技术资料

Cancer cell plasticity contributes significantly to the failure of chemo- and targeted therapies in triple-negative breast cancer (TNBC). Molecular mechanisms of therapy-induced tumor cell plasticity and associated resistance are largely unknown. Using a genome-wide CRISPR-Cas9 screen,we investigated escape mechanisms of NOTCH-driven TNBC treated with a gamma-secretase inhibitor (GSI) and identified SOX2 as a target of resistance to Notch inhibition. We describe a novel reciprocal inhibitory feedback mechanism between Notch signaling and SOX2. Specifically,Notch signaling inhibits SOX2 expression through its target genes of the HEY family,and SOX2 inhibits Notch signaling through direct interaction with RBPJ. This mechanism shapes divergent cell states with NOTCH positive TNBC being more epithelial-like,while SOX2 expression correlates with epithelial-mesenchymal transition,induces cancer stem cell features and GSI resistance. To counteract monotherapy-induced tumor relapse,we assessed GSI-paclitaxel and dasatinib-paclitaxel combination treatments in NOTCH inhibitor-sensitive and -resistant TNBC xenotransplants,respectively. These distinct preventive combinations and second-line treatment option dependent on NOTCH1 and SOX2 expression in TNBC are able to induce tumor growth control and reduce metastatic burden. View Publication

Hematopoietic stem cell transplantation (HSCT) is a curative treatment for many diverse blood and immune diseases. However,HSCT regimens currently commonly utilize genotoxic chemotherapy and/or total body irradiation (TBI) conditioning which causes significant morbidity and mortality through inducing broad tissue damage triggering infections,graft vs. host disease,infertility,and secondary cancers. We previously demonstrated that targeted monoclonal antibody (mAb)-based HSC depletion with anti(α)-CD117 mAbs could be an effective alternative conditioning approach for HSCT without toxicity in severe combined immunodeficiency (SCID) mouse models,which has prompted parallel clinical αCD117 mAbs to be developed and tested as conditioning agents in clinical trials starting with treatment of patients with SCID. Subsequent efforts have built upon this work to develop various combination approaches,though none are optimal and how any of these mAbs fully function is unknown. To improve efficacy of mAb-based conditioning as a stand-alone conditioning approach for all HSCT settings,it is critical to understand the mechanistic action of αCD117 mAbs on HSCs. Here,we compare the antagonistic properties of αCD117 mAb clones including ACK2,2B8,and 3C11 as well as ACK2 fragments in vitro and in vivo in both SCID and wildtype (WT) mouse models. Further,to augment efficacy,combination regimens were also explored. We confirm that only ACK2 inhibits SCF binding fully and prevents HSC proliferation in vitro. Further,we verify that this corresponds to HSC depletion in vivo and donor engraftment post HSCT in SCID mice. We also show that SCF-blocking αCD117 mAb fragment derivatives retain similar HSC depletion capacity with enhanced engraftment post HSCT in SCID settings,but only full αCD117 mAb ACK2 in combination with αCD47 mAb enables enhanced donor HSC engraftment in WT settings,highlighting that the Fc region is not required for single-agent efficacy in SCID settings but is required in immunocompetent settings. This combination was the only non-genotoxic conditioning approach that enabled robust donor engraftment post HSCT in WT mice. These findings shed new insights into the mechanism of αCD117 mAb-mediated HSC depletion. Further,they highlight multiple approaches for efficacy in SCID settings and optimal combinations for WT settings. This work is likely to aid in the development of clinical non-genotoxic HSCT conditioning approaches that could benefit millions of people world-wide. The online version contains supplementary material available at 10.1186/s13287-024-03981-0. View Publication

To enable transmission of information in the brain,synaptic vesicles fuse to presynaptic membranes,liberating their content and exposing transiently a myriad of vesicular transmembrane proteins. However,versatile methods for quantifying the synaptic translocation of endogenous proteins during neuronal activity remain unavailable,as the fast dynamics of synaptic vesicle cycling difficult specific isolation of trafficking proteins during such a transient surface exposure. Here,we developed a novel approach using synaptic cleft proximity labeling to capture and quantify activity-driven trafficking of endogenous synaptic proteins at the synapse. We show that accelerating cleft biotinylation times to match the fast dynamics of vesicle exocytosis allows capturing endogenous proteins transiently exposed at the synaptic surface during neural activity,enabling for the first time the study of the translocation of nearly every endogenous synaptic protein. As proof-of-concept,we further applied this technology to obtain direct evidence of the surface translocation of noncanonical trafficking proteins,such as ATG9A and NPTX1,which had been proposed to traffic during activity but for which direct proof had not yet been shown. The technological advancement presented here will facilitate future studies dissecting the molecular identity of proteins exocytosed at the synapse during activity,helping to define the molecular machinery that sustains neurotransmission in the mammalian brain. View Publication

Patients with chronic myeloid leukemia (CML) respond to tyrosine kinase inhibitors (TKIs); however,CML leukemic stem cells (LSCs) exhibit BCR::ABL kinase-independent growth and are insensitive to TKIs,leading to disease relapse. To prevent this,new therapies targeting CML-LSCs are needed. Rates of mitochondria-mediated oxidative phosphorylation (OXPHOS) in CD34 + CML cells within the primitive CML cell population are higher than those in normal undifferentiated hematopoietic cells; therefore,the inhibition of OXPHOS in CML-LSCs may be a potential cure for CML. NK-128 (C 33 H 61 NO 5 S) is a structurally simplified analog of JCI-20679,the design of which was based on annonaceous acetogenins. NK-128 exhibits antitumor activity against glioblastoma and human colon cancer cells by inhibiting OXPHOS and activating AMP-activated protein kinase (AMPK). Here,we demonstrate that NK-128 effectively suppresses the growth of CML cell lines and that the combination of imatinib and NK-128 is more potent than either alone in a CML xenograft mouse model. We also found that NK-128 inhibits colony formation by CD34 + CML cells isolated from the bone marrow of untreated CML patients. Taken together,these findings suggest that targeting OXPHOS is a beneficial approach to eliminating CML-LSCs,and may improve the treatment of CML. View Publication

Acute myeloid leukemia (AML) is a deadly hematopoietic malignancy. Although many patients achieve complete remission with standard induction therapy,a combination of cytarabine and anthracycline,~40% of patients have induction failure. These refractory patients pose a treatment challenge,as they do not respond to salvage therapy or allogeneic stem cell transplant. Herein,we show that AML patients who experience induction failure have elevated expression of the NF-κB target gene tumor necrosis factor alpha-induced protein-3 (TNFAIP3/A20) and impaired necroptotic cell death. A20 High AML are resistant to anthracyclines,while A20 Low AML are sensitive. Loss of A20 in AML restores sensitivity to anthracycline treatment by inducing necroptosis. Moreover,A20 prevents necroptosis in AML by targeting the necroptosis effector RIPK1,and anthracycline-induced necroptosis is abrogated in A20 High AML. These findings suggest that NF-κB-driven A20 overexpression plays a role in failed chemotherapy induction and highlights the potential of targeting an alternative cell death pathway in AML. Subject terms: Acute myeloid leukaemia,Cancer therapeutic resistance View Publication

The ability to generate natural killer (NK) cells from induced pluripotent stem cells (iPSCs) has given rise to new possibilities for the large-scale production of homogeneous immunotherapeutic cellular products and opened new avenues towards the creation of “off-the-shelf” cancer immunotherapies. However,the differentiation of NK cells from iPSCs remains poorly understood,particularly regarding the ontogenic landscape of iPSC-derived NK (iNK) cells produced in vitro and the influence that the differentiation strategy employed may have on the iNK profile. To investigate this question,we conducted a comparative analysis of two sets of iNK cells generated from the same iPSC line using two different protocols: (i) a short-term,clinically compatible feeder-free protocol corresponding to primitive hematopoiesis,and (ii) a lymphoid-based protocol representing the definitive hematopoietic step. Our work demonstrated that both protocols are capable of producing functional iNK cells. However,the two sets of resulting iNKs exhibited distinct phenotypes and transcriptomic profiles. The lymphoid-based differentiation approach generated iNKs with a more mature and activated profile,which demonstrated higher cytotoxicity against cancer cell lines compared to iNK cells produced under short-term feeder-free conditions suggesting that the differentiation strategy must be considered when designing iNK cell–based adoptive immunotherapies. View Publication

Autism Spectrum Disorder (ASD) is a developmental disorder characterized by impaired social communication and repetitive behaviors. In recent years,a pharmacological mouse model of ASD involving maternal administration of valproic acid (VPA) has become widely used. Newborn pups in this model show an abnormal balance between excitatory and inhibitory (E/I) signaling in neurons and exhibit ASD-like behavior. However,the molecular basis of this model and its implications for the pathogenesis of ASD in humans remain unknown. Using quantitative secretome analysis,we found that the level of leucine-rich repeat and immunoglobulin domain-containing protein 2 (Lingo2) was upregulated in the conditioned medium of VPA model neurons. This upregulation was associated with excitatory synaptic organizer activity. The secreted form of the extracellular domain of Lingo2 (sLingo2) is produced by the transmembrane metalloprotease ADAM10 through proteolytic processing. sLingo2 was found to induce the formation of excitatory synapses in both mouse and human neurons,and treatment with sLingo2 resulted in an increased frequency of miniature excitatory postsynaptic currents in human neurons. These findings suggest that sLingo2 is an excitatory synapse organizer involved in ASD,and further understanding of the mechanisms by which sLingo2 induces excitatory synaptogenesis is expected to advance our understanding of the pathogenesis of ASD. Subject terms: Autism spectrum disorders,Neuroscience View Publication

Mast cells (MCs) are tissue-resident immune cells known to degranulate in response to FcεRI crosslinking or MRGPRX2 engagement. MCs are found close to nerves,but the mechanisms that regulate this privileged localization remain unclear. Here,we investigated MRGPRX2 expression patterns and specific activities in MCs. We show that MRGPRX2 expression is heterogeneous in human MC (hMC) progenitors and mature MCs. Substance P (SP) is a rapid and specific activator of MRGPRX2,and long-term supplementation of MCs with SP expands MRGPRX2-expressing cells. While high concentrations of SP induce rapid MC degranulation,low concentrations prompt immature MC chemotaxis. Lastly,we demonstrate that in inflammatory skin conditions like psoriasis,the number of MRGPRX2 + MCs is increased,and during in vitro skin reinnervation,MRGPRX2 + MCs preferentially reside in proximity to and migrate toward SP + nerve fibers (NFs). This indicates that SP-MRGPRX2 signaling defines MC positioning and relocation within tissues and promotes immune cell-NF communication. Subject areas: Immunology,Molecular biology,Cell biology View Publication

Long-term culture of primary lymphocytes and hematopoietic stem and progenitor cells (HSPCs) is pivotal to their expansion and study. Furthermore,genetic engineering of the above-mentioned primary human cells has several safety needs,including the requirement of efficient in vitro assays for unwanted tumorigenic events. In this work,we tested and optimized the Miniaturized Optically Accessible Bioreactor (MOAB) platform. The MOAB consists of a millifluidic cell culture device with three optically-accessible culture chambers. Inside the MOAB,we inserted a silk-based framework that resembles some properties of the bone marrow environment and cultivated in this device either CD4 + T lymphocytes isolated from healthy donor buffy coat or cord blood-derived hematopoietic CD34 + cells. A fraction of these cells is viable for up to 3 months. Next,we tested the capability of the MOAB to detect tumorigenic events. Serial dilutions of engineered fluorescent tumor cells were mixed with either CD4 + or CD34 + primary cells,and their growth was followed. By this approach,we successfully detected as little as 100 tumorigenic cells mixed with 100,000 primary cells. We found that non-tumorigenic primary cells colonized the silk environment,whereas tumor cells,after an adaptation phase,expanded and entered the circulation. We conclude that the millifluidic platform allows the detection of rare tumorigenic events in the long-term culture of human cells. View Publication

Macrolides are widely used antibiotics for the treatment of bacterial airway infections. Due to its elevated minimum inhibitory concentration in standardized culture media,Pseudomonas aeruginosa is considered intrinsically resistant and,therefore,antibiotic susceptibility testing against macrolides is not performed. Nevertheless,due to macrolides’ immunomodulatory effect and suppression of virulence factors,they are used for the treatment of persistent P. aeruginosa infections. Here,we demonstrate that macrolides are,instead,effective antibiotics against P. aeruginosa airway infections in an Air-Liquid Interface (ALI) infection model system resembling the human airways. Importantly,macrolide treatment in both people with cystic fibrosis and primary ciliary dyskinesia patients leads to the accumulation of uL4 and uL22 ribosomal protein mutations in P. aeruginosa which causes antibiotic resistance. Consequently,higher concentrations of antibiotics are needed to modulate the macrolide-dependent suppression of virulence. Surprisingly,even in the absence of antibiotics,these mutations also lead to a collateral reduction in growth rate,virulence and pathogenicity in airway ALI infections which are pivotal for the establishment of a persistent infection. Altogether,these results lend further support to the consideration of macrolides as de facto antibiotics against P. aeruginosa and the need for resistance monitoring upon prolonged macrolide treatment. Subject terms: Antimicrobials,Clinical microbiology,Pathogens,Antimicrobial resistance View Publication

Venetoclax plus azacitidine treatment is clinically beneficial for elderly and unfit acute myeloid leukemia (AML) patients. However,the treatment is rarely curative,and relapse due to resistant disease eventually emerges. Since no current clinically feasible treatments are known to be effective at the state of acquired venetoclax resistance,this is becoming a major challenge in AML treatment. Studying venetoclax-resistant AML cell lines,we observed that venetoclax induced sublethal apoptotic signaling and DNA damage even though cell survival and growth were unaffected. This effect could be due to venetoclax inducing a sublethal degree of mitochondrial outer membrane permeabilization. Based on these results,we hypothesized that the sublethal apoptotic signaling induced by venetoclax could constitute a vulnerability in venetoclax-resistant AML cells. This was supported by screens with a broad collection of drugs,where we observed a synergistic effect between venetoclax and PARP inhibition in venetoclax-resistant cells. Additionally,the venetoclax-PARP inhibitor combination prevented the acquisition of venetoclax resistance in treatment naïve AML cell lines. Furthermore,the addition of azacitidine to the venetoclax-PARP inhibitor combination enhanced venetoclax induced DNA damage and exhibited exceptional sensitivity and long-term responses in the venetoclax-resistant AML cell lines and samples from AML patients that had clinically relapsed under venetoclax-azacitidine therapy. In conclusion,we mechanistically identify a new vulnerability in acquired venetoclax-resistant AML cells and identify PARP inhibition as a potential therapeutic approach to overcome acquired venetoclax resistance in AML. Subject terms: Acute myeloid leukaemia,Acute myeloid leukaemia View Publication

Despite a record number of clinical studies investigating various anti-myeloma treatments,the 5-year survival rate for multiple myeloma (MM) patients in the US is only 55%,and almost all patients relapse. Poor patient outcomes demonstrate that myeloma cells are “born to survive” which means they can adapt and evolve following treatment. Thus,new therapeutic approaches to combat survival mechanisms and target treatment resistance are required. Importantly,Mcl-1,anti-apoptotic protein,is required for the development of MM and treatment resistance. This study looks at the possibility of KS18,a selective Mcl-1 inhibitor,to treat MM and overcome resistance. Our investigation demonstrates that KS18 effectively induces cell death in MM by dual regulatory mechanisms targeting the Mcl-1 protein at both transcriptional and post-translational levels. Specifically,KS18 suppresses Mcl-1 activation via STAT-3 pathway and promotes Mcl-1 phosphorylation/ubiquitination/proteasome-dependent protein degradation (UPS). Significantly,KS18 triggered caspase-dependent apoptosis in MM patient samples and bortezomib-resistant cells,synergizing with venetoclax to boost apoptosis. KS18 promises to overcome bortezomib and venetoclax resistance and re-sensitize myeloma cells to chemotherapy. Furthermore,the study shows the tremendous impact of KS18 in inhibiting colony formation in bortezomib-resistant cells and demonstrates significant tumor shrinkage in KS18-treated NSG mice without notable toxicity signs after 4 weeks of therapy with a single acceptable dose each week,indicating its powerful anti-neoplastic and anti-resistance characteristics. This study strongly implies that KS18 may treat MM and provide new hope to patients who are experiencing recurrence or resistance. View Publication