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About 70% of all breast cancer tumors are estrogen receptor positive (ER+) and are treated with antiestrogen therapies. While the inevitability of developing resistance to these therapies remains uncontested, little is known about the mechanism and prevention of resistance. Our ultimate goal is to use mathematical modeling to optimize dynamic therapies that decrease proliferation and stave off resistance. In this initial study, we used MCF7 cells as a model of ER+ breast cancer and estrogen deprivation as a surrogate for aromatase inhibitors. We developed long-term estrogen deprived MCF7s (LTEDs) that proliferate similarly to untreated MCF7s but are resistant to antiestrogens. We collected time-course data for simultaneous gene expression and protein levels (NanoString Pan Cancer panel, a non-amplification based digital method) over 6 weeks to capture early molecular adaptations of deprived MCF7s and compare them to those present in LTEDs. PCA analysis of the mRNA and protein data shows a dramatic change in the first component due to estrogen deprivation, and a dramatic change in the second component associated with long-term resistance. Correlation analysis shows a large number of cell cycle genes that are similarly regulated, decreasing with estrogen deprivation and increasing with the onset of resistance. There is also a highly-correlated group of genes associated with resistance. This knowledge allowed us to hypothesize a molecular mechanism for resistance that can be tested experimentally. To begin building a dynamic model, we measured a 7-day time course of estrogen related proteins. The model is built around ER signaling and the cell cycle, and simulates protein and proliferation changes in response to deprivation and antiestrogen (ICI182,780; ICI) treatment. To determine which treatments to model in addition to estrogen deprivation and ICI, we looked for a promising sequential therapy to limit proliferation and, hopefully, stave off resistance compared to a single non-stop therapy. We found that alternating the targeting of CDK4/6 using Palbociclib (clinical anti-cancer therapy) with targeting of RUNX1 (gene that is increased at the onset of resistance in our model), could be a plausible strategy to inhibit endocrine resistance. Future work will involve extending the model to longer time scales and using it for treatment optimization. Citation Format: Wei He, Diane M. Demas, Isabel Conde, Yassi Fallah, William T. Baumann, Ayesha N. Shajahan-Haq. Predicting cellular response to therapy in breast cancer using mathematical modeling [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 674.

Tumor dissemination and relapse are the major problems in Ewing sarcoma (ES) treatment, yet the mechanisms driving these processes are unknown. To elucidate the routes of ES metastatic spread, we used an orthotopic xenograft model. ES cells were injected into the gastrocnemius muscles of SCID/beige mice. Once the primary tumors reached the desired volume, they were excised by limb amputation. Subsequently, tumor dissemination was monitored by MRI and confirmed by histopathologic analysis. Interestingly, aside from typical hematogenous metastases, such as bone and lung lesions, we have also observed frequent perineural tumor dissemination manifested by the presence of migratory ES cells along the nerves adjacent to the primary tumors. This phenomenon was associated with formation of recurrent tumors at the amputation sites, as well as pelvic tumors with spine involvement. Interestingly, the level of perineural invasion (PNI) was dependent on the expression of neuropeptide Y (NPY) in ES cells. NPY is a neuronal protein released from peripheral sympathetic neurons, but also highly expressed in ES cells along with its receptors. The xenografts derived from ES cell lines not releasing endogenous NPY (TC71, TC32) exhibited frequent PNI in tumor-bearing limbs, as well as a high number of recurrent tumors at the surgery site and spine metastases (70% and 100% of mice with evidence of PNI for TC71 and TC32 xenografts, respectively). This phenomenon was less common in ES xenografts derived from NPY-rich SK-ES1 cells (17% of mice with signs of PNI). In line with these observations, NPY knockdown in SK-ES1 xenografts drastically accelerated formation of spinal tumors (60% of mice). Notably, in 40% of mice bearing SK-ES1/NPY shRNA xenografts the spinal tumors developed before the primary tumor growth was detectable at the site of ES cell injection. Thus, our in vivo experiments suggested that a lack of endogenous NPY in ES cells expressing high levels of its receptors triggers chemotactic effects of this peptide released from neighboring peripheral nerves, facilitating PNI. Indeed, in a transwell migration assay, NPY exerted significant chemotactic activity in SK-ES1/NPY shRNA cells, but not in the original SK-ES1 cell line. An even more profound chemotactic effect specific to the SK-ES1/NPY shRNA cells was observed with NPY-rich conditioned media obtained from neuroblastoma cells, which can serve as a model of peripheral sympathetic neurons. Further studies are required to determine which NPY receptors are responsible for its chemotactic properties. If the presence of perineural tumor growth is confirmed in human tumors, factors responsible for PNI in ES, e.g., NPY receptors, may become targets for novel therapies preventing disease dissemination and recurrence. Citation Format: Susana Galli, Sung-Hyeok Hong, Jason U. Tilan, Mina Adnani, Shiya Zhu, Yassi Fallah, Yi-Chien Lee, Olga Rodriguez, Chris Albanese, Ewa Izycka-Swieszewska, Joanna Kitlinska. Perineural invasion in Ewing sarcoma—a novel mechanism and new therapeutic opportunities [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B13.

IT-139 is a novel small-molecule drug that demonstrated a manageable safety profile at the maximum tolerated dose and modest antitumor activity as a monotherapy in a phase I clinical trial. A high affinity for albumin and unique chemistry allow increased uptake and activation of IT-139 within tumor cells, respectively. To date, IT-139 has been reported to inhibit the endoplasmic reticulum stress-sensing protein GRP78. However, different cancer cells show disparate responses to IT-139 and the precise mechanism of action in high-response versus low-response cancer cells remains unclear. In vitro studies have indicated that IT-139 induces cytostatic rather than cytotoxic effects as a monotherapy. In this study, to understand IT-139 mediated signaling mechanism in breast cancer cells, we used estrogen receptor-positive (ER+) MCF7 breast cancer cells to obtain gene-metabolite integrated models following IT-139 treatment (10 or 100 μM for 72 h). Our data showed that IT-139 significantly reduced expression of genes involved in the DNA repair pathway, including BRCA1 and RAD51, following treatment with IT-139 in MCF7 cells. Validation of our findings in other breast cancer cell lines including MDA-MB-175 (ER+), MDA-MB-231 and MDA-MB-468 (both ER-) cells also showed increased phosphorylation of H2AX (Ser139) following IT-139 treatment, suggesting lack of proper DNA surveillance and subsequent accumulation of DNA double-stranded breaks (DSB). Consequently, we investigated the effects of combination treatment approaches with IT-139 and other clinical anticancer therapies that interfere with nucleotide synthesis or DNA damage repair pathways, particularly in ER- breast cancers. Our data showed additive effects of IT-139 in combination with capecitabine (5-FU prodrug) or olaparib (PARP inhibitor) and a synergistic effect with carboplatin in ER- breast cancer cell lines. Further analysis of IT-139 mediated signal transduction in breast cancer cells will reveal whether it can be combined with aforementioned chemotherapies to circumvent resistance. Citation Format: Isabel Conde, Yassi Fallah, Diane Demas, Suzanne Bakewell, Filipa Lynce, Ayesha N. Shajahan-Haq. Inhibition of DNA repair pathways in breast cancer is a potential mechanism of action of IT-139 [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 2862.

About 70% of all breast cancers are estrogen receptor alpha positive (ER+). Anti-hormone therapy such as antiestrogens are often used to treat ER+ breast cancer but breast cancer cells can develop resistance to these drugs (endocrine resistance). Unfortunately, ~50% percent of all antiestrogen treated tumors eventually develop endocrine resistance, and therefore, there is an urgent need to find ways to treat this incurable disease. We have shown that endocrine resistant breast cancer cells show an increased dependence on the amino acid glutamine and this process is regulated by MYC activation via the unfolded protein response (UPR). Metabolites of glutamine such as glutamate and proline are significantly elevated in endocrine resistant cells. Cellular changes in glutamine are sensed by the mammalian target of rapamycin (mTOR) complex, mTORC1, which is known to be deregulated in endocrine resistant breast cancer. In this study, we used human antiestrogen (Fulvestrant/Faslodex or Tamoxifen) sensitive and resistant ER+ breast cancer cells and xenografts to test the efficacy of CB-839, an anti-glutaminase, and everolimus (Afinitor), a mTORC1 inhibitor. Combination of CB-839 and everolimus, but not each drug alone, inhibited growth of antiestrogen resistant tumors compared to vehicle alone at 4 weeks post-treatment. The combination treatment did not significantly inhibit growth of antiestrogen sensitive tumors compared with its respective vehicle alone treatment. Thus, a combination strategy that targets glutamine dependence and increased mTOR activation may be a novel strategy in treating endocrine resistant breast cancer. Citation Format: Yassi Fallah, Diane M. Demas, Susan Demo, Ayesha N. Shajahan-Haq. Combination of CB-839 and everolimus is effective in inhibiting growth of endocrine resistant breast cancer in vivo [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 430. doi:10.1158/1538-7445.AM2017-430

Introduction: Pancreatic ductal adenocarcinoma (PDAC) represents 90% of pancreatic neoplasms and the fourth leading cause of cancer death in the United States. Recently, several research groups have focused on conducting metabolomics based clinical investigations to identify metabolite markers of PDAC. Since a central biomarker repository for PA is lacking, however, it remains challenging to delineate the total number of metabolites reported, and importantly, which bio-signatures overlap between the disparate studies. Approach: We performed an extensive literature search to delineate PDAC case-control studies reporting dysregulated metabolite biomarkers for PDAC utilizing three main sources: PubMed, EDRN and GDOC. We refined our search to 36 publications that reported blood based metabolomics/lipidomic biomarkers. Data from these studies was compiled based on up/down-regulation of specific metabolites in PDAC, study sample size, and other clinical determinants. The final list consisted of 12 metabolites (including amino acids, fatty acids and small organic acids), that were reported by two or more research groups as being dysregulated in PDAC. Of note, these 36 disparate studies were carried out using different methodologies, analyzed on diverse analytical platforms, and utilized different statistical and bioinformatics methodologies. Resultant common features, therefore, were deemed significant as candidate biomarkers for cross-validation. Next, we used stable isotope dilution multiple-reaction monitoring mass spectrometry (SID-MRM-MS) for targeted quantitation of these 12 metabolite markers in plasma samples obtained from patients that were diagnosed with PDAC and compared the same profiles to matched normal controls. Results: We delineated of a sub-set of the original 12 metabolites that showed concordance with those reported in the literature. Statistical analyses to determine the sensitivity and specificity of the resultant panel, and any influence provided by age, gender, and type 2 diabetes, on the predictive performance of the biomarker panel are ongoing. These data will be presented at the AACR meeting. Conclusion: Developing a specific and sensitive panel of blood-derived biomarkers offers a practical approach towards screening and allows increasing overall survival rates for PDAC with early diagnosis. Such disease-specific bio-signatures allow identification of molecular targets for therapeutic development; improve early treatment strategies and thereby clinical outcomes. Creating a compendium of existing biomarker data and performing cross-validation studies represent the first steps for developing clinical assays for the diagnosis and prognosis determination of PDAC. The integration of data generated from multiple analytic platforms and diverse subject cohorts is likely to help identify robust bio-signatures that would then be ready for large scale validation studies. Supported by American Cancer Society Citation Format: Hung-Jen Wu, Khyati Mehta, Smrithi S. Menon, Keith Unger, Massimo S. Fiandaca, Yassi Fallah, Mark Mapstone, Howard J. Federoff, Amrita K. Cheema. Prognostic biomarkers of PDAC - a cross-validation study [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 2517. doi:10.1158/1538-7445.AM2017-2517