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A. Beke et al.
gene body (P<2.2 10-16) and intergenic enhancers (P<0.001) and a significant increase in methylation at promoter regions (P<0.001) and CpG islands (P<0.001) (Figure 7C). Importantly, using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, KRAS(G12D) acqui- sition was shown to induce significant changes in Wnt and Hedgehog signaling pathways, which may indicate a differential role for these pathways in the malignant trans- formation of the different clones (Figure 7D).
Discussion
The reprogramming of CD34+ cells from a CMML patient generated iPSC whose hematopoietic differentia- tion recapitulated the main features of the disease while demonstrating functional heterogeneity between clones (see graphical abstract in Figure 8).
Discrepancies between the functional properties of clones sharing a similar genetic background have been reported in organoids derived from colorectal cancer cells.24 We cannot exclude an effect of recurrent somatic mutations in non-coding regions, as described in some solid tumors,25 but such events have not been identified yet in CMML cells.6 A role for cell reprogramming26,27 in this heterogeneity is also unlikely as control iPSC estab- lished independently from two healthy donors had a sim- ilar and reproducible behavior, in contrast to the differ- ences observed between genetically identical CMML iPSC (Figure 4J). In addition differences sometimes observed between individual clones derived from the same genotype were shown to pre-exist in the tissue of origin rather than being induced by reprogramming.28,29 The distinct behavior of CMML-derived iPSC could also reflect intrinsic heterogeneity in CD34+ cell-priming for differentiation.30,31 If such an intrinsic heterogeneity of CD34+ cell-priming is a general property of CD34+ cells, discrepancies should also have been observed between control iPSC. An alternative explanation is that heteroge- neous priming is a specific feature of CMML progenitor cells, which could be related to intraclonal epigenetic het-
erogeneity. Our epigenetic analyses indicate that, in patient-derived clones, the global pattern of DNA methy- lation correlates with genetic alterations. However, limit- ed differences in their methylation profiles could possibly account for their functional heterogeneity.
iPSC allow the combined investigation of all levels of intratumoral heterogeneity, from genetic, to epigenetic, to phenotypic and functional properties associated with the disease, offering unique opportunities to study diseases in which functional heterogeneity exceeds genetic hetero- geneity, such as CMML.4 These benefits do, however, come at a cost. As recently reviewed,15 the derivation of iPSC from patients with a myeloid malignancy to model their disease has to face the relative refractoriness of malignant progenitors to reprogramming, which is in part related to their genetic background and could preclude the capture of intraclonal heterogeneity as some subclones may be reprogrammed more easily than others. The dys- plastic nature of these cells, which often correlates with an increased sensitivity to apoptosis, is another challenge to overcome. Lastly, in the patients, the heterogeneous behavior of individual cells may be further influenced by clonal interference and non-cell-autonomous factors,32 contributing to the diversity of CMML phenotypic traits, and accounting for the current failure of treatments aiming at eradicating the malignant clone.
Acknowledgments
The authors would like to thank Dr Weiss for kindly providing a control iPSC clone. This work was supported by grants from the Ligue Nationale Contre le Cancer (équipe labelisée), Institut National du Cancer (INCA_8073; PRT-K 16-047), Fondation ARC (to AB), Cancéropole Ile de France (emergence program to LL), Siric SOCRATE (INCa-DGOS-INSERM_12551), Molecular Medicine in Oncology program (Agence Nationale de la Recherche), Gustave Roussy Cancer Center (taxe d’apprentis- sage), the University of Miami Sylvester Comprehensive Cancer Center, the Sylvester Comprehensive Cancer Center Onco- Genomics Shared resource, and the John P. Hussman Institute for Human Genomics, Center for Genome Technology at the University of Miami Miller School of Medicine.
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