技术资料

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

Alzheimer’s disease (AD) involves cognitive decline,amyloid-beta (Aβ) accumulation,tau hyperphosphorylation,and neuroinflammation. CREB1,a key transcription factor for memory,is downregulated in AD,contributing to disease progression. Phosphodiesterases 4 and 10 (PDE4 and PDE10) are key enzymes that degrade cAMP,a second messenger involved in CREB signaling,synaptic plasticity,and neuroprotection. Dysregulation of PDE activity has been implicated in AD and other neurodegenerative disorders. Methods: We used human iPSC-derived cortical neurons and microglia,along with the APP/PS1 mouse model,to investigate the role of CREB1 and assess the therapeutic potential of dual PDE4/10A inhibition in AD. Results: CREB1 deficiency in neurons increased Aβ and p-tau231 accumulation. Dual inhibition of PDE4 and PDE10A activated the cAMP-PKA-CREB pathway,restoring CREB1 activity,reducing Aβ and p-tau231,and mitigating neuroinflammation. This intervention improved synaptic plasticity and cognitive performance in vivo. Conclusions: Our findings demonstrate that dual PDE4/10A inhibition synergistically enhances the cAMP-PKA-CREB signaling,promoting neuroprotection and synaptic remodeling. This approach offers a promising therapeutic strategy for modifying AD pathology and restoring cognitive function. View Publication

A growing body of evidence implicates inflammation as a key hallmark in the pathophysiology of Parkinson’s disease (PD),with microglia playing a central role in mediating neuroinflammatory signaling in the brain. However,the molecular mechanisms linking microglial activation to dopaminergic neuron degeneration remain poorly understood. In this study,we investigated the contribution of the PD-associated LRRK2-G2019S mutation to microglial neurotoxicity using patient-derived induced pluripotent stem cell (iPSC) models. We found that LRRK2-G2019S mutant microglia exhibited elevated activation markers,enhanced phagocytic capacity,and increased secretion of pro-inflammatory cytokines such as TNF-α. These changes were associated with metabolic dysregulation,including upregulated glycolysis and impaired serine biosynthesis. In 3D midbrain organoids,these overactivated microglia resulted in dopaminergic neuron degeneration. Notably,treating LRRK2-G2019S microglia with oxamic acid,a glycolysis inhibitor,attenuated microglial inflammation and reduced neuronal loss. Our findings underscore the link between metabolic targeting in microglia and dopaminergic neuronal loss in LRRK2-G2019S mutation,and highlight a potential strategy that warrants further preclinical evaluation. 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

Respiratory organoids have emerged as a powerful in vitro model for studying respiratory diseases and drug discovery. However,the high-throughput analysis of organoid images remains a challenge due to the lack of automated and accurate segmentation tools. This study presents a semi-automatic algorithm for image analysis of respiratory organoids (nasal and lung organoids),employing the U-Net architecture and CellProfiler for organoids segmentation. The algorithm processes bright-field images acquired through z-stack fusion and stitching. The model demonstrated a high level of accuracy,as evidenced by an intersection-over-union metric (IoU) of 0.8856,F1-score = 0.937 and an accuracy of 0.9953. Applied to forskolin-induced swelling assays of lung organoids,the algorithm successfully quantified functional differences in Cystic Fibrosis Transmembrane conductance Regulator (CFTR)-channel activity between healthy donor and cystic fibrosis patient-derived organoids,without fluorescent dyes. Additionally,an open-source dataset of 827 annotated respiratory organoid images was provided to facilitate further research. Our results demonstrate the potential of deep learning to enhance the efficiency and accuracy of high-throughput respiratory organoid analysis for future therapeutic screening applications. Author summaryIn this study,we developed a semi-automated tool to analyze images of respiratory organoids—3D cell structures that mimic the human respiratory system. These organoids are vital for studying diseases like cystic fibrosis and testing potential drugs,but manually analyzing their images is time-consuming and prone to errors. Our tool uses artificial intelligence (AI) to quickly and accurately measure organoid size and shape from bright-field microscope images,eliminating the need for fluorescent dyes that can harm cells. We trained our AI model on a publicly shared dataset of 827 annotated organoid images,achieving high accuracy in detecting and quantifying organoids. When applied to cystic fibrosis research,the tool successfully measured differences in organoid swelling (forskolin-induced swelling - a key test for drug response) between healthy and patient-derived samples. By making our dataset and method openly available,we hope to support further research into respiratory diseases. Our work bridges the gap between complex lab techniques and practical applications,offering a faster,more reliable way to study human health and disease. View Publication

Objective: To develop a rapid functional assay to validate variants of uncertain significance (VUS) in the MEFV gene. Methods: Overactivity of the pyrin inflammasome pathway and ASC speck oligomerisation in response to stimulation with low concentrations of Clostridium difficile toxin A was directly visualised by immunofluorescence microscopy. A semi‐automated algorithm was developed to count cells and ASC specks. Results: The semi‐automated ASC speck assay is able to discriminate between healthy controls and patients with familial Mediterranean fever (FMF) and pyrin inflammasome overactivity with high sensitivity. It is also able to discriminate pyrin inflammasome overactivity from other autoinflammatory disease controls with high specificity. Conclusion: The semi‐automated ASC speck assay may be a useful test to functionally validate VUS in the MEFV gene and screen for pyrin inflammasome overactivity. A semi‐automated ASC speck assay using machine learning is able to discriminate between healthy controls and patients with familial Mediterranean fever (FMF) with high sensitivity. It is also able to discriminate FMF from other autoinflammatory diseases with high specificity. View Publication

Aggressive non‐Hodgkin lymphoma (aNHL) often relapses after first‐line treatment. Clinical data supports the safety and efficacy of the combination of mosunetuzumab,a CD20×CD3 bispecific antibody,and polatuzumab vedotin,an anti‐CD79b antibody drug conjugate (Mosun‐Pola) in relapsed/refractory aNHL. This study investigated the molecular mechanism behind the combination effect of Mosun‐Pola in human diffuse large B‐cell lymphoma (DLBCL) cell lines. Methods: The in vitro Mosun‐Pola efficacy in DLBCL cells (SU‐DHL‐8 and HT) was evaluated by T cell‐dependent cellular cytotoxicity (TDCC) assay. CD20‐stable‐knockdown SU‐DHL‐8 cells were established using lentiviral short hairpin RNA. Surface and T‐cell activation marker proteins expression were determined by flow cytometry. Human T‐cell‐injected mice or humanized NOD/Shi‐scid,IL‐2Rγnull (huNOG) mice were used for an in vivo study. Results: An in vitro TDCC assay showed a synergistic effect in SU‐DHL‐8 and HT cells. Based on our experimental results of suppressing CD20 expression,it was suggested that this combination effect could be caused by an increase in CD20 expression by polatuzumab vedotin. In addition,examining the effects of CD20 upregulation in tumor cells on T‐cell activation demonstrated that the combination of Mosun‐Pola enhanced T‐cell activation markers in both CD4+ and CD8+ T cells during the TDCC reaction. In vivo studies,using human immune system‐reconstituted mouse models confirmed that polatuzumab vedotin enhanced CD20 expression in tumors,and the combination of Mosun‐Pola showed significantly improved anti‐tumor effects compared with single‐drug treatments. Conclusions: These findings suggest that polatuzumab vedotin‐induced CD20 upregulation provides a molecular rationale to explain the synergistic effect of this combination therapy.Trial RegistrationThe authors have confirmed clinical trial registration is not needed for this submission. View Publication

Tolerogenic dendritic cells (tolDCs) that dampen T cell responses can be induced from blood monocytes in vitro using factors such as Vitamin D3 (VitD),dexamethasone,IL‐10,or rapamycin. However,challenges remain in obtaining robust and efficient generation of cell therapy‐based tolDCs without compromising their viability. We recently reported that CCR2‐dependent recruitment of monocytic cells,with the capacity to dampen T‐helper responses,occurs in mice treated with a single‐stranded oligonucleotide (ssON). Here,we investigated the effects of this immunomodulatory noncoding ssON on differentiating human monocytes towards DC in the presence of IL‐4 and GM‐CSF (moDC). The moDC differentiated in the presence of ssON upregulated CD1a but also increased their expression of PD‐L1. The differentiation of monocytes to moDC in the presence of ssON introduced transcriptomic changes,many of which overlapped with VitD‐moDC and resulted in moDCs with altered lipopolysaccharide (LPS)‐responsiveness. Moreover,ssON‐moDC exhibited a low capacity to stimulate alloreactive T cells in vitro and instead promoted the induction of CD4+FoxP3+CD25+ T cells. Experiments using chemical reagents support a role for PPAR‐γ in the generation of ssON‐moDC. Collectively,our data show that monocytes differentiated with IL‐4,GM‐CSF,and ssON generate cells with phenotypic and functional characteristics of tolDCs. In this article,the authors elucidated the immunoregulatory role of an oligonucleotide (ssON) that favors the induction of human tolerogenic dendritic cells (DC). The tolerogenic profile was evidenced by reduced responsiveness to lipopolysaccharides (LPS) (A). Importantly,the tolerogenic DCs had upregulated PD‐L1 molecules and functionally inhibited the proliferation of alloreactive T cells and induced FoxP3+ Tregs (B). This study envisions the development of ssON as therapeutic for rebalancing overactive T‐helper cell responses. View Publication

Ataxia with oculomotor apraxia type 1 (AOA1) is a rare,autosomal recessive,early-onset,progressive cerebellar ataxia caused by mutations in the APTX gene,which encodes aprataxin,a DNA-adenylate hydrolase involved in DNA damage repair. The pathogenesis of AOA1 remains unclear. The purpose of this study was to investigate the pathogenesis of a novel mutation,p.H201P/H201R,carried by our AOA1 patient and the mechanism of AOA1 in an induced pluripotent stem cells (iPSCs) model. We edited iPSCs derived from a healthy individual to carry the APTX homozygous mutation p.H201P (H201P-iPSCs) or p.H201R (H201R-iPSCs) via CRISPR/Cas9. We found that aprataxin expression was absent in both H201P- and H201R-iPSCs. The capacity of these APTX-mutant iPSCs to differentiate into neural progenitor cells (NPCs) and mature neurons was diminished. We observed an increase in DNA single-strand breaks (SSB) via a comet assay and poly(ADP-ribose) staining,and an increase in the ratio of cleaved PARP-1/total PARP-1 in APTX-mutant NPCs and early immature neurons (EiNs),in addition of a heightened sensitivity to tert-butyl hydroperoxide in APTX-mutant EiNs. Moreover,a decrease of APE1 expression was observed in APTX-mutant NPCs and H201R-EiNs during neural differentiation. Our study established a practical iPSCs model to investigate AOA1 disease. We found that mutant aprataxin leads to defective neural differentiation,accompanied by the accumulation of DNA SSBs with increased cleaved PARP-1 and reduced APE1 expression of the base excision repair pathway. 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

In stem cell biology,a long-held structure–function relationship is the domed colony morphology and naïve pluripotency for mouse or human pluripotent stem cells. This link has provided a convenient way to recognize bona fide naïve pluripotent cells during derivation,passaging and characterization. However,the molecular basis of this link remains poorly understood. Results: We show that a loss of domed morphology may not impact the overall genetic architecture of naïve pluripotency in mouse embryonic stem cells (mESCs). We first generated stable mESC lines by knocking out Myh9 that encodes non-muscle myosin heavy chain IIA,resulting in colonies deprived of the typical domed morphology,but competent to differentiate into the three germ layers and chimeric mice. Modulating cell morphologies with inhibitors against kinases known to regulate myosin pathway also phenocopy the knockout in wild type mESCs. Conclusions: These results provide evidence that the domed morphology and potency can be uncoupled and suggest that domed structure is not a pre-requisite for acquiring and maintaining naïve pluripotency. 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