Kinases Tyk2 Research Articles

The multiple-action allosteric inhibition of TYK2 by deucravacitinib: insights from computational simulations

Participating in the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, TYK2 emerges as a promising therapy target in controlling various autoimmune diseases, including psoriasis and multiple sclerosis. Deucravacitinib (DEU) is a novel oral TYK2-specific inhibitor approved in 2022 that is clinically effective in moderate to severe psoriasis trials. Upon the AlphaFold2 predicted TYK2 pseudokinase domain (JH2) and kinase domain (JH1), we explored the details of the underlined allosteric inhibition mechanism on TYK2 JH2-JH1 with the aid of molecular dynamics simulation. Our results suggest that the allosteric inhibition of DEU on TYK2 is accomplished by affecting the JH2-JH1 interface and hampering the state transition and ATP binding in JH1. Particularly, DEU binding stabilized the autoinhibitory interface between JH2 and JH1 while disrupting the formation of the activation interface. As a result, the negative regulation of JH2 on JH1 was greatly enhanced. These findings offer additional details on the pseudokinase-dependent autoinhibition of the JAK kinase domain and provide theoretical support for the JH2-targeted drug discovery in JAK members.

TYK2 on tau.

The kinase TYK2 facilitates pathological tau assembly through protein-stabilizing modifications.

Targeting Interferon Signalling in Systemic Lupus Erythematosus: Lessons Learned.

The development of new medicines for systemic lupus erythematosus (SLE) has not addressed unmet clinical need, with only three drugs receiving regulatory approval for SLE in the last 60years, one of which was specifically licensed for lupus nephritis. In the last 20years it has become clear that activation of type 1 interferons (IFN) is reproducibly detected in the majority of SLE patients, and the actions of IFN in the immune system and on target tissues is consistent with a pathogenic role in SLE. These findings led to considerable drug discovery activity, first with agents directly targeting IFN family cytokines, with results that were encouraging but underwhelming. In contrast, targeting the type I IFN receptor with the monoclonal antibody anifrolumab, thereby blocking all IFN family members, was effective in a phase II clinical trial. This led to a pair of phase III trials, one of which was negative and the other positive, reflecting the difficulty of obtaining outcomes from trials in this complex disease. Nonetheless, the balance of evidence resulted in approval of anifrolumab in multiple jurisdictions from 2021 onwards. Multiple approaches to targeting the type 1 IFN pathway have subsequently had positive phase II clinical trials, including antibodies targeting cells that produce IFN, and small molecules targeting the receptor kinase TYK2, required for IFN signalling. Despite multiple hurdles, it is clear that IFN targeting in SLE is here to stay. The story of IFN-targeting therapy in SLE has lessons for drug development overall in this disease.

JAK-STAT signaling maintains homeostasis in T cells and macrophages

Immune cells need to sustain a state of constant alertness over a lifetime. Yet, little is known about the regulatory processes that control the fluent and fragile balance that is called homeostasis. Here we demonstrate that JAK-STAT signaling, beyond its role in immune responses, is a major regulator of immune cell homeostasis. We investigated JAK-STAT-mediated transcription and chromatin accessibility across 12 mouse models, including knockouts of all STAT transcription factors and of the TYK2 kinase. Baseline JAK-STAT signaling was detected in CD8+ T cells and macrophages of unperturbed mice—but abrogated in the knockouts and in unstimulated immune cells deprived of their normal tissue context. We observed diverse gene-regulatory programs, including effects of STAT2 and IRF9 that were independent of STAT1. In summary, our large-scale dataset and integrative analysis of JAK-STAT mutant and wild-type mice uncovered a crucial role of JAK-STAT signaling in unstimulated immune cells, where it contributes to a poised epigenetic and transcriptional state and helps prepare these cells for rapid response to immune stimuli.

P105 Mendelian Randomization screening for JAK inhibitor related potential serious adverse events

Abstract Background JAK inhibitors (JAKi) suppress the JAK/STAT signalling pathway through inhibition of the four Janus kinase proteins JAK1, JAK2, JAK3 and TYK2. JAKi are licensed for the treatment of a wide range of immune mediated inflammatory diseases (IMIDs) including inflammatory bowel disease. Concerns have been raised for the potential of this class of medication to associate with serious adverse events, particularly in those older than 65 years of age or with previous cardiovascular comorbidities. Mendelian randomization (MR) uses genetic variants as instrumental variables (IVs) to examine the causal effects of an exposure (e.g. inhibition of a drug target) on an outcome (e.g. the risk of developing a disease and/or potential adverse effects due to alteration of gene activity). We carried out a comprehensive MR evaluation to explore the therapeutic potential of JAKi for a range of IMIDs, and to study the unintended drug effects that might be caused by this therapeutic strategy. Methods The basis of MR relies on the use of genetic variants as IVs that are reliably related to the risk factor and that are not susceptible of reverse causation and confounding. We used genetic variants (SNPs) associated with protein levels and gene expression (pQTLs and eQTLs, respectively) as IVs to study the effects of inhibiting the genes in the JAK/STAT signalling pathway. The underlying assumption is that these IVs serve as proxies of the effects of JAKi. As a source of the effect estimates on the selected outcomes, we used summary statistics from a compiled list of genome-wide association studies. Results We used the MR approach to chart the range of possible outcomes associated with JAKi treatment. First, we explored the therapeutic potential of JAKi for IMIDs. Second, we evaluated a range of potential adverse events associated with JAKi, focusing particularly on cardiovascular, infectious and cancer-related malignancies. Our MR results confirm a general tendency towards JAK/STAT signalling pathway involvement in IMIDs risk, implying the suitability of JAKi for IMIDs. Regarding potential adverse outcomes, we carried out stratification-based analyses to evaluate whether potential adverse events are enhanced in individuals at particular high risk. The MR analyses highlighted potential risk of JAKi for non-IMIDs traits, particularly when targeting JAK2. This includes already known associations with cardiovascular disease and specific cancer types. Conclusion We performed an outcome-wide exploration based on MR to characterize the therapeutic potential and range of adverse effects expected for JAKi. We recapitulate the known potential of these drugs to treat IMIDs, while uncovering specific severe adverse effects associated with this therapeutic target.

Direct and biologically significant interactions of human herpesvirus 8 interferon regulatory factor 1 with STAT3 and Janus kinase TYK2.

Human herpesvirus 8 (HHV-8) encodes four viral interferon regulatory factors (vIRFs) that target cellular IRFs and/or other innate-immune and stress signaling regulators and suppress the cellular response to viral infection and replication. For vIRF-1, cellular protein targets include IRFs, p53, p53-activating ATM kinase, BH3-only proteins, and antiviral signaling effectors MAVS and STING; vIRF-1 inhibits each, with demonstrated or likely promotion of HHV-8 de novo infection and productive replication. Here, we identify direct interactions of vIRF-1 with STAT3 and STAT-activating Janus kinase TYK2 (the latter reported previously by us to be inhibited by vIRF-1) and suppression by vIRF-1 of cytokine-induced STAT3 activation. Suppression of active, phosphorylated STAT3 (pSTAT3) by vIRF-1 was evident in transfected cells and vIRF-1 ablation in lytically-reactivated recombinant-HHV-8-infected cells led to increased levels of pSTAT3. Using a panel of vIRF-1 deletion variants, regions of vIRF-1 required for interactions with STAT3 and TYK2 were identified, which enabled correlation of STAT3 signaling inhibition by vIRF-1 with TYK2 binding, independently of STAT3 interaction. A viral mutant expressing vIRF-1 deletion-variant Δ198-222 refractory for TYK2 interaction and pSTAT3 suppression was severely compromised for productive replication. Conversely, expression of phosphatase-resistant, protractedly-active STAT3 led to impaired HHV-8 replication. Cells infected with HHV-8 mutants expressing STAT3-refractory vIRF-1 deletion variants or depleted of STAT3 displayed reduced vIRF-1 expression, while custom-peptide-promoted STAT3 interaction could effect increased vIRF-1 expression and enhanced virus replication. Taken together, our data identify vIRF-1 targeting and inhibition of TYK2 as a mechanism of STAT3-signaling suppression and critical for HHV-8 productive replication, the importance of specific pSTAT3 levels for replication, positive roles of STAT3 and vIRF-1-STAT3 interaction in vIRF-1 expression, and significant contributions to lytic replication of STAT3 targeting by vIRF-1.

Identification of HHT-9041P1: A novel potent and selective JAK1 inhibitor in a rat model of rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic systemic disease associated with long-term disability and premature mortality. If left untreated, it can seriously affect patients’ quality of life. The JAK-STAT signal transduction process is known to affect the occurrence and development of RA, and small molecule JAK inhibitors, such as tofacitinib, have been identified as treatments for RA. However, tofacitinib is a non-selective JAK inhibitor that was found to be associated with dose-limiting tolerability and safety issues, such as anemia in phase 2 dose-ranging studies. Therefore, we developed a selective JAK1 inhibitor, HHT-9041P1, to overcome target-related adverse reactions. We used enzyme and cytokine potency assays in vitro as well as the collagen-induced arthritis (CIA) model in vivo to explore the efficacy and mechanism. In vitro, HHT-9041P1 was diluted (0.017 nM–1 mM) in DMSO) and mixed with JAK1, JAK2, JAK3 or TYK2 kinases for use in the respective assays for inhibitory activity and selectivity evaluation. Fresh human PBMCs were activated and incubated with 100 ng/mL cytokine IL-6 or 20 ng/mL GM-CSF for use in the investigation of the immune mechanism. In vivo, HHT-9041P1 (1 mg/kg, 3 mg/kg and 10 mg/kg) was administered by oral gavage twice daily to CIA model Lewis rats from Day 8 to Day 29 for paw swelling and arthritis score evaluation. At the end of the experiment, the rats were sacrificed before collection of the hind ankle joint, spleen and blood for analysis of inflammation, arthritis phenotypes, inflammatory cytokine expression and Th1 cell proportions. As expected, HHT-9041P1 showed 10-fold greater selectivity for JAK1 over JAK2, and 23-fold greater selectivity over JAK3 in cellular assays. The high selectivity of HHT-9041P1 was also validated by in vivo safety studies. HHT-9041P1 demonstrated significant efficacy in a rat model of collagen-induced arthritis (CIA) and was associated with reduced helper T Cell 1 (Th1) cell differentiation. HHT-9041P1 also exhibited excellent pharmacokinetics properties. Thus, HHT-9041P1 was identified as a candidate for clinical development with many options for the treatment of RA.

25-OR: ADA Presidents' Select Abstract: Preclinical Evaluation of TYK2 Inhibitors in Type 1 Diabetes

Interferon-α (IFNα) plays a prominent role in type 1 diabetes (T1D) pathogenesis and mediates its effects through the IFN receptor (IFNAR) and the protein tyrosine kinases JAK1 and TYK2. Polymorphisms that decrease TYK2 activity are protective against T1D, and TYK2 inhibitors (TYKi) are being evaluated for therapeutic benefit in other autoimmune conditions. To test whether TYK2 inhibitors BMS-986202 and BMS-986165 have similar efficacy in diverse models of T1D, we evaluated their effect in vitro on human islets, EndoC-βH1 cells, and iPSC-derived islet-like aggregates and monitored diabetes incidence in RIP-LCMV and NOD mice following in vivo treatment with BMS-986202. TYK2i prevented IFNα-mediated upregulation of CXCL10, MX1, and HLA-ABC in human islets, iPSCs, and EndoC-βH1 and decreased IFNα-mediated STAT1/2 phosphorylation and apoptosis in human islets. Importantly, BMS-986202 reduced diabetes incidence in RIP-LCMV mice by 80% (n=18 vehicle/18 TYK2i; p<0.001). Flow cytometry analysis 3 days post LCMV injection revealed a decrease in the percentage of CD11b+F4/80+ macrophages (M1) and CD11b+CD49+ NK cells in pancreatic lymph nodes (PLN) and blood and an increase in circulating CD11b-CD49+ tolerogenic-NK cells in TYK2i-treated RIP-LCMV mice. At days 7 and 14 post LCMV, PDI+CD8+T-cells in the blood, spleen, and PLN and PDI+FOXP3+ Treg cells in the spleen were increased in TYK2i-treated RIP-LCMV mice, indicating T-cell exhaustion. Similarly, treatment with BMS-986202 resulted in a 44% decrease in diabetes incidence in NOD mice (n=32 vehicle/34 TYK2i; p=0.0075) and a significant reduction in insulitis (p<0.05). smFISH analysis revealed decreased β cell expression of STAT1 and MX1 in TYK2i-treated RIP-LCMV mice and NOD mice compared to vehicle-treated mice, while T-cell activation upon co-incubation with target human β cells was inhibited by TYK2i treatment. Taken together, our findings demonstrate that TYK2 inhibition protects against β cell inflammation and T1D development in multiple preclinical models. Disclosure F.Syed: None. M.I.Alvelos: None. G.Chang: None. K.Orr: None. K.Yamada: None. J.Liu: None. A.Zaldumbide: None. D.Scheuner: None. D.L.Eizirik: None. C.Evans-molina: Advisory Panel; Provention Bio, Inc., DiogenX, Avotres Inc., Neurodon, MaiCell Therapeutics, Other Relationship; Isla Technology, Bristol-Myers Squibb Company, Nimbus Therapeutics, Research Support; Lilly, Astellas Pharma Inc. O.Ballew: None. C.Lee: None. J.Rana: Research Support; Bayer Inc. A.Castela: None. S.A.Weaver: Employee; Eli Lilly and Company. S.Thomaidou: None. S.Demine: None. A.Coomans de brachène: None. Funding National Institutes of Health (DK127308, UC4DK104166); JDRF (2-SRA-2019-834-S-B, 5-CDA-2022-1176-A-N)

HDAC4 mediated LHPP deacetylation enhances its destabilization and promotes the proliferation and metastasis of nasopharyngeal carcinoma

Studies have shown that acetylation modification plays an important role in tumor proliferation and metastasis. Phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) is downregulated in certain tumors, as a tumor suppressor role. However, the regulation of LHPP expression and its function in nasopharyngeal carcinoma (NPC) remain unclear. In the present study, we found that LHPP was downregulated in NPC, and overexpression of LHPP inhibited the proliferation and invasion of NPC cells. Mechanistically, HDAC4 deacetylated LHPP at K6 and promoted the degradation of LHPP through TRIM21 mediated K48-linked ubiquitination. HDAC4 was confirmed to be highly expressed in NPC cells and promoted the proliferation and invasion of NPC cells through LHPP. Further research found that LHPP could inhibit the phosphorylation of tyrosine kinase TYK2, thereby inhibiting the activity of STAT1. In vivo, knockdown of HDAC4 or treatment with small molecule inhibitor Tasquinimod targeting HDAC4 could significantly inhibit the proliferation and metastasis of NPC by upregulating LHPP. In conclusion, our finding demonstrated that HDAC4/LHPP signal axis promotes the proliferation and metastasis of NPC through upregulating TYK2-STAT1 phosphorylation activation. This research will provide novel evidence and intervention targets for NPC metastasis.

STAM and Hrs interact sequentially with IFN-α Receptor to control spatiotemporal JAK-STAT endosomal activation.

Activation of the JAK-STAT pathway by type I interferons (IFNs) requires clathrin-dependent endocytosis of the IFN-α and -β receptor (IFNAR), indicating a role for endosomal sorting in this process. The molecular machinery that brings the selective activation of IFN-α/β-induced JAK-STAT signalling on endosomes remains unknown. Here we show that the constitutive association of STAM with IFNAR1 and TYK2 kinase at the plasma membrane prevents TYK2 activation by type I IFNs. IFN-α-stimulated IFNAR endocytosis delivers the STAM-IFNAR complex to early endosomes where it interacts with Hrs, thereby relieving TYK2 inhibition by STAM and triggering signalling of IFNAR at the endosome. In contrast, when stimulated by IFN-β, IFNAR signalling occurs independently of Hrs as IFNAR is sorted to a distinct endosomal subdomain. Our results identify the molecular machinery that controls the spatiotemporal activation of IFNAR by IFN-α and establish the central role of endosomal sorting in the differential regulation of JAK-STAT signalling by IFN-α and IFN-β.

Discovery and Characterization of the Topical Soft JAK Inhibitor CEE321 for Atopic Dermatitis

The JAK kinases JAK1, JAK2, JAK3, and TYK2 play key roles in cytokine signaling. Activation of the JAK/STAT pathways is linked to many diseases involving the immune system, including atopic dermatitis. As systemic JAK inhibitor pharmacology is associated with side effects, topical administration to the skin has been considered to locally restrict the site of action. Several orally bioavailable JAK inhibitors repurposed for topical use have been recently approved or are in clinical development. Here, we disclose our clinical candidate CEE321, which is a potent pan JAK inhibitor in enzyme and cellular assays. In contrast to repurposed oral drugs, CEE321 does not display high potency in blood and has a high clearance in vivo. Therefore, we consider CEE321 to be a "soft drug". When applied topically to human skin that was stimulated with the cytokines IL4 and IL13 ex vivo, CEE321 potently inhibited biomarkers relevant to atopic dermatitis.

Tyk2 is a tumor suppressor in colorectal cancer

ABSTRACT Janus kinase Tyk2 is implicated in cancer immune surveillance, but its role in solid tumors is not well defined. We used Tyk2 knockout mice (Tyk2Δ/Δ) and mice with conditional deletion of Tyk2 in hematopoietic (Tyk2ΔHem) or intestinal epithelial cells (Tyk2ΔIEC) to assess their cell type-specific functions in chemically induced colorectal cancer. All Tyk2-deficient mouse models showed a higher tumor burden after AOM-DSS treatment compared to their corresponding wild-type controls (Tyk2+/+ and Tyk2fl/fl), demonstrating tumor-suppressive functions of Tyk2 in immune cells and epithelial cancer cells. However, specific deletion of Tyk2 in hematopoietic cells or in intestinal epithelial cells was insufficient to accelerate tumor progression, while deletion in both compartments promoted carcinoma formation. RNA-seq and proteomics revealed that tumors of Tyk2Δ/Δ and Tyk2ΔIEC mice were immunoedited in different ways with downregulated and upregulated IFNγ signatures, respectively. Accordingly, the IFNγ-regulated immune checkpoint Ido1 was downregulated in Tyk2Δ/Δ and upregulated in Tyk2ΔIEC tumors, although both showed reduced CD8+ T cell infiltration. These data suggest that Tyk2Δ/Δ tumors are Ido1-independent and poorly immunoedited while Tyk2ΔIEC tumors require Ido1 for immune evasion. Our study shows that Tyk2 prevents Ido1 expression in CRC cells and promotes CRC immune surveillance in the tumor stroma. Both of these Tyk2-dependent mechanisms must work together to prevent CRC progression.

STAT and Janus kinase targeting by human herpesvirus 8 interferon regulatory factor in the suppression of type-I interferon signaling

Human herpesvirus 8 (HHV-8), also known as Kaposi’s sarcoma (KS)-associated herpesvirus, is involved etiologically in AIDS-associated KS, primary effusion lymphoma (PEL), and multicentric Castleman’s disease, in which both viral latent and lytic functions are important. HHV-8 encodes four viral interferon regulatory factors (vIRFs) that are believed to contribute to viral latency (in PEL cells, at least) and/or to productive replication via suppression of cellular antiviral and stress signaling. Here, we identify vIRF-1 interactions with signal transducer and activator of transcription (STAT) factors 1 and 2, interferon (IFN)-stimulated gene factor 3 (ISGF3) cofactor IRF9, and associated signal transducing Janus kinases JAK1 and TYK2. In naturally infected PEL cells and in iSLK epithelial cells infected experimentally with genetically engineered HHV-8, vIRF-1 depletion or ablation, respectively, led to increased levels of active (phosphorylated) STAT1 and STAT2 in IFNβ-treated, and untreated, cells during lytic replication and to associated cellular-gene induction. In transfected 293T cells, used for mechanistic studies, suppression by vIRF-1 of IFNβ-induced phospho-STAT1 (pSTAT1) was found to be highly dependent on STAT2, indicating vIRF-1-mediated inhibition and/or dissociation of ISGF3-complexing, resulting in susceptibility of pSTAT1 to inactivating dephosphorylation. Indeed, coprecipitation experiments involving targeted precipitation of ISGF3 components identified suppression of mutual interactions by vIRF-1. In contrast, suppression of IFNβ-induced pSTAT2 was effected by regulation of STAT2 activation, likely via detected inhibition of TYK2 and its interactions with STAT2 and IFN type-I receptor (IFNAR). Our identified vIRF-1 interactions with IFN-signaling mediators STATs 1 and 2, co-interacting ISGF3 component IRF9, and STAT-activating TYK2 and the suppression of IFN signaling via ISGF3, TYK2-STAT2 and TYK2-IFNAR disruption and TYK2 inhibition represent novel mechanisms of vIRF function and HHV-8 evasion from host-cell defenses.

Characterization of ARTS-011, a potent and selective TYK2 inhibitor for the treatment of inflammatory disease

Abstract The JAK family kinase TYK2 is a key regulator of IL-12 and IL-23 signaling pathways, which have been shown to be involved in the pathogenesis of multiple inflammatory and autoimmune diseases such as psoriasis, lupus and inflammatory bowel diseases (IBD). TYK2 inhibition is an attractive therapeutic strategy for human autoimmune diseases. Here, we report a potent oral TYK2 allosteric inhibitor ARTS-011, with high selectivity and potency through optimized binding to the JH2 regulatory domain of the TYK2 protein. ARTS-011 potently binds to TYK2 JH2 domain in binding assay and inhibits IL-23 and IFNα/β induced reporter activation depending on TYK2 kinase. ARTS-011 also inhibits IFNα triggered pSTAT5 potently in human whole blood assay. In addition, ARTS-011 excellent selectivity against other JAK family kinases and panels of receptors, transporters, ion channels, CYP enzymes and the hERG channel. Furthermore, ARTS-011 was also investigated in multiple animal models including IL12/18-induced serum IFNγ mouse model, IL-23-induced mouse psoriasis model and CD40 antibody-induced mouse inflammatory bowel disease (IBD) model. In vivo pharmacodynamic study indicates that ARTS-011 significantly reverses IL12/18-induced IFNγ production in mouse. ARTS-011 also significantly reduces the ear swelling in a dose-dependent manner in IL-23-induced mouse psoriasis model. Lastly, ARTS-011 was highly efficacious in anti-CD40 antibody induced mouse IBD model. In summary, ARTS-011 is a highly selective, potent and orally available small molecule TYK2 inhibitor with suitable pharmaceutical properties as potential therapeutics in inflammatory disorders.

Tyrosine kinases in KMT2A/MLL-rearranged acute leukemias as potential therapeutic targets to overcome cancer drug resistance.

Aim: The main goal of this study was to elucidate at the transcript level the tyrosine kinase expression profiles of primary leukemia cells from mixed lineage leukemia 1 gene rearranged (KMT2A/MLL-R+) acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients. Methods: We evaluated protein tyrosine kinase (PTK) gene expression profiles of primary leukemic cells in KMT2A/MLL-R+ AML and ALL patients using publicly available archived datasets. Results: Our studies provided unprecedented evidence that the genetic signatures of KMT2A/MLL-R+ AML and ALL cells are characterized by transcript-level overexpression of specific PTK. In infants, children and adults with KMT2A/MLL-R+ ALL, as well as pediatric patients with KMT2A/MLL-R+ AML, the gene expression levels for FLT3, BTK, SYK, JAK2/JAK3, as well as several SRC family PTK were differentially amplified. In adults with KMT2A/MLL-R+ AML, the gene expression levels for SYK, JAK family kinase TYK2, and the SRC family kinases FGR and HCK were differentially amplified. Conclusion: These results provide new insights regarding the clinical potential of small molecule inhibitors of these PTK, many of which are already FDA/EMA-approved for other indications, as components of innovative multi-modality treatment platforms against KMT2A/MLL-R+ acute leukemias.

TYK2 inhibition and its potential in the treatment of chronic inflammatory immune diseases.

Immune-mediated chronic inflammatory diseases have emerged as a leading cause of morbidity and mortality in Western countries over the last decades. Although multiple putative factors have been suspected to be causally related to the diseases, their overarching etiology remains unknown. This review article summarizes the current state of scientific knowledge and understanding of the role of non-receptor tyrosine kinases, with a special focus on the Janus kinase TYK2 in autoimmune and immune mediated diseases as well as on the clinical properties of its inhibition. A panel of experts in the field discussed the scientific evidence and molecular rationale for TYK2 inhibition and its clinical application. Reviewing this meeting, we aim at providing an integrated overview of the clinical profile of TYK2 inhibition and its potential in targeted pharmacological therapy of chronic autoimmune and immune-mediated diseases, with a special focus on inflammatory diseases of the skin.

Repurposing of gastric cancer drugs against COVID-19

Corona Virus Disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has become a global pandemic. Additionally, the SARS-CoV-2 infection in the patients of Gastric Cancer (GC; the third leading cause of death in the world) pose a great challenge for the health management of the patients. Since there have been uncertainties to develop a new drug against COVID-19, there is an urgent need for repurposing drugs that can target key proteins of both SARS-CoV-2 and GC. The SARS-CoV-2-RdRp protein contains the NiRAN domain, which is known to have kinase-like folds. A docking study of the FDA approved drugs against GC was performed using AutoDock 4.2 and Glide Schrodinger suite 2019 against SARS-CoV-2-RdRp protein. MMGBSA and MD simulation studies were performed to investigate the binding and stability of the inhibitors with the target protein. In this study, we have found 12 kinase inhibitors with high binding energies namely Baricitinib, Brepocitinib, Decernotinib, Fasudil, Filgotinib, GSK2606414, Peficitinib, Ruxolitinib, Tofacitinib, Upadacitinib, Pamapimod and Ibrutinib. These FDA approved drugs against GC can play a key role in the treatment of COVID-19 patients along with GC as comorbidity. We also hypothesize that JAK, ITK, Rho-associated kinases, FGFR2, FYN, PERK, TYK2, p38-MAPK and SYK kinases can be considered as key therapeutic targets in COVID-19 treatment. Taken altogether, we have proposed the SARS-CoV-2-RdRp as a potential therapeutic target through in-silico studies. However, further in-vitro and in-vivo studies are required for the validation of the proposed targets and drugs for the treatment of COVID-19 patients already suffering from GC.

Exome Sequencing Of Paired Chronic-Blast Phase Samples In Myeloproliferative Neoplasms Reveals High Frequency Of Rare Germline Sequence Variants In TYK2 and Novel Blast Phase-Restricted Mutations

The molecular underpinnings of leukemic transformation in myeloproliferative neoplasms (MPN) are not well characterized. We used Whole Exome Sequencing (WES) as a screening tool to gain further insight into the genomic origin of MPN progression to blast phase disease. WES analysis was performed in 6 JAK2V617F(+) MPN patients for which paired samples from chronic and blast phase disease were available. Respective clinical data were incorporated into the evaluation, and variations of interest were validated by conventional Sanger sequencing. Single-nucleotide-polymorphism database (dbSNP) was used to exclude/include established germline aberrations. Using WES filtering for non-synonymous variations as predicted deleterious, we detected a significant number of genetic abnormalities in both chronic and blast phase of MPN; these could be functionally linked to “tumorigenesis” (157 genes), “cancers and tumors” (159 genes) and “hematological neoplasia” (48 genes). In both phases of MPN, we could find mutations in genes already known to be linked with the development and/or transformation of MPN (ie, MPL, TET2, EZH2, IDH1, NRAS, LNK, TP53, WT1). Notably, eight genes were identified exclusively at onset of leukemic transformation and could be classified as potential driver mutations with damaging translational impact; these novel mutations could be grouped into “transcriptional regulators” (CUX2, ORC2, KDM6B, CNOT3, TBL1XR1), “NOTCH-signaling pathway” (NOTCH2NL, NOTCH1) and “tyrosine kinases” (STAP2). Moreover, we also identified several single-nucleotide polymorphisms encoding amino acid substitutions (ie, non-synonymous changes) in receptor as well as cytoplasmatic tyrosine kinase genes. Interestingly, the JAK-family member TYK2 showed the largest number of rare non-synonymous polymorphisms (A165T, V362F, G363S, I684S) leading to missense mutations in 67% (4/6) of the MPN patients; in contrast, none of 30 control samples with other types of cancer showed sequence variances in the TYK2 gene. Deficiency of TYK2 has shown to impair tumor surveillance and increase the susceptibility for neoplasia including AML. The present study demonstrates recurrent germline sequence variants in the non-receptor tyrosine kinase TYK2, and novel functionally-relevant somatic mutations with potential pathogenetic contribution to leukemic transformation in MPN. Disclosures:No relevant conflicts of interest to declare.

Abstract 91: FLT3 is a major driver of glucose consumption in squamous cell lung cancer

Abstract Squamous cell lung cancer (SqCLC) represents 30% of all cases of non-small cell lung cancer. Despite the prevalence of SqCLC, there are few molecularly targeted therapies for SqCLC patients. There is an urgent need for new approaches in this area. SqCLC cells consume elevated levels of glucose and are highly dependent on glucose consumption for their survival. Targeting glucose consumption may be a valuable therapeutic strategy in SqCLC but is challenged by the fact that glucose consumption is critical for vital organs. Our ability to selectively target glucose consumption in SqCLC is limited by an incomplete understanding of the shared and distinct mechanisms through which SqCLC and healthy cells drive glucose consumption. We recently developed and validated a high-throughput glucose consumption assay that serves as a platform technology for identifying new proteins and pathways that drive glucose consumption in cancerous and healthy cells. In this project, we screened H520, SK-MES-1, and H596 SqCLC cell lines against 3555 bioactive small molecules including a library of kinase inhibitors and FDA-approved drugs, and measured glucose consumption 24 hours after drug treatment. We discovered and validated 62 compounds that decreased glucose consumption per cell by >50% in at least one cell line. From this list, we searched for compounds that decreased glucose consumption in all three cell lines, by >70% in at least one cell line, and with an EC50 value <~1 µM; that have been used in mice and people; that have not been previously linked to glucose consumption; and that do not significantly decrease cell growth at the time point measured (24 hours post-treatment). This led us to further investigate Pacritinib, a small molecule kinase inhibitor in clinical trials for the treatment of myelofibrosis. Pacritinib reduces glucose consumption in all three SqCLC cell lines with an EC50 value of 1.2 µM in cell culture and blocks glucose consumption in cell line xenografts in vivo without affecting glucose consumption in healthy tissues. In the SqCLC cell lines, Pacritinib decreases protein levels of Hexokinase 1 and Hexokinase 2 as well as hexokinase activity as measured by a FRET-based glucose sensor. Hexokinase 1 overexpression in the SqCLC cell lines blocks Pacritinib from inhibiting glucose consumption. Pacritinib targets JAK2, TYK2, FLT3, and additional kinases at low nanomolar concentrations in vitro. Additional small molecule inhibitors of JAK2 and TYK2 had no effect on SqCLC glucose consumption. However additional inhibitors of FLT3 blocked glucose consumption in all three SqCLC cell lines. FLT3 overexpression increased SqCLC glucose consumption and blocked Pacritinib from inhibiting glucose consumption. In conclusion, using our high-throughput technology, we discovered that FLT3 is a selective and targetable driver of glucose consumption in SqCLC. Our data suggest that FLT3 activates glucose consumption by increasing Hexokinase 1 levels. Citation Format: Chiara Ghezzi, Robert Damoiseaux, Peter M. Clark. FLT3 is a major driver of glucose consumption in squamous cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 91.

Janus kinase signaling as risk factor and therapeutic target for severe SARS-CoV-2 infection.

Cytokine signaling, especially interferon (IFN) signaling is closely linked to several aspects of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection. During initial SARS‐CoV‐2 infection, symptomatic patients present with impaired type I/III IFN‐mediated antiviral responses. Interestingly, IFNs regulate the cellular entry receptor for SARS‐CoV‐2 on epithelial and endothelial cells. As reported recently, critically ill COVID‐19 patients show genetic polymorphisms in one IFN receptor gene (IFNRA2) and in a gene locus near the Janus kinase (JAK) TYK2, which is key for IFN, interleukin (IL)‐12 and IL‐23 signaling, and T helper (Th) 1/Th17 cell‐mediated antiviral immune responses. In the advanced stage of the disease, critically ill COVID‐19 patients develop a cytokine storm where many inflammatory mediators using the JAK/STAT signaling pathway such as IL‐6, IFN‐γ, the granulocyte colony‐stimulating factor (G‐CSF) or IL‐2, and chemokines result in an influx of macrophages and neutrophils damaging the lung tissue. The knowledge on the cytokine and JAK/STAT signaling pathways in severe COVID‐19 disease explains the promising first results with JAK inhibitors like baricitinib, which not only dampen the inflammation but in the case of baricitinib also affect virus replication and endocytosis in target cells. Here, we summarize the current immunological associations of SARS‐CoV‐2 infection with cytokine signaling, the JAK/STAT pathway, and the current clinical stage of JAK inhibitors for improving severe COVID‐19 disease.