Hematopoietic Stem Cells Are the Disease-Initiating Cells in the Myelodysplastic Syndromes

Abstract 789

Myelodysplastic syndrome (MDS) is a disorder of ineffective hematopoiesis presumed to originate from self-renewing clonal hematopoietic stem cells (HSC). Previous work has shown that immunophenotypic HSC from MDS patients harbor characteristic clonal cytogenetic abnormalities such as del(5q) at high levels, strongly suggesting that the HSC is the MDS-initiating cell (Tehranchi R., et al., NEJM, 363:11;1025-37, 2010); however, these studies did not examine other cytogenetic subtypes of MDS, nor did they functionally evaluate the HSC from these patients for their ability to initiate disease. We began a molecular and functional evaluation of FACS-purified HSC (Lin-CD34+CD38−CD90+CD45RA-) from MDS patients. These studies showed that the frequency of HSC in MDS bone marrow is not expanded when compared to normal, age-matched control samples. Annexin V staining also demonstrated no difference in apoptosis levels in MDS HSC compared to normal HSC; however, MDS committed myeloid progenitors (Lin-CD34+CD38+) exhibited increased apoptosis compared with normal progenitors (18% vs 39%, respectively, p <0.05). Transciptome analysis of FACS-purified MDS HSC from 10 low-risk MDS patients compared with HSC from an equal number of normal adults showed dysregulation of 3,258 mRNAs (FDR <0.1) including increased expression of genes positively associated with cell growth and proliferation (p < 0.001) and increased expression of inflammatory response genes (p < 0.015). In addition, there was widespread downregulation of numerous ribosomal protein transcripts in non-5q MDS including RPS6 and RPS19, but not RPS14 (p < 0.05). When FACS-purified HSC from a group of low-risk MDS patients were evaluated for the presence of known FISH abnormalities, the vast majority of HSC in MDS patients with defined cytogenetic abnormalities harbored clonal abnormalities (n=5, range 84–92% of total HSC) but they were not completely replaced, suggesting that non-MDS clones co-exist with MDS clones in MDS patient bone marrows. Finally, we show that FACS-purified MDS HSC can engraft irradiated, immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) pup recipients transplanted with as few as 1000 purified HSC. Long-term engraftment (assessed >12 weeks) was achieved with 50% of MDS samples tested (4/8), and resulted predominantly in myeloid engraftment with 0.8–5% total hCD45+ chimerism in the bone marrow. For each MDS HSC engrafted mouse, engraftment of the MDS clone was verified by FISH by detecting previously characterized cytogenetic abnormalities in FACS-sorted hCD45+ cells. The frequency of FISH positive cells was similar to that seen in the primary samples, suggesting no competitive disadvantage of MDS HSC in the xenotransplantation assay. Interestingly, methylcellulose colony and clonal liquid culture assays initiated from FACS-purified MDS HSC consistently grew poorly, suggesting that in vitro assays of hematopoietic potential may not accurately reflect MDS HSC biology. Together, these studies indicate that while MDS HSC are molecularly and functionally different from normal HSC, they are capable of engrafting immunodeficient NSG pups. Moreover, these data formally demonstrate that the HSC is the disease-initiating cell in MDS. This finding has significant implications for MDS research, as it provides a potential in vivo preclinical model for testing MDS therapeutics – an experimental model previously not available to investigators.