The AKT inhibitor AZD5363 elicits synthetic lethality in ARID1A-deficient gastric cancer cells via induction of pyroptosis.

BACKGROUND: Gastric cancer (GC) is a deadly disease with poor overall survival and limited therapeutic options. Genetic alterations such as mutations and/or deletions in chromatin remodeling gene AT-rich interactive domain 1 A (ARID1A) occur frequently in GC. Although ARID1A mutations/deletions are not a druggable target for conventional treatments, novel therapeutic strategies based on a synthetic lethal approach may be effective for the treatment of ARID1A-deficient cancers. METHODS: A kinase inhibitor library containing 551 compounds was screened in ARID1A isogenic GC cells for the ability to induce synthetic lethality effect. Selected hits' activity was validated, and the mechanism of the most potent candidate drug, AKT inhibitor AD5363 (capivasertib), on induction of the synthetic lethality with ARID1A deficiency was investigated. RESULTS: After robust vulnerability screening of 551 diverse protein kinase inhibitors, we identified the AKT inhibitor AZD5363 as being the most potent lead compound in inhibiting viability of ARID1A-/- cells. A synthetic lethality between loss of ARID1A expression and AKT inhibition by AZD5363 was validated in both GC cell model system and xenograft model. Mechanistically, AZD5363 treatment induced pyroptotic cell death in ARID1A-deficient GC cells through activation of the Caspase-3/GSDME pathway. Furthermore, ARID1A occupied the AKT gene promoter and regulated its transcription negatively, thus the GC cells deficient in ARID1A showed increased expression and phosphorylation of AKT. CONCLUSIONS: Our study demonstrates a novel synthetic lethality interaction and unique mechanism between ARID1A loss and AKT inhibition, which may provide a therapeutic and mechanistic rationale for targeted therapy on patients with ARID1A-defective GC who are most likely to be beneficial to AZD5363 treatment.

Activation of osteoblast ferroptosis via the METTL3/ASK1-p38 signaling pathway in high glucose and high fat (HGHF)-induced diabetic bone loss.

Diabetes mellitus (DM) and osteoporosis are two common diseases that may develop as a cause-and-effect relationship since the incidence of osteoporotic fractures is significantly increased in DM patients. However, the pathophysiology of diabetic osteoporosis is yet to be clearly understood. Iron overload has been reported to lead to bone loss and closely related to osteoporosis. In this study, we hypothesized that high glucose and high fat (HGHF) may induce osteoblastic ferroptosis for the pathogenesis of diabetic osteoporosis and explored the possible molecular mechanisms behind. Using the diabetic rat model established by HGHF feeding with a subsequent intraperitoneal injection of a single low dose of streptozocin, we found that the serum ferritin level (a biomarker for body iron store) was significantly elevated in HGHF-fed rats and the expression of SLC7A11 and GPX4 (inhibitory marker proteins for ferroptosis) was markedly attenuated in the bone tissue of the rats with diabetic bone loss as compared to the normal rats. In an osteoblast cell model, treatment of pre-osteoblastic MC3T3-E1 cells with high glucose and palmitic acid (HGPA) not only suppressed osteoblast differentiation and mineralization but also triggered ferroptosis-related osteoblastic cell death. m6 A-seq revealed that m6 A methylation on ASK1 was 80.9-fold higher in HGPA-treated cells. The expression of p-ASK1 and p-p38 was also significantly elevated in the HGPA-treated cells. Knockout of METTL3 (methyltransferase-like 3), one of the major m6 A methyltransferases, in MC3T3-E1 cells not only abrogated HGPA-induced activation of ASK1-p38 signaling pathway but also attenuated the level of ferroptosis. Therefore, HGHF-induced ferroptosis in osteoblasts may be the main cause of osteoporosis in DM via activation of METTL3/ASK1-p38 signaling pathway, and inhibition of ferroptosis in osteoblasts may provide a potential therapeutic strategy for diabetic osteoporosis.

Suppression of IRF9 Promotes Osteoclast Differentiation by Decreased Ferroptosis via STAT3 Activation.

Osteoporosis is a chronic disease that endangers the health of the elderly. Inhibiting osteoclast hyperactivity is a key aspect of osteoporosis prevention and treatment. Several studies have shown that interferon regulatory factor 9 (IRF9) not only regulates innate and adaptive immune responses but also plays an important role in inflammation, antiviral response, and cell development. However, the exact role of IRF9 in osteoclasts has not been reported. To elucidate the role of IRF9 in osteoclast differentiation, we established the ovariectomized mouse model of postmenopausal osteoporosis and found that IRF9 expression was reduced in ovariectomized mice with overactive osteoclasts. Furthermore, knockdown of IRF9 expression enhanced osteoclast differentiation in vitro. Using RNA sequencing, we identified that the differentially expressed genes enriched by IRF9 knockdown were related to ferroptosis. We observed that IRF9 knockdown promoted osteoclast differentiation via decreased ferroptosis in vitro and further verified that IRF9 knockdown reduced ferroptosis by activating signal transducer and activator of transcription 3 (STAT3) to promote osteoclastogenesis. In conclusion, we identified an essential role of IRF9 in the regulation of osteoclastogenesis in osteoporosis and its underlying mechanism.

Suppression of TLR4 prevents diabetic bone loss by regulating FTO-mediated m6A modification.

Toll-like receptor-4 (TLR4) has been implicated in the development and progression of diabetic osteoporosis. However, the mechanisms underlying TLR4-regulated bone metabolism in diabetes are yet to be fully understood. Epigenetic modifications have been indicated as a possible mechanism leading to increased risk of osteoporosis and bone fracture. As N6-methyladenosine (m6A) is the most common epigenetic modification in eukaryotic mRNAs, we hypothesized that TLR4 regulates m6A modification in bone tissues of diabetic rats, thereby potentially explaining the pathogenesis of diabetic bone loss. m6A sequencing (m6A-seq) was performed in samples of the femur of TLR4-wild type (TLR4WT) and TLR4-knockout (TLR4KO) diabetic rats to identify genes with differential m6A modifications that may be associated with the bone loss phenotype. We found that in TLR4KO rats, the rapid weight loss of diabetic rats was prevented, and bone mineral density (BMD) was significantly increased. m6A-seq and Gene Ontology enrichment analysis revealed that m6A-modified genes in the femur of TLR4KO diabetic rats were associated with regulation of biological processes such as osteoclast differentiation. qRT-PCR analysis on the expression levels of the m6A-modified methyltransferases and demethylases demonstrated that only the m6A demethylase fat mass and obesity-associated protein（FTO）was decreased. Using an osteoclast cell model, we confirmed that TLR4-mediated osteoclast differentiation was induced by glycolipid toxicity via inhibition of FTO expression. Taken together, these results suggest that inhibition of TLR4 may prevent diabetic bone loss via regulation of FTO-mediated m6A modification.

Glycolipid toxicity induces osteogenic dysfunction via the TLR4/S100B pathway.

Diabetes can cause bone metabolism disorders and osteoporosis. The occurrence of both diabetes mellitus and osteoporosis increases the disability and mortality of elderly individuals due to pathological fracture. Abnormal metabolism of nutrientsis considered to be one of the important mechanisms of diabetes mellitus-induced osteoporosis. This study preliminarily explored the roles of TLR4 (Toll-like receptor 4) and S100B in osteogenic dysfunction induced by glycolipid toxicity. In this study, a diabetic rat model and TLR4-knockdown diabetic rat model were used in vivo. MC3T3-E1 cells in a high glucose and palmitic acid environment were used as glycolipid toxicity cell models in vitro. We investigated the effects of TLR4 and S100B on osteogenesis by overexpression or inhibition of TLR4 and S100B in vitro. We found that when TLR4 or S100B was inhibited, ALP and OCN were significantly up-regulated and p-ERK was significantly down regulated in the glycolipid model. These results suggest that TLR4/S100B may play a role in reducing glycolipid toxicity by regulating ERK phosphorylation.

TLR4 knockout can improve dysfunction of ß-cell by rebalancing proteomics disorders in pancreas of obese rats.

PURPOSE: Studies showed that TLR4 knockout (TLR4KO) could mitigate obesity and insulin resistance induced by high-fat diet in rats. In this study, we further investigated the effects of TLR4KO on islet function and pancreatic proteomics in obese rats by high-fat diet. METHODS: PA-induced lipotoxicity ß-cells, SD and TLR4KO rats were used in this study. iTRAQ was used to screen out meaningful differential proteins.The protein expression level was evaluated by Western blotting; the cell apoptosis was detected by TUNEL assay. RESULTS: TLR4KO could reduce inflammatory and regulate body composition in obese rats, and improve ß-cells function. The quantitative analysis of protein revealed that TLR4KO rebalanced proteomics disorders in pancreas of obese rats. In addition, the pathways involved in differential proteins were mainly metabolic pathways, arachidonic acid metabolism, ECM-receptor interaction, pancreatic secretion, PI3K-Akt signaling pathway, and FoxO signaling pathway. Further analysis of protein-protein interaction (PPI) revealed that Stk39 and Ass1 interacting through Mapk14-Ywhae were node proteins and participated in inflammatory response, carboxylic acid metabolic process, and small molecule metabolic process. In vitro experiments we confirmed that silencing TLR4 can inhibit PA-induced ß-cell apoptosis, insulin secretion disorders, and increase Ass1 expression. While, overexpression of Ass1 in ß-cell inhibited PA or LPS-induced ß-cell damage. CONCLUSIONS: Our study confirmed that TLR4KO could improve dysfunction of ß-cell, and the underlying mechanism might be involved in ebalancing proteomics disorders in pancreas, affecting the expression of Ass1.

[Effect of NaCl stress on growth and antioxidant systems of Pogostemon cablin].

OBJECTIVE: To study the effects of salt stress on the growth and resistant-oxidation enzyme activities of Pogostemon cablin. METHOD: Cutting-rooted seedlings were cultivated in the plastic flowerpot irrigated with various concentrations of NaCl (Hoagland solution) for different duration, and the height, fresh/dry weight, root vitality, SOD,POD and CAT activity, membrane permeability and MDA contents were determined. RESULT: The growth and development of P. cablin was restrained after salt stress. The differences of salt stress of lower concentration and for a short time (less than 20 mmol x L(-1) and the 9th day) were not obvious. The fresh/ dry weight, growth rate and increasing rate of dry matter were obviously decreased with the increasing of NaCl concentration and prolonging of the time, but membrane permeability and contents of MDA were increased all along, and lipid peroxidation enhanced, while the root vitality and activities of SOD, POD and CAT were firstly increasing at lower concentration and then decreasing at higher concentration. CONCLUSION: The growth of P. cablin depends on NaCl concentration and the treated duration.

Chromatin remodelling factor BAF155 protects hepatitis B virus X protein (HBx) from ubiquitin-independent proteasomal degradation.

HBx is a short-lived protein whose rapid turnover is mainly regulated by ubiquitin-dependent proteasomal degradation pathways. Our prior work identified BAF155 to be one of the HBx binding partners. Since BAF155 has been shown to stabilize other members of the SWI/SNF chromatin remodelling complex by attenuating their proteasomal degradation, we proposed that BAF155 might also contribute to stabilizing HBx protein in a proteasome-dependent manner. Here we report that BAF155 protected hepatitis B virus X protein (HBx) from ubiquitin-independent proteasomal degradation by competing with the 20S proteasome subunit PSMA7 to bind to HBx. BAF155 was found to directly interact with HBx via binding of its SANT domain to the HBx region between amino acid residues 81 and 120. Expression of either full-length BAF155 or SANT domain increased HBx protein levels whereas siRNA-mediated knockdown of endogenous BAF155 reduced HBx protein levels. Increased HBx stability and steady-state level by BAF155 were attributable to inhibition of ubiquitin-independent and PSMA7-mediated protein degradation. Consequently, overexpression of BAF155 enhanced the transcriptional transactivation function of HBx, activated protooncogene expression and inhibited hepatoma cell clonogenicity. These results suggest that BAF155 plays important roles in ubiquitin-independent degradation of HBx, which may be related to the pathogenesis and carcinogenesis of HBV-associated HCC.