A major project that involves the TIS is the definition of molecular biomarkers for autoimmune and autoinflammatory diseases. The positive results observed with tofacitinib in the animal model of SLE, served as a springboard for a phase 1b/2a, randomized, double blind, placebo-controlled clinical trial. Undertaken in collaboration with Drs. Sarfaraz Hasni and Mariana Kaplan, this study aimed to determine the safety and tolerability of tofacitinib in patients with SLE and mild to moderate disease activity. Over the course of 84 days, tofacitinib was administered to 30 lupus patients with mild to moderate disease activity at a dose of 5 mg twice daily (2:1 drug to placebo ratio). Patients were also stratified by the presence or absence of a STAT4 risk allele which was associated with higher risk of severe manifestations. The TIS performed some of the mechanistic studies that, albeit not the primary endpoint of the trial, could support relevant clinical findings. In particular, we were interested in developing a robust bioassay that could validate target engagement by the drug. To this end, using fluorescent cell barcoding (FCB) multi parametric flow cytometry, we assessed ex vivo cytokine-induced activation of STATs transcription factors. Moreover, we measured changes in gene expression patterns during treatment by using RNASeq as well as NanoString. A manuscript reporting the findings of the trial is currently in preparation whereas another manuscript in which we validated the Fluorescent barcoding approach utilized for the pSTAT assays is currently under review. The TIS is also currently collaborating with Drs. Robert Colbert and Hanna Kim on a natural history study of juvenile dermatomyositis (JDM) paired with a compassionate use of the JAK1/JAK2 inhibitor baricitinib. JDM is the most common form of idiopathic inflammatory myopathy in children and its pathogenesis is thought to be due in part to innate immune dysregulation as well as autoimmunity. It is associated with a high expression of interferon or IFN-regulated genes (IFN signature) in peripheral blood, muscle, and skin. Treatment is empiric with a mainstay of high-dose steroids combined with other immunosuppressive medications often requiring prolonged therapy with significant side effects. Case reports in adults have anecdotally noted some clinic benefit with JAK inhibitor therapy (tofacitinib, ruxolitinib) with suggesting that this may target the IFN-driven pathology in dermatomyositis. In this project our group helps with translational studies to characterize the JDM patients. We collected PAXgene tubes for RNA and gene expression, particularly interferon-regulated gene expression, serum, and urine for cytokine analysis and PBMCs for assessment of detailed immunophenotyping. As in some of the studies highlighted above, we have begun to assess the drug target engagement and pharmacodynamic effects by measuring STAT phosphorylation in different immune cell types at multiple timepoints during the day, which can be correlated with the level of JAK inhibitor in the blood. In collaboration with Dr. Peter Grayson we have started to analyze sera from patients with a diagnosis of Takayasu vasculitis, who received treatment with a TNF blocker. Vasculitis is an immune-mediated disease that manifests as an inflammation of the blood vessels. There are three forms of vasculitis, affecting the small, medium and large vessels. Giant cell arteritis is primarily localized in peripheral, medium-sized arteries, affecting the third to fifth branches of the aorta. Takayasu arteritis preferentially occurs in large vessels, affecting the aorta and its primary branches. These arteritides show similar histological abnormalities and pathogenic pathways, therefore combinations of biological assays can help to further classify similar diseases, and this is what we are currently doing with the Grayson group. Another pathology being investigated by Drs. Peter Grayson and Marcela Ferrada is Relapsing polychondritis (RP), an immune-mediated, inflammatory, systemic disorder characterized by recurrent episodes of inflammation in tissues such as the ear and nose and peripheral joints as well as the tracheobronchial tree. The inflammatory status eventually results in tissue damage and functional loss. Moreover, the disease is frequently associated with RA. In order to better understand the immunological alterations occurring in RP patients, we have initiated a study in which we have extensively immunophenotyped the patients that enrolled in their natural history study. Preliminary results have shown some interesting alterations in the number of CD19+ B cells, which appeared to be significantly decreased in the patients when compared to healthy donors. We are continuing our extremely successful collaborative work with Dr. Aksentijevich on the spectrum of pathomechanisms associated with autoinflammatory diseases that include defects in deubiquitination. In particular, we have been investigating the effects of mutations in the ubiquitin-specific protease USP43. We have shown that immune cells obtained from a patient carrying homozygous mutation in USP43 are hyperresponsive to type I IFN stimulation (assessed as phosphorylated STAT1). Gene expression studies on peripheral blood leukocytes have shown an increased expression of IFN-dependent genes. Interestingly, the patient, who has a clinical presentation similar to the CANDLE interferonopathy, was unresponsive to treatment with several biologics but responded very well to the JAK inhibitor ruxolitinib. Finally, we are also currently collaborating with Drs. Fitzgerald and Antignani at NCI whose group is investigating how tofacitinib potentiates the anti-tumor activity of immunotoxins. Dr. Fitzgeralds group has observed that tofacitinib alters the tumor microenvironment by reducing the number of tumor-associated inflammatory cells and allowing increased delivery of antibody-based agents to malignant cells. Alone, tofacitinib exhibited no antitumor activity, but combination treatments with recombinant immunotoxins or an antibody drug conjugate resulted in increased anti-tumor responses compared to monotherapy with either antibody-based drug, confirming in vivo synergy. Further, fluorescently labeled antibody-based agents accumulated in a higher percentage of malignant cells following tofacitinib treatment. We showed that several cytokines involved in inflammatory cell chemotaxis such as IL-6, CCL2, CCL4, and CXCL2, were reduced . A manuscript describing these results has just been published in JCI Insight. The TIS has also been investigating novel approaches for the treatment of autoimmune diseases. In collaboration with the O'Shea group, the Kaplan group, and Pfizer (via a CRADA), we are currently evaluating the effects of tofacitinib and second-generation JAK-selective inhibitors on T cells and innate lymphoid cells. Since a major limitation of JAK/STAT gene knockout studies in mice is the complete loss of ILC populations (including NK cells), pharmacological manipulation of this signaling cascade with JAK inhibitors is an attractive alternative strategy to study the role of cytokine signaling in ILC biology. We have been investigating the effects of pan- as well as JAK-selective inhibitors on the development and functions of IFN-gamma producing ILCs (both NK and ILC1) by tofacitinib, ruxolitinib (a JAK1/JAK2 inhibitor), or PF-06651600 (a JAK3-TEC kinase inhibitor).
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