Acute Myeloid Leukemia (AML) exhibits significant heterogeneity in clinical outcomes, yet current prognostic stratification systems based on genetic alterations alone cannot fully capture this complexity. This study aimed to develop an integrated epigenetic-based classification system and evaluate its prognostic value.

We performed multi-omics analysis on five independent cohorts totaling 1,103 AML patients. The Cancer Genome Atlas-Acute Myeloid Leukemia (TCGA-LAML) cohort (n = 83) provided comprehensive multi-omics data including DNA methylation profiles (Illumina 450K platform), RNA sequencing (mRNA, lncRNA, and miRNA), and somatic mutation profiles. The BEAT (n = 649), TARGET (n = 156), GSE12417 (n = 79), and GSE37642 (n = 136) cohorts contributed transcriptome data. Molecular subtypes were identified using empirical Bayes-based clustering on the TCGA cohort. LSC17 scores were calculated using a validated 17-gene expression signature. A random survival forest model was developed integrating molecular features with LSC17 scores, validated across all cohorts. Immune microenvironment analysis employed multiple deconvolution methods (ESTIMATE, CIBERSORT, xCell) and pathway analysis (GSVA, GSEA). Drug sensitivity was predicted using the pRRophetic algorithm with GDSC database reference.

Multi-omics integration revealed two molecularly distinct AML subtypes with significant survival differences (CS2 vs CS1, P < 0.001). The random survival forest model, incorporating 20 key epigenetic features (including CPNE8, CD109, and CHRDL1) and LSC17 scores, achieved superior prognostic accuracy (C-index: 0.72-0.78) across validation cohorts. Both epigenetic risk score (HR = 2.45, 95%CI: 1.86-3.24) and LSC17 score (HR = 1.89, 95%CI: 1.42-2.51) maintained independent prognostic value in multivariate analysis. Integration of both scores in a nomogram improved 1-, 3-, and 5-year survival predictions (C-index: 0.81). High-risk patients exhibited distinct immune profiles with elevated M2 macrophages (1.8-fold) and Tregs (2.3-fold), while low-risk patients showed enhanced NK cell activity (2.1-fold). Drug sensitivity analysis identified differential responses to epigenetic regulators (LAQ824, P = 0.000139; MS-275, P = 0.00104) and proteasome inhibitors (Bortezomib, P = 0.00747; MG-132, P = 0.0106) between risk groups.

This integrated classification system combining epigenetic features and stem cell signatures provides new insights into AML heterogeneity and therapeutic targeting. The complementary nature of epigenetic and stem cell-related prognostic factors suggests potential for improved risk stratification in clinical practice. Future prospective validation studies are warranted to confirm these findings.

Acute myeloid leukemia (AML) is an aggressive hematological cancer with a 70% five-year mortality rate. Relapse occurs in approximately half of adults treated with intensive chemotherapy, while responses to targeted therapies are short-lasting. Frequent mutations in signaling pathways, such as FLT3 tyrosine kinase and RAS, lead to dysregulated mammalian target of rapamycin complex 1 (mTORC1)and mitogen-activated protein kinase (MAPK) signaling, increased protein synthesis, enhanced mitochondrial fitness, and metabolic adaptations that drive leukemic cell proliferation and survival. Here, emerging evidence supporting the unique role of eukaryotic initiation factor 4F as a key driver of the expression of proteins regulating leukemic cell metabolism and survival and the potential therapeutic benefit of targeting this pathway pharmacologically in AML are discussed.

Acute myeloid leukemia (AML) is a highly heterogeneous malignancy, presenting significant challenges in accurately predicting patient prognosis. Dysregulation of endoplasmic reticulum (ER) stress and resistance to programmed cell death (PCD) are hallmarks of AML cells. However, the prognostic significance of the interplay between ER stress and cell death pathways in AML remains largely unexplored.

We analyzed RNA sequencing and clinical data from 887 AML patients across 4 cohorts to develop an ER stress-related cell death index (ERCDI) using 10 machine-learning algorithms with 117 unique combinations. Survival and time-dependent Receiver Operating Characteristic Curve (ROC) analyses were performed to assess the model's efficacy. Clinical characteristics, the tumor immune microenvironment, and drug sensitivity differences between the high- and low-risk groups were also analyzed. The CMap database was used to identify potential therapeutic drugs. In vitro and in vivo experiments, including CCK-8, colony formation, flow cytometry, Transwell assays, and xenograft mouse models, were conducted to evaluate the effects of the target genes and candidate drugs.

The ERCDI demonstrated strong prognostic and predictive performance for prognosis in AML patients. Furthermore, the ERCDI effectively predicted immunotherapy and chemotherapy outcomes and was associated with the immune features of the different risk groups. DNA damage-inducible transcript 4 protein (DDIT4), a key gene associated with ERCDI, is related to poor prognosis in AML patients with high expression. Additionally, the knockdown of DDIT4 significantly inhibited AML cell proliferation, induced cell apoptosis, and promoted cell cycle arrest. Chaetocin was subsequently identified as a candidate compound for AML treatment. Subsequent experiments suggested that combining chaetocin and venetoclax is a potentially promising therapeutic strategy for AML.

The ERCDI provides personalized risk assessment and treatment recommendations for individual AML patients. The combined use of chaetocin and venetoclax can potentially be repurposed for AML therapy.

Isocitrate dehydrogenase 1/2 (IDH) mutations are early initiating events in acute myeloid leukemia (AML). The complex clonal architecture and cellular heterogeneity in IDH-mutant AML underlies the heterogeneous clinical presentation and outcomes. Integrating single-cell genotyping and transcriptomics, we demonstrate a stem-like and inflammatory phenotype of IDH-mutant AML and identify clone-specific programs associated with NPM1, NRAS, and SRSF2 co-mutations. Furthermore, these clones had distinct responses to treatment with combination IDH inhibitors and chemotherapy, including elimination, reconstitution of myeloid differentiation, or retention within progenitor populations. At relapse after IDH inhibitor monotherapy, we identify upregulated stemness, inflammation, mitochondrial metabolism, and anti-apoptotic factors, as well as downregulated major histocompatibility complex (MHC) class II antigen presentation. At the pre-leukemic stage, we observe upregulation of IDH2-associated pathways, including inflammation. We deliver a detailed phenotyping of IDH-mutant AML and a framework for dissecting contributions of recurrently mutated genes in AML at diagnosis and following therapy, with implications for precision medicine.

In human acute myeloid leukemia (AML), mutations of isocitrate dehydrogenase-1 (IDH1) often co-occur with NPM1 mutations, and less frequently with FLT3 mutations. To investigate whether the effects of IDH1 mutation differ according to the specific co-occurring mutation, we generated two strains of double knock-in mutant mice. Idh1R132H combined with Npm1c induced overt AML, whereas Idh1R132H plus Flt3ITD resulted in Flt3ITD-driven myelo- or lymphoproliferation that was minimally affected by Idh1R132H and rarely generated AML. Gene expression profiling revealed differences between Idh1R132H;Npm1c cells and Idh1R132H;Flt3ITD cells and suggested altered heme metabolism and immune responses in the former. The profile of Idh1R132H;Npm1c cells corresponded to that of human IDH-mutated AML cells, particularly those resistant to inhibitors of mutant IDH. Compared to treatment with a menin inhibitor, IDH1-targeted therapy of Idh1R132H;Npm1c AML-bearing mice was less efficacious in improving cell differentiation and extending survival. The differential cooperation of Idh1R132H with Npm1c vs. Flt3ITD may have implications for the devising of subtype-specific treatments for human AML.

Stemness-associated cell states are linked to chemotherapy resistance in acute myeloid leukemia (AML). We uncovered a direct mechanistic link between expression of the stem cell transcription factor GATA2 and drug resistance. The GATA-binding protein 2 (GATA2) plays a central role in blood stem cell generation and maintenance. We find substantial intrapatient and interpatient variability in GATA2 expression across samples from patients with AML. GATA2 expression varies by molecular subtype and has been linked to outcome. In a murine model, KMT2A-MLL3-driven AML originating from a stem cell or immature progenitor cell population has higher Gata2 expression and is more resistant to the standard AML chemotherapy agent doxorubicin. Deletion of Gata2 resulted in a more robust induction of p53 after exposure to doxorubicin. Chromatin immunoprecipitation sequencing, RNA sequencing, and functional studies revealed that GATA2 regulates the expression of RASSF4, a modulator of the p53 inhibitor MDM2 (mouse double minute 2). GATA2 and RASSF4 are anticorrelated in human cell lines and in bulk and single-cell expression data sets from patients with AML. Knockdown of Rassf4 in Gata2-low cells resulted in doxorubicin or nutlin-3 resistance. Conversely, overexpression of Rassf4 results in sensitization of cells expressing high levels of Gata2. Finally, doxorubicin and nutlin-3 are synergistic in Gata2-high murine AML and in samples from patients with AML. We discovered a previously unappreciated role for GATA2 in dampening p53-mediated apoptosis via transcriptional regulation of RASSF4, a modulator of MDM2. This role for GATA2 directly links the expression of a stemness-associated transcription factor to chemotherapy resistance.

AML exhibits substantial molecular and genetic heterogeneity. Therefore, identifying key biological processes and related genes involved in the pathogenesis, as well as contributing to therapeutic resistance, is imperative for enhancing clinical outcomes. However, the assessment of mitochondrial function in AML has gradually been acknowledged but has not been widely emphasized. Hence, prioritizing the identification of mitochondrial-related biomarkers is crucial to enhance existing stratification methodologies and guide decisions on risk-adapted therapies.

We systematically integrated and analyzed data from nine online AML transcriptomics sequencing databases, screening the Human.MitoCarta3.0 mitochondrial gene database to identify AML-specific mitochondrial genes. A prognostic mitochondrial score was developed using LASSO regression analysis in the HOVON database as training cohort (n = 618) and validated in another eight publicly available independent cohorts (n = 1,697).

A 19-mitochondrial function gene AML score was further generated and exhibited high prognostic power in 2,315 AML patients, named as MitoScore. MitoScore was an independent survival prognosis biomarker (p < 0.001). The MitoScore effectively distinguishes several genetic abnormalities and significantly improves the ELN (European Leukemia Net) classification. Patients with a high MitoScore demonstrated a notably poor response to induction chemotherapy and related refractory AML (p < 0.001). In the favorable risk gene variant and cytogenetic abnormality group, MitoScore was significantly lower compared to patients without those variants. Conversely, in the adverse group, MitoScore was significantly higher compared to patients with favorable genetic abnormalities.

Our findings underscore the utility of the MitoScore as a powerful tool for refined risk stratification and predicting chemotherapy resistance.

Pathogenic variants of isocitrate dehydrogenase 1 and 2 (IDH1/2) genes are present in approximately 20% of acute myeloid leukemia (AML) cases, resulting in the oncometabolite R-2-hydroxyglutarate (R-2-HG). The accumulation of R-2-HG in leukemic cells and in their niche induces epigenetic modifications, profound rewiring of the cellular metabolism, and microenvironmental remodeling. These changes promote cellular differentiation bias, enhancing the survival and proliferation of leukemic cells, and thus playing a pivotal role in leukemogenesis and resistance to standard AML therapy. This review focuses on the different perspectives offered by studying metabolism and resistance to standard treatments in AML with IDH1 or IDH2 pathogenic variants, for the development of new biomarkers and therapeutic solutions.

Acute myeloid leukemia (AML) is a devastating disease characterized by extensive inter-patient and intra-patient heterogeneity. Despite the introduction of intensive chemotherapy in the 1970s as the standard treatment, the development of mechanism-based targeted therapies since 2017 has been broadening the therapeutic landscape. However, both chemotherapy and targeted therapies continue to face the challenges of primary and secondary resistance. This review summarizes the mechanisms underlying resistance to chemotherapy and targeted therapies in AML and discusses the opportunities and challenges brought by the transition from chemotherapy to precision medicine.

Cancer progression and therapeutic resistance are closely linked to a stemness phenotype. Here, we introduce a protein-expression-based stemness index (PROTsi) to evaluate oncogenic dedifferentiation in relation to histopathology, molecular features, and clinical outcomes. Utilizing datasets from the Clinical Proteomic Tumor Analysis Consortium across 11 tumor types, we validate PROTsi's effectiveness in accurately quantifying stem-like features. Through integration of PROTsi with multi-omics, including protein post-translational modifications, we identify molecular features associated with stemness and proteins that act as active nodes within transcriptional networks, driving tumor aggressiveness. Proteins highly correlated with stemness were identified as potential drug targets, both shared and tumor specific. These stemness-associated proteins demonstrate predictive value for clinical outcomes, as confirmed by immunohistochemistry in multiple samples. The findings emphasize PROTsi's efficacy as a valuable tool for selecting predictive protein targets, a crucial step in customizing anti-cancer therapy and advancing the clinical development of cures for cancer patients.

Acute myeloid leukemia (AML) is a hematopoietic malignancy with poor outcomes and high recurrence. Disulfidptosis, a novel form of programmed cell death driven by aberrant disulfide bonds and F-actin collapse, provides insights into cancer progression and treatment.

We investigated the correlation network and prognostic values of disulfidptosis-related genes (DRGs) in AML. Unsupervised clustering was performed to reveal distinct disulfidptosis-related AML subtypes. We implemented the differential analysis and enrichment analysis to explore the difference of the distinct subtypes in biological processes. Least absolute shrinkage and selection operator (LASSO) Cox model was used to generate a disulfidptosis-related signature. We employed the ESTIMATE, CIBERSORT, and scRNA analyses to assess the tumor microenvironment of AML. Moreover, experiments validated the functions of PTPN6 and CSK in OCI-AML2 cells.

We identified 10 prognostic DRGs and revealed two disulfidptosis subtypes. DRGs significantly affected immune processes like interferon-gamma response and MHC class II antigen presentation. LASSO algorithm was implemented to established a 6-gene signature (HLA-DRB5, CCDC124, PTPN6, HLA-DMA, CSK, ISG15) that predicted prognosis in two validation cohorts more robustly than other signatures. Disulfidptosis was correlated with tumor microenvironment immune cells, especially monocytes. The two risk subgroups differed significantly in susceptibilities of multiple chemotherapy drugs, indicating disulfidptosis as a potential therapeutic target. Knockdown of PTPN6 and CSK inhibited the proliferation of AML cells and increased apoptosis.

Our study provides insights into DRG prognoses and immunomodulation, establishing a robust 6-gene risk model for predicting AML outcomes that may enhance precision medicine and treatment strategies.

Acute myeloid leukemia (AML) is caused by altered maturation and differentiation of myeloid blasts, as well as transcriptional/epigenetic alterations and impaired apoptosis, all of which lead to excessive proliferation of malignant blood cells in the bone marrow. It is these mutations that cause tumor heterogeneity, which is linked to a higher risk of relapse and death and makes anti-AML treatments like HSCT, chemotherapy, and immunotherapy (ICI, CAR T-cell-based therapies, and cancer vaccines) less effective. Single-cell RNA sequencing (scRNA-seq) also makes it possible to find cellular subclones and profile tumors, which opens up new diagnostic and therapeutic targets for better AML management. The HSCT process works better when genetic and transcriptional information about the patient and donor stem cells is collected. This saves time and lowers the risk of harmful side effects happening in the body.

Leukemic stem cells (LSCs) of acute myeloid leukemia (AML) can be enriched in the CD34+CD38- fraction and reconstitute human AML in vivo. However, in acute promyelocytic leukemia (APL), which constitutes 10% of all AML cases and is driven by promyelocytic leukemia-retinoic acid receptor alpha (PML::RARα) fusion genes, the presence of LSCs has long been unidentified because of the difficulty in efficient reconstitution of human APL in vivo. Herein, we show that LSCs of the short-type isoform APL, a subtype of APL defined by different breakpoints of the PML gene, concentrate in the CD34+CD38- fraction and express T cell immunoglobulin mucin-3 (TIM-3). Short-type APL cells exhibited distinct gene expression signatures, including LSC-related genes, compared to the other types of APL. Moreover, CD34+CD38-TIM-3+ short-type APL cells efficiently reconstituted human APL in xenograft models with high penetration, whereas CD34- differentiated APL cells did not. Furthermore, CD34+CD38-TIM-3+ short-type APL cells reconstituted leukemia cells after serial transplantation. Thus, short-type APL was hierarchically organized by self-renewing APL-LSCs. The identification of LSCs in a subset of APL and establishment of an efficient patient-derived xenograft model may contribute to further understanding the APL leukemogenesis and devise individual treatments for the eradication of APL LSCs.

Hematopoietic stem cells (HSCs) are rare cells residing at the top of the haematopoietic hierarchy capable of reconstituting all blood cell populations through their ability of self-renewal and differentiation. Their ability to maintain haematopoiesis can be majorly depleted by chemotherapeutic agents, leading to a long-term bone marrow injury. However, pre-clinical studies have focused on the acute effects of chemotherapy, leaving the lasting impact on healthy cells poorly understood. To study this, we combined rapid ex vivo models to study the long-term/late-stage effects of a cyclin-dependent kinase subunit 1 (CKS1) inhibitor. Inhibition of CKS1 has been shown to protect healthy HSCs from chemotherapy during acute myeloid leukaemia, and here we show a dose-dependent role of long-term CKS1 inhibition on haematopoiesis, either boosting B lymphopoiesis or ablating HSC proliferation capacity, dependent on the context. Mechanistically, low doses of the CKS1 inhibitor (CKS1i) affects AKT-Foxo1 signalling potentiating B-cell differentiation, but impairing HSC proliferation. These results reveal a novel role for CKS1 in boosting B lymphopoiesis and propose the use of rapid ex vivo models to investigate the long-term effects of chemotherapeutic treatments targeting HSCs with the potential of reducing late adverse effects.

Despite advances in outcome, one third of children with acute myeloid leukemia (AML) relapse, and less than half will achieve long-term survival. Relapse in AML has been shown to be driven in part by leukemic stem cells (LSCs), highlighting the unmet medical need to better characterize and target this therapy-resistant cell population. Micro-array profiling of pediatric AML subpopulations (LSCs and leukemic myeloblasts) and their healthy counterparts revealed nidogen-1 (NID1) as expressed in both leukemic subpopulations while absent in the hematopoietic stem cell. Using the TARGET dataset including pediatric AML patients (n = 1025), NID1 expression showed a correlation with worse event-free survival and KMT2A rearrangements. Drug response profiling of a NID1 knockdown model demonstrated differential sensitivity to HSP90 inhibition. RNA sequencing and gene set enrichment analysis between NID1high and NID1low phenotypes showed involvement of NID1 in mitochondrial metabolic pathways known to be enriched in LSCs. Altogether, this study highlights NID1 as a novel oncogene associated with worse EFS and metabolic LSC phenotype in AML. NID1 could serve as a biomarker and aid in further mapping LSCs to establish therapeutic strategies tackling the high relapse rates in pediatric AML.

Targeting the dependency of MLL-rearranged (MLLr) leukemias on menin with small molecule inhibitors has opened new therapeutic strategies for these poor-prognosis diseases. However, the rapid development of menin inhibitor resistance calls for combinatory strategies to improve responses and prevent resistance. Here we show that leukemia stem cells (LSCs) of MLLr acute myeloid leukemia (AML) exhibit enhanced guanine nucleotide biosynthesis, the inhibition of which leads to myeloid differentiation and sensitization to menin inhibitors. Mechanistically, targeting inosine monophosphate dehydrogenase 2 (IMPDH2) reduces guanine nucleotides and rRNA transcription, leading to reduced protein expression of LEDGF and menin. Consequently, the formation and chromatin binding of the MLL-fusion complex is impaired, reducing the expression of MLL target genes. Inhibition of guanine nucleotide biosynthesis or rRNA transcription further suppresses MLLr AML when combined with a menin inhibitor. Our findings underscore the requirement of guanine nucleotide biosynthesis in maintaining the function of the LEDGF/menin/MLL-fusion complex and provide a rationale to target guanine nucleotide biosynthesis to sensitize MLLr leukemias to menin inhibitors.

Acute myeloid leukemia (AML) corresponds to a heterogeneous group of clonal hematopoietic diseases, which are characterized by uncontrolled proliferation of malignant transformed myeloid precursors and their inability to differentiate into mature blood cells. The prognosis of AML depends on many variables, including the genetic features of the disease. Treatment outcomes, despite the introduction of new targeted therapies, are still unsatisfactory. Recently, there have been an increasing number of reports on enzymatic proteins of the sirtuin family and their potential importance in cancer in general. Sirtuins are a group of 7 (SIRT1-7) NAD+-dependent histone deacetylases with pleiotropic effects on metabolism, aging processes, and cell survival. They are not only responsible for post-translational modification of histones but also play various biochemical functions and interact with other proteins regulating cell survival, such as p53. Thus, their role in key mechanisms of tumorigenesis makes them a worthwhile topic in AML. Different sirtuins have been shown to act oppositely depending on the biological context, the mechanism of which requires further exploration. This review provides a comprehensive description of the significance and role of sirtuins in AML in light of the current state of knowledge. It focuses in particular on molecular mechanisms regulated by sirtuins and signaling pathways involved in leukemogenesis, as well as clinical aspects and potential therapeutic targets in AML.

Uncovering early gene network changes of human hematopoietic stem cells (HSCs) leading to differentiation induction is of utmost importance for therapeutic manipulation. We employed single cell proteo-transcriptomic sequencing to FACS-enriched bone marrow hematopoietic stem and progenitor cells (HSPCs) from 15 healthy donors. Pseudotime analysis reveals four major differentiation trajectories, which remain consistent upon aging, with an early branching point into megakaryocyte-erythroid progenitors. However, young donors suggest a more productive differentiation from HSPCs to committed progenitors of all lineages. tradeSeq analysis depicts continuous changes in gene expression of HSPC-related genes (DLK1, ADGRG6), and provides a roadmap of gene expression at the earliest branching points. We identify CD273/PD-L2 to be highly expressed in a subfraction of immature multipotent HSPCs with enhanced quiescence. Functional experiments confirm the immune-modulatory function of CD273/PD-L2 on HSPCs in regulating T-cell activation and cytokine release. Here, we present a molecular map of early HSPC differentiation across human life.

In recent years, remarkable technological advances have illuminated aspects of the pathogenesis of myeloid malignancies-yet outcomes for patients with these devastating diseases have not significantly improved. We posit that a synthesized view of the three dimensions through which hematopoietic cells transit during their healthy and diseased life-clonal evolution, stem cell hierarchy, and ontogeny-promises high yields in new insights into disease pathogenesis and new therapeutic avenues.

Zinc plays a central role in the hematological development. Therapeutic interventions with zinc are shown to improve the health status of patients with malignancies by stimulating the immune system and reducing side effects. Despite the abnormal zinc homeostasis in leukemia, the role and mechanisms of zinc signaling in leukemia development remain poorly understood. Recently, some important breakthroughs are made in laboratory and clinical studies of zinc in leukemia, such as the role of zinc in regulating ferroptosis and the effects of zinc in immunotherapy. Zinc-based strategies are urgently needed to refine the current zinc intervention regimen for side-effect free therapy in chemotherapy-intolerant patients. This review provides a comprehensive overview of the role of zinc homeostasis in leukemia patients and focuses on the therapeutic potential of zinc signaling modulation in leukemia.

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are hematological malignancies characterized by complex genetic alterations, leading to poor clinical outcomes. Despite advances in treatment, there is an urgent need for novel therapeutic approaches. This review outlines recent progress in humanized models of MDS and AML and highlight their role in advancing our understanding of these diseases.

Patient derived xenografts (PDXs) were among the first humanized models for studying MDS and AML, allowing researchers to analyze patient-specific cancer properties in vivo . However, they face challenges related to sample availability and consistent engraftment in mice. New methods, including specialized mouse strains and human tissue scaffolds, have been developed to address these issues. Induced pluripotent stem cells (iPSCs) offer the advantage of indefinite expansion and genetic modification, making them valuable for in vitro research, though protocols to enhance their engraftment in vivo are still being refined. Genetically engineered human primary hematopoietic stem and progenitor cells (HSPCs) provide reliable in vivo models with good engraftment in mice, and recent advancements in culture systems and gene-editing techniques are helping to overcome challenges related to ex vivo expansion and genetic modification.

PDXs, iPSCs, and genetically engineered HSPCs are crucial models for the study of MDS and AML. This review discusses strengths, limitations, and recent advancements of these humanized models, which provide insights into human-specific disease biology and therapeutic development.

In this study, we investigated the measurable residual leukemic stem cell (MR-LSC) population after allogeneic stem cell transplantation (allo-SCT) for high-risk acute myeloid leukemia (AML), utilizing T-cell immunoglobulin mucin-3 (TIM-3) expression as a functional marker of AML leukemic stem cells (LSCs). Analysis of the CD34+CD38- fraction of bone marrow cells immediately after achievement of engraftment revealed the presence of both TIM-3+LSCs and TIM-3- donor hematopoietic stem cells (HSCs) at varying ratios. Genetic analysis confirmed that TIM-3+ cells harbored patient-specific mutations identical to those found in AML clones, whereas TIM-3- cells did not, indicating that TIM-3+CD34+CD38- cells represent residual AML LSCs. In 92 allo-SCT occasions involving 83 AML patients, we enumerated the frequencies of TIM-3+LSCs immediately after achieving hematologic complete remission with complete donor cell chimerism. Notably, only 22.2% of patients who achieved a TIM-3+MR-LSClow status (<60%) experienced relapse, with a median event-free survival (EFS) of 1581 days (median follow-up duration was 2177 days among event-free survivors). Conversely, 87.5% of patients with TIM-3+MR-LSCint/high (≥60%) relapsed, with a median EFS of 140.5 days. Furthermore, MR-LSC status emerged as a significant independent risk factor for relapse (hazard ratio, 8.56; p < 0.0001), surpassing the impact of patient disease status prior to allo-SCT, including failure to achieve complete remission (hazard ratio, 1.98; p = 0.048). These findings suggest that evaluating TIM-3+ MR-LSCs immediately after engraftment, which reflects the competitive reconstitution of residual TIM-3+ LSCs and donor HSCs, may be valuable for predicting outcomes in AML patients undergoing allo-SCT.

Refractory disease and relapse are major challenges in acute myeloid leukemia (AML) therapy attributed to survival of leukemic stem cells (LSC). To target LSCs, antibody-drug conjugates (ADCs) provide an elegant solution, combining the specificity of antibodies with highly potent payloads. We aimed to investigate if FLT3-20D9h3-ADCs delivering either the DNA-alkylator duocarmycin (DUBA) or the microtubule-toxin monomethyl auristatin F (MMAF) can eradicate quiescent LSCs. We show here that DUBA more potently kills cell-cycle arrested AML cells compared to microtubule-targeting auristatins. Due to limited stability of 20D9h3-DUBA ADC in vivo, we analyzed both ADCs in advanced in vitro stem cell assays. 20D9h3-DUBA successfully eliminated leukemic progenitors in vitro in colony-forming unit and long-term culture initiating cell assays, both in patient cells and in patient-derived xenograft (PDX) cells. Further, it completely prevented engraftment of AML PDX leukemia-initiating cells in NSG mice. 20D9h3-MMAF had a similar effect in engraftment assays, but a less prominent effect in colony assays. Both ADCs did not affect healthy stem and progenitor cells at comparable doses providing the rationale for FLT3 as therapeutic LSC target. Collectively, we show that FLT3-directed ADCs with DUBA or MMAF have potent activity against AML LSCs and represent promising candidates for further clinical development.

Neutrophil extracellular traps (NETs) play pivotal roles in various pathological processes. The formation of NETs is impaired in acute myeloid leukemia (AML), which can result in immunodeficiency and increased susceptibility to infection.

The gene set variation analysis (GSVA) algorithm was employed for the calculation of NET score, while the consensus clustering algorithm was utilized to identify molecular subtypes. Weighted gene coexpression network analysis (WGCNA) revealed potential genes and biological pathways associated with NETs, and a total of 10 machine learning algorithms were applied to construct the optimal prognostic model.

Through the analysis of multiomics data, we identified two molecular subtypes with high and low NET scores. The low-NET score subgroup exhibited increased infiltration of immune effector cells. Conversely, the high-NET score subtype presented an abundance of monocytes and M2 macrophages, accompanied by elevated expression levels of immune checkpoint genes. These findings suggest that a pronounced immunosuppressive effect is associated with a significantly worse prognosis for this subtype. The optimal risk score model was selected by employing the C-index as the criterion on the basis of training 10 machine learning algorithms on 9 multicenter AML cohorts. Survival analysis confirmed that patients with high-risk scores had considerably poorer prognoses than those with lower scores. Receiver operating characteristic (ROC) curve and Cox regression analyses further validated the strong independent prognostic value of the risk score model. The nomogram, which was constructed by integrating the risk score model and clinicopathological factors, demonstrated high accuracy in predicting the overall survival of AML patients. Moreover, patients with refractory or chemotherapy-unresponsive AML had significantly higher risk scores. By analyzing drug therapy data from in vitro AML cells, we identified a subset of drugs that demonstrated increased sensitivity in the high-risk score group. Additionally, patients with a high risk score were also predicted to exhibit a favorable response to anti-PD-1 therapy, suggesting that these individuals may derive greater benefits from immunotherapy.

The NET-related signature, derived from a combination of diverse machine learning algorithms, has promising potential as a valuable tool for prognostic prediction, preventive measures, and personalized medicine in patients with AML.

Immunogenic cell death (ICD) is the kind of cell death that triggers the immune system. It affects several tumors, whereas its significance for prognosis in acute myeloid leukemia (AML) remains uncertain. AML categorization by cytogenetic variables is inaccurate. In addition, risk stratification of AML based on cytogenetics is imprecise. The data of AML patients were extracted from 4 databases, a total of 1,537 patients. Univariate and LASSO Cox regression analyses were conducted to construct an ICD risk signature (ICDRS). The ICDRS showed strong prognostic value for AML through Kaplan-Meier, Cox, ROC analyses and nomogram. Combining the ICDRS with the European LeukemiaNet (ELN) classification to redefine the risk stratification can better predict the prognosis of AML. Moreover, the ICDRS was examined to identify gene functional enrichment, immunological characteristics, drug susceptibility, and somatic mutation, which revealed considerable variations among different risk categories. We further validated the expression of ICDRS in the AML bone marrow microenvironment by single-cell RNA (scRNA) analysis. Ultimately, the functional role of CASP1 was proven in AML by a series of in-vitro experiments. Our study highlights the significant impact of ICDRS on AML, which may improve ELN risk classification, predict immune landscapes, and guide personalized therapy.

Stimulator of interferon response cGAMP interactor (STING) is essential for both innate and adaptive immunity. However, a comprehensive molecular characterization of STING expression across hematological malignancies is lacking.

In this study, the pan-blood-cancer landscape related to STING expression was identified using the GTEx, CCLE, Hemap, and TCGA databases, and the potential value for predicting prognosis was investigated. The relationship between STING expression and immune cell enrichment was assessed in the Hemap database. Moreover, the value of STING in predicting the efficacy of immunotherapy was validated using tumor immune dysfunction and exclusion (TIDE) biomarkers and real-world immunotherapy datasets.

STING was found to be relatively highly expressed in acute myeloid leukemia (AML) and chronic myeloid leukemia, with higher STING expression correlated with poorer prognosis in AML. STING expression was positively correlated with immune-related pathways such as IFN-gamma response, IFN-alpha response, and inflammatory response. Cytolytic score and STING expression were positively correlated in some hematological tumors, especially chronic lymphocytic leukemia and mantle cell lymphoma. Interestingly, STING expression was negatively correlated with TIDE biomarkers in AML, suggesting that AML patients with a high STING expression level may benefit from immunologic treatment. Our findings contribute a molecular characterization of STING across hematological malignancies, facilitating the development of individualized prognosis and treatment strategies.

T-cell acute lymphoblastic leukemia (T-ALL) is a hematopoietic malignancy characterized by increased proliferation and incomplete maturation of T-cell progenitors, for which relapse is often of poor prognosis. To improve patient outcomes, it is critical to understand the chemoresistance mechanisms arising from cell plasticity induced by the bone marrow (BM) microenvironment. Single-cell RNA sequencing of human T-ALL cells from adipocyte-rich and adipocyte-poor BM revealed a distinct leukemic cell population defined by quiescence and high CD44 expression (Ki67neg/lowCD44high). During in vivo treatment, these cells evaded chemotherapy, and were further called Chemotherapy-resistant Leukemic Cells (CLCs). Patient sample analysis revealed Ki67neg/lowCD44high CLCs at diagnosis and during relapse, with each displaying a specific transcriptomic signature. Interestingly, CD44high expression in T-ALL Ki67neg/low CLCs was associated with E-selectin binding. Analysis of 39 human T-ALL samples revealed significantly enhanced E-selectin binding activity in relapse/refractory samples compared with drug-sensitive samples. These characteristics of chemoresistant T-ALL CLCs provide key insights for prognostic stratification and novel therapeutic options.