Myelodysplastic Syndrome Stem Cells Research Articles

Transposable Elements Are Differentially Expressed in MDS Stem Cells in a Disease-Stage-Specific Manner

Introduction Transposable elements (TEs) are widely spread across the human genome. Although TEs have been implicated in the pathogenesis of hematological malignancies, their expression in myelodysplastic syndromes (MDS) has not yet received extensive analysis. In the present study, we performed RNA sequence analysis of bone marrow stem and progenitor cells from patients with MDS and secondary acute myeloid leukemia (AML) and profiled differentially expressed TEs. Methods Bone marrow samples were obtained from each patient at the time of diagnosis. Normal bone marrow (NBM) samples from four healthy individuals were purchased from Lonza and processed in the same manner. After separation of CD34 + cells using magnetic beads, we purified lineage marker-negative (Lin -) CD34 + CD38 - stem cells and Lin - CD34 + CD38 + progenitor cells by cell sorting. The sorted cells were subjected to RNA-seq analysis using oligo-dT primers, which can capture poly-adenylated TE transcripts. Each read was aligned to the reference genome, then the annotation of the transcripts was performed using RepeatMasker, a program that screens DNA sequences for interspersed repeats and low-complexity DNA sequences (https://www.repeatmasker.org). Read counts for individual TEs were aggregated to the level of TE class, family, and subfamily according to hierarchies defined by the human RepeatMasker (repClass, repFamily, and repName, respectively). To generate raw counts of individual TE regions in RepeatMasker, the bedtools map function (version 2.27.1) was used with bed files of each sample. Normalization and significant expression differences were detected using DESeq2 (version 2.2.1). Differentially expressed TEs were defined using the cutoff of adjusted P values (q < 0.05). DESeq2 normalized counts were obtained from read counts by considering sequence depth, gene length, and RNA composition. Normalized counts were z-score scaled, and then subjected to uniform manifold approximation and projection (UMAP). Results Thirty-two patients were included in the present study. According to the 2016 revision to the World Health Organization classification (Arber DA, et al. Blood. 2016; 127: 2391-2405), 13 patients (40.6%) were diagnosed with MDS with multilineage dysplasia (MDS-MLD), 15 patients (46.9%) with MDS with excess blasts (MDS-EB), and 4 patients (12.5%) with AML with myelodysplasia-related changes (AML-MRC). We found that 26 and 17 repName TEs were significantly up-regulated in stem and progenitor cells compared with their NBM counterparts, and the majority of them belonged to LTRs, followed by DNA transposons. Since each LTR has multiple individual regions where similar sequences are scattered, LTR expression was further distinguished by the genomic locations of individual LTRs. Comparison of MDS-MLD, MDS-EB, and AML/MRC stem and progenitor cells to NBM counterparts revealed that more LTRs were differentially expressed in stem cells than in progenitor cells. Among them, many of the up-regulated LTRs were shared among different disease types (MDS-MLD, MDS-EB, and AML-MRC) in both stem and progenitor cells. AML-MRC had the largest number of differentially up- and down-regulated LTRs in stem cells. MDS-EB had the largest number of differentially up-regulated LTRs in progenitor cells, while only a small portion of LTRs were down-regulated in progenitor cells. LTRs at individual regions were differentially expressed in a more disease-specific manner, particularly in stem cells. Accordingly, UMAP plots that visualized the expression profiles of LTRs at individual regions showed that AML-MRC stem cells form a unique cluster, discriminating AML-MRC from MDS. To further understand the transcriptional reprogramming of LTRs during the disease progression from MDS to secondary AML, we analyzed the expression of differentially expressed LTRs at individual regions specific to AML-MRC by K-means clustering. They were subdivided into four clusters, of which Clusters 1, 3, and 4 showed gradual upregulation during disease progression, while Cluster 2 showed comparable upregulation at MDS and AML-MRC stages. Conclusions Our results suggest that specific LTRs behave in a disease-status and cell-type-specific manner in MDS and during leukemic transformation, providing fundamental information for understanding the pathogenesis of MDS.

Immunophenotypic aberrant hematopoietic stem cells in myelodysplastic syndromes: a biomarker for leukemic progression

Myelodysplastic syndromes (MDS) comprise hematological disorders that originate from the neoplastic transformation of hematopoietic stem cells (HSCs). However, discrimination between HSCs and their neoplastic counterparts in MDS-derived bone marrows (MDS-BMs) remains challenging. We hypothesized that in MDS patients immature CD34+CD38− cells with aberrant expression of immunophenotypic markers reflect neoplastic stem cells and that their frequency predicts leukemic progression. We analyzed samples from 68 MDS patients and 53 controls and discriminated HSCs from immunophenotypic aberrant HSCs (IA-HSCs) expressing membrane aberrancies (CD7, CD11b, CD22, CD33, CD44, CD45RA, CD56, CD123, CD366 or CD371). One-third of the MDS-BMs (23/68) contained IA-HSCs. The presence of IA-HSCs correlated with perturbed hematopoiesis (disproportionally expanded CD34+ subsets beside cytopenias) and an increased hazard of leukemic progression (HR = 25, 95% CI: 2.9–218) that was independent of conventional risk factors. At 2 years follow-up, the sensitivity and specificity of presence of IA-HSCs for predicting leukemic progression was 83% (95% CI: 36–99%) and 71% (95% CI: 58–81%), respectively. In a selected cohort (n = 10), most MDS-BMs with IA-HSCs showed genomic complexity and high human blast counts following xenotransplantation into immunodeficient mice, contrasting MDS-BMs without IA-HSCs. This study demonstrates that the presence of IA-HSCs within MDS-BMs predicts leukemic progression, indicating the clinical potential of IA-HSCs as a prognostic biomarker.

MiR-103-3p regulates the differentiation of bone marrow mesenchymal stem cells in myelodysplastic syndrome

The pathogenesis of myelodysplastic syndrome (MDS) may be related to the abnormal expression of microRNAs (miRNAs), which could influence the differentiation capacity of mesenchymal stem cells (MSCs) towards adipogenic and osteogenic lineages. In this study, exosomes from bone marrow plasma were successfully extracted and identified. Assessment of miR-103-3p expression in exosomes isolated from BM in 34 MDS patients and 10 controls revealed its 0.52-fold downregulation in patients with MDS compared with controls (NOR) and was downregulated 0.55-fold in MDS-MSCs compared with NOR-MSCs. Transfection of MDS-MSCs with the miR-103-3p mimic improved osteogenic differentiation and decreased adipogenic differentiation in vitro, while inhibition of miR-103-3p showed the opposite results in NOR-MSCs. Thus, the expression of miR-103-3p decreases in MDS BM plasma and MDS-MSCs, significantly impacting MDS-MSCs differentiation. The miR-103-3p mimics may boost MDS-MSCs osteogenic differentiation while weakening lipid differentiation, thereby providing possible target for the treatment of MDS pathogenesis.

Biglycan as a mediator of proinflammatory response and target for MDS and sAML therapy

ABSTRACT Myelodysplastic syndromes (MDS) and their progression to secondary acute myeloid leukemia (sAML) are associated with an altered protein expression including extracellular matrix (ECM) components thereby promoting an inflammatory environment. Since the role of the proteoglycan biglycan (BGN) as an inflammatory mediator has not yet been investigated in both diseases and might play a role in disease progression, its expression and/or function was determined in cell lines and bone marrow biopsies (BMBs) of MDS and sAML patients and subpopulations of MDS stem cells by Western blot and immunohistochemistry. The bone marrow (BM) microenvironment was analyzed by multispectral imaging, patients’ survival by Cox regression. ROC curves were assessed for diagnostic value of BGN. All cell lines showed a strong BGN surface expression in contrast to only marginal expression levels in mononuclear cells and CD34+ cells from healthy donors. In the MDS-L cell line, CD34−CD33+ and CD34+CD33+ blast subpopulations exhibited a differential BGN surface detection. Increased BGN mediated inflammasome activity of CD34−CD33+TLR4+ cells was observed, which was inhibited by direct targeting of BGN or NLRP3. BGN was heterogeneously expressed in BMBs of MDS and sAML, but was not detected in control biopsies. BGN expression in BMBs positively correlated with MUM1+ and CD8+, but negatively with CD33+TLR4+ cell infiltration and was accompanied by a decreased progression-free survival of MDS patients. BGN-mediated inflammasome activation appears to be a crucial mechanism in MDS pathogenesis implicating its use as suitable biomarker and potential therapeutic target. Abbreviations: Ab, antibody; alloSCT, allogenic stem cell transplant; AML, acute myeloid leukemia; BGN, biglycan; BM, bone marrow; BMB, bone marrow biopsy; casp1, caspase 1; CTLA-4, cytotoxic T lymphocyte-associated protein 4; DAMP, danger-associated molecular pattern; ECM, extracellular matrix; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HD, healthy donor; HSPC, hematopoietic stem and progenitor cell; HSC, hematopoietic stem cell; IFN, interferon; IHC, immunohistochemistry; IL, interleukin; MDS, myelodysplastic syndrome; MPN, myeloproliferative neoplasm; MSI, multispectral imaging; NGS, next-generation sequencing; NLRP3, NLR family pyrin domain containing 3; OS, overall survival; PBMC, peripheral blood mononuclear cell; PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1, PFS, progression-free survival; PRR, pattern recognition receptor; SC, stem cell; SLRP, small leucine-rich proteoglycan; TGF, transforming growth factor; TIRAP, toll/interleukin 1 receptor domain-containing adapter protein; TLR, toll-like receptor; Treg, regulatory T cell.

Targeting stem cells in myelodysplastic syndromes and acute myeloid leukemia.

The genetic architecture of cancer has been delineated through advances in high‐throughput next‐generation sequencing, where the sequential acquisition of recurrent driver mutations initially targeted towards normal cells ultimately leads to malignant transformation. Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are hematologic malignancies frequently initiated by mutations in the normal hematopoietic stem cell compartment leading to the establishment of leukemic stem cells. Although the genetic characterization of MDS and AML has led to identification of new therapeutic targets and development of new promising therapeutic strategies, disease progression, relapse, and treatment‐related mortality remain a major challenge in MDS and AML. The selective persistence of rare leukemic stem cells following therapy‐induced remission implies unique resistance mechanisms of leukemic stem cells towards conventional therapeutic strategies and that leukemic stem cells represent the cellular origin of relapse. Therefore, targeted surveillance of leukemic stem cells following therapy should, in the future, allow better prediction of relapse and disease progression, but is currently challenged by our restricted ability to distinguish leukemic stem cells from other leukemic cells and residual normal cells. To advance current and new clinical strategies for the treatment of MDS and AML, there is a need to improve our understanding and characterization of MDS and AML stem cells at the cellular, molecular, and genetic levels. Such work has already led to the identification of promising new candidate leukemic stem cell molecular targets that can now be exploited in preclinical and clinical therapeutic strategies, towards more efficient and specific elimination of leukemic stem cells.

P748: SINGLE CELL GENOTYPING OF MATCHED BONE MARROW AND PERIPHERAL BLOOD CELLS IN TREATMENT NAIVE AND AZA-TREATED MDS AND CMML

Background: Somatic mutations in hematopoietic stem cells (HSCs) are a central pathogenic event in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) where they induce proliferative advantages and impaired differentiation with subsequent cytopenias in peripheral blood (PB). Patients with high-risk disease who are ineligible for allogenic stem cell transplantation are treated with hypomethylating agents, including 5-azacytidine (AZA). AZA treatment can improve PB counts and delay progression to Akute Myeloid Leukemia. However, AZA response does not require eradication of mutated HSCs and may be related to improved differentiation capacity of mutated hematopoietic stem and progenitor cells (HSPCs). However, the contribution of mutated HSPCs to steadystate hematopoiesis in MDS and CMML is unclear. Aims: In this project, we aimed to determine the distribution of individual subclones and even wild-type cells within the HSPC-compartment and the contribution of these subclones and wild-type cells to effective hematopoiesis in patients with MDS or CMML. Methods: We used a combination of index sorting and single cell genotyping to characterize the somatic mutations of individual stem- and progenitor cells and matched high-turnover circulating cells in three patients, one treatment naïve and two AZA-treated. Using this approach, myeloid driver mutations previously detected in a traditional approach were tracked in HSC/multipotent progenitors (MPP), MDS stem cells MDS-SC, progenitor cells (common myeloid progenitor (CMP), granulocyte monocyte progenitor (GMP), megakaryocyte erythroid progenitor (MEP)), as well as in monocytes, neutrophils and naïve B cells (nBC). Results: Patient H198302 and Patient H198303 both had CMML and had been treated with AZA for ̴10 years with complete response. Patient H198304 had MDS-EB1 and has never been treated with hypomethylating agents. For each patient, the figure shows the proportions of cells across the haematopoietic hierarchy carrying zero, one, two, three, or four mutations in the specified alleles. Only very small numbers of HSPCs carrying no mutation could be detected. In all three patients, most stem and progenitor cells carried a majority of tracked mutations, with no major differences between stem cells, progenitor cells and MDS-stem cells. In one patient, H198304, MDS-stem cells had a higher proportion of cells with 3 or 4 mutations detected. Overall, in all three patients, a similar frequency of mutations was observed in differentiated monocytes and neutrophils with a substantial proportion of the circulating cells derived from highly mutated progenitors. One notable exception were nBCs in Patient H198304, which were mostly wild-type, suggesting that the small wildtype HSC population is the predominant origin of nBCs in this individual. Analysis of additional lymphoid populations in PB from this patient revealed that naïve T, but not NK cells were also predominantly wild-type, suggesting specific impairment of B- and T-lineage maturation in the mutated cells. Image:Summary/Conclusion: In conclusion, attrition of highly mutated cells during myeloid maturation was not observed in any of the three patients, irrespective of HMA therapy. This suggests that in vivo, highly mutated stem and progenitor cells retain the capacity to differentiate to mature myeloid and in some patients mature lymphoid cells and contribute significantly to circulating blood cells in MDS and CMML, prior to and following AZA treatment.

Characterization of myelodysplastic syndromes hematopoietic stem and progenitor cells using mass cytometry.

Myelodysplastic syndromes (MDS) at risk of transformation to acute myeloid leukemia (AML) are difficult to identify. The bone marrows of MDS patients harbor specific hematopoietic stem and progenitor cell (HSPC) abnormalities that may be associated with sub-types and risk-groups. Leukemia-associated characteristics of such cells may identify MDS patients at risk of progression to AML and provide insight in the pathobiology of MDS. Bone marrow samples from healthy donors (n =10), low risk (n =12) and high risk (n =13) MDS patients were collected, in addition, AML samples for 5 out of 6 MDS patients that progressed. Mass cytometry was applied to assess expression of stem cell subset and leukemia-associated immunophenotype markers. We analyzed the data using FlowSOM to cluster cells with similar expression of 10 commonly used stem cell markers. Metaclusters (n =20) of these clusters represented populations of cells with a related phenotype, largely resembling known stem cell subsets. Within specific subsets, intra-cellular expression levels of pCREB, IkBα, or pS6 differed significantly between healthy bone marrow (HBM) and MDS or consecutive secondary AML samples. CD34, CD44, and CD49f expression was significantly increased in high risk MDS and AML-associated metaclusters. We identified MDS/sAML cells with aberrant phenotypes when compared to HBM. Such cells were observed in clusters of both primary MDS and secondary AML samples. High-dimensional mass cytometry and computational data analyses enabled characterization of HSPC subsets in MDS and identification of leukemia stem cell populations based on their immunophenotype. Stem cells in MDS that display leukemia-associated features may predict the risk of developing AML.

Unique Metabolic Vulnerabilities of Myelodysplastic Syndrome Stem Cells

Background: The mechanisms that cause the progression of myelodysplastic syndrome (MDS) are poorly understood. Little is known about major signaling networks and energy metabolism in MDS cells as patients progress from low risk (LR) to high risk (HR) disease and from high risk to secondary acute myelogenous leukemia (sAML). As many as 30% of HR MDS patients progress to sAML and a portion of LR MDS patients progress to HR. The goal of this project is preventing progression by identifying MDS-specific targets for therapy. A deeper understanding of the metabolic properties of leukemia stem cells (LSCs) in AML has shown these cells are uniquely vulnerable to venetoclax and azacitidine (Ven/Aza) (Pollyea et al, Nat. Medicine, 2018) and metabolic changes cause resistance to Ven/Aza (Stevens et al, Nat. Cancer, 2021). Little however is known about the contribution of metabolism to the pathogenesis of MDS. The contributing factors to progression including metabolic properties, transcriptional programs, and immunophenotype are examined in this study.Methods: Bone marrow specimens from MDS patients at various disease stages, including serial samples during progression, were obtained. Single cell techniques including mass cytometry, antibody based single cell RNA sequencing (CITE-Seq) and transcriptional profiling with RNA sequencing were used to elucidate novel mechanisms of progression. Selective targeting of primitive MDS cells was tested using several agents.Results: Our previous work characterizing MDS stem cells (MDSC) showed significant similarities between MDSCs and AML LSCs (Stevens et al, Nat. Communications, 2018). However, little is known about lower risk disease. In order to understand transcriptional changes and their relationship to metabolism across pathogenesis, the transcriptome of blasts from patients with LR, intermediate (INT), or HR IPSS scores was investigated. The first major transcriptional difference identified was enrichment of glycolysis pathway at LR and INT stage. In contrast, HR MDS demonstrated enrichment of oxidative phosphorylation. Furthermore, comparison of intermediate to HR MDS showed increased RNA polymerase and Ribosome pathways at the HR stage. These changes demonstrate the progressive alteration of metabolic properties during MDS pathogenesis with cells first relying on mechanisms associated with normal stem cells (i.e. glycolysis) and later transitioning to a state associated with AML stem cells (i.e. reliance on oxidative phosphorylation).Using serial specimens of patients of who progressed from LR to HR MDS we performed CITE-Seq and mass cytometry. CITE-seq in serial specimens showed up-regulation of protein translation and oxidative phosphorylation in a subset of MDS stem and progenitor cells (CD34+ at transcript and antibody level) present at LR stage and conserved at HR stage (Fig 1A-C). MDSCs also acquired surface antigens including CD99 and CD52 upon progression from LR to HR. Analysis of the mass cytometry data showed significant overlap with CITE-Seq data including increased CD123+ and MCL1 expression in MDS stem cells upon progression.In order to understand therapeutic vulnerabilities as they relate to progression, we investigated ex vivo drug response in LR and HR specimens. MDS samples were challenged with two regimens, Ven/Aza, a regimen known to inhibit OXPHOS; and omacetaxine and azacitidine (Oma/Aza), which inhibits translation. CITE-seq showed that MDSC were selectively sensitive to these agents (Fig 1D). Importantly, addition of either drug regimen caused ablation of MDSC at LR and HR stages and these changes were most profound in cells with LSC properties.Based on preclinical findings, we are investigating MDS patients treated with Ven/Aza or Oma/Aza via CITE-seq and metabolomics for correlation of clinical response with properties of MDSC. Preliminary studies show that patients that respond to Oma/Aza present with a population of MDSC with transcriptional signatures of protein translation and LSCs (Fig. 1E). Studies are underway to investigate overlapping properties of ven/aza resistance in AML to resistance in MDS specifically investigating fatty acid metabolism in MDSC.Conclusions: Analysis of MDS patient bone marrow reveals acquisition of aberrant metabolic properties at both low and high risk stages of disease. These distinct aspects of MDSC biology create unique and targetable features. [Display omitted] DisclosuresPollyea: Genentech: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Janssen: Consultancy; Karyopharm: Consultancy; Syndax: Consultancy; Takeda: Consultancy; Daiichi Sankyo: Consultancy; Celgene/BMS: Consultancy; Amgen: Consultancy; AbbVie: Consultancy, Research Funding; Agios: Consultancy; Glycomimetics: Other.

Targeting MDS Stem Cells with Omacetaxine and Azacitidine for Newly Diagnosed High Grade Patients: Phase 1 Trial Results and Preliminary Mechanistic Studies

BackgroundMyelodysplastic syndrome (MDS) arises from and is perpetuated by malignant stem cells. Curative therapy requires eradication of this population, but this is not achieved with currently available medical therapies. MDS stem cells possess unique molecular properties that can be therapeutically exploited, but in general these strategies have not been explored clinically. Our recent in vitro studies showed that combination treatment with the protein synthesis inhibitor omacetaxine (oma) and the hypomethylating agent (HMA) azacitidine (aza) efficiently targeted MDS stem cells with minimal impact on normal stem cells. Based on these findings we conducted a Phase 1 clinical trial for MDS patients with concomitant oma + aza (NCT03564873).MethodsPatients were eligible if they had newly diagnosed MDS with ≥5% blasts. Aza was given at 75 mg/m 2 IV days 1-7 of a 28-day cycle; oma was given subcutaneously twice daily on days 1-7 of a 28-day cycle with 4 dose cohorts (0.75, 1.0 and 1.25 mg/m 2, and a de-escalation cohort of 0.5 mg/m 2). Dose escalation was based on a 3+3 design. Bone marrow biopsies occurred at baseline and day 8 and 28 of cycle 1, and at the conclusion of odd-numbered cycles. The primary endpoint was the maximum tolerated dose (MTD) of oma with aza. Responses were assessed according to 2006 IWG criteria with hematologic improvement (HI). Using pre- and post-treatment patient samples, advanced single cell techniques including mass cytometry and antibody-based single-cell RNA sequencing (CITE-Seq) were used to elucidate mechanisms of sensitivity and resistance. High sensitivity DNA sequencing techniques were used to understand clonal evolution and detect measurable residual disease (MRD).ResultsNine patients were required to complete phase 1. See Table 1 for baseline characteristics. Two patients in cohort 1 experienced dose limiting toxicity (DLT) (grade 3 hypoxia and grade 3 respiratory failure). De-escalation to cohort 0 resulted in one DLT event (grade 4 GI bleed) among the six patients treated in this cohort. The MTD was established as 0.5 mg/m 2 oma days 1-7. Adverse events are detailed in Table 2. Median number of cycles was 2 (1-3). 8/9 patients achieved a marrow CR; 5 had HI. 6 patients proceeded to transplantation and none have relapsed with median time from transplant of 772 (183-1158) days. Median response duration is 587 days (29-938). 5 remain alive.MRD negativity, as measured with droplet digital PCR (sensitivity up to 0.1%), was achieved in 2 out of 4 patients measured. Both MRD negative patients had complete clearance of mutated splice factor genes; we previously showed no ability to clear these mutations (0/13 patients) using venetoclax+aza in AML (Nature Medicine (2018) 24:1859-1866).Baseline analysis of patient specimens by single cell transcriptomics demonstrates a distinct population of MDS stem cells (Figure 1A). This subset has overlapping transcriptional properties with known AML stem cell profiles as highlighted by increased geneset expression. Within the stem cell compartment, we observed up-regulation of protein synthesis pathways (Figure 1B) (e.g. KEGG ribosome pathway in MDS stem cells), indicating that oma/aza treatment may specifically target the malignant stem cell population. Subsequent studies are investigating the use of CITEseq as a means to evaluate drug response, which will provide very high resolution analyses of transcriptional signatures in MDS patients. By specifically monitoring eradication of MDS stem cells during the course of treatment, it should be possible to predict therapeutic efficacy and response duration while elucidating genes that correspond to oma/aza sensitivity and resistance.ConclusionsPhase 2 of this study is ongoing; 23 newly-diagnosed high risk MDS patients will be enrolled with a primary endpoint to determine the overall response rate. In addition a ten-patient HMA failure expansion cohort will be enrolled to determine preliminary toxicity and efficacy assessments. Correlative studies will seek to determine the impact of this regimen on the MDS stem cell population and the mechanism of this impact. [Display omitted] DisclosuresMcMahon: Takeda: Membership on an entity's Board of Directors or advisory committees. Smith: Syros: Research Funding; Kura: Research Funding; Argenx: Research Funding. Pollyea: Foghorn: Honoraria; Gilead: Consultancy, Honoraria; Celgene: Honoraria; Bristol Myers Squibb: Honoraria; Genentech: Consultancy, Honoraria; Jazz: Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Amgen: Honoraria; Astellas: Honoraria; Aprea: Honoraria; Karyopharm: Consultancy, Honoraria; Kiadis: Honoraria; Novartis: Consultancy, Honoraria; Syndax: Honoraria; Syros: Consultancy, Honoraria; Takeda: Honoraria; Teva: Research Funding. OffLabel Disclosure:Omacetaxine for MDS

PI3-Kinase Deletion Dysregulates Autophagy in HSCs and Promotes Myelodysplasia

▪Myelodysplastic Syndrome (MDS) is a heterogeneous clonal malignancy arising in hematopoietic stem cells (HSCs), characterized by ineffective hematopoiesis, cytopenias, and the potential to progress to acute myeloid leukemia (AML). However, the perturbations in HSCs that lead to MDS initiation are poorly understood. It has been reported that HSCs are particularly dependent on autophagy for the maintenance of differentiation and self-renewal. We observed that, compared to healthy donor bone marrow hematopoietic stem and progenitor cells (HSPCs), MDS patient stem and progenitor cells (Lin-CD33-CD34+CD38-) have abnormal levels of autophagic degradation, as demonstrated by abnormal intracellular LC3II and P62 staining (Figure 1A). Autophagy is known to be regulated by the (PI3K)/AKT pathway, which transduces hematopoietic growth factor and cytokine signals in HSCs. PI3K/AKT is frequently activated in AML, but its role in MDS is less clear. Surprisingly, we found that CD34+ cells from a subset of MDS patients have upregulated expression of PTEN, the major negative regulator of the PI3K/AKT pathway, suggesting that PI3K/AKT may be downregulated in MDS stem cells. Therefore, we hypothesized that the Class IA PI3K isoforms (P110α, β, and δ) are required to maintain HSC differentiation and self-renewal.To understand the consequences of PI3K downregulation in HSCs, we generated a triple knockout (TKO) mouse model with conditional deletion of P110α and P110β in hematopoietic cells, and germline deletion of P110δ. Surprisingly, we found that PI3K deletion causes transplantable pancytopenia and decreased survival, despite the abnormal expansion of donor TKO HSCs (Figure 1 B,C). Consistent with this inefficient hematopoiesis, TKO bone marrow cells exhibited dysplastic features in multiple blood lineages and multiple chromosomal abnormalities (Figure 1 E,F), suggesting that PI3K inactivation in HSCs can promote MDS initiation. To determine whether impaired HSC differentiation in TKO mice could be due to dysregulated autophagy, we assessed autophagy in TKO HSCs by flow cytometry and immunofluorescence with the autophagosomal marker, LC3II. Our results showed that, compared to the WT controls, TKO HSCs have inefficient autophagic flux and decreased degradation of the cargo protein P62. We also discovered that TKO HSCs have significantly enlarged autophagic vesicles (Figure 1 G), and impaired fusion of autophagosomes with lysosomes, consistent with a marked defect in autophagic degradation. Treatment of TKO mice with two pharmacologic inducers of autophagy, rapamycin or metformin, improved HSC differentiation with an increase in Flk2+ MPPs (Figure 1 H), reduced dysplasia, and decreased the size of the TKO mutant clone in chimeric mice. Thus, our results uncover an important role for PI3K in regulating autophagy in HSCs to maintain the proper balance between self-renewal and differentiation. Our new mouse model of MDS will be a useful tool to study the mechanisms of MDs initiation. In addition, our findings open exciting avenues for future investigations of autophagy-inducing agents in MDS. [Display omitted] DisclosuresVerma: Celgene: Consultancy; Stelexis: Current equity holder in publicly-traded company; Throws Exception: Current equity holder in publicly-traded company; Acceleron: Consultancy; Novartis: Consultancy; Stelexis: Consultancy, Current equity holder in publicly-traded company; Eli Lilly: Research Funding; Curis: Research Funding; Medpacto: Research Funding; Incyte: Research Funding; BMS: Research Funding; GSK: Research Funding. Gritsman: iOnctura: Research Funding.

A Transgenic Murine Model Expressing Hyperactive STAT3 Recapitulates the Features of MDS/AML

Myelodysplastic syndromes (MDS) are clonal, myeloid malignancies that emerge and progress due to the expansion of disease-initiating aberrant hematopoietic stem cells that can evolve into Acute Myeloid Leukemia (AML). FDA approved therapies such as the recently approved Bcl-2 inhibitor venetoclax, FLT3 inhibitors, among others, have moved the field forward in newly diagnosed MDS/AML. However, relapsed/refractory (R/R) disease, as well as leukemic transformation post-MDS continues to have a poor prognosis. A pool of hematopoietic stem and progenitor cells (HSPCs) escape chemotherapy, proliferate during disease remission, and causes relapse partly in effect due to signaling effector mutations. It is imperative, for future therapeutic agents, to target these HSPCs populations to achieve a durable remission for aggressive myeloid malignancies. There is an urgent need to develop mouse models that recapitulate human disease for the study of pathogenesis and drug development in these disorders.Signal transducer and activator of transcription 3 (STAT3) belongs to the STAT family of transcription factors that are inappropriately activated in several malignancies. Our preliminary data indicates that STAT3 is overexpressed in MDS and AML stem cells and is associated with an adverse prognosis in a large cohort of patients. (Shastri et al, JCI 2018). We have successfully demonstrated that a selective antisense oligonucleotide inhibitor of STAT3, Danvatirsen, is rapidly incorporated into MDS/AML HSPCs and induces selective apoptosis and downregulation of STAT3 in these cells in comparison with healthy control HSPCs.To determine the role of STAT3 in the initiation of myeloid malignancies, a murine model was generated by crossing R26STAT3C stopfl/fl mice with vavCre transgenic mice. In this model, a hyperactive version of STAT3, STAT3C, is knocked into the Rosa26 locus with an upstream floxed stop cassette (R26STAT3C stopfl). Excision of the stop cassette by Cre recombinase leads to expression of a flag-tagged STAT3C protein and concomitant expression of EGFP in hematopoietic cells. GFP expression allows tracking of cells in which the floxed stop/Neo cassette is deleted and STAT3C is expressed. STAT3C-vavCre double transgenic mice were validated by GFP expression in HSPCs and differentiated hematopoietic cells. The STAT3C-vavCre mice developed ruffled fur, a hunched phenotype and weight-loss by five months of age. CBC analysis of STAT3C-vavCre mice shows a proliferative phenotype reminiscent of high-risk MDS/AML with higher WBC & platelet counts and lower hemoglobin (Figure 1A). Review of the peripheral smear showed an increase in granulocytic precursors that are likely leukemic blasts (Fig 1E). In addition, STAT3C-vavCre mice developed massive splenomegaly (Figure 1B). HSC lineage analysis by FACS showed the presence of GFP positive cells (Figure 1C) with increased expansion of the MPP and HSC compartment compared to controls, suggesting a stem and progenitor phenotype (Figure 1D). Murine myeloid colony assays showed larger colonies in the STAT3C-vavCre mice compared to controls. At this time, single cell RNA sequencing, and bulk RNA sequencing are being performed and will be used to further characterize the phenotype of the STAT3C-vavCre transgenic mice in addition to bone marrow and splenic aspirates & biopsies.Through the generation of a STAT3C-vavCre mouse model, that recapitulates the features of MDS/AML, we aim to further our understanding of the molecular mechanisms and pathways that play an important role in MDS to AML transformation and will help us identify downstream mediators of this event that can be therapeutically targeted. We would also like to use this murine model as an ideal substrate for preclinical studies of STAT3 targeting therapies in hematologic malignancies such as previously reported antisense inhibitors of STAT3 and STAT3 degraders. [Display omitted] DisclosuresFrank: Roche Genentech: Research Funding; Kymera: Consultancy, Research Funding; Revitope: Consultancy; Vigeo: Consultancy. Verma: Throws Exception: Current equity holder in publicly-traded company; BMS: Research Funding; GSK: Research Funding; Acceleron: Consultancy; Incyte: Research Funding; Stelexis: Current equity holder in publicly-traded company; Medpacto: Research Funding; Curis: Research Funding; Eli Lilly: Research Funding; Celgene: Consultancy; Stelexis: Consultancy, Current equity holder in publicly-traded company; Novartis: Consultancy. Shastri: Kymera Therapeutics: Research Funding; GLC: Consultancy; Guidepoint: Consultancy; Onclive: Honoraria.

Stem Cells in Myelodysplastic Syndromes and Acute Myeloid Leukemia: First Cousins or Unrelated Entities?

Myelodysplastic syndromes (MDSs) are associated with a significant risk of transformation to acute myeloid leukemia (AML), supported by alterations affecting malignant stem cells. This review focuses on the metabolic, phenotypic and genetic characteristics underlying this dynamic evolution, from myelodysplastic stem cells (MDS-SCs) to leukemic stem cells (LSCs). MDS-SCs are more likely to be derived from healthy hematopoietic stem cells (HSCs), whereas LSCs may originate from healthy progenitors, mostly LMPP (lymphoid-primed multipotential progenitors). Moreover, overexpression of CD123 and CLL1 markers by LSCs and MDS-SCs in high risk-MDS [HR-MDS] has led to exciting therapeutic applications. Single-cell sequencing has suggested that clonal evolution in the stem cell compartment was non-linear during MDS initiation and progression to AML, with pre-MDS-SC acquiring distinct additional mutations in parallel, that drive either MDS blast production or AML transformation. In AML and HR-MDS, common metabolic alterations have been identified in malignant stem cells, including activation of the protein machinery and dependence on oxidative phosphorylation. Targeting these metabolic abnormalities could prevent HR-MDS from progressing to AML. Strikingly, in low risk-MDS-SC, the expression of ribosomal proteins is decreased, which may be accompanied by a reduction in protein synthesis.

Bone Marrow Stem Cells from Patients with Myelodysplastic Syndrome (MDS) Engraft and Migrate in-between the Humanized Niches in Immuno-Compromised Mouse Models

IntroductionMyelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized mainly by ineffective hematopoiesis. Xenograft models of MDS in immunodeficient mice have been instrumental in improving our understanding of clonal origin and evolution of this disease (Mian & Rouault-Pierre et al., Nat Commun. 2015; Rouault-Pierre & Mian et al Haematologica 2016; Leukemia, 2017); however, the low level of engraftment generally observed in these mouse models makes them impractical for testing therapeutic strategies. We used a 3D humanized scaffold in vivo system to test the MDS stem cell engraftment as well as study the cross talk between MDS-Initiating cells (MDS-ICs) and their niche.MethodologyIn this study bone marrow (BM) cells from 31 patients (27 MDS cases covering all WHO subtypes, 1 CMML, 1 MDS/MPD and 2 sAML) were studied. Initially, whole-exome sequencing was performed on all cases. Colony-forming assay was used to assess the colony forming ability of BM CD34+ cells from each patient. Mesenchymal stromal cells (MSCs) were isolated from the BM of the patient samples. We then used 3 immunodeficient mouse models (NSG, NSG-SGM3 and NBSGW) to study the engraftment kinetics and clonal evolution of the transplanted cells in gelatin-based 3D scaffolds coated with patient MSCs (RARS/RCMD, autologous; RAEB, autologous/allogenic).ResultsBM MSCs obtained from the MDS patients showed variable expansion levels, with the ability to differentiate into the adipogenic lineage whereas the osteogenic differentiation was highly aberrant.Next, 0.5x106 to 3x106 patient (n=31) mononuclear cells (MNCs T cell depleted) were seeded into 3D-scaffolds prior to implantation in NSG-SGM3 and/or NSG mice. We observed persistent long-term engraftment of hCD45+ cells ranging from 0.05% to 80% (up to 18 weeks). Importantly, 74% of the cases generated an engraftment level of >20% hCD45+ cells. Similar engraftment levels were observed in mice humanized scaffolds that were injected with BM cells from MDS/MPD, CMML and sAML (hCD45+ 10%, 22% and 86%, respectively). No difference in the hCD45+ cell engraftment between the patients with low-risk or high-risk MDS was observed.Remarkably, our 3D humanized scaffolds generated multi-lineage engraftment, with mice that received BM cells from RAEB cases yielding higher myeloid engraftment compared to RARS and RCMD (RARS hCD33+ 44%; RCMD hCD33+ 46.6%; RAEB hCD33+ 65%). Furthermore, transplantation of BM cells from 5 patients into NBSGW mice gave rise to engraftment levels of hCD45+ cells similar (hCD45+ cells, up to 40%) to the NSG and NSG-SGM3 mice. The MDS cells recovered from the mice maintained their genotypic characteristics, as demonstrated by the presence of the mutations that remained largely unchanged between the pre- and post-transplanted samples.Interestingly, Immuno-staining of the scaffolds revealed colonization by the host murine vasculature (mCD31+ endothelial cells). In line with this finding, mouse hematopoietic cells were also found to have migrated into the implanted humanized scaffolds. Surprisingly, we did not observe hCD45+ cell engraftment in the bone marrow of any of the xenotransplanted mouse strains (NSG or NSG-SGM3 mice with or without Irradiation and non-irradiated NBSGW mice). However, we demonstrated that MDS stem cells migrate by implanting a supplementary scaffold into NSG-SGM3 and NBSGW mice that was only coated with MDS-MSCs. Notably, these MDS-MSC seeded scaffolds were colonized by the circulating human MDS cells.Finally, we tested whether the cells recovered from the primary engrafted 3D humanized scaffolds would be able to generate secondary transplants. Our data shows that these cells were able to engraft in the secondary mice implanted with humanized 3D scaffolds.DiscussionOur model provides evidence that MDS BM cells are highly dependent on humanized MSC niche, confirming the importance of the cross-talk between MDS-ICs and MDS niche. Our data demonstrates that MDS stem cells do migrate out of the 3D Scaffold but require their “own niche” for colonization. Thus, our in vivo 3D model system, can be reliably used to better dissect the cross-talk between MDS stoma and MDS-ICs. This model requiring relatively low number of human MNCs, provides a robust preclinical assessment tool to study clonal evolution as well as to test therapeutic strategies on the MDS clonality prior to treatment of MDS patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Immunophenotypically Aberrant Stem Cells in Myelodysplastic Syndromes: A Biomarker for Leukemic Progression

Introduction: Myelodysplastic syndromes (MDS) are generally referred to as a spectrum of myeloid malignancies that are notorious for their heterogeneity. Subsequently, the question arises as to what is the best method to dissect the heterogeneity of these syndromes, which may range from a normal life expectancy and MDS-unrelated dead to rapid progression to acute myeloid leukemia (AML). Most effort is still being put into the development of morphological classification and prognostic models like the classification of the World Health Organization (WHO) and the revised International Prognostic Scoring System (IPSS-R). However, the need for a more refined classification based on new insights into MDS pathophysiology is emerging.Methods: Hitherto we propose a new approach to predict leukemic progression among MDS patients using hematopoietic stem cell immunoprofiles. In this study, leukemic stem cell associated antigens - CD7, CD11b, CD22, CD33, CD44, CD45RA, CD56, CD123, CD366, CD371 - were analyzed on CD34+CD38- hematopoietic stem cells (HSCs) derived of 130 patients and controls at time of diagnosis.Results: Aberrant expression of these antigens was found in 33% (24/72) and in 20% (1/5) of the MDS and chronic myelomonocytic leukemia (CMML) patients, respectively. Even in patients diagnosed with idiopathic cytopenia of undetermined significance (ICUS) based on inexplicable cytopenia but less than 10% dysplasia, expression of aberrant antigens was found in 50% (4/8). In contrast to MDS, CMML, and ICUS patients, aberrant antigen expression was absent on HSCs of healthy, age-matched controls (0/9) and pathological controls (0/36), i.e. patients with non-myeloid clonal disorders. In MDS patients, the presence of aberrant immunophenotypic HSCs (AIP-HSCs) at diagnosis was negatively associated with the leukemia free and overall survival time (Figure 1a-b, p<.001), and increased the likelihood of leukemic progression and death by 99.5% (HR=0.005, 95%CI: 0-10.0) and 87.2% (HR=0.128, 95%CI: 0.04-0.41). The robust performance of flow cytometric detection of AIP-HSCs as diagnostic test to identify MDS patients prone to AML was illustrated by a high sensitivity of 88% and specificity of 69% at a follow-up time of six months. Although a slide trend toward increased AIP-HSCs prevalence for higher risk categories, MDS patients with AIP-HSCs were found across almost all WHO 2016 and IPSS-R categories (Figure 2a-b). Interestingly, multivariate regression analyses, including AIP-HSC percentage as well as the IPSS-R variables cytogenetic abnormalities, bone marrow blasts, hemoglobin, absolute neutrophil count, and platelets, revealed AIP-HSC percentage to be the only determinant with a significant effect on leukemia free survival. In predicting overall survival, apart from AIP-HSCs, cytogenetic abnormalities rendered statistical significant value.Conclusion: Flow cytometric detection of AIP-HSCs can be used as a biomarker to predict which MDS patients will finally develop AML. As a result, the presence of AIP-HSCs may improve currently used classification models and substitute the 20% blast cut-off to discriminate between MDS and AML. Addition of molecular features will be needed to validate the malignant nature of these AIP-HSCs. In the near feature, we anticipate that flow cytometric detection of AIP-HSCs will not only contribute to the dissection of MDS heterogeneity and an increased understanding of MDS stem cell biology, but also to therapy decision making, disease monitoring, and the clinical outcome of MDS patients. [Display omitted] DisclosuresOssenkoppele:J&J: Consultancy, Honoraria; Celgene: Honoraria, Research Funding; Roche: Honoraria; Karyopharm: Consultancy, Research Funding; Novartis: Research Funding.

A Pilot Study of CX-01 with Azacitidine for Treatment of Hypomethylating Agent-Refractory AML and MDS

Hypomethylating agents are standard first line therapy for older patients with AML and MDS. While these agents can prolong overall survival and disease free survival, they are not curative and only result in a complete response in about 20-25% of patients. For patients that fail these agents, there are no standard treatment options and overall survival is short at 4-6 months.Targeting the bone marrow microenvironment may be a valid strategy for treating refractory disease. CX-01, a 2-O, 3-O desulfated heparin, is a derivative of low molecular weight heparin with minimal anticoagulant activity, and may be potentially therapeutic as it blocks the CXCL12/CXCR4 axis and disrupts the HMGB1 interactions with TLR4 and RAGE, in addition to altering other vascular adhesion molecules.We hypothesize that infusional CX-01 will disrupt the bone marrow microenvironment and increase the cytotoxic effects of azacitidine on MDS and AML hematopoietic stem cells. We designed a pilot study combining a continuous infusion of CX-01 0.25 mg/kg/hr x 7 days with azacitidine 75 mg/m2 daily x 7 days per 28 day cycle. 15 patients were enrolled with MDS INT-1 or higher and AML that were refractory to >4 cycles of prior hypomethylating agent therapy or progressed after a prior response. The primary objective was to assess the overall response rate. The study was approved by the institutional IRB.To date, 13 patients have started treatment with the combination of infusional CX-01 and azacitidine. The median patient age is 73 years and 62% are male. Four patients have secondary AML, 4 have MDS INT-1, 2 have MDS INT-2, 2 have therapy related AML and 1 has de novo AML. Six patients have poor risk cytogenetics and pretreatment bone marrow biopsies revealed a median blast count of 16%. Patients were heavily pretreated, receiving a median of 6 cycles of prior hypomethylating agent therapy (range 4 - 20 cycles) with 7 patients receiving more than 1 prior line of therapy.Three of the 13 patients have reached the first bone marrow assessment following cycle 2. Of these 3 evaluable patients, 2 patients have achieved a bone marrow CR. None of these patients yet have evidence of hematologic improvement per MDS IWG criteria. Treatment has been associated with 6 severe adverse events, including 3 cases of febrile neutropenia, an episode of recurrent fungal pneumonia, a case of cellulitis, and a case of grade 3 transaminitis. All AEs were thought unlikely to be related to therapy with CX-01. There have been several instances of prolonged PTTs causing dose delays or reductions but no cases of bleeding.In conclusion, infusional CX-01 and azacitidine may be an effective treatment for hypomethylating agent-refractory AML and MDS. Based on this preliminary data it has a higher than expected rate of marrow CR, which was achieved in 2 of 3 evaluable patients. This treatment appears feasible with no instances of study-related severe adverse events. This trial is ongoing to evaluate the overall response rate and evaluate correlative data including blast mobilization and expression of CXCR4. DisclosuresUy:GlycoMimetics: Consultancy; Novartis: Consultancy, Other: Travel Suppport; Boehringer Ingelheim: Consultancy.

Activation of SIRT1 Deacetylase As a Therapeutic Approach for Myelodysplastic Syndromes By Restoring TET2 Function

Myelodysplastic syndromes (MDS) which arise from a primitive CD34+ hematopoitic stem/progenitor cell (HSPC), are incurable by non-transplant therapy. Better understanding of MDS HSPC regulation is critical for developing targeted therapies against the fatal disease. Tet methylcytosine dioxygenase 2 (TET2), initiating DNA demethylation through converting 5-methylcytosine to 5-hydromethylcytosine (5hmC), is the most frequently mutated gene in MDS. TET2 alterations lead to DNA hypermethylation and dysregulated gene expression in HSPCs, resulting in enhanced self-renewal and skewed myeloid specific expansion. While TET2's biological function has been well studied, the knowledge of its regulation is limited. We had demonstrated the TET2 c-terminus acetylation was reversed by SIRT1 deacetylase which maintains normal HSPCs activity under stress. We also observed that SIRT1 protein in MDS CD34+CD38- was expressed below normal levels, and that knockdown (KD) of SIRT1 enhanced in vivo growth of MDS-L cells, a patient derived MDS cell line, is associated with TET2 acetylation (Blood. 2016;128:1053). These results indicate an important role for SIRT1 in MDS HSPCs maintenance.To further assess SIRT1 function in MDS, we ectopically expressed SIRT1 in primary MDS CD34+ cells enriched for HSPCs and MDS-L cells, respectively. Overexpression (OE) of SIRT1 resulted in inhibition of colony formation of MDS CD34+ cells (p<0.01, n=3) but not normal counterpart (p>0.05, n=3). SIRT1 OE also suppressed MDSL colony formation (p<0.01) and cell growth (p<0.01). SIRT1 OE significantly reduced MDS-L cell BM engraftment in NSGs mice relative to controls (human CD45+ cells: 5.7±0.8% of BM in SIRT1 OE group vs 31.4 ± 5.8% in control, p<0.01, n=7), resulting in delayed mortality of recipient mice (median survival: 52 days vs 35 days; p<0.05, n=5). Meanwhile, treatment of SIRT1 agonist SRT1720, reduced colony formation of primary MDS CD34+ cells in a dose dependent manner (p<0.01, n=9). Similar results were also achieved in SRT1720 treated MDS-L cells. Furthermore, TET2 was hyperacetylated in primary MDS cells (n=3) compared to normal counterpart (n=3). Next, we tested whether inhibition of MDS HSPCs following SIRT1 activation was related to TET2 activation via protein deacetylation. Indeed, enhanced TET2 activity marked by 5hmC was observed in MDS CD34+ cells upon SRT1720 treatment (P<0.05, n=3). TET2 KD diminished growth inhibition induced by SIRT1 OE or SRT1720, indicating a requirement of TET2 for SIRT1 function. Through mass spectrometry analysis, three conserved acetylated lysine residuals (K1472, K1473, K1478) were identified in TET2 catalytic domain (CD). Engineered lysines to glutamines (TET2 3KQ) mutant, mimicking acetylated TET2, produced lower 5hmC, and led to increased colony growth compared to MDS-L expressing TET2 WT (p<0.01). We further evaluated the effects of SRT1720 using NUP98-HOXD13 (NHD13)MDS mouse. This murine MDS is transferable and secondary recipients receiving primary NHD13 MDS cells succumb to MDS at 4 months, allowing generation of a cohort of MDS mice for preclinical studies of therapeutics against MDS. CD45.2+ BM cells from donor NHD13+ mice showed evidence of dysplasia were harvested, and transplanted into preconditioned congenic recipients expressing CD45.1. SRT1720 treatment (100mg/kg/day for 12 weeks, p.o.) resulted in partial reversal of MDS-like disease developed in recipients receiving MDS cells, including an increase in HGB, PLT, RBC and WBC (p<0.01,n=6). Importantly, significant reduction of NHD13+ CD45.2+ chimerism in LSK and other progenitors from BM were also observed (p<0.01, n=6). As expected, 5hmC level in lineage depleted cells from SRT1720 treated mice was increased (p<0.01). Secondary transplantation was performed to evaluate the effects of SRT1720 on MDS initiating cells. We observed that the mice receiving cells from SRT1720 treated donors showed reduced NHD13+ CD45.2+ cell long-term engraftment (p<0.01, n=5).In summary, we demonstrated that genetic overexpression or pharmacological activation of SIRT1 drastically inhibited MDS maintenance, and that TET2 function was required for the inhibitory effect of SIRT1. These results support the hypothesis that SIRT1 deficiency promotes MDS cell expansion through TET2 inactivation and support the rationale for exploring SIRT1 activation as a promising approach to selectively target MDS stem cells. DisclosuresNo relevant conflicts of interest to declare.

Stem cell concepts in myelodysplastic syndromes: lessons and challenges.

According to the cancer stem cell (CSC) hypothesis, CSCs are the only cancer cells that can give rise to and sustain all cells that constitute a cancer as they possess inherent or acquired self-renewal potential, and their elimination is required and potentially sufficient to achieve a cure. Whilst establishing CSC identity remains challenging in most cancers, studies of low-intermediate risk myelodysplastic syndromes (MDS), other chronic myeloid malignancies and clonal haematopoiesis of indeterminant potential (CHIP) strongly support that the primary target cell usually resides in the rare haematopoietic stem cell (HSC) compartment. This probably reflects the unique self-renewal potential of HSCs in normal human haematopoiesis, combined with the somatic initiating genomic driver lesion not conferring extensive self-renewal potential to downstream progenitor cells. Mutational 'fate mapping' further supports that HSCs are the only disease-propagating cells in low-intermediate risk MDS, but that MDS-propagating potential might be extended to progenitors upon disease progression. The clinical importance of MDS stem cells has been highlighted through the demonstration of selective persistence of MDS stem cells in patients at complete remission in response to therapy. This implies that MDS stem cells might possess unique resistance mechanisms responsible for relapses following otherwise efficient treatments. Specific surveillance of MDS stem cells should be considered to assess the efficiency of therapies and as an early indicator of emerging relapses in patients in clinical remission. Moreover, further molecular characterization of purified MDS stem cells should facilitate identification and validation of improved and more stem cell-specific therapies for MDS.

Senescent Mesenchymal Stem Cells in Myelodysplastic Syndrome: Functional Alterations, Molecular Mechanisms, and Therapeutic Strategies.

Myelodysplastic syndrome (MDS) is a group of clonal hematopoietic disorders related to hematopoietic stem and progenitor cell dysfunction. However, therapies that are currently used to target hematopoietic stem cells are not effective. These therapies are able to slow the evolution toward acute myeloid leukemia but cannot eradicate the disease. Mesenchymal stem cells (MSCs) have been identified as one of the main cellular components of the bone marrow microenvironment, which plays an indispensable role in normal hematopoiesis. When functional and regenerative capacities of aging MSCs are diminished, some enter replicative senescence, which promotes inflammation and disease progression. Recent studies that investigated the contribution of bone marrow microenvironment and MSCs to the initiation and progression of the disease have offered new insights into the MDS. This review presents the latest updates on the role of MSCs in the MDS and discusses potential targets for the treatment of MDS.

Ectopic humanized mesenchymal niche in mice enables robust engraftment of myelodysplastic stem cells.

Myelodysplastic syndrome (MDS) are clonal stem cell diseases characterized mainly by ineffective hematopoiesis. Here, we present an approach that enables robust long-term engraftment of primary MDS stem cells (MDS-SCs) in mice by implantation of human mesenchymal cell-seeded scaffolds. Critically for modelling MDS, where patient sample material is limiting, mononuclear bone marrow cells containing as few as 104 CD34+ cells can be engrafted and expanded by this approach with the maintenance of the genetic make-up seen in the patients. Non-invasive high-resolution ultrasound imaging shows that these scaffolds are fully perfused. Our data shows that human microenvironment but not mouse is essential to MDS-SCs homing and engraftment. Notably, the alternative niche provided by healthy donor MSCs enhanced engraftment of MDS-SCs. This study characterizes a new tool to model MDS human disease with the level of engraftment previously unattainable in mice, and offers insights into human-specific determinants of MDS-SC microenvironment.

NF-κB constitutes a potential therapeutic target in high-risk myelodysplastic syndrome

AbstractMyelodysplastic syndrome (MDS) is a preneoplastic condition that frequently develops into overt acute myeloid leukemia (AML). The P39 MDS/AML cell line manifested constitutive NF-κB activation. In this cell line, NF-κB inhibition by small interfering RNAs specific for p65 or chemical inhibitors including bortezomib resulted in the down-regulation of apoptosis-inhibitory NF-κB target genes and subsequent cell death accompanied by loss of mitochondrial transmembrane potential as well as by the mitochondrial release of the caspase activator cytochrome c and the caspase-independent death effectors endonuclease G and apoptosis-inducing factor (AIF). Bone marrow cells from high-risk MDS patients also exhibited constitutive NF-κB activation similar to bone marrow samples from MDS/AML patients. Purified hematopoietic stem cells (CD34+) and immature myeloid cells (CD33+) from high-risk MDS patients demonstrated the nuclear translocation of the p65 NF-κB subunit. The frequency of cells with nuclear p65 correlated with blast counts, apoptosis suppression, and disease progression. NF-κB activation was confined to those cells that carried MDS-associated cytogenetic alterations. Since NF-κB inhibition induced rapid apoptosis of bone marrow cells from high-risk MDS patients, we postulate that NF-κB activation is responsible for the progressive suppression of apoptosis affecting differentiating MDS cells and thus contributes to malignant transformation. NF-κB inhibition may constitute a novel therapeutic strategy if apoptosis induction of MDS stem cells is the goal.