ALBERT

Description: Key Points Mutational trajectories are defined by complex patterns of molecular heterogeneity in MDS, including lower-risk cases. Therapeutic intervention dynamically reshapes mutational patterns often resulting in branched or independent evolution of MDS clones.

Description: Introduction: Myelodysplastic Syndrome (MDS) can occur in young people but it is mainly a disease of the elderly with a dramatic increase of incidence in the decades above 60 years. Accordingly, the factor age may be an important gateway to the understanding of the molecular pathogenesis of MDS. Insights into the molecular changes of aging hematopoiesis in healthy organisms have found molecular changes, which often parallel the observations in MDS such as increase of clonality with age, change of epigenetic profiles, skewed lineage commitment toward the myeloid compartment and reduced regenerative capacity after stress. The development of MDS is often suggestive of an accelerated extrapolation of molecular changes, which also occur in normal aging hematopoiesis. Beyond this, increasing evidence is suggesting that MDS hematopoiesis is highly dependent on support of the bone marrow (BM) stroma, which has been shown to display aberrant transcriptomic profiles as compared to healthy BM stroma. To this end, we aimed to test the hypothesis whether the emergence of MDS may be associated with a continuity of molecular changes in BM stroma cells during aging. Therefore, we performed explorative RNA sequencing in a set of MSCs collected from healthy young, healthy old and patients with MDS with a highly standardized pre-analytical work-up algorithm. Methods: We collected BM samples from voluntary healthy young adults (age = 24 - 25 years, female n=3, male n=3), healthy old adults (age 66 - 79 years, female n=3, male n=3) and patients with very low - intermediate risk MDS (age 51 - 87 years, female n=3, male n=3). After isolation of BM mononuclear cells by Ficoll gradient centrifugation, 5x106 mononuclear BM cells were seeded into 25cm² flasks and cultured using StemMACS human MSC Expansion Media (Miltenyi Biotec) with weekly media exchange to select for MSCs. These were expanded and harvested in passage 2. Absence of residual hematopoietic cells was controlled by FACS with anti CD45, CD31, and CD146. Whole transcriptome RNA-sequencing on all samples was carried out from 150ng of high quality RNA using the TruSeq stranded total RNA protocol and 100bp paired end sequencing (Illumina). The bio-informatical pipeline consisted of mapping using hisat2 and cufflinks for calculation of differentially expressed genes. Results: RNA-sequencing generated a mean of 94 million reads per sample. Between the groups "healthy young" and "healthy old" 331 differentially regulated genes were identified. Between "healthy old" and "MDS" 514 genes were differentially regulated (fold change 〉 1.5, false discovery rate, FDR 〈 0.05). Among these, 197 genes were differently expressed between all three groups. With these parameters, a total of 17 genes showed a continuous and significant increase of expression from healthy young over healthy old toward MDS. Among these were Kit ligand (KITLG) but also a cluster of membrane based cell adhesion molecules such as Cadherin-6 (CDH6), Laminin Subunit Alpha 2 (LAMA2) and Laminin Subunit Gamma 2 (LAMC2) and others. Conversely, 5 genes showed a continuous and significant decrease of expression from healthy young over healthy old toward MDS, among these Leukocyte-specific protein 1 (LSP1), a gene implicated in regulation of T-cell migration. Gene set enrichment analysis revealed that MDS MSCs exhibited a significant depletion of genes involved in early adipogenic differentiation and enrichment of gene sets associated with extracellular matrix remodeling (FDR 〈 0.05, normalized enrichment score 〉 1.7). Although cells were cultured under normoxic conditions, MDS-MSCs displayed marked intrinsic feature of hypoxia. Conclusion: By integrating transcriptomic data from BM stroma cells from healthy individuals during aging and comparison to BM stroma cells from MDS patients we have identified gene sets that are significantly differentially expressed per continuitatem. On the background of the hypothesis that molecular changes in the microenvironment of MDS are an exacerbation of changes also taking place during normal aging in the bone marrow, these genes, which are accumulated in the context of extracellular matrix and cell adhesion are promising candidates to further elucidate a BM stroma based pathogenesis of MDS. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1694 Introduction: Myelodysplastic syndromes are a heterogeneous group of malignant clonal hematologic disorders characterized by ineffective hematopoiesis, peripheral cytopenias and dysplastic bone marrow cells, with frequent progression to acute myeloid leukemia. Because of its heterogeneous nature, modeling of this disease has proven to be very difficult in cell culture systems as well as mice. In addition, attempts to generate a xenotransplant model in immuno-compromised mice have only achieved very low levels of engraftment that are often transient, making it very difficult to study the biology of this disease in vivo. Recent studies in mice have shown that conditional impairment of the small RNA processing enzyme Dicer in mouse osteolineages induced a stromal niche that promoted myelodysplasia, leading to the hypothesis that abnormal bone marrow stromal cells might provide a “fertile soil“ for the expansion of the malignant clone. Patients and Methods: To the date of writing, a total of 12 primary hematopoietic stem cell- and mesenchymal stroma cell (MSCs) samples selected from patients with MDS have been isolated and xenotransplanted into NOD.Cg-Prkdscid Il2rgtm1Wjl/Szj (NSG) mice: MDS 5q- (n=7), MDS RCMD (n=3), MDS RAEB I (n=1), MDS-U (n=1). Engraftment was monitored by FACS using human specific antibodies to CD45, CD34 and CD38. In addition cell cycle behavior was analyzed by Ki67/Hoechst staining. Mesenchymal stromal cells were characterized using previously described stromal markers: CD105, CD271, CD73, CD166, CD90, CD146 and CD44. To isolate genomic DNA and RNA for molecular analyses, MDS xenografts were flow sorted based on human CD45 expression. Molecular characterization of primary MDS samples and xenotransplants was carried out by serial copy number analysis using Affymetrix SNP 6.0 Arrays, metaphase cytogenetics and direct sequencing of known mutations in the transplanted MDS samples. Results: We show, that the concomitant transplantation of MDS-derived mesenchymal stromal cells with the corresponding hematopoietic patient stem/progenitor cells leads to significant and long-term engraftment (0.1 – 15% for up to 23 weeks) of cells isolated from IPSS low and intermediate risk MDS patients. In addition to the bone marrow, MDS hematopoietic cells also infiltrate other hematopoietic compartments of the mouse including the spleen. Significant engraftment of cells with progenitor (CD34+CD38+) as well as stem cell phenotype (CD34+CD38-) was observed, which is consistent with engraftment of an MDS stem cell that sustains long-term hematopoiesis. SNP array analysis confirmed the clonal origin of the engrafted cells as MDS xenografts harboring the identical genomic lesions as present in the patient disease. Conclusion: We present a robust MDS xenograft model of low risk MDS entities based on the concomitant transplantation of primary MDS hematopoietic cells with MSCs from the same patients. This model does not only allow to study the biology of this disease in vivo but also the molecular and cellular interactions between MSCs and hematopoietic MDS cells. In addition it provides a useful platform to study the effects of new experimental therapeutic agents for the treatment of MDS in molecularly defined MDS cells. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction Myelodysplastic syndromes (MDS) are clonal hematologic disorders characterized by ineffective hematopoiesis, dysplasia and increased risk of progression to acute myeloid leukemia. The development of targeted therapies for MDS has been lagging behind and remains a key clinical challenge that has been hampered, at least in part, by difficulties to establish in vivo model systems that recapitulate disease heterogeneity and complexity. Attempts to generate a xenograft model of lower risk MDS have only achieved low and often transient levels of engraftment. Recent evidence from mouse studies suggests that MDS is a disease in which both the hematopoietic system and the bone marrow microenvironment might be involved. Thus, we hypothesized that a specific MDS microenvironment might be required for the successful modeling of low risk MDS in mice, proposing a dependency of the “disease propagating cells“ on their corresponding niche cells in human MDS. Methods Our study is based on xenotransplantation of material from 19 MDS patients classified as follows: IPSS low risk (n=6), intermediate-1 risk (n=13), WHO 2008 classification: MDS 5q- (n=7), MDS RCMD (n=7), MDS RAEB I (n=3), MDS-U (n=1), MDS RARS (n=1). MDS CD34+ cells were co-injected with patient-derived mesenchymal stromal cells (MSCs) directly in the bone marrow cavity (i.f) of NOD.Cg-Prkdscid Il2rgtm1Wjl/Szj (NSG) or NSGS (NSG mice expressing human SCF, IL3 and GM-CSF) mice. Molecular tracking of MDS cells was carried out by copy number analysis (Affymetrix SNP 6.0 Arrays), metaphase cytogenetics, interphase FISH, Roche 454 deep sequencing and pyrosequencing of known mutations. Mice were analyzed after a minimum of 16 weeks post transplantation. Results We show that co-injection of MDS CD34+ cells with their corresponding MSCs leads to significant and long-term engraftment of over 77% of the MDS patients analyzed, both in NSG (10/13 patients, range hCD45+= 1-18%) and NSGS mice (7/8 patients, range hCD45+=2.2-74%). In contrast, absence of MSCs or co-injection of healthy age-matched MSCs only gave rise to limited engraftment in NSG mice (2/7 patients (hCD45+=1-3.8%) and 1/2 patients (hCD45+=2%), respectively). Transplanted samples exhibited a clear myeloid bias and significant engraftment of cells with progenitor (CD34+CD38+) and stem cell phenotype (CD34+CD38-) that could be serially transplanted. In addition, presence of morphologically dysplastic cells was readily detectable in NSGS mice. Importantly, molecular analysis of the engrafted cells confirmed their “diseased” origin as they carried identical lesions to the ones present in the original MDS patient. Furthermore, we could demonstrate that disease-propagating stem cells in lower risk MDS exclusively reside within the lin-CD34+CD38- stem cell fraction. Finally, RNA sequencing analysis comparing MDS and age-matched healthy control MSCs revealed altered expression of key genes involved in cellular adhesion, extra-cellular matrix (ECM) remodeling and cellular cross-talk in diseased MSCs, strongly supporting the notion of a complex interplay between MDS hematopoietic cells and their corresponding stroma. In addition, patient MSCs exhibited clear molecular features of fibrosis, a clinical feature often associated with MDS. Conclusion In this study we have identified patient-derived MSCs as a critical functional component of lower risk MDS. Together with MDS stem cells, these patient MSCs form a functional stem cell-niche unit, which allows the propagation of the disease in a xenograft recipient. The striking changed expression in diseased MSCs of genes involved in processes like cytokine-cytokine receptor interaction, cellular adhesion, ECM remodeling as well as hypoxia further suggests that diseased MDS cells might alter the function of the normal HSC niche into one that can support the requirement of MDS cells. Studying the interaction of MDS stem cells and MSCs at the cellular and molecular level will provide a platform for unraveling the molecular basis of clonal dominance in MDS as well as allow the design of targeted strategies aimed to disrupt the MDS stem cell-MSC niche interactions. Disclosures: No relevant conflicts of interest to declare.