Journal: Blood cancer discovery

Article Title: Persistence of Drug-Resistant Leukemic Stem Cells and Impaired NK Cell Immunity in CML Patients Depend on MIR300 Antiproliferative and PP2A-Activating Functions

doi: 10.1158/0008-5472.BCD-19-0039

Figure Lengend Snippet: MIR300 loss in leukemic progenitors and differential induction of its cell context–independent tumor suppressor activities in quiescent LSCs. A, (left) MIR300 levels in healthy NBM and CML-CP and -BC CD34+ BM cell fractions. Inset shows MIR300 levels in additional CD38-fractionated CD34+ CML-CP BM cells expressed as n-fold difference in CD34+CD38+ compared with CD34+CD38− samples. B, MIR300 levels in untreated and imatinib (24 hours)-treated CD34+ quiescent (CFSEmax) and dividing (Div.1) CFSE-labeled CML and UCB cells. Asterix on CD34+CD38− cell populations (panel 1a) indicate significance between MIR300 levels CD34+CD38− versus CD34+CD38+ cells. Data are shown as mean ± SEM from at least three independent experiments; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Flow Cytometry and Cell Sorting CD34 + , CD34 + CD38 − , CD34 + CD38 + fractions were magnetic (CD34 MicroBead Kit; Miltenyi Biotec) and/or FACS (αCD34 APC/PE and αCD38 PE/Cy7 Abs, BD Biosciences) purified (purity: >90%–100%).

Techniques: Labeling

Journal: Blood cancer discovery

Article Title: Persistence of Drug-Resistant Leukemic Stem Cells and Impaired NK Cell Immunity in CML Patients Depend on MIR300 Antiproliferative and PP2A-Activating Functions

doi: 10.1158/0008-5472.BCD-19-0039

Figure Lengend Snippet: MIR300 activity in quiescent leukemic stem and progenitor cells. A, Growth (48 hours) and clonogenic potential (CFC) of CpG-scramble- and CpG-miR-300-treated (500 nmol/L) CD34+ CML-BC and UCB cells. B, Effect of CpG-miR-300 and CpG-scramble (500 nmol/L) on spontaneous and IM (18 hours)-induced apoptosis (Annexin V/7-AAD) in CD34+ CML-BC cells (n = 3). Data are reported as mean ± SE (P < 0.01) from three independent experiments inside representative Annexin V/7AAD FACS pseudocolor plots. C, Ki-67/DAPI (left; G0: MIR300 = 46% vs. scr ≅ 10%; G1: MIR300 ≅ 15% vs. scr ≅ 50%; S/G2–M: MIR300 ≅ 4% vs. scr ≅ 27.4%; and sub-G1: MIR300 ≅ 35% vs. scr ≅ 3%) and FUCCI-2BL (right; G1–G0: MIR300 ≅ 40.2% vs. scr ≅ 23.6%; G1–S: MIR300 ≅ 15% vs. scr ≅ 2.85%; S/G2–M: MIR300 ≅ 45% vs. scr ≅ 76.6%) cell-cycle analysis of UBC and Ph+ (primary CD34+ and synchronized LAMA-84) cells exposed to the indicated CpG-ONs. D, Dose-dependent differential regulation of MIR300 antiproliferative and proapoptotic activities on CML qLSC (CFSEmax) and progenitor (Div. 1–2) cell (left) and LTC-IC (right) numbers. Vector transduced and 500 nmol/L CpG-scramble and CpG-anti-miR-300 served as controls. Inset, MIR300 levels in pCDH-MIR300 lentiviral–transduced and 250–500 nmol/L CpG-miR-300–treated Ph+ cells. Data are shown as mean ± SEM from at least three independent experiments; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Range values of controls are reported in Supplementary Table S1.

Article Snippet: Flow Cytometry and Cell Sorting CD34 + , CD34 + CD38 − , CD34 + CD38 + fractions were magnetic (CD34 MicroBead Kit; Miltenyi Biotec) and/or FACS (αCD34 APC/PE and αCD38 PE/Cy7 Abs, BD Biosciences) purified (purity: >90%–100%).

Techniques: Activity Assay, Cell Cycle Assay, Plasmid Preparation

Journal: Blood cancer discovery

Article Title: Persistence of Drug-Resistant Leukemic Stem Cells and Impaired NK Cell Immunity in CML Patients Depend on MIR300 Antiproliferative and PP2A-Activating Functions

doi: 10.1158/0008-5472.BCD-19-0039

Figure Lengend Snippet: C/EBPβ-dependent MIR300 tumor suppressor antiproliferative activity accounts for BMM-induced LSC entry into quiescence. A, Effect of hypoxia on (i) MIR300 levels in CD34+ CML-BC, and BM-derived primary (hMSCs) and HS-5 MSCs (B); and (ii) MIR300 targets in untreated and CpG-anti-miR-300–treated (500 nmol/L, 48 hours) and CML-BC cells. Inset, effect of hypoxia on CFSE+CD34+ CML-BC proliferation. B, Effect of hMSC and HS-5 conditioned medium (CM) and/or exosomes (50–100 μg/mL) from parental, vector (pZIP), and anti-MIR300 (pZIP-MIR300)–transduced primary hMSCs and/or HS-5 cells on: (i) proliferation (% growth inhibition); (ii) qLSC fraction (CFSEmaxCD34+); and (iii) MIR300 levels in CD34+ CML-BC and LAMA-84 cells. Insets, β-catenin and SET levels in anti-MIR300 (pZip-300)-transduced HS-5 (left); MIR300 in HS-5 Alix+CD63+ exosomes (right). E, Effect of hypoxia on primary MIR300 transcripts (pri-miR-300), C/EBPβ (LAP1, LAP2, and LIP isoforms), BCR-ABL1 expression (αABL) and activity (αPY), and GRB2 levels in CD34+ CML-BC cells. (*): nonspecific band. E, MIR300 promoter/enhancer activity in hypoxia- (48 hours) and normoxia-cultured CML-BC CD34+ cells transduced with pGFP/Luc-based MIR300-reported constructs. p109mut is mutated in the −64 and −46 bp C/EBPβ-binding sites. F, Effect of ectopic C/EBPβ (inset) on mature (MIR300) and primary (pri-miR-300) MIR300 levels in CD34+ CML-BC cells. Data are represented as mean ± SEM for at least three experiments. Range values of controls are reported in Supplementary Table S1.

Article Snippet: Flow Cytometry and Cell Sorting CD34 + , CD34 + CD38 − , CD34 + CD38 + fractions were magnetic (CD34 MicroBead Kit; Miltenyi Biotec) and/or FACS (αCD34 APC/PE and αCD38 PE/Cy7 Abs, BD Biosciences) purified (purity: >90%–100%).

Techniques: Activity Assay, Derivative Assay, Plasmid Preparation, Inhibition, Expressing, Cell Culture, Transduction, Construct, Binding Assay

Journal: Blood cancer discovery

Article Title: Persistence of Drug-Resistant Leukemic Stem Cells and Impaired NK Cell Immunity in CML Patients Depend on MIR300 Antiproliferative and PP2A-Activating Functions

doi: 10.1158/0008-5472.BCD-19-0039

Figure Lengend Snippet: MIR300 acts as master PP2A activator and inhibitor of G1–S transition through a dose-dependent target selection mechanism. A, Left:, representative blots show effect of MIR300 on its targets and PP2A activity in UCB and CML-BC CD34+ cells and cell lines exposed to CpG-scramble and CpG-miR-300 (500 nmol/L; 48–72 hours). Right, (top) Dapi/Ki67 cell-cycle analysis of CpG-scramble, -miR-300, and CpG-anti-miR-300 (500 nmol/L; 21 hours)-treated aphidicolin-synchronized K562 cells; (middle) Flag-SET lentiviral constructs with wild-type or a deleted mRNA 3′UTR; (bottom) MIR300-induced downregulation of Flag-SET proteins, and rescue of Ph+ cells from exogenous MIR300-induced growth inhibition (Trypan blue exclusion)/apoptosis (Annexin V+) by Flag-SET cDNAs lacking MIR300-binding site. Similar results were obtained with LAMA-84 cells. B, Hierarchical clustering of statistically significant (P < 0.05 with FDR correction) MIR300 targets using the indicated databases (number of binding sites is indicated in red). Top right, schematic representation of the biological effects of MIR300 dose-dependent target selection activity in qLSCs and leukemic progenitors; (bottom) SET, CDK6, CCND2, and β-actin levels in CpG-MIR300- and CpG-scramble–treated (100–500 nmol/L; 48 hours) CML-BC CD34+ cells.

Article Snippet: Flow Cytometry and Cell Sorting CD34 + , CD34 + CD38 − , CD34 + CD38 + fractions were magnetic (CD34 MicroBead Kit; Miltenyi Biotec) and/or FACS (αCD34 APC/PE and αCD38 PE/Cy7 Abs, BD Biosciences) purified (purity: >90%–100%).

Techniques: Selection, Activity Assay, Cell Cycle Assay, Construct, Inhibition, Binding Assay

Journal: Blood cancer discovery

Article Title: Persistence of Drug-Resistant Leukemic Stem Cells and Impaired NK Cell Immunity in CML Patients Depend on MIR300 Antiproliferative and PP2A-Activating Functions

doi: 10.1158/0008-5472.BCD-19-0039

Figure Lengend Snippet: Selective suppression of MIR300 proapoptotic, but not antiproliferative, activity by TUG1 lncRNA in CML quiescent LSCs. A, BMM-generated signals regulating MIR300-TUG1 interplay and its effect on CML LSC survival and quiescence. B, TUG1 levels in CD34+ quiescent stem (CFSEmax) and dividing progenitors (Div.1, 2) and in untreated and imatinib-treated CD34+ CML cells. C, Effect of anti-TGFβ antibody (Ab) and/or hypoxia (1% O2, 48 hours) on TUG1 lncRNA and FoxM1 levels in CD34+ and CD34+CFSEmax CML-BC cells. D, Dose-dependent differential effect of low (100 nmol/L) and high (500 nmol/L) CpG-TUG1-shRNA and CpG-scramble on Ph+ LAMA-84 cell proliferation and survival (Annexin V). E, Effect of anti-TGFβ Ab, TUG1-shRNA, TUG1 RNA, and control (CpG-scramble or empty vector) on recovery of untreated and CpG-scramble, -miR-300, and/or -anti-miR-300 eFluor+CD34+ CML qLSCs (eFluormax) and dividing (Div.1–2) progenitors relative to input. Inset, TUG1 levels in vector, TUG1 shRNA and scramble-shRNA cells. Data are represented as mean ± SEM for at least three experiments. Range values of controls are reported in Supplementary Table S1.

Article Snippet: Flow Cytometry and Cell Sorting CD34 + , CD34 + CD38 − , CD34 + CD38 + fractions were magnetic (CD34 MicroBead Kit; Miltenyi Biotec) and/or FACS (αCD34 APC/PE and αCD38 PE/Cy7 Abs, BD Biosciences) purified (purity: >90%–100%).

Techniques: Activity Assay, Generated, shRNA, Control, Plasmid Preparation

Journal: Blood cancer discovery

Article Title: Persistence of Drug-Resistant Leukemic Stem Cells and Impaired NK Cell Immunity in CML Patients Depend on MIR300 Antiproliferative and PP2A-Activating Functions

doi: 10.1158/0008-5472.BCD-19-0039

Figure Lengend Snippet: Disruption of MIR300-TUG1 interplay and PAD treatment abrogate the BMM-protective effect on survival of CML qLSCs and BCR-ABL1+ leukemia-initiating cells. A, Xenotransplantation protocol of ex vivo–treated CD34+ chronic (CP), accelerated (AP) and blastic phase (BC) CML cells in NRG-SGM3 mice (n = 4 mice/treatment/patient sample). B and C, Analysis of CpG-MIR300-, CpG-TUG1-shRNA-, CpG-TUG1-shRNA+CpG-MIR300-, and CpG-scramble–treated CML cells from BM aspirates at 2–12 (3D plots) and 10 to 20 weeks posttransplant quantitative analysis of CML cells stained with the indicated antibodies. D, Evaluation at 10 to 20 weeks posttransplant of BCR-ABL1 transcripts by qRT-PCR (left) and of % Ph-negative (Ph−) cells by FISH (right) in total and FACS-sorted hCD45+BM cells, respectively. E, Analyses of BM CML cells at 10 to 20 weeks posttransplant: hCD45+ cells (%) in BM (left) and PB (right) of mice transplanted with CML (CP, AP, and BC) and treated with the indicated CpG-ODNs. Age-matched mice served as controls. Error bars, mean ± SEM. F, Effect of 2.5 μmol/L FTY720 or 1 μmol/L imatinib (IM) on CAFC activity (left) and numbers of CFSEmaxAnnexinVneghCD45+CD34+ CML qLSCs derived from CFSE-labeled CD34+ CML-BC cells cocultured for 7 days on BM-derived HS-5 MSC cells (right). Inset, FACS plot shows gating of CFSEmax CML qLSCs. G, Relative number and representative images of CAFC (red arrows) of CFSE-labeled 32D-BCR-ABL cells cocultured with primary mMSCs in the absence or presence of IM (1 μmol/L, 48 hours) or FTY720 (2 μmol/L, 48 hours; n = 5). Inset: CFSEbright fraction of adherent 32D-BCR-ABL cells in medium and cocultured for 4 days with mMSCs. Range values of controls are reported in Supplementary Table S1.

Article Snippet: Flow Cytometry and Cell Sorting CD34 + , CD34 + CD38 − , CD34 + CD38 + fractions were magnetic (CD34 MicroBead Kit; Miltenyi Biotec) and/or FACS (αCD34 APC/PE and αCD38 PE/Cy7 Abs, BD Biosciences) purified (purity: >90%–100%).

Techniques: Disruption, Ex Vivo, shRNA, Staining, Quantitative RT-PCR, Activity Assay, Derivative Assay, Labeling