A Screening of Antineoplastic Drugs for Acute Myeloid Leukemia Reveals Contrasting Immunogenic Effects of Etoposide and Fludarabine.
ABSTRACT: BACKGROUND:Recent evidence demonstrated that the treatment of acute myeloid leukemia (AML) cells with daunorubicin (DNR) but not cytarabine (Ara-C) results in immunogenic cell death (ICD). In the clinical setting, chemotherapy including anthracyclines and Ara-C remains a gold standard for AML treatment. In the last decade, etoposide (Eto) and fludarabine (Flu) have been added to the standard treatment for AML to potentiate its therapeutic effect and have been tested in many trials. Very little data are available about the ability of these drugs to induce ICD. METHODS:AML cells were treated with all four drugs. Calreticulin and heat shock protein 70/90 translocation, non-histone chromatin-binding protein high mobility group box 1 and adenosine triphosphate release were evaluated. The treated cells were pulsed into dendritic cells (DCs) and used for in vitro immunological tests. RESULTS:Flu and Ara-C had no capacity to induce ICD-related events. Interestingly, Eto was comparable to DNR in inducing all ICD events, resulting in DC maturation. Moreover, Flu was significantly more potent in inducing suppressive T regulatory cells compared to other drugs. CONCLUSIONS:Our results indicate a novel and until now poorly investigated feature of antineoplastic drugs commonly used for AML treatment, based on their different immunogenic potential.
Project description:Cytarabine (Ara-C) and Daunorubicin (Dnr) forms the backbone of acute myeloid leukemia (AML) therapy. Drug resistance and toxic side effects pose a major threat to treatment success and hence alternate less toxic therapies are warranted. NF-E2 related factor-2 (Nrf2), a master regulator of antioxidant response is implicated in chemoresistance in solid tumors. However, little is known about the role of Nrf2 in AML chemoresistance and the effect of pharmacological inhibitor brusatol in modulating this resistance. Primary AML samples with high ex-vivo IC50 to Ara-C, ATO, Dnr had significantly high NRF2 RNA expression. Gene-specific knockdown of NRF2 improved sensitivity to these drugs in resistant AML cell lines by decreasing the expression of downstream antioxidant targets of Nrf2 by compromising the cell's ability to scavenge the ROS. Treatment with brusatol, a pharmacological inhibitor of Nrf2, improved sensitivity to Ara-C, ATO, and Dnr and reduced colony formation capacity. AML cell lines stably overexpressing NRF2 showed increased resistance to ATO, Dnr and Ara-C and increased expression of downstream targets. This study demonstrates that Nrf2 could be an ideal druggable target in AML, more so to the drugs that function through ROS, suggesting the possibility of using Nrf2 inhibitors in combination with chemotherapeutic agents to modulate drug resistance in AML.
Project description:Daunorubicin (DNR) and Cytarabin (Ara-C) are the main chemotherapeutic drugs used for Acute Myeloid Leukemias (AML) treatment. However, their mode of action is not well understood. To decipher if these drug would induce rapid transcriptional reprogramming, we treated HL-60 AML cell line with DNR or Ara-C for 3 hours and carried out a transcriptomic analyses. In addition, we had shown that Reactive Oxigen Species (ROS) produced by NADPH oxidases (NOX) are involved in DNR-induced gene expression. We therefore also performed a transcriptomic analyses in HL-60 cells treated with DNR and VAS2870, an NADPH oxidase inhibitor. HL-60 cells were treated or not for 3 hours with 2 µM Ara-C, 1 µM DNR or 1 µM DNR + 20µM VAS 2870. RNAs were purified from 3 independent experiments and used to probe Affymetrix Human Gene 2.0 ST Genechips
Project description:BACKGROUND: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. METHODS: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. RESULTS: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. CONCLUSION: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.
Project description:PURPOSE:Chemotherapy drug resistance and relapse of the disease have been the major factors limiting the success of acute myeloid leukemia (AML) therapy. Several factors, including the pharmacokinetics (PK) of Cytarabine (Ara-C) and Daunorubicin (Dnr), could contribute to difference in treatment outcome in AML. METHODS:In the present study, we evaluated the plasma PK of Dnr, the influence of genetic polymorphisms of genes involved in transport and metabolism of Dnr on the PK, and also the influence of these factors on clinical outcome. Plasma levels of Dnr and its major metabolite, Daunorubicinol (DOL), were available in 70 adult de novo AML patients. PK parameters (Area under curve (AUC) and clearance (CL)) of Dnr and DOL were calculated using nonlinear mixed-effects modeling analysis performed with Monolix. Genetic variants in ABCB1, ABCG2, CBR1, and CBR3 genes as well as RNA expression of CBR1, ABCB1, and ABCG2 were compared with Dnr PK parameters. RESULTS:The AUC and CL of Dnr and DOL showed wide inter-individual variation. Patients with an exon1 variant of rs25678 in CBR1 had significantly higher plasma Dnr AUC [p = 0.05] compared to patients with wild type. Patients who achieved complete remission (CR) had significantly lower plasma Dnr AUC, Cmax, and higher CL compared to patients who did not achieve CR. CONCLUSION:Further validation of these findings in a larger cohort of AML patients is warranted before establishing a therapeutic window for plasma Dnr levels and targeted dose adjustment.
Project description:Ubiquitin and the ubiquitin-like SUMO are covalently conjugated to thousands of proteins to modulate their function and fate. Many of the enzymes involved in their conjugation are dysregulated in cancers and involved in cancer cell response to therapies. We describe here the identification of biomarkers of the activity of these enzymes and their use to predict acute myeloid leukemias (AML) response to standard chemotherapy (daunorubicin-DNR and cytarabine-Ara-C). We compared the ability of extracts from chemosensitive and chemoresistant AML cells to conjugate ubiquitin or SUMO-1 on 9,000 proteins spotted on protein arrays. We identified 122 proteins whose conjugation by these posttranslational modifiers marks AML resistance to DNR and/or Ara-C. Based on this signature, we defined a statistical score predicting AML patient response to standard chemotherapy. We finally developed a miniaturized assay allowing for easy assessment of modification levels of the selected biomarkers and validated it in patient cell extracts. Thus, our work identifies a new type of ubiquitin-based biomarkers that could be used to predict cancer patient response to treatments.
Project description:Wide inter-individual variation in terms of outcome and toxic side effects of treatment exist among patients with AML receiving chemotherapy with cytarabine (Ara-C) and daunorubicin (Dnr). Drug resistance and relapse are considered major causes of treatment failure. Gene expression profiling was undertaken to address possible mechanisms of Ara-C/Dnr resistance. Based on ex vivo Ara-C cytotoxicity at diagnosis, Ara-C sensitive (IC50 <3uM AraC) and Dnr sensitive samples (IC50 < 0.5 uM) (5 samples each) were included for microarray analysis. These were compared with the samples which were drug resistant ex vivo at diagnosis. Our microarray experiment resulted in indentifying differentially expressed genes under ex vivo Ara-C sensitive as well as Dnr sensitive samples compared to ex vivo Drug resistant samples. One-color experiment,Organism: Homo sapiens, Custom Human Whole Genome 8x60k Array designed by Genotypic Technology Private Limited (AMADID: 27114), Labeling kit: Agilent Quick-Amp labeling Kit (p/n5190-0442)
Project description:To minimize the non-specific toxicity of drug combination during cancer therapy, we prepared a new system synthesized from bacteria to deliver the anticancer drugs cytosine arabinoside (Ara-C) and daunorubicin (DNR). In this study, we selected genipin (GP) and poly-l-glutamic acid (PLGA) as dual crosslinkers. Herewith, we demonstrated the preparation, characterization and in vitro antitumor effects of Ara-C and DNR loaded GP-PLGA-modified bacterial magnetosomes (BMs) (ADBMs-P). The results show that this new system is stable and exhibits optimal drug-loading properties. The average diameters of BMs and ADBMs-P were 42.0 ± 8.6 nm and 65.5 ± 8.9 nm, respectively, and the zeta potential of ADBMs-P (-42.0 ± 6.4 mV) was significantly less than that of BMs (-28.6 ± 7.6 mV). The optimal encapsulation efficiency and drug loading of Ara-C were 68.4% ± 9.4% and 32.4% ± 2.9%, respectively, and those of DNR were 36.1% ± 2.5% and 17.9% ± 1.6%. Interestingly, this system also exhibits long-term release behaviour sequentially, without an initial burst release. The Ara-C drug continued to release about 85% within 40 days, while DNR release lasted only for 13 days. Moreover, similar to free drugs, ADBMs-Ps are strongly cytotoxic to cancer cells in vitro (HL-60 cells), with the inhibition rate approximately 96%. This study reveals that this new system has a potential for drug delivery application in the future, especially for combination therapy.
Project description:Acute myeloid leukemia (AML) is a common adult leukemia often arising from a preexistent myelodysplastic syndrome (MDS). High mortality rates of AML are caused by relapse and chemoresistance; therefore, we analyzed the role of P2X7 receptor (P2X7R) splice variants A and B in AML progression and response to chemotherapy. The expression of P2X7RA and P2X7RB was investigated in samples obtained from MDS and AML untreated subjects or AML patients in relapse or remission after chemotherapy. Both P2X7RA and P2X7RB were overexpressed in AML versus MDS suggesting a disease-promoting function. However, in relapsing patients, P2X7RA was downmodulated, while P2X7RB was upmodulated. Treatment with daunorubicin (DNR), one of the main chemotherapeutics for AML, upregulated P2X7RB expression while reducing P2X7RA mRNA in AML blasts. Interestingly, DNR administration also caused ATP release from AML blasts suggesting that, following chemotherapy, activation of the receptor isoforms via their agonist will be responsible for the differential survival of blasts overexpressing P2X7RA versus P2X7RB. Indeed, AML blasts expressing high levels of P2X7RA were more prone to cell death if exposed to DNR, while those overexpressing P2X7RB were more vital and even protected against DNR toxicity. These data were reproducible also in HEK-293 cells separately expressing P2X7RA and B. P2X7RA facilitation of DNR toxicity was in part due to increased uptake of the drug inside the cell that was lost upon P2X7RB expression. Finally, in an AML xenograft model administration of DNR or the P2X7R antagonist, AZ10606120 significantly reduced leukemic growth and coadministration of the drugs proved more efficacious than single treatment as it reduced both P2X7RA and P2X7RB levels and downmodulated c-myc oncogene. Taken together, our data suggest P2X7RA and P2X7RB as potential prognostic markers for AML and P2X7RB as a therapeutic target to overcome chemoresistance in AML relapsing patients.
Project description:The Acute Myeloid Leukemia cell line HL-60 was rendered resistant to daunorubicin (DNR) or cytarabine (Ara-C) by continuous exposure to the drug up to concentrations of 30nM for DNR and 100nM for Ara-C. Transcriptomic analysis were then performed by RNA-Seq to compare the cell lines Overall design: 3 biological replicates were performed for parental HL-60, Ara-C resistant (HRA) and DNR-resistant (HRD)
Project description:Novel treatment strategies are of paramount importance to improve clinical outcomes in pediatric AML. Since chemotherapy is likely to remain the cornerstone of curative treatment of AML, insights in the molecular mechanisms that determine its cytotoxic effects could aid further treatment optimization. To assess which genes and pathways are implicated in tumor drug resistance, we correlated ex vivo drug response data to genome-wide gene expression profiles of 73 primary pediatric AML samples obtained at initial diagnosis. Ex vivo response of primary AML blasts towards cytarabine (Ara C), daunorubicin (DNR), etoposide (VP16), and cladribine (2-CdA) was associated with the expression of 101, 345, 206, and 599 genes, respectively (p < 0.001, FDR 0.004-0.416). Microarray based expression of multiple genes was technically validated using qRT-PCR for a selection of genes. Moreover, expression levels of BRE, HIF1A, and CLEC7A were confirmed to be significantly (p < 0.05) associated with ex vivo drug response in an independent set of 48 primary pediatric AML patients. We present unique data that addresses transcriptomic analyses of the mechanisms underlying ex vivo drug response of primary tumor samples. Our data suggest that distinct gene expression profiles are associated with ex vivo drug response, and may confer a priori drug resistance in leukemic cells. The described associations represent a fundament for the development of interventions to overcome drug resistance in AML, and maximize the benefits of current chemotherapy for sensitive patients.