FLT3N676K drives acute myeloid leukemia in a xenograft model of KMT2A-MLLT3 leukemogenesis

Activating signaling mutations are common in acute leukemia with KMT2A (previously MLL) rearrangements (KMT2A-R) [1]. When defining the genetic landscape of infant KMT2A-R acute lymphoblastic leukemia (ALL), we identified a novel FLT3 mutation in both infant ALL and non-infant acute myeloid leukemia (AML) [1]. FLT3 was the most common FLT3 mutation in our cohort and we recently showed that it cooperates with KMT2A-MLLT3 in a syngeneic mouse model [1, 2]. To study the ability of FLT3 to cooperate with KMT2AMLLT3 in human leukemogenesis, we transduced human * Anna K. Hagström-Andersson Anna.Hagstrom@med.lu.se

Retroviral supernatants were produced according to standard protocols using transient transfection of HEK293T cells and viral containing medium was harvested 36h later and stored at -80˚C.

Isolation and retroviral transduction of CD34 + CB cells
Collection and use of human CB was performed after informed and written consent in accordance with the Declaration of Helsinki and was approved by the Lund/Malmö Ethical Committee. Mononuclear cells were isolated through density-gradient centrifugation using Lymphoprep (Axis-Shield, Oslo, Norway) and CD34 + cells were enriched by magneticactivated cell sorting (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer's instructions. Cells were pre-stimulated for 48h in DMEM (Thermo Scientific, Logan, UT, USA) supplemented with 100 units/mL Penicillin and 100 g/ml Streptomycin (Thermo Scientific), 10% fetal bovine serum (Thermo Scientific), 100ng/ml SCF, 50ng/ml FLT3-ligand, and 50ng/ml hTPO (all cytokines were purchased from Peprotech, Rocky Hill, NJ, USA). Retroviral co-transduction was performed as previously described 1 .

Validation of somatic mutations
Validation of mutations was performed in matched primary, secondary recipient, and constitutional DNA, using PCR amplicon deep sequencing. PCR was used to amplify the region covering KRAS G13D using the Platinum Taq DNA Polymerase kit (Life Technologies) according to the manufacturer's instructions and products were analyzed using gel electrophoresis (primer sequences are available upon request). PCR amplicons were purified using AMPure XP beads (Beckman Coulter Inc., Brea, CA) and prepared for sequencing using the Nextera XT DNA Sample Preparation Kit and Index Kit (Illumina). 2 × 150 bp paired-end sequencing was performed using the Illumina NextSeq 500 (Illumina). Adapters were trimmed using Trimmomatic (0.32) and the trimmed paired-end reads were aligned to hg19 using BWA (0.7.15) 2 . Variant calling was performed using samtools mpileup (1.3.1) 6 and varscan (2.4.1) 7 .
RNA extraction, RNA sequencing, and analysis of RNA sequencing data RNA extraction and library preparation was performed as previously described 1 and 2x80bp paired-end sequencing was performed on Illumina NextSeq 500 (Illumina). Paired-end reads from RNA sequencing were aligned to the human genome hg19 using STAR 8 . Transcript expression levels were estimated as fragments per kilobase of transcript per million mapped fragments (FPKM) and gene FPKM was calculated and normalized using Cufflink 9 . An FPKM cutoff of ≥0.5 was used to define "expressed" genes and data was log2 transformed. When expression data was combined with data sets generated elsewhere 10,11 , quantile normalization was performed using R (3.4.2) (RStudio, Inc., Boston, MA, USA) and the limma package (3.34.9) 12 . Visualization and statistical analysis of the RNA sequencing data was performed using Qlucore Omics Explorer 3.4 (Qlucore, Lund, Sweden). Gene set enrichment analyses were performed using GSEA v2.2.0 13,14 with pre-ranked gene lists.

Statistical analysis
Differences between groups were assessed by Mann-Whitney U test. Correlation was assessed using Spearman's rank correlation coefficient. Statistical analysis of survival curves was performed using Mantel-Cox log-rank test. All graphs show mean with all individual data points. All analyses were performed with Prism software version 7.0a (GraphPad software).