Whole Genome and Transcriptome Analysis of Genetic Alterations in Context of Busulfan in Vitro Resistance in Pediatric Acute Leukemias

INTRODUCTION: Busulfan (BUS) is bifunctional cell cycle non-specific alkylating antineoplastic agent. High-dose busulfan in combination with cyclophosphamide is an effective preparative regimen for patients undergoing allogeneic or autologous bone marrow transplantation for leukemias and solid tumors. The main mechanism of action involves the interference of DNA replication and RNA transcription (by alkylation and cross-linking of strands), which results in the disruption of nucleic acid functions. Alterations in drug transport and metabolism, increased activity of glutathione S-transferase and aldehyde dehydrogenase activity or enhanced DNA repair may also play a role in the resistance to this alkylator. OBJECTIVE: The aim of this study was to elucidate candidate genes and molecular pathways involved in ex vivo resistance to busufan in childhood acute leukemias. METHODS: In order to determine the in vitro BUS resistance profile, MTT cytotoxicity assay was performed on mononuclear cells, collected from 153 patients. All samples were processed for RNA and DNA extraction, using standard protocols. Templates provided as suitable for hybridization were assessed with 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Oligonucleotide array-CGH was performed using the Agilent SurePrint G3 Human CGH Microarray Kit, 6×60K. Experiments were done according to the standard protocol (Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis). Commercially available genomic DNA (Promega, Madison, WI, USA) was used as the control. Microarray data were extracted with Feature Extraction Software. Data analysis and chromosome segmentation were performed with CytoGenomics Software. Gene expression profiles were prepared on the basis of cRNA hybridization to oligonucleotide arrays of the human genome (Affymetrix, Santa Clara, CA, USA) for 51 patients with ALL and 16 patients with AML. Hierarchical clustering, assignment location and biological function were performed. Real time PCR was performed on a LightCycler^® 480 instrument (Roche, Mannheim, Germany) using specific primers and Universal ProbeLibrary set for 20 target and 3 reference genes. RESULTS: Expression analysis level highlighted some of the most relevant biological processes and molecular functions. These included also the apoptosis (eg. APC, STAT1,3, FGF2, MAPK14, PRKCG, ANGPT2, PDGFC), p53 (CDC25C, CDK2, SFN, GADD45G), WNT signallig (eg. APC, CTBP2, CSNK2A2, TBLX1), EGF receptor signaling (eg. STAT5A, BTC, AREG) and B-cell activation (eg. LYN, BLK, PTPN6) pathways. Three genomic segments were frequently and exclusively changed: del3q26, del9p21 and amp14q23 (Table 1). Deletion of the 3q26 segment was primarily associated with TBL1XR1 decrease expression (data from GEP and qPCR). Homozygous deletions were identified for sensitive cells in 9p21 (extended over a 1.8 Mb region and affecting CDKN2A/CDKN2B). Genomic amplification in 14p23 was seen in 5 cases and encompassing ADAM 6 and long intergenic non-protein coding RNA 226

CONCLUSION: In this study, we employed high-throughput approaches to profile the transcriptome and genome of leukemic blasts and an integrative bioinformatics approach to elucidate the molecular pathways associated with the BUS resistance phenotype. The results suggest that lack of sensitivity is multifactorial and includes constitutively up-regulated expression of genes involved in anti-apoptotis, drug metabolism and DNA repair. By combining genomic and expression profiles we could show that deletion in 3q26 was associated with decreased TBL1XR1 expression level. The presented data indicates TBL1X and its receptor as a possible target genes.

This study was supported by Grant from the National Science Centre No. DEC-2011/03/D/NZ5/05749