Tec Family Of Kinases Research Articles

Abstract 2591: Ibrutinib can reverse established chronic graft-versus-host disease, which is dependent upon IL-2 inducible T-cell kinase (ITK) and Bruton's tyrosine kinase (BTK)-driven lymphocyte activation

Abstract Chronic graft versus host disease (cGVHD) is a life threatening impediment to the otherwise curative potential of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Intensive study has unveiled the lymphoid subsets that drive multifaceted autoimmune and fibrotic cascades which disrupt healthy tissues. Clinical and in-vivo data implicate B-cells and certain subsets of CD4 helper T-cells in the development and progression of cGVHD. These specific immune subsets are unique in their singular dependence upon the TEC-family kinases BTK or ITK for receptor driven activation. Ibrutinib is an irreversible inhibitor of BTK and ITK that has produced durable remissions in B-cell malignancies with minimal toxicity. We sought to examine ibrutinib's potential to attenuate B-cell and CD4 T-cell driven anti-host immunity in the setting of cGVHD. We utilized two complementary murine models independently focused on interrogating sclerodermatous T-cell driven cGVHD (LPJ→C57BL/6) or alloantibody driven bronchiolar obliterans (BO) and multi-organ system disease without skin involvement (C57BL/6→B10.BR). We examined therapeutic administration of 25mg/kg/day ibrutinib initiated when mice first develop signs of cGVHD (25-28 days after allo-HSCT). In the minor-MHC mismatch LPJ→C57BL/6 model, ibrutinib significantly extended median time to cGVHD progression by 14 days and 33% (6 of 18) of ibrutinib treated mice remained progression free as compared to 12% (2 of 18) of mice receiving vehicle and 10% (1 of 11) of mice receiving cyclosporine 10mg/kg/day (p<0.02). Overall survival was also highest in the ibrutinib treatment cohort. Mice receiving ibrutinib showed restoration of external cGVHD criterion including body weight, posture, skin lesions, hair loss, and mobility. There was significant ablation of lymphohistiocytic T- and B-cell pulmonary and renal infiltration in ibrutinib treated animals. Data from the C57BL/6→B10.BR model confirmed the efficacy of ibrutinib in significantly restoring pulmonary compliance, elastance, and resistance to levels indistinguishable from that of a healthy animal and significantly better than cGVHD vehicle treated controls (n=12/group)(p<0.01). These results were associated with significantly decreased alloantibody deposition and decreased fibrosis in the lungs of ibrutinib treated mice. Germinal center (GC) reactions, required for multi-organ system pathology in this model, were also significantly reduced within the spleens of mice receiving therapeutic ibrutinib (p<0.05). Using in-vivo genetic ablation models, we demonstrate the critical nature of BTK and ITK respectively in the development of cGVHD pathognomonic BO. Our data support ibrutinib as a promising therapeutic direction for the treatment of cGVHD by virtue of its ability to suppress BTK and ITK signaling. Citation Format: Jason A. Dubovsky, Ryan Flynn, Jing Du, Bonnie K. Harrington, Yiming Zhong, Carrie Yang, William Towns, Amy Lehman, Amy Johnson, Steven Devine, Samantha Jaglowski, Jonathan S. Serody, William J. Murphy, David H. Munn, Leo Luznik, Geoffrey Hill, Kelli K.P. MacDonald, Ivan Maillard, John Koreth, Corey Cutler, Robert J. Soiffer, Joseph H. Antin, Jerome Ritz, Angela Panoskaltsis-Mortari, John C. Byrd, Bruce R. Blazar. Ibrutinib can reverse established chronic graft-versus-host disease, which is dependent upon IL-2 inducible T-cell kinase (ITK) and Bruton's tyrosine kinase (BTK)-driven lymphocyte activation. [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 2591. doi:10.1158/1538-7445.AM2014-2591

A Conserved Isoleucine Maintains the Inactive State of Bruton's Tyrosine Kinase

Despite high level of homology among non-receptor tyrosine kinases, different kinase families employ a diverse array of regulatory mechanisms. For example, the catalytic kinase domains of the Tec family kinases are inactive without assembly of the adjacent regulatory domains, whereas the Src kinase domains are autoinhibited by the assembly of similar adjacent regulatory domains. Using molecular dynamics simulations, biochemical assays, and biophysical approaches, we have uncovered an isoleucine residue in the kinase domain of the Tec family member Btk that, when mutated to the closely related leucine, leads to a shift in the conformational equilibrium of the kinase domain toward the active state. The single amino acid mutation results in measureable catalytic activity for the Btk kinase domain in the absence of the regulatory domains. We suggest that this isoleucine side chain in the Tec family kinases acts as a “wedge” that restricts the conformational space available to key regions in the kinase domain, preventing activation until the kinase domain associates with its regulatory subunits and overcomes the energetic barrier to activation imposed by the isoleucine side chain.

Targeting Btk/Etk of prostate cancer cells by a novel dual inhibitor.

Btk and Etk/BMX are Tec-family non-receptor tyrosine kinases. Btk has previously been reported to be expressed primarily in B cells and has an important role in immune responses and B-cell malignancies. Etk has been shown previously to provide a strong survival and metastasis signal in human prostate cancer cells, and to confer androgen independence and drug resistance. While the role of Etk in prostate carcinogenesis is well established, the functions of Btk in prostate cancer have never been investigated, likely due to the perception that Btk is a hematopoietic, but not epithelial, kinase. Herein, we found that Btk is overexpressed in prostate cancer tissues and prostate cancer cells. The level of Btk in prostate cancer tissues correlates with cancer grades. Knockdown of Btk expression selectively inhibits the growth of prostate cancer cells, but not that of the normal prostate epithelial cells, which express very little Btk. Dual inhibition of Btk and Etk has an additive inhibitory effect on prostate cancer cell growth. To explore Btk and Etk as targets for prostate cancer, we developed a small molecule dual inhibitor of Btk and Etk, CTN06. Treatment of PC3 and other prostate cancer cells, but not immortalized prostate epithelial cells with CTN06 resulted in effective cell killing, accompanied by the attenuation of Btk/Etk signals. The killing effect of CTN06 is more potent than that of commonly used inhibitors against Src, Raf/VEGFR and EGFR. CTN06 induces apoptosis as well as autophagy in human prostate cancer cells, and is a chemo-sensitizer for docetaxel (DTX), a standard of care for metastatic prostate cancer patients. CTN06 also impeded the migration of human prostate cancer cells based on a ‘wound healing' assay. The anti-cancer effect of CTN06 was further validated in vivo in a PC3 xenograft mouse model.

THU0499 Hm71224, A Novel Oral BTK Inhibitor, Inhibits Human Immune Cell Activation: New Drug Candidate to Treat B-Cell Associated Autoimmune Diseases

BackgroundBruton tyrosine kinase (BTK), a Tec family kinase, is involved in B cell activation and osteoclast formation. As BTK plays a central role in B cell receptor (BCR) signaling and...

FRI0364 Hm71224, A Novel BTK Inhibitor Demonstrates Potent Activities in Rodent Models of Arthritis

BackgroundBTK is a member of the Tec family kinase of non-receptor protein tyrosine kinases1. BTK is mostly expressed in hematopoietic cells such as B cells, mast cells and macrophages2. BTK...

The Accessory Factor Nef Links HIV-1 to Tec/Btk Kinases in an Src Homology 3 Domain-dependent Manner

The HIV-1 Nef virulence factor interacts with multiple host cell-signaling proteins. Nef binds to the Src homology 3 domains of Src family kinases, resulting in kinase activation important for viral infectivity, replication, and MHC-I down-regulation. Itk and other Tec family kinases are also present in HIV target cells, and Itk has been linked to HIV-1 infectivity and replication. However, the molecular mechanism linking Itk to HIV-1 is unknown. In this study, we explored the interaction of Nef with Tec family kinases using a cell-based bimolecular fluorescence complementation assay. In this approach, interaction of Nef with a partner kinase juxtaposes nonfluorescent YFP fragments fused to the C terminus of each protein, resulting in YFP complementation and a bright fluorescent signal. Using bimolecular fluorescence complementation, we observed that Nef interacts with the Tec family members Bmx, Btk, and Itk but not Tec or Txk. Interaction with Nef occurs through the kinase Src homology 3 domains and localizes to the plasma membrane. Allelic variants of Nef from all major HIV-1 subtypes interacted strongly with Itk in this assay, demonstrating the highly conserved nature of this interaction. A selective small molecule inhibitor of Itk kinase activity (BMS-509744) potently blocked wild-type HIV-1 infectivity and replication, but not that of a Nef-defective mutant. Nef induced constitutive Itk activation in transfected cells that was sensitive to inhibitor treatment. Taken together, these results provide the first evidence that Nef interacts with cytoplasmic tyrosine kinases of the Tec family and suggest that Nef provides a mechanistic link between HIV-1 and Itk signaling in the viral life cycle.

Therapeutic treatment of multi-organ system, obstructive pulmonary and scleradermatous chronic graft-versus-host disease with the BTK and ITK inhibitor Ibrutinib (TRAN3P.873)

Abstract Chronic GVHD (cGVHD) is dependent on the ability of donor bone marrow (BM) B cells to produce pathogenic antibody (Ab) deposited in target organs, which is associated with increased germinal centers (GCs). BTK is necessary for B cells to enter GCs and ITK is necessary for T cell activation. We hypothesized that alloreactive GC B cells and T follicular helper cells require BTK or ITK and inhibition by ibrutinib (Ib) would prevent cGVHD. We evaluated the therapeutic effect of Ib in two models of cGVHD: a MHC-disparate (B6→B10.BR) multi-organ model complicated by bronchiolitis obliterans (BO) and a sclerodermatous (SCL) minor-mismatch (LP/J→B6) model. Mice treated with Ib had a decrease in pathogenic Ab, collagen deposition and GC B cells in target organs similar to healthy control mice. In contrast to cGVHD mice, pulmonary function in Ib treated mice was similar to control mice. To confirm that BTK and ITK are required for cGVHD we used donor mice with an ablation of BTK or ITK. Absence of BTK in B cells impeded GCs, Ab deposition and BO. Mice transplanted with T cells lacking ITK and WT BM did not develop BO. In the SCL model, mice treated with Ib did not develop clinical signs of cGVHD including less alopecia and SCL lesions. Furthermore, there was a decrease in lymphocyte infiltration surrounding the bronchioles and nephrons in treated mice. The inhibition of TEC-family kinases with Ib is a novel therapeutic for multi-organ system cGVHD with BO or SCL manifestations.

Treatment of Immunological or Inflammatory Disorders with ITK Kinase Inhibitors.

Title: Pyrazole Carboxamide Compounds, Compositions and Methods of Use Patent Application Number: WO 2014/023258 Al Publication date: 13 February 2014 Priority Application: US 61/682,063 Priority date: 10 August 2012 US 61/764,434 13 February 2013 US 61/764,930 14 February 2013 Inventors: Brookfield, F.; Burch, J.; Goldsmith, R. A.; Hu, B.; Lau, K. H. L.; Mackinnon, C. H.; Ortwine, D. F.; Pei, Z.; Wu, G.; Yuen, P.-W.; Zhang, Y. Assignee Company: F. Hoffmann-La Roche AG, Grenzacherstrasse, 124, CH-4070 Basel (CH) (for all designated States except US) Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA (for US only) Disease Area: Asthma and immunological or inflammatory disorders mediated by ITK kinase Biological Target: Inhibition of ITK kinase Summary: The invention in this patent application relates to pyrazole carboxamide derivatives represented generally by formula (I) which are inhibitors of ITK kinase. The compounds may potentially treat immunological or inflammatory disorders and other diseases responsive to the inhibition of ITK kinase. Interleukin-2-inducible T-cell kinase (ITK) belongs to the Tec family kinases, and it is expressed in T cells, NKT cells, NK cells, and mast cells. Activated ITK kinase mediates T cell receptor (TCR) signals through the phosphorylation and activation of phospholipase C-g (PLCg). Studies show that ITK knockout mice exhibit reduced lung inflammation, mucus production, and airway hyperreactivity in allergic asthma models. The studies also indicated that the kinase activity of ITK is necessary for asthma pathology. Additionally, ITK is found to be expressed at high levels in peripheral blood T cells of human patients with immunological and inflammatory disorders such as atopic dermatitis. Thus, inhibition of ITK kinase presents a viable therapeutic target to potentially treat the immunological or inflammatory disorders mediated by the activity of this kinase.

Itk-mediated integration of T cell receptor and cytokine signaling regulates the balance between Th17 and regulatory T cells

A proper balance between Th17 and T regulatory cells (Treg cells) is critical for generating protective immune responses while minimizing autoimmunity. We show that the Tec family kinase Itk (IL2-inducible T cell kinase), a component of T cell receptor (TCR) signaling pathways, influences this balance by regulating cross talk between TCR and cytokine signaling. Under both Th17 and Treg cell differentiation conditions, Itk(-/-) CD4(+) T cells develop higher percentages of functional FoxP3(+) cells, associated with increased sensitivity to IL-2. Itk(-/-) CD4(+) T cells also preferentially develop into Treg cells in vivo. We find that Itk-deficient T cells exhibit reduced TCR-induced phosphorylation of mammalian target of rapamycin (mTOR) targets, accompanied by downstream metabolic alterations. Surprisingly, Itk(-/-) cells also exhibit reduced IL-2-induced mTOR activation, despite increased STAT5 phosphorylation. We demonstrate that in wild-type CD4(+) T cells, TCR stimulation leads to a dose-dependent repression of Pten. However, at low TCR stimulation or in the absence of Itk, Pten is not effectively repressed, thereby uncoupling STAT5 phosphorylation and phosphoinositide-3-kinase (PI3K) pathways. Moreover, Itk-deficient CD4(+) T cells show impaired TCR-mediated induction of Myc and miR-19b, known repressors of Pten. Our results demonstrate that Itk helps orchestrate positive feedback loops integrating multiple T cell signaling pathways, suggesting Itk as a potential target for altering the balance between Th17 and Treg cells.

Inhibition of IL-2 inducible T-cell kinase alleviates T-cell activation and murine myocardial inflammation associated with CVB3 infection

BackgroundCoxsackievirus B3 (CVB3) infection causes myocarditis, pancreatitis, and aseptic meningitis. Targeting antigen-specific T cell reactions might be a promising way to alleviate the inflammatory response induced by CVB3 infection. IL-2-inducible T-cell kinase (ITK), a member of Tec kinase family expressed mainly in T cells, plays an important role in the activation of T cells. The role of ITK in viral myocarditis induced by CVB3 has not been documented. MethodologyIn this study, we inhibited the ITK expression in Jurkat cells, primary human peripheral blood mononuclear cells (PBMC), and mouse splenocytes by ITK-specific siRNA. The inhibition efficiently suppressed cell proliferation (P<0.05) and T-cell related cytokine secretion (P<0.05). In order to inhibit ITK in vivo, the pGCSIL plasmid containing short hairpin RNAs targeting ITK was constructed and transduced into mice infected with CVB3. ITK-inhibited mice showed reduced cell proliferation (3, 5, and 7 days post-challenge, P<0.05) as well as CD4+ and CD8+ T cells (5 days post-challenge, P<0.05). The altered production of inflammatory cytokines alleviated pathologic heart damage and improved mice survival rate (P<0.05). ConclusionITK played an important role in the T cell development and represented a new target for the modulation of T-cell-mediated inflammatory response by CVB3 infection.

Current understanding of tyrosine kinase BMX in inflammation and its inhibitors.

Tec family kinases, which include tyrosine kinase expressed in hepatocellular carcinoma (TEC), Bruton’s tyrosine kinase (BTK), interleukin (IL)-2-inducible T-cell kinase (ITK), tyrosine-protein kinase (TXK), and bone marrow tyrosine kinase on chromosome X (BMX), are the second largest group of non-receptor tyrosine kinases and have a highly conserved carboxyl-terminal kinase domain. BMX was identified in human bone marrow cells, and was demonstrated to have been expressed in myeloid hematopoietic lineages cells, endothelial cells, and several types of cancers. Significant progress in this area during the last decade revealed an important role for BMX in inflammation and oncologic disorders. This review focuses on BMX biology, its role in inflammation and possible signaling pathways, and the potential of selective BMX inhibitors.

The Influence ofBMXGene Polymorphisms on Clinical Symptoms after Mild Traumatic Brain Injury

Mild traumatic brain injury (mTBI) is one of the most common neurological disorders. Most patients diagnosed with mTBI could fully recover, but 15% of patients suffer from persistent symptoms. In recent studies, genetic factors were found to be associated with recovery and clinical outcomes after TBI. In addition, results from our previous research have demonstrated that the bone marrow tyrosine kinase gene in chromosome X (BMX), a member of the Tec family of kinases, is highly expressed in rats with TBI. Therefore, our aim in this study was to identify the association between genetic polymorphisms of BMX and clinical symptoms following mTBI. Four tagging single nucleotide polymorphisms (tSNPs) of BMX with minimum allele frequency (MAF) >1% were selected from the HapMap Han Chinese database. Among these polymorphisms, rs16979956 was found to be associated with the Beck anxiety inventory (BAI) and dizziness handicap inventory (DHI) scores within the first week after head injury. Additionally, another SNP, rs35697037, showed a significant correlation with dizziness symptoms. These findings suggested that polymorphisms of the BMX gene could be a potential predictor of clinical symptoms following mTBI.

CD28 and ITK signals regulate autoreactive T cell trafficking.

Activation of self-reactive T cells and their trafficking to target tissues leads to autoimmune organ destruction. Mice lacking the coinhibitory receptor CTLA-4 develop fatal autoimmunity characterized by massive lymphocytic invasion into non-lymphoid tissues. Here we demonstrate that the CD28 costimulatory pathway regulates the trafficking of self-reactive Ctla4−/− T cells to tissues. Co-ablation of the CD28-activated Tec family kinase ITK does not block spontaneous T cell activation, but instead causes self-reactive Ctla4−/− T cells to accumulate in secondary lymphoid organs. Despite a fulminant autoimmune process in the lymphoid compartment, Itk−/−Ctla4−/− mice are otherwise healthy and exhibit a long lifespan. We propose that ITK licenses autoreactive T cells to enter tissues to mount destructive immune responses. Importantly, ITK inhibitors mimic the null mutant phenotype and also prevent pancreatic islet infiltration by diabetogenic T cells in mouse models of Type I diabetes, highlighting their potential utility for the treatment of human autoimmune disorders.

Inhibition Of BTK Activity In Myeloma Cells Within a Supportive Microenvironment Promotes Their Growth But Suppresses Metastasis

Bruton’s Tyrosine Kinase (BTK), a TEC family kinase, is expressed by hematopoietic cells and is indispensable for B cell maturation and migration. We and others recently demonstrated that BTK is expressed in MM cells and mediates their homing to bone through chemotaxis to SDF-1, a chemokine highly produced in the bone marrow (BM) (Tai et al,Blood 2012; Bam et al.,AJH 2013). The aim of this study was to unravel the role of BTK in MM cell adhesion, growth and metastasis in our in vitro and in vivo experimental systems. BTK activity was suppressed using lentiviral particles containing BTK or scramble shRNA, or using the clinical BTK inhibitor, ibrutinib. Primary MM cells passaged in our animal model or IL6-dependent INA6 MM line were also infected with EGFP/luciferase construct for tracking purposes and monitoring tumor growth. Knockdown of BTK in INA6 cells diminished their homotypic cell aggregation growth pattern and reduced adhesion to fibronectin compared to control cells by 38±6% (p&lt;0.01). In vivo we exploited our SCID-rab model whereby SCID mice were implanted with a piece of rabbit femur on each flank and luciferase-expressing INA6 MM cells containing scramble or BTK shRNA were injected directly into the right-side implanted bone. We previously demonstrated that MM cells grow restrictively in the implanted bone area and are capable of metastasis to the secondary implanted bone but not murine organs (Yata et al, Leukemia 2004). MM growth in the primary bone site of BTK shRNA group (n=13) was higher than in the scramble shRNA group (n=12) by 4±1 folds (p&lt;0.04) assessed by live-animal imaging. Overall MM burden was also higher in the BTK shRNA group by 4.3 folds (p&lt;0.03) and 2.3 folds (p&lt;0.002) detected by measurement of circulating human immunoglobulins and soluble syndecan-1 levels, respectively. In contrast, metastasis to the secondary implanted bones assessed by ex-vivo imaging 10 weeks after engraftment was lower by 37±11 folds (p&lt;0.002) in the BTK shRNA group compared to control scramble shRNA group. In both groups, metastatic tumor cells were identified only in the secondary implanted bones. MM cells extracted from the primary myelomatous bones retained their differences in BTK expression between the two groups. Cell cycle analysis on extracted MM cells revealed decreased sub-G phase (apoptotic cells) and increased in vivo BrdU labeling index in the BTK knockdown MM cells compared to control MM cells. Gene array analysis revealed BTK target genes including upregulation of factors associated with lymphoid malignancies (e.g. BCL11A, TIAM1, RAB31) and underexpression of genes mediating cell adhesion and migration (e.g. LPXN, DBN1, F11R, CT45) in the BTK knockdown cells compared to control cells. In vitro, we established a novel coculture system of MM cells with healthy adult human BM that first has been cultivated with MM patients’ serum for 7 days. Primary MM cells (n=20) were consistently capable of long-term (7 days) survival and growth in this coculture system. Growth of BTK-expressing MM cells (n=7) in the coculture was significantly increased by 61%-90% in the presence of 5 μM (p&lt;0.02) or 10 μM (p&lt;0.01) ibrutinib. Cocultured MM cells treated with 5 μM Ibrutinib had increased BrdU labeling index compared to control MM cells as assessed by flow cytometry. We also observed reduced expression of BTK in MM cells from focal lesion compared to interstitial marrow in paired clinical samples (n=106, p&lt;3.24E-05). These data suggest that BTK plays a role in MM cell homotypic and heterotypic adhesion, and that inhibition of BTK activity in MM cells within a supportive microenvironment promotes their proliferation and growth but suppresses metastasis. Disclosures: No relevant conflicts of interest to declare.

A Novel Bruton′s Tyrosine Kinase Inhibitor CC-292 In Combination With The Proteasome Inhibitor Carfilzomib Iimpacts Multiple Myeloma Bone Microenviroment With Resultant Anti-Myeloma Activity

A member of the Tec family kinases, Bruton's tyrosine kinase (Btk) modulates B-cell development and activation, and plays an important role in antibody production. Interestingly, Btk and Tec (the other Tec kinase family) regulate osteoclast (OC) differentiation via Receptor Activator of Nuclear Factor κ B (RANK) signaling. Moreover, OCs derived from X-linked agammaglobulinemia (XLA) patients who harbor Btk null mutations have impaired function.Here we show that a potent and specific Btk inhibitor, CC-292 inhibits OC function in multiple myeloma (MM) patients. CC-292 is a highly selective, covalent Btk inhibitor. OC derived from MM patient monocytes were assayed with or without CC-292. Interestingly, OC function was significantly inhibited in the presence of CC-292 (100 nM and 1000 nM) as demonstrated by pit formation assay. However, mRNA expression for TRAP and Cathepsin K, two OC differentiation markers were increased in the presence of CC-292 suggesting that CC-292 inhibits OC function without inhibiting OC differentiation.OC sealing zone contributes to OC bone resorption function. Given the role of c-Src and Proline-rich tyrosine Kinase 2 (Pyk2) signaling in sealing zone formation and OC function we next evaluated CC-292's effect on Pyk2 and c-Src. Pyk2 plays a role in OC activation and localizes to the sealing zone in OC. RANK signaling activates c-Src, which phosphorylates Pyk2. Moreover c-Src controls OC bone resorption by regulating actin organization via cortactin. Interestingly, CC-292 (100 nM) inhibited c-Src total protein, c-Src phosphorylation and Pyk2 phosphorylation. Furthermore, CC-292 (100 nM) inhibited cortactin protein and mRNA expression, and upregulated c-Cbl protein (E3 ubiquitin ligase for c-Src) expression in OC derived from MM patient monocytes with resultant inhibition of OC sealing zone formation. However, at the same low doses (100 nM) CC-292 did not show any direct in vitro effect against MM cell viability.Because carfilzomib, a proteasome inhibitor that binds irreversibly to its target, has potent anti-MM activity and also inhibits OC resorptive activity, we studied CC-292 in combination with carfilzomib. Our data suggests that carfilzomib (1.25 nM) has no impact on OC sealing zone formation but inhibits OC differentiation. CC-292 (100 nM) in combination with carfilzomib (1.25 nM) inhibited not only sealing zone formation but also OC differentiation, resulting in stronger suppression of OC function than carfilzomib alone.The combination of CC-292 (30mg/kg p.o. for 5 days per week for 6 weeks) and carfilzomib (3 mg/kg i.v. x 2 days per week for 4 weeks and 2 mg/kg i.v. x 2 days per week for 2 weeks) significantly inhibited tumor burden and myeloma cell numbers in a diffuse NOD-SCID MM model. The calvarial cells derived from these mice treated with CC-292 alone, carfilzomib alone or the combination showed higher osteocalcin mRNA (osteoblast differentiation marker) expression. A specific bone resorption marker, carboxy-terminal telopeptide collagen crosslinks (CTX) in mouse serum was significantly inhibited in CC-292 and CC-292 in combination with carfilzomib treatment groups in comparison with control mice. Furthermore, 3D microCT reconstructions showed increase in cancellous bone volume in lumbar vertebrae in mice treated with CC-292 or carfilzomib, while the combination treatment resulted in an increase in cancellous bone volume in an additive manner.These data demonstrate that the novel BTK inhibitor CC-292 inhibits OC function through inhibition of OC sealing zone formation. Moreover, CC-292 in combination with carfilzomib augments effects against the bone microenvironment with resultant anti-MM activity. Disclosures:Arastu-Kapur:Onyx Pharmaceuticals, Inc.: Employment. Evans:Celgene Avilomics Research: Employment, Equity Ownership. Singh:Celgene Avilomics Research: Employment, Equity Ownership. Kirk:Onyx Pharmaceuticals, Inc.: Employment. Westlin:Celgene Avilomics Research: Employment, Equity Ownership. Raje:Celgene: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Amgen: Consultancy; Acetylon: Research Funding; Eli Lilly: Research Funding.

Cytokine and T-Cell Phenotypic Changes Upon In Vivo Ibrutinib Therapy For CLL – Targeting Both CLL Cells and The Tumor-Microenvironment

IntroductionProliferation of chronic lymphocytic leukemia (CLL) cells is highly dependent on the microenvironment. B-cell receptor (BCR) signaling and interactions of the tumor cells with elements of the tissue microenvironment including T cells and macrophages appear to be of particular importance (Burger et al, Blood 2009; Herishanu at al, Blood 2011; Bagnara at al, Blood 2011). The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib is highly effective in blocking BCR signaling and leads to impressive clinical responses in CLL (Byrd et al, NEJM 2013). BTK is a member of the TEC kinase family that also includes TEC, IL2-inducible T cell kinase (ITK), and BMX/ETK. BTK is not expressed in T cells; however ITK, which is expressed in T cells, is directly inhibited by ibrutinib, and the drug reduces cytokine secretion from activated T cells without inducing apoptosis (Herman et al, Blood, 2011). Here, we sought to determine the in vivo effect of ibrutinib on T cells and cytokine levels in CLL patients treated with single agent ibrutinib. MethodsThe effect of ibrutinib on T-cell subsets, T-cell activation, and cytokine profiles was assessed in 10 CLL patients treated with 420mg ibrutinib daily in an ongoing phase II trial (NCT01500733). Matched samples of viably frozen peripheral blood mononuclear cells obtained from patients pre-treatment and after 6 months on ibrutinib were analyzed by flow cytometry. Cytokine levels pre-treatment and on days 1, 28, months 2, and 6 on ibrutinib were measured in the same patients using the Milliplex human cytokine assay. ResultsConsistent with inhibition of BCR signaling in CLL cells, CCL3 and CCL4 serum levels were rapidly and significantly decreased by ibrutinib as described previously (Ponader et al, Blood, 2012). In addition, serum levels of a number of inflammatory cytokines including IL6, IL8, IFNg, and TNFα were decreased by > 50% by day 28 of ibrutinib treatment and remained so by 6 months. This is of specific interest as “pseudoexhausted” T cells from CLL patients were recently shown to secrete high amounts of IFNg, and TNFα (Riches et al, Blood 2013). Thus, the decreased levels of inflammatory cytokines may reflect a reversal of T cell “pseduoexhaustion”. Furthermore, the immunosuppressive cytokine IL10, a Th1-type cytokine that is secreted by CLL cells and activated T cells, was also rapidly and significantly reduced. These in vivo data are consistent with previous in vitro data showing decreased secretion of IL6 and IL10 from T cells upon exposure to ibrutinib (Herman et al, Blood, 2011). Thus, ibrutinib appears to reduce cytokine and chemokine secretion from both CLL and T cells resulting in an overall decrease in inflammatory cytokines. While absolute T-cell numbers showed little change on treatment, we found that ibrutinib reduced the frequency of activated CD4+ T cells (Table). Furthermore, for 3 out of 4 patients, the percentage of Ki67 positive T cells in the peripheral blood decreased on ibrutinib therapy (mean decrease 63%). The frequency of the Th17 T-cell subset was also diminished. Consistently, a decrease in serum levels of IL17 was seen in the two patients having detectable IL17 levels pre-treatment. While changes in the cytokine pattern (decrease in IFNg and IL10) might suggest inhibition of a Th1-type response, there was no change in the ratio of Th1 to Th2 T-cell subsets by immunophenotyping.TableSubset of CD4+ T-cells (measured in 10 patients)Pre-ibrutinib (mean %)At 6 months on ibrutinib (mean %)P (paired t test)Activated cells (HLA-DR+, CD39+)3.91.80.003Th17 (CXCr3neg, CCR6+)1.70.90.04Th1 (CXCR3+, CCR6neg)66570.51Th2 (CXCR3neg, CCR6neg)28390.42 ConclusionsWe here demonstrate a decrease in the levels of inflammatory cytokines and in T-cell activation in CLL patients treated with ibrutinib. Whether this is a direct consequence of BTK inhibition in B-cells or, at least in part, results from inhibition of T-cell signaling remains to be determined. Nevertheless, our data indicate that ibrutinib significantly alters the composition of the tumor microenvironment in CLL, affecting soluble as well as cellular elements. These effects may be important for clinical response and the development of combination therapies and therefore deserve further study.Supported by the Intramural Research Program of NHLBI. We thank our patients for participating and acknowledge Pharmacyclics for providing study drug. Disclosures:Off Label Use: Ibrutinib in chronic lymphocytic leukemia. Chang:Pharmacyclics: Employment, Equity Ownership. Buggy:Pharmacyclics: Employment, Equity Ownership.

Mini-Review: Bmx Kinase Inhibitors for Cancer Therapy

Kinase inhibitors are among the fastest growing class of anti-cancer therapies. One family of kinases that has recently gained attention as a target for treating malignant disorders is the Tec kinase family. Evidence has been published that one member of this family; the Bmx kinase, may play a role in the pathogenesis of glioblastoma, prostate, breast and lung cancer. Bmx has also shown potential as an anti-vascular therapy in combination with radiation or as a sensitizer to chemotherapeutic agents. Therefore, several companies such as Pharmacyclics, Avila Therapeutics, Merck and Co., Metaproteomics, IRM, and Moerae Matrix have developed compounds or peptides that function as Bmx kinase inhibitors. These companies have subsequently been issued patents for these inhibitors. Additionally, it has been shown that current clinical stage EGFR inhibitors can irreversibly inhibit Bmx, suggesting these compounds might be rapidly moved to clinical trials for other malignancies. This review will discuss current patents issued since 2009 that contain data specifically on inhibition of the Bmx kinase, and will also discuss the scientific literature that suggests their potential application as therapeutics in the treatment of the aforementioned malignancies.

Cross-talk between T cell receptor and cytokine signaling is mediated by Itk: implications for the balance between Th17 and regulatory T cells (P1141)

Abstract A proper balance between Th17 and T regulatory cells (Tregs) is critical for the generation of immune protective immune responses while minimizing autoimmunity. We show that the Tec family kinase Itk influences the balance between Th17 and T regulatory cells by regulating crosstalk between T Cell Receptor (TCR) and cytokine signaling. Under both Th17 and Tregs differentiation conditions, Itk-/- CD4 T cells develop higher percentages of functional Foxp3+ cells, associated with increased responsiveness to IL-2. Itk-deficient CD4 cells show reduced TCR-induced phosphorylation of mTOR targets, including ribosomal S6 and AKT, accompanied by changes in metabolic signatures that are affected by mTOR pathways. Surprisingly, iTreg Itk-/- cells also show reduced IL-2-induced mTOR pathways, despite increased STAT5 phosphorylation. We find that TCR stimulation in WT CD4+ T cells leads to a dose-dependent repression of expression of Pten; however, at low TCR stimulation or in the absence of Itk, Pten is not effectively repressed, thereby uncoupling STAT5 phosphorylation and PI3K/mTOR pathways and increasing Foxp3 expression. Functional studies show that naïve Itk-/- CD4 T cells also preferentially develop into Tregs in an in vivo colitis model. Furthermore Itk-deficient Tregs are capable to inhibit the development of colitis demonstrating that Treg function is independent of Itk. Our results suggest that Itk is a potential target for altering the balance between Th17 and Treg cells.

Abstract 2161: Targeting Bruton's tyrosine kinase (BTK) in multiple myeloma with novel BTK inhibitors.

Abstract Multiple myeloma (MM) is a malignant disease that is characterized by an excess of monotypic plasma cells in the bone marrow (BM). A key clinical characteristic of MM is the localization of the MM cells to the bone marrow where they promote osteolytic bone destruction and impaired hematopoietic function. As a consequence MM patients experience bone pain, hypocalcaemia, anemia. Although there has been some progress in recent years in the development of novel drugs, such as proteasome inhibitors and derivatives of thalidomide, MM remains incurable and the majority of patients eventually succumb to their cancer. We have recently identified BTK over-expression in cancer cells taken from patients with multiple myeloma. In addition we have observed that BTK is also expressed in the activated osteoclasts of MM patients indicating that targeted inhibition of BTK might not only effect the MM cancer cells but may also influence the activity of osteoclasts and the associated osteolytic lesions. Bruton's tyrosine kinase (BTK) is a cytoplasmic nonreceptor tyrosine kinase belonging to the Tec family of kinases. BTK has been extensively studied for its role in B-cell maturation and activation of B-cells by various ligands is accompanied by the translocation of BTK to the cell membrane where it binds phosphatidylinositol-3,4,5-trisphosphate through its PH domain. Activation of BTK results in downstream signaling through the PI3K/AKT, PLCγ1/2, NFκB, and other signaling pathways important for B-cell development and function. We propose that BTK plays an important role in multiple myeloma pathophysiology and that therapeutically targeting BTK will inhibit the growth of cancer cells and alter the tumor microenvironment in the bone marrow of multiple myeloma cancer patients. Using a structure-based approach we have developed a series of irreversible BTK inhibitors with selective and potent low nanomolar activity. In preclinical studies to date, our compounds have demonstrated promising activity in biochemical and cell-based experiments. Our BTK-targeted agents have shown activity in MM cells and have good pharmacokinetics when delivered IV and oral. MM cells create an adverse microenvironment from a pathophysiologic and clinical perspective because of the disruption of bone remodeling and this disruption of normal bone function has been shown to inhibit the response of MM cell to drug treatment. Therefore, a strategy to develop new drugs for MM must take into account the ability of the new agents to partition and distribute to the bone and function in the bone microenviroment of MM. Our compounds are being optimized for their ability to partition to bone and remain active in the BM microenvironment. By inhibiting BTK we seek to block BTK-dependent growth and migration of multiple myeloma cells and inhibit of the production of differentiated activated osteoclasts thereby disrupting the bone marrow microenvironment in MM. Citation Format: Destinee Bushman, Jared J. Bearss, Venkataswamy Sorna, Hariprasad Vankayalapati, Sunil Sharma, Fenghuang Zhan, David Bearss. Targeting Bruton's tyrosine kinase (BTK) in multiple myeloma with novel BTK inhibitors. [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 2161. doi:10.1158/1538-7445.AM2013-2161

The zinc-binding region of IL-2 inducible T cell kinase (Itk) is required for interaction with Gα13 and activation of serum response factor

Tec family kinases play critical roles in the activation of immune cells. In particular, Itk is important for the activation of T cells via the T cell Receptor (TcR), however, molecules that cooperate with Itk to activate downstream targets remain little explored. Here we show that Itk interacts with the heterotrimeric G-protein α subunit Gα13 during TcR triggering. This interaction requires membrane localization of both partners, and is partially dependent on GDP- and GTP-bound states of Gα13. Furthermore, we find that Itk interacts with Gα13 via the zinc binding regions within its Tec homology domain. The interaction between Itk and Gα13 also results in tyrosine phosphorylation of Gα13, however this is not required for the interaction. Itk enhances Gα13 mediated activation of serum response factor (SRF) transcriptional activity dependent on its ability to interact with Gα13, but its kinase activity is not required to enhance SRF activity. These data reveal a new pathway regulated by Itk in cells, and suggest cross talk between Itk and G-protein signaling downstream of the TcR.