Figure 6. Ovarian Cancer Cells Chronically Exposed to JQ1 Acquire Dependency on RTK, PI3K and/or ERK Signaling and Exhibit Exquisite Sensitivity to Kinase Inhibitors Targeting These Pathways.

(A) Inhibition of FGFR signaling blocks growth in JQ1-R OC cells to a greater extent than parental cells in colony formation assays. Long-term 14-day colony formation assay of JQ1-R or parental cells treated with FGFR inhibitor AZD4547 (1 μM) or DMSO.

(B–C) A1847-R cells show enhanced sensitivity to IGF1R inhibitor (GSK1904529A) or EGFR inhibitor (lapatinib) relative to parental cells. JQ1-R treated cell viabilities normalized to DMSO treated JQ1-R cells. Viability assessed by CellTiter-Glo.

(D) Blockade of RTKs EGFR or IGF1R in A1847-R cells inhibits downstream kinase survival signaling as shown by blot. Cells were treated with lapatinib (1 or 3 μM) or GSK1904529A (1 or 3 μM) for 48 h.

(E–F) A1847-R cells show enhanced sensitivity to trametinib or GDC-0941 relative to parental cells. JQ1-R treated cell viabilities normalized to DMSO treated JQ1-R cells. Cell viability was assessed by CellTiter-Glo.

(G) Acquired dependency on AKT and ERK signaling in A1847-R cells. Parental A1847 cells or JQ1-R A1847 cells were transfected with siRNAs targeting PI3Ks, AKTs and ERK2 and cultured for 72 h. JQ1-R knockdown cells were normalized to JQ1-R cells transfected with non-targeting siRNA.

(H) Treatment of A1847-R cells with trametinib or GDC-0941 represses FOSL1 and MYC protein levels to a greater extent than JQ1 treatment. Parental and JQ1 resistant cells were treated with escalating doses of JQ1, GDC-0941 or trametinib for 48 h and protein levels determined by western blot.

(I–J) Escalating doses of GDC-0941 or trametinib for 48 h induces apoptosis and blocks RNA pol II phosphorylation in A1847-R cells. Protein levels determined by western blot.

(K) Enhanced sensitivity to PI3K inhibition across JQ1-R cells. Parental or JQ1-resistant OC cells were treated with escalating doses of GDC-0941. Viability assessed by CellTiter-Glo.

(L) Escalating doses of GDC-0941 for 48 h induces apoptosis in JQ1-R cells to a greater extent than parental cells. Caspase activity determined by Caspase-Glo 3/7 assay according to manufacturer.

(M) Blockade of PI3K signaling inhibits colony formation of JQ1-R OC cells. Long-term 14-day colony formation assay of JQ1-R OC cells treated with DMSO (500 nM, JQ1), I-BET151 (2 μM) or GDC-0941 (500 nM). The dose of (500 nM) GDC-0941 was selected, as it is sufficient to reduce downstream AKT activity.

Data presented in (B–C), (E–F), (G), (K) and (L) are triplicate experiments SEM. *p ≤0.05 by student’s t-test. Also see Figure S6.