Myelodysplastic syndrome hematopoietic stem cell


Correspondence to: June Li, MD, PhD, Department of Genetics, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Box 1010, Houston, TX 77030, USA, Tel.: +1-832-235-0826, E-mail:


Myelodysplastic syndromes (MDSs) are clonal hematopoietic stem cell (HSC) malignancies that are characterized by ineffective hematopoiesis and frequent progression to acute myeloid leukemia (AML). Thus far, few treatments can actually alter the natural history of this disease. Allogeneic stem-cell transplantation for high-risk MDS is becoming the only curative therapy probably because of the improvement of bone marrow transplant procedures. The lack of other options underscores the urgent need to develop new therapy. The prevailing model suggests that genetic and/or epigenetic alterations that occur in HSCs or HSC niche compromise HSC function, resulting in MDS; therefore, MDS HSCs are likely the ideal targets for MDS treatment. Recent encouraging advances—capturing a molecular portrait of the whole genome of MDS CD34+ cells, including identifying altered signaling pathways and altered microRNAs—have improved our understanding of MDS pathogenesis and provided novel potential clinical targets for MDS. Here, I will briefly review the characteristics of MDS HSCs and discuss the therapeutic promise of targeting MDS HSCs.

Myelodysplastic syndromes (MDSs) are a heterogeneous group of clonal hematopoietic stem cell (HSC) malignancies that are characterized by ineffective hematopoiesis in one or more of the lineages of the bone marrow. They are preleukemic conditions with a high risk of progression to acute myeloid leukemia (AML). MDS can arise de novo or as a consequence of previous chemo- or radiotherapy in cancer patients. MDS is considered a disease related to aging.[1-4] However, there is a pediatric population of MDS patients (such as Fanconi anemia, Shwachman–Diamond syndrome, severe congenital neutropenia, dyskeratosis congenital, and Diamond–Blackfan anemia) in which inherited bone marrow-failure syndromes are related high-risk factors. Many different therapies for MDSs have been developed based on the revealed molecular mechanisms of the diseases, for example, inhibitors of DNA methylation are recently proved to be effective in treating patients with MDS.[5-8] Although various treatments have improved the disease symptoms of some patients, few treatments can alter the natural history of the disease. Patients often die of complications that arise from cytopenia or from the subsequent AML. Allogeneic stem-cell transplantation for high-risk MDS is becoming the only curative therapy probably because of the improvement of bone marrow transplant procedures. The lack of other therapeutic options underscores the urgent need to develop new therapy. Furthermore, the likely increase in the incidence of MDS in the future owing to increased longevity and the increased number of cancer patients who survive chemo- and radio therapy, makes the identification of new targets in MDS for effective drug-treatments more urgent than ever. Recently, Tehranchi et al.[9] identified rare and phenotypically distinct MDS stem cells in patients with MDS with loss of long arm of chromosome 5 (5q-); these MDS stem cells were selectively resistant to therapeutic targeting at the time of complete clinical and cytogenetic remission, indicating their importance not only in the pathogenesis of MDS but also for the treatment of MDS. In this article, I will review the characteristics of MDS HSCs and discuss the therapeutic promise of targeting MDS HSCs.

Evidences that MDS is a stem cell disease

The existence of leukemia stem cells (LSCs) was proposed several years ago[10, 11] and has been confirmed by purification and clonal tracking experiments.[12-14] However, although MDS has been considered to be a clonal stem cell disorder, efforts to verify this hypothesis using xenograft mouse transplantation models (NOD/SCID mice as recipients) have failed, because the MDS cells either engraft poorly or not at all and the mice do not develop any signs of MDS.[15-19] Also, whether MDS occurs in a myeloid progenitor (i.e., that involves megakaryocytic, erythroid and granulocytic/ monocytic lineages) or occurs in a true HSC was open to debate, as dysplasias are rarely found within the lymphoid compartment. Therefore, identification of the cell of origin in MDS should facilitate a better understanding of MDS pathogenesis, and could be of crucial importance to the development of a new therapeutic strategy for MDS.

It is well known that LSCs originate from normal HSCs in many leukemia cases; however, to obtain the proof of leukemic clonal involvement of all blood cell lineages derived from the multipotent HSCs appears to be difficult, since most leukemias are lineage restricted in nature. It is believed that a transforming event in an HSC not only promotes the development of the dominating leukemic lineage but also simultaneously suppress the development of other lineages. This may also be the case for MDS. In addition, although lack of evidence for a normal HSC origin of MDS HSC could mean that MDSs rarely originate from normal HSCs or multipotent progenitors, it may not be feasible to prove that hypothesis through traditional approaches. An alternative approach would be to apply genomics to better identify the origin of MDS HSCs; as normal HSCs have been purified and found to have a distinct gene-expression profile when compared with normal progenitors.[20, 21]

Previously, Nilsson et al.[22] compared global gene expression profiles of CD34+CD38Thy-1+ MDS HSCs from 5q− syndrome patients with those of CD34+ CD38 Thy-1+ normal HSCs and CD34+CD38+Thy-1 progenitor cells from healthy subjects, and demonstrated a much closer identity between normal HSCs and 5q–MDS HSCs than that between 5q–MDS HSCs and normal progenitors, indicating that normal HSCs may be the origin of MDS HSCs. When fluorescence in situ hybridization assays were used to examine CD34+ CD38Thy1+ (CD90) cells from 11 patients with a 5q–cytogenetic abnormality, almost all cells (92% to 100%) in this HSC compartment appeared to be involved in the 5q- clone; however, long-term culture initiating cell activity could be demonstrated in only 50% of patients tested, suggesting that although 5q–MDS HSCs largely outcompete or suppress normal counterparts to grow, they are ineffective in reconstituting hematopoiesis. In another study,[21] among CD34+CD38 cells from 5q–MDS patients, 88–98% had 5q deletions. Furthermore, 5q deletion was frequently detected in the myeloid-committed progenitor cells (CD34+ CD33+) and less frequently detected in the lymphoid-committed progenitors, including pro-B cells (CD34+CD19+) and pro–T cells (CD34+CD7+). Together, these findings indicated that MDS is a stem cell disease. This concept is supported by the NUP98-HOXD13 (NHD13) mouse model, a transgenic MDS mouse model with NHD13 expression in hematopoietic tissues. In a study using NHD13 model,[23] MDS can be transplanted to the lethally irradiated WT recipient mice, as the recipients of MDS bone marrow cells from NHD13 mice displayed all of the critical features of MDS, including peripheral blood cytopenias, dysplasia, and transformation to AML; moreover, NHD13 cells significantly outcompeted the WT cells over a 38-week period in the recipients transplanted with 1:10 ratio of NHD13 bone marrow cells and WT counterparts. The MDS was also transferable to secondary recipients as a premalignant condition. These findings further suggest that MDS originates from a transplantable, premalignant, long-term repopulating, MDS-initiating cells.

An unresolved issue is that T cells and B cells in MDS patients appear not to be derived from the MDS HSC clones in many cases. One possible explanation is that the reconstituting common lymphoid progenitors and lymphoid progenitors can be long-lived[24-27]; thus, the B and T cells may be derived from lymphoid-restricted progenitors generated before the 5q deletion. A second possibility is that the genetic (and epigenetic) abnormalities that occur in the MDS HSCs make the cells either deficient in their ability to differentiate toward mature lymphocytes or subject to efficient deletion, and there is an efficient compensatory mechanism for lymphocyte production by “normal” hematopoietic cells in these patients. In fact, the latter possibility is supported by Sternberg et al.'s study[28] in which the global gene expression profile in CD34+ cells from an MDS patients with a normal karyotype were compared with those from healthy controls and patients with anemia from causes other than MDS. The MDS patients' profiles showed a great decrease in expression of a large set of genes related to B cell development, including VPREB1, IL7R, and PAX5. The lack of B cell differentiation from MDS stem cells would mean that peripheral mature B cells may be derived from residual normal hematopoiesis.

Deregulated signaling pathways in MDS HSCs

The molecular basis of MDS is now being elucidated, finding thus far includes nonrandom chromosomal copy number alterations (amplifications and particularly deletions), such as 5q−, loss of chromosome 7 (−7/7q−), deletion of chromosome 20q (20q−) and gain of chromosome 8; gene mutations in various classes of proteins, such as transcription factors (e.g., RUNX1), tyrosine kinase pathway members (e.g., RAS), cell-cycle inhibitors (e.g., TP53) and antiapoptotic factors; epigenetic abnormalities, such as DNA methylation, histone modifications and RNA interference; and impaired immune system, altered microenvironment as well as spliceosome mutation. It is a prevailing model that MDS is an HSC disorder in which genetic and/or epigenetic alterations compromise the function of HSCs and progenitors[29, 30]; therefore, HSCs are likely the more relevant population as compared with total bone marrow cells when investigating the underlying defects in MDS. Recent encouraging advances—capturing a molecular portrait of the whole genome of MDS CD34+ cells, including identifying altered gene expression profiles and related signaling pathways, as well as altered microRNAs—have improved our understanding of MDS pathogenesis and provided novel potential clinical targets for MDS treatment.

Early studies demonstrated that distinct gene expression profiling by microarray analysis in bone marrow derived HSCs and progenitors (CD34+ cells/AC133+ cells) may facilitate the separation of MDS from healthy controls,[31] MDS with monosomy 7 from MDS with trisomy 8,[32] low-risk MDS from high-risk MDS,[33] and MDS-associated leukemia from de novo AML,[34] indicating that distinct gene expression profiling may reveal distinct molecular mechanisms for each specific MDS subtype. However, this approach has some limitations. First, genes with moderate but meaningful expression changes may not meet the strict cutoff and alternation of molecular processes may be missed.[35, 36] These are particularly important when studying a complex disease, like MDS, that is associated with changes of expression of multiple genes. Second, frequently there is little overlap between lists of genes obtained by different groups investigating the same biological conditions.[37] These limitations could be overcome by signaling pathway analysis. Especially, better understanding of pathways deregulations may lead to improvement of therapeutic strategies.[38]

Pellagatti et al.[39] compared global gene expression profiles in CD34+ cells from 183 MDS patients and 17 healthy controls, and analyzed the related signaling pathways by using Ingenuity Pathway Analysis software, and found that the most significantly deregulated pathways in MDS include interferon signaling, thrombopoietin signaling, and the Wnt pathways (Table 1). Upregulation of interferon-stimulated genes and of the interferon signaling pathway is a major feature of MDS and may be responsible for some of the hematological characteristics of MDS, including cytopenias. “Thrombopoietin signaling” is the second most upregulated pathway in MDS. Thrombopoietin and its receptor (TPOR) have an important function in maintaining production of controlled numbers of megakaryocytes and platelets.[40] Moreover, thrombopoietin signaling has a function in HSC self-renewal.[41] Upregulation of the thrombopoietin pathway in MDS represents a possible mechanism for the observed megakaryocytic abnormalities in MDS and deregulated MDS stem cell self-renewal. The Wnt pathway has a critical function in the regulation of the self-renewal of HSC[42]; the significantly downregulated Wnt canonical pathway may lead to defective self-renewal of HSCs in this disorder. Among the most significantly deregulated gene pathways in early MDS (RA) are immunodeficiency, apoptosis, and chemokine signaling, suggesting that immune deregulation and activation of apoptosis pathways are characteristic of early MDS cells, consistent with clinically observed ineffective hematopoiesis. In contrast, advanced MDS (RARE2) is characterized by deregulation of DNA damage response and checkpoint pathways, such as ATM signaling. Disruption (downregulation) of the DNA damage checkpoints in advanced MDS results in an increase in the error rate of DNA repair with a concomitant increase in genomic instability, leading to evolution to AML (Table 2). For example, RAD51, which mediates homologous recombination repair of DNA double-strand breaks, is down-regulated in advanced MDS compared with healthy controls. Repression of RAD51 gene expression has been described in some tumors and is associated with genomic instability.[43, 44] It suggests that the mechanisms of AML evolved from MDS are distinct with de novo AML which does not result from genomic instability but is rather determined by a small number of oncogenic “driver” mutations.[45] Distinct gene expression profiles and deregulated gene pathways in patients with del(5q), trisomy 8, or −7/del(7q) have also been identified. Patients with trisomy 8 are characterized by deregulation of pathways involved in the immune response, such as T-cell receptor signaling; patients with −7/del(7q) have deregulation of pathways involved in cell survival, such as PI3K/AKT signaling; whereas patients with del(5q) have deregulation of integrin signaling and cell cycle regulation pathways (Table 3). The deregulated pathways identified give new insights into the molecular pathogenesis of this disorder, thereby providing new potential targets for therapeutic intervention in MDS.

Table 1. Some of the deregulated pathways for the upregulated and downregulated genes in MDS CD34+ cells
Ingenuity canonical pathwayRegulated moleculesMDS compared with healthy control
  1. Reprinted by permission from Macmillan Publishers Ltd: Leukemia, Pellagatti et al.,[39] 2010.
Interferon signalingIFIT3, IFIT1, IFITM1, MX1, IRF9, STAT1Upregulation
Thrombopoietin signalingPIK3R3, RRAS2, MPL (includes EG:4352), SOS1, STAT1, PRKCZ, PRKCAUpregulation
IL-3 signalingPIK3R3, PTPN6, RRAS2, SOS1, STAT1, PRKCZ, PRKCAUpregulation
Primary immunodeficiency signalingBLNK, RAG2, IGHM, NR3C1, CD79ADownregulation
B-cell receptor signalingBLNK, CD19, CD79B, GAB1, PAG1, PIK3AP1, MAP3K8, CD79ADownregulation
Wnt/β-catenin signalingTCF4, CD44, TLE4, TLE1, PPP2R2C, LEF1Downregulation
Table 2. Deregulated pathways in CD34+ cells from MDS subtypes RA and RAEB2 compared with in healthy controls
 Ingenuity canonical pathwayRegulated molecules
  1. Reprinted by permission from Macmillan Publishers Ltd: Leukemia, Pellagatti et al.,[39] 2010. Genes in regular font are expressed at lower levels in MDS compared with healthy controls, whereas genes in bold font are expressed at higher levels in MDS compared with healthy controls. Abbreviations: RA, refractory anemia. RAEB, refractory anemia with excess blasts.
RAPrimary immunodeficiency signalingPTPRC, RAG2, RAG1, IGHG1, NR3C1, CD79A, IGLL1
 Apoptosis signalingRRAS, BAX, CASP7, FAS, PRKCA
 Chemokine signalingPLCB2, RRAS, CXCR4, PRKCA
RAEB2Mitotic roles of polo-like kinaseKIF23, CDC20, PTTG1, PPP2R5B, CCNB2, CDC23 (includes EG:8697), SMC1A, CDC25B, PLK4, TGFB1, PPP2R2C, CDC16, CDC25A, CDC25C, ESPL1, CCNB1, PPP2R5C, WEE1, PRC1, ANAPC13, CDC2, PPP2CB, PPP2R4, PKMYT1, PPP2R1B, CDC27, STAG2, KIF11
 Role of CHK proteins in cell-cycle checkpoint controlCDC25C, MRE11A, RFC5, CDC2, CHEK1, E2F6, PCNA, TLK1, RFC4, E2F5, RFC2, E2F2, RFC3, CDC25A
 Role of BRCA1 in DNA damage responseRBBP8 (includes EG:5932), MRE11A, RBL1, RFC5, MLH1, CHEK1, RAD51, E2F6, RB1, FANCD2, RFC4, MSH6, RFC2, E2F5, BRCA2, FANCA, E2F2, RFC3
 Cell cycle: G2/M DNA damage checkpoint regulationCDC25C, CCNB1, WEE1, CCNB2, MDM2, CDC2, SKP2, CHEK1, CDC25B, MYT1, TOP2B, TOP2A, BTRC
 Cell cycle: G1/S checkpoint regulationCCNE2, TFDP1, RBL1, SKP2, HDAC5, RB1, E2F6, CCNE1, MAX, TGFB1, HDAC11, E2F5, BTRC, E2F2, CDC25A
Table 3. Deregulated pathways in CD34+ cells from MDS patients with 5q−,−7/del(7q), or trisomy 8 compared with in healthy controls
 Ingenuity canonical pathwayRegulated molecules
  1. Reprinted by permission from Macmillan Publishers Ltd: Leukemia, Pellagatti et al.,[39] 2010. Genes in regular font are expressed at lower levels in MDS compared with healthy controls, whereas genes in bold font are expressed at higher levels in MDS compared with healthy controls.
5q-Wnt/β-catenin signalingTCF4, PPP2CA, CSNK1G3, CSNK1A1, TLE1, PPP2R2C, LEF1, FZD7
 Role of NFAT in regulation of the immune responseBLNK, GNG11, CD79B, CSNK1G3, MS4A2, CSNK1A1, LYN, CD79A
 Integrin signalingRAP2B, PARVB, WIPF1, ARHGAP26, ARPC1B, ARPC2, CAPN2, CAPN3
7/del(7q)SAPK/JNK signalingMAP3K9, RRAS2, GAB1, PIK3C3, ZAK
 NF-κB signalingCSNK2A2, TRAF3, IL1F9, RRAS2, PIK3C3, MALT1
 PI3K/AKT signalingRRAS2, FOXO1, GAB1, PPP2R2C, RHEB
Trisomy 8Antigen presentation pathwayB2M, HLA-DMB, HLA-G, HLA-F
 Allograft rejection signalingHLA-DMB, IGHG1, HLA-G, HLA-F
 Autoimmune thyroid disease signalingHLA-DMB, IGHG1, HLA-G, HLA-F
 T-cell receptor signalingVAV2, LCK, PAG1, PIK3R1, ZAP70, MAPK12

Deregulated microRNAs (miRNAs) in MDS HSCs

Deregulated miRNA expression in MDS HSCs

Using miRNA arrays, Dostalova Merkerova et al.[46] measured expression of 1,145 miRNAs in CD34+ bone marrow cells obtained from 39 patients with MDS and AML evolved from MDS as compared to in CD34+ cells from six healthy donors. In MDS patients, 13 miRNAs were upregulated (miR-299-3p, miR-299-5p, miR-323-3p, miR-329, miR-370, miR-409-3p, miR-431, miR-432, miR-494, miR-654-5p, miR-665, HS_40 and HS_206), and nine miRNAs were downregulated (miR-196a, miR-423-5p, miR-525-5p, miR-507, miR-583, miR-940, miR-1284, miR-1305 and HS_122.1). Differences in miRNA expression in MDS CD34+ cells were shown between early and advanced MDS, and an apparent dissimilarity between the RAEB-1 and RAEB-2 MDS subtypes was also observed. In another study,[47] miRNA expression profiles were assayed in CD34+ cells from seven 5q− syndrome patients and five controls using high-density TaqMan arrays. Twenty-one miRNAs differed between 5q− syndrome patients and controls. These included 17 overexpressed miRNAs (hsa-miR-10a, hsa-miR-10b, hsa-miR-125a, hsa-miR-125b, hsa-miR-126, hsa-miR-130a, hsa-miR-148a, hsa-miR-151, hsa-miR-199a, hsa-miR-199b, hsa-miR-24, hsa-miR-29c, hsa-miR-335, hsa-miR-34a, hsa-miR-451, hsa-miR-486 and hsa-miR-99b) and 4 underexpressed miRNAs (hsa-miR-128b, hsa-miR-213, hsa-miR-95 and hsa-miR-520c). However, the physiological functions of each of the expression-altered miRNAs, and how each of them contributes to the related MDS-phenotype remain largely unknown. Some other similar studies[48-52] used bone marrow cells from patients and healthy controls; little overlap is seen in the deregulated miRNAs among those studies, as well as compared with those identified in CD34+ cells. This variation suggests that a consistent sample source is necessary to more effectively uncover deregulated miRNA expression signatures in MDS. Because of the heterogeneity of MDS, the small numbers of samples in the studies may also contribute to the discrepancies in the results; future studies should implement larger patient cohorts.

Mechanisms of deregulated miRNAs in the pathogenesis of MDS

Recent studies have demonstrated that miRNAs play a crucial role in normal hematopoiesis through the control of the expression of key regulators of hematopoiesis (i.e., transcription factors, growth factor receptors, chemokine receptors). In particular, miRNAs are known to regulate hematopoietic differentiation in almost every stage such as megakaryocytic differentiation, erythroid differentiation and granulocytic and monocytic differentiation.[53-78] Georgantas et al.[79] explored miRNA expression and function at the level of HSCs and progenitors. They screened 228 miRNAs' expression in CD34+ cells obtained from normal peripheral blood or bone marrow and found that only 33 miRNAs were expressed in these cells. These miRNAs control translation of mRNAs associated with hematopoietic differentiation. In particular, each of these miRNAs targets multiple mRNAs, suggesting that a relatively small subset of miRNAs regulate hematopoiesis. This report suggested a model in which many regulators for hematopoietic differentiation are expressed at undifferentiated stem/progenitor cells, and translation of these regulators appears to be regulated by those small subset of miRNAs. It is plausible that miRNAs are implicated in pathogenesis of such complex and heterogeneous disorders as MDS, a stem cell disease with defective differentiation of one or more hematopoietic lineages.

Lines of evidence that global miRNA deregulation is involved in the pathogenesis of MDS come from Dicer1- knockout mice. In this model, Dicer1, a RNase III endonuclease essential for miRNA biogenesis, was specifically deleted in mouse osteoprogenitors, rather than in the hematopoietic stem/progenitor compartment.[80] These mice developed abnormal hematopoiesis, MDS, and eventually AML. Expression of Sbds, a gene mutated in Shwachman–Diamond syndrome, was reduced in the osteoprogenitors in the Dicer1-deleted mice. These changes in the Dicer1- knockout mice described above appear to be microenvironment dependent, suggesting that certain miRNAs are involved in the regulation of extrinsic regulators responsible for the normal interactions between HSCs and their niche, and loss of functional miRNAs in the absence of Dicer1 would dramatically affect HSC functions, resulting in MDS.

Many miRNA alterations induce MDS in a microenvironment-independent manner. t(2;11) (p21; q23) translocation does not result in a novel gene fusion, but rather contributes to the abnormal elevated expression of miR-125b, which is located near the breakpoint at chromosome 11 and is the only gene in this region with abnormal expression in MDS patients with t(2;11).[81] Ectopic expression of miR-125b is able to interfere with primary human CD34+ cell differentiation, and also inhibit terminal (monocytic and granulocytic) differentiation in HL60 and NB4 leukemic cell lines,[45] suggesting that miR-125b is a causative factor in MDS.

Inappropriate activation of EVI1 was found in 10–15% of MDS. An MDS mouse model has been established by transplantation of murine bone marrow cells with over expression of EVI1, indicating that the activation of EVI1 is an MDS-associated factor.[82] Activation of EVI1 may methylate the CpG island of miR-124 promoter, inducing the downregulation of miR-124, which in turn, ostensibly upregulates genes involved in cell cycling (D3) and self-renewal (Bmi1), leading to MDS.[83]

The 5q- is the most common structural genomic abnormality in MDS, and 13 miRNAs were mapped within the deleted region (5q31–5q35) including miR-143/miR-145 (5q33.1) and miR-146a, which were shown to be enriched in CD34+ cells and expressed at lower levels in 5q- MDS. Knockdown of miR-145 and miR-146a in mouse HSCs and progenitors has been shown to induce thrombocytosis, mild neutropenia, megakaryocytic dysplasia, and progression to a fatal myeloid malignancy in a subset of transplanted mice.[84, 85] Mal/TIRAP and Fli1 are targets of miR-145,[85, 86] and TRAF6 is a target of miR-146a.[85] Both BMal/TIRAP and TRAF6 are proteins that lie in the MyD88-dependent pathway of innate immune signaling upstream of NF-KB.[88, 89] The erythroid defect observed in 5q- syndrome is the result of haploinsufficiency of RPS14, not of reduced miR-145 and miR-146a expression.[89, 90] Combined deletion of RPS14 and miR-145 in a mouse model has been reported to recapitulate nearly the entire spectrum of clinical features of 5q- syndrome, suggesting that both haploinsufficiency of RPS14 and reduced expression of miR-145 and miR-146a contribute to 5q- syndrome symptoms. Based on these observations, mechanisms of deregulated miRNAs in the pathogenesis of MDS are proposed below: genetic and/or epigenetic alterations in HSCs or the HSC niche result in deregulated miRNAs, which in turn induce aberrant expression of HSC intrinsic or extrinsic regulators, compromising HSC functions (Fig. 1).

Figure 1.

Proposed mechanisms of miRNA in MDS pathogenesis. Genetic and epigenetic abnormalities may occur in both osteoblasts and HSCs, which result in the deregulation of certain miRNAs in those cells. MiRNA deregulation is followed by the subsequent abnormal expression of HSC extrinsic regulators in osteoblasts or HSC intrinsic regulators in HSCs, both leading to HSC dysfunction and MDS development.

Spliceosome mutations in MDS

Spliceosomes proteins are part of ribonucleoprotein complexes (RNP) and are involved in the splicing of introns during pre-mRNA maturation. Spliceosome mutations may result in defective spliceosome assembly, deregulated global mRNA splicing and nuclear-cytoplasm export, and altered expression of multiple genes.

Recent studies[91, 92] reported the results of whole-exome sequencing in patients with MDS, and showed that spliceosomal gene mutations play important roles in MDS pathogenesis. Eight spliceosomal genes are mutated with a variable frequency.[92, 93] SF3B1 mutations were found in 20% of individuals with MDS, with particularly high frequency (57–75.3%) among patients whose disease was characterized by ring sideroblasts (RARS),[92, 94-100] indicating a striking association between SF3B1 mutations and RARS. In addition to SF3B1, U2AF35 and SRSF2 are among the most frequently mutated.[101-103] Overexpression of mutant forms of U2AF35 in HSCs (CD34LinSca1+c-Kit+) from WT mice, confers cells a competitive disadvantage in hematopoiesis in long-term competitive transplantation assay. The mechanism study suggests that U2AF35 mutations, and possibly other spliceosomal pathway mutations, function in a dominant-negative manner to inhibit normal splicing.[91]

Unsolved issues: first, most studies identifying mutations in splicesomes used bone marrow cells, whether these mutations occur in HSC level remains unclear. Whole-exome sequencing on different bone marrow cellular compartments including HSC, myeloid and lymphoid progenitors, and peripheral blood samples from the same individual will address this issue. Second, the identification of key target genes and related signaling pathways affected by U2AF35, U2AF2 and SF3B1 mutations will be critical for understanding how these mutations contribute to the pathogenesis of MDS and for identifying rational targets for drug development.

MDS stem cells, a potential target for MDS treatment

Potential application of altered signaling pathways and expression altered miRNA in MDS HSCs

There is still a long way to go; for example, further verification of those altered signaling pathways and expression-altered miRNAs is necessary on a large cohort of patients and healthy controls, and how each altered miRNA contributes to the related MDS phenotype needs to be addressed. It is expected that MDS HSC functions might be rescued by targeting those altered signaling pathways and expression-altered miRNAs in MDS HSCs. A single miRNA has the ability to control a vast network of mRNA targets; therefore, there is a potential that manipulating a few miRNAs may have tremendous clinical benefit.[104] It is possible that aberrant expression of a miRNA in MDS correspondingly results in the deregulation of multiple mRNA targets within a certain signaling pathway, or multiple mRNA targets within different but functionally related signaling pathways, which might synergistically contribute to the phenotype of MDS HSCs in the latter case. Whether there are any links between those altered signaling pathways described above and expression- altered miRNAs in MDS HSCs is completely unknown. We expect that the integration of signaling pathways and miRNA expression might provide more powerful therapeutic strategies for MDS.

Targeting MDS HSC properties

It is hypothesized that leukemia could be eradicated at its root by developing treatments focused on the unique properties of the rare LSCs. MDSs are pre leukemic conditions; if MDS fits into the leukemia stem cell model, MDS could also be eradicated through a similar strategy. Recently, in the patients with 5q-MDS, Tehranchi et al.[9] identified rare and phenotypically distinct MDS stem cells that were selectively resistant to therapeutic targeting at the time of complete clinical and cytogenetic remission, indicating the importance of eliminating MDS HSCs for curing MDS. However, the specific properties of MDS HSCs, including quiescence, self-renewal, and differentiation, are still obscure. As we know, the ability of MDS HSCs to differentiate into one or more lineages is impaired. Most studies suggest an increase in the self-renewal capacity of MDS HSCs, an exception being Pellagatti et al.'s finding of significantly downregulated Wnt signaling in MDS CD34+ cells based on the altered gene expression profile, which likely leads to defective self-renewal of HSCs.[39] In another gene expression profiling study,[66] BMI was found to be preferentially up-regulated in MDS CD34+ cells; BMI1 is an essential regulator of normal HSC self-renewal. More interestingly, secreted frizzled related protein 1 (SFRP1), an extracellular antagonist of the Wnt signaling pathway, has been observed to be transcriptionally downregulated as a common event in AML, acute lymphoblastic leukemia (ALL), and MDS, which correlates with overexpression of the Wnt receptor Frizzled 3, leading to the activation of the Wnt signaling pathway in these hematopoietic diseases.[105] Therefore, altered signaling pathways responsible for impaired self-renewal capacity of MDS HSCs might be promising targets for MDS therapy. However, there has been no report describing quiescence property and the quiescence regulation status of MDS HSCs. As we noted regarding the Dicer1-knockout mouse model within bone progenitors, the MDS phenotype shows microenvironment dependent. Also, observations made from xenotransplantation assays, along with some mouse models, confirm that the pathophysiology of MDS includes in part a non-supportive microenvironment. Abnormalities of the bone marrow stroma dominate in early MDS, where there is disruption in interactions between the marrow stromal cell compartment and the MDS clones. In addition, it is well known that normal interactions between HSC and their niche are critical for the maintenance of HSC quiescence. Taking these data together, it is compelling to speculate about an abnormal quiescence status of MDS HSCs. Characterizing the quiescence status and the quiescence regulator status for MDS HSCs is essential for determining how to efficiently eradicate MDS HSCs.

Taking advantage of MDS mouse models

Mouse models are developed to study, in detail, questions that are difficult or impossible to address in the clinic. In recent years, various MDS mouse models have been developed, such as Evi1 overexpression,[106] Npm1 deletion,[107] Dido1 deficiency,[108] NUP98-HOXD13, SALL4 overexpression,[109] RUNX1 mutations,[110] Arid4a deficiency,[111] DNA polymerase gamma (Polg) mutations[112] as well as miRNA-related mouse models, which have advanced our understanding of the molecular mechanisms of the disease. All of these models have used elegant systems to address abnormalities that result in features observed in MDS. However, none of them are individually able to recapitulate all the major findings of MDS. This fact suggests that the full phenotype of MDS is likely caused by the integration of multiple genetic abnormalities. Despite the difficulties in integrating the various abnormalities in one unified mouse model to model the complete MDS phenotype, these animal models can be used for the development of new therapeutic approaches.[83] We may take advantage of these mouse model systems to address some issues for which using a human system is not feasible. However, when considering miRNA studies, the fact that there are many differences in miRNA expression between human and mouse hematopoietic cells should be taken into consideration.[84]


The author thanks Ms. Sunita C. Patterson for her critical reading and editing the manuscript. This work was supported in part by the NIH grant (HL744409) to Lalitha Nagarajan (L.N).