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.

Objective Vascular calcification is a characteristic feature of atherosclerosis, diabetes and end-stage renal disease. We have demonstrated that activation of AKT upregulates Runx2, a key osteogenic transcription factor that is crucial for calcification of vascular smooth muscle cells (VSMC). Using mice with SMC-specific deletion of PTEN, a major negative regulator of AKT, the present studies uncovered a novel molecular mechanism underlying PTEN/AKT/FOXO-mediated Runx2 upregulation and VSMC calcification. Approaches and Results SMC-specific PTEN deletion mice were generated by crossing PTEN floxed mice with SM22α-Cre transgenic mice. The PTEN deletion resulted in sustained activation of AKT that upregulated Runx2 and promoted VSMC calcification in vitro and arterial calcification ex vivo. Runx2 knockdown did not affect proliferation but blocked calcification of the PTEN deficient VSMC, suggesting that PTEN deletion promotes Runx2-depedent VSMC calcification that is independent of proliferation. At the molecular level, PTEN deficiency increased the amount of Runx2 post-transcriptionally by inhibiting Runx2 ubiquitination. AKT activation increased phosphorylation of FOXO1/3 that led to nuclear exclusion of FOXO1/3. FOXO1/3 knockdown in VSMC phenocopied the PTEN deficiency, demonstrating a novel function of FOXO1/3, as a downstream signaling of PTEN/AKT, in regulating Runx2 ubiquitination and VSMC calcification. Using heterozygous SMC-specific PTEN deficient mice and atherogenic ApoE−/− mice, we further demonstrated AKT activation, FOXO phosphorylation and Runx2 ubiquitination in vascular calcification in vivo. Conclusions Our studies have determined a new causative effect of SMC-specific PTEN deficiency on vascular calcification, and demonstrated that FOXO1/3 plays a crucial role in PTEN/AKT-modulated Runx2 ubiquitination and VSMC calcification.

ChiCTR-TRC-11001530, September 2011.

No abstract