技术资料

Windham et al. discover that APOE in astrocytes can traffic to lipid droplets (LDs),where it modulates LD composition and size. Astrocytes expressing the Alzheimer’s risk variant APOE4 form large LDs with impaired turnover and increased peroxidation sensitivity. View Publication

The peripheral immune system is important in neurodegenerative diseases,both in protecting and inflaming the brain,but the underlying mechanisms remain elusive. Alzheimer’s Disease is commonly preceded by a prodromal period. Here,we report the presence of large Aβ aggregates in plasma from patients with mild cognitive impairment ( n = 38). The aggregates are associated with low level Alzheimer’s Disease-like brain pathology as observed by 11 C-PiB PET and 18 F-FTP PET and lowered CD18-rich monocytes. We characterize complement receptor 4 as a strong binder of amyloids and show Aβ aggregates are preferentially phagocytosed and stimulate lysosomal activity through this receptor in stem cell-derived microglia. KIM127 integrin activation in monocytes promotes size selective phagocytosis of Aβ. Hydrodynamic calculations suggest Aβ aggregates associate with vessel walls of the cortical capillaries. In turn,we hypothesize aggregates may provide an adhesion substrate for recruiting CD18-rich monocytes into the cortex. Our results support a role for complement receptor 4 in regulating amyloid homeostasis. Subject terms: Protein aggregation,Neuroimmunology,Dementia View Publication

One-third of pediatric patients with osteosarcoma (OS) develop lung metastases (LM),which is the primary predictor of mortality. While current treatments of patients with localized bone disease have been successful in producing 5-year survival rates of 65–70%,patients with LM experience poor survival rates of only 19–30%. Unacceptably,this situation that has remained unchanged for 30 years. Thus,there is an urgent need to elucidate the mechanisms of metastatic spread in OS and to identify targetable molecular pathways that enable more effective treatments for patients with LM. We aimed to identify OS-specific gene alterations using RNA-sequencing of extremity and LM human tissues. Samples of extremity and LM tumors,including 4 matched sets,were obtained from patients with OS. Our data demonstrate aberrant regulation of the androgen receptor (AR) pathway in LM and predicts aldehyde dehydrogenase 1A1 (ALDH1A1) as a downstream target. Identification of AR pathway upregulation in human LM tissue samples may provide a target for novel therapeutics for patients with LM resistant to conventional chemotherapy. Subject terms: Bone cancer,Sarcoma,Gene expression View Publication

Wiskott-Aldrich syndrome (WAS) is a severe X-linked primary immunodeficiency resulting from a diversity of mutations distributed across all 12 exons of the WAS gene. WAS encodes a hematopoietic-specific and developmentally regulated cytoplasmic protein (WASp). The objective of this study was to develop a gene correction strategy potentially applicable to most WAS patients by employing nuclease-mediated,site-specific integration of a corrective WAS gene sequence into the endogenous WAS chromosomal locus. In this study,we demonstrate the ability to target the integration of WAS 2-12 -containing constructs into intron 1 of the endogenous WAS gene of primary CD34 + hematopoietic stem and progenitor cells (HSPCs),as well as WASp-deficient B cell lines and WASp-deficient primary T cells. This intron 1 targeted integration (TI) approach proved to be quite efficient and restored WASp expression in treated cells. Furthermore,TI restored WASp-dependent function to WAS patient T cells. Edited CD34 + HSPCs exhibited the capacity for multipotent differentiation to various hematopoietic lineages in vitro and in transplanted immunodeficient mice. This methodology offers a potential editing approach for treatment of WAS using patient’s CD34 + cells. View Publication

Accumulating evidence indicates that various oncogenic mutations interfere with normal myeloid differentiation of leukemogenic cells during the early process of acute myeloid leukemia (AML) development. Differentiation therapy is a therapeutic strategy capable of terminating leukemic expansion by reactivating the differentiation potential; however,the plasticity and instability of leukemia cells counteract the establishment of treatments aimed at irreversibly inducing and maintaining their differentiation states. On the basis of our previous observation that autophagy inhibitor treatment induces the accumulation of cytosolic DNA and activation of cytosolic DNA-sensor signaling selectively in leukemia cells,we herein examined the synergistic effect of cytosolic DNA-sensor signaling activation with conventional differentiation therapy on AML. The combined treatment succeeded in inducing irreversible differentiation in AML cell lines. Mechanistically,cytosolic DNA was sensed by absent in melanoma 2 (AIM2),a cytosolic DNA sensor. Activation of the AIM2 inflammasome resulted in the accumulation of p21 through the inhibition of its proteasomal degradation,thereby facilitating the myeloid differentiation. Importantly,the combined therapy dramatically reduced the total leukemia cell counts and proportion of blast cells in the spleens of AML mice. Collectively,these findings indicate that the autophagy inhibition-cytosolic DNA-sensor signaling axis can potentiate AML differentiation therapy. Clinical effects on AML therapy are closely associated with reactivating the normal myeloid differentiation potential in leukemia cells. This study shows that autophagosome formation inhibitors activate the cytosolic DNA-sensor signaling,thereby augmenting conventional differentiation therapy to induce irreversible differentiation and cell growth arrest in several types of AML cell lines. View Publication

Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis of all cancers. To improve PDAC therapy,we establish screening systems based on organoid and co-culture technologies and find a payload of antibody–drug conjugate (ADC),a bromodomain and extra-terminal (BET) protein degrader named EBET. We select CEACAM6/CD66c as an ADC target and developed an antibody,#84.7,with minimal reactivity to CEACAM6-expressing normal cells. EBET-conjugated #84.7 (84-EBET) has lethal effects on various PDAC organoids and bystander efficacy on CEACAM6-negative PDAC cells and cancer-associated fibroblasts. In mouse studies,a single injection of 84-EBET induces marked tumor regression in various PDAC-patient-derived xenografts,with a decrease in the inflammatory phenotype of stromal cells and without significant body weight loss. Combination with standard chemotherapy or PD-1 antibody induces more profound and sustained regression without toxicity enhancement. Our preclinical evidence demonstrates potential efficacy by delivering BET protein degrader to PDAC and its microenvironment via CEACAM6-targeted ADC. Subject terms: Pancreatic cancer,Drug development,Targeted therapies View Publication

We report a case of Mismatch Repair Deficiency (MMRD) caused by germline homozygous EPCAM deletion leading to tissue-specific loss of MSH2. Through the use of patient-derived cells and organoid technologies,we performed stepwise in vitro differentiation of colonic and brain organoids from reprogrammed EPCAM del iPSC derived from patient fibroblasts. Differentiation of iPSC to epithelial-colonic organoids exhibited continuous increased EPCAM expression and hypermethylation of the MSH2 promoter. This was associated with loss of MSH2 expression,increased mutational burden,MMRD signatures and MS-indel accumulation,the hallmarks of MMRD. In contrast,maturation into brain organoids and examination of blood and fibroblasts failed to show similar processes,preserving MMR proficiency. The combined use of iPSC,organoid technologies and functional genomics analyses highlights the potential of cutting-edge cellular and molecular analysis techniques to define processes controlling tumorigenesis and uncovers a new paradigm of tissue-specific MMRD,which affects the clinical management of these patients. Subject terms: Paediatric cancer,Cancer genetics View Publication

Acute myeloid leukemia (AML) with biallelic ( CEBPA bi ) as well as single mutations located in the bZIP region is associated with a favorable prognosis,but the underlying mechanisms are still unclear. Here,we propose that two isoforms of C/EBPα regulate DNA damage-inducible transcript 3 (DDIT3) transcription in AML cells corporately,leading to altered susceptibility to endoplasmic reticulum (ER) stress and related drugs. Human AML cell lines and murine myeloid precursor cell line 32Dcl3 cells were infected with recombinant lentiviruses to knock down CEBPA expression or over-express the two isoforms of C/EBPα. Quantitative real-time PCR and western immunoblotting were employed to determine gene expression levels. Cell apoptosis rates were assessed by flow cytometry. CFU assays were utilized to evaluate the differentiation potential of 32Dcl3 cells. Luciferase reporter analysis,ChIP-seq and ChIP-qPCR were used to validate the transcriptional regulatory ability and affinity of each C/EBPα isoform to specific sites at DDIT3 promoter. Finally,an AML xenograft model was generated to evaluate the in vivo therapeutic effect of agents. We found a negative correlation between CEBPA expression and DDIT3 levels in AML cells. After knockdown of CEBPA,DDIT3 expression was upregulated,resulting in increased apoptotic rate of AML cells induced by ER stress. Cebpa knockdown in mouse 32Dcl3 cells also led to impaired cell viability due to upregulation of Ddit3,thereby preventing leukemogenesis since their differentiation was blocked. Then we discovered that the two isoforms of C/EBPα regulate DDIT3 transcription in the opposite way. C/EBPα-p30 upregulated DDIT3 transcription when C/EBPα-p42 downregulated it instead. Both isoforms directly bound to the promoter region of DDIT3. However,C/EBPα-p30 has a unique binding site with stronger affinity than C/EBPα-p42. These findings indicated that balance of two isoforms of C/EBPα maintains protein homeostasis and surveil leukemia,and at least partially explained why AML cells with disrupted C/EBPα-p42 and/or overexpressed C/EBPα-p30 exhibit better response to chemotherapy stress. Additionally,we found that a low C/EBPα p42/p30 ratio induces resistance in AML cells to the BCL2 inhibitor venetoclax since BCL2 is a major target of DDIT3. This resistance can be overcome by combining ER stress inducers,such as tunicamycin and sorafenib in vitro and in vivo. Our results indicate that AML patients with a low C/EBPα p42/p30 ratio (e.g.,CEBPA bi ) may not benefit from monotherapy with BCL2 inhibitors. However,this issue can be resolved by combining ER stress inducers. The online version contains supplementary material available at 10.1186/s13046-024-02975-3. View Publication

Chimeric antigen receptor (CAR) T cells have shown remarkable response rates in hematological malignancies. In contrast,CAR T cell treatment of solid tumors is associated with several challenges,in particular the expression of most tumor-associated antigens at lower levels in vital organs,resulting in on-target/off-tumor toxicities. Thus,innovative approaches to improve the tumor specificity of CAR T cells are urgently needed. Based on the observation that many human solid tumors activate epidermal growth factor receptor (EGFR) on their surface through secretion of EGFR ligands,we developed an engineering strategy for CAR-binding domains specifically directed against the ligand-activated conformation of EGFR. We show,in several experimental systems,that the generated binding domains indeed enable CAR T cells to distinguish between active and inactive EGFR. We anticipate that this engineering concept will be an important step forward to improve the tumor specificity of CAR T cells directed against EGFR-positive solid cancers. View Publication

SorLA,encoded by the gene SORL1,is an intracellular sorting receptor of the VPS10P domain receptor gene family. Although SorLA is best recognized for its ability to shuttle target proteins between intracellular compartments in neurons,recent data suggest that also its microglial expression can be of high relevance for the pathogenesis of brain diseases,including glioblastoma (GBM). Here,we interrogated the impact of SorLA on the functional properties of glioma-associated microglia and macrophages (GAMs). In the GBM microenvironment,GAMs are re-programmed and lose the ability to elicit anti-tumor responses. Instead,they acquire a glioma-supporting phenotype,which is a key mechanism promoting glioma progression. Our re-analysis of published scRNA-seq data from GBM patients revealed that functional phenotypes of GAMs are linked to the level of SORL1 expression,which was further confirmed using in vitro models. Moreover,we demonstrate that SorLA restrains secretion of TNFα from microglia to restrict the inflammatory potential of these cells. Finally,we show that loss of SorLA exacerbates the pro-inflammatory response of microglia in the murine model of glioma and suppresses tumor growth. View Publication

Hepatocellular carcinoma (HCC) is widely recognized as a globally prevalent malignancy. Immunotherapy is a promising therapy for HCC patients. Increasing evidence suggests that lncRNAs are involved in HCC progression and immunotherapy. The study reveals the mechanistic role of long non‐coding RNA (lncRNA) FOXD1‐AS1 in regulating migration,invasion,circulating tumor cells (CTCs),epithelial‐mesenchymal transition (EMT),and immune escape in HCC in vitro. This study employed real‐time PCR (RT‐qPCR) to measure FOXD1‐AS1,miR‐615‐3p,and programmed death‐ligand 1 ( PD‐L1 ). The interactions of FOXD1‐AS1,miR‐615‐3p,and PD‐L1 were validated via dual‐luciferase reporter gene and ribonucleoprotein immunoprecipitation (RIP) assay. In vivo experimentation involves BALB/c mice and BALB/c nude mice to investigate the impact of HCC metastasis. The upregulation of lncRNA FOXD1‐AS1 in malignant tissues significantly correlates with poor prognosis. The investigation was implemented on the impact of lncRNA FOXD1‐AS1 on the migratory,invasive,and EMT of HCC cells. It has been observed that the lncRNA FOXD1‐AS1 significantly influences the generation and metastasis of M CTC in vivo analysis. In mechanistic analysis,lncRNA FOXD1‐AS1 enhanced immune escape in HCC via upregulation of PD‐L1,which acted as a ceRNA by sequestering miR‐615‐3p. Additionally,lncRNA FOXD1‐AS1 was found to modulate the EMT of CTCs through the activation of the PI3K/AKT pathway. This study presents compelling evidence supporting the role of lncRNA FOXD1‐AS1 as a miRNA sponge that sequesters miR‐655‐3p and protects PD‐L1 from suppression. View Publication

Myeloperoxidase (MPO) is found almost exclusively in granulocytes and immature myeloid cells. It plays a key role in the innate immune system,catalysing the formation of reactive oxygen species that are important in anti‐microbial action,but MPO also oxidatively transforms the topoisomerase II (TOP2) poison etoposide to chemical forms that have elevated DNA damaging properties. TOP2 poisons such as etoposide are widely used anti‐cancer drugs,but they are linked to cases of secondary acute myeloid leukaemias through a mechanism that involves DNA damage and presumably erroneous repair leading to leukaemogenic chromosome translocations. This leads to the possibility that myeloperoxidase inhibitors could reduce the rate of therapy‐related leukaemia by protecting haematopoietic cells from TOP2 poison‐mediated genotoxic damage while preserving the anti‐cancer efficacy of the treatment. We show here that myeloperoxidase inhibition reduces etoposide‐induced TOP2B‐DNA covalent complexes and resulting DNA double‐strand break formation in primary ex vivo expanded CD34 + progenitor cells and unfractionated bone marrow mononuclear cells. Since MPO inhibitors are currently being developed as anti‐inflammatory agents this raises the possibility that repurposing of these potential new drugs could provide a means of suppressing secondary acute myeloid leukaemias associated with therapies containing TOP2 poisons. View Publication