cd34 microbead ultrapure kit  (Miltenyi Biotec)

Journal: Blood Cancer Discovery

Article Title: Tandem Duplications in UBTF Create XPO1-Dependent Nuclear Export Signals that Reveal a Leukemic Therapeutic Dependency

doi: 10.1158/2643-3230.BCD-25-0112

Figure Lengend Snippet: NES motif in UBTF-TD promotes leukemic proliferation and self-renewal. A, The effects of UBTF-TD NES alanine mutations (NES-M) on cell growth of human donor cbCD34 + cells in liquid culture transduced with viral particles for vector control (EV), UBTF-WT, UBTF-TDs (UBTF-TD48, UBTF-TD54, and UBTF-TD84), and UBTF with NES-M TDs (UBTF-TD48-NES-M, UBTF-TD54-NES-M, and UBTF-TD84-NES-M). For the cell growth assay, we performed a two-way ANOVA using time (weeks) and condition (TD vs. TD-NES-M, i.e., TD54 vs. TD54-NES-M; UBTF-WT vs. EV) as factors. Data were collected from at least three biological replicates and four independent experiments. Post hoc comparisons were made using the Bonferroni multiple comparisons test at each time point, with results specifically shown for the final time point. Mean and SEM are shown. B, The effects of UBTF-TD-NES-M expression on colony-forming potential of cbCD34 cells. A minimum of three cbCD34 + donors per condition were used. A mixed-effects model (restricted maximum likelihood) was used to analyze CFU formation across the different conditions. The same comparisons as in ( A ) were made here, with post hoc analysis (Šidák post hoc test) between UBTF-TD and NES-mutant conditions and UBTF-WT vs. EV shown. Mean and SEM are shown. C, Flow cytometric assessment of cells from three unique cbCD34 + donors after the first round of replating in CFU assay using the stem cell marker CD117. Mean fluorescent intensity (MFI) for CD117 was calculated on live cells/singlets. Significance was calculated by an ANOVA test followed by adjustment with the Tukey method. Right panel depicts a representative flow plot. D, Flow cytometry assessment of myeloid differentiation using CD11b and CD33 from three unique cbCD34 + donors after the first round of replating in CFU assay. Cells are gated on live/singlets and CD117-negative population. Right panel depicts a representative flow plot. Statistical significance was calculated by an ANOVA test followed by adjustment with the Tukey method. E, Principal component analysis of RNA-seq data on three biological replicates per condition on cells from ( A ) at week six in culture. Gray oval highlights samples with normal controls (EV and UBTF-WT) and NES-mutants; pink oval highlights UBTF-TD samples. F, Venn diagram highlighting the common significantly upregulated genes from ( E ) in each UBTF-TD compared with UBTF-WT. Common upregulated genes known to be dysregulated in UBTF -TD AMLs are listed below the diagram. G, Heatmap depicting relative mRNA steady-state levels from RNA-seq analysis of genes dysregulated in UBTF -TD AML. ns, not significant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Article Snippet: Briefly, cbCD34 + cells were isolated using the human CD34 MicroBead Kit UltraPure (Miltenyi Biotec, 130-1-453).

Techniques: Transduction, Plasmid Preparation, Control, Growth Assay, Expressing, Mutagenesis, Colony-forming Unit Assay, Marker, Flow Cytometry, RNA Sequencing

Journal: Blood Cancer Discovery

Article Title: Tandem Duplications in UBTF Create XPO1-Dependent Nuclear Export Signals that Reveal a Leukemic Therapeutic Dependency

doi: 10.1158/2643-3230.BCD-25-0112

Figure Lengend Snippet: Preclinical testing of XPO1 inhibitor eltanexor on UBTF-TD tumors. A, Experimental setup of UBTF-TD PDX model with YFP-luciferase treated with eltanexor at 5 or 10 mg/kg (5 days on, 2 days off). Via tail-vein injection, 200 K cells/mouse were transplanted. Mice were treated for 6 weeks after engraftment was validated via average total flux >1.2E6 photons per second (p/second). All mice were sacrificed at the end of 6-week treatment. B, In vivo imaging system (IVIS) images and quantification of mice treated with vehicle or eltanexor over time. Images were taken before sacrificing mice at the sixth week of treatment. Treatment period is shaded in gray. Statistical analysis was performed using a two-way ANOVA followed by a Dunnett multiple comparisons test comparing each treatment group with vehicle C . Characterization of UBTF-TD PDX at the time of sacrifice. White blood cell counts (WBC), hemoglobin (Hb), and spleen weights were assessed. Blood was collected via terminal cardiac puncture under anesthesia and complete blood counts were determined using a hematology analyzer (Genesis, Oxford Science). D, Differentiation status of hCD45-positive bone marrow cells in mice from ( B ) assessed using stem cell markers (CD117 and CD34) and myeloid markers (CD11b and CD33). Quantification of relative frequency of different cell populations is shown (right). Statistical significance for dosing comparisons (vehicle, eltanexor 5 mg/kg, and eltanexor 10 mg/kg) was determined using ANOVA, followed by post hoc adjustment using a Dunnett multiple comparisons test for pairwise comparisons between vehicle and eltanexor 5 mg/kg and between vehicle and eltanexor 10 mg/kg, as shown in ( C ) and ( D ). E, Histology with hematoxylin and eosin (H&E) and IHC of bone marrow from representative mouse from each treatment group, including those treated with vehicle control or eltanexor at 5 or 10 mg/kg. IHC was performed using antibodies specific for hNuMA1, MAC1, and MPO. All error bars are means with SD. ****, P < 0.0001.

Article Snippet: Briefly, cbCD34 + cells were isolated using the human CD34 MicroBead Kit UltraPure (Miltenyi Biotec, 130-1-453).

Techniques: Luciferase, Injection, In Vivo Imaging, Control

Journal: Blood Cancer Discovery

Article Title: Tandem Duplications in UBTF Create XPO1-Dependent Nuclear Export Signals that Reveal a Leukemic Therapeutic Dependency

doi: 10.1158/2643-3230.BCD-25-0112

Figure Lengend Snippet: Molecular impact of eltanexor in UBTF -TD preclinical models. A, Experimental schema for molecular characterization of UBTF-TD YFP-luciferase–labeled PDX after treatment with the XPO1 inhibitor eltanexor. Briefly, 200 K cells/mouse were transplanted via tail-vein injection. After engraftment was validated (total flux >1.2E6 photons/second), mice were treated with eltanexor at 5 or 10 mg/kg (5 days on, 2 days off) for 3 weeks and then sacrificed for molecular characterization. B, Dot plot depicting scRNA-seq analysis from bone marrow of mice treated with vehicle (gray) or eltanexor at 5 mg/kg (blue) or 10 mg/kg (red) body weight. Two mice/group are included in these figures. C, Dot plot from ( B ) depicting expression of genes associated with UBTF-TD AML ( CD34 , KIT , HOXA9 , and HOXB9 ) or myeloid differentiation ( MPO and CTSG ). D, Violin plots depicting expression of genes in ( C ). In the violin plots, the horizontal line represents the median, whereas the width of the violin body reflects the distribution density. The IQR is also indicated within the shape. Statistical significance was determined using a pairwise Wilcoxon rank-sum test comparing each treatment group with the vehicle control. P values were adjusted using the Benjamini–Hochberg FDR correction (***, adjusted P value < 0.0001). E, Expression of stem cell markers (CD34 and CD117) and myeloid markers (CD33 and CD11b) is shown for live, single, human CD45 + cells as determined by flow cytometry. F, Genomic view of UBTF and XPO1 occupancy of regions dysregulated in AML ( HOXA/HOXB ) gene clusters. UBTF-TD PDX cells were cultured in vitro and treated with DMSO or eltanexor (25 nmol/L) for 24 hours before CUT&RUN assay. P.O., by mouth; UMAP, Uniform Manifold Approximation and Projection; veh, vehicle.

Article Snippet: Briefly, cbCD34 + cells were isolated using the human CD34 MicroBead Kit UltraPure (Miltenyi Biotec, 130-1-453).

Techniques: Luciferase, Labeling, Injection, Expressing, Control, Flow Cytometry, Cell Culture, In Vitro