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Rational design of a JAK1-selective siRNA inhibitor for the modulation of autoimmunity in the skin

ABSTRACT: Inhibition of Janus kinase (JAK) family enzymes has surged as a popular strategy for treating inflammatory and autoimmune skin diseases. In the clinic, current available small molecule JAK inhibitors show distinct efficacy and safety profiles, likely reflecting their variable selectivity for JAK subtypes. Nonselective inhibition of multiple JAK enzymes is associated with increased side effects and has resulted in FDA-mandated black box warnings. Developing therapeutics to enable absolute JAK subtype selectivity is challenging and has not yet been achieved. Here, we harness RNA interference (RNAi) for rationally designing small interfering RNA (siRNA) therapeutics that offer sequence-specific gene silencing of JAK1, thus narrowing the spectrum of action on JAK-dependent cytokine signaling pathways to maintain efficacy and improve safety. We developed a fully chemically modified siRNA that supports efficient silencing of JAK1 expression in human skin explant and functional modulation of JAK1-dependent inflammatory signaling. A single injection of the JAK1 siRNA into mouse skin enables JAK1 silencing for 5 weeks in vivo. In a mouse model of vitiligo, local administration of the JAK1 siRNA significantly reduces skin infiltration of autoreactive CD8+ T cells and prevents epidermal depigmentation. This work outlines a robust framework for designing targeted RNAi-based therapies and establishes a path toward novel siRNA treatments for inflammatory and autoimmune skin diseases.

ORGANISM(S): Homo sapiens

PROVIDER: GSE234070 | GEO | 2023/10/18

REPOSITORIES: GEO

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Selective inhibitors of JAK1 targeting a subtype-restricted allosteric cysteine

Project description:The JAK family of non-receptor tyrosine kinases includes four subtypes (JAK1, JAK2, JAK3, and TYK2) and is responsible for signal transduction downstream of diverse cytokine receptors. JAK inhibitors have emerged as important therapies for immuno(onc)ological disorders, but their use is limited by undesirable side effects presumed to arise from poor subtype selectivity, a common challenge for inhibitors targeting the ATP-binding pocket of kinases. Here, we describe the chemical proteomic discovery of a druggable allosteric cysteine present in the non-catalytic pseudokinase domain of JAK1 (C817) and TYK2 (C838), but absent from JAK2 or JAK3. Electrophilic compounds selectively engaging this site block JAK1-dependent transphosphorylation and cytokine signaling, while appearing to act largely as “silent” ligands for TYK2. Importantly, the allosteric JAK1 inhibitors do not impair JAK2-dependent cytokine signaling and are inactive in cells expressing a C817A JAK1 mutant. Our findings thus reveal an allosteric approach for inhibiting JAK1 with unprecedented subtype selectivity.

2023-07-20 | PXD031384 | Pride

Selective Janus kinase 1 inhibition resolves inflammation and restores hair growth offering a viable treatment option for alopecia areata

Project description:JAK inhibition by means of clinically available pan JAK inhibitors has recently demonstrated great efficacy in both restoring hair growth and resolving inflammation in the skin of patients with Alopecia Areata (AA). These effects are dose dependent and mainly efficacious at ranges close to a questionable risk profile. Given the great responses to JAK inhibition and the current lack of efficacious treatments in AA, it is exciting to explore the possibilities to separate the beneficial and adverse effects in order to provide a successful treatment option for these patients where the medical need is still vastly unmet. Selective JAK1 inhibition mainly affects genes downstream of STAT1, STAT3 & STAT6 in human immune cells. Importantly, pathways like T-cell differentiation and lymphopoiesis are less affected by the selective JAK1 inhibitor versus less specific JAK inhibitors like tofacitinib and ruxolitinib. These findings indicate that it is feasible to develop more selective and specific JAK1 inhibitors that are as efficacious as pan-JAK inhibitors but with a better safety profile as systemic exposure is mandatory for efficacy in this disease.

2023-01-30 | GSE94235 | GEO

RNA-seq of human colonic cancer cell line CACO-2 transfected 48h with siRNA targeting JAK1 (HJ1D8; HJ1D2) or JAK3 (HJ3D41; HMJ3D1) at 100pM final concentration using LipofectamineRNAiMAX or treated 48h with Tofacitinib (final concentration: 1µM) or Filgotinib (final concentration: 5µM) against controls (transfection medium: Opti-MEM, transfection mixt alone: Opti-MEM + Lipof., siMock, DMSO equivalent to volume added for Filgo 5µM).

Project description:Inflammatory bowel diseases are highly debilitating conditions that require constant monitoring and life-long medication. Current treatments are focused on systemic administration of immunomodulatory drugs, but they have a broad range of undesirable side-effects. The RNA interference is a highly specific endogenous mechanism that regulates the expression of the gene at the transcript level, which can be repurposed using exogenous short interfering RNA (siRNA) in order to repress the target gene’s expression. While siRNA therapeutics can offer an alternative to existing therapies, with a high specificity critical for chronically administrated drugs, evidence of their potency compared to chemical kinase inhibitors used in clinics is still lacking in alleviating an adverse inflammatory response. In this experiment, we compared the transcriptomic profile of cells transfected 48hv by siRNA targeting JAK1 and JAK3 (100 pM) to those of cells treated 48h with the most recent and promising kinase inhibitors for Janus kinases (Jakinibs), Tofacitinib (1 µM) and Filgotinib (5 µM).

2023-04-12 | E-MTAB-10348 | biostudies-arrayexpress

Non-clinical and clinical pharmacology of the potent and selective RARγ agonist trifarotene

Project description:Background: First- and third-generation retinoids are the main treatment in acne. Even though efficacious, they lack full selectivity for RARγ expressed in the epidermis and infundibulum. Objectives: To characterize the in vitro metabolism and the pharmacology of the novel retinoid trifarotene. Methods: In vitro assays determined efficacy, potency and selectivity on RARs, as well as the activity on the expression of retinoid target genes in human keratinocytes and ex vivo cultured skin. In vivo studies investigated topical comedolytic, anti-inflammatory and depigmenting properties. The trifarotene-induced gene expression profile was investigated in non-lesional skin of acne patients and compared to ex vivo and in vivo models. Finally, the metabolic stability in human keratinocytes and hepatic microsomes was established. Results: Trifarotene is a selective RARγ agonist with >20-fold selectivity over RAR and RARβ. Trifarotene is active and stable in keratinocytes but rapidly metabolized by human hepatic microsomes, predicting improved safety. In vivo, trifarotene 0.01% applied topically is highly comedolytic and has antiinflammatory and antipigmenting properties. Gene expression studies indicated potent activation of known retinoid-modulated processes (epidermal differentiation, proliferation, stress response, RA metabolism) and novel pathways (proteolysis, transport/skin hydration, cell adhesion) in ex vivo and in vivo models, as well as in human skin after four weeks of topical application of trifarotene 0.005% cream. Conclusion: Based on its RARγ selectivity, rapid degradation in human hepatic microsomes and pharmacological properties including potent modulation of epidermal processes, topical treatment with trifarotene is expected to provide strong efficacy combined with a favourable safety profile in acne and ichthyotic disorders.

2018-04-28 | GSE107232 | GEO

Gene expression profiling upon knockdown of JAK1 in IM9 cells

Project description:Natural Killer (NK) cells are primary effectors of innate immunity directed against transformed cells. In response, tumor cells have developed mechanisms to evade NK cell-mediated lysis but the molecular basis for target cell resistance is not well understood. In the present study, we used a lentiviral shRNA library targeting more than 1000 human genes to identify 83 genes that promote target cell resistance to human NK cells. Many of the genes identified in this genetic screen belong to common signaling pathways, however, none of these genes have previously been known to modulate susceptibility of human tumor cells to immunologic destruction. In particular, gene silencing of two members of the JAK family (JAK1 and JAK2) in a variety of tumor cell targets increased their susceptibility to NK-mediated lysis and induced increased secretion of interferon gamma (IFN-gamma by NK cells. Treatment of tumor cells with JAK inhibitors also induced increased susceptibility to NK cell activity. These findings may have important clinical implications and suggest that small molecule inhibitors of tyrosine kinases being developed as therapeutic anti-tumor agents may also have significant immunologic effects in vivo. IM9 cells were transduced with shRNA-encoding vectors and selected with Puromycin. Two vectors were specifically targeting JAK1 (JAK1-1 and JAK1-3) and one vector encoded an irrelevant control shRNA (CTRL-2). Total RNA was obtained from the parental IM9 cell line, the control-shRNA expressing IM9 cells, the JAK1-1-shRNA and JAK1-3-shRNA expressing IM9 cells in 2 separate experiments (Exp1 and Exp2).

2012-06-27 | E-GEOD-37012 | biostudies-arrayexpress

Treatment of granuloma annulare and suppression of pro-inflammatory cytokine activity with tofacitinib [scRNA-Seq]

Project description:Granuloma annulare (GA) is a common inflammatory cutaneous disorder characterized by macrophage accumulation and activation in the skin. Its pathogenesis is poorly understood and there are no reliably effective treatments. Its potential health implications, if any, are unknown. Using single cell RNA sequencing (scRNAseq) we show that in GA, CD4+ T cells over-produce interferon (IFN)-g resulting in inflammatory polarization of macrophages and in altered extracellular matrix (ECM) production induced by the activity of oncostatin M, an interleukin (IL)-6 family cytokine, on fibroblasts. This mechanism identifies Janus kinase (JAK) inhibition as a potential therapeutic strategy, as both IFN-g and OSM signal via the JAK-STAT pathway. Indeed, treatment of five patients with severe, longstanding GA with tofacitinib (a JAK1/3 inhibitor) resulted in clinical and histologic disease remission in three patients and marked improvement in the other two. Treatment was associated with suppression of pathogenic cytokine activity in the skin and dissolution of macrophages. We also found that severe GA was associated with hypercytokinemia in plasma of patients, and JAK inhibition normalized this hypercytokiemia. Together, our results highlight the constitutive activity of the JAK-STAT pathway in GA as a result of IFN-g and OSM and identify JAK inhibitors as a potential molecularly targeted treatment for this disorder.

2020-12-22 | GSE158924 | GEO

Treatment of granuloma annulare and suppression of pro-inflammatory cytokine activity with tofacitinib [RNA-Seq]

2020-12-22 | GSE158923 | GEO

JAK dependent survival of ALK- ALCL

Project description:Activating JAK and STAT mutations were discovered in many T-cell malignancies including ALK- anaplastic large cell lymphomas (ALCL). However, such mutations often occur in a minority of patients. To investigate the clinical application of targeting Janus Kinase (JAK) for ALK- ALCL, we treated ALK- cell lines of different histologic origins with JAK inhibitors. Interestingly, most exogenous cytokine independent cell lines responded to JAK inhibition regardless of JAK mutation status. JAK inhibitor sensitivity correlated with STAT3 phosphorylation status of tumor cells. Employing retroviral shRNA knockdown, we demonstrated that these JAK inhibitor sensitive cells were dependent on both JAK1 and STAT3 for survival. JAK1 and STAT3 gain-of-function mutations were found in some but not all JAK inhibitor sensitive cells. Moreover, the mutations alone could not explain the JAK1/STAT3 dependency as wild-type JAK1 or STAT3 was sufficient to promote cell survival in the cells that had either JAK1or STAT3 mutations. To investigate whether other mechanisms were involved, we knocked down upstream receptors GP130 or IL-2Rγ. Knockdown of GP130 or IL-2Rγ induced cell death in select JAK inhibitor sensitive cells. High levels of cytokine expression including IL-6 were demonstrated in cell lines as well as in primary ALK- ALCL tumors. Finally, ruxolitinib, a JAK1/2 inhibitor, was effective in vivo in a xenograft ALK- ALCL model. Our data suggest cytokine receptor signaling was required for tumor cell survival in diverse forms of ALK- ALCL even in the presence of JAK1/STAT3 mutations. Therefore, JAK-inhibitor therapy might benefit patients with ALK- ALCL that are pSTAT3+.

2017-08-22 | GSE96048 | GEO

Effect of a JAK inhibitor on gene expression of HUT78 cell line

Project description:Developing targeted therapy for cutaneous T cell lymphoma (CTCL) patients still requires actionable mutated genes and deregulated pathways to be identified. There is increasing evidence that activating mutations in JAK genes and deregulated JAK/STAT signaling are important mechanisms involved in multiple B and T cell malignancies, including CTCL. Therefore, in this study we focused on studying the mutational status of JAK1, JAK2 and JAK3 genes in a series of human CTCL lesions and cell lines using next-generation sequencing (NGS). We found that 7 of 48 (14.7%) of the analyzed cases harbored mutations in the JAK1 and JAK3 genes that mainly affected the pseudokinase domain of the corresponding proteins. On the basis of these results, we used a specific JAK inhibitor (INCB018424) in a series of CTCL cell lines with deregulated JAK/STAT activity. Treatment of CTCL cells with INCB018424 resulted in dose-dependent reduction of activated STAT expression, diminished cell viability, and increased apoptosis. We also studied global changes in gene expression in cells with mutated JAK1 and JAK3 proteins treated with INCB018424 and identified multiple genes that were differentially regulated by JAK/STAT signaling, such as FGF20 (upregulated) and EGR1 (downregulated). Thus, our results show that the detection of deregulated JAK/STAT signaling in CTCL lesions via JAK mutations or other surrogate markers may serve to indicate the clinical use of JAK/STAT inhibitors. 3 replicates of cells treated with DMSO or JAKi during 30 min and 3h

2015-10-31 | E-GEOD-68562 | biostudies-arrayexpress

JAK inhibition ameliorates bone destructions by simultaneously targeting mature osteoclasts and their precursor stages

Project description:Inflammatory bone destruction was induced by locally injecting lipopolysaccharides into fluorescent reporter mice of mature osteoclasts or their precursors. These mice were treated with a JAK inhibitor, ABT-317, which selectively inhibits the activation of JAK1, and intravital imaging with multiphoton microscopy was conducted. We also investigated the molecular mechanism of the effects of the JAK inhibitor on osteoclasts by exhaustive RNA sequence analysis.

2023-03-05 | GSE193104 | GEO

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