Novel Target For Radiosensitization Research Articles

Abstract 692: Uncoupling protein 2 (Ucp2) loss of function enhances mitochondrial ROS and sensitizes PDAC to radiation therapy

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a dependence on highly active mitochondria to grow and metastasize. Normally mitochondria exist in long networks due to mitochondrial fusion but in PDAC mitochondria are fragmented due to excessive fission. We previously found that forcing PDAC cells to increase mitochondrial fusion suppressed tumor growth and metastasis, leading to improved survival in multiple preclinical models. The tumor suppressive mechanisms of mitochondrial fusion remain unclear, and thus we explored the role of redox homeostasis through uncoupling protein 2 (UCP2), a metabolic protein normally upregulated in PDAC that was downregulated upon forced mitochondrial fusion. We hypothesize that UCP2 mediates the oncogenic effects of mitochondrial fragmentation in PDAC. Methods: We explored if Ucp2 is necessary and sufficient for inducing tumor suppression upon mitochondrial fusion. The effects of Ucp2 loss or overexpression were characterized by metabolic flux assays, proliferation, mitochondrial ROS (mROS), membrane potential, and morphology measurements. We also examined if Ucp2 recapitulates mitochondrial fusion-induced tumor suppression via pharmacological inhibition and genetic ablation and assessed the impact on mitochondrial health and cell growth. We utilized the LSL-KRASG12D; Trp53R172H/+; Ptf1⍺ Cre (KPC) cell line as our parental line for all genetic manipulations, with a nontargeting (sgGFP) KPC cell line as a control. Results: Murine PDAC cells with enhanced mitochondrial fusion reduced Ucp2 transcript levels by 70% and was correlated with 3.5-fold increase in mROS, 50% reduction in OXPHOS via oxygen consumption rate assay, and a 4-fold increase in membrane potential compared to controls. Ucp2 re-expression did not rescue these phenotypes, suggesting that Ucp2 alone may not be sufficient for tumor suppression. However, inhibition of Ucp2 in PDAC cells reduced proliferation by 70%, while inducing a 3-fold increase in mROS levels, which was recapitulated with Ucp2 abrogation, thus Ucp2 may be necessary for tumor suppression. Enhanced mROS via Ucp2 inhibition significantly increased radiosensitivity (p-value < 0.0001), suggesting that although it does not recapitulate the mitochondrial fusion phenotype, UCP2 may be a novel target for radiosensitization in PDAC. Conclusions: Mitochondrial fusion may not suppress tumor growth by altering redox homeostasis via UCP2, but forcing mitochondria to fuse elucidated the impacts of UCP2 downregulation on cell metabolism. Thus, we uncovered that UCP2 may be a novel target for radiosensitization. Further insight into UCP2’s role in redox balance, and how it is disrupted with UCP2 modulation enables development of novel therapeutic combinations with UCP2 inhibition that complement and potentially synergize with existing therapeutic strategies such as radiation. Citation Format: Emily G. Caggiano, Meifang Yu, Ariana Acevedo-Diaz, Cullen M. Taniguchi. Uncoupling protein 2 (Ucp2) loss of function enhances mitochondrial ROS and sensitizes PDAC to radiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 692.

Abstract 1430: N-cadherin is a driver of adaptive radioresistance in malignant brain tumors

Abstract Many subtypes of brain tumors are highly malignant and resistant to chemo- and radio- therapy. Tumor cells can shift their phenotype in response to treatments, the so-called adaptive resistance. Adaptive resistance mechanisms in malignant brain tumors are still poorly understood, and effective treatments have not yet been developed. To unveil such mechanisms, we have developed unique new experimental models to identify the adaptive resistance mechanisms to fractionated radiation in malignant brain tumors. We performed repeated irradiation (2-5Gy every 3-4 days, 3-6 weeks) on 6 human Glioma stem cells (GSCs), 2 mouse GSCs and 4 medulloblastomas (MB) cells in vitro and examined how tumor cells adapt to repeated irradiation. Brain tumor cells demonstrated dynamic adaptation to fractionated irradiation. They rapidly altered cell proliferation, intercellular adhesion, and stemness and acquired strong radioresistance. To identify genes responsible for radio-resistance, we performed RNA-seq analysis and CRISPR library screening using primary and radioresistant cells. We found that N-cadherin was upregulated in the majority of radioresistant GSCs. Stably transfecting N-cadherin in parental GSC rendered them radioresistant, reduced their proliferation, and increased their stemness and intercellular adhesive properties. Conversely, radioresistant GSCs lost their acquired phenotypes upon CRISPR/Cas9-mediated knockout of N-cadherin. Mechanistically, elevated N-cadherin expression resulted in the accumulation of b-catenin at the cell surface, which suppressed Wnt/b-catenin proliferative signaling, and reduced neural differentiation. Moreover, N-cadherin increased Clusterin secretion, which protected GSCs against apoptosis after radiation treatment. We also demonstrated that N-cadherin upregulation was induced by radiation-induced IGF1 secretion, which caused an EMT-like phenotype change in GSCs. The N-cadherin-mediated radioresistance phenotype could be reverted with picropodophyllin (PPP), a clinically applicable blood-brain-barrier permeable IGF1 receptor inhibitor. Adjuvant PPP combined with irradiation significantly extended the survival of orthotopically xenografted mice versus irradiation-only or drug-alone controls, supporting clinical translation. In conclusion, our data indicate that IGF1R inhibition can block the N-cadherin-mediated resistance pathway. Our study deepens our understanding of adaptive resistance during repeated irradiation in GBM, and validates the IGF1/N-cadherin/b-catenin/Clusterin signaling axis as a novel target for radio-sensitization, which has direct therapeutic applicability. These findings also confirmed that our radioresistant models effectively identify new adaptive resistance mechanisms in malignant brain tumors.

CBMS-7 IGF1/N-cadherin/Clusterin signaling axis mediates adaptive radioresistance of glioma stem cells

Glioblastoma (GBM) is composed of a variety of tumor cell populations including those with stem cell properties, known as glioma stem cells (GSCs). GSCs are innately less sensitive to radiation than the tumor bulk and are believed to drive GBM formation and recurrence following repeated irradiation. However, it is unclear how GSCs adapt to avoid the toxicity of repeated irradiation used in clinical practice. We established radioresistant human and mouse GSCs by exposing them to repeated rounds of irradiation in order to uncover critical mediators of adaptive radioresistance. Surviving subpopulations acquired strong radioresistance in vivo, which was accompanied by increased cell-cell adhesion, slower proliferation, an elevation of stemness properties and N-cadherin expression. Increasing N-cadherin expression rendered parental GSCs radioresistant, reduced their proliferation, and increased their stemness and intercellular adhesive properties. Conversely, radioresistant GSCs reduced their acquired phenotypes upon CRISPR/Cas9-mediated knockout of N-cadherin. Mechanistically, elevated N-cadherin expression resulted in the accumulation of β-catenin at the cell surface, which decreased Wnt/ β-catenin proliferative signaling, reduced neural differentiation, and protected against apoptosis through Clusterin secretion. Restoration of wild type N-cadherin, but not mutant N-cad lacking β-catenin binding region, led to increased radioresistance in N-cadherin knockout GSCs, indicating the importance of the binding between N-cadherin and β-catenin. We also demonstrated that N-cadherin upregulation was induced by radiation-induced IGF1 secretion, and the radiation resistance phenotype can be reversed with picropodophyllin (PPP), a clinically applicable blood-brain-barrier permeable IGF1 receptor inhibitor, supporting clinical translation. Moreover, the elevation of N-cad and Clusterin are related to prognosis of GBM in the TCGA dataset. In conclusion, our data indicate that IGF1R inhibitor can block the N-cadherin-mediated resistance pathway. Our research provides a deeper understanding of adaptive radioresistance after repeated irradiation, and validates the IGF1/N-cadherin/β-catenin/Clusterin signaling axis as a novel target for radio-sensitization, which has direct therapeutic applicability.

Abstract 3078: IGF1/N-cadherin/b-catenin/Clusterin signaling axis can mediates adaptive radioresistance in glioblastoma

Abstract Glioblastoma (GBM) is composed of heterogeneous tumor cell populations including those with stem cell properties, termed glioma stem cells (GSCs). GSCs are innately less radiation sensitive than the tumor bulk and are believed to drive GBM formation and recurrence after repeated irradiation. However, it is unclear how GSCs adapt to escape the toxicity of repeated irradiation used in clinical practice. To identify important mediators of adaptive radioresistance, we generated radioresistant human and mouse GSCs by exposing them to repeated cycles of irradiation. Surviving subpopulations acquired strong radioresistance in vivo, which was accompanied by increased cell-cell adhesion, slower proliferation, an elevation of stemness properties and N-cadherin expression. Stably transfecting N-cadherin in parental GSC rendered them radioresistant, reduced their proliferation, and increased their stemness and intercellular adhesive properties. Conversely, radioresistant GSCs lost their acquired phenotypes upon CRISPR/Cas9-mediated knockout of N-cadherin. Mechanistically, elevated N-cadherin expression resulted in the accumulation of b-catenin at the cell surface, which suppressed Wnt/b-catenin proliferative signaling, and reduced neural differentiation. Transfection of wild type N-cadherin, but not mutant N-cadherin lacking the b-catenin binding region, restored radioresistance in N-cadherin knockout GSCs, indicating the importance of the binding between N-cadherin and b-catenin. Moreover, N-cadherin increased Clusterin secretion, which protected GSCs against apoptosis after radiation treatment. N-cadherin knockout decreased Clusterin secretion and sensitized the cells to radiation therapy. We also demonstrated that N-cadherin upregulation was induced by radiation-induced IGF1 secretion, which induced an EMT-like phenotype change in GSCs. The N-cadherin-mediated radioresistance phenotype could be reverted with picropodophyllin (PPP), a clinically applicable blood-brain-barrier permeable IGF1 receptor inhibitor. Adjuvant PPP combined with irradiation significantly extended the survival of orthotopically xenografted mice versus irradiation-only or drug alone controls, supporting clinical translation. Moreover, elevated N-cadherin and Clusterin mRNA expression is related to prognosis of GBM in the TCGA dataset. In conclusion, our data indicate that IGF1R inhibition can block the N-cadherin-mediated resistance pathway. Our study deepens our understanding of adaptive radioresistance during repeated irradiation in GBM, and validates the IGF1/N-cadherin/β-catenin/Clusterin signaling axis as a novel target for radio-sensitization, which has direct therapeutic applicability. Citation Format: Satoru Osuka, Dan Zhu, Zhaobin Zhang, Chaoxi Li, Christian T. Stackhouse, Oltea Sampetrean, Jeffrey J. Olson, G. Yancey Gillespie, Hideyuki Saya, Christopher D. Willey, Erwin G. Van Meir. IGF1/N-cadherin/b-catenin/Clusterin signaling axis can mediates adaptive radioresistance in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3078.

STEM-16. IGF1/N-CADHERIN/b-CATENIN/CLUSTERIN SIGNALING AXIS MEDIATES ADAPTIVE RADIORESISTANCE IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is composed of heterogeneous tumor cell populations including those with stem cell properties, termed glioma stem cells (GSCs). GSCs are innately less radiation sensitive than the tumor bulk and are believed to drive GBM formation and recurrence after repeated irradiation. However, it is unclear how GSCs adapt to escape the toxicity of repeated irradiation used in clinical practice. To identify important mediators of adaptive radioresistance, we generated radioresistant human and mouse GSCs by exposing them to repeat cycles of irradiation. Surviving subpopulations acquired strong radioresistance in vivo, which was accompanied by increased cell-cell adhesion, slower proliferation, an elevation of stemness properties and N-cadherin expression. Increasing N-cadherin expression rendered parental GSCs radioresistant, reduced their proliferation, and increased their stemness and intercellular adhesive properties. Conversely, radioresistant GSCs lost their acquired phenotypes upon CRISPR/Cas9-mediated knockout of N-cadherin. Mechanistically, elevated N-cadherin expression resulted in the accumulation of b-catenin at the cell surface, which suppressed Wnt/ b-catenin proliferative signaling, reduced neural differentiation, and protected against apoptosis through Clusterin secretion. Restoration of wild type N-cadherin, but not mutant N-cad lacking b-catenin binding region, led to induce radioresistance in N-cadherin knockout GSCs, indicating the importance of the binding between N-cadherin and b-catenin. We also demonstrated that N-cadherin upregulation was induced by radiation-induced IGF1 secretion, and the radiation resistance phenotype could be reverted with picropodophyllin (PPP), a clinically applicable blood-brain-barrier permeable IGF1 receptor inhibitor, supporting clinical translation. Moreover, the elevation of N-cad and Clusterin are related to prognosis of GBM in the TCGA dataset. In conclusion, our data indicate that IGF1R inhibitor can block the N-cadherin-mediated resistance pathway. Our study deepens our understanding of adaptive radioresistance during repeated irradiation in GBM, and validates the IGF1/N-cadherin/b-catenin/Clusterin signaling axis as a novel target for radio-sensitization, which has direct therapeutic applicability.

Abstract 677: TTK: A novel target for radiosensitization in triple-negative breast cancers

Abstract Background: Increased rates of locoregional recurrence leading to poorer clinical outcomes have been observed in triple-negative breast cancer (TNBC) despite the use of radiation therapy (RT), therefore approaches that result in radiosensitizaton in TNBC are critically needed. Our previous work identified a group of cell cycle kinases differentially expressed in estrogen receptor negative (ER-) versus estrogen receptor positive (ER+) breast cancer (BC). Here we described the impact of elevated expression of one of these identified kinases, TTK, on radiation response and patient outcomes in TNBC. Methods: TCGA breast cancer datasets were used to determine TTK expression in the intrinsic subtypes of BC. TTK RNA and protein levels were measured using qPCR and western blot at baseline and after radiation treatment. Clonogenic survival assays were used to determine the radiosensitization in several cells after TTK inhibition. DNA damage was quantified using γH2AX staining in TNBC cell lines. Kaplan-Meier analysis was used to determine the impact of TTK expression on locoregional recurrence (LRR) and overall survival (OS). A Cox proportional hazards model was constructed to identify potential factors of LRR-free survival in univariate (UVA) and multivariable analyses (MVA). Results: TTK expression is elevated in breast cancer tissue compared to normal breast tissue (Q-VAL: 8.70 E-291) and is most highly expressed in basal-like tumors. TTK is overexpressed in ER- versus ER+ tumors in both breast cancers patient samples (p-value: <0.0001), and cells lines (p-value: <0.0001). TTK expression is significantly correlated with intrinsic radioresistance in a panel of 23 BC cell lines (R=0.58, p-value=0.0035). TTK RNA and protein levels are significantly increased at 12 and 24 hours after RT in TNBC cell lines. Genomic (siRNA knockdown) or pharmacologic (NMS-P715) inhibition of TTK increased radiosensitivity in vitro in 3 different TNBC cell lines. This sensitization is mediated, at least in part, through impaired DNA damage repair. Increased γH2AX foci was found after combination treatment of RT and TTK inhibition compared to only TTK inhibition, or RT. Clinically, breast cancer patients treated with breast conserving surgery and RT whose tumors have higher than median expression of TTK had worse local-recurrence free (LRF) survival and overall survival (HR for local recurrence 1.7 as continuous variable, p-value 0.004) compared to patients with higher than median expression, and this pattern held when considering quartile expression. TTK expression was associated with poorer LRF survival in UVA and in MVA only TTK expression and grade were significantly associated with worse LRF survival in 3 independent datasets. Conclusion: Our results support the rationale for developing translational clinical trials to investigate TTK inhibition as a novel radiosensitizing target in TNBC. Citation Format: Benjamin C. Chandler, Leah Moubadder, Cassie Ritter, Yashar Niknafs, Eric Olsen, Meleah Cameron, Meilan Liu, Kari Wilder-Romans, Shyam Nyati, Powel Brown, Arul Chinnaiyan, Corey Speers. TTK: A novel target for radiosensitization in triple-negative breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 677.

Abstract 3898: Emerging role of Fra-1 as a novel target for radiosensitization in HPV-associated cancers

Abstract Background: Activator protein-1 (AP-1) plays a central role in HPV-mediated cervical and tongue cancer (subset of HNSCC). AP-1, a homo/heterodimeric protein composed of Jun and Fos family proteins, has been implicated in chemoradioresistance but the mechanism(s) remained unexplored. In the present study, AP-1 family protein Fra-1 has been shown to play a critical role in promoting radiosensitization in enriched cervical cancer stem-like cells (CaCxSLCs) and can serve as a target for drug intervention in cervical and other HPV associated cancers. Experimental Procedure: Cervical cancer stem-like cells (CaCxSLCs) were isolated and enriched by sequential gating HPV+ve and HPV-ve human cervical cancer cell lines, SiHa, and C33a using a set of functional and phenotypic markers (ABCG2, CD49f, CD71, CD133) in defined conditioned media (DCM) with intermittent culturing. CaCxSLCs were also assessed for their cervicosphere forming ability, exclusion of DCV dye for generating SP cells, self renewability and quiescenceness. Both CaCxSLCs and Non CaCxSLCs were exposed to different dose of UV irradiation and a herbal compound curcumin, a potent inhibitor of AP-1 was used to examine the role of AP-1 and its family proteins in the radiosensitization of cancer stem cells. Results: Upon UV irradiation, CaCxSLCs showed a dose-dependent higher proliferation and highly increased AP-1 activity whereas it was completely abolished in non-stem cancer cells. CaCxSLCs also showed differential overexpression of c-Fos and c-Jun at transcript as well as in protein level. The loss of AP-1 activity and expression was accompanied by decrease in cell viability and proliferation in UV irradiated non-stem cancer cells. Interestingly, CaCxSLCs treated with curcumin prior to UV irradiation abolished AP-1 activity and a concomitant reduction in SP cells leading to abrogation of sphere forming ability, loss of proliferation, induction of apoptosis and the cells were poorly tumorigenic. The curcumin pre-treatment abolished the expression of c-Fos and c-Jun but upregulated Fra-1 expression in irradiated CaCxSLCs. Conclusion: The study suggests a critical role of AP-1 protein in the manifestation of radioresistance but curcumin pretreatment helps in radiosensitizing CaCxSLCs through upregulation of Fra-1 which may serve as a potential target for elimination of radioresistant cancer stem cells in HPV-associated malignancies. Note: This abstract was not presented at the meeting. Citation Format: Abhishek Tyagi, Shilpi Gupta, Prabhat Kuamr, Alok C. Bharti, Bhudev C. Das. Emerging role of Fra-1 as a novel target for radiosensitization in HPV-associated cancers [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 3898. doi:10.1158/1538-7445.AM2017-3898

Abstract 5839: Androgen receptor (AR): A novel target for radiosensitization in triple-negative breast cancers (TNBC)

Abstract Purpose: Increased rates of local recurrence (LR) have been observed in TNBC despite chemotherapy and radiation (RT). Thus, approaches that result in radiosensitization in TNBC are critically needed. We characterized the RT response of 21 breast cancer cell (BCC) lines using clonogenic survival assays and paired this with high-throughput drug screen data, identifying AR as a top target for radiosensitization. We demonstrate that AR inhibition confers radiosensitization in vitro and in vivo, identified a biomarker of response, and characterize the mechanism of AR-mediated radiosensitization in TNBC. Materials/Methods: Clonogenic survival assays determined the intrinsic RT sensitivity of 21 BCC lines. IC50 values were determined for 130 clinical compounds and correlation coefficients were calculated using IC50 values and SF-2Gy. Gene and protein expression was measured using RNA Seq and RPPA arrays, respectively, in tumor samples (n=2,061) and BCC lines (n=51). AR function was assessed using gene knockdown (KD) or functional inhibition with anti-androgen drugs. 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. A Cox proportional hazards model and MVA were used to determine variables associated with LRF survival. Results: Our unbiased drug radiosensitizer screen nominated bicalutamide as an 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 51 BCC lines and found heterogeneity in AR expression with strongly correlated expression of protein and RNA levels in TNBC (R2=0.89, p-value <0.001). Inhibition of AR, using both KD and drug (MDV3100) induced RT sensitivity with an enhancement ratio (ER) of 1.35-1.42 in AR+ TNBC lines with no effect on controls. Radiosensitization was at least partially dependent on impaired dsDNA break repair mediated by DNAPKcs. AR inhibition with MDV3100 significantly radiosensitized TNBC xenografts in mouse models and markedly delayed tumor tripling time (TTT) and tumor growth (median TTT 17.4 days for RT alone vs. not reached after 50 days for MDV3100+RT, p-value <0.001). Activated DNAPK was identified as a biomarker of response. Clinically, TNBC patients whose tumors had higher than median expression of AR had higher rates of LR after RT (HR for LR ~3, p-value <0.01, 2 independent datasets). In MVA, high AR expression was the variable most significantly associated with worse LRF survival after RT in TNBC patients, outperforming all other variables (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: Benjamin C. Chandler, Corey W. Speers, Shuang G. Zhao, Meilan Liu, Kari Wilder-Romans, Eric Olsen, Shyam Nyati, Daniel Spratt, Daniel Wahl, Daniel Hayes, Felix Y. Feng, Lori J. Pierce. Androgen receptor (AR): A novel target for radiosensitization in triple-negative breast cancers (TNBC) [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 5839. doi:10.1158/1538-7445.AM2017-5839

Abstract 841: UTR analysis identifies Musashi1 as a translational regulator of radiation-induced Golgi-related gene expression

Abstract The cellular radioresponse is regulated by post-translational modification of constitutively expressed proteins and by changes in gene expression. Although the role of constitutively expressed proteins, such as those involved in the DNA damage response and cell cycle checkpoints, is well-understood in the context of the radioresponse, radiation-induced changes in gene expression are not as clearly defined. We have previously demonstrated that glioblastoma stem-like cells (GSCs) alter their gene expression at the translational level in response to ionizing radiation (IR). In particular, the NSC11 GSC line demonstrated thousands of genes differentially translated at one hour after a dose of 2 Gy, while considerably fewer were modified at the transcriptome level. Furthermore, there was little overlap between the transcriptome and the translatome. Genes undergoing translational regulation in response to IR fell into distinct functional categories, including mitochondrial structure and function, Golgi function, and translational regulation. Here, we aimed to elucidate the mechanism by which IR induces translational changes in the NSC11 GSC line. NSC11 cells were either exposed to 2 Gy or mock irradiated, and polysomes were isolated by sucrose gradient fractionation one hour later. Polysome-bound mRNA was purified and analyzed by microarray. The 5’ and 3’ untranslated regions (UTRs) of the top 75 up- and down-regulated genes were analyzed for the presence of regulatory elements. The most frequently occurring element in the UTRs of both the top up- and down-regulated genes was the Musashi1 binding site. Musashi1 is an RNA binding protein that typically binds to 3’ UTRs to regulate mRNA translation. It is a neural stem cell marker, and its expression is correlated with the grade of malignancy in glioma. GO term analysis of known Musashi1 target genes that were translationally regulated in response to IR revealed an enrichment of genes involved in Golgi function and vesicular trafficking. Knockdown of Musashi1 prevented IR-induced Golgi dispersal in NSC11 cells. Musashi1 knockdown also radiosensitized NSC11 cells, indicating a role for the protein in the cellular radioresponse. Together, these data demonstrate that in the NSC11 GSC line, Musashi1 contributes to radiation-induced translational regulation, specifically in the context of Golgi-related genes, and suggests that Musashi1 is a novel target for radiosensitization. Citation Format: Stacey L. Lehman, John W. O'Neill, Kevin Camphausen, Philip J. Tofilon. UTR analysis identifies Musashi1 as a translational regulator of radiation-induced Golgi-related gene expression [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 841. doi:10.1158/1538-7445.AM2017-841

Secretome Signature Identifies ADAM17 as Novel Target for Radiosensitization of Non-Small Cell Lung Cancer.

Ionizing radiation (IR) induces intracellular signaling processes as part of a treatment-induced stress response. Here we investigate IR-induced ADAM17 activation and the role of ADAM17-shed factors for radiation resistance in non-small cell lung cancer. Large-scale secretome profiling was performed using antibody arrays. Secretion kinetics of ADAM17 substrates was determined using ELISA across multiple in vitro and in vivo models of non-small cell lung cancer. Clonogenic survival and tumor xenograft assays were performed to determine radiosensitization by ADAM17 inhibition. On the basis of a large-scale secretome screening, we investigated secretion of auto- or paracrine factors in non-small cell lung cancer in response to irradiation and discovered the ADAM17 network as a crucial mediator of resistance to IR. Irradiation induced a dose-dependent increase of furin-mediated cleavage of the ADAM17 proform to active ADAM17, which resulted in enhanced ADAM17 activity in vitro and in vivo Genetic or pharmacologic targeting of ADAM17 suppressed IR-induced shedding of secreted factors, downregulated ErbB signaling in otherwise cetuximab-resistant target cells, and enhanced IR-induced cytotoxicity. The combined treatment modality of IR with the ADAM17 inhibitor TMI-005 resulted in a supra-additive antitumor response in vivo demonstrating the potential of ADAM17 targeting in combination with radiotherapy. Radiotherapy activates ADAM17 in non-small cell lung cancer, which results in shedding of multiple survival factors, growth factor pathway activation, and IR-induced treatment resistance. We provide a sound rationale for repositioning ADAM17 inhibitors as short-term adjuvants to improve the radiotherapy outcome of non-small cell lung cancer. Clin Cancer Res; 22(17); 4428-39. ©2016 AACR.

Abstract LB-010: Maternal embryonic leucine zipper kinase (MELK): A novel target for radiosensitization that is independently prognostic in triple-negative breast cancers

Abstract Background: Increased rates of locoregional recurrence have been observed in TNBC despite the use of RT, therefore approaches that result in radiosensitizaton in TNBC are critically needed. Our previous work identified one such novel molecular target and we sought to investigate the impact of MELK expression on radiation response and patient outcomes. Methods: MELK expression was measured with RNA-seq and RPPA in 2,061 breast tumor samples and 51 breast cancer cell (BCC) lines. Clonogenic survival assays were used to quantify the degree of radiosensitivity after MELK inhibition. Mass spectrometry was utilized to identify proteins associated with MELK. NHEJ assays utilizing linearized pEYFP vector with FACS and qRT-PCR assessed NEHJ competency. We measured in vivo tumor growth and doubling time using xenografts in mice (16-20 tumors/group) treated with vehicle, RT, MELK-inhibitor, or combination. Kaplan-Meier analysis assessed the clinical impact of MELK on LR and OS. A Cox proportional hazards model was constructed to identify potential factors of LRF-survival in UVA and MVA. Results: MELK expression is absent in normal tissues and only expressed in cancerous tissue, suggesting a favorable therapeutic index. MELK expression was significantly elevated in human TNBC (p-value 7.5e-21) compared to non-TNBC. MELK expression was significantly correlated with radioresistance (R: 0.62, p-value 0.003). Inhibition of MELK using both siRNA and a small molecule inhibitor induced radiation sensitivity in vitro with an enhancement ratio (ER) of 1.5-1.6 through impaired dsDNA repair and cell cycle arrest mechanisms. MELK protein interacts with Ku70 and Ku80 to mediate NHEJ which is significantly impaired at 1, 3, and 6 hours after MELK inhibitor treatment (p-value<0.01). MELK inhibition either with gene knock-down or small molecule inhibitor significantly radiosensitized TNBC xenografts in mouse models and markedly delayed tumor doubling time and tumor growth (median tumor doubling 7.95 vs. 29.1 days for RT vs. MELK inhibition+RT, p-value <0.0001). Breast cancer patients whose tumors have high expression of MELK had markedly higher rates of LR after RT and an overall poorer prognosis than patients with low expression of MELK (HR for LR 1.89-2.23, p-value<0.001; HR for OS 1.46-3.3; p-value<0.001 in 3 independent datasets). In multivariable analysis only MELK expression and grade were significantly associated with worse LRF survival (HR:1.35,p-value<0.01). Conclusion: Our results support the rationale for developing clinical strategies to inhibit MELK as a novel target to increase radiosensitivity in TNBC. Citation Format: Corey Speers, Alyssa Santola, Shuang G. Zhao, Meilan Liu, Vishal Kothari, Kari Wilder-Romans, Theodore S. Lawrence, Powel H. Brown, Lori J. Pierce, Felix Y. Fang. Maternal embryonic leucine zipper kinase (MELK): A novel target for radiosensitization that is independently prognostic in triple-negative breast cancers. [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 LB-010.

712 - Secretome signature identifies ADAM17 as novel target for radiosensitization of non-small cell lung cancer

712 - Secretome signature identifies ADAM17 as novel target for radiosensitization of non-small cell lung cancer

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.

Maternal Embryonic Leucine Zipper Kinase (MELK): A Novel Target for Radiosensitization That is Independently Prognostic in Triple-Negative Breast Cancers

Maternal Embryonic Leucine Zipper Kinase (MELK): A Novel Target for Radiosensitization That is Independently Prognostic in Triple-Negative Breast Cancers

Unbiased Analysis of Pancreatic Cancer Radiation Resistance Reveals Cholesterol Biosynthesis as a Potent Novel Target for Radiosensitization

Unbiased Analysis of Pancreatic Cancer Radiation Resistance Reveals Cholesterol Biosynthesis as a Potent Novel Target for Radiosensitization

Pharmacologic Inactivation of Kinase Suppressor of Ras1 Sensitizes Epidermal Growth Factor Receptor and Oncogenic Ras-Dependent Tumors to Ionizing Radiation Treatment

Selective enhancement of tumor response to radiation therapy is a highly attractive objective, but it has not been met clinically. Gain-of-function Ras (gf) signaling via hyperactivation of receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR), or via oncogenic mutation of Ras is shown to confer radioresistance and requires the engagement of the Raf/MEK/ERK pathway. However, upstream mediators of such interaction in cancer cells that could be targeted for radiosensitization have not been identified and characterized. Here, we provide original observations both in vitro and in vivo that kinase suppressor of Ras1 (KSR1) is a new target for reversing gf Ras-mediated radioresistance. We employed EGFR-dependent A431 squamous cell carcinoma (SCC) and genetically defined the molecular function of KSR1 in irradiation-induced Raf/MEK/ERK activation. In vitro KSR1 inactivation via genetic inhibition of its expression or kinase function abrogated ionizing radiation-induced activation of the Raf/MEK/ERK2 cascade, enhanced the cytotoxic effect of radiation, and achieved radiosensitization associated with inhibition of DNA damage repair and enhancement of clonogenic death. In vivo pharmacologic inactivation of KSR1 by KSR1 AS-ODN infusion leads to radiosensitization in EGFR-dependent A431 SCC and in oncogenic K-Ras-driven A549 human non-small cell lung carcinoma. These observations collectively establish KSR1 as a novel target for radiosensitization and show the feasibility of using KSR1 AS-ODN as a radiosensitizer for treating gf Ras-dependent human malignancies. Identification of such mediators of gf Ras signaling in response to irradiation holds promises for improving the therapeutic efficacy of radiation therapy and our ability to eradicate tumor.

N-linked Glycosylation is a Novel Target for Radiosensitization

Tumor cell proliferation and survival are dependent upon both unique and redundant signaling cascades initiated by receptor tyrosine kinases (RTKs) in response to either growth factors or ionizing radiation. Inhibition of specific receptors (i.e., EGFR) has been shown to enhance cancer cell radiosensitivity in multiple tumor models. However, therapeutic resistance can be mediated by non-targeted receptors coexpressed by tumor cells. We hypothesized that disrupting N-linked glycosylation, a critical and highly regulated post-translational modification, would be an effective strategy to disrupt the function of multiple RTKs over-expressed by tumor cells.