Activating mutations in ALK provide a therapeutic target in neuroblastoma

Abstract

Neuroblastoma, an embryonal tumour of the peripheral sympathetic nervous system, accounts for approximately 15% of all deaths due to childhood cancer. High-risk neuroblastomas are rapidly progressive; even with intensive myeloablative chemotherapy, relapse is common and almost uniformly fatal. Here we report the detection of previously unknown mutations in the ALK gene, which encodes a receptor tyrosine kinase, in 8% of primary neuroblastomas. Five non-synonymous sequence variations were identified in the kinase domain of ALK, of which three were somatic and two were germ line. The most frequent mutation, F1174L, was also identified in three different neuroblastoma cell lines. ALK complementary DNAs encoding the F1174L and R1275Q variants, but not the wild-type ALK cDNA, transformed interleukin-3-dependent murine haematopoietic Ba/F3 cells to cytokine-independent growth. Ba/F3 cells expressing these mutations were sensitive to the small-molecule inhibitor of ALK, TAE684 (ref. 4). Furthermore, two human neuroblastoma cell lines harbouring the F1174L mutation were also sensitive to the inhibitor. Cytotoxicity was associated with increased amounts of apoptosis as measured by TdT-mediated dUTP nick end labelling (TUNEL). Short hairpin RNA (shRNA)-mediated knockdown of ALK expression in neuroblastoma cell lines with the F1174L mutation also resulted in apoptosis and impaired cell proliferation. Thus, activating alleles of the ALK receptor tyrosine kinase are present in primary neuroblastoma tumours and in established neuroblastoma cell lines, and confer sensitivity to ALK inhibition with small molecules, providing a molecular rationale for targeted therapy of this disease.

Figure 1. ALK mutant alleles F1174L and R1275Q are activating in Ba/F3 cells and are sensitive to pharmacologic inhibition

a, Growth of Ba/F3 cells expressing wild-type or mutant ALK in 10-and 100-fold-reduced concentrations of IL-3. The values are means ± standard deviations (SD) of triplicate experiments. b, Western blot analysis of ALK proteins and their downstream effectors in wild-type or mutated ALK-expressing Ba/F3 cells depleted of IL-3 for 6 hours. The mobilities of molecular weight (M.W.) standards are shown on the left. c, Growth of mutated ALK-expressing Ba/F3 cells exposed to TAE684 for 72 hours. The values are means ± SD of triplicate experiments.

Figure 2. Neuroblastoma cell lines harboring the F1174L, but not the R1275Q ALK mutation, are dependent on the altered protein for growth and survival

a, Growth rates of neuroblastoma cell lines with and without ALK mutations after a 3-day exposure to varying concentrations of TAE684. The values are means ± SD of triplicate experiments. b, Induction of apoptosis in the TAE684-sensitive and resistant cell lines as determined by TUNEL assay. c, Growth of ALK-mutant neuroblastoma cell lines Kelly and SH-SY5Y (F1174L) and SMS-KCNR (R1275Q) in which ALK expression was downregulated using shRNA. The inset panel shows western blot analysis of ALK expression in the control and shRNA transduced lines. The mobilities of molecular weight (M.W.) standards are shown on the left. d, Induction of apoptosis by ALK shRNA knockdown as determine by TUNEL assay.

Figure 3. The constitutively activated F1174L ALK protein has a higher turnover rate than wild-type ALK in neuroblastoma cell lines

a, Analysis of ALK expression in ALK wild-type and mutated neuroblastoma cell lines. b, Western blot depicting ALK expression in the mutated cell line Kelly (F1174L), and in the ALK wild-type line IMR-5 following exposure to increasing doses of TAE684. c, Western blot of ALK expression in the ALK mutated cell line Kelly (F1174L), and in IMR-5, harboring wild-type ALK, following treatment with the proteasome inhibitor MG-132.