参考文献

Chimeric antigen receptor (CAR) T cells show suboptimal efficacy in acute myeloid leukemia (AML). We find that CAR T cells exposed to myeloid leukemia show impaired activation and cytolytic function,accompanied by impaired antigen receptor downstream calcium,ZAP70,ERK,and C-JUN signaling,compared to those exposed to B-cell leukemia. These defects are caused in part by the high expression of CD155 by AML. Overexpressing C-JUN,but not other antigen receptor downstream components,maximally restores anti-tumor function. C-JUN overexpression increases costimulatory molecules and cytokines through reinvigoration of ERK or transcriptional activation,independent of anti-exhaustion. We conduct an open-label,non-randomized,single-arm,phase I trial of C-JUN-overexpressing CAR-T in AML ( NCT04835519 ) with safety and efficacy as primary and secondary endpoints,respectively. Of the four patients treated,one has grade 4 (dose-limiting toxicity) and three have grade 1–2 cytokine release syndrome. Two patients have no detectable bone marrow blasts and one patient has blast reduction after treatment. Thus,overexpressing C-JUN endows CAR-T efficacy in AML. Subject terms: Translational research,Leukaemia View Publication

In recent years,the differentiation of bone marrow cells (BMCs) into myocytes has been extensively investigated,but the findings remain inconclusive. The purpose of this study was to determine the conditions necessary to induce myogenic differentiation in short-term cultures of adult BMCs,and to identify the BMC subpopulation responsible for this phenomenon. We report that high-density cultures of murine hematopoietic BMCs gave rise to spontaneous beating cell clusters in the presence of vascular endothelial and fibroblast growth factors. These clusters originated from c-kit(pos) cells. The formation of the clusters could be completely blocked by adding a c-kit/tyrosine kinase inhibitor,Gleevec (imatinib mesylate; Novartis International,Basel,Switzerland,http://www.novartis.com),to the culture. Cluster formation was also blunted in BMCs from c-kit-deficient (Kit(W)/Kit(W-v)) mice. Clustered cells expressed cardiomyocyte-specific transcription factor genes Gata-4 and Nkx2.5,sarcomeric proteins beta-MHC and MLC-2v,and ANF and connexin-43. Immunostaining revealed alpha-sarcomeric actinin expression in more than 90% of clustered cells. Under electron microscopy,the clustered cells exhibited a sarcomeric myofiber arrangement and z-bands. This study defines the microenvironment required to achieve a reproducible in vitro model of beating,myogenic cell clusters. This model could be used to examine the mechanisms responsible for the postnatal myogenic differentiation of BMCs. Our results identify c-kit(pos) bone marrow hematopoietic cells as the source of the myogenic clusters. View Publication

The c-myb transcription factor is highly expressed in immature hematopoietic cells and down-regulated during differentiation. To define its role during the hematopoietic lineage commitment,we silenced c-myb in human CD34(+) hematopoietic stem/progenitor cells. Noteworthy,c-myb silencing increased the commitment capacity toward the macrophage and megakaryocyte lineages,whereas erythroid differentiation was impaired,as demonstrated by clonogenic assay,morphologic and immunophenotypic data. Gene expression profiling and computational analysis of promoter regions of genes modulated in c-myb-silenced CD34(+) cells identified the transcription factors Kruppel-Like Factor 1 (KLF1) and LIM Domain Only 2 (LMO2) as putative targets,which can account for c-myb knockdown effects. Indeed,chromatin immunoprecipitation and luciferase reporter assay demonstrated that c-myb binds to KLF1 and LMO2 promoters and transactivates their expression. Consistently,the retroviral vector-mediated overexpression of either KLF1 or LMO2 partially rescued the defect in erythropoiesis caused by c-myb silencing,whereas only KLF1 was also able to repress the megakaryocyte differentiation enhanced in Myb-silenced CD34(+) cells. Our data collectively demonstrate that c-myb plays a pivotal role in human primary hematopoietic stem/progenitor cells lineage commitment,by enhancing erythropoiesis at the expense of megakaryocyte diffentiation. Indeed,we identified KLF1 and LMO2 transactivation as the molecular mechanism underlying Myb-driven erythroid versus megakaryocyte cell fate decision. View Publication

RNA-binding motif protein 15 (RBM15) is involved in the RBM15-megakaryoblastic leukemia 1 fusion in acute megakaryoblastic leukemia. Although Rbm15 has been reported to be required for B-cell differentiation and to inhibit myeloid and megakaryocytic expansion,it is not clear what the normal functions of Rbm15 are in the regulation of hematopoietic stem cell (HSC) and megakaryocyte development. In this study,we report that Rbm15 may function in part through regulation of expression of the proto-oncogene c-Myc. Similar to c-Myc knockout (c-Myc-KO) mice,long-term (LT) HSCs are significantly increased in Rbm15-KO mice due to an apparent LT-HSC to short-term HSC differentiation defect associated with abnormal HSC-niche interactions caused by increased N-cadherin and beta(1) integrin expression on mutant HSCs. Both serial transplantation and competitive reconstitution capabilities of Rbm15-KO LT-HSCs are greatly compromised. Rbm15-KO and c-Myc-KO mice also share related abnormalities in megakaryocyte development,with mutant progenitors producing increased,abnormally small low-ploidy megakaryocytes. Consistent with a possible functional interplay between Rbm15 and c-Myc,the megakaryocyte increase in Rbm15-KO mice could be partially reversed by ectopic c-Myc. Thus,Rbm15 appears to be required for normal HSC-niche interactions,for the ability of HSCs to contribute normally to adult hematopoiesis,and for normal megakaryocyte development; these effects of Rbm15 on hematopoiesis may be mediated at least in part by c-Myc. View Publication

IntroductionT cells are major components of the immune system. Their activation requires interaction between the T cell receptor and co-stimulatory molecules,crucial during infection,inflammation,and allogeneic rejection. Monomeric CRP (mCRP) is a known modulator of inflammation and particularly the innate immune response,however its interaction with T cells as part of the adaptive immune response remains unclear.MethodsPeripheral blood mononuclear cells (PBMC) and T cells were isolated. Flow cytometric analysis was conducted to evaluate Fcγ receptor CD16 expression on T cells,the binding of CRP to T cells,and its impact on proliferation and apoptosis. T cell activation was assessed after 1,2,3,5 and 7 days by assessing CD69 and CD25 expression,and under various conditions including coculture with monocytes and several inhibitory factors.ResultsT cells express CD16 that binds mCRP in a concentration-dependent manner,and particularly on activated T cells. While mCRP reduces apoptosis and accelerates proliferation in T cells,it does not independently activate them. However,activation of monocytes by mCRP leads to T cell activation,indicating a direct cell to cell interaction during CRP-induced activation. This effect could be alleviated by inhibition of the CD80/CD28 pathway.ConclusionCRP does not activate T Cells directly but via PI3-kinase-dependent activation of monocytes and subsequent CD80/CD28 cell to cell contact. The findings suggest the effects of CRP on T cells depend on their environment and the presence of other proinflammatory agents. 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

Many solid tumors have low levels of cytotoxic CD56dim natural killer (NK) cells,suggesting that CD56dim NK-cell exclusion from the tumor microenvironment (TME) contributes to the decreased response rate of immunotherapy. Complement component 3a (C3a) is known for its tumor-promoting and immunosuppressive roles in solid tumors. Previous reports have implicated the involvement of the C3a receptor (C3aR) in immune cell trafficking into the TME. C3aR is predominantly expressed on the surface of activated cytotoxic NK cells,but a specific role for C3aR in NK-cell biology has not been investigated. Because solid tumors generate elevated C3a and have decreased NK-cell infiltration,we hypothesized that C3aR might play a role in cytotoxic NK-cell recruitment into the TME. Our results indicate that blocking C3aR signaling in NK cells increased NK-cell infiltration into the TME in mouse models and led to tumor regression. Because the critical lymphocyte trafficking integrin LFA-1 orchestrates the migration of activated NK cells,we wanted to gain insight into the interaction between C3aR signaling and LFA-1. Our results demonstrated that direct interaction between C3aR and LFA-1,which led to a high-affinity LFA-1 conformation,decreased NK-cell infiltration into the TME. We propose that approaches to enhance cytotoxic NK-cell infiltration into the TME,through either disrupting C3a and C3aR interaction or inhibiting the formation of high-affinity LFA-1,represent a new strategy to improve the efficiency of immunotherapy for cancer treatment. View Publication

Although Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) have been approved in multiple diseases,including BRCA1/2 mutant breast cancer,responses are usually transient requiring the deployment of combination therapies for optimal efficacy. Here we thus explore mechanisms underlying sensitivity and resistance to PARPi using two intrinsically PARPi sensitive (T22) and resistant (T127) syngeneic murine breast cancer models in female mice. We demonstrate that tumor associated macrophages (TAM) potentially contribute to the differential sensitivity to PARPi. By single-cell RNA-sequencing,we identify a TAM_C3 cluster,expressing genes implicated in anti-inflammatory activity,that is enriched in PARPi resistant T127 tumors and markedly decreased by PARPi in T22 tumors. Rps19/C5aR1 signaling is selectively elevated in TAM_C3. C5aR1 inhibition or transferring C5aR1hi cells increases and decreases PARPi sensitivity,respectively. High C5aR1 levels in human breast cancers are associated with poor responses to immune checkpoint blockade. Thus,targeting C5aR1 may selectively deplete pro-tumoral macrophages and engender sensitivity to PARPi and potentially other therapies. PARP inhibitors (PARPi) have been approved for the treatment of metastatic triple-negative breast cancer (BC),however resistance and recurrence are often observed. Here,in preclinical models of BRCA1/2 wild type and homologous recombination competent BC,the authors show that C5aR1-positive tumor associated macrophages are associated with PARPi-resistance,suggesting targeting C5aR1 as a therapeutic option. View Publication

An expanded hexanucleotide repeat in a noncoding region of the C9orf72 gene is a major cause of amyotrophic lateral sclerosis (ALS),accounting for up to 40% of familial cases and 7% of sporadic ALS in European populations. We have generated induced pluripotent stem cells (iPSCs) from fibroblasts of patients carrying C9orf72 hexanucleotide expansions,differentiated these to functional motor and cortical neurons and performed an extensive phenotypic characterization. In C9orf72 iPSC-derived motor neurons,decreased cell survival is correlated with dysfunction in Ca(2+) homeostasis,reduced levels of the anti-apoptotic protein Bcl-2,increased endoplasmic reticulum (ER) stress and reduced mitochondrial membrane potential. Furthermore,C9orf72 motor neurons,and also cortical neurons,show evidence of abnormal protein aggregation and stress granule formation. This study is an extensive characterization of iPSC-derived motor neurons as cellular models of ALS carrying C9orf72 hexanucleotide repeats,which describes a novel pathogenic link between C9orf72 mutations,dysregulation of calcium signalling and altered proteostasis and provides a potential pharmacological target for the treatment of ALS and the related neurodegenerative disease frontotemporal dementia (FTD). This article is protected by copyright. All rights reserved. View Publication

The blood-brain barrier (BBB) serves as a highly intricate and dynamic interface connecting the brain and the bloodstream,playing a vital role in maintaining brain homeostasis. BBB dysfunction has been associated with multiple neurodegenerative diseases,including amyotrophic lateral sclerosis (ALS); however,the role of the BBB in neurodegeneration is understudied. We developed an ALS patient-derived model of the BBB by using cells derived from 5 patient donors carrying C9ORF72 mutations. Brain microvascular endothelial-like cells (BMEC-like cells) derived from C9ORF72-ALS patients showed altered gene expression,compromised barrier integrity,and increased P-glycoprotein transporter activity. In addition,mitochondrial metabolic tests demonstrated that C9ORF72-ALS BMECs display a significant decrease in basal glycolysis accompanied by increased basal and ATP-linked respiration. Moreover,our study reveals that C9-ALS derived astrocytes can further affect BMECs function and affect the expression of the glucose transporter Glut-1. Finally,C9ORF72 patient-derived BMECs form leaky barriers through a cell-autonomous mechanism and have neurotoxic properties towards motor neurons.Graphical Abstract Supplementary InformationThe online version contains supplementary material available at 10.1186/s12987-024-00528-6. View Publication