A second fusion gene was also detected from this cell line and contains an additional bp fragment derived from an alternatively spliced exon of EML. This alternatively spliced exon was not detected in any of the other fusion variants.A, NSCLC cells were treated with TAE at the indicated concentrations, and viable cells were measured after hof treatment.The percentage of viable cells is shown relative to untreated controls.B, fluorescenceactivated cell sorting analysis of subG fraction without treatment. Significant apoptosis following TAE treatment is only observed in the H cell line.In addition, we confirmed the presence of the EMLALK fusion in the original tumor specimen that gave rise to the DFCI cell line using interphase FISH. We screened NSCLC origin for the EMLALK fusion gene and detected its presence in of. Four tumors contained SDMA variant, two contained variant, and two contained a novel variant. In variant, exon of EML is fused with exon of ALK. Six of the EMLALK containing tumors were detected in NSCLC from U.All eight of the EMLALK containing tumors were adenocarcinomas.Furthermore, the fusion gene was detected significantly with limited smoking history. The tumor from one of the patients had a concurrent EGFR SDMA kinase domain mutation with the EMLALK fusion gene.None of the eight tumors contained a concurrent KRAS or BRAF mutation. Inhibition of ALK kinase activity in EMLALK fusion gene in vitro and in vivo.To determine whether ALK kinase inhibitors may be therapeutically effective in EMLALK containing NSCLC, we evaluated TAE, a highly potent ALK kinase inhibitor. We found that TAE significantly inhibited the growth of only the H cell line, whereas the other two EMLALK containing cell lines, H and DFCI, were as resistant or a KRAS mutation. It should be noted that the IC for the H cells is nmolL and that TAE exhibits its maximal effects in this responsive cell line at nmolL. At these low concentrations, TAE is highly selective for ALK; therefore, the observed response is not likely to be due to offtarget effects. TAE treatment led to significant apoptosis only in the H cell line as detected by fluorescenceactivated cell sorting. No growth arrest or apoptosis was observed in the other cell lines following TAE treatment.To determine why the growth of only one of three of the EMLALK containing cell lines was inhibited by TAE, we examined its effects on phosphorylation of ALK and downstream signaling proteins. Following nmolL TAE treatment, complete inhibition of phosphorylated ALK was observed in all three of the EMLALK positive cell lines. We also examined the effects of TAE treatment on H in vivo using a xenograft model.We compared the effects of TAE with the EGFR kinase inhibitor erlotinib, which did not inhibit the growth of H cells in vitro. We used erlotinib because we detected EMLALK significantly more frequently in never or former light cigarette smokers with NSCLC and because erlotinib is frequently used in clinical trials in this same patient population. Thus, we wished to determine the efficacy of erlotinib in EMLALK containing NSCLC.We also explored the dosing of TAE by examining two different doses in the xenograft studies, both doses of TAE effectively inhibited the growth of H xenografts.

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