Exploiting replication stress induced by IGF blockade in cancer therapy

Thesis

In recent work from our group, we found that genetic or pharmacological inhibition of type 1 insulin-like growth factor receptor (IGF-1R) promotes accumulation of cells in S phase and causes endogenous DNA damage by inducing replication stress. Blocking IGF signalling reduces expression of the regulatory subunit M2 of ribonucleotide reductase (RRM2) and disturbs deoxynucleotide triphosphate (dNTP) supply. The aim of my project is to develop approaches to exploit this effect in therapy. Therefore, with the objective of enhancing replication stress induced by IGF blockade to intolerable levels, a compound screen was conducted in combination with an IGF neutralising antibody drug that is currently being evaluated in clinical trials. Five breast cancer cell lines were screened; top hits include inhibitors of EGFR/HER2, ATM, PARP, RAD51, and CHK1. Low throughput validation focused on inhibition of checkpoint kinase CHK1, which has been reported to induce replication stress through downregulating RRM2. Co-inhibition of IGF and CHK1 significantly suppressed cell viability and cell survival in both 2D cell culture and 3D spheroid culture models. In IGF-1R depleted or inhibited cells, inhibition of CHK1 further downregulated RRM2, reduced dNTP supplies and delayed replication fork progression. Combining IGF blockade with CHK1 inhibition led to accumulation of DNA damage and single-stranded DNA (ssDNA), a replication stress marker, and significant increase in cell death, suggesting replication catastrophe. Exogenous RRM2 expression rescued these replication stress phenotypes, indicating a mechanism in which RRM2 regulation is a critical target of the interaction between IGF blockade and CHK1. Final experiments confirmed that the replication stress was also exacerbated by co-inhibiting IGFs and WEE1, which was a screen hit in one of the five cell lines and had also been reported to regulate RRM2 expression. Combining WEE1 inhibition and IGF-1R depletion induced RRM2 downregulation, delayed replication fork progression, and caused accumulation of DNA damage and ssDNA, resulting in cell death. These results may help to identify novel therapeutic vulnerabilities and provide guidance for future clinical trials of IGF inhibitory drugs.

Authors: Wu, X

• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Replication stress; Breast cancer; WEE1; IGF; Cell cycle checkpoint; CHK1
• Subjects: Cancer biology