Interleukin-2 Inducible T-cell Kinase Inhibitors Research Articles

Conformational Selectivity of ITK Inhibitors: Insights from Molecular Dynamics Simulations.

Interleukin-2-inducible T-cell kinase (ITK) regulates the response to T-cell receptor signaling and is a drug target for inflammatory and immunological diseases. Molecules that bind preferentially to the active form of ITK have low selectivity between kinases, whereas those that bind preferentially to the inactive form have high selectivity for ITK. Therefore, computational methods to predict the conformational selectivity of compounds are required to design highly selective ITK inhibitors. In this study, we performed absolute binding free-energy perturbation (ABFEP) simulations for 11 compounds on both active and inactive forms of ITK, and the calculated binding free energies were compared with experimental data. The conformational selectivity of 10 of the 11 compounds was correctly predicted using ABFEP. To investigate the mechanism underlying the stabilization of the active and inactive structures by the compounds, we performed extensive, conventional molecular dynamics simulations, which revealed that the compound-induced stabilization of the P-loop and linkage of conformational changes in L489, V419, F501, and M410 upon compound binding were critical factors. A guideline for designing inactive-form binders is proposed based on these key structural factors. The ABFEP and the created guidelines are expected to facilitate the discovery of highly selective ITK inhibitors.

Identification of IL-2 inducible tyrosine kinase inhibitors by quantum mechanics and ligand based virtual screening approaches

Interleukin-2-inducible T-cell kinase (ITK) is a crucial intracellular signaling mediator in normal and malignant T-cells and natural killer cells. Selective inhibition of ITK might be useful for treating a variety of disorders including; autoimmune, inflammatory, and neoplastic disorders. Over the past two decades, the clinical management of ITK inhibitors has progressed dramatically. So far, specific inhibitor with no off-target effects against ITK is available. Herein, we aim to discover potential virtual hits to fasten the process of drug design and development against ITK. In this regard, the key chemical characteristics of ITK inhibitors were identified using ligand-based pharmacophore modeling. The validated pharmacophore comprises one hydrogen bond donor and three hydrogen bond acceptors and was utilized as a 3D query in virtual screening using ZINC, Covalent, and in-house databases. A total of 12 hit compounds were chosen on the basis of their critical interactions with the significant amino acids of ITK. The orbital energies such as HOMO and LUMO of the hit compounds were calculated to evaluate the inhibitor’s potencies. Further, molecular dynamics simulation demonstrated the stability of ITK upon binding of selected virtual hits. Binding energy using the MMGBSA method showed the potential binding affinity of all the hits with ITK. The research identifies key chemical characteristics with geometric restrictions that lead to ITK inhibition. Communicated by Ramaswamy H. Sarma

ITK degradation to block T cell receptor signaling and overcome therapeutic resistance in T cell lymphomas

Interleukin (IL)-2-inducible Tcell kinase (ITK) is essential for Tcell receptor (TCR) signaling and plays an integral role in Tcell proliferation and differentiation. Unlike the ITK homolog BTK, no inhibitors of ITK are currently US Food and Drug Administration (FDA) approved. In addition, recent studies have identified mutations within BTK that confer resistance to both covalent and non-covalent inhibitors. Here, as an alternative strategy, we report the development of BSJ-05-037, a potent and selective heterobifunctional degrader of ITK. BSJ-05-037 displayed enhanced anti-proliferative effects relative to its parent inhibitor BMS-509744, blocked the activation of NF-kB/GATA-3 signaling, and increased the sensitivity of Tcell lymphoma cells to cytotoxic chemotherapy both invitro and invivo. In summary, targeted degradation of ITK is a novel approach to modulate TCR signal strength that could have broad application for the investigation and treatment of Tcell-mediated diseases.

ITK tunes the Th17/Treg switch response by controlling calcium dependent signaling.

Abstract Naïve CD4+ T cells can differentiate into pro inflammatory T helper type 17 (Th17) and anti inflammatory T regulatory (Treg) lineages following T cell receptor (TCR) activation. The strength of the TCR signal is tuned by Interleukin-2 inducible T cell Kinase (ITK) and ITK can tune the development of Th17 and Treg cells. ITK signals can also tune a Th17/Treg switch under Th17 conditions. How ITK mediated tuning of TCR signal control naïve CD4+ T cell commitment into Th17/Treg lineages is unclear. Here we used ITK inhibitors, conventional ITK and allele specific ITK (ITKas) IL17 GFP/Foxp3 RFP reporter mice, to track naïve CD4+ T cell commitment into Th17/Treg lineages. Under Th17 conditions, reduced TCR signal strength in absence of ITK activity/expression, tuned naïve CD4+ T cell fate by reducing Th17 differentiation and inducing switch to cells resembling Tregs expressing Foxp3. The ITKas model showed this switch is due to specific ITK inhibition. The switched Treg like cells resemble Tregs by expression of Treg markers (CD25, CTLA4, PD1) and suppression of effector T cell proliferation. Transcriptomic analysis of switched Treg like cells by RNA Seq indicate they resemble Tregs, with reduced Th17 (Rorc, Il17) and increased Treg (Foxp3, Nrp1, Ikzf2) gene expression. Analysis of chromatin accessibility by ATAC Seq indicated closed loci for Th17 (Rorc, Il17) genes. Activating intracellular calcium dependent pathways led to a loss of the Th17/Treg switch response in naïve CD4+ T cell commitment, effectively bypassing ITK mediated tuning of the TCR signal critical for the switch response. In conclusion, we show here ITK tunes a Th17/Treg switch response in naïve CD4+ T cell commitment under Th17 conditions by controlling calcium dependent signaling. Supported by grants from NIH (NIH AI129422).

Hierarchy of signaling thresholds downstream of the T cell receptor and the Tec kinase ITK

The strength of peptide:MHC interactions with the T cell receptor (TCR) is correlated with the time to first cell division, the relative scale of the effector cell response, and the graded expression of activation-associated proteins like IRF4. To regulate T cell activation programming, the TCR and the TCR proximal interleukin-2-inducible T cell kinase (ITK) simultaneously trigger many biochemically separate signaling cascades. T cells lacking ITK exhibit selective impairments in effector T cell responses after activation, but under the strongest signaling conditions, ITK activity is dispensable. To gain insight into whether TCR signal strength and ITK activity tune observed graded gene expression through the unequal activation of distinct signaling pathways, we examined Erk1/2 phosphorylation or nuclear factor of activated T cells (NFAT) and nuclear factor (NF)-κB translocation in naïve OT-I CD8+ cell nuclei. We observed the consistent digital activation of NFAT1 and Erk1/2, but NF-κB displayed dynamic, graded activation in response to variation in TCR signal strength, tunable by treatment with an ITK inhibitor. Inhibitor-treated cells showed the dampened induction of AP-1 factors Fos and Fosb, NF-κB response gene transcripts, and survival factor Il2 transcripts. ATAC sequencing analysis also revealed that genomic regions most sensitive to ITK inhibition were enriched for NF-κB and AP-1 motifs. Specific inhibition of NF-κB during peptide stimulation tuned the expression of early gene products like c-Fos. Together, these data indicate a key role for ITK in orchestrating the optimal activation of separate TCR downstream pathways, specifically aiding NF-κB activation. More broadly, we revealed a mechanism by which variations in TCR signal strength can produce patterns of graded gene expression in activated T cells.

The ITK Inhibitor Cpi-818 Blocks Activation of T Cells from Autoimmune Lymphoproliferative Syndrome (ALPS) Patients and Is Active in a Murine Model of ALPS

Introduction We present preclinical evidence confirming the expression of ITK in double negative (DN) T cells of ALPS patients and demonstrate that CPI-818 inhibits the TCR-induced activation of these cells ex vivo. Furthermore, CPI-818 effectively suppressed the accumulation of DN T cells in the MRL/lpr mice, leading to dramatic improvements in lymphadenopathy, splenomegaly and other pathologies associated with lymphocyte accumulation due to Fas-deficiency. ALPS is a disorder of lymphocyte apoptosis resulting in childhood onset lymphadenopathy, multilineage cytopenias, hypersplenism and autoimmune peripheral destruction. Most ALPS cases are associated with a loss of function mutation in the Fas gene resulting in immune dysregulation such as a marked elevation of abnormal TCR+CD4-CD8-double negative (DN) T cells. ALPS patients are currently treated with immunosuppressive and cytotoxic agents, hence targeted, less toxic treatments are imperative. The critical role of Fas protein in maintaining lymphocyte homeostasis and peripheral immune tolerance to prevent autoimmunity was initially discovered with studies in Fas-deficient MRL/LpJ-Tnfrsf6lpr (MRL/lpr) mice. These mice have dramatically elevated levels of DN T cells and develop many clinical manifestations similar to those observed in ALPS patients. Thus, MRL/lpr mice have been a useful model to assess compounds and therapeutic strategies for the treatment of ALPS. Interleukin-2-inducible T cell kinase (ITK) is a tyrosine kinase critical to T cell receptor (TCR) signal transduction, which modulates T cell proliferation and differentiation. We sought to test whether the effect of selective inhibition of TCR signaling through ITK inhibition could be a strategy to suppress the DN T cell expansion and disease manifestation in MRL/lpr mice. CPI-818 is an orally bioavailable, covalent inhibitor of ITK that potently inhibits TCR signal transduction. It is currently being evaluated in a phase 1/1b clinical trial in T-cell lymphoma (NCT03952078) where it has been well tolerated and resulted in anti-tumor activity. Methods PBMC samples were obtained from healthy donors or ALPS patients collected under NIAID IRB-approved protocol 93-I-0063. Cells were cultured with or without anti-CD3/CD28 activation. The impact of CPI-818 on viability, expression of ITK, and expression of the surface markers CD 25 and CD69 was assessed by flow cytometry. For in vivo studies, MRL/lpr mice received a control diet or a CPI-818 formulated diet (300 mg/kg/day). Lymph nodes were calipered weekly over the course of 22 weeks of disease development. Spleens, lungs and kidneys were harvested at 22 weeks to assess the impact of CPI-818 treatment on T cell subsets and histology. Results Ex vivo studies with PBMC preparations from normal donors and ALPS patients confirmed the expression of ITK in CD4+, CD8+, and DN T cells from ALPS patients. When these T cells were stimulated with anti-CD3/CD28, CPI-818 potently inhibited the upregulation of the activation markers CD25 and CD69 in DN T cells assessed by flow cytometry. In companion experiments, CPI-818 was not cytotoxic to DN T cells when activated through the TCR signaling pathway. Treatment of MRL/lpr mice with CPI-818 prevented the onset or, in a therapeutic design, led to the regression of lymphadenopathy and splenomegaly comparable to the treatment effects observed with cyclophosphamide. Unlike the effects seen with the cytotoxic agent cyclophosphamide which suppressed all T cells, as well as B, NK and myeloid cells in the spleen, CPI-818 selectively depleted DN T cells (see Figure). CPI-818 treatment also resulted in decreased levels of DN T cells in the kidneys and lungs of these mice without impacting the level of normal CD4 and CD8 T cells. CPI-818 limited the increased proteinuria measured in the control animals during the 22-week treatment window. Histological assessment of the kidney revealed reduced inflammatory damage in CPI-818 treated animals. Conclusions The selective, covalent ITK inhibitor, CPI-818, significantly reduced DN T cell numbers, lymphadenopathy and other disease end points in the MRL/lpr murine model of ALPS. This suggests that the dysregulated growth of Fas-deficient DN T cells requires functional TCR signaling. Further, our data suggests that blocking TCR signaling by targeting ITK may be an effective strategy for treating adult and pediatric ALPS. Figure Disclosures Ng: Corvus Pharmaceuticals: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Hill:Corvus Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Miller:Corvus Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Buggy:Corvus Pharmaceuticals: Consultancy, Current Employment, Current equity holder in publicly-traded company. Janc:Corvus Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company.

Cpi-818, an Oral Interleukin-2-Inducible T-Cell Kinase Inhibitor, Is Well-Tolerated and Active in Patients with T-Cell Lymphoma

Cpi-818, an Oral Interleukin-2-Inducible T-Cell Kinase Inhibitor, Is Well-Tolerated and Active in Patients with T-Cell Lymphoma

Inhibition of interleukin-2-inducible T-cell kinase causes reduction in imiquimod-induced psoriasiform inflammation through reduction of Th17 cells and enhancement of Treg cells in mice

Psoriasis is a debilitating chronic skin disease with a worldwide prevalence. Its main features include well-marked silvery scales on the skin of hands and feet and back which arise due to hyperproliferation of keratinocytes and infiltration of immune cells in the skin. Multiple interactions exist between adaptive immune cells such as T cells and innate immune cells such as neutrophils and macrophages which are key players in the pathogenesis of psoriasis. Interleukin-2-inducible T-cell kinase (ITK) plays a key role in Th17 cell development through control of several transcription factors. ITK has been shown to control NFATc1, NFkB and STAT3 in CD4+ T cells. Effect of ITK inhibitor in imiquimod (IMQ)-induced psoriasiform inflammation remains to be explored. In the current examination, role of ITK signaling and its inhibition blockade were evaluated on NFATc1, NFkB and STAT3, IL-17A, TNF-α, IFN-γ, Foxp3, IL-10 in CD4+ T cells in IMQ model. Our data display that ITK signaling is involved in IMQ-induced psoriatic inflammation as paralleled by enhancement of p-ITK, NFATc1, p-NFkB and p-STAT3 in CD4+ T cells. It was associated with enhancement of Th17/Th1 cells and neutrophilic inflammation in the skin. Preventive treatment with ITK inhibitor led to a reduction in Th17/Th1 cells and enhancement of Treg cells. Overall, this study suggests that ITK signaling is an important modulator of transcription factor signaling in CD4+ T cells which is associated with Th17/Th1 cells and psoriasiform inflammation in mice. ITK signaling blockade could be a therapeutic target for the treatment of psoriatic inflammation.

Efficacy of agonist αOX40 and antagonist αCTLA-4 cancer immunotherapy is increased by ibrutinib treatment through enhanced Eomes expression and T-cell response

Abstract Antagonistic monoclonal antibodies targeting individual checkpoint receptors on T cells, such as CTLA-4 and PD-1, are used to treat patients with metastatic disease; however, only a subset of patients respond. Combination therapies, such as CTLA-4 blockade with agonist αOX40 mAb, are synergistic in preclinical models. αOX40/αCTLA-4 therapy synergistically enhances expression of Eomesodermin (Eomes), a transcription factor that drives CD8 T cell differentiation and memory function. We hypothesize that Eomes expression plays a critical role in the synergistic efficacy of αOX40/αCTLA-4 therapy. Compared to Eomeslo CD8 T cells, Eomeshi CD8 T cells induced by αOX40/αCTLA-4 therapy express lower levels of checkpoint receptors (PD-1, TIM-3, LAG-3) and higher levels of tumor trafficking and activation markers (CXCR3, CD44), and intratumoral cytokines (IFN-γ, TNF-α). CD8 T cell-specific Eomes−/− tumor-bearing mice did not respond to αOX40/αCTLA-4 therapy, demonstrating the necessity of Eomes expressing CD8 T cells. Interleukin-2 inducible T cell kinase (ITK) negatively regulates T cell Eomes expression following T cell receptor ligation. Thus, we asked whether enhancing T cell Eomes expression by combining ibrutinib, an FDA-approved ITK inhibitor, with αOX40/αCTLA4 therapy would further improve anti-tumor responses by increasing Eomeshi CD8 T cells. This triple combination increased CD8 T cell-specific Eomes expression, tumor-specific CD8 T cells, Ki-67 (proliferation), and Th1 cytokines (IFN-γ, TNF-α) leading to reduced tumor growth and improved survival over αOX40/αCTLA-4 alone, making ibrutinib/αOX40/αCTLA-4 a potential novel triple combination therapy.

Blockade of interleukin-2-inducible T-cell kinase signaling attenuates acute lung injury in mice through adjustment of pulmonary Th17/Treg immune responses and reduction of oxidative stress

Acute lung injury (ALI) is linked with considerable morbidity and mortality. ALI can be caused by various agents, one of them being sepsis. ALI is characterized by injury to vascular endothelium and alveolar epithelium that results in edema, pulmonary immune cells infiltration and hypoxemia. Neutrophils and T cells particularly play a huge role in amplification of pulmonary inflammation through release of multiple inflammatory mediators. Recent reports suggest a strong involvement of Th17 cells and oxidative stress in initiation/amplification of pulmonary inflammation during ALI. Interleukin-2-inducible T-cell kinase (ITK) plays a key role in Th17 cell development through control of several transcription factors. Therefore, our study explored the role of ITK on airway inflammation (total/neutrophilic cell counts, myeloperoxidase activity, E-cadherin expression, histopathological analyses) and effect of its inhibition on various inflammatory/anti-inflammatory pathways during ALI [phosphorylated-ITK (p-ITK), NFATc1, IL-17A, STAT3, Foxp3, IL-10, iNOS, nitrotyrosine, lipid peroxides). ALI was associated with increased total/neutrophilic cell counts and myeloperoxidase activity, and decreased E-cadherin expression in airway epithelial cells (AECs) which was concurrent with upregulation of p-ITK, NFATc1, IL-17A, STAT3 in CD4+ T cells and iNOS/nitrotyrosine in AECs. Treatment with ITK inhibitor reversed ALI-induced changes in airway inflammation and Th17 cells/oxidative stress. Treatment with ITK inhibitor further expanded Treg cells in mice with ALI. In short, our study proposes that ITK signaling plays a significant role in sepsis-induced ALI through upregulation of Th17 cells and oxidative stress. Further, findings provide evidence that ITK blockade could be a potential treatment strategy to attenuate airway inflammation associated with ALI.

The retinoid derivant ECPIRM selectively exhibited anti-proliferation effects in cutaneous T-Cell lymphoma via ITK-mediated signaling pathway

BackgroundThe treatment of Cutaneous T-cell lymphoma (CTCL) met huge challenges because of the heterogeneity and the scarcity of targeted drugs. ECPIRM derived from isotretinoin exhibited strong anti-proliferation effects in Hut78 and MJ cells rather than Myla cells. However, there was no data regarding the potential target of ECPIRM for its selective activity. ObjectivesTo investigate the potential target of ECPIRM for its selective anti-proliferation activity. MethodsWe evaluated the cell viability of CTCL cells after ECPIRM treatment, and detected the effects of ECPIRM on the biomarker genes of CTCL. Subsequently, the mRNA and protein level of Interleukin-2-inducible T-cell kinase (ITK) was determined. Then the induction of apoptosis triggered by ITK inhibitor BMS-509744 and ITK siRNAs were detected, and the docking of ECPIRM interacted with ITK and the effects of ECPIRM on ITK-mediated signaling pathway were analyzed. Finally, we evaluated the anti-growth activity of ECPIRM in Hut78-xenografted nude mice, and the relative expression of cleaved caspase-3, ITK, p-ERK and p-Akt were determined. ResultsITK was highly expressed in Hut78 and MJ cells rather than Myla cells, and targeted inhibition of ITK triggered cell apoptosis. ECPIRM efficiently bound the hydrophobic active pocket of ITK, and significantly inhibited ITK-mediated signaling pathway. In addition, ECPIRM suppressed tumor growth in Hut78-xenografted model, and upregulated the expression of cleaved caspase 3 and inhibited the expression of ITK, p-ERK and p-Akt in tumor tissues, which was consistent with in vitro study. ConclusionECPIRM might provide a novel strategy for CTCL by inhibiting ITK-mediated signaling pathway.

Design, synthesis and structure–activity relationship of indolylindazoles as potent and selective covalent inhibitors of interleukin-2 inducible T-cell kinase (ITK)

Interleukin-2 inducible T-cell kinase (ITK), a member of the Tec family of tyrosine kinases, plays an important role in T cell signaling downstream of the T-cell receptor (TCR). Herein we report the discovery of a series of indolylindazole based covalent ITK inhibitors with nanomolar inhibitory potency against ITK, good kinase selectivity and potent inhibition of the phosphorylation of PLCγ1 and ERK1/2 in living cells. A computational study provided insight into the interactions between inhibitors and Phe437 at the ATP binding pocket of ITK, suggesting that both edge-to-face π-π interaction and the dihedral torsion angle contribute to inhibitors’ potency. Compounds 43 and 55 stood out as selective covalent inhibitors with potent cellular activity, which could be used as chemical tools for further study of ITK functions.

Discovery of DWP213388, a Potent ITK and BTK Selective Dual Inhibitor with Excellent Efficacy and Safety, for Treating Gvhd

Introduction: Graft-versus-host-disease (GVHD) is a severe complication in patients undergoing hematopoietic stem cell transplantation (HSCT) and one of the major causes of death in patients receiving HSCT despite immunosuppressant drug and intensive therapy. GVHD is a rare disease characterized by excessive activation of T and B cell pathways and drugs such as Ibrutinib and steroids are used to improve the survival rate of GVHD patients by controlling immune cell signaling, yet the use of these drugs is limited due to toxicity. It is necessary to develop new treatment agents for GVHD, because of the limitations of these existing treatment agents. Interleukin-2-inducible T-cell kinase (ITK) and Bruton's tyrosine kinase (BTK) are critical for T and B cells activation, and dysregulation of this process has been known to increase proinflammatory cytokines and cause various inflammatory diseases. Since ITK and BTK signaling pathway play a major role in T and B cell regulation in GVHD, we thought that ITK and BTK selective dual inhibition may act as a promising agent that have beneficial effects on GVHD. So, we developed a novel ITK and BTK selective dual inhibitor, DWP213388, to modulate both T and B cell pathways without any significant off-target effects, thus to ultimately improve survival rate in GVHD without any serious side effect. Methods: Inhibition of ITK and BTK enzyme activity and selectivity against Cys-family kinase group were evaluated by biochemical activity assay. Target occupancy in human T and B cells was measured by quantifying unbound ITK or BTK in ELISA-based assay using biotinylated-DWP213388. To measure occupancy, mouse spleen and thymus were extracted after oral administration of DWP213388. Cellular assays for BCR or TCR stimulation-dependent activation and cytokine secretion were determined in human PBMC. In vitro mixed lymphocyte reaction (MLR) assay, CD4+ T cells were purified from spleens of B6 mice and were co-cultured with irradiated Balb/c splenic APCs as stimulators that were incubated in the presence or absence of DWP213388. In vivo MLR test, recipient BDF1 mouse was lethally irradiated and reconstituted by intravenous injection of splenocytes with B6. The efficacy of DWP213388 was investigated in mouse GVHD model; Recipient BDF1 mouse was lethally irradiated and reconstituted by intravenous injection of T cell depleted bone marrow (TCD-BM) and splenocytes with B6. In CMV study, DWP213388 was administered orally and an inoculum of 1 x 104 PFU/head MCMV in PBS was injected intraperitoneal. into each mouse on day 0. Five and ten days after the MCMV infection, the liver was removed. The NK cell and CD8 T cell was measured by FACs and virus titration was measured by RT-PCR. Results: We developed a novel, potent dual target inhibitor, DWP213388, with ITK and BTK IC50 values of 1.0 nM and 0.4 nM, respectively. More importantly, this compound is highly selective against ITK and BTK, yet has low affinity toward EGFR. DWP213388 potently occupied both of its targets at low concentration, which shows successful inhibition of TCR/BCR-dependent CD69 expression. DWP213388 suppressed T and B cell subsets such as Th1, Th2, Th17 and plasma cells and inhibited T cell proliferation in both in-vitro and in-vivo MLR assays. In mouse GVHD model, DWP213388 at 10 mg/kg improved clinical symptoms and maintains 100% survival rate (vs Ibrutinib 85%). It showed 73% and 62% occupancy of BTK and ITK. To address any potential compromising immune response due to dual inhibition of T, B cell by DWP213388, we conducted a CMV study to evaluate the effects on infection. DWP213388 does not reduce the number of NK cells and CD8+ T cells, and it does not affect virus killing. Therefore, it has a low risk of infection. These results suggest that DWP213388 may serve as a next generation therapeutic agent for GVHD. Conclusion: We developed a novel, highly potent, and selective dual inhibitor of ITK and BTK, DWP213388. We demonstrated that DWP213388 has potent pharmacological activities compared to Ibrutinib in various cellular models and a mouse GVHD model. DWP213388 intends to improve efficacy and safety by specifically inhibiting ITK and BTK without inhibiting EGFR, which is known to cause severe side effects. Therefore, DWP213388 may serve as a next generation therapeutic agent for GVHD. Disclosures No relevant conflicts of interest to declare.

A Phase 1/1b Dose-Escalation Trial Evaluating Cpi-818, an Oral Interleukin-2-Inducible T-Cell Kinase Inhibitor, in Subjects with Relapsed/Refractory T-Cell Lymphoma

A Phase 1/1b Dose-Escalation Trial Evaluating Cpi-818, an Oral Interleukin-2-Inducible T-Cell Kinase Inhibitor, in Subjects with Relapsed/Refractory T-Cell Lymphoma

A Machine Learning Access for Selectionof Influential Variables of Several ITK Inhibitors using Regression Research

Introduction: Interleukin-2 inducible T-cell kinase (ITK) is a tyrosine kinase expressed in T-cells, NK cells and mast cells. Selective ITK inhibitors act as an immunosuppressive and anti-inflammatory agent reduces lung inflammation, eosinophil infiltration, and mucous production in response to induced allergic asthma. Methodology: A dataset of 142 ITK inhibitors as dependent variables with 32 properties of compounds as explanatory variables were studied for their multicollinearity prior multivariate regression analysis. After data normalization, an inter-correlation cutoff value of 0.75 resulted in 15 variables and regression analysis resulted in 0.641 r2 and 0.598 adjusted r2 with RMSE 0.634 respectively. As the statistical parameters are within the limits, outlying data was investigated. Results: The standardized residual analysis resulted in nine data points and a new regression model is attempted with n=133 and p=15 reported improves statistics. Further, stepwise and stepwise AIC regression followed by variance inflation factor analyzed on the dataset revealed only 7 variables as important in defining inhibitory activity of ITK. Permutation and combinations of 7 variables resulted in r2 value >0.6 for 5, 6 and 7 variables. Hence, to select the best model, FIT criterion was employed where a 5-variable model was judged as best model. Conclusion: Finally, it has been emphasized that increase in HOMO, H-Bond Donors and shape index with a concomitant decrease in number of phenyl groups and LUMO parameter favors ITK inhibition

Abstract 1313: CPI-818: A selective inhibitor of interleukin-2-inducible T-cell kinase (ITK) that inhibits T-cell receptor signaling, promotes Th1 skewing, and achieves objective tumor responses when administered to dogs with T cell lymphomas

Abstract Background: ITK is a non-receptor tyrosine kinase that modifies T cell receptor (TCR) signaling. Mice deficient in ITK, but not resting lymphocyte kinase (RLK), exhibit defects in Th2 differentiation while retaining the ability to differentiate into Th1 cells and secrete IFNγ. Combined disruption of ITK and RLK in mice leads to more severe T cell functional defects compared to disrupting ITK alone, and paradoxically allows for normal Th2 responses. Thus, selective pharmacologic inhibition of ITK versus RLK is necessary to inhibit Th2 responses without affecting Th1-dependent immunity. ITK is widely expressed in T cell malignancies, and activation of ITK upregulates GATA-3, a transcription factor that drives Th2 differentiation and is associated with poor survival. Here we report the discovery and characterization of CPI-818, an irreversible inhibitor of ITK. CPI-818 is highly selective for ITK over RLK allowing for an assessment of pure ITK inhibition on normal and malignant T cells. Results: CPI-818 irreversibly inhibited ITK (IC502.3 nM) with >100-fold selectivity over RLK (430 nM) and BTK (850 nM). The mechanism of ITK inhibition involves covalent binding to CYS-442 confirmed by mass spectrometry. Irreversible inhibition of ITK in vitroand in vivowas demonstrated using an active site competitive probe. CPI-818 inhibited anti-CD3/28 induced phosphorylation of ERK (T202/Y204) and PLCγ (Y783) in PMBCs, and inhibited IL2 secretion in Jurkat T cells (IC5075 nM). CPI-818 demonstrated dose dependently inhibition of TCR-induced proliferation of malignant T cells from Sezary Syndrome patients. In mice orally treatedwith CPI-818 an increase in the ratio of IFNγ/IL-4 (p<0.05) upon antigen-specific re-stimulation in an OVA transgenic adoptive transfer model was observed, consistent with Th1-skewing. Additionally, toassess the impact of CPI-818 on Th-biasing, activated human PBMCs were cultured for 6 days with CPI-818 and inhibition of IL-4 production (average 64% inhibition, n=12) was significantly greater than IFNγ production (average 19% inhibition), supportive of Th1 skewing. To assess the potential of CPI-818 to treat human T cell lymphoproliferative disorders, the safety and efficacy of CPI-818 in companion dogs with spontaneously-occurring T cell lymphomas were evaluated. CPI-818 was dosed orally at 20 mg/kg BID for 2 weeks to 5 months. Three animals were treated: 1 with peripheral T cell lymphoma and 2 with cutaneous T cell lymphoma. Full ITK occupancy in peripheral blood was confirmed using a probe assay. Evidence of anti-tumor activity was seen in all dogs including complete and partial responses. CPI-818 was well tolerated with no change in normal lymphocyte counts. These data support evaluation of CPI-818 in clinical trials in patients with T cell malignancies. Citation Format: James W. Janc, Craig M. Hill, Patrick P. Ng, Andrew N. Hoston, Antonett Madriaga, Trang P. Dao-Pick, Kitman S. Yeung, Ryan Hudson, Anne-Marie Beausoleil, Erin Bradley, Erik Verner, Douglas H. Thamm, Richard A. Miller, Joseph J. Buggy. CPI-818: A selective inhibitor of interleukin-2-inducible T-cell kinase (ITK) that inhibits T-cell receptor signaling, promotes Th1 skewing, and achieves objective tumor responses when administered to dogs with T cell lymphomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1313.

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

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.

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.

Pseudo-Progression Among Patients with Follicular Lymphoma Treated with Ibrutinib in the Phase 2 DAWN Study

Pseudo-Progression Among Patients with Follicular Lymphoma Treated with Ibrutinib in the Phase 2 DAWN Study