Abstract
Anaplastic lymphoma kinase (ALK) gene rearrangements are oncogenic drivers in a small subset of patients with non-small-cell lung cancer (NSCLC). The ALK inhibitors are highly effective in NSCLC patients harboring ALK rearrangements; however, most patients acquire resistance to the therapy following an initial response. Mechanisms of acquired resistance are complex. We used LC-MS/MS-based phosphotyrosine-peptide profiling in the EML4-ALK rearranged H3122 and H2228 cells treated with ALK inhibitors, to identify downstream effectors of ALK. We then used Western blot, siRNA experiments, cell proliferation, viability and migration assays to validate our findings. We identified CRKL as a novel downstream effector of ALK signaling. We demonstrated that CRKL tyrosine phosphorylation was repressed by pharmacological inhibition or small interfering RNA (siRNA) knockdown of ALK in the ALK-rearranged cells. More importantly, CRKL knockdown attenuated their cell proliferation, viability, and migration, but it had no effect on ALK phosphorylation and expression in these cells. Furthermore, CRKL tyrosine phosphorylation was inhibited by dasatinib (an inhibitor of ABL and SRC kinases), which in combination with the ALK inhibitor crizotinib displayed a synergistic inhibitory effect in vitro. In conclusion, our study suggests that CRKL is a key downstream effector of ALK, and combined inhibition of ALK and CRKL may represent an effective strategy for treating ALK-rearranged NSCLC patients.
Highlights
Lung cancer remains the leading cause of cancer mortality, accounting for over 150,000 deaths in the US each year, 85% of which are non-small cell lung cancer (NSCLC) [1]
echinoderm microtubule-associated protein-like 4 (EML4)-Anaplastic lymphoma kinase (ALK)-positive lung adenocarcinoma cell lines H3122 and H2228 were sensitive to ALK inhibitors crizotinib and NMS-E628 (Figure 1A)
Among the proteins whose tyrosine phosphorylation status were repressed by ALK inhibitors, we found significant enrichments of integrin signaling, focal adhesion kinase (FAK) signaling and paxillin/talin signaling (Figure 2B); these pathways are highly related to cell migration and actin cytoskeleton modification
Summary
Lung cancer remains the leading cause of cancer mortality, accounting for over 150,000 deaths in the US each year, 85% of which are non-small cell lung cancer (NSCLC) [1]. ALKrearrangements lead to constitutive, ligand-independent activation of the ALK receptor tyrosine kinase, and consequentially aberrant activation of its downstream signaling pathways such as PI3K-AKT, STATs, and RASRAF-MAPK/ERK [6]. Such discovery has led to the successful development of ALK tyrosine kinase inhibitors to treat patients with ALK rearrangements. Crizotinib demonstrated superior efficacy over chemotherapy in ALK-rearranged NSCLC patients, and ceritinib was highly active in patients who had disease progression after crizotinib treatment [79]. Further insights into the molecular underpinnings of response and resistance, and the identification of key downstream effectors of ALK signaling could potentially lead to new treatment strategies in ALK-rearranged NSCLC patients
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