Tec Family Of Kinases Research Articles

The Pharmacokinetics, Metabolism, and Clearance Mechanisms of Ritlecitinib, a Janus Kinase 3 and Tyrosine-Protein Kinase Family Inhibitor, in Humans.

Ritlecitinib is an oral once-daily irreversible inhibitor of Janus kinase 3 and tyrosine-protein kinase family being developed for the treatment of moderate-to-severe alopecia areata. This study examined the disposition of ritlecitinib in male participants following oral and intravenous administration using accelerator mass spectroscopy methodology to estimate pharmacokinetic parameters and characterize metabolite profiles. The results indicated ritlecitinib had a systemic clearance of 43.7 L/h, a steady state volume of distribution of 73.8 L, extent of absorption of 89%, time to maximum plasma concentration of ∼0.5 hours, and absolute oral bioavailability of 64%. An observed long terminal half-life of total radioactivity was primarily attributed to ritlecitinib binding to plasma albumin. Ritlecitinib was the main circulating drug species in plasma (∼30%), with one major pharmacologically inactive cysteine conjugated metabolite (M2) at >10%. Oxidative metabolism (fractional clearance 0.47) and glutathione-related conjugation (fractional clearance 0.24) were the primary routes of elimination for ritlecitinib with the greatest disposition of radioactivity shown in the urine (∼71%). In vitro phenotyping indicated ritlecitinib cytochrome P450 (CYP) fraction of metabolism assignments of 0.29 for CYP3A, 0.09 for CYP2C8, 0.07 for CYP1A2, and 0.02 for CYP2C9. In vitro phenotyping in recombinant human glutathione S-transferases indicated ritlecitinib was turned over by a number of cytosolic and microsomal enzyme isoforms. SIGNIFICANCE STATEMENT: This study provides a detailed understanding of the disposition and metabolism of ritlecitinib, a JAK3 and TEC family kinase inhibitor for alopecia areata in humans, as well as characterization of clearance pathways and pharmacokinetics of ritlecitinib and its metabolites. As an AMS-based ADME study design, we have expanded on reporting the standard ADME endpoints, providing key pharmacokinetic parameters, such as clearance, volume of distribution, and bioavailability, allowing for a more comprehensive understanding of drug disposition.

Interaction of T-cell-specific adapter protein with Src- and Tec-family kinases.

Adapter proteins are flexible and dynamic modulators of cellular signalling that are important for immune cell function. One of these, the T-cell-specific adapter protein (TSAd), interacts with the non-receptor tyrosine kinases Src and Lck of the Src family kinases (SFKs) and Itk of the Tec family kinases (TFKs). Three tyrosine residues in the TSAd C-terminus are phosphorylated by Lck and serve as docking sites for the Src homology 2 (SH2) domains of Src and Lck. The TSAd proline-rich region (PRR) binds to the Src homology 3 (SH3) domains found in Lck, Src and Itk. Despite known interactors, the role TSAd plays in cellular signalling remains largely unknown. TSAd's ability to bind both SFKs and TFKs may point to its function as a general scaffold for both kinase families. Using GST-pulldown as well as peptide array experiments, we found that both the SH2 and SH3 domains of the SFKs Fyn and Hck, as well as the TFKs Tec and Txk, interact with TSAd. This contrasts with Itk, which interacts with TSAd only through its SH3 domain. Although our analysis showed that TSAd is both co-expressed and may interact with Fyn, we were unable to co-precipitate Fyn with TSAd from Jurkat cells, as detected by Western blotting and affinity purification mass spectrometry. This may suggest that TSAd-Fyn interaction in intact cells may be limited by other factors, such as the subcellular localization of the two molecules or the co-expression of competing binding partners.

First Report of Phase 1 Studies of DZD8586, a BBB Penetrant LYN/BTK Dual Inhibitor, in Patients with B-Cell Non-Hodgkin Lymphoma (B-NHL)

Introduction Resistance to covalent (ibrutinib, acalabrutinib and zanubrutinib) and non-covalent BTK inhibitors (pirtobrutinib) is an emerging clinical challenge. DZD8586 was designed as a full blood-brain barrier (BBB) penetrable LYN/BTK dual inhibitor. Preclinical study suggested DZD8586 has a good selectivity against other TEC family kinases (TEC, ITK, TXK, BMX). DZD8586 inhibits BTK-dependent and BTK-independent BCR signaling pathways by targeting LYN and BTK, which could potentially overcome resistance to both covalent and non-covalent BTK inhibitors for the treatment of CLL and MCL, as well as DLBCL. Two phase 1 clinical studies are ongoing in patients with relapsed or refractory (r/r) B-NHL to assess safety, pharmacokinetics (PK), pharmacodynamics (PD) and anti-tumor efficacy of DZD8586. Methods TAI-SHAN1 (NCT05844956; CTR20220558) and TAI-SHAN5 (NCT05824585) are phase 1 dose escalation and expansion studies in patients with r/r B-NHL. Both studies have similar dose escalation strategies. Patients who progressed following or intolerant of prior systemic therapy were enrolled. Pooled safety and efficacy analysis was performed. In addition, modulation of PD biomarkers was also evaluated. Tumor responses were investigator assessed according to Lugano 2014, IPCG 2005, or iwCLL 2018, as appropriate. Results As of June 15, 2023, 17 patients with r/r B-NHL have been enrolled and received DZD8586 at doses ranging from 10 to 100 mg once daily (QD) (10 mg， n=1；25 mg， n=4；50 mg， n=7；100 mg， n=5). The median age was 61 years, 71% were male, and 53% had an ECOG PS of 1. Histological subtypes included CLL, SCNSL, DLBCL, MCL, FL and MZL. The median number of prior therapies was 3 (range 2 - 8). All patients received prior chemotherapy and anti-CD20 therapy. Six patients, including 1 CLL, 2 MCL, 1 MZL, 1 SCNSL and 1 DLBCL had received prior treatment with covalent BTK inhibitors. DZD8586 was well tolerated across the doses investigated. Drug-related treatment emergent adverse events (TEAEs) reported in > 2 patients included thrombocytopenia (10/17), neutropenia (5/17), and upper respiratory tract infection (3/17). The most common ≥ grade 3 TEAE was thrombocytopenia (3/17, all at 100 mg). Other ≥ grade 3 TEAEs, including neutropenia, drug eruption and upper respiratory tract infection, were each reported by one patient. Bleeding, atrial fibrillation, and arthralgia were not reported. Grade 1 diarrhoea was reported in one patient. The maximum tolerated dose was defined as 100 mg QD. All these TEAEs were reversible after dose interruption and/or with supportive care. The longest duration on treatment was 10+ months. Among 13 patients treated at doses ≥ 25 mg QD with at least one tumor assessment, the objective response rate (ORR) was 9/13 (69.2%). Responses were observed across different histological subtypes (1/2 in SCNSL; 4/5 in DLBCL; 1/2 in FL; 1/1 in MCL, 1/2 in MZL and 1/1 CLL carrying C481S mutation). Among the 5 evaluable BTKi treated patients, tumor responses were observed in 3 patients. The longest duration of response (DoR) was 6.4+ months, and the patient was still ongoing with DZD8586 treatment and responding. PK was generally dose-proportional across the dose ranges of 10 to 100 mg. DZD8586 was absorbed with median t max 1.5 hours, and eliminated with mean terminal half-life of ~18 hours. Accumulation of about two folds in AUC was noted at steady state following once daily dosing. Dose dependent pBTK modulation in whole blood cells was detected, and at the doses ≥ 25 mg, sustained pBTK inhibition was observed. The unbound CSF-to-plasma concentration ratio (K puu,CSF) was 1.2 from one CNSL patient with available CSF samples, suggesting excellent CNS penetration of DZD8586 in humans. Conclusion In the preliminary pooled analysis of two ongoing phase 1 trials, DZD8586 has encouraging anti-tumor activity and safety in heavily pre-treated B-NHL patients. The combination of BBB penetration, dual inhibition of LYN/BTK and potential to overcome C481S mutant CLL and other covalent BTKi resistant lymphomas suggest this may be a useful agent. Updated data will be presented at the meeting.

Ritlecitinib (Litfulo) for severe alopecia areata.

The FDA has approved ritlecitinib (Litfulo – Pfizer), an oral JAK and TEC kinase family inhibitor, for treatment of severe alopecia areata in patients ≥12 years old. Ritlecitinib is the second oral drug to be approved in the US for treatment of severe alopecia areata; baricitinib (Olumiant), a JAK inhibitor, is approved only for use in adults.

Characterization and Preclinical Evaluation of AS-1763, an Oral, Potent and Selective Noncovalent BTK Inhibitor, in Chronic Lymphocytic Leukemia

Background: Covalent Bruton's tyrosine kinase (BTK) inhibitors (cBTKi) have transformed treatment landscape of chronic lymphocytic leukemia (CLL). These inhibitors bind to C481 residue in the kinase domain of BTK. This is also the site for the most common mutations rendering cells resistant to cBTKi. To circumvent this limitation of the cBTKi, non-covalent BTKi (ncBTKi) such as pirtobrutinib have been developed. Recently, non-C481 BTK mutations have been reported in patients with CLL at the time of disease progression during pirtobrutinib treatment. These observations underscore the need for ncBTKi that can target C481 as well as non-C481 mutations of BTK. AS-1763 is a potent, highly selective, orally available, and ncBTKi, equipotent against both wild-type and C481S-mutated BTK when tested in biochemical assays. In vivo, AS-1763 demonstrated significant antitumor effects in OCI-LY10 tumor xenograft models harboring wild-type or C481S mutant BTK (Kawahata et al. J Med Chem 64:14129, 2021). Study Design and Methods: In the present project, first we used cell free assay systems to evaluate selectivity and potency of AS-1763 by a kinome-wide profiling and inhibitory effect of AS-1763 in enzyme assays using recombinant mutant BTK. Second, we examined dose- and time-dependent inhibition of mutant BTK that is expressed in HEK293 cell line. Third, we tested biological, biochemical, and molecular impact of AS-1763 in primary CLL cells. Fourth, we determined sensitivity of CLL cells to AS-1763 when combined with other targeted agents. Results: AS-1763 showed a highly selective profile for BTK in a panel of 291 kinase assays with >260-fold selectivity except 3 Tec family kinases (BMX, ITK, and TEC). We have generated a total of 17 recombinant BTK mutant proteins (C481, T474, L528 variants and other BTK mutants; Table 1) reported in the literature or predicted by single nucleotide change in the codon, and established assay methods to measure inhibitory potency of BTKi for those BTK mutants (Table 1). AS-1763 showed potent inhibitory activities for those BTK mutants while inhibitory potencies of other cBTKi and ncBTKi were diminished against some BTK mutants such as T474 and/or L528 mutations. AS-1763 exhibited dose-dependent and slow-off rate inhibitions of BTK autophosphorylation (pY223) in HEK293 cells transfected with various BTK mutants. Furthermore, the observed inhibitory effects of AS-1763 on the BTK autophosphorylation (pY223) in HEK293 cells were continued up to 24 h after washing out of AS-1763. In primary CLL samples, AS-1763, pirtobrutinib or ibrutinib induced a modest apoptosis. AS-1763 effectively inhibited the BCR signaling pathway in a dose dependent manner as evidenced by downregulation of pY223-BTK expression which was also observed in cBTKi and ncBTKi relapsed/refractory CLL samples. In vitro incubations with AS-1763 inhibited CLL cell spontaneous migration, decreased CCL3/CCL4 levels in culture supernatant and was accompanied with the inhibition of intracellular calcium release and B-cell activation, as measured by surface CD86 expression. Besides, AS-1763 incubation for 24 hours was shown to modulate the expression of BCL-2 family proteins with the downregulation of MCL-1 and BCL-xl. Interestingly, in vitro treatment of CLL patient samples with AS-1763 demonstrated a notable elevation in cellular ROS and mitochondrial superoxide levels starting at 1 µM, concurrently impacting SOD1 expression in CLL patient samples. Evaluation of drug interaction models utilizing Compusyn and Synergy Finder applications predominantly indicated additive effects between AS-1763 with BCL-2 inhibitor, venetoclax as well as p53 activator APR-246. Consistent with this data, AS-1763 and venetoclax combinations showed high-rates of apoptosis in samples that were relapsed/refractory to cBTKi and ncBTKi. Conclusions: AS-1763 is a selective ncBTKi that inhibits both wild-type and mutant BTKs listed in Table 1. In CLL cells, AS-1763 was effective in inhibiting BCR pathway signaling and sensitized cells to other agents such as venetoclax. Based on these encouraging data we have initiated a clinical trial to test AS-1763 in patients with CLL and other B cell malignancies who have failed or are intolerant to at least two prior lines of systemic therapy, including cBTKi (NCT05602363 Clinical Trials.gov).

Modeling Gilteritinib Resistance By Enforced cxcl8a Expression in Zebrafish HSPCs

In Acute Myeloid Leukemia (AML), the FMS-like tyrosine kinase III Internal Tandem Duplication (FLT3-ITD) is common and increases the risk of relapse which is only partially mitigated by tyrosine kinase inhibitor (TKI) therapy. Gilteritinib is the current mainstay for treating relapsed FLT3-ITD AML, but therapy is non-curative, and the duration of remission is transient. Mutations in the RAS pathway are known to promote gilteritinib resistance, however non-mutational mechanisms of resistance remain under-explored and could provide new avenues for combination therapy. Our previous work has associated AML cell-autonomous expression of chemokines such as CXCL8 (IL-8) as a mechanism of resistance to gilteritinib therapy. Here we enforce expression of cxcl8a ( cxcl8, ENSDARG00000104795) by hematopoietic stem and progenitor cells (HSPCs) in transgenic zebrafish embryos using a defined Runx1 enhancer element. In the absence of gilteritinib, Runx1:cxcl8(+) animals had greater numbers of HPSCs compared to Runx1:cxcl8(-) animals (4.0 vs 2.1, p = 0.04). When treated with gilteritinib at 100 nM from 48-72 hours post-fertilization (hpf), HSPC numbers were decreased in both groups; however, Runx1:cxcl8(+) animals still had more HSPCs compared to Runx1:cxcl8(-) animals (1.6 vs 0.4, p = 0.02). This suggests that cxcl8 may act as a resistance factor for gilteritinib in vivo and that understanding regulators of cxcl8 expression may provide insight into gilteritinib resistance. Reanalysis of published single cell RNA-seq data in AML patients revealed an association between expression of CXCL8 and BTK and BMX but not other Tec-family kinases such as ITK and TEC itself. Zebrafish do not have an annotated BMX gene; BTK is expressed in HSPCs and neutrophils but not the erythroid lineage. CHMFL is a specific and potent inhibitor of human BMX with an unknown spectrum of activity in zebrafish. In Runx1:cxcl8(-) Runx1:GFP zebrafish, CHMFL treatment reduced HSPC numbers, but this was not rescued in Runx1:cxcl8(+) animals (untreated vs treated, cxcl8(-): 2.9 vs 1.5, p = 0.02, untreated vs treated, cxcl8(+): 4.0 vs 1.4, p = 0.0006). This contrasts with our prior work with the non-specific Tec family kinase inhibitor, ibrutinib, where HSPC numbers were rescued in Runx1:cxcl8(+) animals. Ongoing studies are focused on understanding the targets of ibrutinib and CHMFL in the zebrafish and how these pathways may affect gilteritinib resistance.

Discovery of 2,5-diaminopyrimidine derivatives as the first series of selective monomeric degraders of B-lymphoid tyrosine kinase

B-lymphoid tyrosine kinase (BLK) is an important knot of B cell receptor signaling, and regulates the function and development of B cells subset. Dysfunction of BLK is correlated with autoimmune diseases and cancer. There is an urgent need to develop selective BLK modulators to facilitate the studies of BLK in biological processes. Herein, we report the discovery of a series of 2,5-diaminopyrimidine-based compounds capable of selectively degrading BLK. The optimized compounds 9–11 possess weak biochemical inhibitory activities against BLK, yet they effectively degrade BLK and show high selectivity for BLK over other structurally and functionally related SRC family and TEC family kinases. Furthermore, compounds 9 and 11 demonstrate potent inhibitory activities in several B-lymphoid cell lines. As the first series of effective and selective monomeric BLK degraders, compounds 9–11 serve as valuable tools for further investigation of the functions of BLK.

Stimulation of the catalytic activity of the tyrosine kinase Btk by the adaptor protein Grb2.

The Tec-family kinase Btk contains a lipid-binding Pleckstrin homology and Tec homology (PH-TH) module connected by a proline-rich linker to a 'Src module', an SH3-SH2-kinase unit also found in Src-family kinases and Abl. We showed previously that Btk is activated by PH-TH dimerization, which is triggered on membranes by the phosphatidyl inositol phosphate PIP3, or in solution by inositol hexakisphosphate (IP6) (Wang et al., 2015, https://doi.org/10.7554/eLife.06074). We now report that the ubiquitous adaptor protein growth-factor-receptor-bound protein 2 (Grb2) binds to and substantially increases the activity of PIP3-bound Btk on membranes. Using reconstitution on supported-lipid bilayers, we find that Grb2 can be recruited to membrane-bound Btk through interaction with the proline-rich linker in Btk. This interaction requires intact Grb2, containing both SH3 domains and the SH2 domain, but does not require that the SH2 domain be able to bind phosphorylated tyrosine residues - thus Grb2 bound to Btk is free to interact with scaffold proteins via the SH2 domain. We show that the Grb2-Btk interaction recruits Btk to scaffold-mediated signaling clusters in reconstituted membranes. Our findings indicate that PIP3-mediated dimerization of Btk does not fully activate Btk, and that Btk adopts an autoinhibited state at the membrane that is released by Grb2.

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.

Efficacy of PF-06651600 in alleviating the pro-inflammatory capacity of CD4+ T cells in rheumatoid arthritis patients.

PF-06651600 is a highly specific inhibitor of Janus-activated kinase 3 and the Tec family of kinases. Regarding its dual function in the inhibition of both γc cytokine receptors and T cell receptor signaling, the present study aimed at evaluating the impact of PF-06651600 on the status of T-helper cells (Th) as the central game players in the pathogenesis of rheumatoid arthritis (RA). TCD4+ cells were isolated from 34 RA patients and 15 healthy control individuals and were evaluated after treatment with PF-06651600. RA patients had higher percentages of TCD4+ cells, CD4+ PD-1+ cells, and CD4+ PD-1+ TIGIT+ cells compared to a healthy control group and the TCD4+ cells of these patients showed higher interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-17 secretion along with higher messenger RNA (mRNA) expressions of T-bet. The percentage of CD4+ PD-1+ TIGIT+ cells showed a reverse correlation with the Disease Activity Score of 28 joints of the RA patients. PF-06651600 caused a significant decrease in the mRNA expressions of T-bet and RAR-related orphan receptor γt and the secretion of interferon (IFN)-γ and TNF-α in TCD4+ cells of RA patients. On the other hand, the population of CD4+ PD-1+ TIGIT+ cells was expanded under the influence of PF-06651600. This treatment also reduced the proliferation of TCD4+ cells. PF-06651600 demonstrated a potential to modulate the activity of TCD4+ cells in RA patients and to reduce the commitment of Th cells to the pathogenic Th1 and Th17 subsets. Further, it caused TCD4+ cells to gain an exhausted phenotype which is associated with better prognosis in RA patients.

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

Selective Inhibition of Bruton's Tyrosine Kinase by a Designed Covalent Ligand Leads to Potent Therapeutic Efficacy in Blood Cancers Relative to Clinically Used Inhibitors.

Bruton's tyrosine kinase (BTK) is a member of the TEC-family kinases and crucial for the proliferation and differentiation of B-cells. We evaluated the therapeutic potential of a covalent inhibitor (JS25) with nanomolar potency against BTK and with a more desirable selectivity and inhibitory profile compared to the FDA-approved BTK inhibitors ibrutinib and acalabrutinib. Structural prediction of the BTK/JS25 complex revealed sequestration of Tyr551 that leads to BTK's inactivation. JS25 also inhibited the proliferation of myeloid and lymphoid B-cell cancer cell lines. Its therapeutic potential was further tested against ibrutinib in preclinical models of B-cell cancers. JS25 treatment induced a more pronounced cell death in a murine xenograft model of Burkitt's lymphoma, causing a 30-40% reduction of the subcutaneous tumor and an overall reduction in the percentage of metastasis and secondary tumor formation. In a patient model of diffuse large B-cell lymphoma, the drug response of JS25 was higher than that of ibrutinib, leading to a 64% "on-target" efficacy. Finally, in zebrafish patient-derived xenografts of chronic lymphocytic leukemia, JS25 was faster and more effective in decreasing tumor burden, producing superior therapeutic effects compared to ibrutinib. We expect JS25 to become therapeutically relevant as a BTK inhibitor and to find applications in the treatment of hematological cancers and other pathologies with unmet clinical treatment.

Discovery of JNJ-64264681: A Potent and Selective Covalent Inhibitor of Bruton's Tyrosine Kinase.

Bruton's tyrosine kinase (BTK) is a Tec family kinase that plays an essential role in B-cell receptor (BCR) signaling as well as Fcγ receptor signaling in leukocytes. Pharmacological inhibition of BTK has been shown to be effective in treating hematological malignancies and is hypothesized to provide an effective strategy for the treatment of autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. We report the discovery and preclinical properties of JNJ-64264681 (13), a covalent, irreversible BTK inhibitor with potent whole blood activity and exceptional kinome selectivity. JNJ-64264681 demonstrated excellent oral efficacy in both cancer and autoimmune models with sustained in vivo target coverage amenable to once daily dosing and has advanced into human clinical studies to investigate safety and pharmacokinetics.

The HIV-1 protein Nef activates the Tec family kinase Btk by stabilizing an intermolecular SH3-SH2 domain interaction.

The Nef protein produced by the viruses HIV-1 and SIV drives efficient viral replication partially by inducing constitutive activation of host cell tyrosine kinases, including members of the Src and Tec families. Here, we uncovered the mechanism by which both HIV-1 and SIV Nef enhanced the activity of the Tec family kinase Btk in vitro and in cells. A Nef mutant that could not bind to the SH3 domain of Src family kinases activated Btk to the same extent as did wild-type Nef, demonstrating that Nef activated Src and Tec family kinases by distinct mechanisms. The Btk SH3-SH2 region formed a homodimer requiring the CD loop in the SH2 domain, which was stabilized by the binding of Nef homodimers. Alanine substitution of Pro327 in the CD loop of the Btk SH2 domain destabilized SH3-SH2 dimers, abolished the interaction with Nef, and prevented activation by Nef in vitro. In cells, Nef stabilized and activated wild-type but not P327A Btk homodimers at the plasma membrane. These data reveal that the interaction with Nef stabilizes Btk dimers through the SH3-SH2 interface to promote kinase activity and show that the HIV-1 Nef protein evolved distinct mechanisms to activate Src and Tec family tyrosine kinases to enhance viral replication.

Epigenetic regulation of inflammatory genes involving histone methylation is associated with hyperimmune response in COVID-19 patients

Abstract One of the leading causes of the severe COVID-19 disease symptoms is the production of massive amounts of pro-inflammatory cytokines. Although the precise mechanisms that trigger such hyperimmune response is unclear, epigenetic mechanisms are known to play important roles in the regulation of cytokine production and inflammation. In this study we examined global gene expression and histone methylation profile in the PBMCs of hospitalized COVID-19 patients. Despite individual variations, the expressions of many inflammation-related genes such as Arginase 1 (ARG1) and IL1 receptor 2 (IL1R2) were significantly upregulated in COVID-19 patients. We also found the expressions of coagulation-related genes, VWF and Protein S, were altered in COVID-19 patients. The expression patterns of some genes such as IL1R2 correlated with their histone methylation marks. Pathway analysis revealed that most of these dysregulated genes were in the TGFb, IL1b, IL6 and IL17 pathway. Interestingly, we found that the majority of those altered genes were in the dexamethasone targeting pathway. We also found that the expression of BMX, a member of TEC family kinases, was significantly increased in the PBMCs of COVID-19 patient at both RNA level and protein level. Interestingly, some TEC kinase inhibitors have been used to treat COVID-19 patients in clinical trials. Overall, this study has identified genes in PBMCs of COVID-19 patients with altered histone mark and gene expression profile, providing important information towards identifying potential biomarkers and therapeutic targets for COVID-19 disease. (Supported by NIH grants P01AT003961, P20GM103641, R01AT006888, R01ES030144, R01AI129788 and R01AI123947 to P.S.N and M.N.) Supported by NIH grants P01AT003961, P20GM103641, R01AT006888, R01ES030144, R01AI129788 and R01AI123947 to P.S.N and M.N.

Ibrutinib Inhibits BMX-Dependent Endothelial VCAM-1 Expression In Vitro and Pro-Atherosclerotic Endothelial Activation and Platelet Adhesion In Vivo.

Inflammatoryactivation of the vascular endothelium leads to overexpression of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), contributing to the pro-thromboticstate underpinning atherogenesis. While the role of TEC family kinases (TFKs) in mediating inflammatory cell and platelet activation is well defined, the role of TFKs in vascular endothelial activation remains unclear. We investigated the role of TFKs in endothelial cell activation in vitro and in a nonhuman primate model of diet-induced atherosclerosis in vivo. In vitro, we found that ibrutinib blocked activation of the TFK member, BMX, by vascular endothelial growth factors (VEGF)-A in human aortic endothelial cells (HAECs). Blockade of BMX activation with ibrutinib or pharmacologically distinct BMX inhibitors eliminated the ability of VEGF-A to stimulate VCAM-1 expression in HAECs. We validated that treatment with ibrutinib inhibited TFK-mediated platelet activation and aggregation in both human and primate samples as measured using flow cytometry and light transmission aggregometry. We utilized contrast-enhancedultrasound molecular imaging to measure platelet GPIbα and endothelialVCAM-1 expression in atherosclerosis-prone carotid arteries of obese nonhuman primates. We observed that the TFK inhibitor, ibrutinib, inhibited platelet deposition and endothelial cell activation in vivo. Herein we found that VEGF-A signals through BMX to induce VCAM-1 expression in endothelial cells, and that VCAM-1 expression is sensitive to ibrutinib in vitro and in atherosclerosis-prone carotid arteries in vivo. These findings suggest that TFKs may contribute to the pathogenesis of atherosclerosis and could represent a novel therapeutic target.

Characterization of Altered Gene Expression and Histone Methylation in Peripheral Blood Mononuclear Cells Regulating Inflammation in COVID-19 Patients.

The pandemic of COVID-19 has caused >5 million deaths in the world. One of the leading causes of the severe form of COVID-19 is the production of massive amounts of proinflammatory cytokines. Epigenetic mechanisms, such as histone/DNA methylation, miRNA, and long noncoding RNA, are known to play important roles in the regulation of inflammation. In this study, we investigated if hospitalized COVID-19 patients exhibit alterations in epigenetic pathways in their PBMCs. We also compared gene expression profiles between healthy controls and COVID-19 patients. Despite individual variations, the expressions of many inflammation-related genes, such as arginase 1 and IL-1 receptor 2, were significantly upregulated in COVID-19 patients. We also found the expressions of coagulation-related genes Von Willebrand factor and protein S were altered in COVID-19 patients. The expression patterns of some genes, such as IL-1 receptor 2, correlated with their histone methylation marks. Pathway analysis indicated that most of those dysregulated genes were in the TGF-β, IL-1b, IL-6, and IL-17 pathways. A targeting pathway revealed that the majority of those altered genes were targets of dexamethasone, which is an approved drug for COVID-19 treatment. We also found that the expression of bone marrow kinase on chromosome X, a member of TEC family kinases, was increased in the PBMCs of COVID-19 patients. Interestingly, some inhibitors of TEC family kinases have been used to treat COVID-19. Overall, this study provides important information toward identifying potential biomarkers and therapeutic targets for COVID-19 disease.

Bruton's tyrosine kinase inhibition-An emerging therapeutic strategy in immune-mediated dermatological conditions.

Bruton's tyrosine kinase (BTK), a member of the Tec kinase family, is critically involved in a range of immunological pathways. The clinical application of BTK inhibitors for B‐cell malignancies has proven successful, and there is strong rationale for the potential benefits of BTK inhibitors in some autoimmune and allergic conditions, including immune‐mediated dermatological diseases. However, the established risk‐to‐benefit profile of “first‐generation” BTK inhibitors cannot be extrapolated to these emerging, non‐oncological, indications. “Next‐generation” BTK inhibitors such as remibrutinib and fenebrutinib entered clinical development for chronic spontaneous urticaria (CSU); rilzabrutinib and tirabrutinib are being studied as potential treatments for pemphigus. Promising data from early‐phase clinical trials in CSU suggest potential for these agents to achieve strong pathway inhibition, which may translate into measurable clinical benefits, as well as other effects such as the disruption of autoantibody production. BTK inhibitors may help to overcome some of the shortcomings of monoclonal antibody treatments for immune‐mediated dermatological conditions such as CSU, pemphigus, and systemic lupus erythematosus. In addition, the use of BTK inhibitors may improve understanding of the pathophysiological roles of mast cells, basophils, and B cells in such conditions.

A narrative review on adverse effects of dasatinib with a focus on pharmacotherapy of dasatinib-induced pulmonary toxicities.

Chronic myeloid leukemia (CML), a myeloproliferative disorder caused by the over activity of BCR-ABL1 (breakpoint cluster region-Abelson), has been successfully treated by Tyrosine kinase inhibitors (TKIs). While imatinib is known as the first-line treatment of CML, in some cases other TKIs including dasatinib, nilotinib, bosutinib, and ponatinib may be preferred. Dasatinib, a second-generation TKI, inhibits multiple family kinases including BCR-ABL, SRC family kinases, receptor kinases, and TEC family kinases. It is effective against most imatinib-resistant cases except T315I mutation. Despite the superiority of dasatinib in its hematologic and cytogenetic responses in CML compared to imatinib, its potentially harmful pulmonary complications including pleural effusion (PE) and pulmonary arterial hypertension (PAH) may limit its use. Appropriate management of these serious adverse reactions is critical in both improving the quality of life and the outcome of the patient. In this narrative review, we will scrutinize the pulmonary complications of dasatinib and focus on the management of these toxicities.

Efficacy of Vecabrutinib Treatment in a Murine Model of Sclerodermatous Graft-Versus-Host-Disease

Introduction. Chronic graft-versus-host disease (cGVHD) can manifest as a complication in patients following allogeneic hematopoietic stem cell transplant resulting in morbidity and mortality. Effective treatment strategies for cGVHD are currently lacking. Ibrutinib, an irreversible BTK inhibitor with activity against several other Tec-family kinases, has been clinically developed for cGVHD treatment due to regulation of pathogenetic B cells and T-cell subsets. Vecabrutinib is a more selective, reversible inhibitor of BTK with a distinct kinase domain interaction, which could result in differentiated safety and activity profiles compared to ibrutinib. Vecabrutinib has the capacity to overcome the C481S mutation that mediates resistance to ibrutinib. Vecabrutinib also also demonstrates activity against ITK, which is expressed in T cells. In this study, we investigated the activity and immune modulation of vecabrutinib treatment in a murine model of sclerodermatous cGVHD. Ibrutinib was utilized for comparison.Methods. A murine model of sclerodermatous cGVHD was initiated by adoptive transfer of T-cell depleted bone marrow plus whole splenocytes from B10.D2 donors into BALB/c recipients that were previously subjected to sub-lethal irradiation. Total bone marrow and irradiation only groups were included as controls. Mice with established cGVHD characterized by weight loss and skin irritation symptoms were treated with vecabrutinib once daily at 50mg/kg by oral gavage or ibrutinib at 30mg/kg in drinking water 5 days per week for approximately 3 weeks beginning on day 27 post-adoptive transfer and ending on day 45 (n=10 mice per group). Body weight and clinical symptoms (appearance, activity, skin symptoms, diarrhea, conjunctivitis) were measured throughout the study. Immunophenotyping for B cells and T cells was performed on spleens collected from euthanized mice by flow cytometry at two timepoints (day 40 during treatment and on day 76 post-treatment). Levels of circulating immunoglobulins were measured by multiplex cytokine assay at both timepoints.Results. Clinical symptoms, including appearance, activity, skin irritation, redness, alopecia and diarrhea were significantly reduced in vecabrutinib- and ibrutinib-treated groups. Furthermore, there was a trend toward a more favorable clinical score overall for the vecabrutinib-treated group, however no statistical difference was observed compared to ibrutinib possibly due to small sample size. Weight loss was slightly elevated during vecabrutinib and ibrutinib treatment compared to vehicle (trend), however mice recovered body weight post-treatment and continued to maintain benefit. On day 40 (during treatment) and day 76 (post-treatment) groups of mice were euthanized for immunophenotyping analysis utilizing a 22-color flow cytometry panel. During treatment, both vecabrutinib and ibrutinib-treated mice retained total B cell numbers but exhibited reduced B-cell activation, proliferation, and number of B220+ CD138+ plasma cells. In addition, B cells secreted less IL-10, and IL-4/5. Expression of antigen-presentation molecules CD80 and CD86 on B cells were unchanged. Total CD3+ T cells, activated and proliferating CD4+ and CD8+ T cells, and cytotoxic granzyme-B+ CD8+ T cells were reduced in treated mice. Interestingly, CD4+ CD25+ FoxP3+ Treg number, expression of PD-1 on Tregs and CD4+ CXCR5+ PD-1+ T follicular helper cells were also reduced in both treatment groups. In addition, there were globally reduced numbers of cytokine-secreting CD4+ cells but no differences in Th1/Th2 or Th17/Treg ratios were observed. Post-treatment, proliferation and cytokine secretion of B cells and T cells were still lowered but less impaired than during treatment. Tregs and PD-1 expression were still reduced post-treatment. Finally, circulating levels of IgA were reduced during and post treatment in vecabrutinib-treated mice compared to vehicle, while IgG1, IgG2b were reduced in both treated groups. No changes in IgM levels were observed in either treatment group. In conclusion, vecabrutinib treatment demonstrated efficacy and beneficially regulated B cell and T cell immune subsets in a preclinical murine model of sclerodermatous cGVHD. Studies to further evaluate differences between vecabrutinib and ibrutinib treatment are ongoing. [Display omitted] DisclosuresFox: Sunesis Pharmaceuticals: Current Employment. Taverna: Sunesis Pharmaceuticals: Current Employment. Pinilla Ibarz: Sellas: Other: ), patents/royalties/other intellectual property; MEI, Sunesis: Research Funding; AbbVie, Janssen, AstraZeneca, Takeda: Speakers Bureau; AbbVie, Janssen, AstraZeneca, Novartis, TG Therapeutics, Takeda: Consultancy, Other: Advisory.