Global Increase in H3K79 Dimethylation in Murine and Human MLL-AF4 Lymphoblastic Leukemias.
Authors: Krivtsov Andrei V. , Feng Zhaohui , Lemieux Madeleine , Faber Joerg , Xia Xiaobo , Kung Andrew L. , Armstrong Scott A. 
Published: October 2019

Abstract
The t(4:11) is the most common chromosomal rearrangement associated with acute lymphoblastic leukemia (ALL) in infants and predicts a poor outcome. We created a conditional knock-in mouse model that expresses the Mll-AF4 fusion protein generated by the t(4;11). The mice developed acute leukemia consisting of bone marrow replacement, splenomegaly and variable lymphadenopathy with a latency from 3–6 months depending upon the method of cre recombinase expression. Immunophenotypic analysis demonstrated the development of acute lymphoblastic leukemia (mALL), acute myeloid leukemia (mAML) or occasionally mixed lineage (biphenotypic) leukemia (mMLL). Further immunophenotypic analysis of mALL demonstrated the majority of the cells to be B220(+) CD43(low/−) IgM(−) IgD(−), pre-B cells. Unsupervised gene expression analysis confirmed that mALL cells are more similar to pre-B cells than to pre-pro-B, immature B or mature B-cells. Supervised gene expression analysis demonstrated that mALL cells had increased expression of genes including HoxA3/4/5/7/9/10, Meis1, and Myc, similar to previously described human ALL profiles. Furthermore gene set enrichment analysis demonstrated significant overlap between the murine and human MLL-AF4 leukemia gene expression profiles. As it has been previously shown that MLL-AF10 recruits the K79 methyltransferase DOT1L to HOXA cluster genes, we assessed whether Mll-AF4 mediates gene expression through similar chromatin modifications. We assayed H3K4; H3K27; and H3K79 methylation levels on HoxA cluster genes in mALL and pre-B cells. While H3K4 and H3K27 levels were not significantly different between pre-B and mALL cells, H3K79 was increased on HoxA3 through HoxA10 but not HoxA1/2/11/13. Assessment of genome wide distribution of H3K79 mark demonstrated a dramatic increase in H3K79 methylation on HoxA loci, and hundreds of other loci throughout the mouse genome. To determine whether findings in our mouse model have relevance for human ALL we assessed genome wide distribution of the H3K79 modification in MLL-AF4 rearranged ALL, non MLL-rearranged ALL, and human bone marrow CD34(+) CD19(+) pre-B cells. We found approximately 5000 genes in MLL-AF4 ALL cells possessed enhanced levels of the H3K79 modification as compared to normal human CD34(+) CD19(+) cells whereas MLL-germline ALLs did not possess a global enhancement. Moreover, MLL-AF4 ALL samples possessed the H3K79 modification on the promoters of over-expressed genes that distinguish MLL-rearranged ALL from other ALL. Finally, shRNA mediated knockdown of the K79 methyltransferase DOT1 suppressed growth of MLL-rearranged but not non MLL-rearranged ALL cell lines. These data demonstrate that MLL-AF4 induces genome wide abnormalities in H3K79 chromatin modification, which is likely a critical component of its leukemogenic mechanism. Targeting DOT1 with specific inhibitors may be therapeutically useful for the most common MLL-rearranged leukemias.