KRAS Dimerization Impacts MEK Inhibitor Sensitivity and Oncogenic Activity of Mutant KRAS.

The mechanism by which the wild-type KRAS allele imparts a growth inhibitory effect to oncogenic KRAS in various cancers, including lung adenocarcinoma (LUAD), is poorly understood. Here, using a genetically inducible model of KRAS loss of heterozygosity (LOH), we show that KRAS dimerization mediates wild-type KRAS-dependent fitness of human and murine KRAS mutant LUAD tumor cells and underlies resistance to MEK inhibition. These effects are abrogated when wild-type KRAS is replaced by KRASD154Q, a mutant that disrupts dimerization at the α4-α5 KRAS dimer interface without changing other fundamental biochemical properties of KRAS, both in vitro and in vivo. Moreover, dimerization has a critical role in the oncogenic activity of mutant KRAS. Our studies provide mechanistic and biological insights into the role of KRAS dimerization and highlight a role for disruption of dimerization as a therapeutic strategy for KRAS mutant cancers.

ZAKα Recognizes Stalled Ribosomes through Partially Redundant Sensor Domains.

Impairment of ribosome function activates the MAPKKK ZAK, leading to activation of mitogen-activated protein (MAP) kinases p38 and JNK and inflammatory signaling. The mechanistic basis for activation of this ribotoxic stress response (RSR) remains completely obscure. We show that the long isoform of ZAK (ZAKα) directly associates with ribosomes by inserting its flexible C terminus into the ribosomal intersubunit space. Here, ZAKα binds helix 14 of 18S ribosomal RNA (rRNA). An adjacent domain in ZAKα also probes the ribosome, and together, these sensor domains are critically required for RSR activation after inhibition of both the E-site, the peptidyl transferase center (PTC), and ribotoxin action. Finally, we show that ablation of the RSR response leads to organismal phenotypes and decreased lifespan in the nematode Caenorhabditis elegans (C. elegans). Our findings yield mechanistic insight into how cells detect ribotoxic stress and provide experimental in vivo evidence for its physiological importance.

RICTOR/mTORC2 downregulation in BRAFV600E melanoma cells promotes resistance to BRAF/MEK inhibition.

BACKGROUND: The main drawback of BRAF/MEK inhibitors (BRAF/MEKi)-based targeted therapy in the management of BRAF-mutated cutaneous metastatic melanoma (MM) is the development of therapeutic resistance. We aimed to assess in this context the role of mTORC2, a signaling complex defined by the presence of the essential RICTOR subunit, regarded as an oncogenic driver in several tumor types, including MM. METHODS: After analyzing The Cancer Genome Atlas MM patients' database to explore both overall survival and molecular signatures as a function of intra-tumor RICTOR levels, we investigated the effects of RICTOR downregulation in BRAFV600E MM cell lines on their response to BRAF/MEKi. We performed proteomic screening to identify proteins modulated by changes in RICTOR expression, and Seahorse analysis to evaluate the effects of RICTOR depletion on mitochondrial respiration. The combination of BRAFi with drugs targeting proteins and processes emerged in the proteomic screening was carried out on RICTOR-deficient cells in vitro and in a xenograft setting in vivo. RESULTS: Low RICTOR levels in BRAF-mutated MM correlate with a worse clinical outcome. Gene Set Enrichment Analysis of low-RICTOR tumors display gene signatures suggestive of activation of the mitochondrial Electron Transport Chain (ETC) energy production. RICTOR-deficient BRAFV600E cells are intrinsically tolerant to BRAF/MEKi and anticipate the onset of resistance to BRAFi upon prolonged drug exposure. Moreover, in drug-naïve cells we observed a decline in RICTOR expression shortly after BRAFi exposure. In RICTOR-depleted cells, both mitochondrial respiration and expression of nicotinamide phosphoribosyltransferase (NAMPT) are enhanced, and their pharmacological inhibition restores sensitivity to BRAFi. CONCLUSIONS: Our work unveils an unforeseen tumor-suppressing role for mTORC2 in the early adaptation phase of BRAFV600E melanoma cells to targeted therapy and identifies the NAMPT-ETC axis as a potential therapeutic vulnerability of low RICTOR tumors. Importantly, our findings indicate that the evaluation of intra-tumor RICTOR levels has a prognostic value in metastatic melanoma and may help to guide therapeutic strategies in a personalized manner.

Marine Staurosporine Analogues: Activity and Target Identification in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with high mortality and drug resistance and no targeted drug available at present. Compound 4, a staurosporine alkaloid derived from Streptomyces sp. NBU3142 in a marine sponge, exhibits potent anti-TNBC activity. This research investigated its impact on MDA-MB-231 cells and their drug-resistant variants. The findings highlighted that compound 4 inhibits breast cancer cell migration, induces apoptosis, arrests the cell cycle, and promotes cellular senescence in both regular and paclitaxel-resistant MDA-MB-231 cells. Additionally, this study identified mitogen-activated protein kinase kinase kinase 11 (MAP3K11) as a target of compound 4, implicating its role in breast tumorigenesis by affecting cell proliferation, migration, and cell cycle progression.

Allosteric MEK inhibitors act on BRAF/MEK complexes to block MEK activation.

The RAF/MEK/ERK pathway is central to the control of cell physiology, and its dysregulation is associated with many cancers. Accordingly, the proteins constituting this pathway, including MEK1/2 (MEK), have been subject to intense drug discovery and development efforts. Allosteric MEK inhibitors (MEKi) exert complex effects on RAF/MEK/ERK pathway signaling and are employed clinically in combination with BRAF inhibitors in malignant melanoma. Although mechanisms and structures of MEKi bound to MEK have been described for many of these compounds, recent studies suggest that RAF/MEK complexes, rather than free MEK, should be evaluated as the target of MEKi. Here, we describe structural and biochemical studies of eight structurally diverse, clinical-stage MEKi to better understand their mechanism of action on BRAF/MEK complexes. We find that all of these agents bind in the MEK allosteric site in BRAF/MEK complexes, in which they stabilize the MEK activation loop in a conformation that is resistant to BRAF-mediated dual phosphorylation required for full activation of MEK. We also show that allosteric MEK inhibitors act most potently on BRAF/MEK complexes rather than on free active MEK, further supporting the notion that a BRAF/MEK complex is the physiologically relevant pharmacologic target for this class of compounds. Our findings provide a conceptual and structural framework for rational development of RAF-selective MEK inhibitors as an avenue to more effective and better-tolerated agents targeting this pathway.

TAK1 inhibition elicits mitochondrial ROS to block intracellular bacterial colonization.

Mitogen-activated protein kinase kinase kinase 7 (MAP3K7), known as TAK1, is an intracellular signaling intermediate of inflammatory responses. However, a series of mouse Tak1 gene deletion analyses have revealed that ablation of TAK1 does not prevent but rather elicits inflammation, which is accompanied by elevation of reactive oxygen species (ROS). This has been considered a consequence of impaired TAK1-dependent maintenance of tissue integrity. Contrary to this view, here we propose that TAK1 inhibition-induced ROS are an active cellular process that targets intracellular bacteria. Intracellular bacterial effector proteins such as Yersinia's outer membrane protein YopJ are known to inhibit TAK1 to circumvent the inflammatory host responses. We found that such TAK1 inhibition induces mitochondrial-derived ROS, which effectively destroys intracellular bacteria. Two cell death-signaling molecules, caspase 8 and RIPK3, cooperatively participate in TAK1 inhibition-induced ROS and blockade of intracellular bacterial growth. Our results reveal a previously unrecognized host defense mechanism, which is initiated by host recognition of pathogen-induced impairment in a host protein, TAK1, but not directly of pathogens.

Inflammatory Cytokine-Induced Muscle Atrophy and Weakness Can Be Ameliorated by an Inhibition of TGF-ß-Activated Kinase-1.

Chronic inflammation causes muscle wasting. Because most inflammatory cytokine signals are mediated via TGF-ß-activated kinase-1 (TAK1) activation, inflammatory cytokine-induced muscle wasting may be ameliorated by the inhibition of TAK1 activity. The present study was undertaken to clarify whether TAK1 inhibition can ameliorate inflammation-induced muscle wasting. SKG/Jcl mice as an autoimmune arthritis animal model were treated with a small amount of mannan as an adjuvant to enhance the production of TNF-α and IL-1ß. The increase in these inflammatory cytokines caused a reduction in muscle mass and strength along with an induction of arthritis in SKG/Jcl mice. Those changes in muscle fibers were mediated via the phosphorylation of TAK1, which activated the downstream signaling cascade via NF-κB, p38 MAPK, and ERK pathways, resulting in an increase in myostatin expression. Myostatin then reduced the expression of muscle proteins not only via a reduction in MyoD1 expression but also via an enhancement of Atrogin-1 and Murf1 expression. TAK1 inhibitor, LL-Z1640-2, prevented all the cytokine-induced changes in muscle wasting. Thus, TAK1 inhibition can be a new therapeutic target of not only joint destruction but also muscle wasting induced by inflammatory cytokines.

Transcription factor Foxp1 exerts essential cell-intrinsic regulation of the quiescence of naive T cells.

The molecular mechanisms that underlie T cell quiescence are poorly understood. Here we report that mature naive CD8(+) T cells lacking the transcription factor Foxp1 gained effector phenotype and function and proliferated directly in response to interleukin 7 (IL-7) in vitro. Foxp1 repressed expression of the IL-7 receptor α-chain (IL-7Rα) by antagonizing Foxo1 and negatively regulated signaling by the kinases MEK and Erk. Acute deletion of Foxp1 induced naive T cells to gain an effector phenotype and proliferate in lympho-replete mice. Foxp1-deficient naive CD8(+) T cells proliferated even in lymphopenic mice deficient in major histocompatibility complex class I. Our results demonstrate that Foxp1 exerts essential cell-intrinsic regulation of naive T cell quiescence, providing direct evidence that lymphocyte quiescence is achieved through actively maintained mechanisms that include transcriptional regulation.

TLR-4 Agonist Induces IFN-γ Production Selectively in Proinflammatory Human M1 Macrophages through the PI3K-mTOR- and JNK-MAPK-Activated p70S6K Pathway.

IFN-γ, a proinflammatory cytokine produced primarily by T cells and NK cells, activates macrophages and engages mechanisms to control pathogens. Although there is evidence of IFN-γ production by murine macrophages, IFN-γ production by normal human macrophages and their subsets remains unknown. Herein, we show that human M1 macrophages generated by IFN-γ and IL-12- and IL-18-stimulated monocyte-derived macrophages (M0) produce significant levels of IFN-γ. Further stimulation of IL-12/IL-18-primed macrophages or M1 macrophages with agonists for TLR-2, TLR-3, or TLR-4 significantly enhanced IFN-γ production in contrast to the similarly stimulated M0, M2a, M2b, and M2c macrophages. Similarly, M1 macrophages generated from COVID-19-infected patients' macrophages produced IFN-γ that was enhanced following LPS stimulation. The inhibition of M1 differentiation by Jak inhibitors reversed LPS-induced IFN-γ production, suggesting that differentiation with IFN-γ plays a key role in IFN-γ induction. We subsequently investigated the signaling pathway(s) responsible for TLR-4-induced IFN-γ production in M1 macrophages. Our results show that TLR-4-induced IFN-γ production is regulated by the ribosomal protein S6 kinase (p70S6K) through the activation of PI3K, the mammalian target of rapamycin complex 1/2 (mTORC1/2), and the JNK MAPK pathways. These results suggest that M1-derived IFN-γ may play a key role in inflammation that may be augmented following bacterial/viral infections. Moreover, blocking the mTORC1/2, PI3K, and JNK MAPKs in macrophages may be of potential translational significance in preventing macrophage-mediated inflammatory diseases.

Cell adhesion tunes inflammatory TPL2 kinase signal transduction.

Signaling through adhesion-related molecules is important for cancer growth and metastasis and cancer cells are resistant to anoikis, a form of cell death ensued by cell detachment from the extracellular matrix. Herein, we report that detached carcinoma cells and immortalized fibroblasts display defects in TNF and CD40 ligand (CD40L)-induced MEK-ERK signaling. Cell detachment results in reduced basal levels of the MEK kinase TPL2, compromises TPL2 activation and sensitizes carcinoma cells to death-inducing receptor ligands, mimicking the synthetic lethal interactions between TPL2 inactivation and TNF or CD40L stimulation. Focal Adhesion Kinase (FAK), which is activated in focal adhesions and mediates anchorage-dependent survival signaling, was found to sustain steady state TPL2 protein levels and to be required for TNF-induced TPL2 signal transduction. We show that when FAK levels are reduced, as seen in certain types of malignancy or malignant cell populations, the formation of cIAP2:RIPK1 complexes increases, leading to reduced TPL2 expression levels by a dual mechanism: first, by the reduction in the levels of NF-κΒ1 which is required for TPL2 stability; second, by the engagement of an RelA NF-κΒ pathway that elevates interleukin-6 production, leading to activation of STAT3 and its transcriptional target SKP2 which functions as a TPL2 E3 ubiquitin ligase. These data underscore a new mode of regulation of TNF family signal transduction on the TPL2-MEK-ERK branch by adhesion-related molecules that may have important ramifications for cancer therapy.

Mixed lineage kinase 3 inhibition induces T cell activation and cytotoxicity.

Mixed lineage kinase 3 (MLK3), also known as MAP3K11, was initially identified in a megakaryocytic cell line and is an emerging therapeutic target in cancer, yet its role in immune cells is not known. Here, we report that loss or pharmacological inhibition of MLK3 promotes activation and cytotoxicity of T cells. MLK3 is abundantly expressed in T cells, and its loss alters serum chemokines, cytokines, and CD28 protein expression on T cells and its subsets. MLK3 loss or pharmacological inhibition induces activation of T cells in in vitro, ex vivo, and in vivo conditions, irrespective of T cell activating agents. Conversely, overexpression of MLK3 decreases T cell activation. Mechanistically, loss or inhibition of MLK3 down-regulates expression of a prolyl-isomerase, Ppia, which is directly phosphorylated by MLK3 to increase its isomerase activity. Moreover, MLK3 also phosphorylates nuclear factor of activated T cells 1 (NFATc1) and regulates its nuclear translocation via interaction with Ppia, and this regulates T cell effector function. In an immune-competent mouse model of breast cancer, MLK3 inhibitor increases Granzyme B-positive CD8+ T cells and decreases MLK3 and Ppia gene expression in tumor-infiltrating T cells. Likewise, the MLK3 inhibitor in pan T cells, isolated from breast cancer patients, also increases cytotoxic CD8+ T cells. These results collectively demonstrate that MLK3 plays an important role in T cell biology, and targeting MLK3 could serve as a potential therapeutic intervention via increasing T cell cytotoxicity in cancer.

Five-Year Outcomes with Dabrafenib plus Trametinib in Metastatic Melanoma.

BACKGROUND: Patients who have unresectable or metastatic melanoma with a BRAF V600E or V600K mutation have prolonged progression-free survival and overall survival when receiving treatment with BRAF inhibitors plus MEK inhibitors. However, long-term clinical outcomes in these patients remain undefined. To determine 5-year survival rates and clinical characteristics of the patients with durable benefit, we sought to review long-term data from randomized trials of combination therapy with BRAF and MEK inhibitors. METHODS: We analyzed pooled extended-survival data from two trials involving previously untreated patients who had received BRAF inhibitor dabrafenib (at a dose of 150 mg twice daily) plus MEK inhibitor trametinib (2 mg once daily) in the COMBI-d and COMBI-v trials. The median duration of follow-up was 22 months (range, 0 to 76). The primary end points in the COMBI-d and COMBI-v trials were progression-free survival and overall survival, respectively. RESULTS: A total of 563 patients were randomly assigned to receive dabrafenib plus trametinib (211 in the COMBI-d trial and 352 in the COMBI-v trial). The progression-free survival rates were 21% (95% confidence interval [CI], 17 to 24) at 4 years and 19% (95% CI, 15 to 22) at 5 years. The overall survival rates were 37% (95% CI, 33 to 42) at 4 years and 34% (95% CI, 30 to 38) at 5 years. In multivariate analysis, several baseline factors (e.g., performance status, age, sex, number of organ sites with metastasis, and lactate dehydrogenase level) were significantly associated with both progression-free survival and overall survival. A complete response occurred in 109 patients (19%) and was associated with an improved long-term outcome, with an overall survival rate of 71% (95% CI, 62 to 79) at 5 years. CONCLUSIONS: First-line treatment with dabrafenib plus trametinib led to long-term benefit in approximately one third of the patients who had unresectable or metastatic melanoma with a BRAF V600E or V600K mutation. (Funded by GlaxoSmithKline and Novartis; COMBI-d ClinicalTrials.gov number, NCT01584648; COMBI-v ClinicalTrials.gov number, NCT01597908.).

Therapeutic Potential of Tpl2 (Tumor Progression Locus 2) Inhibition on Diabetic Vasculopathy Through the Blockage of the Inflammasome Complex.

OBJECTIVE: Diabetic retinopathy, one of retinal vasculopathy, is characterized by retinal inflammation, vascular leakage, blood-retinal barrier breakdown, and neovascularization. However, the molecular mechanisms that contribute to diabetic retinopathy progression remain unclear. Approach and Results: Tpl2 (tumor progression locus 2) is a protein kinase implicated in inflammation and pathological vascular angiogenesis. Nε-carboxymethyllysine (CML) and inflammatory cytokines levels in human sera and in several diabetic murine models were detected by ELISA, whereas liquid chromatography-tandem mass spectrometry analysis was used for whole eye tissues. The CML and p-Tpl2 expressions on the human retinal pigment epithelium (RPE) cells were determined by immunofluorescence. Intravitreal injection of pharmacological inhibitor or NA (neutralizing antibody) was used in a diabetic rat model. Retinal leukostasis, optical coherence tomography, and H&E staining were used to observe pathological features. Sera of diabetic retinopathy patients had significantly increased CML levels that positively correlated with diabetic retinopathy severity and foveal thickness. CML and p-Tpl2 expressions also significantly increased in the RPE of both T1DM and T2DM diabetes animal models. Mechanistic studies on RPE revealed that CML-induced Tpl2 activation and NADPH oxidase, and inflammasome complex activation were all effectively attenuated by Tpl2 inhibition. Tpl2 inhibition by NA also effectively reduced inflammatory/angiogenic factors, retinal leukostasis in streptozotocin-induced diabetic rats, and RPE secretion of inflammatory cytokines. The attenuated release of angiogenic factors led to inhibited vascular abnormalities in the diabetic animal model. CONCLUSIONS: The inhibition of Tpl2 can block the inflammasome signaling pathway in RPE and has potential clinical and therapeutic implications in diabetes-associated retinal microvascular dysfunction.

The Map3k12 (Dlk)/JNK3 signaling pathway is required for pancreatic beta-cell proliferation during postnatal development.

Unveiling the key pathways underlying postnatal beta-cell proliferation can be instrumental to decipher the mechanisms of beta-cell mass plasticity to increased physiological demand of insulin during weight gain and pregnancy. Using transcriptome and global Serine Threonine Kinase activity (STK) analyses of islets from newborn (10 days old) and adult rats, we found that highly proliferative neonatal rat islet cells display a substantially elevated activity of the mitogen activated protein 3 kinase 12, also called dual leucine zipper-bearing kinase (Dlk). As a key upstream component of the c-Jun amino terminal kinase (Jnk) pathway, Dlk overexpression was associated with increased Jnk3 activity and was mainly localized in the beta-cell cytoplasm. We provide the evidence that Dlk associates with and activates Jnk3, and that this cascade stimulates the expression of Ccnd1 and Ccnd2, two essential cyclins controlling postnatal beta-cell replication. Silencing of Dlk or of Jnk3 in neonatal islet cells dramatically hampered primary beta-cell replication and the expression of the two cyclins. Moreover, the expression of Dlk, Jnk3, Ccnd1 and Ccnd2 was induced in high replicative islet beta cells from ob/ob mice during weight gain, and from pregnant female rats. In human islets from non-diabetic obese individuals, DLK expression was also cytoplasmic and the rise of the mRNA level was associated with an increase of JNK3, CCND1 and CCND2 mRNA levels, when compared to islets from lean and obese patients with diabetes. In conclusion, we find that activation of Jnk3 signalling by Dlk could be a key mechanism for adapting islet beta-cell mass during postnatal development and weight gain.

The combination of TPL2 knockdown and TNFα causes synthetic lethality via caspase-8 activation in human carcinoma cell lines.

Most normal and tumor cells are protected from tumor necrosis factor α (TNFα)-induced apoptosis. Here, we identify the MAP3 kinase tumor progression locus-2 (TPL2) as a player contributing to the protection of a subset of tumor cell lines. The combination of TPL2 knockdown and TNFα gives rise to a synthetic lethality phenotype via receptor-interacting serine/threonine-protein kinase 1 (RIPK1)-dependent and -independent mechanisms. Whereas wild-type TPL2 rescues the phenotype, its kinase-dead mutant does not. Comparison of the molecular events initiated by small interfering RNA for TPL2 (siTPL2) ± TNFα in treatment-sensitive and -resistant lines revealed that the activation of caspase-8, downstream of miR-21-5p and cFLIP, is the dominant TPL2-dependent event. More important, comparison of the gene expression profiles of all of the tested cell lines results in the clustering of sensitive and resistant lines into distinct groups, providing proof of principle for the feasibility of generating a predictive tool for treatment sensitivity.

A highly selective inhibitor of interleukin-1 receptor-associated kinases 1/4 (IRAK-1/4) delineates the distinct signaling roles of IRAK-1/4 and the TAK1 kinase.

Interleukin-1 receptor-associated kinase-1 (IRAK-1) and IRAK-4, as well as transforming growth factor ß-activated kinase 1 (TAK1), are protein kinases essential for transducing inflammatory signals from interleukin receptors. IRAK family proteins and TAK1 have high sequence identity within the ATP-binding pocket, limiting the development of highly selective IRAK-1/4 or TAK1 inhibitors. Beyond kinase activity, IRAKs and TAK1 act as molecular scaffolds along with other signaling proteins, complicating the interpretation of experiments involving knockin or knockout approaches. In contrast, pharmacological manipulation offers the promise of targeting catalysis-mediated signaling without grossly disrupting the cellular architecture. Recently, we reported the discovery of takinib, a potent and highly selective TAK1 inhibitor that has only marginal activity against IRAK-4. On the basis of the TAK1-takinib complex structure and the structure of IRAK-1/4, here we defined critical contact sites of the takinib scaffold within the nucleotide-binding sites of each respective kinase. Kinase activity testing of takinib analogs against IRAK-4 identified a highly potent IRAK-4 inhibitor (HS-243). In a kinome-wide screen of 468 protein kinases, HS-243 had exquisite selectivity toward both IRAK-1 (IC50 = 24 nm) and IRAK-4 (IC50 = 20 nm), with only minimal TAK1-inhibiting activity (IC50 = 0.5 µm). Using HS-243 and takinib, we evaluated the consequences of cytokine/chemokine responses after selective inhibition of IRAK-1/4 or TAK1 in response to lipopolysaccharide challenge in human rheumatoid arthritis fibroblast-like synoviocytes. Our results indicate that HS-243 specifically inhibits intracellular IRAKs without TAK1 inhibition and that these kinases have distinct, nonredundant signaling roles.

The protein kinase MAP3K19 phosphorylates MAP2Ks and thereby activates ERK and JNK kinases and increases viability of KRAS-mutant lung cancer cells.

Identifying additional mitogen-activated protein kinase (MAPK) pathway regulators is invaluable in aiding our understanding of the complex signaling networks that regulate cellular processes, including cell proliferation and survival. Here, using in vitro kinase assays and by expressing WT or kinase-dead MAPK kinase kinase 19 (MAP3K19) in the HEK293T cell line and assessing activation of the extracellular signal-regulated kinase (ERK) and JUN N-terminal kinase (JNK) signaling pathways, we defined MAP3K19 as a novel regulator of MAPK signaling. We also observed that overexpression of WT MAP3K19 activates both the ERK and JNK pathways in a panel of cancer cell lines. Furthermore, MAP3K19 sustained ERK pathway activation in the presence of inhibitors targeting the RAF proto-oncogene Ser/Thr protein kinase (RAF) and MAPK/ERK kinase, indicating that MAP3K19 activates ERK via a RAF-independent mechanism. Findings from in vitro and in-cell kinase assays demonstrate that MAP3K19 is a kinase that directly phosphorylates both MAPK/ERK kinase (MEK) and MAPK kinase 7 (MKK7). Results from an short-hairpin RNA screen indicated that MAP3K19 is essential for maintaining survival in KRAS-mutant cancers; therefore, we depleted or inhibited MAP3K19 in KRAS-mutant cancer cell lines and observed that this reduces viability and decreases ERK and JNK pathway activation. In summary, our results reveal that MAP3K19 directly activates the ERK and JNK cascades and highlight a role for this kinase in maintaining survival of KRAS-mutant lung cancer cells.

Inhibition of TGFß-activated kinase 1 promotes inflammation-resolving microglial/macrophage responses and recovery after stroke in ovariectomized female mice.

TGFß-activated kinase 1 (TAK1) is a master regulator that drives multiple cell death and proinflammatory signaling pathways, making it a promising therapeutic target to treat ischemic stroke. However, whether targeting TAK1 could improve stroke outcomes has never been tested in female subjects, hindering its potential translation into clinical use. Here we examined the therapeutic effect of 5Z-7-Oxozeaenol (OZ), a selective TAK1 inhibitor, in ovariectomized female mice after middle cerebral artery occlusion (MCAO). OZ significantly reduced neuronal cell death and axonal injury at the acute stage and mitigated neuroinflammation at the subacute stage after MCAO in ovariectomized female mice. Consistent with RNA sequencing analysis that TAK1 activation contributed to microglia/macrophage-mediated inflammatory responses in the post-stroke brain, inhibition of TAK1 with OZ caused phenotypic shift of microglia/macrophages toward an inflammation-resolving state. Furthermore, microglia/macrophage-specific TAK1 knockout (TAK1 mKO) reproduced OZ's effects, causally confirming the role of TAK1 in determining proinflammatory microglial/macrophage responses in post-stroke females. Post-stroke treatment with OZ for 5 days effectively promoted long-term neurological recovery and the integrity of both gray matter and white matter in female mice. Together, the TAK1 inhibitor OZ elicits long-lasting improvement of stroke outcomes in female mice, at least partially through enhancing beneficial microglial/macrophage responses and inflammation resolution. Given its therapeutic efficacy on both male and female rodents, TAK1 inhibitor is worth further investigation as a valid treatment to ischemic stroke.

Reduced MEK inhibition preserves genomic stability in naive human embryonic stem cells.

Human embryonic stem cells (hESCs) can be captured in a primed state in which they resemble the postimplantation epiblast, or in a naive state where they resemble the preimplantation epiblast. Naive-cell-specific culture conditions allow the study of preimplantation development ex vivo but reportedly lead to chromosomal abnormalities, which compromises their utility in research and potential therapeutic applications. Although MEK inhibition is essential for the naive state, here we show that reduced MEK inhibition facilitated the establishment and maintenance of naive hESCs that retained naive-cell-specific features, including global DNA hypomethylation, HERVK expression, and two active X chromosomes. We further show that hESCs cultured under these modified conditions proliferated more rapidly; accrued fewer chromosomal abnormalities; and displayed changes in the phosphorylation levels of MAPK components, regulators of DNA damage/repair, and cell cycle. We thus provide a simple modification to current methods that can enable robust growth and reduced genomic instability in naive hESCs.

Six-Transmembrane Epithelial Antigen of the Prostate 3 Deficiency in Hepatocytes Protects the Liver Against Ischemia-Reperfusion Injury by Suppressing Transforming Growth Factor-ß-Activated Kinase 1.

BACKGROUND AND AIMS: Hepatic ischemia-reperfusion (I/R) injury remains a major challenge affecting the morbidity and mortality of liver transplantation. Effective strategies to improve liver function after hepatic I/R injury are limited. Six-transmembrane epithelial antigen of the prostate 3 (Steap3), a key regulator of iron uptake, was reported to be involved in immunity and apoptotic processes in various cell types. However, the role of Steap3 in hepatic I/R-induced liver damage remains largely unclear. APPROACH AND RESULTS: In the present study, we found that Steap3 expression was significantly up-regulated in liver tissue from mice subjected to hepatic I/R surgery and primary hepatocytes challenged with hypoxia/reoxygenation insult. Subsequently, global Steap3 knockout (Steap3-KO) mice, hepatocyte-specific Steap3 transgenic (Steap3-HTG) mice, and their corresponding controls were subjected to partial hepatic warm I/R injury. Hepatic histology, the inflammatory response, and apoptosis were monitored to assess liver damage. The molecular mechanisms of Steap3 function were explored in vivo and in vitro. The results demonstrated that, compared with control mice, Steap3-KO mice exhibited alleviated liver damage after hepatic I/R injury, as shown by smaller necrotic areas, lower serum transaminase levels, decreased apoptosis rates, and reduced inflammatory cell infiltration, whereas Steap3-HTG mice had the opposite phenotype. Further molecular experiments showed that Steap3 deficiency could inhibit transforming growth factor-ß-activated kinase 1 (TAK1) activation and downstream c-Jun N-terminal kinase (JNK) and p38 signaling during hepatic I/R injury. CONCLUSIONS: Steap3 is a mediator of hepatic I/R injury that functions by regulating inflammatory responses as well as apoptosis through TAK1-dependent activation of the JNK/p38 pathways. Targeting hepatocytes, Steap3 may be a promising approach to protect the liver against I/R injury.