Myelodysplastic Syndromes Patients Research Articles

High Expressions of Checkpoint Receptors in T Cells Subsets of Myelodysplastic Syndromes Patients with TP53 Mutations

Background TP53 mutations were common in MDS patients and associated with adverse prognosis. The pathogenetic mechaminsms of TP53 mutations in MDS were complicated. Recently, TP53mutations were found to confer an immunosuppressive phenotype in MDS and AML. However, the expressions of checkpoint receptors in T cells subsets of MDS patients with TP53 mutations is unclear. Methods Between December 2021 to May 2023, fresh bone marrow mononuclear cells (BM-MNCs) were obtained from 82 de novo MDS patients and 13 healthy donors, and isolated using Ficoll. Then, flow cytometry was performed on BM-MNCs to evaluate the expressions of checkpoint receptors (CTLA4, PD-1, TIM3, LAG3 and TIGIT) in T cells subsets including all T cells (CD3 +), helper T cells (CD3 +, CD4 +), and cytotoxic T cells (CD3 +, CD8 +). TP53 mutations were examined in 67 patients who simultaneously accepted NGS examinations. The differences of the expression of checkpoint receptors in T-cells subsets were assessed by Mann-Whitney U test. Significance was defined as P < 0.05. Results We firstly compared the expressions of checkpoint receptors in T cells subsets of MDS patients with healthy donors. In the whole T cells, MDS patients had increased frequencies of PD-1 + cells (P < 0.05), and TIM-3 + cells (P < 0.05) compared with healthy donors. As for helper T cells subset (Figure 1A), the percentages of PD-1 + cells (P<0.01), TIM-3 + cells (P<0.01), and TIGIT + cells (P<0.05) in MDS patients were higher than healthy donors, while no significant differences of the percentages of CTLA4 + cells or LAG3 + cells were observed between MDS patients and healthy donors. While only considering cytotoxic T cells (Figure 1B), the percentages of CTLA4 + cells, PD-1 + cells, TIM-3 + cells, LAG3 + cell, and TIGIT + cells in MDS patients were as similar as those in healthy donors. Next, we compared the expressions of checkpoint receptors in T-cells subsets between TP53-mutated MDS patients (N=17) and TP53wild-type MDS patients (N=50). Among all T cells, significantly increased frequencies of CTLA4 + cells (P<0.01), PD-1 + cells (P<0.05), TIM-3 + cells (P<0.01), and LAG3 + cells (P<0.01) were found in TP53-mutated MDS patients compared with TP53 wild-type MDS patients. In helper T cells subset (Figure 1C), TP53-mutated MDS patients possessed increased frequencies of TIM-3 + cells (P<0.05) and LAG3 + cells (P<0.05) than TP53 wild-type MDS patients. But the frequencies of CTLA4 + cells, PD-1 + cells and TIGIT + cells in helper T cells of TP53-mutated MDS patients were as similar as those in TP53 wild-type MDS patients. When focusing on cytotoxic T cells (Figure 1D), the percentages of CTLA4 + cells (P<0.01), PD-1 + cells (P<0.05), TIM-3 + cells (P<0.001), LAG3 + cell (P<0.05), and TIGIT + cells (P<0.01) were all significantly greater in TP53-mutated MDS patients than those in TP53 wild-type MDS patients. Also, the frequencies of CTLA4 + cells (P<0.05), PD-1 + cells (P<0.05), TIM-3 + cells (P<0.01), LAG3 + cell (P<0.05), and TIGIT + cells (P<0.05) in cytotoxic T cells of TP53-mutated MDS patients were higher than those in healthy donor (Figure 1D). Conclusion Collectively, the percentages of PD-1 + cells, TIM-3 + cells, and TIGIT + cells in helper T cells subset of MDS patients were higher than those in normal people, while the expressions of checkpoint receptors in cytotoxic T cells of MDS patients had no significant difference compared with those in healthy donors. However, when considering TP53mutation status, the frequencies of CTLA4 + cells, PD-1 + cells, TIM-3 + cells, LAG3 + cell, and TIGIT + cells in cytotoxic T cells of TP53-mutated MDS patients were all significantly higher than those in TP53 wild-type MDS patients or healthy donors. These results indicated T cells exhaustion and immunosuppressive microenvironment existing in MDS especially those with TP53mutations, and suggested that immunomodulatory therapeutic strategies targeting checkpoint receptors may be beneficial for MDS especially those with TP53mutations.

1220. Comparing Breakthrough Fungal Infections in Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS) Patients Receiving Prophylaxis with Different Azoles

Abstract Background Posaconazole is recommended for primary prophylaxis in neutropenic patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) receiving induction chemotherapy. Despite prophylaxis, breakthrough invasive fungal infections (bIFIs) still arise. Initiating posaconazole is not always feasible, and may necessitate use of alternative azoles. The primary objective of this study was to evaluate the rate of bIFIs in patients receiving prophylaxis with posaconazole versus alternative azoles. The secondary objective reviewed reasons for alternative azole use. Methods A retrospective chart review was conducted at University of Washington Medical Center (UWMC) and Fred Hutchinson Cancer Center (FHCC). Patients who received induction chemotherapy for AML/MDS from March 2021 to November 2022 and received a prophylactic azole for at least 7 days during neutropenia were included. Outcomes were evaluated with Chi-Square. The incidence of bIFI was evaluated up to 12 weeks from induction chemotherapy, or until consolidation chemotherapy or hematopoietic stem cell transplant, whichever came first. Results The initial query yielded 175 patients with AML/MDS. Of these, 88 patients were included: 74 received posaconazole and 14 received an alternate azole (Table 1). BIFIs occurred in 9 patients (posaconazole (n=7, 9.5%), alternative azole (n=2, voriconazole, 14.2%); p=0.631) (Figure 1). The most common reason for initiating an alternative azole was insurance restrictions/high cost (Figure 2). Conclusion Numerically, we found reduced incidence of bIFIs among patients receiving posaconazole compared to alternative azoles. Insurance restrictions/cost was the most common reason for alternative azole utilization. This barrier may increase risk of bIFIs, and additional efforts should be considered to use posaconazole over alternative azoles. Disclosures Catherine Liu, MD, Pfizer: Site Investigator|SNIPR BIOME: Advisor/Consultant

Development of MDS in Pediatric Patients with GATA2 Deficiency: Increased Histone Trimethylation and Deregulated Apoptosis as Potential Drivers of Transformation

GATA2 deficiency is a heterogeneous, multisystem disorder associated with a high risk of developing myelodysplastic syndrome (MDS) and the progression to acute myeloid leukemia. The mechanisms underlying malignant transformation in GATA2 deficiency remain poorly understood, necessitating predictive markers to assess an individual’s risk of progression and guide therapeutic decisions. In this study, we performed a systematic analysis of bone marrow biopsies from 57 pediatric MDS patients. Focusing on hematopoiesis and the hematopoietic niche, including its microenvironment, we used multiplex immunofluorescence combined with multispectral imaging, gene expression profiling, and multiplex RNA in situ hybridization. Patients with a GATA2 deficiency exhibited a dysregulated GATA2 transcriptional network. Disease progression (GATA2-EB, n = 6) was associated with increased GATA2 mRNA levels, restored expression of the GATA2 target EZH2, and increased H3K27me3. GATA2-EB was further characterized by the high expression of the anti-apoptotic protein BCL2, a feature absent in children with a GATA2 deficiency and refractory cytopenia of childhood (GATA2-RCC, n = 24) or other pediatric MDS subgroups (RCC, n = 17; MDS-EB, n = 10). The multispectral imaging analysis of additional BCL2 family members revealed significantly elevated Mediators of Apoptosis Combinatorial (MAC) scores in GATA2-EB patients. Taken together, our findings highlight the potential drivers of disease progression in GATA2 deficiency, particularly increased histone trimethylation and dysregulated apoptosis. Furthermore, upregulated BCL2 and EZH2 and increased MAC scores provide a strong rationale for the use of venetoclax and azacitidine in therapeutic regimens for GATA2-EB.

The impact of erythroblast enucleation efficiency on the severity of anemia in patients with myelodysplastic syndrome

Anemia is the most common manifestation in myelodysplastic syndrome (MDS) patients, but the cause of ineffective hematopoiesis is not fully understood. Enucleation is an important event in the maturation process of erythroblasts. According to a series of morphological phenotypes of the pathological development of MDS erythroblasts, we speculate that there may be enucleation disorders. To verify this hypothesis, we cultured MDS bone marrow CD34+ cells in vitro and induced erythroblast development. The results showed that erythroblast enucleation in MDS was significantly lower than that in the normal group, and the rate of enucleation was positively correlated with hemoglobin concentration. Risk stratification of MDS was performed to further analyze the differences in enucleation among the normal group, low-middle risk group and high-risk group. The results showed that the enucleation rate of the high risk group was higher than that of the low-middle risk group but still lower than that of the normal group. Moreover, the expression of pERK and pAKT in MDS erythroblasts in the high risk group was higher than that in the normal group, while the expression of pERK and pAKT in the low-middle risk group was lower than that in the normal group. Furthermore, the enucleation of MDS was positively correlated with the phosphorylation degree of ERK and AKT. In conclusion, this study reveals that the enucleation of erythroblasts is one of the possible causes of anemia in MDS.-kLsQ5yxZq6YLTXHvFfPKmVideo

Prognostic Value of Measurable Residual Disease in High-Risk Myelodysplastic Syndromes with Intensive Chemotherapy Treatment: Analysis of HOVON-SAKK Studies

Diagnosis of myelodysplastic syndromes (MDS) and accurate risk assessment to tailor treatment remains challenging. High-risk MDS (hrMDS) patients who are fit enough may benefit from intensive chemotherapy followed by post-remission consolidation. The HOVON-SAKK (HO) acute myeloid leukemia (AML) trials, designed to test novel drug combinations with an intensive treatment regimen, included hrMDS patients (RAEB and RAEB-T). Since these HO-trials have been executed with large patient cohorts for many years, they offer the unique possibility to study the outcome, risk classification, and relevance of measurable residual disease (MRD) in hrMDS. A pooled database is available from HOVON-SAKK of patients who were treated with intensive chemotherapy (studies containing MDS patients with IPSS ≥ 1.5 HO42A, HO81, HO92, HO102, and IPSS-R > 4.5 HO103, HO132). The database contained 3268 patients; 11% (364/3268) were hrMDS scored according to IPSS and IPPS-R. Three-year overall survival (OS) for hrMDS was 32% (standard error; SE 2%) compared to AML 46% (SE 1%). In total, 1074 AML and 75 hrMDS had reached complete remission (CR/CRi) and received a second induction cycle (C2). MRD after C2 based on multiparameter flow cytometry (Cloos et al., 2018, J. Vis.) with a cut-off of ≥0.1% for MRD positivity was assessed to evaluate the MRD status. We observed a trend of a higher MRD positivity rate for MDS compared to AML patients (MDS vs. AML: 32% (23/73) vs. 22% (232/1064), P = 0.055). Kaplan-Meier OS analysis showed a significant prognostic value of MRD in MDS, but not in the cumulative incidence of relapse (CIR) (log-rank test 0.011, Gray's test P = 0.124). Since the MDS classification has been changed, we re-classified MDS based on the ICC 2022 classification into AML, AML/MDS, and MDS. We reclassified 3035 patients at diagnosis, including 2605 AML, 258 MDS/AML, and 172 MDS. The three-year OS was 47% (SE 1%) for AML, 29% (SE 3%) for MDS/AML, and 33% (SE 4%) for MDS. After C2, 2215 patients had reached CR/CRi. A total of 1074 patients containing 984 AML, 57 AML/MDS, and 33 high-risk MDS patients with CR/CRi after C2, with a suitable bone marrow sample, were included in the MRD analysis. Most patients were treated according to HO132 (40-48%). As expected, MDS patients were older (median age, range; 60, 32-74), had lower bone marrow blast counts, and were predominantly of adverse risk. A trend of a higher proportion of MRD positivity after C2 was observed in the MDS group (33%; 11/33) as compared to AML (22%; 215/984) or MDS/AML (18%; 10/57). Three-year OS was 61% for AML (SE 1.6%), 42% (SE 6.6%), and 49% (8.7%) for AML/MDS and MDS, respectively (Figure 1A). Kaplan-Meier OS analysis and CIR showed a significant prognostic value of MRD for MDS (log-rank test P<0.001, Gray's test P = 0.015) and AML ( P<0.001; P<0.001) but not for MDS/AML patients ( P = 0.5, P = 0.342)(Figure 1B). Remarkably, the three-year OS of MRDpos in MDS was 18% (SE 12%) compared to MRDpos in AML 40% (SE 16%). The limitation of this study is the relatively small number of MDS patients according to the new ICC 2022 guidelines. In summary, MRD detection in the HOVON-SAKK trials shows the potential of MRD as a prognostic value in MDS patients treated on intensive induction regimens with a clinically validated and standardized assay.

A Pilot Multicentric Prospective Study on the Usefulness of Peripheral Blood Immunophenotyping As Erythroid Maturation Response Marker during Luspatercept Treatment in Patients Affected By ß-Thalassemia Major and SF3B1+ Myelodysplastic Syndrome

Introduction Erythropoiesis is a multi-step maturation process; luspatercept proved to be successful on ineffective erythropoiesis acting on the second phase of erythroid maturation reducing the hyper expression of transforming growth factor beta (TGF-beta) ligands via the Smad 2/3 signaling pathway, thus ameliorating the aberrant late stage erythroid maturation featured in the myelodysplastic syndrome (MDS) and thalassemia. MEDALIST and BELIVE studies showed efficacy and safety of Luspatercept in MDS and Thalassemic patients respectively, but, regarding erythroid maturation, a significant correlation has been revealed only between hematological response and the reticulocyte value or medullar erythroblasts counts. In order to avoid for patients extra scheduled bone marrow aspirate , in particular in thalassemic patients, in our pilot study we propose to explore, for the first time, the role of peripheral blood immunophenotyping (IF) as reproducible and more affordable erythroid maturation's marker respect to the medullar immunophenotyping described in the previous published papers. Our hypothesis is that peripheral IF could represent a new tool to detect the ameliorate of inefficacy erythropoiesis during Luspatercept therapy, acting as “minimal residual disease” in patients in response or that loss the response to Luspatercept. Patients, Material and Methods Approval by local Ethic Scientific Commission has be completed. Patients will be enrolling by Hematology Unit and by Hemoglobinophaties Unit from 2 different Institution in Italy (“A. Businco” Oncologic Hospital and “A. Cao” Hemoglobinopathies Hospital). Inclusion criteria: Patients must be affected by MDS with ringed sideroblasts (with or without SF3B1 mutation) very low, low and intermediate revised IPSS stage or affected by ß-thalassemia major; aged ≥ 18 years and able to understand and sign an informant consent; not renal or hepatic failure; EF > 50%. Duration of the study will be scheduled for 1 year. Prevision of 30 patient enrolled in the first 1 year of study. Primary endpoint is to confirm the role of peripheral blood immunophenotyping (IF) as reproducible and affordable erythroid maturation's marker in these setting of patients. Secondary endpoint is to evaluate the role of peripheral IF as “minimal residual disease” in particular during the loss of Lusplatercept response and also the assessments of iron homeostasis in responder's patients. Patients will be assessed for r-IPSS and NGS (only for MDS), endogenous erythropoietin, full blood count and reticulocyte, iron status, transfusion burden in the previous 24 weeks and peripheral IF (centralized in the laboratory of Hematology Unit). All patients will collect these samples at baseline timing and after 12,24,36 and 48 weeks. Peripheral IF is tested by 8-color EuroFlow antibody panels define progressive terminal stages of erythroblasts' maturation (10 health samples have been used as control). Hematological response and Luspatercept's dose escalation are considered as for MEDALIST study criteria for MDS and BELIVE study for thalassemia, as well adverse events. Results At the time of this report 26 patients have been enrolled in the two institution (10 MDS and 16 thalassemic patients). Baseline patients' characteristics are reported in TAB 1 and baseline peripheral blood erythroid maturation (based on centralized IF) are reported in TAB 2 Conclusion Our pilot study aims to explore, for the first time, the role of peripheral blood immunophenotyping (IF) as reproducible and affordable erythroid maturation's marker during Luspatercept therapy in Thalassemic and MDS patients.

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.

Performance of the Molecular International Prognostic Scoring System (IPSS-M) in Hypomethylating Agent-Treated Patients with Myelodysplastic Syndromes

Background: Patients with myelodysplastic syndromes (MDS) undergo risk stratification with various prognostication tools, such as the International Prognostic Scoring System (IPSS) and Revised IPSS (IPSS-R). Recently, the Molecular IPSS (IPSS-M) was developed incorporating genomic data in addition to clinical and cytogenetic characteristics. Though IPSS-M has been shown to improve prognostication in all MDS patients at baseline, its utility in specifically patients undergoing hypomethylating agent (HMA) therapy remains unknown. Here, we assess the efficacy of IPSS-M in HMA-treated patients with MDS. Methods: We retrospectively evaluated all untreated patients with MDS seen at a single tertiary cancer center from July 2017 to July 2021 and identified those who later received HMA therapy. Patient characteristics and bone marrow (BM) data, including morphology, cytogenetics, and mutations, were assessed at diagnosis. Genomic DNA was extracted from whole BM aspirate samples and subject to 81-gene target PCR-based sequencing using a next-generation sequencing platform. Survival data was updated in July 2023. Results: Out of 799 untreated MDS patients, 455 patients (57%) eventually underwent treatment with HMA. At diagnosis, the median age was 69 (range: 22-90) with 289 (64%) male patients. Patients were generally high risk, with therapy-related MDS in 160 (35%), complex cytogenetics in 167 (37%), and TP53 mutations in 168 (37%). By IPSS-R, 259 patients (58%) had high- or very high-risk disease, and 340 patients (75%) had higher-risk disease (moderate high-, high-, and very high-risk) by IPSS-M. A total of 291 patients (64%) were treated with HMA monotherapy, and lower-risk MDS patients received more cycles of HMA (p < 0.001). By IPSS-R, the median overall survival was 60.0 months (95% CI: 46.0, not estimable (NE)) in very low-, 87.4 months (95% CI: 59.9, NE) in low-, 36.2 months (95% CI: 30.0, 58.1) in intermediate-, 23.7 months (95% CI: 19.6, 34.8), in high-, and 12.8 months (95% CI: 11.0, 16.7) in very high-risk patients (p < 0.0001) with a concordance index of 0.669. Figure 1 depicts the reclassification of patients between IPSS-R and IPSS-M. Risk stratification by IPSS-M (Figure 2) provided similar results: not reached (95% CI: 56.8, NE) in very low-, 60.0 months (95% CI: 49.8, NE) in low-, 40.8 months (95% CI: 22.8, NE) in moderate low-, 44.2 months (95% CI: 27.8, NE) in moderate high-, 33.4 months (95% CI: 22.8, 44.5) in high-, and 16.6 months (95% CI: 13.8, 19.4) in very high-risk patients (p < 0.0001). The concordance index of IPSS-M in all HMA-treated MDS patients was 0.648. When analyzing patients with MDS who were more likely to undergo HMA as per standard-of-care treatment (those with IPSS-M moderate low-, moderate high-, high-, and very high-risk disease), the IPSS-M remained statistically significant (p < 0.0001) though less discerning in the patients with moderate-risk MDS, as seen by the concordance index of 0.620. Conclusions: In this group of MDS patients treated with HMA, the IPSS-M did not provide additional prognostic power over the IPSS-R though median overall survival remained inversely proportional to IPSS-M risk category. Concordance indices for survival remained below 0.7, likely due to the overlapping curves in moderate-risk patients by IPSS-M. Further development and validation of prognostic scoring systems in patients treated with HMA are warranted.

The Pyruvate Kinase (PK) Activator AG-946 Improves PK Properties and Red Blood Cell (RBC) Characteristics upon Ex Vivo treatment of RBCs from Patients with Myelodysplastic Syndromes

Background: Myelodysplastic syndromes (MDS) consist of a heterogenous group of clonal myeloid malignancies, characterized by dysplasia, ineffective hematopoiesis and cytopenias. MDS patients frequently suffer from anemia, directly affecting quality of life. The limited therapeutic options often do not result in the desired clinical improvement. Interestingly, in MDS the activity of the red blood cell (RBC) enzyme pyruvate kinase (PK), a key regulatory enzyme of glycolysis, may be decreased. The activation of PK via small molecules is hypothesized to be beneficial in a wide range of anemias. Currently, a phase 2a, open-label, proof of concept trial is running on the use of the PK activator AG-946 in low-risk MDS patients (NCT05490446). In light of these developments, we investigated the effect of ex vivo treatment of MDS RBCs with AG-946. Objectives: To evaluate RBC PK and cellular properties of patients with MDS, and to determine the effect of ex vivo treatment with the PK activator AG-946. Methods: Eleven low-risk non-transfusion dependent MDS patients and six healthy controls (HCs) were studied. Baseline PK activity and PK thermostability were measured in RBC lysates of purified RBCs, under V max conditions (phosphoenolpyruvate (PEP) 5mM final concentration). Hexokinase (HK) activity was included to relate PK activity to mean RBC age. Ex vivo treatment was performed by incubating MDS RBCs for 16 hours at 37 °C in presence or absence of the PK activator AG-946 (10μM or 5μM) using dimethylsulfoxide (DMSO) as a blank. Effect was assessed by measuring PK activity (using a final concentration of PEP 0.5mM) and levels of adenosine triphosphate (ATP) (LC-MS/MS). Functional RBC analysis was performed by osmotic gradient ektacytometry (Lorrca MaxSis). To investigate the effect on PK thermostability, RBC lysates were incubated with AG-946 (2μM or 1μM) or DMSO, after which residual PK activity was measured (PEP 0.5mM). The effect of AG-946 on erythroid development was studied using peripheral blood mononuclear cells, cultured in MethoCult TM H4434 medium for 14 days in the presence or absence of AG-946 (10μM or 625nM, DMSO as blank). Differences between conditions were determined using Unpaired T-test, ANOVA with Dunnett's test or Kruskal-Wallis with Dunn's test via GraphPad Prism. Results: Mean PK/HK ratio was significantly lower in RBCs from MDS patients compared to HCs (6.1 (SD 1.8) versus 10.5 (SD 1.5), p<0.001). Upon ex vivo treatment, PK activity significantly increased (independent of dosage) when compared to DMSO (10μM AG-946, mean increase 30% ( p<0.001); 5μM, 28% ( p<0.0001)) (Figure 1A). MDS RBCs displayed decreased PK thermostability at baseline under V max conditions (HCs 79% residual activity versus 68% in MDS, p<0.01). This was restored by ex vivo treatment with AG-946 (DMSO, 33% residual activity, 2μM AG-946, 85% ( p<0.0001); 1μM, 87% ( p<0.0001)). The increase in PK activity was accompanied by significantly increased ATP levels (N=10, DMSO, mean ATP 14.9 μg/mL RBC; 10μM AG-946, 21.0 μg/mL RBC ( p<0.01); 5μM, 21.1 μg/mL RBC ( p<0.01)) (Figure 1B). Functionally, RBC properties improved upon ex vivo treatment with AG-946 as reflected by the modest yet significant increase in O hyper, indicating improved hydration status (10μM AG-946, mean increase 2.3% ( p<0.001); 5μM, 2.9% ( p<0.0001)). To date, culture assays were performed in 8 patients; 3/8 showed an increase in the number of burst forming units-erythroid (BFU-Es) upon treatment with AG-946 when compared to DMSO. The percentual increases differed per patient: increase of 25% with 10μM AG946 and 150% with 625nM, increase of 40% (10μM) and 39% (625nM), and for the third an increase of 22% (10μM) and 39% (625nM). Conclusion: Our findings show that RBCs from MDS patients have decreased PK activity and thermostability. We demonstrate that ex vivo treatment with AG-946 increases PK activity and ATP levels and restores PK thermostability. Moreover, ex vivo treatment with AG-946 improves RBC hydration, suggesting that improved energy status directly improves RBC functional properties. The preliminary results of the culture assay could indicate that in certain patients, dyserythropoiesis may be ameliorated upon PK activation. To better understand the effects on erythropoiesis additional experiments are required. In conclusion, our data might support a rationale for the use of PK activators as a novel therapeutic option for MDS.

Azacitidine Combination with Lenalidomide in Treating Higher-Risk Myelodysplastic Syndromes: A Single-Center, Phase 2 Trial

Background: Azacitidine, which works through the proteosomic destruction of DNA methyltransferase and resultant chromatin decondensation, is used primarily in patients with higher-risk myelodysplastic syndromes (MDS). Lenalidomide has been approved by the US Food and Drug Administration for the treatment of MDS patients with del(5q) cytogenetic abnormality. It has also shown activity in patients with higher-risk MDS and acute myeloid leukemia. In this trial, we assessed the efficacy and safety of combining azacytidine and lenalidomide in higher-risk MDS. Methods: This is a single-center, phase 2 trial. Aza (75mg/m 2/d on days 1-7 of a 28-day cycle) was administered in combination with lenalidomide (10 mg/day on days 1 to 21 every 28 days) for patients. The primary endpoint was the overall response rate (ORR). Secondary endpoints were overall survival (OS), progression-free survival (PFS), and adverse events (AEs). An MDS targeted-exome-seq panel including 37 driver genes was used for screening. Results: Between Oct 2020 to Apr 2023, a total of 41 MDS patients were enrolled. 32 patients were evaluable for efficacy. 23 (71.875%) responded to therapy: 7 achieved a complete response (CR) (21.875%), 12 a marrow CR (mCR) (37.5%), and 4 a hematologic improvement (HI) (12.5%), of whom 10 of the mCR patients also had a HI. The median follow-up was 5.6 months (1 to 27.4), the median overall survival (OS) was not reached, and the median progression-free survival was 12.2 months. The time to reach CR was 2 months (range, 1 to 4.5), and the median CR duration was 8+ months (range, 5 to 27.4) ( Table 1). 27 patients had at least one mutation, the most common being TET2 (n = 12), ASXL1 (n = 12), TP53 (n = 7), SRSF2 (n = 6), and RUNX1 (n = 6) ( Fig.1A). Compared with patients without mutations, CR/mCR rate was significantly higher for those with mutations in TP53 (100% v 48%; P =0 .025) ( Fig. 1B), ORR were numerically higher for those with TP53 (100% v 64%; P = 0.149) and TET2 (85% v 63%; P = 0.422) ( Fig. 1C). We also observed a reduction of VAF in TP53 and TET2 mutation after the combination treatment ( Fig. 1D & 1E). Common grade 3/4 nonhematologic adverse events included thrombocytopenia (18.75%), infection (12.5%), and febrile neutropenia (9.4%). Conclusion This trial demonstrated that the combination of azacytidine with a lenalidomide regimen is an effective therapy for higher-risk MDS patients, with high efficacy and well-tolerance. Patients with TP53 mutation appear more likely to achieve a CR/mCR.

Myelodysplastic Syndrome Hematopoietic Stem Cells Can Undergo Differentiation Trajectory Rewiring to Escape Venetoclax Vulnerability

Myelodysplastic syndromes (MDS) are incurable neoplasms that arise from early hematopoietic stem cells (HSCs). The standard of care for MDS is hypomethylating agent (HMA) therapy. However, the disease invariably fails these agents, resulting in fatal cytopenias or progression to acute myeloid leukemia (AML) with a dismal prognosis. We previously showed that the cellular hierarchy of MDS HSCs predicts the mechanisms of progression after HMA failure and can guide the design or choice of specific therapeutic approaches targeting these cells (Ganan-Gomez et al., Nature Medicine 2022). MDS HSCs in one of two differentiation states, long-term HSCs or lymphoid-primed multipotent progenitors, have a “common myeloid progenitor (CMP) pattern” or “granulocytic-monocytic progenitor (GMP) pattern” of differentiation. HSCs in these 2 differentiation states persist throughout the disease course and, driven by the recurrent activation of BCL2- or NF-κB-mediated survival pathways, respectively, expand at disease progression. In our preclinical studies, pharmacologically inhibiting these pathways depleted MDS HSCs and reduced tumor burden, which suggests that only patients with “CMP pattern” MDS may benefit from venetoclax-based therapies. Accordingly, patients with “CMP pattern” MDS, whose disease progressed after frontline HMA failure, enrolled in clinical trials of venetoclax-based therapy (NCT04160052, NCT04550442, or NCT04655755; n=28: 12 “CMP pattern” MDS and 16 “GMP pattern” MDS) had a shorter cumulative time to complete remission (P=0.012) and a longer relapse-free survival duration (14.0 months vs 5.2 months) than those with “GMP pattern” MDS did. Consistent with the hypothesis that venetoclax-based therapy selectively targets different HSC populations after HMA therapy failure, bone marrow (BM) HSC frequencies were significantly decreased at the time of remission after venetoclax-based therapy in “CMP pattern” MDS patients, but not in those with “GMP pattern” MDS. These results explain why “GMP pattern” MDS are resistant to venetoclax therapy. However, 8 of the 12 patients with “CMP pattern” MDS relapsed during venetoclax treatment and/or progressed to AML after an initial remission. In 2 patients with “CMP pattern” MDS, sequential flow cytometry and next generation sequencing analyses showed that the HSC hierarchy switched to “GMP pattern” MDS before venetoclax failure. This immunophenotypic hierarchical change was associated with the detection or expansion of STAG2- or RUNX1-mutant clones with isolated trisomy 8. To evaluate whether the immunophenotypic switch resulted from the distinct differentiation potential of early HSCs or from the aberrant expression of cell surface markers, we performed sequential single-cell RNA sequencing analyses of BM mononuclear cells isolated from the 2 “CMP pattern” MDS patients whose disease changed hierarchy before venetoclax failure. Our analyses revealed that after undergoing significant depletion during disease remission, HSCs expanded at therapy failure. Differential expression analysis showed that the expansion of STAG2- or RUNX1-mutant clones in the HSC compartment not only rewired MDS HSCs' differentiation state towards a granulo-monocytic-biased trajectory but also changed HSCs' survival dependency from BCL2-mediated anti-apoptotic pathways to TNFa-induced pro-survival NF-κB signaling, thus enabling HSCs to evade the cytotoxic effects of venetoclax (Fig. 1). The anti-apoptotic gene MCL1, a direct transcriptional target of NF-κB, was also significantly upregulated in HSCs at the time of progression, which suggests that the combination of venetoclax with an MCL1 inhibitor can overcome secondary failure to venetoclax. Together, these results reveal a novel mechanism of venetoclax resistance. HSCs under venetoclax therapy undergo survival pressure, which results in the expansion or acquisition of clones carrying specific mutations that change HSCs' survival dependencies (Fig. 2). Our study suggests that MDS patients receiving venetoclax-based therapy should be monitored at the molecular level and enrolled in clinical trials of other targeted agents before their disease undergoes clonal evolution and HSC hierarchical rewiring.

Clinical Characteristics of Patients with TP53 Multihit MDS and AML. a Single-Center Experience

Introduction TP53 is a tumor suppressor protein encoded by the TP53 gene that plays a pivotal role in maintaining genomic stability in response to DNA damage, activates DNA repair programs and triggers cell-cycle arrest. Mutations in TP53 (TP53-mut) are present in approximately 5-10% of de novo myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML), and confer an extremely poor prognosis, irrespectively of the treatment administered. The frequency of TP53 abnormalities increases up to 25-40% in therapy-related MDS and AML, up to 70%-80% in patients with complex karyotype and in patients with loss of chromosome 17/17p, 5/5q, or 7/7q. “Multihit” TP53-mut present with the loss of both wildtype TP53-alleles, either through point mutations or larger aberrations to chromosome 17, in complex monosomal karyotypes. Recent data strongly support the concept that TP53mut, particularly multi-hit TP53, results in similarly poor clinical outcomes, regardless of its classification as MDS or AML, arguing for a revised TP53 mutant myeloid entity that includes both MDS and AML. The aim of our analysis is to assess the impact of the multihit TP53-mut on clinical outcomes according to the type of treatment received. We will particularly focus on the presence of mono or biallelic mutation status of TP53 mutation. Methods We retrospectively evaluated 42 patients with TP53 mutated MDS or AML diagnosed from October 2016 to June 2023 in our department at the Catalan Institute of Oncology-L'Hospitalet in Spain. We analyze the prognosis of these two diseases, focusing on single-hit or multi-hit status of TP53 mutation and treatments received. Results Median age at diagnosis was 71 years (range, 44-87 years) with a male predominance (67%). Thirty-five (84%) patients were diagnosed of AML and 7 (16%) of MDS. Among AML patients, 17% had a prior diagnosis of dysplasia, 78% were diagnosed of AML with dysplasia-related changes, 13% with therapy-related neoplasms, and 9% were AML with maturation and/or AML with mutated NPM1. Regarding MDS patients, 57% had an excess of blast-2 (EB2) MDS, 43% MDS multilineage dysplasia (MLD) and MDS with excess of blast-1(EB1). Complex karyotype was present in 79% of the patients. The median variant allele frequency (VAF) of TP53mut was 36%. Multi-hit TP53 status was observed in 24 patients (57%). In this group of multi-hit TP53mut median age was 72 years (range,44-81 years) with a male predominance (67%), the median VAF of TP53mut was 40%. Twenty (83%) patients had MDS diagnosis and 4 (17%) had AML diagnosis; all of them presented complex karyotype. In the whole group the most concurrent mutations associated with TP53 were TET2 in 17%, ASXL1 in 14% and DNMT3A, NRAS y CREBBP in 12% of patients. Intensive chemotherapy (IC) was administrated as first-line treatment in 17% of patients, 45% received hypomethylating agents (HMA) monotherapy, 24% venetoclax-based combination with HMA, and 14% exclusively supportive treatment. 16 patients (38%) achieved a complete response (CR), 44%, 31% and 25% with HMA+venetoclax, IC and with HMA monotherapy, respectively. Eight patients (19%) achieved a partial response (PR), 88% with HMA and 12% with IC, 7 patients (17%) stable disease, 86% with HMA monotherapy and 14% with HMA+venetoclax and 2 patients (5%) were refractory to IC. In 2 patients (5%), disease was not evaluable. The treatment related mortality was 4.2%. Median overall survival (OS) was 12.5 months [IC 95 % (0.0, 23.54)] and median progression free survival (PFS) was 9, 2 months [IC 95% (3.2, 32.6)]. Median OS of patients with multihit TP53 was 9.8 months, vs 25.7 months in patients with single-hit TP53-mut (P=0.026) (Figure1). Median PFS of the patients with multi-hit and single-hit TP53 was 9.1 and 11.6 months (P=0.07), respectively. The impact of multi-hit TP53 on OS and PFS according to the treatment received (HMA monotherapy vs HMA + venetoclax vs IC) was not statistically significant, just like in the single-hit TP53 (Figure1). Conclusions The presence of multihit TP53 -mut is a prognostic variable for survival in our group of patients analyzed with TP53-Mut. Survival is poor, around one year, in this group of patients independently on the treatment strategy received; this fact would call into question the benefit of IC with the toxicity it entails, in this group of patients. Further development of new effective therapies for multihit TP53-mut AML and MDS is needed.

Downregulation of Mitochondrial Complex II (MC II) in Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are a group of diseases of hematopoietic stem cells (HSCs) that occur in the aging population and following therapy for unrelated cancers. The standard of care for MDS involves hypomethylating agents (HMAs) that invariably result in resistance and disease progression. We previously determined the role of mitochondrial metabolism in MDS pathophysiology, whereby suppression of mitochondrial activity leads to the accumulation of oncometabolites that competitively inhibit α-ketoglutarate-dependent dioxygenases (2OGDDs). Consequently, hypermethylation of epigenomes and hypoxia-inducible factor 1A (HIF1A) activation ensues a pseudohypoxia state that drives MDS pathogenesis. Our recent informatics analyses of primary MDS samples identified a patient subset with high TGF-β signaling in which mitochondrial complex II (MC II) genes were downregulated. MC II, or succinate dehydrogenase (SDH) is the smallest complex of the oxidative phosphorylation system that oxidizes succinate to fumarate and thereby donates electrons to reduce FAD to FADH 2 and the ubiquinone pool. Its downregulation results in the accumulation of Krebs cycle intermediate metabolites that can act as antagonists for 2OGDDs. To understand whether SDH dysregulation alone can contribute to MDS pathobiology, we generated a doxycycline-inducible and reversible mouse model (SDH-KD) that expresses shRNAs against the SDH subunits (SDHA, B, and D) in HSCs. These mice showed a gradual buildup of intracellular succinate in c-Kit + bone marrow (BM) cells and the peripheral blood, and a corresponding decrease of fumarate levels after induction for 2 weeks, 4 weeks, and 2 months. We found that knocking down Sdh was sufficient to induce pancytopenia and trilineage dysplasia in SDH-KD mice, while intracellular iron granules that surround the nucleus of erythroblasts, similar to ring sideroblasts commonly observed in MDS patients were also detected in BM and spleen smears. Of note, removing doxycycline and restoring Sdh expression rescued macrocytic anemia among other MDS phenotypes. Furthermore, treating SDH-KD mice with HMAs restored mitochondrial respiration and reversed HIF1A activation through the upregulation of SDH subunits. Using another MDS model, MllPTD/WT/ Vav1-Cre/Runx1 Flox/Flox, we found that HMA-treated HSCs differentiate towards CD11b +Gr1 + mature myeloid cells and that this requires Sdh expression. In conclusion, our results indicate that pseudohypoxia- and TGF-β signaling-associated changes in metabolic regulation caused by SDH downregulation underlie MDS pathogenesis. Elucidating the role of MC II in MDS pathobiology may provide alternative targets that can be combined with HMA or luspatercept therapy for MDS.

Daprodustat, a Hypoxia-Inducible Factor-Prolyl Hydroxylase Inhibitor, Improves Hematological Insufficiency without Progression of Myelodysplastic Syndrome with Chronic Kidney Disease

Introduction Hematological insufficiency is a critical issue in the treatment of myelodysplastic syndrome (MDS). The current standard treatment for cytopenia in MDS involves blood transfusion and the administration of erythropoiesis stimulating agents (ESAs). However, ESAs are only effective in certain patients with low erythropoietin levels; patients who are not suitable candidates for allogeneic hematopoietic stem cell transplantation (allo-HSCT) require lifelong blood transfusions. As a result, there is a growing demand for new treatments for cytopenia in MDS, and researchers have been exploring various molecular-targeted drugs in recent years. One promising option is the hypoxia-inducible factor -prolyl hydroxylase inhibitor (HIF-PHi). This inhibitor stabilizes HIF1α, which in turn normalizes endogenous erythropoietin production and iron metabolism, leading to improved renal anemia. It is believed that HIF-PHi could potentially enhance MDS-related anemia by increasing erythropoietin levels. However, despite some case reports, there have been no comprehensive clinical studies evaluating the effectiveness and safety of HIF-PH inhibitors for MDS. Moreover, it is essential to address concerns related to HIF1α overexpression, which has been associated with exacerbating various malignant tumors, including MDS. To assess the safety and effectiveness of HIF-PHi for MDS, we conducted a prospective clinical trial evaluating an oral HIF-PHi called daprodustat. Methods The study included MDS patients with chronic kidney disease (CKD) stage ≥ 3, considering daprodustat's approval in Japan, at Ebina General Hospital. Patients with MDS who were treated with deoxyribonucleic acid methylation inhibitors and/or who underwent allo-HSCT were excluded. Participants were administered daprodustat at the approved dose for renal anemia and underwent regular blood sampling and bone marrow examination. The primary and secondary endpoints were the leukemia rate within 1 year after HIF-PHi administration and hematological improvement (HI), respectively. The study was conducted in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) and was approved by the Institutional Review Board of the Ebina General Hospital (approval number: JMA-388). Written informed consent was obtained from all participants. Results Seventeen patients with MDS, with a median age of 81years, were included in the analysis. Revised International Prognostic Scoring System scores (IPSS-R) were very low, low, intermediate, high, and very high in 1, 11, 3, 1, and 1 patients, respectively. Five cases were changed from ESA treatment. Within 1 year after HIF-PHi administration, no patients developed leukemia, and both peripheral blood Wilms tumor 1 protein concentrations and bone marrow myeloblast counts remained unchanged from pre-treatment levels. HI was observed in 8 patients within 6 months, and the median dose was 6 (range, 2-12) mg for HI achievement. On univariate analysis, pretreatment transferrin saturation (TSAT, P = 0.002, Figure 1) and ferritin concentration ( P = 0.016) were inversely associated with HI achievement. However, pretreatment hepcidin concentration, erythropoietin concentration, previous ESA use, and IPSS-R were not associated with HI achievement. Notably, when a TSAT cutoff of < 45% was used, achieving HI showed a sensitivity and specificity of 87.5% and 100.0%. Moreover, patients with TSAT < 45% showed significant improvement in anemia after 3 months and beyond (Figure 2). No significant differences were observed in neutrophil and platelet counts between the two groups. Throughout the study, eleven adverse events occurred, including 6 infections, and 1 case each of myocardial infarction, eosinophilia, erythema multiforme, organizing pneumonia, and spinal fracture. Conclusion HIF-PHi administration did not lead to MDS progression. Notably, in MDS patients with CKD, low TSAT was a predictor for achieving HI by HIF-PHi.

Mitotic Dysregulation Sensitizes Malignant Stem Cells to CHK1 Inhibition in SF3B1-Mutant Myeloid Neoplasms

Myelodysplastic syndromes (MDS) arise from the acquisition of driver mutations in hematopoietic stem cells (HSCs). Splicing factor SF3B1 mutations are recurrent initiating lesions, found in up to 30% of patients with MDS. SF3B1 mutations are associated with aberrant splicing marked by recognition of alternative 3' splice sites (a3'ss). However, the causal role of mis-spliced genes in disease pathogenesis remains largely unexplored. Although SF3B1 mutations and downstream mis-spliced genes provide attractive therapeutic targets, targeted therapies remain unavailable. Here, we developed a novel method of precise gene editing of CD34 + hematopoietic stem and progenitor cells (HSPCs) to introduce an SF3B1 K700E mutation at the endogenous locus, enabling stoichiometric expression of mutant protein. An intronic fluorescent marker was introduced upstream of the mutation for isolation of edited cells. Global splicing analysis in SF3B1 K700E knock-in HSPCs recapitulated canonical targets identified in SF3B1-mutant (SF3B1m) MDS patients. Annotation of a3'ss mis-spliced genes showed profound enrichment of gene ontology (GO) terms related to cell cycle regulation, mitotic checkpoint signaling, and chromosome segregation. Consistently, cell cycle analysis of SF3B1m HSCs revealed accumulation of cells in G2/M phase, suggesting a previously unidentified stall in G2/M progression driven by SF3B1 mutations. To identify mis-spliced genes that cause this G2/M delay, we compared a3'ss mis-spliced genes in the cell cycle GO category across multiple model systems. BUBR1 and CDC27 were consistently mis-spliced in all datasets, resulting in reduced mRNA and protein levels. BUBR1 is a mitotic kinase involved in the spindle assembly checkpoint and chromosome alignment, and CDC27 is a component of the anaphase-promoting complex. Knockdown of BUBR1 or CDC27 in normal CD34 + HSPCs recapitulated G2/M delay. BUBR1 and CDC27 are thus attractive therapeutic targets to impair cell cycle progression of SF3B1m cells. While there are no direct inhibitors of BUBR1 kinase, BUBR1 is phosphorylated by CHK1 during mitosis, and its expression is highly correlated with response to a selective CHK1 inhibitor prexasertib in a large panel of cancer cell lines (Blosser et al., Oncotarget 2020). Indeed, SF3B1m K562 cells and CD34 + HSPCs were highly sensitized to prexasertib. Knockdown of BUBR1 or CDC27 in wild-type cells induced CHK1 phosphorylation, especially upon prexasertib treatment, and sensitized normal cells to prexasertib. These data indicate that mis-splicing of BUBR1 or CDC27 delays G2/M progression leading to CHK1 activation, which sensitizes SF3B1m cells to prexasertib. To determine if CHK1 inhibition can eradicate SF3B1m HSCs, we leveraged our gene editing system to test drug responses in CD34 +CD133 +CD38 - primary immunophenotypic HSCs. Prexasertib selectively eliminated SF3B1m HSCs, including high-risk SF3B1/ RUNX1 and SF3B1/ STAG2 genotypes, while control AAVS1-edited HSCs were maintained. Furthermore, prexasertib depleted CD34 + cells in primary SF3B1m MDS patient samples. To test prexasertib efficiency in vivo, we transplanted NSGS mice with iPSC-derived HSPCs reprogrammed from a patient with high-risk MDS and SF3B1/ RUNX1 co-mutations, and treated with a standard preclinical regimen. While all the vehicle-treated mice showed consistent engraftment, none of the prexasertib-treated mice were engrafted. Together, these data indicate that prexasertib selectively targets SF3B1m HSCs in vitro and eradicates SF3B1m MDS in vivo. In conclusion, we developed a precise gene editing strategy of human HSCs to identify prexasertib as a promising therapy for SF3B1m myeloid neoplasms, and implicate the mitotic function of CHK1 as a SF3B1m sensitivity. The safety and toxicity profiles displayed in early phase clinical trials make prexasertib a suitable agent for further clinical investigation in SF3B1m MDS and its advanced stages.

Encouraging Efficacy Observed in Bexmab Study: A Phase 1/2 Study to Assess Safety and Efficacy of Bexmarilimab in Combination with Standard of Care in Myeloid Malignancies

Background Clever-1 (common lymphatic endothelial and vascular endothelial receptor-1) constitutes a novel macrophage checkpoint. The receptor contributes to immune tolerance by antigen clearing and T cell inhibition. In multiple models, tumor growth is significantly reduced when Clever-1 function is inhibited. Expression of Clever-1 on bone marrow (BM) cells is a prognostic factor for poor outcome in acute myeloid leukemia (AML) and suppression of Clever-1 signaling inhibits leukemia cell growth. Bexmarilimab (BEX), a humanized IgG4 monoclonal antibody, binds Clever-1 resulting in increased antigen presentation, secretion of proinflammatory cytokines and activation of T cells. Clever-1 is highly expressed on leukemic blasts and monocytes of AML and myelodysplastic syndrome (MDS) patients. Treatment of AML bone marrow cells with BEX alone or in combination with azacitidine/venetoclax results in enhanced antigen presentation capacity and increased activation markers on effector T cells with synergistic effect on cell death. Also, BEX treatment alone lowers blast metabolic fitness via interference with mitochondrial oxidation. The ongoing BEXMAB clinical study investigates safety and efficacy of BEX combined with standard of care (SoC) in patients with myeloid malignancies (NCT05428969). Method This is a Ph1/2 study, investigating BEX combined with azacitidine (doublet) in patients with higher risk MDS frontline or hypomethylating agent (HMA)-failed, and relapsed/refractory (r/r) AML patients. BEX is dosed Q1W in 28-day cycles until disease progression or intolerable toxicity. SoC azacitidine is administered as per label. During Ph1, BEX doses were escalated from 1.0 mg/kg to 6.0 mg/kg (Bayesian optimal interval [BOIN] design). BM was analyzed at the end of Cycle 1 and 3 followed by every three cycles. Ph2 will start with a dose optimization per malignancy followed by a dose expansion for efficacy evaluation (2-Stage design). Results Here we report data from the dose finding Ph1 of the doublet. As of 25 July 2023, 22 patients have been enrolled in the doublet (MDS n=5; MDS HMA failed n=5; r/r AML n=12). Baseline characteristics are shown in Table 1. 8/10 MDS/MDS HMA-failed patients were of high to very high risk based on rIPSS; of the r/r AML 7/12 patients had adverse risk based on ELN 2017. The median number of prior therapies for the MDS HMA-failed patients was 1 (range 1-2). The median number of prior therapies for the r/r AML patients was 2 (range 1-4) with all patients having failed prior HMA-containing therapies and 7/12 having received prior HMA plus venetoclax. The majority of observed adverse events (AE) were Grade 1-2 and no dose limiting toxicities have been reported. As of 25 July 2023, a total of 10 BEX related AEs have been reported (8% of all treatment emergent AEs) with four of these events of ≥ Grade 3, two being immune related (capillary leak syndrome and hemophagocytic lymphohistiocytosis). Sustained target engagement of soluble Clever-1 of up to 75% compared to baseline was seen at all tested dose levels in patient blood. Activity of BEX in patient BM was demonstrated using receptor binding assays with BEX treatment resulting in decreased blast Clever-1 expression at all dose levels of the doublet cohort. Increased immune activation as measured using HLA expression and T/NK cell numbers in patient BM was observed after BEX treatment across indications. Up to 2-3-fold increase of CD8 T and NK cells in the BM of BEX treated patients was observed. Preliminary efficacy shows objective responses in 8/15 evaluable patients across all tested BEX doses. Four were observed in patients with prior HMA-failure and four patients stayed on treatment > 6 months. Clinical activity for frontline MDS was seen as one complete remission (CR), marrow CR (mCR) and hematologic improvement for platelets (HI-P); for MDS with HMA failure as one CR, mCR and partial remission (PR); for r/r AML as two incomplete CRs (CRi). Based on accumulating data, responses are observed between end Cycle 1 and end Cycle 4 for all BEX doses. Conclusion Combining BEX plus azacitidine is well-tolerated and shows efficacy across indications. Additional clinical and pharmacodynamic data of the completed Ph1 of the study will be presented during the meeting. Ph2 of BEX plus azacitidine will open in the 2 nd half of 2023 in HMA-failed r/r AML and/or higher risk MDS patients.

A Phase 2 Study of Canakinumab in Patients with Lower-Risk Myelodysplastic Syndromes or Chronic Myelomonocytic Leukemia

Background Myelodysplastic syndromes (MDS) lie on a continuum of myeloid neoplastic progression between clonal hematopoiesis (CH) and acute myeloid leukemia (AML). Interleukin 1 beta (IL-1β)-induced inflammasome activation is a mechanism of cancer initiation and progression. Given that IL-1β upregulation induces aberrant myeloid differentiation of hematopoietic stem and progenitor cells (HSPCs), we hypothesize that IL-1β plays a critical role in MDS pathogenesis, and targeting IL-1β signaling will overcome aberrant MDS HSPCs' myeloid skewing. Here, we report final results of a phase 2 study evaluating the safety and efficacy of canakinumab, an IL-1β inhibitor, in patients with lower-risk MDS or chronic myelomonocytic leukemia (CMML). Methods We conducted a single center, open-label phase 2 trial at The University of Texas MD Anderson Cancer Center (Houston, TX, USA). Patients aged ≥ 18 with IPSS-R ≤ 3.5 MDS/CMML who have previously been treated were included in the study. Patients received 300 mg of canakinumab on day 1 of a 4-week cycle. The primary objective was to determine the clinical activity of canakinumab in MDS/CMML patients. Response was assessed using the modified International Working Group 2006 criteria for MDS (Cheson et al. 2006). Secondary objectives were to evaluate the safety profile including rate of transfusion independence (TI), duration of response (DoR), progression-free survival (PFS), and overall survival (OS). All patients who received 1 dose of study drug were included in the analyses. This study is registered with ClinicalTrials.gov (NCT04239157). Findings Between August 2020 and May 2023, 27 patients were enrolled in the phase 2 portion of this study, and 2 patients failed screening. The median age was 74 years with 15 (60%) male patients; 20 patients (80%) were post-hypomethylating agent failure with a median number of prior lines of therapy of 2 (1-5). Transfusion dependency was observed in 24 patients (96%) prior to canakinumab initiation. Fourteen (56%) patients showed normal karyotype. IPSS-R stratification revealed an intermediate-2 risk in 12 (48%) patients and a high risk in one (4%). The most common mutations were SF3B1 (40%), TET2 (32%), DNMT3A (28%), and RUNX1 (24%). IPSS -M risk score was calculated for 24 cases showing one (4%) very low (VL), four (16%) low (L), 14 (16%) moderate low (ML), eight (32%) moderate high (MH), five (20%) high (H), and two (8%) very high (VH) risk categories. Canakinumab was well tolerated and no drug-related toxicities were observed. One death due to sepsis, which was deemed not treatment-related, occurred on the study drug. Out of 23 evaluable patients, the overall response rate was 17·4%, with erythroid and platelet hematological improvement (HI-E and HI-P, respectively) confirmed in 3 (13.0%) patients and 1 (4.3%) patient, respectively. Thirteen patients had stable disease (56·5%) and 6 (26·1%) progressed during therapy, 1 of which transformed to AML (Fig. 1). TI was achieved in 3 patients (median DoR 8·53 months (95% CI 0·41-16·1) and 2 of them maintained TI for over 12 months. With a median follow-up time of 22·6 months (95% CI 15·0-29·4), median OS was 17·3 months (95% CI 14·3-not estimable). We performed separate univariate analyses to evaluate any associations between the IPSS-M and OS/PFS. The median OS in patients with higher-risk MDS by IPSS-M (MH, H, VH) was 15·0 months vs 29·4 months in the lower-risk disease by IPSS-M (VL, L, ML) group (p=0·12). Interestingly, statistically significant findings were observed with 1-year PFS when stratifying patients into higher vs lower risk MDS by IPSS-M (64·3% vs 100·0%, respectively; p=0·022, Fig. 2). Conclusion In this cohort of MDS patients who had experienced multiple lines of prior therapy and exhibited high-molecular-complexity, canakinumab showed limited efficacy (HI 17.3%). Nevertheless, canakinumab showed a good safety profile and yielded sustained long-term responses in patients with single somatic driver mutation in TET2 or DNMT3A. This suggests that clonal complexity, and therefore disease burden, may be a determining factor in response to canakinumab. Therefore, we have amended the protocol of a phase 1/2 clinical trial evaluating the safety and activity of canakinumab in clonal cytopenia of unknown significance (CCUS) and low-molecular complexity MDS patients. Our results will clarify the role of IL-1β signaling in MDS initiation and progression.

Zinc As an Erythrocyte Stimulating Agent in Myelodysplastic Syndrome

Background: The anemia highly associated with myelodysplastic syndrome (MDS) has been linked to numerous transcriptional factor regulation abnormalities, specifically SMAD 2/3 upregulation that forms an inhibitory feedback loop to downregulate erythrocyte stimulation (ESA), which can have therapeutic implications. There is evidence that zinc contributes to erythropoiesis through multifactorial mechanisms involving regulation and transportation of necessary micronutrients including copper and iron. Zinc is involved in cell signaling pathways for erythropoiesis including acting as a cofactor in the SMAD 2/3 complex, TGF-β signaling, and incorporation of zinc into GATA-1 (Zinc finger transcription factor). The interaction with zinc in signaling cascades such as the growth hormone and insulin-like growth factor-1 pathways also result in increased erythropoietin transcription and therefore increased erythropoiesis. In addition, zinc is regarded as an important erythroid differentiation factor which progress maturation of erythrocytes. Due to the multifactorial role of zinc in erythropoiesis, including acting as a cofactor in the SMAD 2/3 complex, it may represent an inexpensive, and well tolerated ESA or additional micronutrient to test and supplement in MDS. This descriptive, retrospective study describes zinc deficient patients with MDS, who underwent zinc supplementation, and their changes in hemoglobin (HGB) over a 12-month period in an academic outpatient hematology center in California, United States of America. Objectives: Primary endpoints: determine if zinc supplementation in zinc deficient MDS patients will improve HGB by at least 1.0 g.dL by 1, 3, 6, and 12 month time points. Methods: Eight adult patients with MDS were identified to have zinc deficiency (plasma zinc concentration <65 ug/dL). Patients were not started on ESAs or their treatments were not changed for the management of their MDS. Patients were supplemented with Zinc Sulfate 50 mg daily and their HGB was followed over 12 months. A retrospective analysis for medical history and lab data at time of zinc deficiency diagnosis and at 1, 3, 6 and 12 month following initiation of zinc supplementation was completed. ANOVA, Wilcoxon rank-sum, and unpaired Students and t tests were performed to determine significant differences where p <0.05 is considered statistically significant. Results: Prior to supplementation, average HGB among patients was 8.01 g/dL, WBC was 3.65 g/dL, MCV was 97.6 fl, PLTS were 140g/dL. Following 1 month of supplementation average HGB was 8.5 g/dL (p = 0.5, n = 7), at 3 months average HGB was 9.27 g/dL (p = 0.087, n = 7), at 6 months average HGB was 10.3 g/dL (p= 0.006, n = 7), and at 12 months average HGB was 10.28 g/dL (p = 0.04, n= 7). Primary endpoint of HGB improvement by at least 1.0 g/dL was reached in 29% of patients at 1 month, 43% of patients at 3 months, 86% of patients at 6 months and 71% of patients at 12 months. Resolution of anemia was reached in 1 patient (12.5%) of patients within 12 months. Conclusions: Zinc supplementation improved anemia in MDS patients with incremental improvement in HGB over a 1 year time frame. There was statistically significant improvement from baseline HGB at the 3, 6, and 12 month time markers. Further study is warranted to determine the specific mechanism of zinc deficiency in MDS and its mechanism as an erythrocyte stimulating agent as well as further study in non-zinc deficient patients, various zinc supplementation doses, monitoring, and toxicity.

Myelodysplastic Syndrome Red Blood Cell Physiology Abnormalities and Their Correlation with Disease Risk and Therapeutic Interventions

PV and KT as well as VP and MA contributed equally Introduction: Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders characterized by ineffective hematopoiesis, different degree of cytopenias, and increased risk of transformation to AML. Anemia constitutes a main clinical manifestation contributing to increased morbidity and mortality. It is currently managed with red blood cell (RBC) transfusions, erythropoiesis stimulating agents (EPO), and recently,with luspatercept. The latter enhances late stage erythropoiesis and is indicated for Ring Sideroblasts (RS) MDS resistant to EPO. Several lines of evidence suggest that MDS pathophysiology is associated with structural and functional distortions of mature red cell (RBCs), such as hemolysis that in rare instances include acquired lesions like hemoglobinopathies and membranopathies.These abnormalities add variability to the clinical MDS phenotypes and could serve as biomarkers for early diagnosis or monitoring. However, they have been scarcely investigated. Patients and methods: We examined 40 MDS patients and 12 healthy sex- and age-matched controls. According to the IPSS-Rrisk stratification system, 8 patients were High Risk (HR) and 32 patients Low Risk (LR)MDS, of whom 8 under EPO (EPO-LR) and 4 under luspatercept (luspatercept-LR) treatment. None of them had received RBC transfusions in the past three months. Following full blood count analyses and RBC fractionations, we measured hemolysis (basal, osmotic, mechanical, and oxidative), eryptosis and oxidative stress, as well as the antioxidant capacity of the plasma through spectrophotometry,fluorometry and flow cytometry. Results: Compared to controls, the cohort of MDS patients was characterized by anemia (Hb 10.7 vs 13.6g/dL) and significantly higher HDWand RDW (17.5 vs. 13.4%) indexes. RBCs exhibited signs of eryptosis (RBC calcium concentration 7063±1443 vs. 6027±1541 RFU/mg; PS surface exposure 0.61±0.45 vs. 0.33±0.17%) and oxidative stress (higher metHb/oxyHb ratio; membrane-bound hemichromes 45.0±15.3 vs. 30.2±13.3). About 18% of the patients presented spontaneous hemolysis. Clinical severity correlated with the levels of cytopenias, and oxidative hemolysis. Anemia correlated positively with the age, and the levels of membrane-bound hemichromes, eryptosis and intracellular reactive oxygen species (ROS). According to ROC analysis, the combination of RDW, metHb/oxyHb ratio, and total Hb concentration is predictive of MDS, across all IPSS-R categories(area under the curve: 0.908; Figure 1). We then examined the subgroups of MDS patients. Apart from the RDW, the LR-MDS subgroup exhibited on average normal RBC features, in opposite to the more anemic and thrombocytopenicHR-MDS subgroup that showed pathologically increasedresistance to osmotic hemolysis, intracellular calcium (7946±1917 vs. 6026±1541RFU), and metHb/oxyHb ratio (18.5±7.3 vs. 10.4±3.9%), when compared to controls. Of note, similar abnormalities were observed in the EPO subgroup that further showed reticulocytosis, PS externalization, and membrane-bound hemichromes(p<0.05) Compared to the non-treated subgroup, the EPO-LR had lower WBC counts, but higher RDW, PS exposure (0.75±0.44 vs. 0.41±0.26%) and resistance to osmotic stress, aspreviously reported in other EPO-treated patients.Luspatercept-LR had higher WBC and PLT counts but exhibited EPO-like effects on RBCs, including pathological resistance to osmotic/mechanical lysis, ROS concentration (1347±453 vs. 960±160 RFU), Hb oxidation, and PS exposure (1.05±0.60 vs. 0.41±0.26%), compared to the non-treated LR subgroup. Conclusion: MDS RBCs are highly heterogeneous in terms of volume and Hb concentration and present signs of oxidative stress and eryptosis. Consequently, these easily accessible markers could be an accessory tool to the MDS diagnosis.In the context of ineffective hematopoiesis and increased apoptosis of precursor erythroid cells,the LR-MDS patients exhibit minimal RBC abnormalities. On the other hand, the expansion of BM blasts and the lower level of apoptosis in HR-MDS seem to affect RBCs in a hemoglobinopathy-like manner in terms of Hb oxidation, eryptosis signaling, and membrane ion channels. EPO and luspatercept treated LR patients exhibit improved cytopenias but worse RBC physiology most probably attributed to clinical factors like RS phenotype.

Comprehensive Characterization of Evolution of Genomic Complexity By Structural Variant and Mutational Profiling in Myelodysplastic Syndrome Patients with Hypomethylating Agent Failure

Introduction MDS patients (pts) who develop resistance to hypomethylating agent therapy (HMA-F) have dismal outcomes. Genomic aberrations underlying progression to HMA-F remains poorly understood. By sequencing, studies have demonstrated acquisition of new mutations affecting transcription factors such as FLT3 and RAS-MAPK pathway genes in a subset of cases. Until now, evolution of genomic complexity in HMA-F by acquisition of new structural variants (SVs) has not been systematically investigated. This is due to lack of genome-wide technologies to quantitatively identify all types of SVs at a gene/exon-level resolution (deletions/insertions/inversions/fusions etc..). In this study, we optical genome mapping (OGM), a novel, non-sequencing-based approach for accurate high-resolution SV detection, combined with ultra-deep coverage targeted NGS to comprehensively analyze genome-wide changes affecting both SVs and somatic mutations using paired baseline and progression samples from 21 MDS pts who developed HMA-F. Methods We identified all MDS pts with sufficient (1.5 million) cryopreserved, viable bone marrow (BM) mononuclear cells at both diagnosis (baseline) and HMA-F. All pts underwent BM morphologic exam, traditional banding analysis (TBA), and 81-gene targeted NGS panel (2500X coverage; LOD: 2% VAF; myeloid genes). We excluded pts with AML-defining abnormalities by standard-of-care work-up (per WHO 2022 criteria) and CMML pts. OGM was performed using standard procedures: extraction of ultra-long DNA molecules, sequence-specific fluorophore labeling and stain, followed by loading on to nanochannels and imaging (Saphyr; Bionano Genomics). Rare Variant Pipeline was used for detection of clinically significant SVs [minimum 300X coverage; 5000 bp size cut-off). We used bidirectional paired genome-wide analysis for identifying unique SVs seen exclusively at either baseline or HMA-F. Results There were 21 pts [11 men; 10 women; median age: 73 (43-89)] with available cryopreserved cells for comprehensive mutational and SV profiling. Median BM blast% was 5 (1-18). The median time to HMA failure was 11 (6-21) months. All pts had at least 1 genomic aberration: 14 (66%) by TBA and 20 (95%) by NGS (median: 3 mutations per pt.). Seven (35%) pts had normal karyotype (NK), 6 (30%) pts had complex karyotype (CK). Most frequent gene mutations included TET2, ASXL1 TP53 [each seen in 7 pts, 33%], SRSF2 [6 (30%)], DNMT3A, RUNX1 and STAG2 (5 pts each, 25%). Baseline OGM identified multiple clinically significant SVs, 6 of which, seen across 4 (19%) pts, were cryptic by TBA. These included CBFB::MYH1 and KMT2A partial tandem duplication. OGM at HMA-F showed increased genomic complexity compared to baseline in 9 (43%) pts. Paired analysis comparing genome-wide SV burden at HMA-F with matched baseline sample revealed 306 new unique SVs at HMA-F not seen at baseline ( Fig 1). These included 30 deletions (8 pts), duplications (6 pts), inversions, intrachromosomal and intrachromosomal translocations (5 pts each). Recurrent aberrations included segmental deletions of chr 17, 1, 7, 9, 12 and 21, affecting ETV6, CDKN1B, IKZF1, RUNX1, FHIT and HOXA11 genes, exon-spanning duplication in RUNX1, and FANCA::NF1 and YTHDF3-SETBP1 fusions. Three (14%) pts (2 with CK, 1 with NK), showed a cataclysmic increase in the numbers of SVs (translocations/ inversions) indicative of chromoanagenesis ( Fig 2). In contrast, there were 29 SVs seen exclusively at baseline and not detected at HMA-F [intrachromosomal translocations affecting chr 3 ( FOXP1) and chr 7 ( CUX1), low-level IGH::BCL2, deletions and insertions]. Sequential NGS and TBA was available in 20 pts. At HMA-F, 13 (62%) pts developed additional mutations, most frequent in NRAS, followed by TP53, TET2, CSF3R, KIT, second mutation in CEBPA, DDX41 and WT1 genes. The median number of mutations per pt. was 4. The highest increase in SV burden by OGM was noted in pts who did not acquire new mutations by NGS at HMA-F (5/7; 71%). Seven (33%) pts showed cytogenetic clonal evolution at HMA-F by TBA. OGM identified new SVs at HMA-F in 2 (50%) pts who did not show clonal evolution by NGS or TBA. Conclusions HMA-F MDS is characterized by increased genomic complexity by acquisition of new somatic mutations (NGS) and SVs (OGM), highlighting their complementary role in MDS progression. OGM further highlighted cataclysmic genomic changes by chromoanagenesis in a subset of MDS pts at HMA-F.