Elucidating the Proteomic Interactions of the Mixed Lineage Leukemia Mutant MLL-PTD

Abstract

Acute myeloid leukemia (AML) is a genetically heterogeneous disease with a poor prognosis. A mutation in the mixed lineage leukemia 1 (MLL1) gene that results in partial tandem duplication (PTD) of the N-terminal region is found in 5-10% of cytogenetically normal AML. MLL-PTD is a gain of function mutation and causes increased self-renewal and proliferation of hematopoietic stem and progenitor cells. However, the mechanism through which MLL-PTD induces leukemic transformation is currently unknown. We aimed to identify cofactors that differentially interact with MLL-PTD versus MLL-WT such that inhibitors of these interactions could be tested as a therapeutic strategy for AML. In order to facilitate a comparative study between the wildtype and mutant MLL, we transfected HEK293T cells with FLAG and HA-tagged MLL-WT or MLL-PTD. Expression of the tagged MLL was validated by western blot and RT-qPCR. We performed mass spectrometry analysis on samples from both a FLAG and immunoprecipitation experiment and identified candidate proteins for therapeutic intervention. Inhibitors of two of these interactions, the lysine acetyltransferase KAT2Aand the oncogenic co-factor MEN1, selectively kill leukemic cells with MLL rearrangements. The MLL-PTD cell line EOL-1 is the most sensitive to these small molecule inhibitors. Finally, we show, for the first time, evidence suggesting MLL-PTD retains its ability to undergo proper cleavage in the cytoplasm and interact with WRAD complex members. Therefore, we have uncovered some of the key, previously unknown, protein partners of the leukemogenic protein MLL-PTD.