Strategy For Radiosensitization Research Articles

Inhibiting DNA-PKCS radiosensitizes human osteosarcoma cells

Osteosarcoma survival rate has not improved over the past three decades, and the debilitating side effects of the surgical treatment suggest the need for alternative local control approaches. Radiotherapy is largely ineffective in osteosarcoma, indicating a potential role for radiosensitizers. Blocking DNA repair, particularly by inhibiting the catalytic subunit of DNA-dependent protein kinase (DNA-PKCS), is an attractive option for the radiosensitization of osteosarcoma. In this study, the expression of DNA-PKCS in osteosarcoma tissue specimens and cell lines was examined. Moreover, the small molecule DNA-PKCS inhibitor, KU60648, was investigated as a radiosensitizing strategy for osteosarcoma cells in vitro. DNA-PKCS was consistently expressed in the osteosarcoma tissue specimens and cell lines studied. Additionally, KU60648 effectively sensitized two of those osteosarcoma cell lines (143B cells by 1.5-fold and U2OS cells by 2.5-fold). KU60648 co-treatment also altered cell cycle distribution and enhanced DNA damage. Cell accumulation at the G2/M transition point increased by 55% and 45%, while the percentage of cells with >20 γH2AX foci were enhanced by 59% and 107% for 143B and U2OS cells, respectively. These results indicate that the DNA-PKCS inhibitor, KU60648, is a promising radiosensitizing agent for osteosarcoma.

Abstract 519: Combined inhibition of WEE1 and PARP1 radiosensitizes KRAS mutant non-small cell lung cancers via inhibition of DSB repair

Abstract Mutant KRAS is found in approximately 30% of non-small cell lung cancers (NSCLC) and is associated with poor prognosis. Despite the use of radiation (RT) therapy for the treatment of locally advanced disease, local recurrence remains an issue. The observation that mutations in KRAS lead to both replication stress and DNA double-strand breaks (DSBs) suggests these cancers may have a greater reliance on DNA damage response pathways and therefore may be preferentially radiosensitized by therapies targeting DNA repair. In this study, we investigated the combined inhibition of WEE1 and PARP1 using the small molecule inhibitors, AZD1775 and olaparib, respectively as a radiosensitizing strategy in KRAS mutant NSCLC. We began by comparing radiosensitization by AZD1775+olaparib, in KRAS isogenic H1703 lung cancer cells and found that KRAS mutant cells were preferentially radiosensitized (ER, enhancement ratio 1.8) compared to KRAS wild type cells (ER 1.4). Combined WEE1 and PARP1 inhibition also radiosensitized KRAS mutant Calu-6 and NCI-H23 lung cancer cells (ER 1.9 and 1.5, respectively). These findings were further confirmed in vivo: Calu-6 tumor xenografts were significantly radiosensitized by AZD1775+olaparib, as evidenced by an 11 day delay in tumor volume doubling time relative to RT treatment. Given that WEE1 and PARP1 function to prevent and manage replication stress, respectively, we hypothesized that radiosensitization by AZD1775+olaparib results from persistent replication stress. While replication stress did contribute to AZD1775-mediated radiosensitization in Calu-6 cells, as evidenced by pan-nuclear γH2AX staining, and the ability of exogenous nucleosides to protect cells from radiosensitization by AZD1775 alone, nucleosides had little effect on radiosensitization by AZD1775+olaparib. These results suggest that replication stress is not required for radiosensitization by AZD1775+olaparib. As WEE1 and PARP1 both promote repair of radiation-induced DNA damage, we hypothesized that radiosensitization by AZD1775+olaparib results from persistent, unrepaired DSBs. Assessment of the kinetics of DSB repair by γH2AX flow cytometry demonstrated that while total γH2AX levels in cells treated with RT alone had returned to control levels within 24 h, AZD1775+olaparib treatment significantly delayed the resolution of γH2AX following RT, with 44.8% or 46.2% of Calu-6 or KRAS mutant H1703 cells, respectively remaining γH2AX-positive 24 h post-RT. This delay corresponded with the inhibition of radiation-induced RAD51 foci by AZD1775, indicating inhibition of homologous recombination repair. Taken together these data demonstrate the efficacy of combined inhibition of WEE1 and PARP1 with radiation in KRAS mutant lung cancer. Furthermore, these results suggest that although replication stress occurs in response to AZD1775 and olaparib, persistent DSBs are the cause of radiosensitization. Citation Format: Leslie A. Parsels, David Karnak, Joshua D. Parsels, Zachery Reichert, Jonathan Maybaum, Theodore S. Lawrence, Meredith Ann Morgan. Combined inhibition of WEE1 and PARP1 radiosensitizes KRAS mutant non-small cell lung cancers via inhibition of DSB repair. [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 519.

Overexpression of Inhibitor of Growth 4 Enhances Radiosensitivity in Non-Small Cell Lung Cancer Cell Line SPC-A1.

Inhibitor of growth 4 is a member of the inhibitor of growth family proteins, which is involved in cell apoptosis, migration, invasion, and cell cycle progress. In this study, we investigated the inhibitor of growth 4 level in non-small cell lung cancer tissues and explored the antitumor activity of inhibitor of growth 4 in vitro and in vivo using non-small cell lung cancer cell line SPC-A1 and its underlying molecular mechanisms. We also explored its role on the radiosensitivity in SPC-A1 cells. The level of inhibitor of growth 4 protein was significantly decreased in 28 cases of non-small cell lung cancer tissues in comparison with corresponding noncancerous lung epithelial tissues. Upregulation of inhibitor of growth 4 by plasmid pcDNA3.1-ING4 delivery could suppress proliferation and increase apoptosis of SPC-A1 cells both in vitro and in vivo. Additionally, we found that overexpression of inhibitor of growth 4 in SPC-A1 cell line could lead to a higher Bcl-2/Bax ratio, which might be an important factor in the apoptosis regulation. Furthermore, overexpression of inhibitor of growth 4 enhanced the radiosensitivity of SPC-A1 cells to irradiation. Inhibitor of growth 4 upregulation plus radiotherapy induced synergistic tumor suppression in SPC-A1 xenografts implanted in athymic nude mice. Thus, the restoration of inhibitor of growth 4 function might provide a potential strategy for non-small cell lung cancer radiosensitization.

Dual PI3K- and mTOR-inhibitor PI-103 can either enhance or reduce the radiosensitizing effect of the Hsp90 inhibitor NVP-AUY922 in tumor cells: The role of drug-irradiation schedule.

Inhibition of Hsp90 can increase the radiosensitivity of tumor cells. However, inhibition of Hsp90 alone induces the anti-apoptotic Hsp70 and thereby decreases radiosensitivity. Therefore, preventing Hsp70 induction can be a promising strategy for radiosensitization. PI-103, an inhibitor of PI3K and mTOR, has previously been shown to suppress the up-regulation of Hsp70. Here, we explore the impact of combining PI-103 with the Hsp90 inhibitor NVP-AUY922 in irradiated glioblastoma and colon carcinoma cells. We analyzed the cellular response to drug-irradiation treatments by colony-forming assay, expression of several marker proteins, cell cycle progression and induction/repair of DNA damage. Although PI-103, given 24 h prior to irradiation, slightly suppressed the NVP-AUY922-mediated up-regulation of Hsp70, it did not cause radiosensitization and even diminished the radiosensitizing effect of NVP-AUY922. This result can be explained by the activation of PI3K and ERK pathways along with G1-arrest at the time of irradiation. In sharp contrast, PI-103 not only exerted a radiosensitizing effect but also strongly enhanced the radiosensitization by NVP-AUY922 when both inhibitors were added 3 h before irradiation and kept in culture for 24 h. Possible reasons for the observed radiosensitization under this drug-irradiation schedule may be a down-regulation of PI3K and ERK pathways during or directly after irradiation, increased residual DNA damage and strong G2/M arrest 24 h thereafter. We conclude that duration of drug treatment before irradiation plays a key role in the concomitant targeting of PI3K/mTOR and Hsp90 in tumor cells.

FGFR1 Induces Glioblastoma Radioresistance through the PLCγ/Hif1α Pathway.

FGF2 signaling in glioblastoma induces resistance to radiotherapy, so targeting FGF2/FGFR pathways might offer a rational strategy for tumor radiosensitization. To investigate this possibility, we evaluated a specific role for FGFR1 in glioblastoma radioresistance as modeled by U87 and LN18 glioblastomas in mouse xenograft models. Silencing FGFR1 decreased radioresistance in a manner associated with radiation-induced centrosome overduplication and mitotic cell death. Inhibiting PLCγ (PLCG1), a downstream effector signaling molecule for FGFR1, was sufficient to produce similar effects, arguing that PLCγ is an essential mediator of FGFR1-induced radioresistance. FGFR1 silencing also reduced expression of HIF1α, which in addition to its roles in hypoxic responses exerts an independent effect on radioresistance. Finally, FGFR1 silencing delayed the growth of irradiated tumor xenografts, in a manner that was associated with reduced HIF1α levels but not blood vessel alterations. Taken together, our results offer a preclinical proof of concept that FGFR1 targeting can degrade radioresistance in glioblastoma, a widespread problem in this tumor, prompting clinical investigations of the use of FGFR1 inhibitors for radiosensitization. Cancer Res; 76(10); 3036-44. ©2016 AACR.

AZD2014 Radiosensitizes Oral Squamous Cell Carcinoma by Inhibiting AKT/mTOR Axis and Inducing G1/G2/M Cell Cycle Arrest.

BackgroundOral squamous cell carcinoma (OSCC) is one of the most common malignant neoplasms in Taiwan. Activation of the mTOR signaling pathway has been linked to decreased radiation responsiveness in human oral cancer, thus it limits efficacy of radiotherapy. To address this question, we investigated the effect of AZD2014, a novel small molecular ATP-competitive inhibitor of mTORC1 and mTORC2 kinase, as a radiosensitizer in primary OSCC and OSCC-derived cell line models.MethodsWe isolated primary tumor cells from OSCC tissues and cell lines. AZD2014 was administered with and without ionizing radiation. The radiosensitizing effect of AZD2014 were then assessed using cell viability assays, clonogenic survival assays, and cell cycle analyses. Western blotting was used to detect protein expression.ResultsCombination treatment with AZD2014 and irradiation resulted in significant reduction in OSCC cell line and primary OSCC cell colony formation due to the enhanced inhibition of AKT and both mTORC1 and mTORC2 activity. Pre-treatment with AZD2014 in irradiated oral cancer cells induced tumor cell cycle arrest at the G1 and G2/M phases, which led to disruption of cyclin D1-CDK4 and cyclin B1-CDC2 complexes. Moreover, AZD2014 synergized with radiation to promote both apoptosis and autophagy by increasing caspase-3 and LC3 in primary OSCC cells.ConclusionsThese findings suggest that in irradiated OSCC cells, co-treatment with AZD2014, which targets mTORC1 and mTORC2 blockade, is an effective radiosensitizing strategy for oral squamous cell carcinoma.

Abstract P6-13-05: Androgen receptor (AR): A novel target and mechanism for radiosensitization and treatment in triple-negative breast cancers (TNBC)

Abstract Background: Increased rates of locoregional recurrence have been observed in TNBC despite chemotherapy and radiation (RT). Thus, approaches that result in radiosensitizaton in TNBC are critically needed. We characterized the RT response of 21 breast cancer cell (BCC) lines using clonogenic survival assays. We paired this with high-throughput drug screen data to identify AR as a top target for radiosensitization and assess AR inhibition as a radiosensitization strategy for TNBC. Methods: Clonogenic survival assays were used to determine the intrinsic RT sensitivity of 21 BCC lines. IC50 values were determined for 130 clinically available compounds and correlation coefficients were calculated using IC50 values and SF-2Gy. Gene expression was measured using RNA Seq and protein expression was measured using RPPA arrays in human tumor samples (n=2,061) and BCC lines (n=51). AR function was assessed using siRNA knockdown or functional inhibition with MDV3100 (enzalutamide). We measured in vivo tumor growth with varying control and treatment groups (16-20 tumors/group). Kaplan-Meier analysis was performed to estimate local control and survival. A Cox proportional hazards model was used to identify factors of survival, and MVA was used to determine variables associated with LRF survival. Results: Our radiosensitizer screen nominated bicalutamide as a most effective drug in treating RT-resistant BCC lines (R2= 0.46, p-value <0.01). We interrogated the expression of AR in >2000 human breast tumor samples and found significant heterogeneity in AR expression with enrichment of expression at the protein and RNA level in TNBC. This same heterogeneity was also identified in human BCC lines. There was a strong correlation between AR RNA expression and protein expression (R2= 0.72, p <0.01). Inhibition of AR using both siRNA and MDV3100 (enzalutamide) induced radiation sensitivity in vitro with an enhancement ratio (ER) of 1.35-1.42 in AR-positive TNBC lines. No such radiosensitization was seen in AR-negative TNBC or ER-positive, AR-negative BCC lines. Radiosensitization was at least partially dependent on impaired dsDNA break repair mediated by DNAPKcs. AR inhibition either with MDV3100 significantly radiosensitized TNBC xenografts in mouse models and markedly delayed tumor tripling time and tumor growth (median tumor tripling time 17.4 days for RT alone vs. not reached after 50 days for MDV3100 + RT, p-value <0.001). Biomarker analysis identifies DNAPKcs as a potential biomarker of response. Clinically, analyses of patients with TNBC showed that patients whose tumors had higher than median expression of AR had markedly higher rates of LR after RT (HR for LR 2.9-3.2, p-value <0.01, 2 independent datasets). There was no difference in LR in TNBC patients not treated with RT when stratified by AR expression. In MVA, AR expression was the variable most significantly associated with worse LRF survival after RT (HR of 3.58;p-value < 0.01). Conclusion: Our results implicate AR as a mediator of radioresistance in breast cancer and support the rationale for developing clinical strategies, including clinical trials, to inhibit AR as a novel radiosensitizing target in TNBC. Citation Format: Speers C, Zhao SG, Liu M, Rae JM, Hayes DF, Feng FY, Pierce LJ. Androgen receptor (AR): A novel target and mechanism for radiosensitization and treatment in triple-negative breast cancers (TNBC). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-13-05.

The Role of Apollon Gene Silencing on Viablity and Radiosensitivity of Cervical Cancer Hela Cells

Cervical cancer is the second common cause of cancer deaths in women worldwide. Radioresistancy of cancer is a principal cause of treatment impairing. Inhibitor of apoptosis proteins (IAPs) widely block apoptosis against apoptotic stimuli, including current chemo- and radiation therapies. Apollon, a membrane of IAP, can support cells against apoptosis and is over expressed in some treatment-resistant cancer cells. The aim of this study was to evaluate the effects of apollon knockdown on induction of apoptosis and also its potential for enhancement of radiosensitvity on hela cells. plasmid encoding shRNA which has been confirmed its effect against apollon, transfected into hela cells. Consequent effects on the level of P53 , Bax and BAK analyzed by real time PCR. Apoptotic phenotype of transfected cells was monitored by Tunnel assay. Viability of hela cells after radiotherapy was analyzed by MTT assay. shRNA1 effectively increased transcription of p53, Bax and BAK and induced apoptosis phenotype of treated hela cells. Radiosensitivity of transfected cell was increased after knock-down of apollon obviously. Apollon knockdown induces apoptosis in hela cell . Also it can be as new molecular target for radio-sensitizing strategies in these cells. So, apollon can be a potentially considerable therapeutic object for cervical cancer.

Mouse models of radiation-induced glioblastoma.

Glioblastomas (GBM) are lethal brain tumors that can be triggered by exposure to ionizing radiation (IR), even at low doses from CT scans [1]. High doses of IR are also used to treat GBM, but the irradiated tumors inevitably recur. This raises the possibility that genomic changes induced by radiation may contribute not only to glioma initiation, but also to tumor recurrence. Thus, there is a compelling need for experimental model systems that recapitulate the process of radiation-induced gliomagenesis. Such models could not only help predict GBM-development risks from radiation exposure, but also help identify genetic alterations defining radiation-induced GBM, thereby facilitating the development of rational therapies for treating these recalcitrant tumors. Our study published in the journal Oncogene employed a systematic approach to develop sensitive mouse models that can be used to study radiation-induced gliomagenesis [2]. Ink4a, Ink4b and Arf are key tumor suppressor genes that are deleted in a majority of GBMs [3]. We utilized transgenic mice with brain-restricted deletions of these tumor suppressors, individually and in combination, and examined their susceptibility to IR-induced GBM development. The most deleterious lesion inflicted by IR is the DNA double-strand break (DSB). We have shown previously that accelerated ions (particle radiation) induce complex DSBs that are refractory to repair unlike the simple breaks induced by X-rays (electromagnetic radiation) which are repaired to completion [4]. Therefore, we intra-cranially irradiated these transgenic mice with either X-rays or accelerated Fe ions to understand the process of radiation-induced gliomagenesis, and how this may be influenced by DNA damage complexity. We found that these mice did not develop gliomas spontaneously, but were prone to GBM development after exposure to a single, moderate dose of radiation. Remarkably, we found that Fe ions were at least four-fold more effective than X-rays in inducing these tumors, thereby confirming that complex DSBs triggered by accelerated ions are more harmful than simpler breaks induced by X-rays. This finding has important implications as the use of particle radiation (such as protons and carbon ions) for cancer therapy is steadily increasing. Our work indicates that particle radiation could indeed turn out to be more effective than X-rays for tumor control, but this also raises the specter of increased likelihood of secondary cancers triggered by such radiation. Interestingly, while wild type mice did not develop gliomas upon radiation exposure, loss of Ink4a and Arf was sufficient to render these mice susceptible to IR-induced gliomas; additional loss of Ink4b significantly increased tumor incidence. These observations indicate that Ink4a, Ink4b and Arf act as key barriers to radiation-induced gliomagenesis, and confirms previous results from our laboratory and others implicating Ink4b as an important “backup” tumor suppressor for Ink4a [5]. One of the most interesting findings of our study came from multimodal analyses of the IR-induced tumors and neurosphere cultures derived thereof. We found amplification of the receptor tyrosine kinase Met to be the most prominent oncogenic alteration in these tumors. Met amplification was critical for transformation as well as for the maintenance of a cancer stem cell phenotype via upregulation of the re-programming transcription factor Sox2. Recent studies of other cancers show that MET amplification enables cancer cells to evolve and survive under therapeutic pressure, and that MET amplification confers radioresistance to cancer cells [6]. In light of these studies and our results, we speculate that radiotherapy of GBM could engender clones of MET-amplified cancer cells that drive tumor recurrence. If so, recurrent glioblastomas may be particularly vulnerable to radiosensitization strategies involving the use of MET inhibitors. We are currently validating additional transgenic models with brain-targeted deletions of other GBM-relevant tumor suppressor genes like p53 and Pten. The use of distinct yet complementary mouse models could prove to be very useful for analyzing the mechanistic underpinnings of radiation-induced gliomagenesis. For example, by crossing these mice with DNA repair-deficient mouse models, we hope to understand how specific DSB repair pathways act as barriers to gliomagenesis. In the future, this study could also have “far”-reaching implications for astronauts 100 million miles away on the surface of Mars. These models (along with models of other cancers) are being used in NASA-funded studies to understand cancer risks for Mars-bound astronauts from space radiation which consists of accelerated ions such as the Fe ions used in this study [7]. In sum, validation of these sensitive yet simple mouse models sets the stage for in-depth mechanistic studies of radiation-induced gliomagenesis which could lead to effective approaches for treating radiogenic cancers.

Abstract 3629: Balance shift between error-free and error prone DNA double-strand break repair as a novel mechanism of radiosensitization by nucleoside analogs

Abstract Nucleoside analogs (NAs) represent a large group of anticancer drugs, several members of which have been shown to sensitize cells to ionizing radiation (IR). Among the lesions induced in cells by IR, the DNA double-strand break (DSB) is considered to be the most detrimental. Therefore we focused on interactions between NAs and pathways of DSB repair. We opted for post-irradiation treatment with NAs to avoid complications arising from redistribution of cells throughout the cycle prior to IR exposure. We compared the effects of 4h treatments post-IR with vidarabine (ara-A), fludarabine (F-ara-A) or gemcitabine (dFdC) on the survival of A549 lung carcinoma cells. We found that post-irradiation treatment with ara-A and F-ara-A, but not dFdC, resulted in cell radiosensitization to killing. Radiosensitization by ara-A was superior to that by F-ara-A. We employed immunofluorescence detection of IR-induced foci and I-SceI-based reporter cell lines to identify the pathways of DSB repair that are inhibited by ara-A and cause the observed radiosensitization. We found that the formation of IR-induced Rad51-foci was compromised by ara-A suggesting inhibition of homologous recombination repair (HRR). Repair of I-SceI-induced DSBs by HRR was also suppressed by ara-A. Furthermore, A549 cells deprived of Rad51 by RNAi could not be efficiently radiosensitized by ara-A. On the other hand, we could not detect impairment by ara-A of non-homologous end-joining (NHEJ), as measured by pulsed-field gel electrophoresis (PFGE) at high doses of IR (20 Gy). Notably, we observed an increase in the frequency of mutagenic NHEJ events in several I-SceI-based repair reporter systems, indicative of processing by error-prone repair pathways such as alternative/backup NHEJ (B-NHEJ). In addition, we could show that treatment with ara-A induces a dramatic increase in B-NHEJ in plateau phase Lig4-/- MEFs. Treatment with F-ara-A also inhibited HRR, but was less proficient in promoting mutagenic NHEJ. We conclude that inhibition of HRR and promotion of B-NHEJ represent two important mechanisms of radiosensitization by NAs. The strong impact of ara-A on both of these repair pathways underpins its superior radiosensitizing potential. Targeting of both HRR and B-NHEJ may provide a promising new strategy for radiosensitization. Citation Format: George Iliakis, Simon Magin. Balance shift between error-free and error prone DNA double-strand break repair as a novel mechanism of radiosensitization by nucleoside analogs. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3629. doi:10.1158/1538-7445.AM2015-3629

Abstract B76: Targeting Mcl-1 for radiosensitization of pancreatic cancers

Abstract In order to identify targets whose inhibition may enhance the efficacy of chemoradiation in pancreatic cancer and, thus, improve survival in patients with locally advanced pancreatic cancer, we conducted an siRNA library screen of 8,800 genes. We identified Mcl-1 (myeloid cell leukemia-1), an anti-apoptotic member of the Bcl-2 family, as a target for sensitizing pancreatic cancer cells to gemcitabine-radiation. Thus, we investigated Mcl-1 inhibition by genetic (siRNA) and pharmacological (small molecule) approaches as a radiosensitizing strategy in pancreatic cancer cells. Mcl-1 depletion by siRNA radiosensitized BxPC-3, Panc-1, and MiaPaCa-2 cells with radiation enhancement ratios (RER) of 1.5±0.06, 1.4±0.03, and 1.3±0.01, respectively, and caused Caspase-3 activation and PARP cleavage. We next tested the ability of the recently identified, selective, small molecule inhibitor of Mcl-1, UMI-77 (Abulwerdi F et al., Mol Cancer Ther, 2014), to inhibit the anti-apoptotic functions of Mcl-1 and radiosensitize in pancreatic cancer cells. Consistent with inhibition of Mcl-1 anti-apoptotic function, UMI-77 (1-5uM) caused dissociation of Mcl-1 from the pro-apoptotic protein Bak. Under these same conditions, UMI-77 produced significant radiosensitization in BxPC-3 and Panc-1 cells (RER: 1.5±0.1 and 1.3±0.04, respectively; P<0.05) with minimal radiosensitization of MiaPaCa-2 cells (1.2±0.07). Since Mcl-1 expression levels are highest in BxPC-3 and Panc-1 cells and lowest in MiaPaCa-2 cells, these results suggest that elevated Mcl-1 protein levels may be a biomarker for radiosensitization by Mcl-1 inhibition. In addition, radiosensitization by UMI-77 was associated with Caspase-3 activation, PARP cleavage, and increased sub-G1 DNA content. In contrast, ABT-737, a well-established inhibitor of Bcl-2, Bcl-XL, and Bcl-w, failed to radiosensitize pancreatic cancer cells suggesting the unique importance of Mcl-1 in radiation survival. Taken together, these results validate Mcl-1 as a target for radiosensitization of pancreatic cancer cells and demonstrate the efficacy of small molecules which displace Mcl-1 from Bak/Bax as radiosensitizers in pancreatic cancer. Furthermore, these results support continued investigation of UMI-77 and its analogues in animal models and in combination with gemcitabine-radiation as a strategy for radiation and chemoradiation sensitization of pancreatic cancers. Citation Format: Qiang Zhang, Dongping Wei, Jason Schreiber, Tasneem Kausar, Theodore Lawrence, Yi Sun, Zaneta Nikolovska-Coleska, Meredith A. Morgan. Targeting Mcl-1 for radiosensitization of pancreatic cancers. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B76.

Abstract P6-03-08: Androgen receptor (AR): A novel target for radiosensitization and treatment in triple-negative breast cancers (TNBC)

Abstract Background: Increased rates of locoregional recurrence have been observed in TNBC despite the use of chemotherapy and radiation (RT). Thus, approaches that result in radiosensitizaton of TNBC are critically needed. We have previously characterized the radiation response of 21 breast cancer cell (BCC) lines using clonogenic survival assays. We now pair this data with high-throughput drug screen data available through cancer cell line encyclopedia studies to identify AR as a top target for radiosensitization and assess AR inhibition as a radiosensitization strategy for TNBC. Methods: Clonogenic survival assays were performed to determine the intrinsic RT sensitivity of 21 BCC lines (0-8 Gy RT). IC50 values were determined for 130 clinically available compounds and correlation coefficients were calculated using IC50 values (for drug sensitivity) and SF-2Gy (for radiation sensitivity). Gene expression was measured using Affymetrix microarrays and protein expression was measured using reverse-phase protein lysate arrays (RPPA) of human tumor samples (n=2,061) and BCC lines (n=51). AR function was assessed using siRNA knockdown or inhibition with MDV3100 (enzalutamide). Kaplan-Meier analysis was performed to determine the clinical impact of AR expression on local control and survival. A Cox proportional hazards model was constructed to identify potential factors of survival, and multivariate analysis was used to determine variables most significantly associated with LRF survival. Results: Our radiosensitizer screen nominated bicalutamide as one of the most effective drugs in treating radioresistant BCC lines (R2= 0.46, p-value <0.001). Recognizing that a subgroup of TNBC includes AR expressing tumors, we interrogated the expression of AR in >2000 human breast tumor samples and found signifi[not]cant heterogeneity in AR expression with an increase in TNBC (35% of tumors) compared to non-TNBC (28% of tumors). This same heterogeneity was also identified in human BCC lines. There was a strong correlation between AR RNA expression and protein expression (R2= 0.72, p <0.0001). Inhibition of AR using both siRNA and MDV3100 induced radiation sensitivity in vitro with an enhancement ratio (ER) of 1.35-1.42 in AR-positive TNBC lines. No such radiosensitization was seen in AR-negative TNBC or ER-positive, AR-negative BCC lines. Radiosensitization was at least partially dependent on impaired dsDNA break repair mediated by DNAPKcs. In vivo assessment of tumor growth inhibition with RT and anti-AR strategies are currently underway. Clinically, analyses of patients with TNBC showed that patients whose tumors had high expression of AR had markedly higher rates of LR after RT than patients with low expression of AR (HR for LR 2.9-3.2, p-value <0.01, 2 independent datasets). There was no difference in LR in TNBC patients not treated with RT when stratified by AR expression status. In multivariate analysis, AR expression was the variable most significantly associated with worse LRF survival after RT with a HR of 3.58 (p-value < 0.01). Conclusion: Our results implicate AR as a mediator of radioresistance in breast cancer and support the rationale for developing clinical strategies to inhibit AR as a novel radiosensitizing target in TNBC. Citation Format: Corey Speers, Shuang G Zhao, Meilan Liu, Joseph Evans, Prasanna Alluri, Daniel F Hayes, Felix Y Feng, Lori J Pierce. Androgen receptor (AR): A novel target for radiosensitization and treatment in triple-negative breast cancers (TNBC) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-03-08.

Targeting Mcl-1 for Radiosensitization of Pancreatic Cancers

In order to identify targets whose inhibition may enhance the efficacy of chemoradiation in pancreatic cancer, we previously conducted an RNAi library screen of 8,800 genes. We identified Mcl-1 (myeloid cell leukemia-1), an anti-apoptotic member of the Bcl-2 family, as a target for sensitizing pancreatic cancer cells to chemoradiation. In the present study we investigated Mcl-1 inhibition by either genetic or pharmacological approaches as a radiosensitizing strategy in pancreatic cancer cells. Mcl-1 depletion by siRNA produced significant radiosensitization in BxPC-3 and Panc-1 cells in association with Caspase-3 activation and PARP cleavage, but only minimal radiosensitization in MiaPaCa-2 cells. We next tested the ability of the recently identified, selective, small molecule inhibitor of Mcl-1, UMI77, to radiosensitize in pancreatic cancer cells. UMI77 caused dissociation of Mcl-1 from the pro-apoptotic protein Bak and produced significant radiosensitization in BxPC-3 and Panc-1 cells, but minimal radiosensitization in MiaPaCa-2 cells. Radiosensitization by UMI77 was associated with Caspase-3 activation and PARP cleavage. Importantly, UMI77 did not radiosensitize normal small intestinal cells. In contrast, ABT-737, an established inhibitor of Bcl-2, Bcl-XL, and Bcl-w, failed to radiosensitize pancreatic cancer cells suggesting the unique importance of Mcl-1 relative to other Bcl-2 family members to radiation survival in pancreatic cancer cells. Taken together, these results validate Mcl-1 as a target for radiosensitization of pancreatic cancer cells and demonstrate the ability of small molecules which bind the canonical BH3 groove of Mcl-1, causing displacement of Mcl-1 from Bak, to selectively radiosensitize pancreatic cancer cells.

Nanoparticles for Radiation Therapy Enhancement: the Key Parameters.

This review focuses on the radiosensitization strategies that use high-Z nanoparticles. It does not establish an exhaustive list of the works in this field but rather propose constructive criticisms pointing out critical factors that could improve the nano-radiation therapy. Whereas most reviews show the chemists and/or biologists points of view, the present analysis is also seen through the prism of the medical physicist. In particular, we described and evaluated the influence of X-rays energy spectra using a numerical analysis. We observed a lack of standardization in preclinical studies that could partially explain the low number of translation to clinical applications for this innovative therapeutic strategy. Pointing out the critical parameters of high-Z nanoparticles radiosensitization, this review is expected to contribute to a larger preclinical and clinical development.

Radiosensitization of gliomas by intracellular generation of 5-fluorouracil potentiates prodrug activator gene therapy with a retroviral replicating vector.

A tumor-selective non-lytic retroviral replicating vector (RRV), Toca 511, and an extended-release formulation of 5-fluorocytosine (5-FC), Toca FC, are currently being evaluated in clinical trials in patients with recurrent high-grade glioma (NCT01156584, NCT01470794, NCT01985256). Tumor-selective propagation of this RRV enables highly efficient transduction of glioma cells with cytosine deaminase (CD), which serves as a prodrug activator for conversion of the anti-fungal prodrug 5-FC to the anti-cancer drug 5-fluorouracil (5-FU) directly within the infected cells. We investigated whether, in addition to its direct cytotoxic effects, 5-FU generated intracellularly by RRV-mediated CD/5-FC prodrug activator gene therapy could also act as a radiosensitizing agent. Efficient transduction by RRV and expression of CD was confirmed in the highly aggressive, radioresistant human glioblastoma cell line U87-EGFRvIII and its parental cell line U87MG (U87). RRV-transduced cells showed significant radiosensitization even after transient exposure to 5-FC. This was confirmed both in vitro by a clonogenic colony survival assay and in vivo by bioluminescence imaging analysis. These results provide a convincing rationale for development of tumor-targeted radiosensitization strategies utilizing the tumor-selective replicative capability of RRV, and incorporation of radiation therapy into future clinical trials evaluating Toca 511 and Toca FC in brain tumor patients.

GDC-0032, A Novel p110α Inhibitor, Specifically Radiosensitizes Head and Neck Squamous Cell Carcinomas Harboring Activating PIK3CA Alterations

Recent genomic studies have demonstrated that mutation or amplification of PIK3CA is the most common oncogenic aberration in head and neck squamous cell carcinomas (HNSCCs). p110α, the protein product of the PIK3CA gene, drives downstream activation of PI3K signaling, a signaling pathway that is associated with radioresistance. We hypothesized that pharmacologic inhibition of p110α would be an effective strategy for tumor specific radiosensitization in cancers harboring these alterations.

Whole Genome RNAi Screens Identify USP9X Inhibition as a Strategy for NSCLC Radiosensitization

Whole Genome RNAi Screens Identify USP9X Inhibition as a Strategy for NSCLC Radiosensitization

Deoxycytidine kinase augments ATM-Mediated DNA repair and contributes to radiation resistance.

Efficient and adequate generation of deoxyribonucleotides is critical to successful DNA repair. We show that ataxia telangiectasia mutated (ATM) integrates the DNA damage response with DNA metabolism by regulating the salvage of deoxyribonucleosides. Specifically, ATM phosphorylates and activates deoxycytidine kinase (dCK) at serine 74 in response to ionizing radiation (IR). Activation of dCK shifts its substrate specificity toward deoxycytidine, increases intracellular dCTP pools post IR, and enhances the rate of DNA repair. Mutation of a single serine 74 residue has profound effects on murine T and B lymphocyte development, suggesting that post-translational regulation of dCK may be important in maintaining genomic stability during hematopoiesis. Using [18F]-FAC, a dCK-specific positron emission tomography (PET) probe, we visualized and quantified dCK activation in tumor xenografts after IR, indicating that dCK activation could serve as a biomarker for ATM function and DNA damage response in vivo. In addition, dCK-deficient leukemia cell lines and murine embryonic fibroblasts exhibited increased sensitivity to IR, indicating that pharmacologic inhibition of dCK may be an effective radiosensitization strategy.

PARP-1 inhibitors and radiotherapy sensitivity: future prospects for therapy?

SUMMARY Despite the efficacy of multimodality treatment for women with breast cancer, sustained locoregional control remains a significant issue. Efforts to identify effective targeted therapies for treatment of these patients have intensified in recent years. The PARP family of proteins represents one potential target. PARP-1 is a DNA repair enzyme that plays a critical role in base excision repair, homologous recombination, nonhomologous end joining and transcriptional regulation. In this review, we discuss the rationale for using PARP-1 inhibitors clinically, the role of PARP-1 in DNA damage repair and the potential clinical utility of using PARP-1 inhibitors as a radiosensitization strategy. We will also review the most relevant clinical trials using various PARP-1 inhibitors and the future of biomarker development to predict response to PARP-1 inhibition.

Inhibition of DNA double-strand break repair by the dual PI3K/mTOR inhibitor NVP-BEZ235 as a strategy for radiosensitization of glioblastoma.

Inhibitors of the DNA damage response (DDR) have great potential for radiosensitization of numerous cancers, including glioblastomas, which are extremely radio- and chemoresistant brain tumors. Currently, there are no DNA double-strand break (DSB) repair inhibitors that have been successful in treating glioblastoma. Our laboratory previously demonstrated that the dual phosphoinositide 3-kinase/mTOR inhibitor NVP-BEZ235 can potently inhibit the two central DDR kinases, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia-telangiectasia mutated (ATM), in vitro. Here, we tested whether NVP-BEZ235 could also inhibit ATM and DNA-PKcs in tumors in vivo and assessed its potential as a radio- and chemosensitizer in preclinical mouse glioblastoma models. The radiosensitizing effect of NVP-BEZ235 was tested by following tumor growth in subcutaneous and orthotopic glioblastoma models. Tumors were generated using the radioresistant U87-vIII glioma cell line and GBM9 neurospheres in nude mice. These tumors were then treated with ionizing radiation and/or NVP-BEZ235 and analyzed for DNA-PKcs and ATM activation, DSB repair inhibition, and attenuation of growth. NVP-BEZ235 potently inhibited both DNA-PKcs and ATM kinases and attenuated the repair of ionizing radiation-induced DNA damage in tumors. This resulted in striking tumor radiosensitization, which extended the survival of brain tumor-bearing mice. Notably, tumors displayed a higher DSB-load when compared with normal brain tissue. NVP-BEZ235 also sensitized a subset of subcutaneous tumors to temozolomide, a drug routinely used concurrently with ionizing radiation for the treatment of glioblastoma. These results demonstrate that it may be possible to significantly improve glioblastoma therapy by combining ionizing radiation with potent and bioavailable DNA repair inhibitors such as NVP-BEZ235.