技术资料

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

Pseudouridylation plays a regulatory role in various physiological and pathological processes. A prime example is the mitochondrial myopathy,lactic acidosis,and sideroblastic anemia syndrome (MLASA),characterized by defective pseudouridylation resulting from genetic mutations in pseudouridine synthase 1 (PUS1). However,the roles and mechanisms of pseudouridylation in normal erythropoiesis and MLASA-related anemia remain elusive. We established a mouse model carrying a point mutation (R110W) in the enzymatic domain of PUS1,mimicking the common mutation in human MLASA. Pus1 -mutant mice exhibited anemia at 4 weeks old. Impaired mitochondrial oxidative phosphorylation was also observed in mutant erythroblasts. Mechanistically,mutant erythroblasts showed defective pseudouridylation of targeted tRNAs,altered tRNA profiles,decreased translation efficiency of ribosomal protein genes,and reduced globin synthesis,culminating in ineffective erythropoiesis. Our study thus provided direct evidence that pseudouridylation participates in erythropoiesis in vivo. We demonstrated the critical role of pseudouridylation in regulating tRNA homeostasis,cytoplasmic translation,and erythropoiesis. Subject areas: Molecular biology,Cell biology View Publication

Acute myeloid leukemia (AML) is a highly aggressive hematologic cancer with poor survival across a broad range of molecular subtypes. Development of efficacious and well-tolerable therapies encompassing the range of mutations that can arise in AML remains an unmet need. The bromo- and extra-terminal domain (BET) family of proteins represents an attractive therapeutic target in AML due to their crucial roles in many cellular functions,regardless of any specific mutation. Many BET inhibitors (BETi) are currently in pre-clinical and early clinical development,but acquisition of resistance continues to remain an obstacle for the drug class. Novel methods to circumvent this development of resistance could be instrumental for the future use of BET inhibitors in AML,both as monotherapy and in combination. To date,many investigations into possible drug combinations of BETi with CDK inhibitors have focused on CDK9,which has a known physical and functional interaction with the BET protein BRD4. Therefore,we wished to investigate possible synergy and additive effects between inhibitors of these targets in AML. Here,we describe combination therapy with the multi-CDK inhibitor dinaciclib and the BETi PLX51107 in pre-clinical models of AML. Dinaciclib and PLX51107 demonstrate additive effects in AML cell lines,primary AML samples,and in vivo. Further,we demonstrate novel activity of dinaciclib through inhibition of the canonical/β-catenin dependent Wnt signaling pathway,a known resistance mechanism to BETi in AML. We show dinaciclib inhibits Wnt signaling at multiple levels,including downregulation of β-catenin,the Wnt co-receptor LRP6,as well as many Wnt pathway components and targets. Moreover,dinaciclib sensitivity remains unaffected in a setting of BET resistance,demonstrating similar inhibitory effects on Wnt signaling when compared to BET-sensitive cells. Ultimately,our results demonstrate rationale for combination CDKi and BETi in AML. In addition,our novel finding of Wnt signaling inhibition could have potential implications in other cancers where Wnt signaling is dysregulated and demonstrates one possible approach to circumvent development of BET resistance in AML. The online version contains supplementary material available at 10.1186/s40164-024-00483-w. View Publication

In the adult bone marrow (BM),endothelial cells (ECs) are an integral component of the hematopoietic stem cell (HSC)-supportive niche,which modulates HSC activity by producing secreted and membrane-bound paracrine signals. Within the BM,distinct vascular arteriole,transitional,and sinusoidal EC subtypes display unique paracrine expression profiles and create anatomically-discrete microenvironments. However,the relative contributions of vascular endothelial subtypes in supporting hematopoiesis is unclear. Moreover,constitutive expression and off-target activity of currently available endothelial-specific and endothelial-subtype-specific murine cre lines potentially confound data analysis and interpretation. To address this,we describe two tamoxifen-inducible cre -expressing lines,Vegfr3-creER T2 and Cx40-creER T2,that efficiently label sinusoidal/transitional and arteriole endothelium respectively in adult marrow,without off-target activity in hematopoietic or perivascular cells. Utilizing an established mouse model in which cre -dependent recombination constitutively-activates MAPK signaling within adult endothelium,we identify arteriole ECs as the driver of MAPK-mediated hematopoietic dysfunction. These results define complementary tamoxifen-inducible creER T2 -expressing mouse lines that label functionally-discrete and non-overlapping sinusoidal/transitional and arteriole EC populations in the adult BM,providing a robust toolset to investigate the differential contributions of vascular subtypes in maintaining hematopoietic homeostasis. The online version contains supplementary material available at 10.1007/s12015-024-10703-9. View Publication

Tumor-resident microbiota in breast cancer promotes cancer initiation and malignant progression. However,targeting microbiota to improve the effects of breast cancer therapy has not been investigated in detail. Here,we evaluated the microbiota composition of breast tumors and found that enterotoxigenic Bacteroides fragilis (ETBF) was highly enriched in the tumors of patients who did not respond to taxane-based neoadjuvant chemotherapy. ETBF,albeit at low biomass,secreted the toxic protein BFT-1 to promote breast cancer cell stemness and chemoresistance. Mechanistic studies showed that BFT-1 directly bound to NOD1 and stabilized NOD1 protein. NOD1 was highly expressed on ALDH + breast cancer stem cells (BCSCs) and cooperated with GAK to phosphorylate NUMB and promote its lysosomal degradation,thereby activating the NOTCH1-HEY1 signaling pathway to increase BCSCs. NOD1 inhibition and ETBF clearance increase the chemosensitivity of breast cancer by impairing BCSCs. View Publication

The heart is a metabolic “omnivore” and adjusts its energy source depending on the circulating metabolites. Human cardiac organoids,a three-dimensional in vitro model of the heart wall,are a useful tool to study cardiac physiology and pathology. However,cardiac tissue naturally experiences shear stress and nutrient fluctuations via blood flow in vivo,whilst in vitro models are conventionally cultivated in a static medium. This necessitates the regular refreshing of culture media,which creates acute cellular disturbances and large metabolic fluxes. To culture human cardiac organoids in a more physiological manner,we have developed a perfused bioreactor for cultures in a 96-well plate format. The designed bioreactor is easy to fabricate using a common culture plate and a 3D printer. Its open system allows for the use of traditional molecular biology techniques,prevents flow blockage issues,and provides easy access for sampling and cell assays. We hypothesized that a perfused culture would create more stable environment improving cardiac function and maturation. We found that lactate is rapidly produced by human cardiac organoids,resulting in large fluctuations in this metabolite under static culture. Despite this,neither medium perfusion in bioreactor culture nor lactate supplementation improved cardiac function or maturation. In fact,RNA sequencing revealed little change across the transcriptome. This demonstrates that cardiac organoids are robust in response to fluctuating environmental conditions under normal physiological conditions. Together,we provide a framework for establishing an easily accessible perfusion system that can be adapted to a range of miniaturized cell culture systems. View Publication

The human WDR33 gene encodes three major isoforms. The canonical isoform WDR33v1 (V1) is a well-characterized nuclear mRNA polyadenylation factor,while the other two,WDR33v2 (V2) and WDR33v3 (V3),have not been studied. Here,we report that V2 and V3 are generated by alternative polyadenylation,and neither protein contains all seven WD (tryptophan-aspartic acid) repeats that characterize V1. Surprisingly,V2 and V3 are not polyadenylation factors but localize to the endoplasmic reticulum and interact with stimulator of interferon genes (STING),the immune factor that induces the cellular response to cytosolic double-stranded DNA. V2 suppresses interferon-β induction by preventing STING disulfide oligomerization but promotes autophagy,likely by recruiting WIPI2 isoforms. V3,on the other hand,functions to ncrease STING protein levels. Our study has not only provided mechanistic insights into STING regulation but also revealed that protein isoforms can be functionally completely unrelated,indicating that alternative mRNA processing is a more powerful mechanism than previously appreciated. View Publication

We describe humans with rare biallelic loss-of-function PTCRA variants impairing pre-TCRα expression. Low circulating naïve αβ T cell counts at birth persisted over time,with normal memory αβ and high γδ T cell counts. Their TCRα repertoire was biased,suggesting that noncanonical thymic differentiation pathways can rescue αβ T cell development. Only a minority of these individuals were sick,with infection,lymphoproliferation,and/or autoimmunity. We also report that 1 in 4000 individuals from the Middle East and South Asia are homozygous for a common hypomorphic PTCRA variant. They had normal circulating naïve αβ T cell counts but high γδ T cell counts. Although residual pre-TCRα expression drove the differentiation of more αβ T cells,autoimmune conditions were more frequent in these patients than in the general population. View Publication