ALK mutations confer differential oncogenic activation and sensitivity to ALK inhibition therapy in neuroblastoma

Abstract

Genetic studies have established anaplastic lymphoma kinase (ALK), a cell surface receptor tyrosine kinase, as a tractable molecular target in neuroblastoma. We describe comprehensive genomic, biochemical, and computational analyses of ALK mutations across 1,596 diagnostic neuroblastoma samples. ALK tyrosine kinase domain mutations occurred in 8% of samples--at three hot spots and 13 minor sites--and correlated significantly with poorer survival in high- and intermediate-risk neuroblastoma. Biochemical and computational studies distinguished oncogenic (constitutively activating) from nononcogenic mutations and allowed robust computational prediction of their effects. The mutated variants also showed differential in vitro crizotinib sensitivities. Our studies identify ALK genomic status as a clinically important therapeutic stratification tool in neuroblastoma and will allow tailoring of ALK-targeted therapy to specific mutations.

Figure 1. Distribution of ALK mutations in tumor samples from neuroblastoma patients

The 126 potentially disease-related mutations observed were distributed over the 16 positions marked in the ALK TKD plus R1060 (between the TKD and the transmembrane domain, upper part of figure). Variants with red asterisks (in red text) were also found in germline DNA. Mutations not previously reported in neuroblastoma include R1060H, I1170N, I1183T, L1204F, D1270G, G1286R, and T1343I. See also Table S1 and Figure S1.

Figure 2. Event-free and overall survival based on the presence or absence of an ALK aberration

Kaplan-Meier curves comparing patients with and without an ALK aberration (mutation and/or amplification): (A) event-free survival (EFS) for the entire neuroblastoma cohort, aberration (n=149) vs. no aberration (n=1,199), p<0.0001; (B) overall survival (OS) for the entire neuroblastoma cohort, aberration (n=149) vs. no aberration (n=1,199), p=0.0002; (C) EFS for the high-risk cohort, aberration (n=88) vs. no aberration (n=540), p=0.0043; (D) OS for the high-risk cohort, aberration (n=88) vs. no aberration (n=540), p=0.0018. See also Table S2.

Figure 3. k_cat and K_{M, ATP} values for mutated ALK TKD variants

(A) k_cat values determined for non-phosphorylated ALK TKD variants at saturating (2 mM) ATP, with 2 mM YYY peptide and 10 mM MgCl₂. (B) K_{M, ATP} for non-phosphorylated ALK TKD variants, determined with YYY peptide at 1.0 mM, ATP concentrations from 0.3125 to 2.0 mM, and MgCl₂ at 10 mM. Data for wild-type, F1174L, and R1275Q variants were reported previously (Bresler et al., 2011). (C) k_cat values for phosphorylated ALK TKD variants, determined as in (A). Data (obtained at 25°C) are all shown as mean ± SEM from at least 3 independent experiments. (D) ALK TKD crystal structure from PDB entry 3LCS (Lee et al., 2010), with important structural regions colored as follows: αC/activation loop interface (blue); Phe core (red); P-loop (cyan); N-lobe (green); active site (magenta); and C-lobe (gray). See also Figure S2 and Tables S4 and S5.

Figure 4. Transformation potential of ALK mutants from NIH 3T3 focus formation assays

(A) Representative focus formation assays for NIH 3T3 cells transfected with intact ALK variants or empty vector. (B) Quantitation of data from (A). To correct for transfection efficiency, the number of foci for each transfection was divided by the number of G418-resistant colonies, and transforming ability plotted as foci per G418-resistant colony. Each independent experiment was performed in duplicate and data are presented as mean ± SEM of at least three independent experiments (see Table S6). (C) Plots of ALK transforming ability against in vitro k_cat for non-phosphorylated ALK TKD (left) or autophosphorylated ALK TKD (right). The lines are linear regressions to the data. Correlation coefficients were 0.95 (non-phosphorylated) and 0.095 (phosphorylated), with significant deviation from zero for non-phosphorylated ALK TKD (p<0.0001) but not phosphorylated ALK TKD (p=0.68).