Progression Of Myelodysplastic Syndrome Research Articles

Rapid Disease Progression of Myelodysplastic Syndrome is Reflected in Transcriptomic and Functional Abnormalities of Bone Marrow MSCs.

Bone marrow (BM) mesenchymal stromal cells (MSCs) are important regulators of hematopoietic stem and progenitor cells (HSPCs). When transformed to a dysplastic phenotype, MSCs contribute to hematopoietic diseases such as myelodysplastic syndromes (MDS), but it remains unclear if there are specific properties in MDS-MSCs that contribute to the disease course. To understand this, we investigated MDS-MSCs from fast (MDSfast) vs slow (MDSslow) progressing disease groups and discovered differences between these groups. MDSfast-MSCs secrete more inflammatory factors, support myeloid-skewed differentiation of HSPCs, and importantly, show poorer response to hypomethylation as a key differentiator in GSEA analysis. When exposed to long-term in vivo stimulation with primary MDSfast-MSCs-based scaffolds, healthy donor (HD) HSPCs show elevated NF-κB expression, similar to leukemic HSPCs in MDS. Those "MDSfast-MSCs-primed" HD-HSPCs continue to show enhanced engraftment rates in secondary MDS-MSC-based scaffolds, providing evidence for the microenvironmental selection pressures in MDS towards leukemic HSPCs. Together, our data point towards a degree of co-development between MSCs and HSPCs during the progression of MDS, where changes in MDS-MSCs take place mainly at the transcriptomic and functional levels. These unique differences in MDS-MSCs can be utilized to improve disease prognostication and implement targeted therapy for unmet clinical needs.

Glutaminase 1 plays critical roles in myelodysplastic syndrome and acute myeloid leukemia cells.

Myelodysplastic syndrome (MDS) features bone marrow failure and a heightened risk of evolving into acute myeloid leukemia (AML), increasing with age and reducing overall survival. Given the unfavorable outcomes of MDS, alternative treatments are necessary. Glutamine, the most abundant amino acid in the blood, is metabolized first by the enzyme glutaminase (GLS). To investigate whether GLS is involved in the progression of MDS. The efficacy of GLS inhibitors (CB839 or IPN60090) and BCL2 inhibitor venetoclax was also examined. We employed GLS inhibitors (CB839, IPN60090) and the BCL2 inhibitor venetoclax, prepared as detailed. MDS and AML cell lines were cultured under standard and modified (hypoxic, glutamine-free) conditions. Viability, proliferation, and caspase activity were assessed with commercial kits. RT-PCR quantified gene expression post-shRNA transfection. Mitochondrial potential, ATP levels, proteasome activity, and metabolic functions were evaluated using specific assays. Statistical analyses (t-tests, ANOVA) validated the findings. The glutamine-free medium inhibited the growth of MDS cells. GLS1 expression was higher in AML cells than in normal control samples (GSE15061), whereas GLS2 expression was not. Treatment of MDS and AML cells for 72 h was inhibited in a dose-dependent manner by GLS inhibitors. Co-treatment with the B-cell lymphoma 2 (BCL2) inhibitor venetoclax and GLS inhibitors increased potency. Cells transfected with GLS1 short hairpin RNA showed suppressed proliferation under hypoxic conditions and increased sensitivity to venetoclax. Targeting glutaminolysis and BCL2 inhibition enhances the therapeutic efficacy and has been proposed as a novel strategy for treating high-risk MDS and AML.

The immunobiology of myelodysplastic neoplasms: a mini-review.

This mini review summarizes the immunobiology of myelodysplastic syndromes, specifically focusing on the interactions between immune cells, cytokines, and dysplastic cells within the tumor microenvironment in the bone marrow. We elucidate in detail how immune dysregulation and evasion influence the initiation and progression of myelodysplastic syndromes, as well as resistance to therapy and progression to AML. In addition, we highlight a range of therapeutic strategies, including the most recent breakthroughs and experimental therapies for treating MDS. Finally, we address the existing knowledge gaps in the understanding of the immunobiology of MDS and propose future research directions, promising advancements toward enhancing clinical outcomes and survival for patients with MDS.

Mesenchymal Stem Cells in Myelodysplastic Syndromes and Leukaemia.

Mesenchymal stem cells (MSCs) are one of the main residents in the bone marrow (BM) and have an essential role in the regulation of haematopoietic stem cell (HSC) differentiation and proliferation. Myelodysplastic syndromes (MDSs) are a group of myeloid disorders impacting haematopoietic stem and progenitor cells (HSCPs) that are characterised by BM failure, ineffective haematopoiesis, cytopenia, and a high risk of transformation through the expansion of MDS clones together with additional genetic defects. It has been indicated that MSCs play anti-tumorigenic roles such as in cell cycle arrest and pro-tumorigenic roles including the induction of metastasis in MDS and leukaemia. Growing evidence has shown that MSCs have impaired functions in MDS, such as decreased proliferation capacity, differentiation ability, haematopoiesis support, and immunomodulation function and increased inflammatory alterations within the BM through some intracellular pathways such as Notch and Wnt and extracellular modulators abnormally secreted by MSCs, including increased expression of inflammatory factors and decreased expression of haematopoietic factors, contributing to the development and progression of MDSs. Therefore, MSCs can be targeted for the treatment of MDSs and leukaemia. However, it remains unclear what drives MSCs to behave abnormally. In this review, dysregulations in MSCs and their contributions to myeloid haematological malignancies will be discussed.

Hypermethylation of GSTM5 and its effect on oxidation in myelodysplastic syndrome

AbstractBackgroundHypermethylation of glutathione‐S‐transferase 5 (GSTM5) and its effect on oxidation in the pathogenesis of myelodysplastic syndrome (MDS) were investigated.MethodsGSTM5 methylation was detected in bone marrow (BM) samples from MDS patients, acute myeloid leukemia (AML) patients, and control individuals using methylation‐specific PCR and MassARRAY analysis. Bisulfite sequencing PCR was performed to verify methylation levels, while mRNA levels were determined using reverse transcription polymerase chain reaction. Correlations between GSTM5 methylation and clinical parameters were analyzed. The MDS cell line, SKM‐1, was treated with decitabine, buthionine sulfoximine, or overexpression of GSTM5, and the glutathione level and cell viability were detected.ResultsThe MassARRAY analysis revealed significant differences in GSTM5 methylation levels between the MDS and control groups. GSTM5 methylation levels were significantly increased in the high‐risk subgroup and showed a significant association with MDS progression to AML (hazard ratio = 3.6). Levels of GSTM5 mRNA were significantly decreased in the MDS group, exhibiting a negative correlation with the GSTM5 gene methylation level. Normal BM HS‐5 cells exhibited significantly lower levels of GSTM5 methylation than SKM‐1 cells. Overexpression of GSTM5 in SKM‐1 cells or treatment with buthionine sulfoximine or decitabine resulted in inhibition of proliferation and significantly decreased glutathione levels.ConclusionsGSTM5 plays an anti‐oxidative role in MDS and the tumor suppressor effect of GSTM5 may be mediated by reducing glutathione levels. GSTM5 hypermethylation and low levels of GSTM5 expression may be prognostic markers for MDS.

Analyzing Differences in Hematological and Immunological Characteristics Related to Common Gene Mutations in Myelodysplastic Syndromes.

Genetic mutations play a crucial role in the development and progression of myelodysplastic syndromes (MDS), impacting the immune microenvironment and influencing the choice of treatment regimen, as well as the efficacy and prognosis of patients. The objective of this study was to examine variations in hematological and immunological characteristics associated with common gene mutations in MDS patients and establish a foundation for the precise treatment of MDS. The hematological, immunological, and other clinical features of 71 recently diagnosed MDS patients from January 1, 2019, to July 31, 2023, were retrospectively analyzed. These patients were categorized based on their gene mutations, and the variances in hematological and immunological characteristics among distinct groups were compared. Hematological variances were observed among different gene mutation groups. Specifically, platelet counts in the splicing factor 3B subunit 1 (SF3B1) mutation group were notably higher compared to the wild-type group (p = 0.009). Conversely, in the additional sex combs like 1 (ASXL1) mutation groups, monocyte ratios were significantly elevated in comparison to the wild-type group (p = 0.046), and in the ten-eleven translocation 2 (TET2) mutation group, lymphocyte ratios were significantly lower (p = 0.022). Additionally, the leukocyte (p = 0.005), neutrophil ratio (p = 0.002), and lymphocyte ratio (p = 0.001) were significantly higher in the Runt-related transcription factor 1 (RUNX1) mutation group. Regarding immunological distinctions, the Natural Killer (NK) cell ratio demonstrated a significant increase in the SF3B1 mutation group (p = 0.005). Moreover, the TET2 mutation group exhibited a significantly higher Interleukin-8 (IL-8) level (p = 0.017). In contrast, the U2 small nuclear RNA auxiliary factor 1 (U2AF1) group displayed significantly lower levels of IL-1β (p = 0.033), IL-10 (p = 0.033), and Tumour Necrosis Factor-α (TNF-α) (p = 0.009). Distinct variations exist in the immune microenvironment of MDS associated with different genetic mutations. Further studies are imperative to delve into the underlying mechanisms that drive these differences.

Inflammatory profile of lower risk myelodysplastic syndromes.

The precise link between inflammation and pathogenesis of myelodysplastic syndrome (MDS) is yet to be fully established. We developed a novel method to measure ASC/NLRP3 protein specks which are specific for the NLRP3 inflammasome only. We combined this with cytokine profiling to characterise various inflammatory markers in a large cohort of patients with lower risk MDS in comparison to healthy controls and patients with defined autoinflammatory disorders (AIDs). The ASC/NLRP3 specks were significantly elevated in MDS patients compared to healthy controls (p < 0.001) and these levels were comparable to those found in patients with AIDs. The distribution of protein specks positive only for ASC was different to ASC/NLRP3 ones suggesting that other ASC-containing inflammasome complexes might be important in the pathogenesis of MDS. Patients with MDS-SLD had the lowest levels of interleukin (IL)-1β, tumour necrosis factor (TNF), IL-23, IL-33, interferon (IFN) γ and IFN-α2, compared to other diagnostic categories. We also found that inflammatory cytokine TNF was positively associated with MDS progression to a more aggressive form of disease and IL-6 and IL-1β with time to first red blood cell transfusion. Our study shows that there is value in analysing inflammatory biomarkers in MDS, but their diagnostic and prognostic utility is yet to be fully validated.

Research and clinical updates on IRAK4 and its roles in inflammation and malignancy: themes and highlights from the 1st symposium on IRAK4 in cancer

The intracellular serine/threonine interleukin 1 receptor-associated kinase 4 (IRAK4) is necessary for most signaling by activated Toll-like receptors (TLRs). Activation of IRAK4 drives activation of nuclear factor kappa B (NF-κB) and so promotes cell survival, inflammation, and other aspects of the adaptive immune response. However, the IRAK4 pathway can be coopted by cancers and lead to the survival and proliferation of malignant cells. Inappropriate IRAK4 activity has been linked with the progression of myelodysplastic syndrome (MDS), other hematologic malignancies, and some solid tumors, and preclinical cancer models indicate that IRAK4 inhibition has anti-tumor effects. As such, inhibition of IRAK4 is an emerging and attractive target for tumor suppression. The growing interest in IRAK4 motivated the 1st Symposium on IRAK4 in Cancer held in October 2022 to bring together IRAK4 researchers and clinicians to discuss new insights into the biology of IRAK4 and development of IRAK4 inhibitors. Presentations and discussions at the meeting provided updates on the biology of IRAK4 and its links with mutations in the spliceosome, new outcomes from preclinical models that indicate synergy between inhibitors of IRAK4 and FLT3 and BTK inhibitors, and an update on the clinical development of the investigational IRAK4 inhibitor emavusertib, currently being assessed in ongoing phase 1/2 clinical studies in hematologic cancers and several solid tumors.

Patterns of lower risk myelodysplastic syndrome progression: factors predicting progression to high-risk myelodysplastic syndrome and acute myeloid leukemia.

The patterns of low-risk myelodysplastic syndrome (MDS) progression and the clinical and molecular features of those patterns have not been well described. We divided our low-risk (LR) MDS patients (N=1,914) into 4 cohorts: 1) patients who remained LR-MDS (LR-LR; N=1,300; 68%), 2) patients who progressed from LR to high-risk (HR) MDS (LR-HR) without transformation into acute myeloid leukemia (AML) (N=317; 16.5%), 3) patients who progressed from LR to HR MDS and then AML (LR-HR-AML; N=124; 6.5%), and 4) patients who progressed from LR MDS directly to AML (LR-AML; N=173; 9%). Risk factors for progression included: male gender, low absolute neutrophil count (ANC), low platelet count, high bone marrow (BM) blasts, ferritin >1000 mcg/L, albumin <3.5 g/dL, multi-lineage dysplasia (MLD), and lack of ring sideroblasts. Among patients with marked BM fibrosis (N=49), 18% progressed directly to AML. Somatic mutations (SM) associated with an increased risk of direct or indirect AML progression included SRSF2 and NRAS. SM in IDH1, IDH2 and NPM1 were more common in patients with direct AML transformation. SM associated with progression to higher risk disease only, without AML transformation, were ASXL1, TP53, RUNX1, and CBL. SF3B1 mutation was associated with less progression. About 171 patients (13.1% of all LR-LR patients) died within two years of diagnosis of LR-MDS without disease progression. Among the 61 cases with documented cause of death, 18 patients (29.5%) died from cytopenia and MDS-related complications. Identifying patterns of disease progression of LR MDS patients and their predictive factors will be crucial to be able to tailor therapy accordingly.

Novel therapies for MDS and future prospects

Myelodysplastic syndromes (MDS) are clonal diseases resulting from the accumulation of genetic mutations. In general, MDS is categorized into two risk groups, with management and treatment varying significantly based on this classification. Over the past two decades, allogeneic transplantation and hypomethylating agents (HMAs) have become the standard of care and remain crucial for higher-risk MDS. Unfortunately, no new drugs have emerged to replace HMAs as the standard of care. However, the landscape of practice and research in MDS has evolved. In 2022, the focus of diagnostic classification of MDS shifted significantly from morphology to genetic alterations. As a result, treatment strategies centered on genetic mutations are now already used internationally. Revisions made to the International Working Group (IWG) criteria for assessing treatment response in 2023 are expected to further improve accuracy. Meanwhile, interest has increased in understanding the relationship between inflammation and the development and progression of lower-risk MDS. This year, luspatercept, an anti-anemic agent targeting the TGFβ pathway, became available for clinical use in Japan. Various research initiatives are currently underway to develop new medicines targeting specific molecules within innate immune and inflammasome-signaling pathways, including IL-1β, CD33, TLR, IRAK4, and p38MAPK.

The Mechanism of Setd2 in Mesenchymal Stromal Cell in MDS-Associated Ineffective Hematopoiety

Myelodysplastic syndrome (MDS) is a heterogeneous, clonal, hematopoietic stem progenitor cell malignant disease. Bone marrow microenvironment participates in the occurrence and development of MDS and plays a key role. Histone methyltransferase Setd2 is the only enzyme that can catalyze the methylation of mammalian histone H3K36Me3. Previous studies have confirmed that Setd2 is involved in the the transformation of MDS to leukemia, and the expression of S100A9 decreases after Setd2 knockout. This study is based on NHD13 mice with mesenchymal stem cell (MSC) targeted knockout of Setd2. Through experimental methods such as flow cytometry, peripheral blood routine, Wright staining, HE staining, and Gomori staining, it finds that Setd2 targeted knockout mice undergo faster leukemia transformation compared to NHD13 mice; Peripheral blood white blood cells increase, while hemoglobin and platelets decrease; The differentiation of hematopoietic stem progenitor cells in bone marrow is abnormal, with abnormal hematopoiesis and increased blast cells; Spleen fibrosis aggravates and megakaryocyte infiltrates; and Extramedullary hematopoiesis in liver. Through flow cytometry,β- Galactosidase staining and other experimental methods, we finds that compared with NHD13 mice, the MSC morphology of knockout mice is irregular, and the support function for hematopoietic cells is weakened. This study demonstrates that MSC targeted knockout of Setd2 has a promoting effect on the progression of MDS. We used mass spectrometry to identify proteins that are significantly upregulated in the targeted knockout mouse bone marrow supernatant compared to the NHD13 mouse bone marrow supernatant, and and select the protein Uba5 related to hematopoiesis from them. The expression of Uba5 in the bone marrow supernatant of knockout mice is significantly higher than that in the NHD13 group through WB validation. It is speculated that the hematopoietic changes in Setd2 targeted knockout mice may be related to the downstream target protein Uba5 of Setd2. This study takes the change of bone marrow microenvironment as an entry point to explore the possible mechanism of MDS ineffective hematopoiesis, hoping to provide microenvironment regulation therapy for some MDS patients with genes that hematopoietic stem cell transplantation that cannot cure(such as TP53, NRAS, KRAS), and provide new ideas for MDS treatment. There are still many limitations in this study, further clinical validation and vivo and vitro rescue experiments on downstream target genes and proteins are still needed in the future.

Study on the Changes of Immune Cells in the Evolution of Myelodysplastic Syndromes

ABSTRACT Objective: To analyze the differences of peripheral blood immune cells in healthy people, and patients with myelodysplastic syndromes (MDS) patients or acute myeloid leukemia (AML), and to investigate their correlations with International Prostate Symptom Score(IPSS), blasts and other factors in MDS patients. Methods: 55 patients with MDS(MDS group), 67 patients with AML(AML group) and 25 heathy people(healthy group) were selected as research objects from Dongzhimen Hospital Affiliated with Beijing University of Chinese Medicine from January, 2012 to April, 2022 for this retrospective analysis. By comparing peripheral blood lymphocyte subsets count and peripheral blood lymphocyte subsets percentage, we studied the expression of T lymphocyte subsets (CD3+, CD4+, CD8+, CD4+/CD8+, CD3+CD4+CD25+FoxP3+), B cells (CD19+) and NK cells (CD3-CD16+CD56+). The correlations between the different indexes and IPSS and blasts were analyzed. Results: (1) Peripheral blood lymphocyte subsets count: There were differences among the three groups. Through pairwise comparison, MDS group had lower peripheral blood lymphocyte subsets count(P＜0.025), probably owing to the low peripheral blood lymphocyte count. (2)Peripheral blood lymphocyte subsets percentage: The percentages of Treg cells, B cells and T cells varied among the three groups. Compared with the healthy group, the percentages of T cells ( P＜0.025) and Treg cells ( P＞0.025) in MDS group were higher, and the percentage of B cells ( P＜0.025) was lower. Compared with MDS group, the percentage of Treg cells ( P＜0.025) in AML group was higher and the proportion of B cells was lower ( P＜0.025). (3)Correlations with IPSS and blasts: The percentage of Treg cells was positively correlated with IPSS and blasts. Conclusion: Treg cells showed an increasing trend in turn by healthy people group, MDS group and AML group. The high expression of Treg cells may have great significance in prognosis and progression of MDS. Key words: Acute myeloid leukemia; Blasts; Immune cells; Myelodysplastic syndromes.

Trisomy 8 Remodels Chromatin and Activates Transcription of Runx1-Target Genes in Hematopoietic Stem Cell

Myelodysplastic syndrome (MDS) is a poor prognosis cancer that predominantly affects the elderly, arising from hematopoietic stem cell (HSC), resulting in bone marrow failure and predisposition to acute myelogenous leukemia (AML). It has long been known that numerical chromosome anomalies such as trisomy 8 are critical for the development of MDS. Trisomy 8 is known to be associated with relatively poor prognosis, and is a criterion for the diagnosis of MDS in patients. Systemic inflammation has been implicated in the selective advantage for clonal hematopoietic stem cells and the progression of MDS, such as trisomy 8 MDS associated with Behcet's disease. Next generation sequencing studies revealed that trisomy 8 MDS cells highly mutated RUNX1 transcriptional factor (TF) and ASXL1, an epigenetic modifier, both of which regulate HSC function and the differentiation, relative to MDS cells without trisomy 8, suggesting that those mutations may help trisomy 8 to drive the development of MDS. However, previous clinical studies failed to identify genes on the chromosome 8 commonly amplified among trisomy 8 MDS patients, which may account for the pathogenesis of MDS, the molecular mechanism of development of trisomy 8 MDS remains unclear. Thus, we hypothesized that trisomy 8 initiates the transformation by multiple malfunctions of epigenetic and transcriptional regulators in HSC other than a gene dosage effect due to the extra chromosome 8. In order to determine how trisomy 8 affects hematopoiesis, we established a new trisomy 8 model by introducing a human chromosome 8 to mimic trisomy 8 in mouse ES cells. We succeeded in generating trisomy 8 chimeric mice and confirmed the mice showing emergence of hematopoiesis in fetal liver but significantly decreased the chimerism of trisomy 8 cells than the control mice. Wecompetitively transplanted trisomy 8 fetal liver cells at E14.5 into lethally-irradiated wild-type primary recipient mice, and moreover serial transplanted hematopoietic stem and progenitor cells (HSPCs) which isolated from the primary recipient mice. We found that trisomy 8 reduced the self-renewal capacity of HSPC and impeded the differentiation, but it was not sufficient to develop myeloid malignancies in mice. To elucidate the molecular mechanism underlying the impaired hematopoiesis by trisomy 8, we performed RNA sequencing in HSCs isolated from the primary transplanted mice. Gene set enrichment analysis revealed that in comparison with the control HSCs, trisomy 8 HSCs had significantly positive enrichments in type I and type II interferon response genes, but also the canonical target genes of Runx1 and PU.1 TFs were positively enriched in trisomy 8 HSCs. In addition, ATAC-seq analysis revealed that trisomy 8 HSCs changed chromatin accessibility enriched with the binding motif of Runx TF, compared to control HSCs. Given the changes in chromatin accessibility in trisomy 8 HSC, we performed chromatin conformation capture sequencing (Hi-C) in HSPCs and found that trisomy 8 induced changes in chromatin structures such as topologically associating domain (TAD) and compartment A/B in the other chromosomes, which were partly associated with changes in transcription of genes. Since the Runx1 gene is critical for HSC integrity and differentiation, and loss-of-function mutations of RUNX1 were often found in human myeloid malignancies with trisomy 8, we attempted to determine whether Runx1 deletion cancel the impaired self-renewal capacity of trisomy 8 HSC. We found that Runx1 deletion partially canceled the impaired repopulating capacity of trisomy 8 cells in recipient mice, suggesting the selective advantage of loss of function mutation of the RUNX1 gene in trisomy 8 mosaic people and patients with trisomy 8 MDS. Furthermore, as Janus kinase (JAK) and TANK-binding kinase 1 (TBK1) are critical for interferon signalling and activation of their downstream target genes, we revealed that the inhibition of either JAK or TBK function ameliorated trisomy 8-induced chromatin remodelling in HSCs. Overall, trisomy 8 changes chromatin structure and transcription of genes on other chromosomes in HSC accompanied with the malfunction of Runx1 TF, at least in part, due to the activation of interferon signaling, and results in the impaired hematopoiesis. We demonstrated that trisomy 8 creates the basis for transformation of MDS via remodeling chromatin structures in HSC.

Soluble PD-L1 Predicts Poor Outcome and Disease Progression in Patients with De Novo Myelodysplastic Syndromes

Background: Myelodysplastic syndromes (MDS) consist of a spectrum of myeloid malignancies typified by regular genetic abnormalities, clonal hematopoiesis, dysfunctional myelopoiesis, inefficient blood cell production, peripheral-blood cytopenia, and an elevated risk for progressing to acute myeloid leukemia (AML). Mounting evidence suggests that the dysregulation of immune checkpoints (ICs) plays critical roles in immune evasion of MDS, which thus led to a series of trials using HMAs in combination with immune checkpoints inhibitors (ICIs). Of these, programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) pathway has been studied most extensively. Recently, forms of cell-free soluble PD-1/PD-L1 (sPD-1/sPD-L1) detection in plasm of patients with solid tumors has open a new paradigm for PD-1/PD-L1 investigations. However, within the context of MDS, the potential dysregulation and prognostic impact of sPD-1/sPD-L1 remains uncertain. This study was aimed to assess roles of sPD-1/sPD-L1 in newly diagnosed MDS patients. Method: Between July 2020 and March 2023, 130 MDS/sAML patients matched the inclusion criteria and 55 healthy individuals with no familial or personal history of autoimmune or cancer disorders were included. The criteria for patient inclusion included: (1) a diagnosis of MDS confirmed pathologically according to 2016/2022 World Health Organization (WHO) classification, or sAML patients with a verified prior MDS history; (2) absence of other conditions like autoimmune diseases or different types of tumors; (3) availability of complete clinical information and survival data. MDS patients were categorized by IPSS-R as: lower-risk (very low risk/ low risk; higher-risk (intermediate risk/high risk/very high risk. Follow-up was conducted until June 16th, 2023. The median follow-up was 14.32 months (range 3.2-34.63 months). Profile of sICs (sPD-L1, sPD-1, sCTLA-4, sGITR, sTIM-3, sOX40, sLAG-3, s4-1BB, sICOS) were tested by ELISA, cytokines (IFN-γ, IFN-α2, IL-2, IL-2α, IL-6, IL-7, IL-10, IL-15, IL-17, TNF-α) were detected using Luminex analysis, and the expression levels of PD-1 on T cells (CD3 +, CD4 +, CD8 +, DPT, DNT）were measured using flow cytometry (FCM). Result: In the plasma of 98 newly diagnosed MDS patients, sPD-L1 levels (median 66.80 pg/ml, range 25.00-219.5 pg/ml) were significantly elevated compared to 55 healthy blood donors (median 49.22 pg/ml, range 12.5-218.1 pg/ml). sPD-L1 level was higher in higher-risk IPSS-R groups ( P=0.0380). In addition, higher sPD-L1 levels also showed a correlation with lower hemoglobin concentration, implying a potential link between higher sPD-L1 levels and a more advanced MDS disease state. The AUC for sPD-1 was found to be 0.6333 [95% CI 0.5331-0.7334, P=0.0100] and for sPD-L1, it was 0.6785 [95% CI 0.5836-0.7733, P=0.0002]. The optimal cut-off values were established at 187.5 pg/mL for sPD-1 and 55.08 pg/mL for sPD-L1. Based on these cut-off values, patients with increased sPD-L1 experienced shorter OS ( P=0.0305). By using multivariate Cox model, high expression of sPD-L1 ( P=0.019, HR = 4.172, 95% CI: 1.265-13.755) and high IPSS-R scores ( P=0.030, HR = 2.898, 95% CI: 1.111-7.565) were found to be independent risk factors for newly diagnosed MDS patients. In addition, compared to healthy controls, the PD-1 + T cell subpopulations in MDS patients were significantly reduced, sPD-1 levels were positively correlated with proportion of CD4 + PD-1 + T cells, absolute count of CD4 + PD-1 + T cells and PD-1 + DPT cells, suggesting that CD4 + T cells and DPT cells may be potential sources of sPD-1 in plasma. Subsequently, we analysed the potential roles of cytokines and HMAs in sPD-1/sPD-L1 regulation. It was found that sPD-1 levels had a positive correlation with sPD-L1, IL-2Rα, IP-10, MIG, TNFα, GROα, and TRAIL. sPD-L1 was positively correlated with IL-2Rα, IP-10, MCP-1, MIG, MIP1α, and SCF (Figure 1 A). Notably. We found a reduced sPD-L1 levels in MDS patients who achieved remission after HMA treatment compared to treatment-naïve patients. ( P=0.0302) (Figure 1 B). Conclusions: In conclusion, we found sPD-L1 is an independent risk factors for overall survival in newly diagnosed MDS patients. The elevation of plasma sPD-L1 levels is associated with disease progression in MDS, and may be a potential target for MDS immunotherapy.

Serum Amyloid A1 (SAA1) Secreted By the Stromal Microenvironment Drives Malignant Clonal Proliferation in Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML)

Drivers of malignant hematopoietic stem cell clonal proliferation from age-related clonal hematopoiesis (ARCH) to Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML) are not well understood. However, a pro-inflammatory and immune tolerant aging bone marrow (BM) microenvironment is thought to contribute to disease development. Serum Amyloid A1 (SAA1) has been identified as a novel pro-inflammatory oncoprotein the levels of which progressively increase with MDS and AML progression. SAA1 also selectively promotes growth of patient-derived MDS and AML cells independent of cytogenetic or mutational profile. We aimed to characterize the mechanism by which SAA1 drives oncogenesis within the microenvironment. Herein we further stratified the disease marking properties of SAA1 by clarifying the association of its BM levels with disease progression in MDS and AML. SAA1 levels were significantly elevated in BM aspirates from MDS and AML patients as compared to those from healthy age-matched subjects (p=0.0002 and p=0.0052, respectively). The effects of SAA1 on mutated hematopoietic cells were assessed in a model of age-related clonal hematopoiesis (ARCH). Utilizing a CRISPR-Cas9 system, ASXL1-mutant CD34+ hematopoietic stem cells (HSCs) were generated; the replating capacity of both mutated and healthy CD34+ cells were assessed in the presence or absence of SAA1. Notably, SAA1 increased the replating capability assessed by an increase in the number of colonies formed on serial replating of ASXL1-mutant CD34+ cells, but not those of SAA1-treated healthy CD34+ or vehicle treated ASXL1-mutated CD34+ cells, suggesting that the presence of SAA1 favors stemness of mutated HSCs, and implicating SAA1 as a driver of malignant clonality. To investigate the mechanism of action of SAA1 BM mononuclear cells from AML patients or healthy age-matched subjects were treated with SAA1 or vehicle overnight and single-cell RNA sequencing analysis was performed. Seurat and cell cluster annotation analysis showed that AML samples treated with SAA1 had increased numbers of hematopoietic stem and progenitor cells (HSPCs) and plasmacytoid dendritic cells as compared to matched untreated AML samples. Further, Enrichr analysis showed that pathways upregulated by SAA1 in AML samples included pro-inflammatory molecules, anti-apoptotic, and antiviral defense mechanisms (p&amp;lt;0.0001). Shared genes upregulated in these pathways in HSPCs include interferon-induced signals including JAK2, STAT1, and OAS3, and genes implicated in resistance to chemotherapy, including CD44 and CD36, as well as the anti-phagocytosis signal CD47. Lastly, we initiated the development of a blocking monoclonal antibody against SAA1 as a means of inhibiting its potent oncogenic effects on malignant HSCs and examining its potential therapeutic activity. Utilizing a RAW 264.7 NFκB-luciferase reporter cell line, we screened 51 clones for their ability to selectively block SAA1-mediated NFκB activation. 7 optimal candidates were identified which demonstrated dose-dependent reduction in NFκB activity upon SAA1 challenge. Importantly, these candidate clones were specific for SAA1 and did not affect NFκB activation mediated by lipopolysaccharide (LPS)-mediated activation. We observed up to an 85% reduction of SAA1-mediated activity using supernatants from further subcloning of previously selected candidates. Moreover, cell proliferation of a human AML cell line (OCI-AML3) stimulated by SAA1 was markedly reduced in the presence of candidate antibodies, suggesting anti-proliferative activity. Our findings show that SAA1 levels correlates with disease severity in MDS and AML progression, selectively promotes stemness and proliferative potential of ASXL1-mutant cells, and demonstrates unique immune-related mechanisms selecting for malignant clonality. Further development and purification of a novel inhibitory antibody against SAA1 for future in vivo and ex vivo testing is underway as a promising novel target to reduce SAA1-mediated, niche-driven effects with broad implications in the treatment of hematological malignancies.

Myelodysplastic Syndrome Associated Hemophagocytic Lymphohistiocytosis: A Retrospective Study of 15 Cases in a Single Center

Introduction: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disorder characterized by excessive cytokine secretion, resulting in hyperinflammation and multiorgan dysfunction. It is classified into primary HLH, which is mainly caused by genetic defects, and secondary HLH, triggered by various underlying conditions. Malignancy-associated HLH (M-HLH) is a secondary form commonly observed in patients with hematological malignancies, such as lymphomas, T/NK-cell disorders, acute leukemias, lymphoproliferative diseases, and myelodysplastic syndrome (MDS). M-HLH can occur in up to 1% of patients with hematologic malignancies, with lymphoma being the most frequently associated malignancy. HLH initiated by MDS is a rare entity with only a few cases reported, leading to limited knowledge regarding its pathogenesis, diagnosis and optimal treatment strategies. Methods: In this retrospective study, we analyzed a consecutive series of MDS-HLH patients admitted to our center between March 1, 2019, and March 1, 2023. Bio-clinical data related with MDS and HLH were collected at diagnosis of MDS-HLH. Additional observational indicators encompassed the following: basic clinical characteristics of the patients (age at onset, gender, duration of MDS prior to HLH), correlation between the onset of HLH and MDS, treatment and response, and genetic defects associated with HLH and MDS. MDS risk classification was according to the Revised International Prognosis Scoring System (IPSS-R) and the WHO classification-based Prognosis Scoring System, while patients were categorized into two groups as higher-risk and lower-risk MDS-HLH group. The follow-up period started at the time of MDS-HLH diagnosis and ended at the date of death or last examination. Overall survival (OS) was calculated from the time of HLH diagnosis to the date of death from any cause. Results: A total of 15 patients diagnosed with MDS-HLH were included in the study, with a median age of 66 years (range, 33-83 years). The majority of patients (10/15, 67%) were over 60 years old. All patients exhibited typical features of aggressive HLH: fever, cytopenia and elevated serum ferritin in all patients, hepatomegaly/splenomegaly in 87% (13/15), hypofibrinogenemia/hypertriglyceridemia in 73% (11/15), hemophagocytosis in 73% (11/15), low NK-cell activity in 21% (3/14), and elevated levels of sCD25 in 93% of patients (14/15). Additionally, four patients (27%) had concurrent Epstein-Barr virus (EBV) infection. Further analysis comparing the lower-risk and higher-risk MDS-HLH groups revealed interesting findings. In the lower-risk group, all patients developed HLH concurrently with the diagnosis of MDS. However, in the higher-risk group, the majority of patients (6/8, 62.5%) experienced HLH onset during MDS progression or relapse (p=0.014). Among the 15 patients, 8 patients (53.3%) achieved remission of HLH. During the study period, 8 deaths occurred, all directly attributed to HLH or its complications. The majority of deaths (75%, 6/8) occurred within the first two months after HLH diagnosis. The median overall survival was 12.3 weeks (95%CI, 1.1-23.5). Patients who achieved complete or partial response had significantly prolonged overall survival compared to those who did not respond to treatment (p=0.002). In the multivariate analysis of prognosis, the remission status of HLH was the only factor which was significantly associated with survival (p=0.018, Exp(B)=0.003). Conclusion: This is the first comprehensive study focusing specifically on MDS-HLH, providing valuable insights into the clinical characteristics, treatment outcomes, and prognosis of MDS-HLH. The findings emphasize the need of considering revision of diagnostic criteria specially for MDS-HLH. The pathogenesis of HLH in MDS-HLH differs based on the risk classification of MDS, with higher-risk cases driven by malignancy and lower-risk cases associated with immune dysfunction. The prognosis of MDS-HLH is generally poor. Achieving remission of HLH is crucial for improving the prognosis of MDS-HLH patients. Conventional chemotherapy regimens, including etoposide, have shown limited efficacy and safety in this context. Chemotherapy-free regimens should be preferred considering the features of ineffective hematopoiesis in MDS. The role of EBV infection in MDS-HLH development should be recognized.

Isocitrate Dehydrogenase 2 Mutation Allows Myeloid Differentiation but Impairs Bone Marrow Macrophage Polarization and Function Via Metabolic Dysregulation

Isocitrate dehydrogenase 2 (IDH2) mutations, while less common than other mutations associated with myelodysplastic syndromes (MDS), are also found in clonal hematopoiesis and are associated with increased risk of transformation to leukemia. IDH2 mutations lead to metabolic dysfunction and DNA hypermethylation due to the accumulation of D-2-hydroxyglutarate (2HG), an oncometabolite derived from alpha-ketoglutarate (aKG), which disrupts hematopoiesis. However, the mechanisms that induce clonal progression and transformation remain incompletely understood. Furthermore, the intrinsic and extrinsic impact of IDH mutations on mature myeloid cell functions and the bone marrow microenvironment remains to be elucidated. Previous literature suggested that hypermethylation via this mutant leads to incomplete differentiation and failed mature myeloid cell generation. Our lab previously demonstrated that efferocytic defects in bone marrow macrophages can skew hematopoietic stem and progenitor cells via increased inflammatory mediators. Based on these data, we hypothesized that IDH2 mutations prevent complete polarization down pro-efferocytic phenotypes in macrophages due to blocking of metabolic pathways required for mature macrophage function. Congenic CD45.1+ mice were transplanted with retrovirally infected hematopoietic stem cells containing human WT or R172K IDH2 mutant with an mCherry reporter and showed macrocytic anemia, thrombocytopenia, and elevated reticulocytes by 16 weeks post-transplant. In contrast to prior literature, IDH2 mutants gave rise to mature reporter positive monocytes, macrophages, and neutrophils in the bone marrow and peripheral blood. As expected, we observed myeloid skewed hematopoiesis, a trademark of MDS. When cultured ex vivo, there was loss of efferocytic capacity in IDH2 mutant bone marrow macrophages when fed apoptotic neutrophils. We then generated a genetic murine model using the Vav1 Cre IDH2 R140Q floxed mutant. The genetic model allowed us to study disease progression in the absence of radiation induced stress, and to observe mature myeloid populations while clinically defined MDS was absent. We performed ATACseq and bulk RNAseq analysis on bone marrow macrophages isolated from 3-month-old and 6-month-old mice, which demonstrated a significant decrease in global chromosomal accessibility; however, this had limited impact on the transcriptional program. Based on existing literature, the majority of macrophages in the bone marrow are in a poised state and have not yet activated genes associated with pro-efferocytic/pro-resolution or pro-inflammatory phenotypes. Since aKG is accumulated in macrophages during polarization toward reparative and pro-efferocytic phenotypes and is necessary for metabolic reprogramming for oxidative phosphorylation, we hypothesized that, in the presence of IDH2 mutations, which drain aKG from the system, pro-efferocytic polarized macrophages would be metabolically stunned. To test this theory, we isolated bone marrow macrophages from IDH2 mutants and cultured them to generate pro-efferocytic polarized macrophages. We then tested their efferocytic potential and metabolic capacity by seahorse assay. Continual efferocytosis, a hallmark of pro-efferocytic macrophages, was impaired in IDH2 mutant macrophages as early as 2-3 months of age. Consistent with this finding, mito stress testing by seahorse demonstrated that IDH2 mutant macrophages had severely impaired maximal respiratory capacity compared to WT macrophages. Treatment with global hypomethylating agent azacytidine or a cell permeable aKG analog were unable to restore respiratory capacity or efferocytic activity in the IDH2 mutant. These data strongly suggest that the efferocytic defect in IDH2 mutant macrophages is due to metabolic rather than epigenetic reprogramming. In summary, we identify defects in efferocytic function and oxidative phosphorylation in IDH2 mutant macrophages. This represents a novel mechanism that could alter the bone marrow microenvironment and impact progression of MDS and leukemic transformation. Furthermore, our data suggest that the metabolic dysregulation induced by IDH mutations may play a greater role than epigenetic impairment in IDH2 mutant mature myeloid cells, which could explain why approaches targeting hypermethylation have shown limited efficacy in clinical settings.

MDS-MSC Derived Exosomes Promote MDS Cells Malignant Proliferation through RIG-1/Isgs Pathway Via RN7SL1

The interactions between bone marrow stem cells (BM-MSCs) and myelodysplastic syndromes (MDS) cells perform a crucial role in MDS pathogenesis by secreting cytokines ,growth factors, and exosomes. Although BM-MSCs induced growth of MDS cells has been studied, the role of exosomes from MDS-derived MSC (MDS-MSC-EXO) in this action remains unclear. In our study, we investigate the effect of MDS-MSC-EXO on the viability, proliferation, migration, cellular apoptosis of MDS cell lines, such as MDS-L and SKM-1. Our data identified that MDS-MSC-EXO can promote proliferation, migration, and decrease cell apoptosis. Both LR-MDS-MSC and HR-MDS-MSC-derived exosomes increased MDS cell growth.Exosomes mediate local cell-to-cell communication by transferring mRNAs, miRNAs, and proteins. In our study, we found that MDS-MSC-EXO expressed higher unshielded RN7SL1,an endogenous RNA normally shielded by RNA binding proteins SRP9/14, than that from health controls (HC-MSC-EXO). MDS cloned cells expressed higher retinoic acid inducible gene-I (RIG-I) and interferon-stimulated genes (ISGs), such as ISG15, IFIT1 and IFITM1, which are the receptor and effector molecules of RN7SL1. Both MDS-MSC-EXO and exogenous RN7SL1 can promote MDS cloned cells proliferation and stimulate ISGs expression by activating RIG-1. RIG-I inhibitor can block this effect. We demonstrate that MDS-MSC may promote MDS cloned cell proliferation through RIG-ISGs signal activated by exosomes-derived RN7SL1. Overall, our studies provide new insights that MSC-EXO functions the crucial roles in mediating the progression of MDS via RIG-1/ISGs activation, and demonstrate novel targets of RN7SL1 to the pathogenesis of MDS.This study will provide new ideas for MDS treatment in the aspect of microenvironment.

Forward Genetic Screen Implicates Drivers of Leukemic Progression in a Novel Model of Trp53 R270H myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is characterized by bone marrow failure and a highly variable clinical course. The most catastrophic complication of MDS is transformation to secondary acute myeloid leukemia (sAML). Notably, mutations in TP53 confer the single highest risk of transformation to sAML and death. However, some patients with TP53 mutated MDS do not develop sAML, suggesting that additional genetic events cooperate with TP53 mutations to transform MDS to sAML. Understanding the mechanisms of transformation of MDS to sAML could provide targets for therapeutic intervention. To model the genetics of MDS, we crossed mice bearing Trp53 R270H ( Trp53 is the murine TP53 gene) and deletion of genes syntenic with human chromosome 5q (del(5q)). To discover how additional mutations contribute to disease progression, we utilized Sleeping Beauty (SB) transposon mutagenesis in Trp53 R270H/del(5q) mice. SB transposase mobilized SB mutagenic T2/Onc transposons which randomly insert within the genome. T2/Onc transposons are designed to induce gain or loss of function alterations depending on the site and orientation of insertion with respect to targeted genes. We used the Mx1-Cre transgene to activate SB transposase and T2/Onc transposition in hematopoietic progenitors. Trp53 R270H anddel(5q)(or cytogenetically normal, CN) mice were crossed to SB mice to generate donor mice of the following genotypes: Trp53 R270H/del(5q)/ SB, Trp53 R270H/CN /SB, Trp53 WT/del(5q)/ SB, Trp53 WT/CN/ SB mice, and mice without SB transposition, (no transposition, NT: Trp53 R270H/del(5q) /NT). Bone marrow cells were transplanted into recipients, and SB insertional mutagenesis was activated using pI-pC to activate Cre. Mice receiving Trp53 WT/CN/ SB bone marrow developed more frequent T-cell leukemia (n=3/10) than myeloid leukemia (n=1/10). In contrast, mice receiving Trp53 R270H/del(5q)/SB and Trp53 R270H/CN /SB bone marrow developed predominantly myeloid leukemia (n=14/28) more commonly than T-cell leukemia (1/28). Mixed phenotype leukemia was seen in 7/28 of these mice. Together, these data demonstrate a strong bias towards myeloid disease in SB-mutagenized Trp53 R270H bone marrow. To identify genes with SB insertions, we performed RNA sequencing to detect SB T2/Onc transposon-endogenous genefusion transcripts. Among Trp53 WT/CN/ SB leukemias, the most common recurrent SB fusions involved Notch1 and Ikzf1 as has previously reported for SB-associated T-cell leukemias . Among Trp53 R270H/del(5q)/SB and Trp53 R270H/CN /SB leukemias, the most common recurrent SB-fusions involved Erg, Eras and Il2rb with Erg fusions detected 85% of Trp53 R270H leukemias (n=17/20). SB inserted upstream of Erg promoter indicating that these fusions likely upregulate expression of Erg. Indeed, Erg levels are significantly higher in leukemias that express SB-Erg fusions relative to leukemias that do not (p&amp;lt;0.0023). ERG is not recurrently mutated in human AML, but the ERG gene locus is commonly amplified, especially TP53 mutant AML. ERG is known to support normal hematopoietic stem cell self-renewal. Notably, Erg-insertions were also detected in a model of MDS expressing stabilized cyclin E with SB-mediated progression to erythroleukemia (Loeb 2019). Using gene set enrichment analysis, we found that hematopoietic stem cell and leukemic stem cell signatures are enriched in Erg-SB fusion leukemias. In our analyses of two independent data sets (TCGA and BEAT AML), stem cell signatures are also among the most highly enriched pathways in human AMLs expressing high ERG levels. Furthermore, in a human AML single cell RNA sequencing dataset (van Galen 2019), we found that ERG expression is highest in AML cells with the most immature stem and progenitor-like features. Together, these findings implicate a role for ERG as a driver of progression of MDS to AML by enhancing aberrant self-renewal. In summary, we present a novel murine model of Trp53/del(5q) MDS. In this model, Erg upregulation is associated with progression to AML and upregulation of leukemia stem cell gene expression profiles. These data implicate ERG as a major contributor to progression of MDS to secondary AML in the setting of mutant p53. Understanding the mechanisms of disease progression and self-renewal in myeloid malignancies with p53 mutations is critical to define effective therapeutic strategies in these rapidly fatal, treatment resistant diseases.

CircZBTB46 predicts poor prognosis and promotes disease progression of myelodysplastic syndromes and acute myeloid leukemia.

Circular RNAs (circRNAs) have been recently identified as important regulators of various diseases, especially cancer. However, the roles of circRNAs in hematologic malignancies have been rarely reported. This study aimed to identify a specific circRNA expression profile in patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), and to evaluate the biological roles of circRNA in MDS and AML for understanding their clinical significance. Reverse transcription-quantitative PCR was performed to validate the expression of circZBTB46. Kruskal-Wallis test, Kaplan-Meier curves, and the Cox regression model were employed to analyze the clinical significance of circZBTB46. Two specific shRNAs as well as an expression lentiviral vector of circZBTB46 were constructed to identify the biological function of circZBTB46. The impact of circZBTB46 on leukemia cell proliferation, cell cycle distribution, and apoptosis was confirmed using cell viability assay and flow cytometry analysis. The expression of circZBTB46 gradually increased in patients with higher-risk MDS and AML, as compared to controls. CircZBTB46 expression was significantly correlated with important clinical parameters of MDS, including WHO classification, absolute neutrophil count (ANC), marrow blast, IPSS karyotype, IPSS/IPSS-R risk groups, and AML transformation. CircZBTB46 expression was also associated with ANC, marrow blast, cytogenetic risk groups, FLT3-ITD mutation, and treatment response in AML patients. Furthermore, circZBTB46 overexpression was significantly correlated with shorter overall survival (OS, P = 0.0342, median survival time 18.5 vs. 45.4months) and leukemia-free survival (LFS, P = 0.0421) in MDS, also with the shorter OS in AML (P = 0.0293, median survival time 11.6 vs. 16.9months). Functional studies revealed that silencing circZBTB46 expression significantly inhibited proliferation and induced apoptosis in SKM-1, THP-1, and K562 cell lines, while rescue experiments alleviated the siRNA-mediated growth inhibition and apoptosis in these leukemic cells. The present data suggested the essential oncogenic role of circZBTB46, as a progression and survival indicator in both MDS and AML.