Evaluating Synthetic Lethal Interactions in DNA Damage Signaling for Breast Cancer Therapy

• Author / Creator

  Bukhari, Amirali

• Breast cancer is the most common cancer amongst women in Canada. The current treatment regime for early stage breast cancers is breast-conserving surgery with radiation therapy. However, 10-20% of patients develop local recurrence, and some exhibit metastatic spread, which is associated with a high mortality rate. Treatment resistance is commonly seen in such patients. In this regard, therapies combining two or more therapeutic agents/drugs has become a cornerstone of cancer therapy. Oncogene-induced DNA damage is a common feature of cancer cells, which leads to high levels of replication stress in cancer cells compared to normal proliferating cells.
  To achieve the necessary therapeutic window for a wide range of tumors, in view of tumor heterogeneity, we tested whether increasing genotoxic stress and simultaneously inhibiting an important rescue pathway would lead to cancer cell-selective death by evaluating the efficacy of combined inhibition of the kinases ATR and Wee1. ATR is essential in the DNA damage checkpoint in response to replication stress, whereas Wee1 is an effector kinase required to maintain the G2/M and intra S-phase checkpoints. Our findings suggest that inhibition of Wee1 kinase leads to ATR activation, and combined inhibition of the two kinases promotes synergistic cell killing in a panel of cancer cells, but not in non-tumorigenic epithelial cell lines in vitro. Live cell microscopy experiments monitoring the fate of individual cells showed that combined treatment with ATR and Wee1 inhibitors leads to a significant increase in the number of cancer cells undergoing centromere fragmentation and mitotic catastrophe, eventually resulting in cell death in mitosis. Cell cycle synchronization experiments indicate that combined ATR and Wee1 inhibition leads to a significant delay of S and G2/M phases. Furthermore, 4-day cell survival assays using reversible inhibition of ATR and/or Wee1 for short periods during the cell cycle show that not only do the two drugs act synergistically, but suppression of checkpoint activation and DNA repair in the two cell cycle phases, S and G2, also cooperate to kill cancer cells. In an orthotopic breast cancer model, tumor-selective synergistic lethality between ATR and Wee1 inhibitors led to tumor remission and inhibited metastasis with minimal side effects.
  Furthermore, early identification of non-responders in the clinic could help identify patients that should be put on alternative therapies to minimize unnecessary toxicities. In this regard, we show that [18F]-FLT uptake, measured by positron emission tomography, can be employed as a predictive biomarker to evaluate early response to combined ATR and Wee1 inhibitor treatment. Lastly, we assess the impact of combined ATR and Wee1 inhibitors as adjuvant to radiotherapy or surgery. Our preliminary data suggests that combined ATR and Wee1 inhibitor treatment results in radiosentization of 4T1 tumors. Furthermore, when combined ATR and Wee1 inhibitors were used adjuvant to surgery for advanced tumors, we observed tumor “cure” in a few cases, despite the aggressive nature of the cancer model.
  As these inhibitors of ATR and Wee1 are currently undergoing phase I/II clinical trials, this knowledge could soon be translated into the clinic, especially because we showed that the combination treatment targets a wide range of tumor cells. Particularly the anti-metastatic effect of combined ATR and Wee1 inhibition and the low toxicity of ATR inhibitors compared to Chk1 inhibitors show great clinical potential.

• Subjects / Keywords

  • ATR
  • Wee1
  • Breast cancer
  • Breast cancer metastasis
  • DNA damage response

• Graduation date

  Fall 2021

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-ht4e-9d75

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.