NSCLCs harboring EGFR kinase domain mutations are sensitive to proteasome inhibition

AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA 3261 The ubiquitin-proteasome pathway regulates the intracellular concentration of key proteins including oncogenic kinases. Proteasome inhibition in NSCLC has been tested preclinically, but previous studies have not identified a subset most likely to benefit. Here we report that NSCLC cell lines harboring EGFR kinase domain mutations are routinely sensitive to proteasome inhibition. Activating mutations in the kinase domain of EGFR are most commonly exon 19 deletions and substitutions for L858 in exon 21, and confer sensitivity to the EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib. Over time, resistance universally emerges,associated with a second somatic T790M mutation in approximately 50% of cases. Currently, irreversible EGFR inhibitors are being tested in erlotinib-resistant NSCLCs, with limited efficacy reported in preliminary studies. We propose proteasome inhibition as a novel strategy for NSCLCs resistant to EGFR inhibitors. We have challenged two groups of NSCLC cell lines with bortezomib: one with EGFR mutation and the other with wild-type (WT) EGFR. Annexin V apoptosis assay revealed that IC50s for mutant EGFR NSCLC cells are routinely below 50nM, whereas IC50s for the majority of WT cell lines are above 50nM. Identical results were obtained with other proteasome inhibitors, including MG262 and MG132. In NCI-H1975 cell (EGFR L858R-T790M), 25nM bortezomib treatment caused caspase 3 induction at 16hrs while the induction was not in evident in NCI-H460 cell (EGFR-WT). The induction of apoptosis was partially reversed with a pan-caspase inhibitor Z-VAD-fmk. Proteasome inhibition caused an initial increase in the expression of EGFR, followed by depletion of the protein by 24 hours. Real-time qRT-PCR indicated that EGFR depletion does not occur at the transcription level. The treatment of H1975 or HCC827 (EGFR del) cells with 100nM bortezomib resulted in the induction of Grp78 and Hsp70 at 8 hrs, suggesting the initiation of ER stress. Phosphorylation of eIF2a was evident at 16hrs, which coincided with EGFR depletion, caspase 3 activation, and PARP cleavage. Pretreatment of HCC827 and H1975 cells with a low dose of cyclohexamide reversed the effects of bortezomib. Bortezomib also caused tumor regression in an inducible bitransgenic murine model of adenocarcinoma driven by EGFR harboring compound L858R/T790M mutation. Moreover, HCC827 and H1975 cells were sensitive to Tunicamycin or 2-deoxyglucose, compounds that induce ER stress with a depletion of EGFR. In summary, proteasome inhibition is effective against NSCLCs with EGFR mutations. The mechanism connecting ER stress to apoptosis is under active investigation.