Impairing MLL-fusion gene-mediated transformation by dissecting critical interactions with the lens epithelium-derived growth factor (LEDGF/p75)

Abstract

The lens epithelium-derived growth factor (LEDGF/p75) tethers the mixed-lineage leukemia (MLL1) protein complex to chromatin. Likewise, LEDGF/p75 tethers the HIV-1 pre-integration complex to chromatin. We previously demonstrated that expression of the C-terminal fragment fused to enhanced green fluorescent protein (eGFP) (eGFP-LEDGF(325-530)) impaired HIV-1 replication. Here, we explored this strategy to selectively interfere with the leukemogenic activity of MLL-fusion proteins. We found that expression of LEDGF(325-530) impaired the clonogenic growth of MLL-fusion gene transformed human and mouse hematopoietic cells, without affecting the growth of control cells immortalized by the FLT3-ITD mutant or normal lineage-marker-depleted murine bone marrow cells. Expression of LEDGF(325-530) was associated with downregulation of the MLL target Hoxa9 and impaired cell cycle progression. Structure-function analysis revealed two small eGFP-fused LEDGF/p75 peptides, LEDGF(424-435) and LEDGF(375-386) phenocopying these effects. Both LEDGF(325-530) and the smaller active peptides were able to disrupt the LEDGF/p75-MLL interaction. Expression of LEDGF(325-530) or LEDGF(375-386) fragments increased the latency period to disease development in vivo in a mouse bone marrow transplant model of MLL-AF9-induced AML. We conclude that small peptides disrupting the LEDGF/p75-MLL interface have selective anti-leukemic activity providing a direct rationale for the design of small molecule inhibitors targeting this interaction.

Validation and Structural Characterization of the LEDGF/p75–MLL Interface as a New Target for the Treatment of MLL-Dependent Leukemia

“…1B) containing the IBD, but lacking the PWWP domain, outcompetes endogenous LEDGF/p75 from the MLL-MENIN complex and dramatically reduces clonogenic growth of hematopoietic stem cells immortalized by MLL fusion proteins both in vitro and in a mouse model (23). Similar results were obtained with small molecules targeting the MLL-MENIN interaction (24).…”

“…S1B; ref. 23). Because none of these LEDGF/p75 residues are in direct contact with MLL in the published structure, these data hint toward the existence of an additional important interaction between MLL and LEDGF/p75.…”

“…Huang and colleagues used isothermal titration calorimetry (ITC) to show that the LEDGF/p75 IBD binds the MLL-MENIN complex with high affinity, whereas neither MENIN nor MLL 4-135 DD alone stably associate with LEDGF/p75. In contrast, we were able to detect an interaction between LEDGF/p75 and an Nterminal MLL-derived fragment (aa 1-160) in the absence of MENIN using AlphaScreen technology (23). Moreover, we previously showed that a LEDGF/p75 IBD-derived peptide spanning aa 375 to 386 (LEDGF [375][376][377][378][379][380][381][382][383][384][385][386] ) inhibited the LEDGF/ p75-MLL 1-160 interaction and leukemic transformation (Supplementary Fig.…”

Multiple cellular proteins interact with LEDGF/p75 through a conserved unstructured consensus motif

“…Before this work, the interactions of a diverse set of proteins with the IBD were not fully understood, despite their importance to the development of therapies targeting LEDGF/p75 interactions to block MLL fusionmediated leukaemic transformation and HIV integration 53,54 .…”

An Achilles' Heel for MLL-Rearranged Leukemias: Writers and Readers of H3 Lysine 36 Dimethylation

“…1 , top). LEDGF has been reported to serve as a critical cofactor of MLL fusion proteins ( 9 ) and to recognize H3K36 methylation ( 10 ).…”