CALCRL induces resistance to daunorubicin in acute myeloid leukemia cells through upregulation of XRCC5/TYK2/JAK1 pathway.

Chemotherapy is the main treatment option for acute myeloid leukemia (AML), but acquired resistance of leukemic cells to chemotherapeutic agents often leads to difficulties in AML treatment and disease relapse. High calcitonin receptor-like (CALCRL) expression is closely associated with poorer prognosis in AML patients. Therefore, this study was performed by performing CALCRL overexpression constructs in AML cell lines HL-60 and Molm-13 with low CALCRL expression. The results showed that overexpression of CALCRL in HL-60 and Molm-13 could confer resistance properties to AML cells and reduce the DNA damage and cell cycle G0/G1 phase blocking effects caused by daunorubicin (DNR) and others. Overexpression of CALCRL also reduced DNR-induced apoptosis. Mechanistically, the Cancer Clinical Research Database analyzed a significant positive correlation between XRCC5 and CALCRL in AML patients. Therefore, the combination of RT-PCR and Western blot studies further confirmed that the expression levels of XRCC5 and PDK1 genes and proteins were significantly upregulated after overexpression of CALCRL. In contrast, the phosphorylation levels of AKT/PKCε protein, a downstream pathway of XRCC5/PDK1, were significantly upregulated. In the response study, transfection of overexpressed CALCRL cells with XRCC5 siRNA significantly upregulated the drug sensitivity of AML to DNR. The expression levels of PDK1 protein and AKT/PKCε phosphorylated protein in the downstream pathway were inhibited considerably, and the expression of apoptosis-related proteins Bax and cleaved caspase-3 were upregulated. Animal experiments showed that the inhibitory effect of DNR on the growth of HL-60 cells and the number of bone marrow invasions were significantly reversed after overexpression of CALCRL in nude mice. However, infection of XCRR5 shRNA lentivirus in HL-60 cells with CALCRL overexpression attenuated the effect of CALCRL overexpression and upregulated the expression of apoptosis-related proteins induced by DNR. This study provides a preliminary explanation for the relationship between high CALCRL expression and poor prognosis of chemotherapy in AML patients. It offers a more experimental basis for DNR combined with molecular targets for precise treatment in subsequent studies.

Deucravacitinib is an allosteric TYK2 protein kinase inhibitor FDA-approved for the treatment of psoriasis.

Psoriasis is a heterogeneous, inflammatory, autoimmune skin disease that affects up to 2% of the world's population. There are many treatment modalities including topical medicines, ultraviolet light therapy, monoclonal antibodies, and several oral medications. Cytokines play a central role in the pathogenesis of this disorder including TNF-α, (tumor necrosis factor-α) IL-17A (interleukin-17A), IL-17F, IL-22, and IL-23. Cytokine signaling involves transduction mediated by the JAK-STAT pathway. There are four JAKS (JAK1/2/3 and TYK2) and six STATS (signal transducer and activators of transcription). Janus kinases contain an inactive JH2 domain that is aminoterminal to the active JH1 domain. Under basal conditions, the JH2 domain inhibits the activity of the JH1 domain. Deucravacitinib is an orally effective N-trideuteromethyl-pyridazine derivative that targets and stabilizes the TYK2 JH2 domain and thereby blocks TYK2 JH1 activity. Seven other JAK inhibitors, which target the JAK family JH1 domain, are prescribed for the treatment of neoplastic and other inflammatory diseases. The use of deuterium in the trimethylamide decreases the rate of demethylation and slows the production of a metabolite that is active against a variety of targets in addition to TYK2. A second unique aspect in the development of deucravacitinib is the targeting of a pseudokinase domain. Deucravacitinib is rather specific for TYK2 and its toxic effects are much less than those of the other FDA-approved JAK inhibitors. The successful development of deucravacitinib may stimulate the development of additional pseudokinase ligands for the JAK family and for other kinase families as well.

MEK Inhibition Synergizes with TYK2 Inhibitors in NF1-Associated Malignant Peripheral Nerve Sheath Tumors.

PURPOSE: Malignant peripheral nerve sheath tumors (MPNST) are aggressive sarcomas with limited treatment options and poor survival rates. About half of MPNST cases are associated with the neurofibromatosis type 1 (NF1) cancer predisposition syndrome. Overexpression of TYK2 occurs in the majority of MPNST, implicating TYK2 as a therapeutic target. EXPERIMENTAL DESIGN: The effects of pharmacologic TYK2 inhibition on MPNST cell proliferation and survival were examined using IncuCyte live cell assays in vitro, and downstream actions were analyzed using RNA-sequencing (RNA-seq), qPCR arrays, and validation of protein changes with the WES automated Western system. Inhibition of TYK2 alone and in combination with MEK inhibition was evaluated in vivo using both murine and human MPNST cell lines, as well as MPNST PDX. RESULTS: Pharmacologic inhibition of TYK2 dose-dependently decreased proliferation and induced apoptosis over time. RNA-seq pathway analysis on TYK2 inhibitor-treated MPNST demonstrated decreased expression of cell cycle, mitotic, and glycolysis pathways. TYK2 inhibition resulted in upregulation of the MEK/ERK pathway gene expression, by both RNA-seq and qPCR array, as well as increased pERK1/2 levels by the WES Western system. The compensatory response was tested with dual treatment with TYK2 and MEK inhibitors, which synergistically decreased proliferation and increased apoptosis in vitro. Finally, combination therapy was shown to inhibit growth of MPNST in multiple in vivo models. CONCLUSIONS: These data provide the preclinical rationale for the development of a phase I clinical trial of deucravacitinib and mirdametinib in NF1-assosciated MPNST.

Isorhamnetin Attenuated the Release of Interleukin-6 from ß-Amyloid-Activated Microglia and Mitigated Interleukin-6-Mediated Neurotoxicity.

Alzheimer's disease (AD), characterized by the abnormal accumulation of ß-amyloid (Aß), is the most prevalent type of dementia, and it is associated with progressive cognitive decline and memory loss. Aß accumulation activates microglia, which secrete proinflammatory factors associated with Aß clearance impairment and cause neurotoxicity, generating a vicious cycle among Aß accumulation, activated microglia, and proinflammatory factors. Blocking this cycle can be a therapeutic strategy for AD. Using Aß-activated HMC3 microglial cells, we observed that isorhamnetin, a main constituent of Oenanthe javanica, reduced the Aß-triggered secretion of interleukin- (IL-) 6 and downregulated the expression levels of the microglial activation markers ionized calcium binding adaptor molecule 1 (IBA1) and CD11b and the inflammatory marker nuclear factor-κB (NF-κB). Treatment of the SH-SY5Y-derived neuronal cells with the Aß-activated HMC3-conditioned medium (HMC3-conditioned medium) or IL-6 increased reactive oxygen species production, upregulated cleaved caspase 3 expression, and reduced neurite outgrowth, whereas treatment with isorhamnetin counteracted these neurodegenerative presentations. In the SH-SY5Y-derived neuronal cells, IL-6 upregulated the phosphorylation of tyrosine kinase 2 (TYK2) and signal transducer and activator of transcription 1 (STAT1), whereas isorhamnetin normalized this abnormal phosphorylation. Overexpression of TYK2 attenuated the neuroprotective effect of isorhamnetin on IL-6-induced neurotoxicity. Our findings demonstrate that isorhamnetin exerts its neuroprotective effect by mediating the neuroinflammatory IL-6/TYK2 signaling pathway, suggesting its potential for treating AD.

Cannabinoids Alleviate the LPS-Induced Cytokine Storm via Attenuating NLRP3 Inflammasome Signaling and TYK2-Mediated STAT3 Signaling Pathways In Vitro.

Cannabinoids, mainly cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC), are the most studied group of compounds obtained from Cannabis sativa because of their several pharmaceutical properties. Current evidence suggests a crucial role of cannabinoids as potent anti-inflammatory agents for the treatment of chronic inflammatory diseases; however, the mechanisms remain largely unclear. Cytokine storm, a dysregulated severe inflammatory response by our immune system, is involved in the pathogenesis of numerous chronic inflammatory disorders, including coronavirus disease 2019 (COVID-19), which results in the accumulation of pro-inflammatory cytokines. Therefore, we hypothesized that CBD and THC reduce the levels of pro-inflammatory cytokines by inhibiting key inflammatory signaling pathways. The nucleotide-binding and oligomerization domain (NOD)-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome signaling has been implicated in a variety of chronic inflammatory diseases, which results in the release of pyroptotic cytokines, interleukin-1ß (IL-1ß) and IL-18. Likewise, the activation of the signal transducer and activator of transcription-3 (STAT3) causes increased expression of pro-inflammatory cytokines. We studied the effects of CBD and THC on lipopolysaccharide (LPS)-induced inflammatory response in human THP-1 macrophages and primary human bronchial epithelial cells (HBECs). Our results revealed that CBD and, for the first time, THC significantly inhibited NLRP3 inflammasome activation following LPS + ATP stimulation, leading to a reduction in the levels of IL-1ß in THP-1 macrophages and HBECs. CBD attenuated the phosphorylation of nuclear factor-κB (NF-κB), and both cannabinoids inhibited the generation of oxidative stress post-LPS. Our multiplex ELISA data revealed that CBD and THC significantly diminished the levels of IL-6, IL-8, and tumor necrosis factor-α (TNF-α) after LPS treatment in THP-1 macrophages and HBECs. In addition, the phosphorylation of STAT3 was significantly downregulated by CBD and THC in THP-1 macrophages and HBECs, which was in turn attributed to the reduced phosphorylation of tyrosine kinase-2 (TYK2) by CBD and THC after LPS stimulation in these cells. Overall, CBD and THC were found to be effective in alleviating the LPS-induced cytokine storm in human macrophages and primary HBECs, at least via modulation of NLRP3 inflammasome and STAT3 signaling pathways. The encouraging results from this study warrant further investigation of these cannabinoids in vivo.

Pirfenidone attenuates cardiac hypertrophy against isoproterenol by inhibiting activation of the janus tyrosine kinase-2/signal transducer and activator of transcription 3 (JAK-2/STAT3) signaling pathway.

Cardiovascular risk factors have attracted increasing attention in recent years with the acceleration of population aging, amongst which cardiac hypertrophy is the initiating link to heart failure. Pirfenidone is a promising agent for the treatment of idiopathic pulmonary fibrosis and has recently proven to exert inhibitory effects on the inflammatory response. This study proposes to explore the potential pharmacological action of Pirfenidone in treating cardiac hypertrophy in a rodent model. Four groups of mice were used in the present study: the control, ISO (5 mg/kg/day) for 7 days, Pirfenidone (200 mg/kg/day) for 14 days, and Spironolactone (SPI) (200 mg/kg/day) for 14 days groups. Increased heart weight index, left ventricle (LV) weight index, LV wall thickness, declined LV volume, and elevated serum levels of CK-MB, AST, and LDH were observed in ISO-challenged mice, all of which were dramatically reversed by the administration of Pirfenidone or SPI. Furthermore, an elevated cross-sectional area of cardiomyocytes in the wheat germ agglutinin (WGA) staining of heart cross-sections, upregulated atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), ß Myosin Heavy Chain (ß-MHC), and excessively released tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) in cardiac tissues were observed in the ISO group but greatly alleviated by Pirfenidone or SPI. Lastly, the promoted expression levels of p-JAK-2/JAK-2 and p-STAT3/STAT-3 in the cardiac tissues of ISO-challenged mice were significantly repressed by Pirfenidone or SPI. Collectively, our data reveals a therapeutic property of Pirfenidone on ISO-induced cardiac hypertrophy in mice.

Nicotine Activating α4ß2 Nicotinic Acetylcholine Receptors to Suppress Neuroinflammation via JAK2-STAT3 Signaling Pathway in Ischemic Rats and Inflammatory Cells.

Nicotine plays a role in inhibiting inflammatory factors, which contributes to improving cognitive impairment by activating α4ß2 nAChRs in ischemic rats, but the underlying mechanism has not been fully elucidated. Janus tyrosine kinase 2-signal transducer and activator of transcription 3 (JAK2-STAT3) signaling pathway is involved in cognitive improvement, and there seems to be a relationship between nAChRs and JAK2-STAT3 as well. The aim of this study is to explore the role of JAK2-STAT3 signaling pathway in nicotine-mediated anti-inflammatory effect. Nicotine, DHßE (the strongest competitive antagonist of α4ß2 nAChRs), and AG490 (a specific JAK2-STAT3 blocker) were used to intervene and treat ischemic rats and HEK-293 T-hα4ß2 cells. The Morris water maze (MWM) test and 2-[18F]-A-85380 PET imaging were performed to detect the cognitive function and α4ß2 nAChRs density in ischemic rats. The results demonstrated that nicotine intervention increased the density of α4ß2 nAChRs and improved cognitive impairment, but this effect was blocked by AG490, and the receptors were still upregulated. Essentially, when the JAK2-STAT3 signaling pathway was blocked, nicotine could only upregulate the expression of α4ß2 nAChRs, but not improve the cognitive function. PCR and Western blot analysis further confirmed these results. The cell experiments also showed that nicotine could reduce inflammatory factors stimulated by LPS and upregulate the expression of pJAK2 and pSTAT3 in HEK-293 T-hα4ß2 cells, while AG490 and DHßE reversed the effect of nicotine. To sum up, our work indicated that JAK2-STAT3 signaling pathway played an important role in nicotine-induced cognitive improvement by upregulating α4ß2 nAChRs in ischemic rats.

Lemur tyrosine kinase 2 silencing inhibits the proliferation of gastric cancer cells by regulating GSK-3ß phosphorylation and ß-catenin nuclear translocation.

Previous studies on the mechanism of proliferation and cell cycle progression of gastric cancer cells have shown promising perspectives for the prevention and treatment of gastric cancer. The aim of the present study was to investigate the role of lemur tyrosine kinase 2 (LMTK2) in gastric cancer cell proliferation and cell cycle progression, as well as in tumor-bearing nude mouse models. The expression levels of LMTK2 were determined in gastric cancer cell lines. In addition, the effects of LMTK2 silencing or overexpression on cell proliferation were measured using Cell Counting Kit-8, BrdU and colony formation assays. Cell cycle progression was analyzed using flow cytometry and western blotting. The expression levels of proteins associated with the ß-catenin pathway were assessed using western blot analysis. A tumor-bearing nude mouse model was established by injecting gastric cancer cells, and the effect of LMTK2 knockdown or overexpression on tumor growth was examined. The expression levels of LMTK2 were found to be upregulated in all gastric cancer cell lines. Moreover, LMTK2 knockdown inhibited cell proliferation, colony formation and cell cycle progression. LMTK2 knockdown also inhibited the activation of GSK-3ß/ß-catenin signaling, as evidenced by reduced GSK-3ß phosphorylation and nuclear ß-catenin levels. LMTK2 knockdown also suppressed tumor growth, whereas overexpression accelerated this process. In conclusion, LMTK2 silencing can inhibit the proliferation of gastric cancer cells in vitro and tumor growth in vivo by regulating GSK-3ß phosphorylation and ß-catenin nuclear translocation.

TYK2 licenses non-canonical inflammasome activation during endotoxemia.

The non-canonical inflammasome is an emerging crucial player in the development of inflammatory and neurodegenerative diseases. It is activated by direct sensing of cytosolic lipopolysaccharide (LPS) by caspase-11 (CASP11), which then induces pyroptosis, an inflammatory form of regulated cell death. Here, we report that tyrosine kinase 2 (TYK2), a cytokine receptor-associated kinase, is a critical upstream regulator of CASP11. Absence of TYK2 or its kinase activity impairs the transcriptional induction of CASP11 in vitro and in vivo and protects mice from LPS-induced lethality. Lack of TYK2 or its enzymatic activity inhibits macrophage pyroptosis and impairs release of mature IL-1ß and IL-18 specifically in response to intracellular LPS. Deletion of TYK2 in myeloid cells reduces LPS-induced IL-1ß and IL-18 production in vivo, highlighting the importance of these cells in the inflammatory response to LPS. In support of our data generated with genetically engineered mice, pharmacological inhibition of TYK2 reduced LPS-induced upregulation of CASP11 in bone marrow-derived macrophages (BMDMs) and of its homolog CASP5 in human macrophages. Our study provides insights into the regulation of CASP11 in vivo and uncovered a novel link between TYK2 activity and CASP11-dependent inflammation.

Molecular docking, 3D-QSAR, molecular dynamics, synthesis and anticancer activity of tyrosine kinase 2 (TYK 2) inhibitors.

A therapeutic rationale is proposed by selectively targeting tyrosine kinase 2 (TYK 2) to obtain potent TYK 2 inhibitors by molecular modeling studies. In the present study, we have taken tyrosine kinase (TYK 2) inhibitors and carried out molecular docking, 3 D quantitative structure-activity relationship (3D-QSAR) analysis and molecular dynamics (MD). Based on the 3D-QSAR results thirteen new compounds (R-1 to R-13) were designed and synthesized in good yields. The synthesized molecules were evaluated for their in vitro anticancer activity against LnCap and A549 cell lines. The molecules R-1, R-3, R-5, R-7, and R-10 exhibited considerable anti cancer activity.