Search

Anaplastic large-cell lymphomas (ALCLs) are lymphomas of T or null phenotype often associated with a chromosomal translocation, t(2;5)(p23;q35). This translocation leads to the expression of a hybrid protein consisting of the N-terminal portion of nucleophosmin (NPM) and the intracellular domain of the anaplastic lymphoma kinase (ALK). NPM-ALK possesses a constitutive tyrosine kinase activity responsible for its oncogenic property through activation of downstream effectors such as phospholipase C gamma (PLC-gamma) and the type IA phosphoinositide 3-kinase. Here, we show that the Src-kinases, particularly pp60(c-src), associate with and are activated by NPM-ALK expression in various cells, and in cell lines established from patients with ALCL. The kinase activity and the tyrosine 418 of NPM-ALK are required for its association with Src-kinases. Y418F mutation of NPM-ALK impaired its association with Src-kinases and strongly reduced the proliferation rate of Ba/F3 cells. In agreement, Src-kinase inhibitors or pp60(c-src) siRNA significantly decreased the proliferation rate of NPM-ALK-positive ALCL cell lines. Moreover, using active or inactive forms of pp60(c-src) and NPM-ALK, we provide evidence that NPM-ALK is a potential substrate of pp60(c-src). Overall, our data place Src-kinases as new important downstream effectors of NPM-ALK and as attractive potential therapeutic targets for new ALCL treatment.

In this study, we used subtractive suppression hybridization to compare gene expression between an ALK-positive anaplastic large cell lymphoma (ALCL)-derived cell line and a clinical case of ALK-negative ALCL. Construction and screening of a subtracted library resulted in the cloning of 29 cDNAs which were differentially expressed. Most of these clones corresponded to novel genes with unknown function (EST) or to genes implicated in the differentiation, activation or signalling of T cells such as Ran/TC4, interleukin 1-receptor, thymosin beta4, thymosin beta10, moesin and cytohesin-1. Other genes involved in the regulation of apoptosis, such as human inhibitor of apoptosis-1 (HIAP-1), Bax inhibitor-1 and MCL-1, or DNA repair, such as poly (ADP-ribose) polymerase 1 (PARP-1), X-associated protein-1 (XAP-1), SUMO-1 (sentrin-1) and RanGTPase-activating protein 1 (RanGAP-1), were isolated. Interestingly, we found that both RNA and protein levels of human sterol isomerase (hSI), also referred to as emopamil binding protein (EBP), were overexpressed in ALK+ tumours. This protein is involved in the biosynthesis of cholesterol and may be activated by NPM-ALK. Overall, our results suggest that all the genes described above are upregulated in the NPM-ALK-driven transformation process, and that moesin and cytohesin-1 may be more specifically implicated in a signalling pathway involving PLCgamma and PI3K.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase, initially discovered as part of the NPM-ALK fusion protein, resulting from the t(2;5) translocation that is frequently associated with anaplastic large-cell lymphomas. The native ALK protein is normally expressed in the developing and, at a weaker level, adult nervous system. We recently demonstrated that the oncogenic, constitutively kinase-activated NPM-ALK protein was antiapoptotic when expressed in Jurkat lymphoblastic cells treated with cytotoxic drugs. In contrast, we now show that Jurkat cells overexpressing the wild-type ALK receptor are more sensitive to doxorubicin-induced apoptosis than parental cells. Moreover, the ALK protein is cleaved during apoptosis in a caspase-dependent manner. Mutation of aspartic residues to asparagine allowed us to map the caspase cleavage site in the juxtamembrane region of ALK. In order to assess the role of ALK in neural cell-derived tissue, we transiently expressed ALK in the 13.S.1.24 rat neuroblast immortalized cell line. ALK expression led to apoptotic cell death of the neuroblasts. ALK ligation by specific activating antibodies decreased ALK-facilitated apoptosis in both lymphoid and neuronal cell lines. Moreover, ALK transfection reduced the survival of primary cultures of cortical neurons. Thus, ALK has a proapoptotic activity in the absence of ligand, whereas it is antiapoptotic in the presence of its ligand and when the kinase is intrinsically activated. These properties place ALK in the growing family of dependence receptors.

In this study, we used subtractive suppression hybridization to compare gene expression between an ALK-positive anaplastic large cell lymphoma (ALCL)-derived cell line and a clinical case of ALK-negative ALCL. Construction and screening of a subtracted library resulted in the cloning of 29 cDNAs which were differentially expressed. Most of these clones corresponded to novel genes with unknown function (EST) or to genes implicated in the differentiation, activation or signalling of T cells such as Ran/TC4, interleukin 1-receptor, thymosin beta4, thymosin beta10, moesin and cytohesin-1. Other genes involved in the regulation of apoptosis, such as human inhibitor of apoptosis-1 (HIAP-1), Bax inhibitor-1 and MCL-1, or DNA repair, such as poly (ADP-ribose) polymerase 1 (PARP-1), X-associated protein-1 (XAP-1), SUMO-1 (sentrin-1) and RanGTPase-activating protein 1 (RanGAP-1), were isolated. Interestingly, we found that both RNA and protein levels of human sterol isomerase (hSI), also referred to as emopamil binding protein (EBP), were overexpressed in ALK+ tumours. This protein is involved in the biosynthesis of cholesterol and may be activated by NPM-ALK. Overall, our results suggest that all the genes described above are upregulated in the NPM-ALK-driven transformation process, and that moesin and cytohesin-1 may be more specifically implicated in a signalling pathway involving PLCgamma and PI3K.

This report describes a case of anaplastic large cell lymphoma with the canonical t(2;5)(p23;q35) translocation in association with duplication of the short arm of the non-translocated chromosome 2, as demonstrated by two colour fluorescence in situ hybridisation. Because the tumour cells were tetraploid, these abnormalities were in duplicate, with four copies of the full length ALK gene and two copies of the t(2;5)(p23;q35) translocation. Despite multiple copies of the normal ALK gene, immunohistochemical, reverse transcriptase polymerase chain reaction, and western blot analysis demonstrated that only the fusion gene NPM/ALK was expressed and that normal ALK genes remained silent. Although based on a single case, these data indicate that structural rather than numerical abnormalities of the ALK gene are implicated in the pathogenesis of anaplastic large cell lymphomas.

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.

Adhesion molecules can improve hematopoietic cell survival; however, their role in leukemic cell resistance to drug-induced apoptosis is poorly documented. The CD44 adhesion molecule is strongly expressed on acute myeloid leukemia (AML) blasts. Using 2 myeloid cell lines, HL60 and NB4, evidence is presented that prior incubation with the CD44-specific monoclonal antibody (mAb) A3D8, reported to induce differentiation of AML blasts, significantly decreases apoptosis induced by 3 drugs used in AML chemotherapy: daunorubicin (DNR), mitoxantrone, and etoposide. In addition, in HL60 cells, CD44 ligation with A3D8 mAb fully abrogates the DNR-triggered generation of ceramide, a lipid second messenger involved in the DNR apoptotic signaling pathway. Moreover, results show that the A3D8 mAb and Bcl-2 additively inhibit DNR-induced apoptosis in HL60 cells overexpressing Bcl-2. These results suggest that, to eradicate AML blasts, the differentiation-inducing anti-CD44 mAb A3D8 should not be administered prior to apoptosis-inducing drugs.

Anaplastic lymphoma kinase (ALK)-positive lymphomas are characterized by expression of a hybrid protein, comprising the cytoplasmic portion of the ALK tyrosine kinase fused to a partner protein. This hybrid kinase is often encoded by the nucleophosmin (NPM)NPM-ALK fusion gene resulting from the (2;5)(p23;q35) chromosomal translocation. However, the ALK gene at 2p23 may also be involved in 2 variant translocations, namely t(1;2)(q25;p23) and t(2;3)(p23;q21), which create the TPM3-ALK andTFG-ALK fusion genes, respectively. We report here 2 lymphomas with an unusual finely granular cytoplasmic ALK staining pattern, clearly different from the pattern observed in ALK-positive lymphomas carrying NPM-ALK or its variants. A cloned complementary DNA sequence from 1 of these 2 lymphomas contained the ALK gene fused to the second clathrin heavy chain gene (also referred to as clathrin heavy polypeptide-like gene) (CLTCL). The distinctive granular cytoplasmic staining pattern for ALK was likely to be due to binding of the fusion protein to clathrin-coated vesicles. TheCLTCL gene is constitutively expressed in lymphoid cells and therefore presumably contributes an active promoter for theCLTCL-ALK gene. The fusion protein had a molecular weight (250 kd) that differs from all known ALK products, and it was autophosphorylated in an in vitro kinase assay, confirming that it is constitutively active and hence capable of contributing to malignant transformation. These 2 cases, therefore, represent a hitherto undescribed mechanism of ALK activation in lymphoma and further illustrate the diversity of fusion partners for the ALKgene.