Identification of synthetic lethality of PRKDC in MYC-dependent human cancers by pooled shRNA screening

Abstract

BackgroundMYC family members are among the most frequently deregulated oncogenes in human cancers, yet direct therapeutic targeting of MYC in cancer has been challenging thus far. Synthetic lethality provides an opportunity for therapeutic intervention of MYC-driven cancers.MethodsA pooled kinase shRNA library screen was performed and next-generation deep sequencing efforts identified that PRKDC was synthetically lethal in cells overexpressing MYC. Genes and proteins of interest were knocked down or inhibited using RNAi technology and small molecule inhibitors, respectively. Quantitative RT-PCR using TaqMan probes examined mRNA expression levels and cell viability was assessed using CellTiter-Glo (Promega). Western blotting was performed to monitor different protein levels in the presence or absence of RNAi or compound treatment. Statistical significance of differences among data sets were determined using unpaired t test (Mann–Whitney test) or ANOVA.ResultsInhibition of PRKDC using RNAi (RNA interference) or small molecular inhibitors preferentially killed MYC-overexpressing human lung fibroblasts. Moreover, inducible PRKDC knockdown decreased cell viability selectively in high MYC-expressing human small cell lung cancer cell lines. At the molecular level, we found that inhibition of PRKDC downregulated MYC mRNA and protein expression in multiple cancer cell lines. In addition, we confirmed that overexpression of MYC family proteins induced DNA double-strand breaks; our results also revealed that PRKDC inhibition in these cells led to an increase in DNA damage levels.ConclusionsOur data suggest that the synthetic lethality between PRKDC and MYC may in part be due to PRKDC dependent modulation of MYC expression, as well as MYC-induced DNA damage where PRKDC plays a key role in DNA damage repair.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-944) contains supplementary material, which is available to authorized users.

Comprehensive in vivo identification of the c-Myc mRNA protein interactome using HyPR-MS

“…PRKDC is a serine/threonine-protein kinase involved in DNA double-stranded break repair and cell cycle control, impacting proper chromosome segregation during mitosis (Hsu et al 2012). It has been suggested that PRKDC inhibition causes a down-regulation of c-Myc mRNA expression, decreasing protein levels and reducing c-Myc-driven proliferation (Zhou et al 2014). This functional link is supported by the HyPR-MS results.…”

Driver gene classification reveals a substantial overrepresentation of tumor suppressors among very large chromatin-regulating proteins

“…PRKDC encodes a serine/threonine-kinase involved in DNA repair and recombination, with little current documentation for driver mutations within the gene. However, PRKDC inhibition sensitizes cells to irradiation33 and is synthetic lethal in MYC dependent cancers34 and with the mismatch repair gene MSH3 35. The latter studies suggest non-oncogene addiction to PRKDC .…”

Novel kinase fusion transcripts found in endometrial cancer

“…DNA-PK is also associated with AKT phosphorylation, leading to the activation of the AKT signaling pathway 29 30 . In addition, recent reports have demonstrated synthetic lethal interaction between PRKDC and some genes such as ATM 31 , MYC 32 , or MSH3 33 , suggesting the significance of PRKDC as a druggable target. Although the CPQ-PRKDC fusion is an attractive candidate for a therapeutic target in endometrial cancer, our knock-down experiments and Western blot analysis demonstrated that inhibiting CPQ-PRKDC - positive cell line proliferation was caused by suppression of the wild-type PRKDC expression and not suppression of the fusion transcript, which suggests that this fusion transcript might be a passenger alteration.…”