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Large-scale chromosomal translocations are frequent oncogenic drivers in acute myeloid leukemia (AML). These translocations often occur in critical transcriptional/epigenetic regulators and contribute to malignant cell growth through alteration of normal gene expression. Despite this knowledge, the specific gene expression alterations that contribute to the development of leukemia remain incompletely understood. Here, through characterization of transcriptional regulation by the RUNX1-ETO fusion protein, we have identified Ras-association domain family member 2 (RASSF2) as a critical gene that is aberrantly transcriptionally repressed in t(8;21)-associated AML. Re-expression of RASSF2 specifically inhibits t(8;21) AML development in multiple models. Through biochemical and functional studies, we demonstrate RASSF2-mediated functions to be dependent on interaction with Hippo kinases, MST1 and MST2, but independent of canonical Hippo pathway signaling. Using proximity-based biotin labeling we define the RASSF2-proximal proteome in leukemia cells and reveal association with Rac GTPase-related proteins, including an interaction with the guanine nucleotide exchange factor, DOCK2. Importantly, RASSF2 knockdown impairs Rac GTPase activation, and RASSF2 expression is broadly correlated with Rac-mediated signal transduction in AML patients. Together, these data reveal a previously unappreciated mechanistic link between RASSF2, Hippo kinases, and Rac activity with potentially broad functional consequences in leukemia.

The 8;21 translocation is the most common chromosomal aberration occurring in acute myeloid leukemia (AML). This translocation causes expression of the RUNX1-ETO (AML1-ETO) fusion protein, which cooperates with additional mutations in leukemia development. We report here that interferons (IFNs) and IFN-stimulated genes are a group of genes consistently up-regulated by RUNX1-ETO in both human and murine models. RUNX1-ETO-induced up-regulation of IFN-stimulated genes occurs primarily via type I IFN signaling with a requirement for the IFNAR complex. Addition of exogenous IFN in vitro significantly reduces the increase in self-renewal potential induced by both RUNX1-ETO and its leukemogenic splicing isoform RUNX1-ETO9a. Finally, loss of type I IFN signaling via knockout of Ifnar1 significantly accelerates leukemogenesis in a t(8;21) murine model. This demonstrates the role of increased IFN signaling as an important factor inhibiting t(8;21) fusion protein function and leukemia development and supports the use of type I IFNs in the treatment of AML.

AML1-ETO (RUNX1-ETO) fusion proteins are generated by the 8;21 translocation, commonly found in acute myeloid leukemia, which fuses the AML1 (RUNX1) and ETO (MTG8, RUNX1T1) genes. Previous studies have shown that AML1-ETO interferes with AML1 function but requires additional cooperating mutations to induce leukemia development. In mouse models, AML1-ETO forms lacking the C-terminus have been shown to have greatly enhanced leukemogenic potential. Here, we investigate the role of 2 AML1-ETO C-terminal-interacting proteins, N-CoR, a transcriptional corepressor, and SON, a splicing/transcription factor required for cell cycle progression, in AML1-ETO-induced leukemia development. AML1-ETO-W692A loses N-CoR binding at NHR4, displays attenuated transcriptional repression ability and decreased cellular dysregulation, and promotes leukemia in vivo. These results support the importance of the degree of dysregulation by AML1-ETO in cellular transformation and demonstrate that AML1-ETO-W692A can be used as an effective experimental model for determining which factors compromise the leukemogenic potential of AML1-ETO.