Several TKIs that target ALK, a component of the transforming fusion protein EML4–ALK in NSCLC, have been developed (Christensen et al, 2007
; Galkin et al, 2007
; Soda et al, 2007
). Although most patients with NSCLC positive for EML4–ALK derive benefit from treatment with ALK-TKIs, the clinical efficacy of these drugs varies greatly among such individuals and the molecular mechanism underlying this variability has been unclear. We have now shown that the ALK-TKIs TAE684 and crizotinib exert marked antiproliferative and proapoptotic effects in H3122 cells. In contrast, H2228 cells were resistant to the effects of these agents, consistent with previous observations that TAE684 or EML4–ALK depletion by RNAi failed to induce cell death in H2228 cells (Rikova et al, 2007
; Koivunen et al, 2008
We recently showed that the expression of BIM and that of survivin are independently regulated by ERK and STAT3 signalling pathways, respectively, and that they are implicated in ALK-TKI-induced apoptosis in NSCLC cells positive for EML4–ALK (Takezawa et al, 2011
). Our present results show that TAE684 inhibits STAT3 phosphorylation and downregulates survivin in H2228 cells, but that it fails to inhibit ERK phosphorylation and to upregulate BIM in these cells. We found that the MEK inhibitor AZD6244 inhibits ERK phosphorylation and induces BIM expression within the clinically relevant concentration range in H2228 cells, and that the inhibition of both STAT3 and ERK pathways by the combination of TAE684 and AZD6244 was associated with a marked increase in the number of apoptotic cells. We further found that the combination of AZD6244 with depletion of either STAT3 or survivin also exhibited a pronounced proapoptotic effect in H2228 cells, supporting the notion that simultaneous interruption of STAT3 and ERK signalling pathways mediates ALK-TKI-induced apoptosis.
To investigate the mechanism responsible for the sustained activation of ERK signalling in the presence of TAE684 in H2228 cells, we sequenced full-length cDNAs derived from KRAS, HRAS, NRAS, BRAF, CRAF, MEK1, or MEK2; no mutations were detected in any of these genes (data not shown). We also investigated whether aberrant activation of a cell surface receptor might be responsible for this sustained ERK activation with the use of a phosphorylated receptor tyrosine kinase array, but again no such activated receptor tyrosine kinases were detected in H2228 cells (data not shown). Although the mechanism underlying the sustained activation of the ERK signalling pathway in the presence of an ALK inhibitor in H2228 cells remains unknown, our data suggest that the activity of this pathway is responsible, at least in part, for the poor response of these cells to ALK-TKIs.
In conclusion, our results suggest that interruption of both STAT3-survivin and ERK–BIM signalling is required for the induction of apoptosis in lung cancer cells harbouring EML4–ALK. Additional inhibition of the ERK pathway in the presence of TAE684 thus resulted in a pronounced antitumour effect in EML4–ALK-positive NSCLC cells that are resistant to the ALK-TKI alone. Our results thus provide a rationale for evaluation of combination therapy with ALK and MEK inhibitors in EML4–ALK-positive NSCLC patients for whom ALK inhibitors alone show little effect.