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DNMT3A Haploinsufficiency Transforms FLT3 ITD

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DNMT3A Haploinsufficiency Transforms FLT3 ITD Published OnlineFirst March 25, 2016; DOI: 10.1158/2159-8290.CD-16-0008 RESEARCH BRIEF DNMT3A Haploinsuffi ciency Transforms FLT3 ITD Myeloproliferative Disease into a Rapid, Spontaneous, and Fully Penetrant Acute Myeloid Leukemia Sara E. Meyer 1 , Tingting Qin 2 , David E. Muench 1 , Kohei Masuda 1 , Meenakshi Venkatasubramanian 3 , Emily Orr 1 , Lauren Suarez 4 , Steven D. Gore 5 , Ruud Delwel 6 , Elisabeth Paietta 7 , Martin S. Tallman 8 , Hugo Fernandez 9 , Ari Melnick 10 , Michelle M. Le Beau 11 , Scott Kogan 12 , Nathan Salomonis3 , Maria E. Figueroa 2 , and H. Leighton Grimes 1 , 13 A B S T R A C T Cytogenetically normal acute myeloid leukemia (CN-AML) represents nearly 50% of human AML. Co-occurring mutations in the de novo DNA methyltransferase DNMT3A and the FMS related tyrosine kinase 3 ( FLT3) are common in CN-AML and confer a poorer prognosis. We demonstrate that mice with Flt3 -internal tandem duplication (Flt3 ITD ) and inducible deletion of Dnmt3a spontaneously develop a rapidly lethal, completely penetrant, and transplantable AML of normal karyo- type. AML cells retain a single Dnmt3a fl oxed allele, revealing the oncogenic potential of Dnmt3a hap- loinsuffi ciency. FLT3 ITD/DNMT3A- mutant primary human and murine AML exhibit a similar pattern of global DNA methylation associated with changes in the expression of nearby genes. In the murine model, rescuing Dnmt3a expression was accompanied by DNA remethylation and loss of clonogenic potential, suggesting that Dnmt3a -mutant oncogenic effects are reversible. Dissection of the cellular architecture of the AML model using single-cell assays, including single-cell RNA sequencing, identifi ed clonogenic subpopulations that express genes sensitive to the methylation of nearby genomic loci and responsive to DNMT3A levels. Thus, Dnmt3a haploinsuffi ciency transforms Flt3 ITD myeloproliferative disease by modulating methylation-sensitive gene expression within a clonogenic AML subpopulation. SIGNIFICANCE: DNMT3A haploinsuffi ciency results in reversible epigenetic alterations that transform FLT3 ITD-mutant myeloproliferative neoplasm into AML. Cancer Discov; 6(5); 501–15. ©2016 AACR. 1 Division of Immunobiology, Cincinnati Children’s Hospital Medical Center, Illinois. 12 Department of Laboratory Medicine and Helen Diller Family Com- Cincinnati, Ohio . 2 Department of Pathology, University of Michigan Medical prehensive Cancer Center, University of California, San Francisco, San Fran- School, Ann Arbor, Michigan. 3 Division of Biomedical Informatics, Cincinnati cisco, California. 13 Division of Experimental Hematology and Cancer Biology, Children’s Hospital Medical Center, Cincinnati, Ohio. 4 Department of Oncol- Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio. ogy, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins Note: Supplementary data for this article are available at Cancer Discovery University School of Medicine, Baltimore, Maryland. 5 Division of Hema- Online (http://cancerdiscovery.aacrjournals.org/). tologic Malignancies, Yale Cancer Center, Yale School of Medicine, New Haven, Connecticut. 6 Department of Hematology, and Clinical Trial Center, Corresponding Authors: H. Leighton Grimes, Division of Immunobiology, Erasmus University Medical Center, Rotterdam, the Netherlands. 7 Division Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, MLC of Hemato-Oncology, Department of Medicine (Oncology), Albert Einstein 7038, Cincinnati, OH 45229. Phone: 513-636-6089; Fax: 513-636-5355; College of Medicine/Montefi ore Medical Center, Bronx, New York. 8 Leuke- E-mail: [email protected] ; and Maria E. Figueroa, Department of mia Service, Memorial Sloan Kettering Cancer Center, New York, New York. Pathology, University of Michigan, 109 Zina Pitcher Place, BSRB 2019, 9 Blood and Marrow Transplantation, Moffi tt Cancer Center, Oncologic Sci- Ann Arbor, MI 48109-2200. Phone: 734-763-1865; E-mail: marfi gue@ ences, College of Medicine at University of South Florida, Tampa, Florida. med.umich.edu 10 Department of Medicine, Hematology/Oncology Division, Weill Cornell doi: 10.1158/2159-8290.CD-16-0008 Medical College, New York, New York. 11 Section of Hematology/Oncology, ©2016 American Association for Cancer Research. and the Comprehensive Cancer Center, University of Chicago, Chicago, MAY 2016CANCER DISCOVERY | 501 Downloaded from cancerdiscovery.aacrjournals.org on October 1, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst March 25, 2016; DOI: 10.1158/2159-8290.CD-16-0008 RESEARCH BRIEF Meyer et al. INTRODUCTION RESULTS FLT3 is one of the most frequently mutated genes in To test whether Flt3 and Dnmt3a mutations cooperate to cytogenetically normal acute myeloid leukemia (CN-AML), initiate leukemogenesis, we bred mice to combine Flt3 ITD affecting 37% of cases ( 1–3 ). FLT3 -internal tandem duplica- knock-in and homozygous Dnmt3a fl oxed alleles ( Dnmt3a fl /fl ; tion (ITD) mutations are associated with a worse overall Fig. 1A ) with Mx1-Cre ( MxCre ). We found that Flt3 + /ITD ; Dnmt3a fl /fl survival and constitute an independent prognostic factor for MxCre mice (with one mutant Flt3 allele) had a long median relapse and poor outcome in AML ( 4–7 ). Recently, recurrent survival of 227 days and exhibited splenomegaly (Supple- mutations were found in the epigenetic regulators DNMT3A mentary Fig. S1A). Control Flt3 + /ITD ; Dnmt3a fl /fl mice did not and TET2 ( 2, 3, 8–11 ). It is now recognized that DNA methyl- die in this time period. Conversely, doubling the number of transferase DNMT3A is one of the more commonly mutated Flt3 ITD alleles signifi cantly shortened survival, with a median genes in AML, affecting 23% of cases, and is associated with of 43 days after birth ( Fig. 1B , left and lower table). All an unfavorable prognosis ( 3, 12–14 ). Intriguingly, 36% to Flt3 ITD/ITD ; Dnmt3a fl /fl MxCre mice exhibited splenomegaly 44% of patients with FLT3 -mutant AML also carry DNMT3A ( Fig. 1B, right) and were moribund by 60 days. Secondary mutations. Furthermore, the co-occurrence of DNMT3A and and tertiary transplantation of Flt3 ITD/ITD ; Dnmt3a fl /fl MxCre FLT3 mutants changes the risk classifi cation to a poorer splenocytes resulted in lethality, albeit with delayed latency prognosis ( 3, 12, 13 ). (Supplementary Fig. S1B and data not shown). Because the The mammalian DNA methyltransferases DNMT1, disease occurred spontaneously (pIpC induction of MxCre DNMT3A, and DNMT3B methylate the cytosine residue expression was not required), we examined the Dnmt3a fl /fl at 5 position (5mC) in 5′-C-phosphate-G-3′ (CpG) dinu- alleles ( Fig. 1C ). As expected, tail DNA from 2-week-old cleotides. Methylation of CpG sites at promoter-associated Flt3 ITD/ITD ; Dnmt3a fl /fl MxCre mice showed amplifi cation of CpG islands is thought to silence expression of neighbor- LoxP -fl anked Dnmt3a exon 18 ( Fig. 1C ; “fl oxed” indicates ing genes. In addition, methylation of CpG dinucleotides no recombination; “deleted” indicates recombination). How- can also occur at the gene body or intergenic regions, ever, total or fractionated c-KIT + (CD117) stem/progeni- where its role in gene regulation is less clear ( 15 ). Different tor enriched splenocytes from Flt3 ITD/ITD ; Dnmt3a fl /fl MxCre de molecular subtypes of CN-AML have highly divergent DNA novo or secondary transplant recipients demonstrated both methylation and transcription profiles ( 16, 17 ). Recurring fl oxed and deleted Dnmt3a alleles ( Fig. 1C ). In addition, we missense mutations in DNMT3A identified in CN-AML act plated c-KIT + Flt3 ITD/ITD ; Dnmt3a fl /fl MxCre hematopoietic cells as loss of function or dominant negatives, significantly in methylcellulose, isolated DNA from individual colonies, reducing the methyltransferase activity of DNMT3A by and then analyzed fl oxed Dnmt3a alleles. Of 77 colonies approximately 80% ( 11, 18, 19 ). Although the mechanisms analyzed, 28 lacked detectable fl oxed alleles (36%); thus, the of DNMT3A action in the pathogenesis of AML are still majority of cells spontaneously deleted only one allele of unclear, studies on stem cells demonstrate essential roles Dnmt3a (Supplementary Fig. S1C). In agreement with this, for all DNMT proteins. DNMT1 is required to maintain the mRNA from c-KIT + cells from Flt3 ITD/ITD ; Dnmt3a fl /fl MxCre global DNA methylation through successive rounds of cell mice showed sporadic low-level expression of Cre and approx- division, whereas the de novo methyltransferases DNMT3A imately 50% reduction in Dnmt3a expression ( Fig. 1D ). These and DNMT3B are required for mouse embryonic develop- data suggest that sporadic MxCre expression leads to excision ment ( 20 ). Serial transplantation of Dnmt3a knockout of fl oxed Dnmt3a alleles, and that Flt3 ITD/ITD cells with a single hematopoietic stem/progenitor cells (HSPC) results in Dnmt3a allele may have a selective advantage. impaired differentiation, enhanced proliferation, and dif- Examination of the blood, bone marrow, spleen, and liver ferential methylation of distinct genetic loci, resulting of moribund Flt3 ITD/ITD ; Dnmt3a fl /fl MxCre mice demonstrated in upregulation of stemness genes and proto-oncogenes pathologic features of myeloid leukemia. The peripheral and downregulation of differentiation genes ( 21 ). Despite blood was notable for anemia and for leukocytosis of neu- this, Dnmt3a knockout HSPC-reconstituted mice do not trophilic and monocytic cells, including immature forms develop AML. (Supplementary Table S1 and data not shown). Bone marrow FLT3 ITD-mutant receptors
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