Understanding the oncogenic functions of ENL and AF9 for better leukemia therapies

The National Cancer Institute awarded the Regents of the University of Michigan $622,070 on September 3, 2026, under the Cancer Cause and Prevention Research program (CFDA 93.393, Project Grant R01CA315895) to investigate the oncogenic functions of ENL and AF9 proteins in acute myeloid and lymphoid leukemias.

The research characterizes biochemical and functional differences between ENL and AF9, which are common translocation partners of the MLL (KMT2A) epigenetic regulator in leukemia patients. The study examines how these proteins bind to the DOT1L complex through an ANC1 homology domain (AHD) and develops novel compounds targeting the ENL/AF9 AHD to impair leukemic cell growth. The work determines whether biochemical differences between ENL and AF9 influence patient therapeutic response and whether the AHD domain alone is sufficient for MLL::AF9 leukemia pathogenesis.

Performance occurs at the University of Michigan in Ann Arbor, Michigan, with a period of performance from September 3, 2026, through August 31, 2031. The award is structured as a project grant, an assistance type supporting investigator-initiated research. The ultimate completion date is August 31, 2031.

Abstract

Acute myeloid and lymphoid leukemias (AML and all) arise because of a block in hematopoietic differentiation often due to mutations in epigenetic regulators. Epigenetic proteins deposit, remove and read chemical modifications on genomic regulatory elements that impact gene expression and are commonly misregulated in cancer, including 70% of AML patients. MLL (KMT2A) is an epigenetic writer that is commonly translocated in AML patients and associated with a dismal prognosis (~29% 5-year EFS) necessitating better therapies. MLL translocations fuse the N-terminus of MLL with the C-terminus of a partner protein. Two of the most common translocation partners are the highly homologous proteins ENL and AF9. Both proteins bind chromatin through a yeats domain and associate with the super elongation complex (SEC) or the epigenetic DOT1L complex through an ANC1 homology domain (AHD). However, ENL is critically required in leukemic cells while AF9 is dispensable. We characterized unique translocation events in MLL::ENL and MLL::AF9 patients such that different domains are retained or lost in resultant MLL::ENL and MLL::AF9 fusion proteins. Importantly, we found that while the AHD was invariably retained in all MLL::ENL and MLL::AF9 patients, the AHD of ENL and AF9 possess different oncogenicity. Our biochemical analysis found that the AHD of AF9 binds more tightly to DOT1L than ENL. Among the 12 amino acids that differentiate the ENL and AF9 AHD, we found a single amino acid on AF9 that differentiates AF9 and ENL binding to DOT1L. Further, we have developed novel compounds that bind to the ENL/AF9 AHD leading to impaired cell growth. Importantly, the implications of the biochemical differences between ENL and AF9 and whether they influence patient therapeutic response is not understood. We discovered that fusion of only the AHD is sufficient for MLL::AF9 leukemia, but insufficient for MLL::ENL leukemia. Our objective is to understand how biochemical differences between the AHD of ENL and AF9 impact MLL fusion leukemogenesis. We hypothesize that differential AHD affinity for the SEC and DOT1L complexes between AF9 and ENL affects MLL::fusion mediated activation of leukemia gene programs and leukemogenesis and is an ideal therapeutic target for AML. We will perform the following aims. (1) determine how the AHD of ENL and AF9 affect MLL::ENL and MLL::AF9 mediated epigenetic and transcriptional output. We hypothesize that the fusion of the AHD of ENL or AF9 to MLL will induce unique epigenetic landscapes and transcription. We will test genome wide chromatin condensation, epigenetic reprogramming and epigenetic complex localization. (2) examine how AHD structural differences impact protein interactions with ENL and AF9. We hypothesize that non-conserved residual differences between the ENL and AF9 AHD dictate binding to partner proteins. We will use mutagenesis to identify critical amino acid differences impacting binding affinity and leukemic potential. (3) assess the therapeutic potential of targeting AHD in MLL rearranged leukemia. We hypothesize that targeting the AHD with degrader molecules will display antitumor activity for MLL-fusion leukemias and AML in general. We use mouse models that provide an in vivo system that accurately reflects MLL::fusion patient survival. In vivo studies utilize mouse models driven solely by MLL::fusion proteins, which are ideal for mechanistic studies. We also use mouse syngeneic models and human leukemia cell xenografts to evaluate therapeutic response.