Assessment of DOT1L as a Therapeutic Target In Acute Leukemia

Abstract 3291

Histone modifying enzymes are crucial regulators of hematopoiesis that are commonly disrupted in acute leukemia. DOT1L has emerged as a particularly important methyltransferase in leukemias with Mixed Lineage Leukemia (MLL) rearrangements. Leukemogenic MLL fusion proteins transform primarily through upregulation of A-cluster HOX genes, including HOXA9 and the HOX cofactor MEIS1. Many of the most common MLL translocation partners including the AF4 family members, AF9, ENL, and AF10, form the Elongation Assisting Proteins (EAP) complex that includes DOT1L. DOT1L is the only known histone methyltransferase that methylates histone H3 on lysine 79 (H3K79). Increasing evidence suggests this histone modification, which is generally associated with transcriptional activation, is essential for MLL fusion protein mediated oncogenicity. Chromatin immunoprecipitation (ChIP) on MLL fusion protein containing cell lines shows higher levels of H3K79 methylation across the HOXA9 and MEIS1 loci compared to non-MLL fusion protein containing cell lines. Similarly, patient samples with MLL fusion proteins show elevated H3K79 methylation. Finally, knockdown of DOT1L has been shown to inhibit growth of MLL rearranged cell lines. These findings suggest that DOT1L may be an effective therapeutic target, however further development of DOT1L inhibitors will be dependent on assessing the efficacy of DOT1L disruption in a wider range of leukemic cells as well as determining the potential toxicity and effect on normal hematopoiesis. Given that constitutive Dot1l knock out is early embryonic lethal, we established conditional Dot1l knockout mouse from gene trap sperm obtained from the Knock Out Mouse Project (KOMP). Dot1l targeted animals were crossed to CreER animals so that Dot1l knock out could be induced with 4-OHT or tamoxifen. To determine the effects of Dot1l deletion in transformation ability, mouse were injected with 5-flurouracil and bone marrow cells were transduced with retrovirus expressing oncogenes in the presence or absence of 4-OHT and growth in methocult media was examined. These experiments showed that growth of cells transformed by MLL-AF9 is completely abolished by Dot1l deletion while transformations by HOXA9/MEIS1 (downstream targets of MLL fusion proteins) and E2A-HLF (which expresses very low levels of HOXA9/MEIS1 and transform through other mechanisms) were unaffected. We also examined the toxicity of Dot1l deletion in vivo by treating mice with tamoxifen and monitoring survival and assessing hematopoiesis. Immunohistochemical studies show that loss of Dot1l is associated with widespread loss of lysine 79 di- and tri-methylation in organs including liver, spleen, bone marrow, testis, muscle and gastrointestinal track with some residual methylation retained in the central nervous system. Immunophenotypic analysis of mouse bone marrow 3–4 weeks after tamoxifen treatment revealed reduction in HSCs, GMPs, MEPs, and CLPs with Dot1l excision. Bone marrow transplantations and cell cycle experiments are currently underway to further characterize the hematopoietic defects in Dot1l deficient animals. Additional experiments will be needed to determine if Dot1l activity is required in other leukemias with high level HOX expression. Together these results suggest that Dot1l is a promising therapeutic target as it is specifically required for transformation by MLL fusion leukemia, however bone marrow suppression occurs with Dot1l inhibition.