Abstract 3550: The ENL YEATS domain links leukemic stem cell frequency and enhances YEATS inhibitor sensitivity in MLL-ENL leukemias

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Abstract Chromosome 11q23 translocations are present in ~10% of acute leukemia, which generate the oncogenic MLL fusion (MLL-r hereinafter) proteins and drive a subset of aggressive leukemia. Mechanistic studies of MLL-r leukemias implicated several complexes involved in RNA polymerase II-mediated transcription: the Super Elongation Complex (SEC), the DOT1L Complex (DotCom) and the Polymerase Associated Factor 1 Complex (PAF1c). These protein complexes are dysregulated in MLL-r leukemias and amplify transcription of pro-leukemic target genes. The proteins ENL and AF9 are two common MLL fusion partners and share high homology within their N-terminal YEATS domains that function as epigenetic reader domains. Recently, the importance of the wild type ENL (but not AF9) and its epigenetic reader function has been demonstrated in acute leukemias. However, the importance of the YEATS domain in the context of MLL-ENL fusions has not been explored. In patients, we found that most MLL-ENL fusions (84.1%; N=302 patients), but not MLL-AF9 fusions, retain the YEATS domain. These findings prompted us to investigate 1) how the YEATS domain contributes to MLL-ENL leukemogenesis, 2) whether the YEATS domain affects MLL-ENL fusion protein functions, and 3) if YEATS domain presence in MLL-ENL fusion exposes a vulnerability to YEATS inhibitors. Using published YEATS epigenetic reader mutations, we found that the YEATS epigenetic reader function significantly contributes to MLL-ENL leukemogenesis. Disrupting the YEATS epigenetic reader function in MLL-ENL fusion proteins significantly impacts leukemic stem cell frequency. Using an MLL-ENL construct relevant in patients (ΔYEATS hereinafter), we discovered a subset of MLL-ENL targets with altered expression. GSEA revealed several gene signatures enriched in ΔYEATS cells, most interestingly genes downregulated in leukemic stem cells. Specifically, the MLL-ENL target Eya1 is severely disrupted in MLL-ENL YEATS epigenetic reader mutants and ΔYEATS cells. Our mechanistic data suggest that while MLL-ENL binding at Eya1 is impacted in ΔYEATS, YEATS epigenetic reader mutants do not significantly alter MLL-ENL and PAF1c localization. However, YEATS epigenetic reader mutations severely impact epigenetic modifications associated with active transcription, including H3K4me3, H3K9ac and H3K79me2 at the Eya1 locus. Finally, we tested the YEATS inhibitor sensitivity in AML cell lines. We found that the cell line HB1119, which is driven by MLL-ENL fusion with an intact YEATS domain, is among the most sensitive lines to the YEATS inhibitor SGC-iMLLT. Together, our study provides the biological and mechanistic characterizations of the YEATS domain in MLL-ENL leukemias and contributes to the theoretical framework for YEATS inhibitor development in the majority of MLL-ENL patients. Citation Format: Hsiangyu Hu, Nirmalya Saha, Yuting Yang, Ejaz Ahmad, Lauren Lachowski, Uttar Shrestha, Vidhya Premkumar, James P. Ropa, Lili Chen, Blaine Teahan, Sierrah Grigsby, Rolf Marschalek, Zaneta Nikolovska-Coleska, Andrew G. Muntean. The ENL YEATS domain links leukemic stem cell frequency and enhances YEATS inhibitor sensitivity in MLL-ENL leukemias. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3550.