Spleen Tyrosine Kinase Inhibitor Research Articles

A Phase 3, Randomized, Double-Blind, Placebo-Controlled Study to Assess the Efficacy and Safety of Entospletinib in Combination with Intensive Induction and Consolidation Chemotherapy in Adults with Newly Diagnosed Nucleophosmin 1-mutated Acute Myeloid Leukemia

A Phase 3, Randomized, Double-Blind, Placebo-Controlled Study to Assess the Efficacy and Safety of Entospletinib in Combination with Intensive Induction and Consolidation Chemotherapy in Adults with Newly Diagnosed Nucleophosmin 1-mutated Acute Myeloid Leukemia

An Observational Study of Fostamatinib As Second Line Therapy in Adult Patients with Immune Thrombocytopenia (ITP) in Real-World Clinical Practice

Background: In immune thrombocytopenia (ITP), autoantibody-bound platelets are targeted for phagocytosis by Fcγ-receptors on macrophages in a spleen tyrosine kinase (SYK)-dependent signaling pathway. Fostamatinib is a potent oral SYK inhibitor that demonstrated efficacy for the treatment of ITP in two phase 3 randomized, placebo-controlled, double-blind studies and long-term durable responses in the phase 3 open-label extension study. In these studies, 146 patients with ITP were treated with fostamatinib, and 54% had a platelet response (platelet count ≥50,000/µL), which was maintained over a median of 86% of treatment days. These studies led to approval of fostamatinib in the US, Europe, and Canada as a therapy for chronic ITP in adult patients who have had an insufficient response to a prior treatment. Further, a post-hoc analysis showed that, in those who received fostamatinib as second-line therapy (following steroids ± immunoglobulins), 78% achieved a platelet response, which was maintained over a median of 83% of treatment days. 1 Adverse events (AEs) were reported in 72% of second-line patients and 86% of all patients, and common AEs were similar between second-line patients and the total patient population. 1, 2 Bleeding events were reported in 24% of second-line patients vs. 41% of all patients. The results of the post hoc analysis underscored the necessity for further evaluation of fostamatinib as a second-line therapy for ITP.We are conducting a multi-center, prospective, observational, two cohort study of fostamatinib as second-line therapy for the management of ITP in adult patients who have an insufficient response to prior steroids ± immunoglobulins.Aim: This study will evaluate clinical outcomes and safety of fostamatinib as second-line therapy for ITP in adult patients in real-world clinical practice. Patients will also be assessed for quality of life and utilization of health care resources during the study.Methods: The study (FORTE; NCT04904276; O-FOSTA-901) is being conducted at 10 sites across the US and will enroll 45 patients with ITP into 2 cohorts (see Figure). The study is registered at https://clinicaltrials.gov/ct2/show/NCT04904276. The study aims to enroll adult patients with a confirmed diagnosis of ITP and an inadequate response to prior steroids ± immunoglobulins. Patients who have received any prior ITP treatment other than steroids or immunoglobulins will be excluded. Patients who will initiate fostamatinib as second-line therapy at study entry will be enrolled into Cohort 1; patients currently receiving fostamatinib as second-line therapy with the intent to continue treatment will be enrolled in Cohort 2 (historical platelet counts will be retrospectively collected). Patients will be treated by their clinician at the clinician's discretion (not pre-specified by a study protocol).Primary outcome measures will include platelet count over time, duration of platelet count elevation, use of ITP rescue medication, changes in dosing, use of concomitant ITP medications, incidence of serious and ITP-related AEs, and quality of life. Quality of life assessments will utilize the SF-36v2 and ITP-PAQ. All data will be collected at regularly scheduled clinic visits according to the clinician's normal practice for 12 months or until fostamatinib is discontinued.Summary: This ongoing observational study of adult patients with ITP will enable further characterization of the risk/benefit profile of fostamatinib as a second-line treatment for ITP and will provide insight on how clinicians manage ITP in a real-world setting without the constraints of a clinical trial protocol.

Dual inhibition of reactive oxygen species and spleen tyrosine kinase as a therapeutic strategy in liver fibrosis

Hepatic stellate cells (HSCs) play key roles in liver fibrosis (LF) and hepatocellular carcinoma (HCC). We previously reported that spleen tyrosine kinase (SYK) is critical for HSCs activation, however, the mechanisms are insufficiently understood. In the present study, we found that SYK facilitated autophagy to promote HSCs activation by enhancing reactive oxygen species (ROS) generation. However, SYK inhibitor GS-9973 could efficiently reduce HSCs ROS generation in vitro but not in vivo. Mechanistically, hepatocytes (HCs) would release ROS outside and then diffuse into HSCs to promote autophagy and activation in vitro in the context of inflammation. We then further examined the ROS contents in liver sections and primary liver cells of carbon tetrachloride (CCl4) induced mice treated with or without different doses of Silybin, a natural compound characterized by a well-established antioxidant and hepatoprotective properties, and found that ROS intensities in both liver sections and their deprived primary cells were efficiently inhibited in a dose-dependent fashion. Lastly, we evaluated the rational combination of Silybin and GS-9973 in the treatment of CCl4 induced mice and found that this combination is well tolerated and acts synergistically against HSCs activity, LF and HCC. The combinational use of Silybin and GS-9973 could be a promising therapeutic strategy in patients suffering from LF and even HCC.

Venetoclax-resistant CLL cells show a highly activated and proliferative phenotype.

Venetoclax treatment has demonstrated efficacy and a safety profile in chronic lymphocytic leukemia (CLL) patients, however the emergence of resistant cells is a current complication. We and others, previously reported that the activation of CLL cells by signals that mimic microenvironment stimuli favors the upregulation of anti-apoptotic proteins from B cell lymphoma-2 (BCL-2) family that are not targeted by venetoclax, reducing malignant cell sensitivity to the drug. We here studied venetoclax-resistant CLL cells generated in vitro by autologous activated T lymphocytes, and found that they showed an aggressive phenotype characterized by increased expression of activation and proliferation markers. Moreover, surviving cells expressed high levels of B cell lymphoma-extra-large (BCL-XL) and/or myeloid cell leukemia-1 (MCL-1), and a sustained resistance to a second treatment with the drug. Interestingly, the spleen tyrosine kinase (SYK) inhibitor entospletinib, and the phosphoinositide 3-kinase delta (PI3Kδ) inhibitor idelalisib, reduced T cell activation, impaired the generation of leukemic cells with this aggressive phenotype, and were able to restore CLL sensitivity to venetoclax. Our data highlight a novel combination to overcome resistance to venetoclax in CLL.

Fostamatinib for the Treatment of Hospitalized Adults With Coronavirus Disease 2019: A Randomized Trial.

Coronavirus disease 2019 (COVID-19) requiring hospitalization is characterized by robust antibody production, dysregulated immune response, and immunothrombosis. Fostamatinib is a novel spleen tyrosine kinase inhibitor that we hypothesize will ameliorate Fc activation and attenuate harmful effects of the anti-COVID-19 immune response. We conducted a double-blind, randomized, placebo-controlled trial in hospitalized adults requiring oxygen with COVID-19 where patients receiving standard of care were randomized to receive fostamatinib or placebo. The primary outcome was serious adverse events by day 29. A total of 59 patients underwent randomization (30 to fostamatinib and 29 to placebo). Serious adverse events occurred in 10.5% of patients in the fostamatinib group compared with 22% in placebo (P = .2). Three deaths occurred by day 29, all receiving placebo. The mean change in ordinal score at day 15 was greater in the fostamatinib group (-3.6 ± 0.3 vs -2.6 ± 0.4, P = .035) and the median length in the intensive care unit was 3 days in the fostamatinib group vs 7 days in placebo (P = .07). Differences in clinical improvement were most evident in patients with severe or critical disease (median days on oxygen, 10 vs 28, P = .027). There were trends toward more rapid reductions in C-reactive protein, D-dimer, fibrinogen, and ferritin levels in the fostamatinib group. For COVID-19 requiring hospitalization, the addition of fostamatinib to standard of care was safe and patients were observed to have improved clinical outcomes compared with placebo. These results warrant further validation in larger confirmatory trials. Clinicaltrials.gov, NCT04579393.

Eco-Friendly UPLC-MS/MS Method for Determination of a Fostamatinib Metabolite, Tamatinib, in Plasma: Pharmacokinetic Application in Rats.

Fostamatinib is a prodrug of the active metabolite tamatinib, which is a spleen tyrosine kinase (Syk) inhibitor used in the treatment of primary chronic adult immune thrombocytopenia and rheumatoid arthritis. A highly sensitive, rapid, reliable, and green method was developed and validated using ultra-performance liquid chromatography and tandem mass spectrometry (UPLC–MS/MS) for quantification of tamatinib in rat plasma. Ibrutinib was used as internal standard and liquid–liquid extraction was applied using tert-butyl methyl ether. The analyte was separated on an AcquityTM CSH C18 (2.1 mm × 100 mm, 1.7 µm) column using mobile phase consisting of 10 mM ammonium acetate and acetonitrile (10:90) and the flow rate was 0.25 mL/min. Electrospray ionization (ESI) was carried out in positive mode. Quantitation of tamatinib and the IS was performed using multiple reaction monitoring mode with precursor-to-product transitions of m/z 471.1 > 122.0 and m/z 441.1 > 84.0, respectively. The calibration range was 0.1–1000.0 ng/mL and the linearity of the method was ≥0.997. The developed method greenness was investigated. All principal parameters for the method, including linearity, accuracy, precision, recovery, and stability, were within acceptable ranges. Tamatinib pharmacokinetic study in rats was successfully carried out using the developed method.

Corrigendum] Cryptotanshinone inhibits IgE‑mediated degranulation through inhibition of spleen tyrosine kinase and tyrosine‑protein kinase phosphorylation in mast cells

Following the publication of the above article, an interested reader drew to the authors' attention that they had mentioned that activated PKCδ phosphorylates IKKβ in order that IKKβ is relocated to the plasma membrane, resulting in the induction of mast cell degranulation; however, four references the authors had included did not seem to support this statement. The authors have re-examined their paper, and realized that the four references the reader mentioned were indeed cited incorrectly, and wish to rectify this error through revising the third paragraph in the Discussion section, the References section, and an associated figure (Fig.6C) in order to avoid any further misunderstandings on the part of the readership. First, the authors wish to revise the wording of the third and fourth paragraphs of the Discussion, as featured on pp.1101-1102, to the following (changed text is indicated in bold): 'We showed that CRT exerts anti-AD effect through inhibition of the mast cell degranulation in mast cells. Upon IgE/antigen stimulation, the immunoreceptor tyrosine-based activation motif (ITAM) region of FcεRI receptor which is on the mast cell surface is phosphorylated and the initial signalling protein kinases Lyn and Syk are recruited to the ITAM (28,29). Then, the activated Lyn and Syk leads to phosphorylation of the transmembrane adaptor linker for activation of T cells (LAT). Phosphorylated LAT which is a scaffold for multimolecular signalling complexes and activates PLCγ through phosphorylation. The activated PLCγ hydrolyses phosphatidylinositol biphosphate (PIP2) to generate second signalling molecules IP3 and DAG, which activate PKCs including PKCδ to induce the mast cell degranulation (30,31). On the other hand, cross-linking of FcεRI also activates IKKβ, which moves to the lipid raft fractions and phosphorylates synaptosomal-associated protein23 (SNAP-23) leading to degranulation (7). Since PKCδ phosphorylates IKKα, but not IKKβ (32), it is not likely that two signalling pathways are directly connected. In this study, novel function of CRT on phosphorylations of Lyn/Syk kinases in mast cells is elucidated for the first time. Furthermore, it is likely that this inhibitory effect of CRT on Lyn/Syk kinases negatively affected activities of their downstream signalling molecules including PLCγ, PKCδ, and IKKβ, which leads to decrease in mast cell degranulation by CRT treatment. Besides the inhibitory effect of CRT on mast cell degranulation, here we provide additional evidence that CRT exerts anti-AD effects through inactivation of MAPK and NF‑κB. It has been reported that CRT regulates the activities of MAPK and NF‑κB in various cell types. In rhabdomyosarcoma, hepatoma, and breast carcinoma, CRT activates MAPK p38/JNK and suppresses ERK1/2, followed by caspase-independent apoptosis (10,33,34). In chronic myeloid leukaemia cells, CRT enhances TNF‑α-induced apoptosis through the activation of MAPK p38 (35). In smooth muscle cells, CRT exerts anti-migration/invasion effect as it inhibits TNF‑α/NF‑κB signalling pathway (36).' Secondly, the authors wish to make the following changes to the Reference list: New references 30-32 have been inserted to the list, as follows: 30. Ozawa K, Szallasi Z, Kazanietz MG, BlumbergPM, MischakH, Mushinski JF and Beaven MA: Ca2+-dependent and Ca2+-independent isozymes of protein kinase C mediate exocytosis in antigen-stimulated rat basophilic RBL-2H3 cell. J Biol Chem268: 1749-1756, 1993. 31. Cho SH, Woo CH, Yoon SB and Kim JH: Protein kinaseCδ functions downstream of Ca2+ mobilization in FcεRI signaling to degranulation in mast cells. J Allergy Clin Immunol114: 1085-1092, 2004. 32. Yamaguchi T, Miki Y and Yoshida K: Protein kinaseCδ activates IκB-kinaseα to induce the p53 tumor suppressor in response to oxidative stress. Cell Signal19: 2088-2097, 2007. The addition of these new references means that the former references 30-33 have been accordingly renumbered to references 33-36. Finally, the authors have revised Fig.6C, as it appeared on p.1102, in order to assist the understanding of the readers, and the corrected version of Fig.6 appears on the next page. All these corrections have been approved by all the authors, with the exception of the first author, Sumiyasuren Buyanravjikh, who is no longer uncontactable. The authors regret that these errors were included in the paper, even though they did not substantially alter any of the major conclusions reported in the study, are grateful to the Editor for allowing them this opportunity to publish a Corrigendum, and apologize to the readership for any inconvenience caused. [the original article was published in Molecular Medicine Reports 18: 1095‑1193, 2018; DOI: 10.3892/mmr.2018.9042].

Transitioning From Thrombopoietin Agonists to the Novel SYK Inhibitor Fostamatinib: A Multicenter, Real-World Case Series.

Fostamatinib is an oral spleen tyrosine kinase (SYK) inhibitor used for the treatment of adult patients with chronic immune thrombocytopenia (ITP) who have had an insufficient response to a previous treatment. Clinical and operational barriers may exist that warrant bridging or switching from thrombopoietin receptor agonists (TPO-RAs), such as volatility or unpredictability of platelets, adverse events, and quality of life or patient preference. While fostamatinib demonstrated durable platelet responses, the safety and efficacy in combination, bridging, and/or switching with TPO-RAs is not well documented. The objective of this article is to provide guidance from real-world case studies for a safe and effective strategy for the transitioning of patients from TPO-RAs to fostamatinib, with some degree of overlap between the two agents. Currently, the evidence does not exist to guide the safe and effective use of combination therapy or transition between therapies in ITP. This case series highlights the importance to further understand the complexities of managing this disease, as well as successfully combining, bridging, and/or switching patients over to fostamatinib without the need for rescue therapy or increase in adverse events. The need for real-world evidence that guides providers on the safety and efficacy of short- and long-term combination therapy of fostamatinib and TPO-RAs is crucial. The rationale for combination therapy is to target different pathways, platelet destruction, and production without added toxicities. Additionally, gradual tapering off of TPO-RAs may provide a more favorable clinical outcome when switching to fostamatinib. The need for additional data is necessary to provide clinicians with guidance when managing patients with ITP.

Optimizing drug inhibition of IgE-mediated anaphylaxis in mice

Administering allergens in increasing doses can temporarily suppress IgE-mediated allergy and anaphylaxis by desensitizing mast cells and basophils; however, allergen administration during desensitization therapy can itself induce allergic responses. Several small molecule drugs and nutraceuticals have been used clinically and experimentally to suppress these allergic responses. This study sought to optimize drug inhibition of IgE-mediated anaphylaxis. Several agents were tested individually and in combination for ability to suppress IgE-mediated anaphylaxis in conventional mice, FcεRIα-humanized mice, and reconstituted immunodeficient mice that have human mast cells and basophils. Hypothermia was the readout for anaphylaxis; therapeutic efficacy was measured by degree of inhibition of hypothermia. Serum mouse mast cell protease 1 level was used to measure extent of mast cell degranulation. Histamine receptor 1 (HR1) antagonists, β-adrenergic agonists, and a spleen tyrosine kinase (Syk) inhibitor were best at individually inhibiting IgE-mediated anaphylaxis. ABruton's tyrosine kinase (BTK) inhibitor, administered alone, only inhibited hypothermia when FcεRI signaling was suboptimal. Combinations of these agents could completely or nearly completely inhibit IgE-mediated hypothermia in these models. Both Syk and BTK inhibition decreased mast cell degranulation, but only Syk inhibition also blocked desensitization. Many other agents that are used clinically and experimentally had little or no beneficial effect. Combinations of an HR1 antagonist, a β-adrenergic agonist, and a Syk or a BTK inhibitor protect best against IgE-mediated anaphylaxis, while an HR1 antagonist plus a β-adrenergic agonist± a BTK antagonist is optimal for inhibiting IgE-mediated anaphylaxis without suppressing desensitization.

Cerdulatinib Pharmacodynamics and Relationships to Tumor Response Following Oral Dosing in Patients with Relapsed/Refractory B Cell Malignancies: Results from a Phase I Dose Escalation Study

Preclinical studies suggest that the spleen tyrosine kinase (SYK) and Janus kinase (JAK) family members can contribute to both tumor intrinsic and microenvironment-derived survival signals. A dual inhibitor of these kinases (cerdulatinib) was therefore developed, and a phase I dose-escalation study was completed to understand tolerability and efficacy, and their relationship to pharmacokinetics (PK) and pharmacodynamics (PD). Here, we report on PK/PD relationships and associations with tumor response in patients from the phase I dose-escalation portion of the clinical trial (NCT01994382).Repeat CT scans were obtained from 6 patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL), 12 patients with follicular lymphoma (FL), 12 patients with diffuse large B cell lymphoma, and 6 patients with mantle cell lymphoma. Meaningful clinical responses were observed in patients with CLL/SLL and FL, as previously reported (Hamlin et al, 2016); hence, the correlation between tumor response and PD markers was examined in these patient populations.Complete inhibition of SYK and JAK signaling pathways was achieved in whole blood of dosed patients at tolerated exposures. The level of target inhibition in blood correlated with serum cerdulatinib concentration, and the concentrations at which 50% inhibition was acheived (IC50) against B cell antigen receptor (BCR), interleukin (IL)-2, IL-4, and IL-6 signaling pathways ranged from 0.22-1.08 μM, depending on the phosphorylation event measured. Significant correlations were observed between inhibition of both SYK and JAK signaling pathways and tumor response, most notably the BCR and IL-4 signaling pathways. Serum markers of inflammation were also reduced following cerdulatinib administration, several of which significantly correlated with tumor response, including β-2 microglobulin, C-reactive protein, interferon γ-induced protein 10 (IP10), and macrophage inflammatory protein 3β (MIP3β). Of note, lymphocytosis was observed in CLL, SLL, and FL patients. Evidence of diminished tumor cell surface expression of CD69 and CD86, diminished CD5 expression (a negative regulator of BCR signaling), and enhanced C-X-C chemokine receptor type 4 (CXCR4) expression were observed in 2 patients with CLL, consistent with suppression of BCR and IL-4 signaling.Fresh tumor samples were obtained from the peripheral blood of 6 CLL patients treated with cerdulatinib, as well as archival tumor biopsies obtained from 4 FL patients and 1 MCL patient, which were submitted for next-generation sequencing. Clinical activity was observed in CLL patients bearing mutations to NOTCH1, ATM, p53, and KRAS, which are among the most frequently observed mutations in this disease. One of the responding patients bore a 17p deletion. Importantly, 2 patients with ibrutinib-relapsed CLL who progressed within the first cycle of therapy with cerdulatinib had 3 mutations in common: p53, EP300, and BTK (C481S). Genetic correlates in FL were more limited, with data on 4 patients, 3 of whom had evidence of tumor reduction and 1 which progressed. The 2 best responding patients with whom we have genetic information bore mutations to ZMYM3, KMT2D, FAT4, BCL2, BCL6, CD79B, and STAT6 . Lastly, strong clinical activity was observed in a transformed FL patient who presented with increased MYC, BCL2, and BCL6 expression by immunohistochemistry (“triple-hit” lymphoma).These data demonstrate the ability of cerdulatinib to potently and selectively inhibit SYK and JAK signaling at tolerated exposures in patients with relapsed/refractory B cell malignancies. Moreover, the extent of target inhibition, as well as suppression of inflammation, correlated with tumor response. Cerdulatinib is currently under investigation as part of a phase IIa study to further evaluate efficacy and tolerability in this population. DisclosuresCoffey:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Feng:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Betz:Portola Pharmaceuticals, Inc: Employment, Equity Ownership. Pandey:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Birrell:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Leeds:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Der:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Kadri:Portola Pharmacueticals, Inc.: Research Funding. Lu:Portola Pharmacueticals, Inc.: Research Funding. Segal:Portola Pharmacueticals, Inc.: Research Funding. Wang:Karyopharm: Research Funding; Portola: Research Funding. Michelson:Portola Pharmacueticals, Inc.: Employment. Curnutte:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Conley:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership, Patents & Royalties.

Exposure-Response of Entospletinib (ENTO) for the Treatment of Relapsed or Refractory Chronic Lymphocytic Leukemia

Background: Entospletinib (ENTO) is a selective spleen tyrosine kinase (SYK) inhibitor that has demonstrated evidence of efficacy and tolerability in chronic lymphocytic leukemia (CLL). The relationship between ENTO plasma exposures and clinical efficacy and safety were evaluated in relapsed/refractory (R/R) CLL subjects, following administration of 100, 200, and 400 mg twice daily (BID) in a Phase 2 Study (NCT01799889; study -0102).Methods: ENTO pharmacokinetics (PK) and plasma exposures (Cmax, AUCtau, and Ctau) were characterized in 60 R/R CLL subjects using a population PK model developed from data collected in phase 1/2 clinical studies. ENTO plasma samples were collected on day 1, and day 8 (pre-dose,1.5 hours, and 4 hours post-dose), day 15, and day 22 (pre- and 1.5 hours post-dose) of cycle 1 and on day 1 (pre- and 1.5 hours post-dose) of subsequent 28 day cycles. Data for efficacy and safety endpoints were collected every 8 weeks (for cycles 1-6) followed by every 12 weeks (for cycles 7-18). Efficacy endpoints, including best overall response rate (BOR), progression-free survival (PFS), sum of products of the greatest perpendicular diameters (SPD) of index lesions, maximum change from baseline in lymphocytes, platelets, and hemoglobin were evaluated against ENTO plasma exposure (AUCtau and Ctau). Safety endpoints, including adverse events (all grades) of skin rash, infection, diarrhea, aspartate aminotransferase (AST), alanine aminotransferase (ALT), bilirubin (total), and neutrophils (maximum change from baseline) versus ENTO plasma exposure (AUCtau and Cmax) were evaluated.Results: A dose proportional increase in ENTO exposures was observed across 100, 200, and 400 mg BID regimen. A greater reduction in SPD and lymphocytes and an increase in platelets were associated with higher ENTO exposures. No exposure-response relationship was observed for PFS at Week 24 (i.e., there was an overlap in survival curves across quartiles of ENTO exposures) or hemoglobin. No association between ENTO exposures vs incidence of skin rash, infection, diarrhea, AST, ALT and neutrophils was observed. There was a slight trend toward elevated total bilirubin with higher ENTO exposures.Conclusions:In R/R CLL subjects, following ENTO 100, 200, and 400 mg BID regimen, higher exposures corresponding to 400 mg BID are associated with greater efficacy (reduction in SPD lymphocytes and an increase in platelets). No clinically relevant relationship between ENTO exposures and safety, except total bilirubin, was observed. Overall, the analyses support ENTO 400 mg BID regimen as a starting dose for future phase 2 trials in CLL patients. [Display omitted] DisclosuresGupta:Gilead Sciences, Inc.: Employment, Equity Ownership. Meng:Gilead Sciences, Inc.: Employment, Equity Ownership. Sharma:Gilead Sciences, Inc.: Employment, Equity Ownership. Ye:Gilead Sciences, Inc: Employment, Equity Ownership. Silverman:Gilead Sciences, Inc.: Employment, Equity Ownership. Abella:Gilead Sciences, Inc.: Employment, Equity Ownership. Mathias:Gilead Sciences, Inc.: Employment, Equity Ownership.

Enhanced Efficacy of the SYK Inhibitor Entospletinib and Chemotherapy in Infant Acute Lymphoblastic Leukemia

Background: Chemotherapy resistance and subsequent relapse remain a major cause of childhood cancer mortality, particularly for infants (<12 months of age) with KMT2A -rearranged (R) B-cell acute lymphoblastic leukemia (B-ALL), who have extremely poor event-free survival (EFS). Addition of kinase inhibitors to chemotherapy has markedly improved outcomes for patients with some genetic subsets of B-ALL, such as BCR-ABL1 -R (Ph+) ALL. Unfortunately, addition of the FMS-like tyrosine kinase 3 (FLT3) inhibitor lestaurtinib did not improve 3-year EFS for infants with KMT2A -R and FLT3-overexpressing B-ALL in the Children's Oncology Group trial AALL0631 (Brown et al, SIOP 2016), indicating continued need for alternative therapeutic strategies. We recently observed constitutive phosphorylation (p) of spleen tyrosine kinase (SYK) at tyrosine 323 in infant ALL specimens (Figure 1A) and hypothesized that activated signaling is targetable with the selective SYK inhibitor entospletinib (ENTO). We further hypothesized that combining ENTO with chemotherapy would have superior activity in comparison to single-agent treatments.Methods: To test these hypotheses, we established patient-derived xenograft (PDX) models of KMT2A -R (n=7) and non- KMT2A -R (n=4) infant ALL in NSG mice using diagnostic and relapse bone marrow specimens from patients treated on or as per the AALL0631 trial protocol. Total and phosphorylated (p) SYK and other phosphoprotein levels were assessed by Simple WesternTM analysis of splenic lysates from ALL-engrafted PDX mice to identify leukemias with constitutive SYK signaling activation. Infant ALL PDX models were then treated with control mouse chow or chow containing 0.07% ENTO ad libitum, 0.25 mg/kg vincristine (VCR) dosed intraperitoneally once weekly, or a combination of ENTO and VCR for up to 28 days to determine treatment efficacy. Cohorts of additional infant ALL PDX mice were treated for 72 hours to assess plasma levels and pharmacodynamic effects of ENTO administration.Results: Constitutive SYK signaling was observed in several models of de novo KMT2A -R and non- KMT2A -R B-ALL. ENTO treatment of KMT2A -R ALL PDX models ALL3103 with t(9;11) and ALL135 with t(11;19) inhibited leukemic cell burden in vivo versus control animals by flow cytometric and immunohistochemical (IHC) analyses of murine spleens (representative data in Figure 1B). Importantly, combined ENTO and VCR treatment resulted in a greater reduction in ALL cell number, which was confirmed by human CD19+ IHC analysis of murine spleens and bone marrow. ENTO concentrations were maintained throughout study duration with terminal free fraction plasma levels of 61 and 230 nM (ALL3103) and 89 and 214 nM (ALL135) for ENTO and ENTO/VCR treatment arms, respectively. Evaluation of SYK pathway modulation in vivo demonstrated inhibition of pSYKY323 in human ALL cells within murine spleens that correlated with the level of ENTO exposure of treated mice (ALL3103 PDX; Spearman correlation of -0.63, p<0.05). No alterations in total SYK protein were observed. RNA sequencing of ALL3103 PDX tissues showed that ENTO treatment was associated with downregulation of MYC and of cell cycle and DNA damage gene expression signatures.Conclusions: Constitutive activation of SYK signaling occurs in infant KMT2A -R B-ALL. Entospletinib treatment of infant B-ALL PDX models potently inhibited the SYK pathway and decreased leukemic cell burden in vivo . Superior anti-ALL activity was observed with combined ENTO and VCR compared to ENTO alone. Additional studies are ongoing to determine the activity of ENTO in non- KMT2A -R and other ALL PDX models. [Display omitted] DisclosuresYahiaoui:Gilead Sciences, Inc.: Employment, Equity Ownership. Lopez:Gilead Sciences, Inc.: Employment, Equity Ownership. Mikaelian:Gilead Sciences, Inc.: Employment, Equity Ownership. Tannheimer:Gilead Sciences, Inc.: Employment, Equity Ownership. Forslund:Gilead Sciences, Inc.: Employment, Equity Ownership. Tasian:Gilead Sciences, Inc.: Research Funding; Incyte Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Fostamatinib: A Review in Chronic Immune Thrombocytopenia.

Fostamatinib (Tavalisse®; Tavlesse®) is the first spleen tyrosine kinase (Syk) inhibitor approved for the treatment of chronic immune thrombocytopenia (ITP) in adult patients who have had an insufficient response to previous treatment. By inhibiting Syk activation in macrophages, fostamatinib blocks autoantibody-mediated platelet phagocytosis. In the placebo-controlled phaseIII FIT1 and FIT2 trials, 24weeks of oral fostamatinib therapy increased platelet count in previously treated adults with ITP. A significantly higher proportion of patients achieved stable response with fostamatinib than with placebo in FIT1, but not in FIT2; however, pooled analyses of the two studies showed that fostamatinib produced significantly higher stable and overall response rates than placebo. Interim findings from the ongoing FIT3open-label extension study suggested that the efficacy of fostamatinib was maintained with long-term treatment (up to 62months; median duration 6months), including in patients receiving fostamatinib as second- or later-line treatment. Fostamatinib had a generally manageable tolerability profile in all three FIT studies, with no serious safety risks. Fostamatinib therefore provides an alternative treatment option for chronic ITP in adult patients with an insufficient response to previous treatment.

Mechanosensing of Substrate Stiffness Regulates Effector Functions of Human Neutrophils

During a response to injury or infection, neutrophils (PMNs) navigate through a tissue microenvironment that is densely packed with parenchymal cells and extracellular matrix. Within these tissues, PMNs sense the physical property of elasticity as a regulatory element as it pertains to motility and effector functions. Historically, rigid tissue culture surfaces like glass and plastic have been used to study many key anti-microbial effector functions of PMNs; however, these fail to mimic the elasticity of peripheral tissues. We hypothesize that elasticity plays a key regulatory role for PMN anti-microbial effector functions associated with pathogen clearance such as phagocytosis, NETosis, respiratory burst, and degranulation. When comparing phagocytosis by neutrophils on soft substrates and stiff substrates coated with fibronectin, we found that stiffness is selectively regulatory. While Zymosan and Whole Glucan Particles were more readily phagocytosed on soft substrates, IgG-opsonized latex beads were more efficiently taken up on stiff substrates. Further, C3bi-opsonized latex beads and apoptotic bodies were phagocytosed equivalently on all stiffnesses. In addition to a mechanosensitive response to phagocytosed particles, we also saw differential mechanosensitive responses in their mechanism. For example, on stiff substrates, inhibiting Rho-associated protein kinase reduced phagocytosis of Zymosan, while blocking CD45 reduced phagocytosis of IgG-opsonized latex beads. Further, Zymosan uptake was found to increase on soft substrates when actin-related protein Arp2/3 complex was blocked, but have no effect on stiff substrates, further exaggerating the soft/stiff phenotype; conversely, blocking formin-mediated actin had the opposite effect and lowered phagocytosis of Zymosan on soft substrates, attenuating the soft/stiff phenotype. Finally, not all blocking targets showed mechanosensitive responses; while inhibition of Spleen Tyrosine Kinase had no effect on Zymosan uptake on any stiffness, blocking total actin polymerization reduced uptake on all stiffnesses. Taken together, these findings show a highly complex mechanosensitive response to phagocytosis in regards to both particle type and mechanism of uptake. Further, PMNs seeded on Candida albicans hyphae were mechanosensitive in regards to oxidative burst and NETosis; both ROS production and NETosis increased on stiffer substrates. Finally, in addition to phagocytosis and hyphae, we also looked at cytokine release to secondary stimulation by fMLP. We found both Lactoferrin and IL-8 release to increase on stiffer substrates, while elastase release was not mechanosensitive. Taken together, these findings indicate a strong regulatory role of substrate stiffness during PMN effector responses. While extensive work has been done to elucidate PMN biochemical interactions, understanding important mechanosensitive mechanisms underlying phagocytosis, NETosis, respiratory burst, and cytokine release may provide further therapeutic targets to benefit patients in the future by reducing harmful PMN behaviors and/or promoting behaviors crucial to pathogen clearance.

Inhibition of Syk promotes chemical reprogramming of fibroblasts via metabolic rewiring and H2 S production.

Chemical compounds have recently been introduced as alternative and non-integrating inducers of pluripotent stem cell fate. However, chemical reprogramming is hampered by low efficiency and the molecular mechanisms remain poorly characterized. Here, we show that inhibition of spleen tyrosine kinase (Syk) by R406 significantly promotes mouse chemical reprogramming. Mechanistically, R406 alleviates Syk / calcineurin (Cn) / nuclear factor of activated T cells (NFAT) signaling-mediated suppression of glycine, serine, and threonine metabolic genes and dependent metabolites. Syk inhibition upregulates glycine level and downstream transsulfuration cysteine biosynthesis, promoting cysteine metabolism and cellular hydrogen sulfide (H2 S) production. This metabolic rewiring decreased oxidative phosphorylation and ROS levels, enhancing chemical reprogramming. In sum, our study identifies Syk-Cn-NFAT signaling axis as a new barrier of chemical reprogramming and suggests metabolic rewiring and redox homeostasis as important opportunities for controlling cell fates.

Spleen tyrosine kinase (SYK) inhibitor PRT062607 protects against ovariectomy-induced bone loss and breast cancer-induced bone destruction

Osteolytic diseases, including breast cancer-induced osteolysis and postmenopausal osteoporosis, are attributed to excessive bone resorption by osteoclasts. Spleen tyrosine kinase (SYK) is involved in osteoclastogenesis and bone resorption, whose role in breast cancer though remains controversial. Effects of PRT062607 (PRT), a highly specific inhibitor of SYK, on the osteoclast and breast cancer functionalities are yet to be clarified. This study demonstrated the in vitro inhibitory actions of PRT on the osteoclast-specific gene expression, bone resorption, and osteoclastogenesis caused by receptor activator of nuclear factor kappa B ligand (RANKL), as well as its in vitro suppressive effects on the growth, migration and invasion of breast carcinoma cell line MDA-MB-231, which were achieved through PLCγ2 and PI3K-AKT-mTOR pathways. Further, we proved that PRT could prevent post-ovariectomy (OVX) loss of bone and breast cancer-induced bone destruction in vivo, which agreed with the in vitro outcomes. In conclusion, our findings suggest the potential value of PRT in managing osteolytic diseases mediated by osteoclasts.

BTLA Expression and Function Are Impaired on SLE B Cells.

B- and T-lymphocyte attenuator (BTLA/CD272) is an inhibitory checkpoint molecule expressed on T and B cells. Prior studies reported defective function of BTLA by T cells in patients with systemic lupus erythematosus (SLE), whereas nothing is known about its role on B cells in SLE, a disease with various B cell abnormalities. Peripheral blood mononuclear cells (PBMCs) from 23 healthy donors (HD) and 34 SLE patients were stained for BTLA and its expression on B cells was assessed. PBMCs or CD27-IgD+ naive B cells were stimulated together with an activating anti-BTLA antibody or an inhibitor of spleen tyrosine kinase (SYK) and differentiation as well as the expression of activation markers CD71, PD-1 and CD86 were analyzed. Our phenotypic and functional studies revealed reduced BTLA expression on CD27-IgD+ naïve B cells from SLE patients (p=0.0017) related to anti-dsDNA antibody titers (p=0.0394) and SIGLEC-1/CD169 expression on monocytes (p=0.0196), a type I interferon marker related to disease activity. BTLA engagement was found to control CpG/TLR9 activation limiting plasmablast (p=0.0156) and B cell memory induction (p=0.0078) in normal B cells in contrast to other B cell activation pathways (CD40, BCR). These BTLA functions were impaired in SLE B cells. Inhibition of SYK was found to mimic the effects of BTLA activity in vitro. Thus, is it possible that reduced BTLA expression and function of CD27-IgD+ antigen- and T cell-inexperienced SLE B cells could be overcome by SYK inhibition which should be tested in future studies as potential therapeutic principle.

Basophil activation test: Assay precision and BI 1002494 SYK inhibition in healthy and mild asthmatics.

Application of basophil activation test (BAT) in clinical trials requires assay validity. Whether assay variability differs between healthy and asthmatic subjects is mostly unknown. This study compares basophil stimulation using blood from healthy and asthmatic subjects with or without inhibition of spleen tyrosine kinase (SYK). Whole blood of healthy and mild asthmatic subjects was stimulated with anti-dinitrophenyl (DNP) IgE/DNP bovine serum albumin and anti-IgE. Basophil activation was detected by CD63 and CD203c expression. CD63 expression levels were compared with serum IgE levels. Three operators repeated experiments with three subjects each from both groups at 3 days to observe assay precision. The effect of the SYK inhibitor BI 1002494 was assessed in BAT for both healthy and asthmatic subjects. BAT was reproducible in both groups. Acceptance criteria of <25% CV were mostly fulfilled. Stimulation with anti-DNP (p < 0.001, r=-0.80) but not anti-IgE (p=0.74, r=0.05) was related to serum IgE with levels > 200 IU/ml limiting anti-DNP stimulation. BI 1002494 IC50 values were 497 nM and 1080 nM in healthy and 287 nM and 683 nM in asthmatics for anti-DNP and anti-IgE stimulation, respectively. BAT, performed with blood from healthy or asthmatic subjects, is a robust test for the measurement of a physiological response in clinical trials. Blood from asthmatic donors with serum IgE > 200 IU/ml is less feasible when using anti-DNP stimulation. SYK inhibition was not affected by disease status.

Characterization of the mechanism of action of lanraplenib, a novel spleen tyrosine kinase inhibitor, in models of lupus nephritis

BackgroundB cells are critical mediators of systemic lupus erythematosus (SLE) and lupus nephritis (LN), and antinuclear antibodies can be found in the serum of approximately 98% of patients with SLE. Spleen tyrosine kinase (SYK) is a nonreceptor tyrosine kinase that mediates signaling from immunoreceptors, including the B cell receptor. Active, phosphorylated SYK has been observed in tissues from patients with SLE or cutaneous lupus erythematosus, and its inhibition is hypothesized to ameliorate disease pathogenesis. We sought to evaluate the efficacy and characterize the mechanism of action of lanraplenib, a selective oral SYK inhibitor, in the New Zealand black/white (NZB/W) murine model of SLE and LN.MethodsLanraplenib was evaluated for inhibition of primary human B cell functions in vitro. Furthermore, the effect of SYK inhibition on ameliorating LN-like disease in vivo was determined by treating NZB/W mice with lanraplenib, cyclophosphamide, or a vehicle control. Glomerulopathy and immunoglobulin G (IgG) deposition were quantified in kidneys. The concentration of proinflammatory cytokines was measured in serum. Splenocytes were analyzed by flow cytometry for B cell maturation and T cell memory maturation, and the presence of T follicular helper and dendritic cells.ResultsIn human B cells in vitro, lanraplenib inhibited B cell activating factor-mediated survival as well as activation, maturation, and immunoglobulin M production. Treatment of NZB/W mice with lanraplenib improved overall survival, prevented the development of proteinuria, and reduced blood urea nitrogen concentrations. Kidney morphology was significantly preserved by treatment with lanraplenib as measured by glomerular diameter, protein cast severity, interstitial inflammation, vasculitis, and frequency of glomerular crescents; treatment with lanraplenib reduced glomerular IgG deposition. Mice treated with lanraplenib had reduced concentrations of serum proinflammatory cytokines. Lanraplenib blocked disease-driven B cell maturation and T cell memory maturation in the spleen.ConclusionsLanraplenib blocked the progression of LN-like disease in NZB/W mice. Human in vitro and murine in vivo data suggest that lanraplenib may be efficacious in preventing disease progression in patients with LN at least in part by inhibiting B cell maturation. These data provide additional rationale for the use of lanraplenib in the treatment of SLE and LN.

Novel Treatments for Mantle Cell Lymphoma: From Targeted Therapies to CAR T Cells.

Mantle cell lymphoma is a rare B-cell non-Hodgkin's lymphoma that retains a sobering prognosis despite an extensive research effort. Mantle cell lymphoma remains incurable even with aggressive, and at times toxic, chemoimmunotherapy with early incorporation of autologous stem cell transplantation. Given this, attention has turned to the use of targeted therapies addressing dysregulation of B-cell signaling pathways. Drugs such as immunomodulatory agents, proteasome inhibitors, and Bruton's tyrosine kinase inhibitors have shown success in the relapsed/refractory population, and there is ongoing investigation into the utilization of novel Bruton's tyrosine kinase, B-cell leukemia/lymphoma-2, and spleen tyrosine kinase inhibitors alone or in combination in both the front-line and relapsed settings. Other areas of research in novel immunotherapies include investigations of bispecific T-cell engagers and antibody-drug conjugates. Most recently, chimeric antigen receptor T-cell therapy has been granted US Food and Drug Administration approval as a result of durable remissions even in high-risk patients who have classically done poorly with traditional chemoimmunotherapy. The intent of this article is to review the literature describing these selective therapies and discuss their current and future roles in the treatment of mantle cell lymphoma.