Abstract

AML1/RUNX1 and CBFb encode two critical transcription factors essential for the generation of hematopoietic stem cells (HSC). In acute myeloid leukemia, where leukemic stem cells (LSCs) have been functionally identified, AML1 and CBFb also represent the most commonly mutated targets. While animal models indicate that AML1 fusions per se are not sufficient to induce full-blown leukemia, they enhance self-renewal and expand targeted HSC and early progenitors, a property also reported for other oncogenic transcription factors involved in acute leukemia. Although attempts had been made to identify the critical domains required for AML-ETO (AE) mediated transformation, conflicting results were presented from most of these studies using exploited wellestablished cell lines, which suffer from the pitfall of carrying irrelevant genetic aberrants that may not reflect the normal biology of the disease. The only available structure/ function data on primary cells was limited to NHR2 of the ETO portion of the fusion, but it does not distinguish the functional contribution between homo-oligomerization and hetero-oligomerization. The lack of comprehensive structure/function data has not only significantly impeded the progress of understanding the biology of the disease, but also hinders the development of specific therapeutics. To this end, we performed extensive functional analysis to identify the key components essential for AE-mediated transformation of primary hematopoietic cells. In spite of the critical role of CBFb for wild type AML1 functions and its direct involvement in chromosomal translocation, we demonstrate that multiple AE single point mutants defective in CBFb interaction were still capable of transforming primary hematopoietic cells. Consistently, shRNA mediated knockdown of the endogenous expression of CBFb in primary cells did not compromise the transforming activity of AE, strongly suggesting a dispensable function of CBFb in AE mediated transformation. On the other hand, we demonstrate that NHR2 as the only domain in the ETO portion of the fusion is essential for transformation, but its heterooligomeric function including interaction with transcriptional repressor ETO family proteins is dispensable for the transforming activity. In contrast, synthetic FKBP homooligomerization modules could functionally replace NHR2, indicating that AE mediated transformation is critically dependent on homo-oligomeric property of the resultant fusion. Moreover, the transformation can also be abolished by a small molecule inhibitor that specifically dissociates homo-oligomerization. Together, these results not only identify the essential components and refine potential avenues for therapeutic targeting of AE oncogenic complexes, but also strongly endorse a common homo-oligomerization dependent mechanism shared by the most prevalent leukemia associated transcription factors.

Disclosures: No relevant conflicts of interest to declare.

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