Pathogenesis Of Anaplastic Large Cell Lymphoma Research Articles

ALK Mutants in the Kinase Domain Exhibit Altered Kinase Activity and Differential Sensitivity to Small Molecule ALK Inhibitors

Abnormal expression of constitutively active anaplastic lymphoma kinase (ALK) chimeric proteins in the pathogenesis of anaplastic large-cell lymphoma (ALCL) is well established. Recent studies with small molecule kinase inhibitors have provided solid proof-of-concept validation that inhibition of ALK is sufficient to attenuate the growth and proliferation of ALK (+) ALCL cells. In this study, several missense mutants of ALK in the phosphate anchor and gatekeeper regions were generated and their kinase activity was measured. NPM-ALK L182M, L182V, and L256M mutants displayed kinase activity in cells comparable to or higher than that of NPM-ALK wild type (WT) and rendered BaF3 cells into IL-3-independent growth, while NPM-ALK L182R, L256R, L256V, L256P, and L256Q displayed much weaker or little kinase activity in cells. Similar kinase activities were obtained with corresponding GST-ALK mutants with in vitro kinase assays. With regard to inhibitor response, NPM-ALK L182M and L182V exhibited sensitivity to a fused pyrrolocarbazole (FP)-derived ALK inhibitor comparable to that of NPM-ALK WT but were dramatically less sensitive to a diaminopyrimidine (DAP)-derived ALK inhibitor. On the other hand, NPM-ALK L256M exhibited >30-fold lower sensitivity to both FP-derived and DAP-derived ALK inhibitors. The growth inhibition and cytotoxicity of BaF3/NPM-ALK mutant cells induced by ALK inhibitors were consistent with inhibition of cellular NPM-ALK autophosphorylation. In a mouse survival model, treatment with the orally bioavailable DAP-ALK inhibitor substantially extended the survival of the mice inoculated with BaF3/NPM-ALK WT cells but not those inoculated with BaF3/NPM-ALK L256M cells. Binding of ALK inhibitors to ALK WT and mutants was analyzed using ALK homology models. In summary, several potential active ALK mutants were identified, and our data indicate that some of these mutants are resistant to select small molecule ALK inhibitors. Further characterization of these mutants may help to identify and develop potent ALK inhibitors active against both WT and resistant mutants of ALK.

Frequency and distribution of Epstein-Barr virus infection and its association with P53 expression in a series of primary nodal non-Hodgkin lymphoma patients from South India

This study investigated the prevalence of Epstein-Barr virus (EBV) infection and its association with P53 expression in a panel of 87 previously untreated nodal non-Hodgkin lymphomas (NHLs) from India. Polymerase chain reaction specific for Epstein Barr nuclear antigen 1 (EBNA1) and EBNA-3C was performed on the lymphnode tissue DNA, while P53 expression was analyzed by immunohistochemistry. EBV, predominantly type A strain, was detected in 27/87 (31%) nodal lymphoid malignancies, 11/46 diffuse large B-cell lymphomas, 6/17 follicular lymphoma, 4/6 anaplastic large cell lymphomas (ALCL), 5/11 peripheral T-cell lymphomas (PTCL) and 1/7 lymphoblastic lymphomas. EBV infection was more frequently observed in a specific subset of nodal NHL, suggesting a causative role of EBV infection in the pathogenesis of ALCL and PTCL. There was no significant association between EBV and P53 expression in our series of NHL patients.

NPM-ALK Mutants in Kinase Domain Exhibit Altered Kinase Activity and Various Sensitivity to ALK Inhibitors.

Abnormal expression of constitutively active anaplastic lymphoma kinase chimeric proteins (NPM-ALK) in the pathogenesis of anaplastic large-cell lymphoma (ALCL) is well defined. Recently, small molecule inhibitors have been reported that provide solid proof-of-concept validation that inhibition of ALK is sufficient to attenuate the growth and proliferation of ALK+ALCL cells. Although robust clinical response of ALCL patients to the ALK inhibitors is expected, some of those patients are also anticipated to develop resistance to an ALK inhibitor, most likely associated with single point mutations in the kinase domain of ALK. In this study, the nonsense mutants of NPM-ALK on the phosphate anchor region and the gatekeeper region were generated by site-directed mutagenesis and their kinase activity was measured in cells. NPM-ALK/L182M, L182V and L256M mutants displayed comparable or higher kinase activity in cells relative to NPM-ALK wt, and were able to render BaF3 cells into growth factor-independent growth, while NPM-ALK/L182R, L256R, L256V, L256P and L256Q displayed much weaker or little kinase activity in cells and failed to transform BaF3 cells. While NPM-ALK/L182M and L182V displayed comparable sensitivity to a fused pyrrolocarbazole (FP)-derived ALK inhibitor as NPM-ALK wt, they were >30-fold less sensitive to a diaminopyrimidine (DAP)-derived ALK inhibitor. On the other hand, NPM-ALK/L256M displayed >30-fold less sensitivity to both the FP-derived and the DAP-derived ALK inhibitors. Consistent with NPM-ALK autophosphorylation inhibition, the FP-ALK inhibitor induced growth inhibition and cytotoxicity of BaF3/NPM-ALK/L182M and L182V cells but not L256M cells, and the DAP ALK inhibitor failed to induce growth inhibition and cytotoxicity of all three BaF3/NPM-ALK mutant cell lines in culture. In the absence of an ALK inhibitor, BaF3 cells harboring NPM-ALK mutants did not display growth advantage in culture with 10% serum and in mice over the NPM-ALK wt cells. However, the BaF3/NPM-ALK mutants displayed significant growth advantage in culture with low serum (2%) over the BaF3/NPM-ALKwt cells, suggesting the BAF3/NPM-ALK mutant cells may proliferate better in the stressed conditions. Analyses of binding of ALK inhibitors to ALK wt and mutants in ALK homology models are in progress and the results will be discussed.

Cytoreductive antitumor activity of PF-2341066, a novel inhibitor of anaplastic lymphoma kinase and c-Met, in experimental models of anaplastic large-cell lymphoma

A t(2;5) chromosomal translocation resulting in expression of an oncogenic kinase fusion protein known as nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) has been implicated in the pathogenesis of anaplastic large-cell lymphoma (ALCL). PF-2341066 was recently identified as a p.o. bioavailable, small-molecule inhibitor of the catalytic activity of c-Met kinase and the NPM-ALK fusion protein. PF-2341066 also potently inhibited NPM-ALK phosphorylation in Karpas299 or SU-DHL-1 ALCL cells (mean IC(50) value, 24 nmol/L). In biochemical and cellular screens, PF-2341066 was shown to be selective for c-Met and ALK at pharmacologically relevant concentrations across a panel of >120 diverse kinases. PF-2341066 potently inhibited cell proliferation, which was associated with G(1)-S-phase cell cycle arrest and induction of apoptosis in ALK-positive ALCL cells (IC(50) values, approximately 30 nmol/L) but not ALK-negative lymphoma cells. The induction of apoptosis was confirmed using terminal deoxyribonucleotide transferase-mediated nick-end labeling and Annexin V staining (IC(50) values, 25-50 nmol/L). P.o. administration of PF-2341066 to severe combined immunodeficient-Beige mice bearing Karpas299 ALCL tumor xenografts resulted in dose-dependent antitumor efficacy with complete regression of all tumors at the 100 mg/kg/d dose within 15 days of initial compound administration. A strong correlation was observed between antitumor response and inhibition of NPM-ALK phosphorylation and induction of apoptosis in tumor tissue. In addition, inhibition of key NPM-ALK signaling mediators, including phospholipase C-gamma, signal transducers and activators of transcription 3, extracellular signal-regulated kinases, and Akt by PF-2341066 were observed at concentrations or dose levels, which correlated with inhibition of NPM-ALK phosphorylation and function. Collectively, these data illustrate the potential clinical utility of inhibitors of NPM-ALK in treatment of patients with ALK-positive ALCL.

Complex secondary chromosome abnormalities in advanced stage anaplastic large cell lymphoma of children and adolescents: a report from CCG-E08

Among pediatric non-Hodgkin lymphomas, one of the most distinctive types is anaplastic large cell lymphoma (ALCL). Specific chromosomal abnormalities are associated with prognosis in childhood acute lymphoblastic leukemia, but chromosome abnormalities have not been evaluated for prognostic value in pediatric ALCL. For Children's Cancer Group protocol CCG-E-08 Etiologic Study of Non-Hodgkin Lymphoma in Childhood, three patients were enrolled with cytogenetic analysis of ALCL and simultaneously enrolled on treatment protocol CCG-552. Pathology material and karyotypes at initial diagnosis underwent central review. Demographics included ages of 9, 12, and 14 years, and a male/female ratio of 1:2. All patients had advanced disease (stage III). Disease progressed or relapsed in two patients, and one died. Chromosomal abnormalities, including t(2;5)(p23;q35), the ALK/NPM fusion gene, and complex karyotypes with multiple additional abnormalities, were identified in all three patients. In two patients with progressive disease or relapse, additional chromosomal abnormalities at 1q21 and 10q24, possibly involving MCL1 and HOX11/ TCL3, respectively, may have contributed to worse outcome. Pediatric ALCL cases frequently have complex karyotypes and usually involve ALK/NPM translocations in this limited study. Additional chromosome abnormalities may be involved in the pathogenesis of ALCL. Further studies are warranted in larger cohorts of children and adolescents with ALCL.

C-FLIP confers resistance to FAS-mediated apoptosis in anaplastic large-cell lymphoma

AbstractWe hypothesized that inhibition of the FAS-mediated apoptosis pathway by FLICE-like inhibitory protein (c-FLIP) may contribute to oncogenesis in ALK+ anaplastic large-cell lymphoma (ALCL). Treatment with increasing concentrations of CH-11 (CD95/FAS agonistic antibody) had no effect on cell viability of 2 ALK+ ALCL cell lines, Karpas 299 and SU-DHL1, each expressing high levels of c-FLIP. However, inhibition of endogenous c-FLIP expression by specific c-FLIP siRNA in Karpas 299 and SU-DHL1 cells treated with CH-11 resulted in FAS-mediated cell death associated with increased annexin V binding, apoptotic morphology, and cleavage of caspase-8. In 26 ALK+ ALCL tumors, assessed for expression of DISC-associated proteins, CD95/FAS and c-FLIP were commonly expressed, in 23 (92%) of 25 and 21 (91%) of 23 tumors, respectively. By contrast, CD95L/FASL was expressed in only 3 (12%) of 26 ALCL tumors, although it was strongly expressed by surrounding small reactive lymphocytes. Our findings suggest that overexpression of c-FLIP protects ALK+ ALCL cells from death-receptor-induced apoptosis and may contribute to ALCL pathogenesis. (Blood. 2006;107:2544-2547)

Anaplastic lymphoma kinase activity is essential for the proliferation and survival of anaplastic large-cell lymphoma cells

The roles of aberrant expression of constitutively active ALK chimeric proteins in the pathogenesis of anaplastic large-cell lymphoma (ALCL) have been well defined; nevertheless, the notion that ALK is a molecular target for the therapeutic modulation of ALK+ ALCL has not been validated thus far. Select fused pyrrolocarbazole (FP)-derived small molecules with ALK inhibitory activity were used as pharmacologic tools to evaluate whether functional ALK is essential for the proliferation and survival of ALK+ ALCL cells in culture. These compounds inhibited interleukin 3 (IL-3)-independent proliferation of BaF3/NPM-ALK cells in an ALK inhibition-dependent manner and significantly blocked colony formation in agar of mouse embryonic fibroblast (MEF) cells harboring NPM-ALK. Inhibition of NPM-ALK phosphorylation in the ALK+ ALCL-derived cell lines resulted in significant inhibition of cell proliferation and induction of apoptotic-cell death, while having marginal effects on the proliferation and survival of K562, an ALK- leukemia cell line. ALK inhibition resulted in cell-cycle G1 arrest and inactivation of ERK1/2, STAT3, and AKT signaling pathways. Potent and selective ALK inhibitors may have therapeutic application for ALK+ ALCL and possibly other solid and hematologic tumors in which ALK activation is implicated in their pathogenesis.

In situ detection of hTERT variants in anaplastic large cell lymphoma

The expression of hTERT and its isoforms is difficult to assess in lymphoma tissues with the commonly used reverse transcription-polymerase chain reaction (RT-PCR) methods, because non-neoplastic lymphocytes expressing hTERT are always present in the lymphomatous infiltrates. The present study aimed to investigate hTERT mRNA variants in anaplastic large cell lymphoma (ALCL) (n = 38) with in situ hybridization (ISH), along with the immunodetection of hTERT protein. Probes for the identification of mRNAs containing (Bplus) and lacking (Bdel) exons 7 and 8 of the hTERT mRNA were used. Normal lymphocyte populations equally expressed both Bplus and Bdel mRNAs. Although all ALCL examined were found positive for hTERT expression with RT-PCR, hTERT mRNAs were identified in 68% of these tumors with ISH, with a higher incidence in the group bearing ALK translocations (10 out of 11; 90.9%) compared to the ALK negative group (17 out of 27; 59.3%) (PPearson's = 0.002). The same results were obtained with immunohistochemistry for hTERT. In approximately 50% of cases, only Bplus positive cells were identified, again with a higher incidence in the ALK positive compared to the ALK negative group (PPearson's = 0.016). In conclusion, ISH for hTERT mRNAs appears to be a valuable tool for the investigation of hTERT expression in lymphomas. Aberrations in hTERT variant profiles and a decline in the expression of the B deleted isoform may be associated with the pathogenesis of ALCL, especially with respect to ALK positive tumors.

Activation of α-diacylglycerol kinase is critical for the mitogenic properties of anaplastic lymphoma kinase

AbstractOncogenic rearrangements of the tyrosine kinase receptor anaplastic lymphoma kinase (ALK), most commonly represented by the nucleophosmin/ALK fusion protein (NPM/ALK), are involved in the pathogenesis of anaplastic large-cell lymphomas (ALCLs). In an effort to identify new intracellular transducers operative in ALK-positive malignancies, we have investigated the potential involvement of diacylglycerol kinase (DGK). Here we show that αDGK is constitutively activated in the NPM/ALK-positive ALCL-derived cell line Karpas 299 and in NPM/ALK-infected 32D hematopoietic cells. These results were further validated in fibroblastic NIH-3T3 cells expressing a previously described chimeric epidermal growth factor receptor (EGFR)/ALK molecule that allows dissection of ALK enzymatic function under conditions of controlled ligand-induced activation. In this cell system, we also show that ALK-mediated αDGK activation is dependent on p60src tyrosine kinase, with which αDGK forms a complex. The specific inhibition of αDGK, obtained by cell treatment with R59949, significantly reduced cellular growth in all cell lines. This result was further confirmed in Karpas 299 cells following specific down-regulation of αDGK by RNA interference. Overall, our data indicate that αDGK activation is involved in the control of ALK-mediated mitogenic properties. (Blood. 2005;106: 2175-2182)

Identification and Characterization of an ALK Inhibitor That Attenuates the Proliferation and Survival of Anaplastic Large Cell Lymphoma-Derived Cell Lines and ALK Dependent Cell Lines.

Anaplastic large cell lymphomas (ALCLs) are a unique subgroup of high grade non-Hodgkin lymphomas. Approximately 60% to 70% of ALCLs contain a t (2:5) (p23; q35) chromosomal rearrangement, generating the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion protein. NPM-ALK encodes a constitutively activated tyrosine kinase that has been demonstrated to be directly involved in the pathogenesis of ALCLs. Currently there is no optimal therapeutic regimen for ALK positive ALCLs. Even with high dose CHOP (cyclophophamide, doxorubicin, vincristine and prednisone)-based chemotherapy, a substantial number of patients with ALK positive ALCLs have very poor outcome, either failing to enter remission or relapsing within a few months from the start of treatment. This prompted a search for small molecule ALK inhibitors as potential therapeutic agents for these patients. In this report, we describe the identification and characterization of a potent small molecule ALK inhibitor, cmpd-1. Cmpd-1 inhibited the ALK kinase activity in an in vitro enzymatic assay with an IC50 value of 4 nM. In NPM-ALK transfected BaF3 cells and ALCL-derived cell lines, cmpd-1 potently inhibited the tyrosine phosphorylation of NPM-ALK with a cellular IC50 value of 10–30 nM. Treatment with cmpd-1 inhibited IL-3 independent proliferation of BaF3 cells expressing constitutively active NPM-ALK (more than 95% inhibition at 100 nM) and the addition of IL-3 rescued the growth of these cells, while cmpd-1 had little effect on the parental BaF3 cells. Inhibition of NPM-ALK activity by cmpd-1 in ALK positive ALCL-derived cell lines, Karpas-299, Sudhl-1, Sup-M2 and Sr-786, resulted in inhibition of cell proliferation (50–90% inhibition at 100–300 nM) and induction of apoptotic cell death, while having marginal effects on the proliferation and survival of K562, an ALK negative leukemia cell line. These data suggest that the inhibition of cell proliferation and induction of apoptosis by cmpd-1 is an ALK-dependent event. Several putative downstream targets of NPM-ALK, including ERK1/2, Stat3 and Akt, were hypo- or unphosphorylated when the ALK positive cells were treated with cmpd-1. With the potent ALK inhibitory activity, cmpd-1 can be used as a molecular and pharmacological tool to study the biological roles of ALK and to dissect ALK-mediated signaling pathways in ALCLs and possibly other tumor types in which ALK is implicated in their pathogenesis.

Prevalence and characterization of anaplastic large cell lymphoma and its association with Epstein-Barr virus in Pakistani patients

Anaplastic large cell lymphoma (ALCL), CD30+, is a subtype of T-non-Hodgkin's lymphoma (NHL). Its most common form is a classical systemic type that involves multiple nodal and extranodal sites. In this study, morphologic, immunohistologic, and genetic studies were performed on ALCL cases in Pakistani patients. The median age of the patients in this study was 45 years (age range: 5–70 years), with a male to female ratio of 3.4:1. Thirty-seven (37) patients were diagnosed to have Ki-1 (CD30+) ALCL, which constituted 2% of all NHLs and 12.6% of all T-NHLs, over a period of 11 years (January 01, 1992–December 31, 2002). The tumors were of either T- or null-cell type with constant (100%) expression of CD30 (Ki-1). The majority of the cases (89.2%) expressed EMA, whereas 40.5% of the cases expressed either CD45 (LCA), CD45RO (UCHL1), or ALK. The mean age of ALCL patients with null-cell phenotype was 33.8 years as compared to those with T-cell phenotype having a mean age of 36.3 years. Out of the 37 cases diagnosed as ALCL, amplifiable DNA was isolated from 28 cases, which were further assessed for T-cell clonality for T-cell receptor (TCR)-β, γ, and immunoglobulin heavy chain (IgH) for the FR2 and FR3 regions. The polymerase chain reaction (PCR) technique demonstrated clonal rearrangement of the TCR β, γ, and IgH regions in 15 (53.6%), 11 (39.3%), and 2 (7.1%) ALCL cases, respectively, out of 28 cases. Association of Epstein-Barr virus (EBV) was noted in seven out of 28 cases (25%) of ALCL by PCR, whereas ISH for EBV-encoded nuclear RNA-1 (EBER-1) detected the presence of EBV in two (16.7%) out of 12 cases, where one was T-cell ALCL and the other null-cell ALCL. Immunostaining for LMP-1 could not be performed, because tissue material was not available. In conclusion, our study demonstrated that the prevalence of ALCL in Pakistan is comparable to that reported for some of the Asian communities and by the International Lymphoma Study Group and that EBV could be partly responsible for the pathogenesis of ALCL.

Suppressor of cytokine signaling 3 expression in anaplastic large cell lymphoma.

Using a cDNA microarray, we found that suppressor of cytokine signaling 3 (SOCS3) is highly expressed in anaplastic lymphoma kinase (ALK)+ anaplastic large cell lymphoma (ALCL) cell lines. As SOCS3 is induced by activated signal transducer and activator of transcription 3 (STAT3), and ALK activates STAT3, we hypothesized that SOCS3 may play a role in ALK+ ALCL pathogenesis via the Janus kinase 3 (JAK3)-STAT3 pathway. Using ALCL cell lines, we show by coimmunoprecipitation experiments that SOCS3 physically binds with JAK3 in vitro, and that JAK3 inhibition by WHI-P154 downregulates SOCS3 expression. Western blot analysis confirmed expression of SOCS3 and also showed coexpression of phosphorylated (activated) STAT3 (pSTAT3). Direct sequencing of the SOCS3 gene showed no mutations or alternative splicing. In ALCL tumors that were assessed by immunohistochemistry, nine of 12 (75%) ALK+ tumors were SOCS3 positive and eight (67%) coexpressed pSTAT3. In comparison, 18 of 25 (72%) ALK-- tumors were SOCS3 positive and seven (28%) coexpressed pSTAT3. These results show that SOCS3 is overexpressed in ALCL, attributable to JAK3-STAT3 activation and likely related to ALK in ALK+ tumors. However, SOCS3 is also expressed in tumors that lack STAT3 and ALK suggesting alternative mechanisms of upregulation.

Retinoblastoma Protein Is Frequently Absent or Phosphorylated in Anaplastic Large-Cell Lymphoma

The possible role of retinoblastoma protein (Rb) in the pathogenesis of anaplastic large-cell lymphoma (ALCL) is unknown. We investigated Rb protein expression, both total (phosphorylated and underphosphorylated) and active (underphosphorylated), in four anaplastic lymphoma kinase (ALK)-positive ALCL cell lines (Karpas 299, JB-6, SU-DHL1, and SR-786) by Western blot analysis, and in 67 ALCL tumors (30 ALK-positive, 37 ALK-negative) using immunohistochemical methods. We also used fluorescence in situ hybridization and polymerase chain reaction methods to assess for loss of heterozygosity of the rb gene. The findings were correlated with apoptotic rate assessed by the terminal dUTP nick-end labeling assay. Immunoblots showed high total Rb levels in Karpas 299, SU-DHL1 and SR-786 and relatively lower levels in and JB-6. Underphosphorylated Rb was negative or expressed at low levels in all cell lines. In ALCL tumors, total Rb was detected in 44 (66%) and absent in 23 (34%). The mean apoptotic rate was 3.2% in Rb-negative tumors compared with 2.7%, 2.2%, and 1.2% in tumors with <10%, 10 to 50%, and >50% Rb-positive cells, respectively (P = 0.2, Kruskall-Wallis test). In a subset of 25 total Rb-positive tumors we assessed for underphosphorylated Rb, which was detected in 12 tumors. The detection of only total Rb in the remaining 13 tumors suggests that Rb was phosphorylated. Fluorescence in situ hybridization showed allelic loss of the rb gene in 10 (40%) of 25 tumors analyzed and was significantly associated with absence of Rb expression (P = 0.003). Similar results were obtained for loss of heterozygosity of the 13q14 locus. Five-year progression-free survival for patients with Rb-negative ALCL was 89.4% compared with 47.7% for patients with total Rb-positive ALCL (P = 0.006, log-rank test). Similar trends for progression-free survival held true for patients with ALK-positive and ALK-negative tumors analyzed separately. In conclusion, Rb is absent or phosphorylated in most ALCL cell lines and tumors and absence of Rb expression is associated with better clinical outcome in patients with ALCL.

Overexpression of NPM-ALK induces different types of malignant lymphomas in IL-9 transgenic mice.

Anaplastic large-cell lymphoma (ALCL) comprises approximately 25% of all non-Hodgkin lymphomas (NHL) in children and young adults, and up to 15% of high-grade NHL in older patients. Over 50% of these tumours carry the translocation t(2;5)(p23;q35). The result of this translocation is the fusion of the nucleophosmin (NPM) gene to the anaplastic lymphoma kinase (ALK) gene. The resulting hybrid protein contains the ALK catalytic domain that consequently confers transforming potential, which contributes to the pathogenesis of ALCL. To further analyse the transforming activity in an animal model, a cDNA encoding the protein product, NPM-ALK, was inserted into the retrovirus vector pLXSN and transduced into mouse bone marrow progenitors. These cells were subsequently used in a bone marrow transplant with the aim of reconstituting the haematopoietic compartments of lethally irradiated recipients. IL-9 transgenic mice were chosen as the animal model system, because dysregulated expression of the IL-9 gene in transgenic mice results in the sporadic development of spontaneous thymic lymphomas. Moreover, IL-9 is known to be expressed in cases of human ALCL. We used 15 IL-9 transgenic mice and eight corresponding wild-type mice (FVB/N) and transplanted them with NPM/ALK infected bone marrow cells. Eight IL-9 transgenic mice, serving as a control group, received pLXSN (vector only)-infected marrow. Reconstituted mice developed NPM-ALK-positive lymphomas, including lymphoblastic lymphomas of T-cell type (T-LB), mature and immature plasmacytoma (PC), and plasmoblastic/anaplastic diffuse large-B-cell lymphoma after about 19-20 weeks. The combined overexpression of NPM-ALK and IL-9 led to the transformation of murine lymphoid cells with accelerated and enhanced development of T-LB in 46% of the mice, which only very rarely occurs in IL-9 transgenic mice only. Of the 15 animals, five (33%) developed plasmacytic/plasmoblastic neoplasms, of which the most aggressive tumours share many features with anaplastic/plasmoblastic diffuse large-B-cell lymphoma on the basis of morphology, a characteristic growth pattern and ALK expression.

ALK a Novel Lymphoma-associated Tumor Antigen for Vaccination Strategies

The discovery of Tumor Associated Antigens (TAAs) demonstrated that tumor cells can be specifically recognized by the immune system raising the hypothesis that tumors express antigens that Cytotoxic T Lymphocytes (CTLs) can potentially attack. The identification of immunogenic epitopes led to their use as targets to mediate the specific clearance of neoplastic cells by TAA targeting strategies such as vaccination strategies. One of the critical issues in the development of efficient vaccination protocols is the identification of the appropriate TAAs. The TAA should be effective as a "tumor rejection antigen" able to induce an immune response that will affect tumor growth. A distinct pathologic entity characterized by the expression of the Anaplastic Lymphoma Kinase (ALK) protein and named "ALKoma" has recently emerged within the heterogeneous group of CD30+ Anaplastic Large Cell Lymphoma (ALCL). ALK is a receptor tyrosine kinase whose expression is normally restricted to a few scattered cells in the nervous system. Its pathological expression in lymphoma cells is due to a chromosomal translocation that leads to the formation of an ALK-derived oncogenic fusion proteins. ALK fusion proteins ectopically over-expressed and constitutively activated in lymphoid cells play a key role in the neoplastic transformation by the aberrant phosphorilation of intracellular substrates that likely contributes to the molecular pathogenesis of ALCL. The high level of ALK expression in lymphoma cells and its direct role in lymphomagenesis, combined with the fact that normal ALK is expressed at low levels in the immune privileged nervous system, makes ALK an ideal lymphoma-specific target for immunotherapy of ALK+ALCL.

Nucleophosmin–anaplastic lymphoma kinase associated with anaplastic large-cell lymphoma activates the phosphatidylinositol 3-kinase/Akt antiapoptotic signaling pathway

More than half of anaplastic large-cell lymphomas (ALCLs) have a chromosomal translocation t(2;5) that leads to the expression of a hybrid protein composed of the nucleolar phosphoprotein nucleophosmin (NPM) and the anaplastic lymphoma kinase (ALK) that exhibits an unregulated tyrosine kinase activity. We have previously identified PLC-γ as a crucial downstream signaling molecule of NPM-ALK that contributes to its mitogenic potential. Here, we show that NPM-ALK recruits the C-terminal SH2 domain of the phosphatidylinositol 3-kinase (PI 3kinase) p85 subunit. PI 3-kinase assays revealed that the kinase is activated by NPM-ALK in vivo, in turn activating PKB/Akt in NPM-ALK–expressing cells. The use of 2 specific PI 3-kinase inhibitors, wortmannin and LY294002, demonstrated the requirement of PI 3-kinase for the growth of NPM-ALK–transformed cell lines, as well as a cell line established from a patient with ALCL. Primary murine bone marrow retrovirally transduced with NPM-ALK showed a transformed phenotype that was reversible on treatment with PI 3-kinase inhibitors. Flow cytometric analysis revealed that wortmannin-treated NPM-ALK–transformed cell lines underwent apoptosis. Furthermore, apoptosis induced by overexpression of the proapoptotic molecule Bad could be partially blocked by the overexpression of NPM-ALK. Thus, NPM-ALK activates the antiapoptotic PI 3-kinase/Akt pathway, which likely contributes to the molecular pathogenesis of ALCL.

Nucleophosmin–anaplastic lymphoma kinase associated with anaplastic large-cell lymphoma activates the phosphatidylinositol 3-kinase/Akt antiapoptotic signaling pathway

AbstractMore than half of anaplastic large-cell lymphomas (ALCLs) have a chromosomal translocation t(2;5) that leads to the expression of a hybrid protein composed of the nucleolar phosphoprotein nucleophosmin (NPM) and the anaplastic lymphoma kinase (ALK) that exhibits an unregulated tyrosine kinase activity. We have previously identified PLC-γ as a crucial downstream signaling molecule of NPM-ALK that contributes to its mitogenic potential. Here, we show that NPM-ALK recruits the C-terminal SH2 domain of the phosphatidylinositol 3-kinase (PI 3kinase) p85 subunit. PI 3-kinase assays revealed that the kinase is activated by NPM-ALK in vivo, in turn activating PKB/Akt in NPM-ALK–expressing cells. The use of 2 specific PI 3-kinase inhibitors, wortmannin and LY294002, demonstrated the requirement of PI 3-kinase for the growth of NPM-ALK–transformed cell lines, as well as a cell line established from a patient with ALCL. Primary murine bone marrow retrovirally transduced with NPM-ALK showed a transformed phenotype that was reversible on treatment with PI 3-kinase inhibitors. Flow cytometric analysis revealed that wortmannin-treated NPM-ALK–transformed cell lines underwent apoptosis. Furthermore, apoptosis induced by overexpression of the proapoptotic molecule Bad could be partially blocked by the overexpression of NPM-ALK. Thus, NPM-ALK activates the antiapoptotic PI 3-kinase/Akt pathway, which likely contributes to the molecular pathogenesis of ALCL.