Human Cord Blood-Derived Exosomes Enhance Chemosensitivity in Acute Myeloid Leukemia: A Promising Therapeutic Strategy.

miR-183-5p Inhibits Occurrence and Progression of Acute Myeloid Leukemia via Targeting Erbin.

The interferon consensus sequence-binding protein (ICSBP/IRF8) is an interferon regulatory factor that is expressed in myeloid and B-cells. ICSBP-deficient mice develop a myeloproliferative disorder characterized by cytokine hypersensitivity and apoptosis resistance. To identify ICSBP target genes involved in these effects, we screened a CpG island microarray with chromatin that co-immunoprecipitated with ICSBP from myeloid cells. Using this technique, we identified PTPN13 as an ICSBP target gene. PTPN13 encodes Fas-associated phosphatase 1 (Fap-1), a ubiquitously expressed protein-tyrosine phosphatase. This was of interest because interaction of Fap-1 with Fas results in Fas dephosphorylation and inhibition of Fas-induced apoptosis. In this study, we found that ICSBP influenced Fas-induced apoptosis in a Fap-1-dependent manner. We also found that ICSBP interacted with a cis element in the proximal PTPN13 promoter and repressed transcription. This interaction increased during myeloid differentiation and was regulated by phosphorylation of conserved tyrosine residues in the interferon regulatory factor domain of ICSBP. ICSBP deficiency was present in human myeloid malignancies, including chronic myeloid leukemia. Therefore, these studies identified a mechanism for increased survival of mature myeloid cells in the ICSBP-deficient murine model and in human myeloid malignancies with decreased ICSBP expression.

Increased intracellular cAMP concentration plays a well established role in leukemic cell maturation. We previously reported that U937 cells stimulated by H2 receptor agonists, despite a robust increase in cAMP, fail to mature because of rapid H2 receptor desensitization and phosphodiesterase (PDE) activation. Here we show that intracellular cAMP levels not only in U937 cells but also in other acute myeloid leukemia cell lines are also regulated by multidrug resistance-associated proteins (MRPs), particularly MRP4. U937, HL-60, and KG-1a cells, exposed to amthamine (H2-receptor agonist), augmented intracellular cAMP concentration with a concomitant increase in the efflux. Extrusion of cAMP was ATP-dependent and probenecid-sensitive, supporting that the transport was MRP-mediated. Cells exposed to amthamine and the PDE4 inhibitor showed enhanced cAMP extrusion, but this response was inhibited by MRP blockade. Amthamine stimulation, combined with PDE4 and MRP inhibition, induced maximal cell arrest proliferation. Knockdown strategy by shRNA revealed that this process was mediated by MRP4. Furthermore, blockade by probenecid or MRP4 knockdown showed that increased intracellular cAMP levels induce maturation in U937 cells. These findings confirm the key role of intracellular cAMP levels in leukemic cell maturation and provide the first evidence that MRP4 may represent a new potential target for leukemia differentiation therapy.

Azacitidine Combined with Novel Flavonoid Derivative GL-V9 Demonstrated Synergistic Anti-Leukemia Effect in Acute Myeloid Leukemia By Targeting DDIT4/mTOR Signaling

Nucleophosmin ( NPM1) Mutations in Acute Myeloid Leukemia: An Ongoing (Cytoplasmic) Tale of Dueling Mutations and Duality of Molecular Genetic Testing Methodologies

Acute myelogenous leukemia

Objective: To investigate the effects of miR-23a-3p on proliferation, migration and apoptosis on human acute myeloid leukemia (AML) cells by targeting SMC1A. Methods: Microarray analysis was used to screen differentially expressed microRNAs and mRNAs in human AML cells. Real-time fluorescence quantitative PCR (RT-qRCR) was used to detect the expressions of miR-23a-3p and SMCA in human AML cell line U937. TargetScan database was used to analyze the correlation between miR-23a-3p and SMC1A. Double luciferase reporter gene was used to detect the interaction between miR-23a-3p and SMC1A. The effect of miR-23a-3p expression on the proliferation of U937 cells was detected by clonal assay. The migration, apoptosis, cell cycle and caspase-3 activity of U937 cells regulated by miR-23a-3p were detected by cell scratch assay and flow cytometry, respectively. Western blot was used to detect the expressions of Bax and Bcl-2 in U937 cells. Results: Compared with human normal monocyte SC group (1.00), the expression of miR-23a-3p in U937 cells was up-regulated (2.56±0.78) (P<0.01), while the expression of SMC1A was down-regulated (0.48±0.56, P<0.01). miR-23a-3p specifically bond to SMC1A 3'UTR and regulated the expression activity of SMC1A. Overexpression of miR-23a-3p promoted the proliferation and migration of U937 cells and inhibited the apoptosis of U937 cells, while up-regulation of SMC1A inhibited the proliferation and migration of U937 cells and promoted the apoptosis of U937 cells. The percentages of G(0)/G1 phase, G(2)/M phase and S phase cells in the negative control group were (37.48±0.21)%, (16.78±0.18)% and (45.74±0.15)% respectively, and those in the miR-23a-3p mimics group were (19.96±0.11)%, (41.69±0.24)% and (38.24±0.34)%, respectively. The difference was statistically significant (all P<0.05). The proportions of G(0)/G(1) phase, G(2)/M phase and S phase cells in the group of miR-23a-3p mimics+ pcDNA3.1-SMC1A were (36.88±0.21)%, (30.44±0.33)% and (32.88±0.16)%, respectively, without significant difference when compared with those of the miR-23a-3p mimics group (P>0.05). The relative expression levels of Bax and Bcl-2 protein in the negative control group were 0.55±0.45 and 0.31±0.54, respectively. Overexpression of miR-23a-3p inhibited the expression of Bax protein in U937 cells (0.23±0.13, P<0.001), promoted the expression of Bcl-2 protein (0.50±0.23, P<0.01), while SMC1A increased the expression of Bax protein in U937 cells (0.40±0.11, P<0.01), and inhibited the expression of Bcl-2 protein (0.37±0.15). In the negative control group, caspase-3 activity was (25.82±0.89)%. Overexpression of miR-23a-3p inhibited caspase-3 activity in U937 cells (3.64±0.56)%, P<0.01, while up-regulation of SMC1A promoted caspase-3 activity in U937 cells (15.29±0.85)%, P<0.01. Conclusion: miR-23a-3p can inhibit the proliferation and migration and promote apoptosis of human AML cells by targeting SMC1A.

Background: Although the clinical outcome of pediatric acute myeloid leukemia (AML) has improved over the past few decades, approximately 30% of patients relapse. The overall survival (OS) rate of pediatric patients with primary refractory or relapsed AML is about 30%. Resistance to therapy presents a great challenge for AML patients. Therefore, there is a critical need to understand mechanisms of resistance, as well as to identify novel therapies that will improve OS. The leukemia-initiating cells reside in special hypoxic niches in the bone marrow. Thus, studying drug response of AML cells within a hypoxic environment is essential for more accurate prediction of patients’ response to therapy. We performed functional drug screening using AML cell lines, which demonstrated a differential response of AML cell lines to the survivin-targeting agent YM155 in normoxia versus hypoxia. Survivin (BIRC5), an inhibitor of apoptosis, is overexpressed in a variety of pediatric blood cancers compared to normal cells. We hypothesize that survivin may be an attractive target for therapy in pediatric AML. The overall objective of this study was to study mechanisms of AML response to YM155 in normoxia and hypoxia and survivin’s potential as a therapeutic target in pediatric AML. Purpose: The aims of the present work were (1) to study BIRC5 expression in publicly available databases of pediatric AML including the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative, and (2) to determine the potential mechanisms of hypoxia-altered response to YM155 in AML cells. Methods and Results: In the present study, the TARGET AML gene-expression profiling data available on Affymetrix human gene 1.1 ST arrays (HuGene 1.1) were used to study BIRC5 expression in comparison to HuGene 1.1 datasets from Gene Expression Omnibus (GEO) containing profiling data from adult AML and normal tissues. BIRC5 expression was significantly higher in pediatric AML in comparison to cytogenetically normal (CN) adult AML, and in relapsed pediatric AML in comparison to matched samples at diagnosis. In order to identify novel targeted therapies in AML we used a panel of AML cell lines to screen a library of 50 compounds in normoxia and hypoxia. The survivin-targeting agent YM155 was found to be one of the most cytotoxic compounds in the majority of AML cell lines in normoxia. However, hypoxia conferred resistance to this drug. The IC50 of YM155 in AML cell lines in normoxia ranged from 1.5 nM-8 µM, with an increase of 2-100 fold in hypoxia (IC50 from 22 nM-14 µM). The Mv4-11 cell line that harbors a FLT3-ITD mutation was selected for further mechanistic studies. Hypoxia conferred resistance to YM155 in Mv4-11 cells through inhibition of apoptosis. In order to identify the underlying molecular mechanisms, we performed RNA-seq of YM155-treated Mv4-11 cells in normoxia and hypoxia, and identified a set of over-represented genes that were validated by quantitative RT-PCR. The mRNA expression of HIF2alpha, p21WAF1, and STAT3 was overexpressed after 9h of YM155 treatment, while PIM2 was downregulated in normoxia but not in hypoxia, in comparison to vehicle-treated control. Preliminary data suggest a role of stem cell genes in hypoxia-induced resistance to YM155. Ongoing studies are directed towards determining the functions of the identified genes in resistance. Conclusion: Our data demonstrate that survivin (BIRC5) is overexpressed in pediatric AML in comparison to adult CN-AML, and may be an attractive target for pediatric AML therapy. Additionally, YM155 is highly cytotoxic to AML cell lines in normoxia; however, for its effective use in the clinic, cotargeting hypoxia-induced resistance factors is essential. Citation Format: David W. Lee, Justin Montoya, Kieran Finch, Jack Wilkinson-Dix, Robert J. Arceci, Daniel H. Wai, Eiman Aleem. Overcoming hypoxia-induced resistance is essential for effective survivin targeting in acute myeloid leukemia [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B06.

Countering Development of Resistance to Treatment in AML By Targeting Metabolic Adaptations

A Novel Natural Derivative GL-V9 Inducing Mitotic Catastrophe and Displaying a Synergistic Anti-Tumor Effect with EZH2 Inhibitor in Acute Myeloid Leukemia

Targeting EZH2 Promotes Chemosensitivity of BCL-2 Inhibitor through PIK3IP1-PI3K/AKT Axis in Acute Myeloid Leukemia

BMS-214662 Eliminates Quiescent and Proliferating Acute Myeloid Leukemia Cells through Activation of Protein Kinase Cβ and Enhances the Efficacy of Cytosine Arabinoside

Suicide Gene Therapy with a CD33 Targeted AAV6 Vector Expressing an Inducible Caspase-9 Suicide Gene Is Therapeutic in a Xenotransplantation Model of Acute Myeloid Leukemia

CEBPA-Driven Expression of the Transcriptionally Inactive deltaTP73 Isoform Phenocopies TP53mutated Poor Risk and Drug-Resistant Acute Myeloid Leukemia

The Receptor Tyrosine Kinase Axl Is Required for Resistance to FLT3-Targeted Therapy in Acute Myeloid Leukemia