CHARACTERIZING THE CELLULAR CONSEQUENCES OF CENTROSOME LOSS: DISCOVERY OF A MITOTIC SURVEILLANCE PATHWAY

Centrosomes are the major microtubule-organizing centers of the cell, and play a key role in organizing the bipolar mitotic spindle. Precise regulation of centrosome number is therefore critical for accurate chromosome segregation and the maintenance of genomic integrity. The consequences of centrosome loss, however, have been difficult to study due to a lack of specific tools that allow persistent and reversible centrosome depletion. In the first chapter of this thesis, we combined gene targeting with an auxin-inducible degradation system to achieve rapid, titratable, and reversible control of Polo-like kinase 4 (Plk4), a master regulator of centrosome biogenesis. Depletion of Plk4 led to a failure of centrosome duplication that, unexpectedly, produced an irreversible cell cycle arrest within a few divisions. This arrest was not a result of a prolonged mitosis, chromosome segregation errors or cytokinesis failure. Depleting p53 allowed cells that failed centrosome duplication to proliferate indefinitely, indicating the presence of a p53-dependent surveillance mechanism that protects against genome instability by preventing cell growth following centrosome duplication failure. However, the mechanism by which p53 is activated in response to centrosome loss was unknown. In the second chapter, we describe how we performed a genome-wide CRISPR/Cas9 knockout screens to identify a USP28-53BP1-p53-p21 signaling axis at the core of the centrosome surveillance pathway. We show that USP28 and 53BP1 act to stabilize p53 following centrosome loss and demonstrate this function to be independent of their previously characterized role in the DNA damage response. Surprisingly, the USP28-53BP1-p53-p21 signaling pathway is also required to arrest cell growth following a prolonged prometaphase. We therefore propose that centrosome loss or a prolonged mitosis activate a common signaling pathway that acts to prevent the growth of cells with an increased propensity for mitotic errors. Notably, most cancer cells have a disrupted p53 or p21 pathway, which allows them to proliferate indefinitely in the absence of centrosomes. In the last chapter of this thesis, we illustrate an exception to this trend, identifying a subset of breast cancers that exhibit profound sensitivity to centrosome loss that is independent of the mitotic surveillance pathway. We find this sensitivity to be dependent on the overexpression of TRIM37, an E3 ubiquitin ligase encoded within the 17q23 amplicon found in this subset of breast cancers. Timelapse studies show that centrosome depletion in these cells leads to dramatically prolonged mitoses that largely end in mitotic slippage. Subsequent analyses show that pericentriolar material (PCM) is depleted when TRIM37 is overexpressed, and that this suppresses the ability of these cells to organize a bipolar mitotic spindle through compensating, non-centrosomal pathways. Together, these results reveal a synthetic lethal vulnerability in breast cancers harboring the 17q23 amplicon that could be exploited therapeutically. In summary, this thesis describes the development of precise tools with which to study centrosome depletion, and reports the cellular consequences of centrosome loss in distinct cellular contexts