T24 cells, transduced with CRISPR/dCas9 SAM library containing 70291 sgRNAs were selected for palbociclib resistance. Genomic DNA was extracted and sequenced by NGS. Enrichment of sgRNAs candidates were analyzed using MAGeCK-VISPR. The most significant 10 candidates were validated for mRNA expression, cell cycle distribution, cell proliferation, and colony formation. Bioinformatic analysis of enriched sgRNA candidates was done using DAVID and signaling pathways. Suitable combination therapies were determined with cell viability, cell cycle progression, caspase3/7 activity in vitro and on the CAM xenograft model. Molecular mechanisms of resistance were preliminarily investigated with Western blot.
They identified 3440 genes targeted by 3608 candidate sgRNAs that might confer resistance to Palbociclib. The 10 most significant candidates sgRNAs were all successfully validated. Oncogenic signaling pathways belong to ‘pathways in cancer' containing clinically druggable targets were identified including Cell cycle, PI3K-AKT, MAPK, JAK-STAT, VEGF, Ras, and PPARγ. Combination therapies with Palbociclib and inhibitors against these pathways revealed that targeting FGFRs, VEGFRs and PI3K/Akt pathway exhibited synergism with CDK4/6 inhibition. Further investigation of molecular mechanisms revealed that compensatory activation of PI3K, MAPK, and CDK2 pathways induced resistance. Synergism of combination therapies was achieved via multiple mechanisms including cell cycle arrest and apoptosis.
In conclusion, application of a genome-scale transcriptional activation screen by an engineered CRISPR/dCas9 complex together with bioinformatic analysis are powerful approaches to identify mechanisms of resistance to palbociclib and to develop novel directed combination therapies in bladder cancer.
Presented by: Zhichao Tong, Department of Urology, Klinikum rechts der Isar, Technical University of Munich, Germany
Co-Authors: Anuja Sathe, Benedict Ebner, Pan Qi, Roman Nawroth Department of Urology, Klinikum rechts der Isar, Technical University of Munich
Written by: Stephen B. Williams, M.D., Associate Professor, Division of Urology, The University of Texas Medical Branch, Galveston, TX. and Ashish M. Kamat, M.D. Professor, Department of Urology, Division of Surgery, The University of Texas MD Anderson Cancer Center, Houston, TX at the 16th Annual Meeting of the International Bladder Cancer Network (IBCN) October 11-13, 2018 - the Inntel Hotels Rotterdam Centre, Rotterdam, The Netherlands