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International audience; Most cancers become more dangerous by the outgrowth of malignant subclones with additional DNA mutations that favor proliferation or survival. Using chronic lymphocytic leukemia (CLL), a disease exemplary of this process, and a model for neoplasms in general, we created transgenic mice overexpressing the enzyme, activation-induced deaminase (AID), whose normal function is to induce DNA mutations in B lymphocytes. AID allows normal B lymphocytes to develop more effective immunoglobulin (Ig)-mediated immunity, but also is able to mutate non-Ig genes, predisposing to cancer. In chronic lymphocytic leukemia (CLL), AID expression correlates with poor prognosis suggesting a role for this enzyme in disease progression. Nevertheless, direct experimental evidence identifying the specific genes that are mutated by AID and indicating that those genes are associated with disease progression is not available. To address this point, we overexpressed Aicda in a murine model of CLL (Em-TCL1). Analyses of TCL1/AID mice demonstrate a role for AID in disease kinetics, CLL-cell proliferation, and the development of cancer-related target mutations with canonical AID signatures in non-Igs genes. Notably, our mouse models can accumulate mutations in the same genes that are mutated in human cancers. Moreover, some of these mutations occur at homologous positions, leading to identical or chemically-similar amino acid substitutions as in human CLL and lymphoma.Together, these findings support a direct link between aberrant AID activity and CLL driver mutations that are then selected for their oncogenic effects, whereby AID promotes aggressiveness in CLL and other B-cell neoplasms.

Progression of chronic lymphocytic leukemia (CLL) results from the expansion of a small fraction of proliferating leukemic B cells. When comparing the global gene expression of recently divided CLL cells with that of previously divided cells, we found higher levels of genes involved in regulating gene expression. One of these was the oncogene Musashi 2 (MSI2), an RNA-binding protein that induces or represses translation. While there is an established role for MSI2 in normal and malignant stem cells, much less is known about its expression and role in CLL. Here we report for the first time ex vivo and in vitro experiments that MSI2 protein levels are higher in dividing and recently divided leukemic cells and that downregulating MSI2 expression or blocking its function eliminates primary human and murine CLL and mature myeloid cells. Notably, mature T cells and hematopoietic stem and progenitor cells are not affected. We also confirm that higher MSI2 levels correlate with poor outcome markers, shorter time-to-first-treatment, and overall survival. Thus, our data highlight an important role for MSI2 in CLL-cell survival and proliferation and associate MSI2 with poor prognosis in CLL patients. Collectively, these findings pinpoint MSI2 as a potentially valuable therapeutic target in CLL.

Several studies in chronic lymphocytic leukemia (CLL) patients have reported impaired immune cell functions, which contribute to tumor evasion and disease progression. However, studies on CLL-like monoclonal B-cell lymphocytosis (MBL) are scarce. In the study described here, we characterized the immune environment in 62 individuals with clinical MBL, 56 patients with early-stage CLL, and 31 healthy controls. Gene expression arrays and quantitative reverse transcription polymerase chain reaction were performed on RNA from CD4+ peripheral blood cells; serum cytokines were measured with immunoassays; and HLA-DR expression on circulating monocytes, as well as the percentages of Th1, cytotoxic, exhausted, and effector CD4+ T cells, were evaluated by flow cytometry. In addition, cell cultures of clonal B cells and CD14-enriched or -depleted cell fractions were performed. Strikingly, MBL and early-stage CLL differed in pro-inflammatory signatures. An increased inflammatory drive orchestrated mainly by monocytes was identified in MBL, which exhibited enhanced phagocytosis, pattern recognition receptors, interleukin-8 (IL8), HMGB1, and acute response signaling pathways and increased pro-inflammatory cytokines (in particular IL8, interferon γ [IFNγ], and tumor necrosis factor α). This inflammatory signature was diminished in early-stage CLL (reduced IL8 and IFNγ levels, IL8 signaling pathway, and monocytic HLA-DR expression compared with MBL), especially in those patients with mutations in IGHV genes. Additionally, CD4+ T cells of MBL and early-stage CLL exhibited a similar upregulation of Th1 and cytotoxic genes and expanded CXCR3+ and perforin+ CD4+ T cells, as well as PD1+ CD4+ T cells, compared with controls. Cell culture assays disclosed tumor-supporting effects of monocytes similarly observed in MBL and early-stage CLL. These novel findings reveal differences in the inflammatory environment between MBL and CLL, highlighting an active role for antigen stimulation in the very early stages of the disease, potentially related to malignant B-cell transformation.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of clonal Bcellsin peripheral blood and lymphoid tissues 1. Circulating CLL cells are non-dividing Blymphocytes, but a significant fraction of the clone proliferates in lymphoid tissues wherethey receive a plethora of signals from the microenvironment that promote their survivaland expansion 2. Cathelicidins are a family of proteins with antibacterial functions mainlyexpressed by neutrophils, macrophages and epithelial cells 3. In humans, the only memberof this family, hCAP18, is encoded by the gene CAMP. The cleavage of hCAP18 generatesthe antimicrobial peptide LL-37, which has been recently implicated in the promotion oftumor growth, through direct stimulation of malignant cells, initiation of angiogenesis andrecruitment of immune cells 4. In this study, we investigated the role of hCAP18/LL-37 inCLL. Fil: Podaza, Enrique Arturo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Palacios, Florencia. The Feinstein Institute for Medical Research. Karches Center for Oncology Research; Estados Unidos Fil: Croci Russo, Diego Omar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Risnik, Denise Mariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Yan, Xiao J.. The Feinstein Institute for Medical Research. Karches Center for Oncology Research; Estados Unidos Fil: Almejún, María Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Colado, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Elías, Esteban Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Borge, Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Morande, Pablo Elías. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Bezares, Raimundo F.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Dr. Teodoro Álvarez"; Argentina Fil: Fernández Grecco, Horacio. Sanatorio Municipal Dr. Julio Méndez. Servicio de Hematología; Argentina Fil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Gamberale, Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Chiorazzi, Nicholas. The Feinstein Institute for Medical Research. Karches Center for Oncology Research; Estados Unidos Fil: Giordano, Mirta Nilda. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of clonal B cells arrested in G0/G1 stages that coexist with proliferative B cells. We identified one of these proliferative subsets in the peripheral blood from patients with unmutated disease (UM). Aiming to characterize the molecular mechanism underlying this proliferative behavior, we performed gene expression analysis of the mRNA and microRNAs in this leukemic subpopulation and compared results with those for the quiescent counterpart. Our results suggest that proliferation of this subset mainly depends on microRNA-22 overexpression, which induces phosphatase and tensin homolog (PTEN) down-regulation and phosphoinositide 3-kinase (PI3K)/AKT pathway activation. These results underline the role of the PI3K/AKT pathway at the origin of this proliferative pool in patients with UM CLL and provide additional rationale for the use of PI3K inhibitors.

The enzyme Activation-induced cytidine deaminase (AID) initiates somatic hypermutation (SHM) and class switch recombination (CSR) of the immunoglobulin (Ig) genes, which are critically important for an effective immune response. In addition, AID seems to contribute to B cell tolerance in mice and humans by, in some still undefined way, eliminating developing autoreactive B cells. As a trade-off for the benefits brought about by its physiological roles, AID can also contribute to cellular transformation and tumor progression through its mutagenic activity. AID deaminates deoxycytidines at the Ig genes thereby generating deoxyuridine, which as part of the normal mechanism of SHM and CSR is processed by DNA repair enzymes into a larger spectrum of point mutations and also DNA double- strand breaks. Multiple mechanisms regulate AID function to minimize deleterious or pathogenic DNA damage during antibody gene diversification. Despite this, off-target AID activity still makes point mutations and initiates chromosomal translocations that affect tumor suppressor and proto-oncogenes associated with B-cell lymphoid neoplasms. Through this collateral damage, AID is etiological for the development of lymphoma in several mouse models and is expressed in many human malignancies of mature B-cell origin where it may contribute to tumor clonal evolution. Mounting evidences indicate a role for AID also in disease progression and worsening of the prognosis of Chronic Lymphocytic Leukemia (CLL) and Chronic Myelogenous Leukemia (CML). Since these leukemia are not immediately derived from germinal center B cells, normal AID regulation might not be fully functional in those cases. This review discusses recent findings on the role of AID in lymphomagenesis. We describe the multilevel regulation of AID expression and function in normal compared to tumor B cells, specially focusing on the emerging role of AID in CLL and CML.

We have previously demonstrated that lipoprotein lipase (LPL) is associated to an unmutated immunoglobulin profile and clinical poor outcome in Chronic Lymphocytic Leukemia (CLL). Despite the usefulness of LPL for CLL prognosis, its functional role and the molecular mechanism regulating its expression remain elusive. Since interaction of CLL B-cells with tissue microenvironment favors disease progression by promoting malignant B-cell growth and considering that tissue methylation can be altered by environmental factors, we investigated the methylation status of LPL gene and the possibility that its over-expression could be associated to microenvironment signals. By comparing methylation changes in the LPL-CpG island between unmutated and mutated CLL patients, we could demonstrate a clear association between LPL expression and a demethylation process in the CpG island near the promoter region of the LPL gene. This process can be induced by proliferative and specific stimuli,particularly we found that CLL B-cell activation through the CD40 plus IL-4 pathway led to LPL expression and gene demethylation in LPL negative CLL samples. Overall, these results suggest that an epigenetic mechanism, triggered by the microenvironment, regulates LPL expression in CLL cells. Fil: Moreno, P.. Instituto Pasteur de Montevideo; Uruguay. Universidad de la República; Uruguay Fil: Abreu, C.. Instituto Pasteur de Montevideo; Uruguay Fil: Borge, Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Palacios, F.. Instituto Pasteur de Montevideo; Uruguay Fil: Morande, Pablo Elías. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Pegazzano, M.. Instituto Pasteur de Montevideo; Uruguay Fil: Bianchi, S.. Instituto Pasteur de Montevideo; Uruguay Fil: Landoni, A.I.. Hospital Maciel; Uruguay Fil: Agrelo, R.. Instituto Pasteur de Montevideo; Uruguay Fil: Giordano, Mirta Nilda. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Dighiero, G.. Instituto Pasteur de Montevideo; Uruguay Fil: Gamberale, Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Oppezzo, P.. Universidad de la República; Uruguay. Instituto Pasteur de Montevideo; Uruguay

T-helper 17 (Th17) cells constitute a subset of T cells that characteristically secrete IL-17. In addition to their normal adaptive immune functions, Th17 cells also play roles in supporting dysfunctional immune responses found in autoimmunity and cancer. Several studies suggest that Th17 cells play a protective role in chronic lymphocytic leukemia (CLL). For example, CLL patients exhibit varied levels of circulating Th17 cells, and elevated levels positively correlate with better clinical outcome regardless of IGHV-mutation status. To understand this relationship and elucidate the cellular and molecular mechanisms of Th17 generation in CLL, in particular the role of microRNAs known to affect Th17 development, we investigated cross-talk between naïve CD4+ T cells and CLL B cells. Moreover, since intraclonal leukemic B-cell subpopulations differing in time since cell birth/division can exhibit different functional effects on antigen presentation, we explored the effect of B-cell activation on this T - leukemic B-cell dialogue and how it affects the generation of Th17 cells. To determine potential candidates differentially expressed in CLL, we conducted genome-wide single-cell expression analysis comparing fluorescence activated cell sorting (FACS)-purified mature Th17 cells (CD3+/CD4+/CD45+/CD161+/CCR6+/ CCR4+/CXCR3-) from CLL patients and healthy donors. Selected candidate genes met the criteria of >7-fold increase in expression in CLL, adjusted p-value To determine whether CLL cells directly influence miR-155 levels in naïve CD4+ T cells, co-culture experiments using autologous leukemic or healthy B cells were carried out. FACS-purified peripheral blood naïve CD4+ T cells and B cells from CLL patients and from age-matched healthy controls were co-cultured for 3 days, and expression of T-cell miR-155 was determined by RT-qPCR. In the presence of unstimulated CLL or healthy B cells, miR-155 expression in naïve T cells remained unchanged across all co-cultures. However, upon activation, healthy and leukemic B cells exerted differential effects on miR-155 expression in autologous naïve T cells. In the presence of autologous healthy B cells pre-activated with CpG-ODN2006 and IL-15, miR-155 expression in healthy naïve T cells was significantly increased (P = 0.0313) across all samples. Conversely, CLL naïve T cells co-cultured with autologous, pre-activated leukemic B cells showed heterogeneous modulation of miR-155. Of interest, the magnitude and direction of miR-155 changes in the autologous CLL co-cultures positively correlated not only with circulating Th17 levels (P = 0.019), as determined by flow cytometry, but also with patient time to first treatment (P = 0.0003). Moreover, when samples were divided into 2 groups based on an increase or decrease in miR-155 levels after exposure to activated compared to resting CLL B cells, a significant difference was seen with median survival of 237 months and 67 months, respectively (P = 0.005). Consistent with previous observations from our lab, this correlation was independent of various prognostic markers, including IGHV-mutation status. Our results suggest the existence of a miR-155 modulatory mechanism mediated by CLL B cells that differs based on leukemic B-cell activation state and the degree of change occurring when naïve T cells are exposed to resting vs. activated B cells. Moreover, this variable effect in CLL patients differs from that in normal individuals, and the effect influences number of Th17 cells and patient outcome. Studies are underway to determine the effects that leukemic B cells, unstimulated or CpG-ODN2006 + IL-15 stimulated, have on autologous naïve T-cell maturation into Th17 cells, and the extent that this process depends on the variable miR-155 modulatory capacity of leukemic B cells. Disclosures Chiorazzi: Janssen, Inc: Consultancy; AR Pharma: Equity Ownership.

We studied the subcellular distribution of mitochondria and superoxide dismutase-1 (SOD1) in whole mounts of microdissected motor axons of rats expressing the ALS-linked SOD1-G93A mutation. The rationale was to determine whether physical interactions between the enzyme and mitochondria were linked to the axonopathy of motor fibers occurring in amyotrophic lateral sclerosis (ALS). Mitochondria and SOD1 displayed a homogeneous distribution along motor axons both in nontransgenic rats and in those overexpressing wild-type SOD1. In contrast, axons from SOD1-G93A rats (older than 35 days) showed accumulation of mitochondria in discrete clusters located at regular intervals. Most of SOD1 immunoreactivity was enriched in these clusters and colocalized with mitochondria, suggesting a recruitment of SOD1-G93A to the organelle. The SOD1/mitochondrial clusters were abundant in motor axons but scarcely seen in sensory axons. Clusters also were stained for neuronal nitric oxide synthase, nitrotyrosine, and cytochrome c. The later also was detected surrounding clusters. Ubiquitin colocalized with clusters only at late stages of the disease. The cytoskeleton was not overtly altered in clusters. These results suggest that mutant SOD1 and defective mitochondria create localized dysfunctional domains in motor axons, which may lead to progressive axonopathy in ALS. Antioxid. Redox Signal. 11, 1535–1545.

Among different prognostic factors in chronic lymphocytic leukemia (CLL), we previously demonstrated that lipoprotein lipase (LPL) is associated with an unmutated immunoglobulin profile and clinical poor outcome. Despite the usefulness of LPL for CLL prognosis, its functional role and the molecular mechanism regulating its expression are still open questions. Interaction of CLL B-cells with the tissue microenvironment favors disease progression by promoting malignant B-cell growth. Since tissue methylation can be altered by environmental factors, we investigated the methylation status of the LPL gene and the possibility that overexpression could be associated with microenvironment signals. Our results show that a demethylated state of the LPL gene is responsible for its anomalous expression in unmutated CLL cases and that this expression is dependent on microenvironment signals. Overall, this work proposes that an epigenetic mechanism, triggered by the microenvironment, regulates LPL expression in CLL disease. Fil: Abreu, Cecilia. Instituto Pasteur de Montevideo; Uruguay Fil: Moreno, Pilar. Instituto Pasteur de Montevideo; Uruguay Fil: Palacios, Florencia. Instituto Pasteur de Montevideo; Uruguay Fil: Borge, Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Morande, Pablo Elías. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Landoni, Ana Inés. Hospital Maciel; Uruguay Fil: Gabus, Raul. Hospital Maciel; Uruguay Fil: Dighiero, Guillermo. Hospital Maciel; Uruguay Fil: Giordano, Mirta Nilda. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Gamberale, Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Oppezzo, Pablo. Universidad de la República; Uruguay

Chronic lymphocytic leukemia (CLL) is the commonest form of leukemia in Europe and North America, and mainly, though not exclusively, affects older individuals. It has a very variable course, with survival ranging from months to decades [1]. It is a neoplastic disorder, characterized by progressive accumulation of monoclonal B lymphocytes, expressing CD5 and CD23 molecules and low amounts of surface membrane Ig and CD79b molecules [2]. About one-third of patients never requires treatment, has a long survival and dies of causes unrelated to CLL; in another third an initial indolent phase is followed by progression of the disease; the remaining third of patients has aggressive disease at the onset and requires early treatment [3]

Interaction of chronic lymphocytic leukemia (CLL) B cells with tissue microenvironment has been suggested to favor disease progression by promoting malignant B-cell growth. Previous work has shown expression in peripheral blood (PB) of CLL B cells of activation-induced cytidine deaminase (AID) among CLL patients with an unmutated (UM) profile of immunoglobulin genes and with ongoing class switch recombination (CSR) process. Because AID expression results from interaction with activated tissue microenvironment, we speculated whether the small subset with ongoing CSR is responsible for high levels of AID expression and could be derived from this particular microenvironment. In this work, we quantified AID expression and ongoing CSR in PB of 50 CLL patients and characterized the expression of different molecules related to microenvironment interaction. Our results show that among UM patients (1) high AID expression is restricted to the subpopulation of tumoral cells ongoing CSR; (2) this small subset expresses high levels of proliferation, antiapoptotic and progression markers (Ki-67, c-myc, Bcl-2, CD49d, and CCL3/4 chemokines). Overall, this work outlines the importance of a cellular subset in PB of UM CLL patients with a poor clinical outcome, high AID levels, and ongoing CSR, whose presence might be a hallmark of a recent contact with the microenvironment. © 2010 by The American Society of Hematology. Fil: Palacios, Florencia. Instituto Pasteur de Montevideo; Uruguay Fil: Moreno, Pilar. Instituto Pasteur de Montevideo; Uruguay Fil: Morande, Pablo Elías. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex". Departamento de Inmunología y Medicina Experimental; Argentina Fil: Abreu, Cecilia. Instituto Pasteur de Montevideo; Uruguay Fil: Correa, Agustín. Instituto Pasteur de Montevideo; Uruguay Fil: Porro, Valentina. Instituto Pasteur de Montevideo; Uruguay Fil: Landoni, Ana Ines. Hospital Maciel; Uruguay Fil: Gabus, Raul. Hospital Maciel; Uruguay Fil: Giordano, Mirta Nilda. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex". Departamento de Inmunología y Medicina Experimental; Argentina Fil: Dighiero, Guillermo. Instituto Pasteur de Montevideo; Uruguay Fil: Pritsch, Otto. Instituto Pasteur de Montevideo; Uruguay Fil: Oppezzo, Pablo. Instituto Pasteur de Montevideo; Uruguay

Chronic Lymphocytic Leukemia (CLL) is an incurable disease in which most of the tumor cells in the blood are arrested in G0/G1 stages of the cell cycle with only a minimal number displaying proliferative activity. In this regard, our group has found by gene expression profiling (GEP) that the proliferative fraction (PF) of CLL cells is enriched in the intraclonal subset marked by CXCR4dim CD5brite expression. Indeed, this subset differs by more than 1000 genes from the CXCR4brite CD5dim resting fraction (RF). The genes over-expressed in the PF relate to replication and migration as well as regulation of gene expression. One of these genes is Musashi 2 (MSI2). Of note, MSI2 is expressed at the highest levels in IGHV unmutated CLL (U-CLL) clones and their PFs. Normally, MSI2 binds mRNA and blocks translation of proteins, playing an important role in post-transcriptional regulation. In addition, MSI2 has been linked to proliferation of normal and malignant stem cells, tumorigenesis, and poor prognosis. In CLL, high MSI2 mRNA expression has been identified in patients with worse prognosis. Nevertheless, nothing is known about the function of MSI2 in CLL cells. Therefore, we have begun to study the biological role of MSI2 in B-CLL cells and its possible association with B-cell proliferation and CLL disease progression. Fist, we studied MSI2 protein expression by flow cytometry in CD19+ B cells from healthy donors (HD) and CD5+CD19+ cells from CLL patients, observing an up-regulation in CLL compared to HD. Also, we documented higher MSI2 expression in U-CLL compared to IGHV-mutated (M-CLL) CLL as well as HD. Within the leukemic clone, we observed that MSI2 expression was highest in the PF, lower in the intermediate (INT) fraction (defined as CXCR4int CD5int), and much lower in the RF (PF>INT>RF). The PF expressed 40% more MSI2 than the RF, suggesting that the highest amounts of MSI2 protein is in dividing and recently-divided cells of the clone. Since CLL B-cell proliferation occurs in the microenvironment of lymphoid organs, presumably delivered by external signals, we tested whether such signals could stimulate MSI2 expression. After stimulating CLL cells with TLR9 agonist + IL15 + IL2 in vitro MSI2 protein was up-regulated form 0.3 to 2.5 fold. In addition, the increase in MSI2 protein was associated with an enhancement in Ki-67+ cells and in phosphorylation of MAPK/ERK and AKT signaling components, measured by flow cytometry. These results suggest that signals from the microenvironment that induce cell growth and proliferation lead to MSI2 synthesis in CLL B cells. In order to study a possible association between MSI2 expression and cell division, we labeled CLL PBMCs with a dilutable cell tracer, CFSE, and then stimulated them in vitro with TLR9 agonist + IL15 + IL2. These studies indicated that MSI2 protein synthesis was increased in the activated cells and that MSI2 protein levels increased with each cell division. However, it was also clear that this increase was not directly associated to the extent of cell replication as CLL B cells from only 10% of the patients underwent 4 cycles of cell division. Since we observed an increase in MSI2 and Ki-67 expression after stimulation in all patients' clones but did not detect replication of CLL cells in all patients, we studied the effects of in vitro stimulation on cell cycle entry and completion and how this related to MSI2 expression. Experiments using propidium iodide to evaluate DNA content of PBMCs showed that in vitro stimulation increased the percentage of cells in S phase (5-25%) compared to control cells without activation ( These results, and the facts that MSI2 plays an important role in post-transcriptional regulation and is associated with cell proliferation and poor prognosis in cancer, suggest that a better understanding of the role of MSI2 in CLL patients will provide clues to understanding the birth and growth of CLL B cells and to identifying and designing new therapeutic strategies for the disease. Disclosures No relevant conflicts of interest to declare.