Synthetic lethal targeting of PTEN mutant cells with PARP inhibitors

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1 Report Targeting PTEN deficiency Synthetic lethal targeting of PTEN mutant cells with PARP inhibitors Ana M. Mendes-Pereira 1, Sarah A. Martin 1,2, Rachel Brough 1,2, Afshan McCarthy 1, Jessica R. Taylor 1, Jung-Sik Kim 3, Todd Waldman 3, Christopher J. Lord 1 *, Alan Ashworth 1,2 * Keywords: PTEN; PARP inhibitor; homologous recombination DOI /emmm Received April 6, 2009 Revised July 24, 2009 Accepted July 31, 2009 The tumour suppressor gene, phosphatase and tensin homolog (PTEN), is one of the most commonly mutated genes in human cancers. Recent evidence suggests that PTEN is important for the maintenance of genome stability. Here, we show that PTEN deficiency causes a homologous recombination (HR) defect in human tumour cells. The HR deficiency caused by PTEN deficiency, sensitizes tumour cells to potent inhibitors of the DNA repair enzyme poly(adp-ribose) polymerase (PARP), both in vitro and in vivo. PARP inhibitors are now showing considerable promise in the clinic, specifically in patients with mutations in either of the breast cancer susceptibility genes BRCA1 or BRCA2. The data we present here now suggests that the clinical assessment of PARP inhibitors should be extended beyond those with BRCA mutations to a larger group of patients with PTEN mutant tumours. INTRODUCTION Synthetic lethal approaches to cancer treatment have the potential to deliver relatively large therapeutic windows and significant patient benefit (Kaelin, 2005). We, and others, have previously shown that mutations in either of the tumour suppressor genes, breast cancer 1 (BRCA1) or BRCA2, are synthetically lethal with inhibition of the DNA repair enzyme poly(adp-ribose) polymerase 1 (PARP1) (Bryant et al, 2005; Farmer et al, 2005). The exquisite sensitivity of BRCA1 or BRCA2 mutant cells to PARP inhibitors (PARPi) forms the rationale behind clinical trials that are now assessing the potential of these agents (Ashworth, 2008). The preliminary results from these clinical trials are promising, with favourable toxicity and sustained tumour responses to the drug (Fong et al, 2009). Mutations in the phosphatase and tensin homolog (PTEN) gene (OMIM ) and loss of PTEN expression have both been associated with a wide range of human tumours (Salmena et al, 2008). PTEN encodes a phosphatase whose role in the (1) The Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London, UK. (2) CRUK Gene Function Laboratory, The Institute of Cancer Research, London, UK. (3) Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University School of Medicine, Washington, District of Columbia, USA. *Corresponding author: Tel: þ ; Fax: þ ; [email protected]; [email protected] control of the phosphoinositide 3 kinase (PI3K) signalling pathway is well established (Salmena et al, 2008). A new functional role for PTEN was recently suggested by the observation that mouse embryonic Pten / cells exhibit genomic instability, a phenotype ascribed to either defects in RAD51-mediated DNA double strand break repair (DSBR) (Shen et al, 2007) or defects in cell cycle checkpoints (Gupta et al, 2009). Given that the sensitivity of BRCA mutant cells to PARPi is most likely explained by a defect in RAD51-mediated DSBR by homologous recombination (HR) (Farmer et al, 2005; McCabe et al, 2006), we investigated the possibility that human PTEN mutant cells also display an HR defect and as a consequence, PARPi sensitivity. As PTEN loss of function mutations and loss of PTEN expression are common in a range of hereditary and sporadic cancers (Salmena et al, 2008), we reasoned that such data might significantly extend the utility of this class of drugs. RESULTS PTEN involvement in HR repair To model the effect of PTEN null mutations in human tumour cells, we used isogenically matched wild type and PTEN / HCT116 colorectal tumour cell lines (Lee et al, 2007) as well as isogenic wild type and PTEN / HEC1A endometroid adenocarcinoma cells (Waldman, unpublished work). PTEN deficiency in both HCT116 and HEC1A lines was achieved by targeting a truncating mutation to both copies of PTEN at exon 2, EMBO Mol Med 1, ß 2009 EMBO Molecular Medicine 315

2 Report Targeting PTEN deficiency resulting in an open reading frame encoding only the N-terminal 24 amino acids of the PTEN protein (Lee et al, 2007). First, we confirmed that PTEN / human tumour cells express reduced levels of RAD51 (Fig 1A and Fig S1A of Supporting Information), as previously documented in mouse Pten null cells (Shen et al, 2007). Extending this observation, we demonstrated that PTEN mutant tumour cells also had a reduced capacity to form nuclear RAD51 foci in response to DNA damage, a surrogate marker of HR activity (West, 2003) (Fig 1B, Figs S1B and S2 of Supporting Information). To investigate whether these deficiencies in RAD51 expression and recruitment translated into impaired DSBR by HR, we measured HR using a reporter assay. This comprised a previously validated synthetic DNA substrate that bears an inducible double strand DNA break (DSB; Saeki et al, 2006). PTEN deficient human tumour cells exhibited a 5-fold reduction in DSBR by HR when compared to isogenic wild type cells (Fig 1C and Fig S1C of Supporting Information). PTEN deficiency sensitizes to PARP inhibitors Given the HR repair defect of PTEN deficient cells, we tested the sensitivity of these cells to two DNA-DSB inducers, PARPi and cisplatin. HCT116 PTEN / cells were 20 times more sensitive to the PARP inhibitor KU (Farmer et al, 2005) than their wild type counterparts (compare concentration at which 50% of cells survive (SF 50 ) for HCT116 PTEN þ/þ of MwithSF 50 for HCT116 PTEN / 22 of M, Fig 2A) and up to 25 times more sensitive to the PARP inhibitor KU /AZD2281/ Olaparib (Evers et al, 2008; Fong et al, 2008) (compare SF 50 HCT116 PTEN þ/þ of M versus SF 50 for HCT116 PTEN / 22 of M, Fig 2B). We also observed PTEN selectivity in the HEC1A model (Fig S1D of Supporting Information). These PTEN/PARP synthetic lethal associations were achievable using sub-micromolar concentrations of PARPi, similar to those required to kill BRCA1 deficient cells (Farmer et al, 2005). We also assessed the sensitivity of PTEN / cells to the platinum salt, cisplatin. Cisplatin, although less selective than PARPi, also targets cells with HR deficiency (Tutt et al, 2005). PTEN / cells were five fold more sensitive to cisplatin than their wild type counterparts (compare SF 50 HCT116 PTEN þ/þ of M versus SF 50 for HCT116 PTEN / 22 of M, Fig 2C), consistent with the hypothesis that PTEN deficiency leads to an HR defect that may be targeted. To eliminate the possibility that these observations were reflective of a general sensitivity to chemotherapeutics caused by PTEN deficiency, we Figure 1. PTEN deficiency causes an impairment of DNA repair by HR. A. PTEN deficiency causes a reduction in RAD51 expression. Total cell lysates from isogenic HCT116 colorectal tumour cells were immunoblotted for PTEN and RAD51. Detection of b Tubulin is shown as a loading control. Lysates from parental HCT116 cells were used (HCT116) along with two individually derived PTEN / lines (KO35 and KO22) and also a HCT116-derived line bearing a random integration of the PTEN targeting construct (neo124). Lysates from PTEN þ/ HCT116 cells are also shown (Lee et al, 2007). B. PTEN deficiency causes a reduction in radiation-induced nuclear RAD51 focus formation. Cells were exposed to 10 Gy g-irradiation and nuclear RAD51 foci quantified 8 h later by confocal microscopy (Farmer et al, 2005). Bar chart shows the average number of cells with >5 foci per nucleus. Error bars represent three standard deviations of the mean. p values versus þir HCT116 PTEN þ/þ <0.05 (Student s t-test). All other comparisons returned non-significant (>0.05) p values. C. PTEN deficiency causes a reduction in HR as measured using a synthetic HR substrate. As a measure of HR activity, a reporter plasmid-based assay was used comprising two defective copies of GFP, where one serves as a template to restore an induced DSB in the other. HR between the two GFP coding sequences results in an intact GFP coding sequence and cellular fluorescence (Saeki et al, 2006). Error bars represent three standard deviations of the mean. p values versus HCT116 PTEN þ/þ <0.05 (Student s t-test). 316 ß 2009 EMBO Molecular Medicine EMBO Mol Med 1,

3 Report Ana M. Mendes-Pereira et al. Figure 2. PTEN deficiency sensitizes tumour cells to agents that target HR. A, B. PTEN deficiency sensitizes cells to drug-like PARP inhibitors. Survival curves for HCT116 cells exposed to the PARP inhibitors KU (Farmer et al, 2005) or KU (Evers et al, 2008; Fong et al, 2008). Cells were plated in six-well plates and treated for 15 days, after which SFs were estimated. For both PARP inhibitors, PTEN / SFs were significantly different to both PTEN þ/þ and PTEN þ/ cells (p<0.05 two-way analysis of variance (ANOVA)). No other comparisons returned significant p values. C. PTEN deficiency sensitizes cells to cisplatin (p<0.05 PTEN / lines versus PTEN þ/þ cells, two-way ANOVA). D. PTEN deficiency does not confer sensitization to taxol. assessed sensitivity to the microtubule poison, paclitaxel (taxol). Paclitaxel was not selectively lethal to PTEN deficient cells (Fig 2D), suggesting that the PARPi and cisplatin sensitivities we observed were more likely reflective of HR deficiency rather than of general chemosensitivity. Although PTEN haploinsufficiency effects on tumourigenesis have been reported, complete loss of PTEN is observed in many advanced cancers (Salmena et al, 2008). Given this, we thought it pertinent to assess the effects of PTEN gene dosage upon HR deficiency and PARPi sensitivity. Using the HCT116 isogenic system, we demonstrated that PTEN heterozygosity did not cause the same reduction in RAD51 expression as seen in PTEN / cells (Fig 1B). Consistent with this observation, the induction of damage induced nuclear RAD51 foci was not abrogated in PTEN þ/ cells (Fig 1B) and although a marginal decrease in HR was observed using the synthetic HR assay in heterozygous cells (Fig 1C), this decrease did not translate into PARPi sensitivity (Fig 2B). To assess the generality of our observations, we examined PARPi sensitivity in an additional panel of tumour cell lines (Fig 3, Fig S3 and Tables S1 and S2 of Supporting Information). Although PTEN mutations are not the only genetic variable within this diverse tumour cell panel, we observed a clear distinction in PARPi sensitivity between tumour lines with wild type PTEN expression, such as DU145 and MCF7, and those with PTEN deficiency, such as HCC70, PC3, RPMI-7951, A172, UM-UC3 and MDA-MB-468 (Fig 3A,B and Fig S3 of Supporting Information). Notably, PARPi sensitivity in PTEN deficient lines was of a scale similar to that observed in human tumour lines with BRCA1 silencing (Farmer et al, 2005). With the exception of the glioma line A172, we also observed consistent cisplatin sensitivity in PTEN deficient lines (Fig 3C). EMBO Mol Med 1, ß 2009 EMBO Molecular Medicine 317

4 Report Targeting PTEN deficiency We utilized the PTEN deficient prostate tumour cell line PC3, to assess whether PARPi sensitivity could be abrogated by ectopic expression of PTEN. As expected, infection of PC3 cells with an empty retroviral expression construct did not significantly modify PARPi sensitivity, nor did transfection of an expression construct encoding the p.r55fs 1 PTEN allele normally found in PC3 cells (Fig 4A,B). However, ectopic expression of full length PTEN in PC3 cells elicited PARPi resistance of a scale similar to tumour cells with endogenous wild type PTEN expression (Fig 4A). We also assessed whether PARPi sensitivity by PTEN was independent of the phosphatase activity of PTEN. Ectopic expression of the catalytically inactive C124S PTEN mutant (Maehama & Dixon, 1998; Shen et al, 2007) also caused PARPi resistance in PC3 cells (Fig 4A), suggesting that a non-phosphatase activity of PTEN may determine PARPi sensitivity. We also analysed whether restoring cytoplasmic PTEN expression, while suppressing nuclear PTEN expression could confer PARP inhibitor resistance in a PTEN null cell line, such as PC3. Here we used ectopic expression of a PTEN cdna that bears a mutation (p.k289e) that reduces the shuttling of Figure 3. PARP inhibitor sensitivity in human tumour cells. A. Western blot showing PTEN expression in a panel of human tumour lines. PTEN mutant lines are shown in red and PTEN wild type lines in black. PTEN genotypes are as follows: DU145 (prostate), PTEN wild type; MCF7 (breast), PTEN wild type; PC3 (prostate) p.r55fs 1 homozygous; MDAMB468 (breast), p.a72fs 5 homozygous; A172 (glioma), p.r55fs 1homozygous;UM-UC3 (bladder), p.m1 404del homozygous; RPMI-7951 (melanoma) p.null, c.1-79del79 homozygous; HCC70 (breast), p.f90fs 9 homozygous (see Table S1 of Supporting Information for complete genotypes). B. PARPi sensitivity in the same cell line panel. For each PTEN null line, survival was significantly different than in DU145 and MCF7 cells ( p<0.05, two-way ANOVA) (see Fig S3 of Supporting Information for analysis of an enlarged cell line panel). C. Cisplatin sensitivity in the same cell line panel. With the exception of A172, survival of each PTEN null line at 1 mm was significantly different than in DU145 and MCF7 cells ( p<0.05, two-way ANOVA). Figure 4. PARP inhibitor sensitivity in human tumour cells. A, B. Wild type and catalytically inactive PTEN, both restore PARP inhibitor resistance, as does expression of RAD51; SFs in PC3 cells infected with PTEN wild type, PTEN p.c124s or RAD51 cdna expression constructs was significantly different than in PC3 þ empty vector (p<0.05, two-way ANOVA). PC3 cells infected with p.k289e cdna expression construct did not cause significant resistance. PTEN null PC3 cells were infected with cdna expression constructs as shown and exposed to PARP inhibitor. Western blot and survival curves are shown. 318 ß 2009 EMBO Molecular Medicine EMBO Mol Med 1,

5 Report Ana M. Mendes-Pereira et al. Figure 5. In vivo efficacy of Olaparib in PTEN deficient xenografts. A, B. The clinical PARP inhibitor KU /Olaparib (Evers et al, 2008; Fong et al, 2008) suppresses growth in PTEN / subcutaneous xenografts (A), but not in PTEN þ/þ xenografts (B). PTEN deficient or proficient HCT116 cells were each mixed 2:1 in matrigel and then injected into either bilateral flank of 6 8 week old female athymic nude mice. After two days recovery, mice were randomized into treatment and control groups, (10 animals per cohort, 20 in total) and drug dosing initiated. A treatment regime similar to that known to elicit Brca2 selectivity (Farmer et al, 2005) was used. For five consecutive days, single daily intraperitoneal doses of KU (or vehicle) were administered at a dose of 15 mg/kg in HBC (Farmer et al, 2005). This was followed by two consecutive days of no treatment after which the same treatment cycle was continued until the end of the study. Tumour volumes were measured every four days from the initiation of drug dosing and the results expressed as fold increase in tumour volume relative to that at the first drug administration. Tumour volume vehicle treated vs. drug treated, p ¼ 0.04 for PTEN / xenografts and p ¼ 0.44 for PTEN þ/þ xenografts (two-way repeated measures ANOVA). PTEN into the nucleus (Song et al, 2008) (Fig 4B). Expression of PTEN p.k289e was able to partially but not completely restore PARP inhibitor resistance (Fig 4A), suggesting that an impairment of the nuclear localization of PTEN also modulates sensitivity to PARP inhibitor. Finally, we sought to assess whether the PARPi sensitivity of a PTEN cell line was RAD51 dependent. Ectopic expression of RAD51 caused PARP inhibitor resistance in PC3 cells (Fig 4A and Fig S4 of Supporting Information), supporting the hypothesis that reduced RAD51 expression underlies the PARP inhibitor sensitivity of PTEN deficient cells. To assess PARPi selective efficacy in vivo, we xenografted PTEN deficient HCT116 cells into mice and treated animals with the PARPi KU (Olaparib) (Evers et al, 2008) which is currently undergoing clinical assessment (Fong et al, 2009). Although xenograft growth in this model was extremely rapid, growth from PTEN deficient xenografts was suppressed by KU when compared to vehicle-treated xenografts (Fig 5A). Conversely PARPi did not have a similar effect on the growth of PTEN wild type xenografts (Fig 5B). This provides preliminary evidence to suggest that our in vitro observations may extrapolate to an in vivo setting. DISCUSSION PARP inhibitors are now showing considerable promise in the treatment of cancers characterized by genetic deficiencies that lead to an impairment of HR. For example, observations from a phase I trial of the PARP inhibitor KU /AZD2281/ Olaparib (AstraZeneca) suggest that significant tumour responses can be achieved in ovarian cancer patients carrying BRCA mutations (Fong et al, 2009). These tumour responses, demonstrated by both radiological estimation of tumour size and by the use of biochemical markers of tumour burden, are sustained and can be achieved without the significant toxicity normally associated with standard chemotherapeutic regimes (Fong et al, 2009). These promising results have led to phase II trials of KU /AZD2281/Olaparib for breast and ovarian cancers in BRCA mutation carriers (Tuma, 2007). Here, we show that PARP inhibitors may have utility outside the relatively small proportion of cancer patients carrying BRCA mutations. PTEN mutations are common in both endometrial cancers and glioblastomas and are also found in a significant proportion of malignant melanomas, prostate, breast, lung and colorectal tumours (Keniry & Parsons, 2008). In addition, transcriptional or translational repression of PTEN expression in the absence of PTEN mutation is also a common characteristic of multiple tumour types (Wiencke et al, 2007; Yang et al, 2008). The profound sensitivity of PTEN deficient tumour cells to PARP inhibitors demonstrated here suggests that clinical assessment of these agents in a wider context is warranted and perhaps even in combination with PI3K/AKT inhibitors that have been already been proposed as a means to target PTEN deficient tumours (Knight & Shokat, 2007). Our initial studies suggest that homozygous PTEN mutations that severely truncate the open reading frame sensitize tumour cells to PARP inhibitors. For example, glioblastoma, breast and prostate tumour cell lines bearing homozygous PTEN mutations that truncate the open reading frame before residue c.279 (amino acid residue 93) are as sensitive to KU , as human tumour cells with BRCA1 or BRCA2 deficiencies (Farmer et al, 2005; Turner et al, 2008). Notably, reconstitution of PC3 cells EMBO Mol Med 1, ß 2009 EMBO Molecular Medicine 319

6 Report Targeting PTEN deficiency (p.r55fs 1 homozygous) with ectopic expression of a catalytically inactive PTEN allele (p.c124s) was sufficient to restore PARPi resistance (Fig 4). Therefore, it seems likely that tumourassociated PTEN missense mutations that only modify catalytic activity without ostensibly compromising PTEN expression or non-phosphatase activities may not sensitize tumour cells to PARP. Conversely, PTEN mutations that severely truncate the open reading frame or cause instability of the PTEN protein are more likely to cause PARPi sensitivity. Mutations that truncate the open reading frame of PTEN before residue 93, as in the PARPi sensitive HCC70 cell line (p.f90fs 9 homozygous) are found in a wide variety of tumour types (Table S2 of Supporting Information), suggesting at least one patient subgroup that should be rapidly assessed for response to PARP inhibitors in clinical trials. Tumours that have PTEN deficiency brought about by PTEN mutations in the C-terminus that compromise protein stability (Leslie et al, 2008) or non-genetic alterations that cause changes in PTEN transcription or translation, may also respond to PARP inhibitors, although considerable work is required to establish a threshold of PTEN expression below which PARPi sensitivity occurs. Furthermore, we envisage that the role PTEN plays in determining response to PARP inhibitors, is mediated by a nuclear activity of the protein, suggesting that a complete absence of nuclear PTEN expression, determined by immunohistochemical (IHC) detection in biopsy material, could predict response. This raises the possibility that PARP inhibitors could specifically be assessed in tumourigenic conditions such as the more aggressive forms of oesophageal squamous cell carcinoma (Tachibana et al, 2002), cutaneous melanoma (Whiteman et al, 2002) and colorectal cancer (Trotman et al, 2007; Zhou et al, 2002), where nuclear PTEN expression is absent. Defining thresholds of IHC detection that are predictive of PARPi response will be a key in assessing this possibility, and we propose that combining PTEN IHC with surrogate markers of PARPi sensitivity, such as the inability to form nuclear RAD51 foci (Farmer et al, 2005), may allow further refinement of the use of PARP inhibitors in cancer treatment. MATERIALS AND METHODS Materials PARP inhibitors KU (Farmer et al, 2005) and KU (Evers et al, 2008; Fong et al, 2008) (KuDOS Pharmaceuticals/Astra Zeneca), cisplatin and paclitaxel (Sigma Aldrich) were used as described previously (Edwards et al, 2008; Farmer et al, 2005). Cell culture Wild type, PTEN þ/ and PTEN / HCT116 or HEC1A cells (Kim et al, 2007; Lee et al, 2007) were maintained in McCoy s 5A medium (Invitrogen, Glasgow, UK) supplemented with 10% v/v foetal bovine serum (FBS) (PAA gold, GIBCO). In the case of PTEN / cells, two individually derived lines were used, (KO22 and KO35 for HCT116 and KO22 and KO16 for HEC1A). For the HCT116 experiments, parental cells and a HCT116 derived line bearing a random integration of the PTEN targeting construct (neo124) were used as PTEN wild type comparators. Two HCT116 PTEN þ/ cell lines bearing one targeted allele (130 and 140) were also analysed. All other cells lines were obtained from the ECACC (Teddington, UK) and cultured according to the supplier s instructions. Plasmids The full length coding sequence of PTEN was PCR amplified from a cdna IMAGE clone (Geneservice, Cambridge, UK), using a 5 0 primer harbouring a Kozak consensus sequence (GCCACC prior to initiating ATG). The resultant PCR product was cloned into a tag-free pbabe-puro retroviral expression vector. Mutation of the PTEN cdna sequence was achieved using the Quick Change site-directed mutagenesis kit (Stratagene). Twenty-four hours after infection, cells were selected in 1 mg/l puromycin (InvivoGen) for three days to remove non-infected cells, then seeded in six-well plates (at 1500 cells/well) and treated with drug the following day, as previously described (Edwards et al, 2008; Farmer et al, 2005). Survival assays Long-term survival assays were performed as previously described (Edwards et al, 2008; Farmer et al, 2005). For measurement of sensitivity to PARP inhibitor, cisplatin or paclitaxel (taxol), exponentially growing cells were seeded in six-well plates at a concentration of cells per well. For PARP inhibitor and taxol treatment, cells were continuously exposed to the drug with media and drug replaced every 60 h. For cisplatin treatment, cells were exposed to the drug at the indicated concentrations for 24 h, washed twice with phosphate buffered saline (PBS), then normal media replaced every 3-4 days. After 15 days, cells were fixed and stained with sulphorhodamine-b (Sigma, St. Louis, USA) and a colorimetric assay performed as described previously (Edwards et al, 2008). Surviving fractions (SFs) were calculated and drug sensitivity curves plotted as previously described (Farmer et al, 2005). Protein analysis Whole-cell protein extracts were prepared from cells lysed with radioimmunoprecipitation (RIPA) buffer (Upstate) supplemented with protease inhibitor cocktail tablets (Roche, Burgess Hill, UK). Protein concentrations were measured using BioRad Protein Assay Reagent (BioRad, Hemel Hempstead, UK). For Western blot analysis, lysates were electrophoresed on Novex 4 12% gradient bis tris pre-cast gels (Invitrogen) and immunoblotted overnight at 48C with antibodies targeting the following epitopes: PTEN C-terminus (Cell Signalling, Danvers, USA), RAD51 (H-92, Santa Cruz Biotech, Santa Cruz, USA), ACTIN (Santa Cruz Biotech) or b-tubulin (T4026, Sigma). Incubation with primary antibody was followed by incubation with a horseradish peroxidase-conjugated secondary antibody and chemiluminescent detection of proteins (Amersham Pharmacia, Cardiff, UK). DSB Repair Assay DSBR was quantified using a synthetic repair substrate as previously described (Saeki et al, 2006). Briefly, the DR-green fluorescent protein (GFP) HR reporter plasmid, containing two differentially mutated GFPencoding cassettes, one of which contains a unique ISceI restriction site, was co-transfected into cells alongside a ISceI expression vector, pcbasce (Saeki et al, 2006). Seventy-two hours after transfection, HR competence was estimated by quantifying the number of GFP positive cells from a population of 50,000 cells, using flow cytometry (Saeki et al, 2006). 320 ß 2009 EMBO Molecular Medicine EMBO Mol Med 1,

7 Report Ana M. Mendes-Pereira et al. The paper explained PROBLEM: The tumour suppressor gene, PTEN, is one of the most commonly mutated genes in human cancers. Selectively targeting cancerous PTEN deficient cells would likely make for an efficient and relatively safe cancer therapy applicable to the large number of PTEN deficient cancers. Selective targeting of BRCA1 or BRCA2 deficient tumours has recently been demonstrated with Olaparib, a high-potency PARP inhibitor. This drug can elicit sustained responses in BRCA deficient cancers without causing some of the negative side effects caused by standard chemotherapies. Tumour cells in BRCA mutant patients have a defect in the DNA repair process of HR that repairs DSBs and it is thought that PARP inhibitors target this HR defect by increasing the formation of DSBs. suggesting that drugs that target BRCA mutation could also be used to target PTEN mutation. We demonstrate that PTEN deficiency in human tumour cells also leads to suppression of HR. Using in vitro cell culture and in vivo tumour modelling in mice, we show that, as in BRCA deficient tumours, the HR deficiency in PTEN mutant cells can be exploited therapeutically by Olaparib. IMPACT: Our work suggests that this relatively non-toxic anti-cancer drug can be used to target the large number of PTEN deficient tumours, significantly extending its previously anticipated limited spectrum. RESULTS: Recent evidence suggests that, like BRCA mutant cells, PTEN mutant cells are also characterized by an increase in DSBs, Analysis of RAD51 foci formation Nuclear RAD51 foci were visualized and quantified by confocal microscopy as previously described (Farmer et al, 2005). Detection of gamma (g)-h2ax foci was used a control marker for the formation of DSBs (data not shown). In brief, cells were grown onto L-lysine coated cover slips (BD Biosciences, Oxford, UK) and exposed to 10 Gy ionizing radiation. Eight hours later, cells were fixed, permeabilized and then co-immunostained with primary antibodies targeting RAD51 (rabbit, Santa Cruz Biotech) and gh2ax (mouse, Upstate), detected with fluorescein isothiocyanate (FITC) and Texas red conjugated secondary antibodies respectively Diamidino-2-phenylindole (DAPI) staining was used to detect nuclei. Nuclear foci were visualized by confocal microscopy and a minimum of 100 fields of view used to estimate RAD51 focus formation frequency. Xenografts HCT116 cells were mixed 2:1 in matrigel (BD Biosciences) and then injected into the bilateral flanks of 6 8 week old female athymic nude mice ( cells per animal). After two days recovery, mice were randomized into treatment and control groups, (10 animals per cohort, 20 in total) and drug dosing initiated. We used a treatment regime similar to that known to elicit Brca2 selectivity (Farmer et al, 2005). For five consecutive days, single daily intraperitoneal doses of KU (or vehicle) were administered at a dose of 15 mg/kg in 2-hydroxypropyl-b-cyclodextrin (HBC, Farmer et al, 2005). This was followed by two consecutive days of no treatment after which the same treatment cycle was continued until the end of the study. Tumour volumes were measured every four days from the initiation of drug dosing and the results expressed as fold increase in tumour volume, relative to that at the first drug administration. Author Contributions AMP, CJL and AA conceived and designed the experiments. AMP, SAM, RB, AM and JT performed the experiments. AMP, SAM, RB, AM, JT, CJL and AA analysed the experiments. TW and JSK provided reagents. AMP, CJL and AA wrote the manuscript. CJL and AA devised the project and obtained funding. Acknowledgements We thank Drs. Graeme Smith and Niall Martin of KuDOS Pharmaceuticals for the provision of PARP inhibitors. This work was funded by Breakthrough Breast Cancer and Cancer Research UK. We acknowledge NHS funding to the NIHR Biomedical Research Centre. Supporting information is available at EMBO Molecular Medicine online. The authors declare that they have no conflict of interest. For more information The Breakthrough Research Center: centre/index.html Clinical trials (Olaparib): Cancer Research UK: Cancer Help UK (Olaparib): EMBO Mol Med 1, ß 2009 EMBO Molecular Medicine 321

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9 Supplementary Figure 1 A B HEC1A PTEN +/+ HEC1A PTEN -/- 16 HEC1A PTEN -/- 22 PTEN RAD51 % Cells with RAD51 foci 40 -IR +IR * *! TUBULIN 0 HEC1A PTEN+/+ HEC1A PTEN-/- 16 HEC1A PTEN-/- 22 C D 1.0 HR Efficiency * * Surviving fraction SF50 (M) HEC1A PTEN +/+ >1.0E-05 HEC1A PTEN -/- -/ E-05 HEC1A PTEN -/- -/ E HEC1A PTEN+/+ HEC1A PTEN-/- 16 HEC1A PTEN-/ KU (M) KU (M) 10-5

10 Supplementary Figure 2 "H2AX Merged HCT116 PTEN+/+ Rad51 - IR HCT116 PTEN-/- Nuclei - IR + IR + IR

11 Supplementary Figure 3 Surviving fraction DU145 MCF-7 HeLa SUM159 BT474 MDA-MB-231 HCC70 PC3 RPMI-7951 SW1783 EC50 SF50 (M) > 1E-05 > 1E-05 > 1E-05 > 1E-05 > 1E-05 > 1E E E E E A172 UM-UC3 2.94E E-08 MDA-MB E COLO E-09 KU (M)

12 Supplementary Figure 4 EMPTY VECTOR RAD51 cdna RAD51! Tubulin

13 Supplementary Table 1 Cell Line PTEN PIK3CA TP53 BRAF KRAS MLH1 MSH6 CDKN2A A172 * COLO 829 * * * HCC70 * * MDA-MB-468 * * PC3 * * RPMI-7951 * * * * SW1783 * * UM-UC3 * * * * BT474 * * HEC1A * * * HCT116 * * * * MCF7 * * SUM-159 * MDA-MB-231 * * * * DU145 * * * HeLa Cell Line Mutation PTEN PIK3CA TP53 BRAF KRAS MLH1 MSH6 CDKN2A A172 AA p.r55fs*1 CDS c.165_1212del1048 glioma Zygosity Homozygous COLO 829 AA p.? p.v600e p.a68fs*51 CDS c.493_634del142 c.1799t>a c.203_204delcg melanoma Zygosity Homozygous Heterozygous Homozygous HCC70 AA p.f90fs*9 p.r248q CDS c.270delt c.743g>a breast Zygosity Homozygous Homozygous MDA-MB-468 AA p.? p.r273h CDS c.253+1g>t c.818g>a breast Zygosity Homozygous Homozygous PC3 AA p.r55fs*1 p.k139fs*31 CDS c.165_1212del1048 c.414delc prostate Zygosity Homozygous Homozygous RPMI-7951 AA p.? p.s166* p.v600e p.l16r CDS c.1_79del79 c.497c>a c.1799t>a c.47t>g melanoma Zygosity Homozygous Homozygous Heterozygous Homozygous SW1783 AA p.r233* p.r273h CDS c.697c>t c.818g>a glioma Zygosity Homozygous Heterozygous UM-UC3 AA p.m1_*404del p.f113c p.g12c p.m1_*157del CDS c.1_1212del1212 c.338t>g c.34g>t c.1_471del471 bladder Zygosity Homozygous Homozygous Homozygous Homozygous BT-474 AA p.k111n p.e285k CDS c.333g>c c.853g>a breast Zygosity Heterozygous Homozygous HEC1A AA p.g1049r p.r248q p.g12d CDS c.3145g>c c.743g>a c.35g>a endometrium Zygosity Heterozygous Homozygous Homozygous HCT116 AA p.h1047r p.g13d p.s252* p.r24fs*20 CDS c.3140a>g c.38g>a c.755c>a c.68_69insg colorectal Zygosity Heterozygous Heterozygous Homozygous Homozygous MCF7 AA p.e545k p.m1_*157del CDS c.1633g>a c.1_471del471 breast Zygosity Heterozygous Homozygous SUM-159 AA p.h1047l CDS c.3140a>t breast Zygosity Heterozygous MDA-MB-231 AA p.r280k p.g464v p.g13d p.m1_*157del CDS c.839g>a c.1391g>t c.38g>a c.1_471del471 breast Zygosity Homozygous Heterozygous Heterozygous Homozygous DU145 AA p.p223l, p.v274f p.? p.d84y CDS c.668c>t, c.820g>t c.117-2a>t c.250g>t prostate Zygosity Heterozygous Homozygous Homozygous HeLa AA CDS cervix Zygosity

14 Sample Name COSMIC Sample ID Amino Acid Nucleotide Primary Tissue Histology HCC p.f90fs*9 c.270delt breast carcinoma 5839 cell-line S p.k60fs*9 c.180_181ins? breast carcinoma NS MDA- MB p.l70fs*7 c.208_251del44 breast carcinoma NS p.a39fs*5 c.116_117insg central_nervous_system glioma fresh/frozen S p.a79fs*20 c.237delc central_nervous_system glioma primary SKMG p.c71fs*6 c.212_255del44 central_nervous_system glioma NS S p.d24fs*20 c.70_71insa central_nervous_system glioma primary S p.d24fs*20 c.70_71insa central_nervous_system glioma primary S p.e43fs*11 c.127delg central_nervous_system glioma NS S p.f56fs*2 c.165_492del328 central_nervous_system glioma primary p.k60* c.178a>t central_nervous_system glioma fresh/frozen S p.k66fs*38 c.197_198ins14 central_nervous_system glioma primary S p.k6fs*4 c.17_18delaa central_nervous_system glioma primary S p.l70fs*7 c.210_253del44 central_nervous_system glioma primary S p.l70fs*7 c.210_253del44 central_nervous_system glioma primary SF p.m1_*404del c.1_1212del1212 central_nervous_system glioma cell-line H p.m1_*404del c.1_1212del1212 central_nervous_system glioma cell-line D- 245MG p.m1_*404del c.1_1212del1212 central_nervous_system glioma cell-line D- 336MG p.m1_*404del c.1_1212del1212 central_nervous_system glioma cell-line S p.n12fs*6 c.35_53del19 central_nervous_system glioma primary S p.r14fs*26 c.42_52del11 central_nervous_system glioma primary S p.r15fs*9 c.45_46insga central_nervous_system glioma primary A p.r55fs*1 c.165_1212del1048 central_nervous_system glioma cell-line SW p.r55fs*1 c.165_1212del1048 central_nervous_system glioma cell-line U-87- MG p.v54fs*29 c.160_208del49 central_nervous_system glioma NS S p.y16fs*1 c.47_48insa central_nervous_system glioma NS S p.y46* c.138c>g central_nervous_system glioma primary Pubmed ID Mutation ID Sample Source

15 S p.y65* c.195c>a central_nervous_system glioma primary S p.y65* c.195c>g central_nervous_system glioma primary S p.y76fs*1 c.226_227delta central_nervous_system glioma primary S p.y76fs*1 c.227_228delat central_nervous_system glioma primary S p.y76fs*1 c.227_228delat central_nervous_system glioma primary S p.y88fs*3 c.263_264delat central_nervous_system glioma primary LS p.y88fs*3 c.263_264delat central_nervous_system glioma NS S p.a3fs*14 c.9_30del22 endometrium carcinoma primary S p.c71fs*6 c.213_256del44 endometrium carcinoma primary S p.d24fs*19 c.71_72insc endometrium carcinoma primary S p.d24fs*19 c.71_72insc endometrium carcinoma primary S p.e18fs*5 c.54_57delggat endometrium carcinoma primary p.e73fs*25 c.219_222delaaga endometrium carcinoma fresh/frozen S p.e73fs*4 c.219_220delaa endometrium carcinoma primary p.e7fs*3 c.19_20delga endometrium carcinoma NOS p.f56c c.167t>g endometrium carcinoma fresh/frozen S p.f90fs*9 c.270delt endometrium carcinoma primary S p.h64fs*9 c.190_191delca endometrium carcinoma primary S p.i32fs*22 c.96delt endometrium carcinoma primary S p.i32fs*22 c.96delt endometrium carcinoma primary S p.i33fs*20 c.97_100delattg endometrium carcinoma primary S p.i33fs*21 c.97dela endometrium carcinoma primary S p.i33fs*21 c.97dela endometrium carcinoma metastasis E p.k6fs*4 c.17_18delaa endometrium carcinoma fresh/frozen S p.k6fs*4 c.17_18delaa endometrium carcinoma primary p.k6fs*4 c.17_18delaa endometrium carcinoma fresh/frozen S p.l25fs*28 c.75_78delgacc endometrium carcinoma primary p.l57fs*42 c.166_166delt endometrium carcinoma NOS E p.l57fs*6 c.170_171inst endometrium carcinoma fresh/frozen

16 S p.l57fs*6 c.170_171inst endometrium carcinoma primary p.l57fs*6 c.170_171inst endometrium carcinoma fixed - NOS S p.n48fs*6 c.143dela endometrium carcinoma primary S p.n63fs*10 c.187_188delaa endometrium carcinoma primary p.n63fs*10 c.187_188delaa endometrium carcinoma fresh/frozen p.n63fs*10 c.187_188delaa endometrium carcinoma fresh/frozen S p.n63fs*11 c.188_189insa endometrium carcinoma primary S p.n63fs*36 c.187dela endometrium carcinoma primary S p.n63fs*36 c.188dela endometrium carcinoma primary S p.n63fs*36 c.188dela endometrium carcinoma primary S p.n63fs*36 c.188dela endometrium hyperplasia primary S p.n69fs*30 c.206dela endometrium carcinoma primary S p.p30fs*24 c.89delc endometrium carcinoma primary S p.r14fs*10 c.40dela endometrium carcinoma primary S p.r14fs*29 c.41_42insg endometrium carcinoma primary S p.r15fs*28 c.45_46inst endometrium carcinoma primary S p.r55fs*2 c.165_181del17 endometrium carcinoma primary S p.t26fs*28 c.78delc endometrium carcinoma primary S p.v45fs*10 c.134_135insca endometrium carcinoma primary S p.y16fs*21 c.46_65del20 endometrium carcinoma primary S p.y16fs*28 c.46_47inst endometrium carcinoma primary S p.y27fs*1 c.80_90del11 endometrium carcinoma primary S p.y27fs*16 c.80_81delat endometrium carcinoma primary p.y29fs*25 c.83_83delt endometrium carcinoma fresh/frozen S p.y68fs*5 c.202_203delta endometrium carcinoma primary S p.y76fs*1 c.227_228delat endometrium carcinoma primary S p.y76fs*1 c.227_228delat endometrium carcinoma primary OPM p.m1_*404del c.1_1212del1212 haematopoietic_and_lymphoid_tissue haematopoietic_neoplasm cell-line S p.m1fs*7 c.1_8delatgacagc haematopoietic_and_lymphoid_tissue lymphoid_neoplasm primary KE p.y27fs*1 c.80_1212del1133 haematopoietic_and_lymphoid_tissue haematopoietic_neoplasm cell-line p.e7fs*3 c.21_22delga large_intestine carcinoma

17 p.f90fs*9 c.270delt large_intestine carcinoma p.l57fs*42 c.170delt large_intestine carcinoma p.l57fs*6 c.170_171inst large_intestine carcinoma fresh/frozen fresh/frozen fresh/frozen fresh/frozen S p.c83fs*9 c.247_248inst liver carcinoma primary H p.? c.1-?_492+?del lung carcinoma NS NCI- H p.? c.1-?_492+?del lung carcinoma NS SBC p.m1_*404del c.1_1212del1212 lung carcinoma cell-line NCI- H p.m1_*404del c.1_1212del1212 lung carcinoma cell-line N p.m1_?del fs c.1-?_79+?del lung carcinoma NS N p.m1_?del fs c.1-?_79+?del lung carcinoma NS N p.m1_?del fs c.1-?_79+?del lung carcinoma NS NCI- H p.r55fs*1 c.165_1212del1048 lung carcinoma cell-line LU-134- A p.y27fs*1 c.80_1212del1133 lung carcinoma cell-line S p.l57fs*42 c.170delt meninges meningioma primary p.c71fs*28 c.213_213delt ovary carcinoma fresh - NOS p.f81fs*18 c.241_242tt>g ovary carcinoma NOS RTSG p.k6fs*4 c.17_18delaa ovary carcinoma 4929 cell-line S p.y68fs*5 c.202_203delta ovary carcinoma primary S p.g36fs*18 c.107delg prostate carcinoma primary S p.g44fs*11 c.131_139gcgtataca>acagaaagaca prostate carcinoma primary LNCaP- Clone- FGC p.k6fs*4 c.17_18delaa prostate carcinoma 4929 cell-line LNCaP p.k6fs*4 c.16_17delaa prostate carcinoma NS LNCaP p.k6fs*4 c.16_17delaa prostate carcinoma NS LNCaP p.k6fs*4 c.17_18delaa prostate carcinoma NS S p.q87* c.259c>t prostate carcinoma NS PC p.r55fs*1 c.165_1212del1048 prostate carcinoma cell-line

18 S p.y76fs*1 c.226_227delta prostate carcinoma NS FM p.? c.1-?_492+?del skin malignant_melanoma NS S p.k6fs*4 c.17_18delaa skin malignant_melanoma metastasis G-mel p.m1_?del fs c.1-?_79+?del skin malignant_melanoma NS SW p.m1_*404del c.1_1212del1212 soft_tissue liposarcoma cell-line UM-UC p.m1_*404del c.1_1212del1212 urinary_tract carcinoma cell-line

19 Supplementary Table 1. Genotypes of human tumour cell lines used in this study. Supplementary Table 2. Human tumours and tumour cell lines listed in the COSMIC database (www. bearing PTEN mutations predicted to truncate the open reading frame before amino acid 93. Supplementary Figure 1. Homologous recombination and PARP inhibitor sensitivity in HEC1A tumour cells. A) PTEN deficiency in HEC1A cells causes a reduction in RAD51 expression. Total cell lysates from isogenic HEC1A endometrial tumour cells were immunoblotted for PTEN and RAD51. Detection of ACTIN is shown as a loading control. Parental HEC1A cells are shown (PTEN +/+ ) along with two individually derived PTEN -/- lines (KO16 and KO22). B) PTEN deficiency in HEC1A cells causes a reduction in IR-induced nuclear RAD51 focus formation. * p values vs. +IR PTEN +/+ < 0.05 (Student s t test). No other comparisons were statistically significant. C) PTEN deficiency in HEC1A cells sensitises cells to a druglike PARP inhibitor. p values for both PTEN -/- lines vs. PTEN +/+ cells < (twoway ANOVA). D) PTEN deficiency in HEC1A cells causes a reduction in HR using a synthetic HR substrate. * p values null vs. wild type PTEN cells < 0.05 (Student s t test). Supplementary Figure 2. Decreased RAD51 focus formation upon DNA damage in PTEN deficient cells. γh2ax foci (red), RAD51 foci (green) and nuclei (stained with DAPI blue) are shown. Magnification is 1000X.

20 Targeting PTEN deficiency 2 Supplementary Figure 3. PARP inhibitor sensitivity in human tumour cells. See Supplementary Table 1 for genotypes. PTEN mutant lines are shown in red and PTEN wild type lines in black. For each PTEN null line, survival was significantly different than in DU145 and MCF7 cells (p < 0.05, two-way ANOVA). Supplementary Figure 4. RAD51 expression from a retroviral vector used in Figure 4. PC3 cells were infected with virus and cell lysates analysed by western blotting two days later.