Project description:Quantification of gene expression noise on young versus aged bone marrow HSCs at single-cell resolution revealed Dlk1 increases both noise and expression levels upon aging. Dlk1, encoding a cell surface non-canonical Notch ligand, is partially expressed in the LT-HSC compartment, without generating clusters of individual subpopulations by conventional clustering analysis. We aimed to characterize the trascriptome of aged LT-HSC with positive and negative expression of Dlk1 cells. Dataset created as part of the publication: "VarID2 quantifies gene expression noise dynamics and unveils functional heterogeneity of ageing hematopoietic stem cells"
Project description:The purpose of this RNA sequencing dataset was to analyze transcriptional signaling in young and aged murine hematopoietic stem cells when pulsed ex vivo with dimethyl (dm)PGE2.
Project description:Identification of differentially expressed genes in young (3 month old) versus aged (24 month old) mouse hematopoietic stem cells. Comparison of genes differentially expressed in hematopoietic stem cells of young mice with conditional deletion of mTOR within vascular endothelium.
Project description:Hematopoietic aging is associated with decreased hematopoietic stem cell (HSC) self-renewal capacity and increased risk for myelodysplasia and leukemia. Deficient DNA repair contributes to the decline in HSC self-renewal capacity during aging and it remains unclear whether extrinsic signals can rejuvenate aged HSCs. Here, we demonstrate that augmentation of non-homologous end-joining (NHEJ) DNA repair in aged HSCs via treatment with epidermal growth factor (EGF) rejuvenates HSC function. Seven day culture of BM CD34-ckit+sca-1+lin- (34-KSL) HSCs from aged C57BL/6 mice with EGF suppressed myeloid skewing and increased production of multipotent CFU-granulocyte, erythroid, monocyte and megakaryocyte (CFU-GEMM) colonies. Aged, EGF-treated HSCs displayed increased donor multilineage engraftment in primary competitively transplanted mice and in secondary mice compared to mice transplanted with aged, control HSCs. Donor cell engraftment within the bone marrow (BM) KSL and SLAM+KSL HSC population was > 2-fold increased in mice transplanted with aged, EGF-treated HSCs. Systemic administration of EGF to aged mice for 6 weeks also increased long term – HSC self-renewal capacity as measured by increased donor bone marrow (BM) competitive repopulation in primary and secondary transplanted mice. Conversely, deletion of EGFR in Scl/Tal1+ hematopoietic cells was associated with increased myeloid skewing and depletion of LT-HSCs in middle aged mice. Mechanistically, EGF treatment decreased DNA damage in aged HSCs through activation of DNA PK-cs, Artemis and NHEJ repair. Inhibition of DNA PK-cs blocked EGF-mediated restoration of multipotent differentiation and suppression of myeloid skewing in aged HSCs, suggesting that the restoration of hematopoietic potential in aged HSCs is dependent on EGF-mediated activation of DNA PK-cs. EGF treatment also converted the transcriptome of aged HSCs from enrichment for genes involved in cell death and survival to genes involved in HSC generation and identity. These data suggest that extrinsic activation of EGFR signaling can restore key functional capacities in aged HSCs.
Project description:Phenotypic and functional changes seen in the aged adaptive immune system are primarily driven by aging of hematopoietic stem cells (HSCs), pharmacological rejuvenated aged HSCs were able to reconstituted a youthful immune system
Project description:Hematopoietic aging is associated with decreased hematopoietic stem cell (HSC) self-renewal capacity and increased risk for myelodysplasia and leukemia. Deficient DNA repair contributes to the decline in HSC self-renewal capacity during aging and it remains unclear whether extrinsic signals can rejuvenate aged HSCs. Here, we demonstrate that augmentation of non-homologous end-joining (NHEJ) DNA repair in aged HSCs via treatment with epidermal growth factor (EGF) rejuvenates HSC function. Seven day culture of BM CD34-ckit+sca-1+lin- (34-KSL) HSCs from aged C57BL/6 mice with EGF suppressed myeloid skewing and increased production of multipotent CFU-granulocyte, erythroid, monocyte and megakaryocyte (CFU-GEMM) colonies. Aged, EGF-treated HSCs displayed increased donor multilineage engraftment in primary competitively transplanted mice and in secondary mice compared to mice transplanted with aged, control HSCs. Donor cell engraftment within the bone marrow (BM) KSL and SLAM+KSL HSC population was > 2-fold increased in mice transplanted with aged, EGF-treated HSCs. Systemic administration of EGF to aged mice for 6 weeks also increased long term – HSC self-renewal capacity as measured by increased donor bone marrow (BM) competitive repopulation in primary and secondary transplanted mice. Conversely, deletion of EGFR in Scl/Tal1+ hematopoietic cells was associated with increased myeloid skewing and depletion of LT-HSCs in middle aged mice. Mechanistically, EGF treatment decreased DNA damage in aged HSCs through activation of DNA PK-cs, Artemis and NHEJ repair. Inhibition of DNA PK-cs blocked EGF-mediated restoration of multipotent differentiation and suppression of myeloid skewing in aged HSCs, suggesting that the restoration of hematopoietic potential in aged HSCs is dependent on EGF-mediated activation of DNA PK-cs. EGF treatment also converted the transcriptome of aged HSCs from enrichment for genes involved in cell death and survival to genes involved in HSC generation and identity. These data suggest that extrinsic activation of EGFR signaling can restore key functional capacities in aged HSCs.
Project description:Cancer immunotherapies have produced remarkable results in B-cell malignancies; however, optimal cell surface targets for many solid cancers remain elusive. Here, we present an integrative proteomic, transcriptomic, and epigenomic analysis of tumor specimens along with normal tissues to identify biologically relevant cell surface proteins that can serve as immunotherapeutic targets for neuroblastoma, the often fatal childhood cancer of the developing nervous system. We apply this approach to human-derived cell lines (N=9) and cell/patient-derived xenograft (N=12) models of neuroblastoma. Plasma membrane-enriched mass spectrometry identified 1,461 cell surface proteins in cell lines and 1,401 in xenograft models, respectively. Additional proteogenomic analyses revealed 71 high-confidence candidate immunotherapeutic targets and we prioritized Delta-like canonical notch ligand 1 (DLK1) for further study. High expression of DLK1 directly correlated with the presence of a super-enhancer spanning the DLK1 locus. Robust cell surface expression of DLK1 was validated by immunofluorescence, flow cytometry, and immunohistochemistry. Short hairpin RNA mediated silencing of DLK1 in neuroblastoma cells resulted in increased cellular differentiation. ADCT-701, a DLK1-targeting antibody-drug conjugate (ADC), showed potent and specific cytotoxicity in DLK1-expressing neuroblastoma xenograft models. Finally, we also found that DLK1 is highly expressed in several adult cancer types including adrenocortical carcinoma (ACC), pheochromocytoma/paraganglioma (PCPG) and small cell lung cancer (SCLC) suggesting potential clinical benefit beyond neuroblastoma. A clinical trial has been developed for evaluating ADCT-701 in neuroendocrine tumors in adults (https://www.clinicaltrials.gov/study/NCT06041516?term=adct-701&rank=1). Therefore, comprehensive characterization of cancer surfaceomes can provide biologically relevant immunotherapy targeting strategies.
Project description:Adrenocortical carcinoma (ACC) is characterized by disruption of tissue development and homeostatic pathways. Here, using genetic fate mapping and murine ACC models, we have identified a population of adrenocortical stem cells that express Delta-like non-canonical Notch ligand 1 (DLK1). These cells are active during development, near dormant postnatally but are re-expressed in ACC. DLK1 expression is widespread in human ACC samples and independently influences recurrence-free survival. Spatial transcriptomic analysis has identified DLK1 positive ACC cells to have increased steroidogenic potential, a finding also observed in both human and murine ACC cell lines. Additionally, the cleavable DLK1 ectodomain is detectable in patients’ serum and effectively distinguishes ACC from other adrenal pathologies, offering potential diagnostic and follow-up utility for ACC patients. These data demonstrate a prognostic role for DLK1 in ACC, elucidate its hierarchical expression in homeostasis and cancer and suggest its utility as a biomarker in this malignancy.