Abstract 2966: The epigenetic reader function of the YEATS domain in MLL-ENL fusion critically affects leukemic stem cell frequency in MLL-ENL leukemia

Abstract MLL1 (KMT2A) translocations are found in ~10% of acute leukemia and give rise to an aggressive form of leukemia in infant, pediatric and adult patients. MLL1 fusion driven acute leukemia is characterized by deregulated activity of the Super Elongation Complex (SEC) and the H3K79 methyltransferase DOT1L, which alter the epigenetic landscape and transcription of pro-leukemic MLL1 fusion targets like HoxA9 and Meis1. The Eleven-Nineteen-Leukemia gene (ENL or MLLT1) is a common MLL1 fusion partner and a SEC component. The ENL protein contains a highly conserved N-terminal epigenetic reader YEATS domain that recognizes acetylated H3K9/K18/K27 (H3Kac hereinafter). Wild type ENL was recently found to be essential for leukemic cell growth, which is dependent on its YEATS domain interaction with H3Kac. While this finding highlighted the YEATS domain importance in wild type ENL function in leukemic cells, the inclusion and importance of the YEATS domain in MLL-ENL fusion protein remain to be elucidated. Here, we investigated the clinical relevance and importance of the ENL YEATS domain in MLL-ENL leukemias. We analyzed >300 t(11;19) MLL-ENL leukemia patients for the breakpoint location within the ENL gene and found that the YEATS domain is retained in the resultant MLL-ENL fusion protein in 84.1% of t(11;19) leukemia patients. We tested the importance of the YEATS domain in MLL-ENL mouse models and found that the YEATS domain and downstream sequence is required for MLL-ENL leukemogenesis in vivo. YEATS deletion decreased expression of pro-leukemic targets such as Meis1, an important factor for leukemic stem cells (LSC). To interrogate the contribution of the YEATS epigenetic reader function in MLL-ENL leukemogenesis, we introduced YEATS point mutations rendering the domain defective in interacting with H3Kac and found that this significantly increased leukemia latency in vivo. Further investigation revealed that YEATS point mutations disrupting H3Kac binding significantly decreased MLL-ENL LSC frequency while not affecting homing to the bone marrow. We attribute this LSC frequency change to altered Meis1 expression. Additionally, disruption of the YEATS epigenetic reader function in MLL-ENL leukemia cells does not induce differentiation, apoptosis nor cell cycle arrest. Therapeutically, we predicted the YEATS domain in MLL-ENL would sensitize MLL-ENL leukemia to YEATS domain inhibitors. Indeed, MLL-ENL leukemia cells are more sensitive to the ENL/AF9 YEATS domain inhibitor, SGC-iMLLT, compared to acute leukemia cells driven by other fusions. Together, our results demonstrate that YEATS-H3Kac binding plays an important role in MLL-ENL fusion mediated leukemogenesis. Our data establishes a strong rationale for future exploration of small molecules aimed at disrupting the YEATS-H3Kac interaction as a targeted therapeutics for treating t(11;19) leukemia patients. Citation Format: Hsiangyu Hu, Nirmalya Saha, Ejaz Ahmad, Yuting Yang, Lili Chen, Lauren Lachowski, Blaine Teahan, Sierrah Grigsby, Rolf Marschalek, Zaneta Nikolovska-Coleska, Andrew G. Muntean. The epigenetic reader function of the YEATS domain in MLL-ENL fusion critically affects leukemic stem cell frequency in MLL-ENL leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2966.