Defining the role of RNA binding by the MLL1 (KMT2A) complex in leukemia

About This Grant

Project Summary/Abstract This proposal is focused on the molecular mechanisms at the nexus of the MLL1 (KMT2A) histone modification complex, whose catalytic subunit is the frequently mutated to cause acute myeloid leukemia, and HOTTIP, a long noncoding RNA implicated as a pan-cancer oncogene. A widely accepted model holds that a small set of long noncoding RNAs (including HOTTIP) specifically bind the WDR5 subunit of the MLL1 complex to recruit it to MLL1-rearragend (MLL1-r) target genes, where it acts to install the transcriptionally activating H3K4me3 mark to drive leukemogenesis. The sine qua non of this model is that a specific lncRNA-WDR5 binding interface recruits the MLL1 complex, although this specificity has not been rigorously tested. Instead of specificity, we have accrued extremely strong evidence that WDR5 is promiscuous in its RNA binding, both in vitro, where its Kd scales with the length of the RNA, not its sequence or structure, and in living cells, where RNA binds WDR5 in as a function of abundance in the nucleus and transcript length. What then might RNA binding to the WDR5 subunit of the MLL1 core complex subunit actually do? Our preliminary data provide two possible and non-mutually exclusive hypotheses for the function of RNA interactions with the MLL1 complex that this grant seeks to test. 1.) We have found non-specific RNA binding is a potent inhibitor of the MLL1 complex’s histone methyltransferase activity. Here we endeavor to understand how this novel regulation mechanism may work in leukemia etiology and maintenance. 2.) We suspect a different subunit in the complex, RBBP5, may account for specific recruitment by HOTTIP (and potentially other RNA), as we have mapped a 24-mer motif in HOTTIP that accounts for all of this subunit’s affinity and specificity for this RNA. The overarching goals of this grant are to define the detailed mechanisms of the RNA interfaces within the MLL1 complexe and their functional consequences in leukemia. This grant leverages human cell line and mouse models of MLL1-r leukemia to specifically test how specific disruption of each of these RNA-binding interfaces, both by precision genetic approaches and novel inhibitors, impacts leukemogenesis and maintenance, as well as the molecular underpinnings thereof. The proposed research will examine how multivalent RNA interactions with distinct subunit interfaces act together in the context of the full MLL1 complex, developing new tools to map RNAs to specific complexes that have subunits in common. The science proposed in this grant, founded on our rigorous biochemical and cell-based preliminary data, will represent a paradigm shift in the field of MLL1 complex recruitment and establish the druggability of these new RNA-binding interfaces with tool compounds. Mouse models of leukemia are essential to examine whether insights gained from cultured human cancer cell lines are applicable in a complex, physiologically relevant in vivo context, and to rigorously attribute the effects on leukemia induction in the absence of other confounding genetic alterations that can occur in cell lines.