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A UMI-barcoded in vivo CRISPR-Cas9 screen identifies synthetic lethal targets of Tet2 KO–driven expansion of HSPCs. (A) A nucleotide-encoded UMI linked to an sgRNA expression cassette in a lentiviral vector allows clonal tracing after transduction. Lentiviral infection of WT or Tet2 KO Cas9 expressing murine LSK HSPCs was harvested 14 weeks after transplantation into lethally IR recipients. (B) Analysis strategy to identify genetic dependencies of Tet2 KO HSPC growth after UMI-based in vivo screening. (C-D) Results of the pooled CRISPR-Cas9 KO screen in Tet2 KO and WT HSPCs. (C) Candidate genes result in a reduced number of Tet2 KO clones compared with WT. P value was obtained using a Fisher exact test comparing the number of clones containing nontargeting sgRNAs with the number of clones containing gene-specific sgRNAs in WT vs Tet2 KO. (D) Genes with greater persisting clones in WT (Tet2 KO–WT clone numbers >50) were subsequently ranked by Tet2 KO–specific decrease in clone size (variant allelic frequency [VAF]) at 14 weeks, and VAF differences were assessed by Welch’s t test. Row normalized by z score. All P values were adjusted for multiple hypothesis testing via FDR. (E) VAF and (F) clone numbers for the top Tet2 KO–specific target genes identified in the CRISPR-Cas9 screen. Welch’s t test: n.s., ∗P < .01; ∗∗P < .001; ∗∗∗P < .0001. FDR, false discovery rate; IR, irradiated; LTR, long-terminal repeat; n.s., nonsignificant.
Published: 2025
Figure 1. A UMI-barcoded in vivo CRISPR-Cas9 screen identifies synthetic lethal targets of Tet2 KO–driven expansion of HSPCs. (A) A nucleotide-encoded UMI linked to an sgRNA expression cassette in a lentiviral vector allows clonal tracing after transduction. Lentiviral infection of WT or Tet2... More about this image found in A UMI-barcoded in vivo CRISPR-Cas9 screen identifies synthetic lethal targe...
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Ncoa4 KO impairs Tet2 KO competitive advantage in mouse transplant models. (A) Competitive transplantation of Cas9 expressing WT or Tet2 KO HSCs (CD150+CD135−CD48−lineage−Sca1+Kit+) transduced with sgRNA targeting Ncoa4 (sgNcoa4) or sgRNA nontargeting control (sgNTG). Lentiviral vector expresses a fluorescent protein, enabling tracing of transduced hematopoietic progeny. (B) Serial peripheral blood flow cytometric analysis after transplantation of WT or Tet2 KO cells containing blue fluorescent protein–labeled sgNTG or red fluorescent protein–labeled sgNcoa4. Results were normalized to the percentage of blue fluorescent protein–labeled cells expressing sgNTG. Significance test by 2-way ANOVA. Bars showing mean with standard error of the mean (SEM). N = 9 to 10 mice per arm. (C) Competition experiment between Vav-Cre+;Cd45.1/2 control cells and either Tet2 fl/fl Vav-Cre+;Cd45.2 (Tet2 KO), Ncoa4 fl/fl Vav-Cre+;Cd45.2 (Ncoa4 KO), Tet2 fl/fl Ncoa4 fl/fl Vav-Cre+Cd45.2 (Tet2 KO Ncoa4 KO), or WT Vav-Cre+;Cd45.2 BM cells transplanted at an 80:20 (WT-to-competitor) ratio, injected into lethally IR Cd45.1 recipients. (D) CD45.2 chimerism analysis from peripheral blood at each time point, as a percentage of total donor (CD45.2 and CD45.1/CD45.2) in mice transplanted with Vav-Cre transgenic mice as per panel C. Bars showing mean with SEM. N = 18 to 20 mice per arm. Unpaired t test between Tet2 KO and Tet2 KO Ncoa4 KO. ∗P < .01; ∗∗P < .001. (E) Transplant of whole BM from primary Vav-Cre transgenic mice (from panel C) into secondary recipients. Peripheral blood analysis of whole BM based on CD45 chimerism. Bars showing mean with SEM. N = 5 mice per arm. (F) Competition experiment between Mx-Cre+;Cd45.1/2 control and either Tet2 fl/fl Mx-Cre+;Cd45.2 (Tet2 KO), Ncoa4 fl/fl Mx-Cre+;Cd45.2 (Ncoa4 KO), Tet2 fl/fl Ncoa4 fl/fl Mx-Cre+Cd45.2 (Tet2 KO Ncoa4 KO), or WT Mx-Cre+;Cd45.2 BM cells. BM transplanted at an 80:20 (WT-to-competitor) ratio into lethally IR Cd45.1 recipients. pIpC injections 10 mg/kg intraperitoneally on alternate days for 3 doses. (G) CD45.2 chimerism analysis from peripheral blood at each time point, as a percentage of total donor (CD45.2 and CD45.1/CD45.2) from panel F. Bars showing mean with SEM. N = 17 to 20 mice per arm. Unpaired t test between Tet2 fl/fl and Tet2 fl/fl; Ncoa4 fl/fl. Dashed line demarcates the start of pIpC injections. (H) CD45 chimerism in lineage negative, LK, LSK, and CD150+CD48−LSK (HSC) BM fractions from Mx-Cre transgenic mice from panel F at 11 weeks after transplant. N = 9 to 10 mice per arm. Unpaired t test between genotypes indicated. (I) Flow cytometric analysis of HSC and multipotent progenitor BM populations from mice transplanted with Cas9-expressing WT or Tet2 KO HSCs infected with either sgNcoa4 or sgNTG. Results were normalized to the percentage of cells expressing sgNTG. The flow gating strategy is depicted in supplemental Figure 2F. Unpaired t test, bars showing mean with SEM. Each point represents an individual mouse. N = 7 to 10 mice per arm. (J) Pseudotime trajectory of WT or Tet2 KO Cas9 LSK HSPCs transduced with control or Ncoa4 gRNAs. Analysis was performed using Monocle3.36 Pseudotime trajectory annotation with expression of stem and HSPC markers is highlighted in supplemental Figure 2K. (K) Using cell clusters at the top of the pseudotime trajectory (blue label), which excludes differentiated Fcgcr3+ progenitors, gene set enrichment analysis was performed using all of the C2 Molecular Signatures Database.37Tet2 KO HSPCs transduced with control nontargeting gRNAs were compared with Tet2 KO HSPCs transduced with Ncoa4 gRNAs (gNcoa4). This revealed enrichment of gene sets expressed in stem cell (red) and leukemia (orange) signatures. (L) Highlighted HSPC gene sets enriched in sgNTG compared with sgNcoa4 transduced Tet2 KO HSPCs. ANOVA, analysis of variance; IR, irradiated; LK, lineage−cKit+; pIpC, polyinosinic:polycytidylic acid.
Published: 2025
Figure 2. Ncoa4 KO impairs Tet2 KO competitive advantage in mouse transplant models. (A) Competitive transplantation of Cas9 expressing WT or Tet2 KO HSCs (CD150 + CD135 − CD48 − lineage − Sca1 + Kit + ) transduced with sgRNA targeting Ncoa4 (sgNcoa4) or sgRNA nontargeting control (sgNTG... More about this image found in Ncoa4 KO impairs Tet2 KO competitive advantage in mouse transplant model...
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Expansion of Tet2 KO HSPCs is dependent on lysosome-dependent ferritinophagy. (A) Tet2 fl/fl; Vav-Cre+ (Tet2 KO) compared with Vav-Cre+ (WT) Hoxb8-ER immortalized HSPC have increased growth after 4 days in RPMI + 10% FCS, but not in 1% FCS unless supplemented with FAC (100 μM). N = 6. (B) Immunoblot of WT and Tet2 KO Hoxb8-ER HSPC treated with DFO 50 μM and/or E64D lysosomal protease inhibitor 2 μg/mL for 16 hours. Immunoblot with FTH1 and β-actin antibody. Dashed line, total FTH1 protein; solid line, lysosomal degraded FTH1 product.47 Fold-change quantification of lysosomal degraded FTH1 product, normalized to β-actin and untreated WT cells. N = 3. (C) Cytoplasmic Fe2+ pool in HSC from Tet2 KO compared with WT. Median fluorescent intensity of labile iron stain relative to untreated WT. Cells were treated with either DFO at 50 μM or vehicle control during concurrent staining to demonstrate the specificity of the labile iron stain. Each dot represents an individual mouse. Gating strategy as per supplemental Figure 2F. Representative flow cytometry histogram of HSCs from WT or Tet2 KO mice. N = 7. (D) Schematic of a competition experiment between WT or Tet2 KO CD45.2+ (20% composition) and WT CD45.1+/CD45.2+ control (80% composition) BM cells transplanted into lethally IR CD45.1 recipients. After engraftment at 4 weeks, mice were treated with a 4-week treatment of ironomycin (3 mg/kg intraperitoneally 5 days/wk) vs vehicle before harvest for peripheral blood and BM. (E) Peripheral blood analysis of whole blood and CD11b compartments and (F) BM analysis of CD45.2+Tet2 KO and CD45.1+/CD45.2+ WT donor chimerism after a 4-week treatment of either ironomycin or vehicle control. Each point represents an individual mouse. N = 12 to 15 per arm. Bars show mean with SEM, with unpaired t test used unless otherwise specified. CMP, common myeloid progenitor; DFO, deferoxamine; FAC, ferric ammonium citrate; FCS, fetal calf serum; GMP, granulocyte-monocyte progenitor; IR, irradiated; MEP, megakaryocyte-erythroid progenitor.
Published: 2025
Figure 3. Expansion of Tet2 KO HSPCs is dependent on lysosome-dependent ferritinophagy. (A) Tet2 fl/fl; Vav-Cre + ( Tet2 KO) compared with Vav-Cre + (WT) Hoxb8-ER immortalized HSPC have increased growth after 4 days in RPMI + 10% FCS, but not in 1% FCS unless supplemented with FAC (100 μM... More about this image found in Expansion of Tet2 KO HSPCs is dependent on lysosome-dependent ferritinoph...
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Increased mitochondrial ATP production in Tet2 KO HSPCs is dependent on ferritinophagy. (A) Using cell clusters at the top of the pseudotime trajectory (blue label) (from Figure 2J), which excludes Fcgcr3+ differentiated progenitors, gene set enrichment analysis was performed comparing WT with Tet2 KO HSPCs using all of the C2 Molecular Signatures Database.37 This revealed enrichment of gene sets involved in mitochondrial function (purple) and oxidative phosphorylation (green). (B) Highlighted mitochondrial and oxidative phosphorylation gene sets comparing WT with Tet2 KO HSPCs. (C) Targeted CRISPR screen using mitochondrial and iron homeostasis regulatory genes in WT and Tet2 KO Cas9 LSK HSPCs. Tet2 KO HSPCs exhibit genetic dependency on regulators of iron, mitochondrial respiration, and translation. Highlighted genes have a false discovery rate of <0.25, WT/Tet2 KO persisting clone differences of >2, and WT/Tet2 KO VAF of >1. (D) Doxycycline-inducible Cas9 expressing WT or Tet2 fl/fl Vav1-Cre (Tet2 KO) in Hoxb8 HSPC system transduced with either control sgRNA (NTG) or Steap3 targeting sgRNA (sg1 and sg2) or Idh3g targeting sgRNA (sg1 and sg2). Growth relative to no doxycycline-treated cells at 4 days. N = 5. Bars showing mean ± SEM, unpaired t test. (E) Transmission electron microscopy (TEM) of primary WT and Tet2 KO LSK HSPCs. Cristae number normalized to the length of mitochondria. Scale bar, 500 nm. N = 50 to 57 mitochondria from 5 mice per genotype. Mean and SEM are displayed with results of unpaired t test. (F) TEM of WT and Tet2 KO Hoxb8 with control short hairpin RNA (shRNA), Ncoa4 shRNA, or ironomycin treatment 50 nM for 48 hours. Images in supplemental Figure 4B. Cristae number was normalized to the length of mitochondria. N = 30 to 71 mitochondria per experimental condition. Mean and SEM are displayed with results of unpaired t test. (G) Mitochondrial staining of primary WT and Tet2 KO CD135−CD48−CD34−CD150+ HSCs. Scale bar, 5 μm. Representative slice from a 3-dimensional structured illumination microscopy image shown. Quantification of mean volume divided by counted mitochondria. Bars showing mean ± SEM, with unpaired t test. Each dot represents an analysis of an individual cell from 5 mice per genotype. (H) Representative Seahorse Mito Stress assay of WT and Tet2 KO Hoxb8-ER cells treated with either vehicle (dimethyl sulfoxide [DMSO]) or ironomycin (50 nM) for 48 hours. Quantification of mitochondrial and glycolytic ATP production is shown in supplemental Figure 4D. (I) Primary human CD34+ umbilical cord HSPCs were transfected with either AAVS1 or TET2 (sg1 and sg2) gRNA–CRISPR-Cas9 RNP complexes. Adenovirus encoding mNeonGreen sequence with adjoining DNA sequence homologous to edited loci results in integration at targeted genes. Fluorescent activated cell sorting (FACS) was used to purify successfully edited cells identified via mNeonGreen expression. Purified cells were analyzed with the Seahorse Mito Stress assay. The mitochondrial ATP production rate is derived from OCR and extracellular acidification rate. N = 7. (J) Seahorse Mito Stress assay on primary human CD34 HSPCs transfected with either AAVS1 or TET2 targeting gRNA were treated with either DMSO (vehicle) or ironomycin 200 nM for 48 hours. Quantification of mitochondrial ATP production was summarized from Seahorse assays. N = 2-6. Mean and SEM are displayed with results of unpaired t test. AMA/Rot, antimycin A/rotenone; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; KI, knockin; OCR, oxygen consumption rate; Oligo, oligomycin; RNP, ribonucleoprotein.
Published: 2025
Figure 4. Increased mitochondrial ATP production in Tet2 KO HSPCs is dependent on ferritinophagy. (A) Using cell clusters at the top of the pseudotime trajectory (blue label) (from Figure 2 J), which excludes Fcgcr3 + differentiated progenitors, gene set enrichment analysis was performed com... More about this image found in Increased mitochondrial ATP production in Tet2 KO HSPCs is dependent on f...
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