Edited by Xiao-Fan WangThe centrosome, consisting of two centrioles surrounded by a dense network of proteins, is the microtubule-organizing center of animal cells. Polo-like kinase 4 (PLK4) is a Ser/Thr protein kinase and the master regulator of centriole duplication, but it may play additional roles in centrosome function. To identify additional proteins regulated by PLK4, we generated an RPE-1 human cell line with a genetically engineered "analog-sensitive" PLK4 AS , which genetically encodes chemical sensitivity to competitive inhibition via a bulky ATP analog. We used this transgenic line in an unbiased multiplex phosphoproteomic screen. Several hits were identified and validated as direct PLK4 substrates by in vitro kinase assays. Among them, we confirmed Ser-78 in centrosomal protein 131 (CEP131, also known as AZI1) as a direct substrate of PLK4. Using immunofluorescence microscopy, we observed that although PLK4-mediated phosphorylation of Ser-78 is dispensable for CEP131 localization, ciliogenesis, and centriole duplication, it is essential for maintaining the integrity of centriolar satellites. We also found that PLK4 inhibition or use of a nonphosphorylatable CEP131 variant results in dispersed centriolar satellites. Moreover, replacement of endogenous WT CEP131 with an S78D phosphomimetic variant promoted aggregation of centriolar satellites. We conclude that PLK4 phosphorylates CEP131 at Ser-78 to maintain centriolar satellite integrity.

◥Centrosome amplification (CA), or a numerical increase in centrosomes, is common in human cancers, particularly those with high-risk features. We have discovered that cells with CA have an increased burden of autophagy, a catabolic process whereby autophagosomes engulf damaged organelles and proteins and deliver these contents to the lysosome for degradation and subsequent recycling. Cells with CA demonstrate an accumulation of autophagosomes. We evaluated the alternative hypotheses that CA alters autophagy by modulating microtubule networks and impairing trafficking versus altering lysosome clustering and organization versus chromosome missegregation-induced proteotoxic stress. Using LC3 reporter assays and autophagosome tracking experi-ments, we demonstrate that CA causes an accumulation of autophagosomes by interfering with autophagosome trafficking. To establish whether this was a druggable weakness, we tested autophagy inhibitors in our cell models of CA. Cells with CA are sensitized to chemical and genetic autophagy inhibition. Taken together, our results suggest that autophagy is disrupted by CA and sensitizes cells to inhibition of autophagy. These findings suggest a novel precision medicine strategy, whereby CA increases reliance on autophagy and serves as a biomarker for autophagy inhibitors in high-risk cancers.Implications: Our study suggests that CA could be used as a predictive biomarker for treatment with autophagy inhibitors.

<p>Supp Table 1. Primer sequences for qRT-PCR. Supplemental Figure 1: Validation of centrosome amplification cell models. Supplemental Figure 2: Centrosome amplification induced by cytokinesis failure and STIL overexpression also disrupts autophagy. Supplemental Figure 3: PLK4 inhibition reduces centrosomes and autophagy defect in some cancer cell lines with centrosome amplification. Supplemental Figure 4: Centrosome amplification increases autophagy gene expression but does not significantly alter LC3B-II/I ratio. Supplemental Figure 5: Centrosome amplification does not alter lysosomal hub organization around the centrosomes. Supplemental Figure 6: Centrosome amplification-mediated autophagy disruption does not depend significantly on mitosis. Supplemental Figure 7: Microtubule disruption inhibits autophagy. Supplemental Figure 8: Centrosome amplified cells are less sensitive to mitotic inhibitors.</p>

<p>Supp Table 1. Primer sequences for qRT-PCR. Supplemental Figure 1: Validation of centrosome amplification cell models. Supplemental Figure 2: Centrosome amplification induced by cytokinesis failure and STIL overexpression also disrupts autophagy. Supplemental Figure 3: PLK4 inhibition reduces centrosomes and autophagy defect in some cancer cell lines with centrosome amplification. Supplemental Figure 4: Centrosome amplification increases autophagy gene expression but does not significantly alter LC3B-II/I ratio. Supplemental Figure 5: Centrosome amplification does not alter lysosomal hub organization around the centrosomes. Supplemental Figure 6: Centrosome amplification-mediated autophagy disruption does not depend significantly on mitosis. Supplemental Figure 7: Microtubule disruption inhibits autophagy. Supplemental Figure 8: Centrosome amplified cells are less sensitive to mitotic inhibitors.</p>