Anaplastic Large Cell Lymphoma Cells Research Articles

Complete Response of Relapsed Systemic and Cutaneous Anaplastic Large Cell Lymphoma Using Brentuximab Vedotin: 2 Case Reports

Clinical Practice Points Relapsed and refractory T-cell lymphomas represent a great challenge for clinicians because of their scarce tendency to show a response to further conventional treatments: in particular, for systemic anaplastic lymphoma kinase (ALK)-negative anaplastic large cell lymphoma (ALCL) treatment outcomes remain unknown and their potential benefits are still difficult assess. The conventional therapy in relapsed/refractory ALCL is poorly defined and no standard of care exists in this setting. It is difficult to obtain complete remissions using single-agent therapy. The wide expression of the CD30 antigen in ALCL cells makes this molecule a suitable target for monoclonal antibodies. In a phase II trial, the anti-CD30 monoclonal immunoconjugated brentuximab vedotin has shown an impressive effect in patients with relapsed ALKnegative ALCL, with high overall and complete response rates. Brentuximab vedotin is now approved by the Food and Drug Administration for the treatment of patients with relapsed ALCL, and now represents a valid treatment strategy for heavily pretreated patients.

Aptamer‐Drug Conjugates for Targeted Therapy of CD30‐expressing Lymphomas

CD30 is a biomarker for diagnosis and targeted therapy of Anaplastic Large cell lymphoma (ALCL) and Classical Hodgkin Lymphoma (CHL). Our previous studies have demonstrated specific binding of RNA aptamer to CD30 positive tumor cells. However, for in vivo targeted therapy there is a concern about biostability of the RNA aptamers.To overcome this, an ssDNA aptamer was developed via a hybrid SELEX approach using CD30‐expresing lymphoma cells and purified CD30 protein to yield the aptamer C2NP which specifically bound to CD30 expressing ALCL and CHL cells at concentrations ≤10 nm and did not react to control tumor cells. Importantly, C2NP is highly stable and more than 50% of them remained after incubation in human serum at 37°C for 24 hours, indicating the suitability for in vivo use.For targeted therapy study, C2NP was conjugated with a chemotherapeutic agent and a fluorescent reporter to form an aptamer‐drug conjugate (ADC). The specificity of ADC was confirmed by flow cytometry and intracellular delivery of chemo‐drug was observed by microscopy. Chemical conjugation of drug had no effect on aptamer binding specificity and affinity to lymphoma cells. In addition, potential therapeutic effects of the aptamer‐drug conjugate are under investigation by using cultured ALCL and CHL cell‐lines currently. This is the first study to use an aptamer‐drug conjugate for targeted therapy of CD30‐exprssing lymphomas.

Frequent expression of follicular dendritic cell markers in Hodgkin lymphoma and anaplastic large cell lymphoma

AimsAlthough the tumour cells of Hodgkin lymphoma (HL) are derived from mature B-cells, the lineage infidelity of Hodgkin/Reed–Sternberg cells (HRSs) often causes diagnostic problems. Recently introduced HRS markers are also...

Crizotinib-Resistant NPM-ALK Mutants Confer Differential Sensitivity to Unrelated Alk Inhibitors

The dual ALK/MET inhibitor crizotinib was recently approved for the treatment of metastatic and late-stage ALK+ NSCLC, and is currently in clinical trial for other ALK-related diseases. As predicted after other tyrosine kinase inhibitors' clinical experience, the first mutations that confer resistance to crizotinib have been described in patients with non-small cell lung cancer (NSCLC) and in one patient inflammatory myofibroblastic tumor (IMT). Here, we focused our attention on the anaplastic large cell lymphoma (ALCL), where the oncogenic fusion protein NPM-ALK, responsible for 70% to 80% of cases, represents an ideal crizotinib target. We selected and characterized 2 human NPM-ALK+ ALCL cell lines, KARPAS-299 and SUP-M2, able to survive and proliferate at different crizotinib concentrations. Sequencing of ALK kinase domain revealed that a single mutation became predominant at high crizotinib doses in each cell line, namely L1196Q and I1171N in Karpas-299 and SUP-M2 cells, respectively. These mutations also conferred resistance to crizotinib in Ba/F3 cells expressing human NPM-ALK. The resistant cell populations, as well as mutated Ba/F3 cells, were characterized for sensitivity to two additional ALK inhibitors: the dual ALK/EGFR inhibitor AP26113 and NVP-TAE684. While L1196Q-positive cell lines were sensitive to both inhibitors, cells carrying I1171N substitution showed cross-resistance to all ALK inhibitors tested. This study provides potentially relevant information for the management of patients with ALCL that may relapse after crizotinib treatment.

Wiskott-Aldrich Protein Is Phosphorylated by NPM-ALK and Regulates the Proliferation and Invasion of Anaplastic Large Cell Lymphoma

Abstract 1535 Introduction:Anaplastic large cell lymphoma (ALCL) is a subtype of mature T cell lymphoma. The NPM-ALK fusion protein is expressed in 80% of pediatric ALCL and arises from a chromosomal translocation t(2;5)(p23;q35). NPM-ALK is a constitutively active oncogenic tyrosine kinase that regulates the function of numerous proteins involved in apoptosis and cell cycle progression. In order to better understand the mechanisms of NPM-ALK mediated oncogenesis, we utilized an unbiased mass spectrometry –based phosphoproteomic approach to identify novel downstream signaling targets of the oncogenic NPM-ALK. Approach:The phosphoproteome was enriched using a two-step procedure including immobilized metal affinity chromatography (IMAC) and phospho-tyrosine immunoaffinity purification and subsequently analyzed by liquid chromatography and high mass accuracy tandem mass spectrometry (LC-MS/MS). Western blot analysis with phospho-specific antibodies and in vitro kinase assays were used to validate the MS results. Stable lentiviral transduction of shRNA vectors was used to evaluate the functional consequences. Cell proliferation was measured using the WST-1 assay. Effect on transformation capacity was assessed with colony formation assay using methyl-cellulose agar. Invasive properties were assessed using collagen based transwell assays and adhesion was evaluated by attachment onto fibronectin substratum. Results:Phosphoproteomic analysis identified several proteins involved in actin dynamics to be regulated by ALK tyrosine kinase activity. These include WASP, CRKL, NCK1, ARP2/3 and ITSN2. Pathway network analysis situated WASP at the center of the actin network. Western blot analysis demonstrated that phosphorylation of WASP at Y290 is regulated by NPM-ALK. Expression of wild type NPM-ALK increased the phosphorylation of WASP Y290 in 293T cells compared to a kinase-defective K210R mutant. In vitro kinase assays demonstrated that NPM-ALK phosphorylates WASP at Y290 as well as a novel residue Y102. The interaction between NPM-ALK and WASP was demonstrated using immunoprecipitation of co-transfected 293T cells as well as in ALCL cell lines. Because Y102 residue of WASP is located in the domain involved in interaction with its chaperone WIP, we evaluated whether its phosphorylation regulates the interaction. The interaction of WASP and WIP was decreased when Y102 was phosphorylated as demonstrated by immunoprecipitation of phospho-mutants of WASP (Y102F and Y102E). Surface plasmon resonance studies with recombinant WT or phosphomimetic (Y102E) WASP and WIP confirmed these observations. Furthermore, cycloheximide chase experiments demonstrated that phosphorylation of WASP at Y102 decreased it protein stability. Stable WASP knockdown in SUDHL1 cells led to reduced adhesion to fibronectin (21% ± SEM 0.4% vs 13% ± SEM 0.6%, p<0.01) and invasion through collagen (68% ± SEM 16% vs 12% ± SEM 3%, p<0.05). Interestingly, stable knock down of WASP resulted in increased proliferation (0.79 RFU ± SEM 0.07 vs 0.54 RFU ± SEM 0.019, p<0.01) and colony formation (58 colonies ± SEM 1.2 vs 26 colonies ± SEM 3.3, p< 0.01) of SUDH1L cells. Conclusions:Our results show that NPM-ALK regulates the phosphorylation of a network of proteins involved in actin dynamics, one of which is WASP. NPM-ALK directly phosphorylates WASP at residues Y290 and Y102 and thereby regulates WASP function in actin dynamics critical for cell adhesion, invasion and proliferation. Disclosures:No relevant conflicts of interest to declare.

The dichloromethane extract of the ethnomedicinal plant Neurolaena lobata inhibits NPM/ALK expression which is causal for anaplastic large cell lymphomagenesis

The present study investigates extracts of Neuolaena lobata, an anti-protozoan ethnomedicinal plant of the Maya, regarding its anti-neoplastic properties. Firstly, extracts of increasing polarity were tested in HL-60 cells analyzing inhibition of cell proliferation and apoptosis induction. Secondly, the most active extract was further tested in anaplastic large cell lymphoma (ALCL) cell lines of human and mouse origin. The dichloromethane extract inhibited proliferation of HL-60, human and mouse ALCL cells with an IC50 of ~2.5, 3.7 and 2.4µg/ml, respectively and arrested cells in the G2/M phase. The extract induced the checkpoint kinases Chk1 and Chk2 and perturbed the orchestrated expression of the Cdc25 family of cell cycle phosphatases which was paralleled by the activation of p53, p21 and downregulation of c-Myc. Importantly, the expression of NPM/ALK and its effector JunB were drastically decreased, which correlated with the activation of caspase 3. Subsequently also platelet derived growth factor receptor β was downregulated, which was recently shown to be transcriptionally controlled by JunB synergizing with ALK in ALCL development. We show that a traditional healing plant extract downregulates various oncogenes, induces tumor suppressors, inhibits cell proliferation and triggers apoptosis of malignant cells. The discovery of the 'Active Principle(s)' is warranted.

SY14-5 - Discovery Research of a New Selective ALK Inhibitor CH5424802

ABSTRACT The recent development of targeted protein kinase inhibitors has provided opportunities in cancer treatment. However, certain factors limit the efficacy of cancer therapies, such as a narrow therapeutic index caused by inhibition of multiple kinases, and the emergence of resistant mutants. Anaplastic lymphoma kinase (ALK) is a tyrosine kinase that is constitutively activated in certain cancers, following gene alterations such as chromosomal translocation, amplification, or point mutation. Especially, EML4-ALK has been identified as a fusion oncogene in non-small-cell lung cancer (NSCLC). We have carried out high-throughput screening of enzyme inhibitors against targets of tyrosine kinases, and we finally found CH5424802, a potent, selective, and orally available ALK inhibitor with a new chemical scaffold. In an enzyme inhibitory assay and a binding assay, CH5424802 had a nanomolar affinity with ATP-competitive inhibition mode. CH5424802 showed preferential antitumor activity against cancers with gene alterations of ALK, such as NSCLC cells expressing EML4-ALK fusion and anaplastic large-cell lymphoma cells expressing NPM-ALK fusion in vitro and in vivo. It had a wide therapeutic index due to higher kinase selectivity. Acquired resistance to kinase inhibitors is one of the most serious problems in the long-term cancer treatment. In particular gatekeeper mutations are reported as one of the common resistant mechanisms of kinase inhibitors, such as ABL, EGFR, and KIT. CH5424802 inhibited ALK L1196M, which corresponds to the gatekeeper mutation conferring common resistance to kinase inhibitors, and blocked EML4-ALK L1196M-driven cell growth. Potent ALK inhibitors effective against the gatekeeper mutants may offer clinical advantages in cancer treatment for patients with ALK-driven tumors.

Distinct Motifs in the Intracellular Domain of Human CD30 Differentially Activate Canonical and Alternative Transcription Factor NF-κB Signaling

The TNF-receptor superfamily member CD30 is expressed on normal and malignant lymphocytes, including anaplastic large cell lymphoma (ALCL) cells. CD30 transmits multiple effects, including activation of NF-κB signaling, cell proliferation, growth arrest and apoptosis. How CD30 generates these pleiotropic effects is currently unknown. Herein we describe ALCL cells expressing truncated forms of the CD30 intracellular domain that allowed us to identify the key regions responsible for transmitting its biological effects in lymphocytes. The first region (CD30519–537) activated both the alternative and canonical NF-κB pathways as detected by p100 and IκBα degradation, IKKβ-dependent transcription of both IκBα and the cyclin-dependent kinase inhibitor p21WAF1/CIP1 and induction of cell cycle arrest. In contrast, the second region of CD30 (CD30538–595) induced some aspects of canonical NF-κB activation, including transcription of IκBα, but failed to activate the alternative NF-κB pathway or drive p21WAF1/CIP1-mediated cell-cycle arrest. Direct comparison of canonical NF-κB activation by the two motifs revealed 4-fold greater p65 nuclear translocation following CD30519–537 engagement. These data reveal that independent regions of the CD30 cytoplasmic tail regulate the magnitude and type of NF-κB activation and additionally identify a short motif necessary for CD30-driven growth arrest signals in ALCL cells.

Brentuximab Vedotin and Crizotinib in Anaplastic Large-Cell Lymphoma

Systemic anaplastic large-cell lymphoma (ALCL) is a rare, mature T-cell non-Hodgkin lymphoma. Anaplastic large-cell lymphoma cells express the surface antigen CD30, and more than half express the anaplastic lymphoma kinase (ALK) protein. These 2 proteins provide unique therapeutic targets in ALCL. Remission rates in ALCL with combination chemotherapy are approximately 80%, but relapse after first-line therapy is common. Brentuximab vedotin is a US Food and Drug Administration-approved, antibody-drug conjugate that combines an anti-CD30 antibody with monomethylauristatin E, a potent antimicrotubule agent. Response rates to brentuximab vedotin in patients with relapsed/refractory ALK and ALK ALCL have exceeded 80% with frequent complete responses and a median duration of response greater than 1 year. Brentuximab vedotin in combination with chemotherapy is being explored as a first-line therapy in ALCL. Crizotinib is an inhibitor of ALK tyrosine kinase that has been approved for the treatment of ALK non-small cell lung cancer. Successful treatment of ALK ALCL with crizotinib has been reported in pediatric patients and small case series leading to ongoing trials in relapsed/refractory ALCL. Brentuximab vedotin and crizotinib represent major advances in the treatment of ALK and ALK ALCL and will likely result in marked improvement in prognosis for this subset of aggressive lymphomas.

ALK+ anaplastic large cell lymphoma exhibits phosphatidylinositol‐3 kinase/akt activity with retained but inactivated PTEN—A report from the Children's Oncology Group

ALK+ anaplastic large cell lymphoma (ALCL) is usually a disease of young patients. We investigated phosphatidylinositol-3 kinase (PI3K)/Akt pathway-associated factors in pediatric cases and cell lines. Patient materials consisted of tissue slides of ALK+/CD30+ ALCL from 33 patients treated on Pediatric Oncology Group protocols (9219, n = 8 and 9315, n = 25). Slides were examined by immunohistochemistry for phospho(p)-Akt and PTEN, the primary feedback regulator of the pathway, as well as for p27kip1 and stathmin-1. ALCL cell lines SUDHL-1 and Karpas-299 were examined for ALK, pALK, pAkt, p27/Kip1, PTEN, pPTEN, CD30, pSTAT3, and pSTAT5; ALK inhibition was performed using compound PF-2341066 and PTEN genes were sequenced. A majority of patients expressed pAkt, PTEN, and stathmin, with p27kip1 levels less than controls. Cell lines showed expression of ALK, pALK, pSTAT3, pSTAT5, CD30, pAkt, PTEN, and pPTEN, with p27 slightly less than positive controls, and germline PTEN DNA. There was evidence of phosphorylated PTEN (pPTEN) associated with inhibited function. Pharmacologic inhibition of activated ALK diminished pSTAT3, pSTAT5, and CD30 expression but not pAkt or pPTEN in cultured cell lines. We conclude that the PI3K/Akt pathway is activated in many, though not all, pediatric ALK+ ALCL. Our data suggest that activation of this pathway involves post-translational regulation of PTEN. Pharmacologic inhibition of activated ALK does not reduce modest levels of activated Akt as it does with the more abundant levels of activated STAT3 or STAT5. Future therapy of ALCL might, in selected patients, best combine agents inhibiting PI3K/Akt with those targeting ALK.

Correlation of Seven Biological Factors (Hsp90a, p53, MDM2, Bcl-2, Bax, Cytochrome C, and Cleaved caspase3) with Clinical Outcomes of ALK+ Anaplastic Large-cell Lymphoma

ObjectiveTo explore correlation of seven apoptosis-related proteins (Hsp90a, p53, MDM2, Bcl-2, Bax, Cytochrome C, and Cleaved caspase3) with clinical outcomes of ALK+ anaplastic large-cell lymphoma (ALCL). MethodsUsing immunohistochemistry and immunofluorescence double staining methods, the expressions of these seven apoptosis-associated proteins were studied to clarify their relationship with clinical outcomes of 36 ALK+ and 25 ALK-systemic ALCL patients enrolled between 1996 and 2006. The relationship of these apoptosis-regulating proteins with NPM-ALK status was also evaluated with the tyrosine inhibitor herbimycin A (HA) in vitro by immunocytochemistry, Western blotting and flow cytometric assays. ResultsThe presence of Hsp90α-, MDM2-, Bax-, Cytochrome C, and Cleaved caspase3-positive tumor cells was found significantly different in ALK+ and ALK-ALCLs, which was correlated with highly favorable clinical outcome. The Bcl-2- and p53-positive tumor cells were found in groups of patients with unfavorable prognosis. Inhibition of NPM-ALK by HA could reactivate the p53 protein and subsequent apoptosis-related proteins and therefore induced apoptosis in ALK+ ALCL cells. ConclusionOur results suggest that these seven proteins might be involved in apoptosis regulation and associated with clinical outcome of ALK+ systemic ALCLs. We also reveal a dynamic chain relation that NPM-ALK regulates p53 expression and subsequent apoptosis cascade in ALK+ ALCLs.

Galectin-1-mediated cell death is increased by CD30-induced signaling in anaplastic large cell lymphoma cells but not in Hodgkin lymphoma cells

Endogenous β-galactose-binding lectins have many biological functions, but their biological significance in Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL) remains unclear. By immunohistochemistry, we analyzed the expression of galectin-1 and galectin-3 in HL and ALCL cases as well as in cell lines, and investigated the pharmacological effects of galectin-1 treatment with and without CD30 pre-stimulation of HL and ALCL cell lines. The galectin-3-negative human embryonic kidney cell line (HEK-293) was transfected with galectin-3 cDNA. Galectin-3 is differentially expressed in HL and ALCL. CD30 stimulation of the ALCL cell line Karpas 299 activates NF-κB without induction of apoptosis. Galectin-1 treatment of Karpas 299 induces cell death, which is significantly increased by CD30 pre-stimulation. The CD30-mediated increase of galectin-1-induced cell death is to some extent caspase independent and does not influence the expression of tumor necrosis factor-associated factor 1 (TRAF1), TRAF2, and cellular inhibitor of apoptosis 2 protein (cIAP2), as revealed in Karpas 299 cells. In other cell lines except Karpas 299, CD30 pre-stimulation did not significantly enhance galectin-1-induced cell death. Galectin-3 transfection of HEK-293 cells resulted in cell surface expression of galectin-3, associated with marked cell aggregation. CD30-targeted therapy in combination with galectin-1 treatment may induce effective killing of ALCL cells but not of HL cells.

Consequences of heat shock protein 72 (Hsp72) expression and activity on stress-induced apoptosis in CD30+ NPM–ALK+ anaplastic large-cell lymphomas

Understanding the mechanisms that control stress-induced apoptosis is critical to explain how tumours respond to treatment, as cancer cells frequently escape drug toxicity by regulating stress response through heat shock protein (HSP) expression. The overexpression of Hsp72, in particular, results in increased incidence of cell transformation, and correlates with poor prognosis in a wide range of cancers. We have shown that Hsp72 assists folding of oncogenic NPM-ALK kinase in anaplastic large-cell lymphomas (ALCLs), but its role in the maintenance of the malignant phenotype remains uncertain. Therefore, we assessed Hsp72 expression in ALCLs, investigating more in detail the mechanisms that regulate its status and activity. We found that Hsp72 is unique among the HSPs involved in tumourigenesis to be overexpressed in ALK(+) tumours and cell lines and to be induced by stress. Different from other HSPs, Hsp72 prevents cell injury, Bax activation and death by apoptosis in ALK(+) cells, acting both upstream and downstream of mitochondria. Conversely, Hsp72 is underexpressed in ALK(-) ALCL cells, and it is unable to protect cells from apoptosis under stress. Moreover, when Hsp72 expression is reduced following NPM-ALK inhibition, lymphoma cells undergo apoptosis, demonstrating the importance of Hsp72 in regulating ALCL stress response and drug sensitivity.

Brentuximab vedotin: its role in the treatment of anaplastic large cell and Hodgkin's lymphoma

Brentuximab vedotin is being developed in a joint collaboration between Seattle Genetics and Millennium: The Takeda Oncology Company. In August 2011, it was approved by the FDA for the treatment of patients with Hodgkin's lymphoma (HL) and anaplastic large cell lymphoma (ALCL). Brentuximab vedotin is an antibody-drug conjugate that specifically targets the TNF receptor superfamily member 8 (CD30) antigen on the surface of cancer cells to induce cell death. Brentuximab vedotin has shown efficacy in inducing apoptosis in HL and ALCL cell lines that express CD30 and reducing tumor size in preclinical models. Brentuximab vedotin is under clinical evaluation for the treatment of relapsed or refractory HL and ALCL in both adults and children. It is being investigated for use as a combination agent with pre-existing frontline chemotherapies and as a stand-alone salvage therapy for use prior to autologous stem cell transplant. Treatment with brentuximab vedotin is generally well tolerated although it is associated with grade 1-2 adverse reactions such as neutropenia and there have been reports of grade 3-4 serious adverse events. In particular its use with chemotherapy regimens that include bleomycin is contraindicated because of adverse pulmonary effects.

MiR-135b mediates NPM-ALK–driven oncogenicity and renders IL-17–producing immunophenotype to anaplastic large cell lymphoma

AbstractMany transformed lymphoma cells show immune-phenotypes resembling the corresponding normal lymphocytes; thus, they provide a guide for proper diagnosis and present promising routes to improve their pathophysiologic understanding and to identify novel therapeutic targets. However, the underlying molecular mechanism(s) of these aberrant immune-phenotypes is largely unknown. Here, we report that microRNA-135b (miR-135b) mediates nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)–driven oncogenicity and empowers IL-17–producing immunophenotype in anaplastic large cell lymphoma (ALCL). NPM-ALK oncogene strongly promoted the expression of miR-135b and its host gene LEMD1 through activation of signal transducer and activator of transcription (STAT) 3. In turn, elevated miR-135b targeted FOXO1 in ALCL cells. miR-135b introduction also decreased chemosensitivity in Jurkat cells, suggesting its contribution to oncogenic activities of NPM-ALK. Interestingly, miR-135b suppressed T-helper (Th) 2 master regulators STAT6 and GATA3, and miR-135b blockade attenuated IL-17 production and paracrine inflammatory response by ALCL cells, indicating that miR-135b–mediated Th2 suppression may lead to the skewing to ALCL immunophenotype overlapping with Th17 cells. Furthermore, antisense-based miR-135b inhibition reduced tumor angiogenesis and growth in vivo, demonstrating significance of this “Th17 mimic” pathway as a therapeutic target. These results collectively illuminated unique contribution of oncogenic kinase-linked microRNA to tumorigenesis through modulation of tumor immune-phenotype and microenvironment.

Inhibition of N-linked glycosylation impairs ALK phosphorylation and disrupts pro-survival signaling in neuroblastoma cell lines

BackgroundThe Anaplastic Lymphoma Kinase (ALK) is an orphan receptor tyrosine kinase, which undergoes post-translational N-linked glycosylation. The catalytic domain of ALK was originally identified in the t(2;5) translocation that produces the unglycosylated oncogenic protein NPM-ALK, which occurs in Anaplastic Large Cell Lymphoma (ALCL). Recently, both germline and somatic activating missense mutations of ALK have been identified in neuroblastoma (NB), a pediatric cancer arising from neural crest cells. Moreover, we previously reported that ALK expression is significantly upregulated in advanced/metastatic NB. We hypothesized that ALK function may depend on N-linked glycosylation and that disruption of this post-translational modification would impair ALK activation, regardless the presence of either gene mutations or overexpression.MethodsWe employed tunicamycin to inhibit N-linked glycosylation. The following ALK-positive NB cell lines were used: SH-SY5Y and KELLY (ALK mutation F1174L), UKF-NB3 (ALK mutation R1275Q) and NB1 (ALK amplification). As a control, we used the NB cell lines LA1-5S and NB5 (no ALK expression), and the ALCL cell line SU-DHL1 (NPM-ALK).ResultsTunicamycin treatment of ALK-positive NB cells resulted in a hypoglycosylated ALK band and in decreased amounts of mature full size receptor. Concomitantly, we observed a marked reduction of mature ALK phosphorylation. On the contrary, tunicamycin had no effects on NPM-ALK phosphorylation in SU-DHL1 cells. Moreover, phosphorylation levels of ALK downstream effectors (AKT, ERK1/2, STAT3) were clearly impaired only in ALK mutated/amplified NB cell lines, whereas no significant reduction was observed in both ALK-negative and NPM-ALK-positive cell lines. Furthermore, inhibition of N-linked glycosylation considerably impaired cell viability only of ALK mutated/amplified NB cells. Finally, the cleavage of the Poly-ADP-ribose-polymerase (PARP) suggested that apoptotic pathways may be involved in cell death.ConclusionsIn this study we showed that inhibition of N-linked glycosylation affects ALK phosphorylation and disrupts downstream pro-survival signaling, indicating that inhibition of this post-translational modification may be a promising therapeutic approach. However, as tunicamycin is not a likely candidate for clinical use other approaches to alter N-linked glycosylation need to be explored. Future studies will assess whether the efficacy in inhibiting ALK activity might be enhanced by the combination of ALK specific small molecule and N-linked glycosylation inhibitors.

Ets-1 Activates Overexpression of JunB and CD30 in Hodgkin's Lymphoma and Anaplastic Large-Cell Lymphoma

Overexpression of CD30 and JunB is a hallmark of tumor cells in Hodgkin's lymphoma (HL) and anaplastic large-cell lymphoma (ALCL). We reported that CD30-extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) signaling induces JunB, which maintains constitutive activation of the CD30 promoter. Herein, we localize a cis-acting enhancer in the JunB promoter that is regulated by Ets-1. We show that E26 transformation-specific-1 (Ets-1) (-146 to -137) enhances JunB promoter activation in a manner that is dependent on CD30 or the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-ERK1/2 MAPK pathway. Ets-1 knockdown reduces the expression of both JunB and CD30, and CD30 knockdown significantly reduces JunB expression in HL and ALCL cell lines. NPM-ALK knockdown also reduces JunB expression in ALCL cell lines expressing NPM-ALK. Collectively, these results indicate that CD30 and NPM-ALK cooperate to activate the ERK1/2 MAPK-Ets-1 pathway. Ets-1, constitutively activated by ERK1/2-MAPK, plays a central role in the overexpression of JunB and CD30, which are both involved in the pathogenesis of HL and ALCL.

Interleukin-2 Functions in Anaplastic Large Cell Lymphoma Cells through Augmentation of Extracellular Signal-Regulated Kinases 1/2 Activation

In addition to intrinsic genetic alterations, the effects of the extrinsic microenvironment also play a pathological role in cancer development. Altered chemokine/cytokine networks in the tumor microenvironment may contribute to the dysregulation of cellular functions in cancer cells. Anaplastic large cell lymphoma (ALCL) is an aggressive T-cell lymphoma caused by abnormal expression of anaplastic lymphoma kinase due to a chromosomal translocation. Notably, ALCL cells are also characterized by high-level expression of the high-affinity IL-2 receptor subunit CD25 on the cell surface. However, whether the IL-2/IL-2 receptor functions in ALCL cells and how this signaling affects the tumor remain unclear. In this study, we treated cultured ALCL cells with exogenous IL-2 and examined changes in cellular function and signaling pathways. IL-2 stimulated cell growth and augmented activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) pathway. Additionally, IL-2 enhanced lymphoma cell survival by overcoming kinase inhibitor U0126-induced growth arrest and apoptosis. Subsequently, to identify the potential source of IL-2for lymphoma cells in vivo, we performed gene expression and immunochemical analyses. RT-PCR revealed no IL-2 gene expression in cultured ALCL cells and ruled out the possibility of an IL-2 autocrine loop. Interestingly, immunostaining of lymphoma tumor tissues showed IL-2 protein expression in background cells within tumor tissue, but not in ALCL cells. Our findings demonstrate that IL-2 signaling plays a functional role in ALCL cells, and enhances lymphoma cell survival by increasing activation of the ERK1/2 pathway.

Programmed Death Ligand 1 Is Expressed by Non–Hodgkin Lymphomas and Inhibits the Activity of Tumor-Associated T Cells

Programmed death ligand 1 (PD-L1) is expressed on antigen-presenting cells and inhibits activation of T cells through its receptor PD-1. PD-L1 is aberrantly expressed on some epithelial malignancies and Hodgkin lymphomas and may prevent effective host antitumor immunity. The role of PD-L1 in non-Hodgkin lymphomas (NHL) is not well characterized. PD-L1 expression was analyzed in cell lines and lymphoma specimens by using flow cytometry and immunohistochemistry. Functional activity of PD-L1 was studied by incubating irradiated lymphoma cells with allogeneic T cells with or without anti-PD-L1 blocking antibody; T-cell proliferation and IFN-γ secretion served as measures of T-cell activation. Similar experiments were conducted using cultures of primary lymphoma specimens containing host T cells. PD-L1 was expressed uniformly by anaplastic large cell lymphoma (ALCL) cell lines, but rarely in B-cell NHL, confined to a subset of diffuse large B-cell lymphomas (DLBCL) with activated B-cell features (3 of 28 cell lines and 24% of primary DLBCL). Anti-PD-L1 blocking antibody boosted proliferation and IFN-γ secretion by allogeneic T cells responding to ALCL and DLBCL cells. In autologous cultures of primary ALCL and DLBCL, PD-L1 blockade enhanced secretion of inflammatory cytokines IFN-γ, granulocyte macrophage colony-stimulating factor, interleukin (IL)-1, IL-6, IL-8, IL-13, TNF-α, and macrophage inflammatory protein-1α. In establishing cell lines from an aggressive PD-L1(+) mature B-cell lymphoma, we also noted that PD-L1 expression could be lost under certain in vitro culture conditions. PD-L1 may thwart effective antitumor immune responses and represents an attractive target for lymphoma immunotherapy.

Resveratrol enhances the expression of death receptor Fas/CD95 and induces differentiation and apoptosis in anaplastic large-cell lymphoma cells

Resveratrol (3,5,4′-trihydroxy-trans-stilbene), a phytoalexin found in grapes and other plants, plays a protective role in human atherosclerosis and carcinogenesis. We examined the effects of resveratrol on the anaplastic large-cell lymphoma (ALCL) cell line SR-786. Resveratrol inhibited growth and induced cellular differentiation, as demonstrated by morphological changes and elevated expression of T cell differentiation markers CD2, CD3, and CD8. Resveratrol also triggered cellular apoptosis, as demonstrated by morphological observations, DNA fragmentation, and cell cycle analyses. Further, the surface expression of the death receptor Fas/CD95 was increased by resveratrol treatment. Our data suggest that resveratrol may have potential therapeutic value for ALCL.