Bruton's Tyrosine Kinase Gene Research Articles

Abstract 3112: Transcription activator-like effector nucleases (TALENs) mediated silencing of Bruton's tyrosine kinase (BTK) inhibits phosphorylation of Erk in Burkitt lymphoma

Abstract BACKGROUND: Burkitt lymphoma (BL) is the most common non-Hodgkin lymphoma in children and adolescents (Cairo et al., Blood, 2007; Miles/Cairo et al., BJH, 2012). Children with BL who relapsed or progressed have chemotherapy resistant disease and can rarely be cured after salvage therapy (Cairo et al.,JCO, 2012). Bruton's tyrosine Kinase (BTK) is a major regulator of normal B-cell development and functions through activation of the BCR, MAPK, NF-κB and AKT signaling pathways (Qiu et al., Oncogene, 2000). Chronic active B-cell receptor signaling through activation of the BTK, can be specifically inhibited by a selective BTK inhibitor, ibrutinib (Davis et al., Nature, 2010). However, the mechanism(s) and function of BTK is still poorly understood in B-cells, especially in BL. Transcription Activator-Like Effector Nucleases (TALENs) technologies have been developed for precision targeted genome editing in a variety of organisms and stem cells for new experimental and therapeutic tools (Sander et al, Nat. Biotech., 2011; Carlson et al., Mol Ther., 2012). OBJECTIVES: We hypothesize that 1) TALEN nuclease technology is suitable for the modification of endogenous BTK gene expression in BL cells, and 2) a TALENs-mediated BTK knockout model will provide a useful tool for understanding the mechanism and function of BTK and the development of therapeutic agents that target BTK in BL. METHODS: BTK TALENs were constructed based on REAL (restriction enzyme and ligation) assembly methods (Sander et al., Nat. Biotech, 2011; Lee/Cairo et al., ASH, 2012) for BTK gene disruption. BTK TALENs validities in 293 and Raji cells were confirmed by surveyor assay and sequencing analysis. Western blotting was performed and statistical significance was determined by one-tailed Student t test using GraphPad Prism software. RESULTS: Two pairs of functional BTK TALENs (BTK-T1 and BTK-T2) targeting endogenous BTK gene were constructed and confirmed their validities with gene modification caused by non-homologous end joining (NHEJ) in TALENs mediated 293 cells. We confirmed that entire BTK coding sequences were deleted by PCR genotyping and sequencing analysis in stable BTK-knockout Raji cell clones (BTK+/-). BTK-T12-25 and BTK-T12-29 showed a significant decrease of BTK protein expression (50±1.1% reduction, p<0.0005; 27±0.7% reduction, p<0.0005, respectively) compared to empty vector-transfected Raji cells as mock control. There was a significant decrease in Erk phosphorylation in BTK knockout Raji cells compared to mock control (30±1.6%, p<0.001). CONCLUSIONS: These preliminary data suggest that BTK-TALENs are useful tools for the disruption of endogenous BTK gene expression. This TALEN mediated BTK knockout model will be used for studying BTK function in BL and the efficacy of BTK inhibitors as potential therapeutic agents in BL. Citation Format: Sanghoon Lee, Changhong Yin, Timmy O'Connell, Janet Ayello, Carmella van de Ven, Mitchell S. Cairo. Transcription activator-like effector nucleases (TALENs) mediated silencing of Bruton's tyrosine kinase (BTK) inhibits phosphorylation of Erk in Burkitt lymphoma. [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 3112. doi:10.1158/1538-7445.AM2014-3112

Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model.

X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency that results from mutations within the gene encoding Bruton's tyrosine kinase (BTK). Many XLA-associated mutations affect splicing of BTK pre-mRNA and severely impair B cell development. Here, we assessed the potential of antisense, splice-correcting oligonucleotides (SCOs) targeting mutated BTK transcripts for treating XLA. Both the SCO structural design and chemical properties were optimized using 2'-O-methyl, locked nucleic acid, or phosphorodiamidate morpholino backbones. In order to have access to an animal model of XLA, we engineered a transgenic mouse that harbors a BAC with an authentic, mutated, splice-defective human BTK gene. BTK transgenic mice were bred onto a Btk knockout background to avoid interference of the orthologous mouse protein. Using this model, we determined that BTK-specific SCOs are able to correct aberrantly spliced BTK in B lymphocytes, including pro-B cells. Correction of BTK mRNA restored expression of functional protein, as shown both by enhanced lymphocyte survival and reestablished BTK activation upon B cell receptor stimulation. Furthermore, SCO treatment corrected splicing and restored BTK expression in primary cells from patients with XLA. Together, our data demonstrate that SCOs can restore BTK function and that BTK-targeting SCOs have potential as personalized medicine in patients with XLA.

Clinical and mutational features of Vietnamese children with X-linked agammaglobulinemia.

BackgroundX-linked agammaglobulinemia (XLA) is a primary immune deficiency characterized by recurrent bacterial infections and profoundly depressed serum immunoglobulin levels and circulating mature B cells. It is caused by mutations of the Bruton tyrosine kinase (BTK) gene and is the most common form of inherited antibody deficiency. To our knowledge, this is the first report of XLA from Vietnam.MethodsWe investigated the BTK gene mutations and clinical features of four unrelated Vietnamese children.ResultsThe mean ages at onset and at diagnosis were 2.5 and 8 years, respectively. All patients had a medical history of otitis media, pneumonia, and septicemia at the time of diagnosis. Other infections reported included sinusitis, bronchiectasis, arthritis, skin infections, meningitis, and recurrent diarrhea. We identified one previously reported mutation (c.441G >A) and three novel mutations: two frameshifts (c.1770delG and c.1742 delG), and one nonsense (c.1249A >T).ConclusionsThe delayed diagnosis may be attributable to insufficient awareness of this rare disease on the background of frequent infections even in the immunocompetent pediatric population in Vietnam. Our results further support the importance of molecular genetic testing in diagnosis of XLA.

In vitro Correction of a Novel Splicing Alteration in the BTK Gene by Using Antisense Morpholino Oligonucleotides

A novel sequence variant, c.240+109C>A, in the Bruton's tyrosine kinase (BTK) gene was identified in a patient with X-linked agammaglobulinemia. This alteration resulted in an incorporation of 106 nucleotides of BTK intron 3 into its mRNA. Administration of the 25-mer antisense morpholino oligonucleotide analog in the patient's cultured peripheral blood mononuclear cells was able to restore correctly spliced BTK mRNA, a potential treatment for X-linked agammaglobulinemia.

Clonal Evolution In Patients With Chronic Lymphocytic Leukemia (CLL) Developing Resistance To BTK Inhibition

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib thwarts B cell receptor (BCR) signaling via irreversible inhibition of BTK, and induces durable remissions in relapsed/refractory CLL with a progression-free survival rate of 75% after 26 months of therapy (Byrd JC et al., NEJM 2013). However, a small fraction of patients treated with this targeted therapy develop progressive disease after an initial response. Here, we describe a longitudinal genomic investigation, utilizing whole-exome sequencing (WES) and copy number information of 3 patients who, with daily exposure to ibrutinib, achieved partial remission and later experienced CLL progression, but not Richter's transformation.All 3 patients had advanced stage CLL (Rai stage 3-4) and were enrolled on IRB-approved phase 1/2 trials of ibrutinib (420 mg) as a single agent (Patients 1 and 2) or combined with rituximab (Patient 3). Patients 1 and 3 had relapsed diseased after prior FCR frontline therapy, while Patient 2 had received 4 prior lines of therapy. At treatment initiation, Patients 1 and 2 had known acquired TP53 deletions. Patient 1 additionally had del(13q), and Patient 2 a subclone (11.5% by FISH) carrying trisomy 12 and del(13q). Patient 3 had complex cytogenetics which included del(11q). As the best response to ibrutinib-based therapy, all three experienced partial responses. Patient 1 demonstrated normalization of hematologic parameters but experienced persistent bone marrow disease. Patient 2 achieved a >10-fold reduction but persistently elevated absolute lymphocyte count, and resolution of anemia and thrombocytopenia. Patient 3 had resolution of anemia, thrombocytopenia, and splenomegaly. Progressive disease was observed at days 1022, 554 and 206 following ibrutinib initiation for Patients 1-3, respectively.DNA was extracted from CD19-purified CLL cells before ibrutinib therapy and at the time of disease progression. Matched germline and tumor DNA from 2 timepoints underwent WES (mean coverage depth 170X) and copy number analysis (by SNP 6.0 arrays). Somatic alterations were identified through comparison with germline DNA. To examine clonal populations, we measured the allelic fractions of somatic variants and integrated this information with local copy number and purity information to infer the fraction of cancer cells (CCF) affected by the mutation.Since ibrutinib targets BTK, we searched for resistance-conferring mutations in the BTK gene in the progressing leukemias, such as the previously described C481S BTK mutation in 4 of 13 patients with acquired resistance (Chang et al., ASCO 2013, Abstract 7014). We observed that all three patients lacked mutations in BTK and for the most part, in other genes in the BCR signaling pathway. In Patient 2, we did identify a single nucleotide variant in PLCg2, a substrate of BTK previously reported to be mutated in a patient with ibrutinub resistance. However, the CCF affected by this mutation was smaller than 0.15, and therefore it is unlikely to be the main driver of relapse in this patient. All three CLLs acquired new somatic mutations at the time of progression not observed in the pre-treatment samples, involving recurrent lesions in CLL associated with poor clinical outcome. Patient 1 acquired a new clonal (>0.95 CCF) mutation in SF3B1 (K666T). Patients 2 and 3 revealed clonal deletions in chromosome 8p. Patient 2 additionally demonstrated an increase in a subclone harboring trisomy 12 with an associated MLL2 missense substitution, with CCF rising from 0.12 pre-treatment to 0.5 upon relapse.Our results confirm that clinically evident ibrutinib resistance cannot be uniformly attributed to mutations in BTK or other genes of the BCR signaling pathway. In the 3 CLLs presented herein, progressive disease was associated with the emergence of leukemic populations harboring genetic alterations with putative driver characteristics (del(8p), SF3B1 mutation) arising from a background of pre-existing 17p or 11q deletions. Our findings support the concept that CLL clones persisting during continuous therapeutic pressure can adapt to bypass BTK-related survival signaling. Ongoing studies focus on finer kinetic analysis of clonal dynamics in these patients during the period leading up to progressive disease to elucidate whether these alterations were newly acquired following ibrutinib exposure or represent selective expansions of pre-existing small subclones. Disclosures:Burger:Pharmacyclics: Research Funding. O'Brien:Pharmacyclics: Research Funding. Neuberg:Synta Pharmaceuticals: Trust owns stock; I am a Trustee Other.

Targeting BTK As a Treatment For Multiple Myeloma Stem Cells

BackgroundMultiple myeloma stem cells (MMSCs), characterized by drug-resistance and self-renewal, are very likely responsible for relapse. Though the phenotypic features of MMSCs are still controversial, it is believed that MMSCs are enriched in plasma cell progenitors. Bruton's tyrosine kinase (BTK) plays a central role in B-cell development and plasma cell differentiation. BLAB/c. CBA/N mice carrying the defective BTK gene are resistant to pristine-induced plasmacytomagenesis suggesting activation of BTK is essential for plasma tumor initiation. In this study, we explore the role of BTK in maintaining MMSCs. Materials and methodsReal-time-PCR was used to detect the BTK expression in MMSCs and compared to the bulk cells. BTK-related self-renewal and drug resistance were evaluated by clonogenic formation assays, using 10,000 MM cells from the BTK over-expressing ARP1 and OPM2 MM cell lines, treated with bortezomib (1nM, 10nM), as well as doxorubicin (50nM, 100nM), etoposide (50nM, 100nM), and verapamil (50nM). Flow cytometry was performed to detect the side population (SP) fraction and apoptosis in MM cells. Co-immunoprecipitation assay and western blot were utilized for the mechanistic BTK studies. Effect of BTK on MM progression in vivo was assessed in the 5TGM1 myeloma mice and NOD/SCID mice models. ResultsOur results revealed that BTK, iPS genes, Wnt and Hedgehog signaling pathways were significantly increased in MMSCs (CD138- and SP fraction) compared to bulk myeloma cells in both MM cell lines and primary MM cells. We also demonstrated that over-expression of BTK in the low-expressing MM cell lines, ARP1 and OPM2 increased the SP fraction, clonogenic potential and drug-resistance, while knockdown of BTK in high-expressing MM cell lines, OCI-MY5 and H929 abrogated these characteristics. Our in vivo study indicated that over-expression of BTK in ARP1 cells accelerates tumor formation in NOD/SCID mice compared to the EV control cells. Targeting BTK by shRNA or BTK inhibitor, CGI-1747 delayed MM tumor formation and MM progression in NOD/SCID mice and 5TGM1 model, respectively. Mechanistic studies identified that BTK activates the AKT pathway, inducing the drug-resistant genes, p-Bcl-2 and ABCB1, and inhibiting GSK3β leading to activation of the Wnt/ β-Catenin pathway. Inhibition of AKT by a specific inhibitor decreased Wnt and Hh proteins by western blot. ConclusionBTK plays an important role on maintaining MM stemness and drug-resistance through activating AKT, Wnt and Hh signaling pathways. Targeting BTK in MM may be a promising strategy to eliminate MMSCs. Disclosures:No relevant conflicts of interest to declare.

Index of Suspicion

* ALT: : alanine aminotransferase AST: : aspartate aminotransferase BUN: : blood urea nitrogen CBC: : complete blood count CNS: : central nervous system CSF: : cerebrospinal fluid CT: : computed tomography ECG: : electrocardiography ED: : emergency department EEG: : electroencephalography ESR: : erythrocyte sedimentation rate GI: : gastrointestinal GU: : genitourinary Hct: : hematocrit Hgb: : hemoglobin MRI: : magnetic resonance imaging WBC: : white blood cell An 11-year-old boy presents to the emergency department with a 1-week history of frequent episodes of nonbloody, nonbilious emesis associated with diarrhea and abdominal cramping. He has urgency and fecal incontinence. He has been afebrile and denies any rashes, joint pain, sick contacts, headaches, or other symptoms. On further questioning, his mother reveals that he has had 2 similar episodes of abdominal distension and diarrhea during the past 6 to 7 years. Between these episodes, she states that he normally has nonbloody, hard stools accompanied by straining and chronic abdominal pain and pain with defecation. When asked specifically, his mother does not remember when he passed his first stool after he was born. On physical examination, his temperature is 37.6°C, pulse is 100 beats per minute, respiratory rate is 16 breaths per minute, blood pressure is 113/62 mm Hg, oxygen saturation is 100% on room air, height is 63 inches (95%), and weight is 36.7 kg (25%). He has abdominal distention and diffuse abdominal tenderness with voluntary guarding. There is palpable stool in the left quadrant. There is no perianal erythema or skin tag, and rectal examination reveals a low anal tone. A CT scan of the abdomen (Figure 1) reveals a markedly distended rectosigmoid colon with circumferential, confluent, and contiguous mural thickening. Basic metabolic profile, CBC, and urinalysis results are unremarkable, but the C-reactive protein level is elevated at 131 mg/L (1248 nmol/L). Because of the concern for enterocolitis, he is empirically given broad-spectrum intravenous antibiotics and transferred to a children’s hospital for further evaluation. Figure 1. A CT scan of the abdomen shows a markedly distended rectosigmoid colon with circumferential mural thickening. A 4-year-old boy presents for evaluation of intermittent fevers of 4 weeks’ duration after 2 separate antibiotic courses prescribed for acute otitis media. He has experienced clear rhinorrhea and …

A new mutation that predicted a drastic alteration of the BTK protein function

X-linked agammaglobulinemia (XLA) or Bruton disease is a relatively rare constitutionally immune disorder due to a genetic mutation of BTK (Bruton tyrosine kinase) gene which encodes for BTK protein. BTK is a signal-transducing protein expressed in hematopoietic lineages. The genetic disorder is responsible for B cell lymphocytes' maturation arrest. The humoral immunodeficiency caused by BTK mutation is linked with recurrent bacterial and viral infections. Genetic investigations of the prepositus as well as the other members of the family are necessary to characterize a mutation in BTK gene to confirm the diagnosis and reveal a hereditary transmission or de novo mutation.Authors propose and comment a case report of an 8month old child who suffers from Bruton disease. Initial presentation was neutropenia, but genetic investigation has revealed a previously non-reported mutation in the BTK gene. The same mutation was found in the mother with low expression of BTK in monocytes and normal in B-cells suggesting a lineage specific extinction of BTK which has never been reported before.

Bruton's tyrosine kinase: potential target in human multiple myeloma

Bruton's tyrosine kinase (BTK), a Tec family non-receptor tyrosine kinase that is required for B cell development, is critical for the initiation and maintenance of human B-cell malignancies. However, the expression of BTK and the role that BTK plays in the pathogenesis of multiple myeloma (MM) remain seldom reported. In this study we examined the expression and screened for gene mutations of BTK in MM cells. We showed that BTK was elevated and activated in a dexamethasone-resistant cell line and in two out of nine (22.2%) patients’ cells. Interestingly, patients with higher BTK expression had a poorer prognosis. In addition, a single nucleotide polymorphism (SNP) at cDNA position 2062 (T2062C) in the BTK gene was recorded in six out of eight (75%) patients and in U266 cells. This SNP in MM cells was not detected in other malignant hematopoietic cells of different lineages. These results suggest that the function of BTK warrants further investigation, and BTK expression might be used as a prognostic indicator for patients with MM.

A novel Bruton’s tyrosine kinase gene (BTK) invariant splice site mutation in a Malaysian family with X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA) is a rare genetic disorder caused by mutations in the Bruton's tyrosine kinase (BTK) gene. These mutations cause defects in early B cell development. A patient with no circulating B cells and low serum immunoglobulin isotypes was studied as were his mother and sister. Monocyte BTK protein expression was evaluated by flow cytometry. The mutation was determined using PCR and followed by sequencing. Flow cytometry showed the patient lacked BTK protein expression in his monocytes while the mother and sister had 62% and 40% of the monocytes showing BTK protein expressions respectively. The patient had a novel base substitution in the first nucleotide of intron 9 in the BTK gene, and the mutation was IVS9+1G<C. This mutation resulted in exon 9 skipping. This defect rendered the patient susceptible to asthma, failure to thrive, recurrent pyogenic infections, otitis media and bronchopneumonia. His mother and sister were heterozygous for this mutation. The combination of flow cytometry and genetic study is necessary in the diagnosis of X-linked agammaglobulinemia and may be used for subsequent genetic counseling, carrier detection and prenatal diagnosis.

Role of Bruton's tyrosine kinase in myeloma cell migration and induction of bone disease

Myeloma cells typically grow in bone, recruit osteoclast precursors and induce their differentiation and activity in areas adjacent to tumor foci. Bruton's tyrosine kinase (BTK), of the TEC family, is expressed in hematopoietic cells and is particularly involved in B-lymphocyte function and osteoclastogenesis. We demonstrated BTK expression in clinical myeloma plasma cells, interleukin (IL)-6- or stroma-dependent cell lines and osteoclasts. SDF-1 induced BTK activation in myeloma cells and BTK inhibition by small hairpin RNA or the small molecule inhibitor, LFM-A13, reduced their migration toward stromal cell-derived factor-1 (SDF-1). Pretreatment with LFM-A13 also reduced in vivo homing of myeloma cells to bone using bioluminescence imaging in the SCID-rab model. Enforced expression of BTK in myeloma cell line enhanced cell migration toward SDF-1 but had no effect on short-term growth. BTK expression was correlated with cell-surface CXCR4 expression in myeloma cells (n = 33, r = 0.81, P < 0.0001), and BTK gene and protein expression was more profound in cell-surface CXCR4-expressing myeloma cells. BTK was not upregulated by IL-6 while its inhibition had no effect on IL-6 signaling in myeloma cells. Human osteoclast precursors also expressed BTK and cell-surface CXCR4 and migrated toward SDF-1. LFM-A13 suppressed migration and differentiation of osteoclast precursors as well as bone-resorbing activity of mature osteoclasts. In primary myeloma-bearing SCID-rab mice, LFM-A13 inhibited osteoclast activity, prevented myeloma-induced bone resorption and moderately suppressed myeloma growth. These data demonstrate BTK and cell-surface CXCR4 association in myeloma cells and that BTK plays a role in myeloma cell homing to bone and myeloma-induced bone disease. Am. J. Hematol. 88:463-471, 2013. © 2013 Wiley Periodicals, Inc.

Clinical features and mutation analysis of X-linked agammaglobulinemia in 20 Chinese patients

X-linked agammagobulinemia (XLA) is a primary immunodeficiency caused by Bruton's tyrosine kinase (BTK) gene mutation. XLA patients have an extremely small amount of peripheral B cells and profound deficiency in all immunoglobulin isotypes. We analyzed the clinical, immunologic, and molecular characteristics of children with XLA in an attempt to improve the diagnosis and treatment of XLA in China. Twenty children with XLA-compatible phenotypes from 18 unrelated families were enrolled in this study. The BTK gene was amplified and sequenced, followed by mutation analysis in these children and their female relatives. Eighteen different mutations of the BTK gene were identified in the 20 patients. Eleven mutations had been reported previously including eight missense mutations (c.994C>T, c.1987C>A, c.1885G>T, c.502T>C, c.1085C>T, c.1816C>T, c.214C>T, c.1912G>A) and three nonsense mutations (c.1267T>A, c.1793C>G, c.1618C>T). Seven novel mutations of the BTK gene were also presented and included five missense mutations (c.134T>A, c.1646T>A, c.1829C>G, c.711G>T, c.1235G>A), one splice-site mutation (c.523+1G>A) and one insertion mutation (c.1024-1025in sTTGCTAAAGCAACTGCTAAAGCAAG). Eight out of 18 mutations of the BTK gene were located in the TK domain, 4 in the PH domain, 4 in the SH2 domain and 2 in the TH domain. Genetic study for carrier status was carried out in 18 families with definite BTK gene mutations. Nine carriers with BTK gene mutations were identified. Six families without carriers were detected, and 3 patients were not tested in this study. Our results support that molecular genetic testing represents an important tool for early confirmed diagnosis of congenital agammaglobulinemia and may allow accurate carrier detection and prenatal diagnosis.

Tek Merkezden X'e Bağlı Agamaglobulinemi Hastaların Bruton Tirozin Kinaz Gen Mutasyonları: βTK Geninde Yeni Mutasyonlar

Objective: X-linked agammaglobulinemia (XLA) is caused by a mutation in the Bruton's tyrosine kinase gene and is characterized by a delay in the maturation and differentiation of B lymphocytes. Patients with XLA have either absent or very low levels of circulating mature B cells (<1%), plasma cells, and immunoglobulins of all isotypes. Recurrent bacterial infections are prevalent in patients with XLA. The absence of B cells, agammaglobulinemia and the clinical features of XLA are similar to autosomal recessive forms such as µ heavy chain, λ5, Igα, Igβ and a BLNK gene defects. The aim of this study was to diagnose Turkish patients with suspected XLA by using BTK mutation analysis. Material and Methods: Fourteen male children recruited from 13 different families that were diagnosed with suspected XLA based on recurrent bacterial infections, decreased B cell count in the peripheral blood and hypogammaglobulinemia were included in the study. Clinical and demographic features of patients, serum immunoglobulin levels, peripheral blood B cell counts and BTK gene mutations were reviewed retrospectively. Patients were examined for BTK mutations by polymerase chain reaction. Results: Three of 14 patients were the children of consanguineous marriages and 3 families had a history of immune deficiencies. Most of the patients presented with infections, primarily pneumonia. BTK mutation resulting in XLA was detected in 10 patients. Eight patients had previously been diagnosed and 2 patients had novel mutations. Conclusion: Mutation analysis must be done to differentiate XLA from other autosomal recessive forms such as µ heavy chain, λ5, Igα, Igβ and a BLNK gene defects and this is necessary to design genetic counseling for newly diagnosed patients.

Bruton’s Tyrosine Kinase: Structure and Functions, Expression and Mutations

Bruton’s tyrosine kinase (BTK), a member of the Tec family of protein tyrosine kinases (PTKs), plays a vital and diverse function in many cellular processes. BTK is expressed throughout B cell development, widely participating in multiple signal pathways including PI3K, PLCγ, and PKC. Those pathways play critical functions in cell proliferation, development, differentiation, survival, and apoptosis. The expression of BTK is selectively down-regulated in T lymphocytes and plasma cells and the relative level of BTK expression may be modulated in different developmental populations of B cells. Mutations in the gene for BTK are responsible for X-linked agammaglobulinemia (XLA) in human and X-inked immunodeficiency (xid) in mice. Both of these diseases are characterized by blocks in B-cell development at multiple stages and impaired function of residual mature B cells. To date, more than 1252 mutations have been identified in human BTK gene associating with XLA. Targeting BTK has achieved remarkable efficacy in B cell malignancies, multiple myeloma and related bone disease. The present review discusses the recent data regarding the role of BTK in B cell development and its structure, regulation, functions, expression and mutations.

A Bruton’s Tyrosine Kinase inhibitor prevents antigen-driven B cell activation, germinal center persistence and memory B cell re-activation in vivo (109.10)

Abstract The Tec family kinase, Bruton’s Tyrosine Kinase (BTK), is one of the key intracellular signaling components proximal to the B cell receptor (BCR). In this study, we utilized an orally-accessible BTK inhibitor (BTKi) to study the functional effects blocking this signaling pathway has on antigen-driven B cell activation in vivo. We report that BTK is required for anti-IgD driven B cell activation in vivo as evidenced by suppression of the surface activation markers CD86 and mRNA transcripts (e.g. c-Myc, Bcl-xL, CCL3, CD98, EBI2, EGR1, EGR2 and IRF4) otherwise rapidly induced upon engagement of the BCR with antigen. Using the T-dependent protein PE and SRBCs as model antigens, we tested for a requirement of BTK in generating antigen-specific IgM and IgG antibody titers and germinal centers, and find that BTK is required for germinal center maintenance. Lastly, we tested for a role of BTK in long-lived PE-specific memory B cell re-activation and report that BTKi significantly suppressed the induction of PE-specific IgM and IgG titers upon antigen re-challenge. The use of BTKi allowed us to bypass the impact a genetically inactivated BTK gene has on B cell development and to assess BTK inactivation in both a wild-type setting and in a temporal manner. These results provide insight into the role of BTK in BCR-driven B cell activation in various antigen naïve and experienced B cell populations.

Cell Surface CXCR4 and BTK Expression Are Associated in Myeloma Cells and Osteoclast Precursors and Mediate Myeloma Cell Homing and Clonogenicity, and Osteoclastogenesis

Abstract 884Myeloma cells typically grow in bone, recruit osteoclast precursors and induce their differentiation and activity in areas adjacent to tumor foci. Clonal B-lymphocytes and plasma cells harboring lymphocytic phenotypes have a proposed role in sustaining myeloma. Bruton’s tyrosine kinase (BTK) is expressed in hematopoietic cells and is particularly involved in B-lymphocyte function and osteoclastogenesis. The SDF-1/CXCR4 signaling pathway is reportedly involved in homing to bone of myeloma cells and osteoclast precursors. The aims of the study were to investigate the possible association between CXCR4 and BTK signaling in myeloma cells and osteoclast precursors, and the consequences of BTK inhibition on myeloma cell migration and clonogenicity, and osteoclastogenesis. By global gene expression profiling (GEP), BTK expression was moderately higher in clinical myeloma cells (n=559) than normal plasma cells (n=25, p<0.05). BTK was also detected in IL6–dependent cell lines and myeloma cell lines that can passage in vivo (n=7), using GEP, qRT-PCR and Western Blot analyses. Cell surface CXCR4 determined by flow cytometry was variably expressed in myeloma cells (5%–95%) and was significantly correlated with BTK gene expression (r=0.75, p<0.002, n=14). Most myeloma cell lines grown independently in vitro expressed very low levels of BTK; however, even in such cell lines (e.g. CAG), enrichment of myeloma cells expressing cell-surface CXCR4 by FACS analysis resulted in detectable BTK expression. BTK inhibition by shRNA in IL6–dependent INA6 cells inhibited their migration toward SDF-1 by >50% (p<0.006) and diminished their ability to form colonies in a 2-weeks clonogenic assay (p<0.0002). The BTK small molecule inhibitor LFM-A13 (25–50 μM) consistently reduced SDF-1-induced migration of primary myeloma cells and myeloma lines (n=6, p<0.03), and clonogenicity (colonies number and size) of 3 BTK-expressing myeloma lines by >50% (p<0.03). Using kinase immunoprecipitation assay, SDF-1 rapidly induced BTK phosphorylation (activation) in myeloma cells, an effect that was blocked by LFM-A13. In vivo, pretreatment of luciferase-expressing myeloma cells with LFM-A13 lessened their homing to bone following intravenous injection into SCID-rab mice as determined by live-animal imaging. BTK was highly expressed in osteoclast precursors while cell surface CXCR4 was detected in 5% of this cell population. LFM-A13 also reduced osteoclast precursor migration toward SDF-1 and suppressed osteoclast formation and bone resorption activity on dentine slices (p<0.002). In myeloma-bearing SCID-rab mice, LFM-A13 (n=10, 40 mg/kg, twice daily for 3 weeks, i.p.) reduced osteoclast number by 45% (p<0.004), prevented reduction of bone mineral density (BMD, p<0.04) and attenuated myeloma growth at near significant level (p<0.07). These data indicate that CXCR4 and BTK signaling are linked in myeloma cells and osteoclast precursors and that BTK has potential as a targeted myeloma therapy because of its roles in myeloma cell homing and clonogenicity and myeloma-induced osteolysis. Disclosures:Barlogie:Celgene, Genzyme, Novartis, Millennium: Consultancy, Honoraria, Patents & Royalties. Shaughnessy:Myeloma Health, Celgene, Genzyme, Novartis: Consultancy, Employment, Equity Ownership, Honoraria, Patents & Royalties.

Genetic analysis of contiguous X-chromosome deletion syndrome encompassing the BTK and TIMM8A genes

Patients with X-linked agammaglobulinemia (XLA) can present with sensorineural deafness. This can result from a gross deletion that not only involved the Bruton's tyrosine kinase (BTK) gene, but also TIMM8A, mutations in which underlie the Mohr-Tranebjærg syndrome (MTS). We analyzed the genomic break points observed in three XLA-MTS patients and compared these with deletions break points from XLA patients. Patient 1 had a 63-kb deletion with break points in intron 15 of BTK and 4 kb upstream of TAF7L. Patients 2 and 3 had 149.7 and 196 kb deletions comprising BTK, TIMM8A, TAF7L and DRP2. The break points in patients 1 and 3 were located in Alu and endogenous retrovirus (ERV) repeats, whereas the break points in patient 2 did not show involvement of transposable elements. Comparison of gross deletion sizes and involvement of transposable elements in XLA and XLA-MTS patients from the literature showed preferential involvement of Alu elements in smaller deletions (<10 kb). These results show further insights into the molecular mechanisms underlying gross deletions in patients with primary immunodeficiency.

TEC family kinases in health and disease – loss‐of‐function of BTK and ITK and the gain‐of‐function fusions ITK–SYK and BTK–SYK

The TEC family is ancient and constitutes the second largest family of cytoplasmic tyrosine kinases. In 1993, loss-of-function mutations in the BTK gene were reported as the cause of X-linked agammaglobulinemia. Of all the existing 90 tyrosine kinases in humans, Bruton's tyrosine kinase (BTK) is the kinase for which most mutations have been identified. These experiments of nature collectively provide a form of mutation scanning with direct implications for the several hundred endogenous signaling proteins carrying domains also found in BTK. In 2009, an inactivating mutation in the ITK gene was shown to cause susceptibility to lethal Epstein-Barr virus infection. Both kinases represent interesting targets for inhibition: in the case of BTK, as an immunosuppressant, whereas there is evidence that the inhibition of inducible T-cell kinase (ITK) could influence the infectivity of HIV and also have anti-inflammatory activity. Since 2006, several patients carrying a fusion protein, originating from a translocation joining genes encoding the kinases ITK and spleen tyrosine kinase (SYK), have been shown to develop T-cell lymphoma. We review these disease processes and also describe the role of the N-terminal pleckstrin homology-Tec homology (PH-TH) domain doublet of BTK and ITK in the downstream intracellular signaling of such fusion proteins.

Clinical Characteristics and Molecular Analysis of 21 Chinese Children with Congenital Agammaglobulinemia

Congenital agammaglobulinemia is a humoral primary immunodeficiency and affected patients have extremely low levels of peripheral B cells and profound deficiency of all immunoglobulin isotypes. Mutations of the Bruton's tyrosine kinase (BTK) gene are responsible for most of the congenital agammaglobulinemia. In this study, the phenotypes of congenital agammaglobulinemia were investigated in 21 male children from 21 unrelated Chinese families. Sixteen different mutations of BTK gene were identified in 18 patients, and three patients did not have BTK gene mutations. Nine mutations had been reported previously including one gross deletion (c.722_2041del), one missense mutation (c.1764G>T), three non-sense mutations (c.194C>A, c.895C>T and c.1821G>A) and four invariant splice-site mutations (c.971+2T>C, c.1481+2T>A, c.1482-2A>G, c.1699-2A>G). Seven novel mutations were identified (c.373_441del, c. 504delG, c.537delC, c.851delA, c.1637G>A, c.1879T>C and c. 1482_1882 del). Ten of the eighteen mutations of BTK gene were located in the TK domain, four in the PH domain, three in the SH3 domain and one spanned the TH, SH3, SH2 and TK domain. Candidate genes of autosomal-recessive agammaglobulinemia, including IGHM, CD79a, CD79b and IGLL1, were screened in three patients without mutations in the BTK gene. A compound heterozygosity mutation in the IGHM gene (c.1956G>A, c.175_176insC) was identified in one patient. The results of our study further support that molecular genetic testing represents an important tool for early confirmed diagnosis of congenital agammaglobulinemia and may allow accurate carrier detection and prenatal diagnosis.

Mutations of Bruton's tyrosine kinase gene in Brazilian patients with X-linked agammaglobulinemia

Mutations in Bruton's tyrosine kinase (BTK) gene are responsible for X-linked agammaglobulinemia (XLA), which is characterized by recurrent bacterial infections, profound hypogammaglobulinemia, and decreased numbers of mature B cells in peripheral blood. We evaluated 5 male Brazilian patients, ranging from 3 to 10 years of age, from unrelated families, whose diagnosis was based on recurrent infections, markedly reduced levels of IgM, IgG and IgA, and circulating B cell numbers <2%. BTK gene analysis was carried out using PCR-SSCP followed by sequencing. We detected three novel (Ala347fsX55, I355T, and Thr324fsX24) and two previously reported mutations (Q196X and E441X). Flow cytometry revealed a reduced expression of BTK protein in patients and a mosaic pattern of BTK expression was obtained from mothers, indicating that they were XLA carriers.