Targeting the Dpy30 Subunit of Set1/Mll Complexes to Inhibit MLL-Rearranged Leukemogenesis

Epigenetic modulators have emerged as promising targets for treating cancers, especially blood cancers. As the major histone H3K4 methylation enzymes in mammals, the Set1/Mll complexes represent potential drug targets in epigenetic therapeutics due to the intimate connection of H3K4 methylation with gene expression and their extensive association with multiple cancers including blood cancers. However, the functional role for the Set1/Mll complexes in tumorigenesis remains incompletely understood, and the potential of pharmacologically targeting the H3K4 methylation activity of these complexes has not been fully explored.

The Set1/Mll complexes comprise one of six different catalytic subunits and several shared core subunits including Dpy30. We have previously shown that Dpy30 directly facilitates genome-wide H3K4 methylation, and plays a crucial role in fundamental cellular processes including proliferation and differentiation. Using a conditional Dpy30 KO mouse model that was recently generated in our laboratory, we have established a critical role of Dpy30 in hematopoietic stem cell differentiation and long-term maintenance (Yang et al., J Exp Med, accepted). Although Dpy30 is a global epigenetic modulator, our data show that Dpy30 loss in the hematopoietic system has a much more profound impact on the H3K4me3 at hematopoietic genes than on genes associated with fundamental cellular pathways. Moreover, we have shown that reducing Dpy30 level by heterozygosity significantly suppresses Eμ-myc-driven lymphomagenesis without affecting normal physiology or life span (ASH meeting 2015 Abstract #310), suggesting a role for Dpy30 as a non-oncogene addiction pathway for cancer and a possible therapeutic window for targeting Dpy30 in potential cancer treatment.

As we previously demonstrated that Dpy30 knockdown abolished the growth and colony-formation capacity of several MLL-rearranged leukemia cell lines, we sought to further investigate the role of Dpy30 in leukemogenesis and leukemia stem cell (LSC) function in animals. To this end, we have established a MLL-AF9-driven leukemogenesis mouse model by transducing bone marrow from our conditional Dpy30 KO mice with MLL-AF9 virus and transplanting these cells into recipient mice. Our results clearly demonstrated that inducing Dpy30 deletion in the recipients abrogated leukemia progress. We are currently testing the role of Dpy30 in LSC function by transplantation of MLL-AF9 transduced leukemia cells with the CAG-CreER; Dpy30^F/Fbackground. Mild Dpy30 depletion (~50% reduction) is induced in the secondary recipients to examine the possible effects on LSC function, considering that 50% reduction of Dpy30 level (as seen in our Dpy30 heterozygous mice) does not affect normal physiology.

We next sought to design inhibitors of Dpy30 activity for treating blood cancer. Dpy30 associates with and enhances the methylation activity of Set1/Mll complexes by directly binding to the Ash2l subunit. We have designed and synthesized a cell-permeable peptidomimetic corresponding to the Dpy30-binding region on Ash2l, as well as a mutant peptidomimetic that fails to bind to Dpy30 and serves as a negative control. We have shown that, although both effectively penetrated cells, only the wild-type but not mutant peptidomimetic successfully inhibited Dpy30's biochemical activity in enhancing H3K4 methylation, markedly reduced the intracellular binding of Dpy30 to the Mll complexes, and impaired Dpy30 recruitment to its genomic targets. Continuous treatment of the wild-type, but not the mutant, peptidomimetic also inhibited the growth of leukemia cells. Our results strongly suggest that pharmacological inhibition of Dpy30's activity in Set1/Mll complexes can suppress tumor cell growth. We are currently optimizing peptidomimetics to increase the intracellular stability and improve the inhibitory effect on leukemia growth.

Taken together, our studies have established an important role of Dpy30 in the MLL-rearranged leukemogenesis, and demonstrate a proof-of-principle for pharmacologically targeting Dpy30's activity in potential treatment of hematological malignancies.