Blast cells surviving acute myeloid leukemia induction therapy are in cycle with a signature of FOXM1 activity

Disease relapse remains common following treatment of acute myeloid leukemia (AML) and is due to chemoresistance of leukemia cells with disease repopulating potential. To date, attempts to define the characteristics of in vivo resistant blasts have focused on comparisons between leukemic cells at presentation and relapse. However, further treatment responses are often seen following relapse, suggesting that most blasts remain chemosensitive. Here we characterize in vivo chemoresistant blasts by studying the transcriptional and genetic features of blasts from before and after induction chemotherapy using paired samples from 6 patients with primary refractory AML. Primary human AML were from the Manchester Cancer Research Centre Tissue Biobank (approved by the South Manchester Research Ethics Committee). Their use was authorized by the Tissue Biobank’s scientific sub-committee, with the informed consent of donors. Blasts were flow sorted according to either a CD45low/intCD34+ (n=5) or CD45low/intCD117+ (n=1) immunophenotype. Total RNA was extracted from 10^3-10^5 sorted blasts using QIAshredder spin columns and an RNeasy® Plus Micro kit (Qiagen). Prior to sequencing RNA integrity was checked using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Total RNA yield from the sorted populations ranged from 3.5-475ng. To ensure consistency the sample with the lowest yield was used to define the input (3.5ng) for amplification of all samples. Amplification was performed using a SMARTer Stranded Total RNA-Seq Pico input kit (Clontech, Mountain View, CA). Sequencing was performed using a NextSeq desktop sequencing system (Illumina). A single run (400M reads) of 151bp paired-end sequencing produced a mean of 31.4M reads per sample (range 29.3-33.5M).