Investigation into Tipifarnib as an anti-mitotic agent and Spindly as the mitotic target

• Author / Creator

  Smith, Joanne Dawn

• The mitotic checkpoint is a failsafe mechanism for the cell to ensure correct chromosome segregation. The evolutionarily conserved kinetochore RZZ complex is an essential component of the mitotic checkpoint. Spindly interacts with the RZZ complex in a farnesylation dependent manner and acts as a dynein adaptor at kinetochores. Spindly knockdown results in prometaphase delay, alignment defects, and loss of dynein kinetochore localization. The knockdown of Spindly phenocopies farnesyl transferase inhibitor (FTI) treatment of cells suggesting that Spindly is the mitotic target of FTIs. FTIs were originally developed to target the RAS oncogene, but have been abandoned as inhibitors of the Ras pathway. Our findings indicate a possibility in repurposing FTIs for cancer treatment, especially in combination treatment strategies.
  We found that upon treatment of HeLa cells with Tipifarnib (an FTI) there was loss of Spindly KT localization. This lack of Spindly KT localization also caused a prometaphase arrest and prolonged mitotic duration that increased in a dose dependent manner. This is consistent with other FTIs that have been tested. However, neither Spindly nor Farnesyltransferase beta subunit protein levels corresponded to sensitivity or resistance, and more investigation is required to determine what acts as a marker for sensitivity or resistance to Tipifarnib.
  Adavosertib, a Wee1 kinase inhibitor results in mitotic catastrophe and our lab found that it enhanced the activity of other anti-mitotic agents including an FTI. Based on this we wished to test the combination of Tipifarnib and Adavosertib in a range of cell lines and determine if the combination is synergistic. Our data indicate that the synthetic lethal relationship between Tipifarnib and Adavosertib, both of which are being used in clinical trials for breast cancer individually, is conserved in a subset of breast cancer cells. The combination treatment was synergistic to varying extents depending on the cell line tested and this was not molecular subtype specific. The combination treatment of Tipifarnib and Adavosertib represents a potential new treatment for breast cancers.
  Since neither Spindly nor FNTB protein levels served as a marker for Tipifarnib resistance, we wished to examine the mechanism of resistance, by generating Tipifarnib resistant cells. I found that while the resistant cells had increased Spindly and FNTB levels, this did not correspond to Spindly KT localization in the presence of Tipifarnib. These resistant cells will be invaluable reagents for future examination of Tipifarnib resistance mechanism, and potentially aid in determining how the resistance occurs.

• Subjects / Keywords

  • Spindly
  • Mitosis
  • Tipifarnib
  • Mitotic arrest
  • Adavosertib

• Graduation date

  Fall 2021

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-qf10-wk88

• 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.