High-risk Myelodysplastic Syndrome Research Articles

Altered RNA Export in SF3B1 Mutants Increases Sensitivity to Nuclear Export Inhibition

SF3B1 mutations are the most frequent spliceosomal alterations across cancers, yet no successful therapy exists to target this pathway. Previous findings from a phase 2 clinical trial of the XPO1 inhibitor selinexor in patients with high-risk myelodysplastic syndrome (MDS) relapsed or refractory to hypomethylating agents (HMA) revealed increased activity in patients with SF3B1 mutations. XPO1 (Exportin-1) is responsible for the nuclear export of over 200 proteins, but also plays a role in the transport of multiple RNA species, including small nuclear RNAs (snRNAs), ribosomal RNAs (rRNAs), and select messenger RNAs (mRNAs) out of the nucleus. Given the role of XPO1 in exporting snRNAs, which form the catalytic portion of the spliceosome, we hypothesized that XPO1 inhibition may preferentially affect SF3B1-mutant cells via altered splicing, and that SF3B1-mutant high-risk MDS patients would have a better response to rational targeted drug combinations with next generation XPO1 inhibitors. To evaluate the mechanism underlying the preferential sensitivity of SF3B1-mutant cells to XPO1 inhibition, we performed nuclear and cytoplasmic fractionation followed by RNA sequencing (subcellular RNA-seq) before and after XPO1 inhibition in SF3B1 wildtype and SF3B1 K666N mutant cells. Whole transcriptomic analysis of subcellular RNA-seq data revealed increased global retention of RNA transcripts in the nucleus after XPO1 inhibition in the SF3B1 mutant cell line ( Figure 1). Validation assays using qPCR confirmed nuclear retention of several key transcripts such as Salt Inducible Kinase 1 (SIK1) and Cancer/Testis Antigen Family 45 Member A2 (CT45A2) in SF3B1-mutants (both SF3B1 K666N and K700E). Similarly, we performed subcellular RNA-seq for small RNAs and found snRNAs to be increased in the nucleus after XPO1 inhibition in the SF3B1 mutant cells. We then performed total cellular RNA sequencing to understand the effect of XPO1 inhibition on global RNA expression and RNA splicing. Differential gene expression analysis revealed that XPO1 inhibition had a more pronounced effect on cell cycle in SF3B1 wildtype cells, but differentiation pathways were more significantly affected in SF3B1-mutant cells. Alternative splicing analysis showed increased 3' alternative splicing events in SF3B1 mutant after XPO1 inhibition. These results suggest that the preferential sensitivity of SF3B1 mutant cells to nuclear export inhibition arises through increased nuclear retention of spliceosomal snRNAs and select mRNAs that results in perturbation of differentiation pathways. Despite the promising efficacy of XPO1 inhibition in SF3B1 mutated MDS, toxicity limits its dose escalation. Combination therapies may allow for greater synergism with lower doses of XPO1 inhibitor. In order to identify novel drug combination targets, we employed whole genome CRISPR screens in two myeloid leukemia cell lines treated with eltanexor, a next generation XPO1 inhibitor with lower toxicity than selinexor ( Figure 2). These analyses identified several novel targets, which we validated by testing for synergy in combination with eltanexor in wildtype and SF3B1-mutant cell lines. Using the Bliss independence model to calculate synergy, we identifed two drugs that greatly synergized with eltanexor specifically in the SF3B1 mutant cell lines: venetoclax (a BCL2 inhibitor), and navitoclax (a BCL2/BCL-XL inhibitor). We further validated these combinations in vivo using competitive transplant assays in Sf3b1 K700E conditional knock-in mice. The combination of eltanexor with venetoclax showed a significant decrease in the Sf3b1-mutant burden in the peripheral blood and bone marrow progenitor compartments, suggesting a preferential sensitivity of the combination for SF3B1 mutation. In conclusion, by analyzing the effects of XPO1 inhibition on RNA export, our study provides insight on the mechanisms behind the increased efficacy of XPO1 inhibition in SF3B1-mutant MDS/AML. Our findings may also contribute to the development of potentially synergistic therapeutic combinations. In this regard, recent human data have shown that venetoclax can overcome the poor prognosis of spliceosomal mutant AML patients (Senapati et al, Blood 2023); therefore, combining eltanexor with venetoclax could represent a promising SF3B1-specific therapy.

Phase 1 Study of BXCL701, a Dipeptidyl Peptidase Inhibitor, in Relapsed/Refractory Acute Myeloid Leukemia and High-Risk Myelodysplastic Syndrome

Background and Significance: BXCL701 is an oral small molecule inhibitor of dipeptidyl peptidases (DPP)-primarily DPP8/9-that has been extensively studied in humans with metastatic castration-resistant prostate cancer (both small cell neuroendocrine and adenocarcinoma), non-small cell lung cancer, melanoma, non-Hodgkin's lymphoma, and GI malignancies. Inhibition of DPP8/9 triggers the inflammasome to alert and prime immune cells, leading to induction of IL-18 and IL-1ß, bridging innate and adaptive immunity. Preclinical studies have demonstrated that BXCL701 may have direct induction of apoptosis in AML cells through activation of CARD8 and subsequent activation of inflammatory cytokines and Gasdermin D. Mouse model studies demonstrated a reduction in AML blasts and AML tumor size when treated with BXCL701. We hypothesize that BXCL701 can be safe and effective in patients with relapsed/refractory AML or patients with relapsed/refractory MDS with blasts ≥ 10%. Study Design and Methods: This is a Phase 1 single center investigator-initiated trial of BXCL701 in patients with relapsed/refractory AML or relapsed/refractory MDS with blasts ≥10% (ClinicalTrials.gov Identifier: NCT05703542). In the lead-in stage, we will use a 3 + 3 design with 4 dose levels of BXCL701 (0.2 mg, 0.4 mg, 0.6 mg and 0.8 mg). Doses of BXCL701 will be given on Days 1-3, Days 8-10, Days 15-17, and Days 22-24 on a 28-day cycle. The primary objective is to evaluate the safety of BXCL701 in the AML or MDS with ≥ 10% blasts. Secondary objectives include determining the maximum tolerated dose or recommended phase 2 dose, pharmacokinetics, and to estimate response, duration of response and overall survival. Exploratory endpoints include: pharmacodynamic profiling, T-cell response, inhibitory effect on DPP8/9, and evaluation of potential biomarkers such as Copy Number Variants and mRNA levels of DPP8, DPP9, FAP, Caspase-1, Pro-caspase-1, NLRP1, and CARD8. Major eligibility criteria include: ≥18 years of age, relapsed or refractory AML or relapsed or refractory MDS with ≥10%.refractory to at least 4 cycles of hypomethylating agent, ECOG performance status ≤2, adequate renal function (CrCl ≥30 mL/min), adequate liver function (total bilirubin ≤1.5 x ULN, ALT and AST ≤3 x ULN), WBC <25,000/µL on Day 1 Cycle 1 (hydroxyurea permitted), no symptomatic CNS disease, no active viral or bacterial infections, and >100 days from allogeneic bone marrow transplant with no active graft versus host disease. Patients on gliptin therapy are not permitted on the study due to drug interaction; patients are also excluded if they have a history of orthostatic hypotension or uncontrolled hypertension. There will be a second phase of the study that will evaluate BXCL701 in combination with a hypomethylating agent (decitabine or azacytidine) and venetoclax. Dose selection of BXCL701 in the combination stage will be based on the data from the single agent study. Conclusion: The trial is currently open and continuing to enroll.

Prognostic Impact of Chromosomal Abnormalities in TP53-Mutated Acute Myeloid Leukemia and High-Risk Myelodysplastic Syndromes

Background: Background: Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) exhibit clinical heterogeneity, and genomic profiles play a crucial role in disease characteristics and survival outcomes. Elli Papaemmanuil et al. classified AML into 11 genomic subgroups (NEJM, 2016), where TP53 mutations and aneuploidy predicted dismal survival. Cases with TP53 mutations are often accompanied by complex chromosomal abnormalities (CAs), but the prognostic impact and target genes of CAs have not been fully studied. Methods: To clarify this issue, specimens of AML and MDS with excess blasts (MDS-EB) were collected from 1,001 patients enrolled at collaborating institutes of Kyoto Hematology Study Group between July 2017 and January 2023. Targeted capture sequencing with 1,216 single nucleotide polymorphism sites was used to analyze primary samples, allowing assessment of copy number alteration (CNA) profiles. The target gene panel included common mutations in myeloid neoplasms. Mutation calling was performed using the established pipeline Genomon 2. Prognostic impact of CNA, single nucleotide variants, and insertion/deletion with 10% or more frequency was evaluated, and the frequencies of CNA were calculated for the short and long arms of chromosomes 1-22 and the X chromosome. Univariate and multivariate analyses were conducted using statistical significance criteria of p < 0.05. Results: The average sequencing depth was 548. Among 1,001 patients, 224 (22.4%) harbored TP53 mutations. TP53-mutated cases had a median age of 70 (24-95) years old at diagnosis, with 117 (52.2%) being AML and 107 (47.8%) MDS-EB. TP53 biallelic mutation, defined by 2 or more mutations in TP53 or loss of heterogeneity of TP53 locus, was present in 200 (89.3%) of cases, more commonly associated with 3 or more CAs or CNA (complex karyotype (CK)-like) than monoallelic mutation (99% vs. 42%, p = 1.9x10 -13). The most frequent affected lesions were del(5q), del(7q), del(7p), del(12p), del(18q), gain(11q), and gain(21q) with frequencies of 76%, 59%, 34%, 33%, 28%, 27%, and 26%, respectively. In survival analysis, gain(21q), gain(19p), del(16q), del(3q), del(19p), del(13q), and del(4q), along with diagnosis of AML ( p = 2.0x10 -2), TP53 biallelic mutation ( p = 6.8x10 -3), and CK-like ( p = 3.4x10 -3), were associated with poor outcomes in univariate analysis. Multivariate analysis revealed gain(19p), del(3q), del(13q), del(4q), diagnosis of AML, and CK-like as independent poor prognostic factors. Here we have defined gain(19p), del(3q), del(13q), and del(4q) as “Risk CNA”. A significant difference in outcomes was observed between patients with and without Risk CNA (n = 110 and n = 114, respectively, Hazard ratio = 2.6, p = 6.0x10 -8): The 6-month survival rate was 70 (95% Confidence interval [60-79]) % for patients without Risk CNA and 42 [31-52] % for those with Risk CNA. Patients with two or more Risk CNAs had worse prognosis than those with one ( p = 2.7x10 -2), indicating independent or additive contributions to outcomes. Similar trends were observed when limiting the analysis to TP53-biallelic cases. Further investigation of Risk CNA target genes was performed by identifying commonly amplified/deleted lesions. Target genes of gain(19p) included DNM2, CARM1, SMARCA4, LDLR, and EPOR, while RB1 and TET2 were potential candidates of del(13q) and del(4q), respectively. Identification of candidate genes for del(3q) was challenging due to broad common deleted lesions. Conclusion: Chromosomal abnormalities stratify the prognosis of AML and MDS-EB with TP53 mutation. The number of Risk CNA also impacts survival. Investigation of target genes of Risk CNA suggests potential therapeutic targets for TP53-mutated myeloid neoplasms, warranting further study for treatment optimization.

Reconsidering the Significance of Blast Cut-Offs in Differentiating between MDS and AML Biology

Background The diagnosis of and distinction between acute myeloid leukemia (AML) and high-risk myelodysplastic syndrome (MDS) have long been based on blast counts, with the presence of ≥20% myeloblasts in marrow or peripheral blood required to define AML in World Health Organization classification. Until 2001, blast counts greater than 30% had been used to diagnose AML by the original French-American-British classification system. However, the 2022 International Consensus Classification proposed a new combined category of MDS/AML with 10-19% blasts. Here we report the real-world treatment pattern and outcomes of AML and high-risk MDS patients at an academic institution in the United States, with specific attention to the blast burden at initial disease diagnosis to validate accuracy and limitation of this new proposed change. Methods This is a retrospective study conducted at UCLA Health encompassing all diagnoses of AML and MDS made at academic campus and community practices. We evaluated adult patients with a diagnosis with either AML or MDS from 1/1/2016-7/31/2022. For patients with more than one bone marrow biopsy on record, we recognized the date of diagnosis of MDS or AML with a blast burden ≥ 10% by immunohistochemistry or flow cytometry as the first bone marrow biopsy. In light of the changing diagnostic definitions, we analyzed patients by cohorts of MDS/AML with 10-19% blasts, AML low-blast with 20-29% blasts, and AML high-blast ≥ 30% blasts. Survival analysis by blast count at diagnosis was calculated by Kaplan-Meier estimates. A Multivariable Cox proportional hazard model was used to model survival outcomes by blast cohort, ELN risk 2022, response to first treatment, and receipt of allogeneic hematopoietic cell transplantation (HCT). Results There were 422 patients at UCLA Health with a diagnosis of AML or MDS included in our analysis with blast burden ≥ 10%. Patient demographics are shown in Table 1. Median age at diagnosis for MDS/AML, AML low-blast and AML high-blast cohorts is 67, 66 and 64 respectively (p=0.02). Thirty two (66.7%) patients in MDS/AML cohort received non-intensive first line treatment, whereas 202 (67.8%) AML high-blast patients received intensive first treatment. For AML low-blast patients, 37 (48.7%) received intensive treatment, and 29 (38.2%) received non-intensive treatment. More patients with AML (either low- or high-blast) underwent allogeneic HCT compared to MDS/AML patients. By Kaplain Meier analysis (KM) using blast counts as the sole predictor (Figure 1), no significant difference in OS when following patients for up to five years from diagnosis (p=0.0985) was seen. The p-value for two-way KM comparisons for MDS/AML vs. AML low-blast is 0.9682, and for AML low-blast vs. AML high-blast is 0.112. By multivariable analysis, ELN Risk 2022 (p<.0001), allogeneic HCT (p<.0001), and Complete Response to initial treatment (p<.0001) were predictive of OS, but blast percentages were not (p=0.56). Conclusion In our study, we found similar survival outcomes in high-risk MDS and AML patients regardless of initial blast counts at diagnosis. Though not statistically significant, MDS/AML and AML with low-blast burden appear to share more similar outcomes. This could be due to increased secondary AML seen in the low-blast cohort, and implies potentially different disease biology between high blast burden AML and AML with dysplastic changes. With this observation, we emphasize caution on simply using ICC 2022 to apply traditional AML therapy to the new MDS/AML category. Both WHO and ICC classification in 2022 reflects the increasing potential of molecular profiling to subtype these diseases, which will be the future direction. Rather than moving the blast count cutoff between AML and MDS, it is more critical to emphasize inclusion of high-risk MDS patients on all AML clinical trials and perhaps the converse for low blast burden AML with dysplastic changes, which might allow better understanding of response to therapy and disease biology.

Novel Clinically Useful Inhibitor of Mediator Complex, RVU120, Relives Differentiation Block in MDS/AML

Background: Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemias (AML) are characterized by a block in hematopoietic differentiation that can often be traced to aberrant stage-specific transcription. The kinase module of the mediator complex, consisting of CDK8, its paralog CDK19, Cyclin C, MED12, and MED13, have been identified as a molecular switch that regulates gene expression. A variety of cancers have exploited this mechanism to maintain stemness and an undifferentiated state. RVU120 is a first-in-class, specific, and selective inhibitor of CDK8/19, currently in Phase Ib clinical trial in patients with AML or High-Risk MDS (HR-MDS) (NCT04021368). RVU120 has been shown to stimulate erythroid differentiation in transformed CD34+ cord blood (CB) cells and in CD34+ cells from Diamond-Blackfan Anemia (DBA). To evaluate the further therapeutic potential of CDK8/19 inhibition, we confirmed aberrant expression of mediator complex components and erythroid-stimulating activity in a collection of CD34+ primary samples of AML and MDS. Results: We investigated the expression of mediator complex components in a large database of MDS CD34+ marrow samples and age-matched controls. Our findings revealed that MED12, a critical component of the mediator complex, was significantly overexpressed in MDS samples from refractory anemia with excess blasts (RAEB), a higher risk subset of MDS(p=0.018) (Fig A). Furthermore, we observed that this overexpression of MED12 was associated with a higher rate of transformation to AML. To delve deeper, we examined the expression of MED12 in sorted leukemic stem cells from primary AML samples and found significantly elevated levels in Long Term Hematopoietic Stem Cells from AML samples with both normal karyotype and complex karyotypes compared to healthy controls. Next, we conducted an evaluation of the efficacy of the mediator kinase module inhibition in MDS-derived cell line (MDS-L). Inhibition of CDK8/19 with RVU120 led to increased erythroid differentiation in this model which confirmed our observations in transformed CD34+ CB cells in CB cells, these phenotypic changes were correlated with the inhibition of CDK8-dependent phosphorylation marker S726 on STAT5 and a profound increase in the expression of genes involved in erythroid commitment and hemoglobin metabolism. In order to confirm therapeutic potential of CDK8/CDK19 inhibition in MDS and AML, we performed colony-forming assay on primary samples of MDS and AML (n= 15). The treatment with RVU120 resulted in increased erythroid differentiation in a majority of samples, evident from an augmented number of colonies. Detailed analysis indicated changes in the expression of erythroid differentiation markers, including increased CD71 and Glycophorin A expression on colonies, as assessed by FACS analysis (Figure B, shows representative sample). Conclusion Our data highlight that inhibition of the overexpressed mediator complex proteins can alleviate the differentiation blocks seen in MDS/AML. These results provide a compelling rationale for further developing the CDK8/19 inhibitor RVU120 for transfusion- dependent MDS/AML patients.

Combination of Decitabine and Etoposide Is Highly Effective in Treating p53-Mutated MDS/AML Via Activating Notch Signaling

The elderly MDS/AML patients, especially those with TP53 mutations, remain very difficult to treat and resistant to chemotherapy. A total of 105 elderly (age above 60 years) high risk MDS (n = 84, IPSS-R score > 4.5) or AML-MRC (n = 21) patients' records were collected from three medical centers across China and retrospectively analyzed. Decitabine and low dose etoposide was found to be superior to standard decitabine alone in MDS/AML patients with higher ORR (70.0% vs 52.7%) after two cycles of treatment, the longer median durations of ORR (24.0 months vs 12 months) and CR (38.0 months vs 24.0 months), and longer event-free survival (13 months vs 9 months, p = 0.023), without significant improvement in overall survival (20 months vs 15 months, p = 0.059). Other than the TP53 mutation, no other gene mutation (including NRAS, DNMT3A, TET2, EZH2, ASXL1, RUNX1) in the D+E group could reliably predict OS and EFS when compared to wild type phenotype. Patients with TP53 mutations showed higher response rate after two cycles of D+E therapy and had prolonged overall survival (Mutation vs WT: 31 months vs 9 months, p=0.012) and event-free survival (Mutation vs WT: 24 months vs 5 months, p = 0.022) compared with p53 wild type patients in D+E group from multivariate survival analyses. P53 mutated patients receiving D+E treatment also had significantly improved OS (31 months vs 12 months, p = 0.00) and EFS (24 months vs 8 months, p = 0.00) compared to patients receiving decitabine treatment. Moreover, we found decitabine and etoposide greatly reduced TP53 mutant AML tumor burden in the NSG mice xenografts (tumor burden: 92% reduction in p53-null xenograft mice, 65% reduction in p53-wild-type xenograft mice on day 20 after leukemia cells administration, p < 0.0001). It was found that TP53 mutant cells and p53 gene-editing MOLM13 cells showed terminal neutrophil differentiation in the combo of decitabine and etoposide, demonstrating the expected changes in cell-surface CD11b expression and neutrophil functional assays such as phagocytosis. Combo of decitabine and etoposide showed much higher percentage of differentiation marker expression compared to single drug decitabine or etoposide treatment. However, neither the p53 gene-edited THP-1 cells nor the TP53 wild type cell lines displayed markers of terminal differentiation, such as elevated CD11b expression or phagocytosis. Furthermore, the percentage of CD11b-positive cells increased significantly in p53-mutated primary cells while insignificantly in TP53 wild type cells. Decitabine and etoposide induced significant differentiation thus killing p53 mutated myeloid tumor cells and primary patients' cells in vitro. We also uncovered the underlying mechanism that why D+E could induce significant differentiation in TP53 Mutant while not in TP53 wild type myeloid tumors. Notch1 signaling pathway was activated after D+E treatment in TP53 Mutant myeloid tumors, which was consistently correlated with drug-induced myeloid differentiation. Notch1 knockout or Notch inhibitor incubation abrogated drug induced differentiation in TP53 Mutant myeloid tumors. The activation of the Notch1 signaling pathway has been observed to induce disease regression and decrease leukemia stem cells in AML mice as well as significant apoptosis in the AML patient sample. We also found D+E could upregulate transcription factor transcripts such as ETS1, ETS2, which could bind specifically to PSEN1 (PS1) promoter and activate its transcription directly. PS1 represent catalytic subunit of γ-secretase complexes and is responsible for Notch recognition. While in TP53 wild type AML cells, p53 might interact with p300. p300 acts as a coactivator of Ets1 and Ets2, competition for binding to p300 with p53 may confront p300 recruitment and repress ETS transcription activation directly, which might exert its effect on PS1 repression, thus inhibiting γ-secretase complex activity. In short, integrated p53 could repress PSEN1 transcription thus interfering gamma secretase complex activity, Notch recognition and cleavage as well as abrogating drug induced Notch activation and myeloid differentiation, which could be achieved in p53 mutated state. Combination of decitabine and etoposide regimen is also relatively safe and tolerable in elderly patients, which may open up a new path of therapy in the p53 mutated AML/MDS.

Impact of Post-Transplant Cyclophosphamide (PTCY)-Based Prophylaxis in Matched Sibling Donor Allo-HCT for Patients with Myelodysplastic Syndrome: A Study on Behalf of the Chronic Malignancies Working Party of the EBMT

Background Allogeneic hematopoietic cell transplantation (allo-HCT) remains the only potentially curative treatment option for patients with myelodysplastic syndrome (MDS). The use of PTCY for GVHD prevention is becoming increasingly prevalent across donor types/transplant platforms. However, limited studies have evaluated the efficacy and safety of using PTCY in patients with MDS undergoing matched sibling donor (MSD) allo-HCT. We hereby compare the outcomes of PTCY-based versus (vs.) conventional GVHD prophylaxis in a contemporaneous cohort of MDS patients undergoing MSD allo-HCT from the EBMT registry. Methods A total of 404 MDS patients undergoing first MSD peripheral blood allo-HCT between 2014 and 2020 in 52 participating centers, receiving either PTCY or other GvHD prophylaxis, were included. Primary outcomes were overall survival (OS), progression free survival (PFS), GVHD-free/RFS (GRFS), competing risks analyses for relapse (CIR), non-relapse mortality (NRM), and GVHD. The main comparison was PTCY vs. other types of GvHD prevention. Cox regression analyses with predefined covariates were used to obtain adjusted effect estimates of PTCY in post-transplant results.- Results The median age at allo-HCT was 57 years (IQR 49-62), 66 patients (16.3%) received PTCY, 338 (83.7%) received other prophylaxis. As reported in Figure 1, patient characteristics, disease risk, and pre-transplant status were balanced between the two cohorts. The majority of transplant characteristics were also analogous except for the proportion of adults receiving myeloablative regimens, which was higher in the PTCY group (52.3% vs. 38.2%, P=0.047). PTCY was combined with two additional immunosuppressant agents in 66.7% of the cases, with one additional immunosuppressive drug (generally calcineurin inhibitors (CNI)) in 31.8%, and administered as a single agent in only 1 (1.5%) case. Other prophylaxis mainly combined CNI with either mycophenolate mofetil or methotrexate, and in 162 (47.9%) of the cases, anti-thymocyte globulin was also administered. Post-transplant complications and outcomes are shown in Figure 1. Incidences of Day +28 neutrophil (68% vs. 97%, P=0.011) and platelet (71% vs. 92%, P<0.001) engraftment were lower in patients who received PTCY. The day +100 cumulative incidences (CI) of grade II-IV and III-IV aGVHD, and 5-year CI of extensive cGVHD were 32% (21-43%), 18% (9-27%) and 18% (9-28%) for patients who received PTCY, and 25% (20-30%; p=0.3), 13% (10-17%; p=0.4), 31% (26-36%; p=0.09) for those who did not. The 5-year CIs of cardiac and pulmonary toxicities were similar in both groups (23% (6-40%) vs. 19% (12-25%) (p=0.6) and 23% (8-38%) vs. 27% (21-34%) (p=0.6) in PTCY vs others, respectively). At 5-years after allo-HCT, the estimated OS (51% (39-64%) vs. 52% (46-58%), p=0.6), PFS (48% (36-61%) vs. 46% (40-52%), p=0.9), and GRFS (33% (21-45%) vs. 25% (20-30%), p=0.6), were similar between the two study groups. Nevertheless, patients who received PTCY tended to have lower CIR (20% (10-29%) vs. 33% (28-38%) (p=0.06)), but higher NRM rates (32% (20-43%) vs. 21% (16-25%), p=0.09) than the rest. Next, we performed a multivariable analysis (MVA) of OS, PFS, NRM and CIR (Figure 1). The results observed in the MVA confirmed that using PTCY in patients with MDS undergoing MSD allo-HCT resulted in comparable OS (HR 1.23 (0.77-1.97), p=0.4) and RFS (HR 1.13 (0.73-1.77), p=0.6) to conventional prophylaxis. Furthermore, the diagnosis of a high or very high-risk MDS (according to IPSS-R), and the presence of TP53 mutation at diagnosis were independent predictors of worse post-transplant outcomes. In addition, PTCY-based prophylaxis was associated with a higher NRM (HR 1.79 (1.07-2.98), p=0.03) when adjusted by variables known to be clinically relevant for NRM. Conclusion The use of PTCY in adults undergoing MSD allo-HCT for MDS resulted in comparable OS and RFS rates to conventional GVHD prophylaxis. Allo-HCT performed with PTCY was associated with a longer time to engraftment and comparable incidences of clinically relevant aGVHD. Of note, utilization of PTCY was associated with a non-significant trend to a lower incidence of extensive cGVHD. Furthermore, although a non-significant trend to lower rates of relapse were observed in allo-HCTs performed using PTCY, it use was associated with a higher risk for NRM. Further evaluation of PTCY in the MSD setting for MDS is required.

Molecular Measurable Residual Disease (MRD) Clearance (≤1%) Is Associated with Improved Clinical Outcomes in Patients with Higher-Risk Myelodysplastic Neoplasms (HR-MDS): An Exploratory Analysis of Stimulus-MDS1 in Patients Receiving Sabatolimab or Placebo + Hypomethylating Agent (HMA)

Background: Sabatolimab (MBG453) is a novel immunotherapy targeting TIM-3, an immuno-myeloid regulator expressed on both immune and leukemic stem cells. In the randomized, double-blind, placebo-controlled, Phase (Ph) II STIMULUS-MDS1 study (NCT03946670) in patients (pts) with HR-MDS, although improvements in complete remission (CR) and progression-free survival (PFS) were not statistically significant, sabatolimab + HMA was associated with longer duration of response (DoR), and the data suggest a delayed-onset benefit in the sabatolimab arm, as well as a potential treatment effect in pts with lower disease burden (Zeidan AM, et al. ASH 2022). The clinical significance of MRD in HR-MDS is not well established. We report an exploratory analysis of mutational clearance and MRD by next-generation sequencing (NGS) to evaluate the association with clinical outcomes. Methods: Treatment-naive pts aged ≥18 years with intermediate (+ ≥5% bone marrow [BM] blasts), high or very high risk MDS by Revised International Prognostic Scoring System were randomized to sabatolimab or placebo added to azacitidine/decitabine (Zeidan AM, et al. ASH 2022). Peripheral blood (PBMC) and/or BM mononuclear cell (BMMC) samples were collected and NGS performed on genomic DNA extracted from baseline (BL) and on-treatment samples using a 38-gene NGS-error-corrected panel with sensitivity of 2% and 0.2% variant allelic frequency (VAF), respectively. Concordance and correlation between PBMC and BMMC were assessed. The prognostic value of clonal clearance at different VAF cut-offs (0.2%, 0.5% or 1%) was assessed. MRD-x status was defined as any mutation call above VAF cut-off x, excluding mutations in DNMT3A, TET2 and ASXL1 ( DTA). Association between MRD status and clinical outcomes was analyzed regardless of treatment to increase sample and used best overall response, 6-month landmark analysis, and a time-dependent Cox model to rule out immortal bias. Results: NGS was run on 332 BMMC samples from 112 pts and 439 PBMC samples from 123 pts (127 pts randomized). 112 pts had ≥1 qualifying BL mutation (NGS cohort) and 106 pts (sabatolimab + HMA, N=56; placebo + HMA, N=50) had ≥1 at follow-up (MRD cohort). BL characteristics were balanced between NGS/MRD cohorts and between treatments in the MRD cohort. Overall agreement in mutation calling at variant level between BMMC and PBMC was >97% (8312/8566 total mutation calls), with >88% positive (817/926) and >98% negative agreement (7495/7640). Across reported mutated variants (N=817), there was good correlation (pearson cor=0.88) in VAF between BMMC and PBMC. When considering all paired samples, concordance of best MRD status using PBMC or BMMC results at the pt level (N=95) was: MRD-0.2, 97%; MRD-0.5, 89%; MRD-1, 90%. Given good PBMC and BMMC agreement, results were combined (higher VAF used if both) for outcomes analysis. In the MRD cohort, 11% (N=12), 18% (N=19) and 27% (N=29) of pts achieved MRD-0.2, MRD-0.5 and MRD-1 negativity, respectively. Most pts with MRD-0.2, -0.5 and -1 clearance had documented response of CR/marrow CR [mCR] (>80%) or CR/partial remission [PR]/hematologic improvement [HI] (>75%). Best overall response of MRD-1 negativity achieved anytime on treatment was associated with improved PFS and overall survival (OS) in the overall population and for responding pts (CR/mCR or CR/PR/HI). Similar results were obtained in a landmark analysis at 6 months ( Fig 1). This was supported by a time-dependent Cox-model indicating that MRD-x negativity was associated with a lower hazard ratio for PFS and OS vs MRD-x positivity ( Fig 2). A consistently higher proportion of pts in the sabatolimab (N=56) vs placebo (N=50) arms had on-treatment mutation clearance at different cut-offs: MRD-0.2, 16.1% vs 6.0%; MRD-0.5, 25.0% vs 10.0%; MRD-1, 35.7% vs 18.0%, respectively. Conclusions: We present the first results from a prospective, controlled, randomized study demonstrating the potential prognostic value of MRD for PFS and OS in HR-MDS. Our results demonstrate high NGS concordance between PBMC and BMMC on-treatment samples, indicating that PBMC could represent an alternative for clonal monitoring. Interestingly, more pts treated with sabatolimab + HMA reached MRD negativity while in remission, potentially explaining the longer DoR in pts receiving sabatolimab + HMA vs placebo + HMA. These findings will be further explored in the ongoing Ph III STIMULUS-MDS2 study (NCT04266301).

Sabatolimab in Combination with Hypomethylating Agents (HMAs) Was Safe in Patients (Pts) with Intermediate-, High-, or Very-High-Risk Myelodysplastic Syndrome (MDS)

CONCLUSIONS Complete remission (CR) with standard-of-care HMAs in pts with intermediate-, high-, and very-high-risk MDS is usually infrequent and transient, and prognosis remains poor for these pts, who have limited second-line treatment options. Sabatolimab is a high-affinity, humanized anti-TIM3 IgG4 monoclonal antibody that blocks the TIM3 immune checkpoint receptor, which is preferentially expressed on leukemic stem cells and blasts and plays a key role in regulating adaptive and innate immune responses. In a phase Ib study, sabatolimab in combination with intravenous (IV) HMA was safe and well tolerated in pts with high-risk MDS with overall response rates of 61.1% and 57.9% with decitabine and azacitidine, respectively. We present an interim analysis of the STIMULUS-MDS US trial (NCT04878432) to assess the safety of sabatolimab in combination with not only parenteral but also oral HMAs in pts with intermediate-, high-, and very-high-risk MDS. This is a multicenter, phase II, open-label, single-arm study of sabatolimab in combination with HMAs. Adults aged ≥18 years with intermediate-, high-, or very-high-risk MDS per Revised International Prognostic Scoring System criteria who were not previously treated with HMAs were eligible. Pts received an HMA of investigator's choice (decitabine/cedazuridine 35 mg/100 mg oral days 1-5, decitabine 20 mg/m 2 IV days 1-5, or azacitidine 75 mg/m 2 IV or subcutaneous [SC] days 1-7 or days 1-5 plus days 8 and 9) with sabatolimab 800 mg IV every 4 weeks starting at day 8 or any day between days 5 to 8. The primary objective of this study was to assess the safety of sabatolimab in combination with oral, IV, and SC HMAs. Secondary objectives included assessing the efficacy of the combination. The study includes a core phase for up to 12 months, an extension phase for efficacy and survival status up to 12 months after the core phase, and a post-treatment safety follow-up for 30 days following the last dose of HMA or 150 days following the last dose of sabatolimab, whichever is later. This interim analysis included 33 pts with intermediate-, high-, and very-high-risk MDS at the data cutoff of 6 months after cycle 1 day 1 of treatment with sabatolimab and an HMA. The median age was 69 years (range, 39-86). At data cutoff, 8 pts were in ongoing treatment, and 25 pts had discontinued the core phase. Reasons for discontinuation included new therapy for study indication (n=6), physician decision (n=6), pt decision (n=5), progressive disease (n=4), unsatisfactory therapeutic effect (n=2), adverse event (AE) (n=1), and death (n=1). Of those who discontinued study treatment, 6 pts were in ongoing evaluation in the extension phase and 3 pts were in ongoing evaluation in the post-treatment follow-up phase. The median duration (range) of exposure was 3.9 (0.92-5.91) months for sabatolimab and 4.2 (1.05-6.08) months for HMAs. Among 33 pts evaluable for safety, most experienced all-grade and grade ≥3 AEs, AEs requiring additional therapy, and serious AEs (Figure). However, the most frequent (≥10%) grade ≥3 AEs were expected hematologic AEs, including febrile neutropenia (42.4%), anemia (39.4%), neutrophil count decrease (39.4%), and platelet count decrease (36.4%). Nonhematologic grades ≥3 AEs occurring in >1 pt included hypertension (18.2%), pneumonia (9.1%), hypoxia (6.1%), and fall (6.1%). Only 1 pt was reported with encephalopathy leading to treatment discontinuation, which was not attributed to study treatment. One pt had a fatal AE for which the cause was not identified but not thought to be related to study treatment by the investigator. The best overall response results in up to 6 months of treatment were available for 22 pts. Marrow CR (mCR) with and without hematologic improvement (HI) was reported in 18.2% and 9.1%, respectively. Partial remission (PR) and stable disease (SD) with HI was reported in 4.5%, each. CR + mCR + PR was 31.8% (95% CI, 13.9%-54.9%), CR + PR + HI was 27.3% (95% CI, 10.7%-50.2%), and CR + mCR + PR + HI was 36.4% (95% CI, 17.2%-59.3%). This interim analysis of the STIMULUS-MDS US study demonstrated that sabatolimab in combination with an oral, IV, or SC HMA was safe and well tolerated, with AEs consistent with previous reports for pts with MDS treated with HMAs. Response rates for the combination are preliminary, and more information will be provided with longer follow-ups.

Clinical, Cytogenetic and Molecular Characterization of a 96 MDS and AML Cohort with TP53 Mutation

Introduction The last European LeukamiaNet (ELN) recommendations (Döhner H. et al. Blood. 2022) and the International Consensus Classification (ICC, Hasserjian R.P. et al. Virchows Arch. 2023) described a new entity, called myelodysplastic syndrome (MDS)/ acute myeloid leukemia (AML) with TP53 mutation ( TP53mut), comprising high-risk MDS with an excess of bone marrow blasts cells (more than 10%) and a TP53 mutation (variant allele frequency VAF ≥ 10%). They suggested that MDS and AML represent a biologic continuum rather than two distinct diseases separated by a blast cutoff. Among teams who worked on that topic, T. Grob et al. ( Blood. 2022) studied the molecular characterization of high-risk AML and MDS with TP53mut and suggested that these two types of pathologies should be considered as a single entity, with no difference in molecular characteristics or survival. In this study, we describe the clinical, cytogenetic, and molecular characteristics of a cohort of 96 MDS and AMLs with TP53mut. Patients and methods We included all the patients with AML, MDS/AML, MDS with increased blasts (MDS-IB1) and MDS-low blast (MDS-LB) with at least one TP53mut (VAF ≥ 1%) received between Aug. 2020 and Dec. 2022 at the Institut Paoli-Calmettes (IPC, Marseille, France). Clinical and biological data were extracted retrospectively from the AML IPC database. Morphologic and cytogenetic data were analyzed by standard techniques. NGS was performed using a custom targeted panel of 60 genes (Custom Myeloid Lymphoid Solution, SOPHIA GENETICS). Results We registered 96 patients with a TP53 mutation (VAF ≥ 1%), divided into AML (n=60), MDS/AML (n=14), MDS-IB1 (n=12) and MDS-LB (n=10). Median age was 71 (range, 28-88). Thirty-eight patients had been treated for a prior solid malignancy and 26 patients had a history of myeloid malignancy. TP53mut was detected at diagnosis for 85 patients and at relapse or during follow-up for 12 patients (no NGS data available at diagnosis). We detected 142 mutations in the TP53 gene, mostly missense (73%) between exon 4 and exon 11 with a median VAF of 39% (Figure 1). We found 3 hotspots mutations in positions 273, 248 and 220 with respectively 9, 9 and 8 mutations. Biallelic status (presence of ≥ 2 mutations or a mutation + a deletion) was found in 66 patients (69%). Karyotypes were available for 95 patients. The karyotype was abnormal in 88% of cases, complex in 71% and unfavorable in 80% of cases according to ELN classification or Revised International Prognostic Scoring System (R-IPSS). Complete NGS analysis revealed at least one mutation in 59 of the other genes of the panel in 67 patients (70%), the most frequent being DNMT3A (22%), TET2 (17%), ASXL1 (13%) and PPM1D (11%) (Figure 2). In our cohort of TP53mut LB-MDS (n=10), we found that 9/10 patients had co-mutations in DNMT3A (n=4) , TET2 (n=6) and PPM1D (n=2) genes, 7/10 with biallelic status and 3/10 with complex karyotype. AML and MDS/AML patients with TP53mut detected at diagnosis (n=65) were treated with intensive chemotherapy (n=13), VEN-AZA (n=26), non-intensive therapy (n=14) or best supportive care (BSC, n=12). Seventeen patients were treated with allogeneic stem-cell transplantation (ASCT). With a median follow-up (FU) of 13 months, median overall survival (OS) of the whole cohort was 7 months (ranges, 1-41). Median OS in the AML and MDS/AML group was 6 months (ranges, 1-41). Patients treated with non-intensive approach had the longest median OS (15 months) compared to patients treated with intensive chemotherapy and VEN-based approaches (8 and 6 months, respectively) and patients treated with BSC (1 month, p=0.02). Patients with MDS-IB1 and MDS-LB had 11-months median OS (ranges, 1-28). The four patients treated with upfront ASCT were still alive at the last FU date and have an estimated median OS of 18.5 months (ranges, 6-26). Conclusion Our data confirm that MDS and AML with TP53 mutation are frequently associated with complex karyotypes and have a poor prognosis. nterestingly we identified mutations in PPM1D gene, involved in TP53 pathway, in 11% of cases. Patients with AML or MDS/AML treated with non-intensive approach seem to have the longest OS (15 months). Patients with MDS-IB1 or MDS-LB treated with upfront ASCT seem to have a prolonged OS (18.5 months). Clinical, functional and mechanistic studies are required to complete these findings.

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).

Checkpoint Immunotherapy Is Associated with Preferential Activation of Tumor-Antigen Specific CD4 + T Cells in MDS

Background: Patients with intermediate and higher-risk MDS are generally treated with the DNA hypomethylating agents (HMAs) azacytidine and decitabine. These agents improve survival and cytopenias in ~50% of those treated; but only responders derive clinical benefit, and no pre-treatment response prediction has proven efficacy. There is thus an urgent need to develop new approaches to complement or enhance the response to standard-of-care HMA therapies. Studies suggest that HMAs might act in part via activation of anti-tumor immune mechanisms. Based upon this hypothesis a series of early phase trials combining immune checkpoint inhibitors (ICI: both anti-PD1/PDL1 and CTLA4) with azacitidine in MDS/AML patients were undertaken but clinical and immunological efficacy have proven disappointing. In these studies, the lack of antigen specificity made systematic assessment of the anti-MDS immune response and milieu challenging. Antigen specific assessments are particularly important in MDS patients treated with ICIs because distinct from solid tumors, myeloid stem cell cancers involve the populations of antigen presenting cells that modify the capacity for T-cell response, such as dendritic cells. Our group has shown that MDS/AML patients treated with decitabine show increased expression of NY-ESO-1, a well-characterized tumor antigen whose expression is normally suppressed through dense promoter hypermethylation. In our prior Phase I study, MDS patients received vaccination against NY-ESO-1 in combination with standard decitabine. This combination was not only safe, but it also induced NY-ESO-1 specific CD4 + and CD8 + T cell responses in most patients. We hypothesized that combining anti-NY-ESO-1 vaccination (an antigen specific approach) with decitabine and an ICI would allow us to understand the immune synapse in patients with MDS. Methods: We developed an investigator-initiated Phase 1 trial in transplant-ineligible patients with MDS/low blast count AML adding an anti-PD-1 ICI to our established combination of decitabine + NY-ESO-1 vaccination (#NCT 0335871). This open-label, non-randomized single center Phase 1 study used an HLA unrestricted NY-ESO-1 vaccine (CDX-1401 (1 mg) + poly-ICLC (1.8 mg)) in combination with standard dose decitabine (20mg/m2/d x 5 days) and nivolumab (3 mg/kg every 2 weeks), 4 cycles of combination therapy were planned on study; patients deriving clinical benefit could continue treatment at the discretion of the treating physician. The study was conducted in accordance with the Declaration of Helsinki and approved by the Roswell Park Cancer Institute (RPCI) Internal Review Board. Six patients were enrolled, treated, and completed > 4 cycles of decitabine; serial bone marrow and peripheral blood samples were collected for analysis. The combination was safe. Results: Two patients developed CR and the remainder had stable disease as the best response to therapy (Table 1). The majority of patients mounted NY-ESO-1 specific CD4 + T cell responses associated with up-regulation of anti-PD-1 immunotherapy gene signatures in the CD4 + memory T cell compartment. No responses in CD8 + T cells were seen. In seeking explanation for the disparate findings in CD4 + and CD8 + T-cell compartments, we examined conventional dendritic cell populations associated with activation of CD8 + T cells and identified that our patients had markedly reduced numbers of conventional dendritic cells marked by high expression of CD141 (cDC1). This population is critical for a successful response to immunotherapy. cDC1 from MDS patients also showed reduced expression of genes that are key for optimal T-cell activation and expansion. Conclusions: These results suggest that the use of immunotherapy to induce effective, cytotoxic anti-tumor T cell responses depends upon the myeloid immunologic milieu in MDS patients. The critical importance of cDC1 function has recently been described and appreciated in the context of solid tumor immunotherapy. The observation of both numerical and functional defects of this cell population in patients with myeloid neoplasia provides a possible explanation for the disappointing efficacy of immunotherapies in this disease state. Approaches to augment the number and function of specific cDC1 populations in myeloid disease might overcome this defect and thereby enhance the efficacy of immunotherapy for patients with MDS.

Decider-2: Prospective Randomized Multicenter Phase III Trial of Decitabine and Venetoclax Administered in Combination with All- Trans Retinoic Acid (ATRA) or Placebo in Patients with Acute Myeloid Leukemia Who Are Ineligible for Induction Chemotherapy

Background and Significance DNA-hypomethylating agent (HMA) based therapy is the treatment backbone for older, medically non-fit AML/MDS pts, and studies have been performed with multiple combination partners over the last years (reviewed by Stomper et al., Leukemia 2021). Recently, the combination of azacitidine or decitabine (DEC) with venetoclax (VEN) has become the novel standard for many of these pts. Based on preclinical evidence of priming of AML cells to all- trans retinoic acid (ATRA) by DEC, we have conducted a randomized phase II trial in 200 elderly and medically non-fit AML pts on the effect of ATRA as add-on to DEC (DECIDER-1, Lübbert et al., J. Clin. Oncol 2020). Median overall survival (OS) was 8.2 months with ATRA v 5.1 months without ATRA (hazard ratio, 0.65; 95% CI, 0.48 to 0.89; P = .006). Notably, ATRA did not add toxicity, the combination was active also in AML with adverse genetics, and it appeared to prolong time to secondary treatment resistance. A randomized open-label phase II trial (DEC -/+ ATRA) is also being conducted in higher-risk MDS pts (Zhou et al., ASH 2021, 539). Investigating a triple combination of HMA+VEN+ATRA is a logical next step. We could demonstrate cooperativity between these 3 drugs in AML cell lines irrespective of TP53 mutation status on cell growth inhibition and apoptosis (Bresser et al., ASH 2022, 11537). Study Design and Methods This academic study is a randomized, prospective, double-blind, placebo-controlled, parallel group, multicenter trial. It is planned to randomize 256 pts in a ratio of 1:1 to the treatment arms at ~35 sites in Germany. The primary objective is to compare the efficacy of ATRA v placebo as add-on to the standard treatment DEC+VEN with respect to OS. Secondary objectives are to compare ATRA v placebo as add-on to DAC+VEN with respect to objective best response (i.e. CR/CRi/MLFS/PR), CR with negative measurable residual disease (MRD), probability of survival with objective best response, health-related quality of life and safety. Sample size calculation is based on the primary endpoint OS. The study should have a power of 80% to demonstrate superiority of ATRA vs. placebo with respect to OS at one-sided level alpha of 2.5% under the assumption of a hazard ratio of 0.67. This requires the observation of 191 deaths. With a recruitment period of 3 years and an additional one year follow-up period, it is necessary to include 230 pts. To account for the possibility of up to 10% of the pts with incomplete follow-up, 230/0.9 = 256 pts will be randomized in total (ratio 1:1). Interim safety analyses will be performed after the 2nd cycle of the 10th randomized pt, and after the 3rd cycle of the 50th, the 100th and the 150 th randomized pt, and monitored by an Independent Data Monitoring Committee. Key inclusion criteria are: age ≥18 years; previously untreated AML (WHO 2016); ineligibility for standard induction chemotherapy; ECOG ≤ 2; WBC <25×109/L (prior cytoreduction allowed). Key exclusion criterion is: acute promyelocytic leukemia. Treatment All pts will be treated with DAC+VEN as standard treatment and will additionally either receive ATRA or placebo (Fig. 1). Study treatment is continued until relapse/progression, hematopoietic cell transplantation, death or unacceptable toxicity (whichever occurs first). Correlative investigations include serial MRD measurement, NGS profiling and apoptosis studies. Registration EUDRACT 2020-005495-36; DRKS 00023646 (P001516). Funding Funded by the German Research Foundation (DFG, project no. 367848349/LU 429/13-1); ATRA is provided free of charge by Cheplapharm (Germany). Figure 1 Overview of the study treatment

Phase I/II Clinical Trial Evaluating Azacitidine + Venetoclax + Donor Lymphocyte Infusion in Post-Transplant Relapse Myelodysplastic Syndromes and Acute Myeloid Leukemia: Preliminary Results of Ventograft, a GFM Study

Background: Azacitidine (AZA) is approved for the treatment of patients with high-risk MDS and in combination to Venetoclax (VEN) in AML. Beyond direct cytotoxic/epigenetic effects of AZA and ability to induce remissions even in patients refractory to chemotherapy, AZA has been shown to increase the immunogenicity of AML blasts by reexpression of key antigens [ Schroeder et al. Leukemia 2013]. Moreover, VEN imparts distinct cell death sensitivity and adaptivity patterns in T cells ( Ludwig et al. Cell Death Dis 2021). DLI have shown efficacy to treat or prevent relapse of AML or MDS following allogeneic stem cell transplantation (allo-SCT) in 20-40% of the cases [ Castagna et al. Transfusion and Apheresis 2016]. A german retrospective study showed an overall response rate (ORR) of 33% (including 27% CR and 6% PR), to AZA and DLI following relapse after allo-SCT in AML and MDS [ Schroeder et al. BBMT 2015], a regimen used since in many centers. We present here preliminary results of a phase I/II evaluating the combination of AZA/VEN/DLI in relapse MDS/AML. Patients and Methods: In this open label multicenter phase I/II study, MDS and AML patients relapsing post allo-SCT, without active graft versus host disease (GVHD) and active infections, are treated with VEN during 14 days in combination with AZA at 75 mg/m²/day for 5 days for each cycle (or 50 mg/m²/day for 5 days each cycle if relapse occurs less than 4 months after allo- SCT), and VEN orally at escalating dose (step 1: VEN 100mg/d; step 2: VEN 200mg/d and step 3: VEN 400mg/d) in the phase I part and at maximal tolerated dose (MTD) on days 1-14 of each cycle in the phase II part. Cycles are administered every 28 day (or delayed until day 42 in case of hematological toxicity). DLI are started at least 8 weeks after immunosuppressive therapy discontinuation, if chimerism in <90% recipient, and administered on day 15 every two cycles (C2, C4, C6, C8) in the absence of GVHD. Patients receive at least 8 cycles of AZA/VEN + DLI unless progression occurs. Response is assessed after 4, 6 and 8 cycles. Patients achieving a response receive 12 cycles of AZA + VEN followed by cycles of AZA alone. In the absence of response after 8 cycles, patients stop treatment. Results: From November 2022 to June 2023, 9 patients were included in part 1 of VENTOGRAFT. M/F was 8/1. Median age 57 years (range 18-71). Two patients had MDS and 7 patients had AML at diagnosis. Five patients had MDS (IPSS-R was low (1), intermediate (2) and very high (2)), and 3 patients had AML (ELN2017 was intermediate (1) and unfavorable (2) and molecular relapse (1)) at the time of relapse. Median interval between allo-SCT and relapse was 44 months (range 2-181). At data cut-off, 3 patients had been treated in step 1, 3 patients in step 2 and 3 patients in step 3 (AZA + VEN 400mg/d D1-14)). Median number of AZA + VEN cycles was 3 (range 1-8) and median number of DLI infused was 1 (range 0-3). One dose limiting toxicity (No ANC recovery on D42 and one patient still neutropenic at D28) was observed in step 3 leading to inclusion 3 more patients on this step. Other adverse events observed were grade 1/2 nausea (2 patients), grade 1 skin lesions (1 patient), ALAT increase grade 3 (1 patient), febrile neutropenia grade 3 (2 patients), grade 3 abdominal pain (1 patient) and grade 3 gastrointestinal bleeding (2 patients). GVHD was seen in 1/9 (11%) patients). CMV and EBV reactivation were observed in 0 and 2 patients, respectively. Overall response rate was 6/9 (67%) patients 67% including 22% complete remission (CR), 22% of CR with incomplete recovery (CRi) and 33% of morphologic leukemia-free state (MLFS). Full donor chimerism was obtained in all patients obtaining a response. Conclusion: The combination of AZA at 75mg/m²/d D1-5 and VEN at 100 to 400mg/d D1-D14 (400mg/d ongoing) was safe, showed a promising ORR of 67%. Final results of the phase 1 and first patients treated in this phase 2 will presented at the ASH meeting.

Biallelic Landscape of DNMT3A Mutant Myeloid Neoplasia

The structure of DNMT3A comprises an N-terminal regulatory part promoting nuclear localization and targeting chromatin and a C-terminal domain, mainly involved in DNA binding and methylation catalysis. 1DNMT3A mutations in myeloid neoplasia (MN) and clonal hematopoiesis are mostly heterozygous. Homozygous null Dnmt3amice are born runted and Dnmt3a-/- cells express a hypomethylation phenotype. 2 An invariant detection of heterozygous mutations without homozygous, hemizygous, or biallelic configurations suggests absence of acquisition of more malignant phenotypes by progressive inactivation of DNMT3A. Moreover, residual DNMT3A function is essential for cell survival and potentially suggests that synthetic lethality might be a viable therapeutic strategy whereby cases with two intact alleles would be more resistant allowing a therapeutic window. Consequently, identification of biallelic DNMT3A mutant cases may be extremely instructive for the development of therapeutics targeting DNMT3A. We analyzed targeted NGS sequencing results of 4,895 cases with MN enrolled at our institution from 2002 to 2021 and retrospectively reviewed the clinical characteristics of all patients with deleterious DNMT3A mutations. DNMT3A mutations were identified in 461 patients, of whom 39 had double and 1 triple hits. Most of the mutants had AML (n=236) including biphenotypic AML (n=5), followed by MDS (n=142), MDS/MPN overlap (n=46) and MPN (n=37). In addition, mutations were identified in 9% of CHIP/CCUS (n=117) and 4% of aplastic anemia (n=68). Multiple DNMT3A hits were more often encountered in AML than MDS and MDS/MPN overlap (62.5% vs. 32.5% vs. 2.5%; P = .045). Single hits were represented by missense (73%) within the hotspot R882 (35%), frameshifts (10%), nonsense (9%), splice sites (6%) and in-frame ins/del (2%). Double hits configuration had a constellation of missense/missense (40%), missense/frameshifts (25%), missense/nonsense (13%), and missense/splice sites (10%) ( Fig1). R882 accounted for 10% of the multi hit carriers. Of note is that biallelic involvement was not confined to non-R882. Patients harboring multi hits carriedmore likely IDH2 (27.5% vs. 10.5%; OR 2.73; P = .011) and ETV6 (7.5% vs. 1.7%; OR 4.32 P = .043) mutations compared to patients with single hits DNMT3A by multivariate logistic regression analysis. Since deletion or UPD of DNMT3A locus has not been found, one can assume that all cases with one hit harbor monoallelic rather than biallelic subclones. For carriers of multiple mutations, one can stipulate that when the sum of VAFs exceeds the theoretical 60%, the presence of a biallelic subclone might be suspected. Indeed, 24 cases fulfilled this criterion. However, in 40% of patients with double hits, arithmetic VAF did not allow for unequivocal resolution of the mutational configuration, with either smaller biallelic subclones vs. subclonal mosaicism being possible. Next, we compared clinical features and outcomes of patients with mono vs. biallelic cases. In MDS, compared with single hits DNMT3A, biallelic cases showed significantly higher WBC (median 7.49 vs. 3.15 k/µL; P = .0052), ANC (4.88 vs. 1.52 k/µL; P = .0056), and platelet count (264 vs. 83 k/µL; P = .0036). In high-risk MDS and AML, with a median follow-up of 12.5 months (0.2-201), patients with biallelic DNMT3Ashowed significantly poorer 1-year OS rate than patients with single hits (42.8% vs. 65.8%; P = .0095). Longitudinal molecular analysis of 8 cases (2 MDS at onset and upon progression to sAML; 4 AML at diagnosis and following relapse after conventional intensive chemotherapy-induced remission; 1 AML at diagnosis and CR post SCT; 1 AML and donor-derived AML post SCT) demonstrated an early origin of biallelic hits with leukemogenic potential. Indeed, biallelic clones persisted or expanded in 6 transformed cases. Our study describes the likelihood that biallelic configuration of DNMT3A mutations, while rare, is indeed compatible with clonal expansion and thus questions the applicability of a synthetic lethality strategy. We are currently confirming that, indeed, truly biallelic cases vs. mosaicism are permissive using DNASeq of single sorted progenitor cells. The results of this analysis will be available at the time of ASH meeting.

Do We Still Need to Perform Bone Marrow Examination in All Subjects Suspected of MDS? Evaluation and Validation of Non-Invasive (Web-Based) Algorithm

Background : The gold standard of diagnosing myelodysplastic syndromes (MDS) still requires a bone marrow examination (BME), an old invasive procedure, with possible associated pain and bleeding, and subjective interpretation with 20-25% inter-observer variability. We first developed a logistic regression formula [Oster HS et al., Leuk Res 2018], and then a non-invasive web-based diagnostic algorithm [Oster HS et al., Bld Adv 2021]. The app requires input of 10 readily available clinical and laboratory parameters (age, gender, Hb, MCV, WBC, ANC, monocytes, PLT, glucose, creatinine), resulting in diagnosing or excluding MDS, perhaps obviating the BME (Figure). Here, we performed external validation of the model, using data of MDS patients and non-MDS controls, who had not been included in the development of the model. Methods: The BM registry of Tel Aviv Sourasky Medical Center (1/2017-12/2021) was reviewed. Inclusion criteria for the MDS group were BME diagnosis, and for the controls, age >50yr, and unexplained anemia requiring BME. We excluded patients with other causes of anemia (renal failure; B12/folic acid or iron deficiency), active malignancy and other hematologic diseases. The relevant parameters were entered into the online model (at https://shiny.york.ac.uk/mds/) and the results of both groups were compared. In a sub-analysis, the model performance was tested in patients with lower risk (LR, IPSS-R<3.5) and higher risk (HR, IPSS-R≥3.5) MDS. Results: In total, 204 patients were included and compared, 103 with MDS, and 101 anemic non-MDS control patients, all with BME. The analysis of model performance (Table, upper portion) showed a sensitivity of 85.6%, specificity of 92.3%, positive predictive value (PPV) of 90.3% and a negative predictive value (NPV) of 88.4%. The algorithm was indeterminate in 26.2% of patients with MDS (BME proven), compared with 9.9% in the control group. In the sub-analysis (Table, lower portion), the model performance in the patients with LR-MDS (n=61) demonstrated sensitivity 85.1%, specificity 92.3%, PPV 90.3% and NPV 88.1%. The performance in HR-MDS (n=32), was 89.3%, 92.3%, 90.6% and 91.1%, respectively. Conclusions : This study validates the potential role of this non-invasive, easy-to-use model to assist in diagnosis, and mainly exclusion of MDS, perhaps allowing to avoid bone marrow examination in some patients. This might be especially relevant for patients suspected to have LR-MDS. Emerging technologies with data generated from peripheral blood (genetics, morphometrics) may be incorporated in the model in the future.

A Phase 2, Single-Arm, Multicohort, Open Label, Multicenter Trial of Off-the-Shelf Natural Killer Cells (SAR445419) in Patients with High-Risk Myeloid Malignancies Undergoing Allogeneic HSCT

Background and Significance: Relapse remains the leading cause of treatment failure for patients with high-risk acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although the risk depends on parameters such as age and conditioning intensity, relapse is experienced in 30-50% of patients after conventional HSCT in high-risk AML/MDS. Initial safety and post-HSCT relapse risk reduction results were reported in a phase I study of haploidentical (haplo) donor-derived natural killer (NK) cells expanded ex vivo on feeder cells expressing IL-21 and 4-1BBL in conjunction with haplo-HSCT (Ciurea et al, Leukemia, 2022). This study supported exploring SAR445419 (SAR'419), an off-the-shelf (OTS) product, derived from universal donor NK cells and expanded ex vivo in a feeder cell-free system using plasma membrane particles bearing membrane-bound IL-21 and 4-1BBL. Here, we aim to test whether SAR'419 can mediate an effective anti-leukemia response and reduce post-transplant relapse rate when administered in the peri-transplant period. Study Design and Methods: In this multicenter, multicohort, phase 2, single-arm trial (NCT05726682), patients with high-risk AML/MDS planned for allo-HSCT from matched sibling, matched unrelated or haplo donors will be enrolled in 2 cohorts. Cohort A will comprise patients aged 18‒65 years undergoing myeloablative conditioning with hematopoietic cell transplantation comorbidity index ≤3 and cohort B will comprise patients aged 18‒75 years undergoing reduced intensity conditioning. Patients with prior allogeneic transplantation or AML beyond first complete remission will be excluded from the study. Patients will undergo HSCT conditioning starting on Day ‒7 and will receive a peripheral blood stem cell graft on Day 0. Prophylaxis for Graft-versus-host disease (GVHD) using cyclophosphamide, mycophenolate mofetil, and tacrolimus will start on Day +3. SAR'419 will be administered as adjunctive therapy to the HSCT on Day ‒2, Day +7, and Day +28. To confirm the anticipated safety profile of SAR'419, a safety run-in will be conducted in 6‒12 patients in each cohort prior to open enrollment. Patients will be enrolled until 51 and 44 patients in cohorts A and B, respectively, receive at least the first 2 doses of SAR'419 at the target dose confirmed during safety run-in. In total, approximately 107 patients will be enrolled. The primary endpoint, relapse free survival (RFS), will be assessed at 12 months post-HSCT. Additional primary endpoints include frequency of graft failure, life-threatening infusion-related reactions or cytokine release syndrome, tumor lysis syndrome, immune cell-associated neurotoxicity syndrome, acute GVHD, overall mortality at 100 days post-HSCT, non-relapse mortality, and cytomegalovirus reactivation/infection. Secondary endpoints are safety, tolerability, and the impact of SAR'419 on HSCT outcomes, stem cell engraftment, GVHD, infection, and quality of life. Exploratory assessments include evaluation of the impact of SAR'419 on immune reconstitution, cytomegalovirus seroconversion, minimal residual disease, resource utilization, SAR'419 immunogenicity and persistence and their impact on outcomes in whole blood (including serum and plasma) and bone marrow aspirates. RFS will be reported as the percentage of patients who are relapse free and alive at 12 months from the date of HSCT and who received at least the first 2 planned doses of SAR'419. Adverse events will be graded by the National Cancer Institute Common Terminology Criteria for Adverse Events v5.0 and will be summarized using descriptive statistics.

KPT-8602 Induced Hematopoietic Differentiation in U2AF1 S34F Mutant Cells and Myelodysplastic Syndromes Bone Marrow Stem and Progenitor Cells

Myelodysplastic syndromes (MDS) are a group of clonal bone marrow (BM) neoplasms characterized by ineffective hematopoiesis, cytopenia's, and a high risk of progression to acute myeloid leukemia. Current standard therapies include erythropoiesis stimulating agents for lower risk disease and DNMTi for higher risk MDS; however, less than half of patients respond and even the best responses are transient and non-curative. The majority of MDS patients harbor at least one of ~40 commonly found somatic gene mutations, most of which occur in splicing factor genes such as U2AF1, which also have non-canonical roles in nuclear export. KPT-8602 (Eltanexor) is a novel selective inhibitor of nuclear export (SINE) that acts by disrupting nuclear export of cargo relevant in MDS. We hypothesized that U2AF1 S34 F mutant cells would be selectively sensitive to treatment with KPT-8602. We created immortalized murine hematopoietic progenitor cells by retroviral transduction of stimulated bone marrow (BM) cells isolated from U2AF1 WT or U2AF1 S34F mice with a HOXB8_ER vector. Estrogen drives the hematopoietic transcription factor immortalizing the cells in a progenitor state, however, upon estrogen withdrawal the cells will differentiate and eventually die. This differentiation can be driven by treatment with specific growth factors such as erythropoietin (EPO) for erythroid differentiation or granulocyte macrophage colony stimulating factor (GM-CSF) for myeloid differentiation. KPT-8602 exposure induced differentiation and increased hematopoiesis in murine cell models and primary MDS samples. U2AF1 WT and U2AF1 S34F cells were plated in medium lacking estrogen and treated with GM-CSF (10nM) or EPO (1nM) with or without KPT-8602 (10nM) for 72h, then stained with Ly6C, CD11b, Ter119, and CD71 for flow cytometry analysis. In addition, we performed colony forming capacity assays in both the murine cells and primary MDS BM stem and progenitor cells and age-comparable healthy donors, with 14 day KPT-8602 exposure. Treatment with GM-CSF significantly increased the expression of Ly6C in both U2AF1 WT and U2AF1 S34F cells (WT fold change untreated vs GMCSF =1.9, p=0.03; S34F fold change untreated vs GMCSF =2.3, p<0.01). However, addition of KPT-8602 caused significantly more Ly6C expression than GM-CSF alone in the mutant cells (WT fold change GMCSF vs combination =3.1, p=0.06; S34F fold change GMCSF vs combination =4.0, p<0.01). There were no significant changes in CD11b expression with KPT-8602 treatment. We did not find a statistically significant increase in erythroid markers assessed by Ter-119 and CD71 after EPO treatment, however, KPT-8602 treatment alone showed a trend of increased expression of Ter-119 and CD71 in the mutant cells that fell just below statistical significance (Ter119 p=0.08 for WT, p=0.09 for S34F; CD71 p=0.21 for WT, p=0.06 for S34F). Additionally, we looked at selective toxicity of U2AF1 S34Fby WST-1 reagent and found no differences in toxicity between the WT or mutant cells with KPT-8602. However, U2AF1 WT and U2AF1 S34F progenitor cells showed an increase in colony forming capacity (n=1 with technical duplicates) when plated in the presence of KPT-8602 showing an average fold change in colony growth of 1.4 for U2AF1 WT and 1.3 for U2AF1 S34F underscoring the ability of KPT-8602 to induce differentiation. To confirm these results were not cell line artifacts, we next treated primary BM stem and progenitor cells from MDS patients (n=2) and healthy donors (n=4). MDS patient 1 (age 73y) was diagnosed with MDS-MLD-EB1 and harbored mutations in TP53, ASXL1, BCOR, TET2 and PPM1D. MDS patient 2 (age 79y) was diagnosed with MDS/MPN and harbored an SF3B1 mutation. Healthy donors (n=4) were all male and had a median age of 65y. Treatment with KPT-8602 (10nM) significantly increased burst and colony forming unit-erythroid (BFU-E/CFU-E) in MDS primary cells (p=0.02) with no significant increase in the healthy donors (p=0.57) (BFU-E/CFU-E fold increase was 1.1 for healthy donors and 3.5 for MDS). Collectively, these findings suggest that KPT-8602 promotes myeloid and erythroid differentiation and further validates the use of KPT-8602 as a therapeutic strategy for MDS. Future studies will fully elucidate the specific mechanisms by which KPT-8602 acts in the context of splicing factor mutations, and other commonly found MDS mutation types.

“Transplant Changes Your Life”: Communication Strategies of Transplant Hematologists in High Risk Decision Making Conversations

Introduction: Allogeneic hematopoietic cell transplantation (alloHCT) offers a chance of cure for many patients with blood cancers including higher risk myelodysplastic syndromes (MDS) but carries many risks. In this high risk, high reward situation, shared decision making is essential to empower patients to make goal-concordant decisions. We detail communication strategies hematologists use to discuss treatment options including alloHCT and to facilitate shared decision making with patients. Methods: Our research team recruited hematologists who routinely perform alloHCT in the United States through email. Participants conducted a video-recorded virtual encounter lasting up to one hour with an actor portraying a 67-year-old man with recently diagnosed high risk MDS referred for specialty hematology consultation to discuss treatment options including alloHCT. We transcribed and qualitatively analyzed hematologists' communication approaches. Results: Participants (n=37) from 25 institutions were a median age of 44 years (range 33-73 years) and mostly male (65%) and white (65%). More than half spent >50% of their clinical time in managing alloHCT patients. Hematologists followed a similar conversation structure, though varied in the amount of detail they provided within each of the main sections (Figure 1). Their conversation routinely included an assessment of the patient's perception of his condition, information about MDS, and discussion of treatment options including transplant and chemotherapy and less commonly supportive care or a clinical trial. Many repeatedly highlighted that transplant was the only option with the potential for cure. Their discussion about transplant frequently followed a chronological approach from pretransplant considerations including donor issues, formal fitness testing, and disease control; to the peri-transplant period and hospital experience; and through the posttransplant course including need for dedicated caregiver support. Hematologists discussed risks, complications, and major outcomes at varying lengths. Decision making talk occurred at different points in the visit though was often concentrated in the latter part of the conversation. Hematologists referred to several elements that formed the basis of treatment decision making (Figure 2). While hematologists indicated that multiple factors should inform the patient's and hematologist's decision making, many honed in on transplant's position as the only chance for long-term survival. Although most hematologists acknowledged on a general level the importance of patients' goals and preferences in decision making, they elicited and incorporated the individual patient's specific goals into the discussion to varying degrees. Instead, in several conversations, hematologists' assessment of the patient's fitness and candidacy for transplant was prominent in their discussion about whether the patient could and should consider pursuing the curative option. Hematologists frequently recommended that the patient take time to think and talk with his family about the treatment decision in the immediate future. The strength of their longer-term recommendations ranged from strong recommendations for transplant or chemotherapy to conditional recommendations for transplant or deferral pending further information. These recommendations reflected a range of decision-making strategies, from a physician-led approach to a primarily patient-guided effort. In the middle, some hematologists described a collaborative effort with the patient, in which the patient and hematologist would work together to reach a shared decision. Within a single encounter, many hematologists expressed various statements suggesting that their approach to decision making did not fit tightly within a single domain but rather fell along a continuum which evolved as the encounter proceeded. Conclusions: The transplant decision making discussion is complex. The ways in which hematologists frame information and engage patients affect how they make decisions together. Hematologists consistently discuss similar major content areas as they navigate from beginning to end; identification of these key components can be used as the basis for creation of a communication tool to help hematologists discuss alloHCT with patients.

Somatic Mutations and DNA Hypermethylation at Enhancers and Promoters Identify Distinct Subtypes within Lower-Risk Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are a group of heterogeneous disorders caused by the accumulation of somatic mutations in the hematopoietic stem and progenitor compartment. Besides del(5q), SF3B1 or TP53 mutations, referred to as defining genetic abnormalities, mutation patterning hardly structured the classification of MDS. Mutations in epigenetic factors occur early in the development of clonal hematopoiesis leading to precocious alterations of DNA methylation implicated in oncogenesis. Convergent DNA methylation patterns related to both mutations and microenvironment imprinting may induce specific gene expression profiles and contribute to phenotypic variations. To refine a pathophysiological classification of lower-risk MDS, we combined genetic profiling to DNA methylation and transcriptome data. Methods: We enrolled 543 lower-risk treatment-naïve MDS patients at diagnosis and performed DNA-sequencing of 24 genes. DNA methylation and RNA-sequencing data were generated in a representative subset of 175 cases with available material (75 MDS-SLD/MLD, 40 MDS-EB1, 53 MDS-RS-SLD/MLD, 5 MDS del(5q), 2 MDS-U) and 7 age-matched healthy controls. IPSS-R was very low, low or intermediate in 87.4%. During a median follow-up of 2 years, 16% of MDS cases progressed to AML (MDSp). Infinium EPIC 850K array allowed after filtering the examination of methylation of 723,612 CpG sites with enrichment at enhancers and promoters. Differentially methylated regions with differentially methylated CpG sites (DMR-CpG) between cases and controls were defined with a minimum of 2 probes, a Benjamini-Hochberg-adjusted P-value cut-off of 0.05, a false discovery rate of 0.001 and a mean Δβ-value >20% at CpG sites. Consensus motifs for transcription factors were identified using Homer. Results: Unsupervised clustering on genetic profiling of 543 patients identified distinct subsets of lower-risk MDS with different clinical features: one cluster (group A) was enriched in del(5q), three clusters were characterized by SF3B1 mutations, either isolated (group B) or associated with DNMT3A (group E) or TET2 (group F), while the two remaining clusters were enriched in either complex pattern of mutations (group C) or SRSF2 mutations (group D). Genetic clusters showed significantly different clinical outcomes, clusters C, D and E having lower overall survival and higher risk of progression to high risk MDS or AML compared to other clusters. In the subset of 175 cases, a total of 2,953 unique DMR-CpGs allowed a robust repartition of patient samples in 4 groups using the k-means method. The 4 methylation groups were clinically distinct in terms of age, hemoglobin, neutrophils, monocytes, platelets, BM blasts, WHO classification, IPSS-R, and mutation pattern. A significant correlation was found between genetic and methylation groups ( P<0.001). Hypomethylated CpGs defined group 4 which was enriched in MDS-RSwith low blast count and few co-mutations, while groups 1 and 3 demonstrated hypermethylated profiles driven by TET2/ IDH1-2 mutations and TET2/ SRSF2 mutations, respectively. TET2-mutated cases exhibited an increased proportion of hypermethylated DMR-CpGs at enhancers (DME) significantly enriched in C/EBP and ETS family transcription factor motifs. In MDS without evidence of progression, we identified 1,418 DME and their gene targets of which those significantly deregulated in RNA-seq were involved in the control of translation and response to TGF-β. By contrast, in MDS with rapid progression to AML, DMR-CpGs were significantly enriched at CpG islands and promoters ( P<0.0001) and a set of 10-20 genes downstream of these promoters were specifically deregulated. Conclusion: Somatic mutations and DNA methylation at enhancers and promoters identify distinct subsets of lower-risk MDS with different clinical behavior. Hypermethylation at enhancers with C/EBP or ETS family motifs is a hallmark of MDS and may account for changes in transcription factor recruitment and cell fate. Hypermethylation of promoters with downstream effects on gene expression may indicate a propensity to rapid evolution to AML.