IntroductionWith the establishment of more effective treatments for patients with chronic lymphocytic leukemia (CLL) over the past decade, complete remissions are no longer the exception. 1 Despite these major improvements in CLL treatment, we still consider CLL an incurable disease, because patients generally relapse from minimal residual disease (MRD). 2 There is growing evidence suggesting that CLL cells are protected from conventional drugs in tissue microenvironments, such as the bone marrow and secondary lymphoid organs, with facilitation of residual disease that is drug resistant and ultimately paving the way to clonal evolution and relapses. The complex cellular and molecular contexts in the tissues, collectively referred to as the CLL microenvironment, provide signals for the expansion of the CLL clone and for primary drug resistance. This is largely dependent on direct contact between the malignant B cells and stromal cells, 3 and therefore has been designated as cell adhesion-mediated drug resistance. 4 Disrupting cross talk between leukemia cells and their milieu is an attractive novel but yet incompletely tested strategy for treating CLL. Appropriately, there is growing interest in understanding the biology of CLL-stroma cross talk to find ways to eliminate residual CLL cells that are "hiding" in stromal niches within the marrow and the lymphatic tissues.Importantly, once CLL cells are removed from the in vivo microenvironment and placed in suspension cultures without supportive stroma, they undergo spontaneous apoptosis, highlighting the importance of external signals from accessory cells. 5 Previous studies have shown that CLL cell cocultures with different adherent cell types, collectively referred to as stromal cells, induce leukemia cell survival, migration, and drug resistance. These stromal cells include mesenchymal marrow stromal cells (MSCs), 3,6,7 CD68 ϩ nurselike cells derived from monocytes, 7-10 and follicular dendritic cells. 11 Immunohistochemistry showed that in situ, ␣SMA ϩ mesenchymal stromal cells, 12 the in vivo counterpart of MSCs, are a dominant stromal cell population in the CLL microenvironment, which is in contrast to other B-cell lymphomas, particularly high-grade lymphomas, which harbor larger numbers of CD68 ϩ hemangiogenic cells. 12 MSCs regulate normal hematopoiesis by providing attachment sites and secreted or surface-bound growth factors that constitute the marrow microenvironment. 13 During B-cell development in the marrow, programmed cell death regulates B-cell homeostasis by diverting a large fraction of immature B cells into an apoptotic death pathway to eliminate functionless or potentially harmful cells. 14,15 Critical factors for the survival of selected B cells are interactions with MSCs in the marrow microenvironment, [16][17][18] expression of surface immunoglobulin molecules, and expression of apoptosis-regulatory proteins, such as Bcl-2. 19 In patients with CLL, the marrow invariably is infiltrated with CLL B cells, and the For personal use only. on May 7,...
We investigated the pattern of lipoprotein lipase (LPL) expression in B-cell chronic lymphocytic leukemia (B-CLL) and assessed its prognostic relevance. Expression of LPL mRNA as well as protein was highly restricted to leukemic B cells. The intensity of intracellular immunoreactivity of LPL was higher in samples of patients with unmutated immunoglobulin heavychain variable region genes (IGV H ) compared to those with mutated IGV H genes. LPL mRNA levels in peripheral blood mononuclear cells (PBMNC) from 104 CLL patients differed by 1.5 orders of magnitude between cases with mutated (N ¼ 51) or unmutated (N ¼ 53) IGV H (median: 1.33 vs 45.22 compared to normal PBMNC). LPL expression correlated strongly with IGV H mutational status (R ¼ 0.614; Po0.0001). High LPL expression predicted unmutated IGV H status with an odds ratio of 25.90 (Po0.0001) and discriminated between mutated and unmutated cases in 87 of 104 patients (84%). LPL expression was higher in patients with poor risk cytogenetics. High LPL expression was associated with a shorter treatment-free survival (median 40 vs 96 months, P ¼ 0.001) and a trend for a shorter median overall survival (105 months vs not reached). Our data establish LPL as a prognostic marker and suggest functional consequences of LPL overexpression in patients with B-CLL.
Anaplastic Large Cell Lymphoma (ALCL) is a Non-Hodgkin lymphoma found in children and young adults with poor survival rates. ALCLs frequently carry a chromosomal translocation that results in expression of the oncoprotein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). The key molecular downstream events required for NPM-ALK triggered lymphoma growth are still not entirely clear. Here we show that the AP-1 proteins cJun and JunB promote lymphoma development and tumor dissemination in a murine NPM-ALK lymphomagenesis model via transcriptional regulation of PDGFRB. Therapeutic inhibition of PDGFRB markedly prolonged survival of NPM-ALK transgenic mice and increased the efficacy of an ALK-specific inhibitor in transplanted NPM-ALK tumors. Remarkably, inhibition of PDGFRs in a late stage patient with refractory NPM-ALK-positive ALCL resulted in complete and sustained remission within 10 days of treatment. Our data identify PDGFRB as novel cJun/JunB target that could be utilized for a highly effective therapy to cure ALCL. 3ALCLs are T-cell lymphomas 1,2 that comprise 10-20% of all Non-Hodgkin's lymphoma cases in children and 3% in adults 3 . About half of ALCL cases are positive for NPM-ALK fusion proteins caused by t(2;5)(p23;q35) translocation 4 . ALK translocations or point mutations have also been described in DLBCLs (diffuse large B-cell lymphomas) and in several nonlymphoid neoplasms [5][6][7][8] . Inhibition of ALK fusion proteins by specific compounds such as crizotinib showed promising clinical responses in ALCL and NSCLC (non-small cell lung cancer) 9,10 . However, ALK mutations conferring resistance to crizotinib have also been reported 11 .Recent studies have linked NPM-ALK expression to induction of AP-1 transcription factors JunB and cJun 12,13 . To investigate their role in NPM-ALK-driven T-cell lymphomas, we conditionally deleted cJun and/or JunB in T-cells of transgenic mice carrying the human NPM-ALK fusion-tyrosine-kinase under the control of the murine CD4-promotor 14 (CD4-NPM-ALK) (Fig. 1a). Gene deletion was confirmed by DNA genotyping (data not shown), real-time PCR, Western blotting and immunohistochemistry (IHC) (Suppl. (Fig.1b), however, inactivation of both, cJun and JunB, in CD4-NPM-ALK-CD4 ΔΔJun mice resulted in significantly prolonged survival (Fig. 1b). CD4-NPM-ALK-CD4 ΔΔJun lymphomas showed markedly reduced proliferation and significantly increased apoptosis when compared to CD4-NPM-ALK lymphomas ( Fig. 1c; Suppl. Fig. S2a,b). Consistently, (Fig. 2d) suggesting a transcriptional regulation of PDGFRB by Jun proteins. Consistently, AP-1 consensus sequences were identified within the murine PDGFRB promoter and first intron that were conserved among other species (Suppl. Fig. S3b,c) 15,16 . Moreover, analysis of ENCODE transcription factor binding tracks revealed binding of cJun and JunB to the PDGFRB intronic AP-1 site in the human K562 leukemia cell line (Suppl. Fig. S3d) 15,17 . CD4-NPM-ALK-CD4EMSA analysis of nuclear extracts from NPM-ALK lymphomas demonstrated AP-1 DNA ...
Members of theThe pathomechanisms underlying the disease primarily involve defects that prevent cell turnover because of apoptosis, rather than alterations in cell cycle regulation. 1,2 One of the hallmarks of B-CLL cells is the overexpression of the transmembrane glycoprotein CD23, 1-3 which undergoes spontaneous proteolysis, giving rise to soluble CD23 (sCD23) molecules. 4 The serum concentration of sCD23 can be several hundredfold higher than in healthy individuals and parallels the clinical stage of the disease. 5,6 Two isoforms of CD23 exist, CD23a and CD23b, which are expressed from 2 different promoters. 7 Expression of CD23a is restricted to B lymphocytes, whereas CD23b is found on a number of different hematopoietic cell types, predominantly after interleukin 4 (IL-4) treatment. 8 In B-CLL cells, selective expression of CD23a has been found to be concurrent to a state of cell survival, thereby providing a link between CD23a and the regulation of apoptosis. 9 CD23 is also closely associated with Epstein-Barr virus (EBV)-mediated B-cell immortalization, because a naturally occurring EBV mutant (P3HR1), carrying a deletion of the EBNA2 gene, has lost its ability to induce CD23 expression and to transform primary B cells in vitro. 10-12 EBNA2 activates the CD23a gene through a CBF1 repressor site located in the CD23a proximal promoter. [13][14][15] Several lines of evidence strongly suggest that EBNA2 mimics Notch signaling by acting as a transcriptional activator after binding and masking the repression domain of CBF1. [16][17][18][19] The Notch gene family encodes transmembrane receptors that modulate differentiation, proliferation, and apoptotic programs in response to extracellular ligands expressed on neighboring cells. 20,21 Ligand-mediated stimulation of Notch causes the proteolytic release of the intracellular domain (NotchIC), which then passes into the nucleus where it activates transcription of CBF1 responsive genes. [22][23][24][25] Deregulation of this pathway by overexpression of a constitutively activated form of Notch not only diverts cell fate decisions but is also tumorigenic. For example, truncation of Notch1 found in a subset of human T-cell leukemias leads to the expression of a ligand-independent oncogenic protein lacking the extracellular domain. 26 The truncated Notch proteins consist primarily of the intracellular domain and localize predominantly in the nucleus. Enforced expression of Notch1IC in bone marrow stem cells causes T-cell leukemia in mice, indicating a causative role for Notch1 in T-cell oncogenesis. 27 To elucidate the mechanisms leading to the up-regulation of CD23a in B-CLL cells, we analyzed the CD23a proximal promoter for sequence-specific DNA-protein interactions. By electrophoretic mobility shift assays (EMSAs), we detected a transcription factor For personal use only. on May 10, 2018. by guest www.bloodjournal.org From complex containing Notch2IC that binds to 5 different CBF1 responsive elements. Our data indicate that Notch2 is overexpressed in B-CLL cells, sug...
IntroductionMajor progress has been achieved in the past decade in understanding the biology of B-cell chronic lymphocytic leukemia (CLL), leading to new therapeutic concepts and a trend toward improved survival. However, the disease remains incurable, and many patients develop drug resistance. [1][2][3] Despite the long lifespan of CLL cells in vivo, these cells undergo rapid and spontaneous apoptosis in vitro but can be rescued by marrow stromal cells, [4][5][6] nurse-like cells, 7 and follicular dendritic cells. 8 There are also indications that the stromal cells mediate resistance to chemotherapy in CLL cells. 9 This point to the dependence of CLL cells on antiapoptotic stimuli that could be provided in vivo by marrow microenvironment 10,11 and may have a major effect on disease progression and response or resistance to therapy.Bone marrow microenvironment is a complex structure that comprises accessory cells (stromal cells, adipocytes, reticulum cells, endothelial cells, follicular dendritic cells, T cells, and macrophages), matrix proteins, and soluble factors, including growth factors and cytokines. 11,12 Bone marrow stromal cells (BMSCs) represent a major component of the marrow microenvironment. They originate from the mesenchymal stem cells and have hematopoietic supportive properties. They also produce matrix proteins, express integrin ligands, and release several cytokines and growth factors that are involved in the generation and maturation of normal and leukemic B cells. [11][12][13] Therefore, BMSCs could provide a suitable milieu for the development and survival of CLL cells. However, the antiapoptotic cascades and the molecular events that are activated upon the interaction between CLL cells and BMSCs are not fully identified.Several stimuli that are endogenously produced in the microenvironment were shown to activate the antiapoptotic phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. These include cytokines and growth factors, 14 adhesion molecules and matrix proteins, 15 and involve receptors that are expressed on the surface of CLL cells, including the B-cell receptor, CD19, and CD5. [16][17][18] This strongly suggests that the PI3-K/Akt pathway might play a central role in the interaction between CLL cells and the bone marrow microenvironment. The PI3-K/Akt cascade contributes to the regulation of many cellular processes, including motility, proliferation, apoptosis, and tumorigenesis. 14,19 Class I of PI3-K family is the best characterized and comprises p110␣, p110, p110␥, and The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on May 11, 2018. by guest www.bloodjournal.org From p110␦ isotypes. 14,19 The first generation of pan-PI3-K inhibitors (LY294002 and wortmannin) provided substantial information on the molecular mechanism of action and ...
The clinical course of patients with chronic lymphocytic leukemia (CLL) is heterogeneous with some patients requiring early therapy whereas others will not be treated for years. The evaluation of an individual CLL patient's prognosis remains a problematic issue. The presence or absence of somatic mutations in the IgVH genes is currently the gold-standard prognostic factor, but this technique is labor intensive and costly. Genomic studies uncovered that 70 kDa zeta-associated protein (ZAP-70) expression was associated with unmutated IgVH genes and ZAP-70 protein expression was proposed as a surrogate for somatic mutational status. Among the available techniques for ZAP-70 detection, flow cytometry is most preferable as it allows the simultaneous quantification of ZAP-70 protein expression levels in CLL cells
Key Points• Uridine diphospho glucuronosyltransferase 2B17 (UGT2B17) is overexpressed in poor prognostic chronic lymphocytic leukemia.Uridine diphospho glucuronosyltransferase 2B17 (UGT2B17) glucuronidates androgens and xenobiotics including certain drugs. The UGT2B17 gene shows a remarkable copy number variation (CNV), which predisposes for solid tumors and influences drug response. Here, we identify a yet undescribed UGT2B17 mRNA overexpression in poor-risk chronic lymphocytic leukemia (CLL). In total, 320 CLL patients and 449 healthy donors were analyzed. High (above median) UGT2B17 expression was associated with established CLL poor prognostic factors and resulted in shorter treatment-free and overall survival (hazard ratio ([death] 2.18; 95% CI 1.18-4.01; P ؍ .013). The prognostic impact of mRNA expression was more significant than that of UGT2B17 CNV. UGT2B17 mRNA levels in primary CLL samples directly correlated with functional glucuronidation activity toward androgens and the anticancer drug vorinostat (R > 0.9, P < .001). After treatment with fludarabine containing regimens UGT2B17 was up-regulated particularly in poor responders (P ؍ .030). We observed an exclusive involvement of the 2B17 isoform within the UGT protein family. Gene expression profiling of a stable UGT2B17 knockdown in the CLL cell line MEC-1 demonstrated a significant involvement in key cellular processes. These findings establish a relevant role of UGT2B17 in CLL with functional consequences and potential therapeutic implications. (Blood. 2013;121(7):1175-1183) IntroductionChronic lymphocytic leukemia (CLL) is characterized by a considerable heterogeneity regarding clinical presentation, need for treatment, and outcome. Many prognostic markers have been identified. 1 Although most of them provide information about risk of progression and survival, the functional role of these markers is often unclear and therapeutic consequences are therefore lacking. Apart from the clinical Rai and Binet staging systems and cytogenetics, 2-4 molecular markers, such as immunoglobulin heavy chain variable (IGHV) gene mutational status 5,6 and lipoprotein lipase (LPL) mRNA expression have strong prognostic value. 7,8 In a pilot gene expression study with 20 CLL patients, we identified a significant association of uridine diphospho (UDP) glucuronosyltransferase 2B17 (UGT2B17) with these prognostic factors. 9 Metabolizing phase 2 enzymes of the UGT2B super-family conjugate various endogenous compounds, in particular steroid hormones as well as several pharmaceutical drugs. 10,11 The UGT2B genes and pseudogenes are clustered on chromosome 4q13 and display up to 95% sequence homology among each other, which is reflected in some overlap in substrate specificity but often distinct expression profile. Isoform UGT2B17 is a major androgen inactivating enzyme playing a role in local tissuespecific regulation of it is substrates. 12 Importantly, antileukemic drugs, such as anthraquinones or the histone deacetylase (HDAC) inhibitor vorinostat, are also subject ...
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