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Functional genomics identifies predictive markers and clinically actionable resistance mechanisms to CDK4/6
Zhichao Tong1, Anuja Sathe1, Benedikt Ebner1
1Department of Urology, Klinikum rechts der Isar, Technical University of Munich, Ismaninger Strasse 22, 81675, München, Germany.
Background:
CDK4/6 inhibitors are a promising treatment strategy in tumor therapy but are hampered by resistance mechanisms. 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 membrane 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.
Results:
In the screen, 1024 sgRNAs encoding for 995 genes were significantly enriched indicative of mediators of resistance. 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 in vivo xenografts.
Conclusions:
Identification of potential predictive markers, resistance mechanisms and rational combination therapies could be achieved by applying a CRISPR-dCas9 screening approach in bladder cancer.
Insights
This study used CRISPR screening to identify resistance mechanisms to CDK4/6 inhibitors in bladder cancer. Combining Palbociclib with pathway inhibitors shows promise for personalized treatment strategies.
Area of Science:
- Oncology
- Genomics
- Molecular Biology
Background:
- Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors offer a promising therapeutic strategy for various cancers.
- Resistance mechanisms can limit the efficacy of CDK4/6 inhibitors, necessitating further research into predictive markers and combination therapies.
Purpose of the Study:
- To identify predictive markers and mechanisms of resistance to CDK4/6 inhibitors in bladder cancer.
- To develop rational combination therapies for personalized treatment approaches.
Main Methods:
- A genome-scale CRISPR-dCas9 activation screen was employed to identify genes mediating resistance to Palbociclib in T24 bladder cancer cells.
- Next-generation sequencing and MAGeCK-VISPR were used for sgRNA count analysis.
- In vitro and in vivo validation, including the chicken embryo chorioallantois membrane model, was performed.
Main Results:
- The screen identified 995 genes as potential mediators of resistance.
- Validated genes were associated with Receptor-Tyrosine Kinases, PI3K-Akt, Ras/MAPK, JAK/STAT, and Wnt signaling pathways.
- Combination therapies of Palbociclib with pathway inhibitors demonstrated beneficial effects in both in vitro and in vivo models.
Conclusions:
- CRISPR-dCas9 screening is an effective approach for identifying resistance mechanisms and potential predictive markers in bladder cancer.
- Rational combination therapies targeting specific signaling pathways can overcome resistance to CDK4/6 inhibitors, paving the way for personalized medicine.
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