Direct recruitment of BCOR by AF9 forms a fuzzy complex required for MLL-AF9 leukemogenesis
Schmidt, Charles, Biophysics - School of Medicine, University of Virginia
Bushweller, John, Department of Molecular Phys and Biological Physics, University of Virginia
The Mixed Lineage Leukemia gene (MLL) is a target of chromosomal translocations leading to acute leukemias characterized by poor prognoses and early relapse. The mechanism of MLL leukemogenesis involves constitutive expression of MLL targets, and particularly HOX genes. Though a number of MLL translocation partners have been identified, the three most common (AF4, ENL, and AF9) account for roughly two-thirds of cases. In MLL-AF9 rearrangements, the AF9 C-terminal ANC1 homology domain (AHD) becomes fused in frame to the N-terminal portion of MLL. The AF9 AHD is intrinsically disordered and has the ability to interact with multiple partners, four of which have been identified. These include AF4 and DOT1L, whose importance in the activation and maintenance of MLL-fusion targets is well-documented. The others, CBX8 and BCOR, are traditionally associated with transcriptional repression. The role of CBX8, a component of polycomb repressive complex 1 (PRC1), in these leukemias has been the subject of several studies. The role of the direct BCOR-AF9 interaction remains uninvestigated.
We present the NMR solution structure of the BCOR-AF9 complex. In addition, we show that the minimal AF9-binding motif of BCOR interacts with AF9 only weakly, while an extended BCOR peptide has a much higher affinity for AF9. Based on this structural and binding information, we have designed a point mutation to selectively disrupt BCOR binding to AF9. This mutation, unlike those which affect the other binding partners, has no effect on colony formation, but instead affects proliferation and prevents development of leukemia in vivo. RNAseq analysis demonstrates a change in genes associated with hematopoiesis, cell adhesion, and the ERK cascade, which regulates processes including proliferation, differentiation, and survival.
The C-terminal extension of the BCOR peptide is unassignable in the NMR experiments used for structural studies. We show that this region likely forms an alpha helix that transiently interacts with AF9 to modulate binding affinity. Interruption of the putative helix by a proline mutation recapitulates binding of the shorter peptide in NMR spectra. The structural disorder inherent in the BCOR C-terminus and its requirement for tight AF9 binding makes BCOR-AF9 a textbook example of a fuzzy complex.
PHD (Doctor of Philosophy)