Hsc70 promotes anti-tumor immunity by targeting PD-L1 for lysosomal degradation.

Immune checkpoint inhibition targeting the PD-1/PD-L1 pathway has become a powerful clinical strategy for treating cancer, but its efficacy is complicated by various resistance mechanisms. One of the reasons for the resistance is the internalization and recycling of PD-L1 itself upon antibody binding. The inhibition of lysosome-mediated degradation of PD-L1 is critical for preserving the amount of PD-L1 recycling back to the cell membrane. In this study, we find that Hsc70 promotes PD-L1 degradation through the endosome-lysosome pathway and reduces PD-L1 recycling to the cell membrane. This effect is dependent on Hsc70-PD-L1 binding which inhibits the CMTM6-PD-L1 interaction. We further identify an Hsp90α/ß inhibitor, AUY-922, which induces Hsc70 expression and PD-L1 lysosomal degradation. Either Hsc70 overexpression or AUY-922 treatment can reduce PD-L1 expression, inhibit tumor growth and promote anti-tumor immunity in female mice; AUY-922 can further enhance the anti-tumor efficacy of anti-PD-L1 and anti-CTLA4 treatment. Our study elucidates a molecular mechanism of Hsc70-mediated PD-L1 lysosomal degradation and provides a target and therapeutic strategies for tumor immunotherapy.

Mitophagy defect mediates the aging-associated hallmarks in Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal disease manifested by premature aging and aging-related phenotypes, making it a disease model for aging. The cellular machinery mediating age-associated phenotypes in HGPS remains largely unknown, resulting in limited therapeutic targets for HGPS. In this study, we showed that mitophagy defects impaired mitochondrial function and contributed to cellular markers associated with aging in mesenchymal stem cells derived from HGPS patients (HGPS-MSCs). Mechanistically, we discovered that mitophagy affected the aging-associated phenotypes of HGPS-MSCs by inhibiting the STING-NF-ĸB pathway and the downstream transcription of senescence-associated secretory phenotypes (SASPs). Furthermore, by utilizing UMI-77, an effective mitophagy inducer, we showed that mitophagy induction alleviated aging-associated phenotypes in HGPS and naturally aged mice. Collectively, our results uncovered that mitophagy defects mediated the aging-associated markers in HGPS, highlighted the function of mitochondrial homeostasis in HGPS progression, and suggested mitophagy as an intervention target for HGPS and aging.

Novel mitophagy inducer alleviates lupus nephritis by reducing myeloid cell activation and autoantigen presentation.

Lupus nephritis (LN) is one of the most severe manifestations of systemic lupus erythematosus (SLE), but its mechanism of onset remains unclear. Since impaired mitophagy has been implicated in multiple organs in SLE, we hypothesized that mitophagy dysfunction is critical in the development of LN and that pharmacologically targeting mitophagy would ameliorate this disease. Therefore, lupus-prone MRL/MpJ-Faslpr (MRL/lpr) and NZBWF1/J mice were treated with a novel mitophagy inducer, UMI-77, during their onset of LN. This treatment effectively mitigated kidney inflammation and damage as assessed by histology and flow cytometry. Furthermore, dendritic cell (DC)-T-cell coculture assay indicated that UMI-77 treatment attenuated DC function that would drive T-cell proliferation but did not directly influence the potent T-cell proliferation in lupus mice. UMI-77 also restored mitochondrial function and attenuated proinflammatory phenotypes in lupus DCs. Adoptive transfer of DCs from MRL/lpr mice augmented serum anti-dsDNA IgG, urine protein and T-cell infiltration of the kidney in MRL/MpJ mice, which could be prevented by either treating lupus donors in vivo or lupus DCs directly with UMI-77. UMI-77 also restored mitochondrial function in myeloid cells from patients with LN in vitro as evidenced by increased ATP levels. Thus, enhancing mitophagy in SLE restrains autoimmunity and limits kidney inflammation for LN development. Hence, our findings suggest targeting mitophagy as a tangible pathway to treat LN.

A three-component reaction of cyclobutanone oxime esters, sulfur dioxide and N-alkyl-N-methacryloyl benzamides.

A three-component reaction of cyclobutanone oxime esters, DABCO·(SO2)2 and N-alkyl-N-methacryloyl benzamides is described. This reaction proceeds without the addition of any oxidant or transition metal, affording sulfonyl-containing isoquinoline-1,3-(2H,4H)-diones in moderate to good yields. Various functional groups are tolerated well in this transformation. Mechanistic studies suggest that a radical pathway is involved, including ß-scission, sulfur dioxide insertion, and intramolecular cyclization processes.

TOR-mediated Ypt1 phosphorylation regulates autophagy initiation complex assembly.

The regulation of autophagy initiation is a key step in autophagosome biogenesis. However, our understanding of the molecular mechanisms underlying the stepwise assembly of ATG proteins during this process remains incomplete. The Rab GTPase Ypt1/Rab1 is recognized as an essential autophagy regulator. Here, we identify Atg23 and Atg17 as binding partners of Ypt1, with their direct interaction proving crucial for the stepwise assembly of autophagy initiation complexes. Disruption of Ypt1-Atg23 binding results in significantly reduced Atg9 interactions with Atg11, Atg13, and Atg17, thus preventing the recruitment of Atg9 vesicles to the phagophore assembly site (PAS). Likewise, Ypt1-Atg17 binding contributes to the PAS recruitment of Ypt1 and Atg1. Importantly, we found that Ypt1 is phosphorylated by TOR at the Ser174 residue. Converting this residue to alanine blocks Ypt1 phosphorylation by TOR and enhances autophagy. Conversely, the Ypt1S174D phosphorylation mimic impairs both PAS recruitment and activation of Atg1, thus inhibiting subsequent autophagy. Thus, we propose TOR-mediated Ypt1 as a multifunctional assembly factor that controls autophagy initiation via its regulation of the stepwise assembly of ATG proteins.

On the Dynamic Mechanism of Long-Flexible Fatty Acid Binding to Fatty Acid Binding Protein: Resolving the Long-Standing Debate.

Fatty acids (FAs) are one of the essential energy sources for physiological processes, and they play a vital role in regulating immune and inflammatory responses, promoting cell differentiation and apoptosis, and inhibiting tumor growth. These functions are carried out by FA binding proteins (FABPs) that recognize and transport FAs. Although the crystal structure of the FA-FABPs complex has long been characterized, the mechanism behind FA binding and dissociation from FABP remains unclear. This study employed conventional MD simulations and enhanced sampling technologies to investigate the atomic-scale complexes of heart fatty acid binding proteins and stearic acid (SA). The results revealed two primary pathways for the binding or dissociation of the flexible long-chain ligand, with the orientation of the SA carboxyl head during dissociation determining the chosen path. Conformational changes in the portal region of FABP during the ligand binding/unbinding were found to be trivial, and the overturn of the ″cap″ or the unfolding of the α2 helix was not required. This study resolves the long-standing debate on the binding mechanism of SA with the long-flexible tail to FABP, which significantly improves the understanding of the transport mechanism of FABPs and the development of related therapeutic agents.

ARIH1 activates STING-mediated T-cell activation and sensitizes tumors to immune checkpoint blockade.

Despite advances in cancer treatment, immune checkpoint blockade (ICB) only achieves complete response in some patients, illustrating the need to identify resistance mechanisms. Using an ICB-insensitive tumor model, here we discover cisplatin enhances the anti-tumor effect of PD-L1 blockade and upregulates the expression of Ariadne RBR E3 ubiquitin-protein ligase 1 (ARIH1) in tumors. Arih1 overexpression promotes cytotoxic T cell infiltration, inhibits tumor growth, and potentiates PD-L1 blockade. ARIH1 mediates ubiquitination and degradation of DNA-PKcs to trigger activation of the STING pathway, which is blocked by the phospho-mimetic mutant T68E/S213D of cGAS protein. Using a high-throughput drug screen, we further identify that ACY738, less cytotoxic than cisplatin, effectively upregulates ARIH1 and activates STING signaling, sensitizing tumors to PD-L1 blockade. Our findings delineate a mechanism that tumors mediate ICB resistance through the loss of ARIH1 and ARIH1-DNA-PKcs-STING signaling and indicate that activating ARIH1 is an effective strategy to improve the efficacy of cancer immunotherapy.

Computationally guided discovery of novel non-steroidal AR-GR dual antagonists demonstrating potency against antiandrogen resistance.

As a major class of medicine for treating the lethal type of castration-resistant prostate cancer (PCa), long-term use of androgen receptor (AR) antagonists commonly leads to antiandrogen resistance. When AR signaling pathway is blocked by AR-targeted therapy, glucocorticoid receptor (GR) could compensate for AR function especially at the late stage of PCa. AR-GR dual antagonist is expected to be a good solution for this situation. Nevertheless, no effective non-steroidal AR-GR dual antagonist has been reported so far. In this study, an AR-GR dual binder H18 was first discovered by combining structure-based virtual screening and biological evaluation. Then with the aid of computationally guided design, the AR-GR dual antagonist HD57 was finally identified with antagonistic activity towards both AR (IC50 = 0.394 µM) and GR (IC50 = 17.81 µM). Moreover, HD57 could effectively antagonize various clinically relevant AR mutants. Further molecular dynamics simulation provided more atomic insights into the mode of action of HD57. Our research presents an efficient and rational strategy for discovering novel AR-GR dual antagonists, and the new scaffold provides important clues for the development of novel therapeutics for castration-resistant PCa.

Spatially resolved proteomics via tissue expansion.

Spatially resolved proteomics is an emerging approach for mapping proteome heterogeneity of biological samples, however, it remains technically challenging due to the complexity of the tissue microsampling techniques and mass spectrometry analysis of nanoscale specimen volumes. Here, we describe a spatially resolved proteomics method based on the combination of tissue expansion with mass spectrometry-based proteomics, which we call Expansion Proteomics (ProteomEx). ProteomEx enables quantitative profiling of the spatial variability of the proteome in mammalian tissues at ~160 µm lateral resolution, equivalent to the tissue volume of 0.61 nL, using manual microsampling without the need for custom or special equipment. We validated and demonstrated the utility of ProteomEx for streamlined large-scale proteomics profiling of biological tissues including brain, liver, and breast cancer. We further applied ProteomEx for identifying proteins associated with Alzheimer's disease in a mouse model by comparative proteomic analysis of brain subregions.

Mitophagy induced by UMI-77 preserves mitochondrial fitness in renal tubular epithelial cells and alleviates renal fibrosis.

Renal fibrosis is the final common outcome of chronic kidney disease (CKD), which remains a huge challenge due to a lack of targeted treatment. Growing evidence suggests that during the process of CKD, the integrity and function of mitochondria in renal tubular epithelial cells (TECs) are generally impaired and strongly connected with the progression of renal fibrosis. Mitophagy, a selective form of autophagy, could remove aberrant mitochondria to maintain mitochondrial homeostasis. Deficiency of mitophagy has been reported to aggravate renal fibrosis. However, whether induction of mitophagy could alleviate renal fibrosis has not been stated. In this study, we explored the effect of mitophagy activation by UMI-77, a compound recently verified to induce mitophagy, on murine CKD model of unilateral ureteral obstruction (UUO) in vivo and TECs in vitro. In UUO mice, we found the changes of mitochondrial damage, ROS production, transforming growth factor (TGF)-ß1/Smad pathway activation, as well as epithelial-mesenchymal transition phenotype and renal fibrosis, and these changes were ameliorated by mitophagy enhancement using UMI-77. Moreover, TEC apoptosis, nuclear factor (NF)-κB signaling activation, and interstitial inflammation after UUO were significantly mitigated by augmented mitophagy. Then, we found UMI-77 could effectively and safely induce mitophagy in TECs in vitro, and reduced TGF-ß1/Smad signaling and downstream profibrotic responses in TGF-ß1-treated TECs. These changes were restored by a mitophagy inhibitor. In conclusion, we demonstrated that mitophagy activation protected against renal fibrosis through improving mitochondrial fitness, downregulating TGF-ß1/Smad signaling and alleviating TEC injuries and inflammatory infiltration in kidneys.

Inhibition of RIPK1 by ZJU-37 promotes oligodendrocyte progenitor proliferation and remyelination via NF-κB pathway.

Receptor interacting serine/threonine protein kinase 1 (RIPK1) activation and necroptosis have been genetically and mechanistically linked with human multiple sclerosis and neurodegenerative diseases for which demyelination is a common key pathology. Demyelination can be healed through remyelination which is mediated by new oligodendrocytes derived from the adult oligodendrocyte progenitor cells (OPCs). Unfortunately, the efficiency of remyelination declines with progressive aging partially due to the depletion of OPCs following chronic or repeated demyelination. However, to our knowledge, so far there is no drug which enhances proliferation of OPCs, and it is unknown whether inhibiting RIPK1 activity directly affect OPCs, the central player of remyelination. Using TNFα induced RIPK1-dependent necroptosis in Jurkat FADD-/- cells as a cell death assay, we screened from 2112 FDA-approved drugs and the drug candidates of new RIPK1 inhibitors selected by ourselves, and identified ZJU-37, a small molecule modified by introducing an amide bond to Nec-1s, is a new RIPK1 kinase inhibitor with higher potency than Nec-1s which has the best reported potency. We unveil in addition to protecting myelin from demyelination and axons from degeneration, ZJU-37 exhibits a new role on promoting proliferation of OPCs and enhancing remyelination by inhibiting RIPK1 kinase activity with higher potency than Nec-1s. Mechanistically, ZJU-37 promotes proliferation of OPCs by enhancing the transcription of platelet derived growth factor receptor alpha via NF-κB pathway. This work identifies ZJU-37 as a new drug candidate which enhances remyelination by promoting proliferation of OPCs, paving the way for a potential drug to enhance myelin repair.

Discovery of Novel GR Ligands toward Druggable GR Antagonist Conformations Identified by MD Simulations and Markov State Model Analysis.

Binding of different ligands to glucocorticoid receptor (GR) may induce different conformational changes and even trigger completely opposite biological functions. To understand the allosteric communication within the GR ligand binding domain, the folding pathway of helix 12 (H12) induced by the binding of the agonist dexamethasone (DEX), antagonist RU486, and modulator AZD9567 are explored by molecular dynamics simulations and Markov state model analysis. The ligands can regulate the volume of the activation function-2 through the residues Phe737 and Gln738. Without ligand or with agonist binding, H12 swings from inward to outward to visit different folding positions. However, the binding of RU486 or AZD9567 perturbs the structural state, and the passive antagonist state appears more stable. Structure-based virtual screening and in vitro bioassays are used to discover novel GR ligands that bias the conformation equilibria toward the passive antagonist state. HP-19 exhibits the best anti-inflammatory activity (IC50 = 0.041 ± 0.011 µm) in nuclear factor-kappa B signaling pathway, which is comparable to that of DEX. HP-19 also does not induce adverse effect-related transactivation functions of GR. The novel ligands discovered here may serve as promising starting points for the development of GR modulators.

Deubiquitination and Stabilization of PD-L1 by USP21.

Recent studies have shown that the expression level of PD-L1 in tumor cells positively correlates with tumor metastasis and recurrence rate. The effects of post-translational modifications (PTMs) of PD-L1 are related to immunosuppression. However, the degradation of PD-L1 in cancers has not yet been sufficiently defined. Here, we identified USP21 as a novel deubiquitinase of PD-L1. Overexpression of USP21 significantly increased PD-L1 abundance while its knockdown induced PD-L1 degradation. In vitro deubiquitination assay revealed that USP21-WT, but not USP21-C221A, reduced polyubiquitin chains of PD-L1. These results highlight the role of USP21 in the deubiquitination and stabilization of PD-L1. Furthermore, we show that USP21 is the frequently amplified deubiquitinase in lung cancer, especially in lung squamous cell carcinoma, and its amplification is accompanied by upregulation of PD-L1. This study reveals the mechanism of USP21-mediated PD-L1 degradation, and suggests that USP21 might be a potential target for the treatment of lung cancer.

SC75741, A Novel c-Abl Inhibitor, Promotes the Clearance of TDP25 Aggregates via ATG5-Dependent Autophagy Pathway.

Abnormal accumulation of TDP43-related mutant proteins in the cytoplasm causes amyotrophic lateral sclerosis (ALS). Herein, unbiased drug screening approaches showed that SC75741, a multi-target inhibitor, inhibited inflammation-induced aggregation by inhibiting NF-κB and also degraded already aggregated proteins by inhibiting c-Abl mediated autophagy-lysosomal pathway. We delineate the mechanism that SC75741 could markedly enhance TFEB nuclear translocation by an mTORC1-independent TFEB regulatory pathway. In addition, SC75741 enhanced the interaction between p62 with TDP25 and LC3C, thus promoting TDP25 degradation. Taken together, these findings show that SC75741 has beneficial neuroprotective effects in ALS. Our study elucidates that dual-targeted inhibition of c-Abl and NF-κB may be a potential treatment for TDP43 proteinopathies and ALS.

Delivery of a system xc- inhibitor by a redox-responsive levodopa prodrug nanoassembly for combination ferrotherapy.

A comprehensive understanding of ferroptosis signaling pathways significantly contributes to the advances in cancer ferrotherapy. Herein, we constructed a self-assembled prodrug nanosystem targeting system xc-, a key regulator for ferroptosis, to amplify the therapeutic efficacy of cancer ferrotherapy. The prodrug nanosystem is assembled between sulfasalazine (SSZ, a ferroptosis resistance inhibitor) and disulfide-bridged levodopa (DSSD) that can chelate Fe2+ ions to form SSZ-Fe2+@DSSD, and the resulting nanoassembly can not only inhibit ferroptosis resistance, but also generate ROS in the tumor microenvironment. Whereas the prodrug nanosystem is stable in the physiological environment, it becomes unstable in the tumoral and intracellular reductive microenvironment, where the disulfide linkers are disrupted by high levels of glutathione (GSH), triggering the release of active Fe2+ and SSZ. Under the Fenton reaction, the released Fe2+ thus can induce ferroptosis, which is amplified by SSZ-mediated inhibition of ferroptosis resistance to synergistically improve the therapeutic efficacy of ferroptosis. Our study thus provides an innovative prodrug strategy to advance anticancer ferroptosis.

Mitophagy Regulates Neurodegenerative Diseases.

Mitochondria play an essential role in supplying energy for the health and survival of neurons. Mitophagy is a metabolic process that removes dysfunctional or redundant mitochondria. This process preserves mitochondrial health. However, defective mitophagy triggers the accumulation of damaged mitochondria, causing major neurodegenerative disorders. This review introduces molecular mechanisms and signaling pathways behind mitophagy regulation. Furthermore, we focus on the recent advances in understanding the potential role of mitophagy in the pathogenesis of major neurodegenerative diseases (Parkinson's, Alzheimer's, Huntington's, etc.) and aging. The findings will help identify the potential interventions of mitophagy regulation and treatment strategies of neurodegenerative diseases.

Metformin activates chaperone-mediated autophagy and improves disease pathologies in an Alzheimer disease mouse model.

Chaperone-mediated autophagy (CMA) is a lysosome-dependent selective degradation pathway implicated in the pathogenesis of cancer and neurodegenerative diseases. However, the mechanisms that regulate CMA are not fully understood. Here, using unbiased drug screening approaches, we discover Metformin, a drug that is commonly the first medication prescribed for type 2 diabetes, can induce CMA. We delineate the mechanism of CMA induction by Metformin to be via activation of TAK1-IKKα/ß signaling that leads to phosphorylation of Ser85 of the key mediator of CMA, Hsc70, and its activation. Notably, we find that amyloid-beta precursor protein (APP) is a CMA substrate and that it binds to Hsc70 in an IKKα/ß-dependent manner. The inhibition of CMA-mediated degradation of APP enhances its cytotoxicity. Importantly, we find that in the APP/PS1 mouse model of Alzheimer's disease (AD), activation of CMA by Hsc70 overexpression or Metformin potently reduces the accumulated brain Aß plaque levels and reverses the molecular and behavioral AD phenotypes. Our study elucidates a novel mechanism of CMA regulation via Metformin-TAK1-IKKα/ß-Hsc70 signaling and suggests Metformin as a new activator of CMA for diseases, such as AD, where such therapeutic intervention could be beneficial.

ARIH1 signaling promotes anti-tumor immunity by targeting PD-L1 for proteasomal degradation.

Cancer expression of PD-L1 suppresses anti-tumor immunity. PD-L1 has emerged as a remarkable therapeutic target. However, the regulation of PD-L1 degradation is not understood. Here, we identify several compounds as inducers of PD-L1 degradation using a high-throughput drug screen. We find EGFR inhibitors promote PD-L1 ubiquitination and proteasomal degradation following GSK3α-mediated phosphorylation of Ser279/Ser283. We identify ARIH1 as the E3 ubiquitin ligase responsible for targeting PD-L1 to degradation. Overexpression of ARIH1 suppresses tumor growth and promotes cytotoxic T cell activation in wild-type, but not in immunocompromised mice, highlighting the role of ARIH1 in anti-tumor immunity. Moreover, combining EGFR inhibitor ES-072 with anti-CTLA4 immunotherapy results in an additive effect on both tumor growth and cytotoxic T cell activation. Our results delineate a mechanism of PD-L1 degradation and cancer escape from immunity via EGFR-GSK3α-ARIH1 signaling and suggest GSK3α and ARIH1 might be potential drug targets to boost anti-tumor immunity and enhance immunotherapies.