ara-C Sensitivity Research Articles

Concanavalin A staining: a potential biomarker to predict cytarabine sensitivity in acute myeloid leukemia

Acute myeloid leukemia (AML) is a cancer of the myeloid lineage of blood cells, with an overall 5-year survival rate of 25%, mainly due to therapy-resistant relapses in > 50% of patients. The standard treatment for AML comprises cytarabine (AraC) with anthracyclines. Biomarkers to determine AraC sensitivity are currently lacking, thus hampering the rational choice of optimal treatment protocols, which would be especially warranted in the case of primary refractory disease. In the current study, we hypothesized that AraC-resistant AML cells harbor a different “sugar decoration”, i.e., glycosylation profile, compared with sensitive cells, which could be used as biomarker for AraC sensitivity. Therefore, we analyzed the expression of glycosylation-related genes in publicly available AML datasets, whereby the high expression of mannosylation-related genes (6 out of 13) was significantly associated with a worse survival in patients treated with AraC-based intensive chemotherapy protocols. In line with these data, the AraC-resistant AML cells expressed higher levels of high mannose N-glycans, as detected by mass spectrometry-based glycomics. Concanavalin A (ConA), a lectin that specifically recognizes α-mannoses in N-glycans, bound more strongly to AraC-resistant cells, and the extent of the ConA binding was correlated with AraC sensitivity in a panel of AML cell lines. Furthermore, the ConA staining could discriminate AraC sensitivity in vitro between two patient-derived AML samples taken at diagnosis. Therefore, the ConA staining may be a potential novel biomarker to predict AraC sensitivity in AML.

The m6A modulator-mediated cytarabine sensitivity and immune cell infiltration signature in acute myeloid leukemia.

The study aims to investigate the impact of m6A modulators on drug resistance and the immune microenvironment in acute myeloid leukemia (AML). The emergence of drug resistance is a significant factor that contributes to relapse and refractory AML, leading to a poor prognosis. The AML transcriptome data were retrieved from the TCGA database. The "oncoPredict" R package was utilized to assess the sensitivity of each sample to cytarabine (Ara-C) and classify them into distinct groups. Differential expression analysis was performed to identify m6A modulators differentially expressed between the two groups. Select Random Forest (RF) to build a predictive model. Model performance was evaluated using calibration curve, clinical decision curve, and clinical impact curve. The impacts of METTL3 on Ara-C sensitivity and immune microenvironment in AML were examined using GO, KEGG, CIBERSORT, and GSEA analyses. Seventeen out of 26 m6A modulators exhibited differential expression between the Ara-C-sensitive and resistant groups, with a high degree of correlation. We selected the 5 genes with the highest scores in the RF model to build a reliable and accurate prediction model. METTL3 plays a vital role in m6A modification, and further analysis shows its impact on the sensitivity of AML cells to Ara-C through its interaction with 7 types of immune-infiltrating cells and autophagy. This study utilizes m6A modulators to develop a prediction model for the sensitivity of AML patients to Ara-C, which can assist in treating AML drug resistance by targeting mRNA methylation.

DUSP1 Signaling Pathway Regulates Cytarabine Sensitivity in Acute Myeloid Leukemia.

Objectives: Dual specificity phosphatase 1 (DUSP1) is high-expressed in various cancers and plays an important role in the cellular response to agents that damage DNA. We aimed to investigate the expressions and mechanisms of DUSP1 signaling pathway regulating cytarabine (Ara-C) resistance in acute myeloid leukemia (AML). Methods: Immunohistochemistry was performed on bone marrow biopsy specimens from AML and controls to explore the expression of DUSP1. Western blot and Q-PCR were used to detect the protein and mRNA expression levels. MTT assay was used to detect the proliferation of cells. Cell apoptosis was detected by flow cytometry. The immune protein-protein interaction (PPI) network of DUSP1 was analyzed in the platform of Pathway Commons, and immune infiltration analysis was used to study the immune microenvironment of AML. Results: We found that the expression levels of DUSP1 in AML patients exceeded that in controls. Survival analysis in public datasets showed that AML patients with higher levels of DUSP1 had poor clinical outcomes. Further public data analysis indicated that DUSP1 was overexpressed in NRAS mutated AML. DUSP1 knockdown by siRNA could sensitize AML cells to Ara-C treatments. The phosphorylation level of mitogen-activated protein kinase (MAPK) pathway was significantly elevated in DUSP1 down-regulated NRAS G13D mutated AML cells. The PPI analysis showed DUSP1 correlated with immune gene CREB1 and CXCL8 in NRAS mutated AML. We also revealed a correlation between tumor-infiltrating immune cells in RAS mutated AML microenvironment. Conclusion: Our findings suggest that DUSP1 signaling pathways may regulate Ara-C sensitivity in AML.

Detection of residual and chemoresistant leukemic cells in an immune-competent mouse model of acute myeloid leukemia: Potential for unravelling their interactions with immunity.

Acute myeloid leukemia (AML) is characterized by blocked differentiation and extensive proliferation of hematopoietic progenitors/precursors. Relapse is often observed after chemotherapy due to the presence of residual leukemic cells, which is also called minimal residual disease (MRD). Subclonal heterogeneity at diagnosis was found to be responsible for MRD after treatment. Patient xenograft mouse models are valuable tools for studying MRD after chemotherapy; however, the contribution of the immune system in these models is usually missing. To evaluate its role in leukemic persistence, we generated an immune-competent AML mouse model of persistence after chemotherapy treatment. We used well-characterized (phenotypically and genetically) subclones of the murine C1498 cell line stably expressing the ZsGreen reporter gene and the WT1 protein, a valuable antigen. Accordingly, these subclones were also selected due to their in vitro aracytidine (Ara-c) sensitivity. A combination of 3 subclones (expressing or not expressing WT1) was found to lead to prolonged mouse survival after Ara-c treatment (as long as 150 days). The presence of residual leukemic cells in the blood and BM of surviving mice indicated their persistence. Thus, a new mouse model that may offer insights into immune contributions to leukemic persistence was developed.

Inhibition of microRNA 10a, an Autophagy Modulator, Increases Chemosensitivity to Ara-C in Acute Myeloid Leukemia

Aims: Leukemic relapse due to chemoresistance is a major obstacle in curing acute myeloid leukemia (AML), and the activation of pro-survival autophagy pathways has been implicated in this process. The microRNA-10a (miR-10a), a post-transcriptional regulator of several cell cycle and autophagy-related genes, is highly expressed in normal karyotype (NK) AML, especially those with nucleophosmin1 mutations (NPM1c+ ). This study aimed to investigate whether miR-10a modulation could affect autophagy and chemosensitivity in these cells.Results: Lentiviral transduction of a miR-10a inhibitor into the miR-10a-high NPM1c+ OCI-AML3 cell line significantly inhibited autophagy as signified by a marked decrease in autophagy marker LC3-II accumulation, compared to cells transduced with a scrambled control inhibitor (SCR). This was accompanied by significantly increased sensitivity to Ara-C (IC50 = 8.2µM vs 20.0µM; p <0.0001) characterised by a 2-fold increase in apoptosis (p = 0.035; quantified by Annexin V binding). Conversely, overexpression of miR-10a in the miR-10a-low, NPM1-wildtype MV4-11 AML cell line resulted in increased autophagy as demonstrated by a 3-fold increase in LC3-II (p = 0.001) and conferred pro-survival effects, including reduced apoptosis (p = 0.04) and markedly decreased Ara-C sensitivity (IC50 = 8.7 µM vs 2.7µM; p = 0.03).We previously demonstrated that miR-10a directly targets TFAP2C, a key inhibitor of CDKN1A-encoding cell cycle inhibitor p21. p21 is an important promoter of cell cycle arrest that precedes autophagy in response to stress signalling from the p53 signalling network. miR-10a inhibition was associated with a marked reduction in p21 expression, particularly in those cells in which p53 was activated either by treatment with MDM2 antagonist Nutlin-3a (p = 0.02) or by serum deprivation (p =0.005), and was associated with aberrant cell cycling in response to Ara-C treatment. Inhibition of CDKN1A specifically by siRNA showed similar effects to miR-10a inhibition, with increased cell death in response to Ara-C treatment (p <0.05).The observed increase in chemosensitivity in vitro was replicated in vivo in NSG mice engrafted with miR-10a inhibitor-transduced OCI-AML3 cells and treated with a course of Ara-C (50mg/kg for 3 weeks; n = 6 per treatment group). Treated mice had significantly improved survival (83% of miR-10a inhibited mice surviving at 30 days post-engraftment vs 50% of controls; hazard ratio = 6.6; p = 0.02) and had significantly fewer leukemic cells detectable in the bone marrow 24h after the end of treatment (26.7% of cells compared to 54.5% in animals engrafted with control inhibitor-transduced OCI-AML3 cells; p = 0.04).Conclusion: To our knowledge, this study provides for the first time evidence that miR-10a confers chemoresistance to Ara-C in NK-AML, likely via its effects on the p21/p53-mediated activation of autophagy and may in part explain treatment failure observed in the clinic. DisclosuresStoelzel:medac: Other: Travel support.

GLI1 reduces drug sensitivity by regulating cell cycle through PI3K/AKT/GSK3/CDK pathway in acute myeloid leukemia

Acute myeloid leukemia (AML) is a hematological malignancy with high incidence and recurrence rates. Gene expression profiling has revealed that transcriptional overexpression of glioma‐associated oncogene 1 (GLI1), a vital gene in the Hedgehog (Hh) signaling pathway, occurs in poor-prognosis AML, and high levels of phosphoinositide-3-kinase, regulatory subunit 1 (PIK3R1) and AKT3 predict shorter overall survival in AML patients. In this study, we discovered that GLI1 overexpression promotes cell proliferation and reduces chemotherapy sensitivity in AML cells while knocking down GLI1 has the opposite effect. Moreover, GLI1 promoted cell cycle progression and led to elevated protein levels of cyclins and cyclin-dependent kinases (CDKs) in AML cells. By luciferase assays and co-immunoprecipitation, we demonstrated that the PI3K/AKT pathway is directly activated by GLI1. GLI1 overexpression significantly accelerates tumor growth and upregulated p-AKT, CDK4, and cyclinD3 in vivo. Notably, the GLI1 inhibitor GANT61 and the CDK4/6 inhibitor PD 0332991 had synergistic effects in promoting Ara-c sensitivity in AML cell lines and patient samples. Collectively, our data demonstrate that GLI1 reduces drug sensitivity by regulating cell cycle through the PI3K/AKT/GSK3/CDK pathway, providing a new perspective for involving GLI1 and CDK4/6 inhibitors in relapsed/refractory (RR) patient treatment.

Expression of Chromosome 21-Localized Genes in Acute Myeloid Leukemia: Differences Between Down Syndrome and Non-Down Syndrome Blast Cells and Relationship to In Vitro Sensitivity to Cytosine Arabinoside and Daunorubicin

AbstractThe high event-free survival rates of Down syndrome (DS) children with acute myeloid leukemia (AML) are due, in part, to increased in vitro sensitivity of DS myeloblasts to cytosine arabinoside (ara-C) and daunorubicin and the greater generation of ara-C triphosphate (ara-CTP) from ara-C compared with myeloblasts from non-DS patients (Taub et al,Blood 87:3395, 1996). This study further explores the molecular basis of chemotherapy sensitivity of DS AML patients by examining the expression of chromosome 21-localized genes in myeloblasts from newly diagnosed AML patients. Transcript levels of two chromosome 21-localized genes, cystathionine-β-synthase (CBS) and superoxide dismutase (SOD), measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), were 12.0- and 3.8-fold higher in DS compared with non-DS myeloblasts (P < .0001 and P < .0001, respectively). Conversely, there were no significant increases in transcripts for 2 other chromosome 21-localized genes, carbonyl reductase and the reduced folate carrier. CBS transcript levels correlated with both in vitro ara-C sensitivity measured by the 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium-bromide (MTT) assay (P = .003) and the generation of 3H-ara-C triphosphate (ara-CTP) after in vitro incubations with 5 μmol/L3H-ara-C (P = .0003). Transcripts of deoxycytidine kinase were 2.6-fold higher in DS compared with non-DS cells and may be a factor in the enhanced metabolism of ara-C in DS cells. There was no significant correlation of SOD transcripts with in vitro ara-C and daunorubicin sensitivities. Increased CBS transcripts could result in elevated CBS activity, which modulates ara-C metabolism by altering reduced folate pools, deoxycytidine triphosphate pools, S-adenosylmethionine levels, and/or methylation of the deoxycytidine kinase gene. The further identification of the molecular mechanisms of chemotherapy sensitivity of DS AML patients may lead to significant improvements in the treatment and cure of AML.

TUG1 weakens the sensitivity of acute myeloid leukemia cells to cytarabine by regulating miR-655-3p/CCND1 axis.

Long non-coding RNA taurine upregulated gene 1 (lncRNA TUG1) has been demonstrated to promote malignant phenotypes and Adriamycin resistance in acute myeloid leukemia (AML) cells. However, the function and mechanism of TUG1 in cytarabine (Ara-C) sensitivity in AML remain unclear. Levels of TUG1, microRNA (miR)-655-3p or cyclin D1 (CCND1) mRNA were examined using quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation activity and apoptosis were analyzed using cell counting kit-8 (CCK-8) or flow cytometry, respectively. Western blot was utilized to detect the protein levels of Ki-67, B-cell lymphoma-2-associated X protein (Bax), and CCND1. The interaction between miR-655-3p and TUG1 or CCND1 was confirmed by Dual-Luciferase reporter and pull-down assay. TUG1 and CCND1 were higher expressed, while miR-655-3p was lower expressed in AML cells compared with that in normal cells. Higher expression levels of TUG1 or CCND1, and lower expression levels of miR-655-3p both notably reversed Ara-C-induced proliferation inhibition and apoptosis promotion in AML cells. TUG1 was a sponge of miR-655-3p, and TUG1 knockdown enhanced the sensitivity of AML cells to Ara-C by regulating miR-655-3p. MiR-655-3p directly targeted CCND1, and CCND1 overexpression attenuated miR-655-3p restoration-mediated reinforcement of Ara-C sensitivity in AML cells. Besides that, TUG1 up-regulated CCND1 expression via miR-655-3p. TUG1 weakened the sensitivity of AML cells to Ara-C by up-regulating CCND1 via miR-655-3p, suggesting a new insight into the chemotherapy of AML.

Increased Expression of Micro-RNA-23a Mediates Chemoresistance to Cytarabine in Acute Myeloid Leukemia.

Resistance to chemotherapy is one of the primary obstacles in acute myeloid leukemia (AML) therapy. Micro-RNA-23a (miR-23a) is frequently deregulated in AML and has been linked to chemoresistance in solid cancers. We, therefore, studied its role in chemoresistance to cytarabine (AraC), which forms the backbone of all cytostatic AML treatments. Initially, we assessed AraC sensitivity in three AML cell lines following miR-23a overexpression/knockdown using MTT-cell viability and soft-agar colony-formation assays. Overexpression of miR-23a decreased the sensitivity to AraC, whereas its knockdown had the opposite effect. Analysis of clinical data revealed that high miR-23a expression correlated with relapsed/refractory (R/R) AML disease stages, the leukemic stem cell compartment, as well as with inferior overall survival (OS) and event-free survival (EFS) in AraC-treated patients. Mechanistically, we demonstrate that miR-23a targets and downregulates topoisomerase-2-beta (TOP2B), and that TOP2B knockdown mediates AraC chemoresistance as well. Likewise, low TOP2B expression also correlated with R/R-AML disease stages and inferior EFS/OS. In conclusion, we show that increased expression of miR-23a mediates chemoresistance to AraC in AML and that it correlates with an inferior outcome in AraC-treated AML patients. We further demonstrate that miR-23a causes the downregulation of TOP2B, which is likely to mediate its effects on AraC sensitivity.

MALAT1 knockdown inhibits proliferation and enhances cytarabine chemosensitivity by upregulating miR-96 in acute myeloid leukemia cells

Drug resistance remains a major cause of relapse and therapeutic failure in acute myeloid leukemia (AML). Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been documented to act as an oncogene and is frequently highly expressed in human cancers including AML. However, the function and molecular mechanism of MALAT1 in regulating cytarabine (Ara-C) resistance of AML are largely unknown. The expressions of MALAT1 and miR-96 in AML patients and healthy controls were examined by qRT-PCR. CCK-8 and flow cytometry assay were performed to assess the proliferation and apoptosis of AML cells. The interaction between MALAT1 and miR-96 was investigated by luciferase reporter assay. We found that MALAT1 was upregulated while miR-96 was downregulated in AML patients compared with healthy controls. A negative correlation between MALAT1 and miR-96 expressions was observed in AML patients. Knockdown of MALAT1 inhibited the proliferation, induced apoptosis, and enhanced Ara-C sensitivity of AML cells. Additionally, MALAT1 suppressed miR-96 expression by acting as a molecular sponge of miR-96 in AML cells. miR-96 downregulation abolished the effects of MALAT1 knockdown on the proliferation, apoptosis, Ara-C sensitivity in AML cells. In conclusion, MALAT1 knockdown inhibited proliferation, promoted apoptosis and enhanced Ara-C sensitivity in AML cells by upregulating miR-96, providing novel insights into the critical role of MALAT1 as a miRNA sponge in AML.

Chemotherapy-Resistant Human Acute Myeloid Leukemia Cells Are Not Enriched for Leukemic Stem Cells but Require Oxidative Metabolism.

Chemotherapy-resistant human acute myeloid leukemia (AML) cells are thought to be enriched in quiescent immature leukemic stem cells (LSC). To validate this hypothesis in vivo, we developed a clinically relevant chemotherapeutic approach treating patient-derived xenografts (PDX) with cytarabine (AraC). AraC residual AML cells are enriched in neither immature, quiescent cells nor LSCs. Strikingly, AraC-resistant preexisting and persisting cells displayed high levels of reactive oxygen species, showed increased mitochondrial mass, and retained active polarized mitochondria, consistent with a high oxidative phosphorylation (OXPHOS) status. AraC residual cells exhibited increased fatty-acid oxidation, upregulated CD36 expression, and a high OXPHOS gene signature predictive for treatment response in PDX and patients with AML. High OXPHOS but not low OXPHOS human AML cell lines were chemoresistant in vivo. Targeting mitochondrial protein synthesis, electron transfer, or fatty-acid oxidation induced an energetic shift toward low OXPHOS and markedly enhanced antileukemic effects of AraC. Together, this study demonstrates that essential mitochondrial functions contribute to AraC resistance in AML and are a robust hallmark of AraC sensitivity and a promising therapeutic avenue to treat AML residual disease.Significance: AraC-resistant AML cells exhibit metabolic features and gene signatures consistent with a high OXPHOS status. In these cells, targeting mitochondrial metabolism through the CD36-FAO-OXPHOS axis induces an energetic shift toward low OXPHOS and strongly enhanced antileukemic effects of AraC, offering a promising avenue to design new therapeutic strategies and fight AraC resistance in AML. Cancer Discov; 7(7); 716-35. ©2017 AACR.See related commentary by Schimmer, p. 670This article is highlighted in the In This Issue feature, p. 653.

Enhanced autophagy in cytarabine arabinoside-resistant U937 leukemia cells and its potential as a target for overcoming resistance.

Autophagy is a lysosomal degradation mechanism that is essential for cell survival, differentiation, development, and homeostasis. Autophagy protects cells from various stresses, including protecting normal cells from harmful metabolic conditions, and cancer cells from chemotherapeutics. In the current study, a cytarabine arabinoside (Ara-C)-sensitive U937 leukemia cell line and an Ara-C-resistant U937 (U937/AR) cell line were assessed for baseline autophagy activity by investigating the LC3-I conversion to LC3-II, performing EGFP-LC3 puncta, an acidic autophagolysosome assay, and measuring the expression of various autophagy-related genes. The results demonstrated significantly higher autophagic activity in the U937/AR cells compared with the U937 cells, when the cells were cultured with or without serum. Furthermore, an increase in the autophagic activity in starved U937/AR cells was demonstrated, compared with that in the starved U937 cells. Administration of an autophagy inhibitor demonstrated no change in cell death in the two cell lines when cultured with serum, however, it induced cell death regardless of the Ara-C sensitivity when the cell lines were cultured without serum. In addition, the U937 cells demonstrated an Ara-C resistance when cultured without serum. Co-treatment with Ara-C and the autophagy inhibitor significantly induced cell death in the U937/AR and Ara-C-sensitive U937 cells. In conclusion, autophagy serves an important role in protecting U937 cells from Ara-C and in the development of Ara-C resistance. Inhibition of autophagy combined with the Ara-C treatment in the U937 cells augmented the anti-leukemic effect of Ara-C and overcame Ara-C resistance, suggesting that autophagy may be an important therapeutic target to further improve the treatment outcome in patients with acute myeloid leukemia.

RNA expression of genes involved in cytarabine metabolism and transport predicts cytarabine response in acute myeloid leukemia.

Variation in terms of outcome and toxic side effects of treatment exists among acute myeloid leukemia (AML) patients on chemotherapy with cytarabine (Ara-C) and daunorubicin (Dnr). Candidate Ara-C metabolizing gene expression in primary AML cells is proposed to account for this variation. Ex vivo Ara-C sensitivity was determined in primary AML samples using MTT assay. mRNA expression of candidate Ara-C metabolizing genes were evaluated by RQPCR analysis. Global gene expression profiling was carried out for identifying differentially expressed genes between exvivo Ara-C sensitive and resistant samples. Wide interindividual variations in ex vivo Ara-C cytotoxicity were observed among samples from patients with AML and were stratified into sensitive, intermediately sensitive and resistant, based on IC50 values obtained by MTT assay. RNA expression of deoxycytidine kinase (DCK), human equilibrative nucleoside transporter-1 (ENT1) and ribonucleotide reductase M1 (RRM1) were significantly higher and cytidine deaminase (CDA) was significantly lower in ex vivo Ara-C sensitive samples. Higher DCK and RRM1 expression in AML patient's blast correlated with better DFS. Ara-C resistance index (RI), a mathematically derived quotient was proposed based on candidate gene expression pattern. Ara-C ex vivo sensitive samples were found to have significantly lower RI compared with resistant as well as samples from patients presenting with relapse. Patients with low RI supposedly highly sensitive to Ara-C were found to have higher incidence of induction death (p = 0.002; RR: 4.35 [95% CI: 1.69-11.22]). Global gene expression profiling undertaken to find out additional contributors of Ara-C resistance identified many apoptosis as well as metabolic pathway genes to be differentially expressed between Ara-C resistant and sensitive samples. This study highlights the importance of evaluating expression of candidate Ara-C metabolizing genes in predicting ex vivo drug response as well as treatment outcome. RI could be a predictor of ex vivo Ara-C response irrespective of cytogenetic and molecular risk groups and a potential biomarker for AML treatment outcome and toxicity. Original submitted 22 December 2014; Revision submitted 9 April 2015.

Inhibition of NHE1 Induced Apoptosis in Cytarabine Resistant Leukemia Cell Lines and Primary Leukemia Cells from AML Patients, Which Showed Increased Intracellular pH and NHE1 Activity

Background: Na/H exchanger 1 (NHE1), an important participant in the precise regulation system of intracellular pH (pHi), is known to be involved in pathological processes such as cell transformation, maintenance and active progression of the neoplastic process. Some studies have showed that leukemic cells showed higher pHi than normal cells, and NHE1 inhibitor could induce acidification and apoptosis of the leukemic cells. In this study, we tried to elucidate the role of NHE1 in leukemic cells according to cytarabine (AraC) resistance.Materials and Methods: Two human AML cell lines, AraC sensitive (AS)-OCI-AML2 cells and AraC resistant (AR)-OCI-AML2 cells, primary leukemic cells from AML patients, and normal bone marrow mononuclear cells (BMMNC) from healthy donor were analyzed. The pH-sensitive fluorescent dye, 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) was used to measure pHi and NHE1 activity. The fluorescent ratio of the 490/440 nm was calibrated intracellularly. The expression of NHE1 was measured by qRT-PCR and western blot analysis. To inhibit the NHE1, the amiloride analogue, 5-(N,N-hexamethylene) amiloride (HMA) (10 µM, 20 µM, 30 µM) was used.Results: To confirmed AraC sensitivity, cell lines were treated with 10 µM AraC for 24 hours, and apoptosis fraction in AS-OCI-AML2 cells and AR-OCI-AML2 cells were 53.1±7.2 % and 4.0±0.8 %, respectively. The pHi of AR-OCI-AML2 cells was significantly higher than AS-OCI-AML2 cells (7.839±0.033 vs. 7.589±0.129, P=0.045) and BMMNC (7.839±0.033 vs. 7.578±0.035, P=0.083), and these differences were associated with higher NHE1 activity. Compared AS-OCI-AML2 cells, AR-OCI-AML2 cells showed significantly higher NHE1 expression by western blot analysis (Figure 1), and NHE1 mRNA levels (0.039±0.014 vs. 1.565±0.070, P<.001) by qRT-PCR. Treatment with HMA (20 µM) could induce apoptosis both on AS-OCI-AML2 cells (26.9±2.8%) and AR-OCI-AML2 cells (37.4±18.8%). Interestingly, induction of apoptosis by HMA was dose-dependent both in AS-OCI-AML2 cells and AR-OCI-AML2 cells, and higher concentration of HMA (30 µM) could induce apoptosis on most of AR-OCI-AML2 cells (68.7±20.2%). Co-treatment experiment with 10 µM AraC and 20 µM HMA in AS-OCI-AML2 cells showed additive effect on inducing apoptosis (AraC vs. HMA vs. HMA+AraC = 53.1±12.4 vs. 53.1±12.4 vs. 67.20±4.3%, Figure 2), but in AR-OCI-AML2 cells, co-treatment did not show additional or synergistic effect on inducing apoptosis (AraC vs. HMA vs. HMA+AraC = 4.0±0.1 vs. 27.1±2.2 vs. 28.1±2.0%, Figure 2). As in the cell lines, primary leukemia cells from patients with AraC resistance showing higher pHi and NHE activity than those from patients without. HMA could induce apoptosis on primary cell lines regardless AraC sensitivity.Conclusions: In this study, we first showed that NHE1 inhibition could induce apoptosis in leukemia cells regardless AraC sensitivity. Apoptotic activity was related with higher pHi and NHE activity in AraC resistant cell lines and primary leukemic cells. NHE inhibition induced apoptosis may be independent with AraC induced apoptosis. The heterogeneity in pHi and NHE activity within leukemic cells may be related to alteration in drug delivery machinery or dormant status of leukemia cells. Further experimental and clinical studies are needed to elucidate the therapeutic application of NHE1 inhibitor to AraC resistant AML. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 340: Mitochondrial priming of new targeted agents in acute myeloid leukemia

Abstract As numerous molecularly targeted agents are entering clinical trials, predictive testing is highly desirable. We investigated if response to certain agents correlates with the recently reported method of BH3 profiling (Chonghaile TN et al, Science, 2011), a functional assay developed by Letai's group that measures tumor cell mitochondrial priming by measuring mitochondrial outer membrane permeabilization (MOMP) following exposure to the peptide-mimicking BH3 domains of BH3-only proteins. Mitochondrial priming has been reported to be correlated with clinical responses to conventional chemotherapy in solid tumors and hematological malignancies (Vo TT et al, Cell, 2012, Pierceall W et al, Mol Cancer Ther, 2013). Twenty-two AML lines were tested. Cells were permeabilized with digitonin and exposed to standardized doses of BH3 peptides (BIM, PUMA, NOXA, BAD, BMF, HRK, or PUMA2A). JC-1 was used for detection of MOMP and served as a measure of sensitivity to each peptide (reported as %[BH3 peptide]). Also, BCL-2, BCL-XL and MCL-1 expression levels were determined by Western blot. In addition to studies of untreated cells, treatment effects of different anti-leukemia drugs (AraC, Nutlin-3a, KPT-330 [Selinexor] and ABT-199) were determined over a wide dose range and denoted as ([specific apoptosis] = [(%Annexin V+ cells at each dose) - (%Annexin V+ cells at 0 μM)]/[100- (%Annexin V+ cells at 0 μM)]). Mixed linear models were used for analysis. As expected, ABT-199 sensitivity positively correlated with %[BAD]-%[HRK] (|β| = 3.22, p &amp;lt; 0.001), which is compatible with BCL-2 dependency of ABT-199 (while BAD is binding to BCL-2 and BCL-XL, HRK is binding to BCL-XL only and the difference is therefore BCL-2-specific). This was supported by the observed correlation between %[BAD]-%[HRK] and BCL-2 protein expression levels (r=0.619; p = 0.018). AraC sensitivity showed a similar correlation with %[BAD]-%[HRK] (|β| = 1.61, P &amp;lt; 0.05). KPT-330 sensitivity in p53 wild-type cell lines positively correlated with %[PUMA] (|β| = 0.92, P = 0.054), consistent with the notion that KPT-330 induces PUMA through p53 activation. Unexpectedly, Nutlin-3a activity did not correlate with any of the BH3 peptides. Results indicate that ABT-199, KPT-330 and Nutlin-3a show different modes of action in terms of BH3 peptide dependency, supporting potential combination effects of these agents. For ABT-199, increased MCL1 levels were associated with diminished cytotoxicity (r=0.532; p=0.05), as expected. For Ara-C, a similar correlation with MCL-1 was noted (r=0.505; p=0.06), but no correlations were observed for Nutlin-3a and KPT-330. In conclusion, BH3 profiling is a promising tool to predict the BH3 peptide dependency of the BH3-mimetic ABT-199 and the XPO1-inhibitor KPT-330. Functional BH3-profiling appears to be superior to the static quantitation of Bcl-2 family protein levels in AML. Furthermore, mitochondrial priming may be useful for the rational design of new combination therapies. Citation Format: Jo Ishizawa, Kensuke Kojima, Seshagiri R. Duvvuri, Teresa McQueen, Vivian R. Ruvolo, Graciela M. Nogueras-Gonzalez, Xuelin Huang, William Pierceall, Michael Cardone, Ryan Lena, Camille Doykan, Sharon Shacham, Michael Kauffman, Marina Konopleva, Michael Andreeff. Mitochondrial priming of new targeted agents in acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 340. doi:10.1158/1538-7445.AM2014-340

Using RNA-seq and targeted nucleases to identify mechanisms of drug resistance in acute myeloid leukemia.

The evolution from microarrays to transcriptome deep-sequencing (RNA-seq) and from RNA interference to gene knockouts using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and Transcription Activator-Like Effector Nucleases (TALENs) has provided a new experimental partnership for identifying and quantifying the effects of gene changes on drug resistance. Here we describe the results from deep-sequencing of RNA derived from two cytarabine (Ara-C) resistance acute myeloid leukemia (AML) cell lines, and present CRISPR and TALEN based methods for accomplishing complete gene knockout (KO) in AML cells. We found protein modifying loss-of-function mutations in Dck in both Ara-C resistant cell lines. CRISPR and TALEN-based KO of Dck dramatically increased the IC50 of Ara-C and introduction of a DCK overexpression vector into Dck KO clones resulted in a significant increase in Ara-C sensitivity. This effort demonstrates the power of using transcriptome analysis and CRISPR/TALEN-based KOs to identify and verify genes associated with drug resistance.

Inhibition of miR-17 and miR-20a by oridonin triggers apoptosis and reverses chemoresistance by derepressing BIM-S.

Cancer cell chemoresistance arises in part through the acquisition of apoptotic resistance. Leukemia cells resistant to chemotherapy-induced apoptosis have been found to be sensitive to oridonin, a natural agent with potent anticancer activity. To investigate its mechanisms of action in reversing chemoresistance, we compared the response of human leukemia cells with oridonin and the antileukemia drugs Ara-C and VP-16. Compared with HL60 cells, K562 and K562/ADR cells displayed resistance to apoptosis stimulated by Ara-C and VP-16 but sensitivity to oridonin. Mechanistic investigations revealed that oridonin upregulated BIM-S by diminishing the expression of miR-17 and miR-20a, leading to mitochondria-dependent apoptosis. In contrast, neither Ara-C nor VP-16 could reduce miR-17 and miR-20a expression or could trigger BIM-S-mediated apoptosis. Notably, silencing miR-17 or miR-20a expression by treatment with microRNA (miRNA; miR) inhibitors or oridonin restored sensitivity of K562 cells to VP-16. Synergistic effects of oridonin and VP-16 were documented in cultured cells as well as mouse tumor xenograft assays. Inhibiting miR-17 or miR-20a also augmented the proapoptotic activity of oridonin. Taken together, our results identify a miRNA-dependent mechanism underlying the anticancer effect of oridonin and provide a rationale for its combination with chemotherapy drugs in addressing chemoresistant leukemia cells.

FLT3 Is Involved In Ara-C Transport By Human Equilibrative Nucleoside Transporter (hENT1) In Pediatric Acute Leukemia

IntroductionDespite the remarkable improvement in survival of children with acute leukemia in the last decades, some patients still have a poor outcome. FMS-like tyrosine Kinase 3 (FLT3) is a tyrosine-kinase receptor with a key role in hematopoiesis whose mutations and overexpression have emerged as negative prognostic biomarkers in childhood leukemia. Infant leukemias (those diagnosed at age<1 year) are a special subgroup characterized by distinctive clinico-biological features, including frequent MLL (Mixed Lineage Leukemia) gene rearrangements, high FLT3 expression and high sensitivity to cytarabine (Ara-C), but a dismal prognosis. Ara-C is a deoxycytidine analog known to be transported by the human equilibrative nucleoside transporter 1 (hENT1) across the cell membrane. Once inside the cell Ara-C is activated by deoxycitidine kinase (DCK) to finally exert its cytotoxic action. High hENT1 expression levels have been involved in Ara-C sensitivity in patients with acute leukemia. However, the mechanisms that regulate the expression of hENT1 and its activity, as well as the putative relationship between FLT3 and Ara-C transport and metabolism are scarcely known. AimTo study the role of FLT3 in the regulation of the expression and activity of the main Ara-C transporters and metabolizing enzymes (ME) in pediatric leukemia. Patients, Materials, and MethodsBone marrow samples of 56 pediatric patients diagnosed with acute leukemia in Hospital Sant Joan de Déu and 3 acute leukemia cell lines (MV4-11, SEM, K562) were used for screening of Ara-C transporters and ME expression. In all cases the patients or their parents signed an informed consent. We performed a positive selection of cases with acute lymphoblastic leukemia (ALL) with hyperdiploidy (>50 chromosomes) and of MLL-rearranged cases presenting either as ALL or acute myeloblastic leukemia (AML), as these are the subgroups with highest expression of FLT3 reported in the literature, although other leukemia subtypes were also represented. The mRNA expression levels of FLT3, nucleoside transporters (NT) (hENT1, hENT2, hCNT1, hCNT3) and Ara-C ME, dCK and CNII, were quantified using real-time PCR and analyzed with the 2-ΔΔCt method, with non-neoplastic samples used as controls for the relative quantification. Direct nucleoside and Ara-C uptake measurements were performed using [5,6-3H]-nucleosides. The role FLT3 might play in the expression of NT and ME genes as well as on the activity of Ara-C transporters in these cell lines was addressed by repressing FLT3 function with its specific inhibitor PKC412 (sold only for research purposes). ResultsWe included 56 patients (68% males) diagnosed with acute leukemia. The median age was 5.3 years (range 0-16.2), with 3 cases of infant leukemia. Fifty cases (89%) were precursor B-ALL (24 hyperdiploid cases, 5 MLL rearranged, 3 BCR-ABL+, 4 E2A-PBX1+, 5 TEL-AML1+, 9 other subtypes), 5 cases were AML (4 MLL positive cases and one case with mutated FLT3) and one case was a T-ALL harboring FLT3 mutation. As expected, the FLT3 expression was higher in cases with ALL and MLL rearrangement and, to a less extent, in ALL with hyperdiploidy. Interestingly, a significant positive correlation was found between FLT3 and hENT1 expression (mRNA levels) with all patient samples (figure 1). hENT1 expression and cytarabine-mediated uptake was significantly repressed when MV4-11 and SEM cell lines were exposed to the FLT3 inhibitor PKC412 (figures 2 & 3) without affecting hENT2, CNT1 and CNT3 expression and activity. [Display omitted] [Display omitted] [Display omitted] ConclusionsOur results show a strong correlation between FLT3 and the Ara-C transporter hENT1 in pediatric leukemia patients. This observation was consistent with the in vitro evidence that FLT3 inhibition resulted in hENT1 repression and down-regulation of Ara-C uptake in leukemic derived cell lines. Taken together, our data suggest that FLT3 regulates hENT1, thereby modulating the ability of cancer cells to incorporate Ara-C and promote its cytotoxic action. As FLT3 inhibitors are currently being tested as mono or combined therapy with Ara-C in several clinical trials, based upon our observations we suggest that a better schedule design might eventually be needed when dealing with treatments involving FLT3 inhibitors and Ara-C, thereby improving the outcome of this subset of patients. Disclosures:Off Label Use: The presentation include the results of in vitro studies with the FLT3 inhibitor PKC412. This drug was only used for in vitro studies, exclusively for research purposes.

Abstract C46: Implication of FLT3 in human equilibrative nucleoside transporter 1 (hENT1)-mediated uptake of Ara-C in pediatric acute leukemia.

Abstract Introduction: Infant acute leukemias (those diagnosed at age &amp;lt;1 year) are characterized by high FMS-like tyrosine Kinase 3 (FLT3) expression and high sensitivity to the nucleoside analogue Ara-C. FLT3 is a tyrosine-kinase receptor with a key role in hematopoiesis whose mutations and overexpression have emerged as negative prognostic biomarkers in childhood leukemia. Ara-C is known to be transported by hENT1 undergoing metabolic activation within target cells. In fact, high hENT1 expression levels have been reported to determine Ara-C sensitivity in patients with acute leukemia. However, the mechanisms that regulate the expression of hENT1 and its activity, as well as the putative relationship between FLT3 and Ara-C transport and metabolism are poorly known. Aim: To study the role of FLT3 in the regulation of the expression and activity of the main Ara-C transporters and metabolizing enzymes (ME) in pediatric acute leukemia. Experimental Procedures: Bone marrow samples from 56 pediatric patients diagnosed with acute leukemia in Hospital San Joan de Deu of Barcelona and 3 acute leukemia cell lines (MV4-11, SEM, K562) were used in this study. In all cases an informed consent was signed. The mRNA amounts of FLT3, the putative Ara-C nucleoside transporters (NT), hENT1, hENT2, hCNT1, hCNT3, and the main Ara-C metabolic enzymes, DCK and CNII were quantified by real-time PCR. Direct nucleoside and Ara-C uptake measurements were performed using [5,6-3H]-nucleosides. The role FLT3 might play in the expression of NT and ME genes as well as on the activity of Ara-C transporters was addressed by exposure of cells to the FLT3 inhibitor PKC412. Results: A significant positive correlation was found between FLT3 and hENT1 mRNA levels when analysing the whole cohort of patients. DCK and CNII also showed a positive correlation. As expected, FLT3 expression was higher in cases with acute lymphoblastic leukemia (ALL) and mixed lineage leukemia (MLL) rearranged genes and, to a lesser extent, in ALL with hyperdiploidy (&amp;gt;50 chromosomes), as these are the subgroups with highest expression of FLT3 reported in the literature. Ara-C uptake into cells was mediated mainly by hENT1, hENT2, and hCNT1. The hENT1-mediated uptake of Ara-C was transiently abolished by the FLT3 inhibitor PKC412 reaching its minimum activity after 16 hours with the inhibitor. This down-regulation of hENT1 activity seemed to be dependent on the ability of PKC412 to inhibit hENT1 gene (SLC29A1) expression. Conclusions: The positive correlation between the FLT3 and hENT1 expression levels in leukemic cells from patients, along with the in vitro evidence that FLT3 inhibition represses hENT1 expression and down-regulates Ara-C uptake in leukemic derived cell lines, supports the idea that FLT3 modulates hENT1. This evidence should be taken into account at the therapy schedule level when therapeutic combinations including both AraC and FLT3 inhibitors are designed. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C46. Citation Format: Paula Fernández-Calotti, Albert Català, MarÇal Pastor-Anglada, Roberta Malatesta, Susana Rives, Mireia Camos. Implication of FLT3 in human equilibrative nucleoside transporter 1 (hENT1)-mediated uptake of Ara-C in pediatric acute leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C46.

Overexpression of GATA1 Confers Resistance to Chemotherapy in Acute Megakaryocytic Leukemia

It has been previously shown that acute myeloid leukemia (AML) patients with higher levels of GATA1 expression have poorer outcomes. Furthermore, pediatric Down syndrome (DS) patients with acute megakaryocytic leukemia (AMKL), whose blast cells almost universally harbor somatic mutations in exon 2 of the transcription factor gene GATA1, demonstrate increased overall survival relative to non-DS pediatric patients, suggesting a potential role for GATA1 in chemotherapy response. In this study, we confirmed that amongst non-DS patients, GATA1 transcripts were significantly higher in AMKL blasts compared to blasts from other AML subgroups. Further, GATA1 transcript levels significantly correlated with transcript levels for the anti-apoptotic protein Bcl-xL in our patient cohort. ShRNA knockdown of GATA1 in the megakaryocytic cell line Meg-01 resulted in significantly increased cytarabine (ara-C) and daunorubicin anti-proliferative sensitivities and decreased Bcl-xL transcript and protein levels. Chromatin immunoprecipitation (ChIP) and reporter gene assays demonstrated that the Bcl-x gene (which transcribes the Bcl-xL transcripts) is a bona fide GATA1 target gene in AMKL cells. Treatment of the Meg-01 cells with the histone deacetylase inhibitor valproic acid resulted in down-regulation of both GATA1 and Bcl-xL and significantly enhanced ara-C sensitivity. Furthermore, additional GATA1 target genes were identified by oligonucleotide microarray and ChIP-on-Chip analyses. Our findings demonstrate a role for GATA1 in chemotherapy resistance in non-DS AMKL cells, and identified additional GATA1 target genes for future studies.