Figure 6 from Bcl-xL Is a Key Mediator of Apoptosis Following KRASG12C Inhibition in KRASG12C-mutant Colorectal Cancer

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AZ’1569-acquired resistant cells exhibit increased PD-L1 expression and a proinflammatory phenotype. A, Left: RW7213 parental and AZ’1569-resistant clones (No. 2, No. 3, and No. 4) were treated for 72 hours with indicated concentrations of AZ’1569 or sotorasib, and cell viability was determined using CTG assays. Right: Lysates from RW7213 parental and AZ’1569-resistant clones were analyzed by WB for KRAS, pEGFRY1068, EGFR, pMETY1234/1235, MET, pERK1/2T202/Y204, ERK1/2, pS6S235/6, S6, pAKTS473, AKT, pEphA2S897, pEphA2Y588, pEphA2Y772, and EphA2. Active Raf1-bound Ras was isolated from RW7213 parental and resistant clones using an RAS-GTP assay and basal GTP-bound and total KRAS levels assessed by WB. LE = longer exposure. KRAS mRNA was quantified using RT-PCR. Raw values were normalized to ACTB and GAPDH expression and were analyzed using the ΔΔCT method. A one-way ANOVA was used to calculate statistical significance. Data are representative of three independent experimental repeats. Results of NGS of RW7213 Par and AZ’1569-R clones are shown. B, RW7213 parental and resistant cells were treated with SHP-099, BI-3406, 5-FU, SN-38, oxaliplatin, crizotinib, cetuximab, dasatinib, trametinib, ulixertinib, capivasertib, PF-4708671, AZD1480, ONC206, ABT-737, sabutoclax or entinostat for 72 hours, at the indicated concentrations and cell viability was assessed using CTG assays. Heatmap represents cell viability relative to control. Data are representative of three independent experimental repeats. C, Top left: Human cytokine array using conditioned medium of RW7213 parental and resistant clones. Right: Mean spot pixel density was analyzed using Image J, rZ (relative to parental cells) were calculated using densitometry data and presented in a heatmap. Bottom left: CXCL1, CD274, and IL8 mRNA in parental and resistant clones were quantified using RT-PCR. Raw values were normalized to the expression of housekeeping genes ACTB and GAPDH and were analyzed using the ΔΔCT method. CXCL1 protein levels in the culture media of parental (Par) and resistant subpopulations were measured by ELISA. A one-way ANOVA was used to calculate statistical significance. Data are representative of three independent experimental repeats. D, Left: Dose–response curves for AZ’1569 in RW7213 cells, incubated with conditioned media from parental cells or drug-resistant clones No. 2, No. 3, or No. 4. Cells were treated for 72 hours and cell viability was determined using CTG assay. IC50 values were calculated using a Prism software package. Dashed line indicates 50% cell viability. A representative of three independent experiments is shown. Right: A 24-well 5-μm polycarbonate Transwell insert-plate system was used. 2.5 × 105 PBMCs were resuspended in 2% FCS-supplemented DMEM and were added to the top chamber. The bottom chamber was filled with conditioned medium (medium = 2% FCS-supplemented DMEM) obtained from RW7213 parental and resistant cells. Cells were incubated for 4 hours, following which CellTiter-Glo was used to measure PBMC migration to the bottom chamber (RLU = relative luminescence). Serum-free DMEM was used in the bottom chamber as a negative control (neg CT). Data are representative of three independent experimental repeats.

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