Key Points: METTL3 was upregulated in CKD, leading to increased m ⁶ A modification levels. M ⁶ A modifications were enriched in genes related to the cyclic guanosine monophosphate–AMP synthase-stimulator of IFN genes pathway in CKD. Normalizing heightened METTL3 and m ⁶ A modification levels showed promise as a strategy to combat kidney disease. Background: Chemical modifications on RNA profoundly affect RNA function and regulation. m ⁶ A, the most abundant RNA modification in eukaryotes, plays a pivotal role in diverse cellular processes and disease mechanisms. However, its importance is understudied in human CKD samples regarding its influence on pathological mechanisms. Methods: Liquid chromatography–tandem mass spectrometry and methylated RNA immunoprecipitation sequencing were used to examine alterations in m ⁶ A levels and patterns in CKD samples. Overexpression of the m ⁶ A writer METTL3 in cultured kidney tubular cells was performed to confirm the effect of m ⁶ A in tubular cells and explore the biological functions of m ⁶ A modification on target genes. In addition, tubule-specific deletion of Mettl3 (Ksp-Cre Mettl3 ^f/f ) mice and antisense oligonucleotides inhibiting Mettl3 expression were used to reduce m ⁶ A modification in an animal kidney disease model. Results: By examining 127 human CKD samples, we observed a significant increase in m ⁶ A modification and METTL3 expression in diseased kidneys. Epitranscriptomic analysis unveiled an enrichment of m ⁶ A modifications in transcripts associated with the activation of inflammatory signaling pathways, particularly the cyclic guanosine monophosphate–AMP synthase (cGAS)-stimulator of IFN genes (STING) pathway. m ⁶ A hypermethylation increased mRNA stability in cGAS and STING1 as well as elevated the expression of key proteins within the cGAS-STING pathway. Both the tubule-specific deletion of Mettl3 and the use of antisense oligonucleotides to inhibit Mettl3 expression protected mice from inflammation, reduced cytokine expression, decreased immune cell recruitment, and attenuated kidney fibrosis. Conclusions: Our research revealed heightened METTL3-mediated m ⁶ A modification in fibrotic kidneys, particularly enriching the cGAS-STING pathway. This hypermethylation increased mRNA stability for cGAS and STING1, leading to sterile inflammation and fibrosis.

Current clinical trials of combined EGFR-tyrosine kinase inhibitors (TKI) and immune checkpoint blockade (ICB) therapies show no additional effect. This raises questions regarding whether EGFR-TKIs attenuate ICB-enhanced CD8+ T lymphocyte function. Here we show that the EGFR-TKI afatinib suppresses CD8+ T lymphocyte proliferation, and we identify CAD, a key enzyme of de novo pyrimidine biosynthesis, to be a novel afatinib target. Afatinib reduced tumor-infiltrating lymphocyte numbers in Lewis lung carcinoma (LLC)–bearing mice. Early afatinib treatment inhibited CD8+ T lymphocyte proliferation in patients with non–small cell lung cancer, but their proliferation unexpectedly rebounded following long-term treatment. This suggests a transient immunomodulatory effect of afatinib on CD8+ T lymphocytes. Sequential treatment of afatinib with anti-PD1 immunotherapy substantially enhanced therapeutic efficacy in MC38 and LLC-bearing mice, while simultaneous combination therapy showed only marginal improvement over each single treatment. These results suggest that afatinib can suppress CD8+ T lymphocyte proliferation by targeting CAD, proposing a timing window for combined therapy that may prevent the dampening of ICB efficacy by EGFR-TKIs. Significance: This study elucidates a mechanism of afatinib-mediated immunosuppression and provides new insights into treatment timing for combined targeted therapy and immunotherapy.

Compound 1 is a curcumin di-O-2,2-bis(hydroxymethyl)propionate that shows significant in vitro and in vivo inhibitory activity against MDA-MB-231 cells with eight to ten-fold higher potency than curcumin. Here, we modified the α-position (C-4 position) of the central 1,3-diketone moiety of 1 with polar or nonpolar functional groups to afford a series of 4,4-disubstituted curcuminoid 2,2-bis(hydroxymethyl)propionate derivatives and evaluated their anticancer activities. A clear structure–activity relationship of compound 1 derivatives focusing on the functional groups at the C-4 position was established based on their anti-proliferative effects against the MDA-MB-231 and HCT-116 cell lines. Compounds 2–6 are 4,4-dimethylated, 4,4-diethylated, 4,4-dibenzylated, 4,4-dipropargylated and 4,4-diallylated compound 1, respectively. Compounds 2m–6m, the ester hydrolysis products of compounds 2–6, respectively, were synthesized and assessed for anticancer activity. Among all compound 1 derivatives, compound 2 emerged as a potential chemotherapeutic agent for colon cancer due to the promising in vivo anti-proliferative activities of 2 (IC₅₀ = 3.10 ± 0.29 μM) and its ester hydrolysis product 2m (IC₅₀ = 2.17 ± 0.16 μM) against HCT-116. The preliminary pharmacokinetic evaluation of 2 implied that 2 and 2m are main contributors to the in vivo efficacy. Compound 2 was further evaluated in an animal study using HCT-116 colon tumor xenograft bearing nude mice. The results revealed a dose-dependent efficacy that led to tumor volume reductions of 27%, 45%, and 60% at 50, 100, and 150 mg/kg doses, respectively. The established structure–activity relationship and pharmacokinetic outcomes of 2 is the guidance for future development of 4,4-disubstituted curcuminoid 2,2-bis(hydroxymethyl)- propionate derivatives as anticancer drug candidates.

Gilles de la Tourette's Syndrome (TS) is a childhood-onset disease with clinical features of motor and phonic tics. Yi-Gan-san (YGS) is a traditional Chinese medicine formula that can reduce aggressiveness and agitation and inhibit dopamine function. This study investigated the effects of YGS on the psychiatric behavior of children and adolescents with TS. A double-blind, randomized, controlled preliminary study was conducted. A total of 38 patients with TS were assigned to the control group (CG, 19 patients) who received the oral administration of YGS placebo (90% starch and 10% YGS; 2.5 g thrice daily) or to a treatment group (TG, 19 patients) who received YGS for 4 weeks. The primary outcome measure was the change in Yale Global Tic Severity Scale (YGTSS) overall and subscale scores. The intensity score for phonic tics before oral administration of YGS, and after 2 weeks, 3 weeks and 4 weeks was not significantly different between CG and TG groups (2.94 ± 1.14 vs 2.79 ± 1.08, p = .686; 2.29 ± 1.21 vs 1.95 ± 1.08, p = .370; 2.41 ± 1.18 vs 2.05 ± 1.51, p = .435; and 2.29 ± 1.26 vs 1.84 ± 1.42, p = .323, respectively), while the intensity score for phonic tics after 1-week oral administration of YGS in the TG was 1.89 ± 1.10 lower than 3.06 ± 1.39 in the CG (p = .008). Oral administration of YGS for 1 week only reduced the intensity of phonic tics compared with oral administration of YGS placebo, suggesting that YGS can reduce their intensity for a short period, and the compliance of oral administration of YGS for 4 weeks can be accepted in children and adolescents with Tourette's Syndrome. However, because this study was preliminary, the selection of an appropriate placebo and dosage and long-term observations are crucial areas for future studies.

Because hydrogen sulfide (H₂S) is classified as a gaseous signaling molecule, protein S-sulfhydration is known to be one of the mechanisms by which H₂S signals are conducted. PTP1B, a negative regulator in insulin signaling, has been found to be S-sulfhydrated at Cys215-SH to form Cys215-SSH in response to endoplasmic reticulum (ER) stress. Therefore, we aimed to understand the change in PTP1B S-sulfhydration and cellular redox homeostasis in response to insulin stimulation. We demonstrated a feasible PEG-switch method to determine the levels of PTP1B S-sulfhydration. According to the results obtained from HEK293T and MDA-MB-231 cells, insulin induced a change in PTP1B S-sulfhydration that was similar to the change in Insulin receptor substrate 1 (IRS1) phosphorylation in both cell lines. However, insulin-induced PTP1B S-sulfhydration and IRS1 phosphorylation were only significantly affected by metformin in HEK293T cells. Insulin also induced an increase in reactive oxygen species (ROS) in both cell lines. However, the level of H₂S, GSH, and GSSG was only significantly affected by insulin and metformin in HEK293T cells. HEK293T cells maintained high levels of H₂S and cysteine, but low levels of GSSG and GSH in general compared to MDA-MB-231 cells. From these findings, we suggest that PTP1B activity is modulated by H₂S and redox-regulated S-sulfhydration during insulin signaling.