Functional Genomics Identifies Multiple Clinically Actionable Resistance Mechanisms to CDK4/6 Inhibition in Bladder Cancer

Background: CDK4/6 inhibitors are in combination with chemotherapy or small molecules effective compounds in tumor therapy. This study was performed to reveal predictive markers, mechanisms of resistance and to develop rational combination therapies for a personalized therapy approach in bladder cancer. Methods: A genome-scale CRISPR-dCas9 activation screen for resistance to the CDK4/6 inhibitor Palbociclib was performed in the bladder cancer derived cell line T24. sgRNA counts were analyzed using next generation sequencing and MAGeCK-VISPR. Significantly enriched sgRNAs were cloned and validated on a molecular and functional level for mediating resistance to Palbociclib treatment. Analysis was done in vitro and in vivo in the chorioallantois mebrane model of the chicken embryo. Comparison of screen hits to signaling pathways and clinically relevant molecular alterations was performed using DAVID, Reactome, DGIdb and cBioPortal. Findings: In the screen, 1024 sgRNAs encoding for 995 genes were significantly enriched and 8 random sgRNAs were validated, revealing partial rescue to Palbociclib treatment. Within this gene panel, members of Receptor-Tyrosine Kinases, PI3K-Akt, Ras/MAPK, JAK/STAT or Wnt signaling pathways were identified. Combination of Palbociclib with inhibitors against these signaling pathways revealed beneficial effects in vitro and in vivo xenografts Interpretation: Identification of potential predictive markers, resistance mechanisms and rational combination therapies could be achieved by applying a CRISPR-dCas9 approach in bladder cancer. Funding: This work has been supported by the China Scholarship Council (ZT, QP) and the Fritz-Thyssen foundation (AS). This work was also supported by Pfizer pharma inc. grant IIR WI207703. Declaration of Interest: The authors declare no potential conflicts of interest. Ethical Approval: In accordance with the law of Germany and the District Government of upper Bavaria, the chicken embryos involved in this manuscript were sacrificed at Embryo Day 15, thus no need for additional ethics approval.