技术资料

The long-term maintenance of hematopoietic stem cells (HSCs) relies on the regulation of endoplasmic reticulum (ER) stress at a low level,but the underlying mechanism remains poorly understood. Here,we demonstrate that suppression of ER stress improves the functions of HSCs and protects HSCs against ionizing radiation (IR)-induced injury. We identify epithelial membrane protein 1 (EMP1) as a key regulator that mitigates ER stress in HSCs. Emp1 deficiency leads to the accumulation of protein aggregates and elevated ER stress,ultimately resulting in impaired HSC maintenance and self-renewal. Mechanistically,EMP1 is located within the ER and interacts with ceramide synthase 2 (CERS2) to limit the production of a class of sphingolipids,dihydroceramides (dhCers). DhCers accumulate in Emp1-deficient HSCs and induce protein aggregation. Furthermore,Emp1 deficiency renders HSCs more susceptible to IR,while overexpression of Emp1 or inhibition of CERS2 protects HSCs against IR-induced injury. These findings highlight the critical role played by the EMP1-CERS2-dhCers axis in constraining ER stress and preserving HSC potential. A new study shows EMP1 protects hematopoietic stem cells by suppressing sphingolipid metabolism and ER stress. EMP1 interacts with CERS2 to limit dihydroceramide production,which causes protein aggregation when elevated. View Publication

Gene edited human pluripotent stem cells are a promising platform for developing reparative cellular therapies that evade immune rejection. Existing first-generation hypoimmune strategies have used CRISPR/Cas9 editing to modulate genes associated with adaptive immune responses,but have largely not addressed the innate immune cells,such as neutrophils,that mediate inflammation and rejection processes occurring early after graft transplantation. We identify the adhesion molecule ICAM-1 as a hypoimmune target that plays multiple critical roles in both adaptive and innate immune responses post-transplantation. In our experiments,we find that ICAM-1 blocking or knockout in human pluripotent stem cell-derived cardiovascular therapies imparts significantly diminished binding of multiple immune cell types. ICAM-1 knockout results in diminished T cell proliferation and activation responses in vitro and in longer in vivo retention/protection of knockout grafts following immune cell encounter in NeoThy humanized mice. We also introduce the ICAM-1 knockout edit into existing first-generation hypoimmune human pluripotent stem cells and prevent immune cell binding. This promising hypoimmune editing strategy has the potential to improve transplantation outcomes for regenerative therapies in the setting of cardiovascular pathologies and several other diseases. Hypoimmune gene editing in human pluripotent stem cells (hPSCs) provides a promising platform for cellular therapies. Here,the authors report that CRISPR mediated deletion of ICAM-1 in hPSC-derived grafts reduces immune cell adhesion,dampens T cell activation,and protects against immune rejection. View Publication

Triple-negative breast cancer (TNBC) is an aggressive and clinically challenging subtype defined by the absence of estrogen receptor,progesterone receptor,and HER2 amplification,resulting in poor prognosis and limited therapeutic options. Targeting alternative molecular pathways is urgently needed to overcome resistance and improve patient outcomes. CD147 has emerged as surface marker associated with tumor progression and immune evasion. In this study,CD147 and MHC class I—a key inhibitory ligand for natural killer cells—were analyzed in breast cancer cell lines (MCF7,MDA-MB-453,MDA-MB-231,and HCC38) using flow cytometry. The therapeutic efficacy of a humanized anti-CD147 monoclonal antibody (HuM6-1B9) was evaluated for its capacity to potentiate antibody-dependent cellular cytotoxicity (ADCC). HuM6-1B9 demonstrated the strong binding to MDA-MB-231 (KD = 4.982 nM) and HCC38 (KD = 4.523 nM),which are representative TNBC cell lines. In 3D spheroid models,HuM6-1B9 significantly enhanced PBMC-mediated ADCC,leading to a marked reduction in TNBC spheroid viability. Co-culture of CFSE-labeled MDA-MB-231 and HCC38 cells with primary NK cells confirmed robust ADCC,achieving 50% and 70% cytotoxicity,respectively,despite high MHC class I expression. Live-cell imaging demonstrated caspase-3/7 activation consistent with apoptosis in NK-targeted tumor cells,while CD107a degranulation and IFN-γ secretion confirmed the functional contribution of HuM6-1B9 to ADCC enhancement. Importantly,HuM6-1B9 did not promote migration or invasion in MDA-MB-231 cells,supporting its safety profile regarding metastasis. Collectively,these findings establish HuM6-1B9 as a promising immunotherapeutic candidate that overcomes immune resistance and selectively eliminates TNBC cells through ADCC without enhancing metastatic potential. By integrating mechanistic assays of NK cytotoxicity,apoptosis,and 3D tumor spheroids,this study provides clinically relevant insights underscoring the translational potential of HuM6-1B9 in TNBC immunotherapy. View Publication

EVI1-rearranged acute myeloid leukemia (AML) with inv(3)(q21q26) or t(3;3)(q21q26) represents a distinct and aggressive subtype characterized by poor prognosis and limited treatment options. However,the optimal strategy to overcome resistance to conventional therapy remains elusive. Building upon observations correlating EVI1 overexpression with reduced sensitivity to venetoclax,a BH3-mimetic BCL-2 inhibitor,we investigated the mechanisms of resistance to venetoclax in combination with hypomethylating agents in inv(3)/t(3;3) AML cells. Utilizing novel murine models recapitulating inv(3) AML with concomitant SF3B1 mutations,we conducted comprehensive phenotypic and transcriptomic analyses in the presence or absence of venetoclax-containing therapy. Despite initial therapeutic responses,manifested as partially prolonged survival and myeloid differentiation,resistant leukemic cells demonstrated enhanced dependency on BRD4 and MYB pathways with a dormant phenotype. Notably,inhibition of either BRD4 or MYB significantly augmented the efficacy of venetoclax and hypomethylating agents in both murine and patient-derived AML models harboring inv(3) and SF3B1 mutations. These findings elucidate the transcriptional dynamics underlying venetoclax resistance and propose alternative therapeutic strategies targeting BRD4 and MYB as promising avenues for improving outcomes in patients with EVI1-rearranged AML. Our work highlights the necessity for innovative combination therapies to address the multifaceted mechanisms of resistance in this high-risk leukemia subtype. View Publication

Chagas disease,caused by the parasite Trypanosoma cruzi,affects millions of people globally. Unfortunately,the available treatment options,especially for the chronic stage of the disease,are suboptimal. Given the chronic nature of the disease and the elusive nature of the parasite,there is a high need for new and safer drugs that deliver sterile cure. Posaconazole was a promising lead in the drug discovery pipeline but ultimately failed in clinical trials due to patient relapses. This failure illustrates the need for a drug screening assay that can predict sterile cure by assessing recrudescence after treatment. Here,we used human induced pluripotent stem cell (iPSC)-derived macrophages (iMACs) as host cells for T. cruzi. The iMACs were highly susceptible to infection by the parasites. By combining red fluorescent protein (RFP)-expressing iMACs with mNeonGreen-expressing T. cruzi,we were able to monitor the dynamics of the infection through live cell imaging. The activity of the compounds benznidazole and posaconazole was consistent with the results of an established infection system using mouse primary macrophages. The post-mitotic nature of iMACs makes them suitable host cells for long-term assays needed to assess recrudescence of parasites. Moreover,their human origin,stable genetic background,and capacity for genetic modification make the iMACs excellent host cells for studying host-pathogen interaction. Author summaryThe parasite Trypanosoma cruzi,the causative agent of Chagas disease,is a global health concern affecting millions each year. Infection with T. cruzi can cause chronic disease,often remaining asymptomatic for decades before resulting in severe cardiac or gastro-intestinal pathologies. To date,only benznidazole and nifurtimox are used for treatment of the infection,but both drugs are suboptimal for curing the chronic stage. Posaconazole showed great promise in preclinical studies but failed to achieve sterile cure in clinical trials,causing patient relapses. These disappointing results underline the need for drug screening assays able to predict sterile cure by evaluating recrudescence post-treatment. We used human induced pluripotent stem cell derived macrophages as host cells for T. cruzi and testing of trypanocidal compounds. This model can be used for long-term in vitro screening assays to find new drug candidates against Chagas disease. The human origin of these cells combined with the possibility of upscaling their production make them great host cells for drug screening campaigns. View Publication

Pluripotent cancer stem cells play a pivotal role in inducing phenotypic plasticity across various cancer types,including bladder cancer. This plasticity,crucial for cancer progression,is largely regulated by epigenetic modifications including N6-methyladenosine (m6A) in RNAs. However,the role of the m6A reader protein YTHDC2 in this process remains poorly understood. In this study,we uncovered that the depletion of YTHDC2 significantly increased the pool of bladder cancer stem cells (BCSCs),resulting in a phenotypic shift towards a more invasive subtype of bladder cancer. This shift was characterized by enhanced proliferation,migration,invasion,and self-renewal capabilities of cancer cells,highlighting YTHDC2’s function as a tumor suppressor. Mechanistically,YTHDC2 recognized and bound to m6A-modified SOX2 mRNA,resulting in translational inhibition of SOX2. In conclusion,our study identifies YTHDC2 as a tumor suppressor in bladder cancer through inhibiting SOX2-mediated cell pluripotency and underscores the therapeutic potential of targeting the YTHDC2-SOX2 axis in bladder cancer. View Publication

We examined the role of SLFN12,a member of the Schlafen (SLFN) family of interferon-regulated genes and proteins in leukemogenesis,and its potential as a therapeutic target in acute myeloid leukemia (AML). We explored the effects of velcrins,a class of small molecules able to modulate SLFN12 biological activity,on AML cells. Velcrin treatment of AML cells stabilized SLFN12 and promoted SLFN12 complex formation with phosphodiesterase 3A or phosphodiesterase 3B. Such effects were associated with growth-inhibitory and proapoptotic responses,as well as potent suppressive effects on leukemic cell growth. In addition,velcrin treatment suppressed clonogenic capacity of primitive leukemic progenitors and significantly extended survival in a mouse AML xenograft model. Taken together,these findings establish an important role of SLFN12 in leukemogenesis and raise the potential for the use of velcrins as a therapeutic strategy for AML. Significance: Our studies identify SLFN12 as a potential target in AML with important clinical–translational implications. View Publication

Acute myeloid leukemia (AML) is a genetically heterogeneous malignancy characterized by the clonal proliferation of undifferentiated myeloid precursors in the bone marrow. Although standard induction regimens based on anthracyclines often achieve initial remission,up to 25% of patients exhibit primary refractory disease and nearly 50% relapse,underscoring the urgent need to overcome therapy resistance. Aldehyde dehydrogenase 1 (ALDH1) contributes to leukemic cell survival by maintaining stemness,proliferation,and chemoresistance through aldehyde detoxification and retinoic acid synthesis. Here,we identify two enhancer elements,ALDH1A1‐E3 and ALDH1A2‐E1‐A,that mediate transcriptional activation of ALDH1A1 and ALDH1A2 in response to the anthracycline daunorubicin. These enhancers are regulated by STAT3 and FOS/JUN transcription factors,which cooperatively link drug response to ALDH1 induction. Functional validation in AML cell lines,primary samples,and xenograft models shows that ALDH1 upregulation is part of an adaptive stress response and may contribute to reduced anthracycline sensitivity. Co‐treatment with the ALDH1A1/1A2 inhibitor DIMATE synergistically enhances daunorubicin efficacy across in vitro and in vivo resistant models. Consistently,high ALDH1 expression is associated with adverse genetic risk,prior anthracycline exposure,and inferior OS,particularly in relapsed/refractory AML. These findings uncover a novel enhancer‐mediated mechanism of ALDH1 induction in the context of anthracycline exposure and support the rationale for future clinical trials combining standard treatments with ALDH1‐targeted approaches,including the clinical‐stage inhibitor DIMATE. View Publication

DNA repair mechanisms in human primary cells,including error-free repair,and,recurrent nuclease cleavage events,remain largely uncharacterised. We elucidate gene-editing related repair processes using Cleavage and Lesion Evaluation via Absolute Real-time dPCR (CLEAR-time dPCR),an ensemble of multiplexed dPCR assays that quantifies genome integrity at targeted sites. Utilising CLEAR-time dPCR we track active DSBs,small indels,large deletions,and other aberrations in absolute terms in clinically relevant edited cells,including HSPCs,iPSCs,and T-cells. By quantifying up to 90% of loci with unresolved DSBs,CLEAR-time dPCR reveals biases inherent to conventional mutation screening assays. Furthermore,we accurately quantify DNA repair precision,revealing prevalent scarless repair after blunt and staggered end DSBs and recurrent nucleases cleavage. This work provides one of the most precise analyses of DNA repair and mutation dynamics,paving the way for mechanistic studies to advance gene therapy,designer editors,and small molecule discovery. Quantifying genomic aberrations resulting from designer nucleases activity is essential for gene therapy clinical translation. Here,the authors present a modular digital PCR technique that profiles DNA repair precision and cut-repair cycles at the edited loci,exposing current evaluation biases. View Publication

Since the COVID-19 pandemic,there has been a documented rise in the incidence of neurological manifestations among individuals complicated with encephalitis or myelitis. The spectrum of neurological symptoms associated with HCoVs infections is expanding. However,the infection characteristics and pathogenesis of seasonal HCoVs to the central nervous system remain obscure. No pharmacological agents have demonstrated the capacity to specifically and efficaciously mitigate the neurological symptoms induced by HCoVs infections to date. Methods: We developed human cerebral organoids (HCOs) derived from human induced pluripotent stem cells and established a blood–brain barrier (BBB) HCOs co-culture model. We subjected these models to seasonal human coronavirus (HCoV) infections to investigate the viral characteristics within the central nervous system (CNS). Utilizing RNA sequencing,we conducted a preliminary exploration of the mechanisms underlying virus-induced inflammatory responses in the CNS. Furthermore,we assessed the efficacy of antiviral and anti-inflammatory drugs using the HCO model. Results: Our results showed that among seasonal coronaviruses,HCoV-OC43 replicates efficiently within the organoids,primarily targeting neurons and astrocytes,and disrupts the barrier function of the BBB. RNA sequencing analysis revealed that HCoV-OC43 infection triggers an inflammatory response through the TNF and NF-κB signaling pathways,leading to cell death,impaired neuronal function,and disrupted interneuron signaling. Interestingly,Bardoxolone methyl (CDDO-Me) demonstrated antiviral effects comparable to remdesivir,reducing both inflammation and cell death. Conclusions: Conclusively,HCOs infected with HCoV-OC43 offer valuable insights into the pathogenesis of HCoVs in central nervous system (CNS),and might serve as a tool for developing novel therapeutic strategies for HCoVs infections,including COVID-19,especially on exploring treatment candidates.Graphical abstract View Publication

BackgroundCancer stem cells (CSCs) are considered the ‘seeds’ of recurrence after chemotherapy,but eliminating CSCs remains notoriously challenging. This study aims to examine whether cell cycle checkpoint kinase 1 (CHK1) blockade can abrogate the stemness of ovarian cancer (OC) cells,making them easier targets of anti-tumor immunity. Methods: Prexasertib was used to block CHK1 in OC cell lines and xenografts,and its cytotoxicity was assessed in vitro and in vivo. In vitro tumor-sphere formation assays and stemness markers were used to evaluate cell stemness. PD-L1 expressions were examined via qRT-PCR,Western blot,flow cytometry,and immunohistochemistry. Prexasertib in combination with anti-PD-L1 antibody Atezolizumab was tested in immune-proficient mice bearing OC xenografts in terms of effects on tumor growth,tumor cell stemness,and tumor infiltrating lymphocytes via tumor volume monitoring,immunohistochemistry,and flow cytometry. Results: Prexasertib effectively inhibited CHK1 phosphorylation,exhibited significant anti-tumor effects in vitro and in vivo,accompanied by decreased OC cell stemness. CHK1 was highly expressed in tumor spheres versus tumor cells cultured in 2D system,and Prexasertib treatment suppressed sphere formation and reduced the ALDH+ cell fraction. Unexpectedly,Prexasertib upregulated PD-L1 expression in tumor cells. In vivo,combining Prexasertib with Atezolizumab led to more remarkable remission of tumors,when compared with Prexasertib or Atezolizumab alone. Meanwhile,the tumor-infiltrating CD8+ T cells significantly increased in the combination group,while exhausted T cells decreased; the treatments did not affect CD4+ cell infiltration. Conclusions: Dual targeting of CHK1 and PD-L1 may improve OC treatment by simultaneously suppressing stemness and enhancing anti-tumor immunity. View Publication

Hepatocellular carcinoma (HCC) is the most prevalent primary malignancy of the liver. Characterized by rapid progression and poor overall survival rates,HCC requires effective and streamlined treatment regimens. It predominantly occurs in East Asia and sub-Saharan Africa,where it has historically been managed with herbal formulas. We previously observed that the herbal formula HO-1089 exerts potent anti-HCC effects both in vitro and in vivo. In this study,we investigated the anticancer efficacy and mechanisms of HO-1197,a reconstituted herbal formulation derived from HO-1089. HO-1197 selectively inhibited the viability of HCC cell lines without hepatotoxicity and demonstrated superior anticancer activity compared with both HO-1089 and sorafenib. Mechanistically,HO-1197 induced apoptosis and G2/M arrest through reactive oxygen species-mediated DNA damage,independent of p53 status. Transcriptomic analysis revealed downregulation of mitosis-related genes,particularly those regulated by FOXM1,a key driver of HCC proliferation and metastasis. HO-1197 suppressed FOXM1 expression and nuclear translocation,reducing its downstream targets and diminishing angiogenic and metastatic potential. Furthermore,HO-1197 modulated the tumor immune microenvironment by promoting pro-inflammatory macrophage polarization and enhancing natural killer cell-mediated cytotoxicity. HO-1197 exhibited potent antitumor efficacy,and combination therapy with HO-1197 and sorafenib exhibited synergistic effects in both two-dimensional and immune-activated multicellular spheroid models. These findings suggest that HO-1197 is a promising multifunctional therapeutic candidate with antitumor,antiangiogenic,antimetastatic,and immunomodulatory properties. Its combination with sorafenib may offer effective treatment for HCC. HO-1197,which demonstrated strong efficacy,is a novel herbal medicine developed by H&O Biosis and is referred to as an Integrated Natural Medicine. View Publication