技术资料

BACKGROUND Chimeric antigen receptor (CAR) T cell therapy targeting B cell maturation antigen (BCMA) on multiple myeloma (MM) produces fast but not long-lasting responses. Reasons for treatment failure are poorly understood. CARs simultaneously targeting two antigens may represent an alternative. Here,we (1) designed and characterized novel A proliferation inducing ligand (APRIL) based dual-antigen targeting CARs,and (2) investigated mechanisms of resistance to CAR T cells with three different BCMA-binding moieties (APRIL,single-chain-variable-fragment,heavy-chain-only). METHODS Three new APRIL-CARs were designed and characterized. Human APRIL-CAR T cells were evaluated for their cytotoxic function in vitro and in vivo,for their polyfunctionality,immune synapse formation,memory,exhaustion phenotype and tonic signaling activity. To investigate resistance mechanisms,we analyzed BCMA levels and cellular localization and quantified CAR T cell-target cell interactions by live microscopy. Impact on pathway activation and tumor cell proliferation was assessed in vitro and in vivo. RESULTS APRIL-CAR T cells in a trimeric ligand binding conformation conferred fast but not sustained antitumor responses in vivo in mouse xenograft models. In vitro trimer-BB$\zeta$ CAR T cells were more polyfunctional and formed stronger immune synapses than monomer-BB$\zeta$ CAR T cells. After CAR T cell-myeloma cell contact,BCMA was rapidly downmodulated on target cells with all evaluated binding moieties. CAR T cells acquired BCMA by trogocytosis,and BCMA on MM cells was rapidly internalized. Since BCMA can be re-expressed during progression and persisting CAR T cells may not protect patients from relapse,we investigated whether non-functional CAR T cells play a role in tumor progression. While CAR T cell-MM cell interactions activated BCMA pathway,we did not find enhanced tumor growth in vitro or in vivo. CONCLUSION Antitumor responses with APRIL-CAR T cells were fast but not sustained. Rapid BCMA downmodulation occurred independently of whether an APRIL or antibody-based binding moiety was used. BCMA internalization mostly contributed to this effect,but trogocytosis by CAR T cells was also observed. Our study sheds light on the mechanisms underlying CAR T cell failure in MM when targeting BCMA and can inform the development of improved treatment strategies. View Publication

Erythropoiesis is a crucial process in hematopoiesis,yet it remains highly susceptible to disruption by various diseases,which significantly contribute to the global challenges of anemia and blood shortages. Current treatments like erythropoietin (EPO) or glucocorticoids often fall short,especially for hereditary anemias such as Diamond-Blackfan anemia (DBA). To uncover new erythropoiesis-stimulating agents,we devised a screening system using primary human hematopoietic stem and progenitor cells (HSPCs). We discovered that BRAF inhibitors (BRAFi),commonly used to treat BRAF V600E melanoma,can unexpectedly and effectively promote progenitor cell proliferation by temporarily delaying erythroid differentiation. Notably,these inhibitors exhibited pronounced efficacy even under cytokine-restricted conditions and in patient samples of DBA. Mechanistically,although these BRAFi inhibit the MAPK cascade in BRAF V600E mutant cells,they paradoxically act as amplifiers in wild-type BRAF cells,potently enhancing the cascade. Furthermore,we found that while the oncogenic BRAF V600E mutation disrupts hematopoiesis and erythropoiesis through AP-1 hyperactivation,BRAFi minimally impact HSPC self-renewal and differentiation. In vivo studies have shown that BRAFi can enhance human hematopoiesis and erythropoiesis in severe immunodeficient mouse models and alleviate anemia in the Rpl11 haploinsufficiency DBA model,as well as other relevant anemia models. This discovery underscores the role of the MAPK pathway in hematopoiesis and positions BRAFi as a promising therapeutic option for improving hematopoietic reconstitution and treating anemias,including DBA. Subject terms: Drug screening,Molecular medicine View Publication

BRAF-activating mutations have been reported in several types of cancer,including melanoma ( approximately 70% of cases),thyroid (30-70%),ovarian (15-30%),and colorectal cancer (5-20%). Mutant BRAF has constitutive kinase activity and causes hyperactivation of the mitogen-activated protein kinase pathway. BRAF silencing induces regression of melanoma xenografts,indicating the essential role of BRAF for cell survival. We set up an inducible short hairpin RNA system to compare the role of oncogenic BRAF in thyroid carcinoma versus melanoma cells. Although BRAF knockdown led to apoptosis in the melanoma cell line A375,the anaplastic thyroid carcinoma cell ARO underwent growth arrest upon silencing,with little or no cell death. Reexpression of the thyroid differentiation marker,sodium iodide symporter,was induced after long-term silencing. The different outcome of BRAF down-regulation in the two cell lines was associated with an opposite regulation of p21(CIP1/WAF1) expression levels in response to the block of the BRAF mitogenic signal. These results were confirmed using a specific BRAF small-molecule inhibitor,PLX4032. Restoration of p21(CIP1/WAF1) expression rescued melanoma cells from death. Altogether,our data indicate that oncogenic BRAF inhibition can have a different effect on cell fate depending on the cellular type. Furthermore,we suggest that a BRAF-independent mechanism of cell survival exists in anaplastic thyroid cancer cells. View Publication

Intravenous immunoglobulin (IVIg) is currently evaluated in clinical trials for the treatment of various disorders of the central nervous system. To assess its capacity to reach central therapeutic targets,the brain bioavailability of IVIg must be determined. We thus quantified the passage of IVIg through the blood-brain barrier (BBB) of C57Bl/6 mice using complementary quantitative and qualitative methodologies. As determined by enzyme-linked immunosorbent assay,a small proportion of systemically injected IVIg was detected in the brain of mice (0.009±0.001% of injected dose in the cortex) whereas immunostaining revealed localization mainly within microvessels and less frequently in neurons. Pharmacokinetic analyses evidenced a low elimination rate constant (0.0053% per hour) in the cortex,consistent with accumulation within cerebral tissue. In situ cerebral perfusion experiments revealed that a fraction of IVIg crossed the BBB without causing leakage. A dose-dependent decrease of brain uptake was consistent with a saturable blood-to-brain transport mechanism. Finally,brain uptake of IVIg after a subchronic treatment was similar in the 3xTg-AD mouse model of Alzheimer disease compared with nontransgenic controls. In summary,our results provide evidence of BBB passage and bioavailability of IVIg into the brain in the absence of BBB leakage and in sufficient concentration to interact with the therapeutic targets. View Publication

The glial cell line-derived neurotrophic factor (GDNF) has an important role in neuronal survival through binding to the GFRα1 (GDNF family receptor alpha-1) receptor and activation of the receptor tyrosine kinase Ret. Transient brain ischemia alters the expression of the GDNF signaling machinery but whether the GDNF receptor proteins are also affected,and the functional consequences,have not been investigated. We found that excitotoxic stimulation of cultured hippocampal neurons leads to a calpain-dependent downregulation of the long isoform of Ret (Ret51),but no changes were observed for Ret9 or GFRα1 under the same conditions. Cleavage of Ret51 by calpains was selectively mediated by activation of the extrasynaptic pool of N-methyl-d-aspartate receptors and leads to the formation of a stable cleavage product. Calpain-mediated cleavage of Ret51 was also observed in hippocampal neurons subjected to transient oxygen and glucose deprivation (OGD),a model of global brain ischemia,as well as in the ischemic region in the cerebral cortex of mice exposed to transient middle cerebral artery occlusion. Although the reduction of Ret51 protein levels decreased the total GDNF-induced receptor activity (as determined by assessing total phospho-Ret51 protein levels) and their downstream signaling activity,the remaining receptors still showed an increase in phosphorylation after incubation of hippocampal neurons with GDNF. Furthermore,GDNF protected hippocampal neurons when present before,during or after OGD,and the effects under the latter conditions were more significant in neurons transfected with human Ret51. These results indicate that the loss of Ret51 in brain ischemia partially impairs the neuroprotective effects of GDNF. View Publication

Human brain organoids are emerging as translationally relevant models for the study of human brain health and disease. However,it remains to be shown whether human-specific protein processing is conserved in human brain organoids. Herein,we demonstrate that cell fate and composition of unguided brain organoids are dictated by culture conditions during embryoid body formation,and that culture conditions at this stage can be optimized to result in the presence of glia-associated proteins and neural network activity as early as three-months in vitro. Under these optimized conditions,unguided brain organoids generated from induced pluripotent stem cells (iPSCs) derived from male–female siblings are similar in growth rate,size,and total protein content,and exhibit minimal batch-to-batch variability in cell composition and metabolism. A comparison of neuronal,microglial,and macroglial (astrocyte and oligodendrocyte) markers reveals that profiles in these brain organoids are more similar to autopsied human cortical and cerebellar profiles than to those in mouse cortical samples,providing the first demonstration that human-specific protein processing is largely conserved in unguided brain organoids. Thus,our organoid protocol provides four major cell types that appear to process proteins in a manner very similar to the human brain,and they do so in half the time required by other protocols. This unique copy of the human brain and basic characteristics lay the foundation for future studies aiming to investigate human brain-specific protein patterning (e.g.,isoforms,splice variants) as well as modulate glial and neuronal processes in an in situ-like environment. View Publication

Neurobasal®/B27 is a gold standard culture media used to study primary neurons in vitro. An alternative media (BrainPhys®/SM1) was recently developed which robustly enhances neuronal activity vs. Neurobasal® or DMEM. To the best of our knowledge BrainPhys® has not been explored in the setting of neuronal injury. Here we characterized the utility of BrainPhys® in a model of in vitro mechanical-stretch injury. METHODS/RESULTSPrimary rat cortical neurons were maintained in classic Neurobasal®,or sequentially maintained in Neurocult® followed by BrainPhys® (hereafter simply referred to as BrainPhys® maintained neurons?). The levels of axonal markers and proteins involved in neurotransmission were compared on day in vitro 10 (DIV10). BrainPhys® maintained neurons had higher levels of GluN2B,GluR1,Neurofilament light/heavy chain (NF-L & NF-H),and protein phosphatase 2 A (PP2A) vs. neurons in Neurobasal®. Mechanical stretch-injury (50ms/54% biaxial stretch) to BrainPhys® maintained neurons modestly (albeit significantly) increased 24h lactate dehydrogenase (LDH) levels but markedly decreased axonal NF-L levels post-injury vs. uninjured controls or neurons given a milder 38% stretch-injury. Furthermore,two 54% stretch-injuries (in tandem) exacerbated 24h LDH release,increased α-spectrin breakdown products (SBDPs),and decreased Tau levels. Also,BrainPhys® maintained cultures had decreased markers of cell damage 24h after a single 54% stretch-injury vs. neurons in Neurobasal®. Finally,we tested the hypothesis that lentivirus mediated overexpression of the pro-death protein RBM5 exacerbates neuronal and/or axonal injury in primary CNS cultures. RBM5 overexpression vs. empty-vector controls increased 24h LDH release,and SBDP levels,after a single 54% stretch-injury but did not affect NF-L levels or Tau. CONCLUSIONBrainPhys® is a promising new reagent which facilities the investigation of molecular targets involved in axonal and/or neuronal injury in vitro. View Publication

Here,we show for the first time,that the familial breast/ovarian cancer susceptibility gene BRCA1 activates the Notch pathway in breast cells by transcriptional upregulation of Notch ligands and receptors in both normal and cancer cells. We demonstrate through chromatin immunoprecipitation assays that BRCA1 is localized to a conserved intronic enhancer region within the Notch ligand Jagged-1 (JAG1) gene,an event requiring $$Np63. We propose that this BRCA1/$$Np63-mediated induction of JAG1 may be important the regulation of breast stem/precursor cells,as knockdown of all three proteins resulted in increased tumoursphere growth and increased activity of stem cell markers such as Aldehyde Dehydrogenase 1 (ALDH1). Knockdown of Notch1 and JAG1 phenocopied BRCA1 knockdown resulting in the loss of Estrogen Receptor-$$ (ER-$$) expression and other luminal markers. A Notch mimetic peptide could activate an ER-$$ promoter reporter in a BRCA1-dependent manner,whereas Notch inhibition using a $$-secretase inhibitor reversed this process. We demonstrate that inhibition of Notch signalling resulted in decreased sensitivity to the anti-estrogen drug Tamoxifen but increased expression of markers associated with basal-like breast cancer. Together,these findings suggest that BRCA1 transcriptional upregulation of Notch signalling is a key event in the normal differentiation process in breast tissue. View Publication

Although it is well established that women with germ-line mutations in the BRCA1 gene have a greatly increased lifetime incidence of breast and ovarian cancer,the molecular mechanisms responsible for this tissue-specific carcinogenesis remain undefined. The majority of these breast cancers are of the basal-like phenotype characterized by lack of expression of ER,PR,and ERBB2. Because this phenotype has been proposed to resemble that of normal breast stem cells,we examined the role of BRCA1 in human mammary stem cell fate. Using both in vitro systems and a humanized NOD/SCID mouse model,we demonstrate that BRCA1 expression is required for the differentiation of ER-negative stem/progenitor cells to ER-positive luminal cells. Knockdown of BRCA1 in primary breast epithelial cells leads to an increase in cells displaying the stem/progenitor cell marker ALDH1 and a decrease in cells expressing luminal epithelial markers and estrogen receptor. In breast tissues from women with germ-line BRCA1 mutations,but not normal controls,we detect entire lobules that,although histologically normal,are positive for ALDH1 expression but are negative for the expression of ER. Loss of heterozygosity for BRCA1 was documented in these ALDH1-positive lobules but not in adjacent ALDH1-negative lobules. Taken together,these studies demonstrate that BRCA1 plays a critical role in the differentiation of ER-negative stem/progenitor cells to ER-positive luminal cells. Because BRCA1 also plays a role in DNA repair,our work suggests that loss of BRCA1 may result in the accumulation of genetically unstable breast stem cells,providing prime targets for further carcinogenic events. View Publication

Terminal erythropoiesis is a complex multistep process involving coordination of gene transcription and dramatic nuclear condensation,which leads to the expulsion of nuclei to generate reticulocytes. However,we lack a comprehensive understanding of the key transcriptional and epigenetic regulators involved. We used a high-throughput small molecule screen in primary CD34 + -derived human erythroblasts to identify targets that promoted terminal erythropoiesis,and further confirmed the phenotype in different differentiation systems by inhibitors and shRNAs of different BRD4 isoforms. Then we performed RNA-seq,ATAC-seq,ChIP-qPCR,Co-IP,and reanalyzed previously-published transcriptional data and mass spectrometric data to clarify how BRD4 regulates terminal erythropoiesis. We identified that inhibitors of the bromodomain protein BRD4,an epigenetic reader and transcriptional activator together with CDK9,promoted terminal erythropoiesis from hematopoietic stem/progenitor cells and embryonic stem cells,and enhanced enucleation. Combined analysis of our RNA-seq,ATAC-seq,and previously-published transcriptional data of erythroblast differentiation at different stages confirmed that BRD4 inhibition accelerates erythroblast maturation. Unexpectedly,this BRD4 function was independent of its classical CDK9 interaction and transcriptional activation. Instead,RNA-seq,ATAC-seq,and Cut&Tag upon BRD4 inhibition revealed that BRD4 regulates erythropoiesis by inhibiting the small G protein RhoB and disrupts actin reorganization. ChIP-qPCR,Co-IP,and functional studies revealed that BRD4 acts as a transcriptional repressor by interacting with the histone methyltransferase EHMT1/2. We demonstrate a non-classical role for BRD4 as a transcriptional repressor of RhoB to regulate erythroid maturation,and classical CDK9 dependent role to regulate cell proliferation of erythroblasts. Besides,we clarify RhoB’s activity and function during terminal erythropoiesis. BRD4 inhibition might be a simple method to promote in vitro blood cell production,and a candidate therapeutic target for diseases leading to dyserythropoiesis such as myelodysplastic syndromes. The online version contains supplementary material available at 10.1186/s13045-025-01721-2. View Publication

Bromodomain-containing protein 4 (BRD4) is an important epigenetic reader implicated in the pathogenesis of a number of different cancers and other diseases. Brd4-null mouse embryos die shortly after implantation and are compromised in their ability to maintain the inner cell mass,which gives rise to embryonic stem cells (ESCs). Here we report that BRD4 regulates expression of the pluripotency factor Nanog in mouse ESCs and preimplantation embryos,as well as in human ESCs and embryonic cancer stem cells. Inhibition of BRD4 function using a chemical inhibitor,small interfering RNAs,or a dominant-negative approach suppresses Nanog expression,and abolishes the self-renewal ability of ESCs. We also find that BRD4 associates with BRG1 (brahma-related gene 1,aka Smarca4 (SWI/SNF-related,matrix-associated,actin-dependent regulator of chromatin,subfamily a,member 4)),a key regulator of ESC self-renewal and pluripotency,in the Nanog regulatory regions to regulate Nanog expression. Our study identifies Nanog as a novel BRD4 target gene,providing new insights for the biological function of BRD4 in stem cells and mouse embryos. Knowledge gained from these non-cancerous systems will facilitate future investigations of how Brd4 dysfunction leads to cancers.Cell Death and Differentiation advance online publication,22 August 2014; doi:10.1038/cdd.2014.124. View Publication

Tumors may be initiated and maintained by a cellular subcomponent that displays stem cell properties. We have used the expression of aldehyde dehydrogenase as assessed by the ALDEFLUOR assay to isolate and characterize cancer stem cell (CSC) populations in 33 cell lines derived from normal and malignant mammary tissue. Twenty-three of the 33 cell lines contained an ALDEFLUOR-positive population that displayed stem cell properties in vitro and in NOD/SCID xenografts. Gene expression profiling identified a 413-gene CSC profile that included genes known to play a role in stem cell function,as well as genes such as CXCR1/IL-8RA not previously known to play such a role. Recombinant interleukin-8 (IL-8) increased mammosphere formation and the ALDEFLUOR-positive population in breast cancer cell lines. Finally,we show that ALDEFLUOR-positive cells are responsible for mediating metastasis. These studies confirm the hierarchical organization of immortalized cell lines,establish techniques that can facilitate the characterization of regulatory pathways of CSCs,and identify potential stem cell markers and therapeutic targets. View Publication