Small molecule-mediated knockdown of DOT1L as a new epigenetic therapeutic for leukemia

DOT1L is a histone lysine methyltransferase enzyme made up of 1537 amino acids. It is responsible for the mono-, di-, and tri-methylation of lysine 79 at the core of histone 3, using S-adenosyl methionine, or SAM, as its cofactor and methylating reagent. Under physiological conditions, DOT1L is associated with active transcription, the DNA damage response and cell cycle regulation and plays an important role in haematopoiesis. Abnormal methylation brought about by DOT1L however has been shown to enhance the expression of genes that are responsible for the development of MLL-rearranged leukemia (e.g. HOXA9, MEISI). This type of leukemia occurs in 5-10% of adult acute leukemias and in more than 70% of infant acute lymphoblastic leukemias, and is associated with poor prognosis.
Functional inhibition of the enzyme has been extensively explored, leading to the development of an array of inhibitors, some of which have already progressed into clinical trials. These can be subdivided into two categories: SAM-competitive inhibitors which are derived from the structure of SAM and bind at the SAM binding site, and induced pocket inhibitors which are structurally different from SAM and bind at a different location. The binding of the non-SAM inhibitors induces a conformational change in the enzyme's tertiary structure which as a result abolishes the SAM binding site and prevents histone methylation.
A completely different approach will be undertaken in this project. The development of a proteolysis targeting chimera, or PROTAC reagent, for DOT1L will be investigated. PROTACs are heterobifunctional molecule comprising of an E3 ubiquitin ligase binding ligand on one end, and a ligand which binds the protein of interest at the other end, connected via a linker (Figure 1). As a result of this bifunctionality, the protein of interest comes in close proximity with the ligase and becomes ubiquitinated. After several rounds of ubiquitination, the polyubiquitinated protein unbinds and consequent degradation takes place at the proteasome. This ultimately leads to complete removal of the protein from cells instead of functional inhibition, and stops histone methylation.
PROTAC reagents have been shown to act catalytically. Once the polyubiquitinated protein unbinds, a new protein molecule can bind and the whole cycle can start again. This technology could be considered advantageous compared to functional inhibitors, since a lower dose will need to be administered.