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Activating mutations in ALK provide a therapeutic target in neuroblastoma.

George RE, Sanda T, Hanna M, Fröhling S, Luther W, Zhang J, Ahn Y, Zhou W, London WB, McGrady P, Xue L, Zozulya S, Gregor VE, Webb TR, Gray NS, Gilliland DG, Diller L, Greulich H, Morris SW, Meyerson M, Look AT - Nature (2008)

Bottom Line: 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).Cytotoxicity was associated with increased amounts of apoptosis as measured by TdT-mediated dUTP nick end labelling (TUNEL).

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.

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.

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

Mentions: The functional consequences of four of the mutations, T1151M, F1174L, A1234T, and R1275Q, were determined by testing their abilities to transform interleukin-3 (IL-3)-dependent murine lymphoid Ba/F3 cells to cytokine-independent growth. Reduction in IL-3 concentration by 100-fold to 0.01 ng/ml resulted in a clear difference in cell proliferation, with the Ba/F3 cells expressing F1174L and R1275Q mutations exhibiting much higher cell numbers relative to those transduced with wild-type ALK or the T1151M mutation (Fig 1a). To generate IL3-independent lines, we reduced the IL-3 concentration by half in successive passages of each transduced Ba/F3 line. After 5 passages, the Ba/F3 cells expressing the F1174L and the R1275Q ALK mutations, as well as NPM-ALK, were able to grow in medium completely lacking IL-3, while cells expressing T1151M or wild-type ALK did not survive. Moreover, when expressed in Ba/F3 cells, the F1174L allele, and to a lesser extent, the R1275Q allele, were associated with constitutive phosphorylation of ALK (Fig. 1b). In contrast, neither the T1151M nor A1234T alleles exhibited ALK phosphorylation. Expression of the F1174L ALK protein in IL-3-deprived Ba/F3 cells was also associated with phosphorylation of downstream targets of ALK signaling such as STAT3 and AKT, while R1275Q was associated with phosphorylation of ERK1/2 and AKT (Fig 1b). Together, these studies demonstrate that the ALK mutant proteins F1174L and R1275Q possess gain-of-function kinase activity that can sustain key signaling pathways in the presence of reduced concentrations of IL-3.


Activating mutations in ALK provide a therapeutic target in neuroblastoma.

George RE, Sanda T, Hanna M, Fröhling S, Luther W, Zhang J, Ahn Y, Zhou W, London WB, McGrady P, Xue L, Zozulya S, Gregor VE, Webb TR, Gray NS, Gilliland DG, Diller L, Greulich H, Morris SW, Meyerson M, Look AT - Nature (2008)

ALK mutant alleles F1174L and R1275Q are activating in Ba/F3 cells and are sensitive to pharmacologic inhibitiona, 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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2587486&req=5

Figure 1: ALK mutant alleles F1174L and R1275Q are activating in Ba/F3 cells and are sensitive to pharmacologic inhibitiona, 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.
Mentions: The functional consequences of four of the mutations, T1151M, F1174L, A1234T, and R1275Q, were determined by testing their abilities to transform interleukin-3 (IL-3)-dependent murine lymphoid Ba/F3 cells to cytokine-independent growth. Reduction in IL-3 concentration by 100-fold to 0.01 ng/ml resulted in a clear difference in cell proliferation, with the Ba/F3 cells expressing F1174L and R1275Q mutations exhibiting much higher cell numbers relative to those transduced with wild-type ALK or the T1151M mutation (Fig 1a). To generate IL3-independent lines, we reduced the IL-3 concentration by half in successive passages of each transduced Ba/F3 line. After 5 passages, the Ba/F3 cells expressing the F1174L and the R1275Q ALK mutations, as well as NPM-ALK, were able to grow in medium completely lacking IL-3, while cells expressing T1151M or wild-type ALK did not survive. Moreover, when expressed in Ba/F3 cells, the F1174L allele, and to a lesser extent, the R1275Q allele, were associated with constitutive phosphorylation of ALK (Fig. 1b). In contrast, neither the T1151M nor A1234T alleles exhibited ALK phosphorylation. Expression of the F1174L ALK protein in IL-3-deprived Ba/F3 cells was also associated with phosphorylation of downstream targets of ALK signaling such as STAT3 and AKT, while R1275Q was associated with phosphorylation of ERK1/2 and AKT (Fig 1b). Together, these studies demonstrate that the ALK mutant proteins F1174L and R1275Q possess gain-of-function kinase activity that can sustain key signaling pathways in the presence of reduced concentrations of IL-3.

Bottom Line: 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).Cytotoxicity was associated with increased amounts of apoptosis as measured by TdT-mediated dUTP nick end labelling (TUNEL).

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.

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.

Show MeSH
Related in: MedlinePlus