Unveiling a new role for Wee1 during S-phase entry
File(s)
Author(s)
Morales, Lucas
Type
Thesis
Abstract
Progression through the cell cycle requires temporal and spatial control of cyclin dependent kinase (CDK) activity. Wee1 kinase and Cdc25 phosphatases play a key role in CDK regulation through inhibitory phosphorylation and activating desphosphorylation of CDKs. Wee1 regulates a G2 DNA damage checkpoint, through CDK1 inhibition. Therefore, Wee1 inhibitor in combination with DNA damaging agents, is in clinical trials aiming to promote mitotic catastrophe and cell death in TP53 mutant cancer cells that rely on the Wee1 checkpoint. However, the role of Wee1 in regulating earlier cell cycle transitions and the potential impact of Wee1 inhibitors on cell cycle entry is poorly understood.
I investigated the role of Wee1 in cell cycle entry using chemical inhibitors, siRNA and inducible degradation combined with quantitative single-cell imaging of live and fixed cells. I found that inhibition of Wee1 kinase during cell cycle re-entry from quiescence leads to a delay in S-phase entry, with many cells failing to exit quiescence. Moreover, cells that do enter S-phase after Wee1 inhibition have abnormal DNA replication and the CDK inhibitor protein, p21, abnormally accumulates during DNA replication, ultimately leading to a p21-dependent G2 arrest. My investigations revealed that inhibition of Wee1 leads to the hyperactivation of APC/CCdh1, causing the observed delay in S-phase entry. In addition, using phosphoproteomics analysis following
Wee1 inhibition, I unveiled distinctive alterations in the phosphorylation landscape, providing valuable information for new Wee1 substrates. My work highlights a novel role for Wee1 during cell cycle entry and has implications for the use of Wee1 inhibitors in cancer treatment.
I investigated the role of Wee1 in cell cycle entry using chemical inhibitors, siRNA and inducible degradation combined with quantitative single-cell imaging of live and fixed cells. I found that inhibition of Wee1 kinase during cell cycle re-entry from quiescence leads to a delay in S-phase entry, with many cells failing to exit quiescence. Moreover, cells that do enter S-phase after Wee1 inhibition have abnormal DNA replication and the CDK inhibitor protein, p21, abnormally accumulates during DNA replication, ultimately leading to a p21-dependent G2 arrest. My investigations revealed that inhibition of Wee1 leads to the hyperactivation of APC/CCdh1, causing the observed delay in S-phase entry. In addition, using phosphoproteomics analysis following
Wee1 inhibition, I unveiled distinctive alterations in the phosphorylation landscape, providing valuable information for new Wee1 substrates. My work highlights a novel role for Wee1 during cell cycle entry and has implications for the use of Wee1 inhibitors in cancer treatment.
Version
Open Access
Date Issued
2024-01-11
Date Awarded
01/06/2024
License URL
Advisor
Barr, Alexis
Publisher Department
Institute of Clinical Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)