Synthetic Lethality of PARP Inhibition and Ionizing Radiation is p53-dependent

This article is featured in Highlights of This Issue, p. 1071

PARP inhibitors (PARPi) demonstrate synthetic lethality in cells with impaired homologous recombination (HR)-mediated DNA repair function, particularly BRCA1/2-associated tumors (1, 2). Unfortunately, BRCA1/2-associated HR repair deficiencies occur in only a small percentage of tumors greatly limiting the clinical efficacy of PARPi-based therapies (3, 4). Methods to expand the population of cancer patients that may benefit from PARPi beyond those with BRCA-associated tumors, including the combination of PARPi with other cytotoxic agents, have thus far failed to be efficacious in the clinic. Despite these setbacks, the development of novel paradigms to elicit tumor-specific PARPi synthetic lethality in HR-competent cancers remains an intriguing treatment strategy and recently reported studies in experimental models of HR-proficient cancers have demonstrated an increase in treatment efficacy when PARPi are used in combination with other cytotoxic agents such as ionizing radiation (IR; refs. 5, 6). Existing combinatorial strategies utilize PARPi as an inducer of single-strand DNA breaks, but fail to tap into the synthetic lethality seen in HR-deficient tumors. Recently, we developed a novel combinatorial paradigm in which IR is combined with PARPi (7, 8). Our strategy utilizes IR not just as a cytotoxic agent but more importantly as a means to export BRCA1 from the nucleus to inhibit HR repair and create synthetic PARPi lethality.

The essential roles of BRCA1 in DNA damage response and HR-mediated repair following DNA double strand breaks (DSB) have been well characterized (9–11). While BRCA1 deficiencies are commonly associated with inherited breast and ovarian cancers, BRCA1 status is prognostic of outcome and treatment response in a variety of cancers including glioblastomas (12) and prostate tumors (13). Cells with HR repair deficiencies rely heavily on alternative mechanisms to repair damaged DNA such as single-strand break (SSB) base excision repair. PARP1 is the rate-limiting enzyme in SSB base excision repair, and thus, inhibition of PARP in HR-deficient cancer cells leaves these tumors greatly compromised in their ability to repair damaged DNA accounting for the increased cytotoxicity of PARPi in these tumors (1). While dysfunction of BRCA1 activity due to mutation occurs only in a small subset of cancers, BRCA1 function may be positively or negatively regulated by a variety of mechanisms including subcellular localization in both normal and cancer cells (14–16). Nuclear localization is required for BRCA1 to participate in HR-mediated DNA repair and initiate cell-cycle checkpoints following DNA damage; cytosolic BRCA1, on the other hand, not only is unable to contribute to DNA repair, but is also involved in the induction of apoptosis through a p53-independent mechanism (17–19). Therefore, the nuclear/cytoplasmic localization of BRCA1 is a critical factor in determining whether a cell effectively repairs damaged DNA or dies following genotoxic stress. We have previously demonstrated that nuclear export of BRCA1 suppresses its nuclear function resulting in inhibition of HR-mediated DNA damage repair and, subsequently, increased cytotoxicity to DNA-damaging agents (8, 15, 19, 20). Furthermore, our group and others have demonstrated that IR can induce the export of BRCA1 from the nucleus to the cytoplasm resulting in decreased HR repair and increased cytotoxicity of PARPi in breast tumor models (8, 15, 19).

Like BRCA1 and PARP1, the tumor suppressor p53 is a critical guardian of genome stability (21), and thus, it is not surprising that cross-talk exists between these important factors to coordinate the decision of DNA repair or cell death following genotoxic stress. Recent work (22) has demonstrated that in glioblastoma cell lines the therapeutic efficacy of PARPi plus IR combination therapy is dependent upon p53 status due to the requirement of PARP1 enzymatic activity for p53 nuclear accumulation and transcriptional activity following DNA damage. Work by our laboratory has established an important role for p53 in the subcellular distribution of BRCA1. We have previously reported that p53 dysfunction leads to nuclear accumulation and reduced cytoplasmic translocation of BRCA1 following exposure to IR (15, 20). These data demonstrate that p53-mediated shuttling of BRCA1 is an important mechanism regulating the choice of cell death or DNA repair following genomic damage. We hypothesized that p53 status would be an essential determinant of PARPi sensitivity in our treatment strategy of IR preceding PARPi and, given the high frequency of p53 mutation in solid tumors, sought to elucidate the role of p53 status on the effectiveness of our treatment paradigm to more precisely define those tumors that are likely to benefit.

The results presented herein demonstrate that BRCA1 sequestration to the cytoplasm following exposure to IR can be used to induce HR-mediated DNA repair deficiency and increase PARPi cytotoxicity in multiple tumor models. Importantly, we find that p53 status is a significant determining factor of tumor cell sensitization to PARPi following IR. Together, our results demonstrate that IR can suppress HR-mediated DNA repair in a p53 dependent-manner in variety of cancer types to confer PARPi synthetic lethality, which, if translated to the clinic, may significantly increase the population of cancer patients who benefit from PARPi-based therapies.

Stable MCF7/E6 and their associated control cell line were established from cells purchased from ATCC as described previously (15). The U87 human glioblastoma cell line was purchased from ATCC. SF767 shCtrl and SF767 shp53 human glioma cell lines were a gift from Dr. Jay Fitzgerald Dorsey (Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA) and have been described previously (23). Cell lines were cultured at low passage number and authenticated by morphology and growth characteristics. Cells were tested for Mycoplasma by PCR (ATCC) every three months. All cell lines were maintained in DMEM supplemented with 10% FBS (Sigma) and 1% penicillin/streptomycin. Transient p53 knockdown was accomplished using p53-targeting siRNAs (L-003329-00, Dharmacon) with siRNA A4 (Dharmacon) serving as a nontargeting control and Lipofectamine2000 transfection reagent (Invitrogen) as recommended by the manufacturer. Cells were irradiated as indicated using a RS-2000 biological irradiator (Rad Source). Cells were treated with ABT-888 (Enzo) as described in the text.

IHC staining using antibodies to BRCA1, γH2AX, and Rad51 was performed as described previously (7, 24).

Clonogenic survival assays were performed as described previously (25). Briefly, cells were treated as described in the text and then maintained in culture for approximately three weeks until colonies were of sufficient size to be stained and counted. Colonies were then fixed in a 1:7 mixture of acetic acid and methanol prior to staining with 0.5% crystal violet. Colonies of greater than 50 cells were counted and the survival fraction was calculated as described previously (25).

All animal procedures were approved by the Institutional Animal Care and Use Committee at The Ohio State University (Columbus, OH). A total of 1 × 10⁷ MCF7 or MCF7/E6 cells or 5 × 10⁶ U87, SF767 shCtrl or SF767 shp53 cells were injected into the left flank of 6-week-old female athymic Foxn1 (nu/nu) mice (Harlan Sprague Dawley Inc. or The Jackson Laboratory). For experiments utilizing MCF7 and MCF7/E6 cells, mice were implanted with 17β-estradiol pellets (0.72 mg, 60-day release; Innovative Research of America) 6 days prior to injection of the cells. Mice were randomly assigned to an experimental treatment arm once a tumor volume of approximately 100 mm³ was achieved. As indicated, 25 mg/kg ABT-888 (Enzo) suspended in H₂0 was delivered by oral gavage daily for five days. Mice receiving radiation treatment were anesthetized and ionizing radiation was delivered to the tumor as a single dose of 3 Gy (MCF7 control and E6 tumors) or 4 Gy (SF767 shCtrl and shp53 tumors). Mice receiving a combination of radiation and ABT-888 treatment were irradiated as described and then treated with ABT-888 by oral gavage daily for 5 consecutive days starting 24 hours after radiation.

Whole-cell lysates were prepared as described previously (15) and subjected to SDS-PAGE analysis. Anti-BRCA1 antibody (Ab-1; Calbiochem) was used at a 1:100 dilution while antibodies to β-actin, α-tubulin, and lamin a/c were purchased from Cell Signaling Technology and used at working dilutions of 1:1,000. Protein bands were visualized and photographed by means of a Versadoc Imaging System (Bio-Rad) using HRP-conjugated anti-rabbit and anti-mouse (Cell Signaling Technology) secondary antibodies at working dilutions of 1:5,000 and Immobilon western ECL reagent (Millipore). Quantitation of Western blots was performed using ImageJ software (NIH, Bethesda, MD). Isolation of nuclear and cytoplasmic protein fractions was accomplished using a Cell Fractionation Kit (Cell Signaling Technology) according to the manufacturer's recommendations.

Unsynchronized, subconfluent MCF7 control, or E6 cells were irradiated with 4 Gy or mock treated. Twenty-four hours later, the cells were fixed in 70% ethanol. Cells were then rehydrated in PBS prior to incubation for 30 minutes in a solution containing 0.1% Triton X-100, 0.2 mg/mL DNase-free RNase A, and 0.02 mg/mL propidium iodide prior to sorting using an LSR II (BD Biosciences) and analysis using BD FACSDiva software (BD Biosciences).

MCF7 cells with a stably integrated DR-GFP chromosomal reporter system were utilized for this assay. This reporter system contains two differently mutated GFP genes oriented as direct repeats. The upstream repeat contains a single I-SceI site while the downstream copy is a 5′-3′ truncated GFP fragment. Expression of I-SceI in these cells generates a DSB that, when repaired by HR, results in expression of GFP. Reporter cells were seeded overnight then transfected with control siRNA, p53-targeting siRNA, empty vector, or vector encoding p53R273H, respectively. Sixteen hours later, cells were treated with 4 Gy IR or mock treated. After an additional 4 hours, cells were transfected a control vector or vector encoding I-SceI. Forty-eight hours later, cells were collect and analyzed for GFP expression by flow cytometry.

Statistical analysis between groups was performed by ANOVA while direct comparisons between two groups were done by a two-tailed t test. P < 0.05 was considered statistically significant.

We previously demonstrated that p53 function is required for cytoplasmic translocation of BRCA1 following IR (15, 20). In addition, IR-induced cytoplasmic sequestration of BRCA1 results in inhibition of HR-mediated DNA repair and increased sensitivity to PARPi (7). It remains to be elucidated whether functional p53 is required to render HR-proficient cancer cells sensitive to PARPi subsequent to IR. To begin to address this clinically relevant question, we inhibited p53 function in MCF7 breast cancer cells by stable expression of the HPV16-E6 viral protein that catalyzes the degradation of p53 (MCF7/E6; ref. 26). In agreement with our previously published findings (7), treatment of control MCF7 cells with 4 Gy ionizing radiation resulted in a significant shift in BRCA1 localization from the nucleus to the cytoplasm 24 hours post-IR as determined by immunofluorescence (Fig. 1A; Supplementary Fig. S1A). In contrast, no significant cytoplasmic translocation of BRCA1 was observed following radiation in MCF7/E6 cells (Fig. 1B; Supplementary Fig. S1B).

Subcellular localization of BRCA1 is influenced by the cell cycle (15). During S-phase, BRCA1 is primarily confined to the nucleus and is exported to the cytoplasm during G₂ resulting in an almost exclusively cytoplasmic distribution in M-phase. As cells leave G₁ and reenter S-phase, BRCA1 is trafficked back to the nucleus. Given that p53 is a critical mediator of G₁ and G₂ arrest following DNA damage (27), we next asked whether the differences in BRCA1 localization following DNA damage observed between MCF7 control and E6 cells could be explained by changes in the cell-cycle distribution of these cells following radiation. As expected, radiation elicited a G₁ arrest in MCF7 cells as indicated by increased cells in G₁ and significantly fewer cells in S-phase after radiation (Fig. 1C). In contrast, no G₁ arrest was detected following radiation of MCF/E6 cells (Fig. 1D). The lack of G₁ arrest in MCF7/E6 cells following radiation may account for some of the reduction in BRCA1 nuclear export seen in these cells following radiation. However, the difference in the percentage of the population in S-phase between MCF7 control and E6 cells after radiation was only 7.39% and does not fully account for the almost 25% difference in cells with nuclear BRCA1 after radiation seen between these two cell lines (Fig. 1A and B). This is in agreement with our previous data that demonstrated that, while BRCA1 distribution is dependent upon cell cycle, DNA damage–induced BRCA1 nuclear export is independent of cell cycle and occurs during all phases of the cell cycle (15).

To determine the role of p53 in IR-induced sensitization of MCF7 cells to PARPi, we performed in vitro clonogenic survival assays utilizing MCF7 control and E6-expressing cells treated first with 0 or 4 Gy IR then treated 24 hours later with 0, 5, 10, 15, 20, or 25 μmol/L of the PARP inhibitor ABT-888. In agreement with previously published findings (8), MCF7 cells pretreated with 4 Gy IR demonstrated significantly increased sensitivity to ABT-888 relative to cells that received no radiation (Fig. 1E). However, in MCF7/E6 cells, pretreatment with radiation did not significantly sensitize these cells to ABT-888 relative to unirradiated MCF7/E6 cells (Fig. 1F). These results suggest that p53 function is required for IR-induced sensitization of MCF7 cells to PARPi in vitro.

To analyze effect of p53 on the sensitization of MCF7 control and E6 cells to PARPi subsequent to radiation in vivo, MCF7, and MCF7/E6 tumors were established in nude mice and the animals were randomly divided into one of four treatment arms: control animals receiving no treatment, animals receiving a single dose of 3 Gy radiation, animals receiving 25 mg/kg ABT-888 daily for 5 days, and animals receiving a single dose of 3 Gy radiation followed by daily treatments of 25 mg/kg ABT-888 for 5 days commencing 24 hours after radiation treatment. In animals bearing MCF7 control tumors, the combination treatment of radiation plus PARPi resulted in a significant reduction in tumor volume relative to mice in either monotherapy arm (P < 0.05; Fig. 2A). Treatment with radiation alone or with ABT-888 alone did not result in a significant reduction in tumor volume relative to untreated controls (P < 0.05; Fig. 2A). In mice bearing MCF7/E6 tumors, radiation plus PARPi combination therapy did not result in a significant change in tumor volume relative to untreated animals or animals treated with either of the monotherapy arms suggesting that p53 function is essential for in vivo sensitization of MCF7 tumors to PARPi following IR (Fig. 2B). To determine whether BRCA1 translocation to the cytosol is indeed depends on p53 in tumors in vivo following IR, we harvested representative tumors 24 hours postirradiation and assessed BRCA1 subcellular localization by immunofluorescence. In agreement with our in vitro results, in control MCF7 tumors, treatment with IR resulted in a significant shift in localization of BRCA1 from the nucleus to the cytoplasm 24 hours after treatment (Fig. 2C; Supplementary Fig. S2A). In contrast, cytoplasmic sequestration of BRCA1 following IR treatment was not observed in MCF7/E6 tumors (Fig. 2D; Supplementary Fig. S2B) confirming that p53 function is essential for translocation of BRCA1 to cytosol in response to IR providing a mechanism for subsequent sensitization of MCF7 tumors to PARPi after IR treatment in vivo.

Decreased HR function is a prerequisite for synthetic lethality to PARP1 inhibitors. We hypothesized that p53-depenent nuclear export of BRCA1 following radiation inhibited HR function in these cells to increase their sensitivity to PARPi. To test this hypothesis, the efficiency of HR-mediated DSB repair was analyzed in MCF7 cells carrying a stably chromosomal integrated, HR-specific reporter system that contains in tandem repeat two differently mutated GFP genes. Expression of I-SceI in these cells generates a DSB within the reporter that, when repaired only by HR, will result in expression of GFP that can be detected and quantified by flow cytometry. Knockdown of p53 by siRNA (siP53), which causes BRCA1 nuclear accumulation and resistance to IR-induced BRCA1 nuclear export (15, 20), resulted in a significant increase in HR repair in MCF-siP53 cells with or without 4 Gy radiation (Fig. 3A; Supplementary Fig. S3A). Importantly, MCF7-siP53 cells had approximately 2-fold higher HR efficiency than control MCF7-siCtrl cells regardless of IR treatment. This demonstrated that p53 knockdown promotes HR function even after radiation, most likely due to the accumulation of nuclear BRCA1 (15, 20). Given that p53 is typically mutated, rather than deleted, in cancer, as an additional test, we expressed a tumor-associated, transcriptional activity–deficient mutant form of p53 (R273H) that we have previously demonstrated act in a dominant-negative fashion to inhibit nuclear export of BRCA1 (20). Expression of R273H significantly increased HR repair relative to vector control cells in both cells with or without 4 Gy radiation (Fig. 3B; Supplementary Fig. S3B) and R237H-expressing cells that were treated 4 Gy radiation had equivalent HR activity to unirradiated vector-only cells again demonstrating that p53′s function in BRCA1 nuclear export following DNA damage is important for reduced HR function and increased sensitivity to PARPi.

To determine whether these observed changes in p53-mediated HR activity correlated with sensitivity to PARPi, we performed in vitro clonogenic survival assays utilizing MCF7 treated with control siRNA or siRNA targeting p53. These cells were first treated with 0 or 4 Gy IR then treated 24 hours later with 0, 5, 7.5, or 10 μmol/L of ABT-888 (Fig. 3C). In agreement with our earlier data, knockdown of p53 partially offset the sensitization to ABT-888 achieved by pretreatment with radiation in these cells. In complementary experiments, expression of dominant-negative R273H mutant-p53 also partially, but significantly (P < 0.05) nullified sensitization to ABT-888 following pretreatment with 4 Gy radiation (Fig. 3D).

Our studies to date have examined the sensitization of HR-proficient breast cancer cells to the cytotoxic effects of PARPi following IR-mediated cytosolic sequestration of BRCA1. However, this strategy should be applicable to a wide variety of tumors. Glioblastomas (GBM) are highly aggressive primary brain tumors with poor prognoses and few treatment options. BRCA mutations are rarely reported with malignant gliomas and, while PARPi are currently being evaluated in novel combination therapies for the treatment of GBM and other brain malignancies (28–30), strategies utilizing PARPi have not yet proven effective for treatment of these tumors. To evaluate the role of BRCA1 in HR-mediated repair in malignant glioma cells, we analyzed the expression and function of BRCA1 in the GBM cell line U87. As demonstrated in Fig. 4A, U87 cells express BRCA1 at a level similar to MCF7 breast cancer cells and Western blot analysis of protein lysates from both MCF-7 and U87 show a shift in the molecular weight of BRCA1 following treatment with 4 Gy IR indicative of increased phosphorylation of functional BRCA1 following IR that has been reported previously (15, 31). Next we examined whether knockdown of BRCA1 decreased HR-mediated repair in U87 cells. Similar to our findings in the MCF7 breast cancer cell line, silencing of BRCA1 in U87 cells (Fig. 4B) decreased Rad51 foci (Fig. 4C) and increased persistent γH2AX foci (Fig. 4D) both indicators of impaired HR-mediated repair of radiation-induced double strand breaks. To determine whether deficient HR-mediated DNA repair following silencing of BRCA1 rendered GBM cells more sensitive to PARPi, we compared the surviving fractions of U87 cells treated with either control siRNA or siBRCA1 then treated with 0, 5, 10, or 15 μmol/L ABT-888 (Fig. 4E). Knockdown of BRCA1 significantly increased the sensitivity of U87 cells to ABT-888 confirming that U87 cells can be sensitized to the cytotoxic effects of PARPi after inhibition of HR-mediated DNA repair.

Having established that loss of BRCA1 sensitizes GBM cells to PARPi, we next sought to determine whether GBM cells could be sensitized to PARPi following IR-induced cytoplasmic sequestration of BRCA1. To determine whether radiation initiates translocation of BRCA1 from the nucleus to the cytoplasm in GBM cells, we treated U87 cells with 0 or 4 Gy IR and examined the subcellular localization of BRCA1 by Western blot analysis of nuclear and cytoplasmic protein fractions (Fig. 5A). Treatment with IR caused a significant translocation of BRCA1 from the nucleus to the cytoplasm 24 hours post-IR treatment. Cytoplasmic translocation of BRCA1 in U87 was confirmed by examining the subcellular localization of immunofluorescent stained BRCA1 following IR treatment (Fig. 5B). To determine whether IR-mediated cytoplasmic translocation of BRCA1 decreased DNA repair efficiency and increased PARPi cytotoxicity in U87 GBM cells, we assessed persistent DNA damage by the number of γH2AX foci in cells pretreated with 0 or 4 Gy IR and then treated with 10 μmol/L ABT-888 or vehicle 24 hours postradiation. Pretreatment with IR significantly increased γH2AX foci 24 hours after ABT-888 treatment relative to cells treated with ABT-888 but no radiation (Fig. 5C). To determine more specifically whether HR-mediated DNA repair is compromised in U87 cells following IR-induced BRCA1 cytoplasmic sequestration, we examined the number of Rad51 foci in cells treated initially with 0 or 4 Gy IR and then treated with 10 μmol/L ABT-888 or vehicle 24 hours postradiation. Rad51 foci were tallied 24 hours after treatment with ABT-888 or vehicle. In cells that were not treated with radiation, ABT-888 significantly increased Rad51 foci indicative of active DNA repair in response to PARPi-mediated damage in these cells (Fig. 5D). In contrast, in cells pretreated with radiation, no increase in Rad51 foci is seen following PARPi treatment indicating decreased HR repair capability. These results suggest that, in U87 GBM cells, pretreatment with IR results in increased persistent DNA damage after PARPi due to decreased HR-mediated DNA repair capacity. We next analyzed the change in PARPi toxicity in U87 cells following IR treatment in vitro utilizing colony formation assays. U87 cells were first treated with 0 or 4Gy IR and then 24 hours later treated with 0, 5, 10, or 15 μmol/L PARPi. Pretreatment of U87 cells with IR significantly reduced the surviving fraction of cells at each concentration of ABT-888 tested (Fig. 5E). To test sensitization of U87 cells to PARPi following IR treatment in vivo, we first established U87 tumors in the flanks of Nu/Nu mice. Once the tumors reached an average volume of approximately 100 mm³, the mice were divided into four treatment arms: control animals receiving no treatment, mice receiving a single dose of 4 Gy IR, mice receiving 25 mg/kg ABT-888 daily for 5 consecutive days, or mice receiving a single dose of 4 Gy IR and 25 mg/kg ABT-888 daily for 5 consecutive days starting 24 hours after the radiation treatment. While a single dose of radiation or five daily treatments of ABT-888 alone failed to significantly alter the growth of U87 tumors relative to control tumors, the combination of ABT-888 and radiation significantly reduced the growth of these tumors relative to control tumors or tumors from either single treatment arm (Fig. 5F). These results demonstrate that pretreatment with a suboptimal dose of IR can sensitize GBM cells to PARPi cytotoxicity in vivo.

We demonstrated earlier that p53 function is required for BRCA1 cytosolic sequestration and increased PARPi cytotoxicity in MCF7 breast cancer cells. We thus hypothesized that functional p53 is also required for sensitization of malignant glioma cells to PARPi cytotoxicity following IR. Given the frequency of p53 mutations in cancer, it is critical to evaluate p53 as a universal determinate of HR deficiency following IR to precisely define those tumors may most benefit from PARPi following IR. Rather than utilizing HPV16-E6 to degrade p53 protein, we utilized siRNA against p53 to transiently manipulate p53 expression in U87 cells for this set of experiments. U87 cells were first treated with siRNA to p53 or a nontargeting control siRNA. Cells were then irritated with 0 or 4 Gy IR 24 hours later, and after an additional 24 hours were exposed to 0, 5, 10, 15, or 20 μmol/L ABT-888. The surviving cells were then assessed by their ability to form colonies. In U87 cells treated with nontargeting control siRNA, exposure to 4 Gy IR prior to PARPi significantly sensitized these cells to the cytotoxic effects of PARPi at all doses tested (Fig. 6A). In contrast, in U87 cells treated with siRNA to p53, exposure to 4 Gy IR did not significantly sensitize the cells to subsequent treatment with PARPi at any dose of ABT-888 tested (Fig. 6B). We confirmed these findings utilizing a second malignant glioma cell line, SF767, which, like the U87 line, is wild-type for BRCA1 and p53. In these cells, p53 expression was stably manipulated by shRNA. In agreement with our findings in U87 cells, SF767 cells expressing a control nontargeting shRNA were significantly sensitized to PARPi following a single dose of 4 Gy IR particularly at the lower doses of ABT-888 tested (Fig. 6C). In SF767 cells treated with shRNA to p53, much like our observations in U87 cells treated with siRNA to p53, exposure to 4 Gy IR did not sensitize the cells to subsequent treatment with PARPi at any dose of ABT-888 tested (Fig. 6D). These results demonstrate, in two independent glioma cell lines, that p53 expression is required for increased cytotoxicity to PARPi following IR in vitro. To confirm these results in vivo, SF767 shCtrl or shp53 tumors were established in Nu/Nu mice and grown to a volume of approximately 100 mm³. Tumor-bearing mice were then divided into four treatment arms: control mice receiving no treatment, mice receiving a single dose of 4 Gy IR, mice receiving 25 mg/kg ABT-888 daily for 5 consecutive days, or mice receiving a single dose of 4 Gy IR in combination with 25 mg/kg ABT-888 daily for 5 consecutive days starting 24 hours after radiation treatment. In mice bearing SF767 shCtrl tumors, neither treatment with a single suboptimal dose of IR nor treatment with PARPi alone reduced the relative tumor volume compared with untreated tumors (Fig. 6E). However, in SF767 shCtrl tumor-bearing mice the combination treatment of IR followed by ABT-888 did significantly reduce the relative tumor volume compared with either control tumors or tumors from either single treatment arm (Fig. 6E). In contrast, in mice with SF767 shp53 tumors, IR plus PARPi combination therapy did not result in a significant change in tumor volume relative to control animals or animals in either of the mono-therapy arms (Fig. 6F). These results illustrate that p53 is essential for sensitization of cancer cells to PARPi cytotoxicity following IR.

PARPi are an effective, well-tolerated therapy for treatment of cancers with familial BRCA1/2 mutations (2). However, the clinical efficacy of PARPi has been greatly limited by the relative rarity of BRCA1/2 mutations (3, 4). We previously demonstrated a treatment paradigm utilizing IR to trigger cytoplasmic translocation of BRCA1 and inhibition of HR, which subsequently renders breast cancer cells susceptible to PARPi toxicity (8). Here we demonstrate that this treatment scheme may be applicable to a wide range of HR-proficient tumors including malignant gliomas. Importantly, given the fact that p53 is frequently mutated and dysfunctional in solid tumors, we establish that p53 function is essential for IR-induced inhibition of BRCA1 nuclear function in HR repair and subsequent sensitization of cancer cells to the synthetic lethality of PARPi. These results provide a treatment strategy that potentially increases the population of cancer patients for whom PARPi therapy may be efficacious and provides p53 as a putative biomarker to more precisely define the benefiting population.

As a single-agent therapy, PARPi efficacy is currently limited to tumors with BRCA1/2 mutations or tumors that exhibit “BRCAness,” that is, phenotypes commonly associated with BRCA1/2–mutant tumors (32, 33). PARPi combinatorial therapies have been developed with the goals of increased cytotoxicity in susceptible tumors and to expand the efficacy of PARPi beyond BRCA1/2-associated cancers (34, 35). In pursuit of these goals, PARPi have been combined with a variety of DNA-damaging agents including temozolomide, cisplatin, carboplatin, and topotecan in efforts to determine which combinations best improve tumor cytotoxicity (36–41). While there is evidence, including the results of two recent clinical trials (42, 43) that these combinations may provide benefit to patients, particularly those with HR-deficient tumors, identifying the patients that would most benefit from addition of PARPi remains elusive (44). A common limitation of the aforementioned combinations is the utilization of PARPi primary as a sensitizing agent with the hope that decreased single-strand break repair will increase the cytotoxicity of DNA-damaging agents. The unfortunate short fall of this strategy is that cancer cells with intact HR repair can efficiently repair the ensuing double-strand breaks to circumvent cell death. Recognizing that these strategies fail to tap into the synthetic lethality of PARPi seen in HR-deficient tumors we developed a treatment paradigm to create an artificial HR-deficiency and render increased sensitivity to PARPi. Future studies are warranted to determine whether other DNA-damaging agents such as temozolomide or cisplatin can be combined with our treatment paradigm to further increase the cytotoxic response without unwanted off-target toxicity.

Recent work has expanded the potential use of PARPi through identification of additional mutations in DNA repair pathways such as PTEN, RAD51, and ATM mutations which, like BRCA1/2 mutations, might predict PARPi sensitivity (45–49). The success of this approach was recently highlighted by the finding that mutations in any of 12 DNA repair genes predict response of metastatic castrate-resistant prostate cancer to olaparib (50). While these important studies have succeeded in identifying additional patient populations that are intrinsically sensitive to PARPi toxicity, the benefiting population is still limited to patients harboring a defined set of mutations that compromise HR-mediated DNA repair much like those with BRCA1/2 mutations. Our treatment strategy creates an artificial HR deficiency, and thus, is not constrained to use in tumors with endogenous DNA repair defects that should significantly increase the percentage of patients that may benefit. Additional studies are warranted to determine whether the response to our treatment paradigm is enhanced in tumors that harbor mutations in other DNA repair genes.

Our previous studies revealed that functional p53 is essential for translocation of BRCA1 to the cytoplasm following IR. Here we expand upon this finding to demonstrate that functional p53 is a critical determinant of sensitization to PARPi following IR. We employ several independent and complementary methods including siRNA, shRNA, and E6-mediated p53 knockdown to demonstrate that p53 is required for PARPi sensitization after IR. While p53 is typically mutated rather than lost outright in cancer, we have previously shown that p53 mediates BRCA1 nuclear export via protein–protein binding, rather than by modulation of its transcription. We have also shown that R273H a common p53 dominant-negative mutation demonstrates impaired binding to BRCA1 and is defective in regulating IR-induced BRCA1 cytoplasmic translocation (15, 20). While some p53 mutations, such as H179Q, retain their ability to bind with and export BRCA1 to the cytoplasm (15, 20) the ability of any specific p53 mutant to retain its BRCA1 shuttling function will need to be determined experimentally. It is also possible that p53 mutations that demonstrate impaired p53 binding may play an important role in resistance to PARPi in cancers with wtBRCA1. Future studies will address whether these p53 mutants are enriched in the tumors that grow following our treatment strategy. p53 dysfunction is typically accompanied by increased p53 protein stability and nuclear accumulation and we have demonstrated that p53 nuclear accumulation correlates with BRCA1 nuclear accumulation (15, 20). Future studies will determine whether elevated p53 nuclear accumulation can be used as a biomarker screen to identify tumors that are unlikely to benefit from PARPi following IR due to p53 dysfunction.

PARPi were first introduced to the clinic in 2003 and have since been developed as both a single-agent therapy and as combination therapy in conjunction with other cytotoxic agents (2, 51). PARPi directly inhibit the catalytic function of PARP1/2 and for some PARPi, such as veliparib/ABT-888, this is the major mode of activity (52). However, other PARPis, such as olaparib and niraparib, are also capable of trapping PARP upon DNA interfering with DNA replication leading to a second mechanism of cytotoxicity (52). Veliparib was employed in our studies as it has proven to be efficacious in the treatment of BRCA1/2-associated tumors, can cross the blood–brain barrier making it suitable for the treatment of GBM and brain metastases, and it exhibits little PARP trapping ability simplifying the mechanism of action (52, 53). The results presented here should be representative of those that can be achieved with other PARPi that directly inhibit PARP1/2 catalytic function. Additional studies are needed to determine whether PARPi with greater PARP-trapping ability are more efficacious in our treatment strategy or result in increased unwanted toxicity in vivo.

Combined, our data demonstrate that IR-induced cytoplasmic sequestration of BRCA1 can be used to sensitize a variety of cancer models to PARPi in vitro and in vivo and reveal that functional p53 is necessary for this sensitization. These results have the potential to significantly increase the potential population of patients that may benefit from PARPi. Future studies will focus on the molecular mechanisms of p53 regulation of BRCA1 nuclear/cytoplasmic shuttling and the translation of these findings to the clinical setting and determine the spectrum of p53 mutations that are defective in BRCA1 shuttling may potentially be used as biomarkers to precisely identify the patient population that is most likely to benefit from this treatment paradigm.

No potential conflicts of interest were disclosed.

Conception and design: S.T. Sizemore, A. Chakravarti, F. Xia

Development of methodology: S.T. Sizemore, M.A. Rahman, A. Chakravarti, F. Xia

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S.T. Sizemore, G.M. Sizemore, S. Nowsheen, H. Yu

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S.T. Sizemore, S. Nowsheen, H. Yu, A. Chakravarti, F. Xia

Writing, review, and/or revision of the manuscript: S.T. Sizemore, G.M. Sizemore, M.C. Ostrowski, F. Xia

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S.T. Sizemore, H. Yu, A. Chakravarti, F. Xia

Study supervision: S.T. Sizemore, M.C. Ostrowski, A. Chakravarti, F. Xia

This work was supported by research grants R01 CA188500 (to F. Xia and A. Chakravarti), R01 CA163838 (to F. Xia) from the NIH, and 81XWH-08-1-0571 (to F. Xia) from the Department of Defense.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.