Hematopoietic Cell-specific Lyn Substrate Research Articles

Identification of the immune-related biomarkers in Behcet’s disease by plasma proteomic analysis

BackgroundThis study aimed to investigate the expression profile of immune response-related proteins of Behcet’s disease (BD) patients and identify potential biomarkers for this disease.MethodsPlasma was collected from BD patients and healthy controls (HC). Immune response-related proteins were measured using the Olink Immune Response Panel. Differentially expressed proteins (DEPs) were used to construct prediction models via five machine learning algorithms: naive Bayes, support vector machine, extreme gradient boosting, random forest, and neural network. The prediction performance of the five models was assessed using the area under the curve (AUC) value, recall (sensitivity), specificity, precision, accuracy, F1 score, and residual distribution. Subtype analysis of BD was performed using the consensus clustering method.ResultsProteomics results showed 43 DEPs between BD patients and HC (P < 0.05). These DEPs were mainly involved in the Toll-like receptor 9 and NF-κB signaling pathways. Five models were constructed using DEPs [interleukin 10 (IL10), Fc receptor like 3 (FCRL3), Mannan-binding lectin serine peptidase 1 (MASP1), NF2, moesin-ezrin-radixin like (MERLIN) tumor suppressor (NF2), FAM3 metabolism regulating signaling molecule B (FAM3B), and O-6-methylguanine-DNA methyltransferase (MGMT)]. Among these models, the neural network model showed the best performance (AUC = 0.856, recall: 0.692, specificity: 0.857, precision: 0.900, accuracy: 0.750, F1 score: 0.783). BD patients were divided into two subtypes according to the consensus clustering method: one with high disease activity in association with higher expression of tripartite motif-containing 5 (TRIM5), SH2 domain-containing 1A (SH2D1A), phosphoinositide-3-kinase adaptor protein 1 (PIK3AP1), hematopoietic cell-specific Lyn substrate 1 (HCLS1), and DNA fragmentation factor subunit alpha (DFFA) and the other with low disease activity in association with higher expression of C–C motif chemokine ligand 11 (CCL11).ConclusionsOur study not only revealed a distinctive immune response-related protein profile for BD but also showed that IL10, FCRL3, MASP1, NF2, FAM3B, and MGMT could serve as potential immune biomarkers for this disease. Additionally, a novel molecular disease classification model was constructed to identify subsets of BD.

3D-STED Super-Resolution Microscopy Reveals Distinct Nanoscale Organization of the Hematopoietic Cell-Specific Lyn Substrate-1 (HS1) in Normal and Leukemic B Cells.

HS1, the hematopoietic homolog of cortactin, acts as a versatile actin-binding protein in leucocytes. After phosphorylation, it is involved in GTPase and integrin activation, and in BCR, TCR, and CXCR4 downstream signaling. In normal and leukemic B cells, HS1 is a central cytoskeletal interactor and its phosphorylation and expression are prognostic factors in chronic lymphocytic leukemia (CLL) patients. We here introduce for the first time a super-resolution imaging study based on single-cell 3D-STED microscopy optimized for revealing and comparing the nanoscale distribution of endogenous HS1 in healthy B and CLL primary cells. Our study reveals that the endogenous HS1 forms heterogeneous nanoclusters, similar to those of YFP-HS1 overexpressed in the leukemic MEC1 cell line. HS1 nanoclusters in healthy and leukemic B cells form bulky assemblies at the basal sides, suggesting the recruitment of HS1 for cell adhesion. This observation agrees with a phasor-FLIM-FRET and STED colocalization analyses of the endogenous MEC1-HS1, indicating an increased interaction with Vimentin at the cell adhesion sites. In CLL cells isolated from patients with poor prognosis, we observed a larger accumulation of HS1 at the basal region and a higher density of HS1 nanoclusters in the central regions of the cells if compared to good-prognosis CLL and healthy B cells, suggesting a different role for the protein in the cell types analyzed. Our 3D-STED approach lays the ground for revealing tiny differences of HS1 distribution, its functionally active forms, and colocalization with protein partners.

Identification of microRNAs and their Endonucleolytic Cleavaged target mRNAs in colorectal cancer

BackgroundColorectal cancer (CRC) ranks the third among the most common malignancies globally. It is well known that microRNAs (miRNAs) play vital roles in destabilizing mRNAs and repressing their translations in this disease. However, the mechanism of miRNA-induced mRNA cleavage remains to be investigated.MethodIn this study, high-throughput small RNA (sRNA) sequencing was utilized to identify and profile miRNAs from six pairs of colorectal cancer tissues (CTs) and adjacent tissues (CNs). Degradome sequencing (DS) was employed to detect the cleaved target genes. The Database for Annotation, Visualization and Integrated Discovery (DAVID) software was used for GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis.ResultsIn total, 1278 known miRNAs (clustered into 337 families) and 131 novel miRNAs were characterized in the CT and CN libraries, respectively. Of those, 420 known and eight novel miRNAs were defined as differentially expressed miRNAs (DEmiRNAs) by comparing the expression levels observed in the CT and CN libraries. Furthermore, through DS, 9685 and 202 potential target transcripts were characterized as target genes for 268 known and 33 novel miRNAs, respectively. It was further predicted that a total of 264 targeted genes for the 85 DEmiRNAs are involved in proteoglycans in cancer and the AMP-activated protein kinase signaling pathway. After systemic analysis of prognosis-related miRNA targets in those cancer-related signal pathways, we found that two targets ezrin (EZR) and hematopoietic cell-specific Lyn substrate 1 (HCLS1) had the potential prognostic characteristics with CRC regarding over survival (OS) or recurrence.ConclusionIn total, we found that endonucleolytic miRNA-directed mRNA cleavage occurs in CRC. A number of potential genes targeted by CRC-related miRNAs were identified and some may have the potential as prognosis markers of CRC. The present findings may lead to an improved better appreciation of the novel interaction mode between miRNAs and target genes in CRC.

Hematopoietic cell-specific lyn substrate (HCLS1 or HS1): A versatile actin-binding protein in leukocytes.

Leukocytes are constantly produced in the bone marrow and released into the circulation. Many different leukocyte subpopulations exist that exert distinct functions. Leukocytes are recruited to sites of inflammation and combat the cause of inflammation via many different effector functions. Virtually all of these processes depend on dynamic actin remodeling allowing leukocytes to adhere, migrate, phagocytose, and release granules. However, actin dynamics are not possible without actin-binding proteins (ABP) that orchestrate the balance between actin polymerization, branching, and depolymerization. The homologue of the ubiquitous ABP cortactin in hematopoietic cells is hematopoietic cell-specific lyn substrate-1, often called hematopoietic cell-specific protein-1 (HCLS1 or HS1). HS1 has been reported in different leukocytes to regulate Arp2/3-dependent migration. However, more evidence is emerging that HS1 functions go far beyond just being a direct actin modulator. For example, HS1 is important for the activation of GTPases and integrins, and mediates signaling downstream of many receptors including BCR, TCR, and CXCR4. In this review, we summarize current knowledge on HS1 functions and discuss them in a pathophysiologic context.

Cortactin stabilization of actin requires actin-binding repeats and linker, is disrupted by specific substitutions, and is independent of nucleotide state

The actin-binding protein cortactin promotes the formation and maintenance of actin-rich structures, including lamellipodial protrusions in fibroblasts and neuronal dendritic spines. Cortactin cellular functions have been attributed to its activation of the Arp2/3 complex, which stimulates actin branch nucleation, and to its recruitment of Rho family GTPase regulators. Cortactin also binds actin filaments and significantly slows filament depolymerization, but the mechanism by which it does so and the relationship between actin binding and stabilization are unclear. Here we elucidated the cortactin regions that are necessary and sufficient for actin filament binding and stabilization. Using actin cosedimentation assays, we found that the cortactin repeat region binds actin but that the adjacent linker region is required for binding with the same affinity as full-length cortactin. Using total internal reflection fluorescence microscopy to measure the rates of single filament actin depolymerization, we observed that cortactin-actin interactions are sufficient to stabilize actin filaments. Moreover, conserved charged residues in repeat 4 were necessary for high-affinity actin binding, and substitution of these residues significantly impaired cortactin-mediated actin stabilization. Cortactin bound actin with higher affinity than did its paralog, hematopoietic cell-specific Lyn substrate 1 (HS1), and the effects on actin stability were specific to cortactin. Finally, cortactin stabilized ADP-actin filaments, indicating that the stabilization mechanism does not depend on the actin nucleotide state. Together, these results indicate that cortactin binding to actin is necessary and sufficient to stabilize filaments in a concentration-dependent manner, specific to conserved residues in the cortactin repeats, and independent of the actin nucleotide state.

Evangelia Kranias: The Mother of Phospholamban.

After a sheltered childhood, Evangelia (Litsa) Kranias defied her parents’ wishes and boarded a ship from Athens to the United States, having been accepted at the University of Chicago and cautioned that how she performed would determine whether other foreign undergraduates would be admitted. “When I think about it, I am amazed at the courage and determination of that 18-year-old Greek girl!,” says Kranias. “My mother wrote to me every other day and I did the same. She often said, ‘If we were to lay down our letters, they would form a bridge between Athens and New York.’” Kranias has kept that bridge intact throughout her career, directing the molecular cardiology group of the Biomedical Research Foundation of the Academy of Athens while maintaining multiple positions at the University of Cincinnati—Director of Cardiovascular Biology in the Department of Pharmacology and Cell Biophysics, codirector of the Cardiovascular Center of Excellence, and Hanna Professor of Cardiology. Evangelia Kranias Shortly after joining the faculty in Cincinnati, Kranias recognized the importance of the recently discovered PLN (phospholamban) molecule in the regulation of calcium cycling through the sarcoplasmic reticulum and in the overall regulation of cardiac function. Kranias and John Solaro soon provided the first evidence that PLN is phosphorylated in the intact heart in “fight or flight” situations.1 After generating a series of genetically altered mouse models, her laboratory revealed that, rather than stimulating contractility, PLN inhibits the calcium pump in the sarcoplasmic reticulum and this inhibition is relieved when the molecule is phosphorylated under β-agonist stimulation.2 Surprisingly, PLN-deficient mice exhibited enhanced myocardial performance without changes in heart rate, raising the possibility of PLN as a target to improve calcium cycling and improve function in patients with heart failure. In the clinical arena, Kranias’ laboratory showed that heart failure is associated with …

HS1 deficiency impairs neutrophil recruitment in vivo and activation of the small GTPases Rac1 and Rap1.

Neutrophil extravasation is a critical step of the innate immune system's response to inflammation. This multistep process is tightly regulated by adhesion and signaling molecules in the endothelium and neutrophils. Activation of the β2 integrin LFA-1 is critical for adhesion of leukocytes to postcapillary venules. This step requires coordinated activation of signaling pathways in chemokine-stimulated neutrophils, including GTPase activation and cytoskeletal remodeling, leading to conformational changes in LFA-1. Hematopoietic cell-specific lyn substrate 1 (HS1) is a cortactin-related and leukocyte-specific actin-binding protein (ABP) that regulates several processes in various immune cells. It has been shown in vitro that HS1 is important for neutrophil chemotaxis and transendothelial migration of NK cells, but its role in neutrophil extravasation in vivo has not been investigated yet. Intravital microscopy of CXCL1-stimulated cremaster venules revealed an increased rolling velocity and reduced neutrophil adhesion and transmigration in HS1 knockout (KO) mice. CXCL1-induced rapid neutrophil arrest in vivo and adhesion under flow conditions in vitro were also reduced significantly. Whereas random motility of neutrophils was unaffected, chemotaxis toward a CXCL1 gradient was reduced in the absence of HS1. Further analysis of the underlying mechanisms demonstrated that HS1 controls CXCL1-induced activation of the small GTPases Ras-related C3 botulinum toxin substrate 1 (Rac1) and Ras-related protein 1 (Rap1), thus supporting LFA-1-mediated neutrophil adhesion. Importantly, with the use of Rac1 KO neutrophils, we could show that Rac1 acts upstream of Rap1. Our results establish HS1 as an important regulator of proper Rac1 and Rap1 activation and neutrophil extravasation.

Wnt5a Induces ROR1 to Complex with HS1, Which Undergoes Tyrosine Phosphorylation and Contributes to Planar-Cell-Polarity Migration in Chronic Lymphocytic Leukemia

ROR1 (Receptor tyrosine kinase-like orphan receptor 1) is an evolutionarily conserved, oncoembryonic antigen that is expressed on CLL cells, but not on normal postpartum tissues. In prior studies we found that ROR1 was a receptor for Wnt5a, which could activate RhoGTPases and enhance chemokine-directed leukemia-cell migration. Moreover, Wnt5a-enhanced migration could be inhibited by cirmtuzumab, a first-in-class humanized mAb specific for a functional epitope of the ROR1 extracellular domain; this mAb is undergoing phase I clinical testing in patients with CLL. However, it is not known whether ROR1 contributes to cell migration only through its recruitment/activation of guanine exchange factors (GEFs) or whether it also can complex with proteins that directly interact with the actin cytoskeleton. We performed mass spectrometry-based proteomics to interrogate immune-precipitates of Wnt5a-activated ROR1 from CLL cells and identified HS1 (or HCLS1, hematopoietic cell-specific Lyn substrate 1), a conserved cytoplasmic protein that can interact with the actin cytoskeleton. Moreover, tyrosine phosphorylation of HS1 has been found in prior studies to be associated with enhanced leukemia-cell migration and adverse prognosis in CLL, although the stimuli/mechanism(s) contributing to HS1 activation were unknown. We validated that Wnt5a induced the association of ROR1 with HS1 in primary CLL cells via co-immunoprecipitation studies and immunoblot analyses. Moreover, we found that Wnt5a could induce tyrosine phosphorylation of HS1 in CLL cells previously deprived of Wnt5a. On the other hand, treatment of CLL cells with the anti-ROR1 mAb cirmtuzumab inhibited the capacity of Wnt5a to induce the association of ROR1 with HS1 or tyrosine phosphorylation of HS1. We speculated that the Src-homology 3 (SH3) domain of HS1 allowed it to dock onto the cytoplasmic proline-rich domain (PRD) of ROR1. Consistent with this notion we found that a mutant-form of ROR1 lacking the PRD was unable to complex with HS1 or facilitate F-actin polymerization in response to Wnt5a. Accordingly, we used site-directed mutagenesis to introduce single amino acid substitutions of proline (P) to alanine (A) in the P-X-X-P- motifs of the ROR1 PRD at positions 784, 808, 826, 841, or 850. Each mutant form of ROR1, except for P841A-ROR1, could complex with HS1, permit tyrosine phosphorylation of HS1, and allow for enhanced leukemia-cell motility. Collectively, these data indicate that Wnt5a can induce HS1 to dock at P841 in the PRD of ROR1 and undergo tyrosine phosphorylation, thereby enhancing F-actin polymerization and planar-cell-polarity leukemia-cell migration. Moreover, we found that cirmtuzumab can inhibit activation of HS1 and F-actin polymerization in response to Wnt5a, potentially accounting in part for its capacity to inhibit Wnt5a-enhanced leukemia-cell migration. DisclosuresKipps:Gilead: Consultancy, Honoraria, Speakers Bureau; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria.

Phenotyping and auto-antibody production by liver-infiltrating B cells in primary sclerosing cholangitis and primary biliary cholangitis

Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are immune-mediated biliary diseases that demonstrate prominent and restricted genetic association with human leukocyte antigen (HLA) alleles. In PBC, anti-mitochondrial antibodies (AMA) are specific and used as diagnostic biomarkers. PSC-relevant auto-antibodies remain controversial despite a distinct HLA association that mirrors archetypical auto-antigen driven disorders. Herein, we compared antibody-secreting B cells (ASCs) in PSC and PBC liver explants to determine if liver-infiltrating ASCs represent an opportune and novel source of disease-relevant auto-antibodies. Using enzymatic digestion and mechanical disruption, liver mononuclear cells (LIMCs) were isolated from fresh PSC and PBC explants and plasmablast (CD19+CD27+CD38hiCD138−) and plasma cell (CD19+CD27+CD38hiCD138+) ASCs were enumerated by flow cytometry. We observed 45-fold fewer plasma cells in PSC explants (n = 9) compared to PBC samples (n = 5, p < 0.01) and 10-fold fewer IgA-, IgG- and IgM-positive ASCs (p < 0.05). Liver-infiltrating ASCs from PSC and PBC explants were functional and produced similar concentrations of IgA, IgG and IgM following 2 weeks of culture. Antibody production by PBC ASCs (n = 3) was disease-specific as AMA to pyruvate dehydrogenase complex E2 subunit (PDC-E2) was detected by immunostaining, immunoblotting and ELISA. Antibody profiling of PSC supernatants (n = 9) using full-length recombinant human protein arrays (Cambridge Protein Arrays) revealed reactivities to nucleolar protein 3 (5/9) and hematopoietic cell-specific Lyn substrate 1 (3/9). Array analysis of PBC supernatants (n = 3) detected reactivities to PDC-E2 and hexokinase 1 (3/3). In conclusion, we detected unique frequencies of liver-infiltrating ASCs in PSC and PBC and in so doing, highlight a feasible approach for understanding disease-relevant antibodies in PSC.

Identification of RNA-binding Proteins in Macrophages by Interactome Capture

Pathogen components, such as lipopolysaccharides of Gram-negative bacteria that activate Toll-like receptor 4, induce mitogen activated protein kinases and NFκB through different downstream pathways to stimulate pro- and anti-inflammatory cytokine expression. Importantly, post-transcriptional control of the expression of Toll-like receptor 4 downstream signaling molecules contributes to the tight regulation of inflammatory cytokine synthesis in macrophages. Emerging evidence highlights the role of RNA-binding proteins (RBPs) in the post-transcriptional control of the innate immune response. To systematically identify macrophage RBPs and their response to LPS stimulation, we employed RNA interactome capture in LPS-induced and untreated murine RAW 264.7 macrophages. This combines RBP-crosslinking to RNA, cell lysis, oligo(dT) capture of polyadenylated RNAs and mass spectrometry analysis of associated proteins. Our data revealed 402 proteins of the macrophage RNA interactome including 91 previously not annotated as RBPs. A comparison with published RNA interactomes classified 32 RBPs uniquely identified in RAW 264.7 macrophages. Of these, 19 proteins are linked to biochemical activities not directly related to RNA. From this group, we validated the HSP90 cochaperone P23 that was demonstrated to exhibit cytosolic prostaglandin E2 synthase 3 (PTGES3) activity, and the hematopoietic cell-specific LYN substrate 1 (HCLS1 or HS1), a hematopoietic cell-specific adapter molecule, as novel macrophage RBPs. Our study expands the mammalian RBP repertoire, and identifies macrophage RBPs that respond to LPS. These RBPs are prime candidates for the post-transcriptional regulation and execution of LPS-induced signaling pathways and the innate immune response. Macrophage RBP data have been deposited to ProteomeXchange with identifier PXD002890.

Mesenchymal stromal cells of osteosarcoma patients do not show evidence of neoplastic changes during long-term culture.

BackgroundIn vitro expanded mesenchymal stromal cells (MSCs) are increasingly used as experimental cellular therapy. However, there have been concerns regarding the safety of their use, particularly with regard to possible oncogenic transformation. MSCs are the hypothesized precursor cells of high-grade osteosarcoma, a tumor with often complex karyotypes occurring mainly in adolescents and young adults.MethodsTo determine if MSCs from osteosarcoma patients could be predisposed to malignant transformation we cultured MSCs of nine osteosarcoma patients and five healthy donors for an average of 649 days (range 601–679 days). Also, we compared MSCs derived from osteosarcoma patients at diagnosis and from healthy donors using genome wide gene expression profiling.ResultsUpon increasing passage, increasing frequencies of binucleate cells were detected, but no increase in proliferation suggestive of malignant transformation occurred in MSCs from either patients or donors. Hematopoietic cell specific Lyn substrate 1 (HLCS1) was differentially expressed (fold change 0.25, P value 0.0005) between MSCs of osteosarcoma patients (n = 14) and healthy donors (n = 9).ConclusionsThis study shows that although HCLS1 expression was downregulated in MSCs of osteosarcoma patients and binucleate cells were present in both patient and donor derived MSCs, there was no evidence of neoplastic changes to occur during long-term culture.Electronic supplementary materialThe online version of this article (doi:10.1186/s13569-015-0031-1) contains supplementary material, which is available to authorized users.

The non-surgical management of a patient with Kostmann syndrome-associated periodontitis: a case report

Kostmann syndrome is a rare, congenital immunological disorder caused by a mutation of the hematopoietic cell-specific LYN substrate 1-associated protein X1. These patients pose a unique challenge to the dental practitioner due to the severe oral infections that are often seen in this population. The patient described in this report is a 16-year-old female with Kostmann syndrome-associated periodontitis. The treatment consisted of scaling and root planing performed in conjunction with subgingival irrigation with povidone-iodine solution. This report details how Kostmann syndrome-associated periodontitis can be successfully treated and maintained long-term, using non-surgical treatment modalities and local antimicrobial therapy.

Aberrant G-CSFR Signaling in Congenital Neutropenia

Clinical observations revealed that congenital neutropenia (CN) patients harboring either ELANE or HAX mutations have similar bone marrow morphology, responses to G-CSF therapy, requirements of G-CSF dosages, and the risk of developing leukemia. Therefore, we suggested a common pathomechanism of defective G-CSFR-triggered granulopoiesis downstream of both mutated genes in these patients. We identified severely diminished expression and functions of the transcription factors lymphoid enhancer binding factor-1 (LEF-1) and C/EBPa in myeloid cells of CN patients, in comparison to healthy individuals and patients with cyclic neutropenia (CyN). LEF-1 expression was abrogated in patients harboring either ELANE or HAX1 mutations, which suggested LEF-1 as a possible common candidate factor for defective G-CSFR signaling. We further identified a mechanism of the diminished LEF-1 expression downstream of HAX1 or ELANE mutations. HAX1 is HCLS1-Associated protein X1. HCLS1 is Hematopoietic Cell-Specific Lyn Substrate 1. We found that HCLS1 protein is expressed at high levels in human myeloid cells and is phosphorylated upon stimulation with G-CSF. HCLS1 interacted with LEF-1 protein, inducing nuclear translocation of LEF-1, LEF-1 autoregulation, C/EBPa activation, and granulocytic differentiation. In CN patients with HAX1 mutations, we found profound defects in the G-CSF-triggered phosphorylation of HCLS1, subsequently leading to abrogated nuclear transport and autoregulation of LEF-1. In CN patients with ELANE mutations we detected severely reduced levels of the natural inhibitor of neutrophil elastase (NE), and secretory leukocyte protease inhibitor (SLPI). We demonstrated the important role of SLPI in myeloid differentiation by activation of Erk1/2 phosphorylation and subsequent phErk1/2-triggered tyrosine phosphorylation and activation of the LEF-1 protein. Therefore, the direct link between ELANE mutations and diminished LEF-1 expression was established: in these patients LEF-1 protein expression is diminished due to the reduced levels of SLPI. We further evaluated how G-CSF treatment overcomes maturation arrest of granulopoiesis in CN patients despite the absence of LEF-1 and C/EBPa in myeloid cells. We identified nicotinamide phosphoribosyltransferase (NAMPT) as an essential enzyme mediating G-CSF-triggered granulopoiesis in healthy individuals and in CN patients. Treatment of healthy individuals with G-CSF resulted in upregulation of NAMPT levels in myeloid cells and in plasma. NAMPT and NAD+ amounts were even more dramatically elevated by G-CSF treatment of CN individuals. The molecular events triggered by NAMPT included elevation of NAD+, NAD+-dependent activation of protein deacetylase sirtuin-1 (SIRT1), binding of SIRT1 to the myeloid specific transcription factors C/EBPα and C/EBPβ, and activation of these transcription factors. In CN patients, C/EBPα expression is severely diminished; therefore “steady-state” granulopoiesis could not be activated. G-CSF treatment induces expression of C/EBPβ in these patients via NAMPT and SIRT1 and operated via the “emergency” pathway. We also investigated the patterns of acquisition of leukemia-associated-mutations in 31 CN patients developing leukemia using next-generation DNA deep sequencing. Intriguingly, 20 of the 31 patients (64.5%) demonstrated mutations within RUNX1. The majority of patients with RUNX1 mutations (85%) had acquired CSF3R mutations. Other leukemia-associated mutations in the patients with RUNX1 mutations were found infrequently. Cytogenetics of the leukemic cells revealed that 10 patients with RUNX1 mutations developed monosomy 7, and six patients had trisomy 21. Single cell analysis in two patients revealed that RUNX1 and CSF3R mutations were segregated in the same malignant clone. Functional studies demonstrated proliferative advantage of CD34+ cells transduced with mutated RUNX1 and CSF3R. By analysis of the leukemogenic role of the defective G-CSFR signaling in CN patients we identified a significant and sustained elevation in the levels of phospho-STAT5 in hematopoietic CD34+ cells of CN patients which were even higher in CN/ acute myeloid leukemia patients. The other possible reason for the leukemogenic transformation could be elevated NAMPT/SIRT-triggered deacetylation of tumor supressor protein p53, proto-oncogene FOXO3a and Akt proteins. Disclosures:No relevant conflicts of interest to declare.

Targeting the LYN/HS1 signaling axis in chronic lymphocytic leukemia

AbstractHS1 (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

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.

Interactions among HCLS1, HAX1 and LEF-1 proteins are essential for G-CSF–triggered granulopoiesis

We found that hematopoietic cell-specific Lyn substrate 1 (HCLS1 or HS1) is highly expressed in human myeloid cells and that stimulation with granulocyte colony-stimulating factor (G-CSF) leads to HCLS1 phosphorylation. HCLS1 binds the transcription factor lymphoid-enhancer binding factor 1 (LEF-1), transporting LEF-1 into the nucleus upon G-CSF stimulation and inducing LEF-1 autoregulation. In patients with severe congenital neutropenia, inherited mutations in the gene encoding HCLS1-associated protein X-1 (HAX1) lead to profound defects in G-CSF-triggered phosphorylation of HCLS1 and subsequently to reduced autoregulation and expression of LEF-1. Consistent with these results, HCLS1-deficient mice are neutropenic. In bone marrow biopsies of the majority of tested patients with acute myeloid leukemia, HCLS1 protein expression is substantially elevated, associated with high levels of G-CSF synthesis and, in some individuals, a four-residue insertion in a proline-rich region of HCLS1 protein known to accelerate intracellular signaling. These data demonstrate the importance of HCLS1 in myelopoiesis in vitro and in vivo.

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.

New G-CSF-Dependent Signaling Pathway and Its Role In Patients with Severe Congenital Neutropenia and Acute Myeloid Leukemia

AbstractAbstract 385We demonstrated that stimulation of hematopoietic CD34+ cells with granulocyte-colony stimulating factor (G-CSF) leads to phosphorylation of hematopoietic cell-specific Lyn substrate 1 (HCLS1) protein and HCLS1 interaction with G-CSF receptor-associated tyrosine kinases Lyn and Syk. Activated HCLS1 binds to lymphoid-enhancer factor 1 (LEF-1) protein, inducing LEF-1-dependent autoregulation of the LEF-1 gene promoter. Transcription factor LEF-1 is a downstream effector of Wnt signaling and a master regulator of myeloid differentiation. In patients with severe congenital neutropenia (CN), inherited mutations in the HCLS1-associated protein × 1 (HAX1) lead to profound defects in the G-CSF-triggered expression and phosphorylation of HCLS1 protein and subsequently, to reduced expression of LEF-1 and abrogated granulopoiesis. Using immunoprecipitation assay, we identified endogenous HAX1 protein in the complex with LEF-1 and HCLS1 proteins. HAX1 is known as a mitochondrial protein, however using cell fractionation experiments of different myeloid cell lines (NB4, HL60, THP-1) and of primary CD34+ cells we identified endogenous HAX1 protein in different intracellular compartments. Thus, HAX1 protein was expressed in mitochondria, cytoplasm, nucleus, cytosol and in membranous fraction. Expression levels of HAX1 protein varied between different cell lines and between maturation stage of the cells. In vitro inhibition of HCLS1 or HAX1 by shRNA severely disrupted granulocytic differentiation of CD34+ cells, due to a lack of LEF-1 and C/EBPa protein expression and subsequently abolished activation of LEF-1 and C/EBPa gene promoters. Intriguingly, LEF-1 in turn induced HCLS1 mRNA and protein expression by direct binding to the HCLS1 gene promoter and inhibition of LEF-1 in CD34+ cells leads to severe downregulation of HCLS1 protein levels. These findings demonstrated a reciprocal feed-back regulation between LEF-1 and HCLS1 proteins. Moreover, HCLS1-/- mice are neutropenic due to a lack of LEF-1 protein expression and defective in vitro G-CSF-triggered F-actin rearrangement in bone marrow myeloid progenitor cells, as compared to WT mice. At the same time, in bone marrow biopsies of 88.46 % of tested patients (46 out of 52 patients) with acute myeloid leukemia (AML), we found significantly elevated levels of HCLS1 protein. Inhibition of HCLS1 in the AML cell lines and primary blasts of AML patients resulted in reduced proliferation and increased apoptosis. Taken together, we described a novel G-CSFR downstream signaling pathway, with dose-dependent effects on myelopoiesis: diminished expression of HCLS1 and LEF-1 resulted in a “maturation arrest” of granulopoiesis and severe neutropenia, but hyperactivation of one of these proteins led to a hyperproliferation of myeloid progenitors and AML. Disclosures:No relevant conflicts of interest to declare.