技术资料

Breast cancer stem cell (CSC) expansion results in tumor progression and chemoresistance; however,the modulation of CSC pluripotency remains unexplored. Transmembrane protein 120B (TMEM120B) is a newly discovered protein expressed in human tissues,especially in malignant tissues; however,its role in CSC expansion has not been studied. This study aimed to determine the role of TMEM120B in transcriptional coactivator with PDZ-binding motif (TAZ)-mediated CSC expansion and chemotherapy resistance. Both bioinformatics analysis and immunohistochemistry assays were performed to examine expression patterns of TMEM120B in lung,breast,gastric,colon,and ovarian cancers. Clinicopathological factors and overall survival were also evaluated. Next,colony formation assay,MTT assay,EdU assay,transwell assay,wound healing assay,flow cytometric analysis,sphere formation assay,western blotting analysis,mouse xenograft model analysis,RNA-sequencing assay,immunofluorescence assay,and reverse transcriptase-polymerase chain reaction were performed to investigate the effect of TMEM120B interaction on proliferation,invasion,stemness,chemotherapy sensitivity,and integrin/FAK/TAZ/mTOR activation. Further,liquid chromatography–tandem mass spectrometry analysis,GST pull-down assay,and immunoprecipitation assays were performed to evaluate the interactions between TMEM120B,myosin heavy chain 9 (MYH9),and CUL9. TMEM120B expression was elevated in lung,breast,gastric,colon,and ovarian cancers. TMEM120B expression positively correlated with advanced TNM stage,lymph node metastasis,and poor prognosis. Overexpression of TMEM120B promoted breast cancer cell proliferation,invasion,and stemness by activating TAZ-mTOR signaling. TMEM120B directly bound to the coil-coil domain of MYH9,which accelerated the assembly of focal adhesions (FAs) and facilitated the translocation of TAZ. Furthermore,TMEM120B stabilized MYH9 by preventing its degradation by CUL9 in a ubiquitin-dependent manner. Overexpression of TMEM120B enhanced resistance to docetaxel and doxorubicin. Conversely,overexpression of TMEM120B-∆CCD delayed the formation of FAs,suppressed TAZ-mTOR signaling,and abrogated chemotherapy resistance. TMEM120B expression was elevated in breast cancer patients with poor treatment outcomes (Miller/Payne grades 1–2) than in those with better outcomes (Miller/Payne grades 3–5). Our study reveals that TMEM120B bound to and stabilized MYH9 by preventing its degradation. This interaction activated the β1-integrin/FAK-TAZ-mTOR signaling axis,maintaining stemness and accelerating chemotherapy resistance. The online version contains supplementary material available at 10.1186/s13058-024-01802-z. View Publication

Acute myeloid leukaemia (AML) is a haematological malignancy characterised by the accumulation of transformed myeloid progenitors in the bone marrow. Piplartine (PL),also known as piperlongumine,is a pro-oxidant small molecule extracted from peppers that has demonstrated antineoplastic potential in solid tumours and other haematological malignancies. In this work,we explored the potential of PL to treat AML through the use of a combination of cellular and molecular analyses of primary and cultured leukaemia cells in vitro and in vivo. We showed that PL exhibits in vitro cytotoxicity against AML cells,including CD34 + leukaemia-propagating cells,but not healthy haematopoietic progenitors,suggesting anti-leukaemia selectivity. Mechanistically,PL treatment increased reactive oxygen species (ROS) levels and induced ROS-mediated apoptosis in AML cells,which could be prevented by treatment with the antioxidant scavenger N -acetyl-cysteine and the pancaspase inhibitor Z-VAD(OMe)-FMK. PL treatment reduced NFKB1 gene transcription and the level of NF-κB p65 (pS536),which was depleted from the nucleus of AML cells,indicating suppression of NF-κB p65 signalling. Significantly,PL suppressed AML development in a mouse xenograft model,and its combination with current AML treatments (cytarabine,daunorubicin and azacytidine) had synergistic effects,indicating translational therapeutic potential. Taken together,these data position PL as a novel anti-AML candidate drug that can target leukaemia stem/progenitors and is amenable to combinatorial therapeutic strategies. Subject terms: Acute myeloid leukaemia,Cancer stem cells,Pharmacology 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

Signal regulatory protein-α (SIRPα,SHPS-1,CD172a) expressed on myeloid cells transmits inhibitory signals when it engages its counter-receptor CD47 on an adjacent cell. Elevated CD47 expression on some cancer cells thereby serves as an innate immune checkpoint that limits phagocytic clearance of tumor cells by macrophages and antigen presentation to T cells. Antibodies and recombinant SIRPα constructs that block the CD47-SIRPα interaction on macrophages exhibit anti-tumor activities in mouse models and are in ongoing clinical trials for treating several human cancers. Based on prior evidence that engaging SIRPα can also alter CD47 signaling in some nonmalignant cells,we compared direct effects of recombinant SIRPα-Fc and a humanized CD47 antibody that inhibits CD47-SIRPα interaction (CC-90002) on CD47 signaling in cancer stem cells derived from the MDA-MB- 231 triple-negative breast carcinoma cell line. Treatment with SIRPα-Fc significantly increased the formation of mammospheres by breast cancer stem cells as compared to CC-90002 treatment or controls. Furthermore,SIRPα-Fc treatment upregulated mRNA and protein expression of ALDH1 and altered the expression of genes involved in epithelial/mesenchymal transition pathways that are associated with a poor prognosis and enhanced metastatic activity. This indicates that SIRPα-Fc has CD47-mediated agonist activities in breast cancer stem cells affecting proliferation and metastasis pathways that differ from those of CC-90002. This SIRPα-induced CD47 signaling in breast carcinoma cells may limit the efficacy of SIRPα decoy therapeutics intended to stimulate innate antitumor immune responses. 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

Protein synthesis is frequently deregulated during tumorigenesis. However,the precise contexts of selective translational control and the regulators of such mechanisms in cancer is poorly understood. Here,we uncovered CNOT3,a subunit of the CCR4-NOT complex,as an essential modulator of translation in myeloid leukemia. Elevated CNOT3 expression correlates with unfavorable outcomes in patients with acute myeloid leukemia (AML). CNOT3 depletion induces differentiation and apoptosis and delayed leukemogenesis. Transcriptomic and proteomic profiling uncovers c-MYC as a critical downstream target which is translationally regulated by CNOT3. Global analysis of mRNA features demonstrates that CNOT3 selectively influences expression of target genes in a codon usage dependent manner. Furthermore,CNOT3 associates with the protein network largely consisting of ribosomal proteins and translation elongation factors in leukemia cells. Overall,our work elicits the direct requirement for translation efficiency in tumorigenesis and propose targeting the post-transcriptional circuitry via CNOT3 as a therapeutic vulnerability in AML. Subject terms: Acute myeloid leukaemia,Translation,RNA decay View Publication

Several genetic risk factors for Alzheimer’s disease implicate genes involved in lipid metabolism and many of these lipid genes are highly expressed in glial cells 1 . However,the relationship between lipid metabolism in glia and Alzheimer’s disease pathology remains poorly understood. Through single-nucleus RNA sequencing of brain tissue in Alzheimer’s disease,we have identified a microglial state defined by the expression of the lipid droplet-associated enzyme ACSL1 with ACSL1-positive microglia being most abundant in patients with Alzheimer’s disease having the APOE4/4 genotype. In human induced pluripotent stem cell-derived microglia,fibrillar Aβ induces ACSL1 expression,triglyceride synthesis and lipid droplet accumulation in an APOE-dependent manner. Additionally,conditioned media from lipid droplet-containing microglia lead to Tau phosphorylation and neurotoxicity in an APOE-dependent manner. Our findings suggest a link between genetic risk factors for Alzheimer’s disease with microglial lipid droplet accumulation and neurotoxic microglia-derived factors,potentially providing therapeutic strategies for Alzheimer’s disease. Subject terms: Alzheimer's disease,Microglia,Neuroimmunology 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

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

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

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

Angiotensin (Ang)-(1–7) stimulates vasoprotective functions of diabetic (DB) CD34 + hematopoietic stem/progenitor cells partly by decreasing reactive oxygen species (ROS),increasing nitric oxide (NO) levels and decreasing TGFβ1 secretion. Telomerase reverse transcriptase (TERT) translocates to mitochondria and regulates ROS generation. Alternative splicing of TERT results in variants α-,β- and α-β-TERT,which may oppose functions of full-length (FL) TERT. This study tested if the protective functions of Ang-(1–7) or TGFβ1-silencing are mediated by mitoTERT and that diabetes decreases FL-TERT expression by inducing splicing. CD34 + cells were isolated from the peripheral blood mononuclear cells of nondiabetic (ND,n = 68) or DB (n = 74) subjects. NO and mitoROS levels were evaluated by flow cytometry. TERT splice variants and mitoDNA-lesions were characterized by qPCR. TRAP assay was used for telomerase activity. Decoy peptide was used to block mitochondrial translocation (mitoXTERT). TERT inhibitor or mitoXTERT prevented the effects of Ang-(1–7) on NO or mitoROS levels in DB-CD34 + cells. FL-TERT expression and telomerase activity were lower and mitoDNA-lesions were higher in DB cells compared to ND and were reversed by Ang-(1–7) or TGFβ1-silencing. The prevalence of TERT splice variants,with predominant β-TERT expression,was higher and the expression of FL-TERT was lower in DB cells (n = 25) compared to ND (n = 30). Ang-(1–7) or TGFβ1-silencing decreased TERT-splicing and increased FL-TERT. Blocking of β-splicing increased FL-TERT and protected mitoDNA in DB-cells. The findings suggest that diabetes induces TERT-splicing in CD34 + cells and that β-TERT splice variant largely contributes to the mitoDNA oxidative damage. View Publication