Identification of the Genomic Rearrangements Associated with the Ex Vivo Resistance to Anthracyclines in Childhood Acute Leukemias

Abstract

INTRODUCTION: Resistance to drugs is connected with several genomic changes, but the major mutations are still unknown. Daunorubicin (DNR), idarubicin (IDA), doxorubicin (DOX) and mitoxantrone (MIT) are chemotherapeutics of the anthracycline family that are commonly used to treat acute leukemias (AL). OBJECTIVE: The aim of the study was to identify changes on the genome level and compare them with ex vivo resistance to DNR, IDA, DOX and MIT among children diagnosed with acute lymphoblastic (ALL) or myeloblastic (AML) leukemia. METHODS: The in vitro drug resistance profile was determined in MTT cytotoxicity assay, which was performed on mononuclear cells taken from 44 (aCGH) and 187 (qPCR) patients. The cyanine labeled genomic DNA (Cy3 - reference, Cy5 - patient) was hybridized to comparative genomic hybridization array (SurePrint G3 Human CGH Microarray 8x60K, Agilent). Data were analyzed using bioinformatics tools, like: Feature Extraction and CytoGenomics (Agilent) and databases, such as UCSC Genome Browser and GeneCards. Additionally, we tested gene expression level by qPCR with UPL probes for 4 genes (CASP1, ITGB2, TBLX1R1, TTC28) compared to the expression level of the reference gene ACTB. The relative expression was calculated by Pfaffl method with using a REST-2009 software (Qiagen). Statistical analysis was performed using Pair Wise Fixed Reallocation Randomisation Test. RESULTS: On the basis of the MTT results, we established that leukemic blast were predominantly resistant to IDA. DNR, MIT and DOX profiles were mostly balanced. According to achieved aCGH results, we identified several frequent genomic changes detected among children with AL. Statistically significant rearrangements are presented in Table 1. In most cases, the genes, which are in the area of genetic drug resistance determinates, have regulatory roles in cell death, proliferation signal cascades, cell adhesion, migration, cell cycle and also they interact with tumor suppressors, such as p16 or p53. Table 1. Most frequent genomic rearrangements among children with acute leukemia. Rearrangements DNR p DOX p IDA p MIT p Genes Del5q32-35.3 S 0.033 - - S 0.035 - - FBXO38, ABLIM3, SLC6A7, ARSI, ADAM19 Amp8p12-p11.21 R 0.039 R 0.023 S 0.035 S 0.047 FUT10, TTI2, RNF122, DUSP26, ZNF703, ADAM9, TM2D2, HTRA4, BAG4, LSM1 Del9p21.3 S 0.033 R 0.029 S 0.035 S 0.034 IFNA21, MTAP, CDKN2A, CDKN2B Amp14q32.33 S 0.049 - - S 0.022 S 0.049 ADAM6, KIAA0125 Del15q11.1-11.2 S 0.033 - - S 0.025 S 0.003 HERC2P3, NBEAP1, CHEK2P2 Del21.11.2-p11.1 S 0.019 - - S 0.025 - - TEKT4P2, TPTE, BAGE Amp22q11.22 S 0.049 R 0.027 S 0.035 - - - S - rearrangement correlated with drug sensitivity; R - rearrangement correlated with drug resistance; p - p-value The results of relative expression analysis are presented in Table 2. It is known, that DOX and IDA activate NLRP3/caspase-1/IL-1β signaling cascades, which are required for effective anticancer immunity. In our experiment, we also observed the increased expression of CASP1 in DOX and IDA profiles. Table 2. Relative expression level analysis. Drugs Genes CASP1 ITGB2 TBL1XR1 TTC28 DNR 1,939 ↑ (0,025) 3,009 ↑ (<0,001) 0,968 (ns) 0,594 (ns) IDA 1,645 (ns) 2,239 (ns) 0,768 (ns) 1,115 (ns) DOX 1,934 ↑ (0,008) 2,365 ↑ (0,002) 0,758 (ns) 0,907 (ns) MIT 1,389 (ns) 1,739 ↑ (0,022) 1,044 (ns) 0,628 (ns) ↑ - up-regulated gene; p-value in brackets; ns - not significant CONCLUSION: The analyses showed resistance to DNR, IDA, MIT and DOX are the result of many genomic changes in leukemic blasts. We assume, that rearrangements del9p21.3 and amp22q11.22 may correlate with lack of sensitivity to DOX, and amp8p12-p11.21 with resistance to DNR and DOX. The overexpression of CASP1 is strongly correlated with resistance to DNR and DOX. Expression changes of CASP1 may interfere the immune response in leukemia. Moreover, the up-regulation of ITGB2 expression is characteristic for cells resistant to DNR, DOX and MIT. Integrins participate in cell adhesion as well as cell-surface mediated signaling and plays an important role in immune response. The application of genomic rearrangements and gene expression signatures derived from resistant and sensitive cell lines may allow to develop of new strategies for cancer response prediction after anthracycline therapy. This study was supported by Grant from the National Science Centre No. DEC-2011/03/D/NZ5/05749. Disclosures No relevant conflicts of interest to declare.

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