技术资料

Therapy-related myeloid neoplasms (t-MN) arise as a complication of chemo- and/or radiotherapy. Although t-MN can occur both in adult and childhood cancer survivors,the mechanisms driving therapy-related leukemogenesis likely vary across different ages. Chemotherapy is thought to induce driver mutations in children,whereas in adults pre-existing mutant clones are selected by the exposure. However,selective pressures induced by chemotherapy early in life are less well studied. Here,we use single-cell whole genome sequencing and phylogenetic inference to show that the founding cell of t-MN in children starts expanding after cessation of platinum exposure. In patients with Li-Fraumeni syndrome,characterized by a germline TP53 mutation,we find that the t-MN already expands during treatment,suggesting that platinum-induced growth inhibition is TP53- dependent. Our results demonstrate that germline aberrations can interact with treatment exposures in inducing t-MN,which is important for the development of more targeted,patient-specific treatment regimens and follow-up. Subject terms: Cancer genomics,Cancer genomics,Haematological cancer,Paediatric cancer View Publication

Heme oxygenase-1 (HO-1,HMOX1 ) degrades heme protecting cells from heme-induced oxidative damage. Beyond its well-established cellular functions,heme has emerged as a stabilizer of G-quadruplexes. These secondary DNA structures interfere with DNA replication. We recently revealed that nuclear HO-1 colocalizes with DNA G-quadruplexes and promotes their removal. Here,we investigate whether HO-1 safeguards cells against replication stress. Experiments were conducted in control and HMOX1 -deficient HEK293T cell lines. Immunostaining unveiled that DNA G-quadruplexes accumulated in the absence of HO-1,the effect that was further enhanced in response to δ-aminolevulinic acid (ALA),a substrate in heme synthesis. This was associated with replication stress,as evidenced by an elevated proportion of stalled forks analyzed by fiber assay. We observed the same effects in hematopoietic stem cells isolated from Hmox1 knockout mice and in a lymphoblastoid cell line from an HMOX1 -deficient patient. Interestingly,in the absence of HO-1,the speed of fork progression was higher,and the response to DNA conformational hindrance less stringent,indicating dysfunction of the PARP1-p53-p21 axis. PARP1 activity was not decreased in the absence of HO-1. Instead,we observed that HO-1 deficiency impairs the nuclear import and accumulation of p53,an effect dependent on the removal of excess heme. We also demonstrated that administering ALA is a more specific method for increasing intracellular free heme compared to treatment with hemin,which in turn induces strong lipid peroxidation. Our results indicate that protection against replication stress is a universal feature of HO-1,presumably contributing to its widely recognized cytoprotective activity. View Publication

Breast cancer is the most common cancer in women diagnosed in the U.S. and worldwide. Obesity increases breast cancer risk without clear underlying molecular mechanisms. Our studies demonstrate that circulating adipose fatty acid binding protein (A-FABP,or FABP4) links obesity-induced dysregulated lipid metabolism and breast cancer risk,thus potentially offering a new target for breast cancer treatment. We immunized FABP4 knockout mice with recombinant human FABP4 and screened hybridoma clones with specific binding to FABP4. The potential effects of antibodies on breast cancer cells in vitro were evaluated using migration,invasion,and limiting dilution assays. Tumor progression in vivo was evaluated in various types of tumorigenesis models including C57BL/6 mice,Balb/c mice,and SCID mice. The phenotype and function of immune cells in tumor microenvironment were characterized with multi-color flow cytometry. Tumor stemness was detected by ALDH assays. To characterize antigen-antibody binding capacity,we determined the dissociation constant of selected anti-FABP4 antibodies via surface plasmon resonance. Further analyses in tumor tissue were performed using 10X Genomics Visium spatial single cell technology. Herein,we report the generation of humanized monoclonal antibodies blocking FABP4 activity for breast cancer treatment in mouse models. One clone,named 12G2,which significantly reduced circulating levels of FABP4 and inhibited mammary tumor growth,was selected for further characterization. After confirming the therapeutic efficacy of the chimeric 12G2 monoclonal antibody consisting of mouse variable regions and human IgG1 constant regions,16 humanized 12G2 monoclonal antibody variants were generated by grafting its complementary determining regions to selected human germline sequences. Humanized V9 monoclonal antibody showed consistent results in inhibiting mammary tumor growth and metastasis by affecting tumor cell mitochondrial metabolism. Our current evidence suggests that targeting FABP4 with humanized monoclonal antibodies may represent a novel strategy for the treatment of breast cancer and possibly other obesity- associated diseases. The online version contains supplementary material available at 10.1186/s13058-024-01873-y. View Publication

Chemotherapy-induced peripheral neuropathy (CIPN) is a disabling side effect of cancer chemotherapy that can often limit treatment options for cancer patients or have life-long neurodegenerative consequences that reduce the patient’s quality of life. CIPN is caused by the detrimental actions of various chemotherapeutic agents on peripheral axons. Currently,there are no approved preventative measures or treatment options for CIPN,highlighting the need for the discovery of novel therapeutics and improving our understanding of disease mechanisms. In this study,we utilized human-induced pluripotent stem cell (hiPSC)-derived motor neurons as a platform to mimic axonal damage after treatment with vincristine,a chemotherapeutic used for the treatment of breast cancers,osteosarcomas,and leukemia. We screened a total of 1902 small molecules for neuroprotective properties in rescuing vincristine-induced axon growth deficits. From our primary screen,we identified 38 hit compounds that were subjected to secondary dose response screens. Six compounds showed favorable pharmacological profiles – AZD7762,A-674563,Blebbistatin,Glesatinib,KW-2449,and Pelitinib,all novel neuroprotectants against vincristine toxicity to neurons. In addition,four of these six compounds also showed efficacy against vincristine-induced growth arrest in human iPSC-derived sensory neurons. In this study,we utilized high-throughput screening of a large library of compounds in a therapeutically relevant assay. We identified several novel compounds that are efficacious in protecting different neuronal subtypes from the toxicity induced by a common chemotherapeutic agent,vincristine which could have therapeutic potential in the clinic. The online version contains supplementary material available at 10.1007/s00018-024-05340-x. View Publication

PU.1 ( SPI1 ) is pivotal in hematopoiesis,yet its role in human endothelial-to-hematopoietic transition (EHT) remains unclear. Comparing human in vivo and in vitro EHT transcriptomes revealed SPI1 ’s regulatory role. Knocking down SPI1 during in vitro EHT led to a decrease in the generation of hematopoietic progenitor cells (HPCs) and their differentiation potential. Through multi-omic analysis,we identified KLF1 and LYL1 - transcription factors specific to erythroid/myeloid and lymphoid cells,respectively - as downstream targets of SPI1 . Overexpressing KLF1 or LYL1 partially rescues the SPI1 knockdown-induced reduction in HPC formation. Specifically,KLF1 overexpression restores myeloid lineage potential,while LYL1 overexpression re-establishes lymphoid lineage potential. We also observed a SPI1 - LYL1 axis in the regulatory network in in vivo EHT. Taken together,our findings shed new light on the role of SPI1 in regulating lineage commitment during EHT,potentially contributing to the heterogeneity of hematopoietic stem cells (HSCs). Subject areas: Biological sciences,Molecular biology,Molecular interaction,Cell biology; View Publication

Programmed cell death 1 (PD-1) is a premier cancer drug target for immune checkpoint blockade (ICB). Because PD-1 receptor inhibition activates tumor-specific T-cell immunity,research has predominantly focused on T-cell-PD-1 expression and its immunobiology. In contrast,cancer cell-intrinsic PD-1 functional regulation is not well understood. Here,we demonstrate induction of PD-1 in melanoma cells via type I interferon receptor (IFNAR) signaling and reversal of ICB efficacy through IFNAR pathway inhibition. Treatment of melanoma cells with IFN-α or IFN-β triggers IFNAR-mediated Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling,increases chromatin accessibility and resultant STAT1/2 and IFN regulatory factor 9 (IRF9) binding within a PD-1 gene enhancer,and leads to PD-1 induction. IFNAR1 or JAK/STAT inhibition suppresses melanoma-PD-1 expression and disrupts ICB efficacy in preclinical models. Our results uncover type I IFN-dependent regulation of cancer cell-PD-1 and provide mechanistic insight into the potential unintended ICB-neutralizing effects of widely used IFNAR1 and JAK inhibitors. Subject terms: Melanoma,Cancer immunotherapy,Tumour immunology View Publication

Despite advances in breast cancer screening and treatment,prognosis for metastatic disease remains dismal at 30% five-year survival. This is due,in large,to the failure of current therapeutics to target properties unique to metastatic cells. One of the drivers of metastasis is miR-10b,a small noncoding RNA implicated in cancer cell invasion,migration,viability,and proliferation. We have developed a nanodrug,termed MN-anti-miR10b,that delivers anti-miR-10b antisense oligomers to cancer cells. In mouse models of metastatic triple-negative breast cancer,MN-anti-miR10b has been shown to prevent onset of metastasis and eliminate existing metastases in combination with chemotherapy,even after treatment has been stopped. Recent studies have implicated miR-10b in conferring stem cell-like properties onto cancer cells,such as chemoresistance. In this study,we show transcriptional evidence that inhibition of miR-10b with MN-anti-miR10b activates developmental processes in cancer cells and that stem-like cancer cells have increased miR-10b expression. We then demonstrate that treatment of breast cancer cells with MN-anti-miR10b reduces their stemness,confirming that these properties make metastatic cells susceptible to the nanodrug actions. Collectively,these findings indicate that inhibition of miR-10b functions to impair breast cancer cell stemness,positioning MN-anti-miR10b as an effective treatment option for stem-like breast cancer subtypes. View Publication

DNA hypomethylating agents (HMAs) are used for the treatment of myeloid malignancies,although their therapeutic effects have been unsatisfactory. Here we show that CRISPR-Cas9 screening reveals that knockout of topoisomerase 1-binding arginine/serine-rich protein ( TOPORS ),which encodes a ubiquitin/SUMO E3 ligase,augments the efficacy of HMAs on myeloid leukemic cells with little effect on normal hematopoiesis,suggesting that TOPORS is involved in resistance to HMAs. HMAs are incorporated into the DNA and trap DNA methyltransferase-1 (DNMT1) to form DNA-DNMT1 crosslinks,which undergo SUMOylation,followed by proteasomal degradation. Persistent crosslinking is cytotoxic. The TOPORS RING finger domain,which mediates ubiquitination,is responsible for HMA resistance. In TOPORS knockout cells,DNMT1 is stabilized by HMA treatment due to inefficient ubiquitination,resulting in the accumulation of unresolved SUMOylated DNMT1. This indicates that TOPORS ubiquitinates SUMOylated DNMT1,thereby promoting the resolution of DNA-DNMT1 crosslinks. Consistently,the ubiquitination inhibitor,TAK-243,and the SUMOylation inhibitor,TAK-981,show synergistic effects with HMAs through DNMT1 stabilization. Our study provides a novel HMA-based therapeutic strategy that interferes with the resolution of DNA-DNMT1 crosslinks. Subject terms: Myelodysplastic syndrome,Myelodysplastic syndrome View Publication

The growth factor Neuregulin-1 (NRG1) has pleiotropic roles in proliferation and differentiation of the stem cell niche in different tissues. It has been implicated in gut,brain and muscle development and repair. Six isoform classes of NRG1 and over 28 protein isoforms have been previously described. Here we report a new class of NRG1,designated NRG1-VII to denote that these NRG1 isoforms arise from a myeloid-specific transcriptional start site (TSS) previously uncharacterized. Long-read sequencing was used to identify eight high-confidence NRG1-VII transcripts. These transcripts presented major structural differences from one another,through the use of cassette exons and alternative stop codons. Expression of NRG1-VII was confirmed in primary human monocytes and tissue resident macrophages and induced pluripotent stem cell-derived macrophages (iPSC-derived macrophages). Isoform switching via cassette exon usage and alternate polyadenylation was apparent during monocyte maturation and macrophage differentiation. NRG1-VII is the major class expressed by the myeloid lineage,including tissue-resident macrophages. Analysis of public gene expression data indicates that monocytes and macrophages are a primary source of NRG1. The size and structure of class VII isoforms suggests that they may be more diffusible through tissues than other NRG1 classes. However,the specific roles of class VII variants in tissue homeostasis and repair have not yet been determined. The online version contains supplementary material available at 10.1186/s12864-024-10723-2. View Publication

Astrocytes are essential for the formation and maintenance of neural networks. However,a major technical challenge for investigating astrocyte function and disease-related pathophysiology has been the limited ability to obtain functional human astrocytes. Despite recent advances in human pluripotent stem cell (hPSC) techniques,primary rodent astrocytes remain the gold standard in coculture with human neurons. We demonstrate that a combination of leukemia inhibitory factor (LIF) and bone morphogenetic protein-4 (BMP4) directs hPSC-derived neural precursor cells to a highly pure population of astroglia in 28 d. Using single-cell RNA sequencing,we confirm the astroglial identity of these cells and highlight profound transcriptional adaptations in cocultured hPSC-derived astrocytes and neurons,consistent with their further maturation. In coculture with human neurons,multielectrode array recordings revealed robust network activity of human neurons in a coculture with hPSC-derived or rat astrocytes [3.63 ± 0.44 min −1 (hPSC-derived),2.86 ± 0.64 min −1 (rat); p = 0.19]. In comparison,we found increased spike frequency within network bursts of human neurons cocultured with hPSC-derived astrocytes [56.31 ± 8.56 Hz (hPSC-derived),24.77 ± 4.04 Hz (rat); p < 0.01],and whole-cell patch-clamp recordings revealed an increase of postsynaptic currents [2.76 ± 0.39 Hz (hPSC-derived),1.07 ± 0.14 Hz (rat); p < 0.001],consistent with a corresponding increase in synapse density [14.90 ± 1.27/100 μm 2 (hPSC-derived),8.39 ± 0.63/100 μm 2 (rat); p < 0.001]. Taken together,we show that hPSC-derived astrocytes compare favorably with rat astrocytes in supporting human neural network activity and maturation,providing a fully human platform for investigating astrocyte function and neuronal-glial interactions. View Publication

Recent advances in imaging suggested that spatial organization of hematopoietic cells in their bone marrow microenvironment (niche) regulates cell expansion,governing progression,and leukemic transformation of hematological clonal disorders. However,our ability to interrogate the niche in pre-malignant conditions has been limited,as standard murine models of these diseases rely largely on transplantation of the mutant clones into conditioned mice where the marrow microenvironment is compromised. Here,we leveraged live-animal microscopy and ultralow dose whole body or focal irradiation to capture single cells and early expansion of benign/pre-malignant clones in the functionally preserved microenvironment. 0.5 Gy whole body irradiation (WBI) allowed steady engraftment of cells beyond 30 weeks compared to non-conditioned controls. In-vivo tracking and functional analyses of the microenvironment showed no change in vessel integrity,cell viability,and HSC-supportive functions of the stromal cells,suggesting minimal inflammation after the radiation insult. The approach enabled in vivo imaging of Tet2 + /− and its healthy counterpart,showing preferential localization within a shared microenvironment while forming discrete micro-niches. Notably,stationary association with the niche only occurred in a subset of cells and would not be identified without live imaging. This strategy may be broadly applied to study clonal disorders in a spatial context. View Publication

Reduction of adult hippocampal neurogenesis is an early critical event in Alzheimer’s disease (AD),contributing to progressive memory loss and cognitive decline. Reduced levels of the nucleoporin 153 (Nup153),a key epigenetic regulator of NSC stemness,characterize the neural stem cells isolated from a mouse model of AD (3×Tg) (AD-NSCs) and determine their altered plasticity and gene expression. Nup153-regulated mechanisms contributing to NSC function were investigated: (1) in cultured NSCs isolated from AD and wild type (WT) mice by proteomics; (2) in vivo by lentiviral-mediated delivery of Nup153 or GFP in the hippocampus of AD and control mice analyzing neurogenesis and cognitive function; (3) in human iPSC-derived brain organoids obtained from AD patients and control subjects as a model of neurodevelopment. Proteomic approach identified Nup153 interactors in WT- and AD-NSCs potentially implicated in neurogenesis regulation. Gene ontology (GO) analysis showed that Nup153-bound proteins in WT-NSCs were involved in RNA metabolism,nuclear import and epigenetic mechanisms. Nup153-bound proteins in AD-NSCs were involved in pathways of neurodegeneration,mitochondrial dysfunction,proteasomal processing and RNA degradation. Furthermore,recovery of Nup153 levels in AD-NSCs reduced the levels of oxidative stress markers and recovered proteasomal activity. Lentiviral-mediated delivery of Nup153 in the hippocampal niche of AD mice increased the proliferation of early progenitors,marked by BrdU/DCX and BrdU/PSANCAM positivity and,later,the integration of differentiating neurons in the cell granule layer (BrdU/NeuN + cells) compared with GFP-injected AD mice. Consistently,Nup153-injected AD mice showed an improvement of cognitive performance in comparison to AD-GFP mice at 1 month after virus delivery assessed by Morris Water Maze. To validate the role of Nup153 in neurogenesis we took advantage of brain organoids derived from AD-iPSCs characterized by fewer neuroepithelial progenitor loops and reduced differentiation areas. The upregulation of Nup153 in AD organoids recovered the formation of neural-like tubes and differentiation. Our data suggest that the positive effect of Nup153 on neurogenesis is based on a complex regulatory network orchestrated by Nup153 and that this protein is a valuable disease target. The online version contains supplementary material available at 10.1186/s13287-024-03805-1. View Publication