HOXBLINC long non-coding RNA activation promotes leukemogenesis in NPM1-mutant acute myeloid leukemia

Generation of the HoxBlinc transgenic (Tg) mouse model

All studies were conducted in accordance with the regulatory guidelines by the Institutional Animal Care and Use Committee (IACUC) at the UT Health San Antonio and University of Miami Miller School of Medicine. The full-length of mouse HoxBlinc cDNA was cloned into HS321/45-vav vector31 by SfiI and NotI so the transgene locates between the Vav1 promoter and Vav1 enhancer. This makes the transgene specifically express in hematopoietic system. The circular plasmid was then linearized by SacII into 2 fragments, only the longer fragment (Vav1 promoter-HoxBlincVav1 enhancer, ~14 kb) was extracted and injected into the nuclear of zygotes of C57BL/6 mice while the shorter fragment (pBlueScript II SK backbone, ~3 kb) was discarded. Two HoxBlincTg founder mice were obtained by genotyping the tail genomic DNAs with P1 (forward primer, to detect both endogenous and transgenic HoxBlinc gene) and P2 (reverse primer, to specifically recognize the transgenic HoxBlinc gene), see primer sequences in Supplementary Table 6. Transgenic mice showed a 589 bp band while the WT mice showed no band. Both founder mice were then crossed with WT C57BL/6 mice, the HoxBlinc negative littermates were used as controls and the HoxBlincTg mice were used as experimental group throughout the study. These two lines of HoxBlincTg were analyzed separately. The mHoxBlinc-set1 and mHoxBlinc-set2 were used as real-time PCR primers to recognize both endogenous and exogenous HoxBlinc lncRNA (Fig. 2d, Supplementary Table 6). The transgenic HoxBlinc expression levels were also confirmed by RNA-seq analysis (Fig. 4a).

Mouse housing conditions

A 14-hour light/10-hour dark cycle is used. Researchers do not enter the mouse room during the dark cycle. Room temperatures were set to 21 °C with 40–60% humidity. Immunodeficient mice (NSG) were housed in the same room condition but in a separate room established for immunodeficient mice at the UT Health SA or University of Miami.

Morphological and histological analyses of the hematopoietic organs

Flow cytometry analysis, cell sorting, and colony assay

The peripheral blood WBCs were obtained by treating peripheral blood with red blood cell lysis buffer (QIAGEN 1045722). BM, spleen, and peripheral blood WBC cells were stained with indicated flow antibodies (flow antibodies were listed in Supplementary Table 7, lineage flow antibody was diluted as 1:25, all other flow antibodies were diluted as 1:50). The flow data was collected by BD LSRII or LSR Fortessa and analyzed with FlowJo.V10 software. The LSK cells used for colony and replating assay were purified by flow sorting: total BM cells of 6–8 weeks old mice were pre-purified with lineage depletion beads (MiltenyiBiotec, Bergisch Gladbach, Germany) and then incubated with c-Kit, lineage, and Sca-1 antibodies (see Supplementary Table 7 for antibody information) and then sorted on BD FACS AriaII. IIThe purity of sorted LSK cells were routinely over 98%. The LSKs were incubated in methylcellulose medium (Methocult M3231) which was diluted with RPMI1640, supplemented with 30% FBS, 2% BSA, and a combination of cytokines (mG-CSF, 10 ng/mL; mIL-3, 5 ng/mL; mEPO, 4 U/mL; hTPO, 100 ng/mL; and mSCF, 100 ng/mL) for colony assay, colony numbers were counted at 7 days. For replating assay, the colonies were passaged sequentially every 7 days for 4 times. The gating strategy of Flow analysis and sorting were included in Supplementary Table 9.

Competitive repopulation assay

A total of 1 × 106 BM cells prepared by mixing 5 × 105 CD45.2 (WT or HoxBlinTg) with 5 × 105 CD45.1 (B6.SJL) were injected into the tail veins of lethally irradiated (950 cGy) B6.SJL recipients (CD45.1). The contribution of CD45.1+ vs. CD45.2+ cells in the PB was monitored every month for 6 months after transplantation.

Paired-daughter cell assay

To examine the frequency of HSCs to undergo self-renewal and differentiation, we performed paired-daughter cell assays32. Single CD34LSK cells from BM of WT and HoxBlincTg mice were clone-sorted into 96-well plates. The cells were maintained in RPMI1640 media supplemented with mSCF (100 ng/mL) and hTPO (50 ng/mL). After the first cell division, the two daughter cells were separated, one per well for an additional 12 days in the media supplemented with mSCF, hTPO, mEPO, mIL-3, and mG-CSF. The self-renewal and differentiation capabilities of cultured CD34LSK cells were determined by morphological analyses of the progenies of the two daughter cells microscopically following May-Grünwald-Giemsa staining. A total of 192 single cells (two 96-well plates) were analyzed to calculate the percentage of symmetric/asymmetric cell divisions.

Human AML samples and patient data analysis

All human samples from healthy donors and patients with primary AML were obtained after informed consent following the guidelines of the Institutional Review Board of Pennsylvania State University College of Medicine (IRB protocol #2000-186 and #29252 EP) or the Institute of Hematology and Blood Disease Hospital, Tianjin, China. For HOXBLINC gene expression level analysis, BM low-density mononuclear cells (MNCs) were purified using Ficoll-Hypaque. RNA specimens extracted from MNCs of AML patients, MNCs of healthy controls or CD34+ cells of healthy controls were treated with RNase-free DNase to remove contaminating genomic DNA and first-strand cDNA was then synthesized. Real-time PCR was performed using Fast SYBR Green master mix. PCR amplifications were performed in triplicate with parallel measurements of human GAPDH (internal controls). The expression levels of HOXBLINC lncRNA were also assessed by analyzing a TCGA AML dataset from 179 AML patients (GEO accession number: GSE62944).

RNA isolation, quantitative RT-PCR, as well as RNA-sequencing and data analysis

Total RNAs were extracted and purified with the RNeasy mini-isolation kit according to the manufacturer’s instructions (Qiagen, MD, USA). A total of 2 μg RNA was subjected to reverse-transcription with Superscript II Reverse Transcriptase (Invitrogen) and analyzed by a real-time PCR Detection System (Bio-Rad). Primer sequences for qPCR are listed in Supplementary Table 6 and key reagents are listed in Supplementary Table 7. For RNA-seq library generation, RNA sequencing library was prepared with the Illumina TruSeq mRNA sample preparation system kit (Cat# 20020594). In brief, total RNA samples were purified with purification beads, and then fragmented with the fragmentation buffer mix. Next, First strand cDNA was synthesized with the First Stand Synthesis Act D mix. Then, the second strand cDNA was synthesized with the Second Strand Marking Master Mix. After that, RNA-seq libraries were amplified and indexed with the adapter primers. Then, the quality of the library was tested with Qubit and Agilent Bioanalyzer. Final libraries were submitted to paired-end sequencing of 50 bp length on an Illumina HiSeq 3000. For RNA-seq data analysis, cutadapt (http://cutadapt.readthedocs.io, version 1.2.0) program was used to trim the adaptors and low quality reads from the RNA-seq raw data files33. Then, all of the filtered sequencing reads were processed and aligned to the mouse genome assembly (mm9) or human genome assembly (hg19) using TopHat (version 2.0) and Bowtie234,35,36. Next, FPKM (paired-end fragments per kilobase of exon model per million mapped reads) was calculated for each gene and further normalized. The differential expression was determined according to the FPKM value and processed with the Cufflinks v2.2.1 and Cuffdiff37. Next, differentially expressed genes with greater than 2.0-fold were identified through comparing WT control and experimental groups according to the FPKM values. The scatter plot was generated according to the log2 transformation of the FPKM values, and the upregulated or downregulated genes with more than two folds changes were marked with blue dots, and no change genes were marked with red dots. Gene Ontology analysis was carried out with the Database for Annotation, Visualization and Integrated Discovery (DAVID) tool (https://david.ncifcrf.gov/, Version 6.8)38. Gene set enrichment analysis (GSEA) was conducted according to recommended parameters (http://software.broadinstitute.org/gsea/doc/GSEAUserGuideFrame.html) using gene sets obtained from the Molecular Signatures Database39. The normalized expression data tracks were loaded to the Integrated Genomic Viewer (IGV) for visualization. The sequence reads have been deposited in the NCBI GEO dataset (GSE115096). Key software and algorithms used are listed in Supplementary Table 7.

RNA immunoprecipitation (RIP) assay

The RNA-IP protocol was performed according to the previous reports40,41. In brief, OCI-AML3 and OCI-AML2 cells were harvested and washed with PBS, centrifuged and re-suspended in freshly RIP nuclear isolation buffer (1.28 M sucrose, 40 mM Tris-HCl pH 7.5, 20 mM MgCl2, 4% Triton X-100), and then cell pellets were kept on ice for 20 min (with frequent mixing). Next, nuclei from cells were precipitated by centrifugation at 2,500 g for 15 min, and then re-suspended in freshly RIP lysis buffer (10 mM HEPES-KOH pH7, 150 mM KCl, 5 mM MgCl2, 5 mM EDTA, 0.5% IGEPAL-CA-630, 0.5 mM dithiothreitol, 0.2 mg/mL Heparin, 100 U/mL RNase OUT, 100 U/mL Superase IN, protease inhibitor tablet adding before use). After that, chromatin shearing was performed with sonication. The suspension was centrifuged at 14,000 g at 4 °C for 10 min, and the supernatant was harvested. Next, the nuclear extracts were pre-cleared with rabbit IgG (Sigma) and Dynabeads™ Protein G (Thermo Scientific). Next, the nuclei extracts were incubated with 5 μg antibodies against LSD1 (Millipore, 07-705), Setd1a (Bethyl, A300-289A), and MLL1 (Novus Biologicals, NB600-248) overnight at 4 °C. Immuno-complexes were captured by incubating with 50 μl Dynabeads™ Protein G (Thermo Scientific) for another 2 h at 4 °C. Next, the precipitant was washed with the ice-cold washing buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM MgCl2, 0.05% IGEPAL-CA-630) supplemented with 0.02 mg/mL heparin. The RNA-protein complexes were eluted with 500 μL elution buffer (50 mM Tris pH 8.0, 100 mM NaCl, 10 mM EDTA, 1% SDS) for 10 min at 65 °C. The precipitated RNA was extracted and purified by TRIzol RNA extraction reagent, eluted with nuclease-free water, treated with DNaseI, and then endogenous RNA was subjected to the RT-qPCR analysis.

Chromatin immunoprecipitation (ChIP)

ChIP assay was performed as described previously42. Briefly, cells were cross-linked with 1% formaldehyde for 10 min at room temperature, and then quenched by addition of 125 mM glycine for 5 min at room temperature. The cell pellets were washed with ice-cold 1x PBS. Then, the cell pellets were re-suspended in ChIP lysis buffer (50 mM Tris-HCl, pH 8, 10 mM EDTA, 1% SDS). The lysates were sonicated with the Bioruprtor™ UCD200. After fragmentation, the lysates were centrifuged at 14,000 rpm for 10 min at 4 °C. The supernatant was diluted with ChIP buffer (20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 1% Triton X-100, 150 mM NaCl). The 10% of remaining lysate was used as an input control. Sheared chromatin samples from 5 × 106 AML cells were immunoprecipitated with 5 µg anti-MLL1 (Novus Biologicals, cat# NB600-248) or 2.5 µg anti-H3K4me3 (Millipore, cat#04-745) overnight at 4 °C, separately. Then 50 µL Dynabeads™ Protein G (Thermo Fisher Scientific) was incubated with each ChIP sample for 2 h at 4 °C with rotation. The precipitated complexes were washed with low salt (20 mM Tris, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), high salt (20 mM Tris, pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS) and lithium chloride (10 mM Tris, pH 8.0, 1 mM EDTA, 1% Triton X-100, 250 mM LiCl, 1% sodium deoxycholate) washing buffer, with an additional final wash in TE buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA). Then, elution buffer (100 mM NaHCO3, 1% SDS) was used to dissolve these precipitated complexes, and then were subjected to reverse crosslinking with 2 μL of 10 mg/mL proteinase K overnight at 65 °C. After reverse-crosslinking, the DNA samples were purified and then analyzed by RT-qPCR. The final results represent the percentage of input chromatin and error bars through triplicate experiments. The MLL1 and H3K4me3 ChIP-DNA libraries were prepared using Illumina’s TruSeq ChIP Sample Preparation Kit according to the manufacturer’s instructions (Cat# IP-202-1012). In brief, 10 ng ChIP DNA fragments were performed end repair using the End Repair Mix, and then purified with the AMPure XP beads. After that, 3′ ends of the ChIP DNA fragments were adenylated with the A-Tailing Mix, and then ligated with the adapter indices. After that, ChIP DNA fragments were amplified with the adapter primers. Then, the quality of these DNA libraries were tested with the Qubit and Agilent Bioanalyzer. Final libraries were submitted to paired-end sequencing of 50 bp length on an Illumina HiSeq 3000. The quality of the library was checked with Qubit and Agilent Bioanalyzer. Final libraries were submitted to paired-end sequencing of 100 bp length on an Illumina HiSeq 3000.

Chromatin Isolation by RNA Immunoprecipitation (ChIRP) assay

ChIRP assay was carried out to analyze the HoxBlinc lncRNA distribution in the genome according to our previously described with some modifications43. Briefly, 20 million cells were harvested and cross-linked in 20 ml of PBS buffer containing 1% glutaraldehyde (Sigma, Cat# G5882) at room temperature for 10 min, and then were quenched with a 1/10th volume of 1.25 M glycine at room temperature for 5 min. Next, these cross-linked cells were washed twice with chilled PBS, and per 100 mg of the pellet of cells were lysed in 1 ml lysis buffer (50 mM Tris-HCl pH 7.0, 10 mM EDTA, 1% SDS, PMSF, DTT, P.I. and SUPERase were added before use). Then, these lysates were sonicated 30 min using a Bioruptor (Diagenode) to prepare chromatin in a 4 °C water bath at the highest setting with 30 s ON, 30 s OFF pulse intervals. Next, the sheared chromatin was centrifuged and supernatant was diluted using hybridization buffer (750 mM NaCl, 1%SDS, 50 mM Tris-HCl 7.0, 1.0 mM EDTA, 15% Formamide, add DTT, PMSF, P.I, and SUPERase-in fresh). After that, the diluted supernatant was hybridized with 100 pmol of biotinylated DNA probes targeting HoxBlinc or LacZ (sequences are listed in Supplementary Table 6) and incubated for 4 h at 37 °C with shaking, and then added 100 µl of Streptavidin-magnetic C1 beads (Invitrogen) for each sample for 30 min at 37 °C. Then, these precipitant were washed 5 times with washing buffer (2x SSC, 0.5% SDS). RNA fraction was extracted from ChIRP samples with TRIzol reagent and then subjected to analyze the HoxBlinc retrieval by RT-qPCR, and β-actin gene was used as a negative control. DNA fraction was extracted from ChIRP samples with Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v), and then precipitated with 3 μL of 20 mg/mL glycogen, 1/10th volume of 3 M sodium acetate (pH 5.2), and 2.5 volumes of 100% ethanol. Then ChIRP-DNA library was prepared for ChIRP-seq. Libraries were constructed using Illumina’s TruSeq ChIP Sample Preparation Kit according to the manufacturer’s instructions (Cat# IP-202-1012). In brief, 10 ng ChIRP DNA fragments were performed end repair using the End Repair Mix, and then purified with AMPure XP beads. After that, 3’ ends of the ChIRP DNA fragments were adenylated with A-Tailing Mix, and then ligated with the adapter indices. After that, ChIRP DNA fragments were amplified with the adapter primers. The quality of these ChIRP DNA libraries were examined with Qubit and Agilent Bioanalyzer. Final ChIRP libraries were submitted to the paired-end sequencing of 100 bp length on an Illumina HiSeq 2500.

ChIP-seq and ChIRP-seq data analysis

The cutadapt (http://cutadapt.readthedocs.io, version 1.2.0) program was used to trim the adaptors and low quality reads from the ChIP-seq or ChIRP-seq raw data33. These Cutadapt-filtered reads were aligned to mouse reference genome (mm9) using Bowtie2 with default parameters35, and the quality of these trimmed data was evaluated by the FastQC program44. After alignment, samtools program was employed to convert the SAM files into BAM files and sorted these BAM files45. Next, peak calling was performed using peak calling algorithm MACS246. Peaks were transformed to the visualized files (bigwig format) with deepTools47, including control and experimental datasets. All sequencing tracks were visualized using the Integrated Genomic Viewer software (IGV)48. Peaks were annotated with“annotatePeaks.pl” program with HOMER package49. Differential Peaks calling was performed with getDifferentialPeaks program in HOMER software. For ChIRP-seq binding motif analysis, the de novo motif analysis was performed by the “findmotifsgenome.pl” from the HOMER motif discovery algorithm49. The HoxBlinc bound regions associated genes and pathways were analyzed and annotated by the Gene Ontology (GO) analysis with the Database for Annotation, Visualization and Integrated Discovery (DAVID) tool (https://david.ncifcrf.gov/, Version 6.8)**37. Each GO term with a p-value more than 1 × 10−3 is used for cutoff (threshold: 10−3). All genomics datasets were deposited in the NCBI GEO under accession number (GSE115096).

Circular Chromosome conformation capture (4C) assays

The 4C-seq assay was performed as previously described50 with minor modifications. In brief, 2 × 106 cells were cross-linked with 1% formaldehyde for 10 min and the reaction was quenched with 0.125 M glycine for 5 min at room temperature. Cells were washed twice with cold PBS, and re-suspended in the digestion buffer containing 0.3% SDS overnight at 37 °C with shaking. After that, 2% Triton X-100 was added to sequester SDS and incubated for 1.5 h at 37 °C with shaking. Then, the chromatin was digested with 400U of BglII enzyme (NEB) at 37 °C overnight. 1.6% SDS buffer was added to stop the digestion at 65 °C for 20 min. After that, the digested chromatin was diluted in T4 DNA ligation buffer (NEB) containing 1% Triton X-100 and incubated at 37 °C for 1.5 h with shaking. Next, 800U of T4 DNA ligase (NEB) was used to ligate these digested chromatin at 16 °C for 3 days followed by 1 hr at room temperature. Then, reverse crosslinking was carried out by adding 200 μg of Proteinase K (Invitrogen) and incubated at 65 °C overnight. Next, the 3 C DNA was subjected to phenol:chloroform and extracted with 100% ethanol, and then dissolved with ddH2O. For the second digestion, the first ligates was digested with 300U of NlaIII (NEB) at 37 °C overnight with shaking. The reaction was stopped by adding SDS to a final concentration of 1.6% at 65 °C for 20 min. The digested chromatin/DNA was ligated in ligation buffer containing 6000 U of T4 DNA ligase at 16 °C overnight. After ligation, the 4 C DNA was extracted by phenol-chloroform, and purified by Qiagen PCR kit and amplified by inverse PCR using bait-specific primers. The invert PCR products were cloned into pGEM®-T Easy Vector Systems (Promega) for Sanger sequencing. 4C-seq Libraries were constructed by adding barcoded Illumina adapters to the 5′ end of each primer (Supplementary Table 6). PCR reactions were performed using the Expand Long Template PCR System (Roche), and DNA libraries were purified and quantified before sequencing. The bar-coded DNA libraries were sequenced as 150 bp pair-end reads using the Illumina Nextseq500 platform. For data analysis, cutadapt (http://cutadapt.readthedocs.io, version 1.2.0) program was carried out to trim the 4C-seq raw data and remove the adaptors and low quality reads33. Filtered reads were aligned to the reference mouse genome (build mm9) with Bowtie2 2.2.951. 4C-seq data was analyzed using the 4cseq_pipeline52 and normalized using DESeq253. Statistical analysis for differential interactions between genotypes was performed using DESeq2. Spearman correlation of each genotype was performed using R54. The 4C-sequencing sequence reads have been deposited in the NCBI GEO database (GSE115096).

Transposase-Accessible Chromatin using sequencing (ATAC-seq) assay

ATAC-seq assay was used to analyze the genome chromatin accessibility in different experimental conditions as described previously12,55. In Brief, 5 × 104 cells were collected for library preparation. Cells were washed with pre-cold phosphate buffered saline (PBS) and re-suspended in lysis buffer containing 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40. After washing with cold 1x PBS buffer, the cell pellets were re-suspended in fragment buffer and then fragmented with Tn5 Transposes for transposition reaction at 37 °C for 30 min. Then these DNA fragments were extracted and purified using the MinElute Kit (QIAGEN). The preparation of library fragments were amplified using 1x NEB next PCR master mix and 1.25 μM indexed Nextra PCR primers (Supplementary Table 6) with following PCR conditions: 72 °C for 5 min, 98 °C for 30 s, followed by thermocycling at 98 °C for 10 s, 63 °C for 30 s and 72 °C for 1 min. After amplification, the eluted DNA was used in a quantitative PCR (qPCR) reaction to estimate the optimum number of amplification cycles. Libraries were quantified using qPCR (Kapa Library Quantification Kit for Illumina, Roche), and AMPure XP beads (Beckman Coulter) was employed to purify the libraries, and then the quality of the DNA library was examined by Agilent Bioanalyzer 2100 prior to sequencing with 2 × 100 bp paired-end reads on an Illumina HiSeq 2500. Each sample includes two replicates for statistical analysis.

ATAC-seq analysis

ATAC-seq assay was carried out and analyzed with two biological replicates according to our previous reports12,55. Briefly, all of the raw data files were filtered through cutadapt (http://cutadapt.readthedocs.io, version 1.2.0) to remove adaptors and low quality reads33. These filtered reads were aligned to the mouse genome (mm9) using Bowtie2 with default parameters (version Bowtie 2/2.2.6)35, and the quality of these trimmed data was evaluated by FastQC program (version 0.11.8)44. PCR duplicates were removed using Picard MarkDuplicates (version 2.0.1), and mitochondrial reads were removed with samtools56. ENCODE blacklist regions were filtered (https://sites.google.com/site/anshulkundaje/projects/blacklists). For quality control, 50 million reads with paired-end sequencing was used for each sample. In addition, the alignment rate of each replicate is more than 95% through removing the unaligned reads. Third, the mitochondrial-related reads and PCR duplicates reads were removed from total reads after alignment. Finally, non-uniquely aligned reads were filtered based on MAPQ scores with samtools (MAPQ > 30), and plotPCA from BiocGenerics package in R package (R/3.6.1) was carried out to identify the variance between control and experimental groups. Moreover, fragSizeDist from ATACseqQC package in R package was used to show the fragment size distribution in control and experimental groups. After alignment and trimming, samtools (version 1.8.0) were used to convert the SAM files into BAM files and sorted the BAM files for further analysis45. Peak calling was performed using peak calling algorithm MACS2 with parameters (“-g mm -p 1e-9 –nolambda -f BAMPE –nomodel –shiftsize=100 –extsize 200”)46. The visualizable bigWig files of fragment or read coverages were generated with bedGraphToBigWig program, including control and experimental datasets (https://www.encodeproject.org/software/bedgraphtobigwig/). All sequencing tracks were viewed using the Integrated Genomic Viewer (IGV/2.4.19)48. Peaks were annotated with the command “annotatePeaks.pl” from HOMER package (version 4.10)49 and GREAT57. Next, the differential binding sites between two peak files were calculated with DEseq2 (Benjamini-Hochberg adjusted p58. The de novo motif analysis was performed by the “findmotifsgenome.pl” from the HOMER package49. For each genomic feature (peaks or chromVAR annotation), the chromatin accessibility median deviation z-score (for chromVAR features) or fragment counts (for peaks) were examined in control and experimental groups with chromVAR package in R language59,60. Overall similarity between the replicates of ATAC-seq global chromatin accessibility signatures was carried out with Pearson’s correlation coefficient and Pearson’s χ2-test. All genomics datasets were deposited in the NCBI GEO under accession number (GSE115096).

dCas9-mediated inactivation of HoxBlinc in AML cells

To generate the i sgRNA vector, sgRNA targeting the promoters of HOXBLINC were designed using the Zhang laboratory web tool (http://crispr.mit.edu), and sgRNA was subcloned into the pLKO5.sgRNA.EFS.GFP vector (Addgene#57822). OCI-AML3 AML cells were co-transfected with the gRNA plasmids encoding GFP and the repressive plasmid encoding dCas9-KRAB (pHR-SFFV-dCas9-BFP-KRAB, Addgene plasmid #46911), and then these cells were cultured for another 48 h. After that, OCI-AML3 AML cells were subjected to 2 μg/mL of puromycin for another 48 h, and then live cells were sorted with GFP by FACS. Finally, RNAs from these positive cells were extracted with TRIzol™ reagent, and then the gene expression level was determined by RT-qPCR.

Doxycycline (DOX) inducible shHOXBLINC in AML cells

Human shRNA targeting HOXBLINC was designed using the Thermo Fisher web tool (https://rnaidesigner.thermofisher.com/rnaiexpress/), and shRNA was cloned into the doxycycline (DOX) inducible pTRIPz vector (dharmacon.horizondiscovery.com). Then OCI-AML3 cells were infected with DOX inducible pTRIPz vector and then cultured for 48 h. After that, cells were selected with 2 µg/ml of puromycin for another 48 h, and the live cells were cultured in the standard culture medium of OCI-AML3 cell line. After that, cells were treated with the DOX at a concentration of 2 μg/mL for 3 days. In addition, refresh or add the fresh culture medium to maintain the doxycycline concentration every 48 h. Then, RNAs from these doxycycline inducible cells were extracted and purified with the TRIzol™ reagent, and then proceeded to evaluate the knockdown efficiency by RT-qPCR.

Lentiviral transduction

The RNAi Consortium (TRC)-based short hairpin RNA lentiviral vector (Sigma, Horizon Discovery, or lab-owned) were transfected into HEK 293 T cells (ATCC) together with pMD2.G (Addgene) and psPAX2 (Addgene) to package viruses. The virus supernatant was collected from 16 h to 48 h after transfection and then purified with Lenti-X™ Concentrator (Takara). The Lin cells from mice BMs were transduced with the packaged viruses for 24 h in plain IMDM added with mSCF (100 ng/mL) and then sorted for GFP+ LSK cells. Sorted LSK cells were collected into different dishes for colony-forming assays or transplantation.

Xenotransplantation of OCI-AML3 AML cells or AML patient BM cells

Adult NOD.CgPrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice (6–8 weeks old) were pretreated with 280 cGy total body irradiation, then 5 × 105 viable OCI-AML3 cells (in 300 μl PBS) or 1 × 106 patient BM cells (in 300 μl PBS) were injected into tail veins of the recipient NSG mice for transplantation. After transplantation, the recipients mice were administered with (for OCI-AML3 cells stably expressing inducible shHOXBLINC to induce HoxBlinc KD) or without (for AML cells with dCas9-mediated HoxBlinc inactivation) doxycycline in the drinking water (Sigma D-9891, 1 mg/ml, 1% sucrose, newly prepared every other day) until being sacrificed, and daily monitored for symptoms (ruffled coat, hunched back, weakness, and reduced motility) and survival time. For each set of xenotransplantation, recipient mice of all groups were killed and analyzed on the same day when any group of recipients exhibited a moribund condition. Human CD45 chimerism in the BM, spleen cells, and PB WBC were analyzed by flow cytometry, histological assay, IHC for hCD45 were also performed as described above.

Quantification and statistical analysis

Differences between experimental groups were determined by the Student’s t-test or analysis of variance (ANOVA) followed by Newman-Keuls multiple comparison tests as appropriate. P

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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