Genome editing–induced t(4;11) chromosomal translocations model B cell precursor acute lymphoblastic leukemias with KMT2A-AFF1 fusion

A t(4;11) leukemia model established from CRISPR-engineered chromosomal translocations between the KMT2A and AFF1 genes recapitulate proteomic, epigenomic, and transcriptomic features of primary patient leukemias.

(A) Example of tSNE analysis from the leukemia compartment as identified in (Fig. 1E).

Animals
Gene-edited cells were cultured in vitro (day 0 to day 26) and transplanted (1×10 6 ) by IV injection into sub-lethally irradiated (250 cGy) immune-compromised NOD.Cg PrkdcscidIL2rgtm1Wjl/SzJ (NSG) mice as previously described (1).Four individual primary gene-edited BCP-ALL cells were injected into nine NSG mice to generate the secondary leukemia survival curve.

Human subjects
Human ALL samples were obtained from patients at the Stanford Medical Center with informed consent and institutional review board approval.Human CD34 + HSPCs (three individuals, both male and female) were obtained from Stanford Hospital via the Binns Program for Cord Blood Research under informed consent.Mononuclear cells were purified by Ficoll-density gradient centrifugation enrichment.
Pan et al.

Chromosomal translocations and fusion gene products detection
Total RNA was extracted as described in RNA-seq.cDNA was synthesized using QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany).qRT-PCR was performed using PowerUp SYBR Green Master Mix on CFX96 and quantified using the ddCT.
Fluorescence in situ hybridization (FISH) and karyotyping were performed by the Cytogenetics Laboratory of Stanford Hospital, as previously described (2).

Mass cytometry (CyTOF)
Samples for CyTOF were prepared as described (3) using a 40-parameters antibody panel built in-house.Viably frozen cells were thawed, resuspended at 1 to 2 million cells per milliliter, and fixed in 1.6% paraformaldehyde for 10 minutes at room temperature.Cell Staining Medium (CSM, phosphate-buffered saline [PBS] + 0.5% bovine serum albumin + 0.02% sodium azide), was used to wash cells.Cells were barcoded using 20-plex barcoding plates prepared in-house as previously described (4).To control for staining and batch effect a healthy BM was added to each barcoded plate.Following barcoding, cells were washed with CSM and combined in one single tube.Human TruStain FcX blocking solution was added to the cells at 50 μL/100 μL staining volume.Metal-conjugated antibodies for surface antigens were added to the cells at titrated concentrations and incubated at room temperature on a shaker for 30 minutes.Cells were washed once with CSM before being permeabilized with 100% methanol for 10 minutes at 4°C.Following permeabilization, cells were washed twice and then stained with metal-conjugated antibodies against intracellular antigens for 30 minutes at room temperature on a shaker.After one CSM was, cells were stained overnight with 1:5,000 191Ir/193Ir DNA intercalator (Fluidigm) in PBS with 1.6% PFA at 4°C.Prior to acquisition on a Helios mass cytometer, cells were washed once in CSM, followed by two washes in ultrapure double-distilled H2O.Cells were resuspended in ddH2O with normalization beads (139La/142Pr/159Tb/169Tm/175Lu), maintained at 4°C and acquired on a Helios mass cytometer at a rate of ∼200 cells per second.

Processing and analysis of CyTOF data
CyTOF raw data were normalized using bead normalization (5) and files were debarcorded as described (4).Cytobank software was used to gate Lin-/B-enriched cells excluding dead cells Pan et al.

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(cPARP+), murine cells (mCD45.1+),myeloid (CD33+) and T-cells (CD3+).We then classified each sample using the B-cell developmental classifier previously published (6).The Lin-/Benriched cells obtained from the healthy BM control were gated in 12 populations as previously described except for the progenitor's populations combined in one single population named CLP (common lymphoid progenitor).Each cell was then classified to the closest (Mahalanobis distance) healthy gated population based on the expression of 10 proteins (CD34, CD38, CD24, TdT, CD179b, CD19, CD20, IgMi, IgMs) used for the manual gating of the healthy BM.
Frequency of each classified sample was then plotted using GraphPad Prism software (v 9.3.1).tSNE dimension reduction analysis was performed by Cytobank and cytofkit2 (https://github.com/JinmiaoChenLab/cytofkit2)using all 40 markers measured at single cell level by mass cytometry.

RNA-seq analysis
RNA was extracted from control and leukemia cells with RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.RNA-seq libraries were generated and sequenced by Novogene (Sacramento, CA).Bulk RS4;11 RNA-seq datasets are available from GEO under accession number GSE76931.RNA-seq reads were mapped with STAR against the human genome (hg19).Differential expression analysis and Visualization were carried out with raw counts using DESeq2 (7) and iDEP (8).
Nuclei were centrifuged at 500 × g for 10 min, 4°C.Nuclei extract were then incubated with Nextera Tn5 Transposase, 2× TD buffer, and nuclease free water at 37°C for 30 min with gentle mixing.After DNA purification with the MinElute PCR Purification Kit (Qiagen), PCR was performed to amplify the library for additional cycles distinctively according to a quantitative PCR reaction for optimum cycles.The final libraries were sequenced by Novogene.Bulk RS4;11 ATAC-seq datasets are available through EMBL-EBI ArrayExpress under accession number E-MTAB-8676.Adapter sequences were trimmed and reads were mapped to Hg19 using Bowtie2 Pan et al. 11 (10).ATAC-seq peak calling was performed with Genrich (https://github.com/jsh58/Genrich)with default setting.

Statistics
Data are presented as mean ± standard deviation, unless otherwise indicated.Student t test (unpaired, 2-tailed) was used to assess significance between 2 groups.Survival curves were analyzed by log-rank (Mantel-Cox) test.P < .05were considered significant.Generation of plots and statistical analyses were performed using Prism GraphPad version 8.

Study Approval
Human samples.Fresh human umbilical cord blood (hUCB) was obtained from Stanford

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C) Expression of CD10 and BCL2 from normal and tumor tissues.(D) Histogram of BCL2 expression analyzed in normal and tumor tissues.Pan et al.

Figure S3 :
Figure S3: Chromatin accessibility and transcriptional landscapes of gene-edited HSPCs display leukemic features.
Hospital via the Binns Program for Cord Blood Research under informed consent.ALL patient samples were obtained from the Stanford Medical Center and Lucile Packard Children's Hospital with informed consent and institutional review board approval.Animal studies.All experiments using mice were performed with the approval of, and in accordance with, the Stanford University Administrative Panel on Laboratory Animal Care.