Summary The Nucleoporin 98 gene (NUP98) is fused to a variety of partner genes in multiple hematopoietic malignancies. Here we demonstrate that NUP98 fusion proteins, including NUP98-HOXA9 (NHA9), NUP98-HOXD13 (NHD13), NUP98-NSD1, NUP98-PHF23, and NUP98-TOP1 physically interact with mixed lineage leukemia 1 (MLL1) and the non-specific lethal (NSL) histone-modifying complexes. ChIP-seq illustrates that NHA9 and MLL1 co-localize on chromatin and are found associated with Hox gene promoter regions. Furthermore, MLL1 is required for the proliferation of NHA9 cells in vitro and in vivo. Inactivation of MLL1 leads to decreased expression of genes bound by NHA9 and MLL1 and reverses a gene expression signature found in NUP98-rearranged human leukemias. Our data reveal a molecular dependency on MLL1 function in NUP98-fusion driven leukemogenesis.

K(lysine) acetyltransferase 8 (KAT8, also known as MOF) mediates the acetylation of histone H4 at lysine 16 (H4K16ac) and is crucial for murine embryogenesis. Lysine acetyltransferases have been shown to regulate various stages of normal hematopoiesis. However, the function of MOF in hematopoietic stem cell (HSC) development has not yet been elucidated. We set out to study the role of MOF in general hematopoiesis by using a Vav1-cre-induced conditional murine Mof knockout system and found that MOF is critical for hematopoietic cell maintenance and HSC engraftment capacity in adult hematopoiesis. Rescue experiments with a MOF histone acetyltransferase domain mutant illustrated the requirement for MOF acetyltransferase activity in the clonogenic capacity of HSCs and progenitors. In stark contrast, fetal steady-state hematopoiesis at embryonic day (E) 14.5 was not affected by homozygous Mof deletion despite dramatic loss of global H4K16ac. Hematopoietic defects start manifesting in late gestation at E17.5. The discovery that MOF and its H4K16ac activity are required for adult but not early and midgestational hematopoiesis supports the notion that multiple chromatin regulators may be crucial for hematopoiesis at varying stages of development. MOF is therefore a developmental-stage-specific chromatin regulator found to be essential for adult but not early fetal hematopoiesis.

Chromatin-based mechanisms offer therapeutic targets in acute myeloid leukemia (AML) that are of great current interest. In this study, we conducted an RNAi-based screen to identify druggable chromatin regulator-based targets in leukemias marked by oncogenic rearrangements of the MLL gene. In this manner, we discovered the H4K16 histone acetyltransferase (HAT) MOF to be important for leukemia cell growth. Conditional deletion of Mof in a mouse model of MLL-AF9-driven leukemogenesis reduced tumor burden and prolonged host survival. RNA sequencing showed an expected downregulation of genes within DNA damage repair pathways that are controlled by MOF, as correlated with a significant increase in yH2AX nuclear foci in Mof-deficient MLL-AF9 tumor cells. In parallel, Mof loss also impaired global H4K16 acetylation in the tumor cell genome. Rescue experiments with catalytically inactive mutants of MOF showed that its enzymatic activity was required to maintain cancer pathogenicity. In support of the role of MOF in sustaining H4K16 acetylation, a small molecule inhibitor of the HAT component MYST blocked the growth of both murine and human MLL-AF9 leukemia cell lines. Furthermore Mof inactivation suppressed leukemia development in a NUP98-HOXA9 driven AML model. Taken together, our results establish that the HAT activity of MOF is required to sustain MLL-AF9 leukemia and may be important for multiple AML subtypes. Blocking this activity is sufficient to stimulate DNA damage, offering a rationale to pursue MOF inhibitors as a targeted approach to treat MLL-rearranged leukemias.

Chromosomal rearrangements of the Mixed-Lineage Leukemia (MLL) gene are found in 5-10% of all patients with acute leukemia and associated with a poor prognosis. MLL-rearrangements are more frequently present in pediatric and infant patients where AF9 is one of the most common fusion partners. In order to identify novel druggable targets in MLL-AF9 rearranged leukemia, we conducted a chromatin regulator focused RNAi screen in murine MLL-AF9 leukemia cells and found hairpins targeting (K)Lysine Acetyltransferase 8 (Kat8, also known as Mof) and the previously identified target Bromodomain Containing 4 (Brd4), to be the most potent suppressors of cell growth. MOF is a histone 4 lysine 16 (H4K16) acetyltransferase and member of the MYST family of histone acetyltransferases (HATs). MOF has been shown to be crucial for murine embryogenesis and is a cell-type dependent regulator of chromatin state and various cellular processes such as T-cell differentiation, DNA damage response and cell cycle progression. Using a conditional murine Mof knockout system, we studied the role of MOF in MLL-AF9 leukemogenesis in detail. In vitro inactivation of Mof in MLL-AF9 transformed mouse hematopoietic stem and progenitor cells led to impaired colony-forming capacity. The specificity of this phenotype was shown by expression of exogenous full-length Mof, which fully rescued transformed cells from the dramatic phenotype. Inactivation of Mof in vivo, lead to reduced tumor burden and prolonged survival of mice bearing MLL-AF9 leukemia cells. RNA sequencing data comparing MLL-AF9 cells with homozygous Mof loss to a wild type control, showed a significant enrichment of genes within the apoptosis (NES 1.98, FDR-q <0.0001) and p53 (NES 2.23, FDR-q <0.0001) pathway. These gene expression data suggest that the importance of MOF in MLL-AF9 leukemogenesis may be through interaction with p53, inducing proliferation and suppressing apoptosis. In addition, we found a reduction of actively cycling cells and a loss of global H4K16 acetylation (H4K16ac) upon Mof knockout. In line with this finding of H4K16ac loss, rescue experiments with HAT domain mutated MOF illustrated that the HAT activity of MOF is indispensable for MLL-AF9 leukemia maintenance. Finally, experiments with the selective MYST protein HAT inhibitor MG149, showed a strong anti-proliferative effect on murine, as well as human MLL-AF9 leukemia cell lines, and MG149 inhibition induced global H4K16ac loss in these cells. These results indicate that MOF HAT activity is required for MLL-AF9 leukemia maintenance. Our data further suggest that MOF HAT activity may be a good target for new small molecule inhibitor development for the treatment of patients with MLL-AF9 rearranged leukemia. Citation Format: Daria G. Valerio, Haiming Xu, Chun-Wei Chen, Takayuki Hoshii, Meghan Eisold, Christopher Delaney, Monica Cusan, Aniruddha J. Deshpande, Chun-Hao Huang, Amaia Lujambio, George Zheng, Tej K. Pandita, Scott W. Lowe, Scott A. Armstrong. Histone acetyltransferase activity of MOF is required for MLL-AF9 leukemogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2694.

<p>Extra data to support the central hypothesis.</p>