JAK Inhibitor Tofacitinib Research Articles

Identification of novel off targets of baricitinib and tofacitinib by machine learning with a focus on thrombosis and viral infection

As there are no clear on-target mechanisms that explain the increased risk for thrombosis and viral infection or reactivation associated with JAK inhibitors, the observed elevated risk may be a result of an off-target effect. Computational approaches combined with in vitro studies can be used to predict and validate the potential for an approved drug to interact with additional (often unwanted) targets and identify potential safety-related concerns. Potential off-targets of the JAK inhibitors baricitinib and tofacitinib were identified using two established machine learning approaches based on ligand similarity. The identified targets related to thrombosis or viral infection/reactivation were subsequently validated using in vitro assays. Inhibitory activity was identified for four drug-target pairs (PDE10A [baricitinib], TRPM6 [tofacitinib], PKN2 [baricitinib, tofacitinib]). Previously unknown off-target interactions of the two JAK inhibitors were identified. As the proposed pharmacological effects of these interactions include attenuation of pulmonary vascular remodeling, modulation of HCV response, and hypomagnesemia, the newly identified off-target interactions cannot explain an increased risk of thrombosis or viral infection/reactivation. While further evidence is required to explain both the elevated thrombosis and viral infection/reactivation risk, our results add to the evidence that these JAK inhibitors are promiscuous binders and highlight the potential for repurposing.

Empagliflozin inhibits intermittent hypoxia‐induced TRAF3IP2/NF‐κB/HIF‐1α/IL‐6‐dependent human aortic smooth muscle cell proliferation

Chronic intermittent hypoxia (IH), a characteristic feature of obstructive sleep apnea and chronic obstructive pulmonary disease, contributes to cardiovascular diseases, including atherosclerosis, through persistent oxidative stress and inflammation. During atherogenesis, vascular smooth muscle cells acquire a proinflammatory and a proliferative phenotype. TRAF3IP2 (TRAF3 Interacting Protein 2) is an oxidative stress‐responsive proinflammatory adapter molecule and plays a causal role in a preclinical model of atherosclerosis. Therefore, we tested the hypothesis that IH stimulates human aortic smooth muscle cell (SMC) proliferation via TRAF3IP2, and this effect is inhibited by the SGLT2 (Sodium/Glucose Cotransporter 2) inhibitor empagliflozin. Results show that IH upregulates TRAF3IP2 expression, TRAF3IP2‐dependent superoxide, hydrogen peroxide and nitric oxide production, HIF‐1α and NF‐κB activation, IL‐6 expression, and SMC proliferation. These effects were inhibited by TRAF3IP2, NOX2, NOX4, p65 or IL6R knockdown, or pre‐exposure to potent and specific iNOS inhibitor AMT or 1400W, the JAK inhibitor Tofacitinib, the STAT3‐specific inhibitor HO‐3867, or the gp130 inhibitor SC144. Importantly, SMC express SGLT2, and pretreatment with empagliflozin abrogated IH‐induced TRAF3IP2 induction, nitroxidative stress, HIF‐1α and NF‐κB activation, IL‐6 expression, and SMC proliferation. In summary, these results suggest the therapeutic potential of empagliflozin in IH‐associated inflammatory vascular proliferative diseases.

P099 Invasive listeriosis in patients with immune-mediated inflammatory diseases on biologics and targeted therapies - a review of the literature

Abstract Background/Aims Listeria monocytogenes is a gram-positive bacterium found ubiquitously in the environment. Human infection is relatively rare and usually occurs after consumption of contaminated food. L. monocytogenes can cause an uncomplicated gastroenteritis in the immunocompetent. Vulnerable individuals are at risk of invasive disease. These include the elderly, pregnant women, neonates and people with impaired immunity, including those treated with biologics and targeted therapies for immune-mediated inflammatory diseases (IMIDs). Invasive listeriosis typically causes bacteraemia, meningoencephalitis and maternal-neonatal infection. Other reviews have described an association between TNF-alpha inhibitors and invasive listeriosis, however the impact of newer biologics and the JAK-inhibitors has not been described. Methods The literature was systemically reviewed to assess the incidence of invasive listeriosis in patients with IMIDs treated with biologics or targeted therapies using Ovid Medline, EMBASE and the Cochrane Database of Systematic Reviews. Papers including patients over 16 years of age with rheumatoid arthritis (RA), ankylosing spondylitis (AS), axial spondyloarthritis, psoriatic arthritis, psoriasis, inflammatory bowel disease (IBD), (ulcerative colitis or Crohn’s disease), managed with biologics or targeted therapies were included. Abstracts, case reports, case series, cohort studies, registry studies, randomised controlled trials and meta-analyses were included. Reports not published in English or not detailing the name of the biologic or targeted therapy used were excluded. The primary outcome was incidence of invasive listeriosis defined as isolation of L. monocytogenes from a normally sterile site. Results Thirty-eight case reports, three disease-specific registry analyses, two randomised controlled trials and one meta-analysis were included in the final results. Together these studies reported 75 cases of invasive listeriosis in patients with IMIDs on biologics and targeted therapies. Seventy cases were described in patients treated with TNF-alpha inhibitors (49 with infliximab, nine with adalimumab, one with certolizumab pegol, one with golimumab and ten with etanercept). Two cases were described in patients treated with IL-12/IL-23 inhibitors (one with ustekinumab and one with guselkumab), one case was described in a patient treated with a gut-selective integrin antagonist (vedolizumab), one case in a patient treated with tocilizumab (an IL-6 inhibitor) and one case in a patient treated with a JAK-inhibitor (tofacitinib). Thirty-four patients had RA, 31 had IBD, five had psoriatic arthritis, two had ankylosing spondylitis, one had both psoriasis and psoriatic arthritis, one had psoriasis and another an unknown IMID. The reporting of listeriosis cases with TNF-alpha inhibitors has remained fairly steady over time so this association is not purely an early reporting phenomenon. Conclusion Cases of invasive listeriosis have been reported across a range of different IMIDs, most often in patients with RA and IBD. Invasive listeriosis is not unique to patients treated with TNF-alpha inhibitors. The association between invasive listeriosis and other targeted therapies (e.g. IL-23 inhibition) needs further study. Disclosure V. Allen: None. E. Alveyn: None. J. Galloway: Honoraria; AbbVie, Celgene, Chugai, Gilead, Janssen, Eli Lilly, Pfizer, Roche and UCB.

P602 Tofacitinib treatment prevents post-operative recurrence of Crohn’s Disease modeled by ileocecal resection in HLA-B27 transgenic rats

Abstract Background Post-operative recurrence (POR) after ileocecal resection (ICR) affects most of Crohn’s disease (CD) patients. Immunomodulatory treatments have not proven efficiency in preventing POR except for anti-TNFα biologic agents. We evaluate the efficacy of the JAK inhibitor Tofacitinib to prevent POR after ICR in HLA-B27 transgenic rat model (TgB27). Methods Thirty-three rats (strain F344, 29 TgB27, 4 control non Tg (nTg) rats) were operated on an ICR at the 12th week (W12) of life and were sacrificed at W18. Tofacitinib (15 mg/kg, n=17) or placebo (n=16) were daily administered by oral gavage at W10 until W18. Anastomotic recurrence were blindly analyzed using macroscopic (from 0 to 4) and histologic (from 0 to 6) scores. Luminal microbiome was analyzed by shotgun metagenomic sequencing (Hiseq Illumina) in 9/33 rats before (W9) and after Tofacitinib treatment or placebo (W12 and W18). STAT3 phosphorylation was quantified in ileal samples by western blotting. Data were expressed by median with IQR. Comparisons were performed using not parametric two tailed Mann-Whitney analysis for unmatched data and Wilcoxon matched-pairs test for matched data. Two tailed χ2 test was used for contingency analysis. Spearman rank correlation was used for microbiota analysis. Results 8/14 TgB27 rats receiving placebo (57%) showed signs of POR (macroscopic score ≥ 2) which were significantly prevented in 2/15 TgB27 rats (13%) treated with Tofacitinib (p= 0.013). nTg animals did not show macroscopic signs of POR. Macroscopic lesions were located 2 cm around the anastomosis in the TgB27 placebo group and consisted in ulcerations with a score of 2 (1–2.25). 11/15 TgB27 rats (73%) treated with Tofacitinib did not show macroscopic lesions and their score were significantly improved (0 (0–1), p= 0,0006). Histologic scores were significantly higher in TgB27 placebo group vs TgB27 Tofacitinib-treated rats (3 (2–4) vs 2 (1–3), p=0.036). Tofacitinib did not impact on alpha diversity and the relative abundance of bacterial taxa either before and after surgery. In contrast, surgery led to a significant 47% decrease of alpha diversity (p=0.0008) and loss of specific taxa in both TgB27 and nTg animals. No correlation was found between individual lesion scores and alpha diversity indexes. Ileal pSTAT3/STAT3 ratios were decreased by 80% in TgB27 rats treated with Tofacitinib (n=4) compared to placebo group (n=4) (0.3 ± 0,16 vs 1.58 ± 0,3, p=0,028). Conclusion We develop a reliable model of ICR in TgB27 rats mimicking the POR of CD. Tofacitinib prevents the macroscopic and histologic POR after ICR, without major modifications of the microbioma. Acknowledgments: Charity Foundation DigestScience and Pfizer SAS

Endoscopy as a Diagnostic Tool for Personalized Therapy in IBD: Prediction of Response to Biological Therapy.

Both Crohnʼs disease [1] and ulcerative colitis [2] are chronic relapsing disorders of the gastrointestinal tract that are characterized pathologically by intestinal inflammation. Both inflammatory bowel disease (IBD) entities are progressive conditions that can lead to bowel damage and disability, having a major impact on an individualʼs quality of life. Furthermore, ongoing inflammatory activity is causative for occurrence of strictures, fistula, abscesses, as well as heightened incidence of colitis-associated neoplasia. Optimized anti-inflammatory therapy is therefore essential in the management of IBD patients [1], [2]. Advances in understanding the underlying immunopathogenesis in ulcerative colitis have led to the development of biological therapies, which selectively inhibit crucial pathways of involved inflammatory processes. Blockade of the pro-inflammatory cytokine tumor necrosis factor (TNF) by neutralizing antibodies was the first approved treatment modality, which was complemented by the anti-adhesion antibody vedolizumab directed against the α4β7 integrin. Subsequently, the anti-IL-12/IL-23p40 antibody ustekinumab and the JAK-inhibitor tofacitinib have broadened our therapeutic armamentarium. In spite of their effectiveness, it was found that only subgroups of IBD patients benefit from these therapies. Depending on the duration and the clinical endpoints chosen, only 30 – 60% of patients will show an improvement of active disease. There is therefore the currently unmet clinical need to predict therapeutic responses prior to the initiation of therapy. Reliable prediction would enable the treating physician to directly introduce the patient to the most efficient therapy, which would enable time-efficient control of the disease and prevent the onset of irreversible structural damage. This would also be essential to avoid exposure of patients to associated potential side effects of an inefficient therapy and reduce the substantial costs of an inappropriate therapy for the health care systems [3].

Rationale and design of a mechanistic clinical trial of JAK inhibition to prevent ventilator-induced diaphragm dysfunction

IntroductionVentilator-induced diaphragm dysfunction (VIDD) is an important phenomenon that has been repeatedly demonstrated in experimental and clinical models of mechanical ventilation. Even a few hours of MV initiates signaling cascades that result in, first, reduced specific force, and later, atrophy of diaphragm muscle fibers. This severe, progressive weakness of the critical ventilatory muscle results in increased duration of MV and thus increased MV-associated complications/deaths. A drug that could prevent VIDD would likely have a major positive impact on intensive care unit outcomes. We identified the JAK/STAT pathway as important in VIDD and then demonstrated that JAK inhibition prevents VIDD in rats. We subsequently developed a clinical model of VIDD demonstrating reduced contractile force of isolated diaphragm fibers harvested after ∼7 vs ∼1 h of MV during a thoracic surgical procedure. Materials and methodsThe NIH-funded clinical trial that has been initiated is a prospective, placebo controlled trial: subjects undergoing esophagectomy are randomized to receive 6 preoperative doses of the FDA-approved JAK inhibitor Tofacitinib (commonly used for rheumatoid arthritis) vs. placebo. The primary outcome variable will be the difference in the reduction that occurs in force generation of diaphragm single muscle fibers (normalized to their cross-sectional area), in the Tofacitinib vs. placebo subjects, over 6 h of MV. DiscussionThis trial represents a first-in-human, mechanistic clinical trial of a drug to prevent VIDD. It will provide proof-of-concept in human subjects whether JAK inhibition prevents clinical VIDD, and if successful, will support an ICU-based clinical trial that would determine whether JAK inhibition impacts clinical outcome variables such as duration of MV and mortality.

Evaluation of the effectiveness of Tofacitinib in rheumatoid arthritis in real clinical practice: The relationship between pain relief in the first 4 weeks and disease activity after 3–6 months

The JAK inhibitor tofacitinib (TOFA) blocks the intracellular signaling pathway that activates the synthesis of cytokines and mediators involved in the development of pain and central sensitization (CS), which determines the rapid analgesic effect. However, it is not clear how pain reduction is associated with achieving low activity in rheumatoid arthritis (RA).The aim of the studywas to assess the relationship between the early clinical response to tofacitinib and a decrease in rheumatoid arthritis activity after 3 and 6 months.Material and methods.The study group consisted of 88 RA patients (age – 53±11.5 years; 79.3% of women) who received basic anti-inflammatory drugs (59.5% – methotrexate, 19.8% – leflunomide) and who were prescribed TOFA in a dose 10 mg/day. Seropositivity for rheumatoid factor was 89.8%; the value of the DAS28 index is 5.2±1.2. The severity of pain was assessed using the Brief Pain Inventory questionnaire, the neuropathic component of pain (NCP) – using the PainDETECT questionnaire, signs of CS – using the Central Sensitization Inventory (CSI) questionnaire in the early stages after the administration of TOFA, RA activity – using the DAS28-CRP index after 3 and 6 months.Results.The mean severity of pain at baseline was 5.3±2.0 on the visual analogue scale (VAS); 51.1% of patients had signs of CS (CSI>40), 15.9% had NCP (PainDETECT>18). 7 days after the start of therapy, there was a significant decrease in pain – to 4.1±1.8 according to VAS (p<0.05) and CS – 40.4±13.5 to 36.5±12.5 according to CSI (p=0.01). After 28 days, the effect was even more significant: the level of pain according to the VAS was 2.8±1.6 (p=0.000), the NCP decreased from 11.8±5.6 to 6.8±3.1 (p=0.000), CS – up to 31.6±13.9 (p=0.000). The value of the DAS28-CRP index after 3 and 6 months was 3.7±1.3 and 3.6±1.2, respectively. The number of patients with pain relief ≥50% after 28 days was 59.9%, low RA activity after 3 months. (DAS28-CRP≤3.2) was acieved in 64.4% of patients. There was a clear correlation between the number of patients with a pain reduction of ≥50% at 28 days and the number of patients who achieved low RA activity at 3 and 6 months. (rS=0.548, p=0.000 and rS=0.790, p=0.000). 6 patients dropped out of the study due to inefficiency or social reasons. No serious adverse reactions were noted.Conclusions.The use of the JAK inhibitor TOFA allows achieving a quick analgesic effect and reducing the signs of CS. An early clinical response to TOFA (pain relief) predicts a decrease in RA activity after 3 and 6 months of therapy.

Tofacitinib Ameliorates Lupus Through Suppression of T Cell Activation Mediated by TGF-Beta Type I Receptor.

Autoreactive T cells play a crucial role in the pathogenesis of systemic lupus erythematosus (SLE). TGF-β type I receptor (TGFβRI) is pivotal in determining T cell activation. Here, we showed that TGFβRI expression in naïve CD4+ T cells was decreased in SLE patients, especially in those with high disease activity. Moreover, IL-6 was found to downregulate TGFβRI expression through JAK/STAT3 pathway in SLE patients. In vitro, the JAK inhibitor tofacitinib inhibited SLE T cell activating by upregulating TGFβRI expression in a dose-dependent manner. In MRL/lpr mice, tofacitinib treatment ameliorated the clinical indicators and lupus nephritis, as evidenced by reduced plasma anti-dsDNA antibody levels, decreased proteinuria, and lower renal histopathological score. Consistently, tofacitinib enhanced TGFβRI expression and inhibited T cell activation in vivo. TGFβRI inhibitor SB431542 reversed the effects of tofacitinib on T cell activation. Thus, our results have indicated that tofacitinib can suppress T cell activation by upregulating TGFβRI expression, which provides a possible molecular mechanism underlying clinical efficacy of tofacitinib in treating SLE patients.

The JAK inhibitor Tofacitinib inhibits structural damage in osteoarthritis by modulating JAK1/TNF-alpha/IL-6 signaling through Mir-149-5p.

Osteoarthritis (OA), a common articular bone degenerative disease, is exacerbated by proinflammatory cytokine signaling. Mounting evidence suggests that epigenetic modifiers, namely microRNAs (miRs), are dysregulated in articular chondrocytes (ACs) during OA. An initial database search led to the identification of miR-149-5p, which was downregulated in clinical OA samples and contributed to chronic inflammation, by increasing TNF-α/IL-6 signaling within the synovium, and OA progression. We overexpressed miR-149-5p in the human chondrocyte cell lines C20A4 and C28/I2 to examine its role in chondrocyte hypertrophy and osteoclastogenesis and found a significant decrease in IL-6 expression, an increase in SOX9 expression, and a reduction in chondrocyte hypertrophy. We evaluated the therapeutic effects of tofacitinib (JAK inhibitor) by suppressing inflammation and restoring miR-149-5p expression. Tofacitinib-treated C20A4 and C28/I2 cells had a significantly lower expression of JAK/IL-6/TNF-α and an increased level of miR-149-5p. Notably, tofacitinib treatment reduced AC hypertrophy and secretion of RANKL and IL-6. Finally, an OA mouse model was used to evaluate the therapeutic potential of tofacitinib. Intra-articular injection of tofacitinib significantly lowered arthritis scores and bone degradation in treated mice compared with their control counterparts. We show for the first time that tofacitinib suppresses the expression level of JAK1/TNF-α/IL-6 by upregulating miR-149-5p level. Our findings revealed the functional association between proinflammatory JAK1/TNF-α/IL-6 signaling and ACs development and highlight the therapeutic potential of tofacitinib in OA.

AB0135 A VERY EARLY CLINICAL RESPONSE TO TOFACITINIB IS ASSOCIATED WITH LOW RHEUMATOID ARTHRITIS ACTIVITY AFTER 3 AND 6 MONTHS

Background:JAK inhibitors block intracellular signaling pathways responsible for the synthesis of cytokines and mediators involved in the development of chronic pain and central sensitization (CS). This determines a very rapid clinical response to JAK inhibitors. However, it is not clear how the significant pain reduction in the first weeks of therapy is associated with the achievement of low rheumatoid arthritis (RA) activity.Objectives:to assess the relationship between the early clinical response to tofacitinib and the decrease in RA activity after 3 and 6 months.Methods:Study group included 88 patients with RA, their age was 53±11,5, 79.3% of women, 89.8% of RF “+”, DAS28 5.2±1.2, receiving DMARDs (methotrexate 59.5% and leflunomide 19.8%), who were administered with tofacitinib 5 mg 2 times a day due to inefficacy or intolerance of biological DMARDs. There were assessed the pain severity using Brief pain inventory (BPI) questionnaire, the presence of neuropathic pain component (NPC) using PainDETECT questionnaire and signs of CS using Central Sensitisation Inventory (CSI) questionnaire at early time after tofacitinib administration, RA activity using DAS28 after 3 and 6 months.Results:The mean pain severity at baseline was 5.3±2.0 according to the visual analogue scale (VAS 0-10), 51.1% of patients had signs of central sensitization (CSI ≥ 40), 15.9% had NPC (PainDETECT ≥18). 7 days after tofacitinib intake there was statistically reliable reduction of pain severity – up to 4.1±1.8 (р<0.05) and CS – CSI from 40.4±13.5 to 36.5±12.5 (р=0.01). After 28 days, the effect was higher: the pain level (VAS) was 2.8±1.6 (p=0.000), PainDETECT decreased from 11.8±5.6 to 6.8±3.1 (p=0.000), CSI – to 31.6±13.9 (p=0.000). DAS28 after 3 and 6 months was 3.7±1.3 and 3.6±1.2. The number of patients with pain decrease of ≥50% after 28 days of therapy was 59.9%. Low RA activity after 3 months. (DAS28 ≤3.2) was achieved in 64.4% of patients. There was a clear correlation between the number of patients with significant pain reduction at 28 days and the number of patients with low RA activity after 3 and 6 months (rS=0.548, p=0.000; rS=0.790, p=0.000). Six patients withdrew from the study due to inefficacy or social reasons. There were no serious adverse reactions.Conclusion:The application of JAK inhibitor tofacitinib allows to reach a fast analgesic effect and reduce CS signs. An early clinical response to tofacitinib (pain relief) predicts a decrease in RA activity after 3 and 6 months of the therapy.Limitation: Open-label observatory study.Disclosure of Interests:None declared

Homozygous variant p. Arg90His in NCF1 is associated with early-onset Interferonopathy: a case report

BackgroundBiallelic loss-of-function variants in NCF1 lead to reactive oxygen species deficiency and chronic granulomatous disease (CGD). Heterozygosity for the p.Arg90His variant in NCF1 has been associated with susceptibility to systemic lupus erythematosus, rheumatoid arthritis, and Sjögren’s syndrome in adult patients. This study demonstrates the association of the homozygous p.Arg90His variant with interferonopathy with features of autoinflammation and autoimmunity in a pediatric patient.Case presentationA 5-year old female of Indian ancestry with early-onset recurrent fever and headache, and persistently elevated antinuclear, anti-Ro, and anti-La antibodies was found to carry the homozygous p.Arg90His variant in NCF1 through exome sequencing. Her unaffected parents and three other siblings were carriers for the mutant allele. Because the presence of two NCF1 pseudogenes, this variant was confirmed by independent genotyping methods. Her intracellular neutrophil oxidative burst and NCF1 expression levels were normal, and no clinical features of CGD were apparent. Gene expression analysis in peripheral blood detected an interferon gene expression signature, which was further supported by cytokine analyses of supernatants of cultured patient’s cells. These findings suggested that her inflammatory disease is at least in part mediated by type I interferons. While her fever episodes responded well to systemic steroids, treatment with the JAK inhibitor tofacitinib resulted in decreased serum ferritin levels and reduced frequency of fevers.ConclusionHomozygosity for p.Arg90His in NCF1 should be considered contributory in young patients with an atypical systemic inflammatory antecedent phenotype that may evolve into autoimmunity later in life. The complex genomic organization of NCF1 poses a difficulty for high-throughput genotyping techniques and variants in this gene should be carefully evaluated when using the next generation and Sanger sequencing technologies. The p.Arg90His variant is found at a variable allele frequency in different populations, and is higher in people of South East Asian ancestry. In complex genetic diseases such as SLE, other rare and common susceptibility alleles might be necessary for the full disease expressivity.

JAK3 Is Expressed in the Nucleus of Malignant T Cells in Cutaneous T Cell Lymphoma (CTCL).

Simple SummaryJAK3 plays an important role in the pathogenesis of cutaneous T cell lymphoma. JAK3 belongs to the Janus kinase family of receptor-associated tyrosine kinases located in cytoplasm adjacent to the plasma membrane. In this study, we show that JAK3 can also be ectopically expressed in the nucleus in CTCL cell lines and primary cells from CTCL patients. Importantly, JAK3 interacts with the nuclear protein RNA polymerase II and phosphorylates Histone H3. Thus, our data provide first evidence for nuclear expression of JAK3 and interactions with key nuclear proteins in malignant T cells suggesting a novel non-canonical role in CTCL.Perturbation in JAK-STAT signaling has been reported in the pathogenesis of cutaneous T cell lymphoma (CTCL). JAK3 is predominantly associated with the intra-cytoplasmic part of IL-2Rγc located in the plasma membrane of hematopoietic cells. Here we demonstrate that JAK3 is also ectopically expressed in the nucleus of malignant T cells. We detected nuclear JAK3 in various CTCL cell lines and primary malignant T cells from patients with Sézary syndrome, a leukemic variant of CTCL. Nuclear localization of JAK3 was independent of its kinase activity whereas STAT3 had a modest effect on nuclear JAK3 expression. Moreover, JAK3 nuclear localization was only weakly affected by blockage of nuclear export. An inhibitor of the nuclear export protein CRM1, Leptomycin B, induced an increased expression of SOCS3 in the nucleus, but only a weak increase in nuclear JAK3. Importantly, immunoprecipitation experiments indicated that JAK3 interacts with the nuclear protein POLR2A, the catalytic subunit of RNA Polymerase II. Kinase assays showed tyrosine phosphorylation of recombinant human Histone H3 by JAK3 in vitro—an effect which was blocked by the JAK inhibitor (Tofacitinib citrate). In conclusion, we provide the first evidence of nuclear localization of JAK3 in malignant T cells. Our findings suggest that JAK3 may have a cytokine-receptor independent function in the nucleus of malignant T cells, and thus a novel non-canonical role in CTCL.

Real-world single centre use of JAK inhibitors across the rheumatoid arthritis pathway.

To evaluate real-world efficacy of approved JAK inhibitors (JAKi) tofacitinib and baricitinib in a large, single-centre cohort of RA patients across the treatment pathway, including those refractory to multiple biologic drugs. All RA patients, treated with tofacitinib (from time of compassionate access scheme) or baricitinib since approval in 2017 had DAS28-CRP scores and components recorded at baseline, 3 and 6 months (with retrospective data for compassionate access scheme). Efficacy was evaluated in the total cohort, each treatment group, and subgroups of number of prior biologic classes failed. One hundred and fifteen patients were treated with a JAKi (tofacitinib 54, baricitinib 69, 8 both); 76.4% female; mean (s.d.) age 57.3 (14.3) years. On average patients had received three previous bDMARDs; 11 (9.6%) were bDMARD naïve. Combined group baseline DAS28-CRP (s.d.) 5.62(1.14) improved by 1.49(1.44) and 1.67(1.61) at 3 and 6 months, respectively, comparable in individual JAKi groups; with 24% in at least low disease activity at 3 months. The biggest improvement was observed in the biologic-naïve group (mean DAS28-CRP improved from 5.16-2.14 after 6 months); while those with prior exposure to minimum three bDMARD classes had DAS28-CRP improvement of >1.2. Five out of 8 patients treated with both JAKi sequentially responded. Twelve patients previously unresponsive to IL-6 blockade responded to JAKi. No unexpected safety events were recorded. Two cases of venous thromboembolism were observed. JAK inhibition is effective in a real-world population of RA patients, including in a subset of patients refractory to multiple previous bDMARDs.

Integration of Deep Multi-Omics Profiling Veals New Insights into the Biology of Poor-Risk Acute Myeloid Leukemia

Background: The poor-risk cytogenetic subgroup of acute myeloid leukaemia (AML) includes various chromosomal aberrations and represents a heterogeneous population of patients with a dismal 10-year overall survival. While the success of genetic landscaping studies is encouraging, it is debatable whether genomics, or indeed any single-omics platform alone, is sufficient to capture the biology of a disease that continues to evade our existing treatments so effectively. Instead, we need to develop a much better understanding of the complexity of this subgroup of AMLs: the relationship and interdependencies across biochemical pathways, how these may differ between patients and their impact on the leukemia and normal stem cell compartments. To launch this process, we have completed a multi-omics profiling programme to shed new light on the genetic and biochemical features of poor-risk AML (https://poor-risk-aml.bham.ac.uk/). Aims: Application of multi-omics and integrative approaches to decipher the complexities of cytogenetically poor-risk AML Methods: Sample inclusion criteria were based on cytogenetics and availability of sufficient diagnostic bone marrow or peripheral blood material for analysis. The 50 primary AMLs included 17 cases with complex karyotype, 13 -7/del(7), 11 KMT2A rearrangements (with the exception of t(9;11)), 4 t(6;9), 3 -5/de(5), 1 del(17) and 1 inv(3). Profiles consisted of a combination of genomics (whole genome sequencing (WGS, 60X for tumour and 30X for germ-line controls), targeted sequencing of 54 myeloid loci, and total RNA-seq (100 million reads per bulk sample), mass spectrometry proteomics and phosphoproteomics (with >6,000 proteins and > 25,000 phosphorylation sites detected and quantified), mass cytometry (CyTOF, 39 markers), drug screening (ranging from 200-500 approved or investigational compounds) and the selective generation of patient-derived xenograft (PDX) models. Results: Near complete datasets have been compiled on all 50 primary AMLs, with the exception of WGS analysis where profiling was restricted to cases where corresponding germline DNA was available. Integration of WGS and RNA-seq data identified 122 genes having notable allele-specific expression (ASE) in ≥ 5 samples supported by ≥ 3 SNPs and these included the transcription factor GATA2 and the DNA topoisomerase TOP1MT. Use of RNA fusion capture tools resolved novel inter- and intra- chromosomal gene rearrangements that were confirmed by WGS. The four t(6;9)(p23;q34)/DEK-NUP214 cases, with a mean age of diagnosis of 43.5 years and all harboring FLT3-ITD mutations, arose from the most immature hematopoietic compartment (CD34+CD117+ enrichment) and demonstrated a unique transcriptomic signature, which included upregulation of FOXO3 and GRP56. Collectively, t(6;9) primary samples also showed a selective drug sensitivity to XPO1 (selinexor and eltanexor) and JAK inhibitors (ruxolitinib, tofacitinib and momelotinib) compared to other cytogenetic risk groups. On the other hand, a comparison of in vitro drug sensitivity data with genomic data of our entire cohort of patients demonstrated that TP53 wt AMLs (n=37) were more sensitive to all four MDM2 inhibitors (AMG-232, idasanutlin, SAR405838 and NVP-CGM097) compared to TP53 mutated cases (n=13). Comparisons of transcriptomics with the in vitro sensitivity to drugs included in early/late phase AML clinical trials, identified signatures of response associated with MDM2 and Aurora B kinase (AZD1152-HQPA) inhibitors. Phosphoproteomics analysis and machine learning modeling separated KMT2A rearranged leukemias into 2 discrete groups (group A: MLLT4, MLLT10 and TET1; group B with MLLT6, ELL and SEP9 fusion partners). Functionally, group A presented with elevated HOXA10 protein expression and enhanced in vitro response to genotoxic drugs and cell cycle inhibitors when compared to group B leukemia. Conclusions: Our study demonstrates the feasibility of simultaneously generating omics data from several different platforms and highlights that a combination of genetic and proteomic profiles may help to inform the choice of therapies based on the underlying biology of a patient's AML. Disclosures Wennerberg: Novartis: Research Funding; Pfizer: Honoraria. Heckman:Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding; Orion Pharma: Research Funding; Innovative Mediicines Initiative project Harmony: Research Funding.

Psoriasis-like skin disorder in transgenic mice expressing a RIG-I Singleton-Merten syndrome variant.

Mutations in DDX58 (DExD/H-box helicase 58), which encodes the cytoplasmic RNA sensor retinoic acid-inducible gene I (RIG-I), were recently identified in the rare autoimmune disease Singleton-Merten syndrome (SMS). We report the spontaneous development of psoriasis-like skin lesions as an SMS-like symptom in transgenic mice harboring one of the RIG-I SMS variants, E373A. Histological analysis revealed typical characteristics of psoriasis, including the abnormal proliferation and differentiation of keratinocytes leading to epidermal hyperplasia, and infiltrates consisting of neutrophils, dendritic cells and T cells. Levels of the IL-23/IL-17 immune axis cytokines were high in the skin lesions. Rag2-/- transgenic mice showed partial amelioration of the phenotype, with down-regulation of inflammatory cytokines, including IL-17A, suggesting the importance of lymphocytes for the pathogenesis similar to that of human psoriasis. Of note, IL-17A deficiency abolished the skin phenotype, and treatment using the JAK inhibitor tofacitinib not only prevented onset, but also improved the skin manifestations even after onset. Our study provides further evidence for the involvement of RIG-I activation in the onset and progression of psoriasis via type I interferon signaling and the IL-23/IL-17 axis.

Identifying novel B-cell targets for chronic inflammatory autoimmune disease by screening of chemical probes in a patient-derived cell assay

B-cell secretion of autoantibodies drives autoimmune diseases, including systemic lupus erythematosus and idiopathic inflammatory myositis. Few therapies are presently available for treatment of these patients, often resulting in unsatisfactory effects and helping only some of the patients. We developed a screening assay for evaluation of novel targets suspending B-cell maturation into antibody secreting cells, which could contribute to future drug development. The assay was employed for testing 43 high quality chemical probes and compounds inhibiting under-explored protein targets, using primary cells from patients with autoimmune disease. Probes inhibiting bromodomain family proteins and histone methyl transferases demonstrated abrogation of B-cell functions to a degree comparable to a positive control, the JAK inhibitor tofacitinib. Inhibition of each target rendered a specific functional cell and potential disease modifying effect, indicating specific epigenetic protein targets as potential new intervention points for future drug discovery and development efforts.

The JAK Inhibitor Tofacitinib Rescues Intestinal Barrier Defects Caused by Disrupted Epithelial-macrophage Interactions.

Loss-of-function variants in protein tyrosine phosphatase non-receptor type-2 [PTPN2] promote susceptibility to inflammatory bowel diseases [IBD]. PTPN2 regulates Janus-kinase [JAK] and signal transducer and activator of transcription [STAT] signalling, while protecting the intestinal epithelium from inflammation-induced barrier disruption. The pan-JAK inhibitor tofacitinib is approved to treat ulcerative colitis, but its effects on intestinal epithelial cell-macrophage interactions and on barrier properties are unknown. We aimed to determine if tofacitinib can rescue disrupted epithelial-macrophage interaction and barrier function upon loss of PTPN2. Human Caco-2BBe intestinal epithelial cells [IECs] and THP-1 macrophages expressing control or PTPN2-specific shRNA were co-cultured with tofacitinib or vehicle. Transepithelial electrical resistance and 4 kDa fluorescein-dextran flux were measured to assess barrier function. Ptpn2fl/fl and Ptpn2-LysMCre mice, which lack Ptpn2 in myeloid cells, were treated orally with tofacitinib citrate twice daily to assess the in vivo effect on the intestinal epithelial barrier. Colitis was induced via administration of 1.5% dextran sulphate sodium [DSS] in drinking water. Tofacitinib corrected compromised barrier function upon PTPN2 loss in macrophages and/or IECs via normalisation of: [i] tight junction protein expression; [ii] excessive STAT3 signalling; and [iii] IL-6 and IL-22 secretion. In Ptpn2-LysMCre mice, tofacitinib reduced colonic pro-inflammatory macrophages, corrected underlying permeability defects, and prevented the increased susceptibility to DSS colitis. PTPN2 loss in IECs or macrophages compromises IEC-macrophage interactions and reduces epithelial barrier integrity. Both of these events were corrected by tofacitinib in vitro and in vivo. Tofacitinib may have greater therapeutic efficacy in IBD patients harbouring PTPN2 loss-of-function mutations.

3D model of harlequin ichthyosis reveals inflammatory therapeutic targets.

The biology of harlequin ichthyosis (HI), a devastating skin disorder caused by loss-of-function mutations in the gene ABCA12, is poorly understood, and to date, no satisfactory treatment has been developed. We sought to investigate pathomechanisms of HI that could lead to the identification of new treatments for improving patients’ quality of life. In this study, RNA-Seq and functional assays were performed to define the effects of loss of ABCA12 using HI patient skin samples and an engineered CRISPR/Cas9 ABCA12 KO cell line. The HI living skin equivalent (3D model) recapitulated the HI skin phenotype. The cytokines IL-36α and IL-36γ were upregulated in HI skin, whereas the innate immune inhibitor IL-37 was strongly downregulated. We also identified STAT1 and its downstream target inducible nitric oxide synthase (NOS2) as being upregulated in the in vitro HI 3D model and HI patient skin samples. Inhibition of NOS2 using the inhibitor 1400W or the JAK inhibitor tofacitinib dramatically improved the in vitro HI phenotype by restoring the lipid barrier in the HI 3D model. Our study has identified dysregulated pathways in HI skin that are feasible therapeutic targets.

FRI0134 EFFECT OF JAK INHIBITORS ON PAIN AND QUALITY OF LIFE IN RHEUMATOID ARTHRITIS PATIENTS

Background:Pain control is considered a treatment priority from most patients with Rheumatoid Arthritis (RA). Despite the treat to target approach, residual pain is commonly reported by patients with RA. Treatment with JAK inhibitors (JAKi) has been associated to a rapid control of pain.Objectives:To investigate the effect of JAKi on pain and quality of life in a mono-centric real-life clinical setting.Methods:Patients candidate to baricitinib or tofacitinib were evaluated at baseline and after 12 and 24 weeks of treatment. Disease activity was assessed by Disease Activity Score (DAS)28 with C reactive protein (CRP). A reduction of ≥ 50% of pain visual-analogue scale (VAS) 0-100 mm was recorded as “very much improved, substantially improved” (1). Pain VAS score ≤ 10 mm was considered “no/limited pain” (2). Patients’ satisfaction was assessed by the Patient Acceptable Symptom State question (3). Data were expressed as mean (SD) or median (interquartile range) according to the variables’ distribution. Mann Witney test was use and p values <0.05 were considered statistically significant.Results:Overall 108 patients started a JAK inhibitor (baricitinib n=67, tofacitinib n=41). Eighty-four patients (baricitinib n=51; tofacitinib n=33) were followed-up for at least 3 months and were included in the analysis. Table 1 summarizes demographic and clinical characteristic of the cohort. After 12 and 24 weeks of treatment we detected a significant reduction of DAS28 compared with baseline [from 4.7 (1.5) to 3.2 (1.7) 2.9 (1.5) and 2.7 (1.1), respectively; p<0.001; p<0.00001 and p<0.00001). At week 4, 27% and 51.8% of patients achieved remission and low disease activity, respectively; the percentages increase to 32.1% and 60.7% at week 12 and 42.2% and 70.3% at week 24. When evaluating the extent of reduction of the single items included in the DAS28 composite index we found that number of tender (TJ) and swollen joints (SJ) decreased from 9 (7.8) to 5 (3.5) to 4 (5) and 1 (3) at week 4, 2 (4) and 1 (3) at week 12, and 2 (4) and 1 (3) at week 24, respectively (p<0.00001 for all); the median reduction of TJC and SJC at week 4, 12 and 24 was 60%, 77% and 88%, and 81%, 86% and 100%, respectively. GH decreased from 70(30) to 40(40) at week 4, 40(30) at week 12 and 37(40) at week 24 (p<0.00001) with a median reduction of 37.5%, 44% and 46%. C reactive protein decreased by 54.5% at week 4, 47% at week 12 and 55% at week 24. VAS pain was significantly reduced at week 4, 12 and 24 [from 70(25) to 40(40,)30(40) at the three timepoints, p<0.00001] decreasing by 37.5%, 50% and 54%, respectively. A substantial reduction (≥50%) in VAS pain was reported by 41.3%, 54.4% and 53.9% of patients after 4, 12 and 24 weeks, respectively. Limited/no pain was reported by 21.3%, 24.7% and 36.5% at weeks 4, 12 and 24, respectively. Overall, 81.8% of patients achieved the PASS after a median time of 10 (7-15) days.Conclusion:JAK inhibitors baricitinib and tofacitinib induce a rapid improvement of disease activity driven both by pain and inflammation control. Even if no/limited pain was described only by one third of the patients, most of them reported a rapid and sustained reduction of pain accounting for the achievement of a satisfactory health condition.

Jak Inhibitors Reverse Vitiligo in Mice but Do Not Deplete Skin Resident Memory T Cells

Jak Inhibitors Reverse Vitiligo in Mice but Do Not Deplete Skin Resident Memory T Cells