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Toward personalized therapy in AML: in vivo benefit of targeting aberrant epigenetics in MLL-PTD-associated AML

Leukemia volume 27, pages 2379–2382 (2013)Cite this article

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The MLL gene, encoding a transcription factor with histone H3 lysine 4 (H3K4) methyltransferase activity, is mutated via a gain-of-function intragenic, self-fusion mutation called the Partial Tandem Duplication (MLL-PTD) in ∼5% of patients with cytogenetically normal acute myeloid leukemia (CN-AML) and associates with poor prognosis.1, 2, 3, 4 Although intense therapy such as post-remission autologous or allogeneic stem cell transplant appears to improve their outcome,5 adult patients with MLL-PTD-positive CN-AML still die of the disease, thus suggesting the need for novel ‘personalized’ treatment(s). Unfortunately, no specific inhibitors that target the aberrant MLL H3K4 methyltransferase activity are currently available. Nevertheless, we previously demonstrated an association of MLL-PTD with increased DNA hypermethylation, an epigenetic change mediated by DNA methyltransferases (DNMTs) that leads to aberrant tumor suppressor gene silencing in AML. We also showed that the combination of epigenetic targeting agents, a DNA hypomethylating agent (that is, decitabine) followed by a histone deacetylase inhibitor (HDACi), decreased the transcript ratio of the MLL-PTD-to-MLL-wild type (WT) alleles and concurrently induced apoptosis in MLL-PTD-positive blasts.6 However, these results were obtained only with in vitro cultures, and as such, in vivo validation of this approach is necessary to move it into the clinic. Thus, we aimed herein to validate the epigenetics-targeting approach to MLL-PTD AML in an in vivo mouse model of spontaneous AML that harbors the knocked-in mutations of Mll-PTD and the receptor tyrosine kinase Flt3-internal tandem duplication (Flt3-ITD), hereafter referred to as double knock-in (dKI). This model recapitulates features of the counterpart human disease.7 Indeed, ∼30% of MLL-PTD AML patients also harbor FLT3-ITD and have a poor prognosis.5

The primary dKI mice take ∼50–60 weeks to develop fatal leukemia. To ensure similar leukemia burden at the time of the treatment start, we serially transplanted whole spleen cells from primary leukemic mice into sublethally irradiated syngeneic recipients. This transplant model allowed us to reduce time to moribund state to ∼36 days. Treatment was started on day 21 post transplant, when mice had reached a white blood cell count >10.5 k/ml and engraftment was >70% donor cells in the blood, as measured by flow cytometry. 5AD (0.2 mg/kg daily intraperitoneal injection for a total of four doses) was either given before or after vehicle or AR42 (50 mg/kg delivered every other day via oral gavage for a total of three doses) to ascertain also whether the sequence impacted anti-leukemic activity in vivo. Prior to the last dose of treatment (either AR42 or 5AD, depending on the sequence), three mice were euthanized to assess early cytoreduction and pharmacodynamic endpoints. Because there was no significant difference in survival or pharmacodynamics endpoints between the sequences of 5AD followed by AR42 compared with AR42 followed by 5AD, results from these two studies were combined (5AD plus AR42). A reduction in leukemic load of ∼twofold was measured by spleen weight (P=0.003; Figure 2d), and a decrease in circulating blasts (Figure 2e) was observed in mice treated with individual agents or the combination compared with vehicle-treated controls. No differences in reduction of tumor burden were observed between the different treatments or sequences at this time point. 5AD alone or the 5AD/AR42 combination induced differentiation of the blasts as measured by an increase in surface expression of CD11b (data not shown) and Gr1 (Figure 2f), thereby supporting early anti-leukemia activity of these epigenetic modifiers either alone or in combination, as measured by cell differentiation rather than by cytotoxic effects. Nevertheless, the combination was more effective over time. Whereas mice treated with 5AD alone trended toward increased survival compared with vehicle-treated control mice (median survival 41 vs 36 days; P=0.10), and whereas AR42 showed a statistically significant increase in survival compared with vehicle controls (median survival 46 vs 36 days, P<0.0001; Figure 2g), survival was further enhanced by the 5AD plus AR42 combination (median survival 54 days, P=0.004 for 5AD plus AR42 vs AR42 alone; P<0.0001 for 5AD plus AR42 vs 5AD alone; P<0.0001 for 5AD plus AR42 vs vehicle).

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Acknowledgements

We thank The Ohio State University Comprehensive Cancer Center’s Shared Resources: Biostatistics, Nucleic Acid, Comparative Pathology and Mouse Phenotyping and Analytical Cytometry. This work was supported in part by National Cancer Institute, Bethesda, MD, USA, grants: to MAC (CA089341; CA009338), to GM (CA102031), to MAC and GM (CA140158), to NAZ (T32 GM12453), and to KMB (T32 CA009338). We also thank the Thomas J. Bardos Science Education Award (RFS), the American Society of Hematology Trainee Research Award (RFS), the OSU College of Medicine Fellowship (NAZ), Pelotonia Fellowships (NAZ, RFS, and AMD), Wells Family Fellowship (JSN and DLB) and the American Society of Hematology Minority Medical Student Award Program (MN).

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  1. G Marcucci and M A Caligiuri: These authors contributed equally to this work.

Authors and Affiliations

  1. The Comprehensive Cancer Center and The James Cancer Hospital and Solove Research Institute, Columbus, OH, USA

    K M Bernot, R F Siebenaler, S P Whitman, N A Zorko, G G Marcucci, R Santhanam, E H Ahmed, M Ngangana, K K McConnell, J S Nemer, D L Brook, S K Kulp, C S Chen, D Frankhouser, P Yan, X Zhang, A M Dorrance, K E Dickerson, D Jarjoura, W Blum, G Marcucci & M A Caligiuri

  2. The Ohio State University, Columbus, OH, USA

    K M Bernot, R F Siebenaler, S P Whitman, N A Zorko, G G Marcucci, R Santhanam, E H Ahmed, M Ngangana, K K McConnell, J S Nemer, D L Brook, S K Kulp, C S Chen, D Frankhouser, P Yan, R Bundschuh, X Zhang, A M Dorrance, K E Dickerson, D Jarjoura, W Blum, G Marcucci & M A Caligiuri

  3. College of Pharmacy, The Ohio State University, Columbus, OH, USA

    S K Kulp & C S Chen

  4. Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, OH, USA

    D Frankhouser, P Yan, R Bundschuh, W Blum, G Marcucci & M A Caligiuri

  5. Departments of Physics and Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH, USA

    R Bundschuh

  6. Center for Biostatistics, The Ohio State University, Columbus, OH, USA

    X Zhang & D Jarjoura

  7. Department of Pediatrics, Nationwide Children’s Hospital, The Ohio State University, Columbus, OH, USA

    K E Dickerson

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Correspondence to M A Caligiuri.

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Competing interests

CSC is the inventor of AR42, and has received royalty payments from Arno Therapeutics per the licensing agreement of AR42 between the Ohio State University Research Foundation and Arno Therapeutics. The remaining authors declare no conflict of interest.

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Bernot, K., Siebenaler, R., Whitman, S. et al. Toward personalized therapy in AML: in vivo benefit of targeting aberrant epigenetics in MLL-PTD-associated AML. Leukemia 27, 2379–2382 (2013). https://doi.org/10.1038/leu.2013.147

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