SMARCB1 Deficiency Integrates Epigenetic Signals to Oncogenic Gene Expression Program Maintenance in Human Acute Myeloid Leukemia

Patient cohort

Human AML (n = 67) bone marrow aspirates (1–2 mL each) were obtained from Park Clinic, Kolkata from untreated, freshly diagnosed patients after written, informed consent according to Institutional Human Ethics Committee (HEC) approval and following Indian Institute of Chemical Biology (CSIR-IICB) Institutional Review Board (IRB) set guidelines. Sample collection was part of routine diagnosis and the inclusion criterion for this study was histopathologic confirmation of bone marrow aspirates or biopsies, karyotyping, and immunophenotypic analyses (20). Bone marrow aspirates were also collected from age-matched normal individuals (n = 6) after informed consent, who turned out to be pathologically negative for AML (20). Individual case information is presented in Supplementary Tables S1 and S2. Umbilical cord blood samples (40 mL each) were obtained from Deb Shishu Nursing Home (Howrah, West Bengal, India) from term pregnancies after written, informed consent according to CSIR-IICB HEC approval and following IRB set guidelines. Low density (1.077 gm/cc) nuclear cells from AML bone marrow, normal bone marrow, or cord blood samples were isolated by Ficoll (Sigma) separation and cryopreserved in liquid nitrogen. Normal blood specimens were obtained from age-matched healthy volunteers (n = 3) after written, informed consent and nucleated cells were isolated using RBC lysis (BD Pharmingen).

Reverse transcription and quantitative PCR

Total RNA was isolated by using TRIzol (Life Technologies) according to manufacturer's recommendation. RNase-free DNase treatment were carried out to remove any genomic DNA contamination using DNase I recombinant, RNase free kit (Roche). RNA amount was quantified and cDNA was prepared using TaqMan Reverse Transcription Reagents (Applied Biosystems). Gene expression levels were determined by quantitative PCR performed using cDNA with SYBR Select Master Mix (Applied Biosystems) on the 7500 Fast Real-Time PCR System (Applied Biosystems). GAPDH was used as a housekeeping gene. Relative expression levels were calculated using the 2^−ΔΔC_t method (21–24). qRT-PCR primer details are available in Supplementary Table S3.

Array Comparative Genomic Hybridization and analysis

Array Comparative Genomic Hybridization (CGH) and analysis were carried out by Genotypic Technology Private Limited. Genomic DNA quantity and purity was assessed by the NanoDrop ND-2000 UV-Vis Spectrophotometer (NanoDrop Technologies) and the integrity was assessed on a 0.8% Agarose Gel. Genomic DNA with OD260/OD280 >1.8 and OD260/OD230 ≥ 1.3 was used for microarray experiments. DNA was considered to be of good quality when a single clear band was seen when run against a reference ladder. A total of 0.5 μg of DNA in 10.1 μL was taken into a microfuge tube and digestion master mix containing restriction enzymes (Alu I, 5U and Rsa I, 5U) was added. The samples were incubated at 37°C for 2 hours followed by heat inactivation of enzymes at 65°C for 20 minutes. Samples were labeled using Agilent sure tag DNA Labeling Kit (catalog no: 5190-3399). Control samples were labeled with Cy3 and test sample with Cy5. The labeled samples were cleaned up using Amicon Ultra columns 30-kDa size exclusion filter. DNA yield and incorporation of labeled dye (specific activity) was measured using NanoDrop spectrophotometer. One micrograms each of Cy3- and Cy5-labeled sample was added with human cot-1 DNA (catalog no. 5190-3393), Agilent aCGH/CoC Blocking agent (part number: 5188-6416), and hybridization buffer (part number: 5188-6420). The labeled samples in above hybridization mix were denatured at 95°C for 3 minutes and were incubated at 37°C for 30 minutes. The samples were then hybridized at 65°C for 24 hours. After hybridization, the slides were washed using Agilent aCGH Wash Buffer1 (Agilent Technologies, part number 5188-5221) at room temperature for 5 minutes and Agilent aCGH Wash Buffer 2 (Agilent Technologies, part number 5188-5222) at 37°C for 1 minute. The slides were then washed with acetonitrile (part number: A2094) for 10 seconds. The microarray slides were scanned using Agilent Scanner (Agilent Technologies, part number G2600D). Image analysis was performed using Agilent Feature Extraction software, feature extracted raw data was analyzed using Agilent Genomic Workbench 7.0 software. The data were normalized using Lowess normalization. Significant regions having amplification and deletions were identified among each of the samples. Genomic view and chromosome view of the amplification and deletion region for each sample were generated. Graphical representation has been done using Human UCSC Genome Browser by loading the data in wiggle file format. Details are included as Supplementary array CGH Files.

Methylation-specific PCR

Genomic DNA was isolated using the QIAamp DNA Blood Mini Kit (Qiagen, catalog no. 51104). Isolated genomic DNA was then bisulfite converted, that is, unmethylated cytosines were converted to uracil using EpiTect Bisulfite Kit (Qiagen, catalog no. 59104) according to the manufacturer's protocol. Methylation-specific primers for the gene of interest were made using MethPrimer. Bisulfite converted DNAs were amplified using methylated DNA (M pair)-specific primers. Methylation-specific PCR at SMARCB1 promoter loci in primary AML blasts was compared with normal BMNC. The fold change levels of the methylated DNA were calculated with respect to GAPDH (unrelated control). Relative methylation levels were plotted after normalizing it with GAPDH. qMSP primer details are available in Supplementary Table S3.

Coimmunoprecipitation, histone acid extraction, and immunoblotting

Nuclear extracts for immunoprecipitation experiments were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) and diluted in 1× RIPA (Cell Signaling Technology) containing protease and phosphatase inhibitor cocktails. About 300 μg extracts were incubated with 2.0 μg of respective antibodies against SMARCC1 (clone R-18, sc-9746, Santa Cruz Biotechnology), p300 (A300-358A, Bethyl Laboratories), CBP (clone D6C5, 7389S, Cell Signaling Technology), BRD4 (clone E2A7X, 13440S, Cell Signaling Technology), or rabbit IgG (P120-101; Bethyl Laboratories), and incubated overnight at 4°C with gentle rocking. Fifty microliters of protein A/G agarose beads (Cell Signaling Technology) were added and incubated for 3–4 hours at room temperature. The beads were then washed 6 times with 1× RIPA supplemented with 300 mmol/L NaCl and resuspended in 1× SDS gel loading buffer. The proteins were separated in SDS-PAGE and transferred to PVDF membrane (Millipore) and subsequently probed with respective antibodies. Detailed list of antibody is included in Supplementary Table S4. All antibodies were used at a dilution of 1:1,000 unless specified otherwise. Total cell lysate for immunoblotting was prepared by incubating cells in 1× RIPA for 15 minutes followed by brief sonication. Supernatants were collected following centrifugation at 16,000 × g for 15 minutes at 4°C. Protein concentration was determined using Pierce BCA Protein assay kit (Thermo Fisher Scientific). SDS-PAGE was used to separate proteins, transferred to PVDF membrane, and probed using respective antibodies. Densitometry analyses were performed using NIH Image J software.

Sucrose density gradient centrifugation

A total of 700 μg–1.0 mg nuclear extracts isolated from pooled (n = 5–7) primary AML BMNCs were prepared and diluted in 300 μL of 1× RIPA. The extracts were overlaid on a 10 mL 20%–50% sucrose gradient (in 1× RIPA) in 13 × 89 mm polyallomer tubes (Beckman Coulter). The tubes were then centrifuged in a SW-41 Ti swing out rotor at 30,000 rpm for 12 hours at 4°C. A total of 500-μL fractions were collected and separated in SDS-PAGE, transferred to a PVDF membrane, and subsequently probed with specific antibodies.

ChIP-seq, ChIP-qPCR, and analyses

ChIP-seq experiments were carried out at Core Technologies Research Initiative (CoTeRI), National Institute of Biomedical Genomics (NIBMG, University of Kalyani, Kolkata, West Bengal, India). A total of 1 × 10⁷ primary bone marrow nuclear cells (BMNC), isolated from three independent (biological replicates) AML patients (Supplementary Table S1 case nos. 82, 83, and 80 as AML 01, AML 02, and AML 03, respectively), per chromatin immunoprecipitation (ChIP) set were crosslinked with formaldehyde (Sigma) in culture media. After crosslinking, chromatin was extracted and sonicated to fragment lengths between 150 and 900 bp in chromatin extraction buffer containing 10 mmol/L Tris pH = 8.0, 1 mmol/L EDTA pH = 8.0, 0.5 mmol/L EGTA pH = 8.0. Chromatin was incubated with ChIP-grade antibodies to SMARCC1 [sc-9746 (R-18), Santa Cruz Biotechnology), H3K27Ac (ab4729, Abcam), H3K27Me3 (07-449, Millipore), or rabbit IgG (clone P120-101; Bethyl Laboratories) or mouse IgG (clone G3A1; 5415S; Cell Signaling Technology) or goat IgG (sc-2028, Santa Cruz Biotechnology) during overnight at 4°C with rotation. Six micrograms of antibody was used per immunoprecipitation. All antibodies were used at 1:1,000 dilution. Detailed list of antibody is included in Supplementary Table S4. Protein A/G Agarose beads (Cell Signaling Technology) were then added and incubated for 2 hours at 4°C. The beads were washed with chromatin extraction buffer and by increasing salt concentration for four times. The chromatin was eluted from the beads in chromatin elution buffer at 65°C with gentle vortexing. The eluted chromatin was treated with RNAse for 30 minutes at 37°C. Reverse cross-linking was performed by treating the eluted chromatin with Proteinase K (Sigma) at 65°C for 2 hours. The DNA was finally precipitated by phenol–chloroform extraction; precipitated DNA was dissolved in TE buffer, and subjected to ChIP-seq analyses. For ChIP-qPCR experiments, 4–5 × 10⁶ normal CD34⁺ cells or 293T cells and 2 μg of antibody, or ChIP DNA obtained from primary AML BMNCs were used. ChIP-qPCR primer details are available in Supplementary Table S5.

Size distribution of the ChIP-enriched DNA was checked using High Sensitivity chips in 2100 Bioanalyzer (Agilent) for each sample and quantitation was performed in Qubit Fluorometer (Invitrogen) by picogreen method. ChIP-seq library preparation was performed using TruSeqChIP Sample Prep Kit (Illumina) according to the manufacturer's instructions. Ten nanograms of input ChIP-enriched DNA was used for ChIP-seq library preparation. Final libraries were checked using High Sensitivity chips in 2100 Bioanalyzer (Agilent). Average fragment size of final libraries was found to be 280 ± 8 bp. Paired-end sequencing (2 × 100 bp) of these libraries were performed in HiSeq-2500 (Illumina). Quality control analysis of the raw data using NGS-QC ToolKit was done and HQ reads with filter criteria of bases having ≥20 Phred score and reads with ≥70% were filtered. Paired-end reads (.fastq format) were aligned with Bowtie software using –best and –m 2, that is, mismatches against reference genome Ensembl build GrCh37/hg19 (considering 2% input as the baseline) and saved in SAM format, which was then converted to sorted BAM file using SAMTOOLS. PCR duplicates were removed using SAMTOOLS rmdup. Peak calling was performed using MACS14 model building with P value cutoff of 0.05. Annotation of the identified peaks was performed with PeakAnalyzer.

Functional enrichment analysis (Gene Ontology and Pathway) was done using The Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8. Gene list were uploaded and converted to its respective ENTREZ Gene ID. The converted gene list was submitted to DAVID and Functional Annotation Clustering was carried out which comprises of Gene Ontology and Pathway analysis. R bioconductor package ChipSeeker was used to generate heatmap, average profile distibutions, and pie charts. Unique gene names were used to plot the Venn diagram represented by peaks in the respective samples either upstream or downstream or overlap to the genetic region. Bigwig/bed files were imported into Integrated Genome Viewer (IGV) and snapshots of particular genomic loci were captured. For motif analysis, the MEME ChIP V4.10 was used to analyze the motif using the peak sequences, with default parameters. and for transcription encoding motif, Jaspar database (Jaspar) was used using MEME ChIP tool. Details are included as Supplementary ChIP-seq Files.

RNA-seq and analyses

RNA-seq experiments were performed by Bionivid Technology. Total RNA was isolated from BMNCs from the identical AML cohort (n = 3) and age-matched normal (n = 2) hematopoietic cells using TRIzol (Life Technologies) according to manufacturer's instruction. DNase treatment was carried out to remove any genomic DNA contamination using DNase I recombinant, RNase free kit (Roche). RNA amount was quantified and the sequencing library prepared using TruSeq RNA Sample Prep Kit v2 (Illumina). Paired-end sequencing was performed on HiSeq 4000 using TruSeq 3000 4000 SBS Kit v3 (Illumina).

Raw data resulted in an average of 35.39 × 10⁶ reads in normal hematopoietic cells and 36.86 × 10⁶ reads of 101-bp length in primary AML cells. Quality control using NGSQC toolkit yielded around 34.28 × 10⁶ HQ reads in normal hematopoietic cells and 36.05 × 10⁶ HQ reads in primary AML cells. Around 88.2% of the HQ reads from normal and 85.12% HQ reads from primary AMLs could be mapped to the Homo sapiens (hg 38) genome reference sequence using TOPHAT, suggesting a good quality of RNA sequencing. Transcripts were given a score for their expression by Cufflinks-based maximum likelihood method. A total of 24,784 transcripts were identified using Cuffdiff validation to be expressed in either normal or primary AML cells representing 13,847 genes. Transcript type analysis revealed 95.5% of the transcripts to be of “Full Length” or “Known Transcripts” and 4.5% as “Potentially Novel Isoforms” Transcripts as per Cufflinks Class Code distribution. This indicates a largely complete transcription machinery activity in both normal and AML cells. Significant Biology for Differentially Expressed Transcripts was performed with GO-Elite_v.1.2.5 Software. A cutoff of P value less than 0.05 was considered for filtering the significantly enriched GO pathways. In testing for differential expression, we consider log₂FC > 2 (upregulation) and log₂FC ← 2 (downregulation). For gene-set enrichment analysis (GSEA), differentially expressed genes from individual comparisons were preranked on the basis of fold change such that maximally upregulated genes fall topmost in the list. This was used as an input to perform GSEA (GeneSpring). GSEA was performed on “H: Hallmark gene set” representing well defined biological states or processes available on Molecular Signature Database. Details are included as Supplementary RNA-seq Files.

Gene enrichment and functional annotation analysis

Gene ontology (GO) analysis of the shared gene set (Supplementary ChIP-seq Shared GENELIST) was carried out using DAVID v6.8 (https://david.ncifcrf.gov/). The P value used in the analysis is a modified one, termed as EASE score threshold (maximum probability). The threshold of EASE Score is a modified Fisher exact P value used for gene enrichment analysis. It ranges from 0 to 1. Fisher exact P value = 0 represents perfect enrichment. Usually P value is equal or smaller than 0.05 to be considered strongly enriched in the annotation categories.

Plasmids

shRNA-expressing lentiviral constructs targeting against SMARCB1 (pLKO-shSMARCB1, 39587) and SMARCB1-overexpression vector HA_INI1/BAF47 was a gift from Dr. Olivier Delattre (Institut Curie, Paris, France). shRNA-expressing lentiviral construct targeting against SMARCB1 (pLKO.1-puro-CMV-TGFP, TRCN0000295966) was purchased from Sigma. Empty vector SHC003 was purchased from Sigma. Lentiviral packaging constructs PAX2 (Addgene; 12260) and pMD2.G (Addgene; 12259) were purchased from Addgene.

HSPC isolation, lentivirus preparation, and transduction

CD34⁺ HSPCs were isolated from freshly collected normal BMNCs and cord blood nuclear cells or from cryopreserved specimens using CD34 Microbead positive selection kit (Miltenyi Biotec) following manufacturer's protocol (21, 23). For lentivirus preparation, 293T cells were maintained in DMEM supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mmol/L l-glutamine (all from Gibco) at 37°C with 5% CO₂. Cells were seeded in T-225 flasks at 70% confluence and transfected with the target plasmid DNA, PAX2 (Addgene; 12260), and pMD2.G (Addgene; 12259) using calcium-phosphate transfection method (23, 25). After overnight incubation, butyrate induction was given for 8 hours. Supernatant containing lentiviral particles were collected after 36–40 hours of incubation at 37°C with 5% CO₂, and ultracentrifuged at 25,000 rpm for 90 minutes at 4°C using (Sorvall, Thermo Scientific). Virus pellet was resuspended in X-VIVO (Lonza) and aliquoted in several tubes and stored at −80°C. HL60 and U937 cell lines were stably transduced with lentiviral particles containing mock (sh-Control) or sh-SMARCB1 plasmids in a U-bottom 96-well nontissue-culture–treated plate. A total of 1 × 10⁵ cells were incubated overnight with virus particles at a MoI of 5 and polybrene was added at a final concentration of 8 μg/mL to initiate lentiviral infection.

Cells, drug treatments and survival, and proliferation assays

Human AML cell lines (obtained from Dr. Jose Cancelas, Cincinnati Children's Hospital, Cincinnati, OH) were maintained in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mmol/L l-glutamine (all from Gibco) at 37°C with 5% CO₂. 293T cells (obtained from Dr. Jose Cancelas, Cincinnati Children's Hospital, Cincinnati, OH; refs. 15, 23) were maintained in DMEM supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mmol/L l-glutamine (all from Gibco) at 37°C with 5% CO₂. Adherent cells were transfected at 70% confluency using the calcium phosphate transfection method (16, 22, 23, 25). Cell lines have been freshly authenticated using STR profiling (Supplementary Cell Lines_STR Profiles).

JQ1 (cat SML0974) was purchased from Sigma. For calculating IC₅₀, parental or lentivirus transduced AML cell lines were treated with varying doses of JQ1 from 0.1 to 50 μmol/L. Viable cell counts were taken after 48 hours of drug treatment and the total number of GFP⁺ cells were analyzed by flow cytometry. Cell counts were normalized and plotted against logarithm of the inhibitor concentration using GraphPad Prism5 to measure the IC₅₀. For proliferation assay, lentivirus transduced cell lines were grown in triplicate in regular media for 6 days. Trypan blue–negative cell numbers were determined at respective time points and the total number of GFP⁺ cells were analyzed by flow cytometry.

AML BMNCs were grown in regular media supplemented with cytokines in presence of 500 nmol/L 5-azacytidine (Sigma, catalog no. A1287) or DMSO (vehicle) for 72 hours. Media was calibrated with fresh 5-azacytidine and cytokines after every 24 hours. Posttreatment, total RNA was isolated and gene expression levels were determined by qRT-PCR.

PAK1 pulldown and Rac GTPase activation assay

Cells were lysed using 400 μL of MLB buffer (1×) with repeated pipetting, centrifuged at 14,000 × g for 5 minutes at 4°C and supernatants were used for PAK1 pull down assay (Upstate, Millipore) as described earlier (15, 16, 18, 26, 27). To the supernatant, 10 μL of Rac1-conjugated agarose beads were added and incubated for 45 minutes at 4°C with gentle rocking. The beads were centrifuged at 14,000 × g for 10 seconds at 4°C. After removing the supernatants the beads were washed three times with MLB buffer (1×) and resuspended in 40 μL of protein loading buffer, boiled for 5 minutes, separated in 12% polyacrylamide gel, and transferred to PVDF membrane and probed using respective antibodies. Presence of active Rac GTP was determined by using antibody against Rac GTP in the pulldown fraction and normalized against total Rac present in the lysate. Densitometry analyses were performed using NIH Image J.

Migration assay

HL60 cells were transduced with lentiviral particles expressing sh-SMARCB1 or sh-Control in a U bottom 96-well nontissue culture–treated plate. Cells were incubated overnight with virus particles at a MoI of 5 and polybrene was added at a final concentration of 8 μg/mL. Forty-eight hours posttransduction, 50,000 cells were seeded in duplicate in the top chamber of a 24-well Transwell (Corning Incorporated) in 100 μL of IMDM. After 4 hours of incubation at 37°C with 5% CO₂, the total migrated cells were counted from bottom chamber containing 600 μL of IMDM supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 100 ng/mL CXCL12 (PeproTech). Total number of GFP⁺ cells was analyzed by flow cytometry.

Flow cytometric analysis

AML cell lines were transduced with lentiviral particles expressing sh-SMARCB1 or sh-Control and coexpressing GFP at a MoI of 5. After 48 hours, cells were harvested at 500 × g for 5 minutes and washed two times with cold PBS and resuspended in 500 μL of PBS supplemented with 2% human serum and 7-AAD at a final concentration of 1 μg/mL. 7AAD⁻/GFP⁺ cells were analyzed in LSRFortessa (Becton Dickinson) using FACSDiva software (Becton Dickinson).

Correlation and survival analysis of TCGA AML cohort

Cross-cancer analysis of SMARCB1 and DOCK5 expression, as well as SMARCB1 and DOCK5 expression heatmap cluster of TCGA AML cohort, was derived using cBioPortal for Cancer Genomics interface (28, 29). For correlation analysis, mRNA expression (RNA Seq V2 RSEM) data were obtained from TCGA database and plotted using GraphPad Prism5. For correlation as well as Kaplan–Meier survival analysis, mRNA expression (RNA Seq V2 RSEM) was used for clustering of samples; all samples showing expression level above mean value for the particular gene were considered “hi,” whereas all samples showing expression level below mean value were considered “lo.”

Statistical analyses

Statistical analyses were performed using GraphPad Prism 5. Statistics were calculated with Student's t test. Quantitative data are expressed as mean ± SEM. unless specified otherwise. For IC₅₀ calculation, cell counts were normalized and plotted against logarithm of the respective inhibitor concentration using GraphPad Prism5. Gene expression correlation plots were derived using the TCGA datasets of AML cohort available through cBioPortal. Densitometry analyses were performed using Image J software (NIH). For all statistical analyses, the level of significance was set at 0.05.

Database availability

All sequencing data have been submitted to the database with accession numbers as follows. ChIP-seq: GSE108976. RNA-seq: SRA accession: SRP127783; BioProject ID: PRJNA428149.