Structural and Functional Studies Of The Intrinsically Disordered Protein AF9 In MLL-AF9 Leukemia

Mixed lineage leukemias are characterized by the creation of a chimeric fusion protein where MLL is fused in frame to over 60 different fusion partners, leading to the disruption of HOX gene regulation. AF9 is one of the most common MLL fusion partners, and MLL-AF9 leukemia is acute and aggressive with a poor overall prognosis. The mechanism by which AF9 regulates normal transcription and contributes to dysregulated transcription is poorly understood. We have shown that the C-terminal domain of AF9 binds to four different proteins, two of which (Dot1L – an H3K79 methyltransferase and AF4 – which recruits P-TEFb), are transcriptional activators, whereas the other two (CBX8 – which is a part of the PRC1 repressive complex and BCOR- BCL6 corepressor), are generally transcriptional repressors, suggesting that AF9 acts as a protein signaling hub. We have previously shown that the C-terminal domain of AF9 is an intrinsically disordered protein (IDP) meaning that it is unstructured on its own but undergoes coupled folding and binding upon interacting with its binding partners¹. Our goal is to use structure-function studies to further understand AF9 protein interactions and to elucidate which of these interactions with AF9 are critical for MLL-AF9 leukemogenesis.

We have determined NMR solution structures of complexes of Dot1L and CBX8 with AF9. Both Dot1L and CBX8 complexes form mixed alpha-beta structures, similar to that of our previously solved AF4-AF9 complex structure where AF4, Dot1L, and CBX8 all have a consensus LXVXIXL sequence and form a ß-strand and an extended ß-turn. Intriguingly, Dot1L contains three separate binding motifs for AF9 interaction, including one repeat motif separated by seven amino acids. Our ¹⁵N-¹H HSQC NMR experiments show that each of these three binding sites on Dot1L adopts a similar fold with AF9 yet has vastly different binding affinities with AF9. We have created several structure-guided mutations on AF9 that differentially block interactions of its binding partners. A single charge reversal mutation on AF9 that blocks all protein interactions with AF9 was introduced into MLL-AF9 in vitro colony forming assays, and was able to abrogate the serial replating capacity of MLL-AF9. Other mutations that selectively block the interaction of various proteins with AF9, show differential effects in colony forming assays and HOX gene expression suggesting the importance of certain AF9 protein interactions in MLL-AF9 leukemogenesis.

1. Leach, B. I. et al. Leukemia fusion target AF9 is an intrinsically disordered transcriptional regulator that recruits multiple partners via coupled folding and binding. Structure21, 176-183, doi:10.1016/j.str.2012.11.011 (2013).