Abrogation of MLL-AF10 and CALM-AF10 Mediated Transformation Through Genetic Inactivation or Pharmacological Inhibition of the H3K79 Methyltransferase DOT1L.

Abstract 2384

The t(10;11)(p12;q23) and the t(10;11)(p12;q14), which encode the MLL-AF10 and CALM-AF10 fusion oncoproteins respectively, are two recurrent chromosomal rearrangements observed in patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Patients with AML harboring either MLL-AF10 or CALM-AF10 rearrangements have a particularly poor outcome compared to patients whose leukemia cells do not harbor these translocations. Thus new therapeutic approaches are clearly needed for patients with leukemias bearing rearrangements of the AF10 gene. Previous studies have suggested that the histone H3 lysine 79 (H3K79) methyltransferase DOT1L is recruited by a part of the AF10 protein retained in leukemic MLL-AF10 and CALM-AF10 fusions, suggesting a role of this methyltransferase in transformation mediated by these fusion oncoproteins. The general amenability of enzymes to pharmacological inhibition makes DOT1L an attractive therapeutic target in leukemias bearing AF10-fusions. We decided to test whether transformation mediated by the MLL-AF10 or CALM-AF10 fusions can be impaired by Dot1l inhibition. We transformed bone marrow cells with either the MLL-AF10 or the CALM (400–648)-AF10 (677–758) fusion proteins that have been previously shown to immortalize hematopoietic progenitors in vitro and in vivo. In contrast to the large, highly clonogenic compact colonies observed in methylcellulose culture of wildtype transformed cells, Dot1l excision from the MLL-AF10 or CALM-AF10 immortalized progenitors led to the formation of small, diffuse colonies that had lost their replating capability. In MLL-AF10 or CALM-AF10 transformed cells, Dot1l- inactivation significantly reduced global H3K79 dimethylation as assessed by Western blotting, and Wright Giemsa staining confirmed morphological features consistent with myeloid differentiation. Moreover, the expression of MLL-AF10 and CALM-AF10 targets, such as Hoxa cluster genes and Meis1, decreased significantly after Dot1l inactivation as assessed by quantitative PCR. We then assessed the impact of Dot1l deletion on the in vivo leukemogenic activity of MLL-AF10 and CALM-AF10 transformed bone marrow cells. Ongoing experiments clearly demonstrate that Dot1l excision strongly impairs the initiation and maintenance of both MLL-AF10 as well as CALM-AF10 mediated murine leukemias.

Having established that genetic inactivation of Dot1l inhibits H3K79 methylation and consequently transforming potential of the AF10-fusions, we investigated the efficacy of Dot1l inhibitors against MLL-AF10 or CALM-AF10 transformed murine bone marrow cells. Treatment of MLL-AF10 or CALM-AF10 transformed cells with EPZ004777, the first specific small-molecule inhibitor of Dot1l, suppressed expression of downstream oncogenic targets such as Hoxa cluster genes and Meis1, and selectively impaired the proliferation of MLL-AF10 and CALM-AF10 transformed, but not Hoxa9/Meis1 transformed cells. MLL-AF10 and CALM-AF10 transformed cells underwent differentiation and cell cycle arrest after EPZ004777 treatment. Results from in vivo colony-forming units-spleen (CFU-S) assays showed that pre-treatment of MLL-AF10 or CALM-AF10 transformed cells with the EPZ004777 inhibitor profoundly impaired their spleen-colony forming ability, suggesting that EPZ004777 may show in vivo efficacy against AF10-fusion transformed cells. Taken together, our results demonstrate that Dot1l inhibition impairs the in vitro and in vivo oncogenic activity of the MLL-AF10 and CALM-AF10 fusion oncogenes. These results indicate that patients with leukemias bearing chromosomal rearrangements of the AF10 gene may benefit from small molecule inhibition of H3K79 methylation.