Journal: Nature Communications

Article Title: MOZ/ENL complex is a recruiting factor of leukemic AF10 fusion proteins

doi: 10.1038/s41467-023-37712-5

Figure Lengend Snippet: a Structures required for AF10 fusion-mediated myeloid transformation. Various AF10 fusion constructs were examined to assess their transforming ability of myeloid progenitors. HA-tag (indicated by red triangles) was fused to MTM and MTMT constructs. FLAG-tag (indicated by blue triangles) was fused to other AF10 fusion constructs. Dotted lines indicate protein-protein interaction. A schema of a myeloid progenitor transformation assay is shown at the top. Hoxa9 expression normalized to Gapdh in first-round colonies (left) is shown as the relative value of CALM-AF10 (arbitrarily set at 100%) (mean of two biological replicates). Colony-forming ability at the third- and fourth-round passages (right) is shown with error bars (mean ± SD of biological replicates, n ≥ 3). b Association of NES-AF10 fusion with ENL in the presence or absence of DOT1L. IP-western blotting (WB) analyses of the chromatin fraction of HEK293T cells [the parental clone or a DOT1L-knockout clone (dDOT1L)] transiently expressing the FLAG-tagged (indicated as f) NES-AF10´ fusion (fNES-AF10´) construct and Xpress-tagged (indicated as x) ENL (xENL) were performed. Co-purification of ENL was observed only in the presence of DOT1L. c Leukemogenesis by NES-ENL fusion in vivo. Various AF10 fusion-derivatives including NES-ENL were transduced to c-Kit-positive hematopoietic progenitors and transplanted into syngeneic mice. Primary NES-ENL leukemia cells were harvested from the bone marrow (BM) and transplanted into recipient mice. d Hierarchical clustering analysis of RNA-seq profiles of AF10 fusion-ICs. Normalized count data in various AF10 fusion-ICs was clustered using R ward D2 method. Source data are provided as a Source Data file.

Article Snippet: Human hematopoietic stem cell—CD34 + cells the from fetal liver (purchased from Cell Applications, INC.) were pre-cultured in Hematopoietic Stem Cell Culture Medium (Cell Applications, INC.) for 4 days and plated in methylcellulose-based medium (MethoCult TM H4435 Enriched, STEMCELL Technologies) in the presence of designated drugs for 6 days.

Techniques: Transformation Assay, Construct, FLAG-tag, Expressing, Western Blot, Knock-Out, Copurification, In Vivo, RNA Sequencing

Journal: JNCI Journal of the National Cancer Institute

Article Title: Hematopoietic Stem Cell Gene Therapy for Brain Metastases Using Myeloid Cell–Specific Gene Promoters

doi: 10.1093/jnci/djz181

Figure Lengend Snippet: Myeloid cells in preclinical models of brain metastases. A) Discosoma sp. red fluorescent protein/Fluc (DF)–tagged EO771 and PyMT breast cancer cells give rise to a single large cancer lesion in the brain following intracranial implantation (H&E staining); n = 4. B) Administration of EO771-DF cancer cells into the internal carotid artery gives rise to micro- and macrometastases 2 weeks post-injection (n = 3/6 for EO771-DF/PyMT-DF). Scale bar = 50 µm. C) Macrometastases (intracranial implantation model) and micrometastases (carotid artery model) in the brain are infiltrated by CD11b+ myeloid cells (group sizes as in A and B ). Scale bars = 50 µm (left panels) and 20 µm (right panels). D–F) Bone marrow in C57Bl/6J mice was ablated by irradiation, followed by transplantation of hematopoietic stem cells (HSCs) isolated from the transgenic mice expressing green fluorescent protein (GFP) downstream of the Ubiquitin C (UBC) promoter. Seven weeks later, tumors were generated by intracranial implantation of cancer cells. Infiltration of GFP+ bone marrow–derived myeloid cells (CD11b+) was quantified by flow cytometry at 2 weeks post–cancer cell injection ( E ). Percentage of GFP+ cells within the CD11b+ cell population is shown in ( F ); n = 5. G) Quantification of GFP+ HSC progeny per mm 3 tissue within PyMT brain tumors and normal tumor-adjacent brain tissue (n = 3, P = .03. Statistically significant difference was determined with the two-tailed Student’s t test. H) Representative dot plots of flow cytometry for quantifying the percentage of CD45 high cells within CD11b+F4/80+ population and percentage of GFP+ cells within CD11b+F4/80+CD45 high cell population infiltrating PyMT tumors (n = 3). I) Gating strategy and quantification of GFP+ and CD49d+P2RY12- cells within CD45+CD11b+Ly6C-Ly6G- microglia/macrophages infiltrating PyMT tumors (n = 3).

Article Snippet: Human CD34+ bone marrow–derived HSCs (DV Biologics) were transduced with pFUGW ( ) (MOI 50) overnight and injected intravenously (2×10 5 cells) into sublethally irradiated (225 cGy) NSG mice.

Techniques: Staining, Injection, Irradiation, Transplantation Assay, Isolation, Transgenic Assay, Expressing, Ubiquitin Proteomics, Generated, Derivative Assay, Flow Cytometry, Two Tailed Test

Journal: JNCI Journal of the National Cancer Institute

Article Title: Hematopoietic Stem Cell Gene Therapy for Brain Metastases Using Myeloid Cell–Specific Gene Promoters

doi: 10.1093/jnci/djz181

Figure Lengend Snippet: Genetically modified hematopoietic stem cell (HSC) progeny in the context of macro- and micrometastases in the brain. A) Murine HSCs transduced with lentiviral pFUGW vector were transplanted into lethally irradiated recipient mice, and tumors were generated by intracranial implantation of EO771-DF cells. B) Detection of pFUGW-transduced HSC progeny (green fluorescent protein [GFP]+) in intracranial tumors generated according to the scheme in (A) by immunofluorescence (n = 7). Scale bar = 200 µm. C) Quantification of pFUGW-transduced HSC progeny (GFP+) in different tissues by flow cytometry; n = 3. D) Quantification of immune cell populations within EO771 intracranial tumors by flow cytometry. Total percentage of individual cell populations, as well as the proportion of cells derived from pFUGW-transduced HSCs is shown; n = 7. E) Immunofluorescence staining of intracranial EO771 tumors for macrophages/microglia (F4/80+) and GFP (n = 4). Scale bars = 50 µm. F) Murine HSCs transduced with lentiviral pFUGW vector were transplanted into lethally irradiated recipient mice, followed by administration of EO771-DF cells into the internal carotid artery. G) CD11b+GFP+ progeny of HSCs was observed in close association with EO771-DF micrometastases in the brain. Fluorescence images were obtained by confocal microscopy (n = 3). Scale bars = 50 µm. H) Cancer cells and micrometastases-associated cells belonging to different hematopoietic cell subpopulations were counted on immunofluorescence images; 4–8 micrometastases-containing coronal brain sections per animal (n = 3) were quantified. I) Percentages of GFP+ cells within micrometastases-associated hematopoietic cell populations were quantified using immunofluorescence images and overall percentages of GFP+ cells in matched blood by flow cytometry (group sizes as in H ).

Article Snippet: Human CD34+ bone marrow–derived HSCs (DV Biologics) were transduced with pFUGW ( ) (MOI 50) overnight and injected intravenously (2×10 5 cells) into sublethally irradiated (225 cGy) NSG mice.

Techniques: Genetically Modified, Transduction, Plasmid Preparation, Irradiation, Generated, Immunofluorescence, Flow Cytometry, Derivative Assay, Staining, Fluorescence, Confocal Microscopy

Journal: JNCI Journal of the National Cancer Institute

Article Title: Hematopoietic Stem Cell Gene Therapy for Brain Metastases Using Myeloid Cell–Specific Gene Promoters

doi: 10.1093/jnci/djz181

Figure Lengend Snippet: Human hematopoietic stem cells (HSCs) and their progeny as cellular vehicles targeting brain metastases. A) Tumors were generated in humanized NSG mice (JAX) 3–4 months after engraftment of human HSCs (hHSCs) by intracranial implantation of MDA-MB-231/brain cells. B) The percentage of human hematopoietic cells (hCD45+) in blood and brain tumors was quantified by flow cytometry (n = 3). C) Myeloid cell subpopulations within MDA-MB-231/brain tumors were quantified by flow cytometry (n = 3). D) Percentage of CD49d+ macrophages within human CD45+CD11b+CD16-CD66B-CD14+ population in intracranial MDA-MB-231/brain tumors was quantified by flow cytometry (n = 3). Representative dot plots are shown. E) Lentiviral pFUGW vector–transduced hHSCs were transplanted into sublethally irradiated NSG mice, and tumors were generated by intracranial implantation of MDA-MB-231/brain cells 3–4 months later (n = 4). Detection of green fluorescent protein-positive human hematopoietic cells in intracranial tumors was performed by immunofluorescence. Scale bar = 50 µm. F, G) Hematopoietic cells and macrophages in patient brain metastases specimens originating from breast cancer ( F ) and lung cancer ( G ) were detected by immunofluorescence (n = 3 per cancer type). To visualize cancer cells, adjacent sections were stained for pan-cytokeratin (breast cancer brain metastases [BrM]) or vimentin (lung cancer BrM) and costained for human CD45 (hematopoietic cells) human CD68 (macrophages). Scale bars = 100 µm. H) Percentage of CD49d+ macrophages within CD45+CD11b+CD16-CD66B-CD14+ microglia/macrophage population in patient BrM originating from different primary cancer types as indicated in ( I ) was quantified by flow cytometry (n = 6). Representative dot plots are shown. I) Percentage of CD45+CD11b+CD16-CD66B-CD14+CD49d+ macrophages within total cell population in patient BrM (n = 6). RCC = renal cell carcinoma.

Article Snippet: Human CD34+ bone marrow–derived HSCs (DV Biologics) were transduced with pFUGW ( ) (MOI 50) overnight and injected intravenously (2×10 5 cells) into sublethally irradiated (225 cGy) NSG mice.

Techniques: Generated, Flow Cytometry, Plasmid Preparation, Irradiation, Immunofluorescence, Staining

Journal: JNCI Journal of the National Cancer Institute

Article Title: Hematopoietic Stem Cell Gene Therapy for Brain Metastases Using Myeloid Cell–Specific Gene Promoters

doi: 10.1093/jnci/djz181

Figure Lengend Snippet: Delivery of lentiviral hematopoietic stem cell (HSC) therapy under the control of the MMP14 promoter fragment. A) Detection of murine tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) in cell lysate and cell culture supernatant (s/n) of HEK293 cells transduced with Ubiquitin C (UBC): TRAIL construct. B) Schematic of experiment to confirm the sensitivity of PyMT cells to TRAIL in vitro. HEK293 UBC: TRAIL cells (effector cells; E) were cocultured with PyMT-Fluc cancer cells (target cells; T) at two different effector/target (E/T) ratios as indicated, and bioluminescence signal intensity (corresponding to the live PyMT cells) quantified after 24 and 48 hours. C) Percentage dead PyMT-Fluc cells (% killing) when cocultured with HEK293 UBC: TRAIL cells as quantified by bioluminescence imaging. Bioluminescence signals obtained in coculture of PyMT-Fluc cells with HEK293 UBC: TRAIL cells were normalized to the bioluminescence signals obtained in coculture with HEK293 UBC: green fluorescent protein (GFP) cells (n = 3; 2 independent experiments). Statistical analysis was performed using a two-tailed t test; P = .04 for 0 vs 24 and 48 hours at E/T 1: 10; P < .001 for 0 vs 24 and 48 hours at E/T 1: 1. D) Schematic of the survival experiment: HSCs were lentivirally transduced with MMP14: GFP or MMP14: TRAIL constructs, and injected into lethally irradiated mice. Following bone marrow reconstitution, PyMT cells were injected intracranially, and survival of the animals was monitored. E) Survival of mice reconstituted with lentivirally transduced MMP14: TRAIL HSCs compared with mice reconstituted with lentivirally transduced MMP14: GFP HSCs (19±3.4 vs 15±2.0 days, P = .006, two-sided log-rank test, n = 17/21 for MMP14: GFP/MMP14: TRAIL group, pooled data from two independent experiments). F) Tnfsf10 (murine TRAIL gene) expression in brain tumor (BrT) tissue was detected by quantitative polymerase chain reaction. Tnfsf10 Ct values were normalized to Gapdh Ct values using comparative Ct method (1.80 ± 0.23 vs 0.70 ± 0.14 for MMP14: TRAIL/MMP14: GFP group, P = .04, two-tailed t test, n = 7 per group).

Article Snippet: Human CD34+ bone marrow–derived HSCs (DV Biologics) were transduced with pFUGW ( ) (MOI 50) overnight and injected intravenously (2×10 5 cells) into sublethally irradiated (225 cGy) NSG mice.

Techniques: Control, Cell Culture, Transduction, Ubiquitin Proteomics, Construct, In Vitro, Imaging, Two Tailed Test, Injection, Irradiation, Gene Expression, Real-time Polymerase Chain Reaction