A Predictive model of ‘on target’ drug resistance


Year:

Session type:

Theme:

Daniel O'Neill1,Mike Firth1,Maryam Clausen1,Jonathan Wrigley1,Emanuela Cuomo1
1AstraZeneca

Abstract

Background

Acquired drug resistance represents a major challenge in the development of therapeutics for the treatment of cancer. The problem is exemplified by non-small cell lung cancer (NSCLC) containing activating EGFR mutations. NSCLC tumours driven by EGFR variants are initially sensitive to tyrosine kinase inhibitors (TKI), however patients show renewed disease progression through expansion of tumour cell clones harbouring additional EGFR mutations. Models of drug resistance are therefore necessary to increase our understanding of resistance mechanisms,  predict variants that will arise in patients and improve development strategies.

Method

In the past, this problem has been addressed by screening large panels of gene variants for drug resistance phenotypes in cell culture. Previous methods such as chemical mutagenesis had the advantage to test mutations in the whole genome but did not cover all possible amino acid variants in a specific region of interest. On the other hand, saturation mutagenesis with ‘on-target’ cDNA libraries resulted in expression of all possible variants but in a non-physiological setting, detached from their endogenous regulatory mechanisms.

For the first time, CRISPR/Cas9 allows endogenous modification of genes and facilitates introduction of all possible mutations at a specific locus, whilst maintaining endogenous gene architecture, chromosomal context and epigenetic mileu.

Results

Here we describe the novel CRISPR-based methodology TEMPR (Targeted Endogenous Mapping of Pharmacological Resistance) developed to identify ‘on-target’ mutations in key oncogenes that drive pharmacological resistance. Using EGFR as a target, we show how directed evolution of some residues in the EGFR kinase domain can be predictive of the resistance observed in the clinic in response to known TKIs, such as gefitinib and osimertinib. In fact, in the region analysed, we can recapitulate the variants observed clinically. We also identify amino acids chemically and structurally able to confer resistance to a TKI in structurally conserved tyrosine kinases such as BCR-Abl and Kit.