from-2de-gel-spot-to-protein-function-lesson-learned-from-hs1-chronic

From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a ...

IRCCS, San Raffaele Scientific Institute

King's College London

Stimulation through the B-cell antigen receptor (BCR) is believed to be involved in the natural history of chronic lymphocytic leukemia (CLL). Some cases respond to the in vitro cross-linking of surface immunoglobulin (sIg) with effective activation. In contrast, the remaining cases do not respond to such stimulation, thereby resembling B cells anergized after antigen encounter in vivo. However the biochemical differences between the 2 groups are ill defined, and in humans the term B-cell anergy lacks a molecular definition. We examined the expression and activation of key molecules involved in signaling pathways originating from the BCR, and we report that a proportion of CLL patients (a) expresses constitutively phosphorylated extracellular signal-regulated kinase (ERK)1/2 in the absence of AKT activation; (b) displays constitutive phosphorylation of MEK1/2 and increased nuclear factor of activated T cells (NF-AT) transactivation; and (c) is characterized by cellular unresponsiveness to sIg ligation. This molecular profile recapitulates the signaling pattern of anergic murine B cells. Our data indicate that constitutive activation of mitogen activated protein (MAP) kinase signaling pathway along with NF-AT transactivation in the absence of AKT activation may also represent the molecular signature of anergic human B lymphocytes. CLL cases with this signature may be taken as a human model of anergic B cells aberrantly expanded.

Constitutive activation of distinct BCR-signaling pathways in a subset of CLL patients: a molecular signature of anergy.

Monoclonal B lymphocytosis (MBL) is a frequent phenomenon in the general population. Despite a phenotype similar to chronic lymphocytic leukemia (CLL), the possibility exists that most cases are not necessarily a pre-leukemic condition. This is suggested by the fact that MBL is at least 100 times more frequent than CLL and the diagnosis of CLL is not an inevitable fate, even among MBL cases with lymphocytosis, where it occurs only in 1.1% of the cases per year. The high incidence of MBL, if coupled with the possibility of evolution into a frank leukemic state, poses evident clinical and health system concerns. MBL in the general population usually accounts for a very low number of all circulating B-cells, being <10% of all B lymphocytes. This creates the need for a better characterisation of MBL at molecular level, aiming to identify biological features that may define which cases are more likely to progress towards clinically overt CLL. This approach should also help to avoid unnecessary and prolonged follow-ups in all individuals carrying MBL, excluding those who are extremely unlikely to develop CLL.

Monoclonal B lymphocytosis in the general population.

Easily reproducible animal models that allow for study of the biology of chronic lymphocytic leukemia (CLL) and to test new therapeutic agents have been very difficult to establish. We have developed a novel transplantable xenograft murine model of CLL by engrafting the CLL cell line MEC1 into Rag2(-/-)gamma(c)(-/-) mice. These mice lack B, T, and natural killer (NK) cells, and, in contrast to nude mice that retain NK cells, appear to be optimal recipient for MEC1 cells, which were successfully transplanted through either subcutaneous or intravenous routes. The result is a novel in vivo model that has systemic involvement, develops very rapidly, allows the measurement of tumor burden, and has 100% engraftment efficiency. This model closely resembles aggressive human CLL and could be very useful for evaluating both the biologic basis of CLL growth and dissemination as well as the efficacy of new therapeutic agents.

A novel Rag2-/-gammac-/--xenograft model of human CLL.

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.

Lack of TIR8/SIGIRR triggers progression of chronic lymphocytic leukemia in mouse models.

The emerging role of Toll-like receptors (TLR) in the pathogenesis of chronic lymphocytic leukemia (CLL) led us to ask whether TLR stimulation may protect CLL cells from drug-induced apoptosis.

In vitro sensitivity of CLL cells to fludarabine may be modulated by the stimulation of Toll-like receptors.

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). 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 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 cytoskeletal effector VAV1 and the downstream kinase ERK also in the presence of BCR and CXC chemokine receptor CXCR4 stimulation. Interestingly, targeting the LYN/HS1 axis in vitro leads to the concomitant reduction of cytoskeletal activity, BCR signaling and cell survival in the subset of patients with activated LYN/HS1. In a transplantable mouse model based on the EμTCL1 transgenic mouse, LYN/HS1 signaling inhibition interferes with CLL progression and lymphoid organ infiltration. Thus LYN/HS1 axis marks distinct signaling profiles and cytoskeletal-related features that may represent valuable targets for cytoskeleton-targeted therapeutic intervention in CLL.

Targeting the LYN/HS1 signaling axis in chronic lymphocytic leukemia.

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 2 main subsets of patients and lack of signaling capacity was associated with constitutive activation of extracellular-regulated kinases 1/2 (ERK1/2) and nuclear factor of activated T cells c1 (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 work, we 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 immunoglobulin M and impairment of calcium mobilization after BCR engagement in vitro. Chronic BCR triggering promoted CLL cell survival selectively in phosphorylated ERK1/2 samples and the use of mitogen-activated protein kinase and NF-AT signaling 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 favors the survival of leukemic lymphocytes.

Targeting B-cell anergy in chronic lymphocytic leukemia.

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 fate of B lymphocytes in Siglecg(-/-) mice over time. We demonstrate that in aging animals SIGLEC-G deficiency promotes 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, and medium-to-large B-cell monomorphic lymphoma but surprisingly not chronic lymphocytic leukemia. The tumors express high levels of BCL-2 and are transplantable. In keeping with these findings we have also observed a remarkable down-regulation of the human ortholog SIGLEC10 in human B-cell lymphoma and leukemia cell lines. Taken together, these observations indicate that the down-regulation of negative B-cell receptor regulators such as SIGLEC-G/SIGLEC10 may represent another mechanism relevant to the pathogenesis of B-cell lymphomas.

Article	Year
Constitutive activation of distinct BCR-signaling pathways in a subset of CLL patients: a molecular signature of anergy. Blood\| PubMed ID: 18292287	2008
Monoclonal B lymphocytosis in the general population. Leukemia & lymphoma\| PubMed ID: 19347732	2009
A novel Rag2-/-gammac-/--xenograft model of human CLL. Blood\| PubMed ID: 20018917	2010
Lack of TIR8/SIGIRR triggers progression of chronic lymphocytic leukemia in mouse models. Blood\| PubMed ID: 21652674	2011
In vitro sensitivity of CLL cells to fludarabine may be modulated by the stimulation of Toll-like receptors. Clinical cancer research : an official journal of the American Association for Cancer Research\| PubMed ID: 23224401	2013
Targeting the LYN/HS1 signaling axis in chronic lymphocytic leukemia. Blood\| PubMed ID: 23325840	2013
Targeting B-cell anergy in chronic lymphocytic leukemia. Blood\| PubMed ID: 23460614	2013
SIGLEC-G deficiency increases susceptibility to develop B-cell lymphoproliferative disorders. Haematologica\| PubMed ID: 24859880	2014

Benedetta Apollonio

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