- Despite the genetic heterogeneity of the leukemias studied, transcriptional analysis found concerted changes in gene expression in resistant blasts. - The refractory gene signature was highly enriched with targets of the transcription factor FOXM1. - To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.. - The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.. - Full list of author information is available at the end of the article. - To identify features characteristic of chemoresistant AML cells we developed a protocol to study primary re- fractory disease, thereby facilitating comparison in the same patient of the transcriptome and mutational status of AML blast cells at presentation with those recovered immediately upon induction failure. - The antibody panel (Table S1) was adapted from Freeman et al. - CD38 expres- sion cut-offs for identifying LSCs and progenitors were defined using the upper limit of the red cell fraction (CD38 low ) and the median of the red cell fraction (CD38. - FOXM1 transcrip- tional targets were from ChIPseq experiments performed by Chen et al. - In keeping with the concept that different genetic sub-clones exhibit differ- ent levels of chemosensitivity, molecular genetic analysis revealed the presence of early clonal selection following the first cycle of chemotherapy, with the emergence of new subclones (patients 64, 285, and 349), the loss of pre-treatment subclones (patients 285 and 494) or a re- duction in size of the dominant clone (patients 64 and 285. - We next performed RNA sequencing of the paired pre- and post-chemotherapy CD45 low/int CD34 + or CD45 low/int CD117 + sorted blast cell populations. - As ex- pected, given the genetic heterogeneity of the analysed samples, principal component analysis (PCA) of 7123 expressed protein-coding genes revealed significant tran- scriptional differences between the six leukemias (Fig. - Pre-chemotherapy Post-chemotherapy. - FISH post- chemotherapy. - Evaluation of the pattern of expression of the Molecular Signatures Database Hallmark Gene Set collection, each of which conveys a specific biological state or process and displays coherent expression [22], demonstrated strong up regulation of “E2F target” genes, “G2M cell. - Also shown is the frequency of the indicated leukemia associated immunophenotype (LAIP) for each sample. - Indeed cross-species comparison of the gene set associated with LSC maintenance in murine MLL-AF9 AML cells showed significantly higher expression in post- versus pre-chemotherapy human AML cells, and vice versa for genes whose expression is anti-correlated with leukemia stem cell activity (Table S4 and Fig. - Intriguingly, leading edge analysis of the genes driving enriched expression of the LSC main- tenance signature in post- versus pre-chemotherapy blast cells revealed the presence of MYB, HMGB3 (High Mobility Group Box 3) and CBX5 (Chromo- box 5) the three genes which, when co-expressed, suf- fice for Hox/Meis-independent immortalization of myeloid progenitor cells (Fig. - Next, to identify transcription factor regulators of the refractory blast gene expression signature, which may serve as candidate therapeutic targets, we performed an in silico analysis using Enrichr [7, 28], a bioinformatics resource which facilitates analysis of experimentally de- rived gene sets. - several of the most highly correlated genes were regula- tors or markers of cell cycle progression (CCNB2, MKI67, TPX2 &. - Of the 270 high confidence FOXM1 targets in U2OS cells, 206 occupied positions ± 1kB from a transcription start site. - B Representative flow cytometry scatter plots show the relative size of the immunophenotypic leukemia stem and progenitor cell populations pre- and post-chemotherapy. - The combined score is the product of the logarithm of the adjusted p -value and the z-score (20). - E Expression of the indicated genes in pre- and post-chemotherapy flow sorted AML blast populations ( n = 6). - G Bar charts show expression of the indicated genes in all samples. - These samples enabled us to perform de- tailed genetic and transcriptional characterisation of the blasts that survived chemotherapy.. - 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