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Homology Directed Repair (HDR) enables precise genome editing,but the implementation of HDR-based therapies is hindered by limited efficiency in comparison to methods that exploit alternative DNA repair routes,such as Non-Homologous End Joining (NHEJ). In this study,we develop a functional,pooled screening platform to identify protein-based reagents that improve HDR in human hematopoietic stem and progenitor cells (HSPCs). We leverage this screening platform to explore sequence diversity at the binding interface of the NHEJ inhibitor i53 and its target,53BP1,identifying optimized variants that enable new intermolecular bonds and robustly increase HDR. We show that these variants specifically reduce insertion-deletion outcomes without increasing off-target editing,synergize with a DNAPK inhibitor molecule,and can be applied at manufacturing scale to increase the fraction of cells bearing repaired alleles. This screening platform can enable the discovery of future gene editing reagents that improve HDR outcomes. Subject terms: Targeted gene repair,Homologous recombination,High-throughput screening View Publication

Mutations in mexZ,encoding a negative regulator of the expression of the mexXY efflux pump genes,are frequently acquired by Pseudomonas aeruginosa at early stages of lung infection. Although traditionally related to resistance to the first-line drug tobramycin,mexZ mutations are associated with low-level aminoglycoside resistance when determined in the laboratory,suggesting that their selection during infection may not be necessarily,or only,related to tobramycin therapy. Here,we show that mexZ -mutated bacteria tend to accumulate inside the epithelial barrier of a human airway infection model,thus colonising the epithelium while being protected against diverse antibiotics. This phenotype is mediated by overexpression of lecA,a quorum sensing-controlled gene,encoding a lectin involved in P. aeruginosa tissue invasiveness. We find that lecA overexpression is caused by a disrupted equilibrium between the overproduced MexXY and another efflux pump,MexAB,which extrudes quorum sensing signals. Our results indicate that mexZ mutations affect the expression of quorum sensing-regulated pathways,thus promoting tissue invasiveness and protecting bacteria from the action of antibiotics within patients,something unnoticeable using standard laboratory tests. Subject terms: Antimicrobial resistance,Pathogens,Infection View Publication

Neocortex expansion during evolution is linked to higher numbers of neurons,which are thought to result from increased proliferative capacity and neurogenic potential of basal progenitor cells during development. Here,we show that EREG,encoding the growth factor EPIREGULIN,is expressed in the human developing neocortex and in gorilla cerebral organoids,but not in the mouse neocortex. Addition of EPIREGULIN to the mouse neocortex increases proliferation of basal progenitor cells,whereas EREG ablation in human cortical organoids reduces proliferation in the subventricular zone. Treatment of cortical organoids with EPIREGULIN promotes a further increase in proliferation of gorilla but not of human basal progenitor cells. EPIREGULIN competes with the epidermal growth factor (EGF) to promote proliferation,and inhibition of the EGF receptor abrogates the EPIREGULIN-mediated increase in basal progenitor cells. Finally,we identify putative cis-regulatory elements that may contribute to the observed inter-species differences in EREG expression. Our findings suggest that species-specific regulation of EPIREGULIN expression may contribute to the increased neocortex size of primates by providing a tunable pro-proliferative signal to basal progenitor cells in the subventricular zone. View Publication

Picornaviruses are a leading cause of central nervous system (CNS) infections. While genotypes such as parechovirus A3 (PeV-A3) and echovirus 11 (E11) can elicit severe neurological disease,the highly prevalent PeV-A1 is not associated with CNS disease. Here,we expand our current understanding of these differences in PeV-A CNS disease using human brain organoids and clinical isolates of the two PeV-A genotypes. Our data indicate that PeV-A1 and A3 specific differences in neurological disease are not due to infectivity of CNS cells as both viruses productively infect brain organoids with a similar cell tropism. Proteomic analysis shows that PeV-A infection significantly alters the host cell metabolism. The inflammatory response following PeV-A3 (and E11 infection) is significantly more potent than that upon PeV-A1 infection. Collectively,our findings align with clinical observations and suggest a role for neuroinflammation,rather than viral replication,in PeV-A3 (and E11) infection. Subject terms: Infection,Central nervous system infections,Viral host response,Innate immunity View Publication

The bone marrow adjusts blood cell production to meet physiological demands in response to insults. The spatial organization of normal and stress responses are unknown owing to the lack of methods to visualize most steps of blood production. Here we develop strategies to image multipotent haematopoiesis,erythropoiesis and lymphopoiesis in mice. We combine these with imaging of myelopoiesis 1 to define the anatomy of normal and stress haematopoiesis. In the steady state,across the skeleton,single stem cells and multipotent progenitors distribute through the marrow enriched near megakaryocytes. Lineage-committed progenitors are recruited to blood vessels,where they contribute to lineage-specific microanatomical structures composed of progenitors and immature cells,which function as the production sites for each major blood lineage. This overall anatomy is resilient to insults,as it was maintained after haemorrhage,systemic bacterial infection and granulocyte colony-stimulating factor (G-CSF) treatment,and during ageing. Production sites enable haematopoietic plasticity as they differentially and selectively modulate their numbers and output in response to insults. We found that stress responses are variable across the skeleton: the tibia and the sternum respond in opposite ways to G-CSF,and the skull does not increase erythropoiesis after haemorrhage. Our studies enable in situ analyses of haematopoiesis,define the anatomy of normal and stress responses,identify discrete microanatomical production sites that confer plasticity to haematopoiesis,and uncover unprecedented heterogeneity of stress responses across the skeleton. Subject terms: Bone marrow cells,Haematopoietic stem cells,Ageing,Imaging the immune system,Haematopoietic stem cells View Publication

Breast cancer stem cell (CSC) expansion results in tumor progression and chemoresistance; however,the modulation of CSC pluripotency remains unexplored. Transmembrane protein 120B (TMEM120B) is a newly discovered protein expressed in human tissues,especially in malignant tissues; however,its role in CSC expansion has not been studied. This study aimed to determine the role of TMEM120B in transcriptional coactivator with PDZ-binding motif (TAZ)-mediated CSC expansion and chemotherapy resistance. Both bioinformatics analysis and immunohistochemistry assays were performed to examine expression patterns of TMEM120B in lung,breast,gastric,colon,and ovarian cancers. Clinicopathological factors and overall survival were also evaluated. Next,colony formation assay,MTT assay,EdU assay,transwell assay,wound healing assay,flow cytometric analysis,sphere formation assay,western blotting analysis,mouse xenograft model analysis,RNA-sequencing assay,immunofluorescence assay,and reverse transcriptase-polymerase chain reaction were performed to investigate the effect of TMEM120B interaction on proliferation,invasion,stemness,chemotherapy sensitivity,and integrin/FAK/TAZ/mTOR activation. Further,liquid chromatography–tandem mass spectrometry analysis,GST pull-down assay,and immunoprecipitation assays were performed to evaluate the interactions between TMEM120B,myosin heavy chain 9 (MYH9),and CUL9. TMEM120B expression was elevated in lung,breast,gastric,colon,and ovarian cancers. TMEM120B expression positively correlated with advanced TNM stage,lymph node metastasis,and poor prognosis. Overexpression of TMEM120B promoted breast cancer cell proliferation,invasion,and stemness by activating TAZ-mTOR signaling. TMEM120B directly bound to the coil-coil domain of MYH9,which accelerated the assembly of focal adhesions (FAs) and facilitated the translocation of TAZ. Furthermore,TMEM120B stabilized MYH9 by preventing its degradation by CUL9 in a ubiquitin-dependent manner. Overexpression of TMEM120B enhanced resistance to docetaxel and doxorubicin. Conversely,overexpression of TMEM120B-∆CCD delayed the formation of FAs,suppressed TAZ-mTOR signaling,and abrogated chemotherapy resistance. TMEM120B expression was elevated in breast cancer patients with poor treatment outcomes (Miller/Payne grades 1–2) than in those with better outcomes (Miller/Payne grades 3–5). Our study reveals that TMEM120B bound to and stabilized MYH9 by preventing its degradation. This interaction activated the β1-integrin/FAK-TAZ-mTOR signaling axis,maintaining stemness and accelerating chemotherapy resistance. The online version contains supplementary material available at 10.1186/s13058-024-01802-z. View Publication

Acute myeloid leukaemia (AML) is a haematological malignancy characterised by the accumulation of transformed myeloid progenitors in the bone marrow. Piplartine (PL),also known as piperlongumine,is a pro-oxidant small molecule extracted from peppers that has demonstrated antineoplastic potential in solid tumours and other haematological malignancies. In this work,we explored the potential of PL to treat AML through the use of a combination of cellular and molecular analyses of primary and cultured leukaemia cells in vitro and in vivo. We showed that PL exhibits in vitro cytotoxicity against AML cells,including CD34 + leukaemia-propagating cells,but not healthy haematopoietic progenitors,suggesting anti-leukaemia selectivity. Mechanistically,PL treatment increased reactive oxygen species (ROS) levels and induced ROS-mediated apoptosis in AML cells,which could be prevented by treatment with the antioxidant scavenger N -acetyl-cysteine and the pancaspase inhibitor Z-VAD(OMe)-FMK. PL treatment reduced NFKB1 gene transcription and the level of NF-κB p65 (pS536),which was depleted from the nucleus of AML cells,indicating suppression of NF-κB p65 signalling. Significantly,PL suppressed AML development in a mouse xenograft model,and its combination with current AML treatments (cytarabine,daunorubicin and azacytidine) had synergistic effects,indicating translational therapeutic potential. Taken together,these data position PL as a novel anti-AML candidate drug that can target leukaemia stem/progenitors and is amenable to combinatorial therapeutic strategies. Subject terms: Acute myeloid leukaemia,Cancer stem cells,Pharmacology View Publication

Protein synthesis is frequently deregulated during tumorigenesis. However,the precise contexts of selective translational control and the regulators of such mechanisms in cancer is poorly understood. Here,we uncovered CNOT3,a subunit of the CCR4-NOT complex,as an essential modulator of translation in myeloid leukemia. Elevated CNOT3 expression correlates with unfavorable outcomes in patients with acute myeloid leukemia (AML). CNOT3 depletion induces differentiation and apoptosis and delayed leukemogenesis. Transcriptomic and proteomic profiling uncovers c-MYC as a critical downstream target which is translationally regulated by CNOT3. Global analysis of mRNA features demonstrates that CNOT3 selectively influences expression of target genes in a codon usage dependent manner. Furthermore,CNOT3 associates with the protein network largely consisting of ribosomal proteins and translation elongation factors in leukemia cells. Overall,our work elicits the direct requirement for translation efficiency in tumorigenesis and propose targeting the post-transcriptional circuitry via CNOT3 as a therapeutic vulnerability in AML. Subject terms: Acute myeloid leukaemia,Translation,RNA decay View Publication

Angiotensin (Ang)-(1–7) stimulates vasoprotective functions of diabetic (DB) CD34 + hematopoietic stem/progenitor cells partly by decreasing reactive oxygen species (ROS),increasing nitric oxide (NO) levels and decreasing TGFβ1 secretion. Telomerase reverse transcriptase (TERT) translocates to mitochondria and regulates ROS generation. Alternative splicing of TERT results in variants α-,β- and α-β-TERT,which may oppose functions of full-length (FL) TERT. This study tested if the protective functions of Ang-(1–7) or TGFβ1-silencing are mediated by mitoTERT and that diabetes decreases FL-TERT expression by inducing splicing. CD34 + cells were isolated from the peripheral blood mononuclear cells of nondiabetic (ND,n = 68) or DB (n = 74) subjects. NO and mitoROS levels were evaluated by flow cytometry. TERT splice variants and mitoDNA-lesions were characterized by qPCR. TRAP assay was used for telomerase activity. Decoy peptide was used to block mitochondrial translocation (mitoXTERT). TERT inhibitor or mitoXTERT prevented the effects of Ang-(1–7) on NO or mitoROS levels in DB-CD34 + cells. FL-TERT expression and telomerase activity were lower and mitoDNA-lesions were higher in DB cells compared to ND and were reversed by Ang-(1–7) or TGFβ1-silencing. The prevalence of TERT splice variants,with predominant β-TERT expression,was higher and the expression of FL-TERT was lower in DB cells (n = 25) compared to ND (n = 30). Ang-(1–7) or TGFβ1-silencing decreased TERT-splicing and increased FL-TERT. Blocking of β-splicing increased FL-TERT and protected mitoDNA in DB-cells. The findings suggest that diabetes induces TERT-splicing in CD34 + cells and that β-TERT splice variant largely contributes to the mitoDNA oxidative damage. View Publication

Pseudouridylation plays a regulatory role in various physiological and pathological processes. A prime example is the mitochondrial myopathy,lactic acidosis,and sideroblastic anemia syndrome (MLASA),characterized by defective pseudouridylation resulting from genetic mutations in pseudouridine synthase 1 (PUS1). However,the roles and mechanisms of pseudouridylation in normal erythropoiesis and MLASA-related anemia remain elusive. We established a mouse model carrying a point mutation (R110W) in the enzymatic domain of PUS1,mimicking the common mutation in human MLASA. Pus1 -mutant mice exhibited anemia at 4 weeks old. Impaired mitochondrial oxidative phosphorylation was also observed in mutant erythroblasts. Mechanistically,mutant erythroblasts showed defective pseudouridylation of targeted tRNAs,altered tRNA profiles,decreased translation efficiency of ribosomal protein genes,and reduced globin synthesis,culminating in ineffective erythropoiesis. Our study thus provided direct evidence that pseudouridylation participates in erythropoiesis in vivo. We demonstrated the critical role of pseudouridylation in regulating tRNA homeostasis,cytoplasmic translation,and erythropoiesis. Subject areas: Molecular biology,Cell biology View Publication

Acute myeloid leukemia (AML) is a highly aggressive hematologic cancer with poor survival across a broad range of molecular subtypes. Development of efficacious and well-tolerable therapies encompassing the range of mutations that can arise in AML remains an unmet need. The bromo- and extra-terminal domain (BET) family of proteins represents an attractive therapeutic target in AML due to their crucial roles in many cellular functions,regardless of any specific mutation. Many BET inhibitors (BETi) are currently in pre-clinical and early clinical development,but acquisition of resistance continues to remain an obstacle for the drug class. Novel methods to circumvent this development of resistance could be instrumental for the future use of BET inhibitors in AML,both as monotherapy and in combination. To date,many investigations into possible drug combinations of BETi with CDK inhibitors have focused on CDK9,which has a known physical and functional interaction with the BET protein BRD4. Therefore,we wished to investigate possible synergy and additive effects between inhibitors of these targets in AML. Here,we describe combination therapy with the multi-CDK inhibitor dinaciclib and the BETi PLX51107 in pre-clinical models of AML. Dinaciclib and PLX51107 demonstrate additive effects in AML cell lines,primary AML samples,and in vivo. Further,we demonstrate novel activity of dinaciclib through inhibition of the canonical/β-catenin dependent Wnt signaling pathway,a known resistance mechanism to BETi in AML. We show dinaciclib inhibits Wnt signaling at multiple levels,including downregulation of β-catenin,the Wnt co-receptor LRP6,as well as many Wnt pathway components and targets. Moreover,dinaciclib sensitivity remains unaffected in a setting of BET resistance,demonstrating similar inhibitory effects on Wnt signaling when compared to BET-sensitive cells. Ultimately,our results demonstrate rationale for combination CDKi and BETi in AML. In addition,our novel finding of Wnt signaling inhibition could have potential implications in other cancers where Wnt signaling is dysregulated and demonstrates one possible approach to circumvent development of BET resistance in AML. The online version contains supplementary material available at 10.1186/s40164-024-00483-w. View Publication

In the adult bone marrow (BM),endothelial cells (ECs) are an integral component of the hematopoietic stem cell (HSC)-supportive niche,which modulates HSC activity by producing secreted and membrane-bound paracrine signals. Within the BM,distinct vascular arteriole,transitional,and sinusoidal EC subtypes display unique paracrine expression profiles and create anatomically-discrete microenvironments. However,the relative contributions of vascular endothelial subtypes in supporting hematopoiesis is unclear. Moreover,constitutive expression and off-target activity of currently available endothelial-specific and endothelial-subtype-specific murine cre lines potentially confound data analysis and interpretation. To address this,we describe two tamoxifen-inducible cre -expressing lines,Vegfr3-creER T2 and Cx40-creER T2,that efficiently label sinusoidal/transitional and arteriole endothelium respectively in adult marrow,without off-target activity in hematopoietic or perivascular cells. Utilizing an established mouse model in which cre -dependent recombination constitutively-activates MAPK signaling within adult endothelium,we identify arteriole ECs as the driver of MAPK-mediated hematopoietic dysfunction. These results define complementary tamoxifen-inducible creER T2 -expressing mouse lines that label functionally-discrete and non-overlapping sinusoidal/transitional and arteriole EC populations in the adult BM,providing a robust toolset to investigate the differential contributions of vascular subtypes in maintaining hematopoietic homeostasis. The online version contains supplementary material available at 10.1007/s12015-024-10703-9. View Publication