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Background: Assessing how clinical resistance to PARP inhibitors develops has been challenging, due to the lack of coverage of potential resistance genes in sequencing panels and biopsies being subject to spatial heterogeneity. We studied the development of PARPi and/or platinum resistant disease using both tissue and a novel liquid biopsy assay, in patients treated for BRCA1/2-mutated metastatic breast cancer (BRCA1/2m mBC). Approach: A cohort of 35 mBC patients with germline or somatic BRCA1/2 mutations were identified as having developed PARPi or platinum resistant disease. Tumour biopsies were analysed by exome and transcriptome sequencing whereas ctDNA isolated from plasma sampled across the pre-treatment, response and eventual progression journey were analysed using the Guardant INFINITY platform equipped to detect mutations in >800 genes, and genome wide methylation, including promoter methylation in 398 cancer-related genes. Somatic mutations or regions of methylation that were associated with resistance were identified, including BRCA1/2 reversion mutations. Somatic mutations with the potential to cause PARPi resistance were annotated using CRISPR screen data and other functional analyses describing genes that alter PARPi synthetic lethality. Results: The most common resistance mechanism was BRCA1/2 reversion mutation (51%; n=8 BRCA1m patients, n=10 BRCA2m patients). Most reversions (77%) occurred via deletions (BRCA1, 70%; BRCA2, 79%) and exhibited microhomology use at junctions (> 80% for both BRCA1 and BRCA2). In 14 patients, multiple concurrent reversion mutations were detected with VAFs ranging from 0.1-40% for BRCA1 and 0.05-18% for BRCA2. Changes in VAF over time indicated that different reversions in the same patient may impart different fitness advantages in the face of treatment. Loss-of-function mutations in the 53BP1-Shieldin pathway were identified in two BRCA1m patients, and VUS mutations in the pathway were seen in two further patients. In some patients, reversion mutations co-occurred with either 53BP1-Shieldin pathway mutations or with replication fork stability mutations (PAXIP1 in BRCA2m patients) - indicating concurrent but mechanistically distinct forms of resistance develop in the same patient. We isolated a PDX model from one patient that exhibits resistance via both BRCA1 reversion and loss of 53BP1, allowing this phenomenon to be modelled in more detail. We also detected BRCA1 methylation in ctDNA in a patient with a somatic rearrangement in BRCA1. Conclusions: Liquid biopsy profiling of PARPi-resistance breast cancer patients indicates that many patients develop multiple reversion mutations, and that alterations in the 53BP1-Shieldin pathway are present but less frequent. The co-occurrence of 53BP1-Shieldin pathway mutations and reversion mutations suggests parallel mechanisms of resistance can operate in the same patient. Citation Format: Elizabeth Harvey-Jones, Maya Raghunandan, Luisa Robbez-Masson, Alaguthurai Thanussuyah, Roberta Liccardo, Arielle Yablonovitch, Mingyang Cai, Leylah Drusbosky, Michael Dorschner, Lorena Magraner Pardo, Rebecca Marlow, Asha Konde, Jennifer Trendell, John Alexander, Syed Haider, Chris Starling, Ioannis Roxanis, Jennifer Yen, Stephen J. Pettitt, Christopher J. Lord, Andrew N. Tutt. Longitudinal analysis of PARP inhibitor and platinum resistance in BRCA1/2m breast cancer using liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6094.

Gastric cancer represents the third leading cause of global cancer mortality and an area of unmet clinical need. Drugs that target the DNA damage response, including ATR inhibitors (ATRi), have been proposed as novel targeted agents in gastric cancer. Here, we sought to evaluate the efficacy of ATRi in preclinical models of gastric cancer and to understand how ATRi resistance might emerge as a means to identify predictors of ATRi response. A positive selection genome-wide CRISPR-Cas9 screen identified candidate regulators of ATRi resistance in gastric cancer. Loss-of-function mutations in either SMG8 or SMG9 caused ATRi resistance by an SMG1-mediated mechanism. Although ATRi still impaired ATR/CHK1 signaling in SMG8/9-defective cells, other characteristic responses to ATRi exposure were not seen, such as changes in ATM/CHK2, γH2AX, phospho-RPA, or 53BP1 status or changes in the proportions of cells in S- or G2–M-phases of the cell cycle. Transcription/replication conflicts (TRC) elicited by ATRi exposure are a likely cause of ATRi sensitivity, and SMG8/9-defective cells exhibited a reduced level of ATRi-induced TRCs, which could contribute to ATRi resistance. These observations suggest ATRi elicits antitumor efficacy in gastric cancer but that drug resistance could emerge via alterations in the SMG8/9/1 pathway. Significance: These findings reveal how cancer cells acquire resistance to ATRi and identify pathways that could be targeted to enhance the overall effectiveness of these inhibitors.

Inactivation of Polybromo 1 (PBRM1), a specific subunit of the PBAF chromatin remodeling complex, occurs frequently in cancer, including 40% of clear cell renal cell carcinomas (ccRCC). To identify novel therapeutic approaches to targeting PBRM1-defective cancers, we used a series of orthogonal functional genomic screens that identified PARP and ATR inhibitors as being synthetic lethal with PBRM1 deficiency. The PBRM1/PARP inhibitor synthetic lethality was recapitulated using several clinical PARP inhibitors in a series of in vitro model systems and in vivo in a xenograft model of ccRCC. In the absence of exogenous DNA damage, PBRM1-defective cells exhibited elevated levels of replication stress, micronuclei, and R-loops. PARP inhibitor exposure exacerbated these phenotypes. Quantitative mass spectrometry revealed that multiple R-loop processing factors were downregulated in PBRM1-defective tumor cells. Exogenous expression of the R-loop resolution enzyme RNase H1 reversed the sensitivity of PBRM1-deficient cells to PARP inhibitors, suggesting that excessive levels of R-loops could be a cause of this synthetic lethality. PARP and ATR inhibitors also induced cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) innate immune signaling in PBRM1-defective tumor cells. Overall, these findings provide the preclinical basis for using PARP inhibitors in PBRM1-defective cancers. Significance: This study demonstrates that PARP and ATR inhibitors are synthetic lethal with the loss of PBRM1, a PBAF-specific subunit, thus providing the rationale for assessing these inhibitors in patients with PBRM1-defective cancer.

To target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining (TMEJ), without targeting Non-Homologous End Joining. Moreover, we show that exposure to ART558 can elicit DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumor cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which are a cause of PARP inhibitor resistance, result in in vitro and in vivo sensitivity to Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells. The inhibition of DNA nucleases that promote end-resection, such as Exo1 or Blm-Dna2 reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance. Citation Format: Diana Zatreanu, Helen Robinson, Omar Alkhatib, Marie Boursier, Harry Finch, Lerin Geo, Diego Grande, Vera Grinkevich, Robert Heald, Sophie Langdon, Jayesh Majithiya, Claire McWhirter, Niall Martin, Shaun Moore, Joana Neves, Eeson Rajendra, Marco Ranzani, Theresia Schaedler, Martin Stockley, Kimberley Wiggins, Rachel Brough, Sandhya Sridhar, Aditi Gulati, Nan Shao, Luted Badder, Daniela Novo, Eleanor Knight, Rebecca Marlow, Syed Haider, Elsa Callen, Graeme Hewitt, Joost Schimmel, Remko Prevo, Christina Alli, Amanda Ferdinand, Cameron Bell, Peter Blencowe, Chris Bot, Mathew Calder, Mark Charles, Jayne Curry, Tennyson Ekwuru, Andre Nussenzweig, Marcel Tijsterman, Andrew N. Tutt, Simon Boulton, Geoff Higgins, Stephen J. Pettitt, Graeme C. Smith, Christopher J. Lord. Targeting PARP inhibitor resistance with Polθ inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5697.

Reovirus (RT3D) is a naturally occurring double-stranded RNA oncolytic virus that has shown preclinical efficacy in a wide range of tumor types. Early phase clinical studies have shown that this agent has modest monotherapy efficacy and can safely be combined with cytotoxic chemotherapy regimens. In the current studies, we used a high-throughput drug screen approach of different targeted therapeutic agents with the aim of looking for potential viral sensitizers that could enhance RT3D tumor killing. BMN-673 (talazoparib), a clinically approved poly(ADP)-ribose polymerase 1 (PARP-1) inhibitor was identified as a top hit and found to sensitize profoundly to RT3D both in vitro and in vivo in human xenograft tumors in a nude mouse model. We found that RT3D activated cellular PARP1 and was associated with PARylation of cellular proteins, including components of the DISC-associated cell death machinery. Combined treatment with RT3D and talazoparib enhanced extrinsic apoptosis (amplified by autocrine/paracrine TNF-α and TRAIL signaling), NF-κB pathway activity and pro-inflammatory cytokine production (CCL5/RANTES, CXCL8/IL8, CXCL1/GRO and CXCL10/IP10). Signaling was shown to be dependent on nucleic acid sensing mechanisms mediated by RIG-I and TLR3. We also found anti-tumour efficacy in an immunocompetent mouse model and this correlated with an increase in an immune response following combination treatment of RT3D and talazoparib. Our data provide a strong rationale for the combination of oncolytic RT3D with PARP1 inhibitors to exploit immunogenic response in cancer treatment. Citation Format: Joan N. Kyula, Victoria Roulstone, Richard Elliott, Harriet Whittock, Galabina Bozhanova, Martin McLaughlin, Malin Pedersen, Dragomir Krastev, Stephen Pettitt, Arnaud Legrand, Tencho Tenev, James Wright, Lu Yu, Jyoti Choudhary, Pascal Meier, Christopher J. Lord, Alan Melcher, Grey Wilkinson, Matt Coffey, Kevin J. Harrington. Talazoparib interacts with oncolytic reovirus to enhance death-inducing signaling complex (DISC)-mediated apoptosis and immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1932.

Invasive lobular breast carcinoma (ILC) is a low grade and mostly chemo-refractory luminal-type breast cancer that has been linked to sustained proliferative quiescence and long-term latency relapses (15-20 years). Loss of E-cadherin causes metastatic lobular breast cancer, partly through acquisition of anchorage independence. It is however still unknown how ILC cells control the balance between proliferative indolence and cell cycle re-entry at the metastatic site. We show here that E-cadherin loss leads to upregulation of Id2 through p120-catenin/Kaiso-dependent transcriptional derepression. Anchorage independent conditions further exacerbate p120-driven Id2 expression, leading to a sustained G0/G1 cell cycle arrest through binding of cytosolic Id2 to hypo-phosphorylated Rb. Intriguingly, we find that E-cadherin inactivation causes increased sensitivity to CDK4/6 inhibition in mouse and human breast cancer cell lines and primary tumor organoids. Finally, we find that Id2 expression is elevated in human ILC when compared to ductal breast cancers. Based on these data, we propose that combined E-cadherin loss and cytosolic Id2 expression can be used for the differential diagnosis of ILC and represent a candidate predictive biomarker pair for cell cycle targeting drug efficacy. Citation Format: Max Antonius Klaus Rätze, Thijs Koorman, Thijmen Sijnesael, Blessing Bassey-Archibong, Robert van de Ven, Lotte Enserink, Daan Visser, Sridevi Jaksani, Elvira Bakker, François Richard, Andrew Tutt, Rebecca Steele, Stephen Pettitt, Christopher J. Lord, Amanda Fitzpatrick, Clare Isacke, Paul J. van Diest, Christine Desmedt, Juliet M. Daniel, Patrick W.B. Derksen. E-cadherin loss drives Id2-dependent dampening of cell cycle progression and predicts increased susceptibility to CDK4/6 inhibition in lobular breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB246.

Background: Inactivating mutations in homologous recombination repair (HRR) genes are associated with a response to PARP inhibitors and platinum-based therapies. However, secondary reversion mutations in HRR genes remain a major mechanism of resistance. We sought to characterize the prevalence and spectrum of these reversions in advanced cancer patients in BRCA1/2 and other HRR genes, such as ATM and CDK12. Methods: We analyzed cfDNA from >75,000 late-stage cancer patients previously tested as part of routine clinical care using Guardant360®, a 73- to 74-gene panel. This assay detects single-nucleotide variants (SNVs), indels, fusions, and copy-number alterations in cfDNA with a reportable range of ≥0.04%, 0.02%, 0.04%, and ≥2.12 copies, respectively. Secondary reversion mutations were identified by re-analysis using a bespoke HRR deficiency (HRD) Module, which annotates SNV/indels and calls multi-exon deletions and copy-number loss with a 95% limit of detection of ≥ 0.1% and 10 to 25% tumor fraction, respectively. Results: Among >20,000 breast, ovarian, prostate and pancreatic cancer patients, deleterious germline or somatic mutations were detected in BRCA1 (2.7%), BRCA2 (4.8%), ATM (2.9%) and CDK12 (1.7%). The majority of reversions occurred in BRCA1/2-mut (8.7%), compared to 0.07% of CDK12-mut and none in ATM. Reversions showed selective gene and allele representation. In breast cancer, reversions in BRCA2-mut patients were twice as frequent as those in BRCA1-mut (12.9% vs 5.5%, p-value=0.0046), while the near opposite was true for ovarian (7.6% vs 12.8%, p-value=0.73). In pancreatic cancer (>7,500 patients), reversions presented exclusively in BRCA2-mut (8.4%, n=131 vs 0 in BRCA1-mut, n=63). CDK12 reversions were relatively rare: 1 detected in >5,000 prostate cancer patients. Across all genes, the majority of reversions were small indels while 18% of reversions removed the exon entirely. Pathogenic hotspots BRCA2 codons A938 and S1982 - accounted for ≥10% of reversions and were more likely to revert compared to other deleterious sites (p-value Conclusions: cfDNA has been previously shown to detect BRCA1/2 germline, somatic and polyclonal reversion mutations acquired over PARP and platinum-based therapy. We demonstrate that the Guardant HRD Module can robustly identify these reversions in the cfDNA of metastatic patients. We emphasize that reversions were frequent among BRCA1/2-mut breast, ovarian, prostate and pancreatic cancers, with preferential enrichment based on gene and tumor type. Understanding resistance patterns may aid in the treatment selection for progressing patients. Further work is needed to define these ideal treatment options. Citation Format: Yu Fu, Jennifer Yen, Alexander Aartyomenko, Sante Gnerre, Caroline Weiport, Elena Helman, Arielle Yablonovitch, Stephen Pettitt, Christopher Llord, Andrew Tutt, Rebecca Nagy, Justin Odegaard, Darya Chudova, AmirAli Talasaz. Reversion patterns in homologous recombination repair genes in metastatic cancer patients profiled by cell-free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 25.

Although PARP inhibitors and platinum salts form part of the standard-of-care for cancers with homologous recombination (HR) defects, drug resistance does emerge and is often caused by reversion mutations in BRCA1 or BRCA2. Reversion mutations are secondary mutations that restore functional protein or compensate for frameshift mutations. To better understand the nature and aetiology of these mutations, we collated, codified and analysed over 200 reversion mutations from 24 published studies. Most reversions have been reported as case reports or small clinical series - we have collected all published reports into a single freely-accessible database to which further cases can be submitted. The majority of reported reversion mutations are from BRCA1/2 mutant serous ovarian cancer, reflecting the longer use of platinum and PARP inhibitors in this disease. Our analysis identified reversion “hotspots” and “deserts” in the N- and C-terminal coding regions (respectively) of BRCA2, suggesting that pathogenic mutations in these domains may be at higher or lower chance of reversion, an effect not seen for BRCA1. Missense and splice-site pathogenic mutations in BRCA1/2 also appeared less likely to revert than frameshift-causing mutations. Unexpectedly, whilst most reversions were Many reversions contain novel protein sequence not found in the wild type protein, for example where a stretch of out-of-frame protein sequence is retained between the pathogenic and reversion mutations. We modelled MHC binding affinities for these sequences and found that many reversions were predicted to encode potentially immunogenic neopeptides, suggesting a route to the treatment of reverted disease. These observations have implications for how drug resistance might be managed in BRCA-mutant cancers. Citation Format: Stephen Pettitt, Jessica Frankum, Marco Punta, Stefano Lise, John Alexander, Yi Chen, Syed Haider, Andrew N. Tutt, Christopher J. Lord. Analysis of clinical BRCA1/2 reversions identifies hotspot mutations and predicted neoantigens associated with therapy resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4072.

Synovial sarcoma (SS) is an aggressive soft-tissue malignancy characterised by expression of SS18-SSX fusions, where treatment options are limited. To identify therapeutically actionable genetic dependencies in SS, we performed a series of parallel, high-throughput small interfering RNA (siRNA) screens and compared genetic dependencies in SS tumour cells to those in >130 non-SS tumour cell lines. This identified an unexpected reliance in SS tumour cells upon the DNA damage response kinase, ATR. Clinical ATR inhibitors (ATRi) also elicited a synthetic lethal effect in SS tumour cells and impaired the growth of SS patient-derived xenografts, suggesting this effect might have therapeutic potential. Oncogenic SS18-SSX family fusion genes alter the composition of the BAF chromatin-remodelling complex, causing ejection of wild-type SS18 and the tumour suppressor SMARCB1 from BAF and their eventual degradation. We found that expression of oncogenic SS18-SSX fusion proteins caused profound ATRi sensitivity and a reduction in SS18 and SMARCB1 protein levels but a SSX18-SSX1 Δ71-78 fusion with a C-terminal deletion did not, thus establishing a causative link between oncogenic SS18-SSX fusion genes and ATRi sensitivity. ATRi sensitivity in SS was characterised by an increase in biomarkers of replication fork stress (increased γH2AX, decreased replication fork speed and increased R-loops), an apoptotic response and was found to be dependent upon Cyclin E expression. Finally, we found that combinations with cisplatin or PARP inhibitors enhanced the anti-tumour cell effect of ATRi, suggesting that either single agent ATRi or combination therapy involving ATRi might be further assessed as candidate approaches for SS treatment.

Triple-Negative Breast Cancers (TNBC) are characterized by high levels of structural chromosome alterations and harbor mutational scars of defects in homologous recombination. Importantly, by acting either as drivers, or as tumor specific dependencies mutated genes in these regions represent potential therapeutic targets In search of such genes we identified that knockdown of the PUM3 gene, which is contained in a 9p24 recurrent ampliicon, impaired the growth of a subset of breast cancer but not mammary epithelial cell lines. Furthermore, the growth defects observed upon PUM3 knockdown in SUM149, a BRCA1 deficient cell line, were reversed by reactivation of BRCA1's function suggesting a BRCA1-deficiency associated dependency. PUM3 knockdown led to an increase in gH2Ax and 53BP1 nuclear foci, impaired restart of stalled replication forks, and decreased RAD51 mediated repair of collapsed replication forks. As a result PUM3 depleted cells have reduced DNA replication rate and slowed cell cycle progression. Knockdown of PUM3 also impaired replication stress induced PARylation and PAR foci formation and PUM3 and PARP1 knockdown showed phenotypic epistasis with respect to cell growth suggesting a potential mechanism for PUM3s replication fork related functions. PUM3 forms a complex with gH2Ax and the replication stalling agent hyroxyurea and PARP-inhibitors increases the nuclear concentration of PUM3. Furthermore PUM3 knockdown increased the sensitivity of breast cancer cells to these agents indicating a role for PUM3 in the repair of lesions generating by these DNA damaging agents. By studying PUM3 we have identified a novel player in replication fork restart and repair whose activity may be particularly relevant in homologous recombination deficient TNBCs. A greater understanding of PUM3's function and the pathways that regulate it may identify novel therapeutic targets for TNBCs. Importantly, direct or indirect modulation of PUM's functions may potentiate the effects of replication stress inducing therapies such as PARP-inhibitors. Citation Format: Daniel Weekes, Elodie Noel, Callum Walker, Nick Balan, Vandna Shah, Bhavna Sidhu, Anna Pardix, Stephen Pettitt, Christopher J. Lord, Anita Grigoriadis, Tutt N. Andrew. PUM3 is a triple-negative breast cancer dependency gene that functions in replication fork restart and repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3363.

The E-cadherin (CDH1) tumour suppressor gene encodes a calcium-dependent cell-cell adhesion glycoprotein, which has roles in maintaining cell polarity, differentiation, cell migration and survival. E-cadherin dysfunction is a feature common to many epithelial tumours, with the highest incidence occurring in diffuse gastric cancer (50%) and lobular breast cancer (63%) and can occur via CDH1 mutation, deletion or epigenetic silencing. Although E-cadherin dysfunction is relatively common, precision medicine approaches that exploit this pathogenic alteration are not yet available. Using genetic and small-molecule perturbation screens in breast tumour cells with CRISPR-Cas9 engineered CDH1 mutations, we identified a synthetic lethal interaction between E-cadherin deficiency and inhibition of the tyrosine kinase ROS1. Using data from large-scale genetic screens in molecularly diverse breast tumour cell lines, we found that the E-cadherin/ROS1 synthetic lethality was not only robust in the face of considerable molecular heterogeneity but was also elicited with clinical ROS1 inhibitors including foretinib and crizotinib. ROS1 inhibitors induced mitotic abnormalities and multinucleation in E-cadherin defective cells, phenotypes that were associated with a defect in cytokinesis and aberrant p120-catenin phosphorylation and localisation. In vivo, ROS1 inhibitors produced profound anti-tumour effects in multiple, distinct, models of E-cadherin defective breast cancer. This data therefore provides the pre-clinical rationale for assessing ROS1 inhibitors such as the licensed drug crizotinib in appropriately stratified patients. Citation Format: Ilirjana Bajrami, Rebecca Marlow, Marieke van de Ven, Rachel Brough, Helen N. Pemberton, Jessica Frankum, Fei Fei Song, Rumana Rafiq, Asha Konde, Malini Menon, James Campbell, Aditi Gulati, Rahul Kumar, Stephen J. Pettitt, Mark D. Gurden, Marta Llorca Cardenosa, Irene Chong, Patrycja Gazinska, Fredrik Wallberg, Elinor J. Sawyer, Lesley-Ann Martin, Mitch Dowsett, Spiros Linardopoulos, Colm Ryan, Patrick W. Derksen, Jos Jonkers, Andrew N.J. Tutt, Alan Ashworth, Christopher J. Lord. E-cadherin/ROS1 inhibitor synthetic lethality in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2986.

Poly(ADP-ribosylation) (PARylation) is a reversible post-translational protein modification that regulates a number of cellular processes. It is generated by a family of PAR polymerases (PARP), of which PARP1 has recently been exploited as a target in ovarian cancer carrying BRCA1/2 mutations. In addition, tankyrase (PARP5A and B), with its regulation of Wnt signalling, telomeres and the mitotic apparatus, has attracted much effort in the development of targeted therapies. All this necessitates the identification of PARylation targets and their mode of regulation by the modification. Genetically encoded fluorescent biosensor that can report the PARylation state of a target protein has the potential to report the modulation of the PARylation target in living cells. We used PAR-binding PBZ domains fused to a non-fluorescent fragment of a GFP molecule in combination with a protein of interest fused to the complementing fragment of GFP. When this protein is PARylated, it is recognized by the PBZ domain, which brings in proximity the two GFP fragments, and restores the GFP fluorescence. Known targets of PARylation, e.g. TERF1 and AXIN1, showed specific spatial patterns of PARylation validating our approach. In order to identify novel PARylation targets, we have designed a genetic screen based on the biosensor. We introduced the GFP fragment in a transposon, which was randomly integrated in cellular genome, thus generating cellular libraries of thousands of tagged genes. The introduction of the PAR binding probe lead to GFP fluorescence in clones that harbour putative PARylation targets. We developed a next generation sequencing approach to isolate the transposon integration sites and annotated the target genes. Reassuringly, we have identified NPM1, a well-known PARylation target, as a strongly scoring gene. We focused on one of novel identified hits (CTIF). We showed that CTIF is PARylated in vivo in a tankyrase-dependent manner. Furthermore, the biosensor revealed that this PARylation is predominant in a specific cellular compartment surrounding the centrosome. Functional studies have implicated this protein the regulation of the centrosomal satellites. Collectively, we are presenting a new approach to identify PARylated proteins and study their patterns of PARylation and regulation in vivo. Citation Format: Dragomir B. Krastev, Stephen J. Pettitt, James Campbell, Alan Ashworth, Christopher J. Lord. A PARylation biosensor genetic screen to identify novel PARylation targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1360.

Paediatric glioblastomas (pGBM) are amongst the most common causes of cancer-related deaths in children, and are defined by highly recurrent mutations in H3 histones. Mutations affecting the chromatin remodeling protein ATRX have been reported in 30% of pGBM cases, and are strongly associated with the alternative lengthening of telomeres (ALT) phenotype, but their precise interaction with histone mutations and their role in tumorigenesis remain unclear. We collected sequence data from 262 published and 64 unpublished cases of pGBM and identified somatic ATRX mutations in 54/326 (17%) of cases. ATRX mutations are mainly loss of function mutations, with the majority of frameshift mutations (37/54, 68,5%) found upstream of the helicase domain resulting in truncation of the main functional domain of ATRX. Missense mutations (16/54, 29,6%) reside almost exclusively in the helicase domain (11/54, 20,4%), whereas nonsense mutations are a less common event (7/54, 13%) but present in both the helicase (4/7, 57,1%) and ADD domains (3/7, 42,9%). ATRX mutations commonly co-segregate with H3.3 G34 (16/54) and TP53 (42/54) mutations, and define a subgroup of patients with a longer overall survival (16 months median overall survival in mutant ATRX cases versus 11 months in wild-type ATRX cases, COXPH p = 0.079), though with a greater number of somatic mutations (MWU p = 0.023) and copy number alterations (MWU p = 0.0011) than wild-type cases. We screened a series of 21 primary patient-derived pGBM cell cultures for histone and ATRX mutation status in addition to ATRX protein expression and ALT, and subjected the panel to a high-throughput in vitro cell viability screen of >400 chemotherapeutics and small molecules. We identified a specific genetic dependency for ATRX mutation and sensitivity to distinct PARP inhibitor chemotypes, including olaparib and rucaparib (PARP catalytic inhibitors), and talazoparib (PARP trapper inhibitor). These data were validated using CRISPR-Cas9-engineered ATRX knockout, targeting either the ADD or helicase domain, in SF188 pGBM cells. Gene editing was confirmed by IonTorrent sequencing and Western blot. ATRX mutant clones were also more sensitive to drugs targeting DNA damage response pathways such as bleomycin and sapacitabine. Gene expression analysis of ATRX mutant pGBM samples confirmed an intact homologous recombination pathway and overexpression of PARP1, suggesting an underlying mechanism distinct from that observed in BRCA-mutant breast and ovarian cancers. Ongoing work is aimed at unravelling the specific pathways involved, and evaluating the utility of PARP inhibition in orthotopic pGBM xenografts in vivo. These data suggest a synthetic lethality for PARP inhibitors in ATRX-deficient pGBM cells, and may represent a novel therapeutic strategy for these highly aggressive tumours in children. Citation Format: Janat Fazal-Salom, Mara Vinci, Diana Carvalho, Helen Pemberton, Stephen J. Pettitt, Christopher J. Lord, Alan Mackay, Lynn Bjerke, Chris Jones. Mutations in ATRX increase genetic instability and sensitivity to PARP inhibitors in paediatric glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1932. doi:10.1158/1538-7445.AM2017-1932

We are using genomic approaches to study the mechanism of action of targeted cancer drugs, particularly poly(ADP-ribose) polymerase (PARP) inhibitors. One of these drugs, olaparib was recently approved for use in ovarian cancers with germline BRCA1/2 mutations and many other clinical trials with this and other PARP inhibitors are underway. Experimental study of potential resistance mechanisms can inform the ongoing clinical development of these drugs. We previously carried out a loss of function mutagenesis screen in haploid cells, which showed that PARP1 was required for cellular toxicity of olaparib and other PARP inhibitors. This supported the hypothesis that PARP inhibitor toxicity occurs via a poisoning mechanism where inhibited PARP is tightly bound to DNA, forming a toxic lesion. Recently we carried out a similar screen using genome-wide lentiviral CRISPR-Cas9 mutagenesis in diploid cells. Many PARP inhibitor resistant clones had loss of function Parp1 mutations as expected. However, we also isolated a point mutation affecting a single amino acid in the Parp1 DNA binding domain. This mutant encodes a stable Parp1 protein that cannot bind DNA and does not become trapped in the presence of inhibitors. Thus the CRISPR screen implicated PARP1 DNA binding directly in determination of PARP inhibitor toxicity and was extremely informative about the mechanism of action compared to conventional loss-of-function mutagenesis. We extended this approach by synthesising a high-density focused sgRNA library targeting only PARP1. We developed a reporter cell line that allows us to selectively isolate in-frame mutations that preserve PARP1 protein expression. By deep sequencing mutagenised and appropriately selected cells we identified a series of subtle mutations in PARP1 that result in PARP inhibitor resistance, giving us a detailed insight into structure-function relationships in PARP1. The approach is applicable to other drug targets, providing a framework for understanding drug action and predicting clinical resistance. Citation Format: Stephen Pettitt, Dragomir B. Krastev, Feifei Song, Alan Ashworth, Christopher J. Lord. Finding determinants of PARP inhibitor sensitivity using genome-wide and focused CRISPR screens. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2743.