Abstract

Disruption of the epigenome can contribute to both cancer initiation and tumor progression. The methylcytosine dioxygenase TET2, which is involved in the regulation DNA methylation patterns, is frequently mutated in Acute Myeloid Leukemia (AML) patients and plays a central role in normal hematopoiesis. However, Tet2–deficient mouse models do not readily develop acute leukemia suggesting that additional oncogenic events are necessary for full-blown transformation.

The translocation t(8;21)(q22;q22) results in expression of the AML1-ETO (AE) fusion protein and is present in ∼5-10% of AML patients. Interestingly, the combination of t(8;21)(q22;q22) and TET2 mutations, although rare, have been observed in several studies in patients groups covering both pediatric and adult de novo AML. To confirm a direct role of TET2 mutations in the development of t(8;21)(q22;q22)-rearranged leukemias we expressed the AE fusion protein in hematopoietic stem and progenitor cells (HSPC) isolated from WT or Tet2-deficient animals. Serial replating assays in semisolid media as well as growth in liquid culture suggested a cooperative effect of AE and Tet2 deficiency as indicated by a significant increase in proliferation and colony formation efficiency. We then used a bone marrow transplantation model to assess the in vivo leukemic potential of AE;Tet2-/- HSPCs. The majority of recipient mice transplanted with AE;Tet2-/- (n=5/6) HSPCs succumbed to an aggressive and transplantable AML-like condition with a median latency of 5-6 months, whereas mice receiving AE HSPCs remained healthy over a 12 month observation period (n=6/6). The morbid mice showed many signs of disease including loss of weight, anemia, splenomegaly and large number of leukemic blasts (CD45neg) in circulation. Further analysis of the bone marrow and spleen revealed a massive expansion of a linneg (CD3e, Gr-1, Mac-1, B220, Ter119), c-Kitlow-high, CD16/32pos cell population indicating a block of differentiation and expansion of an immature myeloid progenitor population from AE:Tet2-/- HSPCs. To investigate how Tet2-deficiency contributes to transformation of t(8;21)(q22;q22)-rearranged cells we generated and cultured pre-leukemic AE-transduced cells in vitro from Tet2fl/fl HPSCs harboring the inducible Rosa26-CreERT2 allele. Genome-wide analysis of methylation patterns and gene expression showed that induction of Tet2-deficiency in pre-leukemic cells leads to progressive hypermethylation of gene-regulatory elements and altered expression of several genes implicated in tumorigenesis. Thus, this study illustrates how aberrant DNA methylation patterns can contribute to disease and confirm the role of Tet2 as a tumor suppressor preventing leukemogenesis.

Disclosures:

Helin:EpiTherapeutics : Consultancy, Equity Ownership.

Author notes

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Asterisk with author names denotes non-ASH members.

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