SOX11 is a novel binding partner and endogenous inhibitor of SAMHD1 ara-CTPase activity in mantle cell lymphoma.

ABSTRACT: Sterile alpha motif and histidine-aspartate (HD) domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphate triphosphohydrolase with ara-CTPase activity that confers cytarabine (ara-C) resistance in several hematological malignancies. Targeting SAMHD1's ara-CTPase activity has recently been demonstrated to enhance ara-C efficacy in acute myeloid leukemia. Here, we identify the transcription factor SRY-related HMG-box containing protein 11 (SOX11) as a novel direct binding partner and first known endogenous inhibitor of SAMHD1. SOX11 is aberrantly expressed not only in mantle cell lymphoma (MCL), but also in some Burkitt lymphomas. Coimmunoprecipitation of SOX11 followed by mass spectrometry in MCL cell lines identified SAMHD1 as the top SOX11 interaction partner, which was validated by proximity ligation assay. In vitro, SAMHD1 bound to the HMG box of SOX11 with low-micromolar affinity. In situ crosslinking studies further indicated that SOX11-SAMHD1 binding resulted in a reduced tetramerization of SAMHD1. Functionally, expression of SOX11 inhibited SAMHD1 ara-CTPase activity in a dose-dependent manner resulting in ara-C sensitization in cell lines and in a SOX11-inducible mouse model of MCL. In SOX11-negative MCL, SOX11-mediated ara-CTPase inhibition could be mimicked by adding the recently identified SAMHD1 inhibitor hydroxyurea. Taken together, our results identify SOX11 as a novel SAMHD1 interaction partner and its first known endogenous inhibitor with potentially important implications for clinical therapy stratification.

SOX11 is a novel binding partner and endogenous inhibitor of SAMHD1 ara-CTPase activity in mantle cell lymphoma.

The sterile alpha motif and histidine-aspartate (HD) domain containing protein 1 (SAMHD1) is a deoxynucleoside triphosphate triphosphohydrolase with ara-CTPase activity that confers cytarabine (ara-C) resistance in several haematological malignancies. Targeting SAMHD1's ara-CTPase activity has recently been demonstrated to enhance ara-C efficacy in acute myeloid leukemia. Here, we identify the transcription factor SRY-related HMG-box containing protein 11 (SOX11) as a novel direct binding partner and first known endogenous inhibitor of SAMHD1. SOX11 is aberrantly expressed not only in mantle cell lymphoma (MCL), but also in some Burkitt lymphomas. Co-immunoprecipitation of SOX11 followed by mass spectrometry in MCL cell lines identified SAMHD1 as the top SOX11 interaction partner which was validated by proximity ligation assay. In vitro, SAMHD1 bound to the HMG box of SOX11 with low-micromolar affinity. In situ crosslinking studies further indicated that SOX11-SAMHD1 binding resulted in a reduced tetramerization of SAMHD1. Functionally, expression of SOX11 inhibited SAMHD1 ara-CTPase activity in a dose-dependent manner resulting in ara-C sensitization in cell lines and in a SOX11-inducible mouse model of MCL. In SOX11-negative MCL, SOX11-mediated ara-CTPase inhibition could be mimicked by adding the recently identified SAMHD1 inhibitor hydroxyurea. Taken together, our results identify SOX11 as a novel SAMHD1 interaction partner and its first known endogenous inhibitor with potentially important implications for clinical therapy stratification.

Targeting SAMHD1 with hydroxyurea in first-line cytarabine-based therapy of newly diagnosed acute myeloid leukaemia: Results from the HEAT-AML trial.

BACKGROUND: Treatment of newly diagnosed acute myeloid leukaemia (AML) is based on combination chemotherapy with cytarabine (ara-C) and anthracyclines. Five-year overall survival is below 30%, which has partly been attributed to cytarabine resistance. Preclinical data suggest that the addition of hydroxyurea potentiates cytarabine efficacy by increasing ara-C triphosphate (ara-CTP) levels through targeted inhibition of SAMHD1. OBJECTIVES: In this phase 1 trial, we evaluated the feasibility, safety and efficacy of the addition of hydroxyurea to standard chemotherapy with cytarabine/daunorubicin in newly diagnosed AML patients. METHODS: Nine patients were enrolled and received at least two courses of ara-C (1 g/m2 /2 h b.i.d. d1-5, i.e., a total of 10 g/m2 per course), hydroxyurea (1-2 g d1-5) and daunorubicin (60 mg/m2 d1-3). The primary endpoint was safety; secondary endpoints were complete remission rate and measurable residual disease (MRD). Additionally, pharmacokinetic studies of ara-CTP and ex vivo drug sensitivity assays were performed. RESULTS: The most common grade 3-4 toxicity was febrile neutropenia (100%). No unexpected toxicities were observed. Pharmacokinetic analyses showed a significant increase in median ara-CTP levels (1.5-fold; p = 0.04) in patients receiving doses of 1 g hydroxyurea. Ex vivo, diagnostic leukaemic bone marrow blasts from study patients were significantly sensitised to ara-C by a median factor of 2.1 (p = 0.0047). All nine patients (100%) achieved complete remission, and all eight (100%) with validated MRD measurements (flow cytometry or real-time quantitative polymerase chain reaction [RT-qPCR]) had an MRD level <0.1% after two cycles of chemotherapy. Treatment was well-tolerated, and median time to neutrophil recovery >1.0 × 109 /L and to platelet recovery >50 × 109 /L after the start of cycle 1 was 19 days and 22 days, respectively. Six of nine patients underwent allogeneic haematopoietic stem-cell transplantation (allo-HSCT). With a median follow-up of 18.0 (range 14.9-20.5) months, one patient with adverse risk not fit for HSCT experienced a relapse after 11.9 months but is now in second complete remission. CONCLUSION: Targeted inhibition of SAMHD1 by the addition of hydroxyurea to conventional AML therapy is safe and appears efficacious within the limitations of the small phase 1 patient cohort. These results need to be corroborated in a larger study.

A Small Molecule Targeting the Intracellular Tyrosine Kinase Domain of ROR1 (KAN0441571C) Induced Significant Apoptosis of Non-Small Cell Lung Cancer (NSCLC) Cells.

The ROR1 receptor tyrosine kinase is expressed in embryonic tissues but is absent in normal adult tissues. ROR1 is of importance in oncogenesis and is overexpressed in several cancers, such as NSCLC. In this study, we evaluated ROR1 expression in NSCLC patients (N = 287) and the cytotoxic effects of a small molecule ROR1 inhibitor (KAN0441571C) in NSCLC cell lines. ROR1 expression in tumor cells was more frequent in non-squamous (87%) than in squamous (57%) carcinomas patients, while 21% of neuroendocrine tumors expressed ROR1 (p = 0.0001). A significantly higher proportion of p53 negative patients in the ROR1+ group than in the p53 positive non-squamous NSCLC patients (p = 0.03) was noted. KAN0441571C dephosphorylated ROR1 and induced apoptosis (Annexin V/PI) in a time- and dose-dependent manner in five ROR1+ NSCLC cell lines and was superior compared to erlotinib (EGFR inhibitor). Apoptosis was confirmed by the downregulation of MCL-1 and BCL-2, as well as PARP and caspase 3 cleavage. The non-canonical Wnt pathway was involved. The combination of KAN0441571C and erlotinib showed a synergistic apoptotic effect. KAN0441571C also inhibited proliferative (cell cycle analyses, colony formation assay) and migratory (scratch wound healing assay) functions. Targeting NSCLC cells by a combination of ROR1 and EGFR inhibitors may represent a novel promising approach for the treatment of NSCLC patients.

Clinical and biological impact of SAMHD1 expression in mantle cell lymphoma.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) that restricts viral replication in infected cells and limits the sensitivity to cytarabine by hydrolysing its active metabolite, as recently shown in acute myeloid leukemia. Cytarabine is an essential component in the Nordic mantle cell lymphoma protocols (MCL2 and MCL3) for induction and high-dose chemotherapy treatment before autologous stem cell transplantation for younger patients with mantle cell lymphoma (MCL). We here investigated the expression of SAMHD1 in a population-based cohort of MCL (N = 150). SAMHD1 was highly variably expressed in MCL (range, 0.4% to 100% of positive tumor cells). Cases with blastoid/pleomorphic morphology had higher SAMHD1 expression (P = 0.028) and SAMHD1 was also correlated to tumor cell proliferation (P = 0.016). SAMHD1 expression showed moderate correlation to the expression of the transcriptional regulator SOX11 (P = 0.036) but genetic silencing of SOX11 and SAMHD1 by siRNA in MCL cell lines did not suggest mutual regulation. We hypothesized that expression of SAMHD1 could predict short time to progression in patients treated with Cytarabine as part of high-dose chemotherapy. Despite the correlation with known biological adverse prognostic factors, neither low or high SAMHD1 expression correlated to PFS or OS in patients treated according to the Nordic MCL2 or MCL3 protocols (N = 158).

ROR1 Is Expressed in Diffuse Large B-Cell Lymphoma (DLBCL) and a Small Molecule Inhibitor of ROR1 (KAN0441571C) Induced Apoptosis of Lymphoma Cells.

The receptor tyrosine kinase ROR1 is absent in most normal adult tissues, but overexpressed in several malignancies. In this study, we explored clinical and functional inhibitory aspects of ROR1 in diffuse large B-cell lymphoma (DLBCL). ROR1 expression in tumor cells was more often observed in primary refractory DLBCL, Richter's syndrome and transformed follicular lymphoma than in relapsed and non-relapsed DLBCL patients (p < 0.001). A survival effect of ROR1 expression was preliminarily observed in relapsed/refractory patients independent of gender and stage but not of age, cell of origin and international prognostic index. A second generation small molecule ROR1 inhibitor (KAN0441571C) induced apoptosis of ROR1+ DLBCL cell lines, similar to venetoclax (BCL-2 inhibitor) but superior to ibrutinib (BTK inhibitor). The combination of KAN0441571C and venetoclax at EC50 concentrations induced almost complete killing of DLBCL cell lines. Apoptosis was accompanied by the downregulation of BCL-2 and MCL-1 and confirmed by the cleavage of PARP and caspases 3, 8, 9. PI3Kδ/AKT/mTOR (non-canonical Wnt pathway) as well as ß-catenin and CK1δ (canonical pathway) were inactivated. In zebra fishes transplanted with a ROR1+ DLBCL cell line, KAN0441571C induced a significant tumor reduction. New drugs with mechanisms of action other than those available for DLBCL are warranted. ROR1 inhibitors might represent a novel promising approach.

Ribonucleotide reductase inhibitors suppress SAMHD1 ara-CTPase activity enhancing cytarabine efficacy.

The deoxycytidine analogue cytarabine (ara-C) remains the backbone treatment of acute myeloid leukaemia (AML) as well as other haematological and lymphoid malignancies, but must be combined with other chemotherapeutics to achieve cure. Yet, the underlying mechanism dictating synergistic efficacy of combination chemotherapy remains largely unknown. The dNTPase SAMHD1, which regulates dNTP homoeostasis antagonistically to ribonucleotide reductase (RNR), limits ara-C efficacy by hydrolysing the active triphosphate metabolite ara-CTP. Here, we report that clinically used inhibitors of RNR, such as gemcitabine and hydroxyurea, overcome the SAMHD1-mediated barrier to ara-C efficacy in primary blasts and mouse models of AML, displaying SAMHD1-dependent synergy with ara-C. We present evidence that this is mediated by dNTP pool imbalances leading to allosteric reduction of SAMHD1 ara-CTPase activity. Thus, SAMHD1 constitutes a novel biomarker for combination therapies of ara-C and RNR inhibitors with immediate consequences for clinical practice to improve treatment of AML.

Inhibition of heat shock protein 90 function by 17-allylamino-17-demethoxy-geldanamycin in Hodgkin's lymphoma cells down-regulates Akt kinase, dephosphorylates extracellular signal-regulated kinase, and induces cell cycle arrest and cell death.

PURPOSE: Heat shock protein 90 (HSP90) is a chaperone for several client proteins involved in transcriptional regulation, signal transduction, and cell cycle control. HSP90 is abundantly expressed by a variety of tumor types and has been recently targeted for cancer therapy. The objective of this study was to determine the role of HSP90 in promoting growth and survival of Hodgkin's lymphoma and to determine the molecular consequences of inhibiting HSP90 function by the small-molecule 17-allylamino-17-demethoxy-geldanamycin (17-AAG) in Hodgkin's lymphoma. EXPERIMENTAL DESIGN: HSP90 expression in Hodgkin's lymphoma cell lines was determined by Western blot and in primary lymph node sections from patients with Hodgkin's lymphoma by immunohistochemistry. Cell viability was determined by the 3-(4,5-dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Apoptosis and cell cycle fractions were determined by flow cytometry. Expression of intracellular proteins was determined by Western blot. RESULTS: HSP90 is overexpressed in primary and cultured Hodgkin's lymphoma cells. Inhibition of HSP90 function by 17-AAG showed a time- and dose-dependent growth inhibition of Hodgkin's lymphoma cell lines. 17-AAG induced cell cycle arrest and apoptosis, which were associated with a decrease in cyclin-dependent kinase (CDK) 4, CDK 6, and polo-like kinase 1 (PLK1), and induced apoptosis by caspase-dependent and caspase-independent mechanisms. Furthermore, 17-AAG depleted cellular contents of Akt, decreased extracellular signal-regulated kinase (ERK) phosphorylation, and reduced cellular FLICE-like inhibitory protein levels (FLIP), and thus enhanced the cytotoxic effect of doxorubicin and agonistic anti-tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor antibodies. CONCLUSION: Inhibition of HSP90 function induces cell death and enhances the activity of chemotherapy and anti-tumor necrosis factor-related apoptosis-inducing ligand death receptor antibodies, suggesting that targeting HSP90 function might be of therapeutic value in Hodgkin's lymphoma.

The HSP90 inhibitor 17-AAG synergizes with doxorubicin and U0126 in anaplastic large cell lymphoma irrespective of ALK expression.

OBJECTIVE: Heat shock protein 90 (HSP90) chaperones and maintains the molecular integrity of a variety of signal transduction proteins, including the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) oncogenic protein, a genetic abnormality that is frequently observed in anaplastic large cell lymphoma (ALCL) cells. Here we demonstrate that HSP90 is overexpressed in primary and cultured ALK-positive and ALK-negative ALCL cells, and we evaluate the potential role of the small molecule inhibitor of HSP90, 17-allylamino-17-demethoxygeldanamycin (17-AAG) in treating ALCL. METHODS: The antiproliferative effect of 17-AAG-cultured cells was determined by MTS assay. Apoptosis and cell-cycle arrest were determined by Annexin-V/propidium iodide and propidium iodide staining, respectively, and fluorescein-activated cell sorting analysis. Expression of HSP90 was evaluated by immunohistochemistry, and molecular changes were determined by Western blot. RESULTS: Treatment of cultured ALCL cells with 17-AAG induced cell-cycle arrest and apoptosis, irrespective of ALK expression. At the molecular level, 17-AAG induced degradation of ALK and Akt proteins, dephosphorylated extracellular signal-regulated kinase, and degraded the cell-cycle regulatory protein cyclin D1 and its cyclin-dependent kinases, CDK4 and CDK6, but had a differential effect on p27 and p53 proteins. Inhibition of extracellular signal-regulated kinase phosphorylation by the mitogen activated protein kinase inhibitor U0126 induced cell death in all ALCL cell lines, and sublethal concentration 17-AAG showed synergistic antiproliferative effects when combined with U0126 or doxorubicin. CONCLUSION: Our data demonstrate that targeting HSP90 function by 17-AAG may offer a novel therapeutic strategy for ALCL, either as single-agent activity or by combining 17-AAG with conventional or targeted therapeutic schemes.

Expression of Smac/DIABLO in B-cell non-Hodgkin and Hodgkin lymphomas.

Inhibitor of apoptosis proteins (IAPs) are upregulated in cancers and suppress cell death, in part, through their ability to directly inhibit caspases. Inhibitor of apoptosis proteins are differentially expressed in B-cell lymphomas. The functions of some IAPs are counteracted by the cell death inducer, second mitochondrial-derived activator of caspases/direct IAP binding protein with low pI (Smac/DIABLO). In this study, we investigated the expression levels of Smac/DIABLO in 14 lymphoma cell lines by Western blot analysis. We also assessed 247 B-cell non-Hodgkin's lymphoma (NHL) and 40 Hodgkin's lymphoma (HL) tumors using immunohistochemical methods. Smac/DIABLO was expressed in most NHL and all HL cell lines. In NHL, Smac/DIABLO was expressed in 117 (47%) tumors and was differentially expressed in various NHL types. In most NHLs, from 29% to 68% of tumors were positive; however, Smac/DIABLO was not detected in small lymphocytic lymphoma/chronic lymphocytic leukemia and Burkitt lymphoma, and was rare in extranodal marginal zone B-cell lymphoma. In HL, Smac/DIABLO was positive in 25 (63%) tumors. Unlike NHL, all types of HL were positive for Smac/DIABLO, although nodular sclerosis was least often positive. The differential expression of Smac/DIABLO in NHLs suggests that apoptotic mechanisms are differentially involved in their pathogenesis. These results may also have implications for using Smac/DIABLO or its agonists as therapeutic agents.

Differential expression of CKS-1B in typical and blastoid variants of mantle cell lymphoma.

Mantle cell lymphoma is a distinct type of B-cell lymphoma characterized by the t(11;14)(q13;q32). Mantle cell lymphomas exhibit a spectrum of morphologic findings, of which a subset of tumors is clinically aggressive with a high proliferation rate. These neoplasms are known as aggressive variants of which there are blastoid and pleomorphic subsets. CKS-1B (CDC28 protein kinase regulatory subunit 1B) is essential for the ubiquitination and degradation of p27 and cell cycle progression. We analyzed CKS-1B expression in mantle cell lymphoma cell lines and tumors by Western blot and immunohistochemical analysis. In 4 mantle cell lymphoma cell lines, CKS-1B was expressed at variable levels and correlated inversely with p27 expression. In mantle cell lymphoma tumors, CKS-1B was positive in 10 (28.6%) of 35 typical versus 14 (87.5%) of 16 blastoid/pleomorphic cases (Fisher exact test, P = .0002). Analyzed as a continuous variable, the percentage of CKS-1B-positive cells significantly correlated with blastoid/pleomorphic morphology (Mann-Whitney U test, P = .001). Twelve (23.5%) of 51 mantle cell lymphoma tumors expressed p27. Proliferation rate (Ki-67) was higher in blastoid/pleomorphic variants than in typical mantle cell lymphoma tumors and was inversely associated with p27 levels in typical mantle cell lymphoma. However, CKS-1B expression did not correlate with p27 expression, proliferation rate, or prognosis in the entire study group. Fluorescence in situ hybridization analysis of 10 CKS-1B-positive mantle cell lymphoma tumors showed no evidence of CKS-1B gene amplification. We conclude that CKS-1B is commonly expressed in mantle cell lymphoma, particularly in aggressive histologic variants, and may be involved in pathogenesis.

Overexpression of CXCR4 predicts adverse overall and event-free survival in patients with unmutated FLT3 acute myeloid leukemia with normal karyotype.

BACKGROUND: CXC chemokine receptor 4 (CXCR4) expression in acute myeloid leukemia (AML) is reported to correlate with FLT3 gene mutation and poorer prognosis. The prognostic significance of CXCR4 expression in patients with AML that have a normal karyotype and no evidence of FLT3 gene mutations was examined. METHODS: The prognostic significance of CXCR4 expression in 122 AML patients with normal karyotype and no evidence of FLT3 gene mutation treated at our institution between 1997 and 2003 was analyzed. All patients received intensive chemotherapy according to institutional protocols; 84% received cytarabine-containing regimens. Bone marrow biopsy or clot specimens obtained before treatment were immunostained for CXCR4. RESULTS: There were 70 men and 52 women with a median age of 62 years (range, 22-82 years). Median follow-up was 18 months (range, <1-97 months). Seventy-six patients achieved complete remission (CR); 39 had recurrence. Sixty-six patients died, including 9 with no evidence of disease. CXCR4 was positive in 70 and negative in 52 patients, with CR rates of 58% and 71%, respectively (P = .09). Multivariate analysis demonstrated that CXCR4 expression, presence of multilineage dysplasia, and high creatinine level predicted poorer overall (OS) and event-free (EFS) survival. CONCLUSIONS.: The results suggest that CXCR4 expression is associated with poor prognosis in AML patients with an unmutated FLT3 gene.

HDM4 (HDMX) is widely expressed in adult pre-B acute lymphoblastic leukemia and is a potential therapeutic target.

Human homolog of murine double minute 2 (HDM2) and HDM4 (or HDMX) are negative regulators of p53. HDM4 has not been assessed in precursor B (pre-B) lymphoblastic leukemia (ALL). We examined bone marrow samples obtained at time of diagnosis from 55 adults with pre-B ALL. A tissue microarray composed of 2 cores per specimen was constructed and immunohistochemical techniques were used to assess HDM4, HDM2, p53, and p21. HDM4 was expressed in 39 of 49 (80%) cases. HDM2 was expressed in 14 of 54 (26%). All HDM2-positive cases were also positive for HDM4 (P<0.05). We confirmed expression of HDM4 and HDM4 variants by Western blotting and sequencing of reverse transcription-polymerase chain reaction products in a subset of ALL tumors. Results were correlated with the presence of the Philadelphia chromosome (Ph). p53 (P<0.05) and p21 (P<0.001) were expressed significantly more often in Ph+ pre-B ALL. HDM4 and HDM2 showed no correlation with Ph status. HDM4 expression in most cases of adult pre-B ALL suggests that HDM4 is a potential therapeutic target.

Nucleophosmin gene mutations in acute myeloid leukemia.

CONTEXT: Heterozygous mutation of the nucleophosmin gene (NPM1) has recently been described as one of the most frequent genetic lesions in acute myeloid leukemia (AML). OBJECTIVE: (1) To discuss the clinical, morphologic, immunophenotypic, and genetic features of AML with NPM1 gene mutations, along with various detection methods, (2) To explore the mechanisms by which NPM1 gene mutations contribute to leukemogenesis. DATA SOURCES/EXTRACTION: Data were analyzed from 7 recently published papers. RESULTS: NPM1 gene mutations tend to occur more frequently in women, and also tend to be associated with a higher white blood cell count. There is no significant age difference. NPM1-mutated AML is preferentially associated with AML with monocytic differentiation (in particular FAB M5b), lack of CD34, normal cytogenetics, FLT3 gene mutations, and a trend toward favorable clinical outcome, especially in patients without FLT3 gene mutation. NPM1 gene mutations cause a frame shift in the C-terminus of exon 12, disrupting the NPM nucleolar-localization signal or generating a leucine-rich nuclear export motif, resulting in abnormal cytoplasmic accumulation of NPM. Several methods are suitable for detecting NPM1 gene mutation, including molecular and immunohistochemical studies. These mutations may contribute to leukemogenesis, at least in part, through disruption of the p14(ARF) (alternative reading frame) MDM2-p53 pathway and centrosomal duplication. CONCLUSIONS: Detection of NPM1 gene mutations may allow dissection of the heterogeneous group of AML with normal karyotype into prognostically different subgroups. Exploring the mechanisms may lead to a better understanding of how mutant NPM protein becomes leukemogenic, thereby providing insights for the development of new chemotherapeutic agents.

Expression of inhibitor of apoptosis proteins in B-cell non-Hodgkin and Hodgkin lymphomas.

BACKGROUND: Inhibitor of apoptosis proteins (IAPs) inhibit apoptosis by binding specific caspases, and possibly by other mechanisms. Eight IAPs have been identified in humans, of which cIAP1, cIAP2, and XIAP are well known. IAPs are being investigated as potential treatment targets in cancer patients. METHODS: cIAP1, cIAP2, and XIAP were assessed in lymphoma cell lines, 240 B-cell non-Hodgkin lymphoma (NHL) tumors, and 40 Hodgkin lymphoma (HL) tumors. RESULTS: All IAPs were expressed in most NHL and all HL cell lines. In NHL tumors, cIAP1 was expressed in 174 (73%), cIAP2 in 115 (48%), and XIAP in 37 (15%). cIAP1 was positive in all precursor B-cell lymphoblastic lymphoma/leukemia (LBL) and nodal marginal zone B-cell lymphoma (MZL), over 90% of follicular lymphoma and diffuse large B-cell lymphoma (DLBCL), and approximately 50% to 60% of myeloma, Burkitt lymphoma (BL), lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia (LPL/WM), small lymphocytic lymphoma/ chronic lymphocytic leukemia (SLL/CLL), extranodal marginal zone B-cell lymphoma of mucosa associated lymphoid tissue (MALT-lymphoma), splenic MZL, and mantle cell lymphoma. cIAP2 was positive in all MALT-lymphoma, over 90% of precursor B-cell LBL (94%), most BL (75%), LPL/WM (71%), and SLL/CLL (67%), and approximately 40% to 60% of follicular lymphoma, myeloma, and DLBCL. XIAP was positive most cases of precursor B-cell LBL (57%) and approximately 30% to 40% of nodal MZL, BL, and DLBCL. In HL tumors, cIAP1 was positive in 30 (75%), cIAP2 in 27 (68%), and XIAP in 23 (58%), and did not correlate with histologic type. CONCLUSIONS: Differential expression of IAPs in B-cell lymphomas suggests differences in pathogenesis that may have implications for novel treatment strategies targeting IAPs.

Inhibition of the phosphatidylinositol-3 kinase/Akt promotes G1 cell cycle arrest and apoptosis in Hodgkin lymphoma.

Activation of the phosphatidylinositol 3-kinase (PI(3)K) pathway has been linked with tumour cell growth, survival and resistance to therapy in several cancer types. The active, phosphorylated form of Akt (pAkt) was found to be aberrantly expressed in Hodgkin lymphoma (HL)-derived cell lines and in Hodgkin-Reed-Sternberg (HRS) cells in 27 of 42 (64.3%) of primary lymph node sections of HL, indicative of PI(3)K activity. Akt phosphorylation was not associated with loss of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) expression, but with its phosphorylation in HL-cell lines, suggesting that its biological function is impaired. Akt phosphorylation was further induced by CD30 ligand (CD30L), CD40L and receptor activator of nuclear factor kappa B (RANK) ligand. The PI(3)K inhibitor LY294002 demonstrated antiproliferative effects in a dose- and time-dependent manner, which was associated with Akt dephosphorylation on Thr308 and Ser473 sites and dephosphorylation of the downstream ribosomal protein S6. LY209002 induced cell cycle arrest in the G0/G1 phase and apoptosis, which were associated with upregulation of MDM2, downregulation of cyclin D1, activation of caspase 9 and poly-ADP-ribose polymerase cleavage. The Akt inhibitor QLT394 also demonstrated antiproliferative effects in a dose- and time-dependent manner, dephosphorylated ribosomal S6 and cleaved caspase 9. Collectively, these data suggest that the aberrant activation of the PI(3)K/Akt survival pathway in HRS cells is not because of loss of PTEN expression. Our data suggest that PTEN phosphorylation and activation of CD30, CD40 and RANK may play a role in activating Akt in HRS cells.

Clusterin expression in malignant lymphomas: a survey of 266 cases.

Clusterin expression has been reported to be characteristic of systemic anaplastic large cell lymphoma and usually negative in cutaneous anaplastic large cell lymphoma as well as other lymphoma types. We surveyed clusterin expression using immunohistochemical methods in 266 cases of non-Hodgkin's lymphoma and Hodgkin's disease to further assess the diagnostic utility of this marker. Clusterin immunostaining was observed in 40 of 49 (82%) systemic anaplastic large cell lymphomas and 12 of 29 (41%) cutaneous anaplastic large cell lymphomas. Clusterin also was expressed in 5 of 43 (12%) diffuse large B-cell lymphomas (4 of 5 CD30+), 1 of 14 (7%) peripheral T-cell lymphomas, 1 of 32 (3%) cases of nodular sclerosis Hodgkin's disease, and 1 case of mycosis fungoides in large cell transformation. Clusterin was negative in all other neoplasms assessed including follicular lymphoma of all grades (n = 24), mantle cell lymphoma (n = 13), marginal zone B-cell lymphoma (n = 12), precursor T-cell or B-cell lymphoblastic leukemia/lymphoma (n = 10), mixed cellularity Hodgkin's disease (n = 8), chronic lymphocytic leukemia/small lymphocytic lymphoma (n = 7), Burkitt lymphoma (n = 7), mycosis fungoides (n = 4), nodular lymphocyte predominant Hodgkin's disease (n = 3), lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia (n = 2), and plasmacytoma (n = 2). We conclude that clusterin is a marker of anaplastic large cell lymphoma and that addition of clusterin to antibody panels designed to distinguish systemic anaplastic large cell lymphoma from classical Hodgkin's disease is useful. However, clusterin is also positive in a substantial subset of cutaneous anaplastic large cell lymphomas, a smaller subset of diffuse large B-cell lymphomas, and rarely in cases of peripheral T-cell lymphoma and nodular sclerosis Hodgkin's disease.

Characterization of 4 mantle cell lymphoma cell lines.

CONTEXT: Mantle cell lymphoma (MCL) is a distinct type of B-cell non-Hodgkin lymphoma characterized by t(11;14)(q13;q32) and cyclin D1 overexpression. The pathogenesis of MCL has not been comprehensively studied, which can be attributed in part to the paucity of well-characterized MCL cell lines. OBJECTIVES: We collected 4 previously developed MCL cell lines and performed extensive characterization, including the susceptibly of these cell lines to transduction by adenovirus vectors. Our aim was to facilitate the establishment of an in vitro model that can be reliably used to study the pathogenesis of MCL. METHODS: Standard techniques were used to compare the morphologic, immunophenotypic, and cytogenetic features of the 4 cell lines. In addition, Western blotting was used to investigate the presence of several cell cycle- and apoptosis-related proteins. TP53 DNA sequencing was also performed on the cell lines. The adenoviral transduction efficiency was assessed using an adenoviral vector carrying the gene encoding for the green fluorescence protein (Ad-GFP). RESULTS: All cell lines demonstrated evidence of t(11;14)(q13;q32) and overexpression of cyclin D1. Cyclin D2 was not detectable in all cell lines, whereas cyclin D3 was weakly expressed in JeKo-1 and SP-53. Other abnormalities of the cell cycle G1 phase regulatory pathway were detected, including loss of expression of p53 (JeKo-1) and p16(INK4a) (SP-53 and Granta 519), as well as TP53 mutation (Mino). All cell lines express high levels of cyclin E, c-Myc, Bcl-2, Bax, Bcl-x(L), and Mcl-1. Retinoblastoma protein is hyperphosphorylated in all cell lines. With the exception of Mino, MCL cell lines are highly transducible with adenoviral vectors. CONCLUSION: These cell lines are representative of MCL and can be used as an in vitro model to further explore the pathogenesis of this disease. The susceptibility of these cell lines to gene transfer provides opportunities to evaluate the importance of various oncogenes and tumor suppressor genes that may have an impact on developing effective therapeutic regimens for MCL.