Dasatinib sensitizes KRAS mutant colorectal tumors to cetuximab

Abstract

KRAS mutation is a predictive biomarker for resistance to cetuximab (Erbitux) in metastatic colorectal cancer (mCRC). This study sought to determine if KRAS mutant CRC lines could be sensitized to cetuximab using dasatinib (BMS-354825, Sprycel), a potent, orally bioavailable inhibitor of several tyrosine kinases, including the Src family kinases (SFKs). We analyzed 16 CRC lines for: (1) KRAS mutation status, (2) dependence on mutant KRAS signaling and (3) expression level of epidermal growth factor receptor (EGFR) and SFKs. From these analyses, we selected three KRAS mutant (LS180, LoVo and HCT116) cell lines and two KRAS wild-type cell lines (SW48 and CaCo2). In vitro, using poly-D-lysine/laminin plates, KRAS mutant cell lines were resistant to cetuximab, whereas KRAS wild-type lines showed sensitivity to cetuximab. Treatment with cetuximab and dasatinib showed a greater antiproliferative effect on KRAS mutant lines when compared with either agent alone in vitro and in vivo. To investigate potential mechanisms for this antiproliferative response in the combinatorial therapy, we performed Human Phospho-Kinase Antibody Array analysis, measuring the relative phosphorylation levels of 39 intracellular proteins in untreated, cetuximab, dasatinib or the combinatorial treatment in the KRAS mutant lines LS180, LoVo and HCT116 cells. The results of this experiment showed a decrease in a broad spectrum of kinases centered on the β-catenin pathway, the mitogen-activated protein kinase (MAPK) pathway, AKT/mammalian target of rapamycin (mTOR) pathway and the family of signal transducers and activators of transcription (STATs) when compared with the untreated control or monotherapy treatments. Next, we analyzed tumor growth with cetuximab, dasatinib or their combination in vivo. KRAS mutant xenografts showed resistance to cetuximab therapy, whereas KRAS wild type demonstrated an antitumor response when treated with cetuximab. KRAS mutant tumors exhibited minimal response to dasatinib monotherapy. However, as in vitro, KRAS mutant lines exhibited a response to the combination of cetuximab and dasatinib. Combinatorial treatment of KRAS mutant xenografts resulted in decreased cell proliferation, as measured by Ki67, and higher rates of apoptosis, as measured by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling). The data presented in this study indicate that dasatinib can sensitize KRAS mutant CRC tumors to cetuximab and may do so by altering the activity of several key signaling pathways. Furthermore, these results suggest that signaling via EGFR and SFKs may be necessary for cell proliferation and survival of KRAS mutant CRC tumors. These data strengthen the rationale for clinical trials combining cetuximab and dasatinib in the KRAS mutant CRC genetic setting.

Figure 1. Characterization of colorectal tumor lines

A) Analysis of EGFR and SFK expression in colon cancer lines. CRC tumor lines were grown and whole cell lysates were obtained, fractionated by SDS-PAGE and immunoblotted for the indicated proteins. α-tubulin was used as a loading control. All sixteen-tumor lines were tested for in vivo tumor growth using mouse xenografts. Tumor lines that grew greater than 500mm³ in vivo are denoted by *. Densitometry measurements of EGFR and SFK relative to Colo320DM (1.0) for EGFR and SW48 (1.0) for SFK are shown. B) KRAS and BRAF mutational status was determined via pyrosequencing. C) KRAS mutant lines LS180, LoVo and HCT116 are dependent on KRAS. KRAS mutant lines LS180, LoVo and HCT116 were treated with transfection reagent only, scramble siRNA (10nM), or KRAS siRNA (10nM). Proliferation was measured at 72 hours after treatment using the proliferation assay as described in the experimental procedures and plotted as a percentage of growth relative to the untreated control cells. Data points are represented as mean ± SEM (n = 4). *p < 0.05. Inset denotes confirmation of KRAS knockdown.

Figure 2. Dasatinib sensitizes KRAS mutant colorectal tumors to cetuximab in vitro

(A) KRAS mutant colorectal lines are resistant to cetuximab when compared to KRAS wild type lines. Two KRAS wild type lines (CaCo2 and SW48) and three KRAS mutant lines (LS180, LoVo and HCT116) were tested for response to cetuximab. Cells were plated on Poly D-lysine/laminin 96 well plates, allowed to adhere overnight and treated with vehicle (PBS) or 160 nM of cetuximab for 72 hours. B) Dasatinib sensitizes KRAS mutant lines LS180, LoVo and HCT116 to cetuximab. Cells were plated on Poly D-lysine/laminin 96 well plates and allowed to adhere overnight. Cells were treated with vehicle (PBS), 500 nM of cetuximab, 50 nM dasatinib or the combination (CTX + DSB) for 72 hours. Proliferation was measured at 72 hours after drug treatment using the proliferation assay as described in the experimental procedures and plotted as a percentage of growth relative to the untreated control cells. Data points are represented as mean ± SEM (n = 7). *p < 0.05.

Figure 3. Human Phospho-Kinase array in KRAS mutant LS180, LoVo and HCT116 cells lines

A) AKT/mTOR/p70 S6 kinase pathway (AKT and p70 S6 Kinase), MAPK/RSK (RSK1/2/3), components of the β-catenin pathway (GSK α/β and β-catenin) and STAT family members were downregulated by the combination of dasatinib and cetuximab in LS180 cells. B) Members of the MAPK signaling pathway were downregulated with the combination of dasatinib and cetuximab in LoVo cells. C) AKT/mTOR/p70 S6 kinase pathway (AKT and p70 S6 Kinase), MAPK/RSK (RSK1/2/3), components of the β-catenin pathway (GSK α/β and β-catenin) and STAT family members were downregulated by the combination of dasatinib and cetuximab in HCT116 cells. After treatment with cetuximab, dasatinib and combination of cetuximab and dasatinib, cells were collected and cell extracts were incubated with membrane containing antibodies to 39 individual proteins. The membranes were washed and incubated with a cocktail of biotinylated detection antibodies, streptavidin-HRP and chemiluminescent detection reagents to measure the levels of phosphorylated protein. Quantitation of phosphorylated protein was completed using scanned images from ImageJ software. Data points are represented as mean of duplicate.

Figure 4. KRAS wild type colorectal tumors are sensitive to cetuximab in vivo

For the following series, all mice were randomized to cetuximab or IgG treatments. All mice received 0.3 mg of their respective treatment intraperitoneally twice weekly. A) A non-EGFR expressing line (Colo320DM) was used as a negative control randomizing 20 mice to cetuximab and 20 mice to IgG treatment. Mice received 3 weeks of treatment. B) A known-sensitive non-small cell lung cancer line (H226) was utilized as a positive control randomizing 10 mice to cetuximab and 10 mice to IgG treatment. Mice received 4.5 weeks of treatment. C and D) Mice (n=20) were inoculated with a KRAS wild type line (SW48 and CaCo2, respectively) and randomized to cetuximab or IgG treatment. SW48 mice received 3.5 weeks of treatment. CaCo2 mice received 5.5 weeks of treatment. Statistical significance is denoted by * (P≤0.001).

Figure 5. Dasatinib sensitizes KRAS mutant colorectal tumors to cetuximab

For the following series of experiments, all mice were randomized to treatment or control groups and treated with the following doses: cetuximab or IgG – 0.3 mg/kg intraperitoneally twice per week; dasatinib – 70 mg/kg by oral gavage five days a week. The same doses were used for sequential and combinatorial experiments. Statistical significance is denoted by * (P≤0.001). (A, B, C upper left panel) Cetuximab response was tested by inoculating mice with a KRAS mutant line (LS180, LoVo, and HCT116, respectively) and randomizing to cetuximab or IgG. LS180 mice received 3 weeks of treatment. LoVo mice received 3 weeks of treatment. HCT116 received 2.5 weeks of treatment. (A, B, C upper right panel) Dasatinib response was tested by inoculating mice with a KRAS mutant line and randomizing to dasatinib or vehicle treatment. LS180 mice received 2.5 weeks of treatment. LoVo mice received 3 weeks of treatment. HCT116 mice received 4.5 weeks of treatment. (A, B, C lower left panel) Sequential treatment response of cetuximab then dasatinib was tested by inoculating mice with a KRAS mutant line and randomizing to cetuximab followed by dasatinib or IgG followed by vehicle. Cetuximab was ceased and dasatinib was started the next day once tumors displayed resistance. LS180 mice received one week of cetuximab/IgG and 2.5 weeks of dasatinib/vehicle. LoVo mice received 3 weeks of cetuximab/IgG and 2.5 weeks of dasatinib/vehicle. HCT116 mice received 2 weeks of cetuximab/IgG and 2.5 weeks of dasatinib/vehicle. (A, B, C lower right panel) Concomitant treatment response of cetuximab and dasatinib was tested by inoculating mice with a KRAS mutant line and randomizing to cetuximab plus dasatinib or IgG plus vehicle. LS180 mice received 2.5 weeks of concomitant treatment. LoVo mice received 3.5 weeks of concomitant treatment. HCT116 mice received 4 weeks of concomitant treatment.

Figure 6. Combinatorial cetuximab and dasatinib treatments decrease proliferation and enhance apoptosis

A) Tumor samples from LS180, LoVo and HCT116 in vivo experiments were prepared and analyzed for proliferation (Ki67) and apoptosis (TUNEL). (−) denotes combinatorial IgG and vehicle control and (+) denotes combinatorial cetuximab plus dasatinib treatment. All representative samples are tumors collected three hours after the final dasatinib or vehicle treatment and 24 hours after the final cetuximab or IgG treatment. White arrows denote positive staining nuclei in Ki67 samples. Black arrows denote positive staining on TUNEL assay samples. Hematoxylin eosin stained section (magnification 400×). B) Quantitation of immunohistochemistry positive staining for Ki67 and TUNEL in combinatorial treatments. Graph of the Ki67 (upper) and TUNEL (lower) expression as percent of positive cells (5 random fields, 4 sections for each sample, * P≤0.05). Concomitant treatment samples were obtained from mice euthanized at 3, 12, and 24 hour time points after the last dasatinib or vehicle treatment and 24, 27, 36, and 48 hours after the last cetuximab or IgG treatment. ImageJ was used to quantify the positive staining. CTX, cetuximab; DSB, dasatinib; IgG, immunoglobulin G,