Increase In Tumor Growth Delay Research Articles

BUB1 regulates non-homologous end joining pathway to mediate radioresistance in triple-negative breast cancer

BackgroundTriple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer subtype often treated with radiotherapy (RT). Due to its intrinsic heterogeneity and lack of effective targets, it is crucial to identify novel molecular targets that would increase RT efficacy. Here we demonstrate the role of BUB1 (cell cycle Ser/Thr kinase) in TNBC radioresistance and offer a novel strategy to improve TNBC treatment.MethodsGene expression analysis was performed to look at genes upregulated in TNBC patient samples compared to other subtypes. Cell proliferation and clonogenic survivals assays determined the IC50 of BUB1 inhibitor (BAY1816032) and radiation enhancement ratio (rER) with pharmacologic and genomic BUB1 inhibition. Mammary fat pad xenografts experiments were performed in CB17/SCID. The mechanism through which BUB1 inhibitor sensitizes TNBC cells to radiotherapy was delineated by γ-H2AX foci assays, BLRR, Immunoblotting, qPCR, CHX chase, and cell fractionation assays.ResultsBUB1 is overexpressed in BC and its expression is considerably elevated in TNBC with poor survival outcomes. Pharmacological or genomic ablation of BUB1 sensitized multiple TNBC cell lines to cell killing by radiation, although breast epithelial cells showed no radiosensitization with BUB1 inhibition. Kinase function of BUB1 is mainly accountable for this radiosensitization phenotype. BUB1 ablation also led to radiosensitization in TNBC tumor xenografts with significantly increased tumor growth delay and overall survival. Mechanistically, BUB1 ablation inhibited the repair of radiation-induced DNA double strand breaks (DSBs). BUB1 ablation stabilized phospho-DNAPKcs (S2056) following RT such that half-lives could not be estimated. In contrast, RT alone caused BUB1 stabilization, but pre-treatment with BUB1 inhibitor prevented stabilization (t1/2, ~8 h). Nuclear and chromatin-enriched fractionations illustrated an increase in recruitment of phospho- and total-DNAPK, and KAP1 to chromatin indicating that BUB1 is indispensable in the activation and recruitment of non-homologous end joining (NHEJ) proteins to DSBs. Additionally, BUB1 staining of TNBC tissue microarrays demonstrated significant correlation of BUB1 protein expression with tumor grade.ConclusionsBUB1 ablation sensitizes TNBC cell lines and xenografts to RT and BUB1 mediated radiosensitization may occur through NHEJ. Together, these results highlight BUB1 as a novel molecular target for radiosensitization in women with TNBC.

Synergistic Effect of Ginsenoside Rh2 Combines with Ionizing Radiation on CT26/luc Colon Carcinoma Cells and Tumor-Bearing Animal Model.

The local tumor control rate of colon cancer by radiotherapy is unsatisfactory due to recurrence and radioresistance. Ginsenoside Rh2 (Rh2), a panoxadiol saponin, possesses various antitumor effects. CT26/luc murine colon carcinoma cells and a CT26/luc tumor-bearing animal model were used to investigate the therapeutic efficacy of Rh2 combined with ionizing radiation and the underlying mechanisms. Rh2 caused cell cycle arrest at the G1 phase in CT26/luc cells; however, when combined with ionizing radiation, the cells were arrested at the G2/M phase. Rh2 was found to suppress the activity of NF-κB induced by radiation by inhibiting the MAPK pathway, consequently affecting the expression of effector proteins. In an in vivo study, the combination treatment significantly increased tumor growth delay time and overall survival. Furthermore, the combination treatment significantly reduced NF-κB and NF-κB-related effector proteins, along with PD-1 receptor expression. Additionally, Rh2 administration led to increased levels of interleukin-12, -18, and interferon-γ in the mice's sera. Importantly, biochemical analysis revealed no toxicities associated with Rh2 alone or combined with radiation. The combination of Rh2 with radiation may have potential as an alternative to improve the therapeutic efficacy of colorectal cancer.

Effect of Proton Therapy on Tumor Cell Killing and Immune Microenvironment for Hepatocellular Carcinoma.

Radiotherapy with proton therapy (PT) has dosimetric advantages over photon therapy, which helps to enlarge the therapeutic window of radiotherapy for hepatocellular carcinoma (HCC). We evaluated the response of HCC to PT and examined the underlying mechanisms. The human liver cancer cell lines HepG2 and HuH7 and the murine liver cancer cell line Hepa1-6 were selected for cell and animal experiments to examine the response induced by PT irradiation. Biological changes and the immunological response following PT irradiation were examined. In vitro experiments showed no significant difference in cell survival following PT compared with photon radiotherapy. In a murine tumor model, the tumors were obviously smaller in size 12 days after PT irradiation. The underlying changes included increased DNA damage, upregulated IL-6 levels, and a regulated immune tumor microenvironment. Protein analysis in vitro and in vivo showed that PT increased the level of programmed cell death ligand 1 (PD-L1) expressed in tumor cells and recruited myeloid-derived suppressor cells (MDSCs). The increase in PD-L1 was positively correlated with the irradiation dose. In Hepa1-6 syngeneic mouse models, the combination of PT with anti-PD-L1 increased tumor growth delay compared with PT alone, which was associated with increased tumor-infiltrating T cells and attenuated MDSC recruitment in the microenvironment. Furthermore, when PT was applied to the primary HCC tumor, anti-PD-L1 antibody-treated mice showed smaller synchronous unirradiated tumors. In conclusion, the response of HCC to PT was determined by tumor cell killing and the immunological response in the tumor microenvironment. The combination with the anti-PD-L1 antibody to enhance antitumor immunity was responsible for the therapeutic synergism for HCC treated with PT. Based on our results, we suggest that PT combined with anti-PD-L1 may be a promising therapeutic policy for HCC.

Antibody Drug Conjugate Targeted Radio-Immunosensitization

<h3>Purpose/Objective(s)</h3> Concurrent chemo-radiotherapy is the basis of organ sparing therapy for locally advanced cancers. However, non-targeted chemotherapies cause toxicities. Antibody drug conjugates (ADCs) spatially target drug delivery for biomarker selected cancer patients. The potent anti-tubulin monomethyl auristatin E (MMAE) is the most common ADC drug warhead. In prior studies, MMAE radiosensitized and improved irradiated tumor control of human tumor xenografts in immune-deficient mice. No studies to date have examined MMAE radiosensitization in the tumor immune microenvironment context. We hypothesized MMAE-ADCs achieve spatially precise radiosensitized tumor kill that engages tumor immune responses. <h3>Materials/Methods</h3> ADCs are optimized to bind human receptors. To synthesize a MMAE-ADC recognizing murine isoforms, we conjugated MMAE to anti-HER3 antibody CDX3379 creating CDX3379-MMAE and labeled it with Cy5 for non-invasive tracking. An advantage of CDX3379 is it recognizes both human & murine HER3. MMAE radiosensitization of murine cancer cell lines (B16, LL2, MC38) was tested by clonogenic survival. In vivo tumor targeting and efficacy studies were done in HER3+ murine syngeneic tumors grown subcutaneously (B16, MOC1) or orthotopically in tongues (MOC2) in C57BL/6 mice. CDX3379-MMAE (2.5 nmoles) was given i.v. followed by focal fractionated ionizing radiation (IR) of 5 Gy x 3. Anti-PD-1 antibody was given i.p. (200 ug, BioXCell) with start of CDX3379-MMAE for 3 doses. For CD8 depletion, mice were i.p. injected with anti-CD8 antibody (10 mg/kg, BioXCell) prior to tumor cell implant and then weekly. Statistical analysis was done by ANOVA and log-rank (Mantel-Cox). Fluorescent mouse imaging for Cy5 labeled ADC evaluated tumor targeting. Efficacy was determined by tumor volume and mouse survival. Tumor immune infiltration was assayed by flow cytometry. <h3>Results</h3> In cell culture, MMAE arrested all murine cancer cell lines tested in G₂/M and radiosensitized in the 1-3 nmolar range, P<0.01. In syngeneic models, CDX3379-MMAE accumulated in HER3+ but not HER3 negative tumors by Cy5 imaging. Combining CDX3379-MMAE with IR increased tumor growth delay and mouse survival, P<0.01. Temporal analysis of tumor immune infiltration demonstrated CDX3379-MMAE + IR therapy increased CD8+ T cell tumor immune infiltration, P<0.01. Mechanistically, tumor control by CDX3379-MMAE + IR was attenuated with CD8 T cell depletion. Finally, anti-PD-1 immune checkpoint inhibition potentiated CDX3379-MMAE + IR durable tumor control, P<0.0001. <h3>Conclusion</h3> These studies provide the first demonstration MMAE-ADC radiosensitization produces durable tumor control in a CD8 T cell dependent manner that is potentiated by immune checkpoint inhibition. Our studies have exciting translational implications by providing a foundation for irradiated cancer patients to be concurrently treated with spatially precise ADC radio-immunosensitization.

205 Targeting Homologous Repair to Overcome Genotoxic Therapy Resistance in Pancreatic Cancer

OBJECTIVES/GOALS: Pancreatic ductal adenocarcinoma (PDAC) is a relatively radioresistant disease, and inhibition of DNA homologous recombination (HR) repair in combination with radiation therapy (RT) is a potentially attractive strategy to overcome radioresistance. We have found that the expression of the HR protein RAD18 is upregulated in PDAC cells. METHODS/STUDY POPULATION: Standard clonogenic assays, γH2aX foci staining, HR-GFP reporter assay, and western blot analysis of DNA damage response proteins were performed in MIA-PaCa2 (MP2) and PANC-1 cells following knockdown of RAD18 in cells via short hairpin RNA (shRNA). Drug targeting of RAD18 was achieved through the use of a USP-7 inhibitor, P5091. Cells with or without stable knockdown of RAD18 were implanted orthotopically in the pancreas of athymic nude mice and treated with sham radiation or radiation to a dose of 20 Gy in 5 daily fractions once tumors reached 100-150 mm3. RESULTS/ANTICIPATED RESULTS: Stable knockdown of RAD18 in MP2 and PANC-1 resulted in decreased radiation clonogenic survival in vitro (dose enhancement factor (DEF)=1.52 and 1.51, respectively), decreased DNA repair after radiation as measured by the increased number of γH2aX nuclear foci assay at 6, 12, and 24 hours (all p&lt;0.05), decreased HR activation following DNA damage via an HR-GFP reporter assay (p=0.039), and increased tumor growth delay following radiation in vivo (p&lt;0.001). P5091 treatment of both MP2 and PANC-1 resulted in efficient knockdown of RAD18, which was confirmed through western blotting, qRT-PCR, and luciferase reporter assays. P5091 increased radiosensitization, yH2aX nuclear foci remained elevated at 12 and 24 hours (p&lt;0.05), and HR repair was also reduced (p=0.014). DISCUSSION/SIGNIFICANCE: Herein, we show the HR repair protein RAD18, and that modulation of RAD18 expression correlates with in vitro and in vivo radiosensitization through altered HR-mediated DNA repair. USP7 inhibition successfully reduced RAD18 expression and resulted in enhanced radiosensitization.

A KRAS-RAD18-miR-296-3p Regulated Network Mediates Radioresistance in Pancreatic Cancer Through Homologous Recombination

Pancreatic ductal adenocarcinoma (PDAC) is a relatively radioresistant disease, and inhibition of DNA homologous recombination (HR) repair in combination with radiation therapy (RT) is a potentially attractive strategy to overcome radioresistance. We have found that the expression of the HR protein RAD18 is increased upon induction of KRAS mutation and reduced following KRAS suppression. Therefore, we hypothesized targeting RAD18 in KRAS mutant PDAC with its predicted inhibitor, miR-296-3p, will increase radiosensitization. Standard clonogenic assays, γH2aX foci staining, HR-GFP reporter assay, and western blot analysis of DNA damage response proteins was performed in MIA-PaCa2 (MP2) and PANC-1 cells following knockdown of RAD18 in cells via short hairpin RNA (shRNA). Forced expression of miR-296-3p mimics was performed using lipofectamine transfection. Cells with or without stable knockdown of RAD18 were implanted subcutaneously in the hind limb of athymic nude mice and treated with sham radiation or radiation to a dose of 20 Gy in 5 daily fractions once tumors reached 100-150 mm3. Doxycycline treatment of KRAS inducible human pancreatic ductal epithelial (HPDE) cells increased both RAD18 protein and RNA expression. In contrast, knockdown of KRAS by siRNA reduced RAD18 protein and RNA expression in PANC-1 and MP2 cell lines (3.5 and 4.2-fold, respectively). Stable knockdown of RAD18 in MP2 and PANC-1 resulted in decreased radiation clonogenic survival in vitro (dose enhancement factor (DEF) = 1.52 and 1.51, respectively), decreased DNA repair after radiation as measured by increased number of γH2aX nuclear foci assay at 6, 12, and 24 hours (all p<0.05), decreased HR activation following DNA damage via a HR-GFP reporter assay (p = 0.039), and increased tumor growth delay following radiation in vivo (p<0.001). Overexpression of RAD18 resulted in increased radiation clonogenic survival (DEF = 0.88). We identified that miR-296-3p has a conserved target sequence complimentary to the 3’UTR region of the RAD18 mRNA. MiR-296-3p expression via qRT-PCR was absent in a panel of 6 KRAS mutant PDAC cell lines and highly expressed in 2 normal pancreatic cell lines. MiR-296-3p mimic transfection of the high RAD18 expressing PANC-1 and MP2 cells resulted in efficient knockdown of RAD18, which was confirmed through western blotting, qRT-PCR, and luciferase reporter assays. Following the transfection of miR-296-3p mimics into PANC-1 or MP2 cells, radiosensitization increased (DEF = 1.15 and 1.14, respectively), yH2aX nuclear foci remained elevated at 12 and 24 hours (p<0.05), and HR repair was reduced (p = 0.014). Herein, we show that KRAS mutation in PDAC positively regulates expression of the HR repair protein RAD18, and that modulation of RAD18 expression correlates with in vitro and in vivo radiosensitization through altered HR-mediated DNA repair. MiR-296-3p successfully reduced RAD18 expression and resulted in enhanced radiosensitization.

Abstract 2925: Targeting histone acetyltransferase (HAT) function for synthetic cytotoxicity in CREBBP/EP300 mutant tumors

Abstract CREB-binding protein (CREBBP) and E1A binding protein p300 (EP300) share significant homology and are collectively mutated in ~14% of head and neck cancers (HNSCC). Recently we have identified CREBBP/EP300 mutations being associated with poor outcome. Thus, tumors which harbor these mutations may be an ideal subset to study for agents that lead to synthetic cytotoxicity. We have performed in vivo shRNA screening combined with radiation in tumors derived from human HNSCC cell lines (CREBBP/EP300 mutant: Cal27, UMSCC22A, UMSCC47; CREBBP/EP300 wild type: UPCISCC152, HN31) to identify targets that are synthetically cytotoxic when combined with radiation in the CREBBP/EP300 mutant background. This identified CREBBP and EP300 proteins as the most significant targets. Additionally, inhibition of either CREBBP or EP300 with shRNA, combined with radiation, led to increased γ-H2AX, decreased BRCA1, increased apoptosis and increased cell death in CREBBP/EP300 mutant, but not wild type cells. In vivo studies of CREBBP mutant tumors using both HPV (+) and HPV (-) xenograft models showed dramatically increased tumor growth delay (TGD) following radiation in CREBBP knockdown tumors, but not controls, with 40-80% of tumors cured after low doses of XRT. This was accompanied by increased TUNEL staining in shCREBBP tumors following radiation (p&amp;lt;0.001), as well as an increase in pro-apoptotic proteins on RPPA. We then evaluated chemical inhibitors of CREBBP and EP300. A CREBBP specific inhibitor led to significant in vitro radiosensitization, irrespective of HPV status, but only in CREBBP mutant HNSCC cell lines. A similar finding was seen using shRNA-mediated inhibition, namely that specific inhibition of either CREBBP or EP300 led to synthetic cytotoxicity only in HNSCC lines harboring the individual mutant. We next turned our attention to agents that affect specific functions of EP300 and CREBBP. We initially used a bromodomain inhibitor in vitro in combination with radiation and observed minimal effect at therapeutic doses. We next examined a HAT inhibitor specific for CREBBP and EP300. This agent, but not its inactive sister compound, led to a profound radiosensitization on clonogenic assay in a panel of EP300 and/or CREBBP mutant HNSCC cell lines (p&amp;lt;0.001 for each of 6 HNSCC lines), but not in 3 double wild type lines. This effect was accompanied by increased apoptosis and decreased BRCA1 foci following radiation. We also observed decreased H3K18 and H3K27 acetylation following HAT inhibitor treatment, but only in CREBBP/EP300 mutant cell lines. In conclusion, the observed synthetic cytotoxicity of radiation and CREBBP/EP300 inhibition in mutated background is seen both in vitro and in vivo, largely due to increased apoptosis, and may be primarily due to the HAT function of the protein. As HAT inhibitors are being developed for clinical use, these observations could have a direct positive clinical impact. Citation Format: Curtis Pickering, Manish Kumar, Kathleen Bridges, Tongxin Xie, David Molkentine, Aakash Sheth, Liang Yang, Mitchell Frederick, Tim Heffernan, Sahil Seth, Jeffrey Myers, Heath D. Skinner. Targeting histone acetyltransferase (HAT) function for synthetic cytotoxicity in CREBBP/EP300 mutant tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2925.

Obstruction of BRAFV600E transcription by complementary PNA oligomers as a means to inhibit BRAF-mutant melanoma growth.

Peptide nucleic acid (PNA) oligomers are DNA mimics, which are capable of binding gene sequences 1000-fold more avidly than complementary native DNA by strand invasion and effectively obstruct transcription. Irreversibly obstructing the transcription or replication of a gene sequence, such as BRAFV600E, offers a potential route to specifically target the cancer cell itself. We have employed PNA oligomers to target BRAFV600E in a sequence-specific complementary manner. These PNAs have been modified by appending configurationally stabilizing cationic peptides in order to improve their cellular delivery and target avidity. Our results indicate that exposure of the melanoma cell lines to a modified PNA-peptide conjugate complementary to BRAFV600E mutation sequence results in a concentration-dependent and time-dependent inhibition of cell growth that is specific for the BRAFV600E-mutant melanoma cell lines with inhibition of mRNA and protein expression. Xenograft mouse trials show increased tumor growth delay and necrosis with the BRAFV600E-complementary PNA-peptide conjugates as compared with the saline and scrambled PNA sequence controls. Similarly, quantitative measurement shows a 2.5-fold decrease in Ki67 and a 3-fold increase in terminal deoxynucleotidyl transferase dUTP nick end labeling expression with this approach. PNA-delivery peptide conjugates represent a novel way to target BRAFV600E and represent a new approach in targeting selective oncogenes that induce tumor growth.

Abstract 3096: Mechanisms of synergy of carboplatin and an EphA2-targeted docetaxel antibody-directed nanotherapeutic

Abstract Platinum-taxane combinations are widely used to treat solid tumors either in first or later lines of therapy. While effective in many settings, platinum-taxane combinatorial regimens are limited by toxicities. We have recently developed an antibody directed nanotherapeutic (MM-310) encapsulating a docetaxel prodrug, targeted to Ephrin receptor A2 (EphA2). Preclinical investigation of MM-310 revealed that the liposomal formulation leads to prolonged docetaxel exposure of the tumor with decreased exposure of normal tissues leading to a shift in toxicity profile and potentially enabling more safe and effective combinations with platinum-based chemotherapeutics. In this study, we evaluated the activity of MM-310 in combination with carboplatin in several xenograft tumor models and compared it to the activity of free docetaxel in combination with carboplatin at equitoxic dosing. Tolerability of MM-310 in combination with carboplatin in mice was evaluated, including assessing hepatotoxicity. Biodistribution, microdistribution, in vivo tumor growth, and mouse survival studies were performed in lung and ovarian cell line-derived (CDX) and patient-derived xenograft (PDX) models. MM-310 in combination with carboplatin was found to be well tolerated, enabling dosing of both drugs at high doses with maximum tolerability when the drugs were dosed three days apart. Carboplatin increased nanotherapeutic delivery to the tumor in a CDX model of triple negative breast cancer and in a PDX model of ovarian cancer. In vivo studies in lung and ovarian cancer xenograft models showed significant synergy between MM-310 and carboplatin when compared to the monotherapies, as well as when compared to free docetaxel with carboplatin, leading to a significant increase in tumor growth delay and survival (docetaxel/ carboplatin vs. MM-310/carboplatin, 0 vs 50% complete tumor regression, 24 vs 80 days median time to regrowth). Additionally, in the same studies, MM-310 and carboplatin was better tolerated than free docetaxel and carboplatin. In conclusion, we found that MM-310 in combination with carboplatin was significantly better tolerated and more effective than free docetaxel in combination with carboplatin. Mechanistically, the synergistic anti-tumor activity of MM-310 with carboplatin may be partially due to a carboplatin mediated enhancement of nanotherapeutic delivery. The increased preclinical activity of the MM-310/carboplatin combination, together with the high tolerability following scheduling optimization tested in mice, makes this combination a promising regimen that warrants evaluation in clinical trials. Citation Format: Walid S. Kamoun, Andrew J. Sawyer, Christine Pien, Alexander Koshkaryev, Lia Luus, Samantha Merrigan, Gang Sun, Sergey Kozin, Zhaohua Richard Huang, Suresh K. Tipparaju, Dmitri B. Kirpotin, Hannah Xu, Vasileios Askoxylakis, Patrick C. Reynolds, Daryl C. Drummond. Mechanisms of synergy of carboplatin and an EphA2-targeted docetaxel antibody-directed nanotherapeutic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3096. doi:10.1158/1538-7445.AM2017-3096

Abstract 1110: AsiDNA induce tumor sensitivity to PARP inhibitors in homologous recombination proficient breast cancer AsiDNA induce tumor sensitivity to PARP inhibitors in homologous recombination proficient breast cancer

Abstract Purpose: PARP inhibitors (PARPi) have shown significant benefits in cancer patients with BRCA mutations. However, their major limitations are the necessity of homologous recombination (HR) deficiency and the rapid emergence of resistance. In the current study, we propose a novel therapeutic strategy, based on drug combination to promote sensitivity to PARPi independently of the tumor genetics. Experimental design: We used AsiDNA, a DNA repair pathways agonist (Dbait concept), consisting in small molecules mimicking double-strand DNA breaks to activate ectopic signaling of DNA damage and prevent recruitment at damage sites of HR repair enzymes. We characterized the DNA repair inhibition activity of AsiDNA by monitoring repair foci formation and DNA damage and analyzed the cell survival to AsiDNA monotherapy and combination with the PARPi Olaparib of 21 tumor cell lines, and 3 non-tumor cell lines. Efficacy of the combination treatment was analyzed in tumor xenografts derived from the MDA-MB-231 cell line showing reduced sensitivity to AsiDNA and Olaparib in vitro. We screened for the frequency of clones resistant to two weeks treatment with AsiDNA or Olaparib in the 4NQO-mutagenized haploid KBM7 cells. Results: Molecular analyses of repair foci formed after irradiation demonstrate that AsiDNA inhibits recruitment of RAD51 and 53BP1 whereas Olaparib prevents XRCC1. Combination of both drugs increases the accumulation of unrepaired spontaneous damage resulting in an increase of cell death in all tumor cell lines. The synergy of the association of AsiDNA with PARPi was also confirmed with 6 other PARPi. AsiDNA do not induce any increase in DNA damage or lethality in non-tumor cells when used in monotherapy as well as in association with PARPi. Multi-omics analysis of tumor cells highlighted a correlation between similarity to normal cells profile and AsiDNA resistance. Selection of resistant clones allowed the emergence of resistance to Olaparib (1.45%), Imatinib (0.6%) and 6-Thioguanine (1.6%) but not to AsiDNA (&amp;lt;0.07%), indicating that the drug acts through multiple targets. In the MDA-MB-231 xenograft model, whereas Olaparib failed to prevent tumor growth and AsiDNA provided only a 62% increase in tumor growth delay, the combination of Olaparib and AsiDNA increase 215% days the mean tumor growth delay. Conclusion: Our results highlight the therapeutic interest of combining AsiDNA and PARPi to recapitulate synthetic lethality in all tumors independently of their HR status. Moreover, the low frequency of appearance of resistant clones to AsiDNA suggests a sustained efficacy during treatment unlike most targeted therapies. Citation Format: marie DUTREIX, Wael Jdey, sylvain Thierry, Inna Kuperstein, Graham Dixon. AsiDNA induce tumor sensitivity to PARP inhibitors in homologous recombination proficient breast cancer AsiDNA induce tumor sensitivity to PARP inhibitors in homologous recombination proficient breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1110. doi:10.1158/1538-7445.AM2017-1110

Hedgehog inhibition enhances efficacy of radiation and cisplatin in orthotopic cervical cancer xenografts

Background:The Hedgehog (Hh) pathway is upregulated in cervical cancer and associated with poor outcome. We explored the effects of Hh pathway inhibition in combination with RTCT in a patient derived orthotopic cervical cancer xenograft model (OCICx).Methods:5E1, a monoclonal antibody for SHH, or Sonidegib (LDE225), a clinical SMO inhibitor (Novartis) were added to RTCT. We investigated tumour growth delay, metastasis and GI toxicity using orthotopic cervical cancer xenografts models. The xenografts were treated with radiotherapy (15 × 2 Gy daily fractions over 3 weeks) and weekly cisplatin 4 mg kg−1 concurrently, with or without 5E1 or Sonidegib (LDE225). The Hh inhibitors were administered by subcutaneous injection (5E1; 20 mg kg−1 weekly for 3 weeks), or by oral gavage (Sonidegib; 60 mg kg−1 daily for 3 weeks).Results:We observed that both Hh inhibitors administered with RTCT were well tolerated and showed increased tumour growth delay, and reduced metastasis, with no increase in acute GI-toxicity relative to RTCT alone.Conclusions:Our data suggest Hh can be a valid therapeutic target in cervical cancer and supports data suggesting a potential therapeutic role for targeting Hh in patients undergoing RTCT. This warrants further investigation in clinical trials.

Very low doses of ionizing radiation and redox associated modifiers affect survivin-associated changes in radiation sensitivity

Exposure of cells to a dose of ionizing radiation as low as 5mGy can induce changes in radiation sensitivity expressed by cells exposed to subsequent higher doses at later times. This is referred to as an adaptive effect. We describe a unique survivin-associated adaptive response in which increased radiation resistance or sensitization of cells can be induced by exposure to 5mGy or to the reactive oxygen species (ROS) generating drug Emodin (1,3,8-trihydroxy-6-methylanthraquinone), a naturally occurring anthraquinone. The purpose of this study was to determine the role of ROS generating processes in affecting both the intracellular localization of the inhibitor of apoptosis protein survivin and its subsequent effect on radiation response in the presence or absence of the anti-oxidant N-acetyl-L-cysteine (NAC). Experiments were performed using two well characterized murine sarcomas: SA-NH p53 wild-type (WT) and FSa p53 mutant (Mut), grown either in culture or as solid tumors in the right hind legs of C3H mice. Doses of 5mGy or 50μM Emodin were used to induce changes in the response of these tumor cells to higher radiation exposures using a multi-dosing paradigm. Effects on radiation sensitivity were determined for SA-NH and FSa cells as a function of survivin translocation either to the cytoplasm or nucleus in the presence or absence of 10mM NAC treatment. In vitro survival assays (2Gy per fraction, two once daily fractions) and tumor growth delay (TGD) (5Gy per fraction, five once daily fractions) studies were performed. Intracellular localization of survivin was determined by enzyme-linked immunosorbent assay (ELISA) and correlated to survival response and treatment conditions. 2Gy alone had no effect on intracellular translocation of survivin. When preceded 15min earlier by 5mGy or Emodin exposures, survivin became elevated in the cytoplasm of p53 WT SA-NH as compared to the nuclei of p53 Mut FSa cells. SA-NH cells transfected with p53 small interfering RNA (siRNA), in contrast, responded similarly to p53 Mut FSa cells by becoming more radiation sensitive if exposed to 5mGy prior to each 2Gy irradiation. In contrast to their respective responses to five once daily 5Gy fractions, SA-NH tumors were protected by 5mGy exposures administered 15min prior to each daily 5Gy dose as evidenced by a more rapid growth (1.9 day decrease in TGD, P=0.032), while FSa tumors were sensitized, growing at a much slower rate (4.5 day increase in TGD, P<0.001). Exposure of SA-NH and FSa tumor cells to 10mM NAC inhibited the ability of 5mGy and Emodin to induce intracellular translocation of survivin and the corresponding altered adaptive survival response. The survivin-associated adaptive response can be induced following a multi-dosing scheme in which very low radiation doses are followed shortly thereafter by higher doses consistent with a standard image guided radiotherapy protocol that is currently widely used in the treatment of cancer. While induced by exposure to ROS generating stresses, the ultimate expression of changes in radiation response is dependent upon the bi-functionality of the tumor associated protein survivin and its intracellular translocation.

Abstract 2592: Anti-tumor activity of icotinib (BPI-2009H) and BPI-403B in the BxPC-3 pancreatic cancer cell line

Abstract Introduction: The purpose of this research was to investigate the anti-tumor effect of BPI-2009H and BPI-403B, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) in the human pancreatic cancer cell lines BxPC-3 and Panc-1, in vitro and in vivo and determine the maximum tolerated dose (MTD) of the compound in mouse. Experimental Design: In vitro, human pancreatic cancer cell lines, BxPC-3 and Panc-1 were exposed to varying concentrations of BPI-2009H and BPI-403B, the concentration of compound required to inhibit growth by fifty percent (IC50) was estimated. In vivo, tumor growth inhibition of low dose BPI-2009H was tested in xenografted nude mice with BxPC-3 pancreatic cancer tumors. The maximum tolerated dose (MTD) was established via oral administration of low, mid and high doses to Harlan mice of both BPI-2009H and BPI-403B. Upon determination of the MTD, the in vivo efficacy of high dose BPI-2009H was evaluated via tumor growth inhibition study on xenografted nude mice with BxPC-3 pancreatic cancer tumors. Results: BPI-2009H, as a single agent, inhibited the growth of BxPC-3 cells in a dose dependent manner, with a relative IC50 value of 25.8 μM. BPI-403B, as a single agent, inhibited growth of BxPC-3 cells in a dose dependent manner as well, with a relative IC50 value of 2.7 μM. BPI-2009H and BPI-403B were tested up to 50μM in Panc-1 cells with no apparent effect at the upper limit of the study for either of the compounds. In vivo, the mice treated with BPI-403B demonstrated a statistically significant tumor growth delay, 14.3 days. 20% of the mice experienced tumor regression. The compound was well tolerated with minimal body weight loss. At low doses, BPI-2009H did not show any statistically significant tumor growth delay, but upon increasing the dose to the MTD, significant signs of anticancer activity was demonstrated, with an increase in tumor growth delay. Conclusions: The in vitro and in vivo findings show that both BPI-2009H and BPI-403B have antiproliferative effects against BxPC-3 pancreatic cancer cells. These compounds are both well tolerated and may prove to be an alternative option of EGFR-TKI to erlotinib treatment in pancreatic cancer cases. Citation Format: Victoria Lynn Wilde, Don Zhang, Jirong Peng, Michael A. Green, Michael N. Greco, Michael Costanzo. Anti-tumor activity of icotinib (BPI-2009H) and BPI-403B in the BxPC-3 pancreatic cancer cell line. [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 2592. doi:10.1158/1538-7445.AM2015-2592

O2.04 * TREATMENT WITH OMX-4.80, A TUMOR-PENETRATING TUNABLE OXYGEN CARRIER, REDUCES TUMOR HYPOXIA AND DRAMATICALLY ENHANCES RADIATION THERAPY IN INTRACRANIAL MODELS OF GLIOBLASTOMA

We have developed OMX-4.80, a tunable oxygen carrier from the H-NOX protein family, that preferentially accumulates in tumor tissue, reduces hypoxia and enhances radiotherapy (RT). Tumor hypoxia is associated with poor patient outcomes in multiple solid tumors including glioblastoma (GB) and is thought to be a major contributor to poor cancer responses to chemo and radiation therapy. Here, we show that OMX-4.80 administered i.v. into the tail vein of mice bearing orthotopic GB passes the blood-tumor barrier, penetrates deep into intracranial tumors, and significantly reduces tumor hypoxia in a dose-dependent manner. Hypoxia reduction is demonstrated by a significant decrease in levels of hypoxia inducible factor, HIF1α, and the external hypoxia marker pimonidazole as assessed by IHC and image analysis. Furthermore, hypoxia before and after treatment is compared in the individual tumors by concurrent immunostaining of tumors with a long-term hypoxia marker, Glut1, and short-lived HIF-1α. These data reveal >50% reduction in hypoxic tumor area after OMX-4.80 treatment confirming the effectiveness of OMX-4.80 in reducing hypoxia in individual tumors. When OMX-4.80 treatment was coupled with a single high dose of RT, we observed a 2.7-fold increase in tumor growth delay and 2.9-fold longer survival (p < 0.05, by logrank). Toxicology studies in rodents and dogs with single and multiple supratherapeutic doses of OMX-4.80 revealed a good safety profile affirming its suitability for clinical development. In conclusion, our results indicate that OMX-4.80 is an effective and promising RT enhancer that warrants clinical development in GB patients.

Isolated limb perfusion with melphalan, tumour necrosis factor‐alpha and oncolytic vaccinia virus improves tumour targeting and prolongs survival in a rat model of advanced extremity sarcoma

Isolated limb perfusion (ILP) is a treatment for advanced extremity sarcoma and in-transit melanoma. Advancing this procedure by investigating the addition of novel agents, such as cancer-selective oncolytic viruses, may improve both the therapeutic efficacy of ILP and the tumour-targeted delivery of oncolytic virotherapy. Standard in vitro assays were used to characterise single agent and combinatorial activities of melphalan, tumour necrosis factor-alpha (TNF-α) and Lister strain vaccinia virus (GLV-1h68) against BN175 rat sarcoma cells. An orthotopic model of advanced extremity sarcoma was used to evaluate survival of animals after ILP with combinations of TNF-α, melphalan and GLV-1h68. We investigated the efficiency of viral tumour delivery by ILP compared to intravenous therapy, the locoregional and systemic biodistribution of virus after ILP, and the effect of mode of administration on antibody response. The combination of melphalan and GLV-1h68 was synergistic in vitro. The addition of virus to standard ILP regimens was well tolerated and demonstrated superior tumour targeting compared to intravenous administration. Triple therapy (melphalan/TNF-α/GLV-1h68) resulted in increased tumour growth delay and enhanced survival compared to other treatment regimens. Live virus was recovered in large amounts from perfused regions, but in smaller amounts from systemic organs. The addition of oncolytic vaccinia virus to existing TNF-α/melphalan-based ILP strategies results in survival advantage in an immunocompetent rat model of advanced extremity sarcoma. Virus administered by ILP has superior tumour targeting compared to intravenous delivery. Further evaluation and clinical translation of this approach is warranted.

Monitoring the Effects of Anti-angiogenesis on the Radiation Sensitivity of Pancreatic Cancer Xenografts Using Dynamic Contrast-Enhanced Computed Tomography

<h3>Purpose</h3> To image the intratumor vascular physiological status of pancreatic tumors xenografts and their response to anti-angiogenic therapy using dynamic contrast-enhanced computed tomography (DCE-CT), and to identify parameters of vascular physiology associated with tumor x-ray sensitivity after anti-angiogenic therapy. <h3>Methods and Materials</h3> Nude mice bearing human BxPC-3 pancreatic tumor xenografts were treated with 5 Gy of radiation therapy (RT), either a low dose (40 mg/kg) or a high dose (150 mg/kg) of DC101, the anti-VEGF receptor-2 anti-angiogenesis antibody, or with combination of low or high dose DC101 and 5 Gy RT (DC101-<i>plus</i>-RT). DCE-CT scans were longitudinally acquired over a 3-week period post-DC101 treatment. Parametric maps of tumor perfusion and fractional plasma volume (F_p) were calculated and their averaged values and histogram distributions evaluated and compared to controls, from which a more homogeneous physiological window was observed 1-week post-DC101. Mice receiving a combination of DC101-<i>plus</i>-RT(5 Gy) were imaged baseline before receiving DC101 and 1 week after DC101 (before RT). Changes in perfusion and F_p were compared with alternation in tumor growth delay for RT and DC101-<i>plus</i>-RT (5 Gy)-treated tumors. <h3>Results</h3> Pretreatment with low or high doses of DC101 before RT significantly delayed tumor growth by an average 7.9 days compared to RT alone (<i>P</i> ≤ .01). The increase in tumor growth delay for the DC101-<i>plus</i>-RT-treated tumors was strongly associated with changes in tumor perfusion (Δ<i>P</i>>−15%) compared to RT treated tumors alone (<i>P</i>=.01). In addition, further analysis revealed a trend linking the tumor's increased growth delay to its tumor volume-to-DC101 dose ratio. <h3>Conclusions</h3> DCE-CT is capable of monitoring changes in intratumor physiological parameter of tumor perfusion in response to anti-angiogenic therapy of a pancreatic human tumor xenograft that was associated with enhanced radiation response.

NAD+ depletion by APO866 in combination with radiation in a prostate cancer model, results from an in vitro and in vivo study

BackgroundAPO866 is a highly specific inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), inhibition of which reduces cellular NAD+ levels. In this study we addressed the potential of NAD+ depletion as an anti-cancer strategy and assessed the combination with radiation. MethodsThe anticipated radiosensitizing property of APO866 was investigated in prostate cancer cell lines PC3 and LNCaP in vitro and in PC3 xenografts in vivo. ResultsWe show that APO866 treatment leads to NAD+ depletion. Combination experiments with radiation lead to a substantial decrease in clonogenic cell survival in PC3 and LNCaP cells.In PC3 xenografts, treatment with APO866 resulted in reduced intratumoral NAD+ levels and induced significant tumor growth delay. Combined treatment of APO866 and fractionated radiation was more effective than the single modalities. Compared with untreated tumors, APO866 and radiation alone resulted in tumor growth delays of 14days and 33days, respectively, whereas the combination showed a significantly increased tumor growth delay of 65days. ConclusionsOur studies show that APO866-induced NAD+ depletion enhances radiation responses in tumor cell survival in prostate cancer. However, the in vitro data do not reveal a solid cellular mechanism to exploit further clinical development at this moment.

Enhanced photodynamic efficacy of PLGA-encapsulated 5-ALA nanoparticles in mice bearing Ehrlich ascites carcinoma

Nanoparticles (NPs) fabricated from the biodegradable copolymer poly(lactic-co-glycolic acid) (PLGA) were investigated as a drug delivery system to enhance the photodynamic efficacy of 5-aminolevulinic acid (5-ALA) in mice bearing Ehrlich ascites carcinoma. The PLGA-encapsulated 5-ALA NPs were prepared using binary organic solvent diffusion method and characterized in terms of shape and particle size. The in vivo photodynamic efficiency in Ehrlich ascites-bearing mice was studied. The obtained particles were uniform in size with spherical shape of mean size of 249.5 nm as obtained by particle size analyzer and the in vitro release studies demonstrated a controlled release profile of 5-ALA. Tumor-bearing mice injected with PLGA-encapsulated 5-ALA NPs exhibited significantly smaller mean tumor volume, increased tumor growth delay compared with the control group and the group injected with free 5-ALA during the time course of the experiment. Histopathological examination of tumor from mice treated with PLGA-encapsulated 5-ALA NPs showed regression of tumor cells, in contrast to those obtained from mice treated with free 5-ALA. The results indicate that PLGA-encapsulated 5-ALA NPs are a successful delivery system for improving photodynamic activity in the target tissue.

Hyperthermia-enhanced indocyanine green delivery for laser-induced thermal ablation of carcinomas

Purpose: Intravenous administration of indocyanine green (ICG) dye can effectively convert near-infrared (NIR) laser light into heat and enhance thermal injury of blood vessels; however, there is no selective uptake of ICG by the tumour compared to the other tissues, which impacts the therapeutic ratio of this strategy unless uptake can be selectively increased in tumour tissue. Here we investigated the use of local hyperthermia prior to intravenous ICG administration to enhance ICG uptake in tumour tissue, thereby enhancing laser thermal ablation of solid tumours. Methods: Murine SCK breast or SCCVII head and neck tumours were treated with a 755-nm laser light either alone or with prior intravenous administration of 4 mg/kg ICG and/or local tumour hyperthermia at 42.5 °C for 60 min. Retention of ICG was quantified using a NIR animal imaging system. Treatment effects were assessed by growth delay and histology. Results: ICG accumulation in the heated tumours was 1.23-fold greater on average compared to non-heated tumours, in both models. In SCK tumours, animals receiving either laser irradiation alone or in conjunction with ICG had a 1.86- or 3.91-fold increase in tumour growth delay, respectively. The addition of local hyperthermia before ICG injection resulted in complete regression of SCK tumours. Uptake of ICG increased in SCCVII tumours; however, little change in tumour growth delay was observed. Conclusion: Using local hyperthermia may improve the delivery of ICG to the tumour and thereby increase the extent of laser thermal ablation of smaller superficial malignancies that can be effectively exposed to laser therapy.

In vitro and in vivo radiosensitization induced by hydroxyapatite nanoparticles

Previous study showed that hydroxyapatite nanoparticles (nano-HAPs) inhibited glioma growth in vitro and in vivo; and in a drug combination, they could reduce adverse reactions. We investigated the possible enhancement of radiosensitivity induced by nano-HAPs. In vitro radiosensitization of nano-HAPs was measured using a clonogenic survival assay in human glioblastoma U251 and breast tumor brain metastatic tumor MDA-MB-231BR cells. DNA damage and repair were measured using γH2AX foci, and mitotic catastrophe was determined by immunostaining. The effect of nano-HAPs on in vivo tumor radiosensitivity was investigated in a subcutaneous and an orthotopic model. Nano-HAPs enhanced each cell line's radiosensitivity when the exposure was 1 h before irradiation, and they had no significant effect on irradiation-induced apoptosis or on the activation of the G2 cell cycle checkpoint. The number of γH2AX foci per cell was significantly large at 24 h after the combination modality of nano-HAPs + irradiation compared with single treatments. Mitotic catastrophe was also significantly increased at an interval of 72 h in tumor cells receiving the combined modality compared with the individual treatments. In a subcutaneous model, nano-HAPs caused a larger than additive increase in tumor growth delay. In an orthotopic model, nano-HAPs significantly reduced tumor growth and extended the prolongation of survival induced by irradiation. These results show that nano-HAPs can enhance the radiosensitivity of tumor cells in vitro and in vivo through the inhibition of DNA repair, resulting in an increase in mitotic catastrophe.