Survival and Immunosuppression Induced by Hepatocyte Growth Factor in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL), the most common leukemia among adults in the western world, is characterized by a progressive accumulation of relatively mature CD5+ B cells in peripheral blood, lymph nodes and bone marrow. Despite much recent advancement in therapy, CLL is still incurable. Lymph nodes and bone marrow represent sanctuary sites preserving leukemic cells from spontaneous or drug-induced apoptosis, and infiltration of leukemic cells in these districts correlates with clinical stages and prognosis. The central role played by the microenvironment in the disease has become increasingly clear. Different chemokines (CXCL12, CXCL13, CCL19, CCL21) may in fact participate in attracting CLL cells into bone marrow and lymph nodes, where various factors, such as IL-15 and CXCL12, enhance leukemic cells survival. Recently, we have suggested that hepatocyte growth factor (HGF), produced by microenvironmental stromal cells, can contribute to CLL pathogenesis. We have demonstrated that HGF exerts a double effect on CLL B cells through the interaction with its receptor c- MET; HGF, infact, protects CLL B cells, which are c-MET+, from apoptosis, and also polarizes mono/macrophages towards the M2 phenotype, thus facilitating the evasion of the CLL clone from immune control. This double effect appears mediated by the activation of two major signaling pathways: STAT3TYR705 and AKT. The aim of this review is to summarize data on HGF and c-MET expression in normal B cells and in B cell malignancies, with a particular emphasis on our results obtained in CLL. Altogether, the observations described here suggest that the HGF/c-MET axis may have a prominent role in malignancy progression further indicating novel potential therapeutic options aimed to block HGF-induced signaling pathways in B lymphoproliferative disorders.

Effects of miRNA-15 and miRNA-16 expression replacement in chronic lymphocytic leukemia: implication for therapy.

Chronic lymphocytic leukemia (CLL) clones are characterized by loss of a critical region in 13q14.3, (del(13)(q14)) involving the microRNA (miRNA) cluster miR-15a and miR-16-1. We have investigated the effects of replacement of miR-15a and miR-16-1. CLL cells transfected with these miRNA mimics exhibited a decrease in cell viability in vitro and impaired capacity for engraftment and growth in NOD/Shi-scid,γcnull (NSG) mice. No synergistic effects were observed when the two miRNA mimics were combined. The phenomena were not restricted to CLL with the del(13)(q14) lesion. Similar effects induced by miRNA mimics were seen in cells with additional chromosomal abnormalities with the exception of certain CLL clones harboring TP53 alterations. Administration of miRNA mimics to NSG mice previously engrafted with CLL clones resulted in substantial tumor regression. CLL cell transfection with miR-15a and miR-16-1-specific inhibitors resulted in increased cell viability in vitro and in an enhanced capacity of the engrafted cells to grow in NSG mice generating larger splenic nodules. These data demonstrate that the strong control by miR-15a and miR-16-1 on CLL clonal expansion is exerted also at the level of full-blown leukemia and provide indications for a miRNA-based therapeutic strategy.

lncRNA profiling in early-stage chronic lymphocytic leukemia identifies transcriptional fingerprints with relevance in clinical outcome.

Long non-coding RNAs (lncRNAs) represent a novel class of functional RNA molecules with an important emerging role in cancer. To elucidate their potential pathogenetic role in chronic lymphocytic leukemia (CLL), a biologically and clinically heterogeneous neoplasia, we investigated lncRNAs expression in a prospective series of 217 early-stage Binet A CLL patients and 26 different subpopulations of normal B-cells, through a custom annotation pipeline of microarray data. Our study identified a 24-lncRNA-signature specifically deregulated in CLL compared with the normal B-cell counterpart. Importantly, this classifier was validated on an independent data set of CLL samples. Belonging to the lncRNA signature characterizing distinct molecular CLL subgroups, we identified lncRNAs recurrently associated with adverse prognostic markers, such as unmutated IGHV status, CD38 expression, 11q and 17p deletions, and NOTCH1 mutations. In addition, correlation analyses predicted a putative lncRNAs interplay with genes and miRNAs expression. Finally, we generated a 2-lncRNA independent risk model, based on lnc-IRF2-3 and lnc-KIAA1755-4 expression, able to distinguish three different prognostic groups in our series of early-stage patients. Overall, our study provides an important resource for future studies on the functions of lncRNAs in CLL, and contributes to the discovery of novel molecular markers with clinical relevance associated with the disease.

Prognostic factors in CLL.

Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease, as some patients progress rapidly toward the more advanced studies, whereas others survive for a long period without the need for treatment. This heterogeneity of clinical course was somehow unexplained until studies on the CLL cell features disclosed that the CLL clones were heterogeneous and were characterized by different phenotypic and genotypic features in the different patients. On the basis of these observations, it was determined in retrospective studies that clones characterized by unmutated IGHV genes, and/or CD38 and/or ZAP-70 expression conferred a more severe prognosis to the CLL patients. Here, we present data on prospective studies carried out on Binet A-stage patients, in whom the markers were determined at diagnosis and their predictive value was assessed in comparison with chromosomal abnormalities and gene expression or micro RNA profiles. In addition, hypothesis on the potential pathogenetic role of these markers will be presented.

PNAEmu can significantly reduce Burkitt's lymphoma tumor burden in a SCID mice model: cells dissemination similar to the human disease.

In human Burkitt's Lymphoma (BL) BRG cells, a t(8;14) translocation, placing c-myc near the Emu enhancer of the H chain locus, causes tumor expansion. Earlier, we showed that a peptide nucleic acid complementary to the Emu sequence (PNAEmu), specifically inhibited the expression of translocated c-myc and impaired the growth of BRG cells-induced subcutaneous tumors in mice suffering from severe combined immunodeficiency (SCID). In this study, the therapeutic potential of PNAEmu was evaluated in a systemic mouse model. BRG-BL cells transfected with the luciferase gene were inoculated intravenously into SCID mice resulting in a preferential expansion, similar to the one of human adult patients, in the abdominal cavity, central nervous system and bone marrow. The mice were chronically injected intraperitoneally either with PNAEmu or with control PNA. The treatment was stopped when the control animals developed severe neurological symptoms. As detected both by inspection at necropsy and imaging, overall tumor growth in PNAEmu-treated mice decreased by >80%. Histological and immunohistochemical studies showed, only in PNAEmu-treated mice, a substantially reduced BL cell growth at the major sites of invasion and vast areas of necrosis in the lymphomatous tissues, with concomitant c-myc expression downregulation. Altogether, the data support the therapeutic potential of PNAEmu in human adult BL.

Lack of mutagenicity and clastogenicity of PNAEmu-NLS targeted to a regulatory sequence of the translocated c-myc oncogene in Burkitt's lymphoma.

Peptide nucleic acids (PNAs) are synthetic homolog of nucleic acids in which the phosphate-sugar polynucleotide backbone is replaced by a flexible polyamide. They bind complementary polynucleotide sequences with higher affinity and specificity than their natural counterparts. PNAs linked to the appropriate nuclear localization signal (NLS) peptide have been used to selectively down-regulate the expression of several genes in viable cells. For example in Burkitt's lymphoma (BL) cells the c-myc oncogene is translocated in proximity to the Emu enhancer of the Ig gene locus and upregulated. PNAs complementary to the second exon of c-myc or to the Emu enhancer sequence (PNAEmu-NLS), selectively and specifically block the expression of the c-myc oncogene and inhibit cell growth in vitro and in vivo. PNAEmu-NLS administration to mice did not exhibit toxic effects even at the highest concentration allowed by the experimental conditions. Because of the accumulating data confirming PNAEmu-NLS potential therapeutic value, PNAEmu-NLS was evaluated for the inability to induce mutations in tester strains of Salmonella typhimurium, Escherichia coli, and at the hprt locus in Chinese hamster ovary cells (CHO). Moreover, the induction of chromosomal aberrations in CHO cells and of micronuclei in human lymphocytes were investigated. We may conclude that PNAEmu-NLS neither induces mutations nor has clastogenic effects as detectable by treatment under the standard test conditions.

Inhibition of Burkitt's lymphoma cells growth in SCID mice by a PNA specific for a regulatory sequence of the translocated c-myc.

In Burkitt's lymphoma (BL) cells due to a t(8;14) chromosomal translocation c-myc is often placed in proximity to the Emu enhancer of the Ig locus and upregulated. We demonstrated that in BL cells a peptide nucleic acid (PNA), complementary to intronic Emu sequences (PNAEmuwt), specifically blocks the expression of the c-myc oncogene under the Emu enhancer control and inhibits BL cell growth in culture. Here, we investigated whether PNAEmuwt was also able to block tumor growth in SCID mice inoculated with human BL cell lines. After subcutaneous inoculum in mice BL cells reproducibly form tumors. Both pre-treatment of BL cells with PNAEmuwt before inoculum and chronic intravenous administration of PNAEmuwt to mice already inoculated with BL cells selectively caused increased latency of tumor appearance and decreased final tumor size. Tumors from PNAEmuwt-treated animals showed substantial areas of cell necrosis and of c-myc downregulation. Inhibition of tumor growth was specific and was not observed with PNAEmumut carrying sequence mutations and in BL cell lines where the translocated c-myc is not under the control of the Emu enhancer. These data confirm the potential therapeutic value of PNA targeted to regulatory non-coding regions.

CD10 is a marker for cycling cells with propensity to apoptosis in childhood ALL.

CD10 constitutes a favourable prognostic marker for childhood acute lymphoblastic leukaemia. Since correlations between CD10, cell cycle and apoptotic abilities were demonstrated in various cell types, we investigated whether differences existed in the cycling/apoptotic abilities of CD10-positive and CD10-negative B acute lymphoblastic leukaemia cells. Twenty-eight cases of childhood acute lymphoblastic leukaemia (mean age of 6.8 years) were subdivided into two groups according to high (17 cases, 93.2+/-4.5%, MRFI 211+/-82 CD10-positive cells) or low (11 cases, 11.5+/-6.2%, MRFI 10+/-7 CD10-negative cells) expression of CD10. CD10-positive acute lymphoblastic leukaemia cells were cycling cells with elevated c-myc levels and propensity to apoptosis, whereas CD10-negative acute lymphoblastic leukaemia cells had lower cycling capacities and c-myc levels, and were resistant to apoptosis in vitro. A close correlation between all these properties was demonstrated by the observations that the few CD10-positive cells found in the CD10-negative acute lymphoblastic leukaemia group displayed elevated c-myc and cycling capacities and were apoptosis prone. Moreover, exposure of CD10-positive acute lymphoblastic leukaemia B cells to a peptide nucleic acid anti-gene specific for the second exon of c-myc caused inhibition of c-myc expression and reduced cell cycling and apoptotic abilities as well as decreased CD10 expression.

Apoptotic cells overexpress vinculin and induce vinculin-specific cytotoxic T-cell cross-priming.

Here we show that apoptotic cells overexpress vinculin and are ingested by dendritic cells, which subsequently cross-prime vinculin-specific cytotoxic T lymphocytes (CTLs). Successful cross-priming requires that the apoptotic cells provide maturation signals to dendritic cells through CD40-CD40 ligand (CD40L) interactions. If apoptotic cells are CD40L-, the help of a third T cell is needed for priming, indicating a regulatory role for apoptotic cells in determining priming or tolerance. Vinculin-specific CTL priming is also related to apoptosis in vivo, given that in HIV-seropositive individuals, the frequency of specific CTLs depends on the proportion of peripheral CD40L+ apoptotic cells.

Analysis of stepwise genetic changes in an AIDS-related Burkitt's lymphoma.

In this study, immunoglobulin variable (Ig V) region genes, c-myc re-arrangement and sequence and p53 status were analyzed in clones derived from a Burkitt's lymphoma cell line (LAM) in which it was previously demonstrated that Epstein-Barr virus (EBV) infection occurred late during lymphomagenesis. Such evidence was based on the finding that 2 groups of cellular clones, characterized by the same c-myc re-arrangement but different EBV-fused termini, were obtained from the LAM cell line. The Ig V gene sequences were identical for the 2 groups of clones with different EBV-fused termini. The Ig variable heavy (V(H)) gene sequence displayed a substantial accumulation of point mutations (but no intra-clonal diversification), whereas the productive Ig V lambda (V(lambda)) gene sequence was virtually unmutated. Studies on the Ig V kappa (V(kappa)) locus suggested a receptor revision event (with a switch from kappa to lambda chain production) prior to EBV infection. Likewise, it was determined that the mutations observed in both p53 alleles and in the re-arranged c-myc gene occurred before EBV infection. Based on these findings, we present a model for the various steps of lymphomagenesis. It is proposed that stimulation by an antigen or a superantigen initially favored the clonal expansion and accumulation of other cytogenetic changes, including those involved in receptor editing. These events occurred prior to or during the germinal center (GC) phase of B-cell maturation. Thereafter, possibly upon exit of the cells from the GC, EBV infection occurred, further promoting lymphomagenesis.

Retinoic acid induces persistent, RARalpha-mediated anti-proliferative responses in Epstein-Barr virus-immortalized b lymphoblasts carrying an activated C-MYC oncogene but not in Burkitt's lymphoma cell lines.

We have previously demonstrated that 13-cis-retinoic acid (RA), 9-cis-RA and all-trans-RA (ATRA) powerfully inhibit the proliferation of Epstein-Barr virus-immortalized B-lymphoblastoid cell lines (LCLs). The aim of the present study was to assess whether these compounds are effective at inhibiting the growth of B cells at more advanced stages of lymphomagenesis, including fully transformed B lymphocytes. To this end, c-myc-transfected LCLs (myc-LCLs) and Burkitt's lymphoma (BL) cell lines were used. We report that 13-cis-RA, 9-cis-RA and ATRA also markedly inhibit the proliferation of myc-LCLs by inducing G(0)/G(1) growth arrest as well as enhancing rates of apoptosis. Conversely, all but 1 (DG75) of the 8 BL cell lines investigated were poorly RA-responsive. Moreover, unlike LCLs and myc-LCLs, RA-treated DG75 cells rapidly resumed proliferation upon drug removal. Analysis of cell cycle-regulatory proteins showed that, as in LCLs, strong up-regulation of p27(Kip-1) and increased levels of under-phosphorylated pRb and p130 were detected in RA-treated DG75 cells. While the catalytic activity of all 3 G(1)-associated CDKs (CDK2, CDK4 and CDK6) was strongly inhibited in RA-treated LCLs, only CDK2-associated kinase activity was reduced in DG75 cells arrested in G(0)/G(1) by RA. Moreover, RA-treated DG75 cells failed to show the down-regulation of cyclin D3 observed in LCLs. Use of receptor-selective agonists and antagonists showed that in LCLs and RA-responsive BL cells, RA-induced growth arrest is mainly mediated by RARalpha. The RARalpha-selective agonist Ro 40-6055 was also effective at very low concentrations (10(-10) M). Nevertheless, comparable levels of RARalpha mRNA were found in RA-responsive and -resistant BL cell lines, indicating that mechanisms different from transcriptional deregulation of RARalpha probably underlie the differential responsiveness of BL cells.

Effects in live cells of a c-myc anti-gene PNA linked to a nuclear localization signal.

Peptide nucleic acids (PNA) are synthetic homologs of nucleic acids in which the phosphate-sugar polynucleotide backbone is replaced by a flexible polyamide. In this study, a PNA construct was employed as an anti-gene agent in intact cells in culture. The cell lines studied were derived from Burkitt's lymphomas (BL) that presented a translocated and hyperexpressed c-myc oncogene. A 17-mer anti-myc PNA, complementary to a unique sequence located at the beginning of the second exon of the oncogene, and was covalently linked at its N terminus to a nuclear localization signal (NLS) (PNA-myc(wt)-NLS). When BL cells were exposed to PNA-myc(wt)-NLS, the anti-gene construct was localized predominantly in the cell nuclei and a rapid consequent downregulation of c-myc expression occurred. Under these conditions, both completion of a productive cell cycle and apoptosis were inhibited.

Late Epstein-Barr virus infection of a hepatosplenic gamma delta T-cell lymphoma arising in a kidney transplant recipient.

BACKGROUND AND OBJECTIVE: gd T-cell lymphomas are only exceptionally observed in transplanted patients. Aim of this study was the detailed characterization of one such case. DESIGN AND METHODS: The patient developed spontaneous splenic rupture six years after kidney transplantation. The splenic red pulp was infiltrated by medium-sized and large lymphoid cells with two or more nucleoli. At autopsy, similar lymphoid cells infiltrated the hepatic sinusoids. Histologic, immunologic and molecular studies were carried out. RESULTS: By immunohistochemistry, the atypical lymphoid cells were found to express CD3, CD45 and CD43, indicating their T-lineage origin. Approximately 99% of spleen mononuclear cells (MNC) were CD3(+), gammadelta TcR+, CD4-, CD8-, alphabeta TcR-. A clonal gammadelta TcR rearrangement (Vgamma1-Jgamma1.3/2.3-Cgamma2; Vdelta1-Ddelta2-Jdelta1) was detected. The final diagnosis was peripheral T-cell lymphoma, hepato-splenic gammadelta-type. EBV infection of spleen MNC was documented by molecular studies. However, in situ hybridization for EBER-1 (EBV-RNA) showed that only a minority of malignant lymphoid cells (5-7%) were EBV-infected. INTERPRETATION AND CONCLUSIONS: It is concluded that EBV infection was as a late event involving an already transformed gd T-cell clone.

Expression of CD10 by human T cells that undergo apoptosis both in vitro and in vivo.

This study shows that human postthymic T cells express CD10 when undergoing apoptosis, irrespective of the signal responsible for initiating the apoptotic process. Cells from continuous T-cell lines did not normally express CD10, but became CD10(+) when induced into apoptosis by human immunodeficiency virus (HIV) infection and exposure to CD95 monoclonal antibody, etoposide, or staurosporin. Inhibitors of caspases blocked apoptosis and CD10 expression. Both CD4(+) and CD8(+) T cells purified from normal peripheral blood expressed CD10 on apoptotic induction. CD10 was newly synthesized by the apoptosing cells because its expression was inhibited by exposure to cycloheximide and CD10 mRNA became detectable by reverse transcription-polymerase chain reaction in T cells cultured under conditions favoring apoptosis. To show CD10 on T cells apoptosing in vivo, lymph node and peripheral blood T cells from HIV(+) subjects were used. These suspensions were composed of a substantial, although variable, proportion of apoptosing T cells that consistently expressed CD10. In contrast, CD10(+) as well as spontaneously apoptosing T cells were virtually absent in peripheral blood from normal individuals. Collectively, these observations indicate that CD10 may represent a reliable marker for identifying and isolating apoptosing T cells in vitro and ex vivo and possibly suggest novel functions for surface CD10 in the apoptotic process of lymphoid cells.

Identification of HSP-60 as the specific antigen of IgM produced by BRG-lymphoma cells.

In previous studies we described a patient with Burkitt's lymphoma and AIDS, whose cells recognized a molecule expressed by normal and malignant breast cells. In the present study, we identified this antigen by two-dimensional (2-D) electrophoresis and Western blotting using the antibody produced by lymphoma cells. The antigen so identified consisted of two clusters of spots with a molecular mass (Mr) of 60 and 50 kDa, respectively. Preparative immobilized pH gradient (IPG) was subsequently used to isolate the clusters of spots of higher molecular masses, from which peptide fragments of approximately 10 aa were separated on reverse-phase chromatography and sequenced. This procedure enabled the identification of the antigen recognized by the lymphoma cells as HSP-60. By means of serological analyses it was possible to identify the lower molecular mass cluster of spots as a molecule related to HSP-60. It is hypothesized that this molecule is a membrane form of HSP-60 that differs from HSP-60 in a COOH terminal portion.

Apoptosis induced by crosslinking of CD4 on activated human B cells.

Using immunofluorescence, RT-PCR, and Western blotting, we have demonstrated the ability of human B cells to express CD4. In each of the 10 lymphoblastoid cell lines (LCL) tested there was variable, but definite, proportion of CD4-positive B cells. Expression of CD4 was related to the cell cycle; CD4 was expressed in the G1 phase and continued at later phases of the cell cycle. CD4 was in part internalized and degraded by the LCL B cells. Surface CD4 was associated to lck and its crosslinking resulted in tyrosine phosphorylation. Additional experiments conducted on freshly prepared tonsillar B cells demonstrated that CD4 was expressed by large activated B cells, but not by small resting B cells. However, not all the activated tonsillar B cells had surface CD4 since germinal center cells were CD4-negative. Crosslinking of CD4 on LCL or on tonsillar activated B cells resulted in apoptosis in vitro, a finding that indicates the capacity of CD4 to deliver functional signals to B cells and to play a regulatory function in their physiology. Exposure of CD4 expressing B cells to gp120 under conditions that resulted in CD4 crosslinking also caused apoptosis suggesting some implications for the pathophysiology of AIDS.

Heterogeneous p53 mutations in a Burkitt lymphoma from an AIDS patient with monoclonal c-myc and VDJ rearrangements.

This study investigates the timing of p53 mutations detected in the malignant cells of a Burkitt's lymphoma cell line (BRG-P) with respect to other maturation or transforming events. The BRG-P cell line, derived from an AIDS patient, was of special value since it displayed subclones that had undergone an isotype switch from IgM to IgA1 (BRG-M and BRG-A cells). BRG-M and BRG-A cells were characterized by the same monoclonal c-myc and VDJ rearrangements and by the expression of Ig receptors with specificity for a 45 kDa protein of human breast cells. Analysis of p53 mutations in the different BRG subclones showed that 1) BRG-M cells displayed 2 different p53 mutations in trans; since the original BL cells also showed the same mutations, this finding indicated that both occurred in vivo; 2) one of the p53 alleles of BRG-A cells was lost, while the other showed a mutation different from those seen in BRG-M cells; and 3) all 3 mutations observed in BRG-M or BRG-A cells resulted in the functional inactivation of the transcriptional activation function of p53. Together, our data demonstrate that p53 mutations were relatively late events during lymphomagenesis. Moreover, in view of the role of p53 in cell apoptosis, it is conceivable that BRG cells were subjected to a strong selective pressure that favored p53 inactivation. Such inactivation was possibly required to counterbalance other potentially apoptotic events, including the presence of a deregulated c-myc oncogene and signals delivered by the host environment in situ.