Lymphocyte Kinase Research Articles

LCK as a Potential Therapeutic Target for Acute Rejection after Kidney Transplantation: A Bioinformatics Clue.

Objectives We aim to identify the key biomarker of acute rejection (AR) after kidney transplantation via bioinformatics methods. Methods The gene expression data GSE75693 of 30 samples with stable kidney transplantation recipients and 15 AR samples were downloaded and analyzed by the limma package to identify differentially expressed genes (DEGs). Then, Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were done to explore the biological functions and potential important pathways of DEGs. Finally, protein-protein interactions (PPIs) and literature mining were applied to construct the cocitation network and to select the hub protein. Results A total of 437 upregulated genes and 353 downregulated genes were selected according to P < 0.01 and |log2(fold change)| > 1.0. DEGs of AR are mainly located on membranes and impact the activation of receptors in immune responses. In the PPI network, Src kinase, lymphocyte kinase (LCK), CD3G, B2M, interferon-γ, CD3D, tumor necrosis factor, VAV1, and CD3E in the T cell receptor signaling pathway were selected as important factors, and LCK was identified as the hub protein. Conclusion LCK, via acting on T-cell receptor, might be a potential therapeutic target for AR after kidney transplantation.

The rheumatic disease-associated FAM167A-BLK locus encodes DIORA-1, a novel disordered protein expressed highly in bronchial epithelium and alveolar macrophages.

Triggering of autoimmunity that leads to rheumatic disease has been suggested to depend upon gene-environment interactions occurring in epithelial barriers and associated immune cells. Genetic studies have identified associations of the FAM167A-BLK locus with rheumatoid arthritis, systemic lupus erythematosus (SLE) and Sjögren's syndrome. While BLK (B lymphocyte kinase) has a well-established role in B cells, family with sequence similarity to 167 member A (FAM167A) and its gene family remain uncharacterized. To begin to understand the role of FAM167A in rheumatic disease pathogenesis, we explored this gene family and cloned and investigated the gene products. Expression of quantitative trait locus analysis was performed in immune cells. FAM167A and FAM167B were cloned from human peripheral blood mononuclear cells (PBMC). Gene conservation and protein properties were analysed by online tools, mRNA expression measured in mouse organs by quantitative polymerase chain reaction (qPCR) and protein expression investigated in human tissues by immunohistochemistry. We found that autoimmune risk genotypes within the FAM167A-BLK locus lead to increased expression of FAM167A. The FAM167 gene family includes two members, FAM167A and FAM167B, which are not homologous to any other annotated gene but are evolutionarily conserved. The encoded proteins, which we denote 'disordered autoimmunity' (DIORA)-1 and DIORA-2, respectively, are characterized by a high content of intrinsic disorder. Notably, DIORA-1 has its highest expression in the lung, detectable in both bronchial epithelium and alveolar macrophages with an endosomal localization pattern. In summary, the FAM167A gene is associated with several rheumatic diseases and encodes a novel disordered protein, DIORA-1, which is expressed highly in the lung, consistent with a potential role in disease pathogenesis.

Blood Gene Expression Predicts Bronchiolitis Obliterans Syndrome.

Bronchiolitis obliterans syndrome (BOS), the main manifestation of chronic lung allograft dysfunction, leads to poor long-term survival after lung transplantation. Identifying predictors of BOS is essential to prevent the progression of dysfunction before irreversible damage occurs. By using a large set of 107 samples from lung recipients, we performed microarray gene expression profiling of whole blood to identify early biomarkers of BOS, including samples from 49 patients with stable function for at least 3 years, 32 samples collected at least 6 months before BOS diagnosis (prediction group), and 26 samples at or after BOS diagnosis (diagnosis group). An independent set from 25 lung recipients was used for validation by quantitative PCR (13 stables, 11 in the prediction group, and 8 in the diagnosis group). We identified 50 transcripts differentially expressed between stable and BOS recipients. Three genes, namely POU class 2 associating factor 1 (POU2AF1), T-cell leukemia/lymphoma protein 1A (TCL1A), and B cell lymphocyte kinase, were validated as predictive biomarkers of BOS more than 6 months before diagnosis, with areas under the curve of 0.83, 0.77, and 0.78 respectively. These genes allow stratification based on BOS risk (log-rank test p < 0.01) and are not associated with time posttransplantation. This is the first published large-scale gene expression analysis of blood after lung transplantation. The three-gene blood signature could provide clinicians with new tools to improve follow-up and adapt treatment of patients likely to develop BOS.

Interaction analysis between BLK rs13277113 polymorphism and BANK1 rs3733197 polymorphism, MMEL1/TNFRSF14 rs3890745 polymorphism in determining susceptibility to rheumatoid arthritis

Two pairwise genetic interactions (B cell lymphocyte kinase (BLK) rs13277113,B cell scaffold protein with ankyrin repeats 1 (BANK1) rs3733197and BLK rs13277113 membrane metalloendopeptidase like 1 (MMEL1)/ tumor necrosis factor receptor superfamily member 14 (TNFRSF14) rs3890745) have been demonstrated in determining susceptibility to rheumatoid arthritis (RA) without replication, thus this study was performed to examine whether abovementioned genetic polymorphisms were associated with RA and further tests were performed to see whether aforementioned genetic interactions existed in RA among Chinese population. A total of 328 patients with RA and 449 healthy control subjects were included in the current study. The polymorphisms were genotyped using the ligase detection reaction-polymerase chain reaction (LDR-PCR) technology. The association of RA with each polymorphism was analyzed by multivariate logistic regression model. Interaction analysis was done by multiple methods. Significant difference in genotype distribution of BLK rs13277113 polymorphism between RA patients and healthy controls was found (p = 1.01 × 10−2). The major allele A of BLK rs13277113 polymorphism was significantly increased in RA patients compared with controls (OR = 1.36, 95% CI = 1.08–1.71, p = 9.27 × 10−3). Significant association of RA with the major allele A of BLK rs13277113 polymorphism under dominant model was also detected (OR = 2.74, 95% CI = 1.42–5.29, p = 2.73 × 10−3). However, we did not find significant association between neither BANK1 rs3733197 polymorphism nor MMEL1/TNFRSF14 rs3890745 polymorphism and RA. Non-significant evidence was found for neither additive nor multiplicative interaction for these two pairwise genetic polymorphisms (BLK rs13277113-BANK1 rs3733197; BLK rs13277113-MMEL1/TNFRSF14 rs3890745). Significant association of RA with G allele of BANK1 rs3733197 polymorphism was only found among individuals carrying A/A genotype of the BLK rs13277113 polymorphism (OR = 1.49, 95% CI = 1.01–2.18, p = .04). In summary, our results indicated that the BLK rs13277113 polymorphism was involved in the genetic background of RA in Chinese population and the association of BANK1 rs3733197 polymorphism with RA was dependent on the genotype of BLK rs13277113 polymorphism, highlighting B-cell response implicated in the pathogenesis of RA.

B-cell lymphocyte kinase polymorphisms rs13277113, rs2736340, and rs4840568 and risk of autoimmune diseases: A meta-analysis.

B-cell lymphocyte kinase (BLK) is an inhibitor of B cells that has an important influence on several autoimmune diseases, but there is a lack of comprehensive analysis of its association with autoimmune diseases. Hence, it is meaningful to conduct a comprehensive analysis. A systematic literature search was performed on the PubMed, ScienceDirect, and Web of Science databases up to June 30, 2016. The data were extracted and quality-assessed before conducting the meta-analysis. The odds ratios (ORs) and 95% confidence intervals (95% CIs) were assessed with the STATA version 12.0 software. Subgroup and sensitivity analysis were conducted to explore potential sources of heterogeneity. Altogether, 33 studies with 68,874 cases and 90,684 controls, 24 studies with 31,095 cases and 39,077 controls for rs13277113, 21 studies with 26,388 cases and 40,635 controls for rs2736340, and 4 studies with 11,391 cases and 10,972 controls for rs4840568 were included in this meta-analysis. The results revealed that the BLK rs13277113 and rs2736340 polymorphisms increased the risk of autoimmune diseases in the total analysis (A vs G: OR = 1.33, 95% CI = 1.27-1.39, P < .01; T vs C: OR = 1.34, 95% CI = 1.27-1.41, P < .01), and rs4840568 was positively associated with systemic lupus erythematosus (SLE) (A vs G: OR = 1.32, 95% CI = 1.22-1.43, P = .01). This meta-analysis shows that the BLK (rs13277113, rs2736340, rs4840568) polymorphisms may be a risk factor for developing autoimmune diseases, especially for Asian populations and SLE.

Ibrutinib inhibits pre-BCR+ B-cell acute lymphoblastic leukemia progression by targeting BTK and BLK

AbstractTargeting B-cell receptor (BCR) signaling is a successful therapeutic strategy in mature B-cell malignancies. Precursor BCR (pre-BCR) signaling, which is critical during normal B lymphopoiesis, also plays an important role in pre-BCR+ B cell acute lymphoblastic leukemia (B-ALL). Here, we investigated the activity and mechanism of action of the BTK inhibitor ibrutinib in preclinical models of B-ALL. Pre-BCR+ ALL cells were exquisitely sensitive to ibrutinib at therapeutically relevant drug concentrations. In pre-BCR+ ALL, ibrutinib thwarted autonomous and induced pre-BCR signaling, resulting in deactivation of PI3K/Akt signaling. Ibrutinib modulated the expression of pre-BCR regulators (PTPN6, CD22, CD72, and PKCβ) and substantially reduced BCL6 levels. Ibrutinib inhibited ALL cell migration toward CXCL12 and beneath marrow stromal cells and reduced CD44 expression. CRISPR-Cas9 gene editing revealed that both BTK and B lymphocyte kinase (BLK) are relevant targets of ibrutinib in pre-BCR+ ALL. Consequently, in mouse xenograft models of pre-BCR+ ALL, ibrutinib treatment significantly prolonged survival. Combination treatment of ibrutinib with dexamethasone or vincristine demonstrated synergistic activity against pre-BCR+ ALL. These data corroborate ibrutinib as a promising targeted agent for pre-BCR+ ALL and highlight the importance of ibrutinib effects on alternative kinase targets.

Association between TNFSF4 and BLK gene polymorphisms and susceptibility to allergic rhinitis.

Allergic rhinitis (AR) is a common inflammatory disease of the upper airway. Recent evidence suggests that gene-gene interactions between tumor necrosis factor receptor superfamily 4 (TNFSF4) and B cell lymphocyte kinase (BLK) may have a synergistic effect on T and B cells in determining immunologic aberration, via the nuclear factor-κB pathway. The present study was performed to evaluate the potential association between specific single nucleotide polymorphisms (SNPs) in the TNFSF4 and BKL genes with susceptibility to AR in Chinese subjects. A population-based case-control study was performed in 600 Chinese AR patients and 700 controls. Blood was drawn for DNA extraction, and 9 SNPs (6 in TNFSF4 and 3 in BKL genes) were selected and genotyped. The TNFSF4 SNPs rs1234314 and rs1234315, and the BLK SNPs rs13277113 and rs1600249 were observed to occur in different frequencies between the AR patients and the controls. The CC (rs1234314, rs1234315) and AA (rs1600249, rs13277113) genotypes provided protective effects against AR, whereas the AG (rs13277113) genotype presented a risk factor for AR. The haplotypes ACC in the rs1234313-rs1234314-rs1234315 block and GA in the rs2254546-rs13277113 block significantly decreased the risk of AR, whereas the GGT and AG haplotypes served protective roles. SNP interaction analysis further indicated that there may be synergistic effects among the selected sets of polymorphisms. The present study suggests a novel association between specific TNFSF4 and BLK gene polymorphisms and AR risk, highlighting their potential utility as genetic biomarkers for AR susceptibility in a Chinese Han population.

A Small Molecule Inhibitor of ITK and RLK Impairs Th1 Differentiation and Prevents Colitis Disease Progression.

In T cells, the Tec kinases IL-2-inducible T cell kinase (ITK) and resting lymphocyte kinase (RLK) are activated by TCR stimulation and are required for optimal downstream signaling. Studies of CD4(+) T cells from Itk(-/-) and Itk(-/-)Rlk(-/-) mice have indicated differential roles of ITK and RLK in Th1, Th2, and Th17 differentiation and cytokine production. However, these findings are confounded by the complex T cell developmental defects in these mice. In this study, we examine the consequences of ITK and RLK inhibition using a highly selective and potent small molecule covalent inhibitor PRN694. In vitro Th polarization experiments indicate that PRN694 is a potent inhibitor of Th1 and Th17 differentiation and cytokine production. Using a T cell adoptive transfer model of colitis, we find that in vivo administration of PRN694 markedly reduces disease progression, T cell infiltration into the intestinal lamina propria, and IFN-γ production by colitogenic CD4(+) T cells. Consistent with these findings, Th1 and Th17 cells differentiated in the presence of PRN694 show reduced P-selectin binding and impaired migration to CXCL11 and CCL20, respectively. Taken together, these data indicate that ITK plus RLK inhibition may have therapeutic potential in Th1-mediated inflammatory diseases.

Targeting Interleukin-2-inducible T-cell Kinase (ITK) and Resting Lymphocyte Kinase (RLK) Using a Novel Covalent Inhibitor PRN694

Interleukin-2-inducible T-cell kinase (ITK) and resting lymphocyte kinase (RLK or TXK) are essential mediators of intracellular signaling in both normal and neoplastic T-cells and natural killer (NK) cells. Thus, ITK and RLK inhibitors have therapeutic potential in a number of human autoimmune, inflammatory, and malignant diseases. Here we describe a novel ITK/RLK inhibitor, PRN694, which covalently binds to cysteine residues 442 of ITK and 350 of RLK and blocks kinase activity. Molecular modeling was utilized to design molecules that interact with cysteine while binding to the ATP binding site in the kinase domain. PRN694 exhibits extended target residence time on ITK and RLK and is highly selective for a subset of the TEC kinase family. In vitro cellular assays confirm that PRN694 prevents T-cell receptor- and Fc receptor-induced cellular and molecular activation, inhibits T-cell receptor-induced T-cell proliferation, and blocks proinflammatory cytokine release as well as activation of Th17 cells. Ex vivo assays demonstrate inhibitory activity against T-cell prolymphocytic leukemia cells, and in vivo assays demonstrate durable pharmacodynamic effects on ITK, which reduces an oxazolone-induced delayed type hypersensitivity reaction. These data indicate that PRN694 is a highly selective and potent covalent inhibitor of ITK and RLK, and its extended target residence time enables durable attenuation of effector cells in vitro and in vivo. The results from this study highlight potential applications of this dual inhibitor for the treatment of T-cell- or NK cell-mediated inflammatory, autoimmune, and malignant diseases.

Targeting Interleukin-2-Inducible T-Cell Kinase (ITK) and Resting Lymphocyte Kinase (RLK) Using a Novel Covalent Inhibitor PRN694

▪IL2-inducible T-cell kinase (ITK) and resting lymphocyte kinase (RLK) are two members of the Tec family of non-receptor tyrosine kinases which are essential mediators of intracellular signaling in both normal and neoplastic T and natural killer (NK)-cells. ITK-deficient mice display alterations in T cell receptor (TCR) responses including proliferation, cytokine production and activation of downstream pathways, and these defects are exacerbated by RLK deficiency. Given their critical roles, inhibitors of ITK and RLK have therapeutic potential in a number of human autoimmune diseases, inflammatory conditions, and malignancies. Specific inhibitors targeting both ITK and RLK have not been reported and until very recently it has been challenging to develop specific inhibitors with clinical potential given the homology to many other human kinases. Targeting cysteine residues within the ATP binding pocket of kinases is a safe and effective way to design and optimize highly potent and selective kinase inhibitors. In this study, we describe preclinical mechanism of action studies of a novel covalent ITK/RLK inhibitor, PRN694, which covalently binds to cysteine moieties 442 of ITK and 350 of RLK and blocks their kinase activities.We undertook the design of highly selective ITK/RLK inhibitors using a combination of tailored covalency and structure-based design. Molecular modeling and a synthetic feasibility assessment led to the prioritization of several small focused compound libraries. The structure-based medicinal chemistry optimization of one such library culminated in the identification of PRN694. The in vitro selectivity and potency of PRN694 against a panel of 250 kinases revealed only small number of kinase targets, and in a microfluidic kinase assay, PRN694 exhibited high selectivity and potency against ITK and RLK with IC50 values of 0.3 nM and 1.3 nM, respectively. This selectivity was validated in Jurkat T cells with mutated ITK or overexpressed RLK. As expected, PRN694 suppressed a variety of downstream TCR pathways including nuclear activation of NFAT1 and cytoplasmic PLCγ1 and IKBα in TCR-activated Jurkat cells expressing wild type ITK. However, when the covalent binding site for PRN694 was removed, using ITK-C442A, this suppressive effect was lost, indicating specific covalent interaction with ITK. Furthermore, we demonstrated that only PRN694 was able to ablate TCR-induced activation in Jurkat cells which stably express RLK in addition to ITK. These data confirmed that PRN694 was a dual inhibitor of ITK and RLK and could block downstream TCR-activation in T-cells that express both complementary TEC-kinases. In vitro cellular assays also confirmed that PRN694 could prevent TCR or Fc receptor (FcR) - induced cellular and molecular activation in primary T or NK cells, inhibit TCR-induced T-cell proliferation without direct cytotoxicity, and block pro-inflammatory cytokine (including IL-2, TNFα, and IFNγ) release as well as activation of Th17 cells. Additionally, in vivo assays demonstrated durable pharmacodynamic effects of PRN694 on ITK. At 1 hour, 6 hour and 14 hour time points, the percentages of ITK occupancy were 98%, 95% and 54% respectively, and the concentrations of PRN694 in the plasma were 1530 ng/ml, 359.3 ng/ml and 14.8 ng/ml respectively.Further, we examined potential therapeutic applications of PRN694 in the treatment of T-cell leukemias or T-cell driven inflammation. Our results demonstrated an effective blockade of TCR stimulation by PRN694 in the HH cutaneous T-cell lymphoma cell line and primary T-cell prolymphocytic leukemia (T-PLL) cells, suggesting a potential clinical application of this compound in the treatment of T-cell leukemias. ITK and RLK play a critical role in T-cell driven inflammation. Therefore, we also tested the effect of PRN694 on an oxazolone induced delayed type hypersensitivity (DTH)reaction using a well-established in vivo model of inflammation driven by cell-mediated immunity, and found that PRN694 treatment significantly inhibited the DTH reaction.In conclusion, we designed and characterized a novel covalent ITK/RLK inhibitor, PRN694, which has direct therapeutic potential for the treatment of T or NK cell related diseases including inflammation, autoimmune diseases, and malignancy. DisclosuresPorcu:Actelion: Honoraria; Infinity: Research Funding; Seattle genetics: Research Funding; Celgene: Honoraria; United States Cutaneous Lymphoma Consortium: Membership on an entity’s Board of Directors or advisory committees; Cutaneous Lymphoma Foundation: Membership on an entity’s Board of Directors or advisory committees. Bradshaw:Principia Biopharma: Employment, Equity Ownership. Owens:Principia Biopharma, Inc: Employment, Equity Ownership. Brameld:Principia Biopharma: Employment, Equity Ownership. Funk:Principia Biopharma Inc.: Employment, Equity Ownership. Hill:Principia Biopharma: Employment. Johnson:Principia Biopharma: Research Funding. Dubovsky:Principia Inc.: Research Funding.

Abstract 2557: Immune modulating agent ibrutinib blocks T-helper 17 activation and release of IL-17A while preserving T-regulatory cell function

Abstract T-helper 17 (Th17) cells play a causal role in the pathogenesis of autoimmune and chronic inflammatory diseases. Chronic inflammation has been linked to various malignancies including gastrointestinal cancers, endometrial cancer, and non-Hodgkin lymphoma. Th17 cells secrete effector cytokines that are distinct from other divergent T-cell subsets. The namesake cytokine IL-17A is particularly pro-inflammatory and pro-fibrotic,driving the recruitment, activation, and migration of fibroblasts,endothelial cells, neutrophils, monocytes, and macrophages. Increases in IL-17A have been seen in patients with rheumatoid arthritis, Crohn's disease, multiple sclerosis, systemic lupus erythematosus, psoriasis, and encephalomyelitis. Dysregulated Th17 activity has also been linked to chronic graft versus host disease after allogeneic stem cell transplantation. Studies have shown that Th17 cells and T-regulatory (T-reg) cells are reciprocally regulated in the human body. T-regs produce anti-inflammatory cytokines that play an essential role in the prevention of autoimmune disease. At the molecular level Th17 cells rely upon ITK for T-cell receptor (TCR) induced activation. T-regs, however, utilize both ITK (IL2-inducible kinase) and RLK (resting lymphocyte kinase) establishing compensatory signaling cascades downstream of the TCR. Ibrutinib represents a first-in-class irreversible ITK inhibitor (Dubovsky et al: Blood July 25, 2013) with the established capacity to inhibit the activation of ITK dependent T-cells. Given our understanding of ITK and RLK expression in immune cell subsets we sought to determine whether ibrutinib could block Th17 cellular activation while preserving the functionality of T-regs. Our studies confirm that ibrutinib significantly blocks the TCR-induced activation of Th17 cells by up to 40% (p&amp;lt;0.0001). Moreover, ibrutinib pretreatment significantly limits the production of profibrotic IL-17A after ex-vivo TCR stimulation (p=0.030). Comprehensive and longitudinal murine in-vivo analysis revealed that up to four months of daily ibrutinib at 25mg/kg preserves T-reg numbers. Additional ex-vivo functional analysis demonstrated that the direct immunosuppressive capacity of highly purified(CD4+CD25hiCD127dimFoxP3+) human T-regs to limit activation and proliferation of healthy autologous CD4 or CD8 T-cells was not altered by ibrutinib. Our previously published and currently reported cumulative data along with an impressive clinical safety profile indicate that ibrutinib may therapeutically inhibit Th2 CD4 T-cell function and Th17 driven inflammation and autoimmunity while preserving the anti-inflammatory effects of T-reg cells, thereby justifying its investigation as an immune modulating therapy for cancer and graft versus host disease. Citation Format: Carrie S. Yang, Jason A. Dubovsky, Danielle L. Chappell, Bonnie K. Harrington, Samantha Jaglowski, Jennifer A. Woyach, Amy J. Johnson, Natarajan Muthusamy, John C. Byrd. Immune modulating agent ibrutinib blocks T-helper 17 activation and release of IL-17A while preserving T-regulatory cell function. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2557. doi:10.1158/1538-7445.AM2014-2557

AB0015 Association of BLK Polymorphism with Susceptibility to Rheumatoid Arthritis in an Algerian Population

BackgroundThe B-cell lymphocyte kinase (BLK) is a src family protein tyrosine kinase expressed in B-lineage cells. BLK gene polymorphism was reported as a risk factor in rheumatoid arthritis (RA) and...

Expansion of the IgD class switched B cell population in carriers of the autoimmune disease associated risk haplotype in BLK (HUM2P.332)

Abstract Polymorphisms in BLK are associated with numerous autoimmune diseases including rheumatoid arthritis and lupus. BLK (B lymphocyte kinase) is a tyrosine kinase involved in signaling through the B cell receptor (BCR). The disease risk haplotype is known to be associated with reduced expression of BLK transcript and the most significant association of reduced BLK transcript is with a candidate causal SNP rs922483. We have previously shown that expression of BLK is reduced in cord blood B cells from carriers of rs922483 risk alleles (HMG 2012). B cell development is perturbed in blk transgenic and knockout mice, and B cells from these mice are hyper-responsive to BCR stimulation. To determine if reduced BLK expression affects the function of primary human B cells we measured CD86 expression in cells stimulated by anti-IgM. As in mice, cord blood B cells from risk allele carriers are hyper-responsive to BCR stimulation. We also assessed if BLK risk variants convey any variation on the composition of circulating B cell populations of healthy adults. We observed that subjects with BLK risk variants have an expansion in the proportion and number of IgD class-switched B cells, and an increase in the total number of class-switched memory B cells. These results suggest that BLK risk haplotypes confer autoimmune disease susceptibility by reducing a negative regulatory effect of BLK, making B cells more likely to receive help from CD4 T cells and to undergo class switch recombination.

Abstract 492: Ibrutinib inhibits Interleukin-2 inducible kinase, driving a Th1 selective pressure in human leukemia patients that serves to alleviate tumor-induced immunosupression.

Abstract Interleukin-2 Inducible Kinase (ITK) is an appealing therapeutic target given its critical role in polarizing T-cell immunity against various infectious, autoimmune, and neoplastic diseases. ITK activity promotes Th2 immunity, thereby subverting healthy Th1-based immune surveillance. Th2 CD4 T-cells depend on ITK for activation, whereas Th1 and CD8 T-cells have compensatory resting lymphocyte kinase (RLK) that conducts T-cell receptor activation in the absence of ITK. Similar to many other tumors, chronic lymphocytic leukemia (CLL) directs an aberrant Th2 bias that drives immune evasion, promotes a supportive microenvironment, and cripples adaptive immunity. In CLL, this immune suppression leads to severe infections which are a leading cause of death. Although ITK inhibitors have the promise of alleviating tumor-induced immune suppression, to date no clinically viable ITK inhibitor exists. Ibrutinib is an irreversible inhibitor of Bruton's tyrosine kinase (BTK) with outstanding activity and tolerability in B-cell malignancies. The striking homology between BTK and ITK combined with our in silico docking studies and in vitro kinase inhibition profiles led us to identify ibrutinib as the first clinically viable irreversible ITK inhibitor. We further confirmed both molecular and functional outcomes using Jurkat T-cells and in vitro polarized Th1 and Th2 CD4 T-cells. We found that mutation of the ITK-Cys442 covalent binding residue for ibrutinib alleviated molecular inhibition. We also demonstrated that Th1 and CD8 T-cell restricted expression of RLK provides a compensatory platform for T-cell activation, offering a molecular explanation for the selective outgrowth of cytotoxic Th1-biased immunity. This effect was confirmed using T-cells directly derived from CLL patients. To clearly demonstrate this effect in vivo, we utilized the Leishmania major model of Th2-mediated infection. Ibrutinib enhanced resistance to the parasite by eliciting robust Th1 immunity. To demonstrate a direct clinical relevance in the setting of CLL we utilized a novel listeriosis/leukemia mouse model. In this model, we observed complete recovery of functional immunity, with all ibrutinib-treated mice surviving a potentially lethal Listeria monocytogenes infection. We further confirmed irreversible binding of ITK in patients currently receiving ibrutinib as part of a clinical trial. Finally, we found that ibrutinib inhibited phosphorlyation of ITK's direct downstream target PLCγ1 in CD4 T-cells. Together, our results provide novel molecular insights into the mechanism of action of ibrutinib in the context of Th2-biased immunosuppressive leukemia. We also postulate that ibrutinib's irreversible ITK inhibitory effects may prove beneficial in a number of other autoimmune, inflammatory, parasitic, and viral diseases. Citation Format: Jason A. Dubovsky, Kyle A. Beckwith, Gayathri Natarajan, Jennifer A. Woyach, Samantha Jaglowski, Joshua Hessler, Ta-Ming Liu, Betty Y. Chang, Karilyn M. Larkin, Matthew R. Stefanovski, Frank W. Frissora, Lisa L. Smith, Kelly A. Smucker, Joseph M. Flynn, Jeffrey A. Jones, Leslie A. Andritsos, Kami Maddocks, Amy M. Lehman, Richard Furman, Jeff Sharman, Anjali Mishra, Michael A. Caligiuri, Abhay R. Satoskar, Joseph J. Buggy, Natarajan Muthusamy, Amy J. Johnson, John C. Byrd. Ibrutinib inhibits Interleukin-2 inducible kinase, driving a Th1 selective pressure in human leukemia patients that serves to alleviate tumor-induced immunosupression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 492. doi:10.1158/1538-7445.AM2013-492

Ibrutinib Is an Irreversible Molecular Inhibitor of Interleukin-2 Inducible Kinase: Expanding Therapeutic Potential and Modulating a Th1 Selective Pressure in CD4 T-Cells

AbstractAbstract ▪775▪This icon denotes a clinically relevant abstractIn chronic lymphocytic leukemia (CLL), mounting evidence points to an aberrant tumor associated Th2 bias that drives leukemic cell immune evasion, promotes formation of a supportive niche microenvironment, and functionally cripples innate and adaptive immunity. The end result is a high incidence of infections which is the primary cause of mortality in CLL. This same Th2 bias is induced by many other types of cancer. Th2 CD4 T-cells are singularly dependent upon IL-2-inducible T-cell kinase (ITK) for activation whereas Th1 CD4 and CD8 T-cells have compensatory resting lymphocyte kinase (RLK) which conducts T-cell receptor activation even in the absence of ITK. Thus, a clinically viable ITK inhibitor would be ideal for targeting immune suppression associated with CLL and potentially other types of cancer. Unfortunately, no such therapeutic is currently available. Ibrutinib, a confirmed inhibitor of the Bruton’s tyrosine kinase (BTK) that irreversibly blocks downstream B-cell receptor activation, has demonstrated outstanding clinical activity in phase I/II clinical trials resulting in durable remissions in CLL. Our studies unveiled a previously uncharacterized Th1 cytokine switch in ibrutinib treated CLL patients which could not be attributed to B-lymphocytes. This ibrutinib-induced Th1 T-cell skewing was confirmed using the EμTCL1 mouse model of leukemia. Such alterations in cytokine patterns were reminiscent of mouse studies in which genetic ablation of ITK subverted Th2 immunity, thereby potentiating Th1-based adaptive immunity. The striking homology between BTK and ITK combined with intriguing in silico docking studies and promising in vitro kinase inhibition profiles with ibrutinib led to the hypothesis that this could be the first clinically viable irreversible ITK inhibitor. Cellular probe assays confirmed that the active site of ITK was covalently blocked by ibrutinib at pharmacologically relevant doses. Our comprehensive molecular analyses of T-cell signaling confirmed this in the Jurkat cell line. We further confirmed both molecular and functional outcomes in primary and in vitro polarized Th1 and Th2 CD4 T-cells. We found that mutation of the ITK-Cys442 covalent binding residue for ibrutinib alleviated molecular inhibition. We also demonstrated that Th1 and CD8 T-cell restricted expression of RLK provides a compensatory platform for T-cell activation offering a molecular explanation for the selective outgrowth of cytotoxic Th1 biased immunity. We further confirmed this effect using T-cells directly derived from CLL patients. To demonstrate that ibrutinib-induced ITK inhibition had direct clinical relevance in the setting of CLL we utilized a novel listeriosis/leukemia mouse model. In this model we clearly demonstrated complete recovery of functional immunity and all ibrutinib treated mice survived a potentially lethal Listeria monocytogenes infection. Our results expose novel molecular insights into the mechanism of action of ibrutinib in the context of Th2-biased immunosuppressive leukemia. We also postulate that ibrutinib’s irreversible ITK inhibitory effects may prove effective in a number of other autoimmune, inflammatory, and viral diseases, including influenza A and HIV/AIDS. Disclosures:Jaglowski:Pharmacyclics: Research Funding. Chang:Pharmacyclics, Inc.: Employment. Maddocks:Pharmacyclics: Research Funding. Buggy:Pharmacyclics: Employment, Equity Ownership. Byrd:Pharmacyclics: Research Funding.

Influence of BLK polymorphisms on the risk of rheumatoid arthritis

B cell lymphocyte kinase (BLK) encodes a member of the Src kinase family and thus may influence the proliferation and differentiation of cells. A single nucleotide polymorphism (SNP) located in the first intron of BLK has shown that the risk C allele of rs2248932 is associated with lower levels of messenger RNA expression of BLK. We hypothesized that this polymorphism may contribute to rheumatoid arthritis (RA) susceptibility. We studied BLK rs2248932 T/C gene polymorphisms in 329 patients with RA and 697 controls in a Chinese population. Genotyping was done using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). When the BLK rs2248932 TT homozygote genotype was used as the reference group, the CC genotype was associated with a significantly increased risk of RA. In the recessive model, when the BLK rs2248932 TT/TC genotypes were used as the reference group, the CC homozygote genotype was associated with a significantly increased susceptibility to RA. In stratification analyses, a significantly increased risk for RA associated with the BLK rs2248932 CC genotype was evident among younger patients, CRP-negative patients and anti-CCP-positive patients compared with the BLK rs2248932 TT/TC genotype. The risk was also significantly evident among RF-positive patients, patients with lower ESR levels, patients with lower or higher DAS28 score and patients with a lower functional class. These findings suggested that the functional SNP BLK rs2248932 T/C variant allele was associated with RA development. However, our results were obtained from a moderate-sized sample, and therefore this is a preliminary conclusion. Validation in a larger study from a more diverse ethnic population is needed to confirm these findings.

The autoimmunity-associated BLK haplotype exhibits cis-regulatory effects on mRNA and protein expression that are prominently observed in B cells early in development

The gene B lymphocyte kinase (BLK) is associated with rheumatoid arthritis, systemic lupus erythematosus and several other autoimmune disorders. The disease risk haplotype is known to be associated with reduced expression of BLK mRNA transcript in human B cell lines; however, little is known about cis-regulation of BLK message or protein levels in native cell types. Here, we show that in primary human B lymphocytes, cis-regulatory effects of disease-associated single nucleotide polymorphisms in BLK are restricted to naïve and transitional B cells. Cis-regulatory effects are not observed in adult B cells in later stages of differentiation. Allelic expression bias was also identified in primary human T cells from adult peripheral and umbilical cord blood (UCB), thymus and tonsil, although mRNA levels were reduced compared with B cells. Allelic regulation of Blk expression at the protein level was confirmed in UCB B cell subsets by intracellular staining and flow cytometry. Blk protein expression in CD4(+) and CD8(+) T cells was documented by western blot analysis; however, differences in protein expression levels by BLK genotype were not observed in any T cell subset. Blk allele expression differences at the protein level are thus restricted to early B cells, indicating that the involvement of Blk in the risk for autoimmune disease likely acts during the very early stages of B cell development.

Multifocal motor neuropathy is not associated with genetic variation in PTPN22, BANK1, Blk, FCGR2B, CD1A/E, and TAG‐1 genes

The contribution of genetic heterogeneity to the pathogenesis of multifocal motor neuropathy (MMN) has not been elucidated. We investigated frequencies of single nucleotide polymorphisms (SNPs) in the candidate genes protein tyrosine phosphatase, non-receptor type 22 (PTPN22), B-cell scaffold protein with ankyrin repeats (BANK1), B lymphocyte kinase (Blk), and Fc gamma receptor class IIB (FCGR2B), which have been found to be associated with other autoimmune diseases, CD1A and CD1E, important for antigen presentation of glycolipids, and transient axonal glycoprotein 1 (TAG-1), which is associated with responsiveness to intravenous immunoglobulin in patients with chronic inflammatory demyelinating polyneuropathy. SNP frequencies were determined by means of TaqMan SNP genotyping assay and direct sequencing of candidate genes in 92 Dutch patients with MMN and 1152 healthy controls. SNP frequencies did not differ between patients and controls (all p-values >0.15) and disease characteristics were not associated with SNP genotypes. Our results suggest that allelic variation in these genes does not play a major role in determining MMN susceptibility.

Association of BLK (rs13277113, rs2248932) polymorphism with systemic lupus erythematosus: a meta-analysis

The B-cell lymphocyte kinase (BLK) is a src-family protein tyrosine kinase specifically expressed in B-lineage cells that has been implicated in the pathogenesis of systemic lupus erythematosus (SLE) and has been investigated in numerous ethnically diverse studies. However, genetic association studies that have examined the association between BLK gene variants and SLE have produced conflicting results. To shed further light on this issue, we performed a meta-analysis of the association between rs13277113, rs2248932 polymorphism and SLE in different ethnic groups. An updated literature-based meta-analysis of six original articles involving 20,271 control individuals and 11,796 subjects with SLE was conducted. Crude ORs with 95% CIs were used to assess the strength of association between rs13277113, rs2248932 polymorphism and SLE risk. Publication bias was estimated using Egger's linear regression test. The authors assessed the evidence of genotypic association using STATA Version 10.0. The combined overall odds ratio, calculated for SLE and the risk A-allele of rs13277113 was 1.416 (95% CI: 1.358, 1.477). An odds ratio of 1.264 (95% CI: 1.208, 1.322) was found for the T-allele of rs2248932. Significant associations of rs13277113 and SLE were observed for dominant model (AA + AG vs. GG, OR: 1.518; 95% CI: 1.411, 1.632), and recessive model (AA vs. AG + GG, OR: 1.553; 95% CI: 1.461, 1.651); so were rs2248932 and SLE for dominant model (TT + TC vs. CC, OR: 1.342; 95% CI: 1.233, 1.460), and recessive model (TT vs. TC + CC, OR: 1.338; 95% CI: 1.257, 1.424). All of these were conducted in fixed effects model as heterogeneity was not detected. Tests for bias revealed no evidence of biases. On the assessment of available evidence, the authors concluded that moderate evidence exists for an association between the BLK rs13277113, rs2248932 variants and SLE. Therefore, further research is warranted on the role of BLK polymorphisms in the etiology of SLE.

The Tec kinases Itk and Rlk regulate conventional versus innate T‐cell development

Tec family kinases are important components of antigen receptor signaling pathways in B cells, T cells, and mast cells. In T cells, three members of this family, inducible T-cell kinase (Itk), resting lymphocyte kinase (Rlk), and Tec, are expressed. In the absence of Itk and Rlk, T-cell receptor signaling is impaired, with defects in mitogen-activated protein kinase activation, Ca(2+) mobilization, and actin polymerization. During T-cell development in the thymus, no role has been found for these kinases in the CD4(+) versus CD8(+) T-cell lineage decision; however, several studies indicate that Itk and Rlk contribute to the signaling leading to positive and negative selection. In addition, we and others have recently described an important role for Itk and Rlk in the development of conventional as opposed to innate CD4(+) and CD8(+) T cells. Natural killer T and gammadelta T-cell populations are also altered in Itk- and Rlk/Itk-deficient mice. These findings strongly suggest that the strength of T-cell receptor signaling during development determines whether T cells mature into conventional versus innate lymphocyte lineages. This lineage decision is also influenced by signaling via signaling lymphocytic activation molecule (SLAM) family receptors. Here we discuss these two signaling pathways that each contribute to conventional versus innate T-cell lineage commitment.