Bruton's Tyrosine Kinase Signaling Research Articles

Targeted Therapies in the Treatment of Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) is a rare, heterogeneous B-cell non-Hodgkin's lymphoma. The standard front-line treatment utilizes chemotherapy, often followed by consolidation with an autologous hematopoietic cell transplant; however, in most patients, the lymphoma will recur and require subsequent treatments. Additionally, mantle cell lymphoma primarily affects older patients and is frequently chemotherapy-resistant, which has further fostered the necessity for new, chemotherapy-free treatment options. In the past decade, targeted therapies in mantle cell lymphoma have been practice-changing as the treatment paradigm shifts further away from relying primarily on cytotoxic agents. Here, we will review the pathophysiology of mantle cell lymphoma and discuss the emergence of targeted, chemotherapy-free treatments aimed at disrupting the abnormal biology driving its lymphomagenesis. Treatments targeting the constitutive activation of NF-kB, Bruton's Tyrosine Kinase signaling, and anti-apoptosis will be the primary focus as we discuss their clinical data and toxicities. Our review will also focus primarily on the emergence and use of targeted therapies in the relapsed/refractory setting but will also discuss the emergence of their use in front-line therapy and in combination with other agents.

BTK drives neutrophil activation for sterilizing antifungal immunity.

We describe a previously-unappreciated role for Bruton's tyrosine kinase (BTK) in fungal immune surveillance against aspergillosis, an unforeseen complication of BTK inhibitors (BTKi) used for treating B-cell lymphoid malignancies. We studied BTK-dependent fungal responses in neutrophils from diverse populations, including healthy donors, BTKi-treated patients, and X-linked agammaglobulinemia patients. Upon fungal exposure, BTK was activated in human neutrophils in a TLR2-, Dectin-1-, and FcγR-dependent manner, triggering the oxidative burst. BTK inhibition selectively impeded neutrophil-mediated damage to Aspergillus hyphae, primary granule release, and the fungus-induced oxidative burst by abrogating NADPH oxidase subunit p40phox and GTPase RAC2 activation. Moreover, neutrophil-specific Btk deletion in mice enhanced aspergillosis susceptibility by impairing neutrophil function, not recruitment or lifespan. Conversely, GM-CSF partially mitigated these deficits by enhancing p47phox activation. Our findings underline the crucial role of BTK signaling in neutrophils for antifungal immunity and provide a rationale for GM-CSF use to offset these deficits in susceptible patients.

Novel 5-Substituted Oxindole Derivatives as Bruton's Tyrosine Kinase Inhibitors: Design, Synthesis, Docking, Molecular Dynamics Simulation, and Biological Evaluation.

Bruton's tyrosine kinase (BTK) is a non-RTK cytoplasmic kinase predominantly expressed by hemopoietic lineages, particularly B-cells. A new oxindole-based focused library was designed to identify potent compounds targeting the BTK protein as anticancer agents. This study used rational approaches like structure-based pharmacophore modeling, docking, and ADME properties to select compounds. Molecular dynamics simulations carried out at 20 ns supported the stability of compound 9g within the binding pocket. All the compounds were synthesized and subjected to biological screening on two BTK-expressing cancer cell lines, RAMOS and K562; six non-BTK cancer cell lines, A549, HCT116 (parental and p53-/-), U2OS, JURKAT, and CCRF-CEM; and two non-malignant fibroblast lines, BJ and MRC-5. This study resulted in the identification of four new compounds, 9b, 9f, 9g, and 9h, possessing free binding energies of -10.8, -11.1, -11.3, and -10.8 kcal/mol, respectively, and displaying selective cytotoxicity against BTK-high RAMOS cells. Further analysis demonstrated the antiproliferative activity of 9h in RAMOS cells through selective inhibition of pBTK (Tyr223) without affecting Lyn and Syk, upstream proteins in the BCR signaling pathway. In conclusion, we identified a promising oxindole derivative (9h) that shows specificity in modulating BTK signaling pathways.

A novel Bruton's tyrosine kinase inhibitor JDB175 shows potent efficacy to suppress central nervous system lymphoma.

Patients with central nervous system (CNS) lymphoma face limited treatment options and poor treatment outcomes, emphasizing the urgent need for effective therapeutic strategies. One limiting factor contributing to the suboptimal efficacy is the inadequate penetration of most treatment drugs across the blood-brain barrier (BBB). Recent insights into the pathophysiology of CNS lymphoma have identified the Bruton's tyrosine kinase (BTK) signaling pathway as a potential target. Some clinical trials have shown impressive responses to BTK inhibitors in CNS lymphoma. However, currently approved BTK inhibitors have low BBB penetration rates, limiting their efficacy. In this study, we discovered that JDB175, a novel and highly selective BTK inhibitor, exhibits excellent BBB penetration capabilities and demonstrates favorable activity in a mouse model of CNS lymphoma while showing no significant signs of toxicity. JDB175 effectively inhibits the BTK signaling pathway in human lymphoma cells, suppressing their proliferation, inducing cell cycle arrest, and promoting apoptosis. The significance of this study lies in addressing the critical unmet medical need for effective treatments for CNS lymphoma. This finding indicates a promising avenue for improved treatments in CNS lymphoma, potentially opening doors for further clinical investigation and therapeutic advancements.

Establishment and Characterization of Mouse Models with Primary and Acquired BTK Inhibitor Resistance

Background: The second-generation Bruton's tyrosine kinase (BTK) inhibitor acalabrutinib is effective for treatment of patients with chronic lymphocytic leukemia (CLL), including high-risk and relapsed patients. Resistance to BTK inhibitors can develop not only due to on-target mutations, but also by bypassing the BTK signaling pathway. Mouse models represent useful tools in studying mechanisms of resistance as well as testing the efficacy of alternative therapy options to overcome resistance. Eµ-TCL1-transgenic (TCL1-tg) mice have the advantage of immunocompetence and resemble the human CD49d-high CLL cohort (Szenes et al., 2020). Transplantation of tumors from these mice to wild-type recipients allows acceleration of pathophysiology, selection of specific tumor clones, and more efficient testing of drug combinations. Here, we developed acalabrutinib resistance models by serial transplantation and long-term administration of the drug in drinking water, characterized these models, and compared molecular resistance signatures with those of human patient samples. Methods: Models were developed starting from four primary TCL1-tg mice at overt disease with tumor burden in spleen ranging up to 95% of lymphocytes. Serial retransplantation to wild-type C57BL/6J recipients for two to four rounds and acalabrutinib therapy pressure resulted in several drug resistant pedigrees. Mice were sacrificed at end-stage disease and tumor burden in spleen, bone marrow and blood was analyzed by flow cytometry using anti-CD5/CD19 antibodies. Purified leukemic cells were subjected to RNA-seq, real-time quantitative PCR and mutational analyses. Results: Three response patterns were identified in recipient mice: a) sustained sensitivity (models were stopped after 23 weeks of treatment), b) acquired resistance (with initial response followed by development of resistance under continuous therapy pressure and c) retransplantation-induced resistance in a case of an aggressive tumor clone that was responding to acalabrutinib in the parental mouse. The sensitive models displayed a 63% reduction in spleen weight upon treatment and a concomitant 65% reduction in blood tumor burden. In acquired or primary resistant mice, spleen weight and tumor burden was not significantly different from that of untreated mice, with blood tumor burden in the magnitude of 100,000 leukemic cells/µl and spleen tumor burden of 500-1000 million leukemic cells. Resistance could not be attributed to the mutational landscape of BTK and PLCγ2. Mutations in p53, Myc, Notch1, Atm, and Sf3b1 were detected but found to be intrinsic in individual ancestors and further present in the follow-up transplantations. RNA-seq analysis revealed 863 differentially expressed genes in mice with acquired resistance to acalabrutinib (compared to untreated, Figure 1) and 114 shared differentially expressed genes in mice with acquired or transplantation-based resistance. Gene set enrichment analysis demonstrated the presence of MAPK signaling and NF-kB signaling gene signatures within the resistant tumors. In particular, acquired resistance was accompanied by changes in the B cell receptor downstream components. We also compared the murine 863 gene signature to human samples from acalabrutinib resistant patients (progression on drug versus baseline) and found 13 genes are differently expressed in both, human and mouse. Validation for this gene expression data is ongoing and updated results will be presented. Conclusion: We have developed novel TCL1-tg based murine models of acalabrutinib resistance. Our models provide well-characterized tools to study primary as well as acquired BTK inhibitor resistance and allow the current testing of potential new therapeutic approaches to overcome resistance. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Bruton's Tyrosine Kinase (BTK) Degrader Nx-2127 Exhibits Lethal Activity and Synergy with Venetoclax and BET Protein Inhibitor Against MCL Cells Sensitive or Resistant to Covalent BTK Inhibitors

Bruton's tyrosine kinase (BTK) is a Tec family member, non-receptor, tyrosine kinase, which is constitutively active in Mantle Cell Lymphoma (MCL) cells, downstream of B cell receptor (BCR), Toll like receptors (TLR) and Fc receptors. BTK plays a critical role in cell cycle progression and B cell activation. In murine models, loss of function and kinase-dead mutants of BTK show impaired B cell maturation. BTK activates multiple pro-growth and pro-survival mechanisms, involving PLCγ2 activity, RAS-RAF-MEK-ERK pathway and NFκB transcriptional activity. Covalent BTK inhibitors (BTKis), including ibrutinib and acalabrutinib, irreversibly bind to cysteine 481 in the kinase domain of BTK, inhibit growth, and induce loss of viability of MCL cells. Although treatment with covalent BTKi induces dramatic clinical remissions, intrinsic and acquired resistance to BTKis is observed in majority of patients. BTKi-resistance in MCL cells has been attributed to activation of the alternative NFκB signaling due to TRAF2, BIRC3 or MAP3K14 mutation, gain-of-function CARD11 mutation, CCND1 mutation and MYC activation. Emergence of C481S BTK mutation causes steric hindrance to the covalent BTKi to C481, leading to acquired resistance, followed by clinical relapse in CLL, and less commonly in MCL. However, this situation still underscores dependence on BTK signaling for growth and survival of MCL cells. It also supports the strategy of using a BTK degrader for degrading and depleting BTK, including C481S-BTK, in MCL cells. NX-2127 and NX-5948 are promising, cereblon-based, orally bioavailable, clinical grade BTK degraders, which degrade and deplete BTK levels. In the present studies, we determined that in vitro exposure of human MCL cells, including MCL cell lines: REC1, Mino and JeKo-1, as well as patient-derived (PD) MCL cells, to NX-2127 (10 to 250 nM) for 2 to 24 hours markedly depleted BTK levels via proteasomal degradation, since co-treatment with the proteasome inhibitor carfilzomib restored BTK levels. NX-2127 but not NX-5948 treatment also degraded and depleted IKZF1 and IKZF3 levels. However, both BTK degraders also depleted BTK levels in BTK1-resistant Mino-IR and Jeko1-IR cells (which display induced in vitro resistance after recovery from repeated exposures to high levels of ibrutinib, i.e., 30 μM for 48 hours). NX-2127 treatment reduced the mRNA levels of IL-10 and TNFα in the MCL cells. Additionally, NX-2127 mediated depletion of BTK was associated with downstream reduction in the protein levels of p-PLCγ2 (Y1217), p-AKT, pERK1/2 and SOX11 levels in Mino, Mino-IR as well as in PD MCL cells. Notably, NX-2127 treatment also markedly depleted the protein levels of C481S-BTK that confers clinical resistance to covalent BTKis in CLL cells. Exposure to NX-2127 dose-dependently inhibited growth and survival of REC1, Mino, JeKo-1, Mino-IR, JeKo-1-IR cells, as well as of PD MCL cells. We have previously reported that BET protein inhibitor and/or BCL2 inhibitor (venetoclax) induce in vitro lethality in cellular models of MCL (Blood, 126: 1565, 2015). Based on this, we determined the synergistic lethal activity of NX-2127 and venetoclax or the BD2-selective, BET protein inhibitor ABBV-744 in MCL cells. Co-treatment with NX-2127 (10 to 100 nM) and low nM levels of venetoclax or ABBV-744 was synergistically lethal against MCL cells (Delta synergy score above 1.0 by ZIP method). Collectively, these findings demonstrate that treatment with NX-2127 degrades and attenuates BTK and IKZF1/3 levels, as well as inhibits the downstream pro-growth and pro-survival signaling, resulting in loss of viability of MCL cells, including those resistant to covalent BTKi treatment. These findings also show promising anti-MCL activity of NX-2127-based combination with BCL2 or BET inhibitor which merits further in vivo validation.

Novel 6-amino-1,3,5-triazine derivatives as potent BTK inhibitors: structure-activity relationship (SAR) analysis and preliminary mechanism investigation

Bruton's tyrosine kinase (BTK) is a promising drug target for the treatment of B-cell related malignancies. Irreversible inhibition of BTK by a covalent inhibitor has been proved to be a clinically effective therapy. However, most irreversible BTK inhibitors also inhibit other kinases including JAK3 and EGFR, leading to some adverse events. Herein, we reported the structure-based design and optimization of a series of irreversible BTK inhibitors bearing the 6-amino-1,3,5-triazine scaffold. Most of the synthesized compounds demonstrated considerable BTK inhibition and improved anti-proliferative activity against Raji and Ramos cells. Among them, compound C11 exhibited potent BTK inhibition (BTK IC50 = 17.0 nM) and a desirable selectivity profile especially over EGFR. Moreover, C11 effectively blocked activation of BTK and downstream signaling, arrested the cell cycle in G0/G1 phase and induced apoptosis in Raji cells. Its irreversible binding mode was further investigated by both molecular modeling and a washout experiment. Collectively, C11 is a novel selective irreversible BTK inhibitor worthy of further in-depth research.

Combined Inhibition of mTOR and BTK Signaling Is Required for Optimal Long Term Growth Inhibition in DLBCL Models

Introduction: The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that functions as a key regulator of cell signalling, growth, metabolism and survival. Many B-cell lymphomas exhibit aberrant mTOR activation, coupled with dysregulated B-cell receptor (BCR) signalling. However, clinical trials with allosteric mTORC1 inhibitors (rapamycin and its analogues), have shown only modest efficacy in most contexts. Vistusertib (AZD2014) is an orally active dual mTORC1/2 inhibitor in clinical development for treatment of several malignancies. We investigated the potential for combining vistusertib with acalabrutinib (ACP-196), a highly selective Bruton tyrosine kinase (BTK) inhibitor, in cellular and in vivo preclinical models of diffuse large B cell lymphoma (DLBCL).Methods and Results: We demonstrate that the combination of mTOR and BTK inhibition causes additive growth inhibition in a panel of DLBCL cell lines, but synergistic growth inhibition in a subset of ABC-DLBCL cell lines (such as OCI-LY10 and TMD8 cell lines). In the same cell lines, the combination causes profound effects on BTK and mTOR downstream signalling markers (>50% inhibition of phosphorylation of AKT and S6). We therefore treated a number of mouse models of diffuse large B-cell lymphomas (e.g. OCI-LY10 xenograft model, Figure 1) with a combination of vistusertib (TORC1/2 inhibitor) and acalabrutinib (BTK inhibitor). Dose -response and pharmacodynamic studies in this mouse model confirmed optimal inhibition of both BTK and mTOR signalling pathways. Further in vivo studies using patient derived models of DLBCL have also demonstrated that this combination is highly efficacious.Conclusions: Based on these data, a phase 1/2 trial of vistusertib plus acalabrutinib in patients with relapsed/refractory DLBCL has opened for accrual (NCT03205046) [Display omitted] DisclosuresBrugger:AstraZeneca: Employment, Equity Ownership. Hattersley:AstraZeneca: Employment, Equity Ownership. Bussey:AstraZeneca: Employment, Equity Ownership. Reimer:AstraZeneca: Employment, Equity Ownership. Tron:AstraZeneca: Employment, Equity Ownership. Staniszewska:AstraZeneca: Employment. Delpuech:AstraZeneca: Employment, Equity Ownership. Carnevalli:AstraZeneca: Employment, Equity Ownership. Krejsa:Acerta Pharma: Employment, Equity Ownership; Astra Zeneca: Equity Ownership. Mayo:AstraZeneca: Employment, Equity Ownership. Neveras:AstraZeneca: Employment, Equity Ownership. San Martin:AstraZeneca: Employment, Equity Ownership. Rideout:AstraZeneca: Employment, Equity Ownership. Proia:AstraZeneca: Employment. Willis:AstraZeneca: Employment, Equity Ownership. Pease:AstraZeneca: Employment, Equity Ownership. Hamdy:Acerta Pharma: Employment, Equity Ownership, Patents & Royalties: Acalabrutinib related patents. Cosulich:AstraZeneca: Employment, Equity Ownership.

CG '806, a First-in-Class Non-Covalent Pan-FLT3/BTK Inhibitor, Exerts Superior Potency Against B-Cell Malignant Cells

CG'806 is a non-covalent pan-FMS like tyrosine kinase 3 (FLT3)/Bruton's tyrosine kinase (BTK) multi-kinase inhibitor that potently inhibits wild type (WT) and mutant forms of FLT3 and BTK. As described previously (2017 AACR Hematologic Malignancies abstract #25 and #44), CG'806 inhibits on FLT3-ITD-driven acute myeloid leukemia (AML) and other forms of AML. Because CG'806 targets the WT BTK (IC50 = 8 nM) and the C481S-BTK (IC50 = 2.5 nM), which is a mutant form resistant to ibrutinib and other covalent BTK inhibitors, this small molecule was profiled for its actions on various types of B-cell malignancies in which B cell receptor (BCR) and BTK signaling are reported to play pivotal pathogenic roles. Against 10 malignant B-cell lines, while CG'806 reduced the level of BTK-Y223 phosphorylation, it was less potent than ibrutinib. In contrast, CG'806 was 50-6,000 times more potent (lower IC50) in the ability to kill the malignant B cells across all tested cell lines evaluated by MTS-based assays of cell proliferation and inhibition of colony formation. Hence, the efficacy of killing malignant B cells by CG'806 and by ibrutinib did not correlate with the degree of BTK inhibition. Pathway analyses (KinomeScan and immunoblotting) demonstrated that CG'806, in addition to inhibiting intracellular BTK, potently inhibited the activity of aurora kinases A, B and C, phospho-H3S10, FLT3 and ERK that are differentially expressed and operative in various cell lines representing mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), B-acute lymphocytic leukemia (B-ALL) and Burkitt's lymphoma. Indeed, measuring BrdU incorporation and Annexin V by flow cytometry, CG'806 directly induced polyploidy and triggered apoptosis in malignant B cells at picomolar (pM) to low nanomolar (nM) concentrations, while ibrutinib potently inhibited BTK activity but was unable to induce apoptosis at lower than micromolar (µM) concentrations. These data distinguish CG'806 from ibrutinib and other covalent and non-covalent BTK inhibitors, and illustrate that CG'806 has activity across a wide range of malignant B cell lines including those intolerant, refractory or resistant (due to the presence of a BTK-C481S mutation) to ibrutinib and other BTK inhibitors. [Display omitted] DisclosuresZhang:Aptose Biosciences, Inc.: Employment. Local:Aptose Biosciences, Inc.: Employment. Benbatoul:Aptose Biosciences, Inc.: Employment. Folger:Aptose Biosciences, Inc.: Employment. Sheng:Aptose Biosciences, Inc.: Employment. Howell:Aptose Biosciences, Inc.: Membership on an entity's Board of Directors or advisory committees. Rice:Aptose Biosciences, Inc.: Employment, Equity Ownership, Patents & Royalties.

Preclinical Activity of Novel BET Protein Proteolysis Targeting Chimeras (BETP-PROTACs) and Their Combination with Venetoclax or CDK4/6 Inhibitor Against Human Mantle Cell Lymphoma (MCL) Cells

Among the genetic alterations described in Mantle Cell Lymphoma (MCL) are those involving cyclin D1, deletion of INK4a/ARF, as well as copy number gains of MYC, CDK4 and BCL2 genes. Activated B-cell receptor (BCR) signaling, and the ensuing downstream pro-growth and pro-survival NFkB activity, is also a notable feature of MCL. Collectively, the genetic alterations and the ensuing deregulated signaling and activity of transcription factors, including c-MYC and NFkB, creates the MCL-specific ‘transcriptome’ that promotes growth and survival of MCL cells. Ibrutinib, a covalent inhibitor of Bruton's tyrosine kinase (BTK), exhibits unprecedented single-agent activity in relapsed/refractory MCL, however approximately 40% of patients demonstrate primary resistance and secondary resistance is common, with a poor one-year survival rate of only 22%. Mutations in CARD11/IKBKB/TRAF2/BIRC3/NIK or the C481S mutation in BTK, sustain alternative or classical NFkB signaling despite ibrutinib treatment and confer resistance to ibrutinib in MCL. We hypothesized that the resistance to ibrutinib in MCL cells would be governed by a dysregulated transcriptome dependent on the activity of the chromatin reader BET proteins (BETPs), including BRD4. Therefore, we determined the lethal preclinical activity of BETP PROTACs (proteolysis targeting chimeras) ARV-825, and its pharmacologically superior version ARV-771, that proteasomally degrade BETPs, against the following cellular models of ibrutinib resistance (MCL-IR): in vitro generated cultured MCL Mino-persister/resistant (Mino-P/R) cells (~4-fold resistant compared to parental Mino cells), endogenously resistant cultured Z138 and Maver-1 cells, as well as patient-derived xenograft (PDX) PDX1 and PDX2 cells that had exhibited in vivo resistance to ibrutinib. Protein expression evaluations by RPPA and/or Western analyses demonstrated that MCL-IR cells expressed relatively high levels of p-AKT over AKT, p-S6, p-4EBP1, MYC, BCL2, BCl-xL, XIAP and MCL1. Treatment with BETP-PROTAC dose-dependently (20 to 200 nM) induced significantly more growth inhibition and apoptosis than the BETP inhibitor BETi OTX015 against the cellular models of MCL-IR (p < 0.001), which was associated with marked depletion of BRD4, BRD2, MYC, BCL2, BCL-xL, cyclin D1, CDK4 and pRB (S807/811) levels, as well as of NFkB target gene expressions, including cIAP2, XIAP, cFLIP, TNFAIP3, BCl-xL, IL10, TNFa and BTK. Concomitantly, BETP-PROTAC increased expression of HEXIM1 and p21 levels in the MCL-IR cells. Treatment with the BCL2 inhibitor venetoclax or the CDK4/6 inhibitor abemaciclib dose-dependently induced apoptosis of MCL-IR cell types (p< 0.01). Notably, co-treatment with BETP-PROTAC ARV-771 and venetoclax or abemaciclib synergistically induced apoptosis of Mino-P/R, Maver-1 and PDX1 and PDX2 cells (combination indices < 1.0). Additionally, compared to treatment with vehicle control, treatment of luciferase-transduced PDX2 cells infused by the tail vein and engrafted in immune-depleted NSG mice with ARV-771 (30 mg/kg SQ daily) and venetoclax (15 mg/kg PO daily) for 3 weeks, significantly reduced the MCL burden (at 2 weeks of treatment) and improved survival of the mice. These findings demonstrate that BETP-PROTACs deplete BETPs and important MCL-relevant oncogene levels, including MYC, cyclin D1, CDK4, BCL2, Bcl-xL, as well as NFkB target gene expressions, leading to synergistic lethality in vitro in combination with venetoclax or abemaciclib, as well as exert in vivo activity in combination with venetoclax against PDX model of MCL-IR cells. DisclosuresRaina:4Arvinas, LLC. New Haven, CT: Employment. Qian:4Arvinas, LLC. New Haven, CT: Employment. Crew:Arvinas, Inc: Employment.

Ibrutinib Inhibits cGVHD Pathogenic Pre-Germinal Center B-Cells and Follicular Helper Cells While Preserving Immune Memory and Th1 T-Cells

Background: Chronic graft-versus-host disease (cGVHD) is a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (allo-HCT). Treatment relies upon corticosteroids and immunosuppressive agents with limited efficacy and considerable side-effects. Evolving research points to the complex involvement of immune T, B, and myeloid cells in the pathogenesis of cGVHD. Ibrutinib is an irreversible inhibitor of Bruton's tyrosine kinase (BTK) and interleukin-2-inducible T-cell kinase (ITK) which inhibits the activation of B-cells, specific T-cell subsets, and certain myeloid cells that are implicated in cGVHD. Substantial clinical benefit was demonstrated in an open-label phase 1b/2 study of ibrutinib in patients with cGVHD that had failed frontline therapy (Miklos et al ASH 2016). Based upon this data, ibrutinib is indicated by the US FDA for the treatment of adult patients with cGVHD after failure of one or more lines of systemic therapy.Methods: Blood samples were collected before and multiple timepoints after starting ibrutinib 420mg daily therapy from 42 subjects with cGVHD that had failed frontline therapy (Miklos et al ASH 2016). Correlative analyses included Nanostring gene expression profiling, Luminex and ELISA based plasma testing, CyTOF phospho-immunophenotyping, and ex-vivo T-cell stimulation assays.Results: Initial data from 8 patients using targeted, Nanostring gene expression analysis showed ibrutinib reduced inflammatory genes including NFkB-1 and chemokines CXCL10, CCL7 and CCL3 (2.6, 2.3, 25, and 1.8-fold decrease over first 3 months, respectively). Luminex plasma quantification supported gene expression profiling results, and unveiled several additional chemotactic, inflammatory, and fibrotic factors which were down modulated by ibrutinib therapy including CCL22, CXCL9, CXCL10, IL-8, sCD25, CCL4, and ST2 (69-80% of patients showed decreasing trend). 42-parameter CyTOF single cell analysis revealed a 10-fold reduction in absolute numbers of cGVHD implicated pre-germinal center B-cells (CD19+, CD27+, CD38+, IgD+), and notably diminished Tfh, Th17, and total B cells (2.6, 1.5, and 1.4-fold decrease over first 90 days, respectively) following ibrutinib therapy. Relative numbers of CD4 and CD8 T-cells, class-switched B-cells, and CD14+ monocytes remained unchanged, and iNKT, Th1, and Treg cells increased incrementally (1.8, 1.2, and 1.1-fold at day 90, respectively). IgG plasma levels persisted while IgM significantly decreased corroborating an ibrutinib germinal center effect that did not deplete long-lived plasma cells. Furthermore, T cell reactivity against influenza increased, and antibodies against EBV and tetanus remained unchanged. Strikingly, single cell phosphorylation analysis showed BTK and ITK signaling was attenuated following ibrutinib treatment in defined B and T cell subsets (Figure 1). Specifically, PLCγ1/2 activation was simultaneously diminished in the pre-germinal center and plasmablast B-cell subsets.Conclusions: Our examination of the biologic and cellular mechanisms which support the clinical results of ibrutinib in the setting of cGVHD demonstrate that ibrutinib selectively inhibits cGVHD pathogenic pre-germinal center B-cells and Tfh cells, while preserving immune memory and Th1 T-cells. These data are especially salient given the clinical efficacy of ibrutinib in cGVHD, and the meticulous nature of the correlative studies designed to elucidate the multifactorial mechanism of action in this life-threatening disease. [Display omitted] DisclosuresSahaf:Pharmacyclics: Research Funding; Stanford: Patents & Royalties. Tebaykin:Stanford University: Patents & Royalties. Hopper:Pharmacyclics: Employment. Cheung:Pharmacyclics: Employment; AbbVie: Equity Ownership. Bittencourt:Pharmacyclics: Employment, Research Funding; AbbVie: Equity Ownership, Research Funding. Cutler:Astellas: Consultancy; Incyte: Consultancy; Bristol-Myers Squibb: Consultancy; Kite: Consultancy; Pharmacyclics: Consultancy; Pfizer: Consultancy. Arora:Takeda Oncology: Consultancy. Waller:Cambium Medical Technologies: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Cerus: Equity Ownership; Helocyte: Consultancy; National Institutes of Health: Research Funding; Katz Foundation: Research Funding; AMGEN: Consultancy; PRA: Consultancy; Coulter Foundation: Research Funding; Celldex: Consultancy; Chimerix: Equity Ownership; Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding. Jagasia:Janssen: Consultancy, Research Funding; Mallinckrodt: Consultancy; Therakos: Consultancy, Research Funding. Flowers:Pharmacyclics: Consultancy. Logan:Novartis: Consultancy, Research Funding; Incyte: Consultancy; Kite: Research Funding; Astellas: Research Funding; Jazz: Consultancy; Amgen: Consultancy; Pharmacyclics: Research Funding; Shire: Consultancy. Jaglowski:Pharmacyclics: Consultancy, Research Funding. Lih:AbbVie: Equity Ownership; Pharmacyclics: Employment. Solman:Pharmacyclics LLC, an AbbVie Company: Employment, Other: Travel, Accommodations, Expenses; AbbVie: Equity Ownership. Lal:Gilead Sciences: Employment; AbbVie: Equity Ownership; The Permanente Medical Group (TPMG): Employment; Pharmacyclics LLC, an AbbVie Company: Employment. Styles:Pharmacyclics LLC, an AbbVie Company: Employment; AbbVie: Equity Ownership. Hill:Amgen: Employment, Equity Ownership, Patents & Royalties; Pharmacyclics: Employment; AbbVie: Equity Ownership; Miravant Medical: Patents & Royalties. James:AbbVie: Equity Ownership; Pharmacyclics LLC, an AbbVie Company: Employment. Bendall:Fluidigm Sciences: Consultancy, Other: Travel, Accomodations, Expenses; Bluebird Bio: Other: Travel, Accomodations, Expenses, Research Funding; Ionpath, Inc.: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Boehringer-ingelheim: Consultancy, Other: Travel, Accomodations, Expenses; Pharmacyclics: Research Funding. Dubovsky:Pharmacyclics LLC, an AbbVie Company: Employment; AbbVie: Equity Ownership.

PRN1008, a Reversible Covalent BTK Inhibitor in Clinical Development for Immune Thrombocytopenic Purpura

PRN1008 is an oral, reversible covalent inhibitor of Bruton's tyrosine kinase (BTK) in clinical development for the treatment of multiple autoimmune diseases. BTK is an essential signaling element downstream of the B cell receptor (BCR), Fc-gamma receptor and Fc-epsilon receptor pathways. BTK activation is critical for B cell activation and maturation. BTK also regulates antibody mediated activation of other immune cells, such as macrophages, neutrophils and mast cells through Fc receptor signaling.Treatment with PRN1008 in vitro profoundly inhibited B cell activation and blocked antibody mediated activation of immune cells via Fc receptor signaling. In preclinical studies, PRN1008 demonstrated a significant dose-dependent reduction of platelet-loss in an anti-CD41 induced mouse model of immune thrombocytopenia. In addition, PRN1008 showed rapid and significant anti-inflammatory effects in an antibody driven rat arthus model and in spontaneous autoantibody-mediated canine pemphigus foliaceus. The preclinical data suggest that PRN1008 could reduce platelet destruction via inhibition of autoantibody/Fc-gamma receptor signaling in splenic macrophages, and also affect autoantibody generation through effects on B-cell activation, to diminish platelet loss in ITP.Platelets express high levels of BTK, however alternative signaling pathways exist which bypass BTK signaling to retain normal platelet functions. The effect of PRN1008 on platelet function was assessed in vitro in both normal healthy volunteer and ITP patient platelets using a standard panel of platelet agonists. In contrast to ibrutinib, treatment with PRN1008 at clinically relevant concentrations did not impact collagen-induced platelet aggregation in either normal or ITP patient platelet samples, or interfere with responses to all other platelet agonists tested. In the clinic, PRN1008 has been well tolerated. Thrombocytopenia, bleeding or bruising signals have not been reported. An analysis of platelet counts from the ongoing 12 week study of pemphigus vulgaris (NCT02704429) showed no evidence of an effect of PRN1008 on platelet counts in the normal range.Principia Biopharma is developing PRN1008 for the treatment of autoimmune diseases including ITP, blistering skin diseases such as pemphigus vulgaris and other immunologic disorders where autoantibodies and/or B-cell signaling are pathogenic. DisclosuresLangrish:Principia Biopharma: Employment. Bradshaw:Principia Biopharma: Employment. Owens:Principia Biopharma: Employment. Campbell:Principia Biopharma: Employment. Francesco:Principia Biopharma: Employment. Karr:Principia Biopharma: Employment. Murray:Principia Biopharma: Employment. Quesenberry:Principia Biopharma: Employment. Smith:Principia Biopharma: Consultancy. Taylor:Principia Biopharma: Consultancy. Zhu:Principia Biopharma: Employment. Nunn:Principia Biopharma: Employment. Gourlay:Principia Biopharma: Employment.

Ibrutinib before Apheresis May Improve Tisagenlecleucel Manufacturing in Relapsed/Refractory Adult Diffuse Large B-Cell Lymphoma: Initial Results from a Phase 1b Study

Ibrutinib before Apheresis May Improve Tisagenlecleucel Manufacturing in Relapsed/Refractory Adult Diffuse Large B-Cell Lymphoma: Initial Results from a Phase 1b Study

Contribution of plasma cells and B cells to hidradenitis suppurativa pathogenesis.

Hidradenitis suppurativa (HS) is a debilitating chronic inflammatory skin disease characterized by chronic abscess formation and development of multiple draining sinus tracts in the groin, axillae, and perineum. Using proteomic and transcriptomic approaches, we characterized the inflammatory responses in HS in depth, revealing immune responses centered on IFN-γ, IL-36, and TNF, with lesser contribution from IL-17A. We further identified B cells and plasma cells, with associated increases in immunoglobulin production and complement activation, as pivotal players in HS pathogenesis, with Bruton’s tyrosine kinase (BTK) and spleen tyrosine kinase (SYK) pathway activation as a central signal transduction network in HS. These data provide preclinical evidence to accelerate the path toward clinical trials targeting BTK and SYK signaling in moderate-to-severe HS.

Regulation of B cell receptor-dependent NF-κB signaling by the tumor suppressor KLHL14

The KLHL14 gene acquires frequent inactivating mutations in mature B cell malignancies, especially in the MYD88L265P, CD79B mutant (MCD) genetic subtype of diffuse large B cell lymphoma (DLBCL), which relies on B cell receptor (BCR) signaling for survival. However, the pathogenic role of KLHL14 in DLBCL and its molecular function are largely unknown. Here, we report that KLHL14 is in close proximity to the BCR in the endoplasmic reticulum of MCD cell line models and promotes the turnover of immature glycoforms of BCR subunits, reducing total cellular BCR levels. Loss of KLHL14 confers relative resistance to the Bruton tyrosine kinase (BTK) inhibitor ibrutinib and promotes assembly of the MYD88-TLR9-BCR (My-T-BCR) supercomplex, which initiates prosurvival NF-κB activation. Consequently, KLHL14 inactivation allows MCD cells to maintain NF-κB signaling in the presence of ibrutinib. These findings reinforce the central role of My-T-BCR-dependent NF-κB signaling in MCD DLBCL and suggest that the genetic status of KLHL14 should be considered in clinical trials testing inhibitors of BTK and BCR signaling mediators in DLBCL.

LC-Facseq: A Novel Method for Detecting Rare Resistant Clones in Leukemia

LC-Facseq: A Novel Method for Detecting Rare Resistant Clones in Leukemia

Synergistic Targeting of BTK and E-Selectin/CXCR4 in the Microenvironment of Mantle Cell Lymphomas

Synergistic Targeting of BTK and E-Selectin/CXCR4 in the Microenvironment of Mantle Cell Lymphomas

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.

Modulation of CD81 By Epigenetic Drug Combination Sensitizes Acute Lymphoblastic Leukemia Via Decreased BTK Signaling

Background We previously discovered that the epigenetic drug combination - azacitidine (aza, DNMTi) and panobinostat (pano, HDACi) can sensitize ALL cells to chemotherapy (Quagliano et al., Leuk Res, 44:101, 2017). This sensitization occurred through a decrease in cellular adhesion and a modulation of the cell surface expression of the tetraspanin protein CD81 (Quagliano et al., Blood, 132:3957, 2018). This study aims to elucidate the molecular mechanism by which CD81 surface modulation by pre-treatment with aza/pano leads to chemosensitization. Methods CD81 knockout Nalm6 (CD81KO) cells were generated by CRISPR/Cas9 mutagenesis. IHC was performed using anti-human mitochondria antibody on femurs harvested from NSG-SGM3 mice at 72 h post transplantation with WT or CD81KO cells. For adhesion assay, ALL cells stained with VPD450 were co-cultured with Saos-2 cells for 24 h. Unbound cells were washed, and bound cells were collected for flow cytometry analysis. CD19 surface expression was determined after 48 h treatment with aza/pano (500 nM/1.5 nM) using flow cytometry. For western blot analysis, aza/pano treated cells were transferred on to Saos-2 monolayers and treated with Ara-C (30 nM) for 16 h. Results CD81KO cells not only had decreased homing and engraftment visualized by reduced ALL cell presence in bone marrow (Fig. 1A), but also had 45% reduction in cellular adhesion to osteoblasts compared to WT Nalm6 cells (Fig. 1B), indicating that CD81 downregulation results in reduced bone marrow interactions. To investigate the signaling effects downstream of aza/pano-mediated CD81 modulation, we first tested if CD19 was affected due to its prominent role in the formation of the B-cell co-receptor and because CD19 requires CD81 for proper membrane trafficking (Cherukuri et al., J Immunol, 172:370, 2004). CD19 surface expression was reduced in aza/pano-treated cells and completely knocked out in CD81KO cells (Fig. 1C). CD19 is known to prolong and amplify the activation of Bruton's tyrosine kinase (BTK), so we also analyzed the phosphorylation of BTK in aza/pano-treated cells and found it to be reduced by 63% (Fig. 1D). This reduction in BTK phosphorylation may be responsible for the decrease in cell adhesion following treatment with aza/pano because prior studies observed reduced adhesion following BTK inhibition (Herman et al., Clin Cancer Res, 21:4642, 2015). Taken together, these data suggest that aza/pano induced reduction in cell adhesion was mediated by downregulation of CD81 and BTK dephosphorylation. We studied how the expression of p53 and its target BCL2 associated X protein (BAX), which are known to be induced by Ara-C, were affected following pre-treatment with aza/pano. Cells with Ara-C treatment alone or the aza/pano pre-treatment both had minor induction of p53 expression compared to control, while aza/pano pre-treated cells that received Ara-C had further increase in p53 protein (1.7-fold increase compared to Ara-C alone, Fig. 1E). Bax mRNA and protein were increased 4.2-fold in aza/pano-treated cells following Ara-C treatment compared to Ara-C alone. Increased Bax expression following Ara-C treatment in aza/pano pre-treated cells was accompanied by a 3.9-fold increase in cleavage of caspase-3 compared to cells treated with Ara-C alone, which in turn causes cleavage of Poly-ADP Ribose Polymerase (PARP) (Fig. 1E). Both p53 and Bax protein levels were higher in CD81KO cells than in WT cells (Fig. 1E), suggesting that aza/pano-mediated reduction of CD81 is involved in inducing the expression of p53. CD81KO cells that were treated with Ara-C also had further increase in p53 and Bax expression compared to Ara-C treated WT cells (Fig. 1E), consistent with an increased sensitivity to chemotherapy. To confirm the role of BTK inhibition in the induction of p53, Nalm6 cells were treated with the BTK inhibitor fenebrutinib for 24 h (10 nM). Following treatment, expression of p53 was induced and Bax expression was 2.6-fold higher in treated cells compared to untreated cells (Fig. 1E). Taken together, these data suggest that modulation of the surface expression of CD81 and the phosphorylation of BTK by aza/pano induces sensitization via upregulation of p53 and overexpression of Bax. Conclusion We identify a novel mechanism by which aza/pano treatment induces chemosensitization by reducing cell adhesion via modulating CD81 surface expression, reducing BTK phosphorylation, and inducing p53. Figure 1 Disclosures No relevant conflicts of interest to declare.

Tyrosine Kinase Inhibitors (TKIs) Targeting Syk and BTK Signaling Differentially Affect PI3K Signalosome Organization and Platelet Function

Small molecule tyrosine kinase inhibitors (TKIs) serve increasingly important roles in the treatment of hematologic malignancies and also have shown efficacy in a range of immunological and inflammatory conditions. However, many successful TKI therapies are associated with problematic effects - particularly on platelets - as bleeding complications have been reported for many TKIs, both on and off clinical trials. Bleeding risk is especially apparent for patients treated with ibrutinib and other irreversible inhibitors of Bruton's tyrosine kinase (BTK), several of which now effectively treat multiple B cell malignancies. Intriguingly, other therapeutic TKIs targeting mechanistic partners of BTK are not associated with platelet-related complications. For instance, inhibitors against the BTK-activating kinase Syk - both with selective (i.e., entospletinib) as well as promiscuous (i.e., fostamatinib) target profiles - are not associated with clinically increased bleeding, despite some reported antiplatelet effects. To gain insight into the multifaceted effects of Syk and BTK inhibitors on platelet function, we analyzed the effects of a panel of eight different clinically relevant Syk- and BTK-directed TKIs (i.e., fostamatinib/R406, entospletinib, ibrutinib, acalabrutinib, AVL-292 and others) on essential platelet responses. We first assessed the effects of TKI treatment on Syk and BTK-mediated signaling events downstream of the platelet immunoreceptor tyrosine-based activation motif (ITAM) receptor GPVI. All Syk and BTK TKIs tested inhibited the phosphorylation of phospholipase C (PLCγ2) Y1217, protein kinase Cδ (PKCδ) Y311 and Akt T308, as well as Akt substrate phosphorylation following platelet stimulation with the GPVI receptor agonist, crosslinked collagen-related peptide (CRP-XL). Independent of TKI target profile, Syk and BTK TKIs differentially inhibited the phosphorylation of DAPP1 Y132, phosphoinositide 3-kinase (PI3K) p85α Y458, Akt S473 and PKC substrates. Syk and BTK TKIs also inhibited platelet adhesion to immobilized CRP-XL in a manner matching the effects of each TKI on ITAM-mediated signaling events. However, platelet signaling and adhesion phenotypes did not match the effects of these same TKIs on GPVI-triggered dense granule secretion, which was inhibited by all TKIs tested with the exception of R406. In contrast, all Syk and BTK inhibitors tested inhibited platelet spreading on immobilized fibrinogen. Based on observations above, we hypothesized that TKIs that irreversibly bind to BTK may disrupt critical molecular interactions around BTK within the PI3K signalosome essential to the proper orchestration of platelet activation programs. To test the effects of Syk and BTK inhibitors on the organization of PI3K signaling in activating platelets, platelets were pretreated with TKIs prior to incubation on immobilized fibrinogen and processing for immunofluorescence microscopy. We found that under control conditions, PI3K p85α regulatory subunit localized to phospholipid PI(3,4)P2-rich regions in adherent platelets associated with active PKC signaling. While Syk and PI3K inhibitors abrogated platelet spreading on fibrinogen, PI3K p85α staining remained strong around undeveloped, nascent adhesions in platelets treated with Syk and PI3K inhibitors. In contrast, PI3K p85α staining was diffuse, diminished or absent in platelets adherent to fibrinogen in the presence of all irreversible BTK inhibitors tested. Our results show that clinically effective kinase inhibitors that target Syk-BTK-PI3K signaling systems have varying effects on platelet function that may offer insights to adverse effects of therapeutic TKIs in different contexts. Moreover, our findings suggest that in addition inhibiting protein kinase activities, the discrepant effects of some TKIs may be attributed to, in part, altered protein relations around BTK, PLCγ2 and other components of the PI3K signalosome in activating platelets. Ongoing studies aim to specify protein:protein interactions affected by reversible as well as irreversible TKIs; to examine patient samples for evidence of PI3K mislocalization - especially in cases of BTK inhibitor-associated bleeding; and, to determine if PI3K signalosome disorganization has roles in other physiological, therapeutic and toxic effects of TKIs. Disclosures Shatzel: Aronora, Inc.: Consultancy.