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Despite the success of antiestrogens in extending overall survival of patients with estrogen receptor positive (ER+) breast tumors, resistance to these therapies is prevalent. ER+ tumors that progress on antiestrogens are treated with antiestrogens and CDK4/6 inhibitors. However, 20% of these tumors never respond to CDK4/6 inhibitors due to intrinsic resistance. Here, we used endocrine sensitive ER+ MCF7 and T47D breast cancer cells to generate long-term estrogen deprived (LTED) endocrine resistant cells that are intrinsically resistant to CDK4/6 inhibitors. Since treatment with antiestrogens arrests cells in the G1 phase of the cell cycle, we hypothesized that a defective G1 checkpoint allows resistant cells to escape this arrest but increases their dependency on G2 checkpoint for DNA repair and growth, and hence, targeting the G2 checkpoint will induce cell death. Indeed, inhibition of WEE1, a crucial G2 checkpoint regulator, with AZD1775 (Adavosertib), significantly decreased cell proliferation and increased G2/M arrest, apoptosis and gamma-H2AX levels (a marker for DNA double stranded breaks) in resistant cells compared with sensitive cells. Thus, targeting WEE1 is a promising anti-cancer therapeutic strategy in standard therapy resistant ER+ breast cancer. Public Health ServiceUnited States Department of Health & Human ServicesUnited States Public Health Service [R01-CA201092, 1P30-CA-51008]; Susan G Komen TREND grant [GTDR15330383] Published version This research was partly funded by Public Health Service grant R01-CA201092 to ASH. YF was supported by a Susan G Komen TREND grant (GTDR15330383). Technical services were provided by the following shared resources at Georgetown University Medical Center: Tissue Culture and Flow Cytometry Shared Resources that were funded through Public Health Service award 1P30-CA-51008 (Lombardi Comprehensive Cancer Center Support Grant).

BOLD-100, a ruthenium-based complex, sodium trans-[tetrachloridobis (1H-indazole) ruthenate (III)] (also known as IT-139, NKP1339 or KP1339), is a novel small molecule drug that demonstrated a manageable safety profile at the maximum tolerated dose and modest antitumor activity in a phase I clinical trial. BOLD-100 has been reported to inhibit the upregulation of the endoplasmic reticulum stress sensing protein GRP78. However, response to BOLD-100 varies in different cancer models and the precise mechanism of action in high-response versus low-response cancer cells remains unclear. In vitro studies have indicated that BOLD-100 induces cytostatic rather than cytotoxic effects as a monotherapy. To understand BOLD-100-mediated signaling mechanism in breast cancer cells, we used estrogen receptor positive (ER+) MCF7 breast cancer cells to obtain gene-metabolite integrated models. At 100 &mu, M, BOLD-100 significantly reduced cell proliferation and expression of genes involved in the DNA repair pathway. BOLD-100 also induced reactive oxygen species (ROS) and phosphorylation of histone H2AX, gamma-H2AX (Ser139), suggesting disruption of proper DNA surveillance. In estrogen receptor negative (ER&minus, ) breast cancer cells, combination of BOLD-100 with a PARP inhibitor, olaparib, induced significant inhibition of cell growth and xenografts and increased gamma-H2AX. Thus, BOLD-100 is a novel DNA repair pathway targeting agent and can be used with other chemotherapies in ER&minus, breast cancer.

Homeobox genes are critical in tumor development. An isoform protein of DLX4 called BP1 is expressed in 80% of invasive ductal breast carcinomas. BP1 overexpression is implicated in an aggressive phenotype and poor prognosis. BP1 upregulation is associated with estrogen receptor negativity so those tumors do not respond to antiestrogens. Breast cancer is the second leading cause of death in women. BP1 could serve as both a novel prognostic biomarker for breast cancer and a therapeutic target. In this review, we address the role of BP1 protein in tumorigenesis of breast cancer and four other malignancies. A number of functions of BP1 in cancer are also discussed.

Dependence on the glutamine pathway is increased in advanced breast cancer cell models and tumors regardless of hormone receptor status or function. While 70% of breast cancers are estrogen receptor positive (ER+) and depend on estrogen signaling for growth, advanced ER+ breast cancers grow independent of estrogen. Cellular changes in amino acids such as glutamine are sensed by the mammalian target of rapamycin (mTOR) complex, mTORC1, which is often deregulated in ER+ advanced breast cancer. Inhibitor of mTOR, such as everolimus, has shown modest clinical activity in ER+ breast cancers when given with an antiestrogen. Here we show that breast cancer cell models that are estrogen independent and antiestrogen resistant are more dependent on glutamine for growth compared with their sensitive parental cell lines. Co-treatment of CB-839, an inhibitor of GLS, an enzyme that converts glutamine to glutamate, and everolimus interrupts the growth of these endocrine resistant xenografts. Using human tumor microarrays, we show that GLS is significantly higher in human breast cancer tumors with increased tumor grade, stage, ER-negative and progesterone receptor (PR) negative status. Moreover, GLS levels were significantly higher in breast tumors from African-American women compared with Caucasian women regardless of ER or PR status. Among patients treated with endocrine therapy, high GLS expression was associated with decreased disease free survival (DFS) from a multivariable model with GLS expression treated as dichotomous. Collectively, these findings suggest a complex biology for glutamine metabolism in driving breast cancer growth. Moreover, targeting GLS and mTOR in advanced breast cancer may be a novel therapeutic approach in advanced ER+ breast cancer.

The transcription factor MYC (MYC proto-oncogene, bHLH transcription factor) is an essential signaling hub in multiple cellular processes that sustain growth of many types of cancers. MYC regulates expression of RNA, both protein and non-coding, that control central metabolic pathways, cell death, proliferation, differentiation, stress pathways, and mechanisms of drug resistance. Activation of MYC has been widely reported in breast cancer progression. Breast cancer is a complex heterogeneous disease and treatment options are primarily guided by histological and biochemical evaluations of the tumors. Based on biochemical markers, three main breast cancer categories are ER+ (estrogen receptor alpha positive), HER2+ (human epidermal growth factor receptor 2 positive), and TNBC (triple-negative breast cancer; estrogen receptor negative, progesterone receptor negative, HER2 negative). MYC is elevated in TNBC compared with other cancer subtypes. Interestingly, MYC-driven pathways are further elevated in aggressive breast cancer cells and tumors that display drug resistant phenotype. Identification of MYC target genes is essential in isolating signaling pathways that drive tumor development. In this review, we address the role of MYC in the three major breast cancer subtypes and highlight the most promising leads to target MYC functions.

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