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. Author manuscript; available in PMC: 2018 Jul 25.
Published in final edited form as: Cell Rep. 2017 Jul 25;20(4):999–1015. doi: 10.1016/j.celrep.2017.07.006

Figure 3. Co-inhibition of the MEK/ERK pathway plus SRC induces synergistic apoptosis in KRAS/PIK3CA double mutant colorectal cancers (CRCs) through induction of BIM.

Figure 3

A) At right, relative depletion of SRC across CRC screens. At left, rank ordered relative depletion scores (threescore) plotted for all 378 genes in a KRAS/PIK3CA double mutant and a KRAS mutant/PIK3CA wild-type cell line. B) GI50 for a SRC inhibitor (dasatinib) in the presence of either vehicle or a constant background dose of ERK inhibitor (VX-11e) in CRC240 cells. CI values are calculated for each dose on the curve. C) SRC inhibitor (dasatinib) sensitization score across a panel of CRC cell lines with indicated alterations in KRAS and PIK3CA. Sensitization score is calculated as the log10 ratio of the GI50 values for SRCi (dasatinib) relative to the same quantity in the presence of a constant background dose of 1 μM ERKi (VX-11e). Additive effects center at zero, antagonistic effects are negative, and sensitization effects are positive. D) Apoptosis measurements, reported as the percentage of annexin v+/7-AAD- cells in six CRC cell lines representing various mutational backgrounds treated with vehicle, a SRC inhibitor (dasatinib, 200nM), an ERK inhibitor (VX-11e, 1μM), or the combination of both. E) Immunoblots of P-AKT, T-AKT, P-ERK, T-ERK, and a loading control in four CRC cell lines representing different mutational backgrounds treated with vehicle, a SRC inhibitor (dasatinib, 1μM), a MEK inhibitor (AZD6244, 0.5μM), or the combination of both for 6 hrs. Loading control for CRC240 and LoVo is Histone H3 and control for CRC240 and SW480 is vinculin. Blots are cropped for clarity. F) HCT116 xenografts treated with vehicle, SRCi (dasatinib 15 mg/kg, daily) or AKTi (MK2206 60 mg/kg, daily), MEKi (AZD6244 10 mg/kg, twice daily), or the combination of a MEKi with either SRCi or AKTi for 21 days, shown as tumor size at endpoint (top) or growth curve (bottom). G) Immunoblot of T-BIM and vinculin in four CRC cell lines representing different mutational backgrounds treated with vehicle, a SRC inhibitor (dasatinib, 1μM), a MEK inhibitor (AZD6244, 0.5μM), or the combination of both for 6 hrs. Blots are cropped for clarity. H) Apoptosis (annexin V+/7-AAD- percentage) following ectopic overexpression of BIM in the absence of drug in indicated CRC cell lines. I) Quantification by immunohistochemistry (IHC) for T-BIM in CRC patient samples stratified into WT/WT or KRAS/PIK3CA mutant groups. To the right are representative images of each case, also showing H&E staining. Error bars show data ± SEM. *p<0.05. See also Figure S3 and Table S5.