Additional file 1: Table S1. of Antitumor activity of the polo-like kinase inhibitor, TAK-960, against preclinical models of colorectal cancer

IC50 values and SEM for fifty-five colorectal cancer cell lines treated with TAK-960 as assessed by CyQuant proliferation assay. (TIFF 689 kb

Evaluation of the efficacy of dasatinib, a Src/Abl inhibitor, in colorectal cancer cell lines and explant mouse model

<div><p>Background</p><p>Dysregulation of the Src pathway has been shown to be important at various stages of cancer. Dasatinib is a potent Src/Abl inhibitor and has demonstrated to have anti-proliferative and anti-invasive activity in many preclinical models. The objective of this study was to determine the anti-tumor activity of dasatinib using <i>in vitro</i> and <i>in vivo</i> preclinical colorectal (CRC) models.</p><p>Methods</p><p>CRC cell lines and patient-derived tumor explant (PDX) models were used to investigate the efficacy of dasatinib. We treated 50 CRC cell lines with dasatinib for 72 hours and proliferation was assayed by a sulforhodamine B (SRB) assay; an IC₅₀ ≤ 0.08 μmol/L was considered sensitive. We treated 17 patient-derived CRC explants with dasatinib (50 mg/kg/day, administered once-daily) for 28 days to determine <i>in vivo</i> efficacy. Tumor growth inhibition (TGI) ≥ 50% was considered sensitive.</p><p>Results</p><p>We found that 8 out of 50 CRC cell lines reached an IC₅₀ ≤ 0.08 μmol/L with dasatinib treatment. In addition, of 17 CRC explants grown in the xenograft mouse model, 2 showed sensitivity to dasatinib. The anti-tumor effects observed in this study were a result of G1 cell cycle arrest as the dasatinib sensitive CRC cell lines exhibited G1 inhibition. Moreover, those CRC cell lines that were responsive (0.08 μmol/L) to treatment demonstrated a significant baseline increase in Src and FAK gene expression.</p><p>Conclusion</p><p>Dasatinib demonstrated significant anti-proliferative activity in a subset of CRC cell lines <i>in vitro</i>, especially in those with increased Src expression at baseline, but only showed modest efficacy in CRC explants. Dasatinib is currently being studied in combination with chemotherapy in patients with advanced CRC, as its use as a single agent appears limited.</p></div

Evaluation of baseline pathway gene expression between sensitive and resistant CRC cell lines.

<p>A) Pathway analysis of sensitive and resistant gene expression and KEGG pathway diagram shows that adherens junction is a pathway enriched in sensitive CRC cell lines (red shows the genes that are increased in the sensitive cell lines and include Src and FAK. B) Src and C) FAK gene expression are significantly elevated in sensitive when compared to resistant CRC cell lines.</p

Patient characteristics.

<p>Patient characteristics.</p

Pharmacodynamic effects of dasatinib on the Src pathway.

<p>A) Treatment with dasatinib (0.8 μmol/L) at 0.5h, 1h, 2h, 4h, and 8h significantly reduced the activation of Src, FAK and paxillin at all time points examined in the HCT116 sensitive CRC cell line when compared to control. B) A decrease in Src activity was seen in 1 out of 3 CRC explants treated with dasatinib in the sensitive (CRC036) and resistant CRC explant (CRC027) measured at end of study (day 28). However, in both cases FAK activity appeared to be increased.</p

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

The effects of dasatinib on CRC cell lines <i>in vitro</i>.

<p>Fifty CRC cell lines were treated with dasatinib (dose 0.08–5 μmol/L) and proliferation was determined by an SRB assay. An IC₅₀ ≤ 0.08 μM were deemed sensitive to dasatinib. The CRC cell lines KM20, LS174T, SNU-977, WiDR, HCT-116, SW1417, SNU-796, and SKCO1 demonstrated sensitivity to dasatinib.</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>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

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