Dania Benedetti

CD38/CD31, the CCL3 and CCL4 Chemokines, and CD49d/Vascular Cell Adhesion Molecule-1 Are Interchained by Sequential Events Sustaining Chronic Lymphocytic Leukemia Cell Survival

CD38 and CD49d are associated negative prognosticators in chronic lymphocytic leukemia (CLL). Despite evidence that both molecules are involved in interactions occurring between CLL and normal cells in the context of CLL-involved tissues, a functional link is still missing. Using gene expression profiles comparing CD38(+)CD49d(+) versus CD38(-)CD49d(-) CLL cells, we showed overexpression of the CCL3 and CCL4 chemokines in cells from the former group. These chemokines were also up-regulated by CD38 signals in CLL; moreover, CCL3 was expressed by CLL cells from bone marrow biopsies (BMB) of CD38(+)CD49d(+) but not CD38(-)CD49d(-) cases. High levels of CCR1 and, to a lesser extent, CCR5, the receptors for CCL3 and CCL4, were found in CLL-derived monocyte-macrophages. Consistently, CCL3 increased monocyte migration, and CD68(+) macrophage infiltration was particularly high in BMB from CD38(+)CD49d(+) CLL. Conditioned media from CCL3-stimulated macrophages induced endothelial cells to express vascular cell adhesion molecule-1 (VCAM-1), the CD49d ligand, likely through tumor necrosis factor alpha overproduction. These effects were apparent in BMB from CD38(+)CD49d(+) CLL, where lymphoid infiltrates were characterized by a prominent meshwork of VCAM-1(+) stromal/endothelial cells. Lastly, CD49d engagement by VCAM-1 transfectants increased viability of CD38(+)CD49d(+) CLL cells. Altogether, CD38 and CD49d can be thought of as parts of a consecutive chain of events ultimately leading to improved survival of CLL cells.

The CD49d/CD29 complex is physically and functionally associated with CD38 in B-cell chronic lymphocytic leukemia cells

CD49d and CD38 are independent negative prognostic markers in chronic lymphocytic leukemia (CLL). Their associated expression marks a disease subset with a highly aggressive clinical course. Here, we demonstrate a constitutive physical association between the CD49d/CD29 integrin complex and CD38 in primary CLL cells and B-cell lines by (i) cocapping, (ii) coimmunoprecipitation and (iii) cell adhesion experiments using CD49d-specific substrates (vascular-cell adhesion molecule-1 or CS-1/H89 fibronectin fragments). The role of CD38 in CD49d-mediated cell adhesion was studied in CD49d+CD38+ and CD49d+CD38− primary CLL cells, and confirmed using CD38 transfectants of the originally CD49d+CD38− CLL-derived cell line Mec-1. Results indicate that CD49d+CD38+ cells adhered more efficiently onto CD49d-specific substrates than CD49d+CD38− cells (P<0.001). Upon adhesion, CD49d+CD38+ cells underwent distinctive changes in cell shape and morphology, with higher levels of phosphorylated Vav-1 than CD49d+CD38− cells (P=0.0006) and a more complex distribution of F-actin to the adhesion sites. Lastly, adherent CD49d+CD38+ cells were more resistant to serum-deprivation-induced (P<0.001) and spontaneous (P=0.03) apoptosis than the CD49d+CD38− counterpart. Altogether, our results point to a direct role for CD38 in enhancing CD49d-mediated adhesion processes in CLL, thus providing an explanation for the negative clinical impact exerted by these molecules when coexpressed in neoplastic cells.

CD49d in B-cell chronic lymphocytic leukemia: correlated expression with CD38 and prognostic relevance.

CD49d in B-cell chronic lymphocytic leukemia: correlated expression with CD38 and prognostic relevance

The miR-17 ∼ 92 family regulates the response to Toll-like receptor 9 triggering of CLL cells with unmutated IGHV genes

Chronic lymphocytic leukemia (CLL) cells from clinically aggressive cases have a greater capacity to respond to external microenvironmental stimuli, including those transduced through Toll-like-receptor-9 (TLR9). Concomitant microRNA and gene expression profiling in purified CLL cells (n=17) expressing either unmutated (UM) or mutated (M) IGHV genes selected microRNAs from the miR-17∼92 family as significantly upregulated and in part responsible for modifications in the gene expression profile of UM CLL cells stimulated with the TLR9 agonist CpG. Notably, the stable and sustained upregulation of miR-17∼92 microRNAs by CpG was preceded by a transient induction of the proto-oncogene MYC. The enforced expression of miR-17, a major member from this family, reduced the expression of the tumor suppressor genes E2F5, TP53INP1, TRIM8 and ZBTB4, and protected cells from serum-free-induced apoptosis (P⩽0.05). Consistently, transfection with miR-17∼92 family antagomiRs reduced Bromo-deoxy-uridine incorporation in CpG-stimulated UM CLL cells. Finally, miR-17 expression levels, evaluated in 83 CLL samples, were significantly higher in UM (P=0.03) and ZAP-70high (P=0.02) cases. Altogether, these data reveal a role for microRNAs of the miR-17∼92 family in regulating pro-survival and growth-promoting responses of CLL cells to TLR9 triggering. Overall, targeting of this pathway may represent a novel therapeutic option for management of aggressive CLL.

Expression of Mutated IGHV3-23 Genes in Chronic Lymphocytic Leukemia Identifies a Disease Subset with Peculiar Clinical and Biological Features

Purpose: B-cell chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease whose outcome can be foreseen by investigating the mutational status of immunoglobulin heavy chain variable (IGHV) genes. Moreover, a different prognosis was reported for CLL expressing specific IGHV genes in the context or not of stereotyped B-cell receptors. Here we investigated novel associations between usage of specific IGHV genes and clinical features in CLL. Experimental Design: Among 1,426 CLL-specific IG-rearrangements, stereotyped B-cell receptor clusters never utilized the IGHV3-23 gene. Given this notion, this study was aimed at characterizing the IGHV3-23 gene in CLL, and identifying the properties of IGHV3-23–expressing CLL. Results: IGHV3-23 was the second most frequently used (134 of 1,426) and usually mutated (M; 109 of 134) IGHV gene in our CLL series. In the vast majority of M IGHV3-23 sequences, the configuration of the 13 amino acids involved in superantigen recognition was consistent with superantigen binding. Clinically, M IGHV3-23 CLL had shorter time-to-treatment than other M non–IGHV3-23 CLL, and multivariate analyses selected IGHV3-23 gene usage, Rai staging, and chromosomal abnormalities as independent prognosticators for M CLL. Compared with M non–IGHV3-23 CLL, the gene expression profile of M IGHV3-23 CLL was deprived in genes, including the growth/tumor suppressor genes PDCD4, TIA1, and RASSF5, whose downregulation is under control of miR-15a and miR-16-1. Accordingly, relatively higher levels of miR-15a and miR-16-1 were found in M IGHV3-23 compared with M non–IGHV3-23 CLL. Conclusions: Altogether, expression of the IGHV3-23 gene characterizes a CLL subset with distinct clinical and biological features. Clin Cancer Res; 16(2); 620–8

NOTCH1 mutations associate with low CD20 level in chronic lymphocytic leukemia: evidence for a NOTCH1 mutation-driven epigenetic dysregulation

In chronic lymphocytic leukemia (CLL), NOTCH1 mutations have been associated with clinical resistance to the anti-CD20 rituximab, although the mechanisms behind this peculiar behavior remain to be clarified. In a wide CLL series (n=692), we demonstrated that CLL cells from NOTCH1-mutated cases (87/692) were characterized by lower CD20 expression and lower relative lysis induced by anti-CD20 exposure in vitro. Consistently, CD20 expression by CLL cells was upregulated in vitro by γ-secretase inhibitors or NOTCH1-specific small interfering RNA and the stable transfection of a mutated (c.7541-7542delCT) NOTCH1 intracellular domain (NICD-mut) into CLL-like cells resulted in a strong downregulation of both CD20 protein and transcript. By using these NICD-mut transfectants, we investigated protein interactions of RBPJ, a transcription factor acting either as activator or repressor of NOTCH1 pathway when respectively bound to NICD or histone deacetylases (HDACs). Compared with controls, NICD-mut transfectants had RBPJ preferentially complexed to NICD and showed higher levels of HDACs interacting with the promoter of the CD20 gene. Finally, treatment with the HDAC inhibitor valproic acid upregulated CD20 in both NICD-mut transfectants and primary CLL cells. In conclusion, NOTCH1 mutations are associated with low CD20 levels in CLL and are responsible for a dysregulation of HDAC-mediated epigenetic repression of CD20 expression.

Microenvironmental Interactions in Chronic Lymphocytic Leukemia: The Master Role of CD49d

Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease characterized by the accumulation/expansion of a clonal population of neoplastic cells with the morphologic appearance of small mature B lymphocytes in blood, bone marrow, and lymphoid organs. A combination of genetic lesions is primarily responsible for the first step(s) of neoplastic transformation, along with microenvironmental signals, which concurrently operate by enhancing proliferation and/or inhibiting apoptosis. In this context, CD49d is known to play a pivotal role in mediating both cell-cell and cell-matrix interactions in CLL-involved tissues, eventually delivering pro-survival signals and protecting CLL cells from drug-induced damages. In the present review, we address, in detail, CD49d activities in the CLL microenvironment, CD49d functional and physical interactions with other microenvironmental receptors (including CD38 and B-cell receptor), and the relationship of CD49d expression with specific cytogenetic features in CLL.

Monocytes/macrophages but not T lymphocytes are the major targets of the CCL3/CCL4 chemokines produced by CD38(+)CD49d(+) chronic lymphocytic leukaemia cells.

p110delta function in B cells. Blood, 107, 642–650. Draghetti, C., Salvat, C., Zanoguera, F., Curchod, M.L., Vignaud, C., Peixoto, H., Di Cara, A., Fischer, D., Dhanabal, M., Andreas, G., Abderrahim, H., Rommel, C. & Camps, M. (2009) Functional whole-genome analysis identifies polo-like kinase 2 and poliovirus receptor as essential for neuronal differentiation upstream of the negative regulator alphaB-crystallin. The Journal of Biological Chemistry, 284, 32053–32065. Engelman, J.A., Luo, J. & Cantley, L.C. (2006) The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nature Reviews. Genetics, 7, 606–619. Hamblin, A.D. & Hamblin, T.J. (2008) The immunodeficiency of chronic lymphocytic leukaemia. British Medical Bulletin, 87, 49–62. Jackson, S.P., Schoenwaelder, S.M., Goncalves, I., Nesbitt, W.S., Yap, C.L., Wright, C.E., Kenche, V., Anderson, K.E., Dopheide, S.M., Yuan, Y., Sturgeon, S.A., Prabaharan, H., Thompson, P.E., Smith, G.D., Shepherd, P.R., Daniele, N., Kulkarni, S., Abbott, B., Saylik, D., Jones, C., Lu, L., Giuliano, S., Hughan, S.C., Angus, J.A., Robertson, A.D. & Salem, H.H. (2005) PI 3-kinase p110beta: a new target for antithrombotic therapy. Nature Medicine, 11, 507–514. Manning, B.D. & Cantley, L.C. (2007) AKT/PKB signaling: navigating downstream. Cell, 129, 1261–1274. Marone, R., Cmiljanovic, V., Giese, B. & Wymann, M.P. (2008) Targeting phosphoinositide 3-kinase: moving towards therapy. Biochimica Et Biophysica Acta, 1784, 159–185. Niedermeier, M., Hennessy, B.T., Knight, Z.A., Henneberg, M., Hu, J., Kurtova, A.V., Wierda, W.G., Keating, M.J., Shokat, K.M. & Burger, J.A. (2009) Isoform-selective phosphoinositide 3¢-kinase inhibitors inhibit CXCR4 signaling and overcome stromal cell-mediated drug resistance in chronic lymphocytic leukemia: a novel therapeutic approach. Blood, 113, 5549–5557. Pleyer, L., Egle, A., Hartmann, T.N. & Greil, R. (2009) Molecular and cellular mechanisms of CLL: novel therapeutic approaches. Nature Reviews. Clinical Oncology, 6, 405–418.

CD49d prevails over the novel recurrent mutations as independent prognosticator of overall survival in chronic lymphocytic leukemia

CD49d, the alpha-chain of the integrin heterodimer α4β1, was identified among the strongest predictors of overall survival (OS) in chronic lymphocytic leukemia (CLL), along with IGHV mutational status and deletion of the 17p chromosome involving TP53. In addition to TP53, the clinical relevance of NOTCH1, SF3B1 and BIRC3 gene mutations has been recently emphasized. By analyzing a cohort of 778 unselected CLL patients, we assessed the clinical relevance of CD49d as an OS predictor in subgroups defined by mutation/deletion of the TP53, NOTCH1, SF3B1 and BIRC3 genes. In this context, CD49d emerged as an independent predictor of OS in multivariate Cox analysis (Hazard ratio =1.88, P<0.0001). Consistently, high CD49d expression identified CLL subsets with inferior OS in the context of each category of a previously reported hierarchical risk stratification model. Moreover, by evaluating the relative importance of biological prognosticators by random survival forests, CD49d was selected among the top-ranked OS predictor (variable importance =0.0410), along with IGHV mutational status and TP53 abnormalities. These results confirmed CD49d as an independent negative OS prognosticator in CLL also in comprehensive models comprising the novel recurrent mutations. In this context, TP53 disruption and NOTCH1 mutations retained prognostic relevance, in keeping with their roles in CLL cell immuno-chemoresistance.

Prognostic impact of ZAP-70 expression in chronic lymphocytic leukemia: mean fluorescence intensity T/B ratio versus percentage of positive cells

BackgroundZAP-70 is an independent negative prognostic marker in chronic lymphocytic leukemia (CLL). Usually, its expression is investigated by flow cytometric protocols in which the percentage of ZAP-70 positive CLL cells is determined in respect to isotypic control (ISO-method) or residual ZAP-70 positive T cells (T-method). These methods, however, beside suffering of an inherent subjectivity in their application, may give discordant results in some cases. The aim of this study was to assess the prognostic significance of these methods in comparison with another in which ZAP-70 expression was evaluated as a Mean-Fluorescence-Intensity Ratio between gated T and CLL cells (T/B Ratio-method).MethodsCytometric files relative to ZAP-70 determination according to the three readouts were retrospectively reviewed on a cohort of 173 patients (test set), all with complete clinical and biological prognostic assessment and time-to-treatment (TTT) available. Findings were then validated in an independent cohort of 341 cases from a different institution (validation set).ResultsThe optimal prognostic cut-offs for ZAP-70 expression were selected at 11% (ISO-method) or 20% of positive cells (T-method), as well as at 3.0 (T/B Ratio-method) in the test set; these cut-offs yielded 66, 60 and 73 ZAP-70+ cases, respectively. Univariate analyses resulted in a better separation of ZAP-70+ vs. ZAP-70- CLL patients utilizing the T/B Ratio, compared to T- or ISO-methods. In multivariate analyses which included the major clinical and biological prognostic markers for CLL, the prognostic impact of ZAP-70 appeared stronger when the T/B-Ratio method was applied. These findings were confirmed in the validation set, in which ZAP-70 expression, evaluated by the T- (cut-off = 20%) or T/B Ratio- (cut-off = 3.0) methods, yielded 180 or 127 ZAP-70+ cases, respectively. ZAP-70+ patients according to the T/B Ratio-method had shorter TTT, both if compared to ZAP-70- CLL, and to cases classified ZAP-70+ by the T-method only.ConclusionsWe suggest to evaluate ZAP-70 expression in routine settings using the T/B Ratio-method, given the operator and laboratory independent feature of this approach. We propose the 3.0 T/B Ratio value as optimal cut-off to discriminate ZAP-70+ (T/B Ratio less than 3.0) from ZAP-70- (T/B Ratio more/equal than 3.0) cases.