B Lymphoid Tyrosine Kinase Research Articles

Molecular and clinical profiles of pediatric monogenic diabetes subtypes: comprehensive genetic analysis of 138 patients.

Single gene variants that give rise to neonatal diabetes mellitus (NDM), maturity onset diabetes of the young (MODY) and syndromic forms of diabetes mellitus (SDM) are responsible for 3.1-4.2% of all diabetes cases. This single-center study with a relatively larger sample size aimed to evaluate the clinical and genetic characteristics of Chinese children with suspected monogenic diabetes (MD) using next generation sequencing (NGS) methods. Data were collected from 1550 consecutive children diagnosed with diabetes/hyperglycemia at the Endocrinology Department of Children's Hospital of Nanjing Medical University from 2012 to 2023. The genotype and phenotype of 138 children with suspected MD were retrospectively analyzed. Among 138 children, 16, 97, and 25 patients with NDM, suspected MODY and SDM were assessed by NGS, with a pick-up rate of 87.5%, 57.8%, and 56%, respectively. In total, there was a high pick-up rate of MD, with 58% (80 of 138) among antibody-negative pediatric patients. Pathogenic variants were found in GCK, HNF1A, INS, KCNJ11, INSR, HNF4A, ABCC8, WFS1, ALMS1, HNF1B, BLK and ZFP57 genes with 13 novel variants in addition to 4 patients with CNVs. In this cohort, GCK-MODY was the leading cause and the mildest type of MODY. GCK-MODY displayed favorable lipid profile when compared to non-GCK-MODY and MODYX, which might be cardioprotective. Following an accurate genetic diagnosis of diabetes, 19 patients switched from insulin therapy to oral agents or lifestyle interventions. NGS tests helped to identify the precise etiology of monogenic diabetic patients which has implications for better individualized management.

Association of BLK and BANK1 gene polymorphisms with systemic lupus erythematous in Egyptian patients

This study examined the genotype, allelic frequencies (polymorphisms) in a sample of Egyptian patients and examined the relationship between disease activity in systemic lupus erythematosus (SLE) and the B lymphoid tyrosine kinase (BLK) and B-cell scaffold protein with ankyrin repeats 1 (BANK1) gene. This case control study involved 70 SLE patients and 40 subjects matched for age and sex as a control group. Clinical data were gathered from each participant, including SLE-related clinical activity indicators. Utilizing the restriction fragment length polymorphism (RFLP)-polymerase chain reaction (PCR), the single nucleotide polymorphisms (SNPs) BLK rs13277113G/A and BANK1 rs10516487G/A were assessed. The most prevalent genotype among the study participants was BLK rs13277113; G/G (57.1%), and BANK rs10516487; GG/ genotype (74.3%). There were no substantial variations in the incidence of genotype and allelic polymorphism between patients and controls (p>0.05). In the studied SLE patients, however, there was no significant association between both alleles (BANK rs10516487 gene alleles, G/G and G/A & BLK rs13277113, G/A, G/G &A/A) and the SLE disease activity score. While there was a significant association between BLK rs13277113 genotype alleles and age. In conclusion, BLK rs13277113 G/A and BANK1 rs10516487 G/A alleles showed no difference between SLE Egyptian patients compared to controls with no link between both genes and SLE disease activity.

Nanoformulation of dasatinib cannot overcome therapy resistance of pancreatic cancer cells with low LYN kinase expression

BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the most difficult to treat tumors. The Src (sarcoma) inhibitor dasatinib (DASA) has shown promising efficacy in preclinical studies of PDAC. However, clinical confirmation could not be achieved. Overall, our aim was to deliver arguments for the possible reinitiating clinical testing of this compound in a biomarker-stratifying therapy trial for PDAC patients. We tested if the nanofunctionalization of DASA can increase the drug efficacy and whether certain Src members can function as clinical predictive biomarkers.MethodsMethods include manufacturing of poly(vinyl alcohol) stabilized gold nanoparticles and their drug loading, dynamic light scattering, transmission electron microscopy, thermogravimetric analysis, Zeta potential measurement, sterile human cell culture, cell growth quantification, accessing and evaluating transcriptome and clinical data from molecular tumor dataset TCGA, as well as various statistical analyses.ResultsWe generated homo-dispersed nanofunctionalized DASA as an AuNP@PVA-DASA conjugate. The composite did not enhance the anti-growth effect of DASA on PDAC cell lines. The cell model with high LYN expression showed the strongest response to the therapy. We confirm deregulated Src kinetome activity as a prevalent feature of PDAC by revealing mRNA levels associated with higher malignancy grade of tumors. BLK (B lymphocyte kinase) expression predicts shorter overall survival of diabetic PDAC patients.ConclusionsNanofunctionalization of DASA needs further improvement to overcome the therapy resistance of PDAC. LYN mRNA is augmented in tumors with higher malignancy and can serve as a predictive biomarker for the therapy resistance of PDAC cells against DASA. Studying the biological roles of BLK might help to identify underlying molecular mechanisms associated with PDAC in diabetic patients.

Transcriptomic analysis identifies B-lymphocyte kinase as a therapeutic target for desmoplastic small round cell tumor cancer stem cell-like cells

Desmoplastic small round cell tumor (DSRCT) is an aggressive pediatric cancer caused by the EWSR1-WT1 fusion oncoprotein. The tumor is refractory to treatment with a 5-year survival rate of only 15–25%, necessitating the development of novel therapeutics, especially those able to target chemoresistant subpopulations. Novel in vitro cancer stem cell-like (CSC-like) culture conditions increase the expression of stemness markers (SOX2, NANOG) and reduce DSRCT cell line susceptibility to chemotherapy while maintaining the ability of DSRCT cells to form xenografts. To gain insights into this chemoresistant model, RNA-seq was performed to elucidate transcriptional alterations between DSRCT cells grown in CSC-like spheres and normal 2-dimensional adherent state. Commonly upregulated and downregulated genes were identified and utilized in pathway analysis revealing upregulation of pathways related to chromatin assembly and disassembly and downregulation of pathways including cell junction assembly and extracellular matrix organization. Alterations in chromatin assembly suggest a role for epigenetics in the DSRCT CSC-like state, which was further investigated with ATAC-seq, identifying over 10,000 differentially accessible peaks, including 4444 sphere accessible peaks and 6,120 adherent accessible peaks. Accessible regions were associated with higher gene expression, including increased accessibility of the CSC marker SOX2 in CSC-like culture conditions. These analyses were further utilized to identify potential CSC therapeutic targets, leading to the identification of B-lymphocyte kinase (BLK) as a CSC-enriched, EWSR1-WT1-regulated, druggable target. BLK inhibition and knockdown reduced CSC-like properties, including abrogation of tumorsphere formation and stemness marker expression. Importantly, BLK knockdown reduced DSRCT CSC-like cell chemoresistance, making its inhibition a promising target for future combination therapy.

Exploring the ceRNA Network in Multiple Sclerosis: Implications for B Cell Receptor Signaling Pathway and Therapeutic Targets

Introduction: Multiple sclerosis (MS) is the most common demyelinating disease of the central nervous system and the second cause of disability in adults. The exact cause and pathogenic mechanisms behind this disease have not been yet understood. Besides T cells, B cells play a role in MS pathogenesis which has received less attention. Method: In the current study, the expression of long non-coding RNAs and four genes of the B cell receptor signaling pathway (CR2, BLK, BLNK, and RASGRP3) were investigated in the GSE21942 microarray dataset. Then, miRNAs targeting these genes and lncRNAs were predicted using miRWalk and DIANA-LncBase databases. Finally, the ceRNA network was constructed using desired genes, lncRNAs, and predicted miRNAs. Results: Seven differentially expressed lncRNAs were identified that all had high expression in MS patients compared with control subjects. Moreover, the expression levels of desired genes (CR2, BLK, BLNK, and RASGRP3) were upregulated in MS patients. Finally, the ceRNA network including one lncRNA (MALAT1), two predicted miRNAs (hsa-miR-92a-3p and hsa-miR-548a-3p), and three genes (CR2, RASGRP3, and BLK) was constructed. In this network, hsa-miR-92a-3p and hsa-miR-548a-3p target CR2, RASGRP3, and BLK genes, and MALAT1 constrains the effect of these miRNAs on desired genes through sponging them. Conclusion: The constructed ceRNA network can be considered a novel mechanism of MS pathogenesis and may play a key role in MS development and progression by influencing the B cell receptor signaling pathway. Furthermore, each of the components of this network can be considered a potential therapeutic target.

SAT081 Maturity Onset Diabetes Of The Young (MODY) Due To A New BLK Gene Mutation

Abstract Disclosure: D. Taha: None. R. Thirunagari: None. A. Adhikari: None. Background: The BLK gene is a tyrosine protein kinase involved in cell proliferation and differentiation. It has been identified as a modulator of beta cell function and plays a role in the regulation of insulin synthesis and secretion. We describe the case of a patient presenting with prediabetes associated with a heterozygous c.1366 C>T of the BLK gene. Case Report: We report a 10 year old Asian female who presented with a hemoglobin A1c (HbA1c) level of 6.1% (4.0-5.7), within prediabetes range. Her weight was 44 kg (Z score 1.34), height 148.2 cm (Z score 1.52) and BMI was 20 kg/m2 (85th percentile; Z score 1.06). She had no acanthosis nigricans by exam and she was at early Tanner stage 2 of puberty. Initial evaluation revealed normal C-Peptide level of 2.1 ng/mL (0.9-6.9), normal insulin level of 21.1 mcUnits/mL (3 - 25). She had normal complete blood count and comprehensive metabolic profile, and normal thyroid function tests. Glutamic acid decarboxylase (GAD65) antibodies and islet cell antibodies were negative. She had an oral glucose tolerance test which revealed a fasting glucose level of 101 mg/dL and a 2-hour glucose level of 158 mg/dL. Lipid profile was normal. She was advised regarding healthy lifestyle changes and her HbA1c level was stable. She was lost to follow-up until 16 years of age when she presented with irregular menstrual cycles and was diagnosed with polycystic ovary syndrome. Her HbA1c was elevated at 6.3% with a normal insulin level. Cortisol and thyroid function tests were normal. Pancreatic antibodies including GAD65, IA-2, insulin antibodies, islet cell antibodies, and zinc transporter 8 antibodies, were negative. C-peptide level was normal at 4.3 ng/mL (1.1-4.4). Family history was significant for patient’s father and paternal grandmother having diabetes and taking Metformin. Patient’s father was diagnosed with diabetes at 13 years of age. Maturity onset diabetes of the young (MODY) panel/ sequencing and deletion/duplication analysis was performed at GeneDx and revealed that she is heterozygous for a c.1366 C>T; p.Arg456Cys (R456C) variant in exon 13 of the BLK gene. This variant has not been previously published as a pathogenic or benign variant to our knowledge. No additional variants were identified in any of the MODY genes on this panel (ABCC 8, APPL1, CEL, GCK, GLUD1, HADH, HNF1A, HNF 1B, HNF4A, INS, KCNJ11, KLF11, NEUROD1, PAX4, PDX1). Conclusion: Variants of the BLK gene have been reported in association with autosomal dominant MODY, type 11. We report a pediatric patient presenting with MODY prediabetes associated with a heterozygous c.1366 C>T; p.R456C variant in the BLK gene. To our knowledge, this variant has not been previously reported in MODY patients. We propose that this is a pathogenic variant and review this rare MODY form. Presentation: Saturday, June 17, 2023

Discovery of 2,5-diaminopyrimidine derivatives as the first series of selective monomeric degraders of B-lymphoid tyrosine kinase

B-lymphoid tyrosine kinase (BLK) is an important knot of B cell receptor signaling, and regulates the function and development of B cells subset. Dysfunction of BLK is correlated with autoimmune diseases and cancer. There is an urgent need to develop selective BLK modulators to facilitate the studies of BLK in biological processes. Herein, we report the discovery of a series of 2,5-diaminopyrimidine-based compounds capable of selectively degrading BLK. The optimized compounds 9–11 possess weak biochemical inhibitory activities against BLK, yet they effectively degrade BLK and show high selectivity for BLK over other structurally and functionally related SRC family and TEC family kinases. Furthermore, compounds 9 and 11 demonstrate potent inhibitory activities in several B-lymphoid cell lines. As the first series of effective and selective monomeric BLK degraders, compounds 9–11 serve as valuable tools for further investigation of the functions of BLK.

Pharmacophore based High Throughput Virtual Screening towards the Discovery of Novel BLK (B-lymphocyte kinase)- tyrosine Kinase Inhibitors

Abstract: Aim: LK (B-lymphocyte kinase) is a protein from the family SRC (Proto-oncogene tyrosine-protein kinase), an important cell signaling molecule that influences cellular response. The BLK tyrosine-protein kinase has been a potential target for cancer therapy. As a result, this could be an initial step toward the development of novel inhibitors to fight cancer. Materials and Methods: A homology model of human BLK tyrosine kinase was constructed using Phyre2. Active site prediction was done for the model using the CASTp server. High Throughput virtual screening was performed with the help of a ligand-based pharmacophore model of FDA (Food and Drug Administration) approved SRC tyrosine family kinase inhibitors using the PharmaGist and ZINCPharmer servers. The 250 novel compounds obtained were docked by a Python script-based method with Autodock Vina. To ensure drug safety, ADME/Tox (Absorption, Distribution, Metabolism, Elimination, Toxicity) analysis was performed for the molecules with the lowest binding energy. Six compounds that passed ADME/Tox analysis were again utilized to perform molecular docking with Autodock4. The active residues were then identified using PLIP [protein ligand interaction profiler]. Results and Conclusion: Six compounds passed ADME/Tox analysis. Based on the molecular docking analysis, the compound ZINC57306994 showed an increased binding affinity with the target BLK tyrosine kinase. The compound ZINC57306994 may serve as a lead molecule that could be developed into a potent BLK tyrosine kinase inhibitor. Keywords: Pharmacophore modeling, Molecular docking, BLK tyrosine kinase, Cancer.

B-lymphoid tyrosine kinase-mediated FAM83A phosphorylation elevates pancreatic tumorigenesis through interacting with β-catenin

Abnormal activation of Wnt/β-catenin-mediated transcription is closely associated with the malignancy of pancreatic cancer. Family with sequence similarity 83 member A (FAM83A) was shown recently to have oncogenic effects in a variety of cancer types, but the biological roles and molecular mechanisms of FAM83A in pancreatic cancer need further investigation. Here, we newly discovered that FAM83A binds directly to β-catenin and inhibits the assembly of the cytoplasmic destruction complex thus inhibiting the subsequent phosphorylation and degradation. FAM83A is mainly phosphorylated by the SRC non-receptor kinase family member BLK (B-lymphoid tyrosine kinase) at tyrosine 138 residue within the DUF1669 domain that mediates the FAM83A-β-catenin interaction. Moreover, FAM83A tyrosine 138 phosphorylation enhances oncogenic Wnt/β-catenin-mediated transcription through promoting β-catenin-TCF4 interaction and showed an elevated nucleus translocation, which inhibits the recruitment of histone deacetylases by TCF4. We also showed that FAM83A is a direct downstream target of Wnt/β-catenin signaling and correlates with the levels of Wnt target genes in human clinical pancreatic cancer tissues. Notably, the inhibitory peptides that target the FAM83A-β-catenin interaction significantly suppressed pancreatic cancer growth and metastasis in vitro and in vivo. Our results revealed that blocking the FAM83A cascade signaling defines a therapeutic target in human pancreatic cancer.

IgA Vasculitis: Influence of CD40, BLK and BANK1 Gene Polymorphisms.

CD40, BLK and BANK1 genes involved in the development and signaling of B-cells are identified as susceptibility loci for numerous inflammatory diseases. Accordingly, we assessed the potential influence of CD40, BLK and BANK1 on the pathogenesis of immunoglobulin-A vasculitis (IgAV), predominantly a B-lymphocyte inflammatory condition. Three genetic variants within CD40 (rs1883832, rs1535045, rs4813003) and BLK (rs2254546, rs2736340, rs2618476) as well as two BANK1 polymorphisms (rs10516487, rs3733197), previously associated with inflammatory diseases, were genotyped in 382 Caucasian patients with IgAV and 955 sex- and ethnically matched healthy controls. No statistically significant differences were observed in the genotype and allele frequencies of CD40, BLK and BANK1 when IgAV patients and healthy controls were compared. Similar results were found when CD40, BLK and BANK1 genotypes or alleles frequencies were compared between patients with IgAV stratified according to the age at disease onset or to the presence/absence of gastrointestinal or renal manifestations. Moreover, no CD40, BLK and BANK1 haplotype differences were disclosed between patients with IgAV and healthy controls and between patients with IgAV stratified according to the clinical characteristics mentioned above. Our findings indicate that CD40, BLK and BANK1 do not contribute to the genetic background of IgAV.

Haplotype-specific chromatin looping reveals genetic interactions of regulatory regions modulating gene expression in 8p23.1.

A major goal of genetics research is to elucidate mechanisms explaining how genetic variation contributes to phenotypic variation. The genetic variants identified in genome-wide association studies (GWASs) generally explain only a small proportion of heritability of phenotypic traits, the so-called missing heritability problem. Recent evidence suggests that additional common variants beyond lead GWAS variants contribute to phenotypic variation; however, their mechanistic underpinnings generally remain unexplored. Herein, we undertake a study of haplotype-specific mechanisms of gene regulation at 8p23.1 in the human genome, a region associated with a number of complex diseases. The FAM167A-BLK locus in this region has been consistently found in the genome-wide association studies (GWASs) of systemic lupus erythematosus (SLE) in all major ancestries. Our haplotype-specific chromatin interaction (Hi-C) experiments, allele-specific enhancer activity measurements, genetic analyses, and epigenome editing experiments revealed that: 1) haplotype-specific long-range chromatin interactions are prevalent in 8p23.1; 2) BLK promoter and cis-regulatory elements cooperatively interact with haplotype-specificity; 3) genetic variants at distal regulatory elements are allele-specific modifiers of the promoter variants at FAM167A-BLK; 4) the BLK promoter interacts with and, as an enhancer-like promoter, regulates FAM167A expression and 5) local allele-specific enhancer activities are influenced by global haplotype structure due to chromatin looping. Although systemic lupus erythematosus causal variants at the FAM167A-BLK locus are thought to reside in the BLK promoter region, our results reveal that genetic variants at distal regulatory elements modulate promoter activity, changing BLK and FAM167A gene expression and disease risk. Our results suggest that global haplotype-specific 3-dimensional chromatin looping architecture has a strong influence on local allelic BLK and FAM167A gene expression, providing mechanistic details for how regional variants controlling the BLK promoter may influence disease risk.

A risk variant for Barrett's esophagus and esophageal adenocarcinoma at chr8p23.1 affects enhancer activity and implicates multiple gene targets.

Nineteen genetic susceptibility loci for esophageal adenocarcinoma (EAC) and its precursor Barrett's esophagus (BE) have been identified through genome-wide association studies (GWAS). Clinical translation of such discoveries, however, has been hindered by the slow pace of discovery of functional/causal variants and gene targets at these loci. We previously developed a systematic informatics pipeline to prioritize candidate functional variants using functional potential scores, applied the pipeline to select high-scoring BE/EAC risk loci and validated a functional variant at chr19p13.11 (rs10423674). Here, we selected two additional prioritized loci for experimental interrogation: chr3p13/rs1522552 and chr8p23.1/rs55896564. Candidate enhancer regions encompassing these variants were evaluated using luciferase reporter assays in two EAC cell lines. One of the two regions tested exhibited allele-specific enhancer activity - 8p23.1/rs55896564. CRISPR-mediated deletion of the putative enhancer in EAC cell lines correlated with reduced expression of three candidate gene targets: B lymphocyte kinase (BLK), nei like DNA glycosylase 2 (NEIL2) and cathepsin B (CTSB). Expression quantitative trait locus (eQTL) mapping in normal esophagus and stomach revealed strong associations between the BE/EAC risk allele at rs55896564 (G) and lower expression of CTSB, a protease gene implicated in epithelial wound repair. These results further support the utility of functional potential scores for GWAS variant prioritization, and provide the first experimental evidence of a functional variant and risk enhancer at the 8p23.1 GWAS locus. Identification of CTSB, BLK and NEIL2 as candidate gene targets suggests that altered expression of these genes may underlie the genetic risk association at 8p23.1 with BE/EAC.

Abstract 902: Identification, characterization, and targeting of desmoplastic small round cell tumor cancer stem cell-like cells

Abstract Desmoplastic Small Round Cell Tumor (DSRCT) is a rare and aggressive form of pediatric cancer caused by a translocation between chromosomes 11 and 22, creating the novel EWSR1-WT1 fusion gene. Current therapy including surgery and the P6 chemotherapy regimen is insufficient, leading to a 5-year survival rate of only 15-25%. One potential explanation for this poor prognosis is the existence of a cancer stem cell (CSC) population with the ability to resist chemotherapy and cause both tumor recurrence and metastasis. While a CSC population has been identified in the closely related Ewing Sarcoma, most commonly caused by the EWSR1-FLI1 fusion gene, no CSC population has yet been identified in DSRCT. In this study, novel culture conditions were developed that enabled the formation of tumorspheres in vitro in the two commonly available DSRCT cell lines: JN-DSRCT-1 and BER-DSRCT. These novel culture conditions led to upregulation of stemness genes at the RNA and protein level including NANOG and SOX2, two genes that are associated with poor survival outcomes in TCGA sarcomas data. CCK-8 assay demonstrated an increased chemoresistance for tumorspheres versus normal adherent culture especially for doxorubicin, which was confirmed by reduction in PARP cleavage. This chemoresistance may be partially explained by a more quiescent CSC state as shown by reduced cell proliferation and a lower percentage of cells in the S and G2/M phases. However, cells grown in tumorspheres still robustly formed tumors in vivo, thus demonstrating the two hallmark CSC traits of chemoresistance and tumor formation. RNA-seq was performed to elucidate key differences between the adherent and sphere culture conditions and identify potential targets for the therapeutically important CSC-like population. Overrepresentation analysis and gene set enrichment analysis both showed upregulation of pathways related to chromatin assembly and disassembly, suggesting epigenetic changes as DSRCT cells move to a more stem-like state. RNA-seq further revealed kinases upregulated in tumorspheres including B-lymphocyte kinase (BLK) which previous studies have demonstrated is oncogenic, regulated by EWSR1-WT1, and not necessary for normal survival. BLK knockdown reduced CSC-like properties including abrogation of tumorsphere formation and reduction in the levels of SOX2 and NANOG. Together, this work for the first time identifies a CSC-like population in DSRCT and BLK as a potential DSRCT CSC target. Citation Format: Justin W. Magrath, Alifiani B. Hartono, Sean B. Lee. Identification, characterization, and targeting of desmoplastic small round cell tumor cancer stem cell-like cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 902.

Precision Diagnosis of Maturity-Onset Diabetes of the Young with Next-Generation Sequencing: Findings from the MODY-IST Study in Adult Patients.

Maturity-onset diabetes of the young (MODY) is a highly heterogeneous group of monogenic and nonautoimmune diseases. Misdiagnosis of MODY is a widespread problem and about 5% of patients with type 2 diabetes mellitus and nearly 10% with type 1 diabetes mellitus may actually have MODY. Using next-generation DNA sequencing (NGS) to facilitate accurate diagnosis of MODY, this study investigated mutations in 13 MODY genes (HNF4A, GCK, HNF1A, PDX1, HNF1B, NEUROD1, KLF11, CEL, PAX4, INS, BLK, ABCC8, and KCNJ11). In addition, we comprehensively investigated the clinical phenotypic effects of the genetic variations identified. Fifty-one adult patients with suspected MODY and 64 healthy controls participated in the study. We identified 7 novel and 10 known missense mutations localized in PDX1, HNF1B, KLF11, CEL, BLK, and ABCC8 genes in 29.4% of the patient sample. Importantly, we report several mutations that were classified as "deleterious" as well as those predicted as "benign." Notably, the ABCC8 p.R1103Q, ABCC8 p.V421I, CEL I336T, CEL p.N493H, BLK p.L503P, HNF1B p.S362P, and PDX1 p.E69A mutations were identified for the first time as causative variants for MODY. More aggressive clinical features were observed in three patients with double- and triple-heterozygosity of PDX1-KLF11 (p.E69A/p.S182R), CEL-ABCC8-KCNJ11 (p.I336, p.G157R/p.R1103Q/p.A157A), and HNF1B-KLF11 (p.S362P/p.P261L). Interestingly, the clinical effects of the BLK mutations appear to be exacerbated in the presence of obesity. In conclusion, NGS analyses of the adult patients with suspected MODY appear to be informative in a clinical context. These findings warrant further clinical diagnostic research and development in different world populations suffering from diabetes with genetic underpinnings.

Discovery of selective irreversible inhibitors of B-Lymphoid tyrosine kinase (BLK)

B-lymphoid tyrosine kinase (BLK), a member of the SRC family nonreceptor tyrosine kinase, is involved in the B-cell receptor (BCR) signaling pathway and B cell development and function. Dysregulation of BLK is associated with autoimmune diseases and cancer. However, there is an absence of good tool compounds for BLK, and the molecular mechanisms by which BLK mediates physiological and pathological processes are poorly understood. Herein, we present the discovery of a novel series of selective and irreversible inhibitors of BLK with nanomolar potency against BLK in biochemical and cellular assays. Compound 25 demonstrated potent antiproliferative activities against several B cell lymphoma cell lines. These compounds constitute the first series of selective inhibitors developed for BLK and could help expedite the exploration of BLK functions.

BLK polymorphisms and expression level in neuromyelitis optica spectrum disorder

AimThis study aimed to determine the correlation between B‐lymphoid tyrosine kinase (BLK) polymorphism, mRNA gene expression of BLK, and NMOSD in a Chinese Han population.BackgroundB‐lymphoid tyrosine kinase gene expressed mainly in B cells plays a key role in various autoimmune disorders. However, no studies have investigated the association of BLK polymorphisms with neuromyelitis optica spectrum disorder (NMOSD).MethodsHan Chinese population of 310 subjects were recruited to analyze three single nucleotide polymorphisms (rs13277113, rs4840568, and rs2248932) under allele, genotype, and haplotype frequencies, followed by clinical characteristics stratified analysis. Real‐time PCR was used to analyze mRNA expression levels of BLK in the peripheral blood mononuclear cells of 64 subjects.ResultsPatients with NMOSD showed lower frequencies of the minor allele G of rs2248932 than healthy controls (odds ratio (OR) =0.57, 95% confidence intervals (CI) 0.39–0.83, p = 0.003). The association between minor allele G of rs2248932 and reduced NMOSD susceptibility was found by applying genetic models of inheritance (codominant, dominant, and recessive) and haplotypes analysis. Subsequently, by stratification analysis for AQP4‐positivity, the minor allele G frequencies of rs2248932 in AQP4‐positive subgroup were significantly lower than in the healthy controls (OR =0.46, 95% CI 0.30–0.72, p = 0.001). Notably, the genotype GG of rs2248932 was more frequent in AQP4‐negative subgroup (n = 14) than in AQP4‐positive subgroup (n = 93) (p = 0.003, OR =0.05, 95% CI =0.01–0.57). BLK mRNA expression levels in the NMOSD patients (n = 36) were lower than in healthy controls (n = 28) (p < 0.05). However, the acute non‐treatment (n = 7), who were untreated patients in the acute phase from the NMOSD group, showed BLK mRNA expression levels 1.8‐fold higher than healthy controls (n = 8) (p < 0.05).ConclusionThis study evaluated that the minor allele G of rs2248932 in BLK is associated with reduced susceptibility to NMOSD and protected the risk of AQP4‐positive. BLK mRNA expression in NMOSD was lower as compared to healthy controls while significantly increased in acute‐untreated patients.

Genome-wide Association Study Identified Chromosome 8 Locus Associated with Medication-Related Osteonecrosis of the Jaw.

Medication‐related osteonecrosis of the jaw (MRONJ) is a rare but serious drug‐related adverse event. To identify pharmacogenomic markers of MRONJ associated with bisphosphonate therapy, we conducted a genomewide association study (GWAS) meta‐analysis followed by functional analysis of 5,008 individuals of European ancestry treated with bisphosphonates, which includes the largest number of MRONJ cases to date (444 cases and 4,564 controls). Discovery GWAS was performed in randomly selected 70% of the patients with cancer and replication GWAS was performed in the remaining 30% of the patients with cancer treated with intravenous bisphosphonates followed by meta‐analysis of all 3,639 patients with cancer. GWAS was also performed in 1,369 patients with osteoporosis treated with oral bisphosphonates. The lead single‐nucleotide polymorphism (SNP), rs2736308 on chromosome 8, was associated with an increased risk of MRONJ with an odds ratio (OR) of 2.71 and 95% confidence interval (CI) of 1.90–3.86 (P = 3.57*10−8) in the meta‐analysis of patients with cancer. This SNP was validated in the MRONJ GWAS in patients with osteoporosis (OR: 2.82, 95% CI: 1.55–4.09, P = 6.84*10−4). The meta‐analysis combining patients with cancer and patients with osteoporosis yielded the same lead SNP rs2736308 on chromosome 8 as the top SNP (OR: 2.74, 95% CI: 2.09–3.39, P = 9.65*10−11). This locus is associated with regulation of the BLK, CTSB, and FDFT1 genes, which had been associated with bone mineral density. FDFT1 encodes a membrane‐associated enzyme, which is implicated in the bisphosphonate pathway. This study provides insights into the potential mechanism of MRONJ.

Peripheral blood B cell subset ratios and expression levels of B cell-associated genes are altered in benign multiple sclerosis

The interplay between the immune system, sleep dysfunction and cognitive impairment participates in the progression of disability in multiple sclerosis (MS). Our aim was to identify molecular pathways and B cell associated with separate components of MS disability. Benign MS, non-benign MS patients and healthy controls were recruited. Patients underwent polysomnography and cognitive studies. Microarray and bioinformatics analysis performed using peripheral blood mononuclear cell samples identified B cell-associated genes with the most significantly altered expression. Expression levels of these genes were validated by real-time PCR and peripheral blood cell subsets were examined by flow cytometry. Putative correlations among clinical and laboratory parameters were investigated by correlation network analysis. Sleep and cognitive functions were equally impaired in BMS and NBMS. BMS patients showed significantly reduced memory B cell and increased regulatory B cell percentages than NBMS patients. Among genes that were selected by bioinformatics, levels of BLK, BLNK, BANK1, FCRL2, TGFB1 and KCNS3 genes were significantly different among study subgroups. Correlation network analysis showed associations among physical-cognitive disability and sleep dysfunction measures of MS versus expression levels of selected genes. BMS and NBMS differ by physical disability but not cognitive and sleep dysfunction. Different components of disability in MS are associated with peripheral blood B cell ratios and B cell related gene expression levels. Thus, it is likely that altered B cell functions participate in the progression of disability in MS.

Expression Levels of B Cell Receptor (BCR) Signaling Pathway Factors are Associated with Conversion from Clinically Isolated Syndrome to Multiple Sclerosis

Objective: We have previously shown altered blood B cells in the first attack blood samples of clinically isolated syndrome (CIS) patients, who converted to multiple sclerosis (MS). Our aim was to investigate levels of B cell receptor (BCR)-associated genes expression in converting CIS (CIS-c) and non-converting CIS patients (CISnc) with a 5-year follow-up. Methods: Seven CIS-c patients, 12 CIS-nc patients and 10 agesex matched healthy donors were enrolled in this study. RNA was extracted from frozen peripheral blood cells of subjects that were obtained in the first attack using a RNA isolation kit. Expression levels of B-cell adaptor protein with ankyrin repeats 1 (BANK1), B lymphocyte kinase (BLK) and Fc receptor-like protein 2 (FCRL2) genes were evaluated samples by real-time polymerase chain reaction (RT-PCR). Results: CIS-nc patients showed elevated BANK1, BLK and FCRL2 gene expression levels as compared to CIS-c patients and healthy controls. Conclusions: Expression levels of the genes that inhibit BCR signaling are increased in CIS-nc, suggesting that inhibiting exaggerated B cell response by optimization of B cell proliferation may prevent the progression of clinically isolated syndrome to MS.

Abnormal thymic B cell activation and impaired T cell differentiation in pristane-induced lupus mice

Changes in the thymus and potential mechanisms underlying the pathogenesis in pristane-induced lupus (PIL) mice are poorly understood. This study aimed to systematically and specifically examine changes in the thymus and the potential mechanisms responsible for immunological abnormalities in PIL mice. The results showed that PIL mice exhibit serious thymic hyperplasia, an elevated thymus index, a damaged histopathological structure and increased thymocyte apoptosis. We found that thymic T cell differentiation was impaired as the CD4+ CD8+ double-positive (DP) thymocyte frequency significantly decreased, becoming almost absent at 28 weeks after induction, while CD4 CD8- double-negative (DN) thymocytes and CD4+ CD8- single-positive (CD4+ SP) and CD4 CD8+ single-positive (CD8+ SP) cells were increased. This phenomenon might be explained by an inhibition of the DN-to-DP-cell transition and stimulation of DP cell conversion into CD4+ /CD8+ SP thymocytes. Moreover, we discovered a dramatic and abnormal increase in thymic B cells, that was associated with CD19, Irf8, Ebf1, Pax5, Irf4, Blk, CXCL13, CXCR5, CD79a, CD79b, Lyn, Syk, Btk, and BLNK gene accumulation, which exhibited positive interactions. We further verified that the mRNA expression of these genes was significantly upregulated and consistent with the RNA-seq results. These results suggest a role of these genes in the increase of B cells in the thymus of PIL mice. In summary, our results showed the changes in the thymus in PIL and elucidated the immunologic abnormalities of increased B cells, potentially providing insight into the associated molecular mechanisms and facilitating further research.