81 Sensitization of triple-negative breast cancer to PI3K inhibition by cotargeting IGF1R

Poster Session – Drug Resistance and Modifiers 80 POSTER Determination of an oxidative stress gene signature in inflammatory breast cancer patient tumors and development of a novel redox modulatory strategy in overcoming chemotherapy resistance and mediating anti-tumor efficacy G.R. Devi1 , J.L. Allensworth2 , M. Evans2 , N. Ueno3 , D. McDonnell4 , F. Bertucci5 , S. Van Laere6 . 1 Duke University, Surgery and Duke Cancer Institute, Durham NC, USA; 2 Duke University, Surgery, Durham NC, USA; 3 University of Texas MD Anderson, Oncology, Houston, USA; 4 Duke University, Pharmacology, Durham NC, USA; 5 Institut Paoli-Calmettes, Oncology, Marsellie, France; 6 General Hospital Sint-Augustinus, Oncology, Antwerp, Belgium Background: Cancer cells often have increased levels of reactive oxygen species (ROS); however, acquisition of redox adaptive mechanisms allows for evasion of ROS-mediated cell death. Chemotherapies including the anthracyclines, taxanes, alkylating agents, platinum compounds as well as radiation therapy all rely heavily on the induction of oxidative stress-induced apoptosis for their anti-tumor activities; thus, redox adaptation can confer resistance to many breast cancer therapies. Inflammatory breast cancer (IBC) is an advanced and distinct breast cancer subtype characterized by high rates of residual disease and regional recurrence despite advances in multimodality treatment. We hypothesize that the poor response of IBC tumors to common treatment strategies, many of which rely heavily on ROS induction, is the result of redox adaptation. Experimental Design: Metagene analysis was conducted to identify an oxidative stress response (OSR) gene signature in IBC cells treated with an acute dose of a strong ROS inducer and then allowed to recover. This OSR signature was applied to normal breast samples, pre-treatment IBC and nonIBC patient tumor samples to assess their oxidative stress response (OSR). A dithiocarbamate, disulfiram (DSF), currently approved as an antialcoholism drug was applied to therapeutically resistant IBC cells alone or in combination with copper (Cu); effects on viability, antioxidants, survival factors, and tumor progression were assessed. Results: Analysis of patient samples using the unique OSR metagene generated revealed significantly higher OSR scores in IBC tumor samples compared to normal or non-IBC tissue. In addition, we have identified a novel mechanism of DSF to act as a Cu ionophore in a Cu-transporter Ctr1-independent manner, target the Cu signaling redox axis in inducing oxidative stress-mediated apoptosis. This resulted in a potent inhibition of NF-úB signaling, aldehyde dehydrogenase activity, 3D mammosphere formation, in vivo orthotopic mammary tumor growth in murine IBC models and in overcoming chemotherapy resistance. Conclusions: These results indicate that IBC tumors are highly redox adapted, which may render them resistant to ROS-inducing therapies. DSF, through redox modulation, may be a useful approach to enhance chemoand/or radio-sensitivity for advanced BC subtypes where therapeutic resistance is a major challenge to durable responses to current standard of care. 81 POSTER Sensitization of triple-negative breast cancer to PI3K inhibition by cotargeting IGF1R K. de Lint1 , J.B. Poell1 , J. Vidal Rodriguez1 , H. Soueidan1 , W. Nijkamp1 , L.F. Wessels1 , R.L. Beijersbergen1 . 1 Netherlands Cancer Institute − Antoni van Leeuwenhoek, Molecular Carcinogenesis, Amsterdam, Netherlands Background: Targeted therapies have proven invaluable in the treatment of breast cancer, as exemplified by tamoxifen treatment for hormone receptorpositive tumors and trastuzumab treatment for HER2-positive tumors. In contrast, a subset of breast cancer negative for these markers (triplenegative breast cancer) has met very limited success with pathwaytargeted therapies. A large fraction of triple negative breast cancers depend on the PI3K pathway for proliferation and survival, however, inhibition of PI3K alone generally has limited clinical benefit. It is of interest to identify potential combination therapies that can enhance the response and thereby improve clinical outcome for triple-negative breast cancer. Materials and Methods: We have analyzed the response of a panel of triple-negative breast cancer cell lines to the PI3K inhibitor GDC-0941. We have generated a breast cancer cell line resistant to PI3K inhibition due to an adaptive response. We have applied large-scale pooled shRNA screening for the identification of genes required for cell proliferation under PI3K inhibition in the adapted triple-negative cell line. Results: We have identified IGF1R as crucial component of the adaptive response to PI3K inhibition. Knock-down of IGF1R potently restores the sensitivity of these cells to the PI3K inhibitor GDC-0941. Pharmacological inhibition of IGF1R prevents colony outgrowth of adapted cells. In treatment-naive cells IGF1R inhibition sensitizes cells to PI3K inhibition and induces apoptosis. In a subset of triple-negative breast cancer cell lines,

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IGF1R inhibition shifts the dose–response curve of GDC-041 up to 5-fold. In particular we find that breast cancer cell lines with high expression of IGF2-processing and signaling genes are responsive to the combination of PI3K and IGF1R inhibitors, whereas high expression of ERBB3 or mutated RAS are associated with a limited benefit of IGF1R inhibition. Conclusions: Our results suggest that combination treatment with PI3K and IGF1R inhibitors may prove beneficial over monotherapy in a subset of triple-negative breast cancers. 82 POSTER Regorafenib resistance in colorectal carcinoma is associated with enhanced expression of type II interleukin 1 receptor and reversed by MEK/ERK inhibitor A.C. Mar1 , C.H. Chu2 , C.W. Shiau3 , T.C. Lee1 . 1 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan; 2 Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan; 3 Institute of Biopharmaceutical Science, National Yang-Ming University, Taipei, Taiwan Targeted therapy initially shows promising tumor regression but the development of drug resistance quickly results in failure of treatment. Among a batch of colorectal cancer (CRC) cell lines, we have preliminarily found that the expression levels of type II interleukin 1 receptor (IL1R2), an IL-1 decoy receptor, were closely associated with Regorafenib resistance. In addition, IL1R2 expression was associated with poor prognosis of patients with CRC cancer. Regorafenib was the newly approved multikinase inhibitor used for treatment of late-stage metastatic CRC by US FDA. In this study, we conducted experiments to elucidate the mechanism underlying which IL1R2 is involved in Regorafenib resistance. We first demonstrated that silencing of IL1R2 in HT29 cells overcame its resistance to Regorafenib, whereas ectopic expression of IL1R2 in HCT116 cells reduced its sensitivity to Regorafenib in both in vitro and in vivo systems. In addition, enhanced expression of IL1R2 was observed in Regorafenib-resistant DLD-1 colon cancer cells (DLD-1-R), which were established by growing DLD-1 cells in the presence of Regorafenib. In IL1R2 overexpressing cells, such as HT29, IL1R2-overexpressing HCT116, and DLD-1-R cells, Regorafenib treatment significantly resulted in activation of MEK/ERK signaling, which is crucial for survival. We also revealed that increased p-ERK levels in Regorafenib treated IL1R2overexpressing cells were likely due to decreased expression of MKP-3, one of the phosphatases of ERK. Furthermore, pretreatment of HT29, IL1R2-overexpressing HCT116, and DLD-1-R cells with MEK/ERK inhibitor U0126 significantly reversed their Regorafenib resistance in in vitro and in vivo systems. Taken together, our present study suggested that enhanced IL1R2 plays certain roles on Regorafenib resistance and the combination of Regorafenib and MEK/ERK inhibitor is a rationale regime to overcome Regorafenib resistance in CRC patients. 83 POSTER Acquired resistance to BET bromodomain inhibitors is associated with modulation of the apoptotic signaling network P. Sandy1 , S. Nerle1 , A. Conery2 , C. Hatton3 , B. Bryant3 , R. Sims2 , E. Normant1 . 1 Constellation Pharmaceuticals, Pharmacology, Cambridge, USA; 2 Constellation Pharmaceuticals, Biology, Cambridge, USA; 3 Constellation Pharmaceuticals, Bioinformatics, Cambridge, USA Background: Bromodomain and extraterminal (BET) family proteins function as chromatin readers that recognize and bind acetylated lysine residues on histones and play an essential role in the regulation of transcription of key oncogenes, including c-Myc and Bcl-2. Potent and selective small molecule inhibitors of BET bromodomains (BETi) induce growth arrest and apoptosis in a wide range of tumor types, in both in vitro and in vivo models, and are currently being evaluated in Phase I clinical trials. In this study we sought to model acquired resistance to long term BETi treatment and elucidate the underlying mechanisms in different tumor types. Materials and Methods: We used two orthogonal approaches to generate cell lines with reduced phenotypic sensitivity to BET bromodomain inhibition. In one, A375 melanoma cells were treated in culture with a BETi at a concentration where the majority of cells were eliminated by apoptosis. After several months, colonies able to proliferate in the presence of the inhibitor were obtained. In parallel experiments, mice inoculated with MV4−11 acute myeloid leukemia cells were dosed with a BETi at its maximum tolerated dose and the surviving tumors were serially transplanted into similarly treated recipient animals. Two BETi-resistant clones maintained decreased sensitivity to BETi in vitro when compared to parental cells in a 72-hour viability assay. Results: Detailed phenotypic analysis revealed an attenuated apoptotic response to high-dose BETi in the resistant clones as compared to parental cells. Transcriptional profiling and proteomic analysis revealed altered