviation are represented by the bar and handles. All data presented as mean±SD, ANOVA Tukey’s adjusted p values: *P<0.05 vs. Control; ^†P<0.05 vs. 901; ^‡P<0.05 vs. 502.</p

Effect of single agent the PI3K/mTORi, PF-04691502, the MEKi, PD-0325901 or the combination on Caspase 3/7 activity.

<p>All data presented as mean±SD, ANOVA Tukey’s adjusted p values: *<i>p</i>,0.05, **<i>p</i><0.01, ***<i>p</i><0.001 vs vehicle, #<i>p</i>,0.05, ##<i>p</i><0.01, ###<i>p</i><0.001 vs PF-502, &<i>p</i>,0.05, &&<i>p</i><0.01, &&&<i>p</i><0.001 vs PD-901. All data represents three independent experiments.</p

Proliferative effects on colorectal cancer cell lines plotted as IC₅₀ following exposure to the MEKi, PD-0325901 (0–1 umol/L).

<p>Cells were plated in 96 well plates and allowed to adhere for 24 hours. Cells were exposed to increasing concentrations of compound for 72 hours and proliferation was assessed using SRB assay. Proliferation assays were conducted in triplicate and IC₅₀ values were calculated from the average proliferation. Mutational status of KRAS, BRAF, and PIK3CA are in colored boxes.</p

Proliferative effects on colorectal cancer cell lines plotted as IC₅₀ following exposure to the PI3K/mTORi, PF-04691502 (0–5 umol/L).

<p>Cells were plated in 96 well plates and allowed to adhere for 24 hours. Cells were exposed to increasing concentrations of compound for 72 hours and proliferation was assessed using CyQuant assay. Proliferaion assays were conducted in triplicate and IC₅₀ values were calculated from the average proliferation. Mutational status of KRAS, BRAF, and PIK3CA are in colored boxes.</p