Supplemental Material – Supplemental Material and Methods
Expression of Hedgehog pathway mediator GLI represents a negative prognostic marker in human acute myeloid leukemia and its inhibition exerts anti-leukemic effects
Supplemental Material and Methods
Including
Patients and samples
Cell culture
Enrichment of CD34+ progenitor cells from healthy donors
Enrichment of endothelial cells from bone marrow mononuclear cells
Lentiviral transduction of AML cell lines with GLI1- and GLI2-specific shRNA
Cell growth and apoptosis assay
Colony formation assay
Reverse transcription quantitative, semi-quantitative PCR and SMO mutation analysis
DHH immunofluorescence staining
DHH immunohistochemistry
Xenograft model
Patients and samples
Bone marrow (BM) and peripheral blood (PB) mononuclear cells from 104 leukemic patients with newly diagnosed AML were analyzed (referred to as cohort A). Patients were treated within the AMLSG 07-04 study of the Austrio-German Study Group AMLSG (refer to the supplemental material for trial design). In brief, the trial design was as follows: Patients between 18 and 60 years of age with newly diagnosed de novo or secondary AML [excluding patients with t(15;17)] were entered into the study. All patients received 2 induction courses with cytarabine, idarubicin and etoposide. Patients with complete remission obtained high-dose cytarabine consolidation. At study entry, patients were randomized between additional therapy with valproic acid, ATRA, both or none. After an interim analysis the randomization to the valproic acid and the combination arm was suspended while randomization for ATRA was continued. The trial was conducted between August 2004 and August 2009. The study (ClinicalTrials.gov Identifier: NCT00151242) was approved by the ethics committees of each study site and was conducted in accordance with Austrian and German drug development regulations and the Declaration of Helsinki(1). The mononuclear cell (MNC) fraction was isolated from fresh BM or PB samples using Ficoll-Paque centrifugation and RNA was isolated as indicated below.
The second, independent patient cohort – referred to as cohort B – comprised 290 AML patients of whom microarray-based gene expression data was published by Verhaak et al (data accessible at NCBI GEO database, accession GSE6891) (2). Patients were derived from a clinical study by Löwenberg et al(3). Patients aged between 15 and 60 years with newly diagnosed AML (APL excluded) received 3 cycles of standard chemotherapy. In a 2×2 factorial design patients were randomized to granulocyte-macrophage colony-stimulating factor either during chemotherapy only, after chemotherapy until recovery of blood counts, during both periods or none. Leukemia-specific outcome was independent of randomization.
Cell culture
Primary AML cells, AML cell line UKE-1 and CD34+ progenitor cells from healthy donors were cultured in IMDM medium with 10% fetal bovine serum (FBS), 10% horse serum (HS), and 106 M hydrocortisone. KG-1 and MV4-11 were cultured in RPMI-1640 medium with 10% FBS and OCI-AML5 in α-MEM with 20% FBS and 10 ng/ml GM-CSF. OECs and HUVECs (human umbilical vein endothelial cells, purchased from PromoCell GmbH, Heidelberg, Germany) were cultured in endothelial growth medium (Lonza, Walkersville, MD) and primary osteoblasts in osteoblast growth medium (PromoCell).
Enrichment of CD34+ progenitor cells from healthy donors
CD34+ progenitor cells were isolated from the mononuclear cell fraction of leukapheresis products of G-CSF primed healthy, anonymous donors using the indirect CD34 MicroBead Kit and the VarioMACS Separator (Miltenyi Biotec, Bergisch Gladbach, Germany).
Enrichment of endothelial cells from bone marrow mononuclear cells
MNC from AML bone marrow samples were stained with a phycoerythrin-labeled (PE) anti-CD45 antibody (BD Biosciences, San Jose, CA) followed by incubation with anti-PE MicroBeads in order to deplete the hematopoietic cells with LD depletion columns and the VarioMACS Separator (all Miltenyi Biotec). For enrichment of endothelial cells, the remaining cell fraction was stained with a fluorescein-labeled (FITC) anti-CD146 antibody (R&D Systems, Minneapolis, MN) followed by incubation with anti-FITC MicroBeads and positive selection using LS Columns and the VarioMACS Separator (all Miltenyi Biotec). Enriched endothelial cells were used for mRNA isolation and immunofluorescent staining.
Lentiviral transduction of AML cell lines with GLI1- and GLI2-specific shRNA
pLKO.1-puro vector encoding GLI1 (TRCN0000020485, sequence 5‘-CCGGCCTGATTATCTTCCTTCAGAACTCGAGTTCTGAAGGAAGATAATCAGGTTTTT-3'), GLI2(TRCN0000238361, sequence 5'-CCGGCTGGACAGGGATGACTGTAAGCTCGAGCTTACAGTCATCCCTGTCCAGTTTTTG-3') or scrambled shRNA (SHC002, non-target shRNA vector) were purchased from Sigma-Aldrich (Taufkirchen, Germany). Plasmids pMD2.G-VSV-G and psPAX2-Gag-Pol were purchchased from Addgene (Cambridge, MA).HEK293T cells were seeded at a density of 5×105 cells of a 6-well plate and co-transfected with the plasmids pLKO.1-puro shRNA (1μg), pMD2.G-VSV-G (250 ng) and psPAX2-Gag-Pol (750ng) using the Promega ProFection Mammalian Transfection System (Promega Corporation, Madison, WI). Supernatants containing the lentiviral particles were harvested 24 hours after transfection. 1 ml of virus supernatant was directly added to 2×106UKE-1or OCI-AML5 cells in 2 ml medium with 8 µg/ml polybrene followed by centrifugation at 1000 g and 30°C for one hour. Cells were either transduced with non-targeting shRNA (negative control) or shRNA against GLI1 or GLI2. Lentiviral containing medium was removed the next day. On day 3 after transduction, transduced cells were selected by addition of puromycin (2μg/ml; Sigma-Aldrich) for 4 days prior to functional assays. The knock-down efficiency was analyzed in quantitative PCR analysis for GLI1 and GLI2 on days 4 and 7 of puromycin selection. All work with lentiviral particles was done in a S2 facility after approval according to German law.
Cell growth and apoptosis assay
AML cells were incubated with different concentrations of the GLI inhibitor GANT61 (2,20-[[Dihydro-2-(4-pyridinyl)-1,3(2H,4H)-pyrimidinediyl]bis(methylene)]bis[N,Ndimethylbenzenamine]; Tocris, Ellisville, MO) ranging from 3-90µM or DMSO as solvent control. For shRNA experiments, AML cells with the GLI1 or GLI2 knock-down were compared to the negative control containing non-targeting shRNA.
Induction of apoptosiswas measured after 24 hours by flow cytometry using Annexin-V and propidium iodideand the FACSCalibur and CellQuestPro Software (BD Biosciences).
For proliferation assays, cell numbers were determined every 3 days for up to two weeks using the cell viability analyzer Vi-Cell XR (Beckman Coulter, Brea, CA). Cells were retreated with GANT61 or solvent control every three days.
Colony formation assay
AML cell lines, primary AML cells and CD34+ progenitor cells were incubated with different concentrations of GANT61 or DMSO control and cultured in methylcellulose-based semi-solid media without or supplemented with growth factors, respectively (Methocult H4230 for AML cell lines and Methocult H4435 Enriched for primary AML cells and CD34+cells, Stemcell Technologies, Vancouver, Canada). For shRNA experiments, the colony formation capacity of AML cell lines with the GLI1 or GLI2 knock-down were compared to the negative control containing non-targeting shRNA. After 7 to 12 days, the number of colonies was counted using an inverted microscope (Axiovert 25, Zeiss, Jena, Germany). Contamination of primary AML samples with colonies derived from normal hematopoietic stem cells was excluded since no colonies with hematopoietic differentiation were observed.
Reverse transcription quantitative, semi-quantitative PCR and SMO mutation analysis
Exon-spanning primers were designed with Primer 3 software (Whitehead Institute for Biomedical Research, Boston, MA). RT-qPCR analysis was carried out on the LightCycler 1.2 (Roche, Basel, Switzerland) using the FastStart DNA Master SYBR Green Kit (Roche) over 40 PCR cycles. Relative amount of expressed cDNA was calculated using a standard curve obtained from logdilutions of plasmids containing the corresponding genes. Plasmids were either constructed by cloning the PCR amplification product into the pCRII Vector (TA Cloning Kit, Invitrogen) or were purchased as full-length cDNA clones (Biocat, Heidelberg, Germany). Samples were analyzed in duplicates and averaged. Calculated cDNA amounts of the target genes were normalized to the reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). All data are represented as ratio of the target gene/GAPDH.
Due to low expression and the possible occurrence of primer dimers in primary AML samples, GLI2 PCR products were separated in gel electrophoresis subsequent to LightCycler analysis and GLI2 positivity was evaluated semi-quantitatively on the basis of a distinct band in gel electrophoresis (refer to supplemental figure S1 for representative gel electrophoresis pictures).
Since mutations within exon 9 or 10 of the SMO gene represent a frequent genetic alteration in basal cell carcinoma (4), both SMO exon 9 and 10 were analyzed for mutations. A PCR product of 466 bp (primers in supplemental table S1) spanning whole exons 9 and 10 was amplified with DreamTaq™ PCR Master Mix (Fermentas, St. Leon-Rot, Germany). PCR products were purified using a PCR purification kit and sequenced by Extended HotShot technology (both Seqlab, Göttingen, Germany). Primers and data on PCR specificity and efficiency are listed in supplemental table S1 and S2.
DHH immunofluorescence staining
Primary OECs, osteoblasts, MNCs from healthy donors and the endothelial cell-enriched and whole leukemic MNC fraction of AML bone marrows were spun onto glass slides using the Rotofix 32A centrifuge (Hettich Zentrifugen, Tuttlingen, Germany). Slides were fixed in 4% paraformaldehyde for 20 min and blocked for 30 min with 2% normal donkey serum. The primary DHH antibody (dilution 1:50, #sc-1193; Santa Cruz, Santa Cruz, CA) and secondary Cy3-conjugated AffiniPure donkey anti-goat IgG (1:200, 705-165-003, Jackson ImmunoResearch) were both incubated for 1h each, followedby staining with DAPI. Double staining with CD146 as shown in figure 2C was due to staining of endothelial cells for immunomagnetic separation (see section Enrichment of endothelial cells from bone marrow mononuclear cells for more information).
DHH immunohistochemistry
For immunohistochemical labelling, formalin-fixed paraffin-embedded BM tissue sections of 7 AML patients and 2 patients with solid tumors without BM involvement were pre-treated with REAL Target Retrieval Solution (pH 6.0; Dako GmbH, Hamburg, Germany) followed by heat incubation in a steamer for 20 min. Sections were blocked by 2% normal rabbit serum followed by incubation with the primary DHH antibody (#sc-1193; Santa Cruz). A biotin-labelled rabbit anti-goat antibody was used as secondary antibody and DAB as chromogenic agent (Zytochem HRP-DAB Kit Broadspectrum; Zytomed, Berlin, Germany) and sections were counter-stained withhematoxylin/eosine.
Xenograft model
For the in vivo experiments, the shRNA sequences used for the in vitro experiments (see above) were cloned into the LeGO vector system (a 3rd generation HIV1-derived lentiviral vector) as they carry fluorescent marker proteins allowing easy testing of transduction efficiencies andeasytrackingin murine tissues ( The GLI2 shRNA sequence was cloned into a vector carrying green fluorescent protein as marker (LeGO-G/Puro+) while the GLI1 shRNA was cloned into a vector carrying mCherry as marker protein (LeGOC/Zeo+). The scrambled control shRNA was cloned into both vectors. MV4-11 cells were transduced with the GLI1 and GLI2 shRNA carrying vectors (double knockdown approach) or with the control vectors coding for the scrambled shRNA (control cells). Two days after the transduction, the transduction efficiency was checked using flow cytometry (FACSCalibur, BD Biosciences) and 5 × 105 MV4-11 cells were intravenously transplanted into female, 16- to 20-week-old NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice (10 control and 11 double-knockdown animals). Mice were sacrificed when showing clear symptoms of leukemia such as dramatic loss of weight, hypothermia or apathy. MNCs from the peripheral blood, femoral bone marrow and the spleen were isolated using Ficoll-Paque centrifugation and checked for leukemic infiltration by flow cytometry.
Supplemental References for Supplemental Material and Methods:
1.Schlenk RF, et al. All-Trans Retinoic Acid Improves Outcome in Younger Adult Patients with Nucleophosmin-1 Mutated Acute Myeloid Leukemia – Results of the AMLSG 07-04 Randomized Treatment Trial. Blood. 2011;118 (suppl; abstr 80)(80).
2.Verhaak RG, et al. Prediction of molecular subtypes in acute myeloid leukemia based on gene expression profiling. Haematologica. 2009;94(1):131-134.
3.Lowenberg B, et al. Value of different modalities of granulocyte-macrophage colony-stimulating factor applied during or after induction therapy of acute myeloid leukemia. J Clin Oncol. 1997;15(12):3496-3506.
4.Lam CW, et al. A frequent activated smoothened mutation in sporadic basal cell carcinomas. Oncogene. 1999;18(3):833-836.
5.Weber K, Mock U, Petrowitz B, Bartsch U, and Fehse B. Lentiviral gene ontology (LeGO) vectors equipped with novel drug-selectable fluorescent proteins: new building blocks for cell marking and multi-gene analysis. Gene Ther. 2010;17(4):511-520.
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