Laura A. Tookman

Genomic DNA damage and ATR-Chk1 signaling determine oncolytic adenoviral efficacy in human ovarian cancer cells

Oncolytic adenoviruses replicate selectively within and lyse malignant cells. As such, they are being developed as anticancer therapeutics. However, the sensitivity of ovarian cancers to adenovirus cytotoxicity varies greatly, even in cells of similar infectivity. Using both the adenovirus E1A-CR2 deletion mutant dl922-947 and WT adenovirus serotype 5 in a panel of human ovarian cancer cell lines that cover a 3-log range of sensitivity, we observed profound overreplication of genomic DNA only in highly sensitive cell lines. This was associated with the presence of extensive genomic DNA damage. Inhibition of ataxia telangiectasia and Rad3-related checkpoint kinase 1 (ATR-Chk1), but not ataxia telangiectasia mutated (ATM), promoted genomic DNA damage and overreplication in resistant and partially sensitive cells. This was accompanied by increased adenovirus cytotoxicity both in vitro and in vivo in tumor-bearing mice. We also demonstrated that Cdc25A was upregulated in highly sensitive ovarian cancer cell lines after adenovirus infection and was stabilized after loss of Chk1 activity. Knockdown of Cdc25A inhibited virus-induced DNA damage in highly sensitive cells and blocked the effects of Chk1 inhibition in resistant cells. Finally, inhibition of Chk1 decreased homologous recombination repair of virus-induced genomic DNA double-strand breaks. Thus, virus-induced host cell DNA damage signaling and repair are key determinants of oncolytic adenoviral activity, and promoting unscheduled DNA synthesis and/or impeding homologous recombination repair could potentiate the effects of oncolytic adenoviruses in the treatment of ovarian cancer.

CRISPR/Cas9-Mediated Trp53 and Brca2 Knockout to Generate Improved Murine Models of Ovarian High-Grade Serous Carcinoma

There is a need for transplantable murine models of ovarian high-grade serous carcinoma (HGSC) with regard to mutations in the human disease to assist investigations of the relationships between tumor genotype, chemotherapy response, and immune microenvironment. In addressing this need, we performed whole-exome sequencing of ID8, the most widely used transplantable model of ovarian cancer, covering 194,000 exomes at a mean depth of 400× with 90% exons sequenced >50×. We found no functional mutations in genes characteristic of HGSC (Trp53, Brca1, Brca2, Nf1, and Rb1), and p53 remained transcriptionally active. Homologous recombination in ID8 remained intact in functional assays. Further, we found no mutations typical of clear cell carcinoma (Arid1a, Pik3ca), low-grade serous carcinoma (Braf), endometrioid (Ctnnb1), or mucinous (Kras) carcinomas. Using CRISPR/Cas9 gene editing, we modeled HGSC by generating novel ID8 derivatives that harbored single (Trp53-/-) or double (Trp53-/-;Brca2-/-) suppressor gene deletions. In these mutants, loss of p53 alone was sufficient to increase the growth rate of orthotopic tumors with significant effects observed on the immune microenvironment. Specifically, p53 loss increased expression of the myeloid attractant CCL2 and promoted the infiltration of immunosuppressive myeloid cell populations into primary tumors and their ascites. In Trp53-/-;Brca2-/- mutant cells, we documented a relative increase in sensitivity to the PARP inhibitor rucaparib and slower orthotopic tumor growth compared with Trp53-/- cells, with an appearance of intratumoral tertiary lymphoid structures rich in CD3+ T cells. This work validates new CRISPR-generated models of HGSC to investigate its biology and promote mechanism-based therapeutics discovery. Cancer Res; 76(20); 6118-29. ©2016 AACR.

Failure of Translation of Human Adenovirus mRNA in Murine Cancer Cells Can be Partially Overcome by L4-100K Expression In Vitro and In Vivo

Adaptive immune responses may be vital in the overall efficacy of oncolytic viruses in human malignancies. However, immune responses to oncolytic adenoviruses are poorly understood because these viruses lack activity in murine cells, which precludes evaluation in immunocompetent murine cancer models. We have evaluated human adenovirus activity in murine cells. We show that a panel of murine carcinoma cells, including CMT64, MOVCAR7, and MOSEC/ID8, can readily be infected with human adenovirus. These cells also support viral gene transcription, messenger RNA (mRNA) processing, and genome replication. However, there is a profound failure of adenovirus protein synthesis, especially late structural proteins, both in vitro and in vivo, with reduced loading of late mRNA onto ribosomes. Our data also show that in trans expression of the nonstructural late protein L4-100K increases both the amount of viral mRNA on ribosomes and the synthesis of late proteins, accompanied by reduced phosphorylation of eIF2α and improved anticancer efficacy. These results suggest that murine models that support human adenovirus replication could be generated, thus allowing evaluation of human adenoviruses in immunocompetent mice.

Paclitaxel resistance increases oncolytic adenovirus efficacy via upregulated CAR expression and dysfunctional cell cycle control

Resistance to paclitaxel chemotherapy frequently develops in ovarian cancer. Oncolytic adenoviruses are a novel therapy for human malignancies that are being evaluated in early phase trials. However, there are no reliable predictive biomarkers for oncolytic adenovirus activity in ovarian cancer. We investigated the link between paclitaxel resistance and oncolytic adenovirus activity using established ovarian cancer cell line models, xenografts with de novo paclitaxel resistance and tumour samples from two separate trials. The activity of multiple Ad5 vectors, including dl922‐947 (E1A CR2‐deleted), dl1520 (E1B‐55K deleted) and Ad5 WT, was significantly increased in paclitaxel resistant ovarian cancer in vitro and in vivo. This was associated with greater infectivity resulting from increased expression of the primary receptor for Ad5, CAR (coxsackie adenovirus receptor). This, in turn, resulted from increased CAR transcription secondary to histone modification in resistant cells. There was increased CAR expression in intraperitoneal tumours with de novo paclitaxel resistance and in tumours from patients with clinical resistance to paclitaxel. Increased CAR expression did not cause paclitaxel resistance, but did increase inflammatory cytokine expression. Finally, we identified dysregulated cell cycle control as a second mechanism of increased adenovirus efficacy in paclitaxel‐resistant ovarian cancer. Ad11 and Ad35, both group B adenoviruses that utilise non‐CAR receptors to infect cells, are also significantly more effective in paclitaxel‐resistant ovarian cell models. Inhibition of CDK4/6 using PD‐0332991 was able both to reverse paclitaxel resistance and reduce adenovirus efficacy. Thus, paclitaxel resistance increases oncolytic adenovirus efficacy via at least two separate mechanisms – if validated further, this information could have future clinical utility to aid patient selection for clinical trials.

New drugs for breast cancer

Recently there have been significant advances in rational drug design for the treatment of breast cancer, especially in the area of targeted drug therapy. These include drugs which target the HER2 receptor and angiogenesis and the novel class of drug the PARP inhibitors. Some of these agents, for example, trastuzumab used in the treatment of HER2 positive breast cancer are already established as the standard of care. However, the duration of adjuvant trastuzumab, whether to continue it beyond progression in metastatic disease and the mechanism for developing trastuzumab resistance, remain to be determined. There is also much still to be learnt regarding other targeted therapies; the efficacy of different agents, the optimal duration of use and combination of therapies. Many of these agents are already in clinical trials, the results of which are likely to change clinical practice.

RAD51 and BRCA2 Enhance Oncolytic Adenovirus Type 5 Activity in Ovarian Cancer.

Homologous recombination (HR) function is critically important in high-grade serous ovarian cancer (HGSOC). HGSOC with intact HR has a worse prognosis and is less likely to respond to platinum chemotherapy and PARP inhibitors. Oncolytic adenovirus, a novel therapy for human malignancies, stimulates a potent DNA damage response that influences overall antitumor activity. Here, the importance of HR was investigated by determining the efficacy of adenovirus type 5 (Ad5) vectors in ovarian cancer. Using matched BRCA2-mutant and wild-type HGSOC cells, it was demonstrated that intact HR function promotes viral DNA replication and augments overall efficacy, without influencing viral DNA processing. These data were confirmed in a wider panel of HR competent and defective ovarian cancer lines. Mechanistically, both BRCA2 and RAD51 localize to viral replication centers within the infected cell nucleus and that RAD51 localization occurs independently of BRCA2. In addition, a direct interaction was identified between RAD51 and adenovirus E2 DNA binding protein. Finally, using functional assays of HR competence, despite inducing degradation of MRE11, Ad5 infection does not alter cellular ability to repair DNA double-strand break damage via HR. These data reveal that Ad5 redistributes critical HR components to viral replication centers and enhances cytotoxicity. Implications: Oncolytic adenoviral therapy may be most clinically relevant in tumors with intact HR function. Mol Cancer Res; 14(1); 44–55. ©2015 AACR.

Pharmacological inhibition of β3 integrin reduces the inflammatory toxicities caused by oncolytic adenovirus without compromising anticancer activity

Adenoviruses have been clinically tested as anticancer therapies but their utility has been severely limited by rapid, systemic cytokine release and consequent inflammatory toxicity. Here, we describe a new approach to tackling these dangerous side effects. Using human ovarian cancer cell lines as well as malignant epithelial cells harvested from the ascites of women with ovarian cancer, we show that tumor cells do not produce cytokines in the first 24 hours following in vitro infection with the oncolytic adenovirus dl922-947. In contrast, dl922-947 does induce inflammatory cytokines at early time points following intraperitoneal delivery in mice with human ovarian cancer intraperitoneal xenografts. In these animals, cytokines originate predominantly in murine tissues, especially in macrophage-rich organs such as the spleen. We use a nonreplicating adenovirus to confirm that early cytokine production is independent of adenoviral replication. Using β3 integrin knockout mice injected intraperitoneally with dl922-947 and β3 null murine peritoneal macrophages, we confirm a role for macrophage cell surface β3 integrin in this dl922-947-induced inflammation. We present new evidence that co-administration of a cyclic RGD-mimetic-specific inhibitor of β3 integrin significantly attenuates the cytokine release and inflammatory hepatic toxicity induced by dl922-947 in an intraperitoneal murine model of ovarian cancer. Importantly, we find no evidence that β3 inhibition compromises viral infectivity and oncolysis in vitro or anticancer efficacy in vivo. By enabling safe, systemic delivery of replicating adenoviruses, this novel approach could have a major impact on the future development of these effective anticancer agents.

Abstract AP24: CRISPR/CAS9–MEDIATED TRP53, BRCA1, BRCA2 AND PTEN KNOCKOUT TO GENERATE IMPROVED MURINE MODELS OF OVARIAN HIGH GRADE SEROUS CARCINOMA

BACKGROUND: Ovarian high grade serous carcinoma (HGSC) is the commonest subtype of human ovarian cancer, and prognosis remains poor. Transplantable murine models of HGSC that recreate key mutations seen in the human disease are greatly needed. These models would assist investigation of the relationships between tumor genotype, chemotherapy response and immune microenvironment. ID8 is the most widely-used mouse model of ovarian cancer, but it has not been characterized in light of current understanding of HGSC biology. METHODS: We performed whole exome sequencing of ID8, covering 194,000 exons at a mean depth of 400x with 90% exons sequenced >50x. Using CRISPR/Cas9 gene editing, we have generated novel ID8 derivatives with single (Trp53-/-) and double (Trp53-/-;Brca2-/-, Trp53-/-;Brca1-/- and Trp53-/-;Pten-/-) deletions. We have characterized intra-peritoneal growth of these novel mutants, and investigated platinum and PARP inhibitor sensitivity and immune cell infiltration into the tumor microenvironment. RESULTS: Whole exome sequencing showed no functional mutations in genes characteristic of HGSC (Trp53, Brca1, Brca2, Nf1, Rb1). In addition, mutations typical of clear cell (Arid1A, Pik3ca), low grade serous (Braf), endometrioid (Ctnnb1) and mucinous (Kras) carcinomas were notably absent. ID8 also demonstrated intact p53 function and homologous recombination in functional assays. We show that loss of p53 alone significantly increases tumor growth rate within the peritoneal cavity of C57Bl/6 mice, and has marked effects upon immune microenvironment. In particular, loss of p53 increases CCL2 expression and induces immunosuppressive myeloid cell infiltration into tumor and ascites. Trp53-/-;Brca2-/- and Trp53-/-;Brca1-/- cells show significantly increased sensitivity to the PARP inhibitor rucaparib in vitro compared to parental and Trp53-/- cells. In vivo, Trp53- /-;Brca2-/- tumors are more sensitive to platinum chemotherapy than Trp53-/-. They also exhibit slower intraperitoneal growth, with appearance of intra-tumoral CD3+ cell-rich tertiary lymphoid structures, a phenotype observed in human tumors with high mutational burden. Mice bearing Trp53-/-;Pten-/- tumors have significantly reduced survival compared to Trp53-/-. Full in vivo assessment of the Trp53-/-;Brca1-/- and new tripleTrp53-/-;Brca2-/- ;Pten-/- lines is on-going. CONCLUSIONS: These models represent a new and simple tool to investigate the biology of HGSC. All cells will be made available to other researchers upon request. Citation Format: Josephine Walton, Julianna Blagih, Malcolm Farquharson, Darren Ennis, Elaine Leung, Suzanne Dowson, Laura A. Tookman, Clare Orange, Dimitris Athineos, Susan Mason, David Stevenson, Karen Blyth, Douglas Strathdee, Frances R. Balkwill, Karen Vousden, Michelle Lockley, Iain A. McNeish. CRISPR/CAS9–MEDIATED TRP53, BRCA1, BRCA2 AND PTEN KNOCKOUT TO GENERATE IMPROVED MURINE MODELS OF OVARIAN HIGH GRADE SEROUS CARCINOMA [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP24.

Abstract 3360: Macrophages promote resistance to pegylated arginine deiminase in malignant pleural mesothelioma

Background Approximately 50% of all malignant pleural mesotheliomas (MPM) are deficient in argininosuccinate synthetase (ASS1), the rate-limiting enzyme in arginine biosynthesis, and are therefore sensitive to arginine deprivation. This discovery in MPM has been translated into the clinic using the arginine depletor pegylated arginine deiminase (ADI-PEG20) which showed a halving in the risk of disease progression in a randomized phase II study (Szlosarek et al, ASCO 2014). However, unstudied to date, stromal resistance to ADI-PEG20 may reduce its efficacy. Here, we studied the effect of macrophages, which are abundant in mesothelioma, on the tumor cytotoxicity of ADI-PEG20. Methods ASS1 negative MPM cell lines treated with ADI-PEG20 were analysed using the Affymetrix Human Genome U133 Plus 2.0 array, with validation of genes involved in stromal-tumor cell communication. Additional studies involved using tumor-macrophage co-culture experiments, mass spectrometry and an MPM xenograft model. Results A distinct pro-inflammatory cytokine gene expression signature involved in macrophage recruitment and activation was identified in the ADI-PEG20-treated ASS1 negative MPM cell lines. Notably, a significant increase in ASS1 negative MPM cell viability was seen upon co-culture with macrophages in the presence of ADI-PEG20. This was accompanied by a significant increase in ASS1 expression in co-cultured macrophages, with a corresponding increase in argininosuccinate lyase (ASL) expression in co-cultured tumor cells and a doubling in levels of the arginine precursor, argininosuccinate, in cell supernatant. The addition of argininosuccinate to tumor cell media rescued ASS1 negative MPM cells from ADI-PEG20 cytotoxicity, while the macrophage-mediated resistance to ADI-PEG20 was abrogated following ASL knockdown in MPM cells. Finally, xenograft studies demonstrated a significant reduction in tumor volume in mice treated with ADI-PEG20 in combination with macrophage depletion, compared with ADI-PEG20 monotherapy. Conclusion Collectively, our data indicate that as a result of metabolic ‘cross-talk’ between macrophages and ASS1 negative MPM cells, macrophages mediate MPM resistance to ADI-PEG20 via the provision of argininosuccinate. Our studies provide a rationale for combining ADI-PEG20 with an inhibitor of macrophage recruitment in the treatment of ASS1-deficient mesothelioma. Citation Format: Melissa M. Phillips, Ramsay Khadeir, Laura Tookman, Fiona McCarthy, Jeremy Steele, John Bomalaski, Essam Ghazaly, Peter W. Szlosarek. Macrophages promote resistance to pegylated arginine deiminase in malignant pleural mesothelioma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3360. doi:10.1158/1538-7445.AM2015-3360

106 Beta-3 integrin inhibition reduces inflammatory cytokine release but not anti-cancer activity of oncolytic adenovirus in ovarian cancer

proteins, and a library of 87,000 diverse lead-like molecules was screened. NFK GreenTM was also used to determine the tryptophan metabolizing activity in a collection of human cancer cell lines and was related to the expression levels of IDO1 and TDO by western blot analysis. Results: Biochemical and cell-based screening assays were developed for IDO1 and TDO using a new fluorescent read-out. High-throughput screening of libraries of small chemical compound libraries yielded novel selective inhibitors of either IDO1 or TDO. Side-by-side comparison of published reference compounds revealed significant, previously unnoted cross-reactivity of a widely used hydroxyamidine-based inhibitor of IDO1 (Compound 5l) with TDO. The selectivity of other reference IDO1 or TDO inhibitors was confirmed, leading to definition of a new tool compound set. Biochemical selectivity of compounds correlated with inhibition of cellular tryptophan metabolizing activity and expression of IDO1 or TDO. Conclusions: We have developed new biochemical and cell-based assays for IDO1 and TDO, to enable the identification of novel small molecule inhibitors and to support lead optimization. Side-by-side comparison of published inhibitors revealed novel, unanticipated cross-reactivity of IDO1 inhibitor scaffolds with TDO.