Polo 6 Phase 2

[Mariela Coxon]

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Purpose: The phase III POLO study demonstrated significant progression-free survival (PFS) benefit for active olaparib maintenance therapy versus placebo for patients with metastatic pancreatic adenocarcinoma and a germline BRCA mutation. Here, we report the final analysis of overall survival (OS) and other secondary end points.

Conclusion: Although no statistically significant OS benefit was observed, the HR numerically favored olaparib, which also conferred clinically meaningful benefits including increased time off chemotherapy and long-term survival in a subset of patients.

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As the main microtubule (MT)-organizing center in animal cells, the centrosome plays a pivotal role in various cellular processes, such as spindle formation, chromosome segregation, and cell division. The centrosome is composed of a pair of MT-derived apparatus, called centrioles, embedded in a pericentriolar protein matrix. Centriole duplication occurs precisely once per cell cycle, and tight control of centriole numbers is essential for the maintenance of genomic stability1,2.

Centriole duplication begins by assembling a procentriole in the late G1/early S phase, and timely activation of Polo-like kinase 4 (Plk4) appears to be central for inducing centriole biogenesis2,3. Notably, Plk4 exhibits a ring-like localization pattern (i.e., ring state) around the Cep152 scaffold in early G1, and then subsequently adopt a dot-like morphology (i.e., dot state) at the future procentriole assembly site4,5,6,7. The symmetry breaking from a uniformly distributed ring state to a confined dot state is critical for Plk4 to assume a physically distinct entity specialized for centriole biogenesis.

Next, we systematically mutated the four PC3 residues to negatively charged aspartic or glutamic acid and generated a condensation-proficient (CP) gain-of-function mutant (i.e., the S698E, S700E, T704E, T707D mutant). In U2OS cells where endogenous Plk4 is silenced, the CP mutant exhibited a greatly increased level of Sas6 recruitment to centrosomes (Supplementary Fig. 2a). A low but significant level of Sas6 was also detected in cells expressing the catalytically inactive KM CP mutant, suggesting that the CP mutations at the PC3 motif of CPB can alleviate the essential requirement of the N-terminal catalytic activity in recruiting Sas6. Quantification carried out with cyclin A-positive G2 cells also yielded similar results (Supplementary Fig. 2b), demonstrating that increased Sas6 recruitment is not due to an altered cell cycle.

Since dynamic disorder is considered the major contributor to the B-factor of a protein crystal structure36,37, we carried out comparative B-factor analyses and found several disordered regions in CPB CP_v1 (Supplementary Fig. 5h, i). Sequence analysis using the PONDR software ( ) revealed that, unlike the PB1 wing edge region, the PB2 tip has strong potential to show structural disorder (Supplementary Fig. 5j). Notably, the PB2-tip region is composed of a stretch of mainly hydrophilic residues with four conserved hydrophobic residues (i.e., Y750, L752, V758, and L761) scattered along the disordered region (Supplementary Fig. 5k). A large fraction of these residues, including the four hydrophobic residues, were absent or lacking their side chains in the CPB CP structure (Fig. 5c), a striking deviation from an α-helix-containing ordered loop found in apo-CPB. Therefore, we focused on the PB2-tip region for further analysis. B-factor-based structural analysis has served as a measure of predicting residue flexibility36,37 and is used to identify the region of conformational plasticity in polo-like kinase 138.

Confocal images were acquired using Zeiss LSM780 equipped with a plan-apochromat 40 (NA 1.3) and 63 (NA 1.4) oil-immersion objective lenses, 34-channel GaAsP spectral detector (Carl Zeiss Microscopy, LLC), and 12-bit, 0.5-μm z-steps. To quantify fluorescence signal intensities, images were acquired under the same settings and the images obtained after the maximum intensity projection of z-stacks were analyzed using the Zeiss ZEN v2.1 software (Carl Zeiss Microscopy, LLC).

For the generation of rabbit polyclonal phosphoantibodies, synthetic phosphopeptides were used for immunization (Young In Frontier, South Korea). Phosphoantibodies were affinity-purified using a corresponding phospho-peptide immobilized to SulfoLink resin (Thermo Fisher Scientific). Alexa Fluor 594-conjugated anti-Plk4 pSSTT antibody was generated using the Alexa Fluor 594 protein labeling kit (Molecular Probes). The list of phosphopeptides and their respective nonphospho-peptides used in this study is provided in Supplementary Table 4.

Immunoblotting analysis was performed according to standard procedures using an enhanced chemiluminescence detection system (Thermo Fisher Scientific). To detect specific phosphoepitopes (i.e., Plk4 pSSTT and STIL pS1108), immunoblotting was carried out in the presence of their respective nonphospho-peptide. Immunoblot signal intensities were quantified using the Image J (Fiji) or Image Lab 5.2.1 (Bio-Rad) program. The specific protein intensity was determined after subtracting non-specific background signals. All the antibodies used for this study are listed in Supplementary Table 3. Detailed information will be provided upon request.

Asynchronously growing HEK293T cells transfected with EGFP-Plk4 were subjected to IP with an anti-GFP antibody in the presence of PhosSTOP (Sigma-Aldrich). The resulting immunoprecipitates were separated by 7.5% SDS-PAGE and stained with Coomassie Brilliant Blue. Hyperphosphorylated slow-migrating Plk4 species was clearly detectable, as shown Supplementary Fig. 1b. Phosphorylated Plk4 excised from the gel was in-gel digested with trypsin P, chymotrypsin, LysC, ArgC, GluC, or AspN to extract the peptides58. After desalting by C18 ZipTip (Millipore), the extracted samples were subjected to liquid chromatography-MS (LC-MS) analysis.

The BioWorks software reported multiple phosphorylation site assignments for the peptides within the pS698 through pT707 region and manual sequencing of the spectra was required. As shown in Supplementary Fig. 2c, equivalent evidence existed to support the presence of two phosphopeptides differing in only the localization of the third phosphorylation site.

Diffraction data sets were indexed, integrated, and scaled with HKL-200059. The crystal structure of CPB CP_v1 was solved by molecular replacement method using phaser from the CCP4 software suite60,61. The crystal structure of apo-CPB (PDB code: 4N9J) was used as an initial model. Further refinement was carried out with refmac5 in CCP4 software suite62 and phenix.refine in Phenix suite63. Data collection and refinement statistics are summarized in Table 1.

The structure of the final CPB CP_v1 model was verified using Phenix suite. Water molecules were not identified likely because of a high Wilson B-factor. The Ramachandran plots showed 91.00%, 99.23%, and 0.77% of the residues in the favored, allowed, and outlier regions, respectively. All structural figures were rendered using the PyMOL 2.2.0 software (PyMOL Molecular Graphics System, Schrdinger, LLC).

A final cycle of B-factor refinement without hydrogen was carried out in Phenix, and the final structure of His6-CPB CP was verified using Phenix suite. The Ramachandran plots showed 97.84%, 99.94%, and 0.06% of the residues in the favored, allowed, and outlier regions, respectively.

We are grateful to Lingjun Meng for initial crystallization of CPB CP_v1, Michael Kruhlak and Langston Lim for assisting with time-lapse microscopy, Kunio Nagashima and Louis (Chip) Dye for providing technical service with electron microscopy samples, Ming Zhou and Benjamin C. Orsburn for mass spec data search and analysis, Di Xia for structural analysis, Yanling Liu for generating 3D surface-rendered movies, Richard J. Wheeler (Oxford University, UK) for sharing unpublished data about α-lipoamide, Karen Oegema for providing centrinone (LCR-263), and Raymond Erikson for critical reading of the manuscript. This research was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute (K.S.L.) and an NST grant CAP-16-03-KRIBB of South Korea (J.K.B.).

Onsite amenities include a barn-style clubhouse from the original polo field, playground, pickleball court, fire pit, plus a beautiful lake with fountain. The community is also located within the Rockwood School District.

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