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Early epigenetic changes in KM3-AML
progression by enhancing cell cycle progression.58 Forced expression of DNMT3B delayed KM3 leukemia develop- ment59 whereas the deletion of DNMT1 prevented KM3 AML progression.60 These findings and our own data demonstrate that the loss of de novo methylation, but not necessarily maintenance methylation, is required for the induction of AML.
Analysis of the correlated epigenetic and gene expres- sion changes highlighted a number of genes of interest, including ADCY9, an atypical member of the adenylate cyclase family. ADCY9 shows low but relatively broad expression in most tissues except blood (of all lineages61) (Online Supplementary Figure S12), and is capable of inducing cAMP accumulation,62 although its activation with G protein subunits appears to be cell-type specific. Our functional studies demonstrate that ADCY9 func- tion is critical for the growth of KM3-AML cells, and lev- els of cAMP in leukemias63,64 have been shown to be important. Nevertheless, whether the growth defects seen in response to the loss of ADCY9 expression are mediated through its ability to produce cAMP remains unclear and will require further study. The integration of our data also highlighted the potential importance of the chemokine receptor gene CCR1 and one of its ligands, CCL23. This receptor-ligand pair were noteworthy in part because of their strong level of upregulation, but also because the expression of both increased immedi- ately after the addition of the gene fusion, prior to the complete transformation of the cells. The CCR1 receptor can recognize at least ten different ligands including CCL3 and CCL5, which are both ubiquitously expressed in normal blood cells. In contrast, CCL23 is only expressed at very low levels in promyelocytes65 (Lin- FSChiSSCintCD34-CD15intCD49dhiCD33hiCD11b-CD16-) and granulocyte-monocyte progenitors65 (Lin- CD34+CD38+CD45RA+CD123+) but is highly expressed in KMT2A-AML according to a number of independent datasets. The impact of CCR1 signaling in AML remains poorly defined, although one consequence appears to be activation of the Gai pathway resulting in the inhibition of cAMP production.66 In this context, it is interesting to note that ADCY9 is specifically upregulated in KMT2A- AML (Figure 2F), potentially as counterbalance to the activity of CCR1 and Gai and to maintain cAMP levels.
Perhaps most surprisingly with respect to chromatin structure and accessibility, while we expected to see widespread changes, in fact there were relatively few modifications specific to the model leukemia cells com- pared to normal blood cells. This observation agrees with those of a recent study showing that KMT2A-MLLT3 helps to preserve gene expression of the cellular states in which it is expressed.67 As noted, most of the leukemia- specific regions overlap open chromatin regions and tran- script factor clusters seen in large-scale ENCODE datasets. Although speculative, these observations sug- gest the interesting hypothesis that there may be a finite number of regions in the genome that are competent to be open in vivo across all cell types. The addition of the KM3 fusion, while activating the expression of a subset of genes, may also cause re-localization of chromatin, lead- ing to the opening of regions not normally seen in blood cells. Thus, while chromatin remodeling is required to give access to endogenously expressed transcription fac- tors in some of the novel locations that we characterized in our study, others may simply be a response to this
remodeling. The distribution of ATAC peaks that we observed, largely in introns and intergenic regions (Figure 3D), also raises the question of the potential biological role as novel enhancers. Similar distributions in ATAC- sequencing data have been noted68 and intronic enhancers have been shown to regulate the expression of genes in a tissue-specific manner,69 which may explain why many of the ATAC-sequencing peaks not present in blood cells nevertheless corresponded to open chromatin regions in other cell types. These results open a new avenue to look at some of these regions of potential interest in the con- text of AML, to better understand their role in enabling access to genes which may be essential for leukemic growth.
In conclusion, here we have leveraged the advantages of our model system to characterize the immediate impact of the KM3 fusion without the complicating fac- tor of additional mutations and other genetic abnormali- ties typically found in patients’ genomes. While our results highlight the subtlety of the changes that occur once the oncogene is introduced, they also support the role of previous genes implicated in leukemogenesis and highlight a novel receptor/ligand pair with potential func- tional relevance for KMT2A-AML. The novel insight into the nature and extent of epigenetic reprogramming that occurs in the disease will help to guide future studies to identify novel potential therapeutic targets.
Disclosures
No conflicts of interest to disclose.
Contributions
BTW and TM designed the study, analyzed data and drafted the manuscript. TM and KL performed ChIP-sequencing and ATAC-sequencing, analyzed the data and generated figures. MC performed methyl-sequencing experiments, analyzed data, generated figures and drafted the manuscript. SS-T-H assisted with the ATAC-sequencing analysis and generation of figures. KL, ER, EB, and AB helped with molecular biology work, cloning and cell cultures. EB, AB and FB generated the model on mice from CD34+ cells. SM provided expression and clinical data for patients’ samples and JH and SC collected, character- ized, and banked the local patients’ samples used in this study. All authors discussed the results and participated in the revision of the manuscript.
Acknowledgments
We would like to thank the clinicians and nurses at CHU Sainte-Justine, CHU de Québec, Hotel Dieu de Lévis for cord blood collection and we also wish to acknowledge the contribu- tion of all of the courageous patients who provided samples used in this study. Finally, we would like to acknowledge Jennifer Huber, Sarah Boissel, Raphaëlle Lambert, Danièle Gagné and Annie Gosselin for their expert technical assistance with experi- mental work.
Funding
This work was supported by grants from the Cole Foundation (to TM, fellowship), the Impact Grant of the Canadian Cancer Society in collaboration with The Cole Foundation, Molson Foundation, R. Howard Webster Foundation, Mirella and Lino Saputo Foundation, Fonds de Recherche du Quebec - Santé, Faculté de Médicine, Université de Montréal, Letko Brosseau, Birks Family Foundation, Maryse and William Brock, CHU Sainte-Justine Foundation, Montreal Children's Hospital
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