MLL Fusion Protein Driven AML Is Selectively Inhibited by Targeted Disruption of the MLL-PAFc Interaction

Abstract 56

Balanced chromosomal translocations of the MLL gene located on chromosome 11q23 result in the expression of a chimeric fusion proteins with enhanced transcriptional activity. The HOX genes and their co-factors, such as MEIS1 and PBX2, are critical downstream targets of MLL fusion proteins and essential for transformation. Previously we showed MLL fusion proteins are critically dependent on a direct interaction with the RNA Pol II Associated Factor complex (PAFc). PAFc is a protein complex important for the initiation, elongation and termination of transcription. It is also necessary for histone H2B K120 mono-ubiquitination through the direct recruitment of the BRE1/RAD6 E3 ubiquitin ligase complex. MLL fusion proteins make two direct contacts with the PAF1 and CTR9 subunits of the PAFc that are crucial for MLL fusion protein mediated transformation. Deletion of regions of MLL that interact with PAFc abrogates AML in mouse bone marrow transplantation assays. Here we tested the general requirement for PAFc in AML using a conditional knockout mouse model of one component of PAFc, Cdc73. These studies show that PAFc is necessary for growth of both E2A-HLF and MLL-AF9 transformed cells. Excision of Cdc73 leads to decreased expression of the MLL target genes Hoxa9 and Meis1, decreased colony formation and decreased proliferation of leukemic blasts and ultimately apoptosis. We then performed chromatin immunoprecipitation assays to assess the binding of PAFc and MLL to target loci with and without Cdc73. Excision of Cdc73 leads to a rapid decrease in association of PAFc as well as MLL fusion proteins and wild type MLL at target loci confirming that proper targeting of MLL fusion proteins requires PAFc. A decrease in H3K4me3 and H2Bub is also observed and consistent with a role of PAFc in the deposition of these epigenetic marks.

We then sought to disrupt the MLL-PAFc interaction through expression of a small 40 amino acid fragment of MLL that interacts with the PAF1 subunit of PAFc. As the MLL-PAFc interaction involves interactions between MLL and both CTR9 and PAF1, it was unknown whether targeting one interaction site would be sufficient to disrupt transformation. Indeed, expression of the short fragment encompassing the pre-CxxC region of MLL acts as a dominant negative and disrupts the MLL-PAFc interaction, significantly decreasing Hox gene expression, colony formation and cell proliferation of MLL-AF9 transformed cells. Importantly, expression of the MLL fragment selectively inhibited MLL fusion mediated leukemic transformation and cell growth while the growth and proliferation of E2A-HLF cells is unaffected. Together these data show that targeting the MLL-PAFc interaction with a small MLL fragment can act as a dominant negative and selectively inhibit the growth of AML cells transformed with MLL fusion proteins. These data also suggest the MLL-PAF1 interaction surface is a promising region for therapeutic targeting.