Oncogene database

238

ALK

anaplastic lymphoma receptor tyrosine kinase;ALK;anaplastic lymphoma receptor tyrosine kinase

ALK tyrosine kinase receptor|CD246 antigen|mutant anaplastic lymphoma kinase

2p23

protein-coding

Anaplastic large cell lymphomas (ALCLs) are frequently associated with the t(2;5)(p23;q35) translocation, leading to the expression of NPM-ALK, a fusion protein linking nucleophosmin and anaplastic lymphoma kinase, a receptor tyrosine kinase. In ALCLs, dimerization of NPM-ALK leads to constitutive autophosphorylation and activation of the kinase, necessary for NPM-ALK oncogenicity. To investigate whether NPM-ALK, like other oncogenic tyrosine kinases, can inhibit drug-induced apoptosis, we permanently transfected NPM-ALK into Jurkat T-cells. As in ALCLs, NPM-ALK was expressed as a constitutively kinase-active 80 kDa protein, and could be detected by immunocytochemistry in nucleoli, nuclei and cytoplasm. Doxorubicin-induced apoptosis (assessed by cell morphology and annexin V-FITC binding) was significantly inhibited in two independent NPM-ALK-expressing clones (5.2+/-1.8 and 7.5+/-0.8% apoptosis), compared to control vector-transduced cells (36+/-6.7%). Similar results were observed with etoposide. In contrast, Fas-induced apoptosis was not inhibited. Cytochrome c release into the cytosol was delayed in doxorubicin-, but not anti-Fas-treated transfectant cells, indicating that apoptosis inhibition occurred upstream of mitochondrial events. Using NPM-ALK mutants, we demonstrated that inhibition of drug-induced apoptosis: (1) requires functional kinase activity, (2) does not involve phospholipase C-gamma, essential for NPM-ALK-mediated mitogenicity and (3) appears to be phosphoinositide 3-kinase independent, despite a strong Akt/PKB activation observed in wild type NPM-ALK-expressing cells. These results suggest that the NPM-ALK antiapoptotic and mitogenic pathways are distinct.

Oncogenic rearrangements of the anaplastic lymphoma kinase (ALK) gene, encoding a receptor type tyrosine kinase, are frequently associated with anaplastic large cell lymphomas. Such rearrangements juxtapose the intracellular domain of ALK to 5 -end sequences belonging to different genes and create transforming fusion proteins. To understand how the oncogenic versions of ALK contribute to lymphomagenesis, it is important to analyze the biological effects and the biochemical properties of this receptor under controlled conditions of activation. To this aim, we constructed chimeric receptor molecules in which the extracellular domain of the ALK kinase is replaced by the extracellular, ligand-binding domain of the epidermal growth factor receptor (EGFR). Upon transfection in NIH 3T3 fibroblasts, the EGFR/ALK chimera was correctly synthesized and transported to the cell surface, where it was fully functional in forming high versus low affinity EGF-binding sites and transducing an EGF-dependent signal intracellularly. Overexpression of the EGFR/ALK chimera in NIH 3T3 was sufficient to induce the malignant phenotype; the appearance of the transformed phenotype was, however, conditionally dependent on the administration of EGF. Moreover, the EGFR/ALK chimera was significantly more active in inducing transformation and DNA synthesis than the wild type EGFR when either was expressed at similar levels in NIH 3T3 cells. Comparative analysis of the biochemical pathways implicated in the transduction of mitogenic signals did not show any increased ability of the EGFR/ALK to phosphorylate PLC-gamma and MAPK compared with the EGFR. On the contrary, EGFR/ALK showed to have a consistently greater effect on phosphatidylinositol 3-kinase activity compared with the EGFR, indicating that this enzyme plays a major role in mediating the mitogenic effects of ALK in NIH 3T3 cells.

A t(2;5) (p23;q35) chromosomal translocation can be found in a high percentage of anaplastic large-cell lymphomas (ALCL). This genetic abnormality leads to the expression of the NPM-ALK fusion protein, which encodes a constitutively active tyrosine kinase that plays a causative role in lymphomagenesis. Employing a modified infection/transplantation protocol utilizing an MSCV-based vector, we were able to reproducibly induce two phenotypically different lymphoma-like diseases dependent on the retroviral titers used. The first phenotype presented as a polyclonal histiocytic malignancy of myeloid/macrophage origin with a short latency period of 3-4 weeks. Clinically, the diseased mice showed rapidly progressive wasting, lymphadenopathy and pancytopenia. Mice displaying the second phenotype developed monoclonal B-lymphoid tumors with a longer latency of approximately 12-16 weeks, primarily involving the spleen and the bone marrow, with less extensive lymph node but also histologically evident extranodal organ infiltration by large immature plasmoblastic cells. The described retroviral mouse model will be useful to analyse the role of NPM-ALK in lymphomagenesis in vivo and may contribute to the development of new treatment options for NPM-ALK induced malignancies.

Janus kinase 3 (Jak3) is a tyrosine kinase that activates signal transducer and activator of transcription 3 (Stat3) in response to cytokine stimulation. Stat3 is an oncogene. In previous studies of anaplastic large cell lymphoma (ALCL), we showed that inhibition of Jak3 down-regulates activated/phosphorylated Stat3 (pStat3), decreases anaplastic lymphoma kinase (ALK) enzymatic activity, and induces cell-cycle arrest and apoptosis in ALK-positive ALCL. These findings implicate Jak3 as playing a significant role in the pathogenesis of ALK-positive ALCL; most likely via Stat3 and ALK activation. To assess this possibility, we used immunohistochemical staining to evaluate the frequency of expression of Jak3 and its activated/phosphorylated form (pJak3) in 48 systemic ALCL tumors included in a tissue microarray. pJak3 was detected in 17 (81%) of 21 ALK-positive tumors, compared with 3 (11%) of 27 ALK-negative tumors (P < .0001, Fisher exact test). pStat3 was present in 12 (86%) of 14 ALK-positive tumors and in 10 (40%) of 25 ALK-negative tumors assessed (P = .0078). Of 12 ALK-positive/pStat3-positive tumors, 8 (67%) expressed pJak3, but none of 10 ALK-negative/pStat3-positive tumors expressed pJak3. We conclude that Jak3 activation is predominantly restricted to ALK-positive ALCL tumors. Most likely, Jak3 collaborates with ALK in activating Stat3, leading to cell survival, cell-cycle progression, and tumor growth. In contrast, the mechanism of Stat3 activation in ALK-negative ALCL tumors appears to be independent of Jak3.

Anaplastic large-cell lymphomas (ALCLs) carry chromosome translocations in which the anaplastic lymphoma kinase (ALK) gene is fused to several partners, most frequently, the NPM1 gene. We have demonstrated that the constitutive activation of ALK fusion proteins results in cellular transformation and lymphoid neoplasia. Herein, we specifically down-regulated ALK protein expression by using small hairpin RNA (shRNA) targeting a sequence coding for the catalytic domain of ALK. The ablation of ALK leads to the down-modulation of known ALK downstream effectors, cell growth arrest, and reversion of the transformed phenotype of ALK(+) mouse embryonic fibroblasts in vitro and in vivo. In human ALCL cells lentiviral-mediated ALK knock-down leads to G(1) cell-cycle arrest and apoptosis in vitro and tumor growth inhibition and regression in vivo. Using a specific approach we have demonstrated that the survival and growth of ALK(+) ALCLs are strictly dependent on ALK activation and signaling. Therefore, ALK is a viable target for therapeutic intervention and its inactivation might represent a pivotal approach for the treatment of ALK lymphomas and other ALK-dependent human tumors.

Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is a chimeric protein expressed in a subset of cases of anaplastic large cell lymphoma (ALCL) for which constitutive expression represents a key oncogenic event. The ALK signaling pathway is complex and probably involves functional redundancy between various signaling substrates of ALK. Despite numerous studies on signaling mediators, the molecular mechanisms contributing to the distinct oncogenic features of NPM-ALK remain incompletely understood. The search for additional interacting partners of NPM-ALK led to the discovery of AUF1/hnRNPD, a protein implicated in AU-rich element (ARE)-directed mRNA decay. AUF1 was immunoprecipitated with ALK both in ALCL-derived cells and in NIH3T3 cells stably expressing NPM-ALK or other X-ALK fusion proteins. AUF1 and NPM-ALK were found concentrated in the same cytoplasmic foci, whose formation required NPM-ALK tyrosine kinase activity. AUF1 was phosphorylated by ALK in vitro and was hyperphosphorylated in NPM-ALK-expressing cells. Its hyperphosphorylation was correlated with increased stability of several AUF1 target mRNAs encoding key regulators of cell proliferation and with increased cell survival after transcriptional arrest. Thus, AUF1 could function in a novel pathway mediating the oncogenic effects of NPM-ALK. Our data establish an important link between oncogenic kinases and mRNA turnover, which could constitute a critical aspect of tumorigenesis.

Oncogenic tyrosine kinases play a ever growing role in the pathogenesis of human malignancies. In human non-Hodgkin lymphomas, the NPM-ALK oncogene arising from the t(2;5) chromosomal translocation represents the most important oncogenic tyrosine kinase identified so far. The ALK-kinase is constitutively activated by NPM-induced dimerization and signals through a multitude of growth promoting and antiapoptotic pathways. Murine models have made a significant impact on the elucidation of the molecular pathogenesis and new treatment options of malignant diseases. Here, the latest developments in the analysis of NPM-ALK induced lymphomagenesis by murine models is reviewed.

The t(2;5) chromosomal translocation occurs in anaplastic large-cell lymphoma arising from activated T lymphocytes. This genomic rearrangement generates the nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK) oncoprotein that is a chimeric protein consisting of parts of the nuclear protein NPM and ALK receptor protein-tyrosine kinase. We used yeast two-hybrid screening to identify an adaptor protein Suc1-associated neurotrophic factor-induced tyrosine-phosphorylated target (SNT)-2 as a new partner that interacted with the cytoplasmic domain of ALK. Immunoprecipitation assay revealed that SNT-1 and SNT-2 interacted with NPM-ALK and kinase-negative NPM-ALK mutant. Y156, Y567 and a 19-amino-acid sequence (aa 631-649) of NPM-ALK were essential for this interaction. The interaction through Y156 and Y567 was dependent on phosphorylation of these tyrosines, whereas the interaction through the 19-amino-acid sequence was independent of phosphorylation. NPM-ALK mutant protein mutated at these three binding sites showed significantly reduced transforming activity. This transformation-defective NPM-ALK mutant still interacted with signal transducing proteins such as phospholipase C-gamma and phosphatidylinositol 3-kinase, which were previously reported to be relevant to NPM-ALK-dependent tumorigenesis. These observations indicate that the three SNT-binding sites of NPM-ALK are important for its transforming activity. This raises a possibility that SNT family proteins play significant roles in cellular transformation triggered by NPM-ALK, which though remains to be verified.

NPM/ALK is an oncogenic fusion protein expressed in approximately 50% of anaplastic large cell lymphoma cases. It derives from the t(2;5)(p23;q35) chromosomal translocation that fuses the catalytic domain of the tyrosine kinase, anaplastic lymphoma kinase (ALK), with the dimerization domain of the ubiquitously expressed nucleophosmin (NPM) protein. Dimerization of the ALK kinase domain leads to its autophosphorylation and constitutive activation. Activated NPM/ALK stimulates downstream survival and proliferation signaling pathways leading to malignant transformation. Herein, we investigated the molecular mechanisms of autoactivation of the catalytic domain of ALK. Because kinases are typically regulated by autophosphorylation of their activation loops, we systematically mutated (Tyr --> Phe) three potential autophosphorylation sites contained in the "YXXXYY" motif of the ALK activation loop, and determined the effect of these mutations on the catalytic activity and biological function of NPM/ALK. We observed that mutation of both the second and third tyrosine residues (YFF mutant) did not affect the kinase activity or transforming ability of NPM/ALK. In contrast, mutation of the first and second (FFY), first and third (FYF), or ALL three (FFF) tyrosine residues impaired both kinase activity and transforming ability of NPM/ALK. Furthermore, a DFF mutant, in which the aspartic residue introduces a negative charge similar to a phosphorylated tyrosine, possessed catalytic activity similar to the YFF mutant. Together, our findings indicate that phosphorylation of the first tyrosine of the YXXXYY motif is necessary for the autoactivation of the ALK kinase domain and the transforming activity of NPM/ALK.

Here, we demonstrate that the expression of the dual specificity phosphatase CDC25A, a key regulator of cell cycle progression, is deregulated in Ba/F3 cells expressing the oncogenic protein NPM/ALK and in human cell lines derived from NPM/ALK-positive anaplastic large cell lymphomas (ALCL). Both transcriptional and post-translational mechanisms account for the constitutive expression of the protein, and the PI3K/Akt pathway is essential for this process. Importantly, pharmacological inhibition of CDC25 dramatically inhibits the proliferation of NPM/ALK-expressing cells, while moderately affecting the proliferation of control Ba/F3 cells. RNA interference-mediated downregulation of CDC25A confirmed that NPM/ALK-expressing cells are highly dependent on this protein for their proliferation. Moreover, similar PI3K/AKt-mediated constitutive expression of CDC25A takes place down-stream of other hematological oncogenes, including BCR/ABL in Chronic Myeloid Leukemia and FLT3-ITD in Acute Myeloid Leukemia. Altogether, our data point to the functional link between hematopoietic oncogenic tyrosine kinases and the G(1) cell cycle regulator CDC25A, and we propose that this protein may be a potential therapeutic target in ALCL and other hematological malignancies.

Type I insulin-like growth factor receptor (IGF-IR) tyrosine kinase plays important roles in the pathogenesis of several malignancies. Although it promotes the growth of stimulated hematopoietic cells, a direct role of IGF-IR in malignant lymphoma has not been identified. Anaplastic lymphoma kinase-positive anaplastic large-cell lymphoma (ALK(+) ALCL) is a unique type of T-cell lymphoma. Approximately 85% of ALK(+) ALCL cases harbor the translocation t(2;5)(p23;q35), which generates the chimeric oncogene NPM-ALK. In the present study, we explored a possible role of IGF-IR in ALK(+) ALCL. Our results demonstrate that IGF-IR and IGF-I are widely expressed in ALK(+) ALCL cell lines and primary tumors. Importantly, we identified novel reciprocal functional interactions between IGF-IR and NPM-ALK. Antagonism of IGF-IR decreased the viability, induced apoptosis and cell-cycle arrest, and decreased proliferation and colony formation of ALK(+) ALCL cell lines. These effects could be explained by alterations of cell survival regulatory proteins downstream of IGF-IR signaling. Our findings improve current understanding of the biology of IGF-IR and NPM-ALK and have significant therapeutic implications as they identify IGF-IR signaling as a potential therapeutic target in ALK(+) ALCL and possibly other types of malignant lymphoma.

Deregulated expression of MYC contributes to the genesis of multiple human tumours. The encoded protein, MYC, functions through the transcriptional regulation of large numbers of target genes. Recent publications show that MYC is closely involved in DNA replication and the checkpoint processes that monitor progress through the S phase, and suggest that limiting replication stress is a key function of this protein. These findings could have considerable implications for our understanding of how MYC transforms cells and which mechanisms protect normal cells from transformation by activated oncogenes.

Transformed cells in lymphomas usually maintain the phenotype of the postulated normal lymphocyte from which they arise. By contrast, anaplastic large cell lymphoma (ALCL) is a T-cell lymphoma with aberrant phenotype because of the defective expression of the T-cell receptor and other T-cell-specific molecules for still undetermined mechanisms. The majority of ALCL carries the translocation t(2;5) that encodes for the oncogenic tyrosine kinase NPM-ALK, fundamental for survival, proliferation, and migration of transformed T cells. Here, we show that loss of T-cell-specific molecules in ALCL cases is broader than reported previously and involves most T-cell receptor-related signaling molecules, including CD3epsilon, ZAP70, LAT, and SLP76. We further show that NPM-ALK, but not the kinase-dead NPM-ALK(K210R), downregulated the expression of these molecules by a STAT3-mediated gene transcription regulation and/or epigenetic silencing because this downregulation was reverted by treating ALCL cells with 5-aza-2-deoxycytidine or by knocking down STAT3 through short hairpin RNA. Finally, NPM-ALK increased the methylation of ZAP70 intron 1-exon 2 boundary region, and both NPM-ALK and STAT3 regulated the expression levels of DNA methyltransferase 1 in transformed T cells. Thus, our data reveal that oncogene-deregulated tyrosine kinase activity controls the expression of molecules that determine T-cell identity and signaling.

The mechanisms of malignant cell transformation mediated by the oncogenic anaplastic lymphoma kinase (ALK) tyrosine kinase remain only partially understood. In this study, we report that T-cell lymphoma (TCL) cells carrying the nucleophosmin (NPM)/ALK fusion protein (ALK+ TCL) strongly express hypoxia-induced factor 1alpha (HIF1alpha) mRNA, even under normoxic conditions, and markedly upregulate HIF1alpha protein expression under hypoxia. HIF1alpha expression is strictly dependent on the expression and enzymatic activity of NPM/ALK, as shown in BaF3 cells transfected with wild-type NPM/ALK and kinase-inactive NPM/ALK K210R mutant and by the inhibition of the NPM/ALK function in ALK+ TCL cells by a small-molecule ALK inhibitor. NPM/ALK induces HIF1alpha expression by upregulating its gene transcription through its key signal transmitter signal transducer and activator of transcription 3 (STAT3), which binds to the HIF1alpha gene promoter as shown by the chromatin immunoprecipitation assay and is required for HIF1alpha gene expression as demonstrated by its small interfering RNA-mediated depletion. In turn, depletion of HIF1alpha increases mammalian target of rapamycin complex 1 activation, cell growth and proliferation and decreases vascular endothelial growth factor synthesis. These results identify a novel cell-transforming property of NPM/ALK, namely its ability to induce the expression of HIF1alpha, a protein with an important role in carcinogenesis. These results also provide another rationale to therapeutically target NPM/ALK and STAT3 in ALK+ TCL.

Neuroblastoma is one of the most common solid cancers among children. Prognosis of advanced neuroblastoma is still poor despite the recent advances in chemo/radiotherapies. In view of improving the clinical outcome of advanced neuroblastoma, it is important to identify the key molecules responsible for the pathogenesis of neuroblastoma and to develop effective drugs that target these molecules. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase, initially identified through the analysis of a specific translocation associated with a rare subtype of non-Hodgkin s lymphoma. Recently it was demonstrated that ALK is frequently mutated in sporadic cases with advanced neuroblastoma. Moreover, germline mutations of ALK were shown to be responsible for the majority of hereditary neuroblastoma. ALK mutants found in neuroblastoma show constitutive active kinase activity and oncogenic potentials. Inhibition of ALK in neuroblastoma cell lines carrying amplified or mutated ALK alleles results in compromised downstream signaling and cell growth, indicating potential roles of small molecule ALK inhibitors in the therapeutics of neuroblastoma carrying mutated ALK kinases.

The echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion oncogene represents a molecular target in a small subset of non-small cell lung cancers (NSCLCs). This fusion leads to constitutive ALK activation with potent transforming activity. In a pivotal phase 1 clinical trial, the ALK tyrosine kinase inhibitor (TKI) crizotinib (PF-02341066) demonstrated impressive antitumor activity in the majority of patients with NSCLC harboring ALK fusions. However, despite these remarkable initial responses, cancers eventually develop resistance to crizotinib, usually within 1 y, thereby limiting the potential clinical benefit. To determine how cancers acquire resistance to ALK inhibitors, we established a model of acquired resistance to crizotinib by exposing a highly sensitive EML4-ALK-positive NSCLC cell line to increasing doses of crizotinib until resistance emerged. We found that cells resistant to intermediate doses of crizotinib developed amplification of the EML4-ALK gene. Cells resistant to higher doses (1 muM) also developed a gatekeeper mutation, L1196M, within the kinase domain, rendering EML4-ALK insensitive to crizotinib. This gatekeeper mutation was readily detected using a unique and highly sensitive allele-specific PCR assay. Although crizotinib was ineffectual against EML4-ALK harboring the gatekeeper mutation, we observed that two structurally different ALK inhibitors, NVP-TAE684 and AP26113, were highly active against the resistant cancer cells in vitro and in vivo. Furthermore, these resistant cells remained highly sensitive to the Hsp90 inhibitor 17-AAG. Thus, we have developed a model of acquired resistance to ALK inhibitors and have shown that second-generation ALK TKIs or Hsp90 inhibitors are effective in treating crizotinib-resistant tumors harboring secondary gatekeeper mutations.

Oncogene-induced senescence (OIS) is a barrier for tumor development. oncogene-dependent DNA damage and activation of the ARF/p53 pathway play a central role in OIS and, accordingly, ARF and p53 are frequently mutated in human cancer. A number of leukemia/lymphoma-initiating oncogenes, however, inhibit ARF/p53 and only infrequently select for ARF or p53 mutations, suggesting the involvement of other tumor-suppressive pathways. We report that NPM-ALK, the initiating oncogene of anaplastic large cell lymphomas (ALCLs), induces DNA damage and irreversibly arrests the cell cycle of primary fibroblasts and hematopoietic progenitors. This effect is associated with inhibition of p53 and is caused by activation of the p16INK4a/pRb tumor-suppressive pathway. Analysis of NPM-ALK lymphomagenesis in transgenic mice showed p16INK4a-dependent accumulation of senescent cells in premalignant lesions and decreased tumor latency in the absence of p16INK4a. Accordingly, human ALCLs showed no expression of either p16INK4a or pRb. Up-regulation of the histone-demethylase Jmjd3 and de-methylation at the p16INK4a promoter contributed to the effect of NPM-ALK on p16INK4a, which was transcriptionally regulated. These data demonstrate that p16INK4a/pRb may function as an alternative pathway of oncogene-induced senescence, and suggest that the reactivation of p16INK4a expression might be a novel strategy to restore the senescence program in some tumors.

Here we report that T-cell lymphoma cells carrying the NPM-ALK fusion protein (ALK(+) TCL) frequently express the cell-stimulatory receptor ICOS. ICOS expression in ALK(+) TCL is moderate and strictly dependent on the expression and enzymatic activity of NPM-ALK. NPM-ALK induces ICOS expression via STAT3, which triggers the transcriptional activity of the ICOS gene promoter. In addition, STAT3 suppresses the expression of miR-219 that, in turn, selectively inhibits ICOS expression. ALK(+) TCL cell lines display extensive DNA methylation of the CpG island located within intron 1, the putative enhancer region, of the ICOS gene, whereas cutaneous T-cell lymphoma cell lines, which strongly express ICOS, show no methylation of the island. Treatment of the ALK(+) TCL cell lines with DNA methyltransferase inhibitor reversed the CpG island methylation and augmented the expression of ICOS mRNA and protein. Stimulation of the ICOS receptor with anti-ICOS antibody or ICOS ligand-expressing B cells markedly enhanced proliferation of the ALK(+) TCL cells. These results demonstrate that NPM-ALK, acting through STAT3 as the gene transcriptional activator, induces the expression of ICOS, a cell growth promoting receptor. These data also show that the DNA methylation status of the intronic CpG island affects transcriptional activity of the ICOS gene and, consequently, modulates the concentration of the expressed ICOS protein.

Phosphoinositide-3-kinase catalytic alpha polypeptide (PIK3CA) encodes the p110alpha subunit of the mitogenic signaling protein phosphoinositide 3-kinase (PI3K). PIK3CA mutations in the helical binding domain and the catalytic subunit of the protein have been associated with tumorigenesis and treatment resistance in various malignancies. Characteristics of patients with PIK3CA-mutant lung adenocarcinomas have not been reported. We examined epidermal growth factor receptor (EGFR), Kirsten rate sarcoma viral oncogene homolog (KRAS), v-Raf murine sarcoma viral oncogene homolog B1 (BRAF), human epidermal growth factor receptor 2 (HER2), PIK3CA, v-akt murine thymoma vial oncogene homolog 1 (AKT1), v-ras neuroblastoma viral oncogene homolog (NRAS), dual specificity mitogen-activated protein kinase kinase 1 (MEK1), and anaplastic lymphoma kinase (ALK) in patients with adenocarcinoma of the lung to identify driver mutations. Clinical data were obtained from the medical records of individuals with mutations in PIK3CA. Twenty-three of 1,125 (2%, 95% CI: 1-3) patients had a mutation in PIK3CA, 12 in exon 9 (10 E545K and 2 E542K), and 11 in exon 20 (3 H1047L and 8 H1047R). The patients (57% women) had a median age of 66 at diagnosis (range: 34-78). Eight patients (35%) were never smokers. Sixteen of 23 (70%, 95% CI: 49-86) had coexisting mutations in other oncogenes-10 KRAS, 1 MEK1, 1 BRAF, 1 ALK rearrangement, and 3 EGFR exon 19 deletions. We conclude that PIK3CA mutations occur in lung adenocarcinomas, usually concurrently with EGFR, KRAS, and ALK. The impact of PIK3CA mutations on the efficacy of targeted therapies such as erlotinib and crizotinib is unknown. Given the high frequency of overlapping mutations, comprehensive genotyping should be carried out on tumor specimens from patients enrolling in clinical trials of PI3K and other targeted therapies.

BACKGROUND: The discovery of distinct subsets of nonsmall cell lung cancer (NSCLC) characterized by activation of driver oncogenes has greatly affected personalized therapy. It is hypothesized that the dominant oncogene in NSCLC would be associated with distinct patterns of metastatic spread in NSCLC at the time of diagnosis. METHODS: A total of 209 consecutive patients with stage IV nonsquamous NSCLC with an EGFR (epidermal growth factor receptor) mutation (N = 39), KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) mutation (N = 49), ALK (anaplastic lymphoma receptor tyrosine kinase) gene rearrangement (N = 41), or wild-type for ALL 3 (triple negative, N = 80) were included. The percentage of patients with metastatic disease at a given site was compared between each molecular cohort (EGFR, KRAS, or ALK) and the triple negative cohort. RESULTS: ALK gene rearrangement was significantly associated with pericardial disease (odds ratio [OR] = 4.61; 95% confidence interval [CI] = 1.30, 16.37; P = .02) and pleural disease (OR = 4.80; 95%

BACKGROUND: Anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma (ALK+ ALCL) is a T cell lymphoma defined by the presence of chromosomal translocations involving the ALK tyrosine kinase gene. These translocations generate fusion proteins (e.g. NPM-ALK) with constitutive tyrosine kinase activity, which activate numerous signalling pathways important for ALK+ ALCL pathogenesis. The molecular chaperone heat shock protein-90 (Hsp90) plays a critical role in allowing NPM-ALK and other signalling proteins to function in this lymphoma. Co-chaperone proteins are important for helping Hsp90 fold proteins and for directing Hsp90 to specific clients; however the importance of co-chaperone proteins in ALK+ ALCL has not been investigated. Our preliminary findings suggested that expression of the immunophilin co-chaperone, Cyclophilin 40 (Cyp40), is up-regulated in ALK+ ALCL by JunB, a transcription factor activated by NPM-ALK signalling. In this study we examined the regulation of the immunophilin family of co-chaperones by NPM-ALK and JunB, and investigated whether the immunophilin co-chaperones promote the viability of ALK+ ALCL cell lines. METHODS: NPM-ALK and JunB were knocked-down in ALK+ ALCL cell lines with siRNA, and the effect on the expression of the three immunophilin co-chaperones: Cyp40, FK506-binding protein (FKBP) 51, and FKBP52 examined. Furthermore, the effect of knock-down of the immunophilin co-chaperones, either individually or in combination, on the viability of ALK+ ALCL cell lines and NPM-ALK levels and activity was also examined. RESULTS: We found that NPM-ALK promoted the transcription of Cyp40 and FKBP52, but only Cyp40 transcription was promoted by JunB. We also observed reduced viability of ALK+ ALCL cell lines treated with Cyp40 siRNA, but not with siRNAs directed against FKBP52 or FKBP51. Finally, we demonstrate that the decrease in the viability of ALK+ ALCL cell lines treated with Cyp40 siRNA does not appear to be due to a decrease in NPM-ALK levels or the ability of this oncoprotein to signal. CONCLUSIONS: This is the first study demonstrating that the expression of immunophilin family co-chaperones is promoted by an oncogenic tyrosine kinase. Moreover, this is the first report establishing an important role for Cyp40 in lymphoma.

PURPOSE: EML4-ALK is a lung cancer oncogene, and ALK inhibitors show marked therapeutic efficacy for tumors harboring this fusion gene. It remains unsettled, however, how the fusion gene should be detected in specimens other than formalin-fixed, paraffin-embedded tissue. We here tested whether reverse transcription PCR (RT-PCR)-based detection of EML4-ALK is a sensitive and reliable approach. EXPERIMENTAL DESIGN: We developed a multiplex RT-PCR system to capture ALK fusion transcripts and applied this technique to our prospective, nationwide cohort of non-small cell lung cancer (NSCLC) in Japan. RESULTS: During February to December 2009, we collected 916 specimens from 853 patients, quality filtering of which yielded 808 specimens of primary NSCLC from 754 individuals. Screening for EML4-ALK and KIF5B-ALK with our RT-PCR system identified EML4-ALK transcripts in 36 samples (4.46%) from 32 individuals (4.24%). The RT-PCR products were detected in specimens including bronchial washing fluid (n = 11), tumor biopsy (n = 8), resected tumor (n = 7), pleural effusion (n = 5), sputum (n = 4), and metastatic lymph node (n = 1). The results of RT-PCR were concordant with those of sensitive immunohistochemistry with ALK antibodies. CONCLUSIONS: Multiplex RT-PCR was confirmed to be a reliable technique for detection of ALK fusion transcripts. We propose that diagnostic tools for EML4-ALK should be selected in a manner dependent on the available specimen types. FISH and sensitive immunohistochemistry should be applied to formalin-fixed, paraffin-embedded tissue, but multiplex RT-PCR is appropriate for other specimen types.

INTRODUCTION: Many patients with oncogene-driven non-small-cell lung cancer (NSCLC) treated with tyrosine kinase inhibitors experience limited sites of disease progression. This study investigated retrospectively the benefits of local ablative therapy (LAT) to central nervous system (CNS) and/or limited systemic disease progression and continuation of crizotinib or erlotinib in patients with metastatic ALK gene rearrangement (ALK+) or EGFR-mutant (EGFR-MT) NSCLC, respectively. METHODS: Patients with metastatic ALK+ NSCLC treated with crizotinib (n = 38) and EGFR-MT NSCLC treated with erlotinib (n = 27) were identified at a single institution. Initial response to the respective kinase inhibitors, median progression-free survival (PFS1), and site of first progression were recorded. A subset of patients with either nonleptomeningeal CNS and/or four sites or fewer of extra-CNS progression (oligoprogressive disease) suitable for LAT received either radiation or surgery to these sites and continued on the same tyrosine kinase inhibitors. The subsequent median progression-free survival from the time of first progression (PFS2) and pattern of progression were recorded. RESULTS: Median progression-free survival in ALK+ patients on crizotinib was 9.0 months, and 13.8 months for EGFR-MT patients on erlotinib. Twenty-five of 51 patients (49%) who progressed were deemed suitable for local therapy (15 ALK+, 10 EGFR-MT; 24 with radiotherapy, one with surgery) and continuation of the same targeted therapy. Post-LAT, 19 of 25 patients progressed again, with median PFS2 of 6.2 months. DISCUSSION: oncogene-addicted NSCLC with CNS and/or limited systemic disease progression (oligoprogressive disease) on relevant targeted therapies is often suitable for LAT and continuation of the targeted agent, and is associated with more than 6 months of additional disease control.

Extensive research has been carried out in the past two decades to study the pathobiology of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), which is an oncogenic fusion protein found exclusively in a specific type of T-cell lymphoid malignancy, namely ALK-positive anaplastic large cell lymphoma. Results from these studies have provided highly useful insights into the mechanisms by which a constitutively tyrosine kinase, such as NPM-ALK, promotes tumorigenesis. Several previous publications have comprehensively summarized the advances in this field. In this review, we provide readers with a brief update on specific areas of NPM-ALK pathobiology. In the first part, the NPM-ALK/signal transducer and activator of transcription 3 (STAT3) signaling axis is discussed, with an emphasis on the existence of multiple biochemical defects that have been shown to amplify the oncogenic effects of this signaling axis. Specifically, findings regarding JAK3, SHP1 and the stimulatory effects of several cytokines including interleukin (IL)-9, IL-21 and IL-22 are summarized. New concepts stemming from recent observations regarding the functional interactions among the NPM-ALK/STAT3 axis, beta catenin and glycogen synthase kinase 3beta will be postulated. Lastly, new mechanisms by which the NPM-ALK/STAT3 axis promotes tumorigenesis, such as its modulations of Twist1, hypoxia-induced factor 1alpha, CD274, will be described. In the second part, we summarize recent data generated by mass spectrometry studies of NPM-ALK, and use MSH2 and heat shock proteins as examples to illustrate the use of mass spectrometry data in stimulating new research in this field. In the third part, the evolving field of microRNA in the context of NPM-ALK biology is discussed.

BACKGROUND: EML4-ALK fusion oncogene has emerged as a novel molecular target in non-small cell lung cancer (NSCLC). Although break-apart fluorescent in situ hybridization (FISH) is the standard method for diagnosis, it is expensive, not readily available and sometimes difficult to interpret. In addition, ALK immunohistochemistry (IHC) may miss the diagnosis because of relatively low level of ALK transcription. METHODS: In situ proximity ligation assay (PLA) originally developed for precise detection and quantification of proteins by dual recognition and amplification process was used for sensitive detection of EML4-ALK fusion oncoprotein in NSCLC cell lines (ALK negative cell: PC-9 and H460, ALK positive cell: H3122 and H2228). EML4-ALK oncogene and protein in lung cancer cells were confirmed by multiplex RT-PCR and Western blots. RESULTS: We detected 117 kDa variant 1 of EML4-ALK in H3122 and 90 kDa variant 3 of EML4-ALK in H2228. These cells were more sensitive to crizotinib, an ALK inhibitor compared with PC-9 and H460 cells without EML4-ALK rearrangement. After fixing on glass slides by cytospin centrifuge, in situ PLA test was performed. Among four cell lines, distinct, tiny spots were visible only in H3122 and H2228 cell lines with ALK rearrangement. The same results were also obtained when paraffin-embedded cell blocks were used. CONCLUSIONS: Highly specific and sensitive detection of EML4-ALK fusion oncoprotein is possible by in situ PLA method suggesting its clinical application.

With this study we have demonstrated that in vitro transduction of normal human CD4(+) T lymphocytes with NPM-ALK results in their malignant transformation. The transformed cells become immortalized and display morphology and immunophenotype characteristic of patient-derived anaplastic large-cell lymphomas. These unique features, which are strictly dependent on NPM-ALK activity and expression, include perpetual cell growth, proliferation, and survival; activation of the key signal transduction pathways STAT3 and mTORC1; and expression of CD30 (the hallmark of anaplastic large-cell lymphoma) and of immunosuppressive cytokine IL-10 and cell-surface protein PD-L1/CD274. Implantation of NPM-ALK-transformed CD4(+) T lymphocytes into immunodeficient mice resulted in formation of tumors indistinguishable from patients anaplastic large-cell lymphomas. Our findings demonstrate that the key aspects of human carcinogenesis closely recapitulating the features of the native tumors can be faithfully reproduced in vitro when an appropriate oncogene is used to transform its natural target cells; this in turn points to the fundamental role in malignant cell transformation of potent oncogenes expressed in the relevant target cells. Such transformed cells should permit study of the early stages of carcinogenesis, and in particular the initial oncogene-host cell interactions. This experimental design could also be useful for studies of the effects of early therapeutic intervention and likely also the mechanisms of malignant progression.

Anaplastic lymphoma kinase (ALK) is an important molecular target in neuroblastoma. Although tyrosine kinase inhibitors abrogating ALK activity are currently in clinical use for the treatment of ALK-positive (ALK(+)) disease, monotherapy with ALK tyrosine kinase inhibitors may not be an adequate solution for ALK(+) neuroblastoma patients. Increased expression of the gene encoding the transcription factor MYCN is common in neuroblastomas and correlates with poor prognosis. We found that the kinase ERK5 [also known as big mitogen-activated protein kinase (MAPK) 1 (BMK1)] is activated by ALK through a pathway mediated by phosphoinositide 3-kinase (PI3K), AKT, MAPK kinase kinase 3 (MEKK3), and MAPK kinase 5 (MEK5). ALK-induced transcription of MYCN and stimulation of cell proliferation required ERK5. Pharmacological or RNA interference-mediated inhibition of ERK5 suppressed the proliferation of neuroblastoma cells in culture and enhanced the antitumor efficacy of the ALK inhibitor crizotinib in both cells and xenograft models. Together, our results indicate that ERK5 mediates ALK-induced transcription of MYCN and proliferation of neuroblastoma, suggesting that targeting both ERK5 and ALK may be beneficial in neuroblastoma patients.

Proteins of the echinoderm microtubule (MT)-associated protein (EMAP)-like (EML) family contribute to formation of the mitotic spindle and interphase MT network. EML1-4 consist of Trp-Asp 40 (WD40) repeats and an N-terminal region containing a putative coiled-coil. Recurrent gene rearrangements in non-small cell lung cancer (NSCLC) fuse EML4 to anaplastic lymphoma kinase (ALK) causing expression of several oncogenic fusion variants. The fusions have constitutive ALK activity due to self-association through the EML4 coiled-coil. We have determined crystal structures of the coiled-coils from EML2 and EML4, which describe the structural basis of both EML self-association and oncogenic EML4-ALK activation. The structures reveal a trimeric oligomerization state directed by a conserved pattern of hydrophobic residues and salt bridges. We show that the trimerization domain (TD) of EML1 is necessary and sufficient for self-association. The TD is also essential for MT binding; however, this property requires an adjacent basic region. These observations prompted us to investigate MT association of EML4-ALK and EML1-ABL1 (Abelson 1) fusions in which variable portions of the EML component are present. Uniquely, EML4-ALK variant 3, which includes the TD and basic region of EML4 but none of the WD40 repeats, was localized to MTs, both when expressed recombinantly and when expressed in a patient-derived NSCLC cell line (H2228). This raises the question of whether the mislocalization of ALK activity to MTs might influence downstream signalling and malignant properties of cells. Furthermore, the structure of EML4 TD may enable the development of protein-protein interaction inhibitors targeting the trimerization interface, providing a possible avenue towards therapeutic intervention in EML4-ALK NSCLC.

This report delivers a comprehensive genetic alteration profile of lung adenocarcinomas (LADC) driven by ALK, RET, and ROS1 oncogene fusions. These tumors are difficult to study because of their rarity. Each drives only a low percentage of LADCs. Whole-exome sequencing and copy-number variation analyses were performed on a Japanese LADC cohort (n = 200) enriched in patients with fusions (n = 31, 15.5%), followed by deep resequencing for validation. The driver fusion cases showed a distinct profile with smaller numbers of nonsynonymous mutations in cancer-related genes or truncating mutations in SWI/SNF chromatin remodeling complex genes than in other LADCs (P < 0.0001). This lower mutation rate was independent of age, gender, smoking status, pathologic stage, and tumor differentiation (P < 0.0001) and was validated in nine fusion-positive cases from a U.S. LADCs cohort (n = 230). In conclusion, our findings indicate that LADCs with ALK, RET, and ROS1 fusions develop exclusively via their dependence on these oncogene fusions. The presence of such few alterations beyond the fusions supports the use of monotherapy with tyrosine kinase inhibitors targeting the fusion products in fusion-positive LADCs.

PURPOSE: Therapies targeted to the immune checkpoint mediated by PD-1 and PD-L1 show antitumor activity in a subset of patients with non-small cell lung cancer (NSCLC). We have now examined PD-L1 expression and its regulation in NSCLC positive for the EML4-ALK fusion gene. EXPERIMENTAL DESIGN: The expression of PD-L1 at the protein and mRNA levels in NSCLC cell lines was examined by flow cytometry and by reverse transcription and real-time PCR analysis, respectively. The expression of PD-L1 in 134 surgically resected NSCLC specimens was evaluated by immunohistochemical analysis. RESULTS: The PD-L1 expression level was higher in NSCLC cell lines positive for EML4-ALK than in those negative for the fusion gene. Forced expression of EML4-ALK in Ba/F3 cells markedly increased PD-L1 expression, whereas endogenous PD-L1 expression in EML4-ALK-positive NSCLC cells was attenuated by treatment with the specific ALK inhibitor alectinib or by RNAi with ALK siRNAs. Furthermore, expression of PD-L1 was downregulated by inhibitors of the MEK-ERK and PI3K-AKT signaling pathways in NSCLC cells positive for either EML4-ALK or activating mutations of the EGFR. Finally, the expression level of PD-L1 was positively associated with the presence of EML4-ALK in NSCLC specimens. CONCLUSIONS: Our findings that both EML4-ALK and mutant EGFR upregulate PD-L1 by activating PI3K-AKT and MEK-ERK signaling pathways in NSCLC reveal a direct link between oncogenic drivers and PD-L1 expression.

We have molecularly cloned a cDNA encoding a protein uniquely expressed and hyperphosphorylated at tyrosine residues in a Ki-1 lymphoma cell that contained chromosomal translocation t(2;5). The encoded protein p80 was shown to be generated by fusion of a protein-tyrosine kinase and a nucleolar protein B23/nucleophosmin (NPM). The coding sequence of this cDNA turned out to be virtually identical to that of the fusion cDNA for NPM-anaplastic lymphoma kinase (ALK) previously cloned from the transcript of the gene at the breakpoint of the same translocation. Overexpression of p80 in NIH 3T3 cells induced neoplastic transformation, suggesting that the p80 kinase is aberrantly activated. The normal form of p80 was predicted to be a receptor-type tyrosine kinase on the basis of its sequence similarity to the insulin receptor family of kinases. However, an immunofluorescence study using COS cells revealed that p80 was localized to the cytoplasm. Thus, subcellular translocation and activation of the tyrosine kinase presumably by its structural alteration would cause the malignant transformation. We also showed that a mutant p80 lacking the NPM portion was unable to transform NIH 3T3 cells. Thus, the NPM sequence is essential for the transforming activity, suggesting that the chromosomal translocation is responsible for the oncogenesis. Finally, Shc and insulin receptor substrate 1 (IRS-1) were tyrosine-phosphorylated and bound to p80 in p80-transformed cells. However, mutants of p80 that were defective for binding to and phosphorylation of Shc and insulin receptor substrate 1 could transform NIH 3T3 cells. Association of these mutants with GRB2 was still observed, suggesting that interaction of p80 with GRB2 but not with Shc or IRS-1 was relevant for cell transformation.