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Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the uncontrolled proliferation of white blood cells. Tyrosine kinase inhibitors (TKIs) are the standard treatment; however, resistance to BCR::ABL1 mutations remains challenging. WEE1, a checkpoint kinase involved in mitosis and DNA repair, is a potential therapeutic target for CML treatment. Ponatinib-resistant CML cells were screened to identify candidates for overcoming drug resistance. The efficacy of the ABL TKI asciminib and the WEE1 inhibitor MK-1775 was evaluated using proliferation and colony formation assays. Public database analysis ( GSE100026 ) assessed WEE1/PKMYT1 expression in CML. In vitro screening identified MK-1775 as a promising therapeutic candidate. WEE1/PKMYT1 expression was elevated in CML cells compared to healthy cells. Both asciminib and MK-1775 inhibited CML cell proliferation after 72 h, with enhanced cytotoxicity when combined. Co-treatment reduced colony formation and induced G2/M arrest, whereas an increase in the sub-G1 cell population indicated apoptosis. Furthermore, the combination treatment disrupted the mitochondrial membrane potential. The combination of asciminib and WEE1 inhibition demonstrated greater efficacy than either drug alone, suggesting a novel therapeutic strategy for treating CML. These findings provide insights into overcoming TKI resistance and highlight a promising approach for future clinical applications. The online version contains supplementary material available at 10.1007/s12672-025-03036-7.

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

Duplication 15q (dup15q) syndrome is a leading genetic cause of autism spectrum disorder, offering a key model for studying autism-related mechanisms. Using single-cell and single-nucleus RNA sequencing of cortical organoids from dup15q patient-derived iPSCs and post-mortem brain samples, we identify increased glycolysis, disrupted layer-specific marker expression, and aberrant morphology in deep-layer neurons during fetal-stage organoid development. In adolescent-adult postmortem brains, upper-layer neurons exhibit heightened transcriptional burden related to synaptic signaling, a pattern shared with idiopathic autism. Using spatial transcriptomics, we confirm these cell-type-specific disruptions in brain tissue. By gene co-expression network analysis, we reveal disease-associated modules that are well preserved between postmortem and organoid samples, suggesting metabolic dysregulation that may lead to altered neuron projection, synaptic dysfunction, and neuron hyperexcitability in dup15q syndrome. Subject terms: Autism spectrum disorders, Autism spectrum disorders, Disease model

Neuroblastoma (NB) is the most common extracranial solid tumor in children characterized by poor immune infiltration and resistance to adaptive immunity, contributing to its limited response to immunotherapy. A key mechanism underlying immune evasion in cancer is autophagy, a cellular process that plays many roles in cancer by supporting tumor survival and regulating immune interactions. In this study, we investigate the impact of autophagy inhibition on NB tumor growth, immune modulation, and the efficacy of immunotherapy. Using both murine and human NB cell lines, we demonstrate that genetic and pharmacological inhibition of autophagy significantly reduces 3D spheroid growth and upregulates major histocompatibility complex class I (MHC-I) expression. In vivo studies further confirm that targeting autophagy suppresses tumor progression and promotes immune infiltration into the tumor. Notably, we observe a significant increase in CD8 + T cell recruitment and activation, suggesting that autophagy inhibition reshapes the immune landscape of NB, rendering it more susceptible to immune-mediated clearance. Crucially, autophagy inhibition also sensitizes NB cells to T cell-mediated cytotoxicity and enhances the therapeutic efficacy of GD2.CAR T-cell therapy. In vitro co-culture assays reveal increased CAR T cell-mediated tumor killing upon autophagy blockade, while in vivo models show prolonged tumor control and improved survival in treated mice compared to CAR T-cell therapy alone. These findings highlight autophagy as a key regulator of immune evasion in NB and suggest that its inhibition could serve as a promising therapeutic strategy to enhance immune recognition and improve the efficacy of immunotherapy. The online version contains supplementary material available at 10.1186/s13046-025-03453-0.

R2 elements, a class of non-long terminal repeat (non-LTR) retrotransposons, have the potential to be harnessed for transgene insertion. However, efforts to achieve this are limited by our understanding of the retrotransposon mechanisms. Here, we structurally and biochemically characterize R2 from Taeniopygia guttata (R2Tg). We show that R2Tg cleaves both strands of its ribosomal DNA target and binds a pseudoknotted RNA element within the R2 3′ UTR to initiate target-primed reverse transcription. Guided by these insights, we engineer and characterize an all-RNA system for transgene insertion. We substantially reduce the system’s size and insertion scars by eliminating unnecessary R2 sequences on the donor. We further improve the integration efficiency by chemically modifying the 5′ end of the donor RNA and optimizing delivery, creating a compact system that achieves over 80% integration efficiency in several human cell lines. This work expands the genome engineering toolbox and provides mechanistic insights that will facilitate future development of R2-mediated gene insertion tools. Subject terms: Transferases, Protein design, Genetic engineering

Our previous studies demonstrated that FOSL1 promotes glioblastoma (GBM) progression and stemness through pathways such as STAT3 and NF-κB signaling. Recently, we identified that FOSL1 physically interacts with the nuclear E3 ligase TRIM21. This study investigates the role of TRIM21 in GBM, including its interaction with FOSL1, its regulation of FOSL1 transactivation, and its ubiquitination-mediated degradation of tumor suppressor p27. Immunoprecipitation assays were used to evaluate the interactions between TRIM21, FOSL1, and p27. TRIM21 expression was manipulated through overexpression and siRNA-mediated knockdown to assess its effects on p27 levels and ubiquitination. TCGA and CGGA datasets were analyzed to explore correlations between TRIM21 expression, glioma subtypes, and patient survival. Glioma cell proliferation (MTT and colony formation) and invasion (transwell assays) were evaluated following TRIM21 manipulation. Immunohistochemistry on glioma patient tissue microarray (TMA) assessed TRIM21 expression and its association with FOSL1, IDH status, and glioma grade. The role of nuclear TRIM21 in FOSL1 promoter transactivation was analyzed via AP-1 binding sites. TCGA and CGGA revealed that TRIM21 is highly expressed in GBM, particularly in the mesenchymal subtypes, and correlates with poor survival outcomes. Functional assays demonstrated that TRIM21 enhances glioma cell proliferation and invasion. Immunohistochemistry confirmed elevated TRIM21 levels in gliomas, positively correlating with FOSL1 expression and glioma grade, and inversely correlating with IDH1 wild-type status. Mechanistically, TRIM21 physically interacts with FOSL1 and p27, driving tumorigenesis by transactivating FOSL1 via AP-1 binding sites and promoting p27 ubiquitination and degradation. These functions are mediated through TRIM21’s RING domain for p27 degradation and its PRYSPRY domain for FOSL1 regulation. TRIM21 functions as an oncogene in GBM by degrading the tumor suppressor p27 and promoting FOSL1 transactivation. These findings highlight TRIM21 as a promising therapeutic target in GBM. The online version contains supplementary material available at 10.1186/s12964-025-02325-6.

Terminal erythropoiesis is a complex multistep process involving coordination of gene transcription and dramatic nuclear condensation, which leads to the expulsion of nuclei to generate reticulocytes. However, we lack a comprehensive understanding of the key transcriptional and epigenetic regulators involved. We used a high-throughput small molecule screen in primary CD34 + -derived human erythroblasts to identify targets that promoted terminal erythropoiesis, and further confirmed the phenotype in different differentiation systems by inhibitors and shRNAs of different BRD4 isoforms. Then we performed RNA-seq, ATAC-seq, ChIP-qPCR, Co-IP, and reanalyzed previously-published transcriptional data and mass spectrometric data to clarify how BRD4 regulates terminal erythropoiesis. We identified that inhibitors of the bromodomain protein BRD4, an epigenetic reader and transcriptional activator together with CDK9, promoted terminal erythropoiesis from hematopoietic stem/progenitor cells and embryonic stem cells, and enhanced enucleation. Combined analysis of our RNA-seq, ATAC-seq, and previously-published transcriptional data of erythroblast differentiation at different stages confirmed that BRD4 inhibition accelerates erythroblast maturation. Unexpectedly, this BRD4 function was independent of its classical CDK9 interaction and transcriptional activation. Instead, RNA-seq, ATAC-seq, and Cut&Tag upon BRD4 inhibition revealed that BRD4 regulates erythropoiesis by inhibiting the small G protein RhoB and disrupts actin reorganization. ChIP-qPCR, Co-IP, and functional studies revealed that BRD4 acts as a transcriptional repressor by interacting with the histone methyltransferase EHMT1/2. We demonstrate a non-classical role for BRD4 as a transcriptional repressor of RhoB to regulate erythroid maturation, and classical CDK9 dependent role to regulate cell proliferation of erythroblasts. Besides, we clarify RhoB’s activity and function during terminal erythropoiesis. BRD4 inhibition might be a simple method to promote in vitro blood cell production, and a candidate therapeutic target for diseases leading to dyserythropoiesis such as myelodysplastic syndromes. The online version contains supplementary material available at 10.1186/s13045-025-01721-2.

p18 INK4 C (CDKN2C, encoded by p18 INK4c or Cdkn2c ) is an early G1-phase cyclin-dependent kinase inhibitor protein. Previous studies demonstrated enhanced self-renewal capacity of hematopoietic stem cells (HSCs) in p18 −/− mice compared to wild-type (WT) mice. Given the critical role of bone marrow niche cells-particularly mesenchymal stromal cells (MSCs)-in hematopoiesis, this study investigated the functional alterations of p18 −/− MSCs and their impact on hematopoietic support. Bone marrow derived MSCs were isolated from p18 −/− and WT mice. Their proliferation and differentiation capacities were assessed, followed by evaluation of hematopoietic support using cobblestone area-forming cell assay and long-term culture-initiating cell assay. RNA sequencing was performed to analyze the transcriptional profile of p18 −/− MSCs, with a focus on differentially expressed genes (DEGs). Key pathways associated with hematopoietic support were identified using Ingenuity Pathway Analysis. A candidate protein was quantified by ELISA, and its functional role in hematopoietic support was validated via a modified coculture system. p18 −/− MSCs displayed an increased proliferation rate, preferential differentiation toward osteogenesis over adipogenesis, and enhanced hematopoietic support. RNA sequencing analysis identified 137 DEGs, with secreted phosphoprotein 1 ( Spp1 , encoding osteopontin, Opn) being significantly upregulated in p18 −/− MSCs. Elevated Opn levels were confirmed in both bone marrow and MSC-conditioned media from p18 −/− mice. Functional validation further demonstrated that Opn enhanced the hematopoietic supportive capacity of MSCs in vitro. p18 deficiency promotes osteogenic differentiation and enhances the hematopoietic supportive function of MSCs, likely mediated by Opn upregulation. These findings suggest a potential therapeutic strategy for improving bone regeneration and HSC expansion. The online version contains supplementary material available at 10.1186/s13287-025-04402-6.

Nanoplastics (NPs) are emerging environmental pollutants that pose growing concerns due to their potential health risks. However, the effects of inhaled NP exposure during pregnancy on fetal brain development remain poorly understood. In this study, we investigated the impact of maternal exposure to polypropylene nanoplastics (PP-NPs) on fetal brain development and neurobehavioral outcomes in a mouse model and further explored its mechanism in human cerebral organoids. Maternal exposure to PP-NPs significantly impaired neuronal differentiation and proliferation in the fetal cortex. Neurobehavioral assessments revealed significant deficits in offspring following maternal exposure, including impaired spatial memory, reduced motor coordination, and heightened anxiety-like behavior. Furthermore, human brain organoids exposed to PP-NPs exhibited reduced growth and neuronal differentiation, with significant downregulation of key neuronal markers such as TUJ1, MAP2, and PAX6. Transcriptomic analysis identified alterations in gene expression, particularly in neuroactive ligand-receptor interaction pathway. Molecular docking and fluorescence co-localization analysis further suggested CYSLTR1 and PTH1R as key molecular targets of PP-NPs. These findings provide novel insights into the toxicological effects of NPs on the developing brain and emphasize the need for preventive measures to protect fetal neurodevelopment during pregnancy. The online version contains supplementary material available at 10.1186/s12951-025-03561-1.

Simvastatin, a 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor, was used in cardiovascular diseases and could decrease low-density lipoprotein cholesterol, and may have a repurposed role in cancer therapy. However, the effects of simvastatin on endometrial cancer remain controversial. We aimed to elucidate the role and mechanisms of simvastatin in regulating previously identified endometrial cancer-associated mesenchymal stem cells (EmCaMSCs)-mediated immunosuppressive effects and anti-tumor progression. Coculture of EmCaMSCs and peripheral blood mononuclear cells (PBMC) was used to assay the population of CD8 + T cells, natural killer (NK) cells, and cytotoxicity of NK cells. The mechanisms were elucidated by applying recombinant proteins and inhibitors of candidate proteins, transforming growth factor-beta 2 (TGF-β2). Finally, the humanized mouse model was generated to study the effects of simvastatin-mediated immunotherapy in treating endometrial cancer. The protein expressions of TGF-β2, CD56, CD8, and PD-L1 in xenograft tumors were analyzed by Western blot or immunohistochemistry assay. In this study, simvastatin inhibited the proliferation of endometrial cancer cells (HEC-1 A and RL95-2) and EmCaMSCs, and the half-maximal inhibitory concentration (IC50) values of EmCaMSCs were much higher. Simvastatin rescued the proliferation and the population of CD8 + T cells and natural killer (NK) cells from PBMC coculturing with EmCaMSC. Simvastatin treatment reduced the expression of TGF-β2 in EmCaMSCs at both the gene and protein levels. TGF-β2 activated the downstream SMAD2/3 signaling, and their inhibition by simvastatin could enhance the cytotoxicity of NK cells against endometrial cancer cells in vitro. Additionally, a combination of simvastatin and NK cell therapy inhibited xenograft growth, potentially by reducing TGF-β2 expression. In conclusion, simvastatin could rescue the population of CD8 + T cells and NK cells from PBMC cocultured with EmCaMSCs. Furthermore, simvastatin could enhance the cytotoxicity of NK cells in vitro and inhibit tumor growth in vivo in a humanized mouse model. These results suggested that simvastatin may be considered as a repurposed and combination drug for treating endometrial cancer. The online version contains supplementary material available at 10.1038/s41598-025-08686-9.