ALL Xenograft Model and Primary Patient Samples
The process by which continuous xenografts from ALL biopsies have been established in immunodeficient NOD/SCID interleukin (IL)-2 receptor gamma chain null (NSG) mice has been previously described in detail15,25. ALL biopsies were obtained for xenografting with informed, written consent approved by the Human Research Ethics Committees of the South Eastern Sydney Illawarra Area Health Service and UNSW Sydney. All animal studies had previous approval from the Animal Care and Ethics Committee of UNSW Sydney. The fidelity of all PDXs in our laboratory is routinely validated by high-density SNP (single nucleotide polymorphism) arrays as previously described21.
In Vivo Treatments, Sample Preparation and Analysis
ALL xenograft cells were inoculated by tail-vein injection into NSG mice, and engraftment was monitored weekly as previously described15,25. Mice were randomized and treated with either dexamethasone (15 mg/kg) or vehicle control by intraperitoneal injection when the %huCD45+ cells in the peripheral blood reached certain percentages as follows:
- 1% for in vivo drug efficacy studies. In brief, when huCD45+ cells reached 1% in the peripheral blood, the ALL-engrafted mice were treated with either dexamethasone or vehicle control on weekdays for 4 weeks. The percentage of huCD45+ cells in murine peripheral blood was monitored weekly. The engraftment burden of ALL cells in different mouse organs after drug treatments was calculated based on the average number of cells harvested and normalized as a percent of average control for each experiment27.
- > 70% for in vitro molecular biology experiments. Briefly, mice were treated when > 70% %huCD45+ cells in the peripheral blood, and euthanized 8 hours thereafter. Cell suspensions of spleens were prepared, and mononuclear cells enriched to >97% human cells by density gradient centrifugation. After harvesting, cells were immediately resuspended in fetal calf serum (FCS) containing 10% DMSO, frozen and stored at liquid nitrogen for further use.
Assessment of Dexamethasone Sensitivity
In vitro dexamethasone sensitivity was assessed by mitochondrial activity using Alamar Blue assay as described previously15,25. The half maximal inhibitory concentration (IC50) was calculated from the dose response curves. In vivo dexamethasone sensitivity was determined by the leukemia growth delay (LGD, treated-control). The sensitivities of ALL cells to dexamethasone in vitro and in vivo have been described in our previous publications15,25. The PDXs were stratified into glucocorticoid sensitive or resistant groups based on their in vitro IC50 and in vivo LGD.
Rapid Immunoprecipitation Mass Spectrometry (RIME)
RIME was done with some minor modifications to the original protocol69. Briefly, 1x108 cells were collected from ALL-54S or ALL-50R upon acute treatment with DEX or vehicle control (8 hours), pelleted and initially cross-linked using 2mM disuccinimidyl glutarate (DSG) in PBS for 20 min with shaking at RT. After pelleting the cells, the cells were resuspended in 1% formaldehyde in PBS and incubated at RT with shaking for 10 min to double-cross-linked the samples42. Crosslinking was quenched by the addition of glycine to a final concentration of 0.125M and an incubation at RT for 5 min. The cells were washed with ice-cold PBS, then resuspended in PBS containing protease inhibitors (PI). The nuclear fraction was extracted by resuspending the pellet in 1mL LB1 (50mM HEPES-KOH pH7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% Igepal CA-630, 0.25% Tritox X-100) containing PI, followed by rotating the samples at 4°C for 10 min. After pelleting the cells, cells were resuspended in 1mL LB2 (10mM Tris-HCl pH8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA) containing PI, mixed by rotation at 4°C for 5 min, then pelleted. The cells were then resuspended in 300µl LB3 (10mM Tris-HCl pH8.0, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-laurosylsarcosine) containing PI, and sonicated for 7 cycles (30 seconds on, 30 seconds off). To the sonicated samples, 10% Triton X-100 in LB3 was added to a final concentration of 1%, followed by centrifugation at 20,000xg for 10 mins at 4°C to remove the debris. The supernatant, which is the nuclear fraction, was then collected for immunoprecipitation. Protein A/G magnetic beads (Thermo Fisher Scientific) were firstly blocked with Pierce Protein-Free PBS blocking buffer (Thermo Fisher Scientific) and resuspended in LB3 containig 1% Triton X-100. The blocked beads were incubated with the supernatant containing the nuclear fraction for 1 hour rotating at RT to preclear the samples. The pre-cleared samples were incubated with 10µg anti-NR3C1 antibody (Atlas Antibodies) for overnight at 4°C with rotation. The samples/antibody mixtures were then incubated with blocked beads for 1 hour at RT with rotation. The beads were washed 10 times with LiCl RIPA buffer (50mM HEPES pH7.6, 1mM EDTA, 0.7% Na-Deoxycholate, 1% Igepal CA-630, 0.5M LiCl), followed by washing twice in 100mM ammonium hydrogen carbonate (AMBIC) solution. The beads were further washed three times with 25mM AMBIC solution, before tryptic on-bead digestion was performed by adding 10µl of sequencing grade trypsin (0.02µg/µl in 25mM AMBIC solution) and incubating overnight at 37°C. Additional 10µl of sequencing grade trypsin (0.02µg/µl in 25mM AMBIC solution) was added to the beads, and on-bead digestion was performed for another 1 hour at 37°C. Supernatant containing peptides was collected and combined with TFA to give a final concentration 0.1%. Peptides were loaded onto EvoTip Pure tips for desalting and as a disposable trap column for nanoUPLC using an EvoSep One system. A pre-set EvoSep 100 SPD gradient was used with an 8 cm EvoSep C18 Performance column (150 mm x 1.5 mm). The nanoUPLC system was interfaced to a timsTOF HT mass spectrometer (Bruker) with a CaptiveSpray ionisation source (Source). Positive PASEF-DIA, ESI-MS and MS2 spectra were acquired using Compass HyStar software (version 6.2, Thermo). Instrument source settings were: capillary voltage, 1,500 V; dry gas, 3 l/min; dry temperature; 180°C. Spectra were acquired between m/z 100-1,700. Custom TIMS settings were applied as: 1/K0 0.-1.60 V.s/cm2; Ramp time, 100 ms; Ramp rate 9.42 Hz. PASEF-DIA acquisition specified DIA-windows of 25 Th width between 400-1201 Th and a mobility range of 0.6-1.43 1/K0. The total cycle time was 1.8 s. Collision energy was interpolated between 20 eV at 0.6 V.s/cm2 to 59 eV at 1.6 V.s/cm2. Peak lists in .d format were imported into DIA-NN software (ver. 1.8)70 for identification and relative quantification. An in-silico spectral library was created from the human subset of SwissProt appended with common proteomic contaminants. The two-pass method was used in DIA-NN, first searching against the in-silico library then iterating against the DIA data from these samples for a second search. The search was run at 1% FDR. DIA-NN results in peptide-centric .tsv format were filtered to peptide q-values <0.01, then pivoted on protein accessions before further filtering to require a minimum of 2 unique peptides per protein match and protein q-values <0.01. For statistical comparison between groups, missing values were imputed with minimum values and differential abundance was tested using limma through the FragPipe-Analyst (http://fragpipe-analyst.nesvilab.org/), run as a local installation in R-shiny. Three biological replicates were used for each group and the Hochberg and Benjamini correction was used for multiple test correction.
HiC Library Preparation and Sequencing
HiC was performed using a modified version of a previously described protocol71. Briefly, 10 million cells were collected and cross-linked in 10 ml of PBS containing 1% formaldehyde for 10 minutes at room temperature. The reaction was stopped by 0.125M glycine solution. Cross-linked cell pellet was used to prepare nuclei, which were subjected to digestion with restriction enzyme MboI followed by end-filling to create biotin-labeled blunt ends. Blunt ends were ligated by T4 DNA ligase at room temperature for 4 hours with rotation. Nuclei were digested with proteinase K and 10% sodium dodecyl sulfate (SDS) at 55°C for 30 minutes. Following sodium acetate and pure ethanol precipitation, DNA was isolated and dissolved in Tris buffer. DNA was sonicated using Bioruptor sonicator (Diagenode SA, Belgium) to obtain fragments ranging in size from 150 bp to 300 bp, followed by double-sided size selection using AMPure XP beads at 0.55X and 0.3X volume for HiC. Biotinylated HiC material was then purified using Dynabeads MyOne Streptavidin T1 beads (Life technologies, 65602). While on beads, DNA ends were blunt ended and dA tailed, followed by ligation of the Illumina sequencing adapter with T4 DNA ligase. PCR reactions were performed on beads involving 8 cycles of amplification (95°C for 2 min, followed by 94°C for 80 s, 65°C for 30 s, 72°C for 30 s). DNA library was size selected using 1.5% agarose gel to obtain fragments ranging in size from 400bp to 700bp. Sequencing was performed on the Illumina HiSeq 2500 to obtain 150bp paired-end reads at sequencing depth of 100 million reads per sample.
ChIP-seq
ChIP and ChIP-seq were carried out as previously described15,25. Briefly, frozen PDX cells that were harvested from mice after dexamethasone or vehicle control treatment in vivo were revived from cryostorage, and fixed with 1% formaldehyde for 10 min at room temperature. Nuclei were extracted from fixed cells by 10 min incubation in lysis buffer (0.2% NP40 in 10 mM Tris buffer, pH 8.0) followed by centrifugation at 1250 × g for 5 min at 4 °C. Chromatin was fragmented using a Bioruptor sonicator (Diagenode SA, Belgium) on high power at 4 °C with 30 s on/off for 10 min. Separate immunoprecipitates were produced using immunoglobulin raised against GR (Cell Signaling, Danvers, MA, USA), CTCF (Cell Signaling), H3 histone (Millipore, Billerica, MA, USA), histone marks (H3K4Me3, H3K4Me1, H3K27Ac and H3K27Me3, Cell Signaling) and processed according to the manufacturer’s instructions. DNA from protein-associated complexes and corresponding input samples were recovered using phenol/chloroform/isoamyl alcohol with phase lock gel tubes (5 Prime, Hilden, Germany). The ChIP DNA samples were dissolved in 20 µl water, library amplified, and sequenced using Hiseq2500 platform for 50bp single-end reads at 20 million reads per sample.
RNA-seq
For gene expression studies, RNA was extracted immediately after cell harvesting using the RNeasy Mini Kit (QIAGEN, Valencia, CA, USA), and RNA samples with integrity number (RIN) > 8.0 were sent to Novogene (Beijing, China) for library preparation and sequencing using Hiseq4000 platform for 150bp paired-end reads at 40 million reads per sample.
HiC Data Analysis
HiC data were first trimmed and then mapped against hg19 human reference genome using runHi-C pipeline based on the 4DN consortium. Specifically, Burrows-Wheeler Aligner (BWA) was used for the FASTQ file alignment and low-quality aligned reads and PCR duplicates removed. Aligned reads were then paired and filtered for fragments containing ligations of at least two different restriction fragments. These reads were then binned at 5-kb resolution. To generate the contact matrix at multiple resolutions (5, 10, 25, 40, 50, 100, 250, 500 kb, 1, 2.5, 5, and 10 Mb), we used the run-cool2multirescool script from 4DN consortium, which performed the ICE normalization at the same time. We used coolbox to visualize ICE normalized genomic HiC data. Juicer tool was also used to generate multiresolution .hic files, which can be visualized using Juicebox.
In Figure 1, we divided the whole genome into 60,000 bins at 50kb resolution and grouped them based on the enrichment of DHS domains. We have previously analyzed DHS-seq datasets from 78 different cell types in the ENCODE database15, which identified 0.8 million open chromatin domains (DHS domains) and defined a series of categories of DHS domains based on their chromatin accessibility and histone modification data. Here, in this study we aligned the PCA values derived from HiC contact intensities with the domain categories.
To study DEX induced chromatin conformation changes on a genome-wide scale, we compared HiC contact intensities before and after DEX treatment in ALL-54S and ALL-50R, and defined dynamic bins and stable bins as described in the legend of Figure 1D. To further interrogate the subset of stable bins, we divided these regions into those with high (-0.2 < PCA < 0.2 and values of each group > 0) and low (-0.2 < PCA < 0.2 and values of each group < 0) contact intensities. Next, ΔΔHiC was calculated as ΔHiC of ALL-54S (DEX – Control) - ΔHiC of ALL-50R (DEX – Control), which depicts differential bin activity between control and dexamethasone-treated group in ALL-50R and -54S following dexamethasone treatment in vivo. ΔΔHiC Fold change < -0.24 or > 0.24 and -log10 (adj.p-Value) > 0.2 were considered as significant changed bins.
RNA-seq and ChIP-seq Data Analysis
RNA-seq and ChIP-seq analyses were performed as previously described72, in brief: sequencing reads were aligned to human genome (hg19) using the Burrows-Wheeler Aligner (BWA, default parameters)73 and visualized using the UCSC Genome Browser. Peak calling was performed using three peak calling algorithms, HOMER (REF, FDR 1e-3 and 1e-2)35, MACS74 (p < 1e-5 and p < 1e-9) and SPP75 (FDR 1e-2 and FDR 1e-5). Peaks identified by two or more of these algorithms were kept in the final peak list. These sites were assigned as regulatory regions to specific genes using the genomic regions enrichment of annotations tool (GREAT) analysis package76.
Paired end RNA sequencing reads were aligned to the human genome (hg19) using the software STAR (v2.5.0b)77 with standard parameters. Gene expression levels were quantified using htseq (v0.9)78 and TMM normalized using the software package DESeq (v3.6) in the R statistical analysis software (v3.3.3)79,80. Signal intensity of the regulatory regions was defined by normalizing absolute reads of each peak to total reads of the dataset and region size. Heatmaps of gene expression profiles and epigenetic profiles at related regulatory regions were generated using Genepattern (Broad Institute, USA). Hierarchical clustering was performed using the commercially available package Partek Genomics Suite (version 6.6). The RNA-seq, ChIP-seq, and HiC data have been deposited in NCBI's Gene Expression Omnibus (GSE109949 and GSE236085).
Link Enhancer with Promoter Using HiC Data
To map enhancer with promoter using HiC data, interaction normalization, matrix creation, significant interaction identification at 10-kb resolution were performed by using HOMER tools (v4.11.1)81. Gene annotations (version GeneCode V42lift37) were retrieved from UCSC82,83. Gene TSS regions that are upstream or downstream 1.5 kb of the interactions of GR binding LSOs are extracted based on the significant interactions of the HiC data. Interacting pairs were deduplicated and sorted by HiC scores.
Co-binding Z-Score Calculation
The bootstrapping technique described in our previous publication37 was used to assess the statistical significance of combinatorial binding events involving CTCF, EBF1, PU.1 and GR across all 15 potential binding patterns. In brief, we used a conservative estimate of 80,000 binding sites per protein as described before84,85 to establish a background distribution of combinatorial binding events and then calculated a standardized z-score to measure the deviation between the number of combinatorial binding events (i.e., determined by ChIP-Seq) from the expected mean of the background distribution.
Motif Analysis
Motif analyses were performed based on ChIP-seq data of H3K4Me1, H3K27Ac, H3K4Me3, H3K27Me3 and GR in ALL-50R and -54S before and post DEX treatment in PDX models in vivo. As defined in Figure 4, GR-bound regulatory domains were stratified into 15 categories, i.e., (1) promoters (Pro) or enhancers (Enh) are marked by H3K4Me3 or H3K4Me1, respectively (S); (2) transcriptionally active (A) regions are marked by H3K27Ac; (3) repressed (R) regions are marked by H3K27Me3; (4) bivalent regions (Bi) are marked by H3K27Ac and H3K27Me3; and (5) regions lacking any ChIP enrichment are defined as no signal (NS). Next, GR-bound regions matching the following criteria were selected (Table S4)
- defined as Pro-S or Pr-Enh-S or RO or NS in ALL-50R control, AND
- defined as Pro-S or Pr-Enh-S or RO or NS in DEX-treated ALL-50R, AND
- defined as Pro-S or Pr-Enh-S or RO or NS or Enh-S in ALL-54S control, AND
- defined as Pro-A or Enh-A or Pro-Enh-A or AO in DEX-treated ALL-54S
The selected GR-bound regions were analyzed using an integrative genomics method for the prediction of regulatory features and cis-regulatory modules from i-cisTarget. The regulatory network model was built based on analysis of connectivity between GR, PU.1, EBF1 and TCF3 with their regulated genes by i-cisTarget and was visualized by Cytoscape.
Motif Analysis of DB2313-Induced PU.1 Loss
The extended “DB-sensitive” PU.1 motif (derived from examination of DB2115 displaced PU.1 binding in MOLM13 cells from Taylor et al., under review46) was used to calculate a log-odds score for GR-LSOs and non-GR-LSOs using the annotate peaks function from the Homer package with the -m and -mscore options enabled35.
Luciferase Reporter Assays
Sequences of target regulatory elements were synthesized as dsDNA by Integrated DNA Technologies (Coralville, IA, USA) and inserted into pGL3-promoter (pGL3p) vector (Promega, Madison, Wisconsin, USA) between Sal1 and BamH1 cutting sites downstream of the firefly luciferase gene. The cloned vector was co-transfected with pRL-TK renilla luciferase control reporter vectors (Promega) into Nalm6 cells. The firefly and renilla luminescence were detected in the transfected cells treated with DB2313 (MCE, NJ, USA) in the presence or absence of DEX (Sigma) using the Dual-Glo Luciferase Assay System (Promega). The firefly luminescence was normalized to renilla luminescence for each condition. Fold inductions were then calculated by normalizing to pGL3p control.
Cytotoxicity Assays
Nalm6 cell line was cultured in RPMI 1640 media supplemented with 10% FCS, 1 mmol/L pyruvate, nonessential amino acids, 10 mmol/L 2-mercaptoethanol, and 2 mmol/L L-glutamine. Nalm6 cells were seeded in 96-well U bottomed plates at 20000 cells per 100 μL medium per well. DB2313 or vehicle control was added to a final concentration of 1 μM in triplicate wells. After 48 hr, 10 μM DEX (Sigma) or vehicle control was added to the cells pre-treated with either DB2313 or vehicle control. The cells were incubated for a series of time points. Following the incubation, Cell Counting Kit-8 (Vazyme) was added. After an additional 4 hr incubation, fluorescence was measured (450 nm) and cell viability expressed relative to vehicle-treated control cells. The presumed additive effect of combination treatment (DB2313 and DEX) was shown by viability% of DB2313 × viability% of DEX-treated conditions. Synergy score was calculated as by SynergyFinder (https://synergyfinder.fimm.fi). The p values were calculated as the combination vs. the presumed additive effect at each concentration point.
qRT-PCR
Quantitative real time polymerase chain reaction (qRT-PCR) was carried out as previously described15,25. Briefly, total RNA was isolated using TRIzol (life technologies) and cDNA was synthesized using HiScriptIII All-in-one RT SuperMix kit (Vazyme). Primers and probes for targeted genes were purchased from Life Technologies and qRT-PCR was carried out in triplicate under cycling conditions according to the manufacturer’s instructions. qRT-PCR was conducted using the ChamQ Universal SYBR qPCR Master Mix (Vazyme) on a ViiA 7 qRT-PCR system (Applied Biosystems, Waltham, MA). Relative expression of target genes was calculated using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression as control. The sequences of primers used are as follows:
- BIM forward primer, ‘5-TAAGTTCTGAGTGTGACCGAGA-3’
reverse primer, ‘5-GCTCTGTCTGTAGGGAGGTAGG-3’;
- RASA1 forward primer, ‘5-ACTTGACAGAACGATAGCAGAAG-3’
reverse primer ‘5-GCCTCCGATCACTCTCTCTTA-3’;
- ZBTB16 forward primer, ‘5-GAGATCCTCTTCCACCGCAAT-3’
reverse primer ‘5-CCGCATACAGCAGGTCATC-3’;
- GliZ forward primer, ‘5-AACACCGAAATGTATCAGACCC-3’
reverse primer, ‘5-TGTCCAGCTTAACGGAAACCA-3’;
- GAPDH forward primer, ‘5-GGAGCGAGATCCCTCCAAAAT-3’
reverse primer, ‘5-GGCTGTTGTCATACTTCTCATGG-3’.
CRISPR/Cas9-Mediated In Vivo Gene Knockout (KO)
Stable Cas9-expressing PDX cells were generated using the FU-Cas9-mCherry plasmid (Addgene #70182). In brief, HEK293FT cells (Invitrogen) were transfected using Lipofectamine™ 2000 (Thermo Fisher Scientific 11668019) with FU-CAS9-mCherry and the pMD2.G envelope (Addgene #12259) and psPAX2 packaging plasmids (Addgene #12260) to generate lentiviral particles. PDX cells were revived and transduced ex vivo with lentivirus for 24 hours, washed in PBS and injected into NSG mice (Australian Bioresources) for in vivo expansion (1 million cells per mouse). Mice were sacrificed at high leukemia burden (>50% huCD45+% in mouse peripheral blood) and PDX cells harvested from the spleen. mCherry+ cells were sorted by fluorescence-activated cell sorting (FACS) to generate a purified Cas9-mCherry population and reinjected into naïve NSG mice for in vivo expansion.
For gene KO, single guide RNA (sgRNA) sequences against human SPI1 (PU.1), BCL2L11 (BIM) or non-targeting sgRNA sequences were cloned into the FgH1tUTG (Addgene #70138) GFP vector and lentiviral particles made as described above. Cas9-mCherry+ PDX cells were transduced, in vivo expanded, and sorted by FACS to generate a purified mCherry+ GFP+ population and reinjected into naïve NSG mice for in vivo expansion. sgRNA sequences are as follows:
Targeted Genes
|
sgRNA sequence
|
SPI1
|
AATACTCGTGCGTTTGGCGT
|
BCL2L11
|
GCCCAAGAGTTGCGGCGTAT
|
Human non-targeting sequence
|
ACGGAGGCTAAGCGTCGCAA
|
For in vivo gene KO and drug efficacy testing, Cas9-mCherry+ sgRNA-GFP+ PDX cells were injected into NSG mice (6 mice/treatment group) and mice switched to Doxycycline-impregnated food (600mg/kg Doxycycline) 7 days post-injection to activate sgRNA expression. Leukemia level was tracked weekly by tail vein bleeding and quantifying huCD45+ cells in mouse peripheral blood. At 1% huCD45+, DEX treatment was administered via intraperitoneal injection at 15mg/kg daily, 5 days on/2 days off for 4 weeks. A leukemia event is defined as 25% huCD45+, or when the animal is euthanised due to leukemia-related morbidity, and event-free survival (EFS) was used to assess therapeutic enhancement with statistical significance defined as p<0.01. At leukemia event, mice were sacrificed and PDX cells harvested from the spleen for quantification of CRISPR efficiency.
Evaluation of Indel Mutagenesis
The CRISPR/Cas9-induced indel frequencies were quantified by Tracking of Indels by Decomposition (TIDE) 86. Genomic DNA was extracted using Isolate II Genomic DNA Kit (Bioline BIO-52066) following manufacturer’s instructions. 100 ng genomic DNA was PCR amplified using Q5 polymerase (NEB M0494X) and primers flanking the sgRNA target region. The PCR products are next subjected to standard Sanger sequencing and the sequence data files of the edited samples and the wild-type samples were analyzed by the TIDE webtool (http://tide.nki.nl). Primer sequences used for INDEL quantification by TIDE assay are as follows:
Genes
|
Forward primer (5’-3’)
|
Reverse primer (5’-3’)
|
SPI1
|
GGAAGAAATGAACCCCGCAC
|
GAGGGCTGTAGGTCCAACG
|
BCL2L11
|
GGTTGGAATGTTTTCAGTTCTTGC
|
TCCTTTGCTGCCTCCTACTG
|
Protein Structure Analysis
Mutated PU.1 proteins structures were predicted using Aphafold2 47 based on the following amino acid sequences and the results were viewed with PyMOL.
- Mutant #1: Serine insertion
MLQACKMEGFPLVPPQPSEDLVPYDTDLYQRSQTHEYYPYLSSDGESHSDHYWDFHPHHVHSEFESFAENNFTELQSVQPPQ
LQQLYRHMELEQMHVLDTPMVPPHPSLGHQVSYLPRMCLQYPSLSPAQPSSDEEEGERQSPPLEVSDGEADGLEPGPGLLPGETGSKKKIRL
YQFLLDLLRSGDMKDSIWWVDKDKGTFQFSSKHKEALAHRWGIQKGNRKKMTYQKMARALRNYGKTGEVKKVKKKLTYQFSGEVLGRGGLAERRHPPH
- Mutant #2: Glycine and Valine insertion
MLQACKMEGFPLVPPQPSEDLVPYDTDLYQRGVQTHEYYPYLSSDGESHSDHYWDFHPHHVHSEFESFAENNFTELQSVQPPQLQ
QLYRHMELEQMHVLDTPMVPPHPSLGHQVSYLPRMCLQYPSLSPAQPSSDEEEGERQSPPLEVSDGEADGLEPGPGLLPGETGSKKKIRL
YQFLLDLLRSGDMKDSIWWVDKDKGTFQFSSKHKEALAHRWGIQKGNRKKMTYQKMARALRNYGKTGEVKKVKKKLTYQFSGEVLGRGGLAERRHPPH
Quantification and Statistical Analysis
HiC-seq, RNA-seq and ChIP-seq studies were performed using PDX cells from three randomized ALL-engrafted mice at each condition of treatments. Gene expression and cytotoxicity studies were performed with three independent experiments. Quantitative variables of normally distributed data were compared by the student t test and non-normally distributed data were compared by the Mann-Whitney U test. All statistical tests were two-sided and p values <0.05 were considered statistically significant.
Data and Software Availability
The datasets generated and/or analyzed in this study are deposited in the NCBI's Gene Expression Omnibus repository (GSE109949 and GSE236085). The GSE109949 is publicly available and the GSE236085 can be accessed with a token mravgggutfcpfsv. We also host a UCSC genome browser session for easy access and viewing of genome-wide mapping at:
- https://www.genome.ucsc.edu/s/Sally%20Cao/All%20peaks%20%2D%20NC