Blood NEOPLASIA

Nifuroxazide inhibits survival of multiple myeloma cells by directly inhibiting STAT3

2008-12-08

Constitutive activation of the transcription factor STAT3 contributes to the pathogenesis of many cancers, including multiple myeloma (MM). Since STAT3 is dispensable in most normal tissue, targeted inhibition of STAT3 is an attractive therapy for patients with these cancers. To identify STAT3 inhibitors, we developed a transcriptionally based assay and screened a library of compounds known to be safe in humans. We found the drug nifuroxazide to be an effective inhibitor of STAT3 function. Nifuroxazide inhibits the constitutive phosphorylation of STAT3 in MM cells by reducing Jak kinase autophosphorylation, and leads to down-regulation of the STAT3 target gene Mcl-1. Nifuroxazide causes a decrease in viability of primary myeloma cells and myeloma cell lines containing STAT3 activation, but not normal peripheral blood mononuclear cells. Although bone marrow stromal cells provide survival signals to myeloma cells, nifuroxazide can overcome this survival advantage. Reflecting the interaction of STAT3 with other cellular pathways, nifuroxazide shows enhanced cytotoxicity when combined with either the histone deacetylase inhibitor depsipeptide or the MEK inhibitor UO126. Therefore, using a mechanistic-based screen, we identified the clinically relevant drug nifuroxazide as a potent inhibitor of STAT signaling that shows cytotoxicity against myeloma cells that depend on STAT3 for survival.

Defective synthesis or association of T-cell receptor chains underlies loss of surface T-cell receptor-CD3 expression in enteropathy-associated T-cell lymphoma

2008-12-08

Enteropathy-associated T-cell lymphoma, an often fatal complication of celiac disease, can result from expansion of aberrant intraepithelial lymphocytes in refractory celiac disease type II (RCD II). Aberrant intraepithelial lymphocytes and lymphoma cells are intracellularly CD3⁺ but lack expression of the T-cell receptor (TCR)–CD3 complex on the cell surface. It is unknown what causes the loss of TCR-CD3 expression. We report the isolation of a cell line from an RCD II patient with the characteristic phenotype of enteropathy-associated T-cell lymphoma. We demonstrate that in this cell line the TCR- and -β chains as well as the CD3, CD3, CD3, and -chains are present intracellularly and that assembly of the CD3, CD3, and -dimers is normal. However, dimerization of the TCR chains and proper assembly of the TCR-CD3 complex are defective. On introduction of exogenous TCR-β chains, but not of TCR- chains, assembly and functional cell surface expression of the TCR-CD3 complex were restored. Defective synthesis of both TCR chains was found to underlie loss of TCR expression in similar cell lines isolated from 2 additional patients. (Pre)malignant transformation in RCD II thus correlates with defective synthesis or defective association of the TCR chains, resulting in loss of surface TCR-CD3 expression.

Inherited and acquired variations in the hyaluronan synthase 1 (HAS1) gene may contribute to disease progression in multiple myeloma and Waldenstrom macroglobulinemia

2008-12-08

To characterize genetic contributions toward aberrant splicing of the hyaluronan synthase 1 (HAS1) gene in multiple myeloma (MM) and Waldenstrom macroglobulinemia (WM), we sequenced 3616 bp in HAS1 exons and introns involved in aberrant splicing, from 17 patients. We identified a total of 197 HAS1 genetic variations (GVs), a range of 3 to 24 GVs/patient, including 87 somatic GVs acquired in splicing regions of HAS1. Nearly all newly identified inherited and somatic GVs in MM and/or WM were absent from B chronic lymphocytic leukemia, nonmalignant disease, and healthy donors. Somatic HAS1 GVs recurred in all hematopoietic cells tested, including normal CD34⁺ hematopoietic progenitor cells and T cells, or as tumor-specific GVs restricted to malignant B and plasma cells. An in vitro splicing assay confirmed that HAS1 GVs direct aberrant HAS1 intronic splicing. Recurrent somatic GVs may be enriched by strong mutational selection leading to MM and/or WM.

Chronic lymphocytic leukemia antibodies with a common stereotypic rearrangement recognize nonmuscle myosin heavy chain IIA

2008-12-08

Leukemic B lymphocytes of a large group of unrelated chronic lymphocytic leukemia (CLL) patients express an unmutated heavy chain immunoglobulin variable (V) region encoded by IGHV1-69, IGHD3-16, and IGHJ3 with nearly identical heavy and light chain complementarity-determining region 3 sequences. The likelihood that these patients developed CLL clones with identical antibody V regions randomly is highly improbable and suggests selection by a common antigen. Monoclonal antibodies (mAbs) from this stereotypic subset strongly bind cytoplasmic structures in HEp-2 cells. Therefore, HEp-2 cell extracts were immunoprecipitated with recombinant stereotypic subset-specific CLL mAbs, revealing a major protein band at approximately 225 kDa that was identified by mass spectrometry as nonmuscle myosin heavy chain IIA (MYHIIA). Reactivity of the stereotypic mAbs with MYHIIA was confirmed by Western blot and immunofluorescence colocalization with anti-MYHIIA antibody. Treatments that alter MYHIIA amounts and cytoplasmic localization resulted in a corresponding change in binding to these mAbs. The appearance of MYHIIA on the surface of cells undergoing stress or apoptosis suggests that CLL mAb may generally bind molecules exposed as a consequence of these events. Binding of CLL mAb to MYHIIA could promote the development, survival, and expansion of these leukemic cells.

ASB2 targets filamins A and B to proteasomal degradation

2008-12-08

The ordered series of proliferation and differentiation from hematopoietic progenitor cells is disrupted in leukemia, resulting in arrest of differentiation at immature proliferative stages. Characterizing the molecular basis of hematopoietic differentiation is therefore important for understanding and treating disease. Retinoic acid induces expression of ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2 (ASB2) in acute promyelocytic leukemia cells, and ASB2 expression inhibits growth and promotes commitment, recapitulating an early step critical for differentiation. ASB2 is the specificity subunit of an E3 ubiquitin ligase complex and is proposed to exert its effects by regulating the turnover of specific proteins; however, no ASB2 substrates had been identified. Here, we report that ASB2 targets the actin-binding proteins filamin A and B for proteasomal degradation. Knockdown of endogenous ASB2 in leukemia cells delays retinoic acid-induced differentiation and filamin degradation; conversely, ASB2 expression in leukemia cells induces filamin degradation. ASB2 expression inhibits cell spreading, and this effect is recapitulated by knocking down both filamin A and filamin B. Thus, we suggest that ASB2 may regulate hematopoietic cell differentiation by modulating cell spreading and actin remodeling through targeting of filamins for degradation.

c-Abl kinase inhibitors overcome CD40-mediated drug resistance in CLL: implications for therapeutic targeting of chemoresistant niches

2008-12-08

In lymph node (LN) proliferation centers in chronic lymphocytic leukemia (CLL), the environment protects from apoptotic and cytotoxic triggers. Here, we aimed to define the molecular basis for the increased drug resistance and searched for novel strategies to circumvent it. The situation in CLL LN could be mimicked by prolonged in vitro CD40 stimulation, which resulted in up-regulation of antiapoptotic Bcl-xL, A1/Bfl-1, and Mcl-1 proteins, and afforded resistance to various classes of drugs (fludarabine, bortezomib, roscovitine). CD40 stimulation also caused ERK-dependent reduction of Bim-EL protein, but ERK inhibition did not prevent drug resistance. Drugs combined with sublethal doses of the BH3-mimetic ABT-737 displayed partial and variable effects per individual CD40-stimulated CLL. The antiapoptotic profile of CD40-triggered CLL resembled BCR-Abl–dependent changes seen in chronic myeloid leukemia (CML), which prompted application of c-Abl inhibitors imatinib or dasatinib. Both compounds, but especially dasatinib, prevented the entire antiapoptotic CD40 program in CLL cells, and restored drug sensitivity. These effects also occurred in CLL samples with dysfunctional p53. Importantly, ex vivo CLL LN samples also displayed strong ERK activation together with high Bcl-xL and Mcl-1 but low Bim levels. These data indicate that CLL cells in chemoresistant niches may be sensitive to therapeutic strategies that include c-Abl inhibitors.

Etiologic heterogeneity among non-Hodgkin lymphoma subtypes

2008-12-08

Understanding patterns of etiologic commonality and heterogeneity for non-Hodgkin lymphomas may illuminate lymphomagenesis. We present the first systematic comparison of risks by lymphoma subtype for a broad range of putative risk factors in a population-based case-control study, including diffuse large B-cell (DLBCL; N = 416), follicular (N = 318), and marginal zone lymphomas (N = 106), and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL; N = 133). We required at least 2 of 3 analyses to support differences in risk: (1) polytomous logistic regression, (2) homogeneity tests, or (3) dichotomous logistic regression, analyzing all 7 possible pairwise comparisons among the subtypes, corresponding to various groupings by clinical behavior, genetic features, and differentiation. Late birth order and high body mass index (≥ 35) kg/m²) increased risk for DLBCL alone. Autoimmune conditions increased risk for marginal zone lymphoma alone. The tumor necrosis factor G-308A polymorphism (rs1800629) increased risks for both DLBCL and marginal zone lymphoma. Exposure to certain dietary heterocyclic amines from meat consumption increased risk for CLL/SLL alone. We observed no significant risk factors for follicular lymphoma alone. These data clearly support both etiologic commonality and heterogeneity for lymphoma subtypes, suggesting that immune dysfunction is of greater etiologic importance for DLBCL and marginal zone lymphoma than for CLL/SLL and follicular lymphoma.

Antileukemic effects of the novel, mutant FLT3 inhibitor NVP-AST487: effects on PKC412-sensitive and -resistant FLT3-expressing cells

2008-12-08

An attractive target for therapeutic intervention is constitutively activated, mutant FLT3, which is expressed in a subpopulation of patients with acute myelocyic leukemia (AML) and is generally a poor prognostic indicator in patients under the age of 65 years. PKC412 is one of several mutant FLT3 inhibitors that is undergoing clinical testing, and which is currently in late-stage clinical trials. However, the discovery of drug-resistant leukemic blast cells in PKC412-treated patients with AML has prompted the search for novel, structurally diverse FLT3 inhibitors that could be alternatively used to override drug resistance. Here, we report the potent and selective antiproliferative effects of the novel mutant FLT3 inhibitor NVP-AST487 on primary patient cells and cell lines expressing FLT3-ITD or FLT3 kinase domain point mutants. NVP-AST487, which selectively targets mutant FLT3 protein kinase activity, is also shown to override PKC412 resistance in vitro, and has significant antileukemic activity in an in vivo model of FLT3-ITD⁺ leukemia. Finally, the combination of NVP-AST487 with standard chemotherapeutic agents leads to enhanced inhibition of proliferation of mutant FLT3-expressing cells. Thus, we present a novel class of FLT3 inhibitors that displays high selectivity and potency toward FLT3 as a molecular target, and which could potentially be used to override drug resistance in AML.

Constitutive activation of the Wnt canonical pathway in mantle cell lymphoma

2008-12-08

Aberrations of the Wnt canonical pathway (WCP) are known to contribute to the pathogenesis of various types of cancer. We hypothesize that these defects may exist in mantle cell lymphoma (MCL). Both the upstream and downstream aspects of WCP were examined in MCL cell lines and tumors. Using WCP-specific oligonucleotide arrays, we found that MCL highly and consistently expressed Wnt3 and Wnt10. β-catenin, a transcriptional factor that is a downstream target of WCP, is localized to the nucleus and transcriptionally active in all 3 MCL cell lines examined. By immunohistochemistry, 33 (52%) of 64 MCL tumors showed nuclear localization of β-catenin, which significantly correlated with the expression of the phosphorylated/inactive form of GSK3β (p-GSK3β; P = .011, Fisher). GSK3β inactivation is directly linked to WCP stimulation, since addition of recombinant sFRP proteins (a naturally occurring decoy for the Wnt receptors) resulted in a significant decrease in p-GSK3β. Down-regulation of DvL-2 (an upstream signaling protein in WCP) by siRNA or selective inhibition of β-catenin using quercetin significantly decreased cell growth in MCL cell lines. To conclude, WCP is constitutively activated in a subset of MCL and it appears to promote tumorigenesis in MCL.

Lenalidomide down-regulates the CD20 antigen and antagonizes direct and antibody-dependent cellular cytotoxicity of rituximab on primary chronic lymphocytic leukemia cells

2008-12-08

Lenalidomide, an immunomodulatory agent that enhances antibody-dependent cellular cytotoxicity (ADCC), is currently being investigated as a therapy for chronic lymphocytic leukemia (CLL). The anti-CD20 antibody rituximab is active in CLL and represents a rational agent to combine with lenalidomide. We therefore examined whether lenalidomide combined with rituximab enhances direct apoptosis and ADCC in CLL cells. In contrast to previous reports using CD20-positive lymphoma cell lines, lenalidomide down-regulated CD20 surface antigen expression in CLL patient cells via enhanced internalization, without influencing transcription. The CD20 surface antigen internalization enhanced delivery of an oligonucleotide incorporated into anti-CD20 immunoliposomes. In addition, CD20 surface antigen down-modulation by lenalidomide in CLL was accompanied by diminished rituximab-mediated apoptosis and ADCC. These observations suggest a need for alternative sequencing strategies to avoid antagonism between lenalidomide and rituximab therapy in CLL. In addition, they suggest that lenalidomide therapy might be useful to enhance targeted delivery of RNAi-based therapies using CD20 immunoliposomes in B-cell malignancies.

BCR-ABL fusion transcript types and levels and their interaction with secondary genetic changes in determining the phenotype of Philadelphia chromosome-positive leukemias

2008-12-08

It remains unresolved how different BCR-ABL transcripts differentially drive lymphoid and myeloid proliferation in Philadelphia chromosome–positive (Ph⁺) leukemias. We compared BCR-ABL transcript type and level with kinase domain (KD) mutation status, genotype, and phenotype in 1855 Ph⁺ leukemias. Compared with e1a2/p190 BCR-ABL cases, de novo e13-e14a2/p210 Ph⁺ lymphoid leukemia more frequently showed CML-type background, had higher blast-normalized BCR-ABL transcript levels, and more frequent persistent BCR-ABL transcript in the absence of detectable lymphoblasts. Secondary lymphoid blast transformation of CML was exclusively due to e13/e14a2/p210 BCR-ABL but was associated, at a much higher level than p210 myeloid transformation, with acquisition of new KD mutations and/or Ph genomic amplification. In contrast, myeloid blast transformation was more frequently accompanied by new acquisition of acute myeloid leukemia-type chromosomal aberrations, particularly involving the EVI1 and RUNX1 loci. Therefore, higher kinase activity by mutation, transcriptional up-regulation or gene amplification appears required for lymphoid transformation by p210 BCR-ABL.