Functional genomic identification of predictive biomarkers of everolimus sensitivity and of new molecular targets in ren
Lead Research Organisation:
CANCER RESEARCH UK
Department Name: Cancer Research UK
Abstract
Everolimus is an effective new drug for the treatment of kidney cancer, however not all patients respond to everolimus and even those who respond develop resistance after 6-9 months of treatment. We will use a technique called ‘functional genomics‘ to de-activate each of the 21.000 human genes individually in kidney cancer cells grown in the laboratory, to identify genes that can lead to everolimus resistance. Resistance genes will be further studies to understand how resistance develops and how it can be prevented or treated. We will also analyse kidney cancer samples from patients treated with everolimus to determine whether the identified resistance genes are commonly altered in resistant cancers. This should lead to the development of a test that can determine whether a patient with kidney cancer is likely to respond to everolimus before treatment is actually started. Such a test would spare patients with resistant disease the side effects of the treatment and improve their quality of life. Everolimus is not approved for the use in the NHS because of the high costs. A test that allows targeting the drug to patients with sensitive disease would reduce overall costs and hopefully make it available to NHS patients.
Technical Summary
Background:
The mTOR (mammalian Target Of Rapamycin) inhibitor everolimus is active in renal cell carcinoma (RCC) but intrinsic and acquired drug resistance limits efficacy. Everolimus has direct cytostatic effects on RCC cells and anti-angiogenic activity through the suppression of hypoxia inducible factor 1 (HIF1 ) and its target VEGF in von-Hippel-Lindau (VHL)-gene deficient RCCs, and by inhibiting mTOR in endothelial cells. Mechanisms of everolimus resistance are poorly understood and there are no reliable predictive biomarkers of response.
Aims:
Whole genome siRNA screens will be applied to RCC cell lines to identify genes that mediate resistance to the cytostatic effect of everolimus, the hypoxic environment generated by its anti-angiogenic activity, and to the suppression of the HIF1 -VEGF pathway by mTOR inhibition. These results and their integration with transcriptomic and genomic profiles of pre- and post-treatment tumour samples from a clinical trial of everolimus in patients with RCC will identify:
1. Clinically relevant resistance mechanisms
2. Predictors of everolimus sensitivity and resistance
3. Therapeutic targets leading to synthetic lethality in VHL deficient RCC cell lines
Design and methodology:
A siRNA screen has already been performed in a VHL deficient RCC cell line and identified novel mediators of everolimus resistance and sensitivity. Further immunostaining of this screen for carbonic anhydrase IX as a marker for HIF1 activity will identify genes that contribute to in vivo resistance by rescuing HIF1 activity and -target gene expression in the presence of everolimus. A second siRNA screen will be performed to reveal how RCC cells can develop resistance to hypoxic conditions. Resistance mechanisms will be further characterized in vitro and by systems biology approaches. Screening results will be compared with mRNA expression, copy number variation and whole genome exon capture sequencing datasets obtained from pre- and post- treatment RCC tumour samples to identify which genes are relevant to resistance in patients. Candidate predictive biomarkers will be assessed in a validation cohort. Genes causing synthetic lethality in the VHL-null background when repressed will be identified in isogenic VHL-wt and VHL-null cell lines.
Scientific and clinical opportunities:
This approach should lead to the identification of a predictive biomarker of everolimus response in RCC. It should simultaneously define molecular mechanisms of resistance and potential targets to facilitate the development of new therapeutic strategies. These strategies could reduce the health economic burden associated with therapeutic resistance and improve outcomes in RCC by targeting treatment to patients with molecularly pre-defined drug sensitive disease.
The mTOR (mammalian Target Of Rapamycin) inhibitor everolimus is active in renal cell carcinoma (RCC) but intrinsic and acquired drug resistance limits efficacy. Everolimus has direct cytostatic effects on RCC cells and anti-angiogenic activity through the suppression of hypoxia inducible factor 1 (HIF1 ) and its target VEGF in von-Hippel-Lindau (VHL)-gene deficient RCCs, and by inhibiting mTOR in endothelial cells. Mechanisms of everolimus resistance are poorly understood and there are no reliable predictive biomarkers of response.
Aims:
Whole genome siRNA screens will be applied to RCC cell lines to identify genes that mediate resistance to the cytostatic effect of everolimus, the hypoxic environment generated by its anti-angiogenic activity, and to the suppression of the HIF1 -VEGF pathway by mTOR inhibition. These results and their integration with transcriptomic and genomic profiles of pre- and post-treatment tumour samples from a clinical trial of everolimus in patients with RCC will identify:
1. Clinically relevant resistance mechanisms
2. Predictors of everolimus sensitivity and resistance
3. Therapeutic targets leading to synthetic lethality in VHL deficient RCC cell lines
Design and methodology:
A siRNA screen has already been performed in a VHL deficient RCC cell line and identified novel mediators of everolimus resistance and sensitivity. Further immunostaining of this screen for carbonic anhydrase IX as a marker for HIF1 activity will identify genes that contribute to in vivo resistance by rescuing HIF1 activity and -target gene expression in the presence of everolimus. A second siRNA screen will be performed to reveal how RCC cells can develop resistance to hypoxic conditions. Resistance mechanisms will be further characterized in vitro and by systems biology approaches. Screening results will be compared with mRNA expression, copy number variation and whole genome exon capture sequencing datasets obtained from pre- and post- treatment RCC tumour samples to identify which genes are relevant to resistance in patients. Candidate predictive biomarkers will be assessed in a validation cohort. Genes causing synthetic lethality in the VHL-null background when repressed will be identified in isogenic VHL-wt and VHL-null cell lines.
Scientific and clinical opportunities:
This approach should lead to the identification of a predictive biomarker of everolimus response in RCC. It should simultaneously define molecular mechanisms of resistance and potential targets to facilitate the development of new therapeutic strategies. These strategies could reduce the health economic burden associated with therapeutic resistance and improve outcomes in RCC by targeting treatment to patients with molecularly pre-defined drug sensitive disease.