Tumor Suppressor RASSF2 Is Downregulated By The RUNX1-ETO Fusion Protein In t(8;21)+ Acute Myeloid Leukemia

Abstract

IntroductionThe t(8;21) chromosomal translocation is one of the most common chromosomal translocations associated with acute myeloid leukemia (AML), present in greater than 10% of de novo AML cases. Most of these t(8;21) AML cases are classified as FAB subtype M2. This translocation results in the formation of a stable fusion protein made up of portions of the RUNX1 (aka AML1) and ETO (aka MTG8 and RUNX1T1) proteins. RUNX1 is a transcription factor that is essential for regulating the differentiation of hematopoietic cells, and the fusion protein retains its DNA-binding domain. Additionally, ETO contains four Nervy homology (NH) domains which facilitate a number of protein-protein interactions, notably with the NCOR2/SMRT co-repressor complex. The identification of individual genes or biological pathways which are specifically disrupted in the presence of RUNX1-ETO will provide further molecular insight into the pathogenesis of t(8;21)+ AML and lead to the possibility for improved treatment for these patients. Methods/ResultsWe analyzed publicly available gene expression microarray datasets (Oncomine, TCGA) to search for genes whose expression was significantly altered in the blood of t(8;21)+ AML patients as compared to non-t(8;21) FAB subtype M2 AML and to CD34+ cells in healthy controls. One such gene that was consistently significantly downregulated in t(8;21)+ patients was Ras-association domain family member 2 (RASSF2). RASSF2 is a putative tumor suppressor that is capable of mediating apoptosis (in a Ras dependent manner) through its interactions with the MST1/2 kinases and the cancer-specific apoptotic protein Par-4. RASSF2 has previously been shown to be frequently downregulated via hypermethylation in a wide variety of solid tumors, however little is known about its function in leukemia. Here we demonstrate that RASSF2 is a potentially interesting target for downregulation by the RUNX1-ETO fusion protein. Gene expression analysis by RT-qPCR in leukemia cell lines confirmed that RASSF2 is significantly downregulated in both Kasumi-1 and SKNO t(8;21)+ cell lines as compared to a similar non-t(8;21) HL-60 line. We found that exogenous expression of AML1-ETO in HL-60 leukemia cells induces a rapid downregulation of RASSF2, further supporting that it is a target of this leukemogenic fusion protein. Over-expression of RASSF2 in leukemia cells significantly inhibits their proliferative capability, indicating an important biological effect of RASSF2 in blood cells. Finally, over-expression of RASSF2 significantly inhibits the long-term self-renewal capability of RUNX1-ETO expressing hematopoietic cells as measured by their serial replating ability in a colony formation assay. DiscussionBased on the analysis of patient data and our own experiments it appears that RASSF2 is a direct target for downregulation by the AML1-ETO fusion protein. Due to its potential involvement as a mediator of apoptosis in important oncogenic signaling pathways RASSF2 is a strong candidate for further investigation in the context of t(8;21)+ AML pathogenesis. In particular, it will be interesting to continue to investigate the relationship between RASSF2 and apoptotic protein Par-4, as several lines of evidence suggest Par-4 to be therapeutically relevant due to its ability to selectively induce apoptosis in cancer cells. Disclosures:No relevant conflicts of interest to declare.