PROJECT SUMMARY The mixed-lineage leukemia (MLL) gene rearrangements account for approximately 80% of infant acute lymphoblastic leukemia (ALL) and 35-50% of infant acute myeloid leukemia (AML). Patients bearing rearrangements of the MLL gene are associated with dismal prognosis. To date, no effective therapies have been approved for treating the fatal diseases. Molecularly, inter-chromosomal translocations of MLL lead to in frame fusions of the N-terminus of MLL to the C-terminus of various fusion partners, which are known as the “driver” lesions of the diseases. Among more than 70 MLL fusion partners, a small subset of fusions account for most leukemogenic cases. In ALL, over 90% MLL rearrangements involve only four fusion partners: AFF1, AF9, ENL, and AF10, all of which are components of the super elongation complex (SEC) or the complex of the histone H3K79 methyltransferase DOT1L. It is believed that these MLL fusions share a common pathway by hijacking the SEC or DOT1L complex to promote aberrant activation of the target genes of MLL fusions, leading to the pathogenesis of leukemias. We and others have recently demonstrated that ENL, a component of the SEC and DOT1L complex, is critical for the oncogenic function of the MLL-fusions. ENL contains an evolutionally conserved YEATS domain that we identified as a reader of histone acetylation. We also found that the YEATS domain of ENL, but not AF9, is essential for growth and survival of the MLL-rearranged leukemic cells. These key findings strongly suggest that ENL is a promising therapeutic target for MLL-rearranged leukemias. However, while a recently reported small-molecule inhibitor effectively blocks the interactions between the YEATS domain of ENL/AF9 and acetylated histone H3, this ENL inhibitor has little or no effect on killing ENL-dependent MLL- rearranged leukemia cells, failing to phenocopy the cell killing effect of ENL knockout. We hypothesize that pharmacological degradation of ENL, instead of ENL inhibition that relies on occupancy-driven pharmacology, will provide a novel and effective therapeutic strategy for treating MLL-rearranged leukemias. To test this hypothesis, we propose to develop first-in-class ENL small-molecule degraders as in vivo chemical probes using the proteolysis targeting chimera (PROTAC) technology and evaluate them in MLL-rearranged leukemia cells and mouse models. We have generated promising preliminary results, suggesting that the proposed research is feasible. The ENL degrader in vivo chemical probes generated in this project will not only help us test and validate our therapeutic hypothesis, but can also be further optimized into drug candidates in the future and ultimately translated in the clinic for MLL-rearranged leukemia patients.