Stable Silencing of RUNX1/ETO Induces Expression of a Shortened PU.1 Variant in t(8;21) Kasumi Cells

Abstract

The t(8;21)(q22;q22) found in about 8% of de novo AML fuses the Runx1 (or AML-1) gene to the ETO (or MTG8) gene. The leukemia-initiating chimeric RUNX1/ETO fusion protein is believed to repress transcription of RUNX1 target genes by ETO-mediated recruitment of transcriptional repressors. Although several studies analyzing short-term depletion of RUNX1/ETO reveal both widespread epigenetic reprogramming and altered protein expression of several signaling molecules depending on RUNX1/ETO (e.g. Ptasinska et al. 2012, Spirin et al. 2014) the molecular mechanisms involved are still not completely understood. We analyzed RUNX1/ETO dependent expression of the myeloid transcription factor PU.1 in t(8;21) Kasumi cells after lentiviral transduction with control or RUNX1/ETO specific shRNAs (shCtrl and shRE). PU.1 is a well known target of RUNX1/ETO being inactivated by the chimeric protein. PU.1 expression was analyzed by immunoblotting of cellular lysates from cells transduced to > 99%. RUNX1/ETO is depleted upon shRE expression, and shRE cells exhibit reduced proliferation, less apoptotic cell death and reduced clonal growth in limiting dilution assays as compared to controls. Around day 7 after transduction expression of the full length ~37 kD PU.1 protein is specifically reduced in shRE cells, and a shorter variant of about 22 kD is increasingly expressed starting at day 4. A 20.7 kD PU.1 variant can be predicted from an alternative transcript missing exon 1 encoding the start ATG for full length PU.1. The alternative transcript encodes an ATG in frame in exon 3 with identical 3´-terminal PU.1 sequences. We cloned several PU.1 cDNAs from Kasumi cells and identified and confirmed the alternative transcript missing exon 1. In vitro transcription/translation of cloned PU.1 transcripts generated a protein with C-terminal PU.1 epitopes of the predicted size. However, using PU.1 isoform-specific quantitative real-time RT-PCR we did not find differential changes in isoform-specific mRNA expression in shRE as compared to shCtrl cells with the full length variant always being most abundant. PU.1 expression can be modulated by an antisense RNA regulated by a shared cis-regulatory element (Ebralize et al. 2008) but its expression was similar in shRE and shCtrl cells as determined by quantitative RT-PCR. Since these data suggest that differential protein expression is not due to differential abundance of specific mRNAs we analyzed expression and phosphorylation status of eIF2-α. eIF2-α is involved in translation regulation during cellular stress response by redirecting ribosomes to alternative open reading frames (ORFs) in a phosphorylation dependent manner (for review Young and Wek 2016). eIF2-α phosphorylation increases at ~ 4 days after transduction in shRE but not in shCtrl cells. Interestingly, protein expression of several proteins such as FOXO3, STAT3, STAT5 and C/EBPα is reduced in shRE as compared to shCtrl cells without significant changes in the respective mRNA levels. Furthermore, expression of the RUNX1/ETO targets PU.1, C/EBPα and BCL2 initially increases before final reduction. These data suggest that RUNX1/ETO modulates mRNA translation in addition to its well-known transcriptional and epigenetic effects. Furthermore, RUNX1/ETO targets identified by loss-of function studies may characterize individual candidates as part of a more general response to translational regulation. DisclosuresNo relevant conflicts of interest to declare.