Transcription and methylation analysis of preleukemic promyelocytes indicate a dual role for PML/RARA in leukemia initiation

Abstract

© F e r r a t a S t o r t i F o u n d a t i o nPML/RARA preleukemic myeloid precursors eventually give rise to leukemic promyelocytes. Methods Murine model and bone marrow harvestMRP8-PML/RARA transgenic mice have been described elsewhere. 3 Mice were bred and maintained at the University of California, San Francisco under standard conditions. For each individual sample, bones from one male and one female were pooled, thoroughly cleaned, ground and filtered. Cells were collected after centrifugation on Histopaque-1119 (Sigma #11191) and CKIT enrichment . Staining and flow cytometryCells were stained with a lymphocyte/erythrocyte/stem cell cocktail followed by secondary antibody. After blocking (Sigma #I4131), the suspension was stained with the specific cocktail. Live cells were double-sorted and purity after sorting was assessed. Compensation tubes were prepared by staining FVB/n spleen with fluorophore-conjugated CD45R/B220 antibodies. RNA isolation and microarray analysisCells (25,000-50,000) were double-sorted into Trizol (Life Technologies #15596). Following phenol/chloroform extraction, RNA was purified on PicoPure columns (Life Technologies #KIT0204). Sample preparation, labeling, and array hybridizations were performed according to standard protocols from the University of California, San Francisco Shared Microarray Core Facilities (http://www.arrays.ucsf.edu/protocols/) and Agilent Technologies. The microarray was run on a murine Agilent platform comprising 41,174 probes, including 28,283 targeting protein-coding genes (27,285 known + 998 predicted), and 1,484 targeting non-coding RNA (950 known + 534 predicted). The dataset was quantile normalized 22 without background subtraction. Differential expression analysis using moderated t statistic with Benjamini-Hochberg false discovery rate (adjusted P value) correction was performed using the limma R/Bioconductor package. 23 Below, biological replicates refer to samples harvested from different litters, over the course of several months, while a technical replicate refers to the same biological sample run twice on the array. For the preleukemic versus normal study, data are representative of three or four biological replicates/population and are available on GEO (GSE54474). For the leukemic versus preleukemic study, data are representative of two biological replicates for the preleukemic group (plus 1 technical duplicate) and three biological replicates for the leukemic group (plus 1 biological and 1 technical duplicate) and are also available on GEO (GSE59431).Genomic DNA isolation and genome-wide methylation analysis by enhanced reduced representation bisulfite sequencing Cells (25,000) were double-sorted and genomic DNA was extracted using the PureGene kit (Qiagen). DNA (25 ng) was used to perform the enhanced reduced representation bisulfite sequencing (ERRBS) assay as previously described 24 and sequenced on a HiSeq2000 Illumina sequencer. Reads were aligned against a bisulfite-converted mm9 genome using Bowtie. 25 Downstream analysis was pe...

p53 as an Effector or Inhibitor of Therapy Response

“…PML NBs also modulate E2F signaling (Vernier and Ferbeyre 2014). Thus, although the first models of oncogenic transformation by PML/RARA stressed the importance of the differentiation arrest through transcriptional silencing, recent studies have highlighted the central role of PML/RARA-mediated disruption of NBs and resulting proliferation boost (Occhionorelli et al 2011;Gaillard et al 2015).…”

Identification of IRF8 as a potent tumor suppressor in murine acute promyelocytic leukemia

“…Acute promyelocytic leukemia (APL) is marked by the accumulation of promyelocytes due to a blockade of differentiation coupled with unlimited expansion. Approximately 97% of patients with APL express the promyelocytic leukemia/retinoic acid receptor a (PML/RARA) fusion protein as a result of t(15;17) (q22;q11.2), and although the paradigm has long suggested that PML/RARA is a potent repressor of key myeloid maturation genes, we recently challenged this model, 1 reopening fundamental questions as to the precise contribution of the fusion protein to leukemic transformation.…”

Co-operative leukemogenesis in acute myeloid leukemia and acute promyelocytic leukemia reveals C/EBP as a common target of TRIB1 and PML/RARA

“…18 By dimerizing and associating with an altered set of co-factors the DNA binding specificity and repressive ability of the fusion protein is expanded, 19 a characteristic that is believed to play a key role in the leukemogenic process. PML/RARA has been shown to interact with key transcription factors of the granulocytic differentiation program, such as PU.1 20 and C/EBPα downregulation has been observed in PML/RARA expressing cells, 21,22 both events possibly participating in the transformation process. Interestingly, TRIB1 has been shown to co-localize with RARA/RXR leading to negative regulation of the transcriptional activity of the complex, 23 although the precise mechanism mediating this inhibition is still unclear.…”