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T. Milan et al.
matched patients’ samples confirmed that the expression of both CCR1 and CCL23 is increased dramatically upon the integration of the KM3 fusion, while neither is expressed in normal CD34+ cells (Figure 3C). External RNA-sequencing data from the BloodSpot database (Online Supplementary Figure S9A) or TARGET cohort4 also confirm the KMT2A-specific expression of CCL23 (Online Supplementary Figure S9B) and there is a small but signifi- cant difference in survival of pediatric AML patients with high levels of CCL23 (Online Supplementary Figure S9C). Although previous studies have highlighted the impor- tance of CCR1 in different cancers (e.g., breast49 and prostate50 cancer) no studies have looked at its role in AML. Although we saw an inhibition of growth specifically in KM3-AML cell lines using a potent inhibitor of CCR1 (data not shown), this inhibition was only observed at levels (10- 20 mM) much higher than the reported half maximal inhibitory concentration (IC50) (9 nM) of the inhibitor.51 The very high expression levels of both the receptor and ligand may explain this observation; however, specific genetic models will be required to define the biological importance of this potential autocrine-signaling axis.
KM3-mediated epigenetic changes are minimal rela- tive to normal blood cells
Location analysis of the annotated ATAC-sequencing peaks unique to the model AML cells revealed the expect- ed peaks at promoters of active genes, although the majority of open chromatin regions were either inter- genic or intronic (Figure 3D). As a point of comparison for our ATAC-sequencing data, we downloaded previously published and normalized ATAC-sequencing data gener- ated from normal blood cells24 along with raw ATAC- sequencing data from normal CD34+ cells and monocytes from the BLUEPRINT project.52 Using peaks identified via MACS2 in our ATAC-sequencing data or remapped pub- lished data, or through supervised thresholding of nor- malized UCSC tracks, we compared regions of open chromatin in normal and leukemic blood cells. As with the histone modifications, the majority of peaks identi- fied in the model leukemia samples were shared with one or more normal types of blood cell. Considering the 10,415 peaks identified by MACS2 that were specific to the model AML (Online Supplementary Figure S10), analy- sis of potential transcription factor binding motifs once again identified factors, such as PU.1, that are critical for normal myeloid cell differentiation (Online Supplementary Figure S11). Hierarchical clustering of all ATAC-sequenc- ing peaks from the CD34+KM3 and model AML revealed that the chromatin landscape in the model AML is close to that of normal monocytes and CD34+ HSPC (Figure 3E). The clear separation of CD34+KM3 cells, despite the small absolute number of unique peaks (and more peaks common to CD34+ HPSC than to monocytes), suggests that the chromatin of these cells is subtly affected by their culture in vitro.
Using data from the ENCODE consortium, we further examined the transcription factors that have been experi- mentally observed to bind to leukemia-specific open chromatin regions. For this, we ranked the abundance of individual factors in the entire dataset, and in the leukemia-specific peaks, to account for the difference in frequencies of binding sites for individual factors (Figure 3F). This analysis revealed an enrichment of factors in the leukemia-specific peaks that did not necessarily correlate
with an increase in expression of the factor. Given the ChIP-sequencing data, this suggests that the novel, leukemia-specific peaks may create competition for the binding of a range of expressed transcription factors, potentially co-opting their activity. In other cases, while there was no difference in the ranking, the factor itself was more highly expressed; in cases in which such factors have been demonstrated to be essential for leukemia development (e.g., FOS53), the higher expression level may be important to ensure a high/continuous occupancy at the open regions in order to drive the leukemic tran- scriptional program.
Across the genome, we observed many examples of genomic regions in which ATAC-peaks common to both leukemic and normal cells were seen in the vicinity of other leukemia-specific peaks (Figure 3G). Given our observations, we questioned whether all of the leukemia-specific peaks we observed were potentially functional or whether they might represent stochastic changes in accessibility. To address this question, we compared data for chromatin accessibility (DNaseI hypersensitive sites) and ChIP-sequencing data from the ENCODE consortium to the ATAC-sequencing peaks that were unique to the model leukemias. Interestingly, the peaks determined by either MACS2 or supervised thresholding showed high levels of overlap (65% and 75%, respectively) with both previously defined tran- scription factor clusters, DNaseI hypersensitive sites and overlapped chromatin marks associated with enhancers seen in other cell types. These data argue that despite their unique presence in leukemic cells, the large majori- ty of leukemia-specific open chromatin regions likely represent enhancers.
Discussion
The KMT2A-MLLT3 gene fusion is one of the most common genetic anomalies found in pediatric AML and is an epigenetic regulator that is essential, directly or indi- rectly, for the alterations of chromatin structure13,54,55 and DNA methylation seen in the disease.56 Despite its exten- sive characterization, less is known regarding its role in the initial transformation of HSPC into leukemic cells. Our current study leverages our previously described human model leukemia system to integrate epigenetic and expression changes to better characterize these changes. With respect to DNA methylation changes, our data confirm previously published results57 showing that an aberrant hypomethylation profile is found in KM3 leukemias. Interestingly, relative to the initial CD34+ cells, CD34+KM3 and B-cell ALL have a hypermethylation phenotype, whereas the model leukemia and patients’ AML show the reverse. While the significance of all of these global differences is not yet clear, previous single- cell studies showed that differentially methylated pro- moter regions in normal blood cells were enriched in binding sites for transcription factors promoting myeloid differentiation.44 Our data (e.g., Online Supplementary Figure S7) agree with this finding, which may reflect a default lymphoid differentiation pathway in the absence of demethylation of these regions. Recent studies have highlighted the importance of DNA methyltransferase in KM3-mediated leukemogenesis in which DNMT3A and DNMT3B loss-of-function mutations accelerate leukemia
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haematologica | 2022; 107(1)