技术资料

BCR::ABL1-independent pathways contribute to primary resistance to tyrosine kinase inhibitor (TKI) treatment in chronic myeloid leukemia (CML) and play a role in leukemic stem cell persistence. Here,we perform ex vivo drug screening of CML CD34 + leukemic stem/progenitor cells using 100 single drugs and TKI-drug combinations and identify sensitivities to Wee1,MDM2,and BCL2 inhibitors. These agents effectively inhibit primitive CD34 + CD38 − CML cells and demonstrate potent synergies when combined with TKIs. Flow-cytometry-based drug screening identifies mepacrine to induce differentiation of CD34 + CD38 − cells. We employ genome-wide CRISPR-Cas9 screening for six drugs,and mediator complex,apoptosis,and erythroid-lineage-related genes are identified as key resistance hits for TKIs,whereas the Wee1 inhibitor AZD1775 and mepacrine exhibit distinct resistance profiles. KCTD5,a consistent TKI-resistance-conferring gene,is found to mediate TKI-induced BCR::ABL1 ubiquitination. In summary,we delineate potential mechanisms for primary TKI resistance and non-BCR::ABL1-targeting drugs,offering insights for optimizing CML treatment. View Publication

AML is characterized by mutations in genes associated with growth regulation such as internal tandem duplications (ITD) in the receptor kinase FLT3. Inhibitors targeting FLT3 (FLT3i) are being used to treat patients with FLT3-ITD+ but most relapse and become resistant. To elucidate the resistance mechanism,we compared the gene regulatory networks (GRNs) of leukemic cells from patients before and after relapse,which revealed that the GRNs of drug-responsive patients were altered by rewiring their AP-1-RUNX1 axis. Moreover,FLT3i induces the upregulation of signaling genes,and we show that multiple cytokines,including interleukin-3 (IL-3),can overcome FLT3 inhibition and send cells back into cycle. FLT3i leads to loss of AP-1 and RUNX1 chromatin binding,which is counteracted by IL-3. However,cytokine-mediated drug resistance can be overcome by a pan-RAS inhibitor. We show that cytokines instruct AML growth via the transcriptional regulators AP-1 and RUNX1 and that pan-RAS drugs bypass this barrier. Subject area: Pharmacy,Molecular biology View Publication

Immune tolerance fails in autoimmune polyendocrine syndrome type 1 (APS-1) because of AIRE mutations. We have used single cell transcriptomics to characterize regulatory T cells (Tregs) sorted directly from blood and from in vitro expanded Tregs in APS-1 patients compared to healthy controls. We revealed only CD52 and LTB (down) and TXNIP (up) as consistently differentially expressed genes in the datasets. There were furthermore no large differences of the TCR-repertoire of expanded Tregs between the cohorts,but unique patients showed a more restricted use of specific clonotypes. We also found that in vitro expanded Tregs from APS-1 patients had similar suppressive capacity as controls in co-culture assays,despite expanding faster and having more exhausted cells. Our results suggest that APS-1 patients do not have intrinsic defects in their Treg functionality,and that their Tregs can be expanded ex vivo for potential therapeutic applications. Subject areas: Health sciences,Immunology,Components of the immune system,Proteomics,Transcriptomics View Publication

MicroRNAs (miRNAs) are short non-coding RNAs that play crucial roles in gene regulation,exerting post-transcriptional silencing,thereby influencing cellular function,development,and disease. Traditional loss-of-function methods for studying miRNA functions,such as miRNA inhibitors and sponges,present limitations in terms of specificity,transient effects,and off-target effects. Similarly,CRISPR/Cas9-based editing of miRNAs using single guide RNAs (sgRNAs) also has limitations in terms of design space for generating effective gRNAs. In this study,we introduce a novel approach that utilizes CRISPR/Cas9 with dual guide RNAs (dgRNAs) for the rapid and efficient generation of short deletions within miRNA genomic regions. Through the expression of dgRNAs through single-copy lentiviral integration,this approach achieves over a 90% downregulation of targeted miRNAs within a week. We conducted a comprehensive analysis of various parameters influencing efficient deletion formation. In addition,we employed doxycycline (Dox)-inducible expression of Cas9 from the AAVS1 locus,enabling homogeneous,temporal,and stage-specific editing during cellular differentiation. Compared to miRNA inhibitory methods,the dgRNA-based approach offers higher specificity,allowing for the deletion of individual miRNAs with similar seed sequences,without affecting other miRNAs. Due to the increased design space,the dgRNA-based approach provides greater flexibility in gRNA design compared to the sgRNA-based approach. We successfully applied this approach in two human cell lines,demonstrating its applicability for studying the mechanisms of human erythropoiesis and pluripotent stem cell (iPSC) biology and differentiation. Efficient deletion of miR-451 and miR-144 resulted in blockage of erythroid differentiation,and the deletion of miR-23a and miR-27a significantly affected iPSC survival. We have validated the highly efficient deletion of genomic regions by editing protein-coding genes,resulting in a significant impact on protein expression. This protocol has the potential to be extended to delete multiple miRNAs within miRNA clusters,allowing for future investigations into the cooperative effects of the cluster members on cellular functions. The protocol utilizing dgRNAs for miRNA deletion can be employed to generate efficient pooled libraries for high-throughput comprehensive analysis of miRNAs involved in different biological processes. View Publication

Non-small cell lung cancer (NSCLC) is known for high relapse rates despite resection in early stages. Here,we present the results of a phase I clinical trial in which a dendritic cell (DC) vaccine targeting patient-individual neoantigens is evaluated in patients with resected NSCLC. Vaccine manufacturing is feasible in six of 10 enrolled patients. Toxicity is limited to grade 1–2 adverse events. Systemic T cell responses are observed in five out of six vaccinated patients,with T cell responses remaining detectable up to 19 months post vaccination. Single-cell analysis indicates that the responsive T cell population is polyclonal and exhibits the near-entire spectrum of T cell differentiation states,including a naive-like state,but excluding exhausted cell states. Three of six vaccinated patients experience disease recurrence during the follow-up period of 2 years. Collectively,these data support the feasibility,safety,and immunogenicity of this treatment in resected NSCLC. View Publication

Prenatal lethality associated with mouse knockout of Mettl16,a recently identified RNA N6-methyladenosine (m 6 A) methyltransferase,has hampered characterization of the essential role of METTL16-mediated RNA m 6 A modification in early embryonic development. Here,using cross-species single-cell RNA sequencing analysis,we found that during early embryonic development,METTL16 is more highly expressed in vertebrate hematopoietic stem and progenitor cells (HSPCs) than other methyltransferases. In Mettl16-deficient zebrafish,proliferation capacity of embryonic HSPCs is compromised due to G1/S cell cycle arrest,an effect whose rescue requires Mettl16 with intact methyltransferase activity. We further identify the cell-cycle transcription factor mybl2b as a directly regulated by Mettl16-mediated m 6 A modification. Mettl16 deficiency resulted in the destabilization of mybl2b mRNA,likely due to lost binding by the m 6 A reader Igf2bp1 in vivo. Moreover,we found that the METTL16-m 6 A- MYBL2 -IGF2BP1 axis controlling G1/S progression is conserved in humans. Collectively,our findings elucidate the critical function of METTL16-mediated m 6 A modification in HSPC cell cycle progression during early embryonic development. View Publication

Chemotherapy remains the mainstay treatment for triple-negative breast cancer (TNBC) due to the lack of specific targets. Given a modest response of immune checkpoint inhibitors in TNBC patients,improving immunotherapy is an urgent and crucial task in this field. CD73 has emerged as a novel immunotherapeutic target,given its elevated expression on tumor,stromal,and specific immune cells,and its established role in inhibiting anti-cancer immunity. CD73-generated adenosine suppresses immunity by attenuating tumor-infiltrating T- and NK-cell activation,while amplifying regulatory T cell activation. Chemotherapy often leads to increased CD73 expression and activity,further suppressing anti-tumor immunity. While debulking the tumor mass,chemotherapy also enriches heterogenous cancer stem cells (CSC),potentially leading to tumor relapse. Therefore,drugs targeting both CD73,and CSCs hold promise for enhancing chemotherapy efficacy,overcoming treatment resistance,and improving clinical outcomes. However,safe and effective inhibitors of CD73 have not been developed as of now. We used in silico docking to screen compounds that may be repurposed for inhibiting CD73. The efficacy of these compounds was investigated through flow cytometry,RT-qPCR,CD73 activity,cell viability,tumorsphere formation,and other in vitro functional assays. For assessment of clinical translatability,TNBC patient-derived xenograft organotypic cultures were utilized. We also employed the ovalbumin-expressing AT3 TNBC mouse model to evaluate tumor-specific lymphocyte responses. We identified quercetin and luteolin,currently used as over-the-counter supplements,to have high in silico complementarity with CD73. When quercetin and luteolin were combined with the chemotherapeutic paclitaxel in a triple-drug regimen,we found an effective downregulation in paclitaxel-enhanced CD73 and CSC-promoting pathways YAP and Wnt. We found that CD73 expression was required for the maintenance of CD44 high CD24 low CSCs,and co-targeting CD73,YAP,and Wnt effectively suppressed the growth of human TNBC cell lines and patient-derived xenograft organotypic cultures. Furthermore,triple-drug combination inhibited paclitaxel-enriched CSCs and simultaneously improved lymphocyte infiltration in syngeneic TNBC mouse tumors. Conclusively,our findings elucidate the significance of CSCs in impairing anti-tumor immunity. The high efficacy of our triple-drug regimen in clinically relevant platforms not only underscores the importance for further mechanistic investigations but also paves the way for potential development of new,safe,and cost-effective therapeutic strategies for TNBC. View Publication

Microglia,the brain’s resident immune cells,play vital roles in brain development,and disorders like Alzheimer’s disease (AD). Human iPSC-derived microglia (iMG) provide a promising model to study these processes. However,existing iMG generation protocols face challenges,such as prolonged differentiation time,lack of detailed characterization,and limited gene function investigation via CRISPR-Cas9. Our integrated toolkit for in-vitro microglia functional genomics optimizes iPSC differentiation into iMG through a streamlined two-step,20-day process,producing iMG with a normal karyotype. We confirmed the iMG’s authenticity and quality through single-cell RNA sequencing,chromatin accessibility profiles (ATAC-Seq),proteomics and functional tests. The toolkit also incorporates a drug-dependent CRISPR-ON/OFF system for temporally controlled gene expression. Further,we facilitate the use of multi-omic data by providing online searchable platform that compares new iMG profiles to human primary microglia: https://sherlab.shinyapps.io/IPSC-derived-Microglia/ . Our method generates iMG that closely align with human primary microglia in terms of transcriptomic,proteomic,and chromatin accessibility profiles. Functionally,these iMG exhibit Ca2 + transients,cytokine driven migration,immune responses to inflammatory signals,and active phagocytosis of CNS related substrates including synaptosomes,amyloid beta and myelin. Significantly,the toolkit facilitates repeated iMG harvesting,essential for large-scale experiments like CRISPR-Cas9 screens. The standalone ATAC-Seq profiles of our iMG closely resemble primary microglia,positioning them as ideal tools to study AD-associated single nucleotide variants (SNV) especially in the genome regulatory regions. Our advanced two-step protocol rapidly and efficiently produces authentic iMG. With features like the CRISPR-ON/OFF system and a comprehensive multi-omic data platform,our toolkit equips researchers for robust microglial functional genomic studies. By facilitating detailed SNV investigation and offering a sustainable cell harvest mechanism,the toolkit heralds significant progress in neurodegenerative disease drug research and therapeutic advancement. The online version contains supplementary material available at 10.1186/s13287-024-03700-9. View Publication

Alcohol consumption increases the risk of breast cancer and promotes cancer progression. Alcohol exposure could affect both processes of the mammary carcinogenesis,namely,the cell transformation and onset of tumorigenesis as well as cancer aggressiveness including metastasis and drug resistance/recurrence. However,the cellular and molecular mechanisms underlying alcohol tumor promotion remain unclear. There are four members of the mammalian p38 mitogen-activated protein kinase (MAPK) family,namely,p38α,p38β,p38γ and p38δ. We have previously demonstrated alcohol exposure selectively activated p38γ MAPK in breast cancer cells in vitro and in vivo . Pirfenidone (PFD),an antifibrotic compound approved for the treatment of idiopathic pulmonary fibrosis,is also a pharmacological inhibitor of p38γ MAPK. This study aimed to determine whether PFD is useful to inhibit alcohol-induced promotion of breast cancer. Female adolescent (5 weeks) MMTV-Wnt1 mice were exposed to alcohol with a liquid diet containing 6.7% ethanol. Some mice received intraperitoneal (IP) injection of PFD (100 mg/kg) every other day. After that,the effects of alcohol and PFD on mammary tumorigenesis and metastasis were examined. Alcohol promoted the progression of mammary tumors in adolescent MMTV-Wnt1 mice. Treatment of PFD blocked tumor growth and alcohol-promoted metastasis. It also significantly inhibited alcohol-induced tumorsphere formation and cancer stem cell (CSC) population. PFD inhibited mammary tumor growth and alcohol-promoted metastasis. Since PFD is an FDA-approved drug,the current findings may be helpful to re-purpose its application in treating aggressive breast cancer and alcohol-promoted mammary tumor progression. View Publication

Outer radial glia (oRG) emerge as cortical progenitor cells that support the development of an enlarged outer subventricular zone (oSVZ) and the expansion of the neocortex. The in vitro generation of oRG is essential to investigate the underlying mechanisms of human neocortical development and expansion. By activating the STAT3 signaling pathway using leukemia inhibitory factor (LIF),which is not expressed in guided cortical organoids,we define a cortical organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The oSVZ comprises progenitor cells expressing specific oRG markers such as GFAP,LIFR,and HOPX,closely matching human fetal oRG. Finally,incorporating neural crest-derived LIF-producing cortical pericytes into cortical organoids recapitulates the effects of LIF treatment. These data indicate that increasing the cellular complexity of the organoid microenvironment promotes the emergence of oRG and supports a platform to study oRG in hPSC-derived brain organoids routinely. View Publication

Induced pluripotent stem cell-derived microglia (iMGL) represent an excellent tool in studying microglial function in health and disease. Yet,since differentiation and survival of iMGL are highly reliant on colony-stimulating factor 1 receptor (CSF1R) signaling,it is difficult to use iMGL to study microglial dysfunction associated with pathogenic defects in CSF1R. Serial modifications to an existing iMGL protocol were made,including but not limited to changes in growth factor combination to drive microglial differentiation,until successful derivation of microglia-like cells from an adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) patient carrying a c.2350G > A (p.V784M) CSF1R variant. Using healthy control lines,the quality of the new iMGL protocol was validated through cell yield assessment,measurement of microglia marker expression,transcriptomic comparison to primary microglia,and evaluation of inflammatory and phagocytic activities. Similarly,molecular and functional characterization of the ALSP patient-derived iMGL was carried out in comparison to healthy control iMGL. The newly devised protocol allowed the generation of iMGL with enhanced transcriptomic similarity to cultured primary human microglia and with higher scavenging and inflammatory competence at ~ threefold greater yield compared to the original protocol. Using this protocol,decreased CSF1R autophosphorylation and cell surface expression was observed in iMGL derived from the ALSP patient compared to those derived from healthy controls. Additionally,ALSP patient-derived iMGL presented a migratory defect accompanying a temporal reduction in purinergic receptor P2Y12 ( P2RY12 ) expression,a heightened capacity to internalize myelin,as well as heightened inflammatory response to Pam 3 CSK 4 . Poor P2RY12 expression was confirmed to be a consequence of CSF1R haploinsufficiency,as this feature was also observed following CSF1R knockdown or inhibition in mature control iMGL,and in CSF1R WT/KO and CSF1R WT/E633K iMGL compared to their respective isogenic controls. We optimized a pre-existing iMGL protocol,generating a powerful tool to study microglial involvement in human neurological diseases. Using the optimized protocol,we have generated for the first time iMGL from an ALSP patient carrying a pathogenic CSF1R variant,with preliminary characterization pointing toward functional alterations in migratory,phagocytic and inflammatory activities. The online version contains supplementary material available at 10.1186/s13024-024-00723-x. View Publication

Anti-CD117 monoclonal antibody (mAb) agents have emerged as exciting alternative conditioning strategies to traditional genotoxic irradiation or chemotherapy for both allogeneic and autologous gene-modified hematopoietic stem cell transplantation. Furthermore,these agents are concurrently being explored in the treatment of mast cell disorders. Despite promising results in animal models and more recently in patients,the short- and long-term effects of these treatments have not been fully explored. We conducted rigorous assessments to evaluate the effects of an antagonistic anti-mCD117 mAb,ACK2,on hematopoiesis in wild-type and Fanconi anemia (FA) mice. Importantly,we found no evidence of short-term DNA damage in either setting following this treatment,suggesting that ACK2 does not induce immediate genotoxicity,providing crucial insights into its safety profile. Surprisingly,FA mice exhibited an increase in colony formation after ACK2 treatment,indicating a potential targeting of hematopoietic stem cells and expansion of hematopoietic progenitor cells. Moreover,the long-term phenotypic and functional changes in hematopoietic stem and progenitor cells did not differ significantly between the ACK2-treated and control groups,in either setting,suggesting that ACK2 does not adversely affect hematopoietic capacity. These findings underscore the safety of these agents when utilized as a short-course treatment in the context of conditioning,as they did not induce significant DNA damage in hematopoietic stem or progenitor cells. However,single-cell RNA sequencing,used to compare gene expression between untreated and treated mice,revealed that the ACK2 mAb,via c-Kit downregulation,effectively modulated the MAPK pathway with Fos downregulation in wild-type and FA mice. Importantly,this modulation was achieved without causing prolonged disruptions. These findings validate the safety of anti-CD117 mAb treatment and also enhance our understanding of its intricate mode of action at the molecular level. View Publication