Interleukin-2-inducible T-cell Kinase Research Articles

PB1835 A PHASE 1/1B DOSE‐ESCALATION TRIAL EVALUATING CPI‐818, AN ORAL INTERLEUKIN‐2‐INDUCIBLE T‐CELL KINASE INHIBITOR, IN PATIENTS WITH RELAPSED/REFRACTORY T‐CELL LYMPHOMA

Background:Interleukin‐2‐Inducible T‐Cell Kinase (ITK) is a Tec‐family, non‐receptor tyrosine kinase expressed in T cells that plays a key role in T‐cell receptor (TCR) signaling, which is required for development of T‐cells. In T‐cell lymphoproliferative disorders, expression of the TCR and its downstream signaling components are maintained, which suggests that malignant cells may exploit this growth and survival pathway to their advantage. Professional antigen‐presenting cells abundant in the lymphoma microenvironment may provide antigen to drive TCR signaling through ITK, which is expressed in a variety of T‐cell lymphomas including PTCL, CTCL, ALCL, and in a subgroup of T‐lymphoblastic leukemia/lymphoma. CPI‐818 is a first‐in‐class, irreversible, covalent‐binding ITK inhibitor with a high degree of selectivity for ITK. By inhibiting ITK, CPI‐818 blocks signaling pathways that are essential to inflammatory responses and has shown to inhibit tumor growth in animal models.Aims:To test the effect of CPI‐818 clinically, a phase 1/1b dose‐escalation trial of CPI‐818 is initiated in subjects with relapsed/refractory T‐cell lymphoma.Methods:The trial will include patients with various types of T‐cell lymphoma (PTCL and CTCL) who have progressed on, refractory to, relapsed, or intolerant to at least 2 standard therapies; age ≥ 18 yo; have ECOG status 0‐1; adequate organ function; and without any other condition that would contraindicate the use of the investigational product. Dose Escalation with 3 + 3 (+ optional 3) design will consist of up to 6 ascending dose levels (100,200, 400, 600, 900, and 1200 mg BID) of CPI‐818. In the Dose Expansion phase, there will be 4 disease‐specific expansion cohorts (AITL, PTCL‐NOS, CTCL and other T‐cell lymphoma), and each cohort may enroll up to a maximum of 28 subjects/cohort based on a 2‐stage expansion design‐ Figure.The objectives of the study are to evaluate the safety and tolerability of CPI‐818; establish the maximum tolerated dose or the maximum administered dose of CPI‐818; evaluate pharmacokinetics and pharmacodynamics of CPI‐818 in humans; assess the anti‐tumor activity of CPI‐818 and identify potential biomarker signals. Study schema in the figure.Approximately 151 subjects will be enrolled at approximately 35 sites in the US, South Korea, and Australia.Results:None. Trial in progressSummary/Conclusion:None. Trial in progressimage

ITK regulates antigen-specific CD8+ T cell-derived IL-10 production and modulates influenza-induced pulmonary immunopathology

Abstract Influenza (flu) infection is a leading cause of respiratory disease and death worldwide, causing 3-5 million cases of severe illness and more than 250,000 deaths during an average flu season. Severe life-threatening influenza infections in humans are accompanied by an aggressive pro-inflammatory response and an insufficient anti-inflammatory immunity. The production of the immunosuppressive cytokine IL-10 by virus-specific T cells is critical in limiting the immunopathology during flu infection, however, our understanding of how this immunomodulatory event is regulated at the molecular level is very limited. Using mouse models that report the production of IL-10 through green fluorescent protein, OTI CD8+ T cells and influenza A viruses that express the OTI antigen ovalbumin (OVA), we find that the T cell receptor (TCR) responsive tyrosine kinase, Interleukin-2 Inducible T cell kinase (ITK), is a critical regulator of the ability of CD8+ T cells to develop into IL-10-producing cells in an antigen specific manner. The absence of ITK resulted in increased morbidity and mortality in murine model post influenza A infection, accompanied by a severe impairment of IL-10 production by CD8+ T cells in the airway. ITK kinase activity is required for its role in promoting IL-10-producing CD8+ T cell differentiation, by regulating the levels of expression of transcription factors including IRF4, Blimp1, AHR and cMAF. Our data suggest that ITK regulates CD8+ T cell production of IL-10 during influenza A infection, and modulates the development of pulmonary immunopathology. Targeting ITK signaling maybe a promising strategy in modulating airway inflammation during viral infections.

A negative role for the interleukin-2-inducible T-cell kinase (ITK) in human Foxp3+ TREG differentiation.

The Tec kinases ITK (interleukin-2-inducible T-cell kinase) and RLK (resting lymphocyte kinase) are critical components of the proximal TCR/CD3 signal transduction machinery, and data in mice suggest that ITK negatively modulates regulatory T cell (TREG) differentiation. However, whether Tec kinases modulate TREG development and/or function in human T cells remains unknown. Using a novel self-delivery siRNA platform (sdRNA), we found that ITK knockdown in human primary naïve peripheral blood CD4 T cells increased Foxp3+ expression under both TREG and T helper priming conditions. TREG differentiated under ITK knockdown conditions exhibited enhanced expression of the co-inhibitory receptor PD-1 and were suppressive in a T cell proliferation assay. ITK knockdown decreased IL-17A production in T cells primed under Th17 conditions and promoted Th1 differentiation. Lastly, a dual ITK/RLK Tec kinase inhibitor did not induce Foxp3 in CD4 T cells, but conversely abrogated Foxp3 expression induced by ITK knockdown. Our data suggest that targeting ITK in human T cells may be an effective approach to boost TREG in the context of autoimmune diseases, but concomitant inhibition of other Tec family kinases may negate this effect.

Interleukin-2–inducible T-cell kinase inhibitors modify functional polarization of human peripheral T-cell lymphoma cells

Key Points ITK inhibitors perturb functional changes due to polarizing culture conditions in normal human tonsil CD4+ T cells. Primary human PTCL cells alter their functional properties in culture and ITK inhibitors modify these changes.

Enantioselective synthesis of GNE-6688, a potent and selective inhibitor of interleukin-2 inducible T-cell kinase (ITK)

An enantioselective synthesis of the previously-disclosed ITK inhibitor GNE-6688 is described. Synthesis of the nitropyrazole fragment is highlighted by a Ru-catalyzed transfer hydrogenation using the Wills tethered ligand system. Synthesis of the pyrazole carboxylic acid fragment features an allylboration catalyzed by a chiral diol-SnCl4 complex, followed by a highly diastereoselective directed cyclopropanation.

Management of XLP-1 and ITK deficiency: The challenges posed by PID with an unpredictable spectrum of disease manifestations

The incorporation of next generation sequencing into routine immunological practice has enabled the identification of novel inborn errors of disease, helped define new categories of immune deficiency and extended the clinical spectrum associated with many long-recognised diseases. The family of EBV (Epstein Barr Virus)-sensitive primary immune deficiencies is one such group and in this paper we describe three families: two with X-linked lymphoproliferative disease type-1 (XLP-1) and one with deficiency of Interleukin-2 Inducible T-cell Kinase (ITK). Both diseases have a wide range of clinical manifestations and are united by an exquisite predisposition to EBV, dysgammaglobulinemia, hemophagocytic lymphohistiocytosis, and lymphoma. We detail our approach to diagnosis, treatment, and risk stratification in these diseases where both clinicians and patients must grapple with constant uncertainty.

Intratumoral Injection of CpG-ODN Plus Systemic Ibrutinib Induces an Anti-Tumor Immune Response Affecting T Cell Subsets in the Microenvironment of Both Injected and Non-Injected Tumor Sites in Patients with Low-Grade Lymphoma

BACKGROUND: Low-grade B cell lymphoma is often characterized by an infiltration of immune effector cells including T, NK and dendritic cells. But, despite the presence of effector cells within the tumor, these cells fail to control tumor growth.In a preclinical mouse model, we showed that Ibrutinib, an inhibitor of Bruton's tyrosine kinase (BTK), but also ITK (IL-2-inducible T cell kinase), synergized with intratumoral CpG to facilitate complete regression of tumors at the treated site as well as a distal, non-treated site, curing all the mice. This was accompanied by a potent anti-tumor memory T cell response by both CD4 and CD8 T cells that rejected the tumor on re-challenge. (Sagiv-Barfi I, et al. Blood. 2015 March:125(13):2079-2086)STUDY: In an ongoing clinical trial (NCT02927964), patients with previously treated low-grade lymphoma receive low dose (2Gyx2) radiotherapy to a single tumor site followed by 5 weekly intratumoral injections of 3 mg CpG-ODN (SD-101, Dynavax Technologies) into the same site. 1 day after the second injection, patients begin taking a daily 560 mg dose of Ibrutinib. A fine needle aspirate (FNA) of the injected site and a non-injected site outside the radiation field is performed prior to radiotherapy, one week after the first injection, and at week 6, 1 week after the final injection of CpG. FNA samples are stained for flow cytometry with panels of antibodies to delineate all major cell populations and their subsets. Cellular activation as well as T cell exhaustion, inhibition and function are also characterized. When feasible, a biopsy is performed prior to treatment providing tumor cells to be used in an immune response assay to evaluate induced anti-tumor responses by circulating peripheral blood T cells obtained throughout the study.RESULTS: To date, 12 patients have been entered onto the study. Of these patients, 4 had excisional biopsies and subsequent immune response assays performed. All 4 patients exhibited CD8 anti-tumor responses as determined by an increase in the activation marker CD137 as well as the functional marker granzyme B above pretreatment CD8 T cells. At week 12, 7 weeks after the last CpG injection, the average increase above pretreatment baseline for CD137 and granzyme B was 6.1% and 9.3%, respectively. In 3 of these patients, we observed an increase in CD8 T cells expressing CD137 and granzyme B from the FNA of the non-injected site. The 4th patient did not have adequate number of cells for staining. Two patients exhibited CD4 immune responses as characterized by an up-regulation of CD137 and CD278.FNA of 7 patients produced enough cells to analyze the tumor microenvironment from both the injected and the non-injected sites over all 3 time points; 1 patient was evaluable through week 2 for both sites. In 7 out of these 8 patients, CD3 T cells increased at the injected and non-injected sites by week 6. The proportion of CD4 and CD8 T cells did not stay constant, however, as reflected by the changes in CD4:CD8 ratios. This suggests that the increase in T cells was not purely the result of a loss of tumor B cells in the samples. Notably, we observed a significant increase in the effector CD4 T cells in all patients at the injected site by week 2 (14.1% ± 6.5%, p = 0.005) and 5 of 8 patients in the non-injected site by week 6 (12.4% ± 10.4%). Moreover, the proportion of T follicular helper cells significantly decreased in all patients at the treated site by week 2 (17.7% ± 9.7% of T cells, p = 0.0012) and in 5 of 8 patients at the non-treated site by week 6 (8.1% ± 5.5% of T cells). Tregs were more variable, decreasing in 5 of 8 patients in the treated and 3 of 8 patients in the non-treated site by week 6.CONCLUSION: CpG is known to activate antigen-presenting cells such as dendritic cells and macrophages. Ibrutinib is a small molecule that has been shown to have direct anti-tumor effects in B cell lymphoma and may skew an immune response towards that of a TH1 type. Here we show that together, they can effect changes in the tumor microenvironment in both treated and in untreated sites of disease.This clinical trial is ongoing and open to accrual. DisclosuresKhodadoust:Innate Pharma: Research Funding.

Translocation-generated ITK-FER and ITK-SYK fusions induce STAT3 phosphorylation and CD69 expression

Many cancer types carry mutations in protein tyrosine kinase (PTK) and such alterations frequently drive tumor progression. One category is gene translocation of PTKs yielding chimeric proteins with transforming capacity. In this study, we characterized the role of ITK-FER [Interleukin-2-inducible T-cell Kinase (ITK) gene fused with Feline Encephalitis Virus-Related kinase (FER) gene] and ITK-SYK [Interleukin-2-inducible T-cell Kinase (ITK) gene fused with the Spleen Tyrosine Kinase (SYK)] in Peripheral T Cell Lymphoma (PTCL) signaling. We observed an induction of tyrosine phosphorylation events in the presence of both ITK-FER and ITK-SYK. The downstream targets of ITK-FER and ITK-SYK were explored and STAT3 was found to be highly phosphorylated by these fusion kinases. In addition, the CD69 T-cell activation marker was significantly elevated. Apart from tyrosine kinase inhibitors acting directly on the fusions, we believe that drugs acting on downstream targets could serve as alternative cancer therapies for fusion PTKs.

Identification of inactive conformation-selective interleukin-2-inducible T-cell kinase (ITK) inhibitors based on second-harmonic generation.

Many clinically approved protein kinase inhibitors stabilize an inactive conformation of their kinase target. Such inhibitors are generally highly selective compared to active conformation inhibitors, and consequently, general methods to identify inhibitors that stabilize an inactive conformation are much sought after. Here, we have applied a high‐throughput, second‐harmonic generation (SHG)‐based conformational approach to identify small molecule stabilizers of the inactive conformation of interleukin‐2‐inducible T‐cell kinase (ITK). A single‐site cysteine mutant of the ITK kinase domain was created, labeled with an SHG‐active dye, and tethered to a supported lipid bilayer membrane. Fourteen tool compounds, including stabilizers of the inactive and active conformations as well as nonbinders, were first examined for their effect on the conformation of the labeled ITK protein in the SHG assay. As a result, inactive conformation inhibitors were clearly distinguished from active conformation inhibitors by the intensity of SHG signal. Utilizing the SHG assay developed with the tool compounds described above, we identified the mechanism of action of 22 highly selective, inactive conformation inhibitors within a group of 105 small molecule inhibitors previously identified in a high‐throughput biochemical screen. We describe here the first use of SHG for identifying and classifying inhibitors that stabilize an inactive vs. an active conformation of a protein kinase, without the need to determine costructures by X‐ray crystallography. Our results suggest broad applicability to other proteins, particularly with single‐site labels reporting on specific protein movements associated with selectivity.

A positional candidate gene association analysis of susceptibility to paratuberculosis on bovine chromosome 7

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative pathogen for paratuberculosis, which is a chronic inflammation of the small intestine in ruminants and some wild animals. It affects negatively on the economics of dairy operations worldwide and has a zoonotic concern for its potential relationship to Crohn's disease in humans. In this study, we used different approaches to investigate genetic markers associated with MAP infection on bovine chromosome 7. The interleukin-2-inducible T-cell kinase (ITK) gene is a non-receptor tyrosine kinase expressed in T cells, which also is involved in regulation and development of immune cells and some inflammatory diseases. The gene was suggested in a previous GWAS analysis as a positional candidate gene located on BTA7 for MAP infection. In this study, bovine ITK was sequenced and seventeen identified SNPs were genotyped in 1419 Holstein and Jersey cows. Association analysis revealed no significantly associated SNP in the bovine ITK gene using either single marker or haplotype analyses. In a complementary analysis, Holstein genotypes were imputed from 50 K SNPs to the full genome sequence of BTA7. Single SNP association tests identified two SNPs at 15 Mb (p = 5 × 10−7) significantly associated with MAP infection. Our results suggest an additional region of BTA7 contributes to susceptibility to MAP infection in cattle, relative to our previous report, and further investigations are required.

Systems-Based Interactome Analysis for Hematopoiesis Effect of Angelicae sinensis Radix: Regulated Network of Cell Proliferation towards Hemopoiesis.

To explore the molecular-level mechanism on the hematopoiesis effect of Angelicae sinensis Radix (ASR) with systems-based interactome analysis. This systems-based interactome analysis was designed to enforce the workflow of "ASR (herb)→compound→target protein→internal protein actions→ending regulated protein for hematopoiesis". This workflow was deployed with restrictions on regulated proteins expresses in bone marrow and anemia disease and futher validated with experiments. The hematopoiesis mechanism of ASR might be accomplished through regulating pathways of cell proliferation towards hemopoiesis with cross-talking agents of spleen tyrosine kinase (SYK), Janus kinase 2 (JAK2), and interleukin-2-inducible T-cell kinase (ITK). The hematopoietic function of ASR was also validated by colony-forming assay performed on mice bone marrow cells. As a result, SYK, JAK2 and ITK were activated. This study provides a new approach to systematically study and predict the therapeutic mechanism for ASR based on interactome analysis towards biological process with experimental validations.

A missense mutation impairs ITK function in a patient with severe Epstein-Barr virus disease.

Abstract T cells are critical in controlling EBV. IL-2-inducible T cell kinase (ITK) is an integral component of T cell signaling and modulates cytotoxic function. ITK activation leads to ITK mediated phosphorylation of phospholipase C (PLC)-γ, resulting in an intracellular calcium flux, activation of ERK, and translocation of NF-κB and NFAT into the nucleus. ITK is expressed in cells critical for controlling EBV infection, including T cells, NK cells, and invariant NKT (iNKT) cells. Mutations in ITK have been previously reported in 9 patients, ranging from 5 to 18 years of age who presented with low numbers of CD4 T and iNKT cells and EBV driven B cell lymphoproliferation. Here we identify a patient with a previously unreported homozygous missense mutation (ITK NM_005546 c.1618G>A p.D540N) in a highly conserved residue in the kinase domain of ITK. This patient presented at age 22 with lung nodules due to lymphomatoid granulomatosis with EBV-positive large tumor cells. She was treated with interferon and cytotoxic chemotherapy and her disease ultimately went into remission. She has since had persistent elevation of EBV DNA in the blood, low CD4 T cells, low NK cells, and nearly absent iNKT cells. Stimulation of her T cells resulted in reduced ITK signaling with diminished PLC-γ and Erk activation, and impaired calcium flux. Western blot analysis showed normal levels of ITK, indicating that the mutation affects the function, but not the stability of ITK. The patient’s CD8 T cells were impaired for degranulation, while her NK cells had reduced expression of the NK cell receptor, NKG2D. Our findings highlight the critical role of ITK to modulate T cell activation and control EBV driven B cell lymphoproliferation.

The TCR/ITK signaling pathway regulates the counterbalance of effector and regulatory T cell development

Abstract The mammalian immune system has evolved both effector and regulatory immune axes, and T cells are the key players in both arms. The immune system sorts, responds to and clears invading pathogens through pro-inflammatory activity, which can cause damage to the affected tissues, and eventually have to be resolved to allow tissue remodeling and resume physiological homeostasis. The balance between pro-inflammatory and suppressive immune functions are critical for host health and survival. The T cell antigen receptor (TCR) signals through the downstream non-receptor interleukin-2 inducible T cell kinase (ITK), which is a key signaling modulator of T cell development, differentiation, activation and function. We find that in the face of different inflammatory and nutritional conditions, the TCR/ITK signaling pathway fine-tunes the balance between effector and regulatory T cell development, such as the ratio between Th17/Treg and Th1/Tr1 cells. This balance is regulated by the TCR/ITK signaling via differential downstream signals and metabolic programs. While ITK is not required for T cell activation, it functions as a rheostat of the TCR to regulate signal strength in response to extracellular antigens, and may be a target to allow therapeutically manipulating the balance between effector and regulatory immunity, dependent on the disease conditions.

Pilot trial of ibrutinib in patients with relapsed or refractory T-cell lymphoma

AbstractIbrutinib has previously been shown to inhibit Bruton's tyrosine kinase (BTK) and interleukin-2–inducible T-cell kinase (ITK), which mediate B-cell and T-cell receptor signaling, respectively. BTK inhibition with ibrutinib has demonstrated impressive clinical responses in a variety of B-cell malignancies. Whether ibrutinib inhibition of ITK can lead to clinical response in T-cell malignancies is unknown. We hypothesized that ibrutinib-mediated ITK inhibition in T-cell lymphoma would result in decreased signaling through the T-cell receptor pathway and promote antitumor immune response by driving selective cytotoxic Th1 CD4 effector T-cell differentiation. This pilot clinical trial evaluated 2 dose levels of ibrutinib: 560 and 840 mg orally daily. Fourteen patients with relapsed, refractory peripheral T-cell lymphoma and cutaneous T-cell lymphoma were enrolled. Both dose levels were safe and well tolerated, and no dose-limiting toxicities were observed. One patient achieved a partial response (overall response rate, 8% [1/13]). ITK occupancy studies demonstrated a mean occupancy of 50% (range, 15%-80%). Higher ITK occupancy of more than 50% correlated with higher serum levels of tumor necrosis factor-α and interferon-γ and favored a Th1 phenotype. Our data suggest that ibrutinib inhibition of ITK has limited clinical activity in T-cell lymphoma. This study is registered at www.clinicaltrials.gov as #NCT02309580.

IL-2 Inducible Kinase ITK is Critical for HIV-1 Infection of Jurkat T-cells

Successful replication of Human immunodeficiency virus (HIV)-1 depends on the expression of various cellular host factors, such as the interleukin-2 inducible T-cell kinase (ITK), a member of the protein family of TEC-tyrosine kinases. ITK is selectively expressed in T-cells and coordinates signaling pathways downstream of the T-cell receptor and chemokine receptors, including PLC-1 activation, Ca2+-release, transcription factor mobilization, and actin rearrangements. The exact role of ITK during HIV-1 infection is still unknown. We analyzed the function of ITK during HIV-1 replication and showed that attachment, fusion of virions with the cell membrane and entry into Jurkat T-cells was inhibited when ITK was knocked down. In contrast, reverse transcription and provirus expression were not affected by ITK deficiency. Inhibited ITK expression did not affect the CXCR4 receptor on the cell surface, whereas CD4 and LFA-1 integrin levels were slightly enhanced in ITK knockdown cells and heparan sulfate (HS) expression was completely abolished in ITK depleted T-cells. However, neither HS expression nor other attachment factors could explain the impaired HIV-1 binding to ITK-deficient cells, which suggests that a more complex cellular process is influenced by ITK or that not yet discovered molecules contribute to restriction of HIV-1 binding and entry.

Ibrutinib for chronic graft-versus-host disease after failure of prior therapy

AbstractChronic graft-versus-host disease (cGVHD) is a serious complication of allogeneic stem cell transplantation with few effective options available after failure of corticosteroids. B and T cells play a role in the pathophysiology of cGVHD. Ibrutinib inhibits Bruton tyrosine kinase in B cells and interleukin-2–inducible T-cell kinase in T cells. In preclinical models, ibrutinib reduced severity of cGVHD. This multicenter, open-label study evaluated the safety and efficacy of ibrutinib in patients with active cGVHD with inadequate response to corticosteroid-containing therapies. Forty-two patients who had failed 1 to 3 prior treatments received ibrutinib (420 mg) daily until cGVHD progression. The primary efficacy end point was cGVHD response based on 2005 National Institutes of Health criteria. At a median follow-up of 13.9 months, best overall response was 67%; 71% of responders showed a sustained response for ≥20 weeks. Responses were observed across involved organs evaluated. Most patients with multiple cGVHD organ involvement had a multiorgan response. Median corticosteroid dose in responders decreased from 0.29 mg/kg per day at baseline to 0.12 mg/kg per day at week 49; 5 responders discontinued corticosteroids. The most common adverse events were fatigue, diarrhea, muscle spasms, nausea, and bruising. Plasma levels of soluble factors associated with inflammation, fibrosis, and cGVHD significantly decreased over time with ibrutinib. Ibrutinib resulted in clinically meaningful responses with acceptable safety in patients with ≥1 prior treatments for cGVHD. Based on these results, ibrutinib was approved in the United States for treatment of adult patients with cGVHD after failure of 1 or more lines of systemic therapy. This trial was registered at www.clinicaltrials.gov as #NCT02195869.

Acalabrutinib (ACP-196): A Covalent Bruton Tyrosine Kinase Inhibitor with a Differentiated Selectivity and In Vivo Potency Profile.

Several small-molecule Bruton tyrosine kinase (BTK) inhibitors are in development for B cell malignancies and autoimmune disorders, each characterized by distinct potency and selectivity patterns. Herein we describe the pharmacologic characterization of BTK inhibitor acalabrutinib [compound 1, ACP-196 (4-[8-amino-3-[(2S)-1-but-2-ynoylpyrrolidin-2-yl]imidazo[1,5-a]pyrazin-1-yl]-N-(2-pyridyl)benzamide)]. Acalabrutinib possesses a reactive butynamide group that binds covalently to Cys481 in BTK. Relative to the other BTK inhibitors described here, the reduced intrinsic reactivity of acalabrutinib helps to limit inhibition of off-target kinases having cysteine-mediated covalent binding potential. Acalabrutinib demonstrated higher biochemical and cellular selectivity than ibrutinib and spebrutinib (compounds 2 and 3, respectively). Importantly, off-target kinases, such as epidermal growth factor receptor (EGFR) and interleukin 2-inducible T cell kinase (ITK), were not inhibited. Determination of the inhibitory potential of anti-immunoglobulin M-induced CD69 expression in human peripheral blood mononuclear cells and whole blood demonstrated that acalabrutinib is a potent functional BTK inhibitor. In vivo evaluation in mice revealed that acalabrutinib is more potent than ibrutinib and spebrutinib. Preclinical and clinical studies showed that the level and duration of BTK occupancy correlates with in vivo efficacy. Evaluation of the pharmacokinetic properties of acalabrutinib in healthy adult volunteers demonstrated rapid absorption and fast elimination. In these healthy individuals, a single oral dose of 100 mg showed approximately 99% median target coverage at 3 and 12 hours and around 90% at 24 hours in peripheral B cells. In conclusion, acalabrutinib is a BTK inhibitor with key pharmacologic differentiators versus ibrutinib and spebrutinib and is currently being evaluated in clinical trials.

Response of renal cell carcinoma to ibrutinib, a bruton tyrosine kinase inhibitor, in a patient treated for chronic lymphocytic leukemia.

Ibrutinib is a bruton tyrosine kinase (BTK) inhibitor approved for B cell malignancies. Although there are currently two clinical trials evaluating ibrutinib in combination with nivolumab (programmed cell death protein 1, PD-1, inhibitor) or everolimus (mammalian target of rapamycin, mTOR, inhibitor) for metastatic renal cell carcinoma (RCC), there are no reports of RCC (metastatic or non-metastatic) showing response to a BTK inhibitor in humans. Here we report a 22-month clinical response of biopsy-proven RCC to ibrutinib. This is unexpected, given that BTK is not wellimplicated in RCC pathophysiology. We explore a possible mechanism for the response of RCC to ibrutinib through inhibition of interleukin-2-inducible T-cell kinase (ITK) leading to enhanced antitumour immune responses.

Defective activation of IL2-inducible T cell kinase (ITK) in XMEN disease

Abstract We have previously described a primary immunodeficiency due to mutations in the magnesium transporter MAGT1, named XMEN disease (X-linked Magnesium defect with Epstein-Barr infection and Neoplasia). Loss of MAGT1 results in impaired T cell receptor signaling. Previous analyses suggested that proximal TCR signaling was unaffected in XMEN patients but PLCg1 phosphorylation was delayed. We have further characterized the TCR activation defect in MAGT1 deficient cells and found that the primary cause is the impaired activity of Interleukin-2 inducible T cell kinase (ITK). ITK is phosphorylated normally by LCK and recruited to immunological synapse but fails to phosphorylate downstream targets efficiently. A similar phenotype is apparent when cells from normal controls are activated in the absence of extracellular magnesium. A transient influx of magnesium is sufficient to restore ITK activity and PLCg1 phosphorylation in XMEN T cells. We are in the process of further characterizing the molecular underpinnings of ITK regulation by MAGT1. Acknowledgements: This research was supported by the Intramural Research Program of NIAID, NIH

Abstract B36: Preclinical development of a multikinase targeting molecule with activity against the cancer stem cell phenotype in pancreatic adenocarcinoma

Abstract Background: Pancreatic cancer is highly resistant to existing chemotherapy regimens, with only a minority of patients exhibiting a response to standard therapy. One aspect of treatment resistance is based on the cancer stem cell (CSC) hypothesis, which holds that a small population of highly chemoresistant cells is responsible for initiating and sustaining tumor growth. Compounds with activity against this population are highly desired in the adjuvant setting, where failure to sterilize micrometastases not detected at time of operation results in recurrence in nearly all patients. Methods: A high-throughput drug screen (HTS) was performed in an in vitro model of pancreatic cancer stem-like cells (spheroids). Molecules with high potency against both the cancer stem cell-like spheroid and monolayer cell formats were evaluated for efficacy and mechanism of action in a variety of preclinical models of pancreatic cancer. Unbiased global in situ phosphoproteomic profiling using KiNativ technology was used to identify kinase targets impacted by the top hit. Stemness phenotype was interrogated by qRT-PCR of stemness genes, flow cytometry, colony formation assays, and tumor initiation in immunocompromised mice. Results: The top hit from the HTS was a 1H-benzo[d]imidazol-2-amine-based inhibitor of interleukin-2-inducible T-cell kinase (ITK) (NCGC00188382, 1). 1 effectively mediated growth inhibition, blocked migration and invasion, and reduced clonogenicity of cancer cells with an associated decrease in the expression of several CSC-associated stemness markers at non-lethal concentrations (Sox2, Notch1) compared to gemcitabine used at >10-fold concentration. Metastatic burden in both the liver and lung was significantly reduced in mice orthotopically injected with pancreatic CSCs in the treatment group after 6 weeks. Target deconvolution of 1 demonstrated inhibition of multiple select kinase targets. There were 16 targets identified from an initial screening panel of >400 kinases. The targets were further evaluated for the ability to phenocopy the effect of treatment with 1 via shRNA knockdown. Inhibition of the thousand-and-one amino acid kinase 3 (TAOK3), cyclin-dependent kinase 7 (CDK7), aurora B kinase (AURKB), and ephrin B2 receptor (EPHB2) effectively phenocopied the effect of treatment with 1. Concurrent silencing of two or more targets increased apoptosis induced by 1, suggesting intrinsic additivity of the multikinase agent. Enforced overexpression of TAOK3 increased expression of stemness markers, clonogenicity, and tumor initiation in vivo. Conclusion: A potent inhibitor of ITK with a unique target profile and anti-stemness activities was derived from a differential HTS in a pancreatic spheroid CSC model. The 1H-benzo[d]imidazol-2-amine-derivative targets select characteristics attributed to pancreatic CSCs, including cell migration, colony formation, and metastasis formation with concomitant suppression of stemness traits without inducing cell death at the concentrations used. Target deconvolution of the compound identified multiple kinase targets, including TAOK3, a kinase not previously known to be involved in pancreatic tumor biology. Overexpression of TAOK3 results in an enforced CSC phenotype including increased colony formation and tumor initiation in vivo. The observed inhibitory effect of 1 on stemness phenotype at nanomolar concentrations without affecting cell viability or showing toxicity in vivo supports further preclinical development. Citation Format: Taylor Aiken, Yansong Bian, Yaroslav Teper, Lesley Mathews Griner, Rajarshi Guha, Paul Shinn, Hong-Wu Xin, Holger Pflicke, Jian-kang Jiang, Paresma Patel, Steven Rogers, Jeffery Aube, Marc Ferrer, Craig J. Thomas, Udo Rudloff.{Authors}. Preclinical development of a multikinase targeting molecule with activity against the cancer stem cell phenotype in pancreatic adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B36.