Myelodysplastic Syndromes Cells Research Articles

Acadesine Circumvents Azacitidine Resistance in Myelodysplastic Syndrome and Acute Myeloid Leukemia.

Myelodysplastic syndrome (MDS) defines a group of heterogeneous hematologic malignancies that often progresses to acute myeloid leukemia (AML). The leading treatment for high-risk MDS patients is azacitidine (Aza, Vidaza®), but a significant proportion of patients are refractory and all patients eventually relapse after an undefined time period. Therefore, new therapies for MDS are urgently needed. We present here evidence that acadesine (Aca, Acadra®), a nucleoside analog exerts potent anti-leukemic effects in both Aza-sensitive (OCI-M2S) and resistant (OCI-M2R) MDS/AML cell lines in vitro. Aca also exerts potent anti-leukemic effect on bone marrow cells from MDS/AML patients ex-vivo. The effect of Aca on MDS/AML cell line proliferation does not rely on apoptosis induction. It is also noteworthy that Aca is efficient to kill MDS cells in a co-culture model with human medullary stromal cell lines, that mimics better the interaction occurring in the bone marrow. These initial findings led us to initiate a phase I/II clinical trial using Acadra® in 12 Aza refractory MDS/AML patients. Despite a very good response in one out 4 patients, we stopped this trial because the highest Aca dose (210 mg/kg) caused serious renal side effects in several patients. In conclusion, the side effects of high Aca doses preclude its use in patients with strong comorbidities.

Downregulation of microRNA-144 inhibits proliferation and promotes the apoptosis of myelodysplastic syndrome cells through the activation of the AKAP12-dependent ERK1/2 signaling pathway

BackgroundMyelodysplastic syndromes (MDS) represent a family of hematopoietic stem cell disorders characterized by ineffective hematopoiesis. While the functions of many microRNAs have been identified in MDS, microRNA-144 (miR-144) remains poorly understood. Thus, the aim of the present study was to determine the effects of miR-144 on cell proliferation and apoptosis in MDS cells and mechanism thereof. MethodsMDS-related microarrays were used for screening differentially expressed genes in MDS. The relationship between miR-144 and A-kinase anchoring protein 12 (AKAP12) was determined by a dual luciferase reporter gene assay. Subsequently, gain- and loss-function approaches were used to assess the effects of miR-144 and AKAP12 on cell proliferation, cell cycle and cell apoptosis by MTT assay and flow cytometry. Following the induction of a mouse model with MDS, the tumor tissues were extract for evaluation of apoptosis and the expression of miR-144, AKAP12, and the relevant genes associated with extracellular-regulated protein kinases 1/2 (ERK1/2) signaling pathway and apoptosis. ResultsWe observed significantly diminished expression of AKAP12 in MDS samples. miR-144 directly bound to AKAP12 3′UTR and reduced its expression in hematopoietic cells. Downregulation of miR-144 or upregulation of AKAP12 was observed to prolong cell cycle, inhibit cell proliferation, and induce apoptosis, accompanied by increased expression of AKAP12, p-ERK1/2, caspase-3, caspase-9, Bax, and p53, as well as decreased expression of Bcl-2. The transplanted tumors in mice with down-regulated miR-144 exhibited a lower mean tumor diameter and weight, and increased apoptosis index and expression of AKAP12 and ERK1/2. ConclusionTaken together, these studies demonstrate the stimulative role of miR-144 in MDS progression by regulating AKAP12-dependent ERK1/2 signaling pathway.

B-cell acute lymphoblastic leukemia in an elderly man with plasma cell myeloma and long-term exposure to thalidomide and lenalidomide: a case report and literature review

BackgroundThe advent of the immunomodulatory imide drugs (IMiDs) lenalidomide and thalidomide for the treatment of patients with plasma cell myeloma (PCM), has contributed to more than a doubling of the overall survival of these individuals. As a result, PCM patients join survivors of other malignancies such as breast and prostate cancer with a relatively new clinical problem – second primary malignancies (SPMs) – many of which are a result of the treatment of the initial cancer. PCM patients have a statistically significant increased risk for acute myeloid leukemia (AML) and Kaposi sarcoma. IMiD treatment has also been associated with an increased risk of myelodysplastic syndrome (MDS), AML, and squamous cell carcinoma of the skin. However, within these overlapping groups, acute lymphoblastic leukemia (ALL) is much less common.Case presentationHerein, we describe an elderly man with PCM and a 14-year cumulative history of IMiD therapy who developed persistent pancytopenia and was diagnosed with B-cell acute lymphoblastic leukemia (B-ALL). He joins a group of 17 other patients documented in the literature who have followed a similar sequence of events starting with worsening cytopenias while on IMiD maintenance for PCM. These PCM patients were diagnosed with B-ALL after a median time of 36 months after starting IMiD therapy and at a median age of 61.5 years old.ConclusionsPCM patients with subsequent B-ALL have a poorer prognosis than their de novo B-ALL counterparts, however, the very low prevalence rate of subsequent B-ALL and high efficacy of IMiD maintenance therapy in PCM should not alter physicians’ current practice. Instead, there should be a low threshold for bone marrow biopsy for unexplained cytopenias.

Influence of demethylation on regulatory T and Th17 cells in myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) represents a heterogeneous hematopoietic disorder in which mature blood cells are derived from an abnormal multipotent progenitor cell. The current therapy for MDS involves repeated cycles of DNA methyltransferase (DNMT) inhibitors, particularly the demethylation drug 5-azacytidine (5-azaC) which has been shown to increase the survival of patients with high-risk MDS. The mechanisms behind the therapeutic effects of 5-azaC are not yet clear. In this study the effect of 5-azaC on the development of regulatory T cells (Tregs) and T-helper 17 (Th17) cells was investigated. The numbers of CD4+ T-cell subsets in 30 patients with intermediate-2/high-risk MDS were serially assessed at diagnosis and following 5-azaC treatment. The number of FoxP3+ Tregs was significantly higher after 3 months of therapy. However, there was no statistical difference in the number of Th17 cells following treatment. In vitro, 5-azaC enhanced the overall proportion of Tregs, but not Th17, in CD4+ T cells from patients with MDS. Addition of 5-azaC reduced the proliferative capacity of Tregs, suggesting that the increase in Tregs was due to conversion of conventional CD25− cells, rather than proliferation of CD25+FoxP3+ cells. The FoxP3 expression in 5-azaC-treated T effectors was also increased. Interestingly, while Tbet and RORγT mRNA transcription had no obvious changes, due to the demethylation of the FoxP3 promoter, these findings are important in associating the induction of DNA hypomethylation and the clinical response to 5-azaC.

R-2HG/KDM2B/RIPK1 Signaling Mediates Necroptosis in Myelodysplastic Syndromes

Introduction: Myelodysplastic syndromes (MDS) are a group of heterogeneous hematopoietic stem cell disorders and manifested as ineffective hematopoiesis, refractory cytopenia and a propensity to evolve into acute myeloid leukemia (AML). Isocitrate dehydrogenase 1/2 (IDH1/2) mutations are common in both MDS and AML. Mutated IDH produces R-2-hydroxyglutarate (R-2HG) which inhibits multiple α-ketoglutarate/α-KG-dependent dioxygenases by competing against α-KG binding. Recent studies have demonstrated that R-2-HG can abrogates leukemic growth and induce leukemia cell death. Several nonapoptotic cell death have been identified, including phosphoribosyl pyrophosphate (PPRP)-1 mediated necrosis, pyroptosis, ferroptosis and necroptosis. By now, the specific type of cell death by which R-2-HG exerts anti-tumor effects is still unknown. Results: (1) (2R)-Octyl-2-HG exhibited anti-tumor effect on SKM-1, THP-1, Molm-13, HL-60 cell lines and bone marrow mononuclear cells from MDS and AML patients in a dose- and time-dependent manner. The overexpression of IDH2 mutation induced by doxycycline significantly decreased the viability of AML cell lines, and the inhibition rate was related to the dose of doxycycline. (2R)-Octyl-2-HG promotes apoptosis and causes G0/G1 phase arrest. (2) R-2-HG leads to increased expression of RIPK1 in high-risk MDS cells. The results of gene enrichment analysis indicated that the apoptotic pathway was enriched in (2R)-Octyl-2-HG groups, and the expression of RIPK1 gene was increased in all three (SKM-1, NOMO-1 and MA9.3ITD) (2R)-Octyl-2-HG groups. After treatment with (2R)-Octyl-2-HG, the mRNA and protein expression levels of RIPK1 gene were increased. (3) R-2-HG triggers RIPK1-dependent necroptosis and occurs earlier than apoptosis. Within 10 hours after treatment with (2R)-Octyl-2-HG, cell membrane permeability was disrupted, interactions between RIPK1 and caspase 8 increased, as well as phosphorylated MLKL level. However, caspase activity did not increase significantly, suggesting that necroptosis occurs earlier than apoptosis. With RIPK1 inhibitor Necrostatin-1 and (2R)-Octyl-2-HG co-treating cells, proliferation inhibition was reduced, cell membrane permeability was more stable and RIPK1-caspase8 complex was difficult to form. The same phenomenon occurs in SKM-1 cells stably transfected with RIPK1 shRNA virus, suggesting that RIPK1-dependent necroptosis is involved in cell death caused by (2R)-Octyl-2-HG. (4) In vivo experiments demonstrated that necroptosis by R-2-HG is dependent on the expression of RIPK. In RIPK1 shRNA MDS mice, the tumor burden showed a decreasing trend, but did not show a significant change in the R-2-HG treatment group. Treatment of scramble shRNA MDS mice with R-2-HG resulted in significantly smaller tumors in the spleen and less engrafment of CD45+ cells in bone marrow. (5) Low RIPK1 expression predicts poor prognosis in MDS patients. Data from the TCGA and GEO public databases indicate that RIPK1 expression is reduced in MDS and AML patients compared to healthy controls. Survival analysis showed that patients with lower RIPK1 expression levels had significantly shorter overall survival (OS) than patients with higher RIPK1 expression levels. MDS patients with lower RIPK1 expression levels progress to leukemia more frequently. (6) Inhibition of KDM2B induces necroptosis independently. Western blot assay shows the knockdown of KDM2B, upregulation of RIPK1 and increased levels of p-MLKL. Analysis of cell numbers showed that proliferation ceased from 4 days after doxycycline treatment onwards in shKDM2B cells. Co-IP assay shows the formation of RIPK1-caspase8 complex. Conclusion: This study confirmed that R-2-HG inhibited the viability of MDS and AML cells. R-2-HG increased the expression of RIPK1 in MDS cells, inducing necroptosis. Necroptosis occurred earlier than apoptosis. Inhibition of RIPK1 can alleviate the inhibitory effect of R-2-HG on MDS and AML cells. Clinical studies have shown that low expression of the RIPK1 gene is associated with poor prognosis in patients with MDS and AML. MDS patients with low expression of RIPK1 was more likely to progress to leukemia. Inhibition of KDM2B can induce necroptosis independently. Disclosures No relevant conflicts of interest to declare.

HO-1 Promotes Resistance to EZH2 Inhibitor through the Prb-E2F Pathway: Corelated with the Conversion of Myelodysplastic Syndrome to Acute Myeloid Leukemia

Myelodysplastic syndrome (MDS) patients would have a chance to become acute myeloid leukemia (AML) when undergoing chemotherapy or waiting for the hematopoietic stem cell transplantation. These patients were not sensitive to demethylation therapy and we should explore deeper mechanisms. According to the WPSS scoring system, we divided 58 MDS patients into four different groups. Real-time PCR results showed the expression of EZH2 or HO-1 in MDS patients were higher than that in normal donors (P<0.05). Even HO-1 and EZH2 were simultaneously increased in some patients, especially in high-risk and extremely very high-risk groups. The linear correlation analysis result of them was 0.42 (P<0.05). In addition, Laser scanning confocal microscopy results also indicated that they were both present in the nucleus of tumor cells. Therefore, we speculated that there was a correlation between EZH2 and HO-1 in MDS patients. Using the High-throughput sequencing to analyze MDS cells, we found that the conversion of MDS to AML may be related to EZH2. The EZH2 in converted MDS patients were significantly higher than that of other MDS patients (P<0.05). Among these patients, we also found that HO-1 and EZH2 were also positively correlated. We found the new EZH2 inhibitor JQEZ5 could significantly promote tumor cells apoptosis in this part of patients. When the concentration of JQEZ5 was 10 umol/mL, the apoptosis rate of tumor cells reached 46.7% after 24 hours (P<0.05). Apoptosis rate was positively correlated with the concentration of JQEZ5 (P<0.05). And tumor cells were significantly inhibited in the G0/G1 cell phase. Cell cycle regulatory genes (CDK4 and CDK6) and apoptosis regulatory genes (Caspase-3 and Caspase-9) would changed. The expression of P15 and P53 would also be affected. In order to verify the malignant degree of MDS cells whether be related to the expression of EZH2. We injected MDS cells into 10 mice. Compared to the control group, MDS cells that highly express EZH2 infiltrated the spleen of experimental mice. Interestingly, the spleens of the experimental group were significantly reduced (0.2CM-0.4CM) and the spleens weight of the experimental group was reduced by 0.028g-0.12g compared to the control group spleens weight. These cells also significantly shortened the survival days of mice and reduced their body weight. Although control mice could survived for 30 days without disease, the time of the experimental mice was significantly shortened (18-25 days). Even One of them survived only 15 days. The results of immunohistochemistry indicated that EZH2 was related to the pRB-E2F pathway. Using the E2F inhibitor HLM006474, we proved HO-1 could regulated EZH2 through the pRB-E2F pathway. Our previous experiments indicated that HO-1 could help leukemia cells resistance and proliferation. The effect of JQEZ5 would be affected when we used hemin and zinc protoporphyrin to regulate HO-1 in MDS cells. The EZH2 was significantly inhibited by JQEZ5 after HO-1 was silenced by siRNAs. Also, the apoptosis rate of MDS cells increased and the cell cycle was arrested in the G0/G1 phase. However, when HO-1 expression was up-regulated by lentivirus, the effects of JQEZ5 were attenuated. MDS patients are frequently in a state of HO-1 enrichment during chemotherapy. HO-1 stimulates MDS patients to transcribe and activate excess EZH2 through pRB-E2F pathway, which increases the chances of becoming AML. Therefore, the conversion of MDS may be attributed to EZH2. In addition, considering HO-1 could promote the expression of EZH2, HO-1 may be a target for enhancing the effects of EZH2 inhibitors on MDS and the influence of HO-1 should be considered in the treatment of patients with high-risk and extremely very high-risk MDS. Disclosures No relevant conflicts of interest to declare.

Synergistic Effect of DHODH Inhibitor PTC299 with DNA Demethylating Agent Decitabine in Myelodysplastic Syndromes

Background:Myelodysplastic Syndrome (MDS) is a clonal bone marrow disorder characterized by ineffective and clonal hematopoiesis accompanied by morphological dysplasia and variable cytopenia. There are few treatment options for MDS, and allogenic hematopoietic stem cell transplantation is the only curative option. Dihydroorotate dehydrogenase (DHODH) catalyzes a rate-limiting step in de novo pyrimidine synthesis, the conversion of DHO to orotate. DHODH inhibition has been described recently as a new approach for treating acute myeloid leukemia (AML) by inducing differentiation of diverse AML subclasses. PTC299 represents a novel potent DHODH inhibitor and recently clinical development of PTC299 as a potential treatment option for acute leukemia was initiated. Here, we explored the efficacy of DHODH inhibitor PTC299 for MDS. Methods:Anti-MDS efficacy of PTC299 was studied using human MDS cell lines and primary MDS cells in vitro. PTC299 was synthesized at PTC Therapeutics Inc. Mechanistic studies were conducted via flow cytometric analysis and RNA-sequencing (RNA-seq). Gene expression levels were analyzed by quantitative PCR (qPCR). Results:PTC299 inhibited proliferation of AML cell lines and induced their differentiation. As previously reported in other DHODH inhibitors, upregulation of CD11b was observed after PTC299 treatment in both HL-60 and THP-1 cells. In addition, PTC299 inhibited the proliferation of MDS cell lines, MDSL and SKM-1 cells, with EC50s of 12.6 nM in MDSL cells and 19.7 nM in SKM-1 cells. The inhibitory effect was reversed by the exogenous addition of 100 µM uridine, which bypasses the requirement for de novo pyrimidine synthesis by feeding into the salvage pathway, thereby negating the need for DHODH. Because the basal expression levels of CD11b are high in MDS cells, we examined the expression levels of CD38. Both cell lines showed dose-dependent upregulation of CD38 after PTC299 treatment. To investigate the possible synergism between PTC299 and decitabine, we treated MDSL and SKM-1 cells with increasing concentrations of PTC299 and decitabine as single agents or in combination. After 3 days of culture, cells were analyzed by MTS assays. PTC299 and decitabine exerted a enhanced cytotoxic effect on MDSL and SKM-1 cells. Similar results were obtained with primary MDS samples. In Annexin/PI assays, the percentage of apoptotic cells was increased by combination of PTC299 with decitabine in both cell lines. Mechanistically, treatment with PTC299 induced an intra-S-phase arrestfollowed by entry intoapoptotic cell death. It has also been reported that the expression of p53 is increased in response to the intra-S-phase arrest. To understand the genome-wide effects and target genes of PTC299 and the combination with decitabine, we performed RNA-seq of MDSL and SKM-1 cells treated with PTC299, decitabine, or the combination of both agents versus DMSO-treated cells. Gene set enrichment analysis (GSEA) using our RNA-seq data confirmed that MYC target gene sets were negatively enriched in both PTC299-, decitabine- and combination- treated cells. KEGG pathway enrichment analysis revealed activation of genes associated with apoptosis in both cell lines. To better elucidate a synergistic effect of PTC299 and decitabine, we performed qPCR of CDKN1A, which is a major target of p53 activity. The mRNA expression levels of CDKN1A were upregulated after treatment with PTC299, which was further enhanced by the combination with decitabine. Conclusions:Our result indicate that the DHODH inhibitor PTC299 suppresses the growth of MDS cells in vitro and acts in at least an additive and possibly synergistic manner with decitabine in this process. This combination therapy could be a new therapeutic option for the treatment of MDS. Disclosures Lennox: PTC Therapeutics: Employment. Weetall:PTC Therapeutics: Employment. Sheedy:PTC Therapeutics: Employment.

Decitabine Induces Ferroptosis in Myelodysplastic Syndrome

Decitabine is one of the classical demethylation drugs in the treatment of myelodysplastic syndrome (MDS); however, the exact mechanism of decitabine has not been fully understood. Such knowledge is essential to develop mechanism-based, targeted approaches in the treatment of MDS. Here, we show that decitabine-induced ROS raise leads to ferroptosis in myelodysplastic syndrome cells. To investigate whether decitabine could induce ferroptosis in MDS cells and its mechanism, cell lines SKM-1 and MUTZ-1 were co-cultured with decitabine and ferroptosis inhibitor (ferrostatin-1), respectively. CCK-8 assay was used to detect the effects of drugs on cell viability. At the same time, we observed whether necroptosis inhibitor (necrostatin-1), apoptosis inhibitor (z-vad-fmk) and iron chelating agent (DFO) could reverse the inhibitory effect of decitabine on MDS cells. The results showed that, necrostatin-1 could increase the cell viability significantly. The growth-inhibitory effect of decitabine on SKM-1 and MUTZ-1 could be partially reversed by ferrostatin-1, DFO and necrostatin-1. The effect of ferrostatin-1 is the most significant. Ferroptosis inducer (erastin) could increase the cytotoxicity of decitabine at different concentrations. Flow cytometry was used to detect the ROS level. Biochemical method was used to detect the intracellular glutathione (GSH) level and glutathione peroxidase (GPXs) activity. The results showed that, the level of GSH and the activity of GPXs decreased while the ROS level increased in SKM-1 and MUTZ-1 cell lines when treated with decitabine, which could all be inhibited by ferrostatin-1. The iron overload model of C57BL/6 mice was next constructed to observe whether iron overload could induce ferroptosis. The results showed that, the concentration of hemoglobin in peripheral blood of mice was negatively correlated with intracellular Fe2+level and ferritin concentration. Iron overload led to decreased viability of bone marrow mononuclear cells (BMMNCs), which was negatively correlated with intracellular Fe2+level. Ferrostatin-1 and necrostatin-1 partially reversed the decline of cell viability in iron overload groups, and erastin promoted the proliferation of BMMNCs in iron overload mice. The level of GSH and the activity of GPXs decreased while the ROS level increased in BMMNCs of iron overload mice compared with the control. DFO could increase the level of GSH in iron overload mice. Ferrostatin-1, z-vad-fmk and DFO could increase the GPXs activity of BMMNCs in iron overload mice. Finally, to explore the role of ferroptosis in the pathogenesis of low-risk and high-risk MDS patients respectively, the BMMNCs were obtained from low-risk MDS, high-risk MDS and lymphoma patients respectively and co-cultured with decitabine and above-mentioned inhibitors. The results showed that, ferrostatin-1, necrostatin-1, z-vad-fmk could significantly reverse the inhibitory effect of decitabine of low-risk MDS patients. Necrostatin-1 and Fer-1 could also reverse the inhibitory effect of decitabine of high-risk MDS patients, although the difference was not significant. Decitabine could significantly increase the ROS level in both MDS groups, which could both be inhibited by ferrostatin-1 or promoted by erastin. Ferrostatin-1, necrostatin-1 and z-vad-fmk could significantly reverse the inhibitory effect of decitabine on GSH level in low-risk MDS patients. Ferrostatin-1 and necrostatin-1 could significantly reverse the inhibitory effect of decitabine on GSH level in high-risk MDS patients. Erastin combined with decitabine could further reduce the GSH level, and the difference was significant in high-risk MDS group. For low-risk MDS group, GPXs activity of ferrostatin-1 combined with decitabine and z-vad-fmk combined with decitabine groups were significantly higher than that of decitabine group. For high-risk MDS group, the activity of GPXs of ferrostatin-1 combined with decitabine and necrostatin-1 combined with decitabine groups were significantly higher than that of decitabine group. Erastin could further decrease the activity of GPXs when compared with decitabine group. Our findings reveal a novel therapeutic mechanism of decitabine and may open a new window for therapeutic targeting in the treatment of MDS. Figure Disclosures No relevant conflicts of interest to declare.

Klotho Expression Predicts Poor Prognosis in Myelodysplastic Syndrome

Introduction: An important explanation for the occurrence of Myelodysplastic syndrome (MDS) in the elderly is related to hematopoietic stem-cell (HSC) senescence. Recent studies have identified aging-related alterations in mesenchymal stromal cells in MDS. During the aging process, the different HSC subtypes lose the ability to differentiate into lymphoid and myeloid precursor cells, causing a breakdown in the immune system balance. This process exposes HSC to important genotoxic factors and may induce genetic or epigenetic alterations because exposure to these factors increases the risk of hematological malignancies. A new biomarker for cancer is Klotho (α and β subtypes), which has been identified as an anti-aging gene that extended the lifespan in murine models when it was overexpressed. Several studies have demonstrated that αKlotho (KL) deficiency causes development of aging features, including increased proliferation, reduction in tumor cell apoptosis, and disruption of HSC development. Some studies have established an association between low expression of KL and risks of cancer (i.e. lymphoma) by inducing cell apoptosis, inhibiting tumor growth, and causing a decrease in insulin-like growth factor 1 receptor (IGF-1R) activation. In parallel to these studies, a recent study reported that KL downregulation was associated with the occurrence of anemia, the main clinical feature of MDS, in cancer patients. However, the expression profile of KL and βKlotho (KLB) genes in the bone marrow (BM) of healthy patients with different ages and the way in which the genes modulate the pathogenesis of MDS in the elderly are unknown. Methods: Total BM cell samples from 62 de novo MDS patients with a mean age of 66 ± 16 years old (10 MDS with single lineage dysplasia, 14 MDS with ring sideroblasts, 27 MDS with multilineage dysplasia, and 11 MDS with excess blasts based on World Health Organization's (WHO) 2016 classification) and from eleven healthy volunteer individuals were collected with informed consents. Healthy patients were stratified into young (n=6; mean 25 ± 6 years old) and elderly (n=5; mean 64 ± 9 years old) individuals. Pre-developed TaqMan gene expression assays for KL (Hs00934627_m1) and KLB (Hs00545621_m1) were used to quantify mRNA expression. Each sample was performed in duplicate, and the expression ratios were calculated using the 2−ΔCq method. Results: Our results demonstrate that the KL gene was expressed in the BM samples from all experimental groups. High KL expression was identified in MDS patients when compared to young (p <0.001) and elderly (p <0.001) controls (Figure 1A). In MDS, KL was downregulated in male patients (p=0.042) (Figure 1B), neutropenic MDS cases (p=0.003) (Figure 1C), MDS patients with transfusion dependence (p=0.001) (Figure 1D), and MDS with an excess of blasts (p=0.038) (Figure 1D), representing more aggressive diseases. A multiple regression was performed in order to predict KL expression from gender, absolute neutrophil count (ANC), transfusion dependence, and WHO 2016 classification. These variables significantly predicted KL expression (F [4, 27] = 3.396; p=0.023; r2=0.335). Transfusion dependence (p=0.027) and WHO 2016 classification (p=0.049) variables significantly added to the prediction. The KLB gene was only expressed in a total of 8/62 MDS patients. The feature that stood out in these patients was the presence of dyserythropoiesis (62.5%). No control sample expressed the KLB gene. Conclusions: Our results demonstrated an important expression profile of KL and KLB in BM of healthy young and old individuals and/or in MDS patients for the first time in contrast to a previous study in which it was demonstrated that human BM induces a negligible expression of these genes. Based on absence of differences between controls, our data showed that KL expression not was associated with the MDS pathogenesis-related senescence process, but its lower expression contributed to the poor prognostic MDS features, probably due to its high potential for affecting HSC number, function, and erythropoiesis. Additionally, low KLB expression in MDS patients with dyserythropoiesis suggests a compensatory response of the bone marrow environment to hematopoietic development block via clonal expansion, especially in the erythroid lineage. These approaches may define KL and KLB as new pathological markers of poor prognosis in MDS and improve future potential therapies. Disclosures No relevant conflicts of interest to declare.

Variable Response to BCL2 Inhibition in MDS Is Enhanced across MDS Subtypes with Synergistic Combination of BCL2+MCL1 Inhibition

Myelodysplastic syndromes (MDS) are heterogeneous bone marrow failure neoplasms marked by cytopenias, reduced quality of life and predilection to transform into AML. While several treatments for AML have recently been approved, the available treatments for MDS are lacking, and adaptation of AML therapy to MDS is complicated. This is due, in part, to the heterogeneity of MDS. Despite this heterogeneity, most clonal cells in MDS have an imbalance of mitochondrial-controlled BCL2 family proteins resulting in dysregulated apoptosis. These anti- (or pro-) apoptotic proteins compete for ligand to block (or promote) apoptosis, providing an opportunity to selectively target anti-apoptotic proteins and advance therapy for MDS. Venetoclax (VEN), a newly FDA-approved therapy that specifically inhibits the anti-apoptotic protein BCL2, has yielded response rates of up to 50-70% in elderly AML including impressive responses in transformed MDS which previously failed DNMTi (DiNardo et al, 2019, Wei et al, 2019). Upregulation of the anti-apoptotic protein, induced myeloid cell leukemia-1 (MCL1), is a known resistance mechanism in AML resistant or refractory to BCL2 inhibition (Pan et al, 2014), and MCL1 increases when some MDS samples are treated with BCL2 inhibitors (Jilg et al, 2016). Therefore, strategic inhibition of BCL2 and/or MCL1 is a logical therapeutic approach in MDS. We have shown efficacy of MCL1 inhibitors in the laboratory against AML patient samples that are dependent on MCL1 protein or resistant to BCL2 inhibition, including AML cells that arose from MDS (Ramsey et al, 2018). Here, our goal was to determine the sensitivity of MDS cells to inhibition of specific anti-apoptotic proteins, elucidate the characteristic determinants of response, and investigate synergy with combined BCL2 and MCL1 inhibition. We cultured MDS patient samples and determined the in vitro sensitivity of 35 MDS patient samples to selective BCL2, BCL-XL and MCL1 inhibitors using CellTiter-Glo to determine the relative cell viability concentrations (GI50) for each selective inhibitor after 48 hours of exposure. While there was little sensitivity to BCL-XL inhibition across all samples, we detected a gradient of low to high response to the BCL2 inhibitor with low to higher blast count MDS subtypes; higher blast count MDS (EB1 and EB2) were more sensitive than low blast count subtypes (RS-SLD/MLD and MLD). Interestingly, nearly all MDS subtypes were sensitive to the selective MCL1 inhibitor, S63845. To determine if there were any correlations between sensitivity to specific inhibitors and mutational status, a targeted NGS panel of 37 commonly mutated genes in myeloid disease was conducted on all samples. As expected, we observed an increased number of SF3B1 mutations in the lower blast count MDS-RS patient samples. Likewise, though there were only two samples in this cohort containing mutations in PTPN11, one was completely resistant to BCL2 inhibition.(Stevens et al, ASH 2018) Otherwise, we did not observe any correlation between specific mutations and BH3 dependence. Since upregulation of MCL1 is seen in VEN treated MDS cells and is a known resistance mechanism for VEN treatment in AML, we treated these same patient samples with VEN+S63845 to determine any synergistic benefit of combining these drugs. While there were differential responses to VEN monotherapy between subtypes, all MDS subtypes exhibited response benefit to the addition S63845 to VEN. Drug synergy was confirmed with the Zero Interaction Potency model. These results were further corroborated by increased annexin-V staining and reduced colony formation in methylcellulose. Toxicity experiments in the MISTRG immunocompromised animal model indicate that the combination of selective inhibitors of MCL1 and BCL2 are tolerated, and treatment of MDS within these xenografts is underway and will be presented. Overall, our data suggests that higher blast count MDS subtypes (EB1 and EB2) are more likely to respond to VEN monotherapy than low blast count subtypes, while all MDS subtypes may respond to MCL1 inhibition. Moreover, drug synergy can be obtained across all subtypes of MDS by combining BCL2 and MCL1 inhibitors. BCL2 inhibition is changing the standard of care in AML, thus, refining the design of clinical trials testing BCL2 and MCL1 inhibitors in MDS and the precision of patient selection for therapy is a great priority. Disclosures Savona: Boehringer Ingelheim: Patents & Royalties; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sunesis: Research Funding; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Selvita: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Distinct Bone Marrow Microenvironment Abnormalities in MDS and MPN with Fibrosis

Background: Bone marrow (BM) fibrosis is common in both myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN), its pathogenesis remains unclear. It is also unknown whether and how the pathophysiology of BM fibrosis in MDS and MPN are similar or different, while it is anticipated that altered BM microenvironment plays a key role. Methods: To visualize various BM microenvironmental cells in MDS or MPN patients as compared to those in control subjects, we utilized multicolor immunostaining using a tyramide signal amplification system and a fluorescence spectrum analyzer. Paraffin-embedded BM biopsy samples from 47 patients with MDS (26 without and 21 with fibrosis), 10 with primary myelofibrosis or myelofibrosis secondary to essential thrombocythemia (MPN-F), and 17 with non-Hodgkin lymphoma without BM involvement (as control) were immunostained with antibodies for mesenchymal/neuronal cell markers. Gene mutations were analyzed by targeted sequencing. Results: In control samples, C-X-C motif ligand 12 (CXCL12) was stained mainly in cells at perivascular areas and the periphery of a minor proportion of adipocytes. These patterns did not differ in MDS without fibrosis from control. However, CXCL12-stained non-adipose cells were significantly increased in MDS with fibrosis (MDS-F) [median (range), 5190 (2117-14299) μm2/mm2 as compared to 628 (464-1331) μm2/mm2 in control; p=0.00453]. These cells tended to be increased in also MPN-F though statistical significance was not reached [6096 (1098-24458) μm2/mm2; p=0.06304]. The localization of these cells was not confined to the perivascular areas in both diseases. There were no apparent differences in the degree or the pattern of staining for CXCL12 between MDS-F and MPN-F. We also focused on Schwann cells and Nestin-expressing stromal cells because these cells have been independently reported to function as an important hematopoietic stem cell niche. Neurofilament heavy chain (NFH) or neuron-specific class III beta-tubulin-positive nerve fibers (Tuj1) surrounded by glial fibrillary acidic protein (GFAP)-positive Schwann cells were rarely observed in control samples. This observation was true of MDS without fibrosis. However, GFAP-positive Schwann cells were significantly increased in MDS-F and MPN-F [median (range), 39457 (1325-60654) um2/mm2 and 75951 (19879-326216) um2/mm2, as compared to 6258 (1325-14842) um2/mm2 in control; p=0.0017 and 0.0034, respectively]. When the section was stained for Nestin, Schwann cells in MPN-F did not show positive signals except for one case. In contrast, Nestin was frequently stained in the GFAP-positive Schwann cells of MDS-F (9 of 21 MDS-F, 42.9%); particularly 5 of 7 cases (71.4%) in which fibrosis was judged as severe (MF-3), showed positive signals. One exceptional case of MPN-F that showed Nestin-positivity in increased Schwann cells was a triple-negative case (no mutations in JAK2, CALR, or MPL); instead, mutations were found in NRAS, TET2, BCOR, and SMC1A, indicating that this case may have a feature of MDS. Conclusion: Abnormal increases in CXCL12-expressing mesenchymal cells and nerve fiber-surrounding Schwann cells were visualized by multicolor fluorescence in MDS-F and MPN-F. Schwann cells in MDS-F and MPN-F showed a remarkable difference in the expression of Nestin. It is to be elucidated whether the increase in the Schwann cells has a role in the pathogenesis/pathophysiology, and if so, whether those Schwann cells with distinct phenotypes have differential roles in these two disease conditions. Disclosures Ogawa: ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Kan Research Laboratory, Inc.: Consultancy; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; RegCell Corporation: Equity Ownership; Qiagen Corporation: Patents & Royalties; Asahi Genomics: Equity Ownership.

Cross Talk between the Immune Compartment and the Tumor Cells in Myelodysplastic Syndromes (MDS)

Background: Abnormalities of the immune system and innate immune signaling have been described in patients (pts) with MDS, however the interaction between the MDS tumor cells and the immune compartment is poorly defined. Immune modulatory therapies are being explored in MDS and a better understanding of the interaction and potential cross-talk with immune components and MDS tumor cells is critical in developing new therapeutic strategies. Methods: We developed a multiscale immune profiling strategy to map the immune environment of treatment naïve pts with lower- and higher-risk MDS, using mass cytometry by time of-flight (CyTOF) combined with Olink proteomics analyses. We obtained bone marrow (BM) and peripheral blood (PB) from 33 pts with MDS, (17 higher-risk; 16 lower-) and 10 healthy donors. Pts were representative of the MDS distribution across age, gender, mutational status and histologic subtype. PB and BM were freshly processed and stained with an antibody panel for myeloid cells. For the lymphoid panel, all the pts' samples were stained simultaneously to avoid batch effect. We processed the BM and the PB plasma for Olink. Results: We profiled myeloid cells, NK cells and T cells in pts with MDS to determine differences in their constituents. Phenograph clustering across all pts revealed distinct T lymphoid metaclusters, that corresponded to known immune cell populations. We observed a robust immune response in the BM from MDS patients. T cells and myeloid cells were found in equal proportion in the BM from MDS and healthy patients. However, the B cell population were present at significantly lower frequency in the MDS BM (p<0.05), whereas gamma delta (gd)T cells and NK cells were at significantly higher frequency in the MDS pts (p <0.05). Paired mass cytometric analysis revealed a distinct composition and phenotype of T cell subsets. Among the T cell compartment, regulatory T cells (Treg) (p<0.001), cytolytic CD8 T cells (expressing perforin (p<0.01) or granzyme (p<0.001)) and exhausted CD8 T cells (expressing PD-1 (p= 0.09), LAG-3 (p<0.001) or TIM-3) seemed to accumulate in the MDS BM. Compared to healthy BM Treg in MDS BM expressed higher levels of TIGIT, TIM3, CD27, CD25, CD28 and OX40. Cytolytic NK cells accumulate in MDS BM (p<0.05). In addition to measuring adaptive lymphocyte responses to tumors, we also analyzed the distribution of innate lymphocytes and in particular NK cells in MDS BM. In the NK cell population, the cytotoxic NK cells (CD56dim CD16+) were found in a higher proportion (p<0.05) than the cytokinic counterpart (CD56bright) in the MDS BM. NK cells that accumulate in the MDS BM expressed higher level of CD45RA, CD38, granzyme B, CD56 suggesting their superior cytotoxic potential at a critical developmental stage of retaining enhanced sensitivity to pro-inflammatory cytokines derived from myeloid cells. We also analyzed a panel of 92 unique cytokines, chemokines and inflammatory markers produced in the tumor milieu using OLink Proteomics proximity extension assay. Many chemokines and cytokines were differently expressed in the MDS BM compared to the healthy BM, suggesting an important inflammatory reaction. Conclusion: These data demonstrate new findings suggesting interactions and cross-talk between Tregs, NK cells, CD8 T cells and gdT cells in the BM of pts with untreated MDS. The data also suggest that there is a strong immune response in the MDS BM, particularly an intense cytotoxic response with the accumulation of cytotoxic CD8 T cells, NK cells and gdT cells. These results are are contrary to those observed in most solid tumors. This cytotoxic response seemed to be counteracted by the presence of Treg. Each of the subgroups of T cells (as well as NK cells) have been implicated as possibly promoting immune tolerance and tumor growth in different model systems and other tumor types. NK cells and gdT cells are known to respond to virus-infected and transformed cells rapidly and without the need for prior sensitization. However, anecdotal evidence from numerous independent studies may suggest that a strong but prolonged NK cell- and gdT cell-mediated antiviral response may result in dysregulation and promote tumorigenesis and/or aggravate pathology. Additional studies are underway to further characterize the immune landscape in those untreated and treated and to study the interaction of the immune components with tumor cells. Disclosures Navada: Onconova Therapeutics Inc: Research Funding. Silverman:Onconova Therapeutics Inc: Patents & Royalties, Research Funding; Celgene: Research Funding; Medimmune: Research Funding.

The SF3B1 K700E Mutation Induces R-Loop Accumulation and Associated DNA Damage

The myelodysplastic syndromes (MDS) are common myeloid malignancies. Mutations in genes involved in pre-mRNA splicing (SF3B1, SRSF2, U2AF1 and ZRSR2) are the most common mutations found in MDS. There is evidence that some spliceosomal components play a role in the maintenance of genomic stability. Splicing is a transcription coupled process; splicing factor mutations affect transcription and may lead to the accumulation of R-loops (RNA-DNA hybrids with a displaced single stranded DNA). Mutations in the splicing factors SRSF2 and U2AF1 have been recently shown to increase R-loops formation in leukemia cell lines, resulting in increased DNA damage, replication stress and activation of the ATR-Chk1 pathway. SF3B1 is the most frequently mutated splicing factor gene in MDS, but a role for mutated SF3B1 in R-loop accumulation and DNA damage has not yet been reported in hematopoietic cells. We have investigated the effects of the common SF3B1 K700E mutation on R-loop formation and DNA damage response in MDS and leukemia cells. R-loop signals and the DNA damage response were measured by immunofluorescence staining using S9.6 and anti-γ-H2AX antibodies respectively. Firstly, we studied K562 (myeloid leukemia) cells with the SF3B1 K700E mutation and isogenic SF3B1 K700K wildtype (WT) K562 cells. K562 cells with SF3B1 mutation showed a significant increase in the number of S9.6 foci [Fold change (FC) 2.01, p<0.001] and in the number of γ-H2AX foci (FC 2.32, p<0.001), indicating increased R-loops and DNA damage, compared to SF3B1 WT K562 cells. Moreover, we observed increased Chk1 phosphorylation at Ser345, a hallmark of activation of the ATR pathway, in SF3B1 mutant K562 cells. Next, we analyzed induced pluripotent stem cells (iPSCs) that we generated from the bone marrow cells of one SF3B1 mutant MDS patient and of one healthy control. A significant increase in R-loops and DNA damage response was observed in an iPSC clone harboring SF3B1 mutation compared to another iPSC clone without SF3B1 mutation obtained from same MDS patient (S9.6 mean fluorescence intensity - FC 1.72, p<0.001; γ-H2AX foci - FC 1.34, p=0.052) and to iPSCs from the healthy control (S9.6 mean fluorescence intensity - FC 1.53, p<0.001; γ-H2AX foci - FC 1.61, p=0.006). In addition, bone marrow CD34+ cells from a SF3B1 mutant MDS patient showed increased R-loops (as measured by number of S9.6 foci) compared to CD34+ cells from a MDS patient without splicing factor mutations (FC 1.9) and from a healthy control (FC 2.6). To investigate whether the observed DNA damage and ATR activation in SF3B1 mutant K562 cells result from induced R-loops, we overexpressed RNASEH1 (encoding an enzyme that degrades the RNA in RNA:DNA hybrids) to resolve R-loops in these cells. RNASEH1 overexpression significantly reduced the number of S9.6 (FC 0.51, p<0.001) and γ-H2AX foci (FC 0.63, p=0.035) in SF3B1 mutant K562 cells compared to SF3B1 WT K562 cells. RNASEH1 overexpression also resulted in decreased Chk1 phosphorylation, indicating suppression of ATR pathway activation in SF3B1 mutant K562 cells. To determine the functional importance of ATR activation associated with SF3B1 mutation, we evaluated the sensitivity of SF3B1 mutant cells towards the ATR inhibitor VE-821. SF3B1 mutant K562 cells showed preferential sensitivity towards VE-821 compared to SF3B1 WT K562 cells. Chk1 is a critical substrate of ATR, and we next investigated the effects of Chk1 inhibition in SF3B1 mutant cells. Interestingly, SF3B1 mutant K562 cells demonstrated preferential sensitivity towards the Chk1 inhibitor UCN-1 (IC50 61.8 nM) compared to SF3B1 WT K562 cells (IC50 267 nM), suggesting that ATR activation is important for the survival of SF3B1 mutant cells. SF3B1 mutant K562 cells were preferentially sensitive to the splicing modulator Sudemycin D6 (IC50 53.2 nM) compared to SF3B1 WT K562 cells (IC50 130.7 nM). The effects of VE-821 and UCN-1 on SF3B1 mutant K562 cells were enhanced by Sudemycin D6 (Combination index <1), indicating synergy. In summary, our results show that the SF3B1 mutation leads to accumulation of R-loops and associated DNA damage resulting in activation of the ATR pathway in MDS and leukemia cells. Thus different mutated splicing factors have convergent effects on R-loop elevation leading to DNA damage. Moreover, our data suggest that Chk1 inhibition, alone or in combination with splicing modulators, may represent a novel therapeutic strategy to target SF3B1 mutant cells. Disclosures Schuh: Janssen: Speakers Bureau; Verastem: Speakers Bureau; Kite: Speakers Bureau; Gilead: Speakers Bureau; Seattle Genetics: Speakers Bureau; Jazz Pharmaceuticals: Speakers Bureau; Bristol-Myers Squibb: Research Funding; AbbVie: Consultancy, Speakers Bureau; Genentech: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy, Speakers Bureau. Wiseman:Novartis, Celgene: Consultancy, Honoraria.

Circulating Small Noncoding RNAs As Novel Semi-Invasive Markers of Patient Survival in Myelodysplastic Syndromes

Introduction: Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders with a tendency to transform to acute myeloid leukemia. Several prognostic systems based almost entirely on clinicopathological characteristics have been developed. However, considering large heterogeneity of MDS, there is an ongoing substantial effort to identify new molecular markers, which would improve patient stratification. It has recently been discovered that various types of small noncoding RNAs (sncRNAs) are exported into blood circulation. Encapsulated in extracellular vesicles (EVs) or as a part of various molecular complexes, they play important roles in long distance cell-to-cell communication. Their secretion appears to be controlled and specific and thus may reflect (patho)physiological processes occurring in different cells. As diagnostic procedures move from bone marrow biopsies towards less invasive techniques, circulating sncRNAs have become of particular interest as potential novel semi-invasive biomarkers. Aims: We investigated circulating sncRNAs in MDS on the genome-wide level to better understand MDS pathogenesis and to search specific RNAs applicable as potential biomarkers. We analyzed paired samples from the whole plasma and EV fractions to define which of these two materials is better source of relevant sncRNA-based markers of MDS. Methods: We performed small RNA-seq in paired samples (plasma and EVs) in 42 patients and 17 healthy controls. To associate sncRNA levels with mutational status, Illumina TruSight Myeloid Sequencing Panel Kit examining 54 genes was applied. Overall survival (OS)‐associated sncRNAs were identified by performing univariate Cox regression along with a permutation test using BRB‐ArrayTools. Multivariate Cox regression analysis with backward variable selection was done to identify independent variables associated with patient survival. Results: Data analysis showed that the most abundant category of RNAs were miRNAs (51 %), followed by rRNAs, piRNAs, tRNAs, and mRNAs. Sample clustering revealed that RNA content of EV cargo is more homogenous than of plasma. Differential analysis identified a striking disbalance in RNA content between plasma and EVs in MDS patients (> 400 significantly deregulated sncRNAs) whereas these sncRNA profiles remained similar in controls. Such disproportion may suggest that mechanisms of sncRNA export into blood circulation may be specifically affected in MDS. As a result, these changes might substantially affect cell-to-cell communication of blood cells, contributing to the disease development. Further, we analyzed levels of individual sncRNAs and found that many hematopoiesis-related miRNAs were significantly increased in MDS patients compared to healthy controls, mostly both in plasma and EVs (e.g., miR-34a-5p, miR-125a-5p, and miR-150). Several miRNAs (e.g., miR-103b, miR-107, and miR-221) showed different levels between early and advanced MDS. Interestingly, the majority of miRNAs from the 14q32 miRNA cluster were specifically increased in early MDS. Additionally, we investigated possible impacts of somatic mutations on expression of circulating sncRNAs. However, only low numbers of circulating sncRNAs significantly associated with mutational status, suggesting no fundamental effects of somatic mutations on sncRNA export from MDS cells. Survival analysis identified sncRNAs with the highest level of association with OS (plasma: miR-1260b, miR-3191-3p, and miR-328-3p; EVs: miR-1237-3p, U33, hsa_piR_019420, and miR-548av-5p). To better stratify MDS patients, we defined risk prediction scores (separately for plasma and EVs) that combined effects of the above mentioned sncRNAs. The results showed that EV-combined score significantly increased the predictive power of the survival risk model (Figure 1). Moreover, Cox multivariate analysis revealed that this score is an independent variable the most significantly associated with OS (HR = 2.942, 95% CI 1.786 to 4.849, p < 0.001). Conclusions: Our data demonstrate that profile of circulating sncRNAs is specific in MDS and changes with the disease progression. Monitoring of sncRNA levels in EVs provides better prognostic performance for patient survival (compared to whole plasma), probably due to higher homogeneity of the EV fraction. Supported by grants 16-33617A and 00023736 from the Ministry of Health of the Czech Republic. Disclosures No relevant conflicts of interest to declare.

R-Loops-Induced ATR Signaling As a Potential Therapeutic Target in Myelodysplastic Syndrome

Introduction: Somatic mutations in genes coding for splicing factors (e.g. SF3B1, U2AF1 and SRSF2) are found in about 50% of patients with Myelodysplastic Syndrome (MDS). These mutations have been shown to frequently occur early in the mutational hierarchy of the disease making them particularly attractive therapeutic targets. Recent research has revealed an association of splicing factor mutations (sfm) with elevated levels of DNA:RNA intermediates (R-loops), which induce replication stress and downstream activation of the ataxia telangiectasia and Rad3-related protein (ATR) pathway. The aim of this work was to investigate R-loops-associated ATR signaling as a novel therapeutic concept in primary CD34+ MDS patient cells carrying sfm, and to identify possible novel options for combination therapy. Methods: Using quantitative immunofluorescence microscopy we assessed levels of R-loops in primary CD34+ bone marrow cells isolated from MDS patients (n=23) with and without sfm. In addition, we evaluated the direct association of R-loops with induction of replication stress and activation of associated signaling by analyzing replication fork progression rates and phosphorylation of ATR target proteins. Furthermore, we determined the in vitro sensitivity of mutant (n=12) and non-mutant CD34+ (n=10) cells of MDS patients towards ATR inhibitors (VE-821 and AZD6738) alone and in combination with splicing modulator Pladienolide B and investigated the impact on DNA damage accumulation and apoptosis. We also performed these experiments in cord blood derived CD34+ cells overexpressing SRSF2P95H without other MDS-associated cellular alterations. Results: We found significantly elevated levels of R-loops in CD34+ cells from MDS patients carrying sfm compared to non-splicing factor mutated (non-sfm) MDS cells and healthy controls (mean MFI= 177 (sfm) vs. 78 (non-sfm) vs. 91 (healthy), p<0.0001). These were primarily found in the stem and progenitor as well as myeloid cell compartments, while lymphoid cells showed normal levels. Induced R-loops caused delayed replication fork dynamics as determined by fiber assay (mean fork speed= 0.282 kb/min (sfm) vs. 0.401 kb/min (non-sfm), p ≤0.0001), which in association with elevated levels of single-strand DNA marker replication protein A (RPA) strongly suggests the presence of replication stress in splicing factor mutated CD34+ cells. When exposed to ATR inhibitors in vitro, splicing factor mutant cells showed a significantly elevated sensitivity towards these drugs (normalized mean IC50= 0.89 µM (sfm) vs. 2.84 µM (non-sfm), p ≤0.001) associated with heightened levels of DNA damage and apoptosis, both of which were further increased by the addition of splicing modulator Pladienolide B. We also confirmed a direct correlation of R-loops associated ATR signaling with the presence of mutant SRSF2P95H by lentiviral overexpression in cord blood CD34+ cells. Conclusion: Overall, our results identify ATR as a promising novel therapeutic target in MDS with splicing factor mutations and provide a preclinical rationale for combination therapy with splicing modulator drugs. Disclosures Nolte: Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding.

Expression of CD47 and Calr in Myeloproliferative Neoplasms and Myelodysplastic Syndrome: Potential New Therapeutical Targets

Myelodysplastic neoplasms (MPN) and myelodysplastic syndrome (MDS) are myeloid malignancies characterized by either overproduction of mature blood cells or hyperplastic bone marrow and peripheral cytopenia, but both with a tendency to evolve into acute myeloid leukaemia. In solid tumours, calreticulin (CALR) overexpression produces a pro-phagocytic signal and is counteracted by concomitant expression of anti-phagocytic CD47, reflecting an apoptosis vs survival mechanism. Increases of both CALR and CD47 on the cell membrane have been observed in response to chemotherapy, however their role in myeloid malignancies is poorly understood. Aims: Investigate the expression and cellular localisation of CALR and CD47 in untreated and treated patients with essential thrombocythemia (ET), polycythemia vera (PV) myelofibrosis (MF), and in MDS patients in comparison with healthy controls. Methods: Mononuclear cells were collected by Ficoll separation, from peripheral blood of 27 MPN (8 PV, 16 ET, 3 MF); 14 MPN patients received cyto-reductive therapies (Hydroxyurea, Anagrelide or Ruxolitinib); 10 MDS patients and 4 controls. Cells were fractionised into 4 compartments: membrane, cytoplasm, cytosol and nucleus. Proteins were extracted using TRIzol, with CALR and CD47 protein expression analysed by western blotting. Results: Total CALR and CD47 protein expression increased in MPN samples compared with controls (CALR- 7.9 vs 5.1; CD47- 2.7 vs 2.2 fold, respectively). CD47 showed higher expression of its overall protein on MPN cell membranes when compared with CALR (22% vs 13.9%). We observed a significant reduction of CALR expression in all MPN subtypes when patients were treated with cyto-reductive agents (ET- untreated 43.3% vs treated 2%, PV- 3.6% vs 2.2%, ET- 21% vs 11%). Interestingly we have observed a significant increase in CD47 cell membrane expression after treatment in MF and PV (CD47 in MF- untreated 11.8% vs treated 34.3%, PV-11.4% vs 35.9%), suggesting an anti-phagocytic effect being induced by cytotoxic drugs in MF and PV cells. In ET cell membranes however, CD47 expression is reduced after cyto-reductive treatment (22% vs 16.6%), suggesting instead a prophagocytic effect. Similarly, in MDS cells CD47 is overexpressed compared with controls (CD47- 11.31 vs 2.2 fold, respectively) and it mainly located to the membrane. Interestingly the degree of CD47 expression correlated to patients IPSS-R, increasing from low risk to high risk (low - 15.7%, intermediate 1 - 41.3%, intermediate 2 - 53.9% and high - 67.6%). CALR expression is also reduced in MDS cells comparing with controls when split by IPSS-R risk score (low - 9%, intermediate 1 - 16.9%, intermediate 2 - 17%, high - 17.9% Vs control - 29.1%). Summary/Conclusion: CD47, but not CALR, is overexpressed on the membrane of patients with MPN and MDS. In MDS, we observed a progressive increase in CD47 expression as the MDS evolve in accordance to the IPSS-R risk score. This opposes previous studies in solid tumours, which showed significant increases of both CALR and CD47 as disease progresses, suggesting a role for CD47 as a strong antiphagocytic signal responsible for immune survival in MPN and MDS. All MPN patients showed decreased CALR expression during cyto-reduction therapy, but we observed a significant difference in CD47 expression across different MPN subtypes. In fact, treating MF and PV patients with cyto-reductive agents, increases CD47 expression leading to a stronger anti-phagocytic signal, while in ET we observed a reduction, suggesting a more pro-phagocytic reaction to cytotoxic drugs in ET. A larger cohort of patients is required to confirm our findings and allow a further stratification based of type of cyto-toxic drugs. The use of anti-CD47 antibodies could represent a new strategy to enhance the pro-phagocytic signal via increasing the CALR expression, and in combination with standard cyto-reduction therapy, might represent a new therapeutical strategy in both MPN and MDS. Disclosures No relevant conflicts of interest to declare.

EP300 Suppresses Leukemia Development in Myelodysplastic Syndrome through Myb Repression

Background Epigenetic dysregulation is a hallmark feature of myelodysplastic syndrome (MDS) as epigenetic regulator genes, such as ASXL1, TET2, and SRSF2, are highly mutated in MDS patients. Tet2 (ten-eleven translocation-2, a methylcytosine deoxygenase) mutations are found in ~20% of MDS patients; they are also seen in patients with acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPN). Loss of Tet2 in hematopoietic cells leads hematopoietic defects including enhanced hematopoietic stem cell (HSC) self-renewal, myeloid expansion and an increased propensity to develop MDS or AML. The lysine acetyltransferase (KAT) p300 (encoded by the EP300 gene) plays pivotal roles in essential cellular functions including proliferation, differentiation and signal transduction. However, the function of p300 in hematopoietic malignancies is clearly cell-context dependent, as we have shown that p300 functions as an oncogene in AML1-ETO-driven AML, and a tumor suppressor gene in NUP98-HOXD13-driven MDS. The EP300 and the related CREBBP genes are infrequently mutated in MDS patients. Methods To investigate the role of p300 in MDS driven by lack of Tet2, we generated a mouse model carrying a hematopoietic-specific EP300 conditional knockout on a Tet2-null background and characterized the effect of p300 loss on HSC function and the pathogenesis of MDS and AML. We also explored the therapeutic potential of manipulating p300 KAT activity to affect the course of MDS driven by Tet2 loss. Results Compared to the Tet2-/- mice, the EP300Δ/ΔTet2-/- (DKO) mice had shortened survival and an accelerated onset of AML, with increased blasts in the bone marrow and peripheral blood, anemia, splenomegaly, and universal presence of granulocytic sarcomas, demonstrating that depletion of p300 enhances the leukemogenicity of Tet2-/- HSC. The DKO mice showed enhanced hematopoietic stem/progenitor cell (HSPC, defined as Lin-Sca1+Kit+ cell) proliferation and an increased HSPC pool only 2 weeks after p300 deletion, far in advance of the development of any disease manifestations. In contrast, deletion of p300 in wild type (wt) mice affected neither HSPC proliferation, nor pool size. To further define the mechanisms by which these two genetic events cooperate to drive the development of MDS/AML, we assessed the DNA methylation status and gene expression signatures of HSPCs isolated from wt, Tet2-/- and DKO mice. As expected, loss of Tet2 leads to a genome-wide decrease in 5-hmC, whereas loss of p300 does not change 5-hmC distribution across the genome. Interestingly, DKO HSPCs showed local gains of 5-hmC at a variety of genes (compared to Tet2-null HSPCs), including at the c-Myb gene, which is a known oncogenic driver of AML. Gene Set Enrichment Analysis (GSEA) revealed that p300 loss in Tet2-/- HSPCs interferes with leukocyte differentiation, enhances proliferation and inhibits apoptosis. ChIP-X Enrichment Analysis (ChEA) further also indicated that among the differentially expressed genes, between DKO HSPCs and Tet2-null HSPCs, are potential Myb target genes. Given this data and the RNA-Seq and Q-PCR results that showed elevated levels of Myb mRNA in the DKO HSPCs compared to the Tet2-/- HSPCs, we examined the biological effects of knocking down (KD) Myb. Myb KD reduced the colony-forming capacity of the DKO HSPCs, but not the Tet2 -/- HSPCs, identifying its potential role in mediating the effect of p300 on MDS prone HSPCs. These results indicate that loss of p300 in Tet2-null HSPCs enhances their leukemogenicity through elevated Myb expression and activity. To determine whether increasing p300 KAT activity could have an opposite effect on MDS cells, we utilized I-CBP112, which binds p300 and increases its KAT activity and ability to stimulate H3K18 acetylation. Exposure to I-CBP112 did in fact eliminate the indefinite self-renewal capacity of Tet2-/- HSPCs, as measured by serial replating assays, further highlighting the importance of p300 KAT activity in regulating the behavior of Tet2-/- HSPCs. Additional in vivo studies of the effects of I-CBP112 on the course of MDS/AML driven by the absence of Tet2 are ongoing. Conclusions This work clearly indicates that the EP300 gene can play a tumor suppressor role in MDS/AML driven by loss of Tet2- by repressing Myb expression and activity. It also suggests that the enhancement of p300's KAT activity by use of small molecules, could be an effective therapeutic strategy for MDS/AML. Disclosures Cole: Abbvie: Consultancy; Acylin Therapeutics: Equity Ownership.

Transcriptional Regulation of Hematopoietic Stem Cells in Aging and Myelodysplastic Syndrome Reveals DDIT3 As a Potential Driver of Transformation

Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell (HSC) malignancies characterized by ineffective hematopoiesis. Genetic alterations do not fully explain the molecular pathogenesis of the disease, indicating that other types of lesions, such as transcriptional aberrations, may play a role in its development. Moreover, MDS prevalence is almost exclusive to older patients, suggesting that elderly-related alterations may predispose to the development of this clinical entity. Thus, study of the transcriptional lesions occurring in the aging-MDS axis could shed some light of the molecular bases of the disease. To characterize the transcriptional profile of HSCs in aging and MDS, we isolated CD34+, CD38-, CD90+, CD45RA- cells from 11 untreated MDS patients with unilineage and multilineage dysplasia (median of 75 y/o), as well as from 16 young and 8 elderly healthy donors (median of 21 and 70 y/o, respectively), and their expression profile was analyzed using MARS-seq. Unsupervised principal component analysis demonstrated that the three groups of HSCs clustered separately, indicating that different expression profiles characterize healthy young and elderly, and MDS-associated HSCs. To better understand the gene expression deregulation of HSCs, we analyzed the transcriptional dynamisms along the aging-MDS axis, detecting groups of genes following different patterns of expression. Some gene clusters showed exclusive alteration either in aging or in the progression from elderly HSCs to MDS-HSCs, other groups of genes presented a continuous alteration along the axis, and some displayed opposite regulation in aging and in the transition to MDS (Figure 1). Genes showing specific downregulation in aging were involved in DNA damage sensing and repair, and in cell cycle regulation, whereas genes overexpressed in this process were enriched in apoptosis regulators and in cancer-associated genes, including AML-related factors. These findings indicate that transcriptional changes in aging may predispose for MDS and AML, and potentially other malignancies. Interestingly, we detected a group of genes in which the age-mediated upregulation of gene expression was reversed to that of young HSCs in MDS, indicating a "rejuvenation" profile of malignant HSCs. These genes were involved in response to inflammation, to different types of stress conditions such as hypoxia or radiation, and to cytokines. Elderly HSCs may upregulate such genes in response to the known inflammatory microenvironment of elderly bone marrow. Intriguingly, the decrease in expression detected in MDS suggests that malignant HSCs lose the ability of reacting to such stimuli, possibly favoring their survival in a hostile microenvironment. Finally, the analyses performed allowed for the identification of genes showing MDS-specific deregulation. Genes specifically overexpressed in MDS compared to normal (both young and elderly) HSCs, we enriched in transcriptional and epigenetic regulators, and among them, we detected the presence of DDIT3/CHOP, a member of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors. To determine its potential effects on hematopoietic deregulation, DDIT3 was exogenously overexpressed in healthy HSCs. Notably, its upregulation produced an erythroid bias in an ex-vivo differentiation system, with an increase in the percentage of erythroblasts and a decrease in granulocytes and monocytes compared to HSCs transduced with the empty vector. Transcriptomic analysis of transduced HSCs not subjected to differentiation demonstrated how DDIT3 overexpression produced an erythroid-prone state of HSCs, suggesting it may act as a pioneer factor in MDS-HSCs. Furthermore, gene set enrichment analysis showed that DDIT3 overexpression produced an MDS-like transcriptional profile, suggesting this factor may be key in the acquisition of the disease. Altogether, our results demonstrate that HSCs undergo transcriptional changes in the aging-MDS axis that may alter their intrinsic functions as well as their response to the microenvironment, ultimately contributing to the acquisition of the disease. In particular, our data show that DDIT3 may be a potential driver of MDS transformation. Disclosures Paiva: Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche, and Sanofi; unrestricted grants from Celgene, EngMab, Sanofi, and Takeda; and consultancy for Celgene, Janssen, and Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau. Díez-Campelo:Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

A Novel Chemical Compound Inhibiting Hematopoietic Cell Kinase (iHCK) Has a Synergic Effect with Azacytidine (Aza) or Cytarabine (Ara-C) for Acute Myeloid Leukemia Treatment

Introduction. Hematopoietic cell kinase (HCK) belongs to the Src kinase family (SFK) involved in the oncogenic process and hematological malignancy. Some SFK inhibitors are currently under investigation in clinical trials for leukemia after demonstrating efficacy in patients with solid tumors. We have previously reported that HCK is overexpressed in leukemic cells and its inhibition by lentivirus resulted in reduction of cell growth and increased cell death (Roversi et al. BBA Mol Basis Dis. 2017, 1863(2):450-61). In light of the genomic and molecular diversity of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), the development of chemical compounds specific for new molecular targets is currently an important subject. Aims. To investigate the in vitro and in vivo effects of a new chemical compound targeting HCK inhibition (iHCK), alone or in combination with the most used drugs for treatment of MDS and AML (Azacytidine - Aza - or Cytarabine - Ara-C). Methods. After iHCK development, we tested its activity alone or in combination with Aza or Ara-C in CD34+ cells isolated from AML patients (n=5) as well as in a panel of myeloid leukemia cell lines (KG1, HL-60, HEL and K562). Additionally, we tested the iHCK in normal and malignant cells cultured in a 3D bioscaffold obtained by decellularization of bovine bone marrow (Bianco et al. Biomat Sci 2019, 7(4):1516-28), in order to mimic the bone marrow niche. After informed written consent and approval of the Ethical Committee of University of Campinas (CAAE 1000.0.146.00-11), in accordance to the Helsinki Declaration, CD34+ cells were isolated from bone marrows of healthy donors (HD), MDS and AML patients and were treated with iHCK or vehicle (DMSO) in liquid culture, for three days. Meanwhile, HS-5 mesenchymal cells were cultured into the 3D bioscaffold. iHCK or vehicle treated CD34+ cells were introduced into the 3D bioscaffold containing HS-5 and evaluated after 7 and 14 days, by light microscopy (hematoxilin and eosin regular staining) and immunohistochemistry (expression of CD34 and CD90 antigens). NOD.CB17-Prkdcscid/J mice received 2 Gy irradiation followed by transplantation with caudal intravenous injection of leukemia cells obtained from hCG-PML-RARα transgenic mice. After acute promyelocytic leukemia (APL) establishment, animals were treated or not with intraperitoneally iHCK and peripheral blood was collected for hematological analysis and protein was extracted from spleen and bone marrows for Western Blot analysis. ANOVA and Student's T-Test were used. Results.In leukemia cell lines and primary cells, the combinatory treatment of iHCK and Cytarabine (1μM) or 5-Azacitidine (1μM) demonstrated synergistic effects, compared to either drug alone, on the reduction of growth and induction of cell death (P<0.001; Figure 1). Further, Western blot revealed increased BAX expression and decreased BCL-XL expression. Moreover, iHCK treatment was able to reduce the activation of oncogenic pathways, MAPK/ERK and PI3K/AKT, leading to severe reduction of ERK, AKT and p70S6 phosphorylation. Treatment with iHCK reduced CD34+ MDS and AML cells proliferation cultured into the 3D bioscaffold but had no effect upon normal CD34+cells. In vivo analysis showed that APL mice treated with iHCK (5μM) for 48h had reduced leukocyte number compared to APL mice treated with vehicle (13.2±1.1 vs 49.4±18.8; P<0.001). No alterations in hemoglobin levels and platelet were found. Likewise, the in vivo iHCK (2.5μM, 5.0μM or 10.0μM) treatment decreased the phosphorylation of ERK, AKT and P70S6K proteins of leukemic cells (Figure 2). Conclusion.The iHCK pharmacological inhibitor has an antiproliferative activity in leukemic cells without altering cell death and survival rate of normal cells, demonstrating on-target malignant cell killing activity as a single agent or in combination with Azacytidine (Aza) or Cytarabine (Ara-C). Disclosures No relevant conflicts of interest to declare.

Roxadustat (FG4592; ASP1517; AZD9941) in the Treatment of Anemia in Patients with Lower Risk Myelodysplastic Syndrome (LR-MDS) and Low Red Blood Cell (RBC) Transfusion Burden (LTB)

Background: Roxadustat (FG-4592) is an oral hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor that promotes coordinated erythropoiesis through HIF-mediated transcription. Transient, intermittent HIF activation by roxadustat mimics a physiological response that increases endogenous erythropoietin (EPO) production to near physiologic range and stimulates EPO receptor synthesis. In addition, roxadustat promotes iron metabolism by reducing serum hepcidin to allow absorption of iron from the gut and mobilize iron from cellular storage making more iron available for erythropoiesis. Roxadustat corrected and maintained hemoglobin (Hb) in chronic kidney disease patients in multiple Phase 3 trials irrespective of underlying inflammation. In these studies, roxadustat-treated patients required less IV iron to correct and maintain Hb compared to erythropoiesis-stimulating agent (ESA)-treated patients. MDS is characterized by symptomatic anemia and many patients require RBC transfusion and/or ESA treatment. As response to ESAs varies across sub-populations with limited durability, there is an unmet need for treatment of anemia in LR-MDS. This is the first study to evaluate the effect of roxadustat in treating anemia in primary MDS patients. Methods: This is a two-part study; an open-label (OL), dose-finding segment (N=24) followed by a randomized double-blind (DB) placebo-controlled segment (N=156, 3:2 ratio of roxadustat to placebo). The primary goal of the OL segment is to identify the starting roxadustat dose-level for the DB segment. A total 24 patients in the OL segment have been enrolled in three sequential cohorts with 8 patients in each starting dose cohort (1.5, 2.0, or 2.5 mg/kg). RBC transfusion has been allowed per institutional criteria. Eligible patients were very low, low or intermediate risk primary MDS patients based on the International Prognostic Scoring System Revised classification with <5% bone marrow blasts; had baseline Hb < 10.0 g/dL; were >18 years old; and had LTB defined as receiving 1-4 RBC units per 8 week period. Patients were ineligible if they used an ESA within 8 weeks of the study start; had endogenous EPO levels >400 mIU/mL, or had a del(5q) cytogenetic abnormality. Roxadustat was administered TIW with doses titrated every 8 weeks per a dosing algorithm based on Hb response and transfusion need. Data from the OL segment were evaluated to identify the starting dose for the DB segment (currently enrolling). The primary endpoint is transfusion independence (TI) for ≥56 consecutive days during the first 28 weeks of treatment. The proportion of patients who achieve ≥50% reduction in RBC transfusion over any 8 weeks compared to baseline (8 weeks prior to Day 1) and proportion of patients who achieve TI for ≥20 consecutive weeks are evaluated. Safety and tolerability are assessed by adverse event reporting, percentage of patients progressing to acute myeloid leukemia (AML), and clinical laboratory values. Results: Twenty-four transfusion dependent, LR-MDS patients were enrolled in the OL segment of this global Phase 3 trial. Nine patients (38%) achieved TI for at least 56 consecutive days within the first 28 weeks.Three of 9 patients started at 1.5 mg/kg dose, 1 patient started at 2.0 mg/kg dose and 5 patients started at 2.5 mg/kg dose. At the time of achieving TI, 7 of 9 patients (78%) were on 2.5 mg/kg dose, 1 patient (11%) was on 2.0 mg/kg dose (started with 1.5 mg/kg dose) and one patient (11%) was on 1.5 mg/kg dose. Four patients remained TI for >20 weeks (one at 1.5 mg/kg dose level and three at 2.5 mg/Kg dose-level). One additional patient achieved TI after the initial 28-week dosing period at a dose-level of 3.5 mg/kg (starting dose 2.0 mg/kg). A total of 14 patients (58%) achieved a ≥50% reduction in RBC units in any 8-week period compared to baseline (range of 2-4 RBC units in 8 weeks before dosing); 12 of these patients were at ≥ 2.5 mg/kg dose level when achieving a 50% reduction in transfusion without need for IV iron. The overall safety profile observed is consistent with the patient population under study. Six patients reported 8 treatment-emergent SAEs with none being fatal. No patient has progressed to AML. Full results from the OL segment of the study will be presented. Conclusion: Based on the observed response (TI and transfusion reduction) and safety profile in LR-MDS patients, 2.5 mg/kg was selected as the starting dose for the ongoing 156-patient DB portion of the trial. Disclosures Harrup: Cooperative Trial Group for NeuroOncolog: Other: Collaborative Clinical Trials Group; Cancer Council of Tasmania: Membership on an entity's Board of Directors or advisory committees. Mittelman:Novartis: Honoraria, Research Funding, Speakers Bureau. Bradley:FibroGen Inc.: Employment, Equity Ownership. Saha:FibroGen Inc.: Employment, Equity Ownership. Bartels:FibroGen Inc.: Employment, Equity Ownership. Robert:FibroGen Inc.: Employment, Equity Ownership. Yu:FibroGen Inc.: Employment, Equity Ownership.