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Early epigenetic changes in KM3-AML
variants may play a greater role, is extremely challenging and increases the complexity of identifying novel poten- tial targeted therapeutics. Studies underscore the fact that even though the mutational burden in AML is one of the lowest of any cancer4 there is still tremendous hetero- geneity between leukemic patients which creates chal- lenges when trying to define the genetic determinates of the disease.
As noted, chromosomal fusions are common in pedi- atric AML, particularly those involving the lysine specific methyl transferase 2A gene (KMT2A; also known as the mixed lineage leukemia [MLL] gene). KMT2A is rearranged in approximately 10% of all leukemias6 but the frequency in infant acute lymphocytic leukemia (ALL, >70%7) and AML (>35%7) is much higher. The KMT2A gene encodes a large (500 kDa) and complex 38-exon pro- tein8,9 that can be cleaved by taspase-1 into two separate fragments (KMT2A-C and KMT2A-N).10 The conserved SET domain located at the C-terminus of the protein is responsible for the methylation of the histone H3 at lysine 4 (H3K4). Previous studies have shown that KMT2A is part of a large macromolecular complex com- posed of different proteins11 that function to improve the stability of KMT2A allowing the complex to regulate the transcriptional activation of HOX genes. The KMT2A gene can be fused in-frame with more than 120 different partners, creating fusion proteins that are typically associ- ated with poor prognosis leukemias.6 One of the most common fusion partners is the MLLT3 gene (also known as AF9), which is found in 30% and 13% of KMT2A- rearranged AML and ALL,12 respectively; this fusion is associated with an intermediate risk. The KMT2A- MLLT3 (KM3) fusion protein is part of the DOT1L com- plex (DOTCOM) and leads to aberrant expression of spe- cific target genes marked by H3K79 methylation.13 Despite the identification of several key gene targets required for transformation, the complete molecular mechanisms utilized by this oncogenic fusion are still unclear.
The strong oncogenic potential of KMT2A fusions makes them ideal drivers for in vivo experimental model systems to explore molecular mechanisms involved in leukemogenesis.14 To specifically overcome the problems of patients’ genetic heterogeneity and scarcity of samples, we have developed a single-donor, human model leukemia system using healthy CD34+ cord blood HSPC.15 This adaptation of a prior human model16 uses a retro- virus to deliver the human KM3 fusion gene which we have shown is sufficient to generate a human leukemia.15 Transduced cells are cultured in vitro for 30-40 days prior to injection into immune-deficient mice which develop a leukemia 24-30 weeks later. Because we can sequence the transcriptome/genome/exome of the initial CD34+ cells used to generate the model AML, the genetic background (e.g., single nucleotide variants) of the initial donor is well defined, allowing the potential role of acquired mutations to be assessed precisely. In addition to these advantages, this model also allows the study of genetic mechanisms involved in the initiation of the disease, when the fusion is first introduced but before the complete transformation of the cells. Our previous genomic analyses demonstrated that the oncogenic fusion alone is sufficient for the devel- opment of leukemias, without the requirement for any recurrent secondary mutations,15 despite the presence of these in pediatric patients with KMT2A-mutated AML.
We have also used these data to identify a number of genes that represent novel biomarkers in patients with KMT2A fusions.17 Interestingly, while approximately one- third of these biomarker genes were expressed in our model leukemia system shortly after cells were trans- duced with the KM3 fusion, the majority were only expressed after xenotransplantation. This observation, coupled with the limited potential for in vitro growth of these cells, suggests that cells partially transformed (or “primed”) by KM3 may require additional in vivo signals (e.g., from the bone marrow niche) to complete their leukemic transformation. The single-donor model leukemias therefore not only recapitulate the behavior and phenotype of the disease, but also provide an exper- imental system to study genetic and epigenetic mecha- nisms involved in the disease, and a unique insight into early stages of transformation.
In the present study, we leverage these advantages to perform a detailed epigenetic analysis of the various stages of the model and, through correlation with model and expression data from patients with leukemia, define the epigenetic changes driven by the oncogenic fusion that contribute to leukemia development. Analysis of changes in the patterns of DNA methylation confirmed previous observations of profound hypomethylation in KMT2A-translocated AML. Interestingly, however, B-cell ALL driven by the same fusion show a smaller number of differentially methylated cytosines relative to HSPC methylation, which are predominately hypermethylated. Despite the generally poor global correlation between DNA methylation and gene expression, we identified ADCY9, a member of the adenylyl cyclase family, as a gene essential for KM3-AML growth that also exhibits coordinated changes in DNA methylation and gene expression. We further characterized changes in chro- matin accessibility through an assay for transposase- accessible chromatin with high-throughput sequencing (ATAC-sequencing), along with alterations in histone modifications including H3K4me3 and H3K79me2. Interestingly, our ATAC-sequencing analysis revealed that the vast majority of regions of open chromatin are shared with normal HSPC and monocytes, with very few being specific to the leukemic cells. Even those regions that are unique to the leukemia show a high degree of overlap with regions of open chromatin seen in other cell types and are also characterized by the presence of known clusters of transcription factor binding sites. Integration of the epigenetic data with expression data highlighted a role for the histone demethylase KDM4B during the initial stages of leukemic transformation through its impact on S100A8/S100A9 expression levels, whose relative ratios have been demonstrated to be criti- cal for blocking differentiation of leukemic cells.18 Collectively, these results suggest that the leukemic trans- formation of normal HSPC by KM3 involves a very subtle epigenetic shift that also implicates co-option of normal transcriptional networks.
Methods
Patients’ samples and model leukemia generation
All pediatric and adult AML patients’ samples and additional clinical information used in this study were collected by the Banque de Cellules Leucémiques du Québec (BCLQ) with
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