技术资料

JAK (Janus Kinase) inhibitors,such as ruxolitinib,were introduced a decade ago for treatment of myeloproliferative neoplasms (MPN). To evaluate ruxolitinib’s impact on MPN clonal evolution,we interrogate a myelofibrosis patient cohort with longitudinal molecular evaluation and discover that ruxolitinib is associated with clonal outgrowth of RAS pathway mutations. Single-cell DNA sequencing combined with ex vivo treatment of RAS mutated CD34 + primary patient cells,demonstrates that ruxolitinib induces RAS clonal selection both in a JAK/STAT wild-type and hyper-activated context. RAS mutations are associated with decreased transformation-free and overall survival only in patients treated with ruxolitinib. In vitro and in vivo competition assays demonstrate increased cellular fitness of RAS- mutated cells under ruxolitinib or JAK2 knock-down,consistent with an on-target effect. MAPK pathway activation is associated with JAK2 downregulation resulting in enhanced oncogenic potential of RAS mutations. Our results prompt screening for pre-existing RAS mutations in JAK inhibitor treated patients with MPN. Subject terms: Myeloproliferative disease,Cancer therapeutic resistance,Tumour heterogeneity,Cancer genetics View Publication

Schwann cells (SCs) play a crucial role in peripheral nerve repair by supporting axonal regeneration and remyelination. While extensive research has been conducted using rodent SCs,increasing attention is being directed toward human SCs due to species-specific differences in phenotypical and functional properties,and accessibility of human SCs derived from diverse sources. A major challenge in translating SC-based therapies for nerve repair lies in the inability to replicate human SC myelination in vitro,posing a significant obstacle to drug discovery and preclinical research. In this study,we compared the myelination capacity of human,rodent,and porcine SCs in various co-culture conditions,including species-matched and cross-species neuronal environments in a serum-free medium. Our results confirmed that rodent and porcine SCs readily myelinate neurites under standard culture conditions after treatment with ascorbic acid for two weeks,whereas human SCs,at least within the four-week observation period,failed to show myelin staining in all co-cultures. Furthermore,we investigated whether cell culture manipulation impairs human SC myelination by transplanting freshly harvested and predegenerated human nerve segments into NOD-SCID mice for four weeks. Despite supporting host axonal regeneration into the grafts,human SCs exhibited very limited myelination,suggesting an intrinsic species-specific restriction rather than a cell culture-induced defect. These observations suggest fundamental differences between human and rodent SCs and highlight the need for human-specific models and protocols to advance our understanding of SC myelination. View Publication

Reticular dysgenesis (RD) is a rare genetic disorder caused by mutations in the adenylate kinase 2 ( AK2 ) gene. It is characterized by a T − B − severe combined immunodeficiency,agranulocytosis,and sensorineural deafness. We established and characterized a haematopoiesis‐specific conditional Ak2 ‐knockout mouse model to provide a model system to study the molecular pathophysiology of RD. As expected from the human phenotype of RD,haematopoiesis‐specific AK2‐deficient embryos had a small,atrophic thymus consisting mainly of epithelial cells. No recognizable T‐cell component was observed,but B‐cell lineage precursor cells were present in the foetal liver. The effects of AK2 deficiency on myelopoiesis were less severe in mice than in humans. The absolute numbers of monocytes,macrophages,granulocytes and megakaryocytes in foetal liver as well as colony‐forming precursors were not reduced. In contrast to humans,haematopoiesis‐specific Ak2 ‐knockout mice exhibit embryonic lethality between E13 and E15 due to severe anaemia caused by an early block in definitive erythropoiesis. Murine erythroid progenitors mainly express AK2 and only low levels of functionally related kinases,which are unable to compensate for AK2 deficiency,in contrast to human erythroid progenitors. View Publication

Rotavirus (RV),a primary cause of diarrhea-related mortality in 2021,has been shown to damage intestinal epithelial cells while upregulating intestinal stem cells (ISCs) activities. ISCs within the crypt niche drive the continuous self-renewal of intestinal epithelium,preserving its barrier functions. Paneth cells secrete antimicrobial peptide and signaling molecules within the intestine crypt,thereby playing a crucial role in intestinal immune defense and providing ISCs functional support. However,the regulatory function of Paneth cells under pathological conditions,such as RV infection,remains unclear. To determine the impact of RV infection on Paneth cells and how Paneth cells regulate ISCs during intestinal injury repair. We constructed a reference genome for the RV enteric cytopathogenic human orphan virus strain and reanalyzed published single-cell RNA sequencing data to investigate Paneth cell responses to RV-induced intestinal injury. We derived Paneth-ISC communication networks using CellChat,tracked ISC differentiation with pseudotime analysis,and validated our findings in leucine-rich repeat-containing G protein-coupled receptor 5-enhanced green fluorescent protein-internal ribosomal entry site-Cre recombinase estrogen receptor variant 2 mice and organoids via immunofluorescence,flow cytometry,and reverse transcription quantitative polymerase chain reaction. We found that RV directly infects Paneth cells,leading to a reduction in mature Paneth cells and an increase in kallikrein 1-high immature Paneth cells. Paneth-ISC communication was significantly enhanced. In particular,the bone morphogenic protein 7 (BMP7)-activin A receptor type 2B/BMP receptor type 1A-Smad pathway was upregulated post-infection,suggesting that Paneth cells suppress excessive ISC proliferation. Functional validation confirmed activation of this pathway. Paneth cells regulate ISC proliferation during RV infection by activating BMP7 signaling,limiting excessive stem cell expansion and preserving crypt homeostasis for effective epithelial repair. View Publication

uveal melanoma (UM) is the most common primary intraocular tumor in adults,with metastasis being the leading cause of death. However,effective treatments for metastatic UM remain limited. Emerging evidence suggests that cholesterol metabolism plays a role in cancer progression,but its impact on UM metastasis is not well understood. we investigated the effects of miR-181a on UM metastasis using multiple UM cell lines and a suprachoroidal injection mouse model. Functional assays,including migration,invasion,and cancer stem-like cell (CSC) formation,were performed. The target of miR-181a was identified through bioinformatics,luciferase assays,and western blotting. Cholesterol levels were measured,and in vitro and in vivo studies assessed the therapeutic potential of combining miR-181a with crizotinib. miR-181a significantly decreases UM cell migration,invasion,and metastasis. Mechanistically,miR-181a was found to target sterol regulatory element-binding protein 2 (SREBP2),thereby inhibiting cholesterol biosynthesis. This decrease in cholesterol levels hindered reduced epithelial-to-mesenchymal transition (EMT) and led to a decline in cancer stem-like cell (CSC) populations in UM. Furthermore,elevated cholesterol or overexpression of SREBP2 abrogated the anti-metastatic effects of miR-181a. Additionally,a combination of miR-181a and crizotinib significantly inhibited metastasis,both in vitro and in vivo. miR-181a inhibits UM metastasis by targeting SREBP2 and reducing cholesterol biosynthesis. Its combination with crizotinib may provide a promising therapeutic strategy for metastatic UM. The online version contains supplementary material available at 10.1186/s13046-025-03459-8. View Publication

Resistance to chemotherapy remains a major clinical challenge in triple-negative breast cancer (TNBC),an intrinsic subtype with limited available therapeutic options. The expression of moesin (MSN) is upregulated in TNBC patients,but little is known about the role of MSN in breast carcinogenesis. We investigated the MSN-dependent autocrine loop between extracellular interleukin 6 (IL-6) and NF-κB,along with a signaling cascade involving GTPase-mediated STAT3 phosphorylation. Various in vitro and in vivo assays were used to evaluate tumor initiation,growth,and stemness properties in TNBC models. High MSN expression was correlated with shorter overall and disease-free survival in TNBC patients. In vivo,MSN promotes tumor initiation and growth. Mechanistically,MSN-mediated IL-6/NF-κB autoregulatory feedback enhances IL-6 transcription. IL-6 binding to LPAR1 activated MSN phosphorylation,which then sequentially phosphorylated the CDC42-PAK4 complex,triggering nuclear translocation of the pSTAT3-MSN complex. This led to pSTAT3-mediated activation of cancer stemness genes (IGFN1,EML1,and SRGN),contributing to Adriamycin resistance. Notably,combination treatment with the FDA-approved STAT3 inhibitor Atovaquone and Adriamycin restored drug sensitivity. Our findings uncover the critical role of MSN in regulating STAT3-mediated cancer stemness via the IL-6/NF-κB signaling axis. These results provide a strong rationale for repositioning STAT3 inhibitors such as Atovaquone as a therapeutic strategy in Adriamycin-resistant TNBC patients exhibiting pSTAT3-MSN complex upregulation. The online version contains supplementary material available at 10.1186/s13058-025-02072-z. View Publication

Background: Human cytomegalovirus (CMV) is a major pathogen that poses significant risks to immunocompromised individuals and neonates. The tegument protein pp71,encoded by the UL82 gene,plays a pivotal role in initiating viral lytic replication and evading host immune responses. Despite its clinical relevance,standardized monoclonal antibodies (mAbs) for pp71 remain limited,prompting the need to expand the available repertoire of antibodies targeting this critical protein. Methods: In this study,we constructed a diverse human single-chain variable fragment (scFv) library using RNA derived from the B cells of four healthy donors. The library was expressed in Saccharomyces cerevisiae,and iterative rounds of magnetic-activated cell sorting (MACS) were performed against recombinant pp71. Clonal enrichment was monitored using flow cytometry. Results: Among the isolated clones,one designated ID2 exhibited high sensitivity and specificity for pp71,as demonstrated by flow cytometry,immunofluorescence,an enzyme-linked immunosorbent assay (ELISA),and biolayer interferometry (BLI). Conclusions: Collectively,these findings establish a novel pp71-specific mAb and underscore the utility of yeast surface display combined with MACS for expanding the antibody toolkit available for CMV research and diagnostics. View Publication

We have developed the HybriSeq method for single-cell RNA profiling,which utilizes in situ hybridization of multiple probes for targeted transcripts,followed by split-pool barcoding and sequencing analysis of the probes. We have shown that HybriSeq can achieve high sensitivity for RNA detection with multiple probes and profile entire transcripts without an end bias. The utility of HybriSeq is demonstrated in characterizing cell-to-cell heterogeneities of a panel of 196 genes in peripheral blood mononuclear cells and the detection of missed annotations of transcripts. Subject terms: Gene expression profiling,RNA sequencing View Publication

Epigenetic modulators of the histone deacetylase (HDAC) family control key biological processes and are frequently dysregulated in cancer. There is superior activity of HDAC inhibitors (HDACi) in patients with myeloproliferative neoplasms (MPNs) that carry the Janus kinase-2 point mutant JAK2 V617F . This constitutively active tyrosine kinase activates signal-transducer-and-activator-of-transcription (STAT) transcription factors to promote cell proliferation and inflammatory processes. We reveal that the inhibition of HDAC1/HDAC2 with the clinically advanced HDACi romidepsin,the experimental HDACi entinostat and MERCK60,and genetic depletion of HDAC1/HDAC2 induce apoptosis and long-term growth arrest of primary and permanent MPN cells in vitro and in vivo. This treatment spares normal hematopoietic stem cells and does not compromise blood cell differentiation. At the molecular level,HDAC1 and HDAC2 control the protein stability of SIAH2 through acetylation. Genetic knockout experiments show that SIAH2 accelerates the proteasomal degradation of JAK2 V617F in conjunction with the E2 ubiquitin-conjugating enzyme UBCH8. SIAH2 binds to the surface-exposed SIAH degron motif VLP1002 in the catalytic domain of JAK2 V617F . At the functional level,SIAH2 knockout MPN cells are significantly less sensitive to HDACi. Global RNA sequencing verifies that JAK-STAT signaling is a prime target of SIAH2. Moreover,HDAC1 is an adverse prognostic factor in patients with acute myeloid leukemia ( n = 150,p = 0.02),being a possible complication of MPNs. These insights reveal a previously unappreciated link between HDAC1/HDAC2 as key molecular targets,the still undefined regulation of cytoplasmic-to-nuclear signaling by HDACs,and how HDACi kill JAK2 V617F -positive cells from MPN patients and mice with JAK2 V617F in vitro and in vivo. Subject terms: Haematological cancer,Oncogenes,Target identification,Haematopoietic stem cells View Publication

Metformin rejuvenates adult rat oligodendrocyte progenitor cells (OPCs) allowing more efficient differentiation into oligodendrocytes and improved remyelination,and therefore is of interest as a therapeutic in demyelinating diseases such as multiple sclerosis (MS). Here,we test whether metformin has a similar effect in human stem cell derived-OPCs. We assess how well human monoculture,organoid and chimera model culture systems simulate in vivo adult human oligodendrocytes,finding most close resemblance in the chimera model. Metformin increases myelin proteins and/or sheaths in all models even when human cells remain fetal-like. In the chimera model,metformin leads to increased mitochondrial area both in the human transplanted cells and in the mouse axons with associated increase of mitochondrial function/metabolism transcripts. Human oligodendrocytes from MS brain donors treated pre-mortem with metformin also express similar transcripts. Metformin’s brain effect is thus not cell-specific,alters metabolism in part through mitochondrial changes and leads to more myelin production. This bodes well for clinical trials testing metformin for neuroprotection. Subject terms: Oligodendrocyte,Multiple sclerosis,Multiple sclerosis,Regeneration and repair in the nervous system View Publication

Chronic myeloid leukemia (CML) is a clonal malignancy propelled by the BCR::ABL1 fusion gene originating from the Philadelphia chromosome. This gene activates ABL tyrosine kinase,which enhances the survival of leukemic cells. Although tyrosine kinase inhibitors (TKIs) have significantly advanced the treatment of CML,resistance to these inhibitors presents a substantial hurdle. Consequently,novel therapeutic strategies targeting resistance mechanisms independent of BCR::ABL1 are urgently needed. This study investigated the potential impact of combining WEE1 inhibitors,particularly MK‐1775,with vitamin K2 (VK2) in treating CML. To analyze differentially expressed and spliced transcripts in CML,we examined mRNA profiles from peripheral blood mononuclear cells of five patients with CML (during chronic and blast phases) and five healthy controls. The samples were analyzed using deep sequencing. Differential expression analyses were performed using RaNA‐Seq and Heatmapper,the latter of which was designed for complex data set visualizations. WEE1 controls the G2/M checkpoint to prevent early mitosis,and blocking it increases the cytotoxicity of agents that damage deoxyribonucleic acid,especially in cancers lacking p53. VK2,a micronutrient,exerts anticancer effects against various malignancies. Gene expression studies have indicated that PKMYT1 expression is elevated in CML but not WEE1 cells. MK‐1775 successfully halted the growth of both standard and TKI‐resistant CML cell lines by triggering apoptosis via caspase 3/7 activation. VK2 reduced the viability of CML cells and increased cytotoxicity. A combined regimen of MK‐1775 and VK2 markedly decreased colony growth,disrupted mitochondrial membrane potential,and increased death in CML cells,including those resistant to TKIs. The results suggest that a combination of MK‐1775 and VK2 represents a potentially effective treatment strategy for CML,especially in drug‐resistant cases. View Publication

Suspension bath bioprinting,whereby bioinks are extruded into a yield stress bath with rapid recovery from shearing,has enabled the printing of low viscosity bioinks into constructs with high geometric complexity. Previous studies have often relied upon external stabilisation of the suspension bath (e.g. collagen) in order to culture soft materials without loss of printed structure. Here,we report a systematic investigation of suspension bath properties that support the printing,fusion,and culture of spheroid-based bioinks without added stabilisation. Specifically,agarose fluid gels of varied polymer concentrations and dilutions were produced and characterised morphologically and rheologically. Juvenile bovine chondrocytes or mesenchymal stromal cells (MSCs) were formed into spheroids of ∼150 µ m in diameter and investigated within agarose suspension baths either for their fusion in hanging drop cultures or as jammed bioinks. MSC spheroids were also printed when mixed with hydrogel microparticles to demonstrate additional versatility to the approach. Suspension baths of lower polymer concentrations and increased dilution enabled faster spheroid fusion; however,the most heavily diluted suspension bath was unable to maintain print fidelity. Other formulations supported the printing,fusion,and culture of spheroid-based inks,either as simple lines or more complex patterns. These findings help to inform the design of suspension baths for bioprinting and culture. View Publication