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The introduction of antibody–drug conjugates represents a significant advancement in targeted therapy of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Our study aims to investigate the role of the DNA damage response pathway and the impact of PARP1 inhibition,utilizing talazoparib,on the response of AML and ALL cells to Gemtuzumab ozogamicin (GO) and Inotuzumab ozogamicin (INO),respectively. AML and ALL cells were treated with GO,INO and γ-calicheamicin in order to induce severe DNA damage and activate the G2/M cell-cycle checkpoint in a dose- and time-dependent manner. The efficacy of PARP1 inhibitors and,in particular,talazoparib in enhancing INO or GO against ALL or AML cells was assessed through measurements of cell viability,cell death,cell cycle progression,DNA damage repair,accumulation of mitotic DNA damage and inhibition of clonogenic capacity. We observed that both ALL and AML cell lines activate the G2/M cell-cycle checkpoint in response to γ-calicheamicin-induced DNA damage,highlighting a shared cellular response mechanism. Talazoparib significantly enhanced the efficacy of INO against ALL cell lines,resulting in reduced cell viability,increased cell death,G2/M cell-cycle checkpoint override,accumulation of mitotic DNA damage and inhibition of clonogenic capacity. Strong synergism was observed in primary ALL cells treated with the combination. In contrast,AML cells exhibited a heterogeneous response to talazoparib in combination with GO. Our findings suggest a potential link between the differential responses of ALL and AML cells to the drug combinations and the ability of talazoparibto override G2/M cell-cycle arrest induced by antibody–drug conjugates. PARP1 emerges as a key player in the response of ALL cells to INO and represents a promising target for therapeutic intervention in this leukemia setting. Our study sheds light on the intricate interplay between the DNA damage response pathway,PARP1 inhibition,and response of γ-calicheamicin-induced DNA damages in AML and ALL. These findings underscore the importance of targeted therapeutic strategies and pave the way for future research aimed at optimizing leukemia treatment approaches. The online version contains supplementary material available at 10.1186/s12967-024-05838-9. View Publication

Transplantation of engineered hematopoietic stem/progenitor cells (HSPCs) showed curative potential in patients affected by neurometabolic diseases treated in early stage. Favoring the engraftment and maturation of the engineered HSPCs in the central nervous system (CNS) could allow enhancing further the therapeutic potential of this approach. Here we unveil that HSPCs haplo-insufficient at the Cx3cr1 (Cx3cr1 −/+ ) locus are favored in central nervous system (CNS) engraftment and generation of microglia-like progeny cells (MLCs) as compared to wild type (Cx3cr1 +/+ ) HSPCs upon transplantation in mice. Based on this evidence,we have developed a CRISPR-based targeted gene addition strategy at the human CX3CR1 locus resulting in an enhanced ability of the edited human HSPCs to generate mature MLCs upon transplantation in immunodeficient mice,and in lineage specific,regulated and robust transgene expression. This approach,which benefits from the modulation of pathways involved in microglia maturation and migration in haplo-insufficient cells,may broaden the application of HSPC gene therapy to a larger spectrum of neurometabolic and neurodegenerative diseases. Subject terms: Targeted gene repair,Haematopoietic stem cells,Microglial cells View Publication

The effect of mechanical cues on cellular behaviour has been reported in multiple studies so far,and a specific aspect of interest is the role of mechanotransductive proteins in neuronal development. Among these,yes-associated protein (YAP) is responsible for multiple functions in neuronal development such as neuronal progenitor cells migration and differentiation while myocardin-related transcription factor A (MRTFA) facilitates neurite outgrowth and axonal pathfinding. Both proteins have indirectly intertwined fates via their signalling pathways. There is little literature investigating the roles of YAP and MRTFA in vitro concerning neurite outgrowth in mechanically confined microenvironments. Moreover,our understanding of their relationship in immature neurons cultured within engineered confined microenvironments is still lacking. In this study,we fabricated,via two-photon polymerization (2PP),2.5D microgrooves and 3D polymeric microchannels,with a diameter range from 5 to 30 μm. We cultured SH-SY5Y cells and differentiated them into immature neuron-like cells on both 2.5D and 3D microstructures to investigate the effect of mechanical confinement on cell morphology and protein expression. In 2.5D microgrooves,both YAP and MRTFA nuclear/cytoplasmic (N/C) ratios exhibited maxima in the 10 μm grooves indicating a strong relation with mechanical-stress-inducing confinement. In 3D microchannels,both proteins’ N/C ratio exhibited minima in presence of 5 or 10 μm channels,a behaviour that was opposite to the ones observed in the 2.5D microgrooves and that indicates how the geometry and mechanical confinement of 3D microenvironments are unique compared to 2.5D ones due to focal adhesion,actin,and nuclear polarization. Further,especially in presence of 2.5D microgrooves,cells featured an inversely proportional relationship between YAP N/C ratio and the average neurite length. Finally,we also cultured human induced pluripotent stem cells (hiPSCs) and differentiated them into cortical neurons on the microstructures for up to 2 weeks. Interestingly,YAP and MRTFA N/C ratios also showed a maximum around the 10 μm 2.5D microgrooves,indicating the physiological relevance of our study. Our results elucidate the possible differences induced by 2.5D and 3D confining microenvironments in neuronal development and paves the way for understanding the intricate interplay between mechanotransductive proteins and their effect on neural cell fate within engineered cell microenvironments. View Publication

Liver cancer stem cells (LCSCs) are thought to drive the metastasis and recurrence,however,the heterogeneity of molecular markers of LCSCs has hindered the development of effective methods to isolate them. This study introduced an effective approach to isolate and culture LCSCs from human primary liver cancer (HPLC),leveraging mouse embryonic fibroblasts (MEFs) as feeder cells in conjunction with using defined medium. Isolated LCSCs were further characterized by multiple approaches. Transcriptome sequencing data analysis was conducted to identify highly expressed genes in LCSCs and classify different subtypes of liver cancers. Total sixteen cell strains were directly isolated from 24 tissues of three types of HPLC without sorting,seven of which could be maintained long-term culture as colony growth on MEFs,which is unique characteristics of stem cells. Even 10 of cloned cells formed the tumors in immunodeficient mice,indicating that those cloned cells were tumorgenic. The histologies and gene expression pattern of human xenografts were very similar to those of HPLC where these cloned cells were isolated. Moreover,putative markers of LCSCs were further verified to all express in cloned cells,confirming that these cells were LCSCs. These cloned LCSCs could be cryopreserved,and still maintained the feature of colony growth on MEFs after the recovery. Compared to suspension culture as conventional approach to culture LCSCs,our approach much better maintained stemness of LCSCs for a long time. To date,these cloned cells could be cultured on MEFs over 12 passages. Moreover,bioinformatics analysis of sequencing data revealed the gene expression profiles in LCSCs,and liver cancers were classified into two subtypes C1 and C2 based on genes associated with the prognosis of LCSCs. Patients of the C2 subtype,which is closely related to the extracellular matrix,were found to be sensitive to treatments such as Cisplatin,Axitinib,JAK1 inhibitors,WNT-c59,Sorafenib,and RO-3306. In summary,this effective approach offers new insights into the molecular landscape of human liver cancers,and the identification of the C2 subtype and its unique response to the treatment pave the way for the creation of more effective,personalized therapeutic strategies. The online version contains supplementary material available at 10.1186/s12967-024-05870-9. View Publication

Pericytes play a crucial role in controlling inflammation and vascular functions in the central nervous system,which are disrupted in Parkinson’s disease (PD). Still,there is a lack of studies on the impact of pericytes on neurodegenerative diseases,and their involvement in the pathology of PD is unclear. Our objective was to investigate the molecular and functional differences between healthy pericytes and pericytes with the LRRK2 G2019S mutation,which is one of the most common mutations associated with PD. Our study employed pericyte-like cells obtained from induced pluripotent stem cells produced from PD patients with the LRRK2 G2019S mutation as well as from healthy individuals. We examined the gene expression profiles of the cells and analyzed how the alterations reflect on their functionality. We have shown differences in the expression of genes related to inflammation and angiogenesis. Furthermore,we observe modified migration speed in PD pericyte-like cells as well as enhanced secretion of inflammatory mediators,such as soluble VCAM-1 and MCP-1,in these pericyte-like cells following exposure to proinflammatory stimuli. In summary,our findings support the notion that pericytes play a role in the inflammatory and vascular changes observed in PD. Further investigation of pericytes could provide valuable insight into understanding the pathogenesis of PD. The online version contains supplementary material available at 10.1186/s12987-024-00592-y. View Publication

Ex vivo activation is a prerequisite to reaching adequate levels of gene editing by homology-directed repair (HDR) for hematopoietic stem and progenitor cell (HSPC)-based clinical applications. Here,we show that shortening culture time mitigates the p53-mediated DNA damage response to CRISPR-Cas9-induced DNA double-strand breaks,enhancing the reconstitution capacity of edited HSPCs. However,this results in lower HDR efficiency,rendering ex vivo culture necessary yet detrimental. Mechanistically,ex vivo activation triggers a multi-step process initiated by p38 mitogen-activated protein kinase (MAPK) phosphorylation,which generates mitogenic reactive oxygen species (ROS),promoting fast cell-cycle progression and subsequent proliferation-induced DNA damage. Thus,p38 inhibition before gene editing delays G1/S transition and expands transcriptionally defined HSCs,ultimately endowing edited cells with superior multi-lineage differentiation,persistence throughout serial transplantation,enhanced polyclonal repertoire,and better-preserved genome integrity. Our data identify proliferative stress as a driver of HSPC dysfunction with fundamental implications for designing more effective and safer gene correction strategies for clinical applications. View Publication

Global dissemination of SARS-CoV-2 Omicron sublineages has provided a sufficient opportunity for natural selection,thus enabling beneficial mutations to emerge. Characterisation of these mutations uncovers the underlying machinery responsible for the fast transmission of Omicron variants and guides vaccine development for combating the COVID-19 pandemic. Through systematic bioinformatics analysis of 496,606 sequences of Omicron variants,we obtained 40 amino acid substitutions that occurred with high frequency in the S protein. Utilising pseudoviruses and a trans -complementation system of SARS-CoV-2,we identified the effect of high-frequency mutations on viral infectivity and elucidated the molecular mechanisms. Finally,we evaluated the impact of a key emerging mutation on the immune protection induced by the SARS-CoV-2 VLP mRNA vaccine in a murine model. We identified a proline-to-leucine substitution at the 1263rd residue of the Spike protein,and upon investigating the relative frequencies across multiple Omicron sublineages,we found a trend of increasing frequency for P1263L. The substitution significantly enhances the capacity for S-mediated viral entry and improves the immunogenicity of a virus-like particle mRNA vaccine. Mechanistic studies showed that this mutation is located in the FERM binding motif of the cytoplasmic tail and impairs the interaction between the S protein and the Ezrin/Radixin/Moesin proteins. Additionally,this mutation facilitates the incorporation of S proteins into SARS-CoV-2 virions. This study offers mechanistic insight into the constantly increasing transmissibility of SARS-CoV-2 Omicron variants and provides a meaningful optimisation strategy for vaccine development against SARS-CoV-2. This study was supported by grants from the National Key Research and Development Plan of China (2021YFC2302405,2022YFC2303200,2021YFC2300200 and 2022YFC2303400),the National Natural Science Foundation of China (32188101,32200772,82422049,82241082,32270182,82372254,82271872,82341046,32100755 and 82102389),Shenzhen Medical Research Fund (B2404002,A2303036),the Shenzhen Bay Laboratory Startup Fund (21330111),Shenzhen San-Ming Project for Prevention and Research on Vector-borne Diseases (SZSM202211023),Yunnan Provincial Science and Technology Project at Southwest United Graduate School (202302AO370010). The New Cornerstone Science Foundation through the New Cornerstone Investigator Program,and the Xplorer Prize from Tencent Foundation. View Publication

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Induction of fetal hemoglobin (HbF) has been shown to be a viable therapeutic approach to treating sickle cell disease and potentially other β-hemoglobinopathies. To identify targets and target-modulating small molecules that enhance HbF expression,we engineered a human umbilical-derived erythroid progenitor reporter cell line (HUDEP2_HBG1_HiBiT) by genetically tagging a HiBiT peptide to the carboxyl (C)-terminus of the endogenous HBG1 gene locus,which codes for γ-globin protein,a component of HbF. Employing this reporter cell line,we performed a chemogenomic screen of approximately 5000 compounds annotated with known targets or mechanisms that have achieved clinical stage or approval by the US Food and Drug Administration (FDA). Among them,10 compounds were confirmed for their ability to induce HbF in the HUDEP2 cell line. These include several known HbF inducers,such as pomalidomide,lenalidomide,decitabine,idoxuridine,and azacytidine,which validate the translational nature of this screening platform. We identified avadomide,autophinib,triciribine,and R574 as novel HbF inducers from these screens. We orthogonally confirmed HbF induction activities of the top hits in both parental HUDEP2 cells as well as in human primary CD34+ hematopoietic stem and progenitor cells (HSPCs). Further,we demonstrated that pomalidomide and avadomide,but not idoxuridine,induced HbF expression through downregulation of several transcriptional repressors such as BCL11A,ZBTB7A,and IKZF1. These studies demonstrate a robust phenotypic screening workflow that can be applied to large-scale small molecule profiling campaigns for the discovery of targets and pathways,as well as novel therapeutics for sickle cell disease and other β-hemoglobinopathies. View Publication

The lack of a permissive cell culture system has limited high-resolution structures of parvovirus B19 (B19V) to virus-like particles (VLPs). In this study,we present the atomic resolution structure (2.2 Å) of authentic B19V purified from a patient blood sample. There are significant differences compared to non-infectious VLPs. Most strikingly,two host protease inhibitors (PIs),inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) and serpinA3,were identified in complex with the capsids in all patient samples tested. The ITIH4 binds specifically to the icosahedral fivefold axis and serpinA3 occupies the twofold axis. The protein-coated virions remain infectious,and the capsid-associated PIs retain activity; however,upon virion interaction with target cells,the PIs dissociate from the capsid prior to viral entry. Our finding of an infectious virion shielded by bound host serum proteins suggests an evolutionarily favored phenomenon to evade immune surveillance and escape host protease activity. Subject terms: Cryoelectron microscopy,Virology View Publication

The application of organoids has been limited by the lack of methods for producing uniformly mature organoids at scale. This study introduces an organoid culture platform,called UniMat,which addresses the challenges of uniformity and maturity simultaneously. UniMat is designed to not only ensure consistent organoid growth but also facilitate an unrestricted supply of soluble factors by a 3D geometrically-engineered,permeable membrane-based platform. Using UniMat,we demonstrate the scalable generation of kidney organoids with enhanced uniformity in both structure and function compared to conventional methods. Notably,kidney organoids within UniMat show improved maturation,showing increased expression of nephron transcripts,more in vivo-like cell-type balance,enhanced vascularization,and better long-term stability. Moreover,UniMat’s design offers a more standardized organoid model for disease modeling and drug testing,as demonstrated by polycystic-kidney disease and acute kidney injury modeling. In essence,UniMat presents a valuable platform for organoid technology,with potential applications in organ development,disease modeling,and drug screening. Subject terms: Nanofabrication and nanopatterning,Biomaterials,Stem-cell biotechnology View Publication

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