Investigating the role of CDC25B and PRKDC in CHK1 inhibitor resistance

Abstract

To cope with replication stress and genomic instability, tumours become dependent on DNA damage checkpoint pathways, such as the kinase CHK1. This has led to the development of drugs targeting these pathways, like CHK1 inhibitors (CHK1i). Despite there being clinical trials using CHK1is, mechanisms of resistance to these inhibitors are still unclear. We have shown that development of CHK1i resistance is achieved through loss or alteration of CHK1 activity, together with the activation of compensatory bypass pathways. To learn more about CHK1i resistance, targeted exome sequencing using circulating free DNA from patient serum obtained through the SRA737 CHK1i clinical trial was performed. Mutations arising in genes associated with the ATR/CHK1 pathway following SRA737 treatment were identified. Following SRA737 treatment, three patients developed novel loss-of-function mutations within CDC25B, a regulator of the G2/M cell cycle checkpoint while four patients developed mutations within PRKDC, a critical component of double strand break repair. To determine whether these mutations confer resistance to CHK1i treatment, a CRISPR/Cas9 knock-in protocol was used to introduce the individual CDC25B and PRKDC mutations into the U2OS osteosarcoma cell line. Cells were then subjected to long-term CHK1i treatment to establish whether loss of CDC25B or PRKDC function was associated with increased survival and the development of resistance. Following this protocol, a high percentage of surviving cells were found to have inactivated CDC25B due to the presence of frameshifting insertions/deletions (INDELS). To investigate this further, siRNA knockdown of CDC25B followed by CHK1i treatment in U2OS cells was performed. This indicated that loss of CDC25B leads to CHK1i resistance but does not inhibit CHK1 activity under conditions of DNA replication stress. For PRKDC, there was a high percentage of cells containing the desired R2704fs mutation before CHK1i treatment. After the treatment regime, this mutation was completely lost indicating that U2OS cells undergoing CHK1i treatment may require a functioning PRKDC to cope with the pressures. Further investigation using inhibitors against PRKDC, DNA-PKi, were used in combination with CHK1i but did not have any synergistic effects. Although it seemed that PRKDC may not have a significant role in CHK1i resistance, it is hoped that by understanding how loss of CDC25B leads CHK1i resistance, potentially new therapeutic targets can be discovered and used in combination with SRA737 treatment.