ABSTRACT
AF9 (MLLT3) and its paralog ENL(MLLT1) are members of the YEATS family of proteins with important role in transcriptional and epigenetic regulatory complexes. These proteins are two common MLL fusion partners in MLL-rearranged leukemias. The oncofusion proteins MLL-AF9/ENL recruit multiple binding partners, including the histone methyltransferase DOT1L, leading to aberrant transcriptional activation and enhancing the expression of a characteristic set of genes that drive leukemogenesis. The interaction between AF9 and DOT1L is mediated by an intrinsically disordered C-terminal ANC1 homology domain (AHD) in AF9, which undergoes folding upon binding of DOT1L and other partner proteins. We have recently reported peptidomimetics that disrupt the recruitment of DOT1L by AF9 and ENL, providing a proof-of-concept for targeting AHD and assessing its druggability. Intrinsically disordered proteins, such as AF9 AHD, are difficult to study and characterize experimentally on a structural level. In this study, we present a successful protein engineering strategy to facilitate structural investigation of the intrinsically disordered AF9 AHD domain in complex with peptidomimetic inhibitors by using maltose binding protein (MBP) as a crystallization chaperone connected with linkers of varying flexibility and length. The strategic incorporation of disulfide bonds provided diffraction-quality crystals of the two disulfide-bridged MBP-AF9 AHD fusion proteins in complex with the peptidomimetics. These successfully determined first series of 2.1-2.6 Å crystal complex structures provide high-resolution insights into the interactions between AHD and its inhibitors, shedding light on the role of AHD in recruiting various binding partner proteins. We show that the overall complex structures closely resemble the reported NMR structure of AF9 AHD/DOT1L with notable difference in the conformation of the ß-hairpin region, stabilized through conserved hydrogen bonds network. These first series of AF9 AHD/peptidomimetics complex structures are providing insights of the protein-inhibitor interactions and will facilitate further development of novel inhibitors targeting the AF9/ENL AHD domain.
Subject(s)
Peptidomimetics , Peptidomimetics/chemistry , Peptidomimetics/metabolism , Humans , Intrinsically Disordered Proteins/chemistry , Intrinsically Disordered Proteins/metabolism , Intrinsically Disordered Proteins/genetics , Intrinsically Disordered Proteins/antagonists & inhibitors , Histone-Lysine N-Methyltransferase/chemistry , Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Histone-Lysine N-Methyltransferase/metabolism , Histone-Lysine N-Methyltransferase/genetics , Models, Molecular , Oncogene Proteins, Fusion/chemistry , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Crystallography, X-Ray , Protein Domains , Myeloid-Lymphoid Leukemia Protein/chemistry , Myeloid-Lymphoid Leukemia Protein/metabolism , Myeloid-Lymphoid Leukemia Protein/genetics , Myeloid-Lymphoid Leukemia Protein/antagonists & inhibitorsABSTRACT
MLL (KMT2a) translocations are found in ~10% of acute leukemia patients, giving rise to oncogenic MLL-fusion proteins. A common MLL translocation partner is ENL and associated with a poor prognosis in t(11;19) patients. ENL contains a highly conserved N-terminal YEATS domain that binds acetylated histones and interacts with the PAF1c, an epigenetic regulator protein complex essential for MLL-fusion leukemogenesis. Recently, wild-type ENL, and specifically the YEATS domain, was shown to be essential for leukemic cell growth. However, the inclusion and importance of the YEATS domain in MLL-ENL-mediated leukemogenesis remains unexplored. We found the YEATS domain is retained in 84.1% of MLL-ENL patients and crucial for MLL-ENL-mediated leukemogenesis in mouse models. Mechanistically, deletion of the YEATS domain impaired MLL-ENL fusion protein binding and decreased expression of pro-leukemic genes like Eya1 and Meis1. Point mutations that disrupt YEATS domain binding to acetylated histones decreased stem cell frequency and increased MLL-ENL-mediated leukemia latency. Therapeutically, YEATS containing MLL-ENL leukemic cells display increased sensitivity to the YEATS inhibitor SGC-iMLLT compared to control AML cells. Our results demonstrate that the YEATS domain is important for MLL-ENL fusion protein-mediated leukemogenesis and exposes an "Achilles heel" that may be therapeutically targeted for treating t(11;19) patients.
Subject(s)
Histones , Leukemia, Myeloid, Acute , Mice , Animals , Histones/metabolism , Myeloid-Lymphoid Leukemia Protein/genetics , Myeloid-Lymphoid Leukemia Protein/metabolism , Leukemia, Myeloid, Acute/genetics , Translocation, Genetic , Epigenesis, Genetic , Stem Cells/metabolism , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolismABSTRACT
Based on past research findings with college students, we tested whether perceived burdensomeness and thwarted belongingness represent important mediators of the association between future orientation and suicide risk (namely, depression and suicidality) in primary care patients. We conducted a multiple mediator test in a sample of 97 primary care patients. Our results indicated that perceived burdensomeness and thwarted belongingness accounted for the negative associations present between future orientation and measures of suicide risk. These findings provide evidence for perceived burdensomeness and thwarted belongingness as mechanisms accounting for the association between future orientation and suicide risk in primary care patients.
