Chromosomal Instability (CIN) is regarded as a unifying feature of heterogeneous tumor populations, driving intratumoral heterogeneity. Polo-Like Kinase 1 (PLK1), a serine-threonine kinase that is often overexpressed across multiple tumor types, is one of the key regulators of CIN and is considered as a potential therapeutic target. However, targeting PLK1 has remained a challenge due to the off-target effects caused by the inhibition of other members of the polo-like family. Here we use synthetic dosage lethality (SDL), where the overexpression of PLK1 is lethal only when another, normally non-lethal, mutation or deletion is present. Rather than directly inhibiting PLK1, we found that inhibition of PP2A causes selective lethality to PLK1-overexpressing breast, pancreatic, ovarian, glioblastoma, and prostate cancer cells. As PP2A is widely regarded as a tumor suppressor, we resorted to gene expression datasets from cancer patients to functionally dissect its therapeutic relevance. We identified two major classes of PP2A subunits that negatively correlated with each other. Interestingly, most mitotic regulators, including PLK1, exhibited SDL interactions with only one class of PP2A subunits (PPP2R1A, PPP2R2D, PPP2R3B, PPP2R5B and PPP2R5D). Validation studies and other functional cell-based assays showed that inhibition of PPP2R5D affects both levels of phospho-Rb as well as sister chromatid cohesion in PLK1-overexpressing cells. Finally, analysis of clinical data revealed that patients with high expression of mitotic regulators and low expression of Class I subunits of PP2A improved survival. Overall, these observations point to a context-dependent role of PP2A that warrants further exploration for therapeutic benefits.

Application of tumor genome sequencing has identified numerous loss-of-function alterations in cancer cells. While these alterations are difficult to target using direct interventions, they may be attacked with the help of the synthetic lethality (SL) approach. In this approach, inhibition of one gene causes lethality only when another gene is also completely or partially inactivated. The EPHB6 receptor tyrosine kinase has been shown to have anti-malignant properties and to be downregulated in multiple cancers, which makes it a very attractive target for SL applications. In our work, we used a genome-wide SL screen combined with expression and interaction network analyses, and identified the SRC kinase as a SL partner of EPHB6 in triple-negative breast cancer (TNBC) cells. Our experiments also reveal that this SL interaction can be targeted by small molecule SRC inhibitors, SU6656 and KX2-391, and can be used to improve elimination of human TNBC tumors in a xenograft model. Our observations are of potential practical importance, since TNBC is an aggressive heterogeneous malignancy with a very high rate of patient mortality due to the lack of targeted therapies, and our work indicates that FDA-approved SRC inhibitors may potentially be used in a personalized manner for treating patients with EPHB6-deficient TNBC. Our findings are also of a general interest, as EPHB6 is downregulated in multiple malignancies and our data serve as a proof of principle that EPHB6 deficiency may be targeted by small molecule inhibitors in the SL approach.

RAD51 is a critical component of the homologous recombination pathway, forming a nucleoprotein filament that enables strand exchange and templated error-free DNA repair. The tumor suppressors BRCA1 and BRCA2 interact with RAD51 to control its activity on DNA. Defects in homologous recombination in tumors are clinically relevant, with evidence of synthetic lethality of such cancers to poly ADP ribose polymerase (PARP) inhibitors. Mutations in RAD51 are uncommon in cancer, but aberrant over-expression of RAD51 has been reported as a mechanism to overcome a recombination defect in in-vitro models. However there are no large scale studies on RAD51 expression in clinically annotated data sets. Here we report a series of experiments to study RAD51 protein expression in ovarian cancer, a tumor type where recombination defects are prominent and being evaluated in several clinical trials of PARP inhibition. Analysis of DNA repair protein expression in formalin fixed clinical tissue is challenging, and our experiments highlight recent advances in quantitative microscopy that are generalizable to other clinical scenarios as well. We first evaluated several commercially available monoclonal antibodies to RAD51 using siRNA depleted cell blocks, to identify an appropriate reagent and staining conditions for formalin fixed paraffin embedded (FFPE) material. We then analyzed RAD51 expression in a collection of 600 ovarian cancer samples obtained through the British Columbia Cancer Agency (BCCA) OvCare consortium, using a combination of multiplexed immunofluorescence staining and automated spectral microscopy to quantify staining on a per-cell basis. We find that RAD51 expression displays a Gaussian distribution in this cohort of high grade epithelial cancers, with no obvious correlation to BRCA mutation status. RAD51 sub-nuclear foci, which are a commonly used measure of homologous recombination in-vitro, did not prove to be accurately quantifiable in FFPE material. We then stained for RAD51 in a collection of approximately 250 ovarian cancer samples obtained from the SCOTROC4 trial. This clinical trial enrolled women with ovarian cancer into two treatment arms with fixed dose and escalated doses of carboplatin. Platinum sensitivity has been shown to correlate with sensitivity to PARP inhibitors, due to the requirement of homologous recombination for repair of platinum adducts. Therefore this trial offers a unique opportunity to study the predictive significance of biomarkers of homologous recombination in-vivo. In our analysis of this data set, we find that samples in the lowest quartile of RAD51 expression displayed a significantly improved survival after platinum chemotherapy, consistent with decreased HR in these cases. These are the first data on RAD51 in a clinical trial dataset of platinum sensitivity. Our study highlights methods and technical challenges for the quantitative analysis of DNA repair proteins in human clinical trial specimen, and describes a potential biomarker for synthetic lethal approaches targeting homologous recombination deficiency. Citation Format: Michal M. Hoppe, David SP Tan, Diana GZ Lim, Anthony Karnezis, David Huntsman, Jennifer Steel, Xinxue Liu, James Paul, Liz-Anne Lewsley, Nadeem Siddiqui, Robert Brown, Anand D. Jeyasekharan. RAD51 expression as a biomarker of homologous recombination deficiency in ovarian cancer [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B04.

aged 5!) years, a gentleman of a nervo-sanguineous temperament; active, industrious, and temperate in his habits ; had enjoyed perfect and uninterrupted health, with the exception of slight and occasional attacks of diarrhoea, for the last year of bis life.On tho evening of Wednesday, the 19th of March last, he returned homo from his place of business, much fatigued in body and in mind ; complaining of weariness and aching of the limbs, chilliness, sore throat, headache, and some febrile excitement. His wife persuaded him to bathe bis feet in hot water, with the addition of mustard, and to take some magnesia and Epsom salts, which be did, and retired to bed. During the night be experienced great restlessness, pain in the bead, nausea, and some vomiting of bilious fluid, with a sensation of distress at tho epigastrium ; but these symptoms were partially relieved by the operation of the purgative medicine he had taken, and towards morning he felt himself bolter, and slept.