Abrogation of MLL-AF10 and CALM-AF10-mediated transformation through genetic inactivation or pharmacological inhibition of the H3K79 methyltransferase Dot1l

Abstract

The t(10;11)(p12;q23) translocation and the t(10;11)(p12;q14) translocation, which encode the MLL (mixed lineage leukemia)-AF10 and CALM (clathrin assembly lymphoid myeloid leukemia)-AF10 fusion oncoproteins, respectively, are two recurrent chromosomal rearrangements observed in patients with acute myeloid leukemia and acute lymphoblastic leukemia. Here, we demonstrate that MLL-AF10 and CALM-AF10-mediated transformation is dependent on the H3K79 methyltransferase Dot1l using genetic and pharmacological approaches in mouse models. Targeted disruption of Dot1l using a conditional knockout mouse model abolished in vitro transformation of murine bone marrow cells and in vivo initiation and maintenance of MLL-AF10 or CALM-AF10 leukemia. The treatment of MLL-AF10 and CALM-AF10 transformed cells with EPZ004777, a specific small-molecule inhibitor of Dot1l, suppressed expression of leukemogenic genes such as Hoxa cluster genes and Meis1, and selectively impaired proliferation of MLL-AF10 and CALM-AF10 transformed cells. Pretreatment with EPZ004777 profoundly decreased the in vivo spleen-colony-forming ability of MLL-AF10 or CALM-AF10 transformed bone marrow cells. These results show that patients with leukemia-bearing chromosomal translocations that involve the AF10 gene may benefit from small-molecule therapeutics that inhibit H3K79 methylation.

Fig.1. Cre-mediated deletion of Dot1l leads to loss of H3K79me2 in MLL-AF10 and CALM-AF10 immortalized murine bone marrow cells

(A) Schematic representation of experimental design. (B) Western blot showing the expression of MLL-AF10 using anti-MLL antibody and FLAG-CALM-AF10 using anti-FLAG antibody in transformed bone marrow cells. Total H3 was used as a control. (C) Western blot showing the loss of H3K79 methylation 7 days after transduction with Cre or MIT.

Fig.2. Loss of Dot1l leads to decreased colony forming potential and increased differentiation of MLL-AF10 or CALM-AF10 transformed cells

(A) Blast and differentiated colony count of Dot1l-deleted MLL-AF10 (left) or CALM-AF10 (right) transformed cells in methylcellulose 9 days after transduction with Cre in comparison to controls (n=3 independent experiments). (B) Morphologic changes (10X image of colony morphology in methylcellulose, 40X image of Wright-Giemsa stain) and H3K79me2 immunofluorescence (20X image, Alexa 674-H3K79me2 and DAPI nuclear stain) in MLL-AF10 (left) or CALM-AF10 (right) transformedpreleukemia cells 9 days after transduction with Cre. (C) Relative expression levels of Hoxa5, Hoxa7, Hoxa9, Hoxa10, Meis1, and Hoxb4 on cells 5 days after transduction with Cre or MIT. Expression levels were normalized to Gapdh and expressed relative to MIT-transduced cells (set to 100%). Error bars indicate the SEM (n=3 independent experiments). (D) Genotyping of transduced bone marrow cells on day 3, day 9, and day 16 after transduction of Cre. f: floxed allele. Δ: deleted allele.

Fig.3. EPZ004777 selectively inhibits proliferation of MLL-AF10 and CALM-AF10 transformed murine bone marrow cells

(A) Growth of MLL-AF10, CALM-AF10, MLL-AF9, and Hoxa9/Meis1a transformed bone marrow cells during several days’ incubation with 10 µM EPZ004777. Viable cells were counted and replated at equal cell numbers in fresh media with fresh compound every 3–4 days. Results were plotted as percentage of split-adjusted viable cells in the presence of 10 µM EPZ004777 compared to DMSO vehicle control. Results are representative of three independent experiments. (B) Dosage effect of EPZ004777 treatment on MLL-AF10, CALM-AF10, MLL-AF9, and HoxA9/Meis1a transformed bone marrow cells. Cells were counted and replated at equal cell numbers in fresh media with fresh compound every 3–4 days. Results were plotted as percentage of split-adjusted viable cells on day 17 in media with 0.1 µM, 1 µM, 10 µM of EPZ004777 compared to DMSO control (set as 100%). Results are representative of three independent experiments. (C) Time course of Hoxa9 and Meis1 mRNA expression in MLL-AF10 and CALM-AF10 transformed cells over 10 days of incubation with 10 µM EPZ004777 as measured by quantitative real-time PCR. Expression levels were normalized to Gapdh and expressed relative to those at day 0 (set to 100%). Error bars indicate the SEM (n=3 independent experiments). (D) Quantitative real-time PCR analysis of Hoxa9, Meis1, and β-Actin mRNA levels in MLL-AF10 and CALM-AF10 transformed cells following 7 days of incubation with EPZ004777. Relative mRNA expression levels are plotted as a percentage of those in vehicle-treated control cells. Error bars represent SEM (n=3 independent experiments). (E) Inhibition of cellular H3K79me2 levels in MLL-AF10, CALM-AF10, MLL-AF9 or Hoxa9-Meis1a transformed bone marrow cells following 7 days of treatment with the indicated concentrations of EPZ004777 as measured by immunoblot analysis of extracted histones with an anti-H3K79me2 antibody.

Fig.4. EPZ004777 causes cell cycle arrest and apoptosis in MLL-AF10 and CALM-AF10 transformed bone marrow cells

(A) Cell cycle changes (BrdU/7-AAD flow cytometry) in MLL-AF10 transformed bone marrow cells after being treated with 10 µM EPZ004777 for 0, 4, 8, or 10 days. Results are representative of two independent experiments. (B) Annexin V staining in MLL-AF10 transformed bone marrow cells 10 days after treatment with 10 µM EPZ004777 or DMSO control (n = 2 independent experiments). Error bars represent standard error of the mean (SEM). (C) Cell cycle changes (BrdU/7-AAD flow cytometry) in CALM-AF10 transformed bone marrow cells after being treated with 10 µM EPZ004777 for 0, 4, 8, or 10 days. Results are representative of two independent experiments. (D) Annexin V staining in CALM-AF10 transformed bone marrow cells 10 days after treatment with 10µM EPZ004777 or DMSO control (n = 2 independent experiments). Error bars represent standard error of the mean (SEM).

Fig. 5. EPZ004777 decreased the colony forming potential and induced differentiation in MLL-AF10 and CALM-AF10 transformed bone marrow cells in vitro and EPZ004777 pretreatment diminished spleen-colony forming potential in vivo

(A) Blast and differentiated colony count of MLL-AF10 or CALM-AF10 transformed cells cultured in methylcellulose based medium in the presence of 10uM EPZ004777 or DMSO vehicle control for 1 week or 2 weeks. *: p<0.05; ns: not significant (n=2 independent experiments). (B) Morphologic changes (10X image of colony morphology in methylcellulose, 40X image of Wright-Giemsa stain) in MLL-AF10 (left) or CALM-AF10 (right) transformed cells cultured in methylcellulose based medium containing EPZ004777 for 7 days. (C) Schematic representation of CFU-S experimental design. (D) The morphology of spleen dissected from mice injected with pretreated MLL-AF10 or CALM-AF10 transformed cells.

Figure 6. Dot1l is required for initiation and maintenance of MLL-AF10-driven leukemia in vivo

(A) Survival curves for mice injected with 5X10⁵ MLL-AF10 transformed bone marrow cells 2 days after transduction with Cre or MIT-control retrovirus and sorting for GFP⁺/tdTomato⁺ cells. (B) Survival curves for secondary recipient mice that received 2X10⁵ MLL-AF10 leukemia cells 2 days after transduction with Cre or MIT and sorting for GFP⁺/tdTomato⁺ cells. (C) Immunophenotype of spleen cells of secondary MLL-AF10 leukemic mice (Mac1⁺Gr1⁺CD3⁻B220⁻). (D) Spleen picture and spleen size in mice injected with Cre or MIT transduced Dot1l ^f/f MLL-AF10 cells (n=5.*: p<0.01). (E) Morphology of peripheral blood smear (40X image of Wright-Giemsa stain) and pathology of organs from secondary MLL-AF10 leukemic mice (10X H&E stain). (E) Peripheral blood chimerism in mice 102 days after injection of MIT or Cre transduced MLL-AF10 preleukemic cells. Donor cells are GFP⁺/tdTomato⁺ and recipient cells are GFP⁻/tdTomato⁻.