Response In Acute Myeloid Leukemia Research Articles

Chemical Modifications to Enhance the Drug Properties of a VIP Receptor Antagonist (ANT) Peptide.

Antagonist peptides (ANTs) of vasoactive intestinal polypeptide receptors (VIP-Rs) are shown to enhance T cell activation and proliferation in vitro, as well as improving T cell-dependent anti-tumor response in acute myeloid leukemia (AML) murine models. However, peptide therapeutics often suffer from poor metabolic stability and exhibit a short half-life/fast elimination in vivo. In this study, we describe efforts to enhance the drug properties of ANTs via chemical modifications. The lead antagonist (ANT308) is derivatized with the following modifications: N-terminus acetylation, peptide stapling, and PEGylation. Acetylated ANT308 exhibits diminished T cell activation in vitro, indicating that N-terminus conservation is critical for antagonist activity. The replacement of residues 13 and 17 with cysteine to accommodate a chemical staple results in diminished survival using the modified peptide to treat mice with AML. However, the incorporation of the constraint increases survival and reduces tumor burden relative to its unstapled counterpart. Notably, PEGylation has a significant positive effect, with fewer doses of PEGylated ANT308 needed to achieve comparable overall survival and tumor burden in leukemic mice dosed with the parenteral ANT308 peptide, suggesting that polyethylene glycol (PEG) incorporation enhances longevity, and thus the antagonist activity of ANT308.

A novel immunogenic cell death-related classification indicates the immune landscape and predicts clinical outcome and treatment response in acute myeloid leukemia

BackgroundImmunogenic cell death (ICD) is closely related to anti-tumor therapy and regulates the tumor microenvironment (TME). This study aims to explore the molecular characteristics of ICD in acute myeloid leukemia (AML) and to analyze the value of ICD-related biomarkers in TME indication, prognosis prediction, and treatment response evaluation in AML.MethodsSingle-sample gene set enrichment analysis was used to calculate the ICD score. LASSO regression was used to construct a prognostic risk score model. We also analyzed differences in clinical characteristics, immune landscape, immunotherapy response, and chemotherapy sensitivity between high-risk and low-risk patients.ResultsThis study identified two ICD-related subtypes and found significant heterogeneity in clinical prognosis, TME, and immune landscape between different ICD subtypes. Subsequently, a novel ICD-related prognostic risk score model was developed, which accurately predicted the prognosis of AML patients and was validated in nine AML cohorts. Moreover, there were significant correlations between risk scores and clinicopathological factors, somatic mutations, TME characteristics, immune cell infiltration, immunotherapy response, and chemosensitivity. We further validated the model gene expression in a clinically real-world cohort.ConclusionsThe novel ICD-related signatures identified and validated by us can serve as promising biomarkers for predicting clinical outcomes, chemotherapy sensitivity, and immunotherapy response in AML patients, guiding the establishment of personalized and accurate treatment strategies for AML.Graphical

Abstract 732: Toward optimizing CXCR4 inhibition with beta adrenergic blockade to enhance chemotherapy response in acute myeloid leukemia

Abstract Introduction: CXCR4 is a chemokine receptor for CXCL12 that plays a critical role in homing of hematopoietic stem cells (HSCs) to the bone marrow microenvironment. The CXCR4 inhibitor plerixafor is used to increase mobilization of peripheral blood stem cells in combination with filgrastim. This pathway has also been investigated for applications in hematologic malignancies such as acute myeloid leukemia (AML), including clinical trials, with the goal of disrupting AML from the protective marrow environment. These have included use of plerixafor with 7+3 or MEC, BL-8040 with cytarabine, and ulocuplumab with MEC. For the ulocuplumab trial, at the study site of the authors, CR/CRi correlated with >10% CXCR4 expression (Becker et al. ASH 2014). The objective of this preclinical study is to identify the AML patient population most likely to respond to CXCR4 inhibition, and the role of combined beta-adrenergic blockade, as the latter has been shown pre-clinically to augment mobilization of HSCs when combined with a CXCR4 inhibitor, GPC-100 (Sukhtankar et al. PLoS One, 2023). Procedures: AML patient samples (N=124 including 90 blood and 34 bone marrow samples, and 21 with both) were obtained with informed consent on an IRB approved protocol. Multicolor flow cytometry was performed with sequential gating on blasts (by FSC/SSC, CD45/SSC, CD34 or phenotype), and leukemia stem cells (LSCs:CD34+CD38−,CD123+), then analysis for CXCR4 and beta-2 adrenergic receptor (ADRB2) surface expression. Correlations between CXCR4 expression and clinical characteristics including age, gender, new diagnosis vs. relapse, ELN2022 risk, cytogenetics, FLT3/NPM1 mutation status, CR1 duration, and overall survival were performed. GraphPad Prism9 was used for statistical analysis, and Mann Whitney test (two groups) and ordinary one-way ANOVA (>2 groups) were used for comparisons. Results: The range of CXCR4 expression by AML blasts is 0.05 to 99.7% (median 13.1%; mean 16.2%), and by AML LSCs 0 to 99% (median 2%, mean 15.7%). The expression by blasts and LSCs for each patient exhibited tight correlation, R2=0.88 p=1.23 × 10e-25). There was higher % expression of CXCR4 by both blasts and LSCs for new diagnosis, as compared to relapsed, patient samples (p=0.002). There was no correlation between level of CXCR4 expression and the other clinical features. For a subset of 20 patient samples analyzed for both CXCR4 and ADBR2 expression by AML blasts, ADBR2 expression ranged from 0.4 to 20.1%. For the samples that express ADBR2, there was high level expression of CXCR4, range 35.9 to 98.7% (median 78.4%, mean 76%). Conclusion: These studies support further investigation of whether simultaneous blockade of CXCR4 and ADBR2 may potentiate chemotherapy response in AML by limiting microenvironment mediated chemotherapy protection and reveal that patients with new diagnosis may be more susceptible to this approach. Citation Format: Brenda Loera, Chunxiao Zhang, Pina M. Cardarelli, Niña G. Caculitan, Sylvia Chien, Jin Dai, Vivian G. Oehler, Jianying Zhang, Guido Marcucci, Pamela S. Becker. Toward optimizing CXCR4 inhibition with beta adrenergic blockade to enhance chemotherapy response in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 732.

Epigenetic control over the cell-intrinsic immune response antagonizes self-renewal in acute myeloid leukemia

AbstractEpigenetic modulation of the cell-intrinsic immune response holds promise as a therapeutic approach for leukemia. However, current strategies designed for transcriptional activation of endogenous transposons and subsequent interferon type-I (IFN-I) response, show limited clinical efficacy. Histone lysine methylation is an epigenetic signature in IFN-I response associated with suppression of IFN-I and IFN-stimulated genes, suggesting histone demethylation as key mechanism of reactivation. In this study, we unveil the histone demethylase PHF8 as a direct initiator and regulator of cell-intrinsic immune response in acute myeloid leukemia (AML). Site-specific phosphorylation of PHF8 orchestrates epigenetic changes that upregulate cytosolic RNA sensors, particularly the TRIM25-RIG-I-IFIT5 axis, thereby triggering the cellular IFN-I response-differentiation-apoptosis network. This signaling cascade largely counteracts differentiation block and growth of human AML cells across various disease subtypes in vitro and in vivo. Through proteome analysis of over 200 primary AML bone marrow samples, we identify a distinct PHF8/IFN-I signature in half of the patient population, without significant associations with known clinically or genetically defined AML subgroups. This profile was absent in healthy CD34+ hematopoietic progenitor cells, suggesting therapeutic applicability in a large fraction of patients with AML. Pharmacological support of PHF8 phosphorylation significantly impairs the growth in samples from patients with primary AML. These findings provide novel opportunities for harnessing the cell-intrinsic immune response in the development of immunotherapeutic strategies against AML.

Utilizing exosomes as sparking clinical biomarkers and therapeutic response in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a malignant clonal tumor originating from immature myeloid hematopoietic cells in the bone marrow with rapid progression and poor prognosis. Therefore, an in-depth exploration of the pathogenesis of AML can provide new ideas for the treatment of AML. In recent years, it has been found that exosomes play an important role in the pathogenesis of AML. Exosomes are membrane-bound extracellular vesicles (EVs) that transfer signaling molecules and have attracted a large amount of attention, which are key mediators of intercellular communication. Extracellular vesicles not only affect AML cells and normal hematopoietic cells but also have an impact on the bone marrow microenvironment and immune escape, thereby promoting the progression of AML and leading to refractory relapse. It is worth noting that exosomes and the various molecules they contain are expected to become the new markers for disease monitoring and prognosis of AML, and may also function as drug carriers and vaccines to enhance the treatment of leukemia. In this review, we mainly summarize to reveal the role of exosomes in AML pathogenesis, which helps us elucidate the application of exosomes in AML diagnosis and treatment.

Mapping the proteogenomic landscape enables prediction of drug response in acute myeloid leukemia

Acute myeloid leukemia is a poor-prognosis cancer commonly stratified by genetic aberrations, but these mutations are often heterogeneous and fail to consistently predict therapeutic response. Here, we combine transcriptomic, proteomic, and phosphoproteomic datasets with exvivo drug sensitivity data to help understand the underlying pathophysiology of AML beyond mutations. We measure the proteome and phosphoproteome of 210 patients and combine them with genomic and transcriptomic measurements to identify four proteogenomic subtypes that complement existing genetic subtypes. We build a predictor to classify samples into subtypes and map them to a "landscape" that identifies specific drug response patterns. We then build a drug response prediction model to identify drugs that target distinct subtypes and validate our findings on cell lines representing various stages of quizartinib resistance. Our results show how multiomics data together with drug sensitivity data can inform therapy stratification and drug combinations in AML.

Deep Sequencing Is a Widely Applicable Tool for Relapse Prediction in Acute Myeloid Leukemia with Mutated NPM1

Analysis of measurable residual disease (MRD) is a powerful tool for assessment of treatment response in acute myeloid leukemia (AML). In patients with mutation in NPM1, reverse transcription quantitative polymerase chain reaction (RT-qPCR) is recommended by European Leukemia Net for refinement of risk stratification and monitoring after treatment. However, since RT-qPCR is mutation-specific and requires stringent quality control, many clinical laboratories restrict NPM1 monitoring to the type A mutation (c.860_863dupTCTG) although >50 mutations have been reported in exon 12. Deep sequencing is an alternative method, performed on DNA, that covers all NPM1 exon 12variants in the same assay. Guidelines for its clinical use are however lacking. We here performed a retrospective analysis in a population-based cohort of AML patients to evaluate if deep sequencing MRD analysis of NPM1 during chemotherapy provides prognostic information. Adult patients with AML with mutated NPM1 treated with curative intent in our region during 2006-2016 who achieved complete remission (CR) or CR with incomplete count recovery (CRi) were eligible for the study. This time period was chosen to avoid inclusion of patients treated based on molecular MRD status. Through review of the national AML registry, clinical charts, and screening of diagnostic samples, 99 patients with mutated NPM1 were identified. Of these, 97 patients (60 women and 37 men, median age 64 years, range 19-82) had bone marrow slides for isolation of DNA from at least one time point during treatment. DNA from 257 bone marrow aspirate slides were assessed for MRD using deep sequencing of NPM1. Based on previous comparison with RT-qPCR (Pettersson et al, Int J Lab Hematol 2021;43:664-674), variant allele frequency (VAF) of ≥0.05% was chosen as cut-off to define deep sequencing MRD positivity (MRD pos). For subgroup analyses, patients were divided into groups of treatment intensity; high-dose (cytarabine- and daunorubicin-based, n=67) or low-dose (cytarabine- and daunorubicin-, or azacytidine-based, n=30) treatment regimens. Thirty-one patients underwent allogeneic stem cell transplantation (alloSCT). We first evaluated if deep sequencing MRD status during consolidation (n=70, analyzed after 2 or 3 cycles) had an impact on prognosis. Both 3-year relapse-free survival (RFS) (26.7±11.4% vs 71.6±6.2%, p=<0.001, Figure) and overall survival (OS) (33.3±12.2% vs 72.7±6%, p=0.004) were lower for patients that were MRD pos at any time point during consolidation than for MRD neg patients. Among MRD pos patients, 12/15 (80%) relapsed and 13/15 (87%) died, compared to 19/55 (35%) and 23/55 (42%) MRD neg patients. In univariate analyses, also age and treatment intensity, but not alloSCT or DNMT3A mutation status, predicted RFS and OS. FLT3-ITD predicted OS but not RFS, and there was no effect of FLT3-ITD allelic ratio. In Cox regression multivariate analysis including MRD, age, FLT3-ITD and treatment intensity, MRD pos was the only significant predictor of RFS (Hazard ratio (HR): 2.54, p=0.019). In the multivariate analysis, FLT3-ITD (HR: 2.43, p=0.014) and treatment intensity (HR: 0.43, p=0.035) were significant for OS (MRD pos HR: 1.75, p=0.13). When dividing the cohort based on treatment intensity, the effect of MRD status was most obvious in patients treated with low-dose regimens. When deep sequencing MRD analysis was performed post first cycle (n=96), it had no statistically significant effect on either RFS or OS. At the end of treatment (n=51), deep sequencing MRD status had a strong prognostic value with 3-year RFS and OS for MRD pos patients 30±14.5% and 40±15.5%, compared to 73.2±6.9% and 78±6.5% for MRD neg patients ( p=0.003 and p=0.006, respectively). In summary, this population-based study shows that MRD status by deep sequencing of NPM1 is predictive of both RFS and OS when assessed during or after consolidation. The study confirms the threshold of deep sequencing MRD pos of VAF 0.05% previously obtained by comparison with RT-qPCR. Since deep sequencing can be used for all NPM1 mutations, it is widely applicable and extends the use of molecular MRD analysis and risk refinement to patients with rare mutations in NPM1.

A Single Phenotypic Biomarker of Gemtuzumab Ozogamicin (GO) Response in Acute Myeloid Leukemia Using Ex-Vivo Testing

Background or Introduction: Gemtuzumab Ozogamicin (GO), a therapeutic antibody targeting CD33, gained FDA approval in 2017 for the treatment of patients with Acute Myeloid Leukemia (AML). Biomarkers to predict GO response were historically based on multiple indicators: CD33 expression levels, favorable cytogenetics and specific genetic alterations (NPM1, FLT3-internal tandem duplications, other mutations). Moreover, comprehensive analyses including stemness expression (LSC17 score) and molecularly derived scores such as the CD33_PGx6_Score (a composite score derived from six CD33 SNP of prognostic significance) have further refined the biomarkers to identify potential responders (Fenwarth et al, nt. J. Mol. Sci. 2020, 21, 5626). However, due to its toxicity profile and elevated cost, the identification of robust biomarkers of GO response in AML remains a challenge to optimize its therapeutic efficacy and worldwide clinical adoption. Methods: In this study, we utilized an innovative ex-vivo functional platform of Patient Micro Avatars (PMAs) developed at OncoPrecision (García et al, Blood 2022 140 (Supplement 1): 13027-13028) to assess the response to GO in a small cohort of AML patients. Patient-Derived Cells (PDCs) were obtained from five newly diagnosed AML patients and GO activity was assessed using high-throughput flow cytometry. The correlation between CD33 expression and the ex-vivo response to GO was also examined. Results: Our findings revealed a clear differential response to GO in AML patients, with contrasting outcomes observed within a dose-range that does not exceed its theoretical plasma concentration (Figure - Upper Panel). Interestingly, despite significant variations in CD33 expression levels among the evaluated patients, no obvious association was found between CD33 expression and the ex-vivo response to GO (Figure - Lower Panel). Conclusions: This study presents an innovative approach with the potential to predict GO responders versus non-responders in AML. A single phenotypic biomarker overcomes the need to determine CD33 expression levels in each patient or to assess the presence of specific polymorphic variants, as well as additional molecular biomarkers. Patient Micro Avatars present a compelling alternative to comprehensively identify patients who are most likely to benefit from GO treatment.

Identification and validation of necroptosis-related gene signatures to predict clinical outcomes and therapeutic responses in acute myeloid leukemia.

Necroptosis is a tightly regulated form of necrotic cell death that promotes inflammation and contributes to disease development. However, the potential roles of necroptosis-related genes (NRGs) in acute myeloid leukemia (AML) have not been elucidated fully. We conducted a study to identify a robust biomarker signature for predicting the prognosis and immunotherapy efficacy based on NRGs in AML. We analyzed the genetic and transcriptional alterations of NRGs in 151 patients with AML. Then, we identified three necroptosis clusters. Moreover, a necroptosis score was constructed and assessed based on the differentially expressed genes (DEGs) between the three necroptosis clusters. Three necroptosis clusters were correlated with clinical characteristics, prognosis, the tumor microenvironment, and infiltration of immune cells. A high necroptosis score was positively associated with a poor prognosis, immune-cell infiltration, expression of programmed cell death 1/programmed cell death ligand 1 (PD-1/PD-L1), immune score, stromal score, interferon-gamma (IFNG), merck18, T-cell dysfunction-score signatures, and cluster of differentiation-86, but negatively correlated with tumor immune dysfunction and exclusion score, myeloid-derived suppressor cells, and M2-type tumor-associated macrophages. Our observations indicated that a high necroptosis score might contribute to immune evasion. More interestingly, AML patients with a high necroptosis score may benefit from treatment based on immune checkpoint blockade. Consequently, our findings may contribute to deeper understanding of NRGs in AML, and facilitate assessment of the prognosis and treatment strategies.

Development and validation of a disulfidptosis-related scoring system to predict clinical outcome and immunotherapy response in acute myeloid leukemia by integrated analysis of single-cell and bulk RNA-sequencing.

Background: Disulfidptosis is a metabolically relevant mode of cell death, and its relationship with acute myeloid leukemia (AML) has not been clarified. In this study, disulfidptosis scores were computed to examine the potential biological mechanisms. Methods: Consensus clustering was applied to detect disulfidptosis-related molecular subtypes. The least absolute shrinkage and selection operator (LASSO) regression analysis was used to construct a DRG prognostic model. Results: DRGs are upregulated in AML and associated with poor prognosis. The higher the disulfidptosis activity score, the worse the clinical outcome for patients, accompanied by increased immune checkpoint expression and tumor marker pathway activity. The two molecular subtypes exhibited distinct prognoses and tumor microenvironment (TME) profiles. A prognostic risk score model was established using six DRGs, and the AML cohort was divided into high- and low-risk score groups. Patients in the high-risk group experienced significantly worse prognosis, which was validated in seven AML cohorts. Receiver Operating Characteristic (ROC) curve analysis indicated that the area under the curve values for risk score prediction of 1-, 3-, and 5-year survival were 0.779, 0.714, and 0.778, respectively. The nomogram, in conjunction with clinicopathological factors, further improved the accuracy of prognosis prediction. The high-risk score group exhibited a higher somatic mutation frequency, increased immune-related signaling pathway activity, and greater immune checkpoint expression, suggesting a certain degree of immunosuppression. Patients with advanced age and higher cytogenetic risk also had elevated risk scores. According to drug prediction and AML anti-PD-1 therapy cohort analysis, the low-risk score group displayed greater sensitivity to chemotherapy drugs like cytarabine and midostaurin, while the high-risk score group was more responsive to anti-PD-1 therapy. Finally, clinical samples were collected for sequencing analysis, confirming that the progression of myeloid leukemia was associated with a higher risk score and a negative disulfidptosis score, suggesting that the poor prognosis of AML may be associated with disulfidptosis resistance. Conclusion: In conclusion, a systematic analysis of DRGs can help to identify potential disulfidptosis-related mechanisms and provide effective new biomarkers for prognosis prediction, TME assessment, and the establishment of personalized treatment plans in AML.

Identification of cellular senescence-related signature for predicting prognosis and therapeutic response of acute myeloid leukemia.

Cellular senescence is closely related to the occurrence, development, and immune regulation of cancer. However, the predictive value of cellular senescence-related signature in clinical outcome and treatment response in acute myeloid leukemia (AML) remains unexplored. By analyzing the expression profile of cellular senescence-related genes (CSRGs) in AML samples in the TCGA database, we found that cellular senescence is closely related to the prognosis and tumor microenvironment of AML patients, and compared with normal samples, the overall expression level of senescent inducing genes in AML samples was down-regulated, while inhibitory genes were up-regulated. The risk score model further constructed and verified based on CSRGs could be used as an independent prognostic predictor for AML patients, and the overall survival (OS) of high-risk patients was significantly shortened. The area under ROC curve (AUC) values for the prediction of 1-, 3- and 5-year OS were 0.759, 0.749, and 0.806, respectively. In addition, patients with high-risk scores are more sensitive to treatment with cytarabine and may benefit from anti-PD-1 immunotherapy. In conclusion, our results suggest that the cellular senescence-related signature is a strong biomarker of immunotherapy response and prognosis in AML.

Harnessing Immune Response in Acute Myeloid Leukemia.

Despite the results achieved with the evolution of conventional chemotherapy and the inclusion of targeted therapies in the treatment of acute myeloid leukemia (AML), survival is still not satisfying, in particular in the setting of relapsed/refractory (R/R) disease or elderly/unfit patients. Among the most innovative therapeutic options, cellular therapy has shown great results in different hematological malignancies such as acute lymphoblastic leukemia and lymphomas, with several products already approved for clinical use. However, despite the great interest in also expanding the application of these new treatments to R/R AML, no product has been approved yet for clinical application. Furthermore, cellular therapy could indeed represent a powerful tool and an appealing alternative to allogeneic hematopoietic stem cell transplantation for ineligible patients. In this review, we aim to provide an overview of the most recent clinical research exploring the effectiveness of cellular therapy in AML, moving from consolidated approaches such as post- transplant donor's lymphocytes infusion, to modern adoptive immunotherapies such as alloreactive NK cell infusions, engineered T and NK cells (CAR-T, CAR-NK) and novel platforms of T and NK cells engaging (i.e., BiTEs, DARTs and ANKETTM).

Tuning the Organ Tropism of Polymersome for Spleen-Selective Nanovaccine Delivery to Boost Cancer Immunotherapy.

The past few decades have witnessed explosive development in drug delivery systems. However, in vivo delivery suffers from non-specific distribution in non-targeted organs or tissues, which may cause undesired side effects and even genotoxicity. Here, a general strategy that enables tuning the tropism of polymersomes for liver- and spleen-selective delivery is reported. By using a library screening approach, spleen-targeted polymersome PH9-Aln-8020 and liver-targeted polymersome PA9-ZP3-5050 are identified accordingly. Meanwhile, the second near-infrared (NIR-II) fluorescence imaging allows for in vivo dynamic evaluation of their spatial and temporal accumulation in specific tissues. O ur findings indicate that both polymer composition and protein corona on the surface are essential to determine the in vivo fate of polymersomes and tendency for specific organs. Importantly, PH9-Aln-8020 is employed as a systemic nanocarrier to co-deliver the antigen and adjuvant, which remarkably boost splenic immune responses in acute myeloid leukemia, melanoma, and melanoma lung metastasis mouse models. This study may open a new frontier for polymersomes in organ-selective delivery and other biomedical applications.

Role of Intracellular Drug Disposition in the Response of Acute Myeloid Leukemia to Cytarabine and Idarubicin Induction Chemotherapy.

Despite its often low efficacy and high toxicity, the standard treatment for acute myeloid leukemia (AML) is induction chemotherapy with cytarabine and idarubicin. Here, we have investigated the role of transporters and drug-metabolizing enzymes in this poor outcome. The expression levels (RT-qPCR) of potentially responsible genes in blasts collected at diagnosis were related to the subsequent response to two-cycle induction chemotherapy. The high expression of uptake carriers (ENT2), export ATP-binding cassette (ABC) pumps (MDR1), and enzymes (DCK, 5-NT, and CDA) in the blasts was associated with a lower response. Moreover, the sensitivity to cytarabine in AML cell lines was associated with ENT2 expression, whereas the expression of ABC pumps and enzymes was reduced. No ability of any AML cell line to export idarubicin through the ABC pumps, MDR1 and MRP, was found. The exposure of AML cells to cytarabine or idarubicin upregulated the detoxifying enzymes (5-NT and DCK). In AML patients, 5-NT and DCK expression was associated with the lack of response to induction chemotherapy (high sensitivity and specificity). In conclusion, in the blasts of AML patients, the reduction of the intracellular concentration of the active metabolite of cytarabine, mainly due to the increased expression of inactivating enzymes, can determine the response to induction chemotherapy.

Immune cell proportions correlate with clinicogenomic features and ex vivo drug responses in acute myeloid leukemia.

The implementation of small-molecule and immunotherapies in acute myeloid leukemia (AML) has been challenging due to genetic and epigenetic variability amongst patients. There are many potential mechanisms by which immune cells could influence small-molecule or immunotherapy responses, yet, this area remains understudied. Here we performed cell type enrichment analysis from over 560 AML patient bone marrow and peripheral blood samples from the Beat AML dataset to describe the functional immune landscape of AML. We identify multiple cell types that significantly correlate with AML clinical and genetic features, and we also observe significant correlations of immune cell proportions with ex vivo small-molecule and immunotherapy responses. Additionally, we generated a signature of terminally exhausted T cells (Tex) and identified AML with high monocytic proportions as strongly correlating with increased proportions of these immunosuppressive T cells. Our work, which is accessible through a new "Cell Type" module in our visualization platform (Vizome; http://vizome.org/), can be leveraged to investigate potential contributions of different immune cells on many facets of the biology of AML.

Prediction model for drug response of acute myeloid leukemia patients

Despite some encouraging successes, predicting the therapy response of acute myeloid leukemia (AML) patients remains highly challenging due to tumor heterogeneity. Here we aim to develop and validate MDREAM, a robust ensemble-based prediction model for drug response in AML based on an integration of omics data, including mutations and gene expression, and large-scale drug testing. Briefly, MDREAM is first trained in the BeatAML cohort (n = 278), and then validated in the BeatAML (n = 183) and two external cohorts, including a Swedish AML cohort (n = 45) and a relapsed/refractory acute leukemia cohort (n = 12). The final prediction is based on 122 ensemble models, each corresponding to a drug. A confidence score metric is used to convey the uncertainty of predictions; among predictions with a confidence score >0.75, the validated proportion of good responders is 77%. The Spearman correlations between the predicted and the observed drug response are 0.68 (95% CI: [0.64, 0.68]) in the BeatAML validation set, –0.49 (95% CI: [–0.53, –0.44]) in the Swedish cohort and 0.59 (95% CI: [0.51, 0.67]) in the relapsed/refractory cohort. A web-based implementation of MDREAM is publicly available at https://www.meb.ki.se/shiny/truvu/MDREAM/.

Longitudinal single-cell profiling of chemotherapy response in acute myeloid leukemia

Acute myeloid leukemia may be characterized by a fraction of leukemia stem cells (LSCs) that sustain disease propagation eventually leading to relapse. Yet, the contribution of LSCs to early therapy resistance and AML regeneration remains controversial. We prospectively identify LSCs in AML patients and xenografts by single-cell RNA sequencing coupled with functional validation by a microRNA-126 reporter enriching for LSCs. Through nucleophosmin 1 (NPM1) mutation calling or chromosomal monosomy detection in single-cell transcriptomes, we discriminate LSCs from regenerating hematopoiesis, and assess their longitudinal response to chemotherapy. Chemotherapy induced a generalized inflammatory and senescence-associated response. Moreover, we observe heterogeneity within progenitor AML cells, some of which proliferate and differentiate with expression of oxidative-phosphorylation (OxPhos) signatures, while others are OxPhos (low) miR-126 (high) and display enforced stemness and quiescence features. miR-126 (high) LSCs are enriched at diagnosis in chemotherapy-refractory AML and at relapse, and their transcriptional signature robustly stratifies patients for survival in large AML cohorts.

Ex vivo venetoclax sensitivity testing predicts treatment response in acute myeloid leukemia.

The BCL-2 inhibitor venetoclax has revolutionized the treatment of acute myeloid leukemia (AML) in patients not benefiting from intensive chemotherapy. Nevertheless, treatment failure remains a challenge, and predictive markers are needed, particularly for relapsed or refractory AML. Ex vivo drug sensitivity testing may correlate with outcomes, but its prospective predictive value remains unexplored. Here we report the results of the first stage of the prospective phase II VenEx trial evaluating the utility and predictiveness of venetoclax sensitivity testing using different cell culture conditions and cell viability assays in patients receiving venetoclax-azacitidine. Participants with de novo AML ineligible for intensive chemotherapy, relapsed or refractory AML, or secondary AML were included. The primary endpoint was the treatment response in participants showing ex vivo sensitivity and the key secondary endpoints were the correlation of sensitivity with responses and survival. Venetoclax sensitivity testing was successful in 38/39 participants. Experimental conditions significantly influenced the predictive accuracy. Blast-specific venetoclax sensitivity measured in conditioned medium most accurately correlated with treatment outcomes; 88% of sensitive participants achieved a treatment response. The median survival was significantly longer for participants who were ex vivo-sensitive to venetoclax (14.6 months for venetoclax-sensitive patients vs. 3.5 for venetoclax-insensitive patients, P<0.001). This analysis illustrates the feasibility of integrating drug-response profiling into clinical practice and demonstrates excellent predictivity. This trial is registered with ClinicalTrials.gov identifier: NCT04267081.

Triple combination targeting methyltransferase, BCL-2, and PD-1 facilitates antileukemia responses in acute myeloid leukemia.

A recent breakthrough therapy combining the BCL-2 inhibitor venetoclax with hypomethylating agents (HMAs) targeting DNA methyltransferase has improved outcomes for patients with acute myeloid leukemia (AML), but the responses and long-term survival in older/unfit patients and in patients with relapsed/refractory AML remain suboptimal. Recent studies showed that inhibition of BCL-2 or DNA methyltransferase modulates AML T-cell immunity. By using flow cytometry and time-of-flight mass cytometry, the authors examined the effects of the HMA decitabine combined with the BCL-2 inhibitor venetoclax (DAC/VEN therapy) on leukemia cells and T cells in patients with AML who received DAC/VEN therapy in a clinical trial. The authors investigated the response of programmed cell death protein 1 (PD-1) inhibition in the DAC/VEN-treated samples in vitro and investigated the triple combination of PD-1 inhibition with HMA/venetoclax in the trial patients who had AML. DAC/VEN therapy effectively targeted leukemia cells and upregulated the expression of the immune checkpoint-inhibitory receptor PD-1 in T cells while preserving CD4-positive and CD8-positive memory T cells in a subset of patients with AML who were tested. In vitro PD-1 inhibition potentiated the antileukemia response in DAC/VEN-treated AML samples. The combined use of azacitidine, venetoclax, and nivolumab eliminated circulating blasts and leukemia stem cells/progenitor cells and expanded the percentage of CD8-positive memory T cells in an illustrative patient with relapsed AML who responded to the regimen in an ongoing clinical trial. Immunomodulation by targeting PD-1 enhances the therapeutic effect of combining an HMA and venetoclax in patients with AML.

Establishment and validation of a carbohydrate metabolism-related gene signature for prognostic model and immune response in acute myeloid leukemia.

The heterogeneity of treatment response in acute myeloid leukemia (AML) patients poses great challenges for risk scoring and treatment stratification. Carbohydrate metabolism plays a crucial role in response to therapy in AML. In this multicohort study, we investigated whether carbohydrate metabolism related genes (CRGs) could improve prognostic classification and predict response of immunity and treatment in AML patients. Using univariate regression and LASSO-Cox stepwise regression analysis, we developed a CRG prognostic signature that consists of 10 genes. Stratified by the median risk score, patients were divided into high-risk group and low-risk group. Using TCGA and GEO public data cohorts and our cohort (1031 non-M3 patients in total), we demonstrated the consistency and accuracy of the CRG score on the predictive performance of AML survival. The overall survival (OS) was significantly shorter in high-risk group. Differentially expressed genes (DEGs) were identified in the high-risk group compared to the low-risk group. GO and GSEA analysis showed that the DEGs were mainly involved in immune response signaling pathways. Analysis of tumor-infiltrating immune cells confirmed that the immune microenvironment was strongly suppressed in high-risk group. The results of potential drugs for risk groups showed that inhibitors of carbohydrate metabolism were effective. The CRG signature was involved in immune response in AML. A novel risk model based on CRGs proposed in our study is promising prognostic classifications in AML, which may provide novel insights for developing accurate targeted cancer therapies.