Minimizing the transfusion burden is one of the primary clinical goals for most patients with myelodysplastic syndromes (MDS), which are incurable hematopoietic stem cell neoplasms. In some patients, supportive red blood cell transfusions can lead to clinical improvement, but frequent transfusions induce iron overload, which can have detrimental effects on cardiac and hepatic function, affect patients' quality of life, and incur additional healthcare costs. In this review, we summarize how erythropoiesis is regulated under steady-state conditions, dissect the molecular mechanisms underlying anemia in MDS, and review therapeutic approaches to overcome ineffective erythropoiesis in patients with these diseases. SIGNIFICANCE:A better understanding of the biological mechanisms underlying anemia in MDS is needed to develop targeted therapies for a personalized treatment approach. Anemia, a hallmark of MDS, highly affects patients' quality of life and contributes to overall morbidity. Current knowledge of the mechanisms of action of the available drugs targeting anemia in MDS is limited.

Acute myeloid leukemia (AML) is an aggressive myeloid malignancy with a poor prognosis. Venetoclax (Ven), a BCL2 inhibitor, has shown promising results but often leads to relapse due to mitochondrial dysregulation, particularly due to upregulation of the anti-apoptotic protein MCL1. Overexpression of the transcription factor STAT3 has been linked to poor survival in AML patients. Overexpression of STAT3 in a transgenic murine model induces a myeloid malignancy with a short latency period and inflammatory upregulation. The current study identifies STAT3 upregulation as a key mechanism of Ven resistance. A clinically relevant STAT3 degrader effectively reduces both total and phosphorylated STAT3, corrects mitochondrial structural and functional dysregulation, and induces apoptosis in Ven-resistant AML cell lines. KT-333 significantly decreases STAT3 and MCL1 protein levels and improves survival in Ven-resistant (Ven-Res) AML murine models. In summary, STAT3 hyperactivation is leukemogenic, is further potentiated in Ven-resistance and can be clinically targeted with a novel and specific STAT3 degrader. Pictorial representation depicting upregulation of STAT3 and MCL1 in venetoclax resistant myeloid malignancies such as MDS and AML causing mitochondrial structural abnormalities and dysfunction. By using specific STAT3 degrader, STAT3 inhibition, and thereby indirect downregulation of MCL1 can be a promising therapeutic intervention to target drug resistant clones in MDS and AML.

The mechanism(s) driving selective expansion of mutant hematopoietic stem and progenitor cells (HSPC) in clonal hematopoiesis (CH) are incompletely understood. Here, we address the role of metabolism in selection for HSPC with loss of function mutations in TET2 . Loss of Tet2 in murine HSPC triggers overexpression of glycolysis and oxidative phosphorylation genes and increased oxidative metabolism via an enlarged mitochondrial network. However, Tet2 -deficient HSPC maintain a normal redox state. Strikingly, compound loss of the rate-limiting pentose phosphate pathway (PPP) enzyme glucose-6-phosphate dehydrogenase (G6PD) triggers increased reactive oxygen species and impairs the fitness of Tet2 -deficient HSPC. We find that aberrant oxidative metabolism is also a feature of HSPC in human CH and clonal cytopenia of unknown significance (CCUS). Overall, our data point to aberrant metabolism as a critical and conserved driver of selection in TET2 -deficient CH and identify the PPP as a crucial compensatory pathway needed to maintain their selective advantage. Statement of Significance:This study identifies oxidative metabolism as a critical driver of selection for TET2 -deficient HSPC in clonal hematopoiesis (CH). It also demonstrates that cellular redox state is a vulnerability that impairs their fitness. These insights establish targetable metabolic pathway(s) that could be exploited in the setting of TET2 mutant CH.

Myelodysplastic Syndromes (MDS) present an increased risk of progression to Acute Myeloid Leukemia (AML). The complex interactions between neoplastic clone, bone marrow (BM) microenvironment and immune cells during disease evolution remain poorly understood. We used multi-omics single-cell approach to define patterns of clonal expansion and microenvironment shifts associated with MDS disease progression. We analyzed paired BM samples at diagnosis and at time of AML transformation from 20 MDS patients who had not received disease-modifying treatments before progression. Single-cell analysis was performed by CITE-seq, integrating transcriptomic and protein expression data from hematopoietic stem and progenitor cells (HSPC), myeloid, T and NK cells, in combination with single-cell genotyping (TAPESTRI). To study longitudinal dynamics of cell states, we projected each cell into gene expression space and quantified the fold-enrichment of transcriptionally similar cells between diagnosis and AML by k-nearest neighbor analysis. Differential gene/protein expression analyses were performed by linear mixed-effects models accounting for inter-patient variability. We identified two evolution patterns in HSPC compartment. In 9 patients (pts), progression was marked by emergence of novel HSPC clusters with leukemic stem cell (LSC)-like phenotype (absent/minimally detectable at diagnosis), showing upregulation of LSC markers (CD99, CD44) and immune evasion proteins (CD47, CD276) and downregulation of TGF-β and interferon (INF) response programs. In the remaining pts, progression was associated with expansion of a multipotent progenitor (MPP)-like population (already present at diagnosis). MPP-like cells exhibited increased activity of proliferative and INF-related inflammatory pathways, as well as downregulation of HLA molecules, suggesting the involvement of distinct immune escape mechanisms. Patients with NPM1, RUNX1, or TP53 mutations were more likely to show emergence of LSC-like clusters, whereas MDS with spliceosome gene mutations had heterogeneous patterns of HSPC evolution. Notably, pts showing LSC-like cluster emergence progressed more rapidly to AML (p=0.01). Considering BM microenvironment, across all pts, disease progression was associated with increased inflammatory monocytes (CD14⁺ CD86⁺ and high expression of INF-related genes) and neutrophils, suggesting that mature myeloid cells contribute to shape a pro-inflammatory marrow niche. Longitudinal analysis of immune cell states in all pts revealed widespread remodeling from diagnosis to evolution: 1) NK cells reduced their cytotoxic activity (GZMK/B-, PRF1-) and upregulated pro-inflammatory programs (NF-kb, IFN-γ); 2) T-regs acquired a highly immunosuppressive phenotype, with increased ICOS expression, downregulation of BACH2, and a switch to CD45RO⁺; 3) CD4⁺ effector memory T cells showed lower cytotoxic potential reducing GZMB/GZMK expression. Notably, in a subset of pts, small populations of these dysfunctional immune subsets—particularly highly suppressive T-regs—were already detectable at diagnosis and were associated with a shorter time to progression (p = 0.02). When comparing immunological changes based on the type of HSPC expansion, pts with LSC-like cluster displayed a more exhausted immune microenvironment, characterized by reduced frequencies of naïve T cells and increased terminally differentiated effector memory T cells, potentially supporting the selective advantage of LSC-like clones. Conversely, pts with MPP-like expansion showed increased IFN signaling across multiple immune cell populations. TP53-mutated MDS exhibited a distinct inflammatory signature, independent of IFN signaling, in both mature myeloid cells and T-regs. These myeloid cells showed HLA downregulation, while T-regs were enriched for a CD161⁺ subset with enhanced suppressive function—indicating a specific pattern of immune dysregulation driven by myeloid inflammation and impaired antigen presentation. MDS follow distinct evolutionary trajectories within the HSPC compartment. Consistent alterations in the BM microenvironment emerged as a potential common driver of disease progression. Early detection of rare, aberrant myeloid and immune cell populations at diagnosis may help identify pts at higher risk of rapid transformation to AML. TP53-mutated MDS exhibited a unique immunosuppressive profile, which may be a driver of their poor prognosis.

Approximately 30% of patients with chronic myelomonocytic leukemia (CMML) undergo transformation to a chemo-refractory blastic phase (BP-CMML). Seeking novel therapeutic approaches, we profiled blast transcriptomes from 42 BP-CMMLs, observing extensive transcriptional heterogeneity and poor alignment to current acute myeloid leukemia (AML) classifications. BP-CMMLs display distinctive transcriptomic profiles, including enrichment for quiescence and variability in drug response signatures. Integrating clinical, immunophenotype, and transcriptome parameters, Random Forest unsupervised clustering distinguishes immature and mature subtypes characterized by differential expression of transcriptional modules, oncogenes, apoptotic regulators, and patterns of surface marker expression. Subtypes differ in predicted response to AML drugs, validated ex vivo in primary samples. Iteratively refined stratification resolves a classification structure comprising five subtypes along a maturation spectrum, predictive of response to novel agents including consistent patterns for receptor tyrosine kinase (RTK), cyclin-dependent kinase (CDK), mechanistic target of rapamycin (MTOR), and mitogen-activated protein kinase (MAPK) inhibitors. Finally, we generate a prototype decision tree to stratify BP-CMML with high specificity and sensitivity, requiring validation but with potential clinical applicability to guide personalized drug selection for improved outcomes.

Dissecting the preneoplastic disease states' biological mechanisms that precede tumorigenesis can lead to interventions that can slow down disease progression and/or mitigate disease-related comorbidities. Myelodysplastic syndromes (MDS) cannot be cured by currently available pharmacological therapies, which fail to eradicate aberrant hematopoietic stem cells (HSCs), most of which are mutated by the time of diagnosis. Here, we sought to elucidate how MDS HSCs evade immune surveillance and expand in patients with clonal cytopenias of undetermined significance (CCUS), the pre-malignant stage of MDS. We used multi-omic single-cell approaches and functional in vitro studies to show that immune escape at disease initiation is mainly mediated by mutant, dysfunctional natural killer (NK) cells with impaired cytotoxic capability against cancer cells. Preclinical in vivo studies demonstrated that injecting NK cells from healthy donors efficiently depleted CCUS mutant cells while allowing normal cells to regenerate hematopoiesis. Our findings suggest that early intervention with adoptive cell therapy can prevent or delay the development of MDS.

Background: Apoptosis resistance drives treatment failure in relapsed/refractory (R/R) acute myeloid leukemia (AML). Targeting non-apoptotic forms of regulated cell death, such as ferroptosis, presents a promising strategy to circumvent this resistance. Ferroptosis is characterized by iron-dependent lipid peroxidation (LP), with glutathione peroxidase 4 (GPX4) known as a major negative regulator among other factors. In AML, we previously reported that ferroptosis uniquely relies on mitochondrial LP, termed “mitochondrial ferroptosis” (Leukemia, 2024). Given that both apoptosis and ferroptosis are regulated by mitochondria in AML cells, we sought to explore the molecular crosstalk between these two distinct forms of cell death. Results: To test whether ferroptosis bypasses apoptotic resistance, we first treated venetoclax (VEN)-resistant AML cells including BAX/BAK double-knockout (DKO) AML cells with the selective GPX4 inhibitor ML210. ML210 effectively induced cell death in these models, confirming that ferroptosis involves a distinct, apoptosis-independent pathway. However, combining ML210 with VEN triggered synergistic cell death in both VEN-sensitive MOLM13 and VEN-resistant OCI-AML3 cells, with combination indices of 0.53 and 0.71, respectively, suggesting hitherto unknown molecular crosstalk between the two cell death pathways. The synergy was also observed in CD34⁺CD38⁻ leukemic stem/progenitor cells from VEN-resistant AML patients (combination indices < 0.5). Consistently, combined treatment with GPX4 inhibition and VEN significantly reduced peripheral leukemic burden in a patient-derived xenograft mouse model established from an R/R AML case previously treated with decitabine and VEN. To investigate the synergistic mechanisms between apoptosis and ferroptosis, we investigated whether ferroptosis induction supports apoptosis activation. The co-treatment with ML210 and VEN (ML210/VEN) reduced caspase 3 activation (cleaved caspase 3) compared to VEN alone. Notably, this suppression was reversed by the ferroptosis inhibitor ferrostatin-1 (Fer-1), indicating that ferroptosis inhibits apoptosis induction. In contrast, VEN significantly enhanced LP when combined with ML210. Remarkably, the synergistic effects of ML210/VEN were completely abrogated by Fer-1, indicating that the ferroptotic component is the dominant driver of the observed synergy. These findings suggest a unidirectional interaction: BCL2 inhibition promotes ferroptosis, whereas ferroptosis suppresses apoptosis. Interestingly, the mitochondria-targeted antioxidant MitoTEMPO completely blocked both LP and cell death induced by ML210/VEN, suggesting that mitochondrial ROS is a key trigger for the synergistic effects. Although VEN did not affect the transcriptional or protein expression of canonical ferroptosis regulators (e.g., GPX4, FSP1, ACSL4), metabolomics analysis revealed reduced glutathione levels. This suggests that BCL2 inhibition metabolically primes AML cells into a pro-ferroptotic state. Based on these findings, we optimized treatment using a sequential approach with VEN followed by ML210, which further enhanced LP and cell death while preserving caspase activation, compared to concomitant treatment. In vivo, VEN followed by doxycycline-inducible GPX4 knockdown significantly reduced leukemic burden and prolonged the survival in a xenograft mouse model. Strikingly, this sequential combinatory effect was completely abolished in BAX/BAK-DKO AML cells, suggesting a novel non-apoptotic role of BAX/BAK in ferroptosis regulation. Finally, given the current lack of clinically translatable GPX4 inhibitors, we tested FDA-approved repurposing agents as ferroptosis inducer and enhancer, demonstrating that this strategy is also operational by targeting alternative ferroptosis regulators besides GPX4. This finding further supports the clinical translatability of our therapeutic concept. Conclusion: Synergistic AML cell death caused by dual induction of mitochondrial ferroptosis and apoptosis involves paradoxical molecular interactions between the two cell death pathways: BCL2 inhibition enhances ferroptosis, which ultimately dominates the synergy despite the apoptosis-suppressive effects of GPX4 inhibition. Sequential induction of apoptosis followed by ferroptosis maximizes this synergy and may provide a promising future therapeutic strategy for R/R AML.

The impact of exogenous stressors, such as cancer chemotherapies, on the genomic integrity and clonal dynamics of normal hematopoiesis is not well defined. We conducted whole-genome sequencing on 1,276 single-cell-derived hematopoietic stem and progenitor cell (HSPC) colonies from ten patients with multiple myeloma treated with chemotherapies and six normal donors. Melphalan treatment significantly increased the mutational burden, producing a distinctive mutation signature, whereas other chemotherapeutic agents had minimal effects. Consequently, the clonal diversity and architecture of post-treatment HSPCs resemble those observed in normal elderly individuals, particularly through the progression of oligoclonal hematopoiesis, thereby suggesting that chemotherapy accelerates clonal aging. Integrated phylogenetic analysis of matched therapy-related myeloid neoplasm samples traced their clonal origin to a single-HSPC clone among multiple competing clones, supporting a model of oligoclonal to monoclonal transformation. These findings underscore the need for further systematic research on the long-term hematological consequences of cancer chemotherapy.

TET2 mutations cause clonal expansion of hematopoietic stem and progenitor cells (HSPCs) and chimeric antigen receptor (CAR)-expressing T cells, driving clonal hematopoiesis (CH) and CAR+ T cell lymphomas, respectively. TET2-mutated CH has been linked to hyperinflammation, but the mechanisms remain poorly understood. We developed human isogenic induced pluripotent stem cell (iPSC)-based models of the 3 main CH mutations – DNMT3A R882H, TET2 haploinsufficiency and ASXL1 truncations –and performed integrated transcriptome, chromatin accessibility and DNA methylation (whole genome bisulfite sequencing) analyses in CD34+ HSPCs. Expectedly, DNMT3A- and TET2-mutant HSPCs showed a global decrease and increase, respectively, in DNA methylation, compared to isogenic wild-type (WT) controls. Single-cell RNA-seq analyses and immunoblotting experiments, unexpectedly, showed cell-autonomous activation of the type I interferon (IFN) induction pathway and secretion of bioactive type I IFN by TET2-mutated HSPCs. Furthermore, genomic and functional experiments in iPSC- and cord blood (CB)- HSPCs with TET2 shRNA knockdown or CRISPR/Cas9-mediated knockout demonstrated that, while type I IFN suppresses the expansion of WT CD34+/CD38- cells in a dose-dependent manner, TET2-mutant HSPCs downregulate their downstream IFN response and are thus protected. Importantly, while TET2-deficient CD34+/CD38- cells outgrew co-cultured WT cells, blockade of type I IFN signaling by a IFN alpha and beta receptor subunit 1 (IFNAR1) antibody mitigated their selective advantage. To understand the mechanism, we searched for hypermethylated genes with concomitant loss of chromatin accessibility and gene expression specifically in TET2-mutant HSPCs. These analyses, followed by knockdown and knockout (KO) validation experiments, identified silencing of the E3 ubiquitin ligase tripartite motif containing 4 (TRIM4) gene as a putative effector of TET2 deficiency. Corroborating findings from scATAC-seq and TARGET-seq+ datasets of HSPCs from individuals with TET2-mutated CH, clonal cytopenia of undetermined significance (CCUS) and healthy donors, revealed decreased promoter accessibility and expression of TRIM4 in primary TET2-mutant HSPCs. Importantly, engineering TRIM4 deficiency by CRISPR in iPSC- and CB- HSPCs phenocopied TET2 deficiency phenotypes, including monocytic bias and cell-autonomous type I IFN induction. In addition, TRIM4-deficient iPSC- and CB- HSPCs outcompeted WT HSPCs in liquid culture and serial replating assays in semisolid media, recapitulating the clonal advantage of TET2-deficient HSPCs. Furthermore, ectopic TRIM4 re-expression in TET2-deficient HSPCs, rescued their clonal expansion both in vitro and in competitive repopulation assays in NSGS mice. While TRIM proteins have known pleiotropic roles in immune signaling, TRIM4 is not well-characterized and lacks a mouse homolog. To identify substrates of TRIM4 we first performed integrated quantitative proteomics and ubiquitinome profiling in TET2+/-, TRIM4 KO, and isogenic WT iPSC-HSPCs. These analyses nominated 190 proteins with decreased ubiquitination and concomitant increased abundance by both TET2 and TRIM4 deficiency. Knockout of key components of the 3 main innate sensing pathways – TRIF/MYD88 (Toll-like receptor signaling pathway), MAVS (cytosolic RNA sensing) and STING (cytosolic DNA sensing) – identified MAVS as the dominant pathway. DExH-box helicase 58(DHX58), one of the 190 proteins identified by our proteomics analyses and a positive regulator of the MAVS pathway, was confirmed by knockout experiments to mediate the aberrant IFN production by TET2-deficient HSPCs. These results support a model whereby TET2 deficiency and consequent TRIM4 silencing activate type I IFN production through decrease in proteasome-mediated degradation of DHX58. Finally, we show that IFNAR1 blockade by the FDA-approved monoclonal antibody anifrolumab alleviates the clonal advantage of TET2-deficient T cells expressing a CD19-targeted CAR. In summary, we report secretion of type I IFN by TET2-mutant HSPCs as the cause of a pro-inflammatory local microenvironment that favors the expansion of mutant cells; we discover TRIM4 silencing as a critical effector of TET2 deficiency; we characterize TRIM4 as a novel negative regulator of type I IFN induction through degradation of DHX58; and we propose a new pharmacologic approach to mitigate the adverse clinical consequences of TET2 mutations.

ABSTRACT:Immunodeficiency in telomere biology disorders (TBDs) has been described in pediatric patients with severe phenotypes, but is less characterized within the broader TBD spectrum. We collected complete blood counts, lymphocyte subsets, and infection history from 88 consecutive patients with TBD with a median age of 38 years (range, 6-76). Most patients were >18 years old (80/88; 90%) and harbored either a TERT (45%) or TERC germ line mutation (32%). Thirty-two patients (36%) experienced significant infections (opportunistic, recurrent, and/or requiring hospitalization); 47% had lymphopenia, and 3% severe neutropenia. Absolute lymphocyte counts (ALCs) of <0.96 and <1.1 × 103/μL, but not severe neutropenia, were associated with increased infection risk and lower overall survival, respectively. Decreased CD3+ T cells, both CD4+ and CD8+, were associated with bone marrow failure, increased infection risk, and reduced survival. Low CD3+ and CD4+ T cells were associated with solid cancers. Telomere length was shortened across the cohort without correlation with ALC or lymphocyte subsets. In a predominantly adult cohort of TBDs, immunodeficiency was marked by T-cell lymphopenia, possibly a consequence of accelerated aging in the hematopoietic compartment. An ALC cutoff of <1.1 × 103/μL may be a useful biomarker to identify patients with an increased risk of infection, a major cause of death in patients with TBD.

The widespread use of platinum-based chemotherapy has been associated with an increased incidence of therapy-related myeloid neoplasms (t-MNs). Among the three most commonly used platinum-cisplatin, carboplatin, and oxaliplatin-oxaliplatin presents a distinctive clinical usage and toxicity. This distinction is supported by a previous study, which postulated that oxaliplatin-induced cytotoxicity primarily results from ribosomal stress as opposed to a DNA-damage response. Additionally, anecdotal data suggest a lower incidence of t-MNs associated with oxaliplatin. To investigate the differential risk of t-MNs among the various platinum chemotherapies, we analyzed data from 52,179 patients with diverse cancer types who received one of the platinum chemotherapies at our hospital. Of these patients, 22,652 (44%) received carboplatin alone, 15,358 (30%) received cisplatin alone, 8,805 (17%) received oxaliplatin alone, and 5,364 (9%) received multiple combinations of platinums. A total of 171 patients developed t-MNs, with only 6% of these patients having been exposed to oxaliplatin. There was no significant difference in the prevalence of TP53 mutations among t-MNs associated with different platinum exposures (56%, 60%, and 60% for cisplatin, carboplatin, and oxaliplatin, respectively). The cumulative incidence risk of t-MNs, using the Fine and Gray model, was significantly lower for patients treated with oxaliplatin alone compared to those receiving cisplatin alone (HR 0.39 [95% CI: 0.2-0.75], P = 0.005) or carboplatin alone (HR 0.42 [95% CI: 0.22-0.78], P = 0.01). Multivariate Fine and Gray models adjusting for other chemotherapy exposures also indicated that oxaliplatin treatment was associated with a significantly lower risk of t-MNs (HR 0.49 [95% CI: 0.26-0.9], P = 0.02). To explore the mechanisms underlying the protective effect of oxaliplatin on t-MN risk, we hypothesized that oxaliplatin treatment does not confer a selective advantage to mutant hematopoietic stem cells (HSCs), given that the selective expansion of mutant HSCs (such as TP53 or PPM1D) under chemotherapy has been implicated in the development of t-MNs. To test this hypothesis, we generated a chimeric mouse transplant model with Trp53-/- or Ppm1d truncating mutant cells (Ppm1d Tr/+) transplanted with wild-type cells in a 1:9 ratio. Recipient mice were treated with either vehicle, cisplatin, or oxaliplatin. Contrary to our hypothesis, both Trp53-/- and Ppm1d Tr/+ cells showed significant clonal expansion after both cisplatin and oxaliplatin treatment compared to vehicle. Thus, the reduced risk of t-MNs with oxaliplatin treatment cannot be attributed to reduced selective pressure of oxaliplatin compared to other platinums. We then hypothesized that oxaliplatin exerts less genotoxic effect on HSCs compared to other platinums. To test this hypothesis, we generated single-cell-derived HSC colonies from the recipient mice described above and performed whole-genome sequencing of individual colonies to measure platinum-induced somatic mutations in HSCs. Oxaliplatin-treated HSCs exhibited significantly fewer total somatic mutations compared to cisplatin-treated HSCs (mean: 524 vs. 891 mutations, P < 0.0001) and were comparable to vehicle-treated HSCs (mean: 524 vs. 485 mutations, P = 0.49). Mutation signature analysis revealed that a median of 74% (range: 52-100) of somatic mutations were attributed to known platinum signatures (SBS31 and SBS35) in cisplatin-treated HSCs, whereas a median of 34% (range: 25-41) of mutations in oxaliplatin-treated HSCs were platinum-associated (P < 0.001). In summary, while oxaliplatin treatment imposed a similar degree of selective pressure and promoted the expansion of TP53 and PPM1D mutated HSCs compared to other platinums, it induced significantly fewer somatic mutations in HSCs. These data align with clinical findings that the risk of t-MNs is significantly lower for patients treated with oxaliplatin. Nevertheless, t-MNs that do develop after oxaliplatin treatment still exhibit a high prevalence of TP53 mutations, consistent with our findings that oxaliplatin confers a similar selective advantage to TP53-mutated HSCs. These insights enhance our understanding of t-MN pathogenesis and aid in the development of strategies to mitigate this risk.

BACKGROUND:Hypomethylating agents are approved in higher-riskmyelodysplastic syndromes. The combination of a hypomethylating agent with venetoclax is standard of care in acute myeloid leukaemia. We investigated the safety and activity of the first totally oral combination of decitabine plus cedazuridine and venetoclax in patients with higher-risk-myelodysplastic syndromes and chronic myelomonocytic leukaemia. METHODS:We did a single-centre, dose-escalation and dose-expansion, phase 1/2, clinical trial. Patients with treatment-naive higher-risk-myelodysplastic syndromes or chronic myelomonocytic leukaemia (risk level categorised as intermediate-2 or higher by the International Prognostic Scoring System) with excess blasts (>5%). Treatment consisted of oral decitabine 35 mg plus cedazuridine 100 mg on days 1-5 and venetoclax (variable doses of 100-400 mg, day 1 to 14, 28-day cycle). The primary outcomes were safety for the phase 1 part and the overall response for the phase 2 part of the study. The trial is ongoing and this analysis was not prespecified. This study is registered with ClinicalTrials.gov, NCT04655755, and is currently enrolling participants. FINDINGS:Between Jan 21, 2021, and Jan 20, 2023, we enrolled 39 patients (nine in phase 1 and 30 in phase 2). The median age was 71 years (range 27-94), 28 (72%) patients were male, and 11 (28%) were female. The maximum tolerated dose was not reached, and the recommended phase 2 dose was established as oral decitabine 35 mg plus cedazuridine 100 mg for 5 days and venetoclax (400 mg) for 14 days. The most common grade 3-4 adverse events were thrombocytopenia (33 [85%] of 39), neutropenia (29 [74%]), and febrile neutropenia (eight [21%]). Four non-treatment-related deaths occurred on the study drugs due to sepsis (n=2), lung infection (n=1), and undetermined cause (n=1). The median follow-up time was 10·8 months (IQR 5·6-16·4). The overall response rate was 95% (95% CI 83-99; 37/39). 19 (49%) patients proceeded to hematopoietic stem-cell transplantation. INTERPRETATION:This early analysis suggests that the combination of oral decitabine plus cedazuridine with venetoclax for higher-risk-myelodysplastic syndromes and chronic myelomonocytic leukaemia is safe in most patients, with encouraging activity. Longer follow-up will be needed to confirm these data. FUNDING:MD Anderson Cancer Center, MDS/AML Moon Shot, Genentech/AbbVie, and Astex Pharmaceuticals.

Escape from immune surveillance is a hallmark of cancer. Immune deregulation caused by intrinsic and extrinsic cellular factors, such as altered T -cell functions, leads to immune exhaustion, loss of immune surveillance, and clonal proliferation of tumoral cells. The T -cell immune system contributes to the pathogenesis, maintenance, and progression of myelodysplastic syndrome (MDS). Here, we comprehensively reviewed our current biological knowledge of the T -cell compartment in MDS and recent advances in the development of immunotherapeutic strategies, such as immune checkpoint inhibitors and T -cell - and antibody -based adoptive therapies that hold promise to improve the outcome of patients with MDS.

Understanding the dynamic changes in T-cell subtypes during therapy is essential for developing effective therapeutic combinations to improve clinical outcomes. To assess whether the immune system contributes to the therapeutic effect of venetoclax, the most promising agent for patients with higher-risk myelodysplastic syndromes (MDS) that progress after hypomethylating agent (HMA) failure, we performed cytometry by time-of-flight analysis (with a panel of 59 antibodies) of sequential bone marrow (BM) samples from MDS patients enrolled in trials of venetoclax. A subset of CD4+ T cells expanded only in patients with marrow complete remission at venetoclax response. To validate these results, we used flow cytometry to analyze the frequencies of naïve (CD45RA+CD45RO- CD95-CCR7++), stem cell-like memory (SCM; CD45RA+CD45RO-CD95+CCR7+), central memory (CM; CD45RA-CD45RO+CD95+CCR7+), effector memory (EM; CD45RA-CD45RO+CD95+CCR7-) and terminal effector (TE; CD45RA+CD45RO-CD95+CCR7-) CD8+ or CD4+ T cells and CD4+CD25+CD127lowFOXP3+ T regulatory cells (Tregs) in sequential BM samples isolated from 25 patients who received venetoclax. These analyses confirmed that naïve, SCM, and EM CD4+ T-cells significantly expanded at response, whereas Tregs expanded at disease progression. The T-cell subsets' dynamics were specific to venetoclax alone or in combination with HMAs, as they were not observed in patients treated with HMAs alone. No significant changes in the CD8+ T-cell compartment during venetoclax treatment were noted. In contrast, only a slight increase of CD8+ TE cells was observed (P= 0.055). To explore the contribution of T-cell phenotypic states to venetoclax-induced responses and assess clonality and antigen recognition prediction, we performed transcriptomic and T-cell receptor (TCR) analyses using different single-cell multi-omics platforms. Single-cell proteo-genomic analyses (with a panel of 32 antibodies) of CD3+ cells isolated at different times during venetoclax therapy (15 samples from 5 patients; 34,229 sequenced T cells) allowed us to develop specific transcriptomic signatures for each T-cell subtype, which we applied to interrogate a larger cohort of sequential samples analyzed by scRNA- and/or scTCR-sequencing analyses (64 CD3+ samples from 12 patients; 124,871 sequenced T cells). This comprehensive approach confirmed the significant expansion of SCM CD4+ T cells at treatment response and the reduction of these cells at disease relapse, previously observed by flow cytometry analysis. SCM CD4+ T cells had significantly upregulated gene expression programs associated with cytokine secretion and cytotoxicity at disease response but expressed increased levels of exhaustion-related genes at disease relapse. MDS patients whose disease responded to venetoclax-based therapy showed a higher count of CD4+ EM T-cell clonotypes and greater T-cell diversity at baseline, whereas a reduction in these clonotypes during therapy predicted disease progression. SCM and EM CD4+ T cells had the highest rates of TCR sharing, which suggests a close interplay between these subsets. Cytokine secretion assays showed that compared with naïve and CM CD4+ T cells, SCM CD4+ T cells cocultured with MDS cells secreted higher levels of IFN-γ, TNF-α, and granzyme B (GZMB), a cytokine profile resembling that of EM and TE cells. Upon tumoral challenge, venetoclax also significantly increased IFN-γ, TNF-α, and GZMB levels in SCM and EM CD4+ T cells. Our study demonstrates that a subset of CD4+ T cells with SCM features contributes to the outcomes of MDS patients enrolled in trials of venetoclax-based therapy. SCM cells are transcriptionally and functionally similar to EM and TE cells and undergo the greatest improvement in cytotoxic capabilities and pro-inflammatory cytokine secretion upon venetoclax exposure. Thus, adoptive immunotherapy strategies have a potential role in enhancing venetoclax efficacy and preventing immune-mediated venetoclax resistance.

6567 Background: Response to therapy prolongs survival in patients with myelodysplastic syndromes (MDS). The aim of this study is to evaluate the impact of complete cytogenetic response (CCyR) on survival in patients with MDS and abnormal cytogenetics. Methods: We reviewed 2311 consecutive patients with MDS and cytogenetic abnormalities who were treated at our institution from 2006 to 2023. Results: CCyR was observed in 330 patients (14%), complete response (CR) in 208 (9%), CR with bilineage recovery (CRbi) in 255 (11%), CR with unilineage recovery (CRuni) in 151 (7%), CR with incomplete hematologic recovery (CRh) in 4 (0.2%), and non-CR in 1363 (59%). With a median follow-up of 59 months, the median overall survival (mOS) was 14 months. Ten months for non-CR, 19 months for CRh/CRbi/CRuni, 21 months for CR, and 26 months for patients with CCyR (p < 0.001). With stem cell transplant (SCT) censoring, mOS was 15 months. Eleven months in non-CR, 19 months in CRh/CRbi/CRuni, 19 months in CR, and 27 months for patients with CCyR (p < 0.001). In low-risk MDS by the International Prognostic Scoring System (IPSS), mOS with SCT censoring was 87 months for CCyR, compared to 40 months for non-CR, 38 months for CRh/CRbi/CRuni, and 36 months for CR (p < 0.001). In intermediate/high-risk MDS mOS with SCT censoring mOS was 25 months in CCyR, compared to 9 months in non-CR, 15 months in CRh/CRbi/CRuni, and 17 months in CR (p < 0.001). In a multivariate regression analysis, age > 75 years (HR 3.1, p < 0.001), complex karyotype (HR 2.15, p < 0.001) performance status 3-4 (HR 1.48, p < 0.001), hemoglobin < 8 g/dL (HR 1.48, p=0.003), creatinine > 1.3 g/dL (HR 1.44, p < 0.001), therapy-related AML (HR 1.41, p < 0.001), infection at diagnosis (HR 1.34, p =0.003), white blood cells > 50 x109/L (HR 1.31, p <0.001), cardiac comorbidities (HR 1.26, p < 0.001), platelets < 20 x109/L (HR 1.26, p=0.001), pneumonia at diagnosis (1.22, p =0.006), and core-binding factor cytogenetics (HR 0.48, p < 0.001) were independently prognostic for survival in this select group. Conclusions: Achievement of CCyR in patients with MDS and abnormal cytogenetic abnormalities leads to improved survival. Given poor outcomes in older patients with MDS, CCyR can be used as a valid surrogate for long-term outcomes.