HS1 Protein Research Articles

Molecular background of Orai1 accumulation in the immunological synapse

Abstract CRAC channels have a vital role in the activation of T lymphocytes, through their calcium influx. CRAC is composed of the ER-membrane resident STIM1 (stromal interaction molecule 1) and the plasma membrane Orai1 pore-forming subunit. Both are accumulated at the interface of the immunological synapse (IS) between a T cell and an antigen-presenting cell (APC). CRAC channelopathies due to mutations in either Orai1 or STIM1 are characterized by severe combined immunodeficiency (SCID)-like disease, autoimmunity, muscular hypotonia, and ectodermal dysplasia. The down-regulation of actin-binding protein expression (e.g. HS1), which may be responsible for the immobilization of ion channels, hampers Ca2+-signaling and IS formation of T lymphocytes. We hypothesized that actin-binding proteins modulate the trafficking of the CRAC channels to the IS, which is crucial for the Ca2+-signaling. By means of GST-affinity assay we could show that HS1, which is an actin-regulatory adaptor protein, binds to the N-terminus of the Orai1. N-terminal truncated Orai1 channels (ΔN1: 1–74, ΔN2: 1–88) were created and expressed in Jurkat cells. Then Orai1 channel expressing Jurkat cells were conjugated to CD3/CD28 beads as an APC and Orai1 polarization was determined: the Orai1-ΔN1 mutant similar to the wild-type Orai1 redistributed to the IS but the Orai1-ΔN2 did not. Furthermore, the HS1 protein did not show IS-accumulation in cells expressing the Orai1-ΔN2 subunits unlike for those transfected with the Orai1-ΔN1 or the wild-type constructs. Overall, these data indicate that Orai1-HS1 interaction could contribute to the anchoring of the Orai1 in the IS and can affect the Ca2+-signaling upon T cell activation.

Ibrutinib Differentially Interferes With Surface IgM and IgD BCR Signaling Kinetics In Chronic Lymphocytic Leukemia

B cell receptor (BCR) signaling is a driving event in Chronic Lymphocytic Leukemia (CLL) progression. Most CLL cells express both surface IgM (sIgM) and sIgD molecules, and previous reports showed heterogeneous responses to anti-IgM stimulation: CLL cells carrying IGHV unmutated-BCRs are highly responsive in terms of intracellular signaling activation, while cells with IGHV-mutated receptors are usually anergic to BCR cross-linking. In contrast, it has been described that almost all CLL cells retain responsiveness to sIgD, suggesting that sIgD signaling may not be the most relevant event in disease progression and heterogeneity. As the clinical significance of these mechanisms still needs to be unraveled, in the present study we analyzed the kinetics of BCR signaling activation following either sIgM or sIgD stimulation in CLL cells. The activation of HS1 protein, along with BCR proximal kinases (i.e. SRC/LYN and BTK) and the downstream effector ERK, was tested by Western Blot and flow-cytometry analysis up to 60 minutes following receptor engagement. HS1 protein was chosen as an early marker of BCR signaling and cytoskeletal activation (ten Hacken E et al., Blood 2013), also in the light of its prognostic relevance, as HS1 hyperphosphorylation correlates with a dismal clinical outcome of patients.Purified B cells from 16 primary CLL samples were stimulated with either soluble anti-IgM or anti-IgD and the level of HS1 activating tyrosine (Y)397 phosphorylation was analyzed at different time points (2, 5, 15, 30, 60 minutes) by densitometric analysis of Western Blot results or Mean Fluorescent Intensity analysis of flow cytometry data. HS1 activation peaked after 2-5 minutes following stimulation, and then returned to baseline at later time points with different patterns. In 13/16 samples, we observed a protracted HS1 phosphorylation after anti-IgM stimulation. In contrast, 6/16 samples showed protracted HS1 phosphorylation after anti-IgD stimulation, while 10/16 samples showed a very transient HS1 activation that rapidly returned to baseline. The activation kinetics of the other signaling molecules (i.e. SRC/LYN, BTK and ERK) was in line with that of HS1. As protracted BCR signaling activation is linked to cell cycle entry and proliferation, the protracted IgD signaling observed in some patients suggests that not only IgMs, but also IgDs may have pathogenetic relevance in CLL progression and heterogeneity.We then analyzed how the BTK inhibitor ibrutinib affects BCR signaling activation kinetics and analyzed phosphoprotein activation patterns in 10 CLL samples after 1 hour pretreatment with ibrutinib and up to 60 minutes following anti-IgM and anti-IgD stimulation. Both IgM and IgD-induced phosphorylation of HS1, SRC/LYN, BTK and ERK was reduced by ibrutinib treatment, demonstrating that ibrutinib is effective in blocking signaling pathways downstream not only of IgM receptors, as previously demonstrated, but also downstream of IgDs. Interestingly, in 7/10 samples, HS1 partially retained the ability to be phosphorylated after BCR triggering following 1-hour ibrutinib treatment and 5-minute anti-IgM or anti-IgD stimulation, suggesting that HS1 activation can bypass BTK kinase inhibition at least at early time points following receptor stimulation. A broad analysis on a large cohort of patients will further allow the correlation between BCR signaling molecule activation and cell viability with molecular/cytogenetic prognostic factors.Overall, these results show that sIgM stimulation of CLL cells promotes protracted activation of BCR signaling, which can only be observed in a restricted set of patients after sIgD stimulation. These observations support the notion that sIgM signaling may be more relevant for CLL progression, but also suggest that sIgD signaling may contribute to CLL pathogenesis in selected patients. Both signaling pathways can be inhibited by ibrutinib, further supporting the effectiveness of ibrutinib in inhibiting BCR signaling in CLL. Disclosures:O'Brien:Pharmacyclics: Research Funding. Burger:Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Targeting the LYN/HS1 Signaling Axis in Chronic Lymphocytic Leukemia

AbstractAbstract 928B-cell receptor (BCR) signaling and cytoskeletal activation represent critical steps in the pathogenesis of Chronic Lymphocytic Leukemia (CLL). We previously demonstrated that Hematopoietic cell specific Lyn substrate 1 (HS1) i) is a potential prognostic marker as CLL patients whose leukemic cells carry hypo-phosphorylated (hypo-p) HS1 have a better prognosis than patients with the hyper-phosphorylated (hyper-p) form; ii) is a central interactor of several cytoskeletal components and iii) has a profound effect on the development and progression of CLL in the Eμ-TCL1 transgenic mouse suggesting that the hyper-phosphorylation of the molecule may be responsible of HS1 inactivation rather than its activation.To better define the role of HS1 phosphorylation in CLL cell biology we dissected the BCR signaling pathway in cells with differential phosphorylation of the HS1 protein. Taking advantage of the CLL cell line MEC1 where we silenced the expression of HS1, we observed that the phosphorylation status of several BCR signalling molecules, including LYN kinase, is reduced in the absence of HS1. A similar pattern of modifications was confirmed in primary cells from 62 patients with different phosphorylation status of HS1 (Figure A n=35 HS1hypo-p vs n=27 HS1hyper-p, p=0,01), thereby identifying a distinct phospho-signature associated to HS1. LYN phosphorylation correlates with clinical parameters as phosphoLYN+ patients showed significantly increased (Figure B n=28 phosphoLYN+ vs n=28 phosphoLYN− p=0,0002) Progression-free survival and Treatment-free survival (Figure C n=27 phosphoLYN+ vs n=26 phosphoLYN− p=0,0002) as compared to phosphoLYN−patients.We further investigated the relationship between HS1 and LYN phosphorylations and showed that LYN kinase activation- Tyrosine (Y)396 phosphorylation- is associated with the phosphorylation of the active site Y397 on HS1 protein (Spearman correlation n=40, p<0,0001). Patients may thus be categorized into two subgroups based on the phosphorylation status of the two molecules as “active-HS1” - HS1 hypo-p/phosphoLYN+ and “inactive-HS1” - HS1 hyper-p/phosphoLYN−-.These different patterns correlated with distinct cytoskeletal functionality in terms of migration (active-HS1 n=12 vs inactive-HS1 n=9, p= 0,009), spontaneous adhesion (active-HS1 n=12 vs inactive-HS1 n=9, p=0,03), adhesion to an HS5 stromal cell layer (active-HS1 n=9 vs inactive-HS1 n=10 p=0,006) and F-actin polymerization (active-HS1 n=12 vs inactive-HS1 n=11, p=0,001). Targeting the LYN/HS1 axis in vitro with the tyrosine kinase inhibitor Dasatinib significantly affected primary CLL cell viability with a direct correlation with basal LYN phosphorylation (Sperman correlation n=32, p=0,04) and interfered with cytoskeleton activity, with a preference for patients carrying the “active-HS1” signature. Accordingly, 100nM Dasatinib markedly reduced migration of active-HS1 samples (active-HS1 n=10, p=0,003 vs inactive-HS1 n=7, p>0,05), with no significant effect on inactive-HS1 samples. F-actin polymerization (active-HS1 n=9 p=0,003, vs inactive-HS1 n=8 p=0,01) was reduced in both groups by Dasatinib treatment, while spontaneous (active-HS1 n=7, p=0,03 vs inactive-HS1 p>0,05) and HS5-mediated cell adhesion (active-HS1 n=8, p=0,03 vs inactive-HS1 n=8 p>0,05) were affected by the treatment only in the active-HS1 group.In the adoptive transfer mouse model based on the EmTCL1 transgenic mouse, a 4-week Dasatinib treatment reduced the percentage of CD19+CD5+Igk+ CLL cells in the blood as soon as one week after treatment initiation and throughout the whole treatment period (p(Day6)=0,007, p(Day13)=0,005, p(Day20)=0,004, p(day of killing)=0,01). At sacrifice, a significant reduction in the absolute number of leukemic cells in the spleen (p=0,05), bone marrow (p=0,03) and lymph nodes (p=0,04) was observed in treated mice as compared to controls. Consistent with the biochemical action of Dasatinib, significant reduction in SRC-Y416 phosphorylation was observed after intracellular staining of leukemic B cells (p=0,04) at the end of treatment.These observations suggest that the phosphorylations status of LYN and HS1 as a biological indicator of respectively distinct signaling pathway and cytoskeletal-related features that can be selectively targeted for therapeutic intervention in a sizable fraction of CLL patients. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

HS1, a Lyn kinase substrate, is abnormally expressed in B-chronic lymphocytic leukemia and correlates with response to fludarabine-based regimen.

In B-Chronic Lymphocytic Leukemia (B-CLL) kinase Lyn is overexpressed, active, abnormally distributed, and part of a cytosolic complex involving hematopoietic lineage cell-specific protein 1 (HS1). These aberrant properties of Lyn could partially explain leukemic cells’ defective apoptosis, directly or through its substrates, for example, HS1 that has been associated to apoptosis in different cell types. To verify the hypothesis of HS1 involvement in Lyn-mediated leukemic cell survival, we investigated HS1 protein in 71 untreated B-CLL patients and 26 healthy controls. We found HS1 overexpressed in leukemic as compared to normal B lymphocytes (1.38±0.54 vs 0.86±0.29, p<0.01), and when HS1 levels were correlated to clinical parameters we found a higher expression of HS1 in poor-prognosis patients. Moreover, HS1 levels significantly decreased in ex vivo leukemic cells of patients responding to a fludarabine-containing regimen. We also observed that HS1 is partially localized in the nucleus of neoplastic B cells. All these data add new information on HS1 study, hypothesizing a pivotal role of HS1 in Lyn-mediated modulation of leukemic cells’ survival and focusing, one more time, the attention on the BCR-Lyn axis as a putative target for new therapeutic strategies in this disorder.

High expression of hematopoietic cell specific Lyn substrate-1 (HS1) predicts poor survival of B-cell chronic lymphocytic leukemia patients

B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in adults in western countries. HS1 protein regulates leukemic cell migration and homing, and can indirectly promote disease progression and influence patient survival.The aim of this study was to evaluate HS1 expression in CLL patients in connection with other known prognostic factors and patients’ survival. Methods90 untreated CLL patients were included into the study. The control group consisted of 28 healthy matched people. HS1 detection was performed by western-blotting. Mutational status of IgVH, as well as CD38 and ZAP70 expression was also analyzed. ResultsHS1 expression was significantly higher in CLL patients comparing to controls. Positive correlation was shown between HS1 and: age (p=0.0454), Rai stage (p=0.0412), leukocytosis (p=0.0129) and beta-2-microglobulin (p=0.0342). Patients with lymphocyte doubling time shorter or equal to 6 months had higher expression of HS1. Patients with higher HS1 expression had shorter survival than those with lower HS1 expression (p=0.0329). ConclusionsWe showed, that high HS1 expression predicts poor survival of chronic lymphocytic leukemia patients.

High Expression of Haematopoietic Cell Specyfic Lyn Substrate-1 (HS1) Predicts Survival of B-Cell Chronic Lymphocytic Leukemia Patients

Abstract 2853B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in adults in western countries. It is characterized by B lymphocyte accumulation in peripheral blood, bone marrow, lymph nodes and other lymphatic organs. Leukemic cells derive most commonly from B lymphocytes, rarely from T or NK cells. B-CLL is known from its heterogeneous clinical course from indolent to very aggressive. In spite of many known prognostic factors (such as immunoglobulin heavy chain gene mutational status – IgVH, expression of ZAP70 and CD38), is still difficult to classify a single patient to particular risk group and to predict CLL clinical course. That is why new prognostic factors are still needed.HS1 (hematopoietic cell specific Lyn substrate-1) is an intracellular protein, which expression occurs mainly in hematopoietic cells. HS1 plays an important role in regulating T cell immune synapse and affects many functions of NK cells, including the lysis of target cells, adhesion, chemotaxis and clustering of actin in the lytic synapse. The role of HS1 in B cells is poorly understood. This protein was identified in B cells as the primary receptor substrate for phosphorylation by BCR after antigenic stimulation. Other studies have confirmed the role of HS1 in the process of clonal expansion and deletion induced by antigen-receptor interaction in B cells and T. HS1 is rapidly phosphorylated in B cells in the vicinity of tyrosine residues and is a substrate for tyrosine kinases: the Src family and Syk, including Lyn, FGR, Fyn and Lck. It has been shown that HS1 interacts with the cell cytoskeleton in both: normal and leukemic B cells. HS1 protein is an important regulator of motility, migration and adhesion of leukemic cells and is involved in cytoskeleton rearrangement. HS1 can have impact on homing and migration of CLL cells. It can indirectly promote disease progression and influence patient survival. The aimof this study was to evaluate HS1 expression in CLL patients in connection with other known prognostic factors and patient survival. Material and methods:92 untreated CLL patients (45 women and 47 men), aged between 42 and 88 years (median age 67 years), were included into the study. Diagnosis was made basing on typical clinical, hematological and immunophenotypical picture. The control group was consist of 28 healthy matched people (11 men and 17 women), aged between 36 and 79 years (median age 59 years). HS1 protein expression was determined by western blot. Comparative semi-quantitative indication of the degree of saturation of the bands analyzed by densitometry using the gel documentation system Gel-Doc (Bio-Rad) and a computer program to analyze the 1-D Quantity One (Bio-Rad). Assuming conventional units [AU - arbitrary units], depending on the saturation band, patients were divided into four groups with the expression of HS1 protein expressed in value from 0 to 3. Lack of expression was expressed as 0 [AU], and expression of the strongest, with the highest saturation band measured as 3 [AU]. Mutational status of IgVH, as well as CD38 and ZAP70 expression were also analyzed. Results:HS1 expression was significantly higher in CLL patients comparing to controls. Positive correlation was shown between HS1 and: age (p=0.0454), Rai stage (p=0.0412), leukocytosis (p=0.0129) and β2-microglobulin (p=0.0342). There was negative correlation between HS1 and hemoglobin level (p=0.0464) and platelet count (p=0.0310). Patients with lymphocyte doubling time shorter or equal to 6 months had higher expression of HS1. Expression of HS1 significantly influenced survival of CLL patients. Patients with higher HS1 expression had shorter survival than those with lower HS1 expression (p=0.0329). Conclusions:1. Higher HS1 expression is observed in more advanced CLL stages.2. Expression of HS1 in CLL cells is matched with shorter patient survival [Display omitted] The relationship between expression of HS1 and survival of patients with B-CLL. Disclosures:No relevant conflicts of interest to declare.

2.3 HS1 Protein Defines a Distinct Signaling Pathway and Influences the Cell Homing and Migratory Capacity in CLL Primary Cells

We previously demonstrated that the phosphorylation status of hematopoietic cell-specific Lyn substrate 1 (HS1) has a potential prognostic significance in chronic lymphocytic leukemia (CLL), as patients whose leukemic cells carry phosphorylated HS1 have a better prognosis than patients with the hyper-phosphorylated form. We more recently showed in the CLL-prone E-TCL1 transgenic mouse that HS1 is a pivotal molecule in the signal transduction pathway triggered by the B-cell receptor (BCR), interacting with severalcytoskeletalcomponentsandbeinginvolvedintissuetrafficking and homing. Moreover, its inactivation has a profound effect on the development and progression of murine CLL and has a functional parallel with the hyper-phosphorylation status shown by some patients. On this basis our aim was to define if and how the different phosphorylation status of HS1 present in CLL patients may affect the signaling pathway originating from the BCR and the leukemic cell migratory capacity. Initially, we took advantage of the CLL cell line MEC1 silenced for the expression of HS1 and we dissected the signaling pathway downstream from the BCR by western blot and immunoprecipitation. Interestingly, we found that the phosphorylation status of several BCR signaling molecules, including Lyn kinase, SH2-containing tyrosine phosphatase (SHP), extracellular signal-regulated kinases (ERK) and the cytoskeleton-related proteins Vav, Rac and HIP-55, is directly affected by the absence of HS1. We then confirmed a similar pattern of modifications in primary cells from 40 CLL patients showing a different phosphorylation status of HS1. These findings indicate that the pattern of HS1 phosphorylation is associated with a specific biochemical signature characterized by the loss of phosphorylation in several BCR downstream signaling molecules, reflecting a different cytoskeleton activity. Based on this evidence and in order to elucidate possible changes in cell homing and adhesion, we investigated in vivo the migration capacity of human leukemic cells from patients showing different levels of HS1 phosphorylation. CLL cells purified from 8 patients were labeled with different concentrations of carboxyfluorescein succinimidyl ester (CFSE), were then paired into 4 couples according to their HS1 phosphorylation status and injected intravenously into Rag2-/-ϒc-/recipient mice. Each couple included a case with phosphorylated HS1 and a case with hyper-phosphorylated HS1 CLL cells. In 3/4 couples of differentially phosphorylated paired patients, CLL cells with phosphorylated HS1 had a consistent homing rate to the spleen while CLL cells with hyper-phosphorylated HS1 had a preferential homing to the bone marrow (BM). Similar results were obtained after injecting HS1-silenced MEC1 cells into Rag2-/-ϒc -/- mice. Taken together, these observations indicate that: a) the presence of hyperphosphorylatedHS1equalstheabsenceoftheproteinandthus the hyperphosphorylation of HS1 is functionally inhibitory and b) there is a correlation between the status of HS1 phosphorylation and the propensity of human CLL cells to accumulate in distinct environments in vivo. Having this in mind, we investigated the expressionpatternofHS1proteinintheBMandPBofCLLpatientseither lacking or carrying the hyperphosphorylated form of the molecule. Preliminary observations revealed that in the same patient HS1 is differentially expressed in the BM as compared with the PB; specifically BM CLL cells lack HS1 expression when the protein is hyperphosphorylated in PB cells. These data suggest that the expression of HS1 is finely tuned in different compartments and that cells which have switched off the protein expression preferentially accumulate in the BM. These findings strengthen our previous observation that HS1 phosphorylation has an important role in controlling cell migration and homing of leukemic B cells, likely through its involvement in cytoskeleton organization and tissue homing. The fact that HS1 protein expression appears to be finely modulated in CLL patients suggests its crucial role in the development and progression of the disease.

The SH3 domain of HS1 protein recognizes lysine-rich polyproline motifs

HS1 is a protein involved in erythroid proliferation and apoptotic cell death, containing several structurally significant motifs including a C-terminal SH3 domain. HPK1 is a member of the Ste20-related kinase family, which contains four proline-rich sequences and is constitutively associated with HS1 in hematopoietic cells. Recombinant fusion protein GST-SH3(HS1) was expressed to assess the binding properties of 16 peptides derived from the HPK1 proline-rich regions. The binding affinities were determined by non-immobilized ligand interaction assay by circular dichroism. Our results revealed that the classical PxxPxK class II binding motif is not sufficient to induce the interaction with the GST-SH3(HS1) domain, an event dependent on the presence of additional basic residue(s) located at the C-terminus of the PxxPxK motif: Lys(-5) in P2 peptide and Lys(-8) in P4c peptide. Lys replacement by Arg residues decreases the ligand binding affinity. The finding that both SH3(HS1) domain and full-length HS1 protein bind to P2 peptide with similar affinity demonstrates that the whole protein sequence does not affect the interaction properties of the domain. In silico models of SH3(HS1) as a complex with P2 or P4c highlight the domain residues that interact with the recognition determinants of the peptide ligand and that cooperate in the complex stabilization.

Cell-Penetrating Peptides: A Comparative Study on Lipid Affinity and Cargo Delivery Properties.

A growing number of natural and/or synthetic peptides with cell membrane penetrating capability have been identified and described in the past years. These molecules have been considered promising tools for delivering bioactive compounds into various cell types. Although the mechanism of uptake is still unclear, it is reasonable to assume that the relative contribute of each proposed mechanism could differ for the same peptide, depending on experimental protocol and cargo molecule composition. In this work we try to connect the capability to interact with model lipid membrane and structural and chemical characteristics of CPPs in order to obtain a biophysical classification that predicts the behavior of CPP-cargo molecules in cell systems. Indeed, the binding with cell membrane is one of the primary step in the interaction of CPPs with cells, and consequently the studies on model membrane could become important for understanding peptide-membrane interaction on a molecular level, explaining how CPPs may translocate a membrane without destroying it and how this interactions come into play in shuttling CPPs via different routes with different efficiency. We analyzed by CD and fluorescence spectroscopies the binding properties of six different CPPs (kFGF, Nle54-Antp and Tat derived peptides, and oligoarginine peptides containing 6, 8 or 10 residues) in absence or presence of the same cargo peptide (the 392-401pTyr396 fragment of HS1 protein). The phospholipid binding properties were correlated to the conformational and chemical characteristics of peptides, as well as to the cell penetrating properties of the CPP-cargo conjugates. Results show that even if certain physico-chemical properties (conformation, positive charge) govern CPP capability to interact with the model membrane, these cannot fully explain cell-permeability properties.

HS1 (HCLS1) Protein, a Binding Partner of HAX1, Induces G-CSF-Triggered Granulopoiesis Via LEF-1 Transcription Factor.

Abstract 548Mutations in HS1 (HCLS1) associated protein × 1 (HAX1) have been described in patients with severe congenital neutropenia (CN). HAX1 is a ubiquitously expressed and until now it was unclear how HAX1 mutations cause isolated neutropenia in CN patients. So we were interested in investigating G-CSF-dependent HS1 (HCLS1) protein expression and phosphorylation in CN patients compared to healthy individuals, since HS1 (HCLS1) is a hematopoietic-specific protein and may link HAX1 to the G-CSFR signaling via the tyrosine kinases lyn and syk. Indeed, we found that treatment of CD34+ hematopoietic cells with G-CSF leads to phosphorylation of HS1 (HCLS1) protein and recruitment of receptor thyrosine kinases lyn and syk. In CN patients harboring HAX1 mutations, G-CSF failed to upregulate expression and phosphorylation of HS1 (HCLS1), suggesting that for HS1 phosphorylation functional HAX1 protein is required. Indeed, inhibition of HAX1 in CD34+ hematopoietic cells from healthy individuals significantly abrogated G-CSF-triggered HS1 (HCLS1) phosphorylation. Previously, we found that LEF-1 is a master regulator of granulopoiesis and is severely diminished in myeloid cells of CN patients with HAX1 mutations. Moreover, these cells also exhibited severe defects in F-actin assembly. Using immunoprecipitation assay of endogenous proteins, we found that upon G-CSF stimulation of CD34+ hematopoietic cells, HS1 (HCLS1) protein additional to HAX1, also binds to LEF-1 protein modulating its expression, transcriptional activity and subsequently granulopoietic differentiation. Using CFU, qRT-PCR and reporter gene assays we demonstrated that inhibition of HS1 (HCLS1) severely disturbed G-CSF-triggered granulocytic differentiation in line with downregulation of LEF-1 expression and functions, as well as with pathological redistribution of F-actin.Taken together, in this study we demonstrated for the first time the connection between G-CSF receptor, HAX1 mutations, downregulation of HS1 (HCLS1) and LEF-1, abnormal F-actin assembly and the defective granulocytic differentiation in CN patients. Moreover, we demonstrated for the first time an ultimate direct connection between Wnt signaling and G-CSF cytokine cascade in granulopoiesis. Disclosures:No relevant conflicts of interest to declare.

Temporal Proteomic Analysis of CLL B Cell Response to Antigen Receptor Stimulation.

Unraveling the molecular basis for the different functional consequences of BCR cross-linking is essential to understand the leukemogenesis process of CLL. There is increasing evidence that an antigen driven process is crucial in CLL proliferation, based on restriction of the IgVH repertory as well as shared antigen-binding motifs used by mutated or unmutated CLL B-cells. BCR activation leads to a signaling cascade reinforced in the more aggressive CLL form by the ZAP70 protein tyrosine kinase and constitutive phosphorylation of HS1 protein. We have previously shown, using gene expression profiling over time, that crosslinking of the BCR induces a specific temporal gene expression program in the leukemic cells, leading to a complex balance disorder between proliferation and cell death. These results demonstrated a core BCR gene expression shared among all B cell (and across species). Furthermore, each cell category (healthy B-cells, indolent and aggressive CLL cells) also showed a specific temporal gene expression after BCR cross-linking. This analysis has revealed complex expression disorder of multiple genes coding proteins involved in proliferation and death regulation (Vallat et al. Blood 07). To further investigate the functional consequences of BCR cross-linking we also examined modulation of the BCR proteome, since a dynamic description of the BCR functional consequences in CLL cells would not be complete without parallel temporal proteomic analysis. We now have cross-linked the BCR in freshly isolated B-cells from CLL patients. We have isolated total proteins at 2 early, one intermediate and a later time point 30 min later than the time points selected in the previous mRNA experiments. At each analysis time point both stimulated and control unstimulated cells were examined. A 2D electrophoresis analysis (pH 3–10, 150–20 kDa) revealed between 1100 and 1800 (mean 1400) polypetidic spots at each time point after BCR engagement. A total of 600 different polypeptides show a specific pattern of expression over time after stimulation, up- (450 polypeptides) or down-regulated (150 polypeptides). We are currently identifying these different proteins of interest by mass spectrometry (Maldi-TOF and LC-MS/MS) and learning these results in parallel of the previous genomic results. Preliminary results show the complementarity of the two analyses. Many of the proteins identified correspond to changes in mRNA belonging to the temporal gene program previously described after BCR engagement, thereby validating the gene expression data at the protein level, but some proteins show a specific temporal pattern of expression whereas their corresponding coding mRNA were not present in the temporal gene expression. Such complementarities in this dynamical approach allow us to refine the molecular basis for the functional BCR cross-linking consequences in CLL and to identify putative therapeutic targets for intervention in this disease. Ongoing experiments are examining the consequences of intervention to alter expression of critical components within the functional consequences of BCR cross-linking in CLL, and potentially the consequences of antigen driven leukemogenesis.

Hematopoietic lineage cell–specific protein 1 (HS1) is a functionally important signaling molecule in platelet activation

Collagen activates platelets through an intracellular signaling cascade downstream of glycoprotein VI (GPVI). We have investigated the contribution of hematopoietic lineage cell-specific protein 1 (HS1) downstream of GPVI in platelet activation. Stimulation of GPVI leads to tyrosine phosphorylation of HS1, which is blocked by Src-family kinase inhibitors. Coimmunoprecipitation experiments revealed that HS1 associates with Syk and phosphatidylinositol 3-kinases. HS1-null mice displayed increased bleeding times and increased time to occlusion in the FeCl(3) in vivo thrombosis model compared with their wild-type littermates. In addition, aggregation and secretion responses were diminished in HS1-null mouse platelets after stimulation of GPVI and protease-activated receptor 4 (PAR-4) agonists compared with wild-type littermate mouse platelets. Finally, Akt phosphorylation was diminished after GPVI or PAR-4 stimulation in platelets from HS1-null mice compared with their wild-type littermates. These results demonstrate that phosphorylation of the HS1 protein occurs downstream of GPVI stimulation and that HS1 plays a significant functional role in platelet activation downstream of GPVI and PARs.

HS1 protein is differentially expressed in chronic lymphocytic leukemia patient subsets with good or poor prognoses

We used a proteomic approach for identifying molecules involved in the pathogenesis of chronic lymphocytic leukemia (CLL). We investigated 14 patients who were completely concordant for IgV(H) mutational status (unmutated vs. mutated), CD38 expression (positive vs. negative), and clinical behavior (progressive vs. stable); these patients were characterized as having either poor or good prognoses. The 2 patient subsets differed in the expression of hematopoietic lineage cell-specific protein 1 (HS1). In patients with poor prognoses, most HS1 protein was constitutively phosphorylated, whereas only a fraction was phosphorylated in patients with good prognoses. This difference was investigated in a larger cohort of 26 unselected patients. The survival curve of all 40 patients analyzed revealed that patients with predominately phosphorylated HS1 experience a significantly shorter median survival time. As HS1 is a protein pivotal in the signal cascade triggered by B cell receptor (BCR) stimulation, we studied its pattern of expression following BCR engagement. Normal mature B cells stimulated by anti-IgM shifted the non- or less-phosphorylated form of HS1 toward the more phosphorylated form. Naive B cells showed both HS1 forms while memory B cells expressed mainly the phosphorylated fraction. These data indicate a central role for antigen stimulation in CLL and suggest a new therapeutic target for patients with aggressive disease.

Antennapedia/HS1 chimeric phosphotyrosyl peptide: conformational properties, binding capability to c-Fgr SH2 domain and cell permeability.

With the aim of interfering with the signaling pathways mediated by the SH2 domains of Src-like tyrosine kinases, we synthesized a tyrosyl-phospho decapeptide, corresponding to the sequence 392-401 of HS1 protein, which inhibits the secondary phosphorylation of HS1 protein catalyzed by the Src-like kinases c-Fgr or Lyn. This phospho-peptide was modified to enter cells by coupling to the third helix of Antennapedia homeodomain, which is able to translocate across cell membranes. Here we present CD and fluorescence studies on the conformational behavior in membrane-mimicking environments and on lipid interactions of Antennapedia fragment and its chimeric phosphorylated and unphosphorylated derivatives. These studies evidenced that electrostatic rather than amphiphilic interactions determine the peptide adsorption on lipids. Experiments performed with recombinant protein containing the SH2 domain of c-Fgr fused with GST and with isolated erythrocyte membranes demonstrated that the presence of the N-terminal Antennapedia fragment only slightly affects the binding of the phospho-HS1 peptide to the SH2 domain. In fact, it has been shown that in isolated erythrocyte membranes, both phospho-HS1 peptide and its chimeric derivative greatly affect either the SH2-mediated recruitment of the c-Fgr to the transmembrane protein band 3 and the following phosphorylation of the protein catalyzed by the Src-like kinase c-Fgr. The ability of the chimeric phospho-peptide to enter cells has been demonstrated by confocal microscopy analysis.

Src Homology-2 Domains Protect Phosphotyrosyl Residues against Enzymatic Dephosphorylation

The SH2 domain of c-Fgr (class 1A) has been expressed inE. colias GST fusion protein and tested for its ability to prevent the dephosphorylation of a variety of phosphotyrosyl (poly)peptides by three distinct protein tyrosine phosphatases (TC-PTPase, YOP, and Low Mr PTPase). Dephosphorylation of HS1 protein and of a derived phosphopeptide, HS1(388-402), exhibiting the motif selected by class 1A SH2 domains is inhibited in a dose dependent manner with full inhibition promoted by a 2- to 3-molar excess of GST/SH2 domain irrespective of either the nature or the amount of phosphatase used. The IC50values for inhibition of these and other phosphotyrosyl substrates roughly correlates with their expected affinity for class 1A SH2 domain. Inhibition is partially reversed by the addition of D-myo-inositol 1,4,5-triphosphate, which competes for the binding to the SH2 domains. Our data on one side show that additional mechanism(s) besides mere competition must assist PTPases to dissociate SH2-PTyr complexes and on the other suggest a role for SH2 domains in protecting phosphotyrosyl residues from premature dephosphorylation.

Specific Stimulation of c‐Fgr Kinase by Tyrosine‐Phosphorylated (Poly)Peptides

Hematopoietic lineage cell-specific HS1 protein is converted into a substrate for c-Fgr by previous Syk-mediated phosphorylation, at site(s) that bind to the SH2 domain of c-Fgr [Ruzzene, M., Brunati, A. M., Marin, O., Donella-Deana, A. & Pinna, L. A. (1996) Biochemistry 35, 5327-5332]. Here we show that a phosphopeptide derived from one such site, HS1-(320-329)-phosphopeptide (PEGDYpEEVLE), enhances up to tenfold, in a dose-dependent manner, the catalytic activity of c-Fgr either assayed with peptide substrates or evaluated as intermolecular autophosphorylation of c-Fgr itself. The dephosphorylated HS1-(320-329)-peptide is totally ineffective, while the stimulatory efficacy of other phosphopeptides derived from the polyoma virus middle T antigen-(393-402) sequence, c-Src, and c-Fgr autophosphorylation sites, and the C-terminal c-Src site (Tyr527) is variable and correlates reasonably well with the predicted affinity for the c-Fgr SH2 domain. Stimulation of c-Fgr catalytic activity is also promoted by the full-length HS1 protein, previously tyrosine phosphorylated by Syk, and is accounted for by an increased Vmax while the Km values are unchanged. If the normal activator of c-Fgr kinase, Mg2+, is replaced by Mn2+, stimulation by HS1-(320-329)-phosphopeptide is still observable with peptide substrates, while autophosphorylation is, in contrast, inhibited by the phosphopeptide. These findings, in conjunction with the ability of previously autophosphorylated c-Fgr to be stimulated by HS1-(320-329)-phosphopeptide, support the view that stimulation of c-Fgr by phosphopeptide is not or is not entirely a consequence of increased autophosphorylation. Interestingly, neither Syk and C-terminal Src kinase nor three other members of the Src family (Lyn, Lck, and Fyn) are susceptible to stimulation by phosphopeptide, as observed with c-Fgr. These data support the notion that c-Fgr undergoes a unique mechanism of activation promoted by tyrosine-phosphorylated polypeptide that binds to its SH2 domain. This suggests that such a mode of regulation is peculiar of protein-tyrosine kinases committed to the secondary phosphorylation of sequentially phosphorylated proteins.

Role of tyrosine phosphorylation of HS1 in B cell antigen receptor-mediated apoptosis.

The 75-kD HS1 protein is highly tyrosine-phosphorylated during B cell antigen receptor (BCR)-mediated signaling. Owing to low expression of HS1, WEHI-231-derived M1 cells, unlike the parental cells, are insensitive to BCR-mediated apoptosis. Here, we show that BCR-associated tyrosine kinases Lyn and Syk synergistically phosphorylate HS1, and that Tyr-378 and Tyr-397 of HS1 are the critical residues for its BCR-induced phosphorylation. In addition, unlike wild-type HS1, a mutant HS1 carrying the mutations Phe-378 and Phe-397 was unable to render M1 cells sensitive to apoptosis. Wild-type HS1, but not the mutant, localized to the nucleus under the synergy of Lyn and Syk. Thus, tyrosine phosphorylation of HS1 is required for BCR-induced apoptosis and nuclear translocation of HS1 may be a prerequisite for B cell apoptosis.

Restoration of surface IgM-mediated apoptosis in an anti-IgM-resistant variant of WEHI-231 lymphoma cells by HS1, a protein-tyrosine kinase substrate.

The HS1 protein is one of the major substrates of non-receptor-type protein-tyrosine kinases and is phosphorylated immediately after crosslinking of the surface IgM on B cells. The mouse B-lymphoma cell line WEHI-231 is known to undergo apoptosis upon crosslinking of surface IgM by anti-IgM antibodies. Variants of WEHI-231 that were resistant to anti-IgM-induced apoptosis expressed dramatically reduced levels of HS1 protein. Expression of the human HS1 protein from an expression vector introduced into one of the variant cell lines restored the sensitivity of the cells to apoptosis induced by surface IgM crosslinking. These results suggest that HS1 protein plays a crucial role in the B-cell antigen receptor-mediated signal transduction pathway that leads to apoptosis.

Antigen-receptor induced clonal expansion and deletion of lymphocytes are impaired in mice lacking HS1 protein, a substrate of the antigen-receptor-coupled tyrosine kinases.

HS1, an intracellular protein expressed specifically in hematopoietic cells, is rapidly tyrosine phosphorylated after cross-linking of antigen receptors on B and T lymphocytes, implicating involvement of this molecule in the signal transduction pathways from the antigen receptors as a substrate of membrane-associated tyrosine kinase(s). The development of lymphoid cells in HS1-deficient mice, generated through gene targeting, appeared normal. However, antibody production to T-independent antigen and proliferative responses of splenic B and T cells after cross-linking of the antigen receptors were impaired in these mutant mice. Furthermore, B cells in the peritoneal cavity of the mutant mice were resistant to multivalent cross-linking of the antigen receptor, which causes apoptosis of such cells in normal mice. Crossing the HS1-deficient mice with the mice harboring transgenes encoding alpha and beta chains of T-cell antigen receptor against a male H-Y antigen resulted in a progeny that demonstrated a significantly impaired ability of thymic negative selection. These results indicate that HS1 is a novel molecule involved in the antigen-receptor-derived signaling pathways and plays important roles not only in clonal expansion, but also in clonal deletion of B and T cells.

Identification of HS1 protein as a major substrate of protein-tyrosine kinase(s) upon B-cell antigen receptor-mediated signaling.

Crosslinking of membrane-bound immunoglobulins, which are B-cell antigen receptors, causes proliferation and differentiation of B cells or inhibition of their growth. The receptor-mediated signaling involves tyrosine phosphorylation of cellular proteins and rapid activation of Src-like kinases. The amino acid sequences of five proteolytic peptides of p75, a major substrate of protein-tyrosine(s) in the signaling, showed that p75 is the human HS1 gene product. The HS1 gene is expressed specifically in hematopoietic cells and encodes p75HS1, which carries both helix-turn-helix and Src homology 3 motifs. p75HS1 showed rapid tyrosine phosphorylation and association with a Src-like kinase, Lyn, after crosslinking of membrane-bound IgM. Thus, p75HS1 may be an important substrate of Lyn and possibly other protein-tyrosine kinases upon B-cell antigen receptor-mediated signaling.