Differential Inhibitor Sensitivity of Anaplastic Lymphoma Kinase Variants Found in Neuroblastoma

Abstract

Activating mutations in the anaplastic lymphoma kinase (ALK) gene were recently discovered in neuroblastoma, a cancer of the developing autonomic nervous system that is the most commonly diagnosed malignancy in the first year of life. The most frequent ALK mutations in neuroblastoma cause amino acid substitutions (F1174L and R1275Q) in the intracellular tyrosine kinase domain of the intact ALK receptor. Identification of ALK as an oncogenic driver in neuroblastoma suggests that crizotinib (PF-02341066), a dual-specific inhibitor of the ALK and Met tyrosine kinases, will be useful in treating this malignancy. Here, we assessed the ability of crizotinib to inhibit proliferation of neuroblastoma cell lines and xenografts expressing mutated or wild-type ALK. Crizotinib inhibited proliferation of cell lines expressing R1275Q-mutated ALK and a cell line with amplified and overexpressed wild-type ALK. By contrast, cell lines harboring F1174L-mutated ALK were relatively resistant to crizotinib. Biochemical analyses revealed that this reduced susceptibility of F1174L-mutated ALK to crizotinib inhibition results from an increased ATP-binding affinity (as also seen in acquired resistance to EGFR inhibitors), and should be surmountable with higher doses of crizotinib and/or with higher affinity inhibitors.

The R1275Q Neuroblastoma Mutant and Certain ATP-competitive Inhibitors Stabilize Alternative Activation Loop Conformations of Anaplastic Lymphoma Kinase

“…All of the mutations cluster around the active site of ALK. One of the most commonly mutated residues, Phe 1174 , sits in a hydrophobic pocket at the base of the ␣C-helix, where it is involved in hydrophobic stacking interactions with other phenylalanine residues from the N-terminal ␤-turn region (Phe 1098 ), the activation loop (Phe 1271 ), and C-terminal kinase domain (Phe 1245 ) as described by others (35,38,39). The second commonly mutated residue, Arg 1275 , is found in the activation loop where it is part of the ␣-helix formed in the unphosphorylated ALK kinase domain structures.…”

“…The predominant mutations identified from patient samples and neuroblastoma cell lines are F1174L and R1275Q (34). As with the ALK fusion proteins, the neuroblastoma activating mutants are amenable to inhibition by small molecule inhibitors of the ALK kinase activity, although differential sensitivity has been observed depending on the particular inhibitor and mutant (33,35). Interestingly, the F1174L variant and the related F1174C variant have been independently identified in the clinic as a mutations conferring resistance to crizotinib treatment (36,37).…”

“…Models of Crizotinib Binding to the F1174L and R1275Q Neuroblastoma Mutants-The F1174L and R1275Q ALK neuroblastoma mutants can be inhibited by ATP-competitive inhibitors including crizotinib (33,35). Literature IC 50 values for crizotinib inhibition of the F1174L and R1275Q ALK mutants range from 89 -130 and 67-85 nM in in vitro enzyme assays, respectively (35).…”

“…Literature IC 50 values for crizotinib inhibition of the F1174L and R1275Q ALK mutants range from 89 -130 and 67-85 nM in in vitro enzyme assays, respectively (35). In cellular assays, it is generally accepted that the F1174L mutant displays reduced sensitivity to crizotinib relative to the R1275Q mutant or to wild-type enzyme, although the reported level of reduced sensitivity varies (33,35,36,54).…”

“…Literature IC 50 values for crizotinib inhibition of the F1174L and R1275Q ALK mutants range from 89 -130 and 67-85 nM in in vitro enzyme assays, respectively (35). In cellular assays, it is generally accepted that the F1174L mutant displays reduced sensitivity to crizotinib relative to the R1275Q mutant or to wild-type enzyme, although the reported level of reduced sensitivity varies (33,35,36,54). In the clinical setting, recent reports also identify the F1174L variant and the related F1174C variant as secondary mutations conferring resistance to crizotinib therapy in patients harboring an oncogenic ALK fusion protein (36,37).…”