Although targeting of cell metabolism is a promising therapeutic strategy in acute myeloid leukemia (AML), metabolic dependencies are largely unexplored. We aimed to classify AML patients based on ...their metabolic landscape and map connections between metabolic and genomic profiles. Combined serum and urine metabolomics improved AML characterization compared with individual biofluid analysis. At intracellular level, AML displayed dysregulated amino acid, nucleotide, lipid, and bioenergetic metabolism. The integration of intracellular and biofluid metabolomics provided a map of alterations in the metabolism of polyamine, purine, keton bodies and polyunsaturated fatty acids and tricarboxylic acid cycle. The intracellular metabolome distinguished three AML clusters, correlating with distinct genomic profiles: NPM1-mutated(mut), chromatin/spliceosome-mut and TP53-mut/aneuploid AML that were confirmed by biofluid analysis. Interestingly, integrated genomic-metabolic profiles defined two subgroups of NPM1-mut AML. One was enriched for mutations in cohesin/DNA damage-related genes (NPM1/cohesin-mut AML) and showed increased serum choline + trimethylamine-N-oxide and leucine, higher mutation load, transcriptomic signatures of reduced inflammatory status and better ex-vivo response to EGFR and MET inhibition. The transcriptional differences of enzyme-encoding genes between NPM1/cohesin-mut and NPM1-mut allowed in silico modeling of intracellular metabolic perturbations. This approach predicted alterations in NAD and purine metabolism in NPM1/cohesin-mut AML that suggest potential vulnerabilities, worthy of being therapeutically explored.
Proton beam therapy is considered a step forward with respect to electromagnetic radiation, thanks to the reduction in the dose delivered. Among unwanted effects to healthy tissue, cardiovascular ...complications are a known long-term radiotherapy complication. The transcriptional response of cardiac tissue from xenografted BALB/c nude mice obtained at 3 and 10 days after proton irradiation covering both the tumor region and the underlying healthy tissue was analyzed as a function of dose and time. Three doses were used: 2 Gy, 6 Gy, and 9 Gy. The intermediate dose had caused the greatest impact at 3 days after irradiation: at 2 Gy, 219 genes were differently expressed, many of them represented by zinc finger proteins; at 6 Gy, there were 1109, with a predominance of genes involved in energy metabolism and responses to stimuli; and at 9 Gy, there were 105, mainly represented by zinc finger proteins and molecules involved in the regulation of cardiac function. After 10 days, no significant effects were detected, suggesting that cellular repair mechanisms had defused the potential alterations in gene expression. The nonlinear dose-response curve indicates a need to update the models built on photons to improve accuracy in health risk prediction. Our data also suggest a possible role for zinc finger protein genes as markers of proton therapy efficacy.
The contribution of cell-extrinsic factors in Acute Myeloid Leukemia (AML) generation and persistence has gained interest. Bitter taste receptors (TAS2Rs) are G protein-coupled receptors known for ...their primary role as a central warning signal to induce aversion toward noxious or harmful substances. Nevertheless, the increasing amount of evidence about their extra-oral localization has suggested a wider function in sensing microenvironment, also in cancer settings. In this study, we found that AML cells express functional TAS2Rs. We also highlighted a significant association between the modulation of some TAS2Rs and the poor-prognosis AML groups, i.e.,
- and
-mutated, supporting a potential role of TAS2Rs in AML cell biology. Gene expression profile analysis showed that TAS2R activation with the prototypical agonist, denatonium benzoate, significantly modulated a number of genes involved in relevant AML cellular processes. Functional assay substantiated molecular data and indicated that denatonium reduced AML cell proliferation by inducing cell cycle arrest in G0/G1 phase or induced apoptosis via caspase cascade activation. Moreover, denatonium exposure impaired AML cell motility and migratory capacity, and inhibited cellular respiration by decreasing glucose uptake and oxidative phosphorylation. In conclusion, our results in AML cells expand the observation of cancer TAS2R expression to the setting of hematological neoplasms and shed light on a role of TAS2Rs in the extrinsic regulation of leukemia cell functions.
Abstract
Hematopoietic stem cells (HSC) and leukemic stem cells (LSC) develop in the hypoxic bone marrow (BM) niche. Hypoxia contributes to the development and maintenance of LSC and might influence ...chemosensitivity in acute myeloid leukemia (AML). In this study we analyzed the transcriptomic and metabolomic profile of 2 AML cell lines with diverse genomic background, in order to better understand the impact of hypoxia in AML. Gene Expression Profiling (GEP) and Liquid Chromatography-Tandem Mass Spectroscopy were performed on OCI-AML3 (FAB M4,NPM1 and DNMT3A mutations) and KASUMI-1 (FAB M2, t(8;21) and KITmutation) cell lines, after 20 hours of incubation under normoxia or hypoxia (1% O2). GEP analysis was performed by Transcriptome Analysis Console (Affymetrix), DAVID tool and Gene Set Enrichment Analysis (GSEA). Hypoxia alters the transcriptional level of 1301 and 276 genes in OCI-AML3 and KASUMI-1, respectively. Hypoxia induces MYC down-regulation in both lines, combined with increased expression of several HIF-1α-related genes (e.g. ARNT, CXCR4, S100A4). Moreover, hypoxia upregulates HIF-1α target genes associated with the glycolytic-pathway, as HK2, GPI, PFKP, PKM, LDHA, ALDOA and ENO1 (p<0.01). Differentially expressed transcripts were significantly enriched for genes involved in metabolic pathways including regulation of cellular amino acid metabolic process, canonical glycolysis, gluconeogenesis, pyruvate metabolism and glycolytic process (p<0.01). Conversely, gene sets related to tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OxPhos), cytochrome complex assembly and mitochondrial respiratory chain were enriched in normoxia. Hypoxia forced metabolic adaptation that was dependent on the cell type. In OCI-AML3 cells, deep hypoxia induced metabolic changes associated to the Warburg effect, with increased glycolysis and low efficiency of OxPhos. The metabolic hypoxia-related profile was characterized by an increased conversion of pyruvate to lactate and alanine with high levels of fumarate and succinate, which are intermediates of the TCA cycle, elevated 2-hydroxiglutarate and glycerol 3-phospate, suggesting a reduction of energy production by OxPhos. The observed increase of glutamine levels and the reduction of glutamate, that is catabolized by TCA cycle, was associated with down-regulation of MYC expression, induced by hypoxia. Kasumi-1 showed an increase of lysolipid and fatty acid metabolism, with low impact on TCA intermediates and increased levels of alanine, glutamine and glutamate. The observed cell-line specific metabolic response to hypoxia suggests that a deep characterization of stem cells residing in the hypoxic BM niche is required in each patient. Moreover, the analysis of the metabolic profile may help define specific vulnerabilities and guide personalized therapeutic approaches.Supported by: ELN, AIL, AIRC, FP7 NGS-PTL
Note: This abstract was not presented at the meeting.
Citation Format: Samantha Bruno, Martina Pazzaglia, Claudio Cerchione, Simona Soverini, Michele Cavo, Lorenzo Montanaro, Giorgia Simonetti, Giovanni Martinelli. Deep hypoxia and the genomic background cooperate to shape the metabolic profile of acute myeloid leukemia cells abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2651.
The stromal and immune bone marrow (BM) landscape is emerging as a crucial determinant for acute myeloid leukemia (AML). Regulatory T cells (Tregs) are enriched in the AML microenvironment, but the ...underlying mechanisms are poorly elucidated. Here, we addressed the effect of IFN-γ released by AML cells in BM Tregs induction and its impact on AML prognosis.
BM aspirates from AML patients were subdivided according to IFNG expression. Gene expression profiles in INFGhigh and IFNGlow samples were compared by microarray and NanoString analysis and used to compute a prognostic index. The IFN-g release effect on the BM microenvironment was investigated in mesenchymal stromal cell (MSC)/AML cell co-cultures. In mice, AML cells silenced for IFN-γ expression were injected intrabone.
IFNGhigh AMLsamples showed an upregulation of inflammatory genes, usually correlated with a good prognosis in cancer. By contrast, in AML patients, high IFNG expression associated with poor overall survival. Notably, IFN-g release by AML cells positively correlated with a higher BM suppressive Tregs' frequency. In co-culture experiments, IFNGhigh AML cells modified MSC transcriptome by up-regulating IFN-γ-dependent genes related to Treg induction, including indoleamine 2,3-dioxygenase 1 (IDO1). IDO1 inhibitor abrogated the effect of IFN-γ release by AML cells on MSC-derived Treg induction. Invivo, the genetic ablation of IFN-γ production by AML cells reduced MSC IDO1 expression and Treg infiltration, hindering AML engraftment.
IFN-g release by AML cells induces an immune-regulatory program in MSCs and remodels BM immunological landscape toward Treg induction, contributing to an immunotolerant microenvironment.
Abstract
Acute Myeloid Leukemia (AML) is a clonal disease developing from a rare population of leukemic stem cells. Alongside the identification of disease-specific alleles harbored by AML clones, ...the contribution that cell-extrinsic factors have in AML generation and persistence by influencing AML cell genomic landscape and therapy-resistance is gaining increasing interest. In the cross-talk between AML cells and their microenvironment, several membrane receptors sense the external changes by triggering intracellular signals. Among these, the largest group belongs to the family of G protein-couple receptors (GPCRs). Bitter taste receptors (T2Rs) comprise 25 distinct members of the GPCR family. Initially described in the oral cavity, T2Rs are actually widely expressed in different tissues and in various cancer cell types. In the present work, we showed that AML cells expressed fully functional T2R subtypes. Their activation substantially modified the AML cell transcriptomic profile and deregulated relevant cellular processes, resulting in inhibited clonogenic capacity, proliferation and cell motility and induced apoptosis. In addition, T2R stimulation with agonist altered mitochondrial bioenergetic capacity and made AML cells more prone to oxidative and metabolic stress. Given the effect of T2R activation on crucial AML cell function, we tested the therapeutical potential of targeting T2Rs. Interestingly, we observed that T2R stimulation had a synergistic effect with cytarabine, reducing leukemia cell viability and allowing to reach high toxicity using lower doses of chemotherapic agent. Then we analyzed T2Rs expression and function on normal hematopoietic stem cells (HSCs). HSCs expressed several T2R subtypes with some differences compare to AML cells. T2Rs activation didn't affect HSCs viability and motility, conversely to AML cells, but significantly inhibited clonogenic capacity, by reducing the frequency of precursors. Our data may suggest a role for microenvironment “bitter” molecules in regulating normal and leukemic hematopoiesis. Notably, many common drugs, such as antibiotics, chloroquine, haloperidol, procainamide, are bitter tasting and are thus effective ligands for T2Rs. For this reason, they could exhibit an off-target effect in T2R expressing cells, including leukemic and normal hematopoietic cells. Our results may have implications in understanding the off-target actions of diverse drugs and could reveal potential new therapeutic targets
Citation Format: Valentina Salvestrini, Valentina Pensato, Marilena Ciciarello, Giorgia Simonetti, Samantha Bruno, Martina Pazzaglia, Elena De Marchi, Dorian Forte, Stefania Orecchioni, Giovanni Martinelli, Francesco Bertolini, Simon Mendez-Ferrer, Elena Adinolfi, Michele Cavo, Antonio Curti. Bitter taste receptors system is expressed and functional in both HSCs and leukemic cells abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4946.
Abstract
Introduction: PPM1D (wild-type p53-inducible protein phosphatase WIP1) is a member of the PP2C family serine/threonine phosphatase involved in negative regulation of cell stress response ...pathways, leading to suppression of p53 after stress. To restore p53 function through MDM2 inhibition (Nutlin-3a) is a promising approach in AML. Promising results of the combination of MDM2 inhibitors and WIP1 inhibitor (WIP1i), obtained in many preclinical studies of solid tumors, paved the way for their application in AML. We investigated whether the inhibition of WIP1 (GSK2830371) could sensitize AML cell lines and primary cells to Nutlin-3a (Nut-3a) in order to obtain a novel therapeutic strategy for AML patients, based on restoration of p53 activity.
Methods: In vitro viability (by WST-1 reagent) and Annexin-V/PI apoptosis assay were performed on a panel of TP53-wt AML cells (MOLM-13, MV-4-11 and OCI-AML3), on TP53-mutated NOMO-1 AML cells and on primary AML samples. Gene expression profile (GEP) and Western Blot (WB) analyses were performed on MV-4-11 and NOMO-1 after 16h.
Results: Combined inhibition of increasing dosage of Nut-3a (0.5 to 5 uM) and WIP1i (5 to 20 uM) synergistically reduces TP53-wt AML cells viability, while NOMO-1 resulted to be insensitive to the combination. The combination index analyses showed a synergistically effect of the combination of both compounds on TP53-wt AML cells. Annexin V/PI staining showed that WIP1i sensitizes TP53-wt AML cell lines and primary samples to Nut-3a-induced apoptosis, when compared with single treatment. No effect was seen in NOMO-1. GEP demonstrated that MV-4-11 cells exhibits a major response to drug combination with an upregulation of cell cycle control genes, a downregulation of DNA repair machinery genes, upregulation of MDM2 and of TP53-downstream genes (eg.CDKN1A), confirming the activation of p53 pathway. NOMO-1 cells showed upregulation of resiliency-mechanism and confirmed insensitivity to both treatment. WB analysis confirmed GEP data showing an increased expression of p53 and p21 in wt-TP53 cell line after 16h of combined-treatment.
Conclusions: We identified a novel synergistic drug combination between Nut-3a and WIP1i that induces apoptosis in AML cell lines and primary samples. GEP and WB of TP53-wt MV-4-11 and TP53-mutated NOMO-1 cells showed mechanisms underlying drug sensitivity and resistance giving novel insights on potential markers of response and novel drugable targets. In vivo studies are needed to confirm these preclinical data. Supported by: AIRC, FP7-NGS-PTL, Fondazione del Monte, HARMONY.
Citation Format: Maria Chiara Fontana, Jacopo Nanni, Giovanni Marconi, Martina Pazzaglia, Matteo Bocconcelli, Antonella Padella, Simona Soverini, Ilaria Iacobucci, Cristina Papayannidis, Anna Ferrari, Maria Teresa Bochicchio, Enrica Imbrogno, Michele Cavo, Andrea Ghelli Luserna di Rora, Giorgia Simonetti, Giovanni Martinelli. Pharmacological inhibition of WIP1 by GSK2830371 sensitizes AML cells to MDM2 inhibitor Nutlin-3a abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2964.
Abstract Background Severely ill patients with coronavirus disease 2019 (COVID‐19) show an increased risk of new‐onset atrioventricular blocks (AVBs), associated with high rates of short‐term ...mortality. Recent data suggest that the uncontrolled inflammatory activation observed in these patients, specifically interleukin (IL)‐6 elevation, may play an important pathogenic role by directly affecting cardiac electrophysiology. The aim of our study was to assess the acute impact of IL‐6 changes on electrocardiographic indices of atrioventricular conduction in severe COVID‐19. Methods We investigated (1) the behavior of PR‐interval and PR‐segment in patients with severe COVID‐19 during active phase and recovery, and (2) their association with circulating IL‐6 levels over time. Results During active disease, COVID‐19 patients showed a significant increase of PR‐interval and PR‐segment. Such atrioventricular delay was transient as these parameters rapidly normalized during recovery. PR‐indices significantly correlated with circulating IL‐6 levels over time. All these changes and correlations persisted also in the absence of laboratory signs of cardiac strain/injury or concomitant treatment with PR‐prolonging drugs, repurposed or not. Conclusions Our study provides evidence that in patients with severe COVID‐19 and high‐grade systemic inflammation, IL‐6 elevation is associated with a significant delay of atrioventricular conduction, independent of concomitant confounding factors. While transient, such alterations may enhance the risk of severe AVB and associated short‐term mortality. Our data provide further support to current anti‐inflammatory strategies for severe COVID‐19, including IL‐6 antagonists.
Abstract
Functional profiling is an emerging trend in precision medicine. Manual laboratory methods proved to be effective in predicting the response to drug treatments both in patients stratified ...according to genetic profiling and in patients without any specific molecular aberration. In this work, we validated the predictive power of an innovative functional profiling platform, based on lab-on-a-chip technology, which allows testing the response of live tumor cells exposed to anticancer drugs in a fully-automated process, requiring only a Ficoll stratification as a manual step.
15 AML patients (Age: 40% <60 years, 46% in the 60-70 years range, 14% >70 years; Gender: 40% Female; ELN Classification: 20% Favorable, 26% Intermediate-I, 33% Intermediate-II, 14% Adverse; AML stage: 53% De novo, 7% Relapse, 40% Refractory; Treatment: 20% FLAI-3, 40% FLAI-5, 26% Decitabine; 7% Cytarabine, 7% 5-azacitidine) were treated at the Policlinico Sant'Orsola in Bologna. At the same time, bone marrow samples were challenged with the same drugs at scaled concentrations in the functional profiling platform developed by CellPly and an output score was extracted from time-lapse fluorescence image analysis of patient tumor cells exposed to the drug or drug combination for 24 hours. Clinical follow up resulted in the following classification: complete hematologic response (CR) was found in 6/15 patients (6/6 with a De Novo disease), while the remaining 9/15 patients (2/9 de novo; 1/9 Relapse; 6/9 Refractory) were classified as stable disease (SD).
In-vitro functional profiling of 14 patients provided a correlation with the clinical outcome (p=0.01) irrespective of the stage of the disease. 100% of the patients identified as responders by functional profiling (n = 5), resulted in a CR outcome. 89% of the patients identified as non-responders (n = 9) resulted in an SD outcome.
The same patient samples were also investigated for the molecular assessment of the most relevant genes commonly analyzed in AML patients. FLT3-ITD mutation was found in 2/14 patients, both in SD. Negative FLT3 was not correlated to clinical outcome. FLT3-TKD mutation was found in 3/14 patients, 2/3 resulted in CR, 1/3 in SD. TP53 mutation was found in 1/13 patients, classified as SD. Intronic variant rs1625895 was found in 6/13 patients equally distributed in CR and SD. NPM1 mutation was identified in 4/13 patients, 2/4 resulted in CR, 2/4 in SD indicating no direct correlation. IDH2 was found in 1/11 patients (with R172K mutation) with an SD.
As a conclusion, functional profiling proved to be highly correlated with clinical outcome irrespective of patient stage and demonstrated a superior predictive power compared to molecular profiling.
Citation Format: Laura Rocchi, Andrea Faenza, Viviana Guadagnuolo, Laura Rambelli, Giovanni Marconi, Maria Chiara Fontana, Martina Pazzaglia, Cristina Papayannidis, Giorgia Simonetti, Nicola Pecorari, Luca Giulianelli, Dario Biscarini, Marco Bettelli, Michele Federici, Rita Ruggiano, Giovanni Martinelli, Roberto Guerrieri, Massimo Bocchi. Lab-on-a-chip-based in-vitro functional profiling proves to be effective in predicting therapy outcome in AML patients abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3613.
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