Inducible T-cell Kinase Research Articles

Mapping the role of ITK (interleukin 2 inducible T cell kinase) and TCR (T cell receptor) signal strength in determining the early fate of CD8 T cells using Nr4a3 “timed” fluorescent reporter

Abstract ITK plays a key role in the rate and magnitude of TCR-dependent transcription factor activity and early genetic programming that determines the extent of CD8 T cell effector and memory function. To study the extent of this role we crossed an OT1 transgenic ITK germline KO to the Nr4a3-Tocky timer model which produces a time dependent fluorescent reporter. Nr4a3 is highly expressed following TCR activation and thus is a good metric for TCR signal strength and duration. The Tocky protein fluoresces blue, at λ 466 and then, after ~7hrs, degrades to a red fluorescence at λ 583. OT-1 Rag1KO ITK (WT, +/-, and -/-) Nr4a3-Tocky bulk splenocytes were stimulated with the altered peptide ligand T4 Ova peptide at varying concentrations for 12/24 hours and sorted on Tocky+ Blue/Red and negative events. ITK -/- CD8s showed elevated CD69 and CD25 but were variable for Tocky expression. Both Tocky Negative and Tocky Blue sorted ITK -/- CD69+ CD8s expressed higher IRF8, cFos, cMyc, Bcl2, and TCF1/7 as well as lower IRF4 versus WT at 12 hours post-stimulation. Analyses of Tocky Blue/Red or persistently active CD8s showed WT having increased expression of TCF1 and similar expression of IRF8, cFos, Bcl2, Eomes and Egr2 compared to ITK -/-. Bcl2 and PDL1 are also highly expressed through all stages of TCR signaling in ITK -/- CD8s. These data indicate that differences in TCR signal strength with and without ITK can affect the time at which transcriptional networks arise after TCR stimulation.

Impact of ibrutinib on inflammation in a mouse model of Graves' orbitopathy.

Bruton's tyrosine kinase (BTK) and interleukin (IL)-2 Inducible T-cell Kinase (ITK) inhibitors have anti-inflammatory properties. We investigated the therapeutic effect of ibrutinib, an orally bioavailable BTK/ITK inhibitor, in a mouse model of Graves' orbitopathy (GO). Genetic immunization was performed through intramuscular administration of the recombinant plasmid, pCMV6-hTSHR cDNA, to 8-week-old female BALB/c mice. Serum levels of T3, T4, and thyroid-stimulating hormone receptor (TSHR) antibodies (TRAbs) were quantified using enzyme-linked immunosorbent assay. Histopathological changes in orbital tissues were examined using immunohistochemistry (IHC) staining for TSHR and various inflammatory markers. Following successful genetic immunization, ibrutinib was orally administered daily for 2 weeks in the GO model mice. After treatment, the mRNA and protein expression levels of BTK, ITK, IL-1β, and IL-6 in orbital tissues were evaluated using real-time PCR and Western blotting. In total, 20 mice were sacrificed to confirm successful genetic immunization. The GO mouse group exhibited significantly increased serum T3, T4, and TRAb levels. IHC revealed increased expression of TSHR, IL-1β, IL-6, transforming growth factor-β1, interferon-γ, CD40, CD4, BTK, and ITK in the GO mouse model. The orbital inflammation was significantly attenuated in ibrutinib-treated mice. The mRNA and protein expression levels of BTK, ITK, IL-1β, and IL-6 in orbital tissue were lower in ibrutinib-treated GO mouse group compared to the phosphate-buffered saline-treated GO mouse group. The GO mouse model demonstrated enhanced BTK and ITK expression. Ibrutinib, a BTK/ITK inhibitor, suppressed the inflammatory cytokine production. These findings highlight the potential involvement of BTK/ITK in the inflammatory pathogenesis of GO, suggesting its role as a novel therapeutic target.

Dynamic Single-Cell Profiling Reveals Novel Immune Regulatory Mechanism of ITK Inhibitor Soquelitinib in Refractory T Cell Lymphoma

Introduction Interleukin 2 inducible T cell kinase (ITK) plays an important role in the T cell receptor (TCR) signaling pathway. The highly selective covalent ITK inhibitor, soquelitinib (formerly known as CPI-818), is being evaluated as a monotherapy in an ongoing Phase 1/1b trial in patients with refractory T-cell lymphoma (TCL) (NCT03952078). Single cell RNA-sequencing and flow cytometry were performed on patient samples to identify potential prognostic biomarkers and to study immune regulatory mechanisms. Methods Paired peripheral blood and tumor tissue samples were collected from 3 responding TCL patients treated with soquelitinib. Dynamic single cell RNA-seq and flow cytometry studies were performed and changes in gene expression profiling and signature gene enrichment were analyzed at baseline and on treatment. Results Single cell RNA-seq of mRNA expression demonstrated that soquelitinib induced expression of cytotoxic genes (PRF1, GZMB, GZMK, GZMA, GZMH, GNLY and NKG7) and effector genes (IFNG, EOMES, CST7), and reduced exhaustion genes (PDCD1, LAG3, CTLA4, HAVCR2) in normal CD8+ cells. KEGG and GSEA enrichment analysis revealed that Interferon-Gamma, IL-2-STAT5 and TNF-alpha related signaling pathways were upregulated; the protein-protein interaction network analysis revealed that the hub gene TBX21 might play a central regulatory role. KEGG enrichment analysis also demonstrated that soquelitinib treatment significantly decreased DNA replication and cell cycle signaling in malignant T cells, and that normal CD4+ Treg cells within tumor microenvironment and peripheral blood were significantly reduced post soquelitinib treatment. Flow cytometry analysis of the peripheral blood and intratumoral T cell subpopulations confirmed that the normal CD4+ and CD8+ T cells, especially Th1 cells and CD8+ terminally differentiated T effector memory cells were significantly increased and CD4+ Treg cells were decreased compared to baseline levels following soquelitinib treatment. In addition, human T cell differentiation studies and EL4 syngeneic tumor bearing mice studies confirmed the immunomodulatory effect of soquelitinib in the tumor microenvironment in vitro and in vivo. Similar results were also observed in the murine syngeneic B cell lymphoma A20, breast cancer 4T1 and colon cancer CT26.WT models. Conclusions Our findings reveal a novel immune regulatory mechanism by which the ITK inhibitor soquelitinib induced normal CD4+ Th1 cells and CD8+ TEMRA cells and reduced CD4+ Treg cells in the tumor microenvironment in responding patients. These findings demonstrate the potential of soquelitinib as a novel immunotherapy for the treatment of T-cell lymphomas and solid tumors.

Abstract 1813: Selective ITK blockade induces antitumor responses and enhances efficacy to immune checkpoint inhibitors in preclinical models

Abstract Interleukin 2 inducible T cell kinase (ITK) plays a role in both T cell receptor (TCR) signaling and T helper cell differentiation. ITK-/- mice exhibit defects in Th2 differentiation while retaining the ability to differentiate into Th1 cells that secrete IFNγ. We generated CPI-818, a covalent inhibitor of ITK (KD 2.5nM) with > 100 fold selectivity over resting lymphocyte kinase (RLK) and other TEC family kinases. This drug is now being evaluated as a single agent in an ongoing Phase 1 clinical trial in refractory T cell lymphoma where objective tumor responses have been observed. In the studies reported here, we have investigated the immunomodulatory effects of specific ITK blockade of human T cells with CPI-818 in vitro and in vivo in murine tumor models. Analysis of the cytokine profiles in human CD4 T cells differentiated under non-polarizing, Th1 or Th2 polarizing conditions showed that CPI-818 inhibited generation of Th2-associated cytokines whereas, production of Th1 cytokines such as IFNγ were largely unaffected. Suppression of IL-2-mediated proliferative response occurred only at high concentrations of CPI-818 (>1uM). The effect of CPI-818 on anti-tumor immunity was evaluated in both murine syngeneic CT26 colon cancer and EL4 T cell lymphoma models. In vivo treatment of animals with established tumors showed single-agent anti-tumor activity in both models. Combining CPI-818 with suboptimal doses of anti-PD1 and anti-CTLA4 synergistically inhibited the growth of established CT26 tumors, leading to complete tumor elimination in 100% treated animals. Furthermore, triple combination therapy elicited durable anti-tumor immune memory after animals were rechallenged with a new engraftment of CT26 cells. Enhancement of anti-tumor activity required CD8+ T cells as in vivo antibody-mediated depletion of CD8, but not CD4, abolished the efficacy of CPI-818 monotherapy. Consistent with that, we found increased tumor-infiltrating CD8+ T cells and increased ratio of CD8: Treg in the responding tumors of CPI-818 treated animals. Levels of several exhaustion makers were down-regulated by treatment with CPI-818, suggesting that inhibition of ITK by CPI-818 produces favorable changes in the tumor microenvironment. Our findings provide insights into the effects of selective ITK blockade on tumor immunity and its potential role in immunotherapy. Citation Format: Lih-Yun Hsu, Rahul D. Pawar, Dan Li, Poorva Ghosh, Kevin Nguyen, Carlene Williams, Yuqin Song, Ning Ding, Erik Verner, Richard A. Miller. Selective ITK blockade induces antitumor responses and enhances efficacy to immune checkpoint inhibitors in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1813.

ITK Inhibitor Induces Dose-Dependent Th1 Skewing in Normal T Cells and Is Active in Refractory T Cell Lymphomas

Introduction Interleukin 2 inducible T cell kinase (ITK) is a TEC family tyrosine kinase expressed in T cells with an important role both in T cell receptor (TCR) signaling and T helper cell differentiation. Resting lymphocyte kinase (RLK), a closely related kinase, is redundant to ITK and is involved in the differentiation of naïve T cells into Th1 T cells. ITK-/- mice exhibit defects in Th2 differentiation while retaining the ability to differentiate into Th1 cells that secrete IFNγ; whereas, double ITK/RLK knockout mice exhibit profound defects in T cell function. Th1 cells are needed for various cytotoxic T cell functions such as destruction of tumor cells. CPI-818 is a covalent inhibitor of ITK (KD 2.5nM) with > 100 fold selectivity over RLK and other TEC family kinases. CPI-818 is being evaluated in an ongoing phase 1 trial in patients (pts) with refractory T cell lymphomas (TCL). We now report that selective ITK blockade with CPI-818 induces Th1 skewing of normal T cells and demonstrates anti-tumor activity. Methods Normal human peripheral blood mononuclear cells (PBMC) were stimulated in vitro for 6 days with anti-CD3/CD28 in the presence of CPI-818. Th1 cells were measured by flow cytometry based on ratio of IFNγ:IL4 staining cells. Sezary cells were similarly stimulated in the presence of CPI-818. Peripheral blood and tumor samples were stained with antibodies to CD3, 4, 8, 183, 196, 197 and 45RA to enumerate Th1 and other T cell subsets using multicolor flow cytometry. Successive cohorts of TCL pts were treated in a phase 1 trial until disease progression with continuous oral dosing of CPI-818 100mg, 200mg, 400mg or 600mg given BID. Results T cell receptor stimulated PBMC from 12 healthy donors incubated in vitro with CPI-818 demonstrated Th1 skewing at concentrations of 0.1 to < 10uM (Figure 1). Maximal skewing was seen at 1uM; cell viability was unaffected but, proliferation was inhibited at 10uM. Similar studies using Th2 Sezary cells showed inhibition of proliferation with CPI-818 (0.5-2.5uM). Thirty-three pts with TCL with a median age of 62 yrs and median of 4 prior therapies were enrolled. Histologies included: PTCL-NOS (17), AITL (4), and CTCL (12). The most common (≥ 10%) AEs were nausea, chills, vomiting, fatigue, pyrexia, decreased appetite, pruritus, and rash. Treatment-related Grade 3+ AEs were: anemia (1), lymphocytosis (1), WBC decreased (1) and neutropenia (1). The swimmer plot (Figure 2) shows a dose-dependent relationship with an optimum dose of 200mg (N=10) producing 1 CR 25 mo; 1 PR 2+ mo; 1 nodal CR 16 mo (CTCL); 2 SD; 1 PD; 4 not yet evaluated; 6 pts remain on treatment. Pharmacokinetic and target occupancy studies for the 200mg dose showed mean plasma Cmax of 2.3uM (SD 1.9uM) and ITK target occupancy of >75%. Analysis of blood in 4 of 4 pts treated in this cohort showed increases in Th1 cells (CD3+/CD4+/CD8-/CD183+/CD196-) compared to baseline and increases in terminally differentiated T effector memory cells (TEMRA CD3+/CD4-/CD8+/CD197-/CD45RA+). Tumor biopsy from 1 patient taken during response demonstrated an increase in Th1 (0 to 40%) and TEMRA CD8+ T cells (2.83% to 18.6%). CPI-818 reduced blood eosinophils (Eos) in 3 of 3 pts in this cohort with elevated Eos count, consistent with inhibition of Th2 function. Conclusions This study demonstrates that selective inhibition of ITK, while sparing RLK, induces Th1 skewing. As expected, the effects on T cell differentiation and proliferation are dose dependent: lower doses induce Th1 skewing and higher concentrations in vitro and in vivo lead to inhibition of TCR signaling and proliferation. Tumor responses were observed in several patients receiving 200mg BID, which achieves drug plasma levels consistent with Th1 skewing observed in vitro. The increases in Th1 cells and TEMRA CD8+ cells suggest that anti-tumor activity is due to induction of a host anti-tumor response. This raises the possibility that immunomodulation with ITK blockade could be used for immunotherapy of other cancers. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Effects of ibrutinib on T-cell immunity in patients with chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL), a highly heterogeneous B-cell malignancy, is characterized by tumor microenvironment disorder and T-cell immune dysfunction, which play a major role in the proliferation and survival of CLL cells. Ibrutinib is the first irreversible inhibitor of Bruton’s tyrosine kinase (BTK). In addition to targeting B-cell receptor (BCR) signaling to kill tumor cells, increasing evidence has suggested that ibrutinib regulates the tumor microenvironment and T-cell immunity in a direct and indirect manner. For example, ibrutinib not only reverses the tumor microenvironment by blocking cytokine networks and toll-like receptor signaling but also regulates T cells in number, subset distribution, T-cell receptor (TCR) repertoire and immune function by inhibiting interleukin-2 inducible T-cell kinase (ITK) and reducing the expression of inhibitory receptors, and so on. In this review, we summarize the current evidence for the effects of ibrutinib on the tumor microenvironment and cellular immunity of patients with CLL, particularly for the behavior and function of T cells, explore its potential mechanisms, and provide a basis for the clinical benefits of long-term ibrutinib treatment and combined therapy based on T-cell-based immunotherapies.

Effects of Prior Exposure to Tec Kinase(BTK/ITK) Inhibitors on Kte-X19 Products

Introduction:Frequent and durable responses were recently reported in relapsed or refractory (R/R) mantle cell lymphoma (MCL) patients treated with KTE-X19, an autologous CD19-targeted chimeric antigen receptor-engineered T-cell (CAR-T) product (Wang et al. N Engl J Med. 2020). Most patients enrolled had received at least one line of Tec kinase inhibitor prior to KTE-X19 manufacturing, either in the form of ibrutinib, a Bruton's tyrosine kinase (BTK) and Inducible T cell kinase (ITK) inhibitor, or acalabrutinib, a more selective BTK inhibitor. Pharmacokinetic expansion of KTE-X19 was higher in ibrutinib-treated patients relative to acalabrutinib-treated patients. We previously showed that prolonged exposure to ibrutinib enhanced T cell effector function and proliferation in patients with CLL (Fraietta et al, Blood, 2016). To assess the impact of Tec kinase inhibitor on KTE-X19 products and downstream clinical outcomes, we examined the phenotype, transcriptional profile and cytokine production of KTE-X19 infusion products and post-infusion lymphocytes from patients with R/R MCL treated on the Zuma-2 study.Study Design and Methods:We evaluated biospecimens from MCL patients who enrolled on the Zuma-2 clinical trial (NCT02601313) and who were previously treated with ibrutinib (n=14) or acalabrutinib (n=6). Samples analyzed consisted of KTE-X19 CAR T products and peripheral blood mononuclear cells (PBMC) collected 7 days after infusion. Lymphocytes were assessed for CAR expression, T cell phenotype, transcriptional profile and cytokine production. In addition, CAR T cell phenotypes and cytokines were profiled following co-culture of KTE-X19 with CD19 + Toledo cells (DLBCL).Results:Flow cytometric analysis of KTE-X19 demonstrated similar distributions of CD4+ and CD8+ T cells and comparable frequencies of central and effector memory populations in the CAR+ T cells derived from patients with prior exposure to ibrutinib vs. acalabrutinib. T helper subset analysis trended towards enrichment of Th1/Th17 populations within the CAR+ CD4+ cells of the ibrutinib cohort. This finding was further supported by transcriptional profiling of sorted CAR+ T cells from infusion products, where Th1/Th17, Jak/STAT and activation-related genes were enriched in the cohort with prior ibrutinib exposure. In addition, the Th1 phenotype was more frequent in PBMC of ibrutinib-exposed patients (8/14) compared to acalabrutinib-exposed patients (1/4). Interestingly, a shift from a central memory-dominant product towards an effector memory phenotype was observed in peripheral CD4+ and CD8+ CAR T cells in the ibrutinib cohort, whereas acalabrutinib post-infusion CAR T cells maintained a central memory phenotype. In vitro stimulation of KTE-X19 CAR-T infusion products with tumor cells resulted in a significant enrichment of the Th1 population in patients who had received ibrutinib compared to those that received acalabrutinib (p=0.0058). Following stimulation, CAR-T cells from the acalabrutinib cohort produced higher levels of Th2 cytokines, including IL-4, IL-5, and IL-13 as well as GM-CSF compared to the ibrutinib cohort.Conclusions:Analysis of KTE-X19 infusion products and day 7 post-infusion PBMC demonstrated that CAR T cells from patients with prior ibrutinib exposure have a Th1 predominant phenotype, suggesting that ibrutinib but not acalabrutinib promotes Th1 differentiation and effector function, potentially through the inhibition of ITK. Furthermore, our data suggest that inhibition of non-BTK targets such as ITK may play a role in driving a Th17 phenotype. Prior exposure to ibrutinib may increase CAR T cell effector function to a greater extent than exposure to acalabrutinib to enhance clinical outcome in patients with MCL. DisclosuresBudka: Kite Pharma: Current Employment. Sowrirajan: Kite Pharma: Current Employment. Nguyen: Kite Pharma: Current Employment. Shen: Gilead Sciences: Current equity holder in publicly-traded company; Kite, a Gilead Company: Current Employment, Other: Leadership role, Patents & Royalties; Atara: Current Employment, Current equity holder in publicly-traded company, Other: Leadership role, Patents & Royalties. Bot: Kite, a Gilead Company: Current Employment; Gilead Sciences: Consultancy, Current equity holder in publicly-traded company, Other: Travel support. Maus: Agenus: Consultancy; Arcellx: Consultancy; Astellas: Consultancy; AstraZeneca: Consultancy; Atara: Consultancy; Bayer: Consultancy; BMS: Consultancy; Cabaletta Bio (SAB): Consultancy; CRISPR therapeutics: Consultancy; In8bio (SAB): Consultancy; Intellia: Consultancy; GSK: Consultancy; Kite Pharma: Consultancy, Research Funding; Micromedicine: Consultancy, Current holder of stock options in a privately-held company; Novartis: Consultancy; Tmunity: Consultancy; Torque: Consultancy, Current holder of stock options in a privately-held company; WindMIL: Consultancy; Adaptimmune: Consultancy; tcr2: Consultancy, Divested equity in a private or publicly-traded company in the past 24 months; century: Current equity holder in publicly-traded company; ichnos biosciences: Consultancy, Current holder of stock options in a privately-held company.

Bortezomib enhances the anti-cancer effect of the novel Bruton's tyrosine kinase inhibitor (BGB-3111) in mantle cell lymphoma expressing BTK.

BGB-3111, a novel Bruton’s tyrosine kinase (BTK) inhibitor, shows promising anti-cancer effects in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), mantle cell lymphoma (MCL), and Waldenstrom macroglobulinemia (WM). This study aimed to investigate the anti-cancer effects of BGB-3111 combined with bortezomib (BTZ) against the BTK-expressing MCL. We found that BTK, which was overexpressed in 59.4% of patients with MCL, was mainly characterized by high Ki67 and elevated MIPI scores. BGB-3111 strongly inhibited cell proliferation, induced cell cycle arrest in the G1/G0-phase, and promoted cell apoptosis in the MCL cells expressing BTK. BGB-3111 provides better safety than another BTK inhibitor, ibrutinib as ibrutinib inhibits the inducible T-cell kinase (ITK) as an off-target effect but BGB-3111 does not inhibit ITK. Low doses of BTZ enhanced the anti-cancer effect induced by the low dose of BGB-3111 by downregulating the expression levels of PARP and Bcl-2 and increasing the expression levels of cleaved PARP and cleaved caspase-9. In addition, low doses of BGB-3111, but not of BTZ, inhibited BTK phosphorylation. However, low-doses of BTZ strengthened the anti-cancer effect induced by the low-doses of BGB-3111 via synergistically suppressing the IκBα and P65 phosphorylation. Taken together, our findings validate that BGB-3111 is a novel and effective BTK inhibitor for MCL-expressing BTK. Hence, it can be harnessed as a potential therapeutic strategy through a combinatorial treatment comprising low-dose BGB-3111 and low-dose BTZ to gain strong anti-cancer effects and better safety for MCL patients.

Interleukin-2 inducible T-cell kinase: a potential prognostic biomarker and tumor microenvironment remodeling indicator for hepatocellular carcinoma.

Background: The heterogeneous tumor microenvironment (TME) contributes to poor prognosis of hepatocellular carcinoma (HCC). However, determining the modulation of TME during HCC progression remains a challenge.Methods: Herein, the stromal score and immune score of HCC samples from The Cancer Genome Atlas database were calculated using the ESTIMATE algorithm and differentially expressed genes (DEGs) were obtained. Key DEGs were identified based on a protein-protein interaction network and survival analysis. Immunohistochemistry was carried out using primary samples to evaluate key DEGs expression. The CIBERSORT algorithm was applied to evaluate immune components. Gene Set Enrichment Analysis (GSEA) and correlation analysis were carried out to determine the relationship between key DEGs and tumor-infiltrating immune cells (TICs).Results: The stromal score, immune score and estimate score correlated significantly with 1-year recurrence-free survival of patients with HCC. Interleukin-2 inducible T-cell kinase (ITK) was identified as the most prognostic DEG for patients with HCC. GSEA revealed that genes in the high ITK subgroup were enriched in inflammatory-immunological terms. CIBERSORT analysis identified nine TIC subsets that correlated with ITK expression.Conclusion: We identified ITK as a novel indicator for early post-surgery tumor recurrence and microenvironment remodeling in HCC, providing a potential therapeutic target to treat HCC.

SY-1530, a highly selective BTK inhibitor, effectively treats B-cell malignancies by blocking B-cell activation.

Objective:B-cell antigen receptor (BCR) signaling is required to maintain the physiological functions of normal B cells and plays an important pathogenic role in B-cell malignancies. Bruton tyrosine kinase (BTK), a critical mediator of BCR signaling, is an attractive target for the treatment of B-cell malignancies. This study aimed to identify a highly potent and selective BTK inhibitor.Methods:Homogeneous time-resolved fluorescence assays were used to screen BTK inhibitors. Typhoon fluorescence imaging and Western blot analysis were used to confirm the effects of SY-1530 on the BCR signaling pathway. Additionally, the anti-tumor activities of SY-1530 were evaluated in TMD8 xenografts and spontaneous canine B-cell lymphoma.Results:We found a novel irreversible and non-competitive inhibitor of BTK, SY-1530, which provided dose-dependent and time-dependent inhibition. SY-1530 selectively bound to BTK rather than inducible T-cell kinase; consequently, it did not significantly affect T-cell receptor signaling and caused limited off-target effects. SY-1530 blocked the BCR signaling pathway through down-regulation of BTK activity, thus leading to impaired phosphorylation of BTK and its downstream kinases. Moreover, SY-1530 induced apoptosis in a caspase-dependent manner and efficaciously inhibited tumor growth in mouse xenograft models of B-cell malignancy (P < 0.001). SY-1530 also induced positive clinical responses in spontaneous canine B-cell lymphoma.Conclusions:SY-1530 is an irreversible and selective BTK inhibitor that shows inhibitory effects on B-cell malignancies by blocking the BCR signaling pathway. Therefore, it may be a promising therapeutic approach for the treatment of B-cell malignancies.

Possible Therapeutic Strategy Involving the Purine Synthesis Pathway Regulated by ITK in Tongue Squamous Cell Carcinoma.

Simple SummaryWe identified overexpression of interleukin-2–inducible T-cell kinase (ITK) as a novel biomarker for predicting the prognosis of tongue squamous cell carcinoma patients with poor outcomes. Oral cancer cell lines overexpressing ITK exhibited significantly increased proliferation in three-dimensional culture assays and murine inoculation models as compared with mock control cells. Phosphorylation proteomics analysis revealed that ITK expression induces the phosphorylation of a novel tyrosine residue of the trifunctional purine biosynthetic protein adenosine-3 (GART), an enzyme in the purine biosynthesis pathway. A significant increase in de novo purine biosynthesis was observed in cells expressing ITK. These results suggest that ITK is involved in enhancing the proliferation of cancer cells in the malignant phenotype by activating de novo purine biosynthesis through phosphorylation of GART. In this study, we report a possible therapeutic strategy involving the purine synthesis pathway regulated by ITK in tongue squamous cell carcinoma.The epidermal growth factor receptor is the only available tyrosine kinase molecular target for treating oral cancer. To improve the prognosis of tongue squamous cell carcinoma (TSCC) patients, a novel molecular target for tyrosine kinases is thus needed. We examined the expression of interleukin-2–inducible T-cell kinase (ITK) using immunohistochemistry, and the biological function of ITK was investigated using biochemical, phosphoproteomic, and metabolomic analyses. We found that ITK is overexpressed in TSCC patients with poor outcomes. The proliferation of oral cancer cell lines expressing ITK via transfection exhibited significant increases in three-dimensional culture assays and murine inoculation models with athymic male nude mice as compared with mock control cells. Suppressing the kinase activity using chemical inhibitors significantly reduced the increase in cell growth induced by ITK expression. Phosphoproteomic analyses revealed that ITK expression triggered phosphorylation of a novel tyrosine residue in trifunctional purine biosynthetic protein adenosine-3, an enzyme in the purine biosynthesis pathway. A significant increase in de novo biosynthesis of purines was observed in cells expressing ITK, which was abolished by the ITK inhibitor. ITK thus represents a potentially useful target for treating TSCC through modulation of purine biosynthesis.

BTK inhibitors, irrespective of ITK inhibition, increase efficacy of a CD19/CD3-bispecific antibody in CLL

AbstractBruton tyrosine kinase inhibitors (BTKis) are a preferred treatment of patients with chronic lymphocytic leukemia (CLL). Indefinite therapy with BTKis, although effective, presents clinical challenges. Combination therapy can deepen responses, shorten treatment duration, and possibly prevent or overcome drug resistance. We previously reported on a CD19/CD3-bispecific antibody (bsAb) that recruits autologous T-cell cytotoxicity against CLL cells in vitro. Compared with observations with samples from treatment-naïve patients, T cells from patients being treated with ibrutinib expanded more rapidly and exerted superior cytotoxic activity in response to the bsAb. In addition to BTK, ibrutinib also inhibits interleukin-2 inducible T-cell kinase (ITK). In contrast, acalabrutinib, does not inhibit ITK. Whether ITK inhibition contributes to the observed immune effects is unknown. To better understand how BTKis modulate T-cell function and cytotoxic activity, we cultured peripheral blood mononuclear cells (PBMCs) from BTKi-naive and ibrutinib- or acalabrutinib-treated CLL patients with CD19/CD3 bsAb in vitro. T-cell expansion, activation, differentiation, and cytotoxicity were increased in PBMCs from patients on treatment with either BTKi compared with that observed for BKTi-naïve patients. BTKi therapy transcriptionally downregulated immunosuppressive effectors expressed by CLL cells, including cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and CD200. CTLA-4 blockade with ipilimumab in vitro increased the cytotoxic activity of the bsAb in BTKi-naïve but not BTKi-treated PBMCS. Taken together, BTKis enhance bsAb-induced cytotoxicity by relieving T cells of immunosuppressive restraints imposed by CLL cells. The benefit of combining bsAb immunotherapy with BTKis needs to be confirmed in clinical trials.

Addition of BTK inhibitor orelabrutinib to rituximab improved anti-tumor effects in B cell lymphoma

Bruton tyrosine kinase (BTK) inhibitor ibrutinib has been validated as an effective drug to treat B cell malignancies. Combined therapies comprising ibrutinib and anti-CD20 antibodies like rituximab were designed as a backbone in many clinical trials. However, the off-target inhibition of ibrutinib on interleukin-2 (IL-2)-inducible T cell kinase (ITK) may reduce rituximab’s antibody-dependent cellular cytotoxicity (ADCC) efficacy. Orelabrutinib (Orel), a novel BTK inhibitor, was designed with high selectivity to BTK. In our study, we demonstrated in preclinical models that orelabrutinib in combination with rituximab could preserve NK-cell-mediated ADCC induced by rituximab and enhanced the apoptosis of tumor cells in vitro. The addition of orelabrutinib to rituximab had produced promising combined anti-tumor effects in B cell lymphomas in vivo. Collectively, combination therapy of orelabrutinib with rituximab would benefit patients with B cell lymphoma, especially those with relapsed or refractory disease.

A comprehensive analysis of the expression and regulation network of lymphocyte-specific protein tyrosine kinase in breast cancer.

BackgroundLymphocyte-specific protein tyrosine kinase (LCK), an encoded Src family protein tyrosine kinase, performs a pivotal molecular signaling role in the selection and maturation processes during T-cell development. Although aberrant LCK expression is known to have a significant association with carcinogenesis, the underlying role of LCK in breast cancer (BC) is still obscure.MethodsAn analysis of the levels of LCK mRNA expression in BC was performed, and the value of LCK expression for predicting the prognosis of patients with BC was studied using various online data resources, which included Oncomine, Gene Expression Profiling Interactive Analysis (GEPIA), and UALCAN. The web-based NetworkAnalyst tool was utilized to investigate the functional network of differentially expressed LCK. LinkedOmics was employed to identify the genes with which LCK has correlations in BC, together with the kinases, microRNAs, and transcription factors (TFs) potentially targeted by LCK in BC. The expression levels of LCK and its significantly correlated genes in BC were investigated with the Human Protein Atlas (HPA).ResultsWe observed a significant difference in the level of LCK mRNA expression between BC patients and healthy individuals, and a higher LCK expression was associated with poor overall survival (OS). The functional enrichment results revealed that the differential expression of LCK was mainly involved in the regulation of immune response and inflammatory response in BC. The expression of significantly related genes, such as inducible T-cell kinase (ITK), CD5, CD96, CD247, SH2 domain containing 1A (SH2D1A), phosphatidylinositol-4,5-bisphosphate 3-kinase (PIK3CD), Src-like-adaptor 2 (SLA2), and interleukin 2 receptor (IL2RG), was associated with poor OS in patients with BC. Regulatory network analysis found that LCK regulated immune cells, cancer progression, apoptosis, and cell cycle signal transduction through cancer-related kinases (ITK and MAPK3), miRNAs (miR-345 and miR-524), and TFs (AP1, SRF, and E2F1).ConclusionsThis study presents new perspectives on the differential expression and prognostic value of LCK in BC. Our observations will provide a basis for further study on the oncogenic and regulatory roles of LCK in BC.

Enhancing the Generation of Eomeshi CD8+ T Cells Augments the Efficacy of OX40- and CTLA-4-Targeted Immunotherapy.

CTLA-4 blockade in combination with an agonist OX40-specific monoclonal antibody synergizes to augment antitumor immunity through enhanced T-cell effector function, leading to increased survival in preclinical cancer models. We have shown previously that anti-OX40/anti-CTLA-4 combination therapy synergistically enhances the expression of Eomesodermin (Eomes) in CD8+ T cells. Eomes is a critical transcription factor for the differentiation and memory function of CD8+ T cells. We hypothesized that EomeshiCD8+ T cells were necessary for anti-OX40/anti-CTLA-4 immunotherapy efficacy and that further enhancement of this population would improve tumor-free survival. Indeed, CD8+ T cell-specific deletion of Eomes abrogated the efficacy of anti-OX40/anti-CTLA-4 therapy. We also found that anti-OX40/anti-CTLA-4-induced EomeshiCD8+ T cells expressed lower levels of checkpoint receptors (PD1, Tim-3, and Lag-3) and higher levels of effector cytokines (IFNγ and TNFα) than their Eomeslo counterparts. Eomes expression is negatively regulated in T cells through interleukin-2-inducible T-cell kinase (ITK) signaling. We investigated the impact of modulating ITK signaling with ibrutinib, an FDA-approved tyrosine kinase inhibitor, and found that anti-OX40/anti-CTLA-4/ibrutinib therapy further enhanced CD8+ T cell-specific Eomes expression, leading to enhanced tumor regression and improved survival, both of which were associated with increased T-cell effector function across multiple tumor models. Taken together, these data demonstrate the potential of anti-OX40/anti-CTLA-4/ibrutinib as a triple therapy to improve the efficacy of immunotherapy.

Topical Application of BMS-509744, a Selective Inhibitor of Interleukin-2-Inducible T Cell Kinase, Ameliorates Imiquimod-Induced Skin Inflammation in Mice.

Psoriasis is an immune disorder-related inflammatory skin disease. Recent studies have suggested a contribution of T cell activation in the pathogenesis of psoriasis. Interleukin-2 (IL-2)-inducible T cell kinase (ITK) regulates T cell activation, including proliferation, and cytokine production. In this study, we investigated the effect of the topically administered selective ITK inhibitor BMS-509744 on imiquimod (IMQ)-induced psoriasis-like skin inflammation in mice. Topically administered BMS-509744 ameliorated IMQ-induced psoriasis-like skin inflammation as shown by decreased skin lesions, epidermal thickening, and cell infiltration into the dermis. These suppressive effects occurred with lower numbers of cluster of differentiation antigen-3+ (CD3+) T cells and T helper subset 17 (Th17)-related cytokine expression in IMQ-treated skin. IMQ-induced upregulation of proinflammatory cytokine expression was also inhibited by topical application of BMS-509744 in IMQ-treated skin. Our report showed for the first time that topical application of BMS-509744 ameliorated psoriasis-like skin inflammation in mice, which is likely mediated by the inhibition of T cell activation in the skin lesions.

Interleukin-2 Inducible T-Cell Kinase: A Potential Prognostic Biomarker and Tumor Microenvironment Remodeling Indicator for Hepatocellular Carcinoma

Background: Hepatocellular carcinoma (HCC) is the dominant type of primary liver cancer, which leads to the fourth-ranked common cause of cancer-related death worldwide. The highly heterogeneous tumor microenvironment (TME) contributes to poor prognosis, particularly tumor recurrence of HCC. There is still a great challenge to illuminate the complicated modulation of TME during HCC progression. Methods: In the current study, RNA expression data and clinical-pathological data of HCC patients were downloaded from The Cancer Genome Atlas (TCGA) database. The immune and stromal scores of the samples were calculated using ESTIMATE algorithm and DEGs were obtained by comparison of low- and high-score groups. Intersection of hubgenes in protein-protein interaction (PPI) network and factors correlated with survival from univariate COX regression analysis were applied to screen out the key DEGs. The proportion of tumor-infiltrating immune cells (TICs) and stromal components were evaluated using CIBERSORT algorithm. Further Gene Set Enrichment Analysis (GSEA) and correlation analysis were carried out to elucidate the relation between ITK and TIC composition. Findings: The Immune score and Estimate score were proved to be significantly correlated with long-term recurrence-free survival (RFS) of HCC patients. 197 DEGs were carried out based on the Immune and Stromal scores. Two factors including interleukin-2 inducible T-cell kinase (ITK) and HLA Class II histocompatibility antigen DRB5 (HLA-DRB5) were identified as key DEGs. Further analysis proved that ITK was the most powerful DEG to evaluate the clinical-pathological characteristics and outcomes of HCC patients.The results of GSEA revealed that genes in the high ITK subgroup were enriched in inflammatory-immunological terms. CIBERSORT analysis for the proportion of TICs indicated that activated CD4+ memory T cells, CD8+ T cells, and M1 Macrophages were positively correlated with ITK expression, while plasma cells, resting natural killer (NK) cells, activated dendritic cells, activated NK cells, naive CD4+ T cells and resting mast cells were negatively correlated with ITK expression. Interpretation: Our work identified ITK as a novel indicator for tumor recurrence and microenvironment remodeling in HCC, and provide a potential therapeutic target for HCC treatment. Funding Statement: National ST National Natural Science Funds for Distinguished Young Scholar of China (No. 81625003); Key Program, National Natural Science Foundation of China (No. 81930016); Key Research & Development Plan of Zhejiang Province (No. 2019C03050); Youth Program of National Natural Science Foundation of China (No.81702858); Medical and health science and technology program of Zhejiang Province (No.2018256286) Declaration of Interests: None.

Multi-omics analysis of mantle cell lymphoma reveals an immune-cold tumor microenvironment associated with ibrutinib resistance.

8055 Background: Mantle cell lymphoma (MCL) is an aggressive and incurable B-cell lymphoma. Although the role of the MCL tumor microenvironment (TME) in survival and therapeutic response has been studied, greater knowledge regarding the tumor-immune interaction is needed to develop MCL immunotherapeutic strategies. Methods: Whole exome sequencing (WES; n = 41) and RNA-seq (n = 93) were performed on fresh peripheral blood, apheresis, or biopsy MCL patient primary samples. Joint WES and RNA-seq mutation calling, expression analysis were performed by BostonGene. Results: Both tumor and TME molecular signatures were characterized based on ibrutinib response. Concurrent analysis of MCL biopsy samples with an additional previously published cohort (n = 122; Scott et al., JCO, 2017) identified 4 MCL microenvironment signatures (Nomie et al., Blood, 2019) in which the ibrutinib-resistant MCL samples primarily belonged to the “stroma-enriched” subtype (29%; 6/8 resistant, non-immune with increased stromal signature and tumor-promoting cytokines), whereas most of the ibrutinib-sensitive samples were assigned to the “immune-hot” subtype (53%, 9/9 sensitive; anti-tumor infiltration, high immune and checkpoint molecule expression with low stromal expression, Chi-square test p-value = 0.001). NOTCH1 gain-of-function mutations (25%, 3/12 resistant) in the PEST domain were found exclusively in the ibrutinib-resistant cohort associated with the microenvironment-depleted subtype. Frequent recurring inactivating mutations in the epigenetic modifier KMT2D (30%) were identified in MCL cells associated with the ‘immune-suppressed” subtype (p &lt; 0.05). Loss-of-function mutations in epigenetic modifiers have been tied to immune evasion. PD-L1 was significantly downregulated in the ibrutinib-resistant MCL tumors (p = 0.03), indicating that targeting the PD-L1 and PD-1 immune checkpoint axis may not be beneficial. Conclusions: Ibrutinib sensitivity and resistance were defined by immune-hot and immune-cold TME portraits, respectively, suggesting that the TME has a prominent role in mediating ibrutinib response. Ibrutinib has been suggested to activate the immune TME through its off target inhibition of interleukin 2–inducible T-cell kinase (ITK). The immune activation by ibrutinib suggests that anti-tumor activity of ibrutinib may be better harnessed by combining ibrutinib with immunotherapy.

Targeting of ITK-SYK fusion oncogene through in-silico generated and biologically validated ITK-PH domain modulators

Abstract A subset of human Peripheral T cell Lymphomas, Not Otherwise Specified (PTCL-NOS), harbors a chromosomal translocation of interleukin-2 (IL-2) inducible T cell kinase (ITK) and spleen tyrosine kinase (SYK), giving rise to the composite ITK-SYK protein. ITK, a TEC family member kinase, plays an essential role in TCR and CD28 signaling. SYK signaling pathway is more broadly linked to activation of diverse lymphoid and myeloid cell types. ITK-SYK fusion protein consists of the PH domain of ITK and the SYK kinase domain allowing localization to the signalosome complex at the membrane via the PH domain and constitutive activity in T cells. Given the ubiquitous activity of SYK, it is predicted that targeting the ITK-PH domain of the fusion protein with small molecules can be a precise and less toxic approach to suppress ITK-SYK+PTCL-NOS in vivo. To this end, we performed a computational screen of small molecule libraries to identify candidate ITK-PH domain interacting compounds. A thermal shift assay was used to verify the specificity of binding of candidates to the target protein domain. Cellular assays tested small molecule activity in human and mouse T cells constitutively expressing the ITK-SYK fusion protein. We showed that the first-pass small molecule candidates specific to ITK-PH domain inhibit cytokine production driven by ITK-SYK fusion protein in human and mouse T cells. Our results demonstrate the efficiency of the virtual screening to identify drug candidates with biological activity in lymphocytes. Development of drugs that target ITK-PH domain is a rationale strategy towards first-in-class therapeutics for this subset of PTCL-NOS with very poor prognoses.

Combining ibrutinib and checkpoint blockade improves CD8+ T-cell function and control of chronic lymphocytic leukemia in Em-TCL1 mice.

Ibrutinib is a Bruton’s tyrosine kinase (BTK) inhibitor approved for the treatment of multiple B-cell malignancies, including chronic lymphocytic leukemia (CLL). In addition to blocking B-cell receptor signaling and chemokine receptor-mediated pathways in CLL cells, that are known drivers of disease, ibrutinib also affects the microenvironment in CLL via targeting BTK in myeloid cells and IL-2–inducible T-cell kinase (ITK) in T cells. These non-BTK effects were suggested to contribute to the success of ibrutinib in CLL. By using the Eμ-TCL1 adoptive transfer mouse model of CLL, we observed that ibrutinib effectively controls leukemia development, but also results in significantly lower numbers of CD8+ effector T cells, with lower expression of activation markers, as well as impaired proliferation and effector function. Using CD8+ T cells from a T-cell receptor (TCR) reporter mouse, we verified that this is due to a direct effect of ibrutinib on TCR activity, and demonstrate that co-stimulation via CD28 overcomes these effects. Most interestingly, combination of ibrutinib with blocking antibodies targeting PD-1/PD-L1 axis in vivo improved CD8+ T-cell effector function and control of CLL. In summary, these data emphasize the strong immunomodulatory effects of ibrutinib and the therapeutic potential of its combination with immune checkpoint blockade in CLL.