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1 Department of Biochemistry, Western University, London, Ontario, Canada. 2 Department of Oncology, Western University, London, Ontario, Canada. 3 London Regional Cancer Program, London, Ontario, Canada. 4 Chil-dren’s Health Research Institute, London, Ontario, Canada. Corresponding Author: Frederick A. Dick, Western University, Cancer Research Labs, 790 Commissioners Road East, London, Ontario N6A 4L6, Canada. Phone: 519-685-8500, ext. 53027; Fax: 519-685-8616; E-mail: fdick@uwo.ca doi: 10.1158/2159-8290.CD-18-1448 ©2019 American Association for Cancer Research.

IN THE SPOTLIGHT

Drugging RB1 Defi ciency: Synthetic Lethality with Aurora Kinases Frederick A. Dick 1 , 2 , 3 , 4 and Shawn Shun-Cheng Li 1 , 2 , 4

Summary: Two recent reports describe pharmacologic approaches to specifi cally treat RB1 -mutant cancers. The basis of this approach is a synthetic lethal relationship between RB1 mutations and inhibition of Aurora kinases A or B.

See related article by Oser et al., p. 230 (1).See related article by Gong et al., p. 248 (2).

Targeted cancer therapies, directed against specifi c onco-genic driver mutations, have been successfully deployed in the clinic. These agents are inhibitors of enzymes that are mutationally activated and directly antagonize the underly-ing molecular etiology of the cancer. They often cause rapid regression of cancer with limited side effects, and for this reason have become fi rst-line treatment options for many patients. A conceptual challenge in broadening this approach is the relative scarcity of cancers with actionable driver muta-tions. Frequent loss-of-function mutations in tumor sup-pressor genes such as RB1 and TP53 have historically been untargetable. The best example of targeting a cancer-specifi c vulnerability that is created through tumor suppressor gene loss is that of BRCA1 mutations and PARP inhibitors. Loss of BRCA1 compromises DNA damage repair through the homologous recombination pathway and creates a depend-ence on nonhomologous end joining (NHEJ) to repair DNA breaks. Consequently, inhibition of NHEJ by PARP inhibitors is specifi cally toxic to BRCA1 -mutant cancer cells. Two arti-cles in this issue of Cancer Discovery describe agents and their molecular targets for a similar synthetic lethal approach to treating RB1 -deleted cancers ( 1, 2 ).

APPROACHES TO TARGETING DEFECTIVE RB1 FUNCTION IN CANCER

Loss of function of the RB1 pathway is a common event in cancer ( 3 ). Typically, activating mutations in G 1 cyclin–dependent kinase (CDK) complexes phosphorylate and inac-tivate the RB1 protein. Alternatively, inactivating mutations of CDK inhibitors, such as CDKN2A , can have a similar effect on activation of G 1 CDKs, leading to constitutively phosphoryl-ated RB1. Direct mutation of the RB11 gene itself is less com-

mon than upstream RB1 pathway mutations. RB1 deletion is common in retinoblastoma and small cell lung cancer (SCLC), and deep deletion of RB1 is found sporadically in most disease sites ( 1, 2 ). Loss of RB1 function creates a number of distinct but interrelated cellular phenotypes including deregulated pro-liferation, altered metabolism, and genome instability. Each of these phenotypes can be induced by RB1 pathway mutations leading to hyperphosphorylated RB1 and deregulated E2F tran-scription. Using fruit fl y genetics and mammalian cell culture, Du and colleagues have identifi ed synthetic lethal connec-tions between RB1 pathway disruption and mutation of TSC2 and TORC1, as proliferation and stress from disrupted energy metabolism are incompatible ( 4 ). Approaches to exploit mis-regulated E2F transcription and its effects on mitochondrial biogenesis and function have also been suggested to create an RB1 pathway–dependent vulnerability in cancer. This has been noted by studies that observed synergy between CDK4/6 inhibi-tors and inhibitors of PI3K, IGF1, and others ( 4 ). Efforts to fi nd compounds that selectively kill or inhibit proliferation of RB1 pathway–disrupted cancer cells have also identifi ed PLK1 and CHK1 as targets ( 5 ). This study identifi ed misregulated tran-scription as fueling replication stress and mitotic checkpoint abnormalities that create potentially targetable vulnerabilities.

In a related but distinct manner, rational approaches to treating retinoblastoma have been developed. A genomic study suggested that epigenetic alterations occur in response to RB1 loss ( 6 ). This study highlights elevated expression of SYK and demonstrates that SYK inhibitors can slow progression in a patient-derived xenograft model. In a similar effort, Sangwan and colleagues identifi ed deregulated E2F transcription and abnormal CDK2 activity as downstream consequences of RB1mutation in a mouse model of retinoblastoma ( 7 ). Pretreat-ment of pregnant murine mothers was shown to prevent retinoblastoma formation in offspring, suggesting a means by which targeting E2Fs and CDK2 can offer a cancer prevention approach. Collectively, these studies highlight ongoing efforts to exploit RB1 loss in new cancer treatments.

IDENTIFICATION OF AURORA KINASES FOR TREATING RB1-DEFICIENT CANCERS

As highlighted above, there are many similarities between RB1 pathway mutations that misregulate CDKs and genetic

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Figure 1.  Model of synthetic effects of RB1 deletion and Aurora kinase inhibition. A, In cells with a functional RB1 gene and Aurora A and B kinases, normal mitotic progression and checkpoints ensure high fidelity of chromosome separation and cell division. B, In the absence of RB1, chromosome condensation and cohesion defects lead to misshapen centromeres, misaligned chromosomes in metaphase, syntelic and merotelic microtubule attach-ments, and an activated spindle assembly checkpoint. In this circumstance, defective mitosis is tolerated, in part, through augmented activity of Aurora kinases. C, When RB1 is deleted and Aurora A or B kinase is inhibited pharmacologically, cells attempt to transit mitosis, but fail to enter anaphase and rapidly undergo apoptosis. D, Xenografted SCLC tumors treated with Aurora kinase inhibitors display evidence of mitotic catastrophe and regress in response to treatment.

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Aurora BAurora A

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Unaligned chromosomes

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DIncreased mitotic errors

and apoptosis

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RB1- cancer withmitotic errors

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deletion of the RB1 gene. Remarkably, the studies by Oser and Gong in this issue identify vulnerabilities that are specific for RB1 gene deletion (1, 2). This emphasizes that loss of RB1 causes additional targetable vulnerabilities beyond those that are created by CDK misregulation. CDK-independent nonca-nonical functions of RB1 in mitosis and chromosome structure have been reported, but remain relatively unexplored (3). In the article by Gong and colleagues, the authors devise a synthetic lethal screen approach in which a panel of cell lines with known genetic alterations is challenged by a collection of 36 targeted inhibitors, and agents that cause cytotoxicity are selected for further evaluation. This approach specifically identified RB1-mutant cell lines, but not those with RB1 pathway mutations, as being sensitive to compounds known to target functions in mitosis. In particular, compounds that inhibit Aurora kinases A and B had a much more pronounced effect than others in this mitotic category. Oser and colleagues searched for synthetic lethal vulnerabilities by devising a cell line system in which an RB1-mutant SCLC line, NCI-H82, was used. Conditional reex-pression of RB1 in these cells did not inhibit proliferation, and as a consequence the authors searched for RB1-dependent vul-nerabilities using a CRISPR/Cas9 screen approach in which they focused on hits from non–RB1-expressing cells compared with those that reexpress RB1. They used a custom gRNA library that was enriched for cell-cycle, epigenetic, and cancer-related genes, and their screen identified Aurora kinase B (the library did not contain guides to Aurora A).

Both teams utilized highly specific inhibitors for Aurora A or B in their experiments and Gong and colleagues also described the generation of a novel Aurora kinase A–specific compound. Aurora kinase A is best known for its role in cen-trosome separation and mitotic spindle assembly, whereas Aurora kinase B coordinates microtubule attachments to centromeres and phosphorylates histone H3 in mitosis (8). Because Aurora kinase A can phosphorylate and activate B, it is possible that some interdependence of synthetic lethal-ity exists. For this reason, it is important that both research teams generated drug-resistant mutant forms of their respec-tive kinases to demonstrate on-target dependence of syn-thetic lethality with RB1 deletion.

It is difficult to conceptualize precisely why Aurora kinase inhibition is synthetically lethal with RB1 deficiency, and this will be an important area of future research. However, we envision the model in Fig. 1 to represent a possible expla-nation. In normal cells, RB1 contributes to mitotic fidel-ity through mechanisms that are both E2F-dependent and related to coordinating Cohesin and Condensin II function (4, 9). Aurora kinases also contribute to cell-cycle progression and mitotic fidelity through centrosomes, mitotic spindle formation, and chromosome attachment and separation (8). This suggests mitotic fidelity is simultaneously dependent on all of RB1, Aurora A, and Aurora B kinases (Fig. 1A). In RB1-deleted cells, the centromere structure and function is compromised, leading to misaligned chromosomes in mitosis as well as syntelic and merotelic chromosome attachments. Through an E2F-dependent effect of RB1 loss, numerous components of the spindle assembly checkpoint become overexpressed, leading to a hyperactivated checkpoint (10). Investigation of Aurora kinase expression and function in the current studies suggests that both Aurora A and B kinase

activity and function are augmented in an RB1-deficient background, suggesting these kinases offer some compensa-tion for effects caused by RB1 loss (Fig. 1B). Finally, when Aurora A or B kinases are inhibited, centrosome separation and initial spindle formation appear intact (1, 2), but the formation of a metaphase plate is difficult to observe and chromosomes disburse quickly followed by apoptosis (Fig. 1C). The distinct nature of Aurora A and B inhibitors, and the roles of these kinases in mitosis, suggests that slightly dif-ferent effects on mitosis can elicit this synthetic lethal effect. Both groups sought further explanations for the mechanism through screening approaches. Oser and colleagues point out that there are also synthetic lethal hits with components of Condensin II and other mitotic components such as PLK1 and INCENP. Gong and colleagues carried out an shRNA loss-of-function screen to identify genes that rescue synthetic lethality between RB1 mutation and Aurora kinase A inhibi-tors. They identified mitotic checkpoint components BUB1B and BUB3 as essential for synthetic lethality, further under-scoring the importance of a hyperactivated spindle assembly checkpoint. These findings collectively emphasize that RB1 loss and Aurora kinase inhibition converge on mitotic pro-gression and fidelity.

PRECLINICAL EVALUATION OF AURORA A AND B KINASE INHIBITORS

The study led by Gong and colleagues from Eli Lilly developed and tested a lead compound, LY3295668, that they demonstrate is highly specific for Aurora A. Oser and colleagues make use of AZD2811, an inhibitor of Aurora B that was developed by AstraZeneca. Both groups tested these compounds in multiple cancer models ranging from patient-derived xenografts of SCLC to cell lines, and a geneti-cally engineered mouse model. The inhibitors induced tumor regression in most cases and outperformed the standard-of-care for SCLC where evaluated. Oser and colleagues investi-gated the mechanism of action through histologic analysis of tumors from treated mice, and confirmed that treatment induced mitotic catastrophe in vivo (Fig. 1D). Historically, experimental treatment using Aurora kinase inhibitors has caused bone marrow toxicity that has limited dosing of these agents and compromised their performance in clinical trials. AZD2811 efficacy was greatly enhanced by new delivery using encapsulated nanoparticles, suggesting that past limitations with this class of inhibitor can be overcome. In addition, LY3295668 was tested for toxicity by culturing with bone marrow cells, which indicates that effective treatment doses with this new compound have limited toxicity. On the basis of these results, Eli Lilly and AurKa have initiated a phase I/II clinical trial for RB1-deficient solid tumors (NCT03092934).

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

AcknowledgmentsResearch in the authors’ laboratories on RB1 function, kinase

signaling, and experimental cancer therapeutics is supported by the Canadian Institutes of Health Research and the Cancer Research Society. F.A. Dick is the Wolfe Senior Fellow in tumor suppressor

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genes at Western University, and S.S-C. Li is a Canada Research Chair in molecular and epigenetic basis of cancer.

Published online February 8, 2019.

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2019;9:169-172. Cancer Discov   Frederick A. Dick and Shawn Shun-Cheng Li  KinasesDrugging RB1 Deficiency: Synthetic Lethality with Aurora

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