Differential protein stability of EGFR mutants determines responsiveness to tyrosine kinase inhibitors.

Paramita Ray,Ranjit Mehta,Theodore S Lawrence,Mukesh K Nyati,Yee Sun Tan,Alnawaz Rehemtulla,Dipankar Ray,Aarif Ahsan,Lili Zhao,John P Naughton,Shyam Nyati,Vishal Somnay,Merna Sitto,Alexander Bridges

doi:10.18632/oncotarget.11860

Abstract

Non-small cell lung cancer (NSCLC) patients carrying specific EGFR kinase activating mutations (L858R, delE746-A750) respond well to tyrosine kinase inhibitors (TKIs). However, drug resistance develops within a year. In about 50% of such patients, acquired drug resistance is attributed to the enrichment of a constitutively active point mutation within the EGFR kinase domain (T790M). To date, differential drug-binding and altered ATP affinities by EGFR mutants have been shown to be responsible for differential TKI response. As it has been reported that EGFR stability plays a role in the survival of EGFR driven cancers, we hypothesized that differential TKI-induced receptor degradation between the sensitive L858R and delE746-A750 and the resistant T790M may also play a role in drug responsiveness. To explore this, we have utilized an EGFR-null CHO overexpression system as well as NSCLC cell lines expressing various EGFR mutants and determined the effects of erlotinib treatment. We found that erlotinib inhibits EGFR phosphorylation in both TKI sensitive and resistant cells, but the protein half-lives of L858R and delE746-A750 were significantly shorter than L858R/T790M. Third generation EGFR kinase inhibitor (AZD9291) inhibits the growth of L858R/T790M-EGFR driven cells and also induces EGFR degradation. Erlotinib treatment induced polyubiquitination and proteasomal degradation, primarily in a c-CBL-independent manner, in TKI sensitive L858R and delE746-A750 mutants when compared to the L858R/T790M mutant, which correlated with drug sensitivity. These data suggest an additional mechanism of TKI resistance, and we postulate that agents that degrade L858R/T790M-EGFR protein may overcome TKI resistance.

Highlights

The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases (TK) regulates major developmental and metabolic processes
We propose an additional mechanism of differential responsiveness of tyrosine kinase inhibitors (TKIs) sensitive (L858R, delE746-A750) and resistant (T790M) EGFR mutants to erlotinib treatment
Our data suggest a differential effect of erlotinib on destabilizing various EGFR mutants which, along with differential ATP affinity and drug binding of EGFR mutants, is responsible for differential therapeutic outcomes observed in subsets of Non-small cell lung cancer (NSCLC) patients

Summary

Introduction

The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases (TK) regulates major developmental and metabolic processes. There are two activating mutations of the EGFR gene that together constitute about 90% of all EGFR activating mutations: in-frame deletions in exon 19 (delE746-A750) and a point mutation in exon 21 that substitutes an arginine for a leucine at codon 858 (L858R) In addition to these frequent mutations about 5% of lung cancer patient www.impactjournals.com/oncotarget tumors contain an insertion in exon 20 between amino acids 767 to 774 [3]. These activating mutations lead to an equilibrium shift with ATP that favors the activated TK state leading to an increase in kinase activity, and, the tumor cells displaying these mutations have growth and survival advantages [4]. This threonine at residue 790 is considered the gatekeeper residue which controls ATP recruitment by the kinase domain of EGFR

Methods

Results

Conclusion