Identification of a Dynamic Core Transcriptional Network in t(8;21) AML Regulating Differentiation Block and Self-Renewal

One of the best-characterized chromosomal rearrangements found in AML is the t(8;21) translocation which accounts for approximately 10% of all AMLs and fuses the DNA-binding domain of the hematopoietic master regulator RUNX1 to the almost the entire ETO protein. The resulting RUNX1/ETO fusion protein lacks the transactivation domain of RUNX1 resulting in major differences in the biological activities of RUNX1 and RUNX1/ETO. RUNX1 normally recruits transcriptional activators and binds to DNA as a heterodimer with CBFb which is important for high-affinity DNA binding. The RUNX1/ETO fusion protein also interacts with CBFb but functions as a RUNX1/ETO tetramer, and like ETO itself, it also interacts with NCOR and SIN3A co-repressors. The leukemogenic fusion protein RUNX1/ETO forms a complex with other transcription factors and the binding of this complex to RUNX1 target genes causes a block in myeloid differentiation.

Here we investigate the dynamic changes in global transcription factor binding patterns that occur immediately following depletion of RUNX1/ETO. To that end, we combined digital footprinting, ChIP-sequencing for multiple factors and transcriptome analysis to identify the core transcriptional network of t(8;21) AML cells, and then characterized changes in these networks upon RUNX1/ETO knockdown. These analyses revealed a dynamic equilibrium between RUNX1/ETO and RUNX1 complexes competing for the same genomic sites. We show by sequential ChIP that both complexes have similar transcription factor compositions, but differ in their preference for the recruitment of co-activators and co-repressors. Using a novel digital DNaseI footprinting approach we show that both t(8;21)-positive cell lines and patient-derived primary AML cells share the same pattern of binding site occupancy. Within this core transcriptional network, RUNX1/ETO-bound loci are predominantly associated with gene repression. Furthermore, loss of RUNX1/ETO establishes a new differentiation-associated transcriptional network dominated by de novo binding of C/EBPa resulting from the up-regulation of CEBPA gene expression. Our results demonstrate that the block in myeloid differentiation in t(8;21) is caused by the dynamic interference of RUNX1/ETO with cis-regulatory elements destined to bind alternate factor assemblies during myeloid differentiation including both RUNX1 and C/EBPa.