The Role of the Intrinsically Disordered and Multifunctional AF9 C-Terminal Domain in MLL-AF9 Leukemia,

Abstract 3464

Mixed Lineage Leukemia (MLL) fusion proteins disrupt HOX gene regulation through recruitment of transcriptional elongation factors, leading to acute leukemia. AF9 is one of the most common MLL fusion partners and has roles in normal regulation of HOX genes. AF9 has also been shown to interact with multiple transcriptional regulators, including AF4 family proteins which recruit and activate P-TEFb, suggesting a pivotal role in regulation of transcriptional elongation at HOX loci. The mechanism by which AF9 regulates normal transcription and contributes to dysregulated transcription in MLL-AF9 leukemia is poorly understood. We have recently shown that the AF9 ANC1 Homology Domain (AHD), which is retained in MLL fusion proteins, is intrinsically disordered, i.e. it is unstructured on its own, and recruits AF4 family members by coupled folding and binding. In order to further understand this behavior, we have determined the solution structure of the AF9 AHD in complex with AF4 and identified a point mutation which selectively disrupts the AF4-AF9 interaction. Introduction of this mutation into MLL-AF9 results in loss of HOXA9 upregulation and loss of transformation of hematopoietic progenitor cells, establishing the importance of this interaction for MLL-AF9 leukemia. We propose that AF9 functions as a signaling hub which regulates transcriptional elongation by alternative recruitment of transcriptional regulators. Indeed, the AF9 ANC1 homology domain (AHD) has been shown to also bind the H3K79 methyltransferase Dot1L, human polycomb 3 (hPC3), and the BCL6 corepressor, necessitating a carefully orchestrated choreography of binding partners to regulate transcription. Intrinsic disorder may facilitate dynamic exchange between binding partners to mediate rapid changes of HOX gene expression in both normal tissues, as required for specific differentiation programs, and in leukemia. Our results also validate the AF9-AF4 interaction as a potential target for therapeutic development.