Treatment Of Myelodysplastic Syndromes Research Articles

Elesclomol-copper synergizes with imidazole ketone erastin by promoting cuproptosis and ferroptosis in myelodysplastic syndromes

Myelodysplastic syndromes (MDS) encompass a collection of clonal hematopoietic malignancies distinguished by the depletion of peripheral blood cells. The treatment of MDS is hindered by the advanced age of patients, with a restricted repertoire of drugs currently accessible for therapeutic intervention. In this study, we found that ES-Cu strongly inhibited the viability of MDS cell lines and activated cuproptosis in a copper-dependent manner. Importantly, ferroptosis inducer IKE synergistically enhanced ES-Cu-mediated cytotoxicity both in vitro and in vivo. Of note, the combination of IKE and ES-Cu intensively impaired mitochondrial homeostasis with increased mitochondrial ROS, MMP hyperpolarized, down-regulated iron-sulfur proteins and declined oxygen consumption rate. Additionally, ES-Cu/IKE treatment could enhance the lipoylation-dependent oligomerization of the DLAT. To elucidate the specific order of events in the synergistic cell death, inhibitors of ferroptosis and cuproptosis were utilized to further characterize the basis of cell death. Cell viability assays showed that the glutathione and its precursor N-acetylcysteine could significantly rescue the cell death under either mono or combination treatment, demonstrating that GSH acts at the crossing point in the regulation network of cuproptosis and ferroptosis. Significantly, the reconstitution of xCT expression and knockdown of FDX1 cells have been found to contribute to the tolerance of mono treatment but have little recovery impact on the combined treatment. Collectively, these findings suggest that a synergistic interaction leading to the induction of multiple programmed cell death pathways could be a promising approach to enhance the effectiveness of therapy for MDS.

Construction and experimental validation of a novel ferroptosis-related gene signature for myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders characterized by morphological abnormalities and peripheral blood cytopenias, carrying a risk of progression to acute myeloid leukemia. Although ferroptosis is a promising target for MDS treatment, the specific roles of ferroptosis-related genes (FRGs) in MDS diagnosis have not been elucidated. MDS-related microarray data were obtained from the Gene Expression Omnibus database. A comprehensive analysis of FRG expression levels in patients with MDS and controls was conducted, followed by the use of multiple machine learning methods to establish prediction models. The predictive ability of the optimal model was evaluated using nomogram analysis and an external data set. Functional analysis was applied to explore the underlying mechanisms. The mRNA levels of the model genes were verified in MDS clinical samples by quantitative real-time polymerase chain reaction (qRT-PCR). The extreme gradient boosting model demonstrated the best performance, leading to the identification of a panel of six signature genes: SREBF1, PTPN6, PARP9, MAP3K11, MDM4, and EZH2. Receiver operating characteristic curves indicated that the model exhibited high accuracy in predicting MDS diagnosis, with area under the curve values of 0.989 and 0.962 for the training and validation cohorts, respectively. Functional analysis revealed significant associations between these genes and the infiltrating immune cells. The expression levels of these genes were successfully verified in MDS clinical samples. Our study is the first to identify a novel model using FRGs to predict the risk of developing MDS. FRGs may be implicated in MDS pathogenesis through immune-related pathways. These findings highlight the intricate correlation between ferroptosis and MDS, offering insights that may aid in identifying potential therapeutic targets for this debilitating disorder.

Abstract 2081: Biomarkers for CLK inhibitor CTX-712 treatment response in myeloid neoplasms: Paving the way toward clinical trials

Abstract Dysregulated RNA splicing is a molecular feature that characterizes almost all tumor types. Splicing is altered in cancer owing to both recurrent mutations and deregulated expression of trans-acting factors that catalyze and regulate splicing. Therefore, splicing can be targeted for the development of new drugs to treat cancers, particularly those with mutations in splicing factors. In myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), genes that encode splicing factors are among those with the highest mutation rates. These mutations are heterozygous and largely mutually exclusive, suggesting the possibility of synthetic lethality due to multiple splicing factor mutations. Such synthetic lethality can be applied as an innovative strategy to develop drugs for the treatment of MDS and AML. We have reported the development of a CLK kinase inhibitor, CTX-712, and evaluated its anti-leukemic activities both in vitro and in vivo. The mechanism of action of CTX-712 involves the suppression of the phosphorylation of multiple SR proteins, including SRSF2/3/4/6, alteration of nuclear speckle morphology, and switching of binding partners of SRSF2. RNA-seq analysis revealed that CTX-712 induced global alterations in splicing, typically resulting in exon exclusion (exon skipping) of cassette exons. Notably, the anti-leukemic effects of CTX-712 positively correlated with the degree of altered splicing, suggesting that CTX-712 inhibits RNA splicing. [Cancer Res (2022) 82 (12_Supplement): 5494., Blood (2022) 140 (Supplement 1): 489-490]. However, patient stratification markers for CTX-712 based on the above mechanism are still undefined. To identify biomarkers for CTX-712, we performed a comprehensive analysis by integrating both wet and dry DNA/RNA sequencing on more than 60 ex vivo primary samples from MDS/AML patients and 23 MDS/AML-derived xenografts (PDX). Unexpectedly, regardless of their splicing factor-mutation status, various types of primary AML cells and PDX models were highly sensitive to CTX-712. Both the primary cells and PDX models showed significant responses to CTX-712 and greater changes in splicing. Additionally, we performed biomarker analysis by comparing gene mutations and RNA splicing patterns between the sensitive and insensitive models and associating them with the anti-tumor effect. We identified several potential biomarkers that can indicate sensitivity to CTX-712. Mechanistic studies that aim to determine why these biomarkers could make cancer more sensitive to splicing perturbation by CTX-712 are ongoing. In conclusion, our results demonstrated a significant effect of CTX-712 on MDS/AML-derived models regardless of their splicing factor-mutation status. Additionally, we present novel biomarkers that could predict sensitivity to CTX-712. Citation Format: Akinori Yoda, Daisuke Morishita, Yotaro Ochi, Akio Mizutani, Hirokazu Tozaki, Yoshihiko Satoh, Takuto Mori, June Takeda, Hideki Makishima, Masahiro Nakagawa, Yasuhito Nannya, Seishi Ogawa. Biomarkers for CLK inhibitor CTX-712 treatment response in myeloid neoplasms: Paving the way toward clinical trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2081.

Abstract 248: HSPA9/mortalin regulates erythroid maturation through a TP53-dependent mechanism in human CD34+ hematopoietic progenitor cells

Abstract Mortalin, encoded by the HSPA9 gene, is a highly conserved heat-shock chaperone belonging to the HSP70 family. It is predominantly presented in the mitochondria, and is critical in regulating a variety of cell physiological functions such as response to cell stress, control of cell proliferation, and inhibition/prevention of apoptosis. Myelodysplastic syndromes (MDS) are a group of hematopoietic stem cell malignancies characterized by ineffective hematopoiesis, increased apoptosis of bone marrow cells, and anemia. Up to 25% of MDS patients harbor an interstitial deletion on the long arm of chromosome 5, also known as del(5q), creating haploinsufficiency for multiple genes including HSPA9. Our prior study showed that knockdown of HSPA9 induces TP53-dependent apoptosis in human CD34+ hematopoietic progenitor cells, consistent with cytopenia observed in MDS patients. Since anemia is another featured symptom of MDS, we hypothesize that HSPA9 plays a role in regulating erythroid maturation. To test our hypothesis, we inhibited the expression of HSPA9 using various methods and measured the erythroid maturation in human CD34+ cells. We used siRNA targeting HSPA9 and found that HSPA9 siRNA significantly inhibited the cell growth, increased cell apoptosis, inhibited erythroid maturation (using CD71 as a surrogate marker), and increased p53 expression (p<0.01) compared to control siRNA in human CD34+ cells. Pharmacologic inhibition of HSPA9 by the chemical MKT-077, an inhibitor of HSP70 protein family members including mortalin, also increased p53 expression and inhibited erythroid maturation in human CD34+ cells. In addition, knockdown of HSPA9 by shRNA showed significant inhibition of erythroid maturation in human CD34+ cells compared to control shRNA. In order to test whether the regulation of erythroid maturation by HSPA9 is TP53-dependent or not, we constructed shRNAs targeting TP53 genes and simultaneously transduced lentivirus containing shRNAs targeting HSPA9 and TP53 respectively in human CD34+ cells using double antibiotics selection (puromycin for shRNA targeting HSPA9 and hygromycin for shRNA targeting TP53). We found that TP53 knockdown partially rescued the erythroid maturation defect caused by HSPA9 inhibition, suggesting that erythroid maturation inhibition by HSPA9 knockdown is partly mediated through a TP53 mechanism. Collectively, our results suggest that the increased apoptosis and reduced erythroid maturation observed in del(5a)-associated MDS is TP53-dependent. HSPA9/mortalin may be a potential target to treat anemia in del(5q) MDS patients, although simultaneous loss of multiple genes on del(5q) likely contributes to the complex phenotypes observed in MDS. Thus, our study not only uncovers some underlying mechanisms of del(5q) MDS, but also provides potential therapeutic indications through gene targeting in clinical MDS treatment. Citation Format: Tuoen Liu, Christopher Butler, Morgan Dunmire, Gabor Szalai, Anissa Johnson, Jaebok Choi, Matthew Walter. HSPA9/mortalin regulates erythroid maturation through a TP53-dependent mechanism in human CD34+ hematopoietic progenitor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 248.

Impaired binding affinity of YTHDC1 with METTL3/METTL14 results in R-loop accumulation in myelodysplastic neoplasms with DDX41 mutation.

DEAD box helicase 41 (DDX41) mutations are the most prevalent predisposition to familial myelodysplastic syndrome (MDS). However, the precise roles of these variants in the pathogenesis of MDS have yet to be elucidated. Here, we discovered a novel mechanism by which DDX41 contributes to R-loop-induced DNA damage responses (DDR) in cooperation with the m6A-METTL complex (MAC) and YTHDC1 using DDX41 knockout (KO) and DDX41 knock-in (KI, R525H, Y259C) cell lines as well as primary samples from MDS patients. Compared to wild type (WT), DDX41 KO and KI led to increased levels of m6A RNA methylated R-loop. Interestingly, we found that DDX41 regulates m6A/R-loop levels by interacting with MAC components. Further, DDX41 promoted the recruitment of YTHDC1 to R-loops by promoting the binding between METTL3 and YTHDC1, which was dysregulated in DDX41-deficient cells, contributing to genomic instability. Collectively, we demonstrated that DDX41 plays a key role in the physiological control of R-loops in cooperation with MAC and YTHDC1. These findings provide novel insights into how defects in DDX41 influence MDS pathogenesis and suggest potential therapeutic targets for the treatment of MDS.

Clinical significance of IL-18 and IL-18-binding protein in bone marrow of patients with myelodysplastic syndrome

Objective: To analyze the level and clinical significance of IL-18 and IL-18-binding protein (BP) in the bone marrow of patients with myelodysplastic syndrome (MDS) . Methods: A total of 43 newly diagnosed patients with MDS who were admitted to the Department of Hematology, Tianjin Medical University General Hospital, from July 2020 to February 2021 were randomly selected. The control group consisted of 14 patients with acute myeloid leukemia (AML) and 25 patients with iron-deficiency anemia (IDA). The levels of IL-18 and IL-18 BP in the bone marrow supernatant were measured, and their correlations with MDS severity, as well as the functionality of CD8(+) T cells and natural killer cells, was analyzed. Results: The levels of IL-18, IL-18 BP, and free IL-18 (fIL-18) in the bone marrow supernatant of patients with MDS were higher than in the IDA group. The level of fIL-18 was linearly and negatively correlated with the MDS-International Prognostic Scoring System (IPSS) score. IL-18 receptor (IL-18Rα) expression on CD8(+) T cells in the MDS group was lower than in the IDA group, and the levels of fIL-18 and IL-18Rα were positively correlated with CD8(+) T-cell function in the MDS group. Conclusion: IL-18 BP antagonizes IL-18, leading to a decrease in fIL-18 in the bone marrow microenvironment of patients with MDS, affecting CD8(+) T-cell function, which is closely related to MDS severity; therefore, it may become a new target for MDS treatment.

DNA methylation profiling of myelodysplastic syndromes andclinical response to azacitidine: A multicentre retrospective study.

Real-world data have revealed that a substantial portion of patients with myelodysplastic syndromes (MDS) does not respond to epigenetic therapy with hypomethylating agents (HMAs). The cellular and molecular reasons for this resistance to the demethylating agent and biomarkers that would be able to predict the treatment refractoriness are largely unknown. In this study, we shed light on this enigma by characterizing the epigenomic profiles of patients with MDS treated with azacitidine. Our approach provides a comprehensive view of the evolving DNA methylation architecture of the disease and holds great potential for advancing our understanding of MDS treatment responses to HMAs.

Diagnosis and Treatment of Myelodysplastic Syndrome in the Era of Genetic Testing

Myelodysplastic syndrome (MDS) is a heterogeneous disorder with diverse prognoses influenced by cytopenias, genetic variants, and myeloblast proportions in the bone marrow. Accurate prognosis prediction and tailored treatment plans are essential. The International Prognostic Scoring System-Molecular (IPSS-M), which additionally reflects the impact of MDS-related genetic mutations to the clinical and laboratory information, is anticipated to offer superior prognostic accuracy compared to existing systems like the Revised International Prognostic Scoring System (IPSS-R). Despite its statistical complexity, its web-based calculation and ease of discussing results with patients using intuitive data sets provide notable advantages. Progress in MDS treatment, exemplified by effective anemia correction with an erythropoiesis-maturation agent in <i>SF3B1</i>-mutated cases and efforts to refine poor prognoses in <i>TP53</i>-mutated cases, reflects the evolving landscape of genetic-based interventions in MDS. Advancements in genetic diagnostic technology, combined with enhanced knowledge of the bone marrow niche, are anticipated to lead to significant improvement in MDS treatment outcomes in the future.

Effects of Black Raspberry Supplementation on Methylation Pathways in Vav-creAsxl1fl/flTet2fl/fl Double Knockout Mice with Early-stage Myelodysplastic Syndrome.

Myelodysplastic syndromes (MDS) are a subset of myeloid malignancies defined by clonality of immature hematopoietic stem cells that leads to faulty blood cell development. These syndromes can lead to an increased risk of infection and may transform into acute myeloid leukemia, making it critical to determine effective treatments for the condition. While hypomethylating agents such as azacitidine and decitabine, as well as stem cell transplants, have been delineated as favored treatments for MDS, not all patients are physiologically receptive to these treatments. However, black raspberries (BRBs) have been shown to exert hypomethylating effects in various malignancies, with minimal adverse effects and thus a broader range of potential candidacies. This study aimed to investigate the potential of BRBs to exert such effects on MDS using Addition of Sex Combs Like/Tet Methylcytosine Dioxygenase 2 (Asxl1/Tet2) double knockout mice (Vav-cre Asxl1fl/fl Tet2fl/fl), which typically manifest symptoms around 25 weeks of age, mirroring genetic mutations found in humans with MDS. Following a 12-week dietary supplementation of Vav-cre Asxl1fl/fl Tet2fl/fl mice with 5% BRBs, we observed both hyper- and hypomethylation at multiple transcription start sites and intragenic locations linked to critical pathways, including hematopoiesis. This methylation profile may have implications for delaying the onset of MDS, prompting a need for in-depth investigation. Our results emphasize the importance of exploring whether an extended BRB intervention can effectively alter MDS risk and elucidate the relationship between BRB-induced methylation changes, thus further unlocking the potential benefits of BRBs for MDS patients.

How to classify risk based on clinical and molecular modeling: integrating molecular markers in the risk assessment of myelodysplastic syndrome.

Myelodysplastic syndrome (MDS), also known as "myelodysplastic neoplasm," is a heterogeneous group of clonal myeloid neoplasms that typically affects older adults. The clinical phenotype, symptoms, and complications relate to the depth of cytopenia and progression to acute myeloid leukemia (AML). The diagnosis of MDS relies on morphologic criteria, such as evidence of dysplasia, disordered maturation, and increasing blast counts, which separate the disease into histologic subtypes with different probabilities for progression to AML. The treatment of MDS is often risk-adapted depending on the prognostic profile of each patient's disease. There has been a coevolution of diagnostic and prognostic systems for MDS developed over the past 40 years, both of which have now incorporated molecular markers. The new International Prognostic Scoring System-Molecular (IPSS-M) improves partitioning of patients compared to prior versions with resultant upgrading of 34% of patients into higher-risk groups due to the presence of mutations. The new IPSS-M also more accurately distinguishes intermediate-risk patients separating them into two tiers. The two new diagnostic classifications include MDS defined by mutations in SF3B1 and TP53, though there are differences in diagnostic criteria. Future efforts to refine MDS prognostication could investigate the interface between MDS and clonal cytopenia of undetermined significance, expand access to genomic testing, obtain results in a less invasive manner, and develop treatment-response predictors and dynamic risk models.

Azacitidine Treatment in MDS: A Systematic Literature Review and Meta-Analysis Comparing the Efficacy of Real World Data with Randomized Controlled Trials

Background: Myelodysplastic Syndromes (MDS) are a group of myeloid malignancies affecting the hematopoietic stem and progenitor cells and occurring mainly in elderly individuals. Two hypomethylating agents (HMA), Azacitidine (AZA) and Decitabine (DAC), have shown efficacy in treatment of MDS patients and received approval based on randomized controlled trials (RCTs) more than a decade ago. Since then, many publications have reported real world experience on HMA treatment in MDS patients, which showed a reduced efficacy of HMA than the RCT. The reasons for this difference have been extensively discussed but a systematic review never performed. Aim: We performed a systematic literature review and meta-analysis to compare the efficacy of real world data with randomized controlled trials and to identify potential cofactors for different outcomes. Material and Methods: We performed a systematic literature search from observational studies (OBSTs) describing real world experience of AZA treatment in MDS patients using Embase, Ovid Medline, and Web of Science databases. Patient characteristics, IPSS/IPSS-R, and outcome (response rates, overall survival) were collected from OBSTs that were performed between 2003 and 2023. Kaplan-Meier curves with log-rank tests were used to assess differences in survival outcomes. An inverse variance weighted meta-analysis was performed by using restricted mean survival times or proportions (response rate outcome). The restricted mean survival time and the standard error at 12, 24 and 36 months was calculated by using the R package survival. The pooled effect and prediction intervals from random effect models were reported. Results: In our systematic literature search, data from OBSTs describing real world experience of AZA treatment in MDS patients could be extracted and compared to the only RCT published with AZA (Fenaux P et al., Lancet Oncology 2009). Patient and disease characteristics were similar for AZA-RCT and OBST (respect of gender ratio, median age, IPPS/IPSS-R). Restricted mean survival time and standard error of the OBSTs and AZA-RCT showed no significant difference for the time points at 12 months (10.0 [95%-CI: 9.6; 10.3] months (OBSTs) vs 10.0 [95%-CI: 9.5; 10.5] months (AZA-RCT); figure part A) and 24 months (15.5 [95%-CI: 14.6; 16.5] months (OBSTs) vs. 16.9 [95%-CI: 15.7; 18.2] months (AZA-RCT); figure part B). At 36 months, the restricted mean survival time of OBSTs was 18.7 [95%-CI: 17.3; 20.1] months compared to 22.3 [95%-CI: 20.2; 24.5] months of the AZA-RCT (figure part C), which was different in favor of the RCT. Using random effect models, the AZA treatment response rate was 0.51 [95%-CI: 0.47; 0.56] for the OBSTs and 0.49 [95%-CI: 0.42; 0.57] for the AZA-RCT (figure part D). Conclusion: Compared to AZA-RCT data, restricted mean survival time (for time points 12 months and 24 months) and response rate of OBSTs describing real world data of AZA treatment in MDS patients are comparable. Regarding the observation time point at 36 months, the lower restricted mean survival time of the OBSTs compared to AZA-RCT may be most likely explained by study patient selection bias.

Venetoclax and Azacytidine Treatment for High Risk Myelodysplastic Syndromes and Chronic Myelomonocytic Leukemia As a Bridge Therapy to Transplant. a GESMD Study

Introduction Allogeneic hematopoietic cell transplantation (alloHCT) is the only potentially curative treatment for myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML). Most common approach to bridge higher-risk MDS to alloHCT is to reduce tumor burden before alloHCT with azacytidine (AZA). Currently with this strategy less than 50% of patients achieve overall response rate (ORR) and less than 20% complete response (CR). Moreover, it is associated with a considerable rate of drop outs (approximately 50%) due to progression disease and mortality prior to alloHCT. Therefore, we need more effective treatments with faster and higher responses to facilitate more patients to undergo alloHCT. Venetoclax with AZA (VEN/AZA) is being investigated mostly in unfit MDS patients, it has shown higher ORR and early responses. Recently, a study suggested promising activity in 13 high-risk MDS patients who received VEN/AZA and proceeded to alloHCT (Komrokji et al, BCJ, 2022). The objective of our study is to analyze the feasibility, toxicity, and efficacy of VEN/AZA treatment in MDS and CMML patients who are intended to undergo alloHCT. Methods This is a retrospective multicenter study that includes high-risk MDS and CMML patients treated with VEN/AZA who are intended to undergo alloHCT. The response rate was assessed according to the IWG 2006, IWG 2023, ELN 2022. Savona criteria were used in CMML. Results A total of 23 patients were included, with a median age of 61 years (41-74), including 19 MDS and 4 CMML patients. Eight patients received VEN/AZA in a relapsed/refractory (R/R) setting, while 15 in first line (1L). Most patients received a 14-day scheme of VEN (n=16). The administration of VEN/AZA and other characteristics are described in Table 1. Grade 3/4 cytopenias were frequent (83% neutropenia 65% thrombocytopenia, 59% anemia), 17% experienced febrile neutropenia and 17% required hospitalization. G-CSF was used in 44% of patients, antifungal prophylaxis in 43%, and cycle delays due to cytopenias happened in half of them (47%). Thirty-day mortality was 0%, and one patient (4%) died within the first 60 days due to disease progression. The response rate following IWG 2023 criteria was: 7 CR (30%), 6 CR with limited count recovery (CR L, 26%), 4 CR with partial hematologic recovery (CRh, 17%), 3 partial remissions (PR, 13%), 1 hematologic improvement (HI, 4%), and 2 no response (9%). The ORR was 91.3% (21/23), and the rate of composite complete responses (CRc) including CR, CR L and CRh was 73.9% (17/23) . These responses occurred rapidly, all patients achieved the first response in 1 cycle and the median to best response was 1 cycle (range 1-3). In R/R patients the ORR was 87% and the CRc was 62%. Using the ELN 2022 versus IWG 2023 response criteria results in similar CRc, higher rate of CR (34% vs 22%) and lower ORR (86 vs 91%). The median follow-up since the start of treatment was 13.6 months (95% CI: 5.2-21). The 2-year overall survival (OS) was 63% (95% CI ± 24%). Currently, there are 2 patients awaiting alloHCT. Among the 21 evaluable patients, 19 underwent alloHCT, resulting in a alloHCT rate of 91%. The 2 patients who were not transplanted included one CMML patient with disease progression prior alloHCT and one refractory MDS. AlloHCT was performed with a median of 3 prior cycles (range: 1-9), and the median time from the start of VEN/AZA to transplantation was 4 months (range: 2.5-10). One patient who did not respond to VEN/AZA was treated with chemotherapy prior alloHCT achieving CR. Two patients progressed before alloHCT, one achieved CR after chemotherapy and the other one has received a sequential alloSCT. The two-year post-transplant OS was 67% (95% CI ± 28%) (Figure 1). Four patients relapsed after alloHCT, resulting in a two-year cumulative incidence of relapse (CIR) of 37% (95% CI ± 28%). One of these patients was treated with VEN/AZA post-transplant and he is currently in CR for 1.5 years. 2-year transplant-related mortality was 6% (95% CI ± 12%). Conclusion Treatment with VEN/AZA as a bridge to alloHCT in MDS and CMML patients is feasible with a high rate of patients undergoing alloHCT. It provides high and quick responses, even in R/R patients, with low treatment-related toxicity. Funding This study was supported by grants from the Institute of Health Carlos III (ISCIII), with grant numbers PI19/01476 and PI22/01660, co-financed by the European Union. Additional support was provided by Emili-Letang (HCPB).

Decitabine in Combination with Aclacinomycin, Cytarabine and Granulocyte Colony-Simulating Factor (CAG) Had a High Response Rate in Children with Advanced Myelodysplastic Syndrome and Acute Myeloid Leukemia with Myelodysplasia-Related Changes

Advanced myelodysplastic syndrome (MDS) is a rare disease in children with poor prognosis. Allogeneic transplantation is the only curative option. However, there has been no established treatment prior to transplant. We conducted a retrospective study exploring the response rates and safety of decitabine combined with aclacinomycin, cytarabine and granulocyte colony-stimulating factor (CAG) for induction treatment of advanced MDS and acute myeloid leukemia with myelodysplasia-related changes (MDR-AML) in children. Patients from 1 to 14 years administrated in Pediatric Blood Disease Center of CAMS Blood Disease Hospital between January 1the 2019 and December 31th 2022 were screening. All diagnosed with MDS-EB or MDR-AML according to WHO 2016 criteria and treated with decitabine combined with CAG were identified. The efficacy of one-cycle treatment of MDS-ED was evaluated based on The International Working Group Response Criteria 2006. The response for MDR-AML was evaluated according to the criteria for AML published by ELN. The response of MDR-AML was defined as complete remission (CR) or CR with incomplete count recovery (CRi). The response of MDS-EB was defined as CR or bone marrow CR with hematological improvement (HI). A total of 13 patients, including MDS-EB (n=7) and MDR-AML (n=6), were enrolled for analysis. 5-day decitabine was administrated, combined with modified CAG regimen for 7 days. Eventually 12 patients of those were available for evaluation. For patients with MDS-EB, four achieved CR and three reached marrow CR with HI. Four patients with MDR-AML, three achieved CR and two reached CRi. The overall response rate was 100%(12/12). The most frequent adverse events were myelosuppression and suppression-induced infections, but no infection-related mortality was recorded. There had been no cardiotoxic event happened, either. Only one patient experienced fatal bleeding in early stage after treatment. The median follow-up time was 16 months (range 1-48). At last follow-up, 10 patients who had underwent allo-HCT were alive without disease. The remaining 2 patients died of disease progression. The 2-year estimated overall survival was 62.9%. Decitabine combined with modified CAG had a high response rate for children with advanced MDS and MDR-AMl, and it can bridge patients to curative allogeneic transplantation.

Impaired Efficacy and Durability of Hematopoietic Stem Cell Engraftment Caused By Samd9l-Deficiency within Bone Marrow Niches

Germline mutations in the SAMD9 and SAMD9L (SAMD9/L) genes on chromosome 7q drive both childhood myelodysplastic syndrome (MDS)/secondary acute myeloid leukemia (sAML) associated with monosomy 7, and adult-onset MDS/sAML associated with distinct somatic genetic alterations. The ortholog for SAMD9/L in mice is Samd9l. Gain of Function (GoF) and Loss of Function (LoF) Samd9l alleles predispose to MDS at distinct ages. LoF models show that Samd9l-deficient mice, like humans, are more likely to develop MDS during adulthood, while GoF models result in early onset bone marrow (BM) dysfunction and MDS/sAML. Hematopoietic stem cell transplantation (HSCT) is the only curative treatment for bone marrow failure (BMF), MDS, and AML arising from germline genetic syndromes. Our previous research demonstrated that deficits in host BM microenvironment (ME) niches contribute to hematopoietic failure and poor HSC engraftment in murine models of many MDS predisposition syndromes associated with BMF. As we have observed clinical cases where poor engraftment impaired outcomes in patients with SAMD9/L mutations, we recently hypothesized, BM niche functions critical to facilitating HSC engraftment may be impaired due to niche-intrinsic deficits caused by SAMD9/L mutations. Herein, we present data testing this hypothesis, demonstrating Samd9l deficiency alters BM ME composition, extracellular matrix (ECM) regulation, and proinflammatory cytokine signaling resulting in impaired donor HSC engraftment after HSCT. We performed primary (1 ry) and secondary (2 ry) HSCT to model impacts of SAMD9/L deficiency on BM niche function during HSCT using WT ( Samd9l+/+), Samd9l+/-, and Samd9l-/- mice as HSCT recipients of healthy WT GFP + donor BM to measure the capacity of Samd9l LoF niches to engraft donor HSCs. To assess changes in niche composition and gene expression during HSCT, flow cytometry, multiplex ELISA, and RNASeq analysis were performed on WT, Samd9+/- and Samd9-/- mice. Compared to WT control niches, Samd9+/- and Samd9-/- niches exhibited impaired efficiency of donor HSC engraftment at 3 weeks post-1 ry HSCT, as measured by competitive 2 ry-HSCT performed with 1 ry recipient BM harvested at this timepoint. Beyond 30 weeks post-2 ry-HSCT in this model, GFP + reconstitution of GR1 + myeloid, RBC's, and platelets was reduced by 25%, 18%, and 35% in 2 ry recipients of Samd9+/-1 ry recipient BM and by 26%, 28%, and 32% in 2 ry recipients of Samd9-/- 1 ry recipient BM. Durability of HSC engraftment by 8 months was even more significantly impaired in Samd9+/- and Samd9-/- niches, with Samd9-/- mice exhibiting low survival by this timepoint. Competitive 2 ry HSCT performed 8 months post-1 ry HSCT showed reduction in trilineage GFP + blood cells out to 24 weeks post 2 ry-HSCT, including reductions in GFP + Gr1 + myeloid cells by 50% and 83% in 2 ry recipients of Samd9l+/-and Samd9l-/-1 ry recipient BM, respectively, compared to control 1 ry recipient BM. Similar reductions in BM long-term HSC and Lin -Sca-1 +cKit + HSC/progenitor cells in 2 ry recipients of Samd9l-/-compared to Samd9l+/+ 1 ryrecipient BM 8 months post 1 ry-HSCT. Following TBI, Samd9l+/-and Samd9l-/- BM niches contained similar percentages of osteoprogenitors, mesenchymal stem cells, and endothelial cells compared to WT controls. Multiplex ELISA on BM plasma collected 48hrs post-TBI revealed that Samd9l+/-and Samd9l-/-deletion in the BM ME led to markedly reduced expression of IFN-γ (43% and 55% reduction in Samd9l+/-and Samd9l-/-), Eotaxin (33%/36%), GM-CSF (47%/47%), and IL-1a (48%/65%). In contrast, expression of niche cell extracts showed markedly increased levels of IL-17 (4 fold) and CXCL11 (1.4 fold) in Samd9l+/-and of IL-17 (5.3 fold) IL-12p70 (1.3 fold), and CXCL11 (1.5 fold) in Samd9l-/-, suggesting that aberrantly active Th17 responses caused by niche Samd9l deficiency may be responsible for impairing HSC function and maintenance post-HSCT. RNASeq analysis showed downregulation of ECM genes in both Samd9l-/-and Samd9l+/-models. Our data demonstrates BM niche function is impaired by Samd9l deficiency in a gene dosage-dependent manner. These deficits are associated with increased expression of a specific inflammatory cytokine pathway, suggesting that aberrant growth factor receptor recycling due to Samd9l deficiency may drive stress hematopoiesis and alter HSC cell fate decisions leading to failure of durable donor HSC maintenance post-HSCT.

Improved Quality of Life upon Shifting from Darbepoetin to Luspatercept in Patients with Low-Risk Myelodysplastic Syndrome (MDS)

Introduction: Low-risk MDS is characterized by refractory anemia due to Dyserythropoiesis 1. Standard of care treatment is mainly Erythropoiesis stimulating agents (ESA) or blood transfusion or both 1. Response to ESA is achieved in some patients whose erythropoietin (EPO) level is less than 500 mu/ml 2. Luspatercept is a red cell maturing agent that may lead to an increase in Hb of up to 3 g/dL 3 in responders. Response to ESA can be enhanced with the addition of Luspatercept 4. Hereby we are reporting successful shifting of two patients from Darbepoietin to Luspatercept with significant increase in Hb level and improvement in quality of life. Case 1: A 62-year-old man without previous illness presented in April 2018 with progressive symptomatic anemia. His body weight was 107 kg, and his physical examination was unremarkable other than pallor and tachycardia. The laboratory investigation revealed WBC, 5.62 x 10 3/uL, RBC 2.2 x 10 3/uL, Hb 7.8 g/dL, MCV 110.4 fl, platelets 706 x 10 3/uL, and reticulocytes 1%. Blood film showed reduced RBC count with numerous macrocytes and no other abnormalities. Bone marrow revealed 80% cellularity with Dyserythropoiesis, 16% ring sideroblasts, and 2% blasts. The Ferritin level, B12 levels were elevated (table). EPO level was elevated but less than 500 mlU/ml and the cytogenetic analysis showed normal karyotype. However his molecular profiling demonstrated SF3B1 and TET2 mutations. At the time of diagnosis, the Revised International Prognostic Scoring System (IPSS-R) was 2.0, indicating low risk MDS. He was given darbepoetin 300 mcg SC weekly for four weeks without any improvement, then darbepoetin dose was escalated to 500 mcg weekly with a good response. Hb level remained at 8.5 - 9.5 g/dl for three years. The patient's physical performance improved moderately, with an increase in walking distance on flat surfaces from 20 to 35 minutes. In August 2022, he was switched to Luspatercept 100 mcg SC on a three-weekly schedule. Within three months, his hemoglobin level increased to 10.8 g/dl and he experienced significant improvement in physical performance and mental well-being, along with a significant reduction in fatigue, enabling him to increase his walking distance to 76 minutes. Case 2: A 77-year-old man with type 2 diabetes mellitus and mild renal impairment who developed an unexplained progressive anemia. His body weight was 79 kg, and his physical examination was unremarkable other than pallor. His laboratory investigations showed WBC 4.5 x 10 3/uL, RBC 1.5 x 10 6/uL, Hb 5.2 g/dL, MCV 96 fl, platelets 706 x 10 3/uL, and reticulocytes 0.8 %. Blood film showed reduced RBC count, normocytic anemia, and no other abnormalities. Bone marrow revealed 60% cellularity with Dyserythropoiesis, 17% ring sideroblasts, and 1% blasts. His baseline serum creatinine was 1.8 mg/dl, and HbA1C 10.0. Two units of packed red blood cells were transfused every 2- 4 week to keep his Hb level around 8 g/dL. Darbepoetin 80 mcg SC weekly for four weeks,150 mcg weekly for 4 weeks and 300 mcg weekly for further 4 weeks without any response. Eventually, he responded to 500 mcg of Darbepoetin per week. As a result, his Hb increased to the range of 8.6-9.4 g/dl for one year, with moderate improvements in performance status. Darbepoetin was switched to Luspatercept 75 mcg three-weekly in January 2023, resulting in an increase in his hemoglobin level to 10.3 g/dl, with significant improvement in his energy level and overall well-being. Discussion: Blood transfusion and ESA have traditionally been used as standard treatments for low-risk MDS. Recently, Luspatercept in combination with ESA has shown enhanced effectiveness in elevating Hb levels. Both of our patients had significant increases in Hb levels along with a noticeable improvement in quality of life because of Luspatercept alone. Conclusion: Following the switch from Darbepoetin to Luspatercept, there was a significant improvement in Hb level and in quality of life in our 2 patients. This finding needs to be confirmed with larger studies.

Community-Based Practice Patterns and Perceptions of Challenges Integrating New Therapies for High-Risk Myelodysplastic Syndrome

BACKGROUND : With a limited number of approved therapies for myelodysplastic syndrome (MDS), first-line treatment options for patients with high-risk MDS remain an unmet need. As many patients can become refractory to treatment or relapse, it is imperative that they receive the most suitable and effective therapy upfront based on their risk stratification. Several treatment strategies, including immunotherapy, are being investigated and have shown encouraging results with the potential to transform care for this patient population. To create effective education on new therapies for MDS, a two-phase educational initiative was designed. METHODS : The first phase consisted of a deep-dive survey evaluating current practice patterns, barriers, attitudes, and challenges in management of newly-diagnosed high-risk MDS which was launched in February 2023 and closed in March 2023. The survey was disseminated via a partnership with IQVIA with criteria for inclusion set as: treating physicians confirmed by ICD-10 code matching and at least one prescription for any pharmaceutical product for MDS during the prior 3 months from survey launch. The second phase of the initiative was a live and endured CME-certified activity led by two experts in MDS. This was hosted on OMedLive.com and remains on-demand through June 2024. Microlearning clips were disseminated via LinkedIn. The expert panel addressed the identified challenges from the survey results and discussed practice-changing clinical data. Knowledge and competence questions were administered pre-, immediate post-activity, and during the program as interactive polling. Attitude, perceptions, and practice patterns associated with management of MDS were also assessed. RESULTS : The phase one survey was completed by 23 hematologists/oncologists currently treating patients with MDS. The insights from the survey showed a preference for use of the IPSS-R classification system (96%); however, only 52% ‘always’ utilize a risk stratification scoring system. The top three factors influencing treatment decisions for newly-diagnosed MDS were: molecular and genetic prognostic factors (78%), comorbidities (70%), and long-term evidence/real-world data (48%). Across the continuum of care, the most challenging treatment setting was identified as intermediate/high-risk relapsed/refractory MDS. The top three challenges were requirements to follow specific treatment algorithms, access to investigational agents in clinical trial, and managing patients relapsed or are refractory to approved therapies. Clinical perceptions of emerging therapies revealed that 17% of survey respondents would not consider an investigational therapy for a newly diagnosed high-risk patient. To date, 1,371 clinicians have participated in the educational initiative, 91% of whom were physicians and 89% identified their specialty as hematology/oncology. Low baseline knowledge and competence (<40%) was observed across assessment questions. Insights gathered throughout the activity indicated intermediate/low and intermediate/high as the MDS risk categories that are most difficult to manage. Mixed reports of classifications systems was observed with IPSS-R as the highest at 35%, IPSS reported by 28%, and IPSS-M used by 20%. Participants reported equivalent difficulty in treatment of lower vs higher-risk MDS and newly diagnosed vs relapsed/refractory MDS. Following the educational activity, intended practice changes were reported as applying the latest guidelines, improve referral of patients, and refine treatment/management approaches. CONCLUSIONS: This initiative provided insights into provider current practices and perceptions on management of newly diagnosed high-risk MDS. These adequately informed the educational initiative with some differences in perceptions seen post activity compared with the survey analysis from phase one. There are gaps remaining for clinicians to align on use of risk stratification scoring systems and improve algorithmic approaches based on treatment setting and risk status. Educational interventions are needed to address these practice gaps, employ strategies to effectively integrate new therapies, and stay abreast of the rapidly changing MDS landscape.

Risk Factors Associated to Severe Episodes of Infection in Myelodysplastic Syndromes

INTRODUCTION. Mortality differs according to the Revised International Prognostic Scoring System (IPSS-R) risk categories in patients afflicted with myelodysplastic syndromes (MDS). Historically, high-risk (HR)-MDS (IPSS-R >3.5 points) is related to death caused by progression to acute myeloid leukemia (AML), while low-risk (LR)-MDS is associated with mortality due to severe infections (Nachtkamp K, et al. 2016), probably caused by neutrophile dysfunction and hindered oxidative burst. Studies of causes of death (COD) in MDS are scarce. A MD Anderson study in LR-MDS patients identified infections as the leading COD, followed by progression (Dayyani F, et al, 2010), while the Düsseldorf registry recognized infections as the main COD across all MDS categories (Neukirchen, J. et al, 2011). Considering that information in low- and medium-income countries is especially limited, this study aims to describe the characteristics and identify the risk factors for severe infectious events in patients afflicted by MDS in a third-level center in Mexico. METHODS. In this single-center retrospective study, we analyzed severe infection episodes, associated risk factors, and the COD in MDS patients.We included patients diagnosed with MDS from January 2000 to December 2021, according to the 2016 WHO criteria. We registered baseline characteristics and described the episodes of severe infection, defined as hospitalization events due to infectious diseases complicated with septic shock and/or respiratory failure requiring orotracheal intubation. We conducted uni- and multivariate analyses based on the following variables: neutropenia (≤0.5x10 9/L), monocytopenia (≤0.2x10 9/L), lymphopenia (≤1.0x10 9/L), ferritin (≥1,000ng/mL), autoimmune disease, MDS comorbidity index, COPD, granulocyte dysplasia, and treatment with azacytidine or pharmacological immunosuppression. RESULTS. We included 156 patients with MDS treated at our institution. Median age at diagnosis was 65 years (IQR; 55-75 years), 53% of which were women. Multilineage dysplasia was the most prevalent MDS type (59%), followed by excess blasts type 2 (11.5%). LR-MDS represented 68% (n=106), while HR-MDS, 32% (n=50). Hypocellularity in bone marrow biopsy was found in 24.4%. Eighty-seven patients developed 156 infectious episodes, 64.7% of which were severe. A median of 1 serious event (IQR: 0.75-1 event) occurred in each patient, with a median time to diagnosis of 13 months (IQR: 3.5-34.5 months). Infection sites and etiologies are described in Figure 1. Of note, we identified 14 fungal infections, 8 of them by Aspergillus, with a 37.5% death rate. Risk factors associated with increased severe infection risk were neutropenia during follow-up (OR 3.2; 95% CI 1.5-6.4; p=0.001), neutrophil hypogranularity at diagnosis (OR 2.1; CI 95% 1.1-4.1; p=0.029), hyperferritinemia (OR 2.46; CI 95% 1.2-4.9; p=0.011), and azacitidine treatment (OR 3.1; CI 95% 1.01-9.6; p=0.047). Fifty patients (32%) died. In both HR-MDS and LR-MDS groups, the main COD was infection (13 in HR group, 48.1%, vs 10 in LR group, 43.5%; p=0.782), followed by progression to AML (3 in HR group, 11.1%, vs 4 in LR group, 21.7%; p=0.444). Median overall survival was lower in patients with severe neutropenia (62 vs. 210 months; p=0.009) and hyperferritinemia (62 months vs. non-reached, p<0.001) (Figure 2). CONCLUSIONS In our MDS population severe infections represent the leading COD, regardless of risk category. More than half developed at least one infectious episode, 64.7% of which were severe. Fungal infections were highly lethal, suggesting antifungal prophylactic interventions could prove helpful. We found neutropenia during follow-up and hypogranularity at diagnosis to be significant risk factors, the latter perhaps reflecting neutrophil disfunction which should be further evaluated. Hyperferritinemia's association with increased infection and mortality urges to further understand its role in MDS pathophysiology, and could potentially prove a therapeutic target in infections disease prevention. Additionally, there is currently no consensus on infectious disease for use of azacitidine, which might add to a worsening risk of infection.

Combination Therapy with Luspatercept and the Ferroportin Inhibitor Vamifeport Is Superior to Either Drug Alone in Improving Anemia and Reducing Myeloid Skewing in MDS

Background: Patients with myelodysplastic syndromes (MDS) are prone to develop iron overload as a consequence of ineffective erythropoiesis and chronic transfusion therapy. Although iron overload is a common feature in MDS, it has remained unclear whether and how iron excess is detrimental for MDS pathophysiology. Recently, we have shown that iron restriction by the oral ferroportin (FPN) inhibitor vamifeport improves anemia and reduces myeloid skewing, being of benefit for MDS as single treatment. Luspatercept is a recombinant fusion protein acting as TGF-β superfamily ligands trap which has recently gained FDA approval for the treatment of anemia in transfusion-dependent low-risk MDS patients. Luspatercept promotes erythropoietin (EPO)-independent maturation of late-stage erythroid cells, resulting in increased hemoglobin and red blood cells (RBCs) count. However, this drug - due to a lack of improvement in myeloid skewing and AML evolution - is considered a non-disease-modifying therapy. Aims: In this study we investigated the effect of a combined therapy using luspatercept and the FPN inhibitor vamifeport in a preclinical MDS mouse model, with the hypothesis that both drugs could have additive effects with further benefit for the disease. Methods: To this end, we administered the FPN inhibitor vamifeport in NUP98-HOXD13 MDS mice for 3 months (0.5 mg/ml into drinking water), combined with luspatercept for the last 2 months (5 mg/kg twice a week, subcutaneous injection), starting at 3 months of age. Mice receiving the combined treatment were compared to those untreated or receiving single treatments. Results: At steady-state MDS mice develop anemia, neutropenia and lymphopenia, together with an iron overload phenotype, hallmarked by inappropriately low hepcidin levels, elevated serum iron and transferrin saturation, non-transferrin-bound iron (NTBI) formation and tissue iron deposition. Vamifeport administration in MDS mice reduced serum iron and NTBI formation and prevented tissue iron loading, either as a single or combined treatment with luspatercept. Luspatercept and vamifeport, as single-agent treatment, ameliorated anemia and the maturation of bone marrow and splenic RBCs in MDS mice. The combined therapy further improved anemia compared to the treatments with either drugs alone, as suggested by the higher hemoglobin levels, hematocrit and RBC count, and the decreased number of early erythroid precursors, sign of more effective erythropoiesis. Importantly, vamifeport, but not luspatercept, significantly altered myeloid skewing in MDS, revealing a major role of the iron status in myeloid expansion. Myeloid bias, monitored as percentage of CD11b + Gr1 + myeloid cells in the bone marrow was attenuated by iron restriction in vamifeport-treated MDS mice compared to control and luspatercept-treated MDS animals. The combined treatment merkedly reduced myeloid skewing in MDS mice, similarly to the single vamifeport treatment. Furthermore, the number of immature cKit + myeloid blasts in the peripheral blood of MDS animals were decreased by vamifeport alone and in combination with luspatercept, but not by luspatercept alone. Finally, splenomegaly was reduced in MDS mice treated with vamifeport alone or in combination with luspatercept, confirming that iron restriction improves ineffective erythropoiesis and limits myeloid skewing. Overall, the combination therapy with vamifeport and luspatercept showed superior effects in improving anemia and myeloid bias as compared to single treatments. Conclusion: In conclusion, our results show that iron restriction by vamifeport enhances the erythroid maturation action of luspatercept, likely by improving iron utilization in erythroid cells and ameliorating their survival. Furthermore, vamifeport improves the myeloid skewing in the MDS model, suggesting disease-modifying activity as single agent as well as combined therapy with luspatercept. Together, these data prove that combo therapies aimed at restricting iron and boosting erythroid maturation may offer additive beneficial effects in MDS and provide pre-clinical evidence for combining iron restriction and TFG-β superfamily ligand-trap approaches as more effective therapeutic strategies for the treatment of MDS.

Inhibition of Toll-like Receptor 4 (TLR4) Promotes Erythroid and Myeloid Differentiation in Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML)

Introduction: The hallmark of Myelodysplastic Syndrome (MDS) is a block in differentiation of hematopoietic precursors resulting in single or multilineage cytopenias. Evaluation of regulatory pathways that are inappropriately overactive and lead to a blockade in erythroid and myeloid differentiation is an important process in determining future therapeutic targets for the treatment of MDS. The Toll-like receptor (TLR) pathways play an important role in the innate immune response and are also highly expressed on hematopoietic stem cells. Aberrant overactivation of these pathways can stimulate inflammation and cytokine production in the marrow microenvironment. Also, while the overactivation of interleukin-1 receptor-associated kinase 4 (IRAK4), tumor necrosis factor receptor associated factor 6 (TRAF6) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways has been seen in Myelodysplastic Syndrome and Acute Myeloid Leukemia (AML), the upstream signals that drive their activation are not fully identified. Results: We investigated the expression of all TLRs in a large database of MDS CD34+ marrow samples and age-matched controls. Our findings revealed that TLR6 and TLR4 were the most significantly overexpressed receptors in MDS samples (FDR<0.001). Furthermore, we observed that the TLR4 cofactor lymphocyte antigen 96 (LY96), also known as myeloid differentiation factor 2 (MD-2), was also overexpressed in MDS samples when compared to controls. Furthermore, the S100A8 and S100A9 proteins are ligands for TLR4 and were found to be positively associated with severity of anemia in the cohort of MDS samples. These data provided the rationale for determining the functional role of the TLR4 pathway in MDS/AML models. To obtain a potent and specific antibody against TLR4, we inoculated rabbits with recombinant human TLR4 and obtained high affinity antibodies that neutralized TLR4 function at low nano-molar range of doses. The TLR4 polyclonal antibody was able to inhibit lipopolysaccharide (LPS)-induced NF-κb activation in leukemic THP-1 cells. The TLR4 antibody also led to a decrease in proliferative phosphorylated extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase in leukemic cells. We then treated samples from MDS/AML patients (n=14) with the TLR4 antibody (1 nM dosing) and assessed for erythroid and myeloid growth and differentiation. Fluorescence activated cell sorting (FACS) analysis for erythroid differentiation showed increased expression of either CD71, CD235a or both after treatment with the TLR4 antibody. FACS analysis for myeloid differentiation showed increased expression of either CD11b, CD14 or both after treatment with the TLR4 antibody. This effect was more pronounced in the intermediate/high risk group than in the low-risk group of patients. Conclusion: Inhibition of the TLR4 pathway with a potent TLR4 antibody promotes erythroid and myeloid maturation and relieves the differentiation block on these lineages in MDS patient samples in the in vitro setting. Our preclinical data supports further evaluation of TLR4 inhibition as a therapeutic target in patients with MDS, as anemia and neutropenia are hallmarks of the disease.

An Open-Label, Multicenter, Single-Arm, Phase I Dose Escalation Study of OP-2100, an Orally Bioavailable Prodrug of Decitabine, for Patients with Relapsed or Refractory Higher-Risk Myelodysplastic Syndromes and Chronic Myelomonocytic Leukemia

CONCLUSIONS Hypomethylating agents (HMA) are commonly used to treat high-risk myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML).Decitabine (DEC), one of the HMAs, is S phase-specifically incorporated into DNA to exert its effect, and sustained plasma concentration may increase its efficacy (Saunthararajah et al. J Clin Invest. 2015). Therefore, we developed OP-2100, an orally bioavailable prodrug of DEC. It showed equal efficacy to conventional HMAs in various blood cancer mouse models and exhibited a favorable safety profile in healthy mice (Watanabe et al. Blood. 2020; Ureshino et al. Mol Cancer Ther. 2021). In addition, it exhibited a sustained-release pharmacokinetic (PK) profile when orally administered to monkeys. In a phase I study, OP-2100 monotherapy showed the expected PK profile on patients with relapsed or refractory higher-risk MDS and CMML, and we reported the preliminary result. We designed a single-arm, multicenter phase I study for Japanese patients with MDS or CMML with intermediate-risk, high-risk, or very high-risk by Revised International Prognostic Scoring System (IPSS-R) and relapsed, refractory, or intolerant to standard therapy. The primary objective was to determine the maximum tolerated dose following the incidence of dose-limiting toxicity (DLT) in the first cycle, whereas the secondary objectives were to evaluate the safety, PK, and efficacy. Treatment-emergent adverse events (TEAEs) and response to therapy were assessed per NCI CTCAE v5.0 and IWG 2018 response criteria, respectively. Oral administration of OP-2100 as a single agent was continued for the patients daily on days 1-5 of each 28-day cycle until meeting discontinuation criteria, including disease progression. The dose level was defined as 30, 50, 80, and 120 mg/day. The initial dose of 50 mg/day was determined based on the highest non-severely toxic dose of OP-2100 in monkeys and the PK of DACOGEN TM in humans. This study was implemented in the 3+3 design according to DLT incidence. The plasma concentration of DEC and OP-2100 was measured on days 1 and 5. Furthermore, the LINE-1 methylation, a common HMA pharmacodynamics marker, was measured. This study is registered in the Japan Registry for Clinical Trials (jRCT2071220035). In total 3 patients with a history of azacitidine treatment for MDS were registered in the 50-mg initial cohort. These patients had been categorized as intermediate to high-risk in the IPSS-R, respectively. Table 1 shows patient characteristics. Out of 3, 2 patients did not experience DLT, while the 3rd one suffered from Grade 4 neutropenia, which was confirmed as DLT. As of April 30, the most common TEAEs were cytopenia of leukopenia, neutropenia, and thrombocytopenia, all of which were Grade 3 or 4. Other adverse events of Grade 3 or 4 include anemia, lymphopenia, COVID-19, and fall. In terms of efficacy, 2 patients had stable disease, while one had progressive, according to the IWG2018 response criteria. However, the 2 patients with stable disease still received administration up to 9 and 5 cycles, respectively (Figure 1). The PK profile of DEC after OP-2100 administration indicated sustained release, with an area under the curve (AUC) similar to that of DACOGEN TM (5-day regimen, 1-h continuous intravenous administration at 20 mg/m 2) and C max approximately 1/5. The LINE-1 methylation exhibited the similar behavior as conventional HMAs. The PK profile of DEC released from the prodrug after 50 mg of OP-2100 oral administration in 3 patients showed a similar AUC to that of the 5-day regimen of DACOGEN TM, and then C max showed a low with sustained plasma concentration. Among the 3 patients, OP-2100 could be administered for at least 5 cycles in 2 patients. To determine maximum tolerated dose, enrollment for this study is ongoing.