Receptor Anaplastic Lymphoma Kinase Research Articles

Anaplastic Lymphoma Kinase (Alk) Positivity in Non Small Cell Lung Cancer (Nsclc) Has Similar or Worse Prognosis for Clinical Outcome As Alk Wild Type: a Systematic Review of Published Literature

ABSTRACT Aim: Anaplastic lymphoma kinase (ALK) positive NSCLC identifies a unique subset of patients who respond to treatment with crizotinib (Pfizer Inc, NY), an ALK receptor tyrosine kinase inhibitor. However, it is not well established whether ALK status is predictive of a better or a worse outcome for those who do not receive crizotinib. We performed a systematic review of recent literature on ALK positive NSCLC to answer this question. Methods: Ovid Medline and Embase databases were searched for publications on ALK-positive NSCLC until December 2013. Studies were included if outcomes for crizotinib naive patients were presented for either Progression-Free Survival (PFS) or Overall Survival (OS). For each study identified, 2 investigators independently extracted and tabulated relevant findings. Demographics, ALK status, type of diagnostic test, treatment received and response to treatment were noted. To determine if ALK status was prognostic, efficacy outcomes (PFS and / or OS) for ALK-positive patients were compared to ALK wild type (WT) patients. Results: Twenty one studies published between 2009 to 2013, with nearly 8000 screened NSCLC patients and 700 ALK-positive patients, met the selection criteria. All publications were retrospective in nature, majority reported single institution experiences, with a mix of completely resected early stage NSCLC and recurrent or advanced disease. The number of ALK-positive patients in studies determined by Fluorescent In Situ Hybridisation (FISH) ranged from 7 to 121 (mean = 32) and the number of ALK-positive patients in studies determined by non-FISH methods ranged from 3 - 39 (mean = 18). Enrichment strategies were commonly employed with predominantly adenocarcinomas being selected for ALK testing. PFS and / or OS for ALK-positive NSCLC patients did not demonstrate a favourable outcome and was similar or worse compared to EGFR WT/ ALK WT patients. Conclusions: This analysis of published retrospective studies in ALK-positive NSCLC, suggest that ALK-positivity is not a positive prognostic factor and does not confer a favourable outcome in absence of crizotinib therapy. Disclosure: J. Binko: Employee of Pfizer Oncology and holder of Pfizer stock; P. Delahoy: Employee of Pfizer Oncology and holder of Pfizer stock. M. Alam: Employee of Pfizer Oncology and holder of Pfizer stock.

Mechanistic insight into ALK receptor tyrosine kinase in human cancer biology

The burgeoning field of anaplastic lymphoma kinase (ALK) in cancer encompasses many cancer types, from very rare cancers to the more prevalent non-small-cell lung cancer (NSCLC). The common activation of ALK has led to the use of the ALK tyrosine kinase inhibitor (TKI) crizotinib in a range of patient populations and to the rapid development of second-generation drugs targeting ALK. In this Review, we discuss our current understanding of ALK function in human cancer and the implications for tumour treatment.

Phosphoproteomic analysis of anaplastic lymphoma kinase (ALK) downstream signaling pathways identifies signal transducer and activator of transcription 3 as a functional target of activated ALK in neuroblastoma cells

Activation of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase is a key oncogenic mechanism in a growing number of tumor types. In the majority of cases, ALK is activated by fusion with a dimerizing partner protein as a result of chromosomal translocation events, most studied in the case of the nucleophosmin–ALK and echinoderm microtubule-associated protein-like 4–ALK oncoproteins. It is now also appreciated that the full-length ALK receptor can be activated by point mutations and by deletions within the extracellular domain, such as those observed in neuroblastoma. Several studies have employed phosphoproteomics approaches to find substrates of ALK fusion proteins. In this study, we used MS-based phosphotyrosine profiling to characterize phosphotyrosine signaling events associated with the full-length ALK receptor. A number of previously identified and novel targets were identified. One of these, signal transducer and activator of transcription 3 (STAT3), has previously been observed to be activated in response to oncogenic ALK signaling, but the significance of this in signaling from the full-length ALK receptor has not been explored further. We show here that activated ALK robustly activates STAT3 on Tyr705 in a number of independent neuroblastoma cell lines. Furthermore, knockdown of STAT3 by RNA interference resulted in a reduction in myelocytomatosis neuroblastom (MYCN) protein levels downstream of ALK signaling. These observations, together with a decreased level of MYCN and inhibition of neuroblastoma cell growth in the presence of STAT3 inhibitors, suggest that activation of STAT3 is important for ALK signaling activity in neuroblastoma.

Crizotinib - Molekulare Therapie des Lungenkarzinoms

The anaplastic lymphoma kinase (ALK) can act as a key oncogenic driver after activation by means of processes such as gene rearrangement. In approximately 5 % of patients with advanced non-small cell lung cancer (NSCLC), an oncogenic fusion gene of echinoderm microtubule-associated protein-like 4 (EML4) and ALK has been detected using fluorescence in situ hybridisation (FISH). Moreover, various methods including immunohistochemistry and PCR-based assays can be used for analysing ALK expression. Clinical data have been generated for crizotinib, a small molecule inhibitor of the ALK receptor tyrosine kinase, demonstrating a substantial improvement of objective response rate and prolonged progression-free survival (PFS) compared to standard chemotherapy in pretreated NSCLC patients harbouring EML4-ALK fusion genes. In the current review, recent data on the detection and inhibition of ALK in advanced NSCLC are summarised.

Control of ALK (wild type and mutated forms) phosphorylation: Specific role of the phosphatase PTP1B

Phosphorylation of proteins on tyrosine residues is regulated by the activities of protein tyrosine kinases and protein tyrosine phosphatases. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) essentially and transiently expressed during development of the central and peripheral nervous systems. ALK has been identified as a major neuroblastoma predisposition gene and activating mutations have been identified in a subset of sporadic neuroblastoma tumors. We previously established that the mutated receptors were essentially retained in the endoplasmic reticulum/Golgi compartments due to their constitutive activity. Intriguingly we demonstrated a stronger phosphorylation for the minor pool of receptor addressed to the plasma membrane. We decided to investigate the potential involvement of tyrosine phosphatase in dephosphorylation of this intracellular pool. In this study we first showed that general inhibition of tyrosine phosphatases resulted in a dramatic increase of the tyrosine phosphorylation of the wild type but also of the mutated receptors. This increase not only required the intrinsic kinase activity of the ALK receptor but also involved the Src tyrosine kinase family. Second we provided strong evidences that the endoplasmic reticulum associated phosphatase PTP1B is key player in the control of ALK phosphorylation. Our data shed a new light on the biological significance of the basal phosphorylation levels of both wild type and mutated ALK receptors and could be essential to further understand their roles in malignancies.

Cell culture andDrosophilamodel systems define three classes of anaplastic lymphoma kinase mutations in neuroblastoma

SUMMARYNeuroblastoma is a childhood extracranial solid tumour that is associated with a number of genetic changes. Included in these genetic alterations are mutations in the kinase domain of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (RTK), which have been found in both somatic and familial neuroblastoma. In order to treat patients accordingly requires characterisation of these mutations in terms of their response to ALK tyrosine kinase inhibitors (TKIs). Here, we report the identification and characterisation of two novel neuroblastoma ALK mutations (A1099T and R1464STOP), which we have investigated together with several previously reported but uncharacterised ALK mutations (T1087I, D1091N, T1151M, M1166R, F1174I and A1234T). In order to understand the potential role of these ALK mutations in neuroblastoma progression, we have employed cell culture-based systems together with the model organism Drosophila as a readout for ligand-independent activity. Mutation of ALK at position 1174 (F1174I) generates a gain-of-function receptor capable of activating intracellular targets such as ERK (extracellular signal regulated kinase) and STAT3 (signal transducer and activator of transcription 3) in a ligand-independent manner. Analysis of these previously uncharacterised ALK mutants and comparison with ALKF1174 mutants suggests that ALK mutations observed in neuroblastoma fall into three classes. These classes are: (i) gain-of-function ligand-independent mutations such as ALKF1174l, (ii) kinase-dead ALK mutants, e.g. ALKI1250T (Schönherr et al., 2011a) and (iii) ALK mutations that are ligand-dependent in nature. Irrespective of the nature of the observed ALK mutants, in every case the activity of the mutant ALK receptors could be abrogated by the ALK inhibitor crizotinib (Xalkori/PF-02341066), albeit with differing levels of sensitivity.

Pleiotrophin (PTN) Expression and Function and in the Mouse Mammary Gland and Mammary Epithelial Cells

Expression of the heparin-binding growth factor, pleiotrophin (PTN) in the mammary gland has been reported but its function during mammary gland development is not known. We examined the expression of PTN and its receptor ALK (Anaplastic Lymphoma Kinase) at various stages of mouse mammary gland development and found that their expression in epithelial cells is regulated in parallel during pregnancy. A 30-fold downregulation of PTN mRNA expression was observed during mid-pregnancy when the mammary gland undergoes lobular-alveolar differentiation. After weaning of pups, PTN expression was restored although baseline expression of PTN was reduced significantly in mammary glands of mice that had undergone multiple pregnancies. We found PTN expressed in epithelial cells of the mammary gland and thus used a monoclonal anti-PTN blocking antibody to elucidate its function in cultured mammary epithelial cells (MECs) as well as during gland development. Real-time impedance monitoring of MECs growth, migration and invasion during anti-PTN blocking antibody treatment showed that MECs motility and invasion but not proliferation depend on the activity of endogenous PTN. Increased number of mammospheres with laminin deposition after anti-PTN blocking antibody treatment of MECs in 3D culture and expression of progenitor markers suggest that the endogenously expressed PTN inhibits the expansion and differentiation of epithelial progenitor cells by disrupting cell-matrix adhesion. In vivo, PTN activity was found to inhibit ductal outgrowth and branching via the inhibition of phospho ERK1/2 signaling in the mammary epithelial cells. We conclude that PTN delays the maturation of the mammary gland by maintaining mammary epithelial cells in a progenitor phenotype and by inhibiting their differentiation during mammary gland development.

ALK receptor activation, ligands and therapeutic targeting in glioblastoma and in other cancers.

The intracellular anaplastic lymphoma kinase (ALK) fragment shows striking homology with members of the insulin receptor family and was initially identified as an oncogenic fusion protein resulting from a translocation in lymphoma and more recently in a range of cancers. The full-length ALK transmembrane receptor of ~220 kDa was identified based on this initial work. This tyrosine kinase receptor and its ligands, the growth factors pleiotrophin (PTN) and midkine (MK) are highly expressed during development of the nervous system and other organs. Each of these genes has been implicated in malignant progression of different tumor types and shown to alter phenotypes as well as signal transduction in cultured normal and tumor cells. Beyond its role in cancer, the ALK receptor pathway is thought to contribute to nervous system development, function, and repair, as well as metabolic homeostasis and the maintenance of tissue regeneration. ALK receptor activity in cancer can be up-regulated by amplification, overexpression, ligand binding, mutations in the intracellular domain of the receptor and by activity of the receptor tyrosine phosphatase PTPRz. Here we discuss the evidence for ligand control of ALK activity as well as the potential prognostic and therapeutic implications from gene expression and functional studies. An analysis of 18 published gene expression data sets from different cancers shows that overexpression of ALK, its smaller homolog LTK (leukocyte tyrosine kinase) and the ligands PTN and MK in cancer tissues from patients correlate significantly with worse course and outcome of the disease. This observation together with preclinical functional studies suggests that this pathway could be a valid therapeutic target for which complementary targeting strategies with small molecule kinase inhibitors as well as antibodies to ligands or the receptors may be used.

Con: The Case for Expanded Lung Cancer Research Support

Con: The Case for Expanded Lung Cancer Research Support

The Neuroblastoma ALK(I1250T) Mutation Is a Kinase-Dead RTK In Vitro and In Vivo

Activating mutations in the kinase domain of anaplastic lymphoma kinase (ALK) have recently been shown to be an important determinant in the genetics of the childhood tumor neuroblastoma. Here we discuss an in-depth analysis of one of the reported gain-of-function ALK mutations—ALKI1250T—identified in the germ line DNA of one patient. Our analyses were performed in cell culture-based systems and subsequently confirmed in a Drosophila model. The results presented here indicate that the germ line ALKI1250T mutation is most probably not a determinant for tumor initiation or progression and, in contrast, seems to generate a kinase-dead mutation in the ALK receptor tyrosine kinase (RTK). Consistent with this, stimulation with agonist ALK antibodies fails to lead to stimulation of ALKI1250T and we were unable to detect tyrosine phosphorylation under any circumstances. In agreement, ALKI1250T is unable to activate downstream signaling pathways or to mediate neurite outgrowth, in contrast to the activated wild-type ALK receptor or the activating ALKF1174S mutant. Identical results were obtained when the ALKI1250T mutant was expressed in a Drosophila model, confirming the lack of activity of this mutant ALK RTK. We suggest that the ALKI1250T mutation leads to a kinase-dead ALK RTK, in stark contrast to assumed gain-of-function status, with significant implications for patients reported to carry this particular ALK mutation.

Abstract 4385: Effect on retinal function as a mechanism for vision disorders with crizotinib (PF-02341066)

Abstract Crizotinib is a small-molecule receptor tyrosine kinase inhibitor of anaplastic lymphoma kinase (ALK) and c-Met. Crizotinib has been shown to be effective in patients with ALK-positive non-small cell lung cancer. One common adverse event that has been observed is a grade 1 vision disorder (∼45% of patients) characterized as seeing shadows or streaks during changes in lighting conditions. Onset occurs during the first weeks of treatment, with most cases improving with continued treatment and not requiring treatment discontinuation. Such vision disorders were not predicted from preclinical toxicological evaluation of crizotinib. To further understand the possible mechanism of this vision disorder, a rat electroretinography (ERG) study was conducted to determine if retinal function is altered following repeat-dose administration of crizotinib. Male Long-Evans rats (8/group) were administered crizotinib by oral gavage at a dose of 100 mg/kg/day for up to 29 days, during which an evaluation of in-life parameters (clinical signs, body weight, and food consumption), ophthalmic examinations, and ERG were performed. As part of the ERG assessment, five light-stimulation protocols were applied to test scotopic (dark-adapted) and photopic (light-adapted) luminance responses, photopic and scotopic adaptometry, and photopic flicker responses. Terminal plasma and vitreous fluid were examined for crizotinib concentrations in main study animals, and a toxicokinetic profile was obtained from a satellite group of 6 animals. Results of the ERG measurements identified a statistically significant (∼30%) reduction in mean b-wave amplitude relative to control during the initial 16 and 32 minutes of dark adaptation on Days 15 (p<0.05) and 29 (p<0.01), respectively. The magnitude of the reduction did not worsen between Days 15 and 29. Following 2 hours of dark adaptation, b-wave amplitudes were comparable between control and crizotinib-treated animals on both Days 15 and 29. There were no crizotinib-related effects observed for any other ERG parameters or during ophthalmologic examinations. Plasma exposures were ∼6-fold those associated with vision disorders in patients, and vitreous humor concentrations were 0.66-fold those in plasma at an equivalent time post-dose. Results from this preclinical model show that crizotinib causes significant reductions in the rate of retinal dark adaptation, but not the ability to achieve full dark adaptation. Vision disorders reported in the clinical setting may thus be due to direct effects on the retina rather than a central CNS effect. Whether this is a pharmacologically mediated effect at ALK or c-Met receptors expressed in retina or an off-target effect is not clear. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4385. doi:10.1158/1538-7445.AM2011-4385

Targeting Oncogenic ALK: A Promising Strategy for Cancer Treatment

Recently, the anaplastic lymphoma kinase (ALK) has been found to be altered in several solid and hematologic tumors. Novel drugs targeting this tyrosine kinase receptor are under development, and early clinical trials are showing promising activity in non-small cell lung cancer patients with ALK+ tumors. Here, we review the structure and function of the ALK receptor, the mechanisms associated with its deregulation in cancer, methods for ALK detection in tumor samples, its potential as a new marker for candidate patient selection for tailored therapy, and novel drugs under development that target ALK.

Lung Cancer and the Future of Pathology

Molecular diagnostics and molecular therapy have an escalating role in patient management and the implications for pathologists have not always been clear. 1–4 Of course, pathologists, with their command of laboratory methods, test standardization and validation, quality assurance, and laboratory regulations are the obvious choice to direct molecular diagnostic laboratories. Nevertheless, for the foreseeable future, there will be many more pathologists practicing conventional surgical pathology than supervising molecular diagnostic laboratories. And most residents and fellows in pathology continue to emphasize traditional diagnostic skills in their training. 4 At first glance, these observations might appear to suggest that most pathologists will be less valuable in the world of personalized health care. However, if pathologists are willing to adapt, the role of the surgical pathologist has not been diminished by personalized health care, but rather, it has been enhanced, and it is important that other medical specialties, government agencies, third-party payers, and pathologists themselves recognize this. No current field of personalized health care better illustrates the value of the surgical pathologist’s traditional training and expertise than molecular-targeted therapy of lung cancer. Histologic classification by the surgical pathologist using familiar tools is uniquely intertwined with the new molecular diagnosis and targeted therapy of lung cancer. The pathologist’s active discourse with oncologists and other specialists about a patient’s case is more essential than ever. For decades, care of patients with lung cancer was mired in limited treatment options, generally with dismal outcome. The contribution of the surgical pathologist to patient care was largely restricted to differentiating small cell carcinoma from non–small cell carcinoma on small biopsies and cytology specimens and to staging of non– small cell carcinoma resections. 5 In the early 1990s, there were predictions that molecular classification of lung cancers would one day be essential, but without successful molecular-targeted therapies to provide impetus, that idea had no substantive meaning in clinical practice. 6,7 Molecular-targeted therapies involve drugs that ‘‘target’’ specific cell receptors and signaling pathways in lung cancers. So far, most of the drugs for lung cancer that have been approved, are pending approval, or are under investigation target tyrosine kinase receptors or their ligands. 8–13 These include epidermal growth factor receptor (EGFR) 1; anaplastic lymphoma kinase receptor (ALK), primarily as the echinoderm microtubule-associated proteinlike 4-ALK fusion gene (EML4-ALK); vascular endothelial growth factor (VEGF) and the VEGF receptor (VEGFR); and epidermal growth factor receptor 2 (HER2/ ERBB2). Drugs are also under investigation for targets in signaling pathways downstream of the tyrosine kinase receptors, including BRAF, KRAS, MEK, PI3K, and mTOR. The presence of EGFR mutations are the best predictor of response to EGFR tyrosine kinase inhibitors. EGFR mutations occur in lung cancers with adenocarcinoma histology. Indeed, there is an association with specific subtypes of adenocarcinoma, such as lepidic (bronchioloalveolar), papillary, and micropapillary patterns. The EML4-ALK fusion gene is also reported to be associated with adenocarcinoma histology, in particular the signetring pattern and solid subtype. To date, most targeted therapies for lung cancer are effective in adenocarcinoma compared with other cell types, and the corresponding mutations are often more frequently associated with specific subtypes of adenocarcinoma. 14–19

Lung cancer and the future of pathology.

Molecular diagnostics and molecular therapy have an escalating role in patient management and the implications for pathologists have not always been clear. 1–4 Of course, pathologists, with their command of laboratory methods, test standardization and validation, quality assurance, and laboratory regulations are the obvious choice to direct molecular diagnostic laboratories. Nevertheless, for the foreseeable future, there will be many more pathologists practicing conventional surgical pathology than supervising molecular diagnostic laboratories. And most residents and fellows in pathology continue to emphasize traditional diagnostic skills in their training. 4 At first glance, these observations might appear to suggest that most pathologists will be less valuable in the world of personalized health care. However, if pathologists are willing to adapt, the role of the surgical pathologist has not been diminished by personalized health care, but rather, it has been enhanced, and it is important that other medical specialties, government agencies, third-party payers, and pathologists themselves recognize this. No current field of personalized health care better illustrates the value of the surgical pathologist’s traditional training and expertise than molecular-targeted therapy of lung cancer. Histologic classification by the surgical pathologist using familiar tools is uniquely intertwined with the new molecular diagnosis and targeted therapy of lung cancer. The pathologist’s active discourse with oncologists and other specialists about a patient’s case is more essential than ever. For decades, care of patients with lung cancer was mired in limited treatment options, generally with dismal outcome. The contribution of the surgical pathologist to patient care was largely restricted to differentiating small cell carcinoma from non–small cell carcinoma on small biopsies and cytology specimens and to staging of non– small cell carcinoma resections. 5 In the early 1990s, there were predictions that molecular classification of lung cancers would one day be essential, but without successful molecular-targeted therapies to provide impetus, that idea had no substantive meaning in clinical practice. 6,7 Molecular-targeted therapies involve drugs that ‘‘target’’ specific cell receptors and signaling pathways in lung cancers. So far, most of the drugs for lung cancer that have been approved, are pending approval, or are under investigation target tyrosine kinase receptors or their ligands. 8–13 These include epidermal growth factor receptor (EGFR) 1; anaplastic lymphoma kinase receptor (ALK), primarily as the echinoderm microtubule-associated proteinlike 4-ALK fusion gene (EML4-ALK); vascular endothelial growth factor (VEGF) and the VEGF receptor (VEGFR); and epidermal growth factor receptor 2 (HER2/ ERBB2). Drugs are also under investigation for targets in signaling pathways downstream of the tyrosine kinase receptors, including BRAF, KRAS, MEK, PI3K, and mTOR. The presence of EGFR mutations are the best predictor of response to EGFR tyrosine kinase inhibitors. EGFR mutations occur in lung cancers with adenocarcinoma histology. Indeed, there is an association with specific subtypes of adenocarcinoma, such as lepidic (bronchioloalveolar), papillary, and micropapillary patterns. The EML4-ALK fusion gene is also reported to be associated with adenocarcinoma histology, in particular the signetring pattern and solid subtype. To date, most targeted therapies for lung cancer are effective in adenocarcinoma compared with other cell types, and the corresponding mutations are often more frequently associated with specific subtypes of adenocarcinoma. 14–19

209 Monoclonal antibody targeting of the anaplastic lymphoma kinase (ALK) receptor

In adults, glioblastomas are the most lethal and most frequent malignant brain tumors, and the poor prognosis despite aggressive treatment indicates the need to establish novel targets for molecular intervention. The secreted growth factor pleiotrophin (PTN, HB-GAM, HBNF, OSF-1) shows mitogenic, chemotactic, and transforming activity. Whereas PTN expression is tightly regulated during embryogenesis and is very limited in normal adult tissues, a marked PTN up-regulation is seen in tumors including glioblastomas. Likewise, the PTN receptor anaplastic lymphoma kinase (ALK) has been shown previously to be upregulated and functionally relevant in glioblastoma. In this study, we explore the antitumorigenic effects of the simultaneous ribozyme-mediated knockdown of both receptor and ligand. Various glioblastoma cell lines are analyzed for PTN and ALK expression. Beyond the individual efficacies of several specific ribozymes against PTN or ALK, respectively, antiproliferative and proapoptotic effects of a single gene targeting approach are strongly enhanced on double knockdown of both genes in vitro. More importantly, this results in the abolishment of tumor growth in an in vivo subcutaneous tumor xenograft model. Finally, the analysis of various downstream signaling pathways by antibody arrays reveals a distinct pattern of changes in the activation of signal transduction molecules on PTN/ALK double knockdown. Beyond the already known ones, it identifies additional pathways relevant for PTN/ALK signaling. We conclude that double targeting of PTN and ALK leads to enhanced antitumorigenic effects over single knockdown approaches, which offers novel therapeutic options owing to increased efficacy also after prolonged knockdown.

Involvement of Grb2 Adaptor Protein in Nucleophosmin-Anaplastic Lymphoma Kinase (NPM-ALK)-mediated Signaling and Anaplastic Large Cell Lymphoma Growth

Most anaplastic large cell lymphomas (ALCL) express oncogenic fusion proteins derived from chromosomal translocations or inversions of the anaplastic lymphoma kinase (ALK) gene. Frequently ALCL carry the t(2;5) translocation, which fuses the ALK gene to the nucleophosmin (NPM1) gene. The transforming activity mediated by NPM-ALK fusion induces different pathways that control proliferation and survival of lymphoma cells. Grb2 is an adaptor protein thought to play an important role in ALK-mediated transformation, but its interaction with NPM-ALK, as well as its function in regulating ALCL signaling pathways and cell growth, has never been elucidated. Here we show that active NPM-ALK, but not a kinase-dead mutant, bound and induced Grb2 phosphorylation in tyrosine 160. An intact SH3 domain at the C terminus of Grb2 was required for Tyr(160) phosphorylation. Furthermore, Grb2 did not bind to a single region but rather to different regions of NPM-ALK, mainly Tyr(152-156), Tyr(567), and a proline-rich region, Pro(415-417). Finally, shRNA knockdown experiments showed that Grb2 regulates primarily the NPM-ALK-mediated phosphorylation of SHP2 and plays a key role in ALCL cell growth.

Anaplastic lymphoma kinase activates the small GTPase Rap1 via the Rap1-specific GEF C3G in both neuroblastoma and PC12 cells

Many different types of cancer originate from aberrant signaling from the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (RTK), arising through different translocation events and overexpression. Further, activating point mutations in the ALK domain have been recently reported in neuroblastoma. To characterize signaling in the context of the full-length receptor, we have examined whether ALK is able to activate Rap1 and contribute to differentiation/proliferation processes. We show that ALK activates Rap1 via the Rap1-specific guanine-nucleotide exchange factor C3G, which binds in a constitutive complex with CrkL to activated ALK. The activation of the C3G/Rap1 pathway results in neurite outgrowth of PC12 cells, which is inhibited by either overexpression of Rap1GAP or siRNA-mediated knockdown of Rap1 itself or the guanine nucleotide exchange factor C3G. Significantly, this pathway also appears to function in the regulation of proliferation of neuroblastoma cells such as SK-N-SH and SH-SY5Y, because abrogation of Rap1 activity by Rap1-specific siRNA or overexpression of Rap1GAP reduces cellular growth. These results suggest that ALK activation of Rap1 may contribute to cell proliferation and oncogenesis of neuroblastoma driven by gain-of-function mutant ALK receptors.

Another Targeted Therapy for Non–Small-Cell Lung Cancer?

Some patients with lung cancer have abnormalities in the anaplastic lymphoma kinase (ALK) protein. Investigators conducted a phase I trial of an orally delivered small-molecule inhibitor — crizotinib (PF-02341066) — that preferentially inhibits the ALK and c-met (hepatocyte growth factor) receptor tyrosine kinases in solid tumors. The drug's manufacturer partially funded the study. In the dose-escalation phase of the trial (increasing from 50 mg once daily to 300 mg twice daily), two patients with non–small-cell lung cancer (NSCLC) and an ALK rearrangement exhibited response to the drug. That prompted screening of an expanded cohort of about 1500 NSCLC patients for ALK …

Pleiotrophin Regulates Lung Epithelial Cell Proliferation and Differentiation during Fetal Lung Development via β-Catenin and Dlk1

The role of pleiotrophin in fetal lung development was investigated. We found that pleiotrophin and its receptor, protein-tyrosine phosphatase receptor beta/zeta, were highly expressed in mesenchymal and epithelial cells of the fetal lungs, respectively. Using isolated fetal alveolar epithelial type II cells, we demonstrated that pleiotrophin promoted fetal type II cell proliferation and arrested type II cell trans-differentiation into alveolar epithelial type I cells. Pleiotrophin also increased wound healing of injured type II cell monolayer. Knockdown of pleiotrophin influenced lung branching morphogenesis in a fetal lung organ culture model. Pleiotrophin increased the tyrosine phosphorylation of beta-catenin, promoted beta-catenin translocation into the nucleus, and activated T cell factor/lymphoid enhancer factor transcription factors. Dlk1, a membrane ligand that initiates the Notch signaling pathway, was identified as a downstream target of the pleiotrophin/beta-catenin pathway by endogenous dlk1 expression, promoter assay, and chromatin immunoprecipitation. These results provide evidence that pleiotrophin regulates fetal type II cell proliferation and differentiation via integration of multiple signaling pathways including pleiotrophin, beta-catenin, and Notch pathways.

1060 The anaplastic lymphoma kinase receptor inhibits the apoptotic effect of the dependence receptor unc5

1060 The anaplastic lymphoma kinase receptor inhibits the apoptotic effect of the dependence receptor unc5