In:
Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 4195-4195
Abstract:
The popular concept that human cancers might be driven by rare self-renewing cancer stem cells (CSCs) has extensive implications for cancer biology and modelling, as well for development of more efficient and targeted therapies. However, experimental support for the existence of distinct and rare CSCs in human malignancies remain contentious, particularly in light of compelling evidence that cancer-propagating cells frequently fail to read out in existing human stem cell assays. Therefore, to unequivocally establish the existence and identity of human CSCs, the challenge is first to identify candidate CSCs, and to establish their unique ability to self-renew and replenish molecularly and functionally distinct non-tumorigenic progeny followed by functional in situ validation within the patients themselves. Methods We have in the hematological malignancy myelodysplastic syndromes (MDS) characterize candidate hematopoietic stem and progenitor stages in the bone marrow of low-intermediate risk MDS patients by flow cytometry. Distinct cell populations were functionally characterised for lineage commitment in standard colony forming cell (CFC) assays, and for self-renewal potential in long-term culture initiating cell (LTC-IC) assays and in immune-deficient (NSG) mice. Moreover, we tracked the cellular origin of all identified somatic genetic lesions identified in each patient by targeted next-generation sequencing of genomic DNA isolated from each purified MDS stem and progenitor cell population. Results In low-intermediate risk MDS patients, regardless whether they were del(5q) (n=19) or non-del(5q) (n=11), we could identify rare but distinct Lin-CD34+CD38-CD90+CD45RA- candidate stem cells, granuclocyte-monocyte progenitors (GMPs) and megakaryocyte-erythroid progenitors (MEPs) with frequencies within total BM similar to that of normal age-matched controls. Global gene expression analysis by RNA sequencing of MDS stem cells, GMPs and MEPs suggested that these are molecularly distinct populations. Myeloid and erythroid gene expression signatures were restricted to the GMPs and MEPs, respectively, whereas a transcriptional stem cell signature was restricted to the MDS stem cells. GMPs and MEPs isolated from del(5q) (n=12) and non-del(5q) (n=8) MDS patients displayed lineage-restricted myeloid and erythroid differentiation potentials, respectively. Self-renewal in LTC-IC assay was restricted exclusively to MDS Lin-CD34+CD38-CD90+CD45RA- stem cells in del(5q) (n=11) and non-del(5q) (n=8) MDS patients. Xenotransplantation into NSG mice also confirmed that only Lin-CD34+CD38-CD90+CD45RA- MDS stem cells have in vivo self-renewal potential, and these experiments also demonstrated their ability to replenish downstream CMPs, GMPs and MEPs, establishing the hierarchical relationship of MDS stem and progenitor cells. Targeted DNA sequencing of 88 genes recurrently mutated in MDS and other myeloid malignancies was pursued to identify somatic genetic lesions within the bulk bone marrow of MDS patients (n=13). In total we identified 30 presumed genetic driver lesions, including del(5q) and mutations in key transcription factors (RUNX1), signalling pathways (JAK2, CSF3R), epigenetic regulators (TET2, ASXL1), apoptosis regulators (TP53), and spliceosome components (SF3B1, SRSF2, U2AF2, SRSF6). Importantly, in support of their unique ability to self-renew and replenish lineage-restricted MDS progenitors, all stable somatic genetic lesions identified could in each MDS patient be backtracked to the rare stem cell population as defined phenotypically by flow cytometry and functionally by LTC-IC or xenograft potential, unequivocally establishing their unique stem cell identity within the malignant clone. Conclusions These findings provide definitive evidence for the existence of rare and distinct stem cells in MDS, a finding with extensive implications for therapeutic strategies in MDS and other cancers whose existence might also strictly depend on the persistence of rare CSCs. MDS stem cells typically acquire multiple driver mutations, together conferring a competitive advantage over normal stem cells, while even in combination failing to inflict self-renewal ability on MDS myelo-erythroid progenitor cells. Disclosures: No relevant conflicts of interest to declare.
Type of Medium:
Online Resource
ISSN:
0006-4971
,
1528-0020
DOI:
10.1182/blood.V122.21.4195.4195
Language:
English
Publisher:
American Society of Hematology
Publication Date:
2013
detail.hit.zdb_id:
1468538-3
detail.hit.zdb_id:
80069-7
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