[Show abstract][Hide abstract] ABSTRACT: Immortalized cell lines representative of chronic lymphocytic leukemia (CLL) can assist in understanding disease pathogenesis and testing new therapeutic agents. At present, very few representative cell lines are available. We here describe the characterization of a new cell line (PCL12) that grew spontaneously from the peripheral blood (PB) of a CLL patient with progressive disease and EBV infection. The CLL cell origin of PCL12 was confirmed after the alignment of its IGH sequence against the "original" clonotypic sequence. The IGH gene rearrangement was truly unmutated and no CLL-related cytogenetic or genetic lesions were detected. PCL12 cells express CD19, CD20, CD5, CD23, low levels of IgM and IgD and the poor-outcome-associated prognostic markers CD38, ZAP70 and TCL1. In accordance with its aggressive phenotype the cell line is inactive in terms of LYN and HS1 phosphorylation. BcR signalling pathway is constitutively active and anergic in terms of p-ERK and Calcium flux response to α-IgM stimulation. PCL12 cells strongly migrate in vitro in response to SDF-1 and form clusters. Finally, they grow rapidly and localize in all lymphoid organs when xenotrasplanted in Rag2-/-γc-/- mice. PCL12 represents a suitable preclinical model for testing pharmacological agents.
[Show abstract][Hide abstract] ABSTRACT: While multiple myeloma (MM) is almost invariably preceded by asymptomatic monoclonal gammopathy of undetermined significance (MGUS) and/or smoldering MM (SMM), the alterations of the bone marrow (BM) microenvironment that establish progression to symptomatic disease are circumstantial. Here we show that in Vk*MYC mice harboring oncogene-driven plasma cell proliferative disorder, disease appearance associated with substantial modifications of the BM microenvironment, including a progressive accumulation of both CD8+ and CD4+ T cells with a dominant T helper type 1 (Th1) response. Progression from asymptomatic to symptomatic MM was characterized by further BM accrual of T cells with reduced Th1 and persistently increased Th2 cytokine production, which associated with accumulation of CD206+Tie2+ macrophages, and increased pro-angiogenic cytokines and microvessel density (MVD). Notably, MVD was also increased at diagnosis in the BM of MGUS and SMM patients that subsequently progressed to MM when compared with MGUS and SMM that remained quiescent. These findings suggest a multistep pathogenic process in MM, in which the immune system may contribute to angiogenesis and disease progression. They also suggest initiating a large multicenter study to investigate MVD in asymptomatic patients as prognostic factor for the progression and outcome of this disease.
[Show abstract][Hide abstract] ABSTRACT: The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease's biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as "spots" on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.
Full-text · Article · Oct 2014 · Journal of Visualized Experiments
[Show abstract][Hide abstract] ABSTRACT: Mouse models that recapitulate human malignancy are valuable tools for the elucidation of the underlying pathogenetic mechanisms and for pre-clinical studies. Several genetically-engineered mouse models have been generated, either mimicking genetic aberrations or deregulated gene expression in chronic lymphocytic leukemia (CLL). The usefulness of such models in the study of the human disease may potentially be hampered by species-specific biological differences in the target cell of the oncogenic transformation. Specifically, do the genetic lesions or the deregulated expression of leukemia-associated genes faithfully recapitulate the spectrum of lymphoproliferations in humans? Do the CLL-like lymphoproliferations in the mouse have the phenotypic, histological, genetic, and clinical features of the human disease? We here compare the various CLL mouse models with regard to disease phenotype, penetrance and severity. We discuss similarities and differences of the murine lymphoproliferations compared to human CLL. We propose that the Eμ-TCL1 transgenic and 13q14-deletion models that have been comprehensively studied at the levels of leukemia phenotype, antigen-receptor repertoire and disease course, show close resemblance to the human disease. We conclude that modelling CLL-associated genetic dysregulations in mice can provide important insights into the molecular mechanisms of disease pathogenesis and generate valuable tools for the development of novel therapies.
[Show abstract][Hide abstract] ABSTRACT: The sialic-acid-binding immunoglobulin-like lectin SIGLEC-G is a negative regulator of B-cell receptor-mediated calcium signaling. Its deficiency leads to reduced turnover and increased proliferation and survival of murine B-1a cells. Siglecg(‒/‒) mice show a premature expansion of polyclonal CD5(+) B cells in the spleen and the peritoneal cavity. Here we studied the B-lymphocyte fate in Siglecg(‒/‒) mice over time. We demonstrate that in aging animals SIGLEC-G deficiency promotes the progressive accumulation of monoclonal B lymphocytes and increases the susceptibility to develop B-cell lymphoproliferative disorders. Lymphoid tumors arising in aged Siglecg(‒/‒) mice are monoclonal and histologically heterogeneous as they include diffuse large B-cell lymphoma, follicular lymphoma, medium-to-large B-cell monomorphic lymphoma but surprisingly not Chronic Lymphocytic Leukemia. They express high levels of BCL-2 and are transplantable. In keeping with these findings we have also observed a remarkable downregulation of the human orthologue SIGLEC10 in human B-cell lymphoma and leukemia cell lines. Taken together, these observations indicate that the downregulation of negative B-cell receptor regulators such as SIGLEC-G/SIGLEC10 may represent another mechanism relevant for the pathogenesis of B-cell lymphomas.
[Show abstract][Hide abstract] ABSTRACT: Oncogene-induced DNA damage elicits genomic instability in epithelial cancer cells, but apoptosis is blocked through inactivation of the tumor suppressor p53. In hematological cancers, the relevance of ongoing DNA damage and the mechanisms by which apoptosis is suppressed are largely unknown. We found pervasive DNA damage in hematologic malignancies, including multiple myeloma, lymphoma and leukemia, which leads to activation of a p53-independent, proapoptotic network centered on nuclear relocalization of ABL1 kinase. Although nuclear ABL1 triggers cell death through its interaction with the Hippo pathway coactivator YAP1 in normal cells, we show that low YAP1 levels prevent nuclear ABL1-induced apoptosis in these hematologic malignancies. YAP1 is under the control of a serine-threonine kinase, STK4. Notably, genetic inactivation of STK4 restores YAP1 levels, triggering cell death in vitro and in vivo. Our data therefore identify a new synthetic-lethal strategy to selectively target cancer cells presenting with endogenous DNA damage and low YAP1 levels.
[Show abstract][Hide abstract] ABSTRACT: Leukemia is one of the leading journals in hematology and oncology. It is published monthly and covers all aspects of the research and treatment of leukemia and allied diseases. Studies of normal hemopoiesis are covered because of their comparative relevance.
No preview · Article · Aug 2013 · Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K
[Show abstract][Hide abstract] ABSTRACT: The investigation on the mechanisms that govern the development and progression of cancer is constantly swaying between "seed" and "soil". Chronic lymphocytic leukemia (CLL) makes no exception. Its natural history, including response to treatment and drug resistance, is determined both by causal and influential genes and by the relationships that leukemic cells entertain with their supportive microenvironments. Therefore dissecting the role of microenvironment may provide new strategies of diagnosis and treatment. CLL, though phenotypically homogeneous, is clinically heterogeneous and despite major therapeutic advances remains incurable. Conceivably the host of new non-genotoxic drugs that operate at the forefront between tumor cells and their milieu will modify the present therapeutic perspective by re-shaping the tumor cell/microenvironment cross talk.
No preview · Article · Jul 2013 · Seminars in Cancer Biology
[Show abstract][Hide abstract] ABSTRACT: B-Cell Receptor (BCR) triggering and responsiveness have a crucial role in the survival and expansion of Chronic Lymphocytic Leukemia (CLL) clones. Analysis of in vitro response of CLL cells to BCR triggering allowed the definition of two main subsets of patients and lack of signalling capacity was associated with constitutive activation of ERK1/2 and NF-ATc1, consistent with the idea that at least one group of CLL patients derives from the abnormal expansion of anergic B cells. In the present paper we have further investigated the anergic subset of CLL (defined as the one with constitutive ERK1/2 phosphorylation) and found that it is characterized by low levels of surface IgM and impairment of calcium mobilization after BCR engagement in vitro. Chronic BCR triggering promoted CLL cell survival selectively in pERK(+) samples and the use of MAPK and NF-AT signalling inhibitors specifically induced apoptosis in this group of patients. Apoptosis induction was preceded by an initial phase of anergy reversal consisting in the loss of ERK phosphorylation and NF-AT nuclear translocation, and by the restoration of BCR responsiveness, reinforcing the idea that the anergic program favours the survival of leukemic lymphocytes.
[Show abstract][Hide abstract] ABSTRACT: HS1 (Hematopoietic cell-specific Lyn substrate-1) is a cytoskeletal interactor in the B-cell receptor (BCR) signaling pathway whose phosphorylation correlates with prognosis in Chronic Lymphocytic Leukemia (CLL) patients. The differentially phosphorylated sites and the kinases that regulate HS1 activity in CLL remain poorly understood. We demonstrate that HS1 activity is differentially regulated by LYN kinase that, in a sizable subset of patients, phosphorylates HS1 on Tyrosine (Y)397, resulting in its activation. This correlates with increased cytoskeletal functionality in terms of migration, adhesion and F-actin polymerization. In these patients, LYN is also activated on Y396 residue and its inhibition with the Tyrosine Kinase Inhibitor Dasatinib abrogates HS1-Y397 phosphorylation. This results in the reduction of HS1 activation along with that of VAV1 and ERK also in the presence of microenvironmental stimuli including BCR and CXCR4 stimulation. Interestingly, targeting LYN/HS1 axis in vitro with Dasatinib leads to the concomitant reduction of the cytoskeletal activity, BCR signaling and cell survival in the selected subset of patients with activated LYN/HS1. Moreover in a transplantable mouse model based on the EμTCL1 transgenic mouse, LYN/HS1 signaling pharmacological inhibition interferes with CLL progression and lymphoid organ infiltration. These data suggest that the LYN/HS1 axis marks distinct signaling profiles and cytoskeletal-related features that may represent valuable targets for cytoskeleton-targeted therapeutic intervention in a sizable fraction of CLL patients.
[Show abstract][Hide abstract] ABSTRACT: Xenotransplantation of human tumor cells into immunodeficient mice has been a powerful preclinical tool in several hematological malignancies, with the notable exception of chronic lymphocytic leukemia (CLL). For several decades, this possibility was hampered by the inefficient and/or short-term engrafment of CLL cells into available animals. The development of new generations of immunocompromised mice has allowed to partially overcome these constraints. Novel humanized animal models have been created that allow to recapitulate the pathogenesis of the disease and the complex in vivo relationships between leukemic cells and the microenvironment. In this review we discuss the development of xenograft models of CLL, how they may help elucidating the mechanisms that account for the natural history of the disease and facilitating the design of novel therapeutic approaches.Leukemia advance online publication, 5 October 2012; doi:10.1038/leu.2012.268.
Full-text · Article · Sep 2012 · Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K
[Show abstract][Hide abstract] ABSTRACT: Inflammation is involved in the initiation and progression of several chronic lymphoid malignancies of B-cell type. Toll-like receptors (TLR) are transmembrane inflammatory receptors that on recognition of pathogen-associated molecular patterns trigger an innate immune response and bridge the innate and adaptive immune response by acting as costimulatory signals for B cells. Fine tuning of TLR and IL-1R-like (ILR) activity is regulated by TIR8 (SIGIRR), a transmembrane receptor of the TLR/ILR family which inhibits other family members. To test the hypothesis that TLR and/or ILR may play a role in the natural history of chronic B-cell tumors, we crossed Eμ-TCL1 transgenic mice, a well established model of chronic lymphocytic leukemia (CLL), with mice lacking the inhibitory receptor TIR8 that allow an unabated TLR-mediated stimulation. We here report that in the absence of TIR8 the appearance of monoclonal B-cell expansions is accelerated and mouse life span is shortened. The morphology and phenotype of the mouse leukemic expansions reproduce the progression of human CLL into an aggressive and frequently terminal phase characterized by the appearance of prolymphocytes. This study reveals an important pathogenetic implication of TLR in CLL development and progression.
[Show abstract][Hide abstract] ABSTRACT: Chronic lymphocytic leukemia (CLL) is characterized by an accumulation of mature CD19(+)CD5(+)CD20(dim) B lymphocytes that typically express the B-cell activation marker CD23. In the present study, we cloned and expressed in T lymphocytes a novel chimeric antigen receptor (CAR) targeting the CD23 antigen (CD23.CAR). CD23.CAR(+) T cells showed specific cytotoxic activity against CD23(+) tumor cell lines (average lysis 42%) and primary CD23(+) CLL cells (average lysis 58%). This effect was obtained without significant toxicity against normal B lymphocytes, in contrast to CARs targeting CD19 or CD20 antigens, which are also expressed physiologically by normal B lymphocytes. Moreover, CLL-derived CD23.CAR(+) T cells released inflammatory cytokines (1445-fold more TNF-β, 20-fold more TNF-α, and 4-fold more IFN-γ). IL-2 was also produced (average release 2681 pg/mL) and sustained the antigen-dependent proliferation of CD23.CAR(+) T cells. Redirected T cells were also effective in vivo in a CLL Rag2(-/-)γ(c)(-/-) xenograft mouse model. Compared with mice treated with control T cells, the infusion of CD23.CAR(+) T cells resulted in a significant delay in the growth of the MEC-1 CLL cell line. These data suggest that CD23.CAR(+) T cells represent a selective immunotherapy for the elimination of CD23(+) leukemic cells in patients with CLL.
[Show abstract][Hide abstract] ABSTRACT: The function of the intracellular protein hematopoietic cell-specific Lyn substrate-1 (HS1) in B lymphocytes is poorly defined. To investigate its role in migration, trafficking, and homing of leukemic B lymphocytes we have used B cells from HS1(-/-) mice, the HS1-silenced human chronic lymphocytic leukemia (CLL) MEC1 cell line and primary leukemic B cells from patients with CLL. We have used both in vitro and in vivo models and found that the lack of expression of HS1 causes several important functional effects. In vitro, we observed an impaired cytoskeletal remodeling that resulted in diminished cell migration, abnormal cell adhesion, and increased homotypic aggregation. In vivo, immunodeficient Rag2(-/-)γ(c)(-/-) mice injected with HS1-silenced CLL B cells showed a decreased organ infiltration with the notable exception of the bone marrow (BM). The leukemic-prone Eμ-TCL1 transgenic mice crossed with HS1-deficient mice were compared with Eμ-TCL1 mice and showed an earlier disease onset and a reduced survival. These findings show that HS1 is a central regulator of cytoskeleton remodeling that controls lymphocyte trafficking and homing and significantly influences the tissue invasion and infiltration in CLL.
[Show abstract][Hide abstract] ABSTRACT: Chronic lymphocytic leukemia (CLL) is characterized by the accumulation in primary and secondary lymphoid tissues of CD5+ B cells that have the same B cell receptor (BCR) rearrangement. Genetic alterations and different stimuli originating from the microenvironment cooperate in the selection and expansion of the malignant clone. Molecular and functional analyses suggest that stimulation through the BCR affects the destiny of leukemic cells in terms of life or death. Microenvironmental signals are crucial for this process, inducing proliferation and leading to the survival and accumulation of leukemic cells within lymphoid organs. Nevertheless, a number of major biological issues still remain to be solved, including the relationships between cell proliferation and cell accumulation within lymphoid organs as well as the mechanisms that regulate CLL cell migration and recirculation between peripheral blood and lymphoid tissues. We focused on the role played by the cytoskeleton, given its relevance in controlling cellular shape, mobility, and homing. We hypothesize that hematopoietic cell-specific Lyn substrate 1 (HS1), a putative prognostic marker in CLL that interacts with distinct cytoskeleton adapters in leukemic B-lymphocytes, could regulate the CLL cell cytoskeleton.
Full-text · Article · Aug 2010 · Leukemia & lymphoma