Burns, et al HEDGEHOG PATHWAY MUTATIONS DRIVE T-ALL
Supplementary Information for:
Hedgehog pathway mutations drive oncogenic transformation in
high-risk T-cell acute lymphoblastic leukemia
Authors:
Melissa A. Burns1,2, Zi Wei Liao1, Natsuko Yamagata1, Gayle P. Pouliot1,2, Kristen E. Stevenson3, Donna S. Neuberg3, Aaron R. Thorner4, Matthew Ducar4, Emily A. Silverman1, Stephen P. Hunger5, Mignon L. Loh6, Stuart S. Winter7, Kimberly P. Dunsmore8, Brent Wood9, Meenakshi Devidas10, Marian H. Harris11,Lewis B. Silverman1,2, Stephen E. Sallan1,2, and Alejandro Gutierrez1,2*
Affiliations:
1Division of Hematology/Oncology, 11Department of Pathology, Boston Children’s Hospital, Boston, MA 02115, USA.
2Department of Pediatric Oncology, 3Department of Biostatistics and Computational Biology, 4 Center for Cancer Genome Discovery, Dana-Farber Cancer Institute, Boston, MA 02215, USA.
5Division of Oncology and the Center for Childhood Cancer Research, The Children’s Hospital of Philadelphiaand the Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA.
6Department of Pediatrics, University of California San Francisco, San Francisco, CA 94158, USA.
7Department of Pediatrics, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA.
8Division of Oncology, University of Virginia Children’s Hospital, Charlottesville, VA 22903, USA.
9Department of Laboratory Medicine, University of Washington, Seattle, WA, USA.
10Department of Biostatistics, University of Florida, Gainesville, FL 32611, USA.
Corresponding author:
Alejandro Gutierrez
Division of Hematology/Oncology
Boston Children’s Hospital
300 Longwood Avenue, Boston, MA, USA 02115
Phone: 617-919-3660; Fax: 617-730-0934
Email:
orcid.org/0000-0002-0249-9007
SUPPLEMENTARY MATERIALS AND METHODS
Study design and patient samples
The main goal of our study was to discover targetable oncogenic alterations in childhood T-ALL, particularly for patients with chemotherapy-resistant subsets of the disease. The sample size for our study was determined by sample availability. The sample size for our study was determined by sample availability. Adequate tumor material from 109 patients with newly diagnosed T-ALL, with annotated clinical information, was available to us from patients enrolled on Dana-Farber Cancer Institute Study 05-001 ( (1), or Children’s Oncology Group Study AALL0434 ( (2)clinical trials. No stopping rules for data collection were applied for this study, beyond those prospectively defined in these clinical trials. Criteria for data inclusion included availability of adequate tumor material for sequencing analysis, and appropriate informed consent and Institutional Review Board (IRB) approval of the respective institutions, in accordance with the Declaration of Helsinki. No samples that met these criteria were excluded from the sequencing analysis. Of 109 patients in this cohort, 3 were unevaluable for induction response because of death in induction (2 patients) or no outcome data available (1 patient).
Clinical endpoints for the study were based on the events defined in the COG AALL0434 and DFCI 05-001 clinical trials, which included induction failure, induction death, relapse, death, or second malignancy; however, there were no deaths in remission or second malignancies among the samples available for this study. Because the study definition of induction failure differed between these two studies (≥5% leukemic blasts on end-induction bone marrow on DFCI 05-001, versus >25% on COG AALL0434), for the purposes of this manuscript we defined a poor response to induction chemotherapy as ≥5% leukemic blasts in the bone marrow at the end of the first month of induction chemotherapy, as assessed by morphology on DFCI 05-001 or flow cytometry on COG AALL0434(2). Early T-cell precursor T-ALL was defined by flow cytometry immunophenotyping, as originally described(3).
Targeted exon sequencing of T-ALL patient samples
T-ALL diagnostic specimens were purified using Ficoll-Paque reagent and viably frozen. Genomic DNA (gDNA) was extracted using the AllPrep DNA/RNA mini kit (OPv1 cohort; Qiagen, Venlo, Netherlands) or the DNeasy kit (OPv3mod cohort; Qiagen) according to the manufacturer’s instructions. Targeted exon sequencing was performed at the Center for Cancer Genome Discovery at the Dana-Farber Cancer Institute using an Illumina sequencing platform, for all protein-coding exons of the genes shown in Supplementary Table 3. Briefly, 200 ng of double-stranded gDNA was fragmented to an average of 150 bp or 250 bp using Covaris ultrasonication (LE220 Focused-ultrasonicator, Covaris, Woburn, MA, USA). Fragmented DNA was purified using Agencourt AMPure XP beads (Beckman Coulter, Inc. Indianapolis, IN, USA). Size-selected DNA was then ligated to sequencing adaptors using sample-specific barcodes and libraries were constructed (SPRIworks HT, Beckman-Coulter) and quantified using qPCR (Kapa Biosystems, Wilmington, MA, USA) or MiSeq (Illumina Inc., San Diego, CA, USA). For targeted exon enrichment, libraries were pooled in equal mass to a total of 500 ng, and regions of interest were captured using custom-designed baits (SureSelect Target Enrichment system, Agilent Technologies, Santa Clara, CA). All captures were sequenced on the HiSeq 2500 platform (Illumina Inc., San Diego, CA, USA) in Rapid Run Mode.
Pooled sample reads were deconvoluted and sorted using Picard tools ( Reads were aligned to the reference sequence b37 edition from the Human Genome Reference Consortium using bwa ( using parameters “-q 5 -l 32 -k 2 -o 1.” Duplicate reads were removed using the Picard tools (4). The alignments were further refined using the GATK tool for localized realignment around indel sites ( Recalibration of the quality scores was also performed using GATK tools ( (5, 6).
Mutation analysis for single nucleotide variants (SNV) was performed using MuTect v1.1.4 (7) and annotated by Oncotator ( or Variant Effect Predictor (VEP) (8). MuTect was run in single or paired mode using internal control CEPH as the “matched” normal in paired mode. We used the SomaticIndelDetector tool that is part of the GATK for indel calling, as described previously (9). Mutation calls were made for those variants predicted to result in a non-synonymous amino acid alteration, frameshift mutation, stop codon, or alter a splice site, and for variants with at least ten reads of the mutant allele.
Germline variant filters were applied. Variants were filtered against the 6,500 exome release of the Exome Sequencing Project (ESP) database ( and the Genome Aggregation Database (gnomAD v.2.0; Variants represented in either database at > 0.1% frequency were excluded from further analysis. Catalogue Of Somatic Mutations In Cancer (COSMIC v.80; annotations were added to the variant calls, if available.
Array CGH analysis of T-ALL patient samples
Thirty-seven of the 109 T-ALL patient samples were profiled for DNA copy number analysis on SurePrint G3 Human 4×180K CGH Microarrays. Patient and control genomic DNAs (gDNAs) were labeled with Cy3 and Cy5 dyes (PerkinElmer, Waltham, MA, USA) and hybridization was performed according to the manufacturer’s instructions (Agilent Technologies). Data analysis was performed using the arrayCGHbase tool (10), and segmentation was conducted with the BioConductor DNAcopy package ( DNAcopy.html), as previously described (11). Log2 copy number ratio for heterozygous deletion was defined as -0.5 to -1.5 (corresponding to 35-70% of normal copy number), and log2 copy number ratio for homozygous deletion was defined as less than -1.5 (corresponding to <35% of normal copy number).
RNA Sequencing of T-ALL patient samples
RNA was extracted from T-ALL patient samples using the AllPrep DNA/RNA Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA samples were then treated with Ambion Turbo DNAse (Thermo Fisher Scientific, Waltham, MA), and DNA contamination was confirmed to be <10% for all samples. RNA quantity was determined using the Qubit RNA Assay Kit (Thermo Fisher Scientific) and RNA quality was determined on an Agilent Bioanalyzer using the RNA Pico Kit (Agilent, Santa Clara, CA). Using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA), 50-100 ng of total RNA was converted into a DNA library following the manufacturer’s protocol. Following library construction, DNA libraries were assessed for quality control. Library quantity was determined using the Qubit High Sensitivity DNA Kit (Thermo Fisher Scientific) and library size was determined using the Bioanalyzer High Sensitivity Chip Kit (Agilent). Finally, libraries were put through qPCR using the Universal Library Quantification Kit for Illumina (Kapa Biosystems, Wilmington, MA) and run on the 7900HT Fast qPCR machine (Applied Biosystems, Foster City, CA). Libraries passing quality control were diluted to 2nM using sterile water and then sequenced on the HiSeq 2000 (Illumina, San Diego, CA) at a final concentration of 12pM on a single read flowcell with 50 sequencing cycles, following all manufacturer protocols. Alignments were performed with STAR aligner (version 2.3.1z4)(12) against the hg19 w ERCC92 genome (ftp://ftp.ensembl.org/pub/release-75/ fasta/homo_sapiens/dna/). Aligned files were processed to primary (raw) read counts using featureCounts (13), and primary read counts were normalized using DESeq2 (14). Primary read counts from the RNA sequencing data are available as Supplementary Table 4, and sequencing data are available as dbGaP ( entry 27118.
Quantitative DNA PCR Analysis
Quantitative DNA PCR (Q-DNA-PCR) analysis for absence of biallelic TCR deletions were performed on genomic DNA using primers targeting the intron between the most 3’ V exon (V11) and the most 5’ J exon (JP1) of the TCR(TRG@) gene) and control Q-PCR primers targeting the ANLN locus, as previously described (11).
For Q-DNA-PCR for PTCH1 exon 2 copy number, genomic DNA was isolated from Jurkat T-ALL cells or normal PBMCs using the DNeasy kit (Qiagen). Q-DNA-PCR analysis was performed for gDNA copy number analysis of PTCH1 exon 2 using primers targeting exon 2 of the PTCH1 transcript, NM_000264. Control primers targeting centromeric and telomeric regions on chromosome 9q22 were utilized. None of the control genomic regions targeted by quantitative PCR were involved by known germline copy number alterations based on the Database of Genomic Variants ( (15). Results are reported as relative DNA copy number compared to control primers. Q-DNA-PCR was performed using 50ng of gDNA, 3l of 1M forward + reverse primer mix, 2L DEPC-treated water, and 10l of Power SYBR green mastermix (Applied Biosystems, Foster City, CA, USA) using an Applied Biosystems 7300 Real Time PCR System instrument. The Q-PCR reaction conditions were as follows: Initial denaturation step 94oC x 10 min; Forty PCR cycles at 94oC x 10 secs, then 60 oC x 60 sec; 94 oC x 15 minutes. Data was acquired at the end of each 60 oC step. All PCR reactions were performed in triplicate.
Q-DNA-PCR primer sequences used were as follows:
Centromeric Control ForwardTTGGCTCCACAAGGTTGATGT
Centromeric Control ReverseTTAAAAGCCCCCTGCAGAGTT
Telomeric Control ForwardCACATCTCCTGTGCCCCAAT
Telomeric Control ReverseGTCTCACCGTGCAACTACCA
PTCH1 exon 2 i ForwardTGTGGCTGAGAGCGAAGTT
PTCH1 exon 2 i ReverseCCACAACCAAGAACTTGCCG
PTCH1 exon 2 ii ForwardTGGCTGAGAGCGAAGTTTCA
PTCH1 exon 2 ii ReverseGAGGCCCACAACCAAGAACT
Sanger Sequencing
Mutations identified from diagnostic T-ALL specimens using next generation sequencing were validated by Sanger sequencing following PCR amplification of the region of interest. DNA from remission samples for 5 of 9 PTCH1 mutations was also assessed by Sanger sequencing to identify somatic mutations. All Sanger sequencing was performed at Genewiz (South Plainfield, NJ, USA).
PCR primers are as follows:
PTCH1 G43E PCRFCAGGAGggctgtgctgata
PTCH1 G34E PCRRctggctgcactcaccatag
PTCH1 D436N PCRF ACATTGGAAAGAGCCTGCAC
PTCH1 D436N PCRR TCGCACTCTTGCCTTCTTTT
PTCH1 R893L PCRF GCCGCTGCATTTCTAACATT
PTCH1 R893L PCRR AACGGTTAATGACCCTGGTG
PTCH1 T1106M PCRF ACCCAGGCTGGAGTGTAGTG
PTCH1 T1106M PCRR GAATCCTGTGCTGTGGGAAT
PTCH1 D1128Y PCRF GAGCAATTCTCTGCCTCAGC
PTCH1 D1128Y PCRR AATTGCTTGGGACACTGAGC
PTCH1 S1331C PCRFACTGTGGTCCATCCCGAAT
PTCH1 S1331C PCRRTGCAGCTCAATGACTTCCAC
PTCH1 G1343R PCRF GGACACATCAGCCTTGCTCT
PTCH1 G1343R PCRR GCTTTTCTTTTGTGGGTGGA
PTCH1 R1442Q PCRF GGGAAGAGAGCAGTGTGGAG
PTCH1 R1442Q PCRR AGGTGGTCCATCCCGAAT
GLI1 Q208 PCRFTGGTTTCCTCTTCCTTCTGC
GLI1 Q208 PCRRGATTCCACCATGTCCCACTC
GLI1 G274C PCRFTTCCTCCTTGAGGTGGAGTC
GLI1 G274C PCRRCCTTGAAGGAGAGCCCTGAT
GLI2 T286S PCRFGGGCTAGCAACATGCTCATC
GLI2 T286S PCRRTGAACAACCCTCATGTGTCC
GLI3 G1217W PCRFCCTTACAGGGCTGTTCATGG
GLI3 G1217W PCRRCCCATTCAGTGGAACGAAGT
GLI3 R1537H PCRFTTTCTTCCGCTAGGGAGGTC
GLI3 R1537H PCRRCTTGCTCTGCAGTCAGGACA
SUFU K460R PCRFTGGGCAATCTCTGGAAAGAC
SUFU K460R PCRRCTGATGGGTCACAAGGCTTAG
Sequencing primers are as follows:
PTCH1 G43E SeqFctgacaggtcctgcctatgg
PTCH1 G34E SeqRctcctccgtcttctcccagt
PTCH1 D436N SeqF AAACGGCAAATGGGAAAAAT
PTCH1 D436N SeqR GCCCTGGAATCACGTAGAAC
PTCH1 R893L SeqF TCACCCAGAAAGCAGACTACC
PTCH1 R893L SeqR CTTTGTCGTGGACCCATTCT
PTCH1 T1106M SeqF ACCCAGGCTGGAGTGTAGTG
PTCH1 T1106M SeqR GAATCCTGTGCTGTGGGAAT
PTCH1 D1128Y SeqF GGCCCAATCACAATGATTTC
PTCH1 D1128Y SeqRGGAAACTTCGGGGTGAGTATC
PTCH1 S1331C SeqFcatcacccaccctcgaac
PTCH1 S1331C SeqRCAGAAGCCGTCACAGTGGT
PTCH1 G1343R SeqF GAAGCCGTCACAGTGGTGAT
PTCH1 G1343R SeqR TTTCTTTTGTGGGTGGAAGG
PTCH1 R1442Q SeqF TAAAAGGTCACTGGGGTCCA
PTCH1 R1442Q SeqR ACCACTGTGACGGCTTCTG
GLI1 Q208 SeqFGGGAAAGGTGAAGGAAGGAC
GLI1 Q208 SeqRTTCCTCTTCCACGACTCCAC
GLI1 G274C SeqFGAGTGGGACATGGTGGAATC
GLI1 G274C SeqRCCACGCCCAGCTAATTTTTA
GLI2 T286S SeqFGGGAGCAGCGATACAGACAT
GLI2 T286S SeqRAGGAGACGGCAGAATCAAGA
GLI3 G1217W SeqFAGGTACCCCTGTCCCACTG
GLI3 G1217W SeqRGAGAACGTCACCCTGGAGTC
GLI3 R1537H SeqFGGGTTTTTCAGAGTCCTTTTCC
GLI3 R1537H SeqRCTTGCTCTGCAGTCAGGACA
SUFU K460R SeqFCAGAGGCAGAACCAAGGAAG
SUFU K460R SeqRGCCAGAGGAGCTTGAGTAGC
Cell lines and cell culture
T-ALL cell lines, NIH 3T3, BJ, and 293T cells were obtained from ATCC (Manassas, VA, USA) or the A. Thomas Look laboratory (Dana-Farber Cancer Institute) and cultured in RPMI 1640 (Invitrogen, Carlsbad, CA) or DMEM (Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (Sigma-Aldrich, Saint Louis, MO, USA) and 1% penicillin/streptomycin (Thermo Fisher Scientific). The ASZ001 basal cell carcinoma cell line was obtained from the Yoon-Jae Cho laboratory (Oregon Health & Science University, Portland, OR) and cultured in 154 CF Medium with supplemental CaCl2 (Invitrogen, Carlsbad, CA), 1% HKGS (Thermo Fisher Scientific), 2% chelated fetal bovine serum (Sigma-Aldrich, Saint Louis, MO, USA), and 1% penicillin/streptomycin (Thermo Fisher Scientific). All cultured cells were maintained at 37 °C, 5% CO2. T-ALL cell line identity was validated using STR profiling at the Dana-Farber Cancer Institute Molecular Diagnostics Laboratory, and mycoplasma contamination was excluded for all cell lines using the PCR Mycoplasma Test Kit I/C (Promokine).
Primary human T-ALL samples were cultured in reconstituted alpha-minimum essential media supplemented with 10% fetal bovine serum (Sigma-Aldrich), 10% human AB serum (Invitrogen), 1% L-glutamine (Thermo Fisher Scientific), 1% penicillin/streptomycin (Thermo Fisher Scientific) in the presence of recombinant human cytokines stem cell factor (50 ng/mL), Flt3-L (20 ng/mL) and IL-7 (10 ng/mL) (R&D Systems, Minneapolis, MN, USA) at 37° under 5% CO2, as previously described (16).
For drug treatment experiments for T-ALL cells, NIH 3T3 cells, and human BJ fibroblasts, cells were maintained in media containing drug or vehicle control for 48 hours following which functional studies were carried out as described.
5’ Rapid Amplification of cDNA Ends (RACE)
Gene-specific primers (GSP) targeting exon 3 of PTCH1 (RefSeq NM_000264) were designed in accordance with manufacturer’s instructions for use in the SMARTer RACE 5’/3’ kit (Clontech, Mountain View, CA, USA). The PTCH1 GSP, including the 15bp overhang located at the 5’ end to facilitate In-fusion cloning with the provided vector, used is as follows:GATTACGCCAAGCTTTGTAGGAGCGCTTCTGTGGTCAGGA
RNA was isolated from Jurkat cells according to manufacturer’s instructions provided with the RNeasy Mini Kit (Qiagen, Venlo, Netherlands). First strand cDNA synthesis and the RACE reaction using 5’ RACE-ready cDNA and touchdown PCR was carried out using the SMARTer RACE 5’/3’ kit, according to manufacturer’s instructions (Clontech). 5’ RACE products were then characterized on a 1% agarose gel and purified using the NuceloSpin Gel and PCR Clean-Up Kit (Clontech). Finally, the purified RACE product was cloned into the linearized pRACE vector provided with the SMARTer RACE 5’/3’ kit using the In-Fusion HD cloning kit (Clontech), and identification of isolated colonies was achieved by Sanger sequencing.
Lentiviral expression plasmids, gateway cloning, and site-directed mutagenesis
Lentiviral overexpression plasmids for human wild type PTCH1 (EX-T0559-Lv105), human wild type GLI1 (EX-F0407-Lv105), human wild type GLI2 (EX-Y4001-Lv105), human wild type GLI3 (EX-Z3091-Lv105), human wild type SUFU (EX-U0852-Lv105), and EGFP control (EX-EGFP-Lv105) were obtained from Genecopoeia ( Rockville, MD, USA). A lentiviral destination vector, pLenti-CMV-Puro-Dest (w118-1) for use in gateway cloning was a gift from Eric Campeau and Paul Kaufman (Addgene plasmid # 17452) (17).
Generation of a mammalian expression vector containing the p.Gly68fsX5 mutant allele resulting from the identified exon 2 microdeletion of PTCH1 in Jurkat cells was achieved using Gateway Technology with Clonase II, according to manufacturer’s instructions (Invitrogen, Carlsbad, CA). A p.Gly68fsX5 PTCH1 entry clone was generated using the following attB-flanked double stranded DNA oligonucleotide from Eurofins Genomics LLC (Louisville, KY):
GGGGACAAGTTTGTACAAAAAAGCAGGCTTGAAGGAATTCGGTACCATGGCGTCTGCAGGTAACGCCGCCGAGCCCCAGGATCGCGGTGGAGGCGGATCCGGTTGTATTGGGGCTCCTGGGAGGCCTGCAGGTGGTGGTAGGAGACGACGCACCGGGGGACTGCGGCGGGCCGCAGCCCCCGATCGTGACTATCTCCACAGACCAAGCTACTGCGACGCCGCTTTCGCTCTTGAGCAAATCTCAAAGCTGGAAGACGAATAACTCGAGTGCGGCCGCAACCCAGCTTTCTTGTACAAAGTGGTCCCC
A control EGFP entry vector was generated using the EX-EGFP-Lv105 expression vector, which contains attB sites, following restriction digestion with BamH1. BP and LR reactions were carried using the “One-Tube” protocol for cloning attB products directly into destination vectors, according to manufacturer’s instructions (Invitrogen, Carlsbad, CA). Final expression vectors were validated by Sanger sequencing.
Site-directed mutagenesis of wild type expression plasmids to generate pReceiver-Lv105 plasmids containing the PTCH1, GLI, or SUFU patient mutations identified by targeted exon sequencing was performed using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions.
Mutagenic primers designed according to manufacturer’s instructions are as follows:
PTCH1 D436N SenseCCTGAAATCCTTCTCTAACGTCAGTGTCATCCGCG
PTCH1 D436N AntisenseCGCGGATGACACTGACGTTAGAGAAGGATTTCAGG
PTCH1 R893L SenseGCAAACCGGCAGCGATAAGCCCATCG
PTCH1 R893L AntisenseCGATGGGCTTATCGCTGCCGGTTTGC
PTCH1T1106M SenseCAGGCCTTTCTGATGGCCATCGGCGAC
PTCH1 T1106M AntisenseGTCGCCGATGGCCATCAGAAAGGCCTG
PTCH1 D1128Y SenseGCACCCGTCCTGTATGGCGCCGTGT
PTCH1 D1128Y AntisenseACACGGCGCCATACAGGACGGGTGC
PTCH1 S1331C SenseTGAAATTTCTACTGAAGGGCATTGTGGCCCTAGCAA
PTCH1 S1331C AntisenseTTGCTAGGGCCACAATGCCCTTCAGTAGAAATTTCA
PTCH1 G1343R SenseAGAACGGGCCCTGCGAGGGCCCC
PTCH1 G1343R AntisenseGGGGCCCTCGCAGGGCCCGTTCT
GLI1 G274C SenseGCCACTGGGGGTGCTGCTCCAGG
GLI1 G274C AntisenseCCTGGAGCAGCACCCCCAGTGGC
GLI2 T286S SenseAGCCCAGCCTTCTCCTTCCCCCACC
GLI2 T286S AntisenseGGTGGGGGAAGGAGAAGGCTGGGCT
GLI3 R292C SenseCAGCCAGGCCGAGCCGAAAATGTACAC
TGTCCATATCACC
GLI3 R292C AntisenseGGTGATATGGACAGTGTACATTTTCGGCTC
GGCCTGGCTG
GLI3 G1217W SenseGGCTATCAGACCCTCTGGGAGAACAGCAACC
GLI3 G1217W AntisenseGGTTGCTGTTCTCCCAGAGGGTCTGATAGCC
GLI3 R1537H SenseCATAGCTCCTCCCACCTCACCACGCCT
GLI3 R1537H AntisenseAGGCGTGGTGAGGTGGGAGGAGCTATG
SUFU K460R SenseCAGAGGAATTCAAACTTCCCAGAGAGT
ACAGCTGGCCTG
SUFU K460R AntisenseCAGGCCAGCTGTACTCTCTGGGAAGTT
TGAATTCCTCTG
For the PTCH1 R1442Q (c.4325G>A) missense mutation, site-directed mutagenesis of the EX-T0559-Lv105 plasmid was performed by Genewiz (South Plainfield, NJ, USA).
Sanger sequencing (Genewiz) was performed to verify the correct insert for each plasmid of interest.
Lentiviral production and infection
Lentivirus generated by co-transfecting lentiviral plasmids of interest with packaging vectors psPAX2 and pMD2.G using Fugene (Promega, Madison, WI, USA) in 293T cells. Lentiviral infection was performed by spinoculating T-ALL cell lines with viral supernatant (1500 rpm x 90 minutes) and incubating for 24H at 37 °C, 5% CO2 in the presence of 6 μg/ml polybrene (Merck Millipore, Darmstadt, Germany). Selection with puromycin at a concentration of 2 μg/ml commenced 48 hours after spinoculation. After 48 hours of selection, RNA extraction and quantitative RT-PCR analysis was performed and cells were used for functional experiments of viability, apoptosis, and cell cycle analysis.
Transfections
For assessment of Hedgehog pathway activity of the GLI and SUFU mutations, NIH 3T3 cells were transfected with EGFP, wild type expression vector (human GLI1, GLI2, GLI3, or SUFU), or pReceiver-Lv105 mutant vectors generated using site-directed mutagenesis, as described above, using Fugene 6 (Promega, Madison, WI, USA), according to manufacturer’s instructions. Twenty-four hours after transfection, cells were placed in puromycin selection media. After 72 hours of selection, RNA extraction and quantitative RT-PCR analysis was performed.
Small molecules
Smoothened Agonist (SAG) was obtained from Caymen Chemical (Ann Arbor, MI, USA). Cyclopamine was obtained from LC Laboratories (Woburn, MA, USA), and was dissolved in ethanol (Sigma-Aldrich, St. Louis, MO, USA). Vismodegib was obtained from LC Laboratories.
Dimethyl sulfoxide (DMSO) was obtained from Sigma-Aldrich (St. Louis, MO, USA).
RNA extraction and quantitative RT-PCR analysis
For quantitative reverse-transcriptase polymerase chain reaction (Q-RT-PCR) analysis of mRNA transcript levels, total RNA was extracted using the RNeasy Mini Kit (Qiagen) for human T-ALL cells, ASZ001 mouse cells, NIH 3T3 mouse cells, and human BJ fibroblasts, according to the manufacturer’s instructions. cDNA was synthesized using the SuperScript III First-Strand Synthesis system (Invitrogen) according to the manufacturer’s instructions. Q-RT-PCR analysis was performed with the SYBR Green PCR Core Reagents kit (Applied Biosystems, Life Technologies, Grand Island, NY, USA) and a 7500 Real Time PCR System instrument (Applied Biosystems) according to the manufacturer’s instructions. All Q-RT-PCR reactions were performed in triplicate using primers listed below. Cycle threshold (CT) for each condition, in triplicate, was compared to β-actin control according to the 2CT comparative method.