Defect in DNA Double Strand Break Response in Paediatric ALL Is Caused by Upregulation of Multiple Pro-Survival Pathways That Can Serve as Therapeutic Targets.

Abstract

The molecular basis of clinical heterogeneity in paediatric ALL is poorly understood. Defects in cellular responses to DNA double strand breaks (DSBs) have been associated with resistance to DNA damaging agents and poor clinical response in patients with leukaemia. We have previously shown that one third of paediatric ALL tumours exhibit in vitro defect in ionising radiation (IR)-induced apoptosis, despite retained integrity of p53 pathway. In this study we addressed molecular basis of defective (DSB) response in paediatric ALL with the view of identifying novel therapeutic targets.We analysed differences in transcriptional and posttranscriptional in vitro responses to IR in 22 ALL patients, stratified as either apoptotic resistant or apoptosis sensitive. Analysis of coordinate IR induced expression of functionally related genes, by two independent methods (gene set enrichment analysis, GSEA, and functional module analysis) revealed in resistant cases differential upregulation of multiple pro-survival pathways involved in EGF, PDGF, IGF, PI3 and MAPK signalling. In response to IR resistant ALLs also showed differentially induced phosphorylation of Akt and S6, downstream targets in PI-3 pathway. Array analysis of 42 receptor tyrosine kinases (TRKs), revealed differential phosphorylation of TRKs that act upstream of the PI3 including ErbB3,4, FGF R4,2a, VEGF R1,2 as well as IGF-I R. Furthermore, we were able to demonstrate that in apoptotic resistant leukaemias PI3/Akt upregulation during IR response lead to differential phosphorylation of DNA-PK catalytic subunit, a protein involved in non homologous end joining (NHEJ) repair of DNA DSBs. Consequently, increased repair of DSBs was observed in apoptosis resistant ALL cases and this was documented by faster resolution of IR induced intranuclear foci of γH2AX, a marker of DNA DSBs. Finally, pharmacological inhibition of pro-survival pathways PI3-Akt, IGF and MAPK in resistant leukaemias restored sensitivity to IR.We suggest that abnormal activation of pro-survival pathways during DSB response may represent one of the mechanisms of resistance to DNA damaging agents in paediatric ALLs and that targeting these pathways should be considered as therapeutic approach in aggressive form of disease.