Journal: Nature Communications

Article Title: Targeting miR-126 in inv(16) acute myeloid leukemia inhibits leukemia development and leukemia stem cell maintenance

doi: 10.1038/s41467-021-26420-7

Figure Lengend Snippet: a Relative levels of miR-126 in CD34 + and CD34 − cells from healthy (HL; black circle; n = 6; HL CD34 + vs. HL CD34 − p = 0.0036) donors and inv(16) AML patients (red square; n = 10; inv(16) CD34 + vs. HL CD34 + p = 0.024; inv(16) CD34 − vs. HL CD34 − p = 0.0053; inv(16) CD34 + vs. inv(16) CD34 − p = 0.0002). b Relative levels of EGFL7 in CD34 + and CD34 − cells from HL donors (black circle; n = 6; HL CD34 + vs. HL CD34 − p = 0.016) and inv(16) AML patients (red square; n = 10; inv(16) CD34 + vs. HL CD34 + p = 0.006; inv(16) CD34 + vs. inv(16) CD34 − p < 0.0001). c Counts per million (CPM) reads for miR-126-3p in PB based on miRNA-seq of control (Ctrl; black line; n = 7) and CM (red line; n = 6) mice over time until moribund with leukemia (two-way ANOVA analysis showed CM vs. Ctrl p < 0.0001). d Normalized reads per kilobase per million of transcript (RPKM) for Egfl7 in PB based on RNA-seq of Ctrl (black line; n = 7) and CM (red line; n = 6) mice over time (two-way ANOVA analysis showed Ctrl vs. CM p = 0.0003). e Relative levels of miR-126 in CM (red square) preleukemic (6 weeks after induction; left; n = 6) or leukemic (right; n = 6) vs. Ctrl (black circle; n = 6) HSPC populations, including LSK (preleukemic p = 0.0074; leukemic p = 0.049), GMP (preleukemic p = 0.0064; leukemic p = 0.043), Pre-Meg/E (preleukemic p = 0.00015; leukemic p = 0.0012), Pre-GM (preleukemic p < 0.0001; leukemic p = 0.0002), and EP (leukemic p < 0.0001). f Western blot for CBFβ and CM using anti-CBFβ in 32D cells transduced with CBFB vs. CM (left). Relative levels of primary pri-miR-126 , precursor pre-miR-126 , mature miR-126, and Egfl7 in 32D cells expressing CM (red dots/bars) vs. CBFβ (black dots, gray bars). g Western blot analysis for CM in 32D-CM non-silencing (NS) control vs. CM shRNA (A3, D4) cells (left). Relative levels of mature miR-126, pri-miR-126, pre-miR-126 and Egfl7 expression in NS (red), CM shRNA-A3 (blue) or CM shRNA-D4 (gray) cells (right). h Relative Gata2 expression in BM samples of control (black/gray; n = 5) vs. CM leukemia (red; n = 4; p = 0.0002) mice. i Relative level of Gata2 in leukemia BM cells transduced with NS control (red), Gata2 shRNA #2 (dark blue), Gata2 shRNA #4 (light blue). j Relative expression of miR-126 in leukemia BM cells transduced with NS control (red), Gata2 shRNA #2 (dark blue), Gata2 shRNA #4 (light blue). k EGFL7 promoter-firefly luciferase reporter activity normalized with renilla luciferase as internal control in 293T cells co-transfected with or without GATA2, RUNX1, CBFβ or CM expression vectors as indicated (gray: none; green: GATA2; light blue: RUNX1 + CBFβ; dark blue: RUNX1 + CBFβ + GATA2; orange: RUNX1 + CM; Red: RUNX1 + CM + GATA2). Each dot represents results from an individual sample and data are presented as the mean ± SEM in a , b , e , h ; each line represents the trajectory of an individual mouse in c , d . Representative data of at least three independent experiments are shown in f , g , i , j and data are presented as the mean ± SD. Q-PCR data in a , b , e , f for pri-miR-126 , pre-miR-126 , Egfl7 , and Gata2 were normalized to Beta-2-microglobulin ( B2M ) and miR-126 data were normalized to RNU44 (human) or snoRNA234 (mouse). The statistical significance for all comparisons shown was determined using two-tailed Student’s T tests (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The cell number and viability were assessed and CD34 + cells were enriched using StemSepTM Human CD34 Positive Selection reagents (Stemcell Technologies, 14756).

Techniques: Control, RNA Sequencing, Western Blot, Transduction, Expressing, shRNA, Luciferase, Activity Assay, Transfection, Two Tailed Test

Journal: Nature Communications

Article Title: Targeting miR-126 in inv(16) acute myeloid leukemia inhibits leukemia development and leukemia stem cell maintenance

doi: 10.1038/s41467-021-26420-7

Figure Lengend Snippet: a Relative levels of miR-126 in inv(16) AML CD34 + cells transduced with shCtrl (black) or shmiR-126 (red; p = 0.006), as assessed by qPCR and normalized with internal levels of RNU44. b Frequency (%) of G 0 , G 1 or S/G 2 /M phases of cell cycle in shCtrl (black) or shmiR-126 (red) transduced inv(16) AML CD34 + (G 0 p = 0.0073; G 1 p = 0.046; S/G 2 /M p = 0.0094), CD34 + CD38 − (G 0 p = 0.0069; G 1 p = 0.0184; S/G 2 /M p = 0.0138), and CD34 + CD38 + (G 0 p = 0.0072; G 1 p = 0.0015; S/G 2 /M p = 0.0259) populations. c Frequency (%) of apoptotic cells defined by Annexin V + in shCtrl (black) vs. shmiR-126 (red) transduced inv(16) AML CD34 + ( p = 0.0057), CD34 − ( p = 0.0261), CD34 + CD38 + ( p = 0.0027), and CD34 + CD38 − ( p = 0.0331) populations. d Representative FACS plots showing gating strategy and frequency of Annexin V/DAPI staining in CD34 + and CD34 − fractions of inv(16) AML samples transduced with shCtrl or shmiR-126. e Western blot of p-MYC (S62), MYC, BCL2, BAX in CD34 + cells from inv(16) AML patients (AML1298, 987, 1069) 2 days after transduction with shCtrl or shmiR-126. f Western blot of PARP and Cleaved Caspase 3 in CD34 + cells from inv(16) AML patients (AML1298, 987, 1069) 2 days after transduction of shCtrl or shmiR-126. g Relative levels of BCL2 , BAX , BAK1 in CD34 + cells from inv(16) AML patients (AML#1298, 1069, 987) transduced with shCtrl (black) or shmiR-126 (red), as assessed by qPCR and normalized using internal levels of B2M . Data are presented as the mean ± SD. Two-way ANOVA showed p < 0.0001 for BCL2 ; p < 0.0001 for BAX , and p = 0.006 for BAK1 . In a –c, each dot represents result from an individual patient and data are presented as the mean ± SEM; statistical significance shown was determined using two-tailed Student’s T tests (* p < 0.05; ** p < 0.01; *** p < 0.001). Representative of two independent experiments with similar results are shown in e , f .

Article Snippet: The cell number and viability were assessed and CD34 + cells were enriched using StemSepTM Human CD34 Positive Selection reagents (Stemcell Technologies, 14756).

Techniques: Transduction, Staining, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Targeting miR-126 in inv(16) acute myeloid leukemia inhibits leukemia development and leukemia stem cell maintenance

doi: 10.1038/s41467-021-26420-7

Figure Lengend Snippet: a Schematic of experimental design. Inv(16) AML PDX was established by directly injecting T cell-depleted primary AML cells (1 × 10 6 cells/mouse) i.f. into irradiated NSGS and subsequently expanded into larger cohorts by secondary transplantation via i.v. injection. After 8 weeks, PDX mice were treated with SCR control (20 mg/kg/dose, i.v., daily) or miRisten (20 mg/kg/dose, i.v., daily) for 3 weeks and followed by assessment of human cell engraftment in BM and spleen. b Representative FACS plots showing gating strategy and frequency of hCD45 + cells in BM of mice treated with SCR control (top) or miRisten (bottom) for 3 weeks. c Representative FACS plots showing gating and frequency of hCD34 + /CD45 + cells in BM of mice treated with SCR (top) or miRisten (bottom) for 3 weeks. d Frequency of hCD45 + AML cells in SCR (black) vs. miRisten (red) treated mice ( n = 9/group) BM ( p = 0.0222) or spleen (SP). e Frequency of hCD34 + /CD45 + AML cells in SCR (black) vs. miRisten (red) treated mice ( n = 9/group) BM ( p = 0.01) or SP ( p = 0.0385). f The Kaplan–Meier survival curve of mice treated with SCR (black line; n = 6; median survival 117 days) or miRisten (red line; n = 6; median survival 136 days; p = 0.0161) for 3 weeks starting at 8 weeks after transplant. Dotted line with gray shade indicates treatment window. g The Kaplan–Meier survival curve of second transplant recipients treated with SCR (black line; n = 5; median survival 137 days) or miRisten (red line; n = 5; median survival 199 days; p = 0.0127). The statistical significance for f , g was determined using log-rank (Mantel–Cox) test. Each dot in d , e represents result from an individual mouse and data are presented as the mean ± SEM; statistical significance was determined using two-tailed Student’s T- tests (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The cell number and viability were assessed and CD34 + cells were enriched using StemSepTM Human CD34 Positive Selection reagents (Stemcell Technologies, 14756).

Techniques: Irradiation, Transplantation Assay, Injection, Control, Two Tailed Test

Journal: Oncotarget

Article Title: A tyrosine kinase-STAT5-miR21-PDCD4 regulatory axis in chronic and acute myeloid leukemia cells

doi: 10.18632/oncotarget.19192

Figure Lengend Snippet: (A) miR-21 and PDCD4 mRNA were quantified by RT-qPCR on CD34 + cells prepared from four CML donors (#1 to 4) and treated (5μM, 24h) or not with imatinib. For each CML donor, histograms represent the base 2 logarithm of the ratio (treated/not treated cells). (B) CD34 + cells from a CML donor were transduced with a lentivirus carrying a firefly luciferase coding sequence cloned downstream of the miR-21 promoter, then treated or not treated with imatinib. Luciferase activity was measured after 4, 8 or 24 hours. (C) phospho-STAT5 and STAT5 were detected by western blot of protein extracts from CML CD34 + not treated or treated with imatinib.

Article Snippet: They were labeled with the StemSepTM Human CD34 Positive Selection Cocktail (Stemcell Technologies) and isolated on MS column (Miltenyi Biotec).

Techniques: Quantitative RT-PCR, Transduction, Luciferase, Sequencing, Clone Assay, Activity Assay, Western Blot