Volume 71, Issue 6, Pages 858-862 (June 2017) A Functional Genetic Screen Identifies the Phosphoinositide 3-kinase Pathway as a Determinant of Resistance to Fibroblast Growth Factor Receptor Inhibitors in FGFR Mutant Urothelial Cell Carcinoma  Liqin Wang, Tonći Šuštić, Rodrigo Leite de Oliveira, Cor Lieftink, Pasi Halonen, Marieke van de Ven, Roderick L. Beijersbergen, Michel M. van den Heuvel, René Bernards, Michiel S. van der Heijden  European Urology  Volume 71, Issue 6, Pages 858-862 (June 2017) DOI: 10.1016/j.eururo.2017.01.021 Copyright © 2017 European Association of Urology Terms and Conditions

Fig. 1 PIK3CA suppression enhances sensitivity to fibroblast growth factor receptor (FGFR) inhibition in UCC. (A) RT112 cells were treated with AZD4547 (80nM) and viability was followed using an incucyte assay. Error bars represent four biological replicates. (B) Outline of synthetic lethality short hairpin RNA (shRNA) screen for enhancers of AZD4547 sensitivity. Human kinome shRNA library polyclonal virus was produced to infect RT112 cells, which were then left untreated (Ctrl.) for 10 d or treated with 30nM AZD4547 for 14 d. After selection, shRNA inserts from both arms were recovered by polymerase chain reaction and their abundance was quantified by deep sequencing. (C) Representation of the relative abundance of the shRNA barcode sequences from the shRNA screen. The y-axis shows log2 of the fold change of shRNA abundance of the treated and untreated samples. The x-axis indicates the log10 of the average read count of each shRNA in the untreated sample. shPIK3CA, shPIK3R1, shAKT3, and shEGFR identified as the top hit according to the presence of at least three independent shRNAs in three biological screen replicates. (D) The level of knockdown of PIK3CA by three different shRNAs was measured by PIK3CA protein levels by western blot. HSP90 protein expression is used for normalization. (E) The functional phenotypes of independent shPIK3CA vectors are indicated by colony formation assay in 50nM AZD4547. The cells were fixed, stained, and photographed after 10 d. European Urology 2017 71, 858-862DOI: (10.1016/j.eururo.2017.01.021) Copyright © 2017 European Association of Urology Terms and Conditions

Fig. 2 Functional and biochemical interaction between fibroblast growth factor receptor (FGFR) and phosphoinositide 3-kinase (PI3K) inhibition in urothelial cell carcinoma. (A,B) Synergistic response of RT112 and JMSU1 to combinations of FGFR (AZD4547) and PI3K (BKM120) inhibitors. RT112 and JMSU1 cells were cultured in increasing concentration of FGFR inhibitor AZD4547 alone, PI3K inhibitor BKM120 (0.5μM) alone, or their combination. The cells were fixed, stained, and photographed after 14 d. (C,D) Biochemical analysis of combination of FGFR and PI3K inhibitors. RT112 and JMSU1 cells were harvested after 24h of drug treatment. Phosphorylated-AKT (p-AKT), total AKT (AKT), cleaved-PARP (c-PARP), phosphorylated-FRS2 (p-FRS2), total FRS2 (FRS2), phosphorylated-ERK (p-ERK), and total ERK (ERK) were measured. HSP90 served as a loading control. (E) RTK blot analysis of FGFR inhibitor (100nM, 7 d) treated RT112. AZD4547 induced feedback activation of EGFR and ERBB3. f, Biochemical analysis indicated the reactivation of EGFR and ERBB3 in association with elevated PI3K-AKT signaling, but not MAPK signaling after treating RT112 cells for 1 wk with 50mM AZD4547. Phosphorylated-EGFR (p-EGFR), total EGFR (EGFR), phosphorylated-ERBB3 (p-ERBB3), total HER3 (HER3), phosphorylated-FGFR3 (p-FGFR3), phosphorylated-AKT (p-AKT), total AKT (AKT), phosphorylated-FRS2 (p-FRS2), total FRS2 (FRS2), phosphorylated-ERK (p-ERK), and total ERK (ERK) were measured. β-actin served as a loading control. (G) FGFR inhibitor (AZD4547) in combination with PI3K inhibitor (BKM120) significantly suppresses tumor growth in RT112 xenograft model. (H) FGFR altered tumors initially respond to FGFR inhibition. However, the tumor cells are able to upregulate other RTKs that result in enhanced signaling through the PI3K-AKT pathway, leading to drug resistance. Combining FGFR inhibition with a PI3K inhibitor eliminates the PI3K-AKT activity and synergistically kills cancer cells. c-PARP=poly (adenosine diphosphate-ribose) polymerase; ERBB3=Erb-B2 receptor tyrosine kinase 3; MAPK=mitogen-activated protein kinase; RTK=receptor tyrosine kinases. * p<0.01 analysis of variance. ** p<0.0001. European Urology 2017 71, 858-862DOI: (10.1016/j.eururo.2017.01.021) Copyright © 2017 European Association of Urology Terms and Conditions