Myelodysplastic Syndromes Stage Research Articles

Bone marrow macrophages are involved in the ineffective hematopoiesis of myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are a group of heterogeneous myeloid clonal disorders characterized by ineffective hematopoiesis. Accumulating evidence has shown that macrophages (MΦs) are important components in the regulation of tumor progression and hematopoietic stem cells (HSCs). However, the roles of bone marrow (BM) MΦsin regulating normal and malignant hematopoiesis in different clinical stages of MDS are largely unknown. Age-paired patients with lower-risk MDS (N = 15), higher-risk MDS (N = 15), de novo acute myeloid leukemia (AML) (N = 15), and healthy donors (HDs) (N = 15) were enrolled. Flow cytometry analysis showed increased pro-inflammatory monocyte subsets and a decreased classically activated (M1) MΦs/alternatively activated (M2) MΦs ratio in the BM of patients with higher-risk MDS compared to lower-risk MDS. BM MФs from patients with higher-risk MDS and AML showed impaired phagocytosis activity but increased migration compared with lower-risk MDS group. AML BM MΦs showed markedly higher S100A8/A9 levels than lower-risk MDS BM MΦs. More importantly, coculture experiments suggested that the HSC supporting abilities of BM MΦs from patients with higher-risk MDS decreased, whereas the malignant cell supporting abilities increased compared with lower-risk MDS. Gene Ontology enrichment comparing BM MΦs from lower-risk MDS and higher-risk MDS for genes was involved in hematopoiesis- and immunity-related pathways. Our results suggest that BM MΦs are involved in ineffective hematopoiesis in patients with MDS, which indicates that repairing aberrant BM MΦs may represent a promising therapeutic approach for patients with MDS.

Subunit-Specific Analysis of Cohesin-Mutant Myeloid Malignancies Reveals Distinct Ontogeny and Outcomes

Introduction:Loss of function mutations in the cohesin complex ring components and modulators STAG2, RAD21, SMC1A, SMC3, and PDS5B are recurrent genetic drivers in myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), and MDS/myeloproliferative neoplasm (MPN) overlap syndromes. However, whether different cohesin subunit mutations share disease characteristics and prognostic impact is not well understood. Methods: We assembled a cohort of 4,439 patients with hematologic neoplasms at the Dana-Farber Cancer Institute (DFCI) and the Munich Leukemia Laboratory (MLL), and identified a subset of 790 cohesin-mutant patients, the largest cohesin-mutant cohort to date. We performed a retrospective analysis of the incidence, clinical presentation, genomic landscape, and clinical outcomes in a cohesin subunit-specific way. Results: STAG2 mutations were most common and present in 610 cases (diagnosed as MDS (50%), AML (36%), MDS/MPN (10%)), followed by RAD21 in 104 (20% MDS, 64% AML, 2% MDS/MPN), SMC3 in 26 (35% MDS, 42% AML, no MDS/MPN), SMC1A in 22 (27% MDS, 55% AML, 5% MDS/MPN), PDS5B in 16 (13% MDS, 38% AML and 6% MDS/MPN), and 12 cases with more than one mutated cohesin gene (50% MDS, 33% AML, 8% MDS/MPN). Within the DFCI cohort, 256 (82%) patients were diagnosed with a myeloid malignancy and 55 (18%) patients with a non-myeloid hematologic malignancy. We focused our subsequent analysis on 735 patients diagnosed with MDS, MDS/MPN or AML, and compared them to 3,649 cohesin-wildtype (WT) patients in the DFCI and MLL cohorts. We found that STAG2 mutations were acquired at the MDS stage and were associated with MDS with increased blasts (75% cases with MDS-IB1 or IB2) and secondary AML. We also noted their co-occurrence with secondary AML-type mutations, such as ASXL1 (64%, OR=5.5, FDR=5e-29), SRSF2 (45%, OR=7.4, FDR=5-e31) and RUNX1 (37%, OR=3.5, FDR=2e-13), and trisomy 8 (OR=10.6, FDR= 2.2e-50). In contrast, mutations in RAD21, SMC1A, SMC3 and PDS5B shared features with de novo AML, including co-occurrence with NPM1 (32%, OR 4.3, FDR=1e-6) and FLT3 (23%, OR= 2.8, FDR= 0.01), and association of RAD21 and SMC1A with t(8;21) AML (OR= 131;134; FDR= 3.8e-5;4.1e-19). Of note, both STAG2 and RAD21 mutations shared a mutational exclusivity with TP53 mutations (OR=0.04-0.06, FDR < 0.001, Figure 1A). We next assessed the impact of different cohesin mutations on clinical outcomes. We conducted independent analyses of overall survival (OS) and progression free survival (PFS) in MDS and AML. Given our observations that cohesin mutations were mutually exclusive with TP53 mutations, and the established prognostic impact of TP53 mutations, we compared the overall survival (OS) and AML-free survival (PFS) of patients with STAG2-mutant MDS (n=119) to TP53-mutant MDS (n=280) and cohesin/ TP53-WT MDS (n=1,023). With a median follow-up (FU) of 51.1 months we observed a significantly worse OS of STAG2-mutant MDS compared to cohesin and TP53-WT MDS (HR 1.44, CI= 1.10-1.89, p=0.007) and a similar risk of leukemic transformation as TP53-mutant MDS cases (median AML-PFS of 15.4 months for STAG2, and 12.1 months for TP53, p=0.3). Furthermore, STAG2-mutant AML (n=148) had significantly worse OS than AML without myelodysplasia related changes (MR) (n=1384) (HR= 0.62, 95% CI=0.5-0.76, p<0.001, median FU 42.8 months), and similar outcomes as AML-MR without TP53 mutations (n=567), (HR=1.25, 95% CI= 1.01-1.55, p=0.04). Importantly, this pattern was distinct from outcomes of RAD21-mutant AML (n=39), which was almost identical to AML-non-MR and significantly better than STAG2-mutant AML OS (HR= 0.56, 95% CI= 0.34-0.93, p=0.024) ( Figure 1B). The effects of STAG2 and RAD21 mutations on OS remained significant when censored for allogeneic stem cell transplantation. Our findings suggest that only STAG2 mutations confer a negative impact on MDS and AML, and this is attributed to secondary ontogeny. Conclusions: In summary, our study establishes a role for different cohesin subunit mutations in distinct subtypes of MDS and AML, which has significant prognostic implications and expands our current understanding of this important group of driver genes. The findings from our subunit-specific analysis demonstrate that while STAG2 mutations are characterized by secondary AML ontogeny arising from MDS and associated with a worse prognosis, RAD21 mutations are associated with de novo AML and better survival.

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.

Understanding the Continuum between High-Risk Myelodysplastic Syndrome and Acute Myeloid Leukemia

Myelodysplastic syndrome (MDS) is a clonal hematopoietic neoplasm characterized by bone marrow dysplasia, failure of hematopoiesis and variable risk of progression to acute myeloid leukemia (AML). Recent large-scale studies have demonstrated that distinct molecular abnormalities detected at earlier stages of MDS alter disease biology and predict progression to AML. Consistently, various studies analyzing these diseases at the single-cell level have identified specific patterns of progression strongly associated with genomic alterations. These pre-clinical results have solidified the conclusion that high-risk MDS and AML arising from MDS or AML with MDS-related changes (AML-MRC) represent a continuum of the same disease. AML-MRC is distinguished from de novo AML by the presence of certain chromosomal abnormalities, such as deletion of 5q, 7/7q, 20q and complex karyotype and somatic mutations, which are also present in MDS and carry crucial prognostic implications. Recent changes in the classification and prognostication of MDS and AML by the International Consensus Classification (ICC) and the World Health Organization (WHO) reflect these advances. Finally, a better understanding of the biology of high-risk MDS and the mechanisms of disease progression have led to the introduction of novel therapeutic approaches, such as the addition of venetoclax to hypomethylating agents and, more recently, triplet therapies and agents targeting specific mutations, including FLT3 and IDH1/2. In this review, we analyze the pre-clinical data supporting that high-risk MDS and AML-MRC share the same genetic abnormalities and represent a continuum, describe the recent changes in the classification of these neoplasms and summarize the advances in the management of patients with these neoplasms.

Clinical and Biological Effects of Canakinumab in Lower-Risk Myelodysplastic Syndromes (MDS): Results from a Phase 2 Clinical Trial

Interleukin 1 beta (IL-1B) induced inflammasome activation is a mechanism of cancer initiation and progression (Schroder K, Cell, 2010). Our previous studies (Ganan-Gomez I, Blood 2021) showed that IL-1B was one of the most overexpressed pro-inflammatory cytokines in the hematopoietic stem and progenitor cells (HSPCs) of patients with clonal cytopenia of undetermined significance (CCUS) carrying DNMT3a and TET2 mutations. Given that IL-1B upregulation induces aberrant myeloid differentiation of HSPCs (Pietras EM, Nat Cell Biol., 2016), we hypothesize that IL-1B plays a critical role in MDS pathogenesis, and that targeting IL-1B signaling overcomes aberrant MDS HSPCs' myeloid skewing. To test this hypothesis, we designed a clinical trial to evaluate the IL-1B inhibitor canakinumab's (NCT04239157) safety profile and its clinical and biological effects in patients with lower-risk MDS. Patients and Methods: Patients aged ≥ 18 with IPSS-R ≤ 3.5 MDS, normal renal and hepatic functions, and performance status, who had previously received at least 1 line of other therapy were included in the study (Table1). Patients received 300 mg of canakinumab on day 1 of a 4-week cycle. Bone marrow (BM) samples were obtained before therapy and after 1, 2, and 4 cycles of therapy during the trial and after every 3 or 4 cycles of therapy thereafter. Single-cell RNA sequencing (scRNA-seq; 10X Genomics) analyses were performed in lineage negative (Lin-)CD34+ HSPCs and mononuclear cells (MNCs) isolated at different therapy timepoints from the BM of 1 patient who achieved hematological improvement after canakinumab treatment, 2 patients who achieved stable disease, and 2 patients who did not respond to this therapy. The clinical characteristics of the 5 patients whose samples were analyzed by scRNA-seq analyses are included in Table 1. Results: Twenty-six patients have been enrolled in the clinical trial. Patients' clinical characteristics are included in Table 1. Twenty-three patients were evaluable for response. Among the 22 out of the 23 patients who were transfusion dependent at baseline, one achieved transfusion independence, and another had platelet count improvement. Three out of the 23 had progressive disease, and 18 had stable disease. Treatment was well tolerated with no severe adverse events. One patient died of sepsis unrelated to therapy. Overall, we did not observe any improvement in the blast counts and cytogenetic and/or mutational burden. To evaluate the biological effects of canakinumab, we performed scRNA-seq of Lin-CD34+ HSPCs (Figure 1) and MNCs at therapy baseline and after 2, 4, 5, 8, and 11 cycles of treatment. Consistent with our initial hypotheses, these analyses revealed that canakinumab decreased the frequencies of hematopoietic stem cells (HSCs) and multipotent myeloid progenitors (MPPs). Differential analysis between the samples showed that HSCs and MPPs significantly downregulated the expression of genes involved in cytokine signaling (e.g., IL-1B, CXCL2, and CXCL8), TNFα-induced activation of NF-κB, and hypoxia pathways, which demonstrated efficient target engagement of canakinumab on MDS cells. Similar results were also observed in downstream monocytes and B cells. In contrast, we did not observe any significant change in the HSPC architecture in the BM of patients with stable disease or whose disease did not respond to canakinumab. Consistent with our previous results in early stage MDS, the patient who achieved hematological improvement after canakinumab therapy was the only one among the cohort who carried a single mutation affecting DNMT3A (Table1). Conclusion. This study demonstrates that canakinumab is safe in MDS and efficiently targets IL-1B signaling. However, the biological effect of canakinumab has not yet translated in clinical responses in the subset of patients enrolled in this clinical trial. The study will accrue transfusion independent patients and patients with early stage MDS as CCUS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Polyclonal evolution of Fanconi anemia to MDS and AML revealed at single cell resolution

BackgroundFanconi anemia (FA) is a rare disease of bone marrow failure. FA patients are prone to develop myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, the molecular clonal evolution of the progression from FA to MDS/AML remains elusive.MethodsHerein, we performed a comprehensive genomic analysis using an FA patient (P1001) sample that transformed to MDS and subsequently AML, together with other three FA patient samples at the MDS stage.ResultsOur finding showed the existence of polyclonal pattern in these cases at MDS stage. The clonal evolution analysis of FA case (P1001) showed the mutations of UBASH3A, SF3B1, RUNX1 and ASXL1 gradually appeared at the later stage of MDS, while the IDH2 alteration become the dominant clone at the leukemia stage. Moreover, single-cell sequencing analyses further demonstrated a polyclonal pattern was present at either MDS or AML stages, whereas IDH2 mutated cell clones appeared only at the leukemia stage.ConclusionsWe thus propose a clonal evolution model from FA to MDS and AML for this patient. The results of our study on the clonal evolution and mutated genes of the progression of FA to AML are conducive to understanding the progression of the disease that still perplexes us.

Bone marrow CD3+ CD56+ regulatory T lymphocytes (TR3 -56 cells) are inversely associated with activation and expansion of bone marrow cytotoxic T cells in IPSS-R very-low/low risk MDS patients.

BackgroundEmergence of dysplastic haematopoietic precursor/s, cytopenia and variable leukaemia risk characterise myelodysplastic syndromes (MDS). Impaired immune‐regulation, preferentially affecting cytotoxic T cells (CTL), has been largely observed in MDS. Recently, we described the TR3−56 T cell subset, characterised by the co‐expression of CD3 and CD56, as a novel immune‐regulatory population, able to modulate cytotoxic functions. Here, we address the involvement of TR3−56 cells in MDS pathogenesis/progression.ObjectivesTo analyse the relationship between TR3−56 and CTL activation/expansion in bone marrow (BM) of very‐low/low‐risk MDS subjects.MethodsPeripheral blood and BM specimens, obtained at disease onset in a cohort of 58 subjects, were analysed by immune‐fluorescence and flow cytometry, to preserve the complexity of the biological sample.ResultsWe observed that a trend‐increase of BM TR3−56 in high/very‐high MDS stage, as compared with very‐low/low group, associates with a decreased activation of BM resident CTL; significant correlation of TR3−56 with BM blasts has been also revealed. In addition, in very‐low/low‐risk subjects the TR3−56 amount in BM inversely correlates with the presence of activated BM CTL showing a skewed Vβ T‐cell repertoire.ConclusionsThese data add TR3−56 to the immune‐regulatory network involved in MDS pathogenesis/progression. Better knowledge of the immune‐mediated processes associated with the disease might improve MDS clinical management.

Endothelial Cell Dysfunction Is Involved in the Progression of Myelodysplastic Syndromes

Background:Myelodysplastic syndromes (MDS) are a group of heterogeneous myeloid clonal disorders characterized by ineffective hematopoiesis, refractory anemia, and a tendency to transform to acute myeloid leukemia (AML). Ineffective hematopoiesis progression and immune deregulation are dominating pathophysiological process of MDS. Emerging evidences showed the role of bone marrow (BM) microenvironment in MDS. In MDS murine model, integral BM microenvironment contributes to inferior hematopoietic function and disease progression. As an important component of BM microenvironment, the relationship between endothelial cells (ECs) and MDS progression remains largely unknown. Although ECs from MDS patients have been identified to have decreased supporting ability to normal hematopoietic stem cells (HSCs), the supporting ability of ECs in different clinical stages of MDS remains to be elucidated. In addition, the role of BM ECs from MDS patients in supporting leukemia cells and their immunomodulatory ability remains unclear.Aims:To determine the number and functions of BM ECs in different subtypes of MDS patients. Moreover, to explore the correlation between BM ECs and MDS progression, which may represent a potential therapeutic target for MDS patients.Methods:In the prospective cohort study, patients with multilineage dysplasia (MDS-MLD, N=15), MDS with excess blasts (MDS-EB, N=15), or AML(N=15) and healthy donors (HD, N=15) were enrolled. BM ECs were analyzed in HD and patients by flow cytometry and in situ histological analyses. The functions of BM ECs were analyzed by migration, angiogenesis capacities, levels of apoptosis and reactive oxygen species (ROS). To evaluate the supporting abilities of BM ECs on HSCs, leukemia cells and T cells, in vitro co-culture strategies were used. The levels of apoptosis, ROS and colony-forming unit-plating (CFU) efficiency of CD34+ and HL-60 cells were investigated. T cell subsets were analyzed by flow cytometry as previously reported. To further investigate the underlying mechanism of dysfunctional ECs, RNA sequencing (RNA-Seq) analyses and real time-PCR (qRT-PCR) were performed in BM ECs from HD and MDS patients with different subtypes.Results:In the current study, gradually increased BM ECs were observed from MDS-MLD, MDS-EB to AML patients. Furthermore, dysfunctional BM ECs were found with MDS progression, characterized by increased levels of migration, angiogenesis capacities, apoptosis and ROS. More importantly, BM ECs from MDS patients exhibited decreased supporting ability of HSCs whereas increased supporting ability of leukemia cells in vitro with MDS progression. After coculture with ECs, levels of apoptosis and ROS in CD34+ cells were increased whereas their CFU efficiency reduced. On the other hand, levels of apoptosis and ROS of HL-60 cells were decreased. The proliferation capacity and leukemia CFU efficiency of HL-60 cells after co-cultured with ECs were enhanced with MDS progression. Furthermore, following coculture with BM ECs, deregulated differentiation was demonstrated in T cell subsets, characterized by elevating proportion of Th2 and Treg and decreasing proportion of Th1 and Th17 with MDS progression. RNA-Seq showed that the expression profile of BM ECs from MDS-EB was closer to MDS-MLD, whereas that of MDS-EB was closer to AML. Different gene expression profiles indicated the expression of hematopoiesis and immune related genes increased in BM ECs with MDS progression. Mechanistically, the mRNA levels of CX CL12, SCF and NFKB of ECs were increased with MDS progression.Summary/Conclusion:In summary, the number of BM ECs gradually increased, BM EC dysfunction more and more severe, and the supporting abilities of BM ECs on HSCs decreased, whereas on leukemia cells increased with MDS progression. Moreover, ECs regulated the differentiation of T cells into immune tolerant cells with MDS progression. Although further validation is required, these findings indicated that the improvement of BM ECs may represent a potential therapeutic approach for MDS patients.Keywords: Myelodysplastic syndromes, endothelial cells, disease progression, ineffective hematopoiesis, immune deregulation DisclosuresNo relevant conflicts of interest to declare.

The ability of bone marrow progenitor cells to form colonies in ex vivo culture of MDS patients

The results of in vitro hematopoiesis studies have provided most of the knowledge about the organization, regulation, and development of the human hematopoietic system over the past three to four decades. However, due to the impossibility of an appropriate assessment of hematopoietic stem cells (HSC) in humans and because of the shortcomings of methodological approaches to determining the role of hematopoietic progenitor cells in the pathogenesis of MDS and to predicting the course of the pathological process, semiliquid agar cultures of bone marrow from patients with myelodysplastic syndrome were used. Myelodysplastic syndrome (MDS) refers to a clinically, morphologically, and genetically heterogeneous group of diseases characterized by clonalism and arising from mutations at the level of hematopoietic progenitor cells. Proliferation of such a mutated stem cell progenitors leads to ineffective maturation of myeloid lineage cells and dysplastic changes in the bone marrow (BM). The aim of the study was to establish the relationship between the functional activity of hematopoietic progenitor cells in the ex vivo culture and the activity of the pathological process in the myelodysplastic syndrome. We studied bone marrow samples from patients with the myelodysplastic syndrome, namely refractory anemia with excess blasts I (MDS RAEB I) and refractory anemia with excess blasts II (MDS RAEB II) and AML under conditions in vitro, as well as their clinical laboratory data. It was found that the percentage of blasts and myeloblasts in the samples of patients with AML and MDS RAEB II increased, compared to the samples of patients with MDS RAEB I (63.5±3.9 %, 18.05±1.01 % and 9.49±1.53 % respectively). An increase in the number of erythrocytes and hemoglobin content was noted in the group of patients with MDS RAEB I compared with MDS RAEB II (2.9±1.4×1012 / l and 105.04±3.6 g / l versus 9±0.8×1012 / l and 84.5±4.8 g / l, respectively). The analysis of the results of BM studies of patients with MDS in in vitro culture indicated a significant lag in the formation of cell aggregates during cultivation and a pronounced inhibition of the colony-forming ability of progenitor cells, compared to the control. A noticeable decrease in the colony-forming ability was observed in patients with MDS RAEB I, MDS RAEB II and AML in this sequence – 4.1±1.2 per 1×105 explanted cells, 3.2±0.9 per 1×105 explanted cells and 2.0±0.6 per 1×105 explanted cells, respectively. The analysis of hematological parameters and the results of BM cells cultivation at different stages of MDS indicates that the colony-forming ability of progenitor cells correlates with the depth of the pathological process.

Modeling ASXL1 Mutation Revealed Myelodysplasia Caused By Derepression of p16Ink4a through Aberrant PRC1-Mediated Histone Modification

Clonal hematopoiesis of indeterminate potential (CHIP) is a premalignant condition in patients with blood cells that possess somatic mutations of genes known to be recurrently mutated in hematologic malignancies. Among them is additional sex combs like 1 (ASXL1), whose mutation is frequent in various myeloid malignancies and associated with poor prognosis, and ASXL1 is known to act as an epigenetic modulator. Despite the clinical impact of ASXL1 mutations in myeloid malignancies, lack of animal models expressing mutated Asxl1 at endogenous levels mimicking human ASXL1-mutated diseases hinders from dissecting precise molecular mechanisms of mutated Asxl1. Here in this study, with newly generated knock-in mice carrying a frameshift mutation at the exon 13 of the Asxl1 gene (Asxl1G643WfsX12), which is identical to the most frequent human ASXL1 G646WfsX12 mutation, we extensively investigated the biological effects of the mutant.Asxl1G643fs/+ mice showed decreased body size and weight, resembling hypomorphic features in patients with Bohring-Opitz syndrome carrying de novo constitutive ASXL1 mutation.Asxl1G643fs/+ mice developed slowly progressive peripheral leukopenia, and Wright-Giemsa stain of bone marrow (BM) cells from Asxl1G643fs/+ mice showed dysplastic myeloid cells represented by hypersegmented neutrophils, apoptotic neutrophils and hyposegmented neutrophils with fine nuclear bridging consistent with pseudo-Pelger-Huët anomaly. Flow cytometric analyses of Asxl1G643fs/+ BM cells confirmed aberrant myeloid differentiation with significant increase of granulocytic and monocytic cell fraction, significant decrease of hematopoietic stem cell (HSC) pool (c-kit+ Sca-1+ Lin- (LSK) cells and CD34- CD135- LSK cells), increased apoptosis in hematopoietic stem/progenitor cells (HSPC) and significant accumulation of G1-phase HSC in the cell cycle analysis, all of which were reminiscent of human myelodysplastic syndromes (MDS). Interestingly, Asxl1G643fs/+ mice became moribund about 600 days after birth when compared to Asxl1+/+ littermates with anemia, leukocytosis, neutrophilia/monocytosis and hepatosplenomegaly, suggesting the development of MDS/myeloproliferative neoplasm (MPN)-like disease, most relevant to human cases with chronic myelomonocytic leukemia.RNA sequencing and gene set enrichment analysis in Asxl1G643fs/+ mice identified derepression of sets of genes that are normally repressed by BMI1, a critical regulator of histone modification in polycomb repressive comprex 1 (PRC1). Among these genes were p16Ink4a, p21, and p15Ink4b all of which were significantly upregulated in Asxl1G643fs/+ LSK cells. We performed chromatin immunoprecipitation (ChIP) assay with an anti-H2AK119ub1 antibody using LSK cells from Asxl1G643fs/+ mice and found that H2AK119ub1 level around the promoter region of p16Ink4a was significantly decreased in Asxl1G643fs/+ LSK cells, suggesting perturbation of Bmi1-driven H2AK119ub1 histone modification by mutated Asxl1. Co-immunoprecipitation assay and in vitro pull-down assay revealed that the mutant form of ASXL1 had no ability to interact with BMI1 as opposed to wild type ASXL1 protein. In addition, we performed ChIP assay with an anti-BMI1 and anti-FLAG antibody using a cell line stably expressed FLAG-tagged wild-type (WT) ASXL1 or mutant form (MT), and found that BMI1 level around the promoter region of p16Ink4a was significantly decreased in the ASXL1 MT-expressed cell line, whereas no significant change in the FLAG level between WT and MT. Furthermore, we intercrossed Asxl1G643fs/+ mice with p16Ink4a heterozygous knock out mice to elucidate the effect of p16Ink4a up-regulation in Asxl1G643fs/+ mice, and found restoration of HSC pool and amelioration of increased apoptosis in HSPC obtained from Asxl1G643fs/+p16Ink4a+/- mice. These results indicated that loss of protein interaction between Asxl1 mutant and Bmi1 affected the activity of PRC1, and subsequent derepression of p16Ink4a by aberrant histone ubiquitination could induce cellular senescence, resulting in MDS-like phenotypes represented by decreased HSC pool and aberrant myeloid differentiation in Asxl1G643fs/+ mice.Taken together, our Asxl1 G643fs mutation knock-in mouse line is useful as a model of CHIP or early stage MDS, and our detailed analysis highlights the functional role of ASXL1 mutation on MDS and MDS /MPN development. DisclosuresMasamoto:Astellas Pharma Inc.: Research Funding; Nippon Shinyaku Co., Ltd.: Other: Scholarship donations for the laboratory; Pfizer Inc.: Research Funding. Arai:Bristol-Myers Squibb: Research Funding. Kurokawa:Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Celgene Corporation: Speakers Bureau; Pfizer Inc.: Research Funding; Bristol-Myers Squibb: Speakers Bureau; Astellas Pharma Inc.: Research Funding; Nippon Shinyaku Co., Ltd.: Other: Scholarship donations for the laboratory, Speakers Bureau.

Understanding Early Stage Myelodysplastic Syndrome Pathobiology

Understanding myelodysplastic-syndrome (MDS) pathobiology is critical to derive and evaluate new therapeutic interventions. Transforming growth factor-beta (TGFβ) signaling is abnormally active in early-stage MDS patient hematopoietic stem and progenitors, and the level of TGFβ-signal-induced transcriptional changes may have prognostic value. However, the mechanism of TGFβ signaling resulting in ineffective hematopoiesis in MDS is not known. TGFβ signaling is tightly controlled by antagonists (e.g. SMAD7) which block promiscuous activity. Upon TGFβ receptor signaling, these antagonists are transiently eliminated to amplify the signal; however, they are re-induced by downstream signaling and subsequently terminate the TGFβ signal (a negative feedback loop). The Verma lab demonstrated that human MDS marrow cells have elevated levels of microRNA-21 (miR-21) that target SMAD7. Their work identifies miR-21 as one factor in MDS that interferes with the TGFβ negative feedback loop to generate a chronic TGFβ signal. We used transient transcription assays to reveal that miR-21 targets the SKI corepressor, a TGFβ-signaling antagonist that binds to R-SMADS to block downstream SMAD-target gene activation. Immunofluorescence staining of primary bone-marrow samples from early-stage MDS patients (with elevated miR-21 and chronic TGFβ) shows significant reduction of not only SMAD7 but also SKI protein. TGFβ signaling induces ubiquitin mediated degradation of SKI, so low SKI expression in MDS is the sum of miR-21 and chronic TGFβ effects. To determine the functional impact of low SKI we examined Ski-/- mice. We show that SKI loss of function is itself sufficient to impair hematopoietic stem cell (HSC) fitness. While flow cytometric evaluation of Ski-/- hematopoiesis indicates little phenotypic change in HSC fractions, and Ski-/- HSC engraft lethally irradiated recipients, competitive HSC transplantation reveals profound Ski-/- HSC fitness defects. Ski-like (Sno/Skil) encodes a SKI-related compressor that is not a miR-21 target. Ski-/- defects are not phenocopied in Skil-/- HSC; underscoring the specificity of the HSC defects to the hypothesized miR-21-SKI axis. In agreement with this, retroviral or lentiviral vector forced expression of miR-21 in HSC is counter-selected within 60 days after transplantation. The Growth factor independent-1 (GFI1) transcriptional repressor is required for HSC fitness and is an independent prognostic indicator in AML/MDS. We previously demonstrated GFI1 transcriptional repression of miR21 . Subsequently, SKI protein levels are significantly decreased in Gfi1-/- bone marrow cells (which express elevated miR-21). SKI expression is rescued in Gfi1-/-miR-21-/- knockout mice but Gfi1-/- HSC fitness is not restored. Thus, it is unlikely that loss of SKI downstream of Gfi1 loss of function explains Gfi1-/- HSC fitness defects. Instead, we show that the SKI corepressor binds the GFI1 transcriptional repressor. Native GFI1 and SKI proteins are in the same complex by immunoprecipitation, and synthetic-protein pull-down assays validated direct interaction and mapped the interacting domains. ChIP Seq reveals that Ski is coincident with Gfi1 on a subset of common peaks. We conclude that: 1) in normal cells GFI1 repression of miR-21 protects expression of SKI, an HSC-relevant GFI1 corepressor, and 2) chronic TGFβ signaling in early stage MDS directly affects HSC fitness by targeting SKI. Disclosures No relevant conflicts of interest to declare.

Genome-wide methylation sequencing identifies progression-related epigenetic drivers in myelodysplastic syndromes

The potential mechanism of myelodysplastic syndromes (MDS) progressing to acute myeloid leukemia (AML) remains poorly elucidated. It has been proved that epigenetic alterations play crucial roles in the pathogenesis of cancer progression including MDS. However, fewer studies explored the whole-genome methylation alterations during MDS progression. Reduced representation bisulfite sequencing was conducted in four paired MDS/secondary AML (MDS/sAML) patients and intended to explore the underlying methylation-associated epigenetic drivers in MDS progression. In four paired MDS/sAML patients, cases at sAML stage exhibited significantly increased methylation level as compared with the matched MDS stage. A total of 1090 differentially methylated fragments (DMFs) (441 hypermethylated and 649 hypomethylated) were identified involving in MDS pathogenesis, whereas 103 DMFs (96 hypermethylated and 7 hypomethylated) were involved in MDS progression. Targeted bisulfite sequencing further identified that aberrant GFRA1, IRX1, NPY, and ZNF300 methylation were frequent events in an additional group of de novo MDS and AML patients, of which only ZNF300 methylation was associated with ZNF300 expression. Subsequently, ZNF300 hypermethylation in larger cohorts of de novo MDS and AML patients was confirmed by real-time quantitative methylation-specific PCR. It was illustrated that ZNF300 methylation could act as a potential biomarker for the diagnosis and prognosis in MDS and AML patients. Functional experiments demonstrated the anti-proliferative and pro-apoptotic role of ZNF300 overexpression in MDS-derived AML cell-line SKM-1. Collectively, genome-wide DNA hypermethylation were frequent events during MDS progression. Among these changes, ZNF300 methylation, a regulator of ZNF300 expression, acted as an epigenetic driver in MDS progression. These findings provided a theoretical basis for the usage of demethylation drugs in MDS patients against disease progression.

SET Domain Containing 2 Deficiency in Myelodysplastic Syndrome

Recent studies have shown that myelodysplastic syndrome’s (MDS) progression to acute myeloid leukemia (AML) is associated with gene mutations. SET domain containing 2 (SETD2) variants were reported as a risk factor of poor prognosis in patients with AML. However, little is known about the potential contribution of the SETD2 gene in MDS. In this study, we investigated the roles of SETD2 gene mutations/variants on clinical features and prognosis in patients with MDS. A 43-gene panel was used for next-generation sequencing in 203 patients with primary MDS, and then the effects of SETD2 mutation on Wnt/β-catenin signaling was investigated during the different stages of MDS. At a median follow up of 33 months, 65 (32.0%) deaths and 94 (46.3%) leukemic transformations were recorded. The most frequent mutations/variants included TET2, DNMT3A, and ASXL1 mutations/variants. 37 patients had SETD2 gene mutations/variants, and these patients exhibited a significantly increased frequency of TP53 mutations. Multivariate survival analyses indicated that SETD2 mutations/variants were closely associated with overall survival (OS), and they were identified as risk factors for progression-free survival (PFS), especially with low expression of SETD2 gene. Further, we found that SETD2 loss could promote MDS progression via upregulation DVL3 mRNA level in BM cells and it could also cause genomic instability. Secondary mutations, such as TP53 and FLT3 mutations, were acquired at the time of progression to AML. In conclusion, we showed that SETD2 deficiency was associated with poor outcomes in patients with MDS. Moreover, SETD2 deficiency may upregulate DVL3 expression and modulate genomic stability that caused AML transformation.

Hsp70 Trap Assay for Detection of Misfolded Subproteome Related to Myelodysplastic Syndromes.

The onset and progression of numerous serious diseases (e.g., various types of malignancies, neurodegenerative diseases, and cardiac diseases) are, on a molecular level, associated with protein modifications and misfolding. Current methods for the detection of misfolded proteins are not able to detect the whole misfolded subproteome and, moreover, are rather laborious and time consuming. Herein, we report on a novel simple method for the detection of misfolded proteins employing a surface plasmon resonance (SPR) biosensor and heat shock protein 70 (Hsp70) that recognizes and traps misfolded proteins in a nucleotide-dependent manner. We use this method for the detection of misfolded proteins in blood plasma of patients with various subtypes of myelodysplastic syndromes (MDS) and healthy donors. Our results reveal significantly elevated levels of misfolded proteins in the two stages of MDS that are most affected by oxidative stress: low-risk (RARS) and intermediate-risk (RCMD) patients. This approach can be extended to a variety of diseases and provides unique insights into the thus far unexplored area of blood proteome.

PS1327 PRO‐NECROPTOTIC RIPK3 AS A STAGE‐DEPENDENT MARKER IN THE BONE MARROW OF PATIENTS WITH MDS AND CMML

Background:Chronic inflammation plays a central role in the development of hematological neoplasms. In Myelodysplastic syndromes (MDS) pro‐inflammatory microenvironment is suspected to drive disease development. Several studies have shown increased rates of programmed cell death in marrow cells of patients with early stage MDS. In these patients improvement of cytopenia is a major therapeutic approach. Overcoming resistance to programmed cell death is a central therapeutic aim in patients with high‐risk MDS/sAML. RIPK3 is a crucial player of regulated necrosis (necroptosis). Necroptosis has a strong inflammatory capacity and pro‐inflammatory processes trigger necroptosis.Aims:The aim of this project is to identify pro‐necroptotic proteins in the bone marrow of patients with MDS, CMML and sAML and to correlate the necroptotic capacity with the stage of the disease. By pharmacological modulation of necroptosis we would like to evaluate the effects of necroptotic signaling on inflammation, cell death and cell differentiation.Methods:We therefore analyzed the impact of pro‐necroptotic signaling on bone marrow bulk and the subset of CD34+stem/progenitor cells of patients with MDS and chronic myelomonocytic leukemia (CMML) in vitro. Bone marrow mononuclear cells (BMMNCs) were isolated from BM aspirates of 5 patients with early MDS (MDS‐MLD, MDS‐RS‐SLD and MDS‐RS‐MLD), 5 patients with late MDS (MDS‐EB1 and MDS‐EB2), 5 patients with CMML‐1, 7 patients with CMML‐2 and 4 patients with sAML after a history of MDS and 4 patients with sAML after a history of CMML. Bulk of BMMNCs and purified CD34+stem/progenitor cells were stained intracellular for RIPK3 and analyzed by flow cytometry. The patient samples were compared to 12 age‐matched BM samples obtained from hip replacement surgery from otherwise healthy individuals. Furthermore, immunohistochemistry was performed in an enlarged cohort of patients. Therefore paraffin embedded bone marrow biopsies of individuals with MDS or CMML‐1/2 were analyzed over time and disease progression. Additionally BMMNCs of patients with MDS were treated with specific RIPK1 (Nec1 s) and RIPK3 (GSK 843A) inhibitors for 72 h. Short term viability analysis by flow cytometry were performed and long‐term analysis using growth factor enriched methylcellulose.Results:Expression of RIPK3 was significantly increased in sAML after a history of MDS compared to late MDS and compared to healthy controls. Furthermore patients with sAML after a history of CMML showed a significantly higher expression of RIPK3 than patients with CMML‐1, CMML‐2 and healthy controls. These findings were confirmed by immunohistochemistry in an enlarged cohort of patients. Here paraffin embedded bone marrow biopsies of individuals with MDS or CMML‐1/2 were analyzed over time and disease progression.Additionally BMMNCs of patients with MDS or CMML were treated with specific RIPK1 (Nec1 s) and RIPK3 (GSK 843A) inhibitorsfor 72 h. Short term viability analysis by flow cytometry showed no impact of inhibitor treatment. However after culture in growth factor enriched methylcellulose for 10–14 days colony forming capacity was significantly increased in low‐risk MDS patients after inhibition of pro‐necroptotic signaling. Here increase in colony numbers was not restricted to a specific lineage.Summary/Conclusion:We conclude that RIPK3 plays a critical stage‐dependent role in patients with MDS and CMML. Disease progression to sAML is associated with an increased RIPK3‐Expression. We further hypothesize that pro‐inflammatory processes like necroptosis have an impact on aberrant myeloid homeostasis.

Oral and dental management of people with myelodysplastic syndromes and acute myeloid leukemia: A systematic search and evidence-based clinical guidance.

To systematically search all studies that discussed dental procedures in patients diagnosed with myelodysplastic syndromes (MDS) and/or acute myeloid leukemia (AML) and to provide an evidence-based clinical guidance on oral and dental management of people with MDS and/or AML. The systematic search followed the Preferred Reporting Item for Systematic Review and Meta-analyses Protocols (PRISMA-P) guideline. Two databases systems were used (MEDLINE and EMBASE). PROSPERO was searched for ongoing or recently completed systematic reviews. The International Clinical Trials Registry Platform Search was searched for ongoing or recently completed trials. Level of evidence was evaluated based on the Oxford Level of Evidence. The Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach was used to summarize the evidence. Only 18 articles were selected and included in data synthesis and analysis. The level of evidence and recommendation ranged from 1 to 5 and A to D, respectively. All the included studies in the data synthesis (n=18) showed no specific guidelines were followed; however, all reflected the importance of liaison with the patient's hematoncologist at all stages of MDS and/or AML therapy. Oral and dental assessment is crucial prior to MDS therapy to help reduce anticipated complications. Dental treatment prior to hematopoietic stem cell transplantation and/or active stage of MDS therapy is tricky and always required liaison with the hematoncologist. MDS can progress to AML; hence, dental care providers are in a good position to spot any changes and refer early to the hematoncologist for further assessment.

Myeloid Cell Leukemia-1 Gene Expression and Clinicopathological Features in Myelodysplastic Syndrome

Background and Aims: Myeloid cell leukemia-1 (Mcl-1) plays a pivotal role in the survival of hematologic and solid tumors, and is known as a substantial oncogene. Studies have demonstrated the altered expression of Mcl-1 has been linked to malignancy development and poor prognosis. In this research, we have studied the expression of Mcl-1 mRNA in myelodysplastic syndrome (MDS) patients and determined association with clinico-pathological factors, MDS subgroups as well as international prognostic scoring system.
 Materials and Methods: The relative level of Mcl-1 was determined by real time quantitative real-time polymerase chain reaction and gene expression normalized to Glyceraldehyde-3-phosphate dehydrogenase.
 Results: Results indicated amplification of mRNA encoding Mcl-1 in 100% of the cases. The higher level of Mcl-1 existed in MDS patients compared with the healthy controls but there was no statistically difference of Mcl-1 expression between these groups. Fold change in gene expression was higher in advanced stage MDS, high risk MDS, cases with >5% blast and LDH >400 to their corresponding groups. In addition, the correlation between gene expression and cytogenetic prognostic subgroups was statistically significant (p=0.043).
 Conclusions: In the present study, we showed that Mcl-1 is expressed in MDS independent of the World Health Organization subgroup and international prognostic scoring system. Therefore, Mcl-1 may be up-regulated already in early stages of leukemogenesis.

Myelodysplastic syndrome progression to acute myeloid leukemia at the stem cell level.

Myelodysplastic syndromes (MDS) frequently progress to acute myeloid leukemia (AML); however, the cells leading to malignant transformation have not been directly elucidated. As progression of MDS to AML in humans provides a biological system to determine the cellular origins and mechanisms of neoplastic transformation, we studied highly fractionated stem cell populations in longitudinal samples of patients with MDS who progressed to AML. Targeted deep sequencing combined with single-cell sequencing of sorted cell populations revealed that stem cells at the MDS stage, including immunophenotypically and functionally defined pre-MDS stem cells (pre-MDS-SC), had a significantly higher subclonal complexity compared to blast cells and contained a large number of aging-related variants. Single-cell targeted resequencing of highly fractionated stem cells revealed a pattern of nonlinear, parallel clonal evolution, with distinct subclones within pre-MDS-SC and MDS-SC contributing to generation of MDS blasts or progression to AML, respectively. Furthermore, phenotypically aberrant stem cell clones expanded during transformation and stem cell subclones that were not detectable in MDS blasts became dominant upon AML progression. These results reveal a crucial role of diverse stem cell compartments during MDS progression to AML and have implications for current bulk cell-focused precision oncology approaches, both in MDS and possibly other cancers that evolve from premalignant conditions, that may miss pre-existing rare aberrant stem cells that drive disease progression and leukemic transformation.

Genomic Instability and a Preferential Involvement of Ras Pathway in the Myelodysplastic Syndromes Evolution to Secondary Acute Myeloid Leukemia

Myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) are hematological disorders at high risk of progression to acute myeloid leukemia (sAML). Previous high-throughput sequencing studies have provided insight into the mutational dynamics and clonal evolution underlying disease progression. However, large serial sequencing studies are still required to define which type of mutations alone or in combination contribute to leukemic transformation.To assess the mutational profiles and mutational dynamics underlying progression from MDS to sAML, a targeted-deep sequencing (TDS) of 117 MDS/AML related-genes was performed in 110 bone marrow serial samples from 50 MDS/CMML patients who evolved to sAML and 5 patients who did not evolved (controls), at two different time-points: at the time of diagnosis and at sAML progression or after a median of 3 year follow-up, respectively.A total of 269 mutations in 57 different genes were identified at second sampling. At diagnosis, all patients, progressing and not progressing (controls), presented similar number of mutations (p=0.15). Moreover, patients evolving to sAML were then divided by FAB/WHO subtypes at diagnosis (CMML, low-risk and high-risk MDS subgroups) and no differences were observed in the number of mutations (p=0.71) and variant allele frequency (VAF) between each group (p=0.63). It should be noted that mutations in the splicing pathway were significantly more frequent in low-risk MDS patients (89% low-risk MDS vs. 56% high risk MDS, p=0.038). However, after progression, those patients who evolved to sAML displayed a statistically significant increase of mutations (p=0.001) at the leukemic phase, while controls did not at the follow-up sample (p=0.88). This higher number of mutations at second sampling in patients who evolved to sAML, independently of their diagnostic subtype, may be indicative of a higher genomic instability during disease evolution.To study the mutational dynamics and what mutations could be important during disease evolution, the VAFs of mutations detected at both time-points in each patient of transformation cohort were compared. We observed that some mutations identified at the sAML stage (163 mutations) were already present at the MDS stage, at clonal or subclonal levels, and were retained during evolution, for example in genes such as SRSF2 and DNMT3A. However, 106 mutations increased in clonal size or were newly acquired. Interestingly, most of mutations in Ras signaling pathway showed a same pattern: they were not present at time of diagnosis and appeared at sAML. In fact, mutations in this pathway were detected in 25 of 50 patients (50%) included in this cohort and in 22 of them (88%) mutations displayed this dynamic. Therefore, in this study, Ras signaling was the most common pathway involved in the progression from MDS to sAML. Of note, 9 of these patients (18% of the whole cohort) presented, independently of diagnosis, a co-occurring cohesin mutation, that was already present at diagnosis and, in most cases, markedly increased in clonal size at sAML. Thus, the combination of mutations in these two pathways could play an important role during disease evolution.In addition, 22 of 50 patients were treated with a disease-modifying agent (18 azacytidine and 4 lenalidomide) before they progressed to sAML, while the remaining 28 patients received no treatment or supportive care and were considered as non-treated. Thus, we studied the effect of disease-modifying therapy on mutational dynamics in this cohort of patients progressing to sAML. In the treated patients, a higher proportion of newly acquired or increasing mutations at sAML in chromatin modifiers was observed, while in non-treated patients most mutations remained stable (61% vs. 28.6%, p=0.013). By contrast, regarding treatment, no differences were detected in the mutational dynamics of cohesin (p=0.56) or Ras pathway (p=1.00).MDS progression to sAML was characterized by a higher genomic instability, independently of MDS subtypes of patients at diagnosis. Ras signaling was the most frequent affected pathway during disease evolution in this cohort and, interestingly, the co-occurrence of Ras signaling and cohesin mutations could play an important role in the progression. Moreover, mutations in chromatin modifiers genes could be related to the evolution of patients who received disease-modifying treatment before progression to sAML. DisclosuresOlivier:Celgene: Honoraria; Jassen: Honoraria. Díez-Campelo:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Loss-of-Function Mutation in tet2 in Zebrafish Leads to Early MDS like Phenotype

Myelodysplastic syndrome (MDS) is a pre-leukemic state characterized by the failure of the bone marrow to produce mature and functional blood cells. Nearly one-third of MDS patients progress to acute myeloid leukemia (AML). AML is the most common form of acute leukemia in adults and accounts for a high level of mortality in pediatric leukemia.Loss-of-function mutations in the Ten-11 Translocation 2 (TET2) gene are implicated in MDS and AML, but evidence that TET2 mutations are also found in healthy individuals as a result of clonal hematopoiesis calls into question the contribution of TET2 to leukemogenesis. Many studies have tried to mutate the DNA binding domain in TET2 both in mouse and zebrafish, but the blood phenotypes observed have been inconsistent.We created a tet2 zebrafish mutant using CRISPR-Cas9 technology, where we deleted 2.1 kb from exon 2, confirmed by cDNA analysis. A recent publication suggests the importance of Ser99 in the stability of the TET2 protein (Wu et al., 2018) and this conserved serine residue is contained in the region of deletion in our zebrafish tet2 mutant.These tet2 mutant fish were incrossed to create a maternal-zygotic mutant with no tet2 expression. Importantly, quantitative PCR demonstrated that there was no compensatory effect from tet1 and tet3 in the context of tet2 loss.Whole mount in situ hybridization performed on 24-48h tet2 mutant zebrafish embryos provided evidence of a significant reduction of early and mature myeloid and erythroid cells through staining for spi1/pu.1 (myeloid), gata1 (erythroid) myeloperoxidase (mpx; neutrophils), carboxypeptidase A5 (cpa5; mast cells) and l-plastin (lcp1; macrophages). The stem cell compartment was not impacted, as shown by wild-type runx1/c-myb expression. Further, o-dianisidine staining revealed low hemoglobin content in tet2 mutants compared with control embryos. Overall, the early embryos elucidate a refractory anemia like phenotype.Flow cytometry analysis of kidney marrow from 3-month-old adult tet2 mutant fish similarly demonstrated a decrease in the myeloid and erythroid compartments, in keeping with characteristics typical of low stage MDS. We are currently employing RNAseq and MeDIP analysis on blood cell populations from tet2 mutants to determine the genetic and epigenetic signatures associated with this mutation.This zebrafish model of tet2 loss uniquely provides with a dynamic phenotype that starts with refractory anemia with progression through low stage MDS. Thus this model will be beneficial in elucidating the mechanisms underlying how tet2 contributes to MDS and AML and provide a preclinical platform to screen for therapeutic interventions. DisclosuresNo relevant conflicts of interest to declare.