Overcoming Acquired Resistance to AZD9291, A Third-Generation EGFR Inhibitor, through Modulation of MEK/ERK-Dependent Bim and Mcl-1 Degradation

Abstract

Purpose The mechanisms accounting for anticancer activity of AZD9291 (osimertinib or TAGRISSO™), an approved third generation EGFR inhibitor, in EGFR-mutant non-small cell lung cancer (NSCLC) cells and particularly for the subsequent development of acquired resistance are unclear and thus are the focus of this study. Experimental design AZD9219-resistant cell lines were established by exposing sensitive cell lines to AZD9291. Protein alterations were detected with Western blotting. Apoptosis was measured with annexin V/flow cytomentry. Growth-inhibitory effects of tested drugs were evaluated in vitro with cell number estimation and colony formation assay and in vivo with mouse xenogtaft models. Protein degradation was determined by comparing protein half-lives and inhibiting proteasome. Gene knockdown were achieved with siRNA or shRNA. Results AZD9291 potently induced apoptosis in EGFR-mutant NSCLC cell lines, in which ERK phosphorylation was suppressed accompanied with Bim elevation and Mcl-1 reduction likely due to enhanced Mcl-1 degradation and increased Bim stability. Blocking Bim elevation by gene knockdown or enforcing Mcl-1 expression attenuated or abolished AZD9291-induced apoptosis. Moreover, AZD9291 lost its ability to modulate Bim and Mcl-1 levels in AZD9291-resistant cell lines. The combination of a MEK inhibitor with AZD9291 restores the sensitivity of AZD9291-resistant cells including those with C797S mutation to undergo apoptosis and growth regression in vitro and in vivo. Conclusions Modulation of MEK/ERK-dependent Bim and Mcl-1 degradation critically mediates sensitivity and resistance of EGFR-mutant NSCLC cells to AZD9291 and hence is an effective strategy to overcome acquired resistance to AZD9291.

ERK inhibition effectively overcomes acquired resistance of epidermal growth factor receptor‐mutant non–small cell lung cancer cells to osimertinib

“…Because osimertinib inhibits MEK/ERK signaling, as do MEK or ERK inhibitors, a concern is whether their combination may accordingly increase toxicity while enhancing anticancer activity against osimertinib‐resistant cells. Consistent with our previous findings, the combination of osimertinib with an ERK inhibitor or the combination of an MEK inhibitor with either erlotinib or afatinib apparently did not decrease mouse body weights, suggesting favorable tolerability of these combinations in mice. Hence, targeting MEK/ERK signaling is a safe and very effective strategy for overcoming acquired resistance to osimertinib, at least in preclinical settings.…”

“…ERK inhibition, like MEK inhibition in our previous report, elevated Bim levels concurrently with enhanced reduction of Mcl‐1 levels when combined with osimertinib in osimertinib‐resistant cell lines. Both Bim elevation and enhanced Mc‐1 reduction are critical for the enhanced induction of apoptosis by the combination of ERK inhibition and osimertinib in osimertinib‐resistant cells because either Bim‐KO or Mcl‐1 overexpression significantly attenuated or abolished the ability of the combination to augment apoptosis in these cell lines.…”

“…Our previous study demonstrated that MEK inhibition effectively overcomes acquired resistance to osimertinib both in vitro and in vivo . We were interested in the effect of MEK/ERK inhibition combined with a first‐generation or second‐generation EGFR‐TKI in overcoming osimertinib resistance in the current study.…”

“…Pelitinib, GDC0994 (ravoxertinib), and VRT752271 (ulixertinib or BVD‐523) were purchased from MedChem Express. All antibodies used in this study were the same as described in our previous studies …”

“…Low densities of tested cells were seeded in 12‐well cell culture plates and were repeatedly treated with the tested agents every 3 days. After 12 days, cell colonies were stained, photographed, and counted, as described previously …”