Expression of Human nPTB Is Limited by Extreme Suboptimal Codon Content

Background: The frequency of synonymous codon usage varies widely between organisms. Suboptimal codon content limits expression of viral, experimental or therapeutic heterologous proteins due to limiting cognate tRNAs. Codon content is therefore often adjusted to match codon bias of the host organism. Codon content also varies between genes within individual mammalian species. However, little attention has been paid to the consequences of codon content upon translation of host proteins. Methodology/Principal Findings: In comparing the splicing repressor activities of transfected human PTB and its two tissue-restricted paralogs–nPTB and ROD1–we found that the three proteins were expressed at widely varying levels. nPTB was expressed at 1–3 % the level of PTB despite similar levels of mRNA expression and 74 % amino acid identity. The low nPTB expression was due to the high proportion of codons with A or U at the third codon position, which are suboptimal in human mRNAs. Optimization of the nPTB codon content, akin to the ‘‘humanization’ ’ of foreign ORFs, allowed efficient translation in vivo and in vitro to levels comparable with PTB. We were then able to demonstrate that all three proteins act as splicing repressors. Conclusions/Significance: Our results provide a striking illustration of the importance of mRNA codon content in determining levels of protein expression, even within cells of the natural host species

Regulation of Retention of FosB Intron 4 by PTB

One effect of stressors such as chronic drug administration is that sequence within the terminal exon of the transcription factor FosB is recognized as intronic and removed by alternative splicing. This results in an open-reading-frame shift that produces a translation stop codon and ultimately a truncated protein, termed ΔFosB. In vitro splicing assays with control and mutated transcripts generated from a fosB mini-gene construct indicated a CU-rich sequence at the 3′ end of intron 4 (I4) plays an important role in regulating fosB pre-mRNA splicing due to its binding of polypyrimidine tract binding protein (PTB). PTB binding to this sequence is dependent upon phosphorylation by protein kinase A and is blocked if the CU-rich sequence is mutated to a U-rich region. When this mutated fosB minigene is expressed in HeLa cells, the splicing efficiency of its product is increased compared to wild type. Moreover, transient transfection of PTB-1 in HeLa cells decreased the splicing efficiency of a wild type fosB minigene transcript. Depletion of PTB from nuclear extracts facilitated U2AF65 binding to wild type sequence in vitro, suggesting these proteins function in a dynamic equilibrium to modulate fosB pre-mRNA alternative splicing. These results demonstrate for the first time that phosphorylated PTB promotes intron retention and thereby silences the splicing of fosB I4

Clinical Trial to Evaluate an Approved ITP Therapy Targeting Spleen Tyrosine Kinase (SYK) for Prevention and Treatment of COVID-19 Related Complications

Fostamatinib is a potent SYK inhibitor that targets activation of both the innate and acquired immune systems caused by tissue damage or pathogen signals via C-type lectin receptors (CLR) as well as by antibody-antigen immune complexes via Fc receptors (Figure 1). SYK is also a key modulator of pathways involved in thrombosis formation. Fostamatinib is FDA-approved for chronic immune thrombocytopenia (ITP) and has been evaluated in over 4000 patients for treatment of allergic, autoimmune, and neoplastic disorders. Fostamatinib is well tolerated, with a consistent safety profile across a number of diseases. In rheumatoid arthritis patients, fostamatinib reduced the plasma levels of IL-6.1 The active metabolite (R406) was protective in mouse models of LPS-induced acute lung injury (ALI)2 and LPS- or antibody-induced acute kidney injury (AKI).3 SYK inhibition decreased the incidence of thrombosis in mouse models of thromboembolism,4 and the incidence of thromboembolic events was decreased in patients treated with fostamatinib compared with other treatments for ITP. These immunomodulatory effects suggest that SYK inhibition represents a new therapeutic strategy for the treatment of COVID-19, which led to design of a clinical study of fostamatinib in hospitalized COVID-19 patients. COVID-19 patients are at risk for a potentially fatal acute respiratory distress syndrome (ARDS), cytokine storm, severe systemic capillary leak syndrome, thromboembolic events, and multi-organ dysfunction/failure. SYK is a master regulator of signal transduction pathways implicated in these COVID-19 associated complications, which involves hyperactivation of both innate and acquired immune systems. Targeting SYK could prevent the cytokine storm, downstream activation of endothelial cells and platelets, and influx of neutrophils and monocytes into lungs or kidney leading to ALI and AKI, respectively. Methods: A randomized, double-blind, placebo-controlled, multi-center, Phase 3 study will evaluate the efficacy and safety of fostamatinib in adult, hospitalized, high-risk COVID-19 patients. Patients will be treated with fostamatinib 150mg BID for up to 28 days plus standard of care. Improvement in clinical status will be assessed using the 8-point ordinal scale. References 1. Weinblatt ME et al.Arth Rheum2008;58:3309-18 2. Nadeem A et al.Int Immunopharm2019;68:39-47 3. Al-Harbi NO, et al.Biochimie2019;158: 102-10 4. Van Eeuwijk JMM et al.Arterioscler Thromb Vasc Biol2016;36:1247-53 Results and Conclusions: Trial in Progress Disclosures Dummer: Rigel:Current Employment, Current equity holder in publicly-traded company.Markovtsov:Rigel:Current Employment, Current equity holder in publicly-traded company.Tong:Rigel:Current Employment, Current equity holder in publicly-traded company.Masuda:Rigel:Current Employment, Current equity holder in publicly-traded company. </jats:sec

AB0058 CELL-TYPE SPECIFIC REGULATION OF IL-1R SIGNALING BY R835, A DUAL IRAK1/4 INHIBITOR

Background:Interleukin-1 beta (IL-1b) is a key mediator of the inflammatory response and is known to exacerbate damage during chronic disease and acute tissue injury. Through association with the adaptor protein Myd88, interleukin receptor associated kinases (IRAK)1 and 4 initiate signaling downstream of IL-1Rs resulting in the activation of the NFkB and MAPK pathways and the production of proinflammatory cytokines (1). IL-1Rs are broadly expressed across cell types and little is known about differences in signaling between cell types and the role of IRAK1 and IRAK4 kinase activity.Objectives:We have identified a potent and selective IRAK1/4 inhibitor, R835, that substantially suppressed the elevation of LPS (TLR4 agonist)-induced serum cytokines in healthy human volunteers in a recent phase 1 study. The aim of this study was to evaluate the effect of R835 on IL-1R signaling in primary human fibroblasts and endothelial cells.Methods:Human dermal fibroblasts, lung fibroblasts or endothelial cells were stimulated with IL-1b and the effect of R835 on the signaling pathway was evaluated by western blotting. Human dermal fibroblasts were stimulated with different amounts of IL-1b to evaluate both the signaling pathways activated and the cytokines produced. The ability of R835 to inhibit cytokine production induced by high or low amounts of IL-1b in dermal fibroblasts was assessed.Results:In human endothelial cells, inhibition of IRAK1/4 kinases with R835 resulted in a block of IL-1b-induced IRAK4 phosphorylation, IRAK1 degradation and downstream NFkB, p38 and JNK activation. In contrast, in both human primary dermal and lung fibroblasts stimulated with IL-1b, we observed potent inhibition of IRAK4 phosphorylation, IRAK1 degradation, and downstream JNK phosphorylation, but no inhibition of NFkB pathway proteins and only weak inhibition of p38. Upon titration of IL-1b we observed that dermal fibroblasts produced IL-8 and GRO in response to low levels of IL-1b (20pg/ml), and produced additional cytokines including G-CSF and GM-CSF with higher levels of IL-1b (400pg/ml). In the presence of low levels of IL-1b (20pg/ml), we observed a weak activation of NFkB pathway proteins and p38, compared to a very robust NFkB, p38 and additional JNK activation in the presence of higher levels of IL-1b (400pg/ml). Consistent with these results, in dermal fibroblasts, R835 showed little to no inhibition of IL-8 and GRO induced by low levels of IL-1b, but potently inhibited G-CSF and GM-CSF induced by high levels of IL-1b where JNK was activated.Conclusion:This study has elucidated signaling differences between cell types downstream of the IL-1R. In endothelial cells, as in myeloid cells, the kinase activity of IRAK1 and IRAK4 is required for the activation of all downstream signaling. Unexpectedly, in human fibroblasts, IRAK1/4 kinase activity appears to primarily regulate the JNK pathway, and not the NFkB pathway. Concomitant with that, only the cytokines induced by the additional activation of JNK in fibroblasts are regulated by a dual IRAK1/4 inhibitor. Clinically, an IRAK1/4 inhibitor may show select inhibition of IL-1b-induced cytokines depending on the tissue and cell type involved in inflammation.References:[1]Flannery S, Bowie A G. The interleukin-1 receptor-associated kinases: Critical regulators of innate immune signaling. Biochemical Pharmacology, Volume 80, Issue 12, 15 December 2010, Pages 1981-1991.Disclosure of Interests:Sylvia Braselmann Shareholder of: Shareholder of Rigel Pharmaceuticals, Employee of: Employee of Rigel Pharmaceuticals, Ernest Tai Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Roy Frances Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Chi Young Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Vadim Markovtsov Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Esteban Masuda Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Vanessa Taylor Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals</jats:sec

Prediction of Response to Fostamatinib Based on the Presence of Plasma Platelet Autoantibodies in Adult Patients with Immune Thrombocytopenia (ITP) — Exploratory Analyses from Phase 3 Studies

Abstract Background: Initial results of an analysis of anti-platelet autoantibodies in patients with ITP from two phase 3 studies showed that patients were more likely to respond to treatment with the SYK inhibitor fostamatinib if they were positive for anti-platelet autoantibodies. Stable platelet responses to fostamatinib were reported in 36% of 28 patients who tested positive for anti-platelet autoantibodies and in 9% of 32 patients who tested negative (p&lt;0.02). This analysis has been expanded to include 85 patients and to evaluate various platelet antigens. Methods: Plasma samples from 85 patients in the phase 3 studies were collected with informed consent at 2 weeks after the first dose of fostamatinib. Samples were analyzed for anti-platelet autoantibodies via Luminex beads-based PakLx assayand FACS-based indirect platelet immunofluorescence test (iPIFT) using platelets from healthy volunteers. Results: Of 85 patient samples, 38 (45%) were positive for one or more anti-platelet autoantibodies and 19 (22%) were from patients with a stable platelet response to fostamatinib. Of 38 patients positive for autoantibodies, 13 (34%) had stable platelet responses. Of 47 patients negative for autoantibodies, 6 (13%) had stable platelet responses. Conclusions: The presence of anti-platelet autoantibodies appeared to be associated with a stable platelet response to fostamatinib. This exploratory analysis provides support for the hypothesis that fostamatinib prevents platelet loss through inhibition of autoantibody-directed platelet destruction in ITP. Further studies are ongoing. Disclosures Markovtsov: Rigel: Employment, Equity Ownership. Yi:Rigel: Employment, Equity Ownership. Young:Rigel: Employment, Equity Ownership. Duliege:Rigel: Employment, Equity Ownership. Masuda:Rigel Pharmaceuticals: Employment, Equity Ownership. </jats:sec

Potential Anti-Thrombotic Effect without Accompanying Hemorrhage with Fostamatinib Use in Patients with Immune Thrombocytopenia

Background Immune thrombocytopenia (ITP) results from autoimmune antibody-mediated destruction of platelets. Bruising and bleeding are hallmarks of ITP, but thromboembolic events (TEEs) are also observed in ITP patients, even thrombocytopenic ones. In population-based cohort studies, chronic ITP patients had two-fold higher risk of a venous TEE compared to the general population.1 The risk of venous or arterial TEE is further elevated in splenectomized ITP patients and those on thrombopoietin receptor agonists (TPO-RAs). Other risk factors include: age &gt; 60 years, prolonged corticosteroid treatment, diabetes, hypertension, coronary disease, history of TEEs, presence of anti-phospholipid antibodies, chronic kidney disease, and male sex. Fostamatinib, an inhibitor of spleen tyrosine kinase (SYK), is approved for treatment of thrombocytopenia in adults with ITP. Extensive preclinical data suggest that inhibition of SYK signaling may also have an anti-thrombotic effect. In a model of acute stroke, mice with platelet-specific SYK deficiency were protected from aortic thrombosis and also had diminished size of brain infarction with only marginal effect on hemostasis.2 These results were reproduced by a SYK inhibitor in this model.2 We assessed rates of thrombosis among ITP patients treated with fostamatinib compared to those reported with TPO-RAs. Methods We reviewed the incidence of TEEs during fostamatinib clinical trials in ITP, including 2, randomized, double-blind, placebo-controlled, phase 3, multicenter clinical trials and an open-label extension study. The incidence of TEEs with TPO-RAs during published phase 3 clinical trials and post-phase 3 extension studies was also reviewed. Results The study population comprised 145 ITP patients who received fostamatinib and included 37 (26%) patients &gt;65 years, 51 (35%) splenectomized patients, and 58 (40%) male patients. The 3 fostamatinib clinical trials represent 163 patient-years of fostamatinib exposure. The only reported TEE was a transient ischemic attack (0.7%), which resolved spontaneously in a patient with pre-existing atherosclerosis. The rate of TEEs reported in ITP patients receiving TPO-RAs in multiple studies ranged from 0 to 9% (Table). Discussion The rate of TEEs observed in ITP patients receiving fostamatinib was very low (&lt; 1%) in comparison with the rates of TEEs reported in ITP patients receiving a TPO-RA (up to 9%). ITP patients have higher levels of prothrombin fragments 1 + 2, D-dimer, PAI-1 and soluble P-selectin, which contribute to a procoagulant profile. TPO-RA treatment may cause an increase in PAI-1, P-selectin, and increase microparticles and platelet apoptosis, all of which predispose to onset of TEEs.3,4 What explains the apparent paradox of fostamatinib? SYK plays a key role in signaling of the ITAM receptors GPVI and CLEC-2 in platelets. Both GPVI and CLEC-2 play an important role in thrombosis but are dispensable for normal hemostasis. Consequently, SYK inhibition likely reduces the incidence of TEEs by abrogating the GPVI and CLEC-2 pathways in platelets. R406, the active metabolite of fostamatinib, diminished GPVI and CLEC-2 mediated platelet aggregation with marginal effect on normal hemostasis in vivo.2,5 These mechanisms potentially explain why ITP patients treated with fostamatinib are protected from developing TEEs despite often substantial platelet increases. SYK also plays a key role in Fcγ receptor dependent phagocytosis of auto-antibody coated platelets, the primary disease mechanism in ITP. Thus, SYK inhibition in ITP may simultaneously decrease platelet destruction (increasing platelet count) and decrease the incidence of TEEs. Suumary Current clinical trial experience with fostamatinib in ITP patients demonstrates much lower rates of thrombosis than would be expected in ITP patients compared to other agents. Future work will prospectively evaluate this and confirm in vivo downregulation of platelet activation via inhibition of SYK signaling in platelets. 1. Rodeghiero F. Am J Hematol, 2016. 91: 39-45. 2. van Eeuwijk JM, et al. Arterioscler Thromb Vasc Biol, 2016. 36: 1247-53. 3. Garabet, L., et al. Platelets, 2019. 30: 206-12. 4. Justo Sanz, R., et al. Thromb Haemost, 2019. 119: 645-59. 5. Spalton, J.C., et al. J Thromb Haemost, 2009. 7: 1192-9. Disclosures Altomare: Novartis: Consultancy; Amgen: Consultancy; Rigel: Consultancy; Incyte: Consultancy, Speakers Bureau. Markovtsov:Rigel: Employment, Equity Ownership. Todd:Rigel: Employment, Equity Ownership. Weerasinghe:Rigel: Employment. Numerof:Rigel: Employment, Equity Ownership. Tong:Rigel: Employment, Equity Ownership. Masuda:Rigel: Consultancy, Employment, Equity Ownership. Bussel:Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; 3S Bio: Speakers Bureau; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees; UCB: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; argenx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; RallyBio: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Regeneron: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Kezar Life Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Physician Education Resource: Speakers Bureau; Tranquil: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Dova Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Momenta Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Fostamatinib is a tyrosine kinase (SYK) inhibitor for the treatment of adult patients with chronic immune thrombocytopenia (ITP) who have had an insufficient response to a previous treatment. The use of fostamatinib in other diseases is off-label. </jats:sec

OP0133 PRECLINICAL EFFICACY OF R835, A NOVEL IRAK1/4 DUAL INHIBITOR, IN RODENT MODELS OF JOINT INFLAMMATION

Background:Interleukin receptor associated kinases (IRAK) 1 and 4 are kinases involved in Toll-Like Receptor (TLR) and Interleukin-1 Receptor (IL-1R) signaling pathways, which regulate innate immunity and inflammation. Dysregulation of IRAK1/4 signaling can lead to a variety of inflammatory conditions including rheumatoid and gouty arthritis. As a result, IRAK1/4 are promising therapeutic targets for rheumatic diseases (1). We have identified a potent and selective IRAK1/4 inhibitor, R835, that substantially suppressed the elevation of LPS (TLR4 agonist)-induced serum cytokines in healthy human volunteers in a recently completed phase 1 study.Objectives:The aim of our study was to investigate the effect of IRAK1/4 selective inhibition as a potential therapeutic approach for rheumatological diseases. We evaluated the inhibition by our clinical candidate, R835, on TLR-, IL-1R- and NLRP3 inflammasome-induced cytokine production, as well as in preclinical models of arthritis.Methods:The effect of R835 on TLR- or IL-1R-induced cytokine production was evaluated in vitro using THP-1, human primary endothelial cells and human primary dendritic cells. The activity of R835 on the NLRP3 inflammasome was also tested in vitro using THP-1 cells. The pharmacokinetic-pharmacodynamic relationship of R835 was evaluated in a mouse model of IL-1b-induced cytokine release. Mice were pre-treated orally with vehicle or R835 prior to challenge; serum cytokine and plasma compound levels were determined. The efficacy of IRAK1/4 inhibition by R835 in rodent models of joint inflammation was evaluated in a mouse model monosodium (MSU)-induced peritonitis, in rat model of MSU-induced gouty arthritis and in a rat model of collagen-induced arthritis (CIA).Results:In human cells, R835 blocked proinflammatory cytokine production in response to TLR, IL-1R and NLRP3 inflammasome activation. In mice, R835 dose-dependently decreased serum cytokines in response to administration of IL-1b. Mice pre-treated with R835 demonstrated dose-dependent reductions in MSU crystal-induced serum and peritoneal cytokine levels, as well as neutrophil influx in the peritoneal cavity. Prophylactic and therapeutic treatment with R835 also resulted in significant inhibition of MSU crystal-induced knee edema and pain in a rat model of human gouty arthritis. In the rat model of CIA, R835 blocked both onset and progression of disease, by reducing inflammation, cartilage degeneration and synovial inflammation.Conclusion:R835 is a promising clinical candidate for the treatment of a range of cytokine-driven rheumatological diseases. R835 has proven to have desirable pharmacokinetic properties, was well tolerated and suppressed LPS-induced serum cytokines in healthy volunteers in a recent phase 1 study.References:[1]Bahia M S, Kaur M, Silakari P, Silakari O. Interleukin-1 receptor associated kinase inhibitors: Potential therapeutic agents for inflammatory- and immune-related disorders. Cellular Signalling 27 (2015) 1039–1055.Disclosure of Interests:Chrystelle Lamagna Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Meagan Chan Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Ernest Tai Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Stacey Siu Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Roy Frances Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Sothy Yi Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Chi Young Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Vadim Markovtsov Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Yan Chen Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Lu Chou Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Gary Park Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Esteban Masuda Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Vanessa Taylor Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals</jats:sec