Identification of Prognostic Genes in Acute Myeloid Leukemia Microenvironment: A Bioinformatic and Experimental Analysis.

Acute myeloid leukemia (AML) is a lethal hematologic malignancy with a variable prognosis that is highly dependent on the bone marrow microenvironment. Consequently, a better understanding of the AML microenvironment is crucial for early diagnosis, risk stratification, and personalized therapy. In recent years, the role of bioinformatics as a powerful tool in clarifying the complexities of cancer has become more prominent. Gene expression profile and clinical data of 173 AML patients were downloaded from the TCGA database, and the xCell algorithm was applied to calculate the microenvironment score (MS). Then, the correlation of MS with FAB classification, and CALGB cytogenetic risk category was investigated. Differentially expressed genes (DEGs) were identified, and the correlation analysis of DEGs with patient survival was done using univariate cox. The prognostic value of candidate prognostic DEGs was confirmed based on the GEO database. In the last step, real-time PCR was used to compare the expression of the top three prognostic genes between patients and the control group. During TCGA data analysis, 716 DEGs were identified, and survival analysis results showed that 152 DEGs had survival-related changes. In addition, the prognostic value of 31 candidate prognostic genes was confirmed by GEO data analysis. Finally, the expression analysis of FLVCR2, SMO, and CREB5 genes, the most related genes to patients' survival, was significantly different between patients and control groups. In summary, we identified key microenvironment-related genes that influence the survival of AML patients and may serve as prognostic and therapeutic targets.

Evaluation of the therapeutic effect of very small stem cells from peripheral blood on the treatment of Premature Ovarian Failure: A pilot study.

OBJECTIVE: Premature ovary failure (POF) is a severe health condition with multiple negative outcomes, which deteriorate a patient's life. The current study aimed to evaluate the therapeutic effect of mesenchymal stem cells (MSCs) derived from peripheral blood in the treatment of women with the POF background. METHODS: The current study was a pilot study carried-out on women younger than 40 with premature ovarian failure. Study participants underwent 4-months cell therapy using Mesenchymal stem cells extracted from peripheral bloods. Serum level of Follicle-stimulating hormone (FSH), Estradiol (E2), Anti-mullerian hormone (AMH), and Antral follicle count (AFC) were the main investigated outcomes that were assessed at baseline, month two and month four of the very small stem cell intervention. RESULTS: Average serum level of FSH was 45.0 (12.1) mIU/mL at baseline and continually decreased during the study and reached 33.2 (12.4) mIU/mL in the fourth month. The average AMH level was 0.10 ng/mL prior to the intervention and increased to 0.13 ng/mL in the 2nd month and 0.15 ng/mL in the fourth month. The level E2 was 85.7 (23.6) pg/ml on average at baseline, while the average E2 reduced to 77.2 (25.6) pg/ml in the fourth month. Average number of AFC was 2.0 (0.8) at baseline. We observed a gradual increase in the second month (Mean AFC=2.2) and after four months it increased to 3.1 (1.8) as the highest menstrual restoration and pregnancy was observed in 10% of our study participants. CONCLUSIONS: MSCs could significantly improve hormone secretion in women with POF. Implantation of MSCs in women with POF background was associated with an increase in AMH and AFC, while it downed the serum level of E2 and FSH. MSCs could also lead to menstrual restoration and pregnancy in women with POF.

High yield killing of lymphoma cells by anti-CD22 CAR-NK cell therapy.

Chimeric antigen receptors (CARs) offer a promising new approach for targeting B cell malignancies through the immune system. Despite the proven effectiveness of CAR T cells targeting CD19 and CD22 in hematological malignancies, it is imperative to note that their production remains a highly complex process. Unlike T cells, NK cells eliminate targets in a non-antigen-specific manner while avoiding graft vs. host disease (GvHD). CAR-NK cells are considered safer than CAR-T cells because they have a shorter lifespan and produce less toxic cytokines. Due to their unlimited ability to proliferate in vitro, NK-92 cells can be used as a source for CAR-engineered NK cells. We found that CARs created from the m971 anti-CD22 mAb, which specifically targets a proximal CD22 epitope, were more effective at anti-leukemic activity compared to those made with other binding domains. To further enhance the anti-leukemic capacity of NK cells, we used lentiviral transduction to generate the m971-CD28-CD3ζ NK-92. CD22 is highly expressed in B cell lymphoma. To evaluate the potential of targeting CD22, Raji cells were selected as CD22-positive cells. Our study aimed to investigate CD22 as a potential target for CAR-NK-92 therapy in the treatment of B cell lymphoma. We first generated m971-CD28-CD3ζ NK-92 that expressed a CAR for binding CD22 in vitro. Flow cytometric analysis was used to evaluate the expression of CAR. The 7AAD determined the cytotoxicity of the m971-CD28-CD3ζ NK-92 towards target lymphoma cell lines by flow cytometry assay. The ELISA assay evaluated cytokine production in CAR NK-92 cells in response to target cells. The m971-CD28-CD3ζ NK-92 cells have successfully expressed the CD22-specific CAR. m971-CD28-CD3ζ NK-92 cells efficiently lysed CD22-expressing lymphoma cell lines and produced large amounts of cytokines such as IFN-γ and GM-CSF but a lower level of IL-6 after coculturing with target cells. Based on our results, it is evident that transferring m971-CD28-CD3ζ NK-92 cells could be a promising immunotherapy for B cell lymphoma.

WT1 and TP53 as valuable diagnostic biomarkers for relapse after hematopoietic stem cell transplantation in acute myeloid leukemia.

BACKGROUND: Relapse following hematopoietic stem cell transplantation (HSCT) occurs relatively frequently and is a significant risk factor for mortality in patients with acute myeloid leukemia (AML). Early diagnosis is, therefore, of utmost importance and can provide valuable guidance for appropriate and timely intervention. Here, the diagnostic value of two molecular markers, Wilms tumor 1 (WT1) and tumor suppressor protein p53 (TP53), were studied. METHODS AND RESULTS: Twenty AML patients undergoing HSCT participated in this investigation. Some had relapsed following HSCT, while others were in remission. Peripheral blood (PB) and bone marrow (BM) samples were collected following relapse and remission. WT1 and TP53 messenger RNA (mRNA) expression was evaluated using reverse transcription-quantitative polymerase chain reaction (RT‒qPCR). The diagnostic value of genes was evaluated by utilizing receiver-operating characteristic (ROC) curve analysis. ROC analysis showed WT1 and TP53 as diagnostic markers for relapse after HSCT in AML patients. The mRNA expression level of WT1 was elevated in individuals who experienced relapse compared to those in a state of remission (p value < 0.01). Conversely, the expression level of TP53 mRNA was lower in individuals who had relapsed compared to those in remission (p value < 0.01). CONCLUSIONS: WT1 and TP53 possess the potential to serve as invaluable biomarkers in the identification of molecular relapse after HSCT in patients with AML. Further studies for a definitive conclusion are recommended.

The Potential of Cannabidiol for Acute Respiratory Distress Syndrome in COVID-19.

COVID-19 disease manifests itself in a wide range of signs and symptoms, beginning with mild symptoms, such as fever, cough, and dyspnea, progressing to acute respiratory distress syndrome (ARDS) and death in some cases. The cytokine storm, or an excess of cytokines released locally, is assumed to be the primary cause of ARDS and mortality in COVID-19 patients. To enhance the survival rate of COVID-19 patients, early management of the cytokine storm with immunomodulators is crucial. Although the effectiveness of some immunosuppressants, such as corticosteroids and tocilizumab, has been studied in clinical trials, the administration of these drugs should be exercised cautiously. Cannabidiol (CBD) is a non-psychotropic phytocannabinoid from Cannabis sativa extracts with anti-inflammatory properties. This review is intended to discuss the possible utility of CBD for the management of COVID-19 patients, particularly those with ARDS.

Mechanisms of Action of Noninvasive Brain Stimulation with Weak Non-Constant Current Stimulation Approaches.

Objective: Non-constant current stimulation (NCCS) is a neuromodulatory method in which weak alternating, pulsed or random currents are delivered to the human head via scalp or earlobe electrodes. This approach is widely used in basic and translational studies. However, the underlying mechanisms of NCCS, which lead to biological and behavioral effects in the brain, remain largely unknown. In this review, we characterize NCCS techniques currently being utilized in neuroscience investigations, including transcranial alternating current stimulation (tACS), transcranial pulsed current stimulation (tPCS), transcranial random noise stimulation (tRNS), and cranial electrotherapy stimulation (CES). Method: We unsystematically searched all relevant conference papers, journal articles, chapters, and textbooks on the biological mechanisms of NCCS techniques. Results: The fundamental idea of NCCS is that these low-level currents can interact with neuronal activity, modulate neuroplasticity and entrain cortical networks, thus, modifying cognition and behavior. We elucidate the mechanisms of action for each NCCS technique. These techniques may cause microscopic effects (such as affecting ion channels and neurotransmission systems) and macroscopic effects (such as affecting brain oscillations and functional connectivity) on the brain through different mechanisms of action (such as neural entrainment and stochastic resonance). Conclusion: The appeal of NCCS is its potential to modulate neuroplasticity noninvasively, along with the ease of use and good tolerability. Promising and interesting evidence has been reported for the capacity of NCCS to affect neural circuits and the behaviors under their control. Today, the challenge is to utilize this advancement optimally. Continuing methodological advancements with NCCS approaches will enable researchers to better understand how NCCS can be utilized for the modulation of nervous system activity and subsequent behaviors, with possible applications to non-clinical and clinical practices.

MicroRNA-125b as a valuable predictive marker for outcome after autologous hematopoietic stem cell transplantation.

BACKGROUND: Relapse is a frequent occurrence in autologous hematopoietic stem cell transplantation (AHSCT), and early relapse after AHSCT results in poor survival and low quality of life. Predictive marker determination for AHSCT outcomes could be helpful in the prevention of relapse through personalized medicine. Here the predictive value of circulatory microRNAs (miRs) expression for AHSCT outcomes was studied. METHODS: 50 MM and lymphoma candidates for AHSCT participated in this study. Two plasma samples were obtained before AHSCT from each candidate; one before mobilization and the other after conditioning. Extracellular vesicles (EVs) were isolated by ultracentrifugation. miR-125b, miR-126, miR-150, and miR-155 expression were analyzed in both plasma and EVs using real time polymerase chain reaction analysis. Other data related to AHSCT and its outcomes were also collected. The predictive value of miRs and other factors for outcomes was assessed by multi-variant analysis. RESULTS: By 90 weeks follow up after AHSCT, multi-variant and ROC analysis showed miR-125b as a predictive marker for relapse, high lactate dehydrogenase (LDH), and high erythrocyte sedimentation rate (ESR). The cumulative incidence of relapse, high LDH, and high ESR increased with an increase in circulatory miR-125b expression. CONCLUSION: miR-125b could be applicable in prognosis evaluation and also create a possible new targeted therapy opportunity for enhanced outcomes and survival after AHSCT. TRIAL REGISTRATION: The study was retrospectively registered. Ethic code No: IR.UMSHA.REC.1400.541.

The effect of microvesicles derived from K562 cells on proliferation and apoptosis of human bone marrow mesenchymal stem cells.

Objectives: Microvesicles (MVs) are small membrane-bound particles that act as a vehicle to transfer their contents, such as proteins, RNAs, and miRNAs, to the target cells, making them undergo several changes. Depending on the origin and the target cell, MVs may cause cell survival or apoptosis. This study investigated the effects of MVs released from the leukemic K562 cell line on the human bone marrow mesenchymal stem cells (hBM-MSCs) to evaluate changes in the survival or apoptosis of the cells in an in vitro system. Materials and Methods: In this experimental study, we added the isolated MVs from the K562 cell line to hBM-MSCs, and after three and then seven days, subsequently cell count, cell viability, transmission electron microscopy, tracing MVs by carboxyfluorescein diacetate, succinimidyl ester (CFSE) solution, flow cytometry analysis for Annexin-V/PI staining and qPCR for the evaluation of BCL-2, KI67, and BAX expression were carried out. On the 10th day of the culture, hBM-MSCs were examined by Oil red O and Alizarin Red staining to evaluate their differentiation into adipocytes and osteoblasts. Results: There was a significant decrease in cell viability and KI67 and BCL-2 expression; however, BAX was significantly upregulated in the hBM-MSCs compared to control groups. Annexin-V/PI staining results also showed the apoptotic effects of K562-MVs on hBM-MSCs. Moreover, the differentiation of hBM-MSCs into adipocytes and osteoblasts was not observed. Conclusion: MVs from the leukemic cell line could affect the viability of normal hBM-MSCs and induce cell apoptosis.

Effect of Acute Myeloid Leukemia-derived Extracellular Vesicles on Bone Marrow Mesenchymal Stromal Cells: Expression of Poor Prognosis Genes.

OBJECTIVE: Acute myeloid leukemia (AML) is a heterogeneous clonal disorder resulting from a complex interplay between leukemic cells and supporting factors from their microenvironment. In this context, extracellular vesicles (EVs) have been shown to play an essential role in forming a tumor-protective microenvironment. In this study, we examined the influence of AML-derived EVs on cellular and molecular characterization of bone marrow mesenchymal stromal cells (BM-MSCs), particularly alteration in the expression of genes (IL-6, Gas-6, and Galectin-3) relating to relapse and chemoresistance. METHODS: MSCs were co-cultured with different concentrations of AML-EVs. Our data has been achieved by MTT assay, ROS assay, proliferation assay and apoptosis assay. RT-qPCR was also performed for gene expression analysis. RESULTS: Our results demonstrated that AML-EVs impact the MSCs characterization in a concentration-dependent manner. We revealed higher viability, increased Ki-67 and BCL-2, and lower ROS levels in MSCs treated with a 40 µg/mL dose of EVs. On the other hand, the rate of MSCs apoptosis and BAX expression exposed to a 60 µg/mL dose of EVs were increased compared with the control group. In addition, RT-qPCR results showed that the expression of IL-6, Gas-6, and Galectin-3 was significantly up-regulated in treated MSCs with a 40 µg/mL dose of EVs. CONCLUSION: Because the overexpression of IL-6, Gas-6, and Galectin-3 has contributed to chemoresistance and relapse, our findings suggest that AML-EVs propel MSCs to express these genes, which in turn could guard leukemic cells from chemotherapy-inflicted damages and eventually lead to relapse.

Comparable effects of circuit and traditional resistance exercise on platelet α2bß3 receptor and platelet activation and function.

BACKGROUND: Resistance exercise induces thrombocytosis and increases platelet activation and function. These changes might be related to exercise variables including exercise intensity and type. OBJECTIVE: We compared the effects of traditional resistance exercise (TRE) and circuit resistance exercise (CRE) on cellular markers of platelet activation and function. METHODS: In this crossover study ten healthy male (mean±SD: age, 25.6±2.4 years) subjects performed TRE encompassed 3 sets of 10 repetitions at 100% of 10-RM (10 repetition maximum) for 6 exercises, and CRE protocols included 3 sets of 10 repetitions at 100% of 10-RM for all 6 exercises consecutively, in two separate weeks. To measure platelet indices, PAC1, CD41a, CD42b and CD62P three blood samples were taken before, immediately after exercise, and after 30 min recovery. RESULTS: Lactate concentration, blood pressure, platelet count (PLT), and mean platelet volume (MPV) were significantly (p < 0.05) increased following both resistance exercise trials. Significant increases in PAC1, and CD62P; and significant reductions for CD42b and CD41a were detected following both REs (p < 0.05). However, changes in PAC1 and CD62P were significantly different between the two protocols (p < 0.05), with higher increases detected following CRE. CONCLUSIONS: Acute RE increases platelet indices and platelet activation; and that CRE results in higher platelet activation than TRE, probably due to exercise-induced increases in shear stress.

Investigating the multifaceted cooperation of autophagy, PI3K/AKT signaling pathways, and INPP4B gene in de novo acute myeloid leukemia patients.

BACKGROUND: Acute myeloid leukemia (AML) has been the most prevalent form of acute leukemia among adults, and it has been associated with poor survival rates over the last four decades. Understanding the processes involved in leukemogenesis, particularly autophagy and signaling pathways, can provide critical insights into their roles in disease development, risk assessment, and potential therapeutic interventions. This study investigated gene expression changes, focusing on MAP1LC3B and BECN1, related to autophagy, as well as PI3KCA and AKT1 in the PI3K-AKT pathway, and INPP4B, which regulates this signaling cascade. METHODS: We collected blood samples from 21 AML patients and 9 healthy volunteers. Gene expression was analyzed through qPCR following RNA extraction and cDNA synthesis. Statistical analysis encompassed t-tests, ANOVA, and correlation coefficients. RESULTS: AML patients exhibited significantly increased MAP1LC3B gene expression (****P < 0.0001; fold change = 11.9) and significantly reduced levels of INPP4B (****P < 0.0001; fold change = 0.026), AKT1 (*P < 0.05; fold change = 0.59), and PI3KCA (****P < 0.0001; fold change = 0.16) compared to healthy controls. However, BECN1 gene expression did not significantly differ between the two groups. Additionally, noteworthy correlations were observed between INPP4B and BECN1 (r = 0.57; P = 0.006) and BECN1 and PI3KCA (r = 0.61; P = 0.003) in AML patients. CONCLUSIONS: This study highlights variations in leukemogenesis pathways, exemplified by increased MAP1LC3B expression and diminished expression of regulatory genes in specific AML cases. These findings contribute to our comprehension of the molecular mechanisms underlying AML and may inform future diagnostic and therapeutic approaches.

Toward Hierarchical Assembly of Aligned Cell Sheets into a Conical Cardiac Ventricle Using Microfabricated Elastomers.

Despite current efforts in organ-on-chip engineering to construct miniature cardiac models, they often lack some physiological aspects of the heart, including fiber orientation. This motivates the development of bioartificial left ventricle models that mimic the myofiber orientation of the native ventricle. Herein, an approach relying on microfabricated elastomers that enables hierarchical assembly of 2D aligned cell sheets into a functional conical cardiac ventricle is described. Soft lithography and injection molding techniques are used to fabricate micro-grooves on an elastomeric polymer scaffold with three different orientations ranging from -60° to +60°, each on a separate trapezoidal construct. The width of the micro-grooves is optimized to direct the majority of cells along the groove direction and while periodic breaks are used to promote cell-cell contact. The scaffold is wrapped around a central mandrel to obtain a conical-shaped left ventricle model inspired by the size of a human left ventricle 19 weeks post-gestation. Rectangular micro-scale holes are incorporated to alleviate oxygen diffusional limitations within the 3D scaffold. Cardiomyocytes within the 3D left ventricle constructs showed high viability in all layers after 7 days of cultivation. The hierarchically assembled left ventricle also provided functional readouts such as calcium transients and ejection fraction.

Studying the potential of upregulated PTGS2 and VEGF-C besides hyper-methylation of PTGS2 promoter as biomarkers of Acute myeloid leukemia.

Hereby, we aimed to investigate the expression of prostaglandin-endoperoxide synthase 2 (PTGS2) and Vascular Endothelial Factor-C (VEGF-C) besides the methylation of PTGS2 in AML patients. VEGF-C and PTGS2 expression analysis were evaluated in newly diagnosed AML patients and healthy controls by quantitative Reverse Transcriptase PCR method. Also, PTGS2 methylation status was evaluated by Methylation-Sensitive High-Resolution Melting Curve Analysis (MS-HRM). While 34% of patients were female, the mean age of the patients was 43.41 ± 17.60 years suffering mostly from M4 (48.21%) type of AML. Although methylation level between patients and controls was not significantly different, none of the normal controls showed methylation in the PTGS2 promoter. PTGS2 and VEGF-C levels were elevated in AML cases and correlated with WBC, Platelet, and Hemoglobin levels. The survival of patients with overexpressed VEGF-C and PTGS2 was poorer than others. It can be concluded that PTGS2 and especially VEGF-C expression but not PTGS2 methylation can be considered as diagnostic biomarkers for AML.

Engineering Models of the Heart Left Ventricle.

Despite capturing the imagination of scientists for decades, the goal of creating an artificial heart for transplantation proved to be significantly more challenging than initially anticipated. Toward this goal, recent ground-breaking studies demonstrate the development of functional left ventricular (LV) models. LV models are artificially constructed 3D chambers that are capable of containing liquid within the engineered cavity and exhibit the functionality of native LV including contraction, ejection of fluid, and electrical impulse propagation. Various hydrogels and polymers have been used in manufacturing of LV models, relying on techniques such as electrospinning, bioprinting, casting, and molding. Most studies scaled down the models based on the dimensions of the human or rat ventricle. Initially, neonatal rat cardiomyocytes were the cell type of choice for construction the LV models. Yet, as the stem cell biology field advanced, recent studies focused on the use of cardiomyocytes derived from human induced pluripotent stem cells. In this review, we first describe the physiological characteristics of the human heart, to establish the parameter space for modeling. We then elaborate on current advances in the field and compare recently developed LV models among themselves and with the native human left ventricle. Fabrication methods, cell types, biomaterials, functional properties, and disease modeling capability are some of the major parameters that have distinguished these models. We also highlight some of the current challenges in this field, such as vascularization, cell composition and fidelity, and discuss potential solutions to overcome them.

Britannin, a sesquiterpene lactone induces ROS-dependent apoptosis in NALM-6, REH, and JURKAT cell lines and produces a synergistic effect with vincristine.

BACKGROUND: Britannin, a Sesquiterpene Lactone isolated from Inula aucheriana, has recently gained attraction in the therapeutic fields due to its anti-tumor properties. This study was designed to evaluate the effect of this agent on Acute Lymphoblastic Leukemia (ALL) cell lines, either as a monotherapy or in combination with Vincristine (VCR). METHODS AND RESULTS: To determine the anti-leukemic effects of Britannin on ALL-derived cell lines and suggest a mechanism of action for the agent, we used MTT assay, Annexin-V/PI staining, ROS assay, and real-time PCR analysis. Moreover, by using a combination index (CI), we evaluated the synergistic effect of Britannin on Vincristine. We found that unlike normal Peripheral Blood Mononuclear Cells (PBMCs) and L929 cells, Britannin reduced the viability of NALM-6, REH, and JURKAT cells. Among tested cells, NALM-6 cells had the highest sensitivity to Britannin, and this agent was able to induce p21/p27-mediated G1 cell cycle arrest and Reactive Oxygen Specious (ROS)-mediated apoptotic cell death in this cell line. When NALM-6 cells were treated with Nacetyl-L-Cysteine (NAC), a scavenger of ROS, Britannin could induce neither apoptosis nor reduce the survival of the cells suggesting that the cytotoxic effect of Britannin is induced through ROS-dependent manner. Moreover, we found that a low dose of Britannin enhanced the effect of Vincristine in NALM-6 cells by inducing apoptotic cell death via altering the expression of apoptotic-related genes. CONCLUSIONS: Overall, our results proposed a mechanism for the cytotoxic effect of Britannin, either as a single agent or in combination with Vincristine, in NALM-6 cells.

IFNG-AS1 and MAF4 long non-coding RNAs are upregulated in acute leukemia patients who underwent bone marrow transplantation.

PURPOSE OF THE STUDY: Acute graft versus host disease (aGVHD) is an immune-mediated reaction that results in impaired immune and body function after allogeneic hematopoietic stem cell transplantation (allo-HSCT). lncRNAs have been discovered as particular T cell regulators, and alloreactive T cells have been known as a critical factor in aGVHD. As a result, we investigated the importance of lnc-MAF4 and IFNG-AS1 expression levels in aGVHD patients versus non-aGVHD patients. MATERIAL AND METHODS: This research included 38 patients with hematological disorders who were undergoing primary allo-HSCT. Human identical siblings or unrelated donors were used to collect stem cell. Samples were taken within days 0, 7, 14, 28, and 52±8 after transplantation. The expression of lncRNA levels was measured using the QRT-PCR technique. And the data were analyzed using GraphPad Prism 6 RESULTS: Our data revealed that LncRNA MAF4 and INFG-AS1 expression levels in aGVHD were not significantly different compared to the non-GVHD group immediately after transplantation, nor at day 7 or 14. However, the aGVHD group showed an overt up-regulation of the two lncRNAs on samples taken at day 28 and 52±8 compared to non-GVHD patients. DISCUSSION: Since the intracellular pathway of these lncRNAs shows a direct relationship with the IFNγ cytokine production resulting in differentiation to TH1 cells and inhibition of differentiation to TH2 cells, they can be, therefore, considered as suitable molecular candidates for the prediction of aGVHD in patients receiving HSCT.

Evaluation of Sestrin 2, Adiponectin, AMPK, and mTOR Genes Expression in Acute Myeloid Leukemia Patients.

BACKGROUND: Effective treatment of acute myeloid leukemia (AML) is still controversial, therefore; a comprehensive understanding regarding the impaired cellular signaling pathways in AML can be useful in designing new therapeutic approaches. Among signaling pathways involved in AML, the mammalian target of rapamycin (mTOR) signaling pathway is of particular importance. While dysregulation of mTOR signaling has been reported in a wide range of patients with AML, but most studies have focused on mTOR downstream targets, and mTOR upstream targets have been overlooked. OBJECTIVE: In this study, expression of mTOR genes and three upstream targets (5' adenosine monophosphate-activated protein kinase (AMPK, adiponectin, and sestrin 2) involved in mTOR signaling was investigated. MATERIALS AND METHODS: In this study, expression of mTOR, AMPK, sestrin 2, and adiponectin genes in 60 patients with AML were evaluated compared to those of 30 healthy individuals as controls using the Real-Time polymerase chain reaction (Real-Time RT-PCR) method. RESULTS: According to the results, there was a significant difference in the expression of all the studied genes in patients in comparison to the normal control group (P <0.05). Expression of the mTOR gene was increased, while expression of AMPK, sestrin 2, and adiponectin genes was decreased in the patients with AML. Mean expression of the genes (2-ΔCt) (AMPK, sestrin 2, adiponectin, and mTOR) was equal to 7.9, 3.2, 3.74, and 1.49 for controls and 6, 2.1, 2.83, and 2.64 for patients with AML, respectively. CONCLUSIONS: Given the decreased expression levels of sestrin 2, adiponectin, and AMPK genes as tumor inhibitors and the increased expression level of the mTOR gene as an oncogene in the patients with AML in our study, it is thought that disruption of this pathway may be involved in leukemogenesis and can be considered as an effective factor in the progression of cancer.

Transcription analysis of a histones modifiers panel coupled with critical tumor suppressor genes displayed frequent changes in patients with AML.: mRNA levels of histones modifiers and TSGs in AML.

Epigenetic alterations could cause leukemia through the activation of normally silent loci or silencing of normally active loci. We herein aimed to compare the expression patterns of a histone modifiers panel consisted of SUV39H1, PRDM16, UHRF2, KDM2B, and KDM3C between acute myeloid leukemia(AML) cells and normal cells and to assess the correlation of these genes with the expression of vital tumor suppressor genes, including p16INK4A and p53. Bone marrow or peripheral blood samples of 50 AML patients at diagnosis and also 18 subjects with a normal hematopoietic system as a control group were obtained after informed consent. Then, qRT-PCR was performed to determine the expression levels of the aforementioned genes. We found a broad alteration in the expression signature of five out of seven studied genes in AML patients as compared with the control group. UHRF2 and p53 were remarkably downregulated in AML patients (P<0.001), while SUV39H1, PRDM16, and KDM3C were significantly overexpressed (P<0.01). Based on the Spearman rank correlation, SUV39H1 and KDM2B negatively regulated both p16INK4A and p53 expression. Taken together, our findings provided preliminary evidence regarding the pervasive mRNA perturbation of histone modifiers and their plausible influences on critical tumor suppressor genes. Future studies in this area would be required to assist in establishing these results in the clinical practice of AML patients.

AML-derived Extracellular Vesicles Confer De Novo Chemoresistance to Leukemic Myeloblast Cells by Promoting Drug Export Genes Expression and ROS Inhibition.

In spite of successful initial remission, chemo-resistance and relapse are still concerning points in acute myeloid leukemia (AML) treatment strategies. Multidrug resistance (MDR) appears to be the major contributor of chemo-resistance, arising in some sub-clones of cancers and could be developed in others. The aim of this study was to investigate the role of extracellular vesicles (EVs) derived from AML patients on the transmission of chemo-resistance phenotype. Ultracentrifugation was employed to isolate EVs from healthy controls, new cases, and relapsed AML patients. The EVs size, morphology, and immunophenotype were determined by dynamic light scattering, TEM, and flow cytometry respectively. Bradford assay was performed to measure the protein content of EVs. MTT assay and flow cytometry analysis were also used to determine the viability index, induction of apoptosis, and ROS generation in U937 cells. The expression level of two efflux pumps was assessed using qRT-PCR analysis. Findings of TEM, DLS, and flow cytometry confirmed that EVs had a desirable shape, size, and surface markers. EVs derived from both new cases and relapsed AML patients significantly reduced idarubicin-induced apoptosis in the U937 cells. The analysis of drug efflux pumps gens revealed that EVs over-express MRD1 and MRP1 in the target cells. These findings suggested a novel role of EVs in mediating the acquired chemo-resistance in AML patients by inducing the expression of the drug efflux pumps; however, further investigations will be required to elucidate other underlying mechanisms of resistance that are mediated by EVs.

Acute promyelocytic leukemia derived extracellular vesicles conserve PML-RARα transcript from storage-inflicted degradation: a stable diagnosis tool in APL patients.

The early death, which is more common in acute promyelocytic leukemia (APL) patients rather than other types of acute myelocytic leukemia (AML) highlights the importance of appropriate diagnostic method for early detection of this disease. The low sensitivity of the conventional methods, low tumor burden in some patients, and the need for bone marrow sampling are some of the diagnostic challenges on the way of proper detection of APL. Given these, we aimed to compare the efficacy of extracellular vesicles (EVs), as a diagnostic tool, with the existing methods. RT-PCR, qPCR, and flow cytometry were applied on EVs and their corresponding associated cellular component collected from 18 APL new cases, 23 patients with minimal residual disease (MRD), and NB4 cell line. RT-PCR results were positive in both cellular and vesicular components of all new cases, NB4 cells, and EVs in contrary to MRD cases. Normalized copy numbers (NCN) of PML-RARα were 5100 and 3950 for cell and EVs, respectively (p < 0.05). There was a significant difference in the NCN of PML-RARα between cells and EVs in BM samples. Investigating the effect of storage at room temperature revealed that PML-RARα level was retained near to the baseline level in EVs, but there was a significant reduction in its copy number in the cellular component during 7 days. Taken together, given to the acceptable stability, EVs could be introduced as a non-invasive liquid biopsy that alongside existing methods could remarkably change the paradigm of APL diagnostic approaches.