bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3744: Repurposing the neuroprotective agent dimethyl fumarate against white matter damage and cognitive decline after radiotherapy
Authors: Taraboletti Alexandra , Fornace Albert J.
Published: August 2019

Radiation therapy (RT) is a critical tool for the treatment of metastatic brain tumors, most commonly glioblastoma multiforme (GBM), however, patients can have significant cognitive impairment due to the exposure to ionizing radiation. Up to 50%-90% of adult patients that survive these brain tumors will develop radiation-induced cognitive dysfunction within 3-6 months. Brain injury from IR is characterized by white matter damage from the loss of myelin-producing oligodendrocyte cells, subsequent demyelination, and vascular inflammation. While neuro-oncology outcomes are often concerned with survival, strategies to ameliorate and understand radiation-induced demyelination after IR treatment are needed to preserve and improve patient quality of life (QOL). Here we investigate the ability of dimethyl fumarate (DMF), an established neuroprotective agent, to amend damage and demyelination caused by RT in oligodendrocyte cells versus glioma cells. Our in vitro study confirms that DMF acts opposingly in each cell type at lower doses (10 nM), but proves toxic in both the tumor and normal model at high concentrations (100 µM). DMF also increased survival rates in oligodendrocytes after radiation. Using metabolomics, we also noted that oligodendrocyte cells upregulated TCA cycle intermediates in response to DMF treatment with RT. This work provides a basis for in vivo dosing and timing strategies using DMF to cause cessation of ROS-driven oligodendrocyte loss and inflammation after RT. Ultimately, DMF is a promising therapy that could be used to ameliorate radiation-induced demyelination, promoting patient QOL.

Citation Format: Alexandra Taraboletti, Albert J. Fornace. Repurposing the neuroprotective agent dimethyl fumarate against white matter damage and cognitive decline after radiotherapy [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 3744.
Abstract 3745: Mechanisms of HIF2-mediated small intestine radioprotection
Authors: Garcia Carolina J. Garcia , Govindaraju Suman , Bonilla Marimar de la Cruz , Taniguchi Cullen M.
Published: August 2019

Pancreatic cancer is the fourth-leading cause of cancer-related deaths in the United States, and it is projected to become the second by 2030. The 5-year overall survival rate for locally advanced pancreatic cancer (LAPC) remains < 5%. Chemotherapy regimens often fail due to poor drug delivery and penetration into the tumor. Because surgery is not feasible by definition in LAPC, radiation therapy (RT) is the only option to achieve local control. However, irradiating the pancreas is challenging because potentially curative doses are limited by the neighboring small intestine, which can only tolerate a maximum of 50Gy. A selective radioprotector of the intestinal tract could allow pancreatic cancer patients to receive a higher, and potentially definitive, dose of radiation by increasing the duodenum’s tolerance to survive radiation injury. The EGLN prolyl hydroxylases are cellular oxygen sensors that regulate cell survival and metabolism through the degradation of hypoxia-inducible factors (HIFs) in the presence of oxygen. Our group has shown that HIF2 stabilization through genetic or pharmacologic inhibition of the EGLN family prevents death from radiation-induced gastrointestinal toxicity in mice without protecting the primary tumor. To understand the mechanism by which HIF2 mediates this protection, we generated intestinal organoids from mice with a Cre-inducible allele that expresses a non-degradable form of human HIF1 or HIF2. Whole transcriptomic analysis revealed HIF1 and HIF2 have distinct gene responses in the small intestinal crypt. HIF2 induced the expression of known radiation modulators and genes involved in oxidative damage response, tissue healing, and intestinal homeostasis such as the non-canonical Wnt5a. We validated HIF2 drives Wnt5a expression in multiple organoid model systems. We then generated knockouts (KO) of Wnt5a or Ror2, its cognate receptor, using the Cre/lox system to test if Wnt5a is necessary and sufficient for radioprotection. By using the spheroid formation assay, a modified clonogenic assay, we found Wnt5a KO organoids had decreased crypt regeneration following RT. Our preliminary data suggest Wnt5a protects the duodenum from RT by increasing intestinal stem cell survival.

Citation Format: Carolina J. Garcia Garcia, Suman Govindaraju, Marimar de la Cruz Bonilla, Cullen M. Taniguchi. Mechanisms of HIF2-mediated small intestine radioprotection [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 3745.
Abstract 3746: Fasting in mice enables abdominal radiation dose escalation in the setting of pancreatic cancer by mitigating small intestinal toxicity
Authors: Bonilla Marimar de la Cruz , Stemler Kristina M. , Jeter-Jones Sabrina , Fujimoto Tara N. , Molkentine Jessica , Torres Gabriela M. Asencio , Taniguchi Cullen M. , Piwnica-Worms Helen
Published: August 2019

Surgical resection is the only potentially curative treatment for pancreatic cancer, but only 15-20% of patients have resectable tumors. In unresectable cases, stereotactic body radiotherapy (SBRT) may be used to give tumor-directed radiotherapy (RT). Unfortunately, this can cause severe gastrointestinal (GI) toxicity due to proximity of the pancreatic head to the duodenum. Protecting the intestine from the toxic side-effects of radiation may enable dose-escalation that could achieve more effective local control of disease. We and others have previously shown that a fast of 24 hours protects mice from lethal doses of the DNA-damaging agent etoposide. In this study, we demonstrate that a 24 hour fast also protects mice from lethal doses of total-abdominal radiation. Histologic analyses using the Withers-Elkind microcolony assay show that fasting protected small intestinal (SI) stem cells from radiation damage and promoted early regeneration. To show a proof-of-principle for the use of this radioporotective maneuver in cancer therapy, we used an orthotopic model of pancreatic cancer using KPC tumor cells syngeneic to C57BL/6. Here, we show that fasting-mediated intestinal protection enabled dose escalated SBRT for treatment of these orthotopic tumors. RT with fasting-mediated radioprotection delayed tumor growth and improved survival compared to controls. Given this robust phenotype, we developed a 3D culture ex vivo assay using intestinal stem cell-enriched epithelial spheroid cultures. We modified these intestinal spheroids with a bioluminescent reporter and used these cells to develop a modified clonogenic assay for 3D culture that can be used to identify novel radioprotectors, such as a fasting mimetic. Taken together, these results suggest that fasting protects small intestinal stem cells, allowing animals to receive potentially curative doses of abdominal radiation that would otherwise be lethal. Future work will aim to identifying the mechanisms by which fasting confers intestinal protection and drug candidates that can be used to mimic this fasting-mediated protection.

Citation Format: Marimar de la Cruz Bonilla, Kristina M. Stemler, Sabrina Jeter-Jones, Tara N. Fujimoto, Jessica Molkentine, Gabriela M. Asencio Torres, Cullen M. Taniguchi, Helen Piwnica-Worms. Fasting in mice enables abdominal radiation dose escalation in the setting of pancreatic cancer by mitigating small intestinal toxicity [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 3746.
Abstract 3758: Targeting ovarian cancer induced peritoneal carcinomatosis by inhibition of signaling axis between Interleukin-6 (IL-6) and serine protease inhibitor Kazal type 1 (SPINK1)
Authors: Mehner Christine , Miller Erin , Coban Mathew A. , Hockla Alexandra , Radisky Derek C. , Radisky Evette S.
Published: August 2019

Ovarian cancer remains a challenging disease, with an average 5 year survival rate of 46%. At time of diagnosis, over 70% of patients have advanced metastatic disease, for which there are no effective treatment options. Of patients with metastatic disease, more than 70% of patients develop peritoneal carcinomatosis, making the peritoneum one of the main areas of dissemination.

We have previously found that expression of the serine protease inhibitor Kazal type 1 (SPINK1) by ovarian cancer cells drives anoikis resistance, allowing the tumor cells to circumvent apoptosis protocols and facilitating tumor cell metastasis. For this new study we focused on ovarian clear cell carcinoma (OCCC), a histotype of ovarian cancer that has particularly poor prognosis if diagnosed in late stage. Using OCCC cell lines, we found that knockdown of SPINK1 reduces cell survival and stimulates apoptotic pathways in OCCC cells, when cultured under attachment-free conditions to mimic metastatic disease. We then identified Interleukin-6 (IL-6) as an upstream regulator of SPINK1 expression and a potential therapeutic target for metastatic OCCC: we showed that IL-6 silencing reduced SPINK1 mRNA expression and cell survival, while treatment with recombinant IL-6 increased SPINK1 expression and attachment-free survival. We developed a new OCCC

mouse model and showed that knockdown of SPINK1 results in significant reduction of metastatic lesions, highlighting the impact of SPINK1 on OCCC metastasis. In ongoing studies we are testing intraperitoneal injections of FDA approved IL-6 inhibitors in a preclinical model to assess impact on abdominal dissemination of tumor cells.

By targeting the IL-6 – SPINK1 signaling axis, we aim to develop therapeutic approaches to reduce the metastatic burden and peritoneal carcinomatosis of late stage disease, resulting in better prognosis and survival of patients with ovarian clear cell carcinoma.

Citation Format: Christine Mehner, Erin Miller, Mathew A. Coban, Alexandra Hockla, Derek C. Radisky, Evette S. Radisky. Targeting ovarian cancer induced peritoneal carcinomatosis by inhibition of signaling axis between Interleukin-6 (IL-6) and serine protease inhibitor Kazal type 1 (SPINK1) [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 3758.
Abstract 376: Therapeutic role of ESR1-CCDC170 gene fusion in breast cancer endocrine resistance
Authors: Li Li , Veeraraghavan Jamunarani , Hu Yiheng , Wang Xian , Tan Ying , Schiff Rachel , Wang Xiaosong
Published: August 2019

Background: Gene fusions resulting from genomic rearrangements are important drivers for cancer initiation and progression. Estrogen receptor-positive (ER+) breast cancer is the most common type of breast cancer, and luminal B confers a more aggressive phenotype and higher risk of early relapse and prone to resistance to endocrine therapy. Identify new druggable genetic driver will be critical to improve clinical outcome of aggressive luminal B tumors. Our lab has identified a recurrent rearrangement between the estrogen receptor gene ESR1 and its neighbor gene CCDC170, in 6-8% of luminal B tumors. The exon 2 of ESR1 fused with the exon 6, 7, 8 or 10 of CCDC170, which enables the expression of different-sized N-terminally truncated CCDC170 (ΔCCDC170) under ESR1 promoter. Ectopic expression of ΔCCDC170 led to malignant transformation phenotypes in ER+ breast cancer cells, such as increase the cell migration, invasion and anchor-independent cell growth, as well as enhance xenograft tumor growth in mice.

Methods: The effect of ESR1-CCDC170 on endocrine resistance was examined by cologenic assay in vitro, and by xenograft mouse model in vivo. Bioinformatics approaches, Reverse Phase Protein Array (RPPA) analysis and Bimolecular Fluorescence Complementation assay were used to elucidate the potential molecular mechanism.

Results: This study aimed to determine the role of ESR1-CCDC170 in breast cancer endocrine resistance and elucidate the potential mechanisms as well as the therapeutic effect thereof. Our results show that ectopic expression of fusion variants in ER+ T47D cell line sustains the cell proliferation ability under estrogen deprivation (ED), and decreases the sensitivity to 4-OH Tamoxifen treatment. While silencing of ESR1-CCDC170 in fusion positive HCC1428 cell line increases the sensitivity to 4-OHTamoxifen and Fulvestrant treatment. Results from T47D xenograft tumor models show that ESR1-CCDC170 fusion induces endocrine resistance in vivo. Further mechanism studies show that ESR1-CCDC170 possesses a potential ATP binding pocket, has the ability to form homodimers.

Conclusion: These results demonstrate the important role of ESR1-CCDC170 fusions in breast cancer endocrine resistance, and suggest that ESR1-CCDC170 fusion could be a potential therapeutic target for the treatment of breast cancer.

Citation Format: Li Li, Jamunarani Veeraraghavan, Yiheng Hu, Xian Wang, Ying Tan, Rachel Schiff, Xiaosong Wang. Therapeutic role of ESR1-CCDC170 gene fusion in breast cancer endocrine resistance [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 376.
Abstract 3760: Identifying site-specific disparities in the tumor microenvironment of primary and metastatic disease
Authors: Vance Krysten E. , Hollingsworth Micheal A. , Grandgenett Paul
Published: August 2019

The tumor microenvironment (TME), or all cellular and molecular components in and around the tumor, has a well-documented impact on cancer cell proliferation, chemoresistance, and overall patient survival. However much of the research into the TME has been limited to the primary site. How the TME of metastases compares to the primary lesions remains poorly characterized, even though metastasis is responsible for 90% of all cancer deaths. Understanding these differences is critical to treating advanced cancer patients. To study this our lab examined the TME in 22 matched sets primary pancreatic tumors and liver metastasis obtained from end-stage pancreatic cancer patients via our rapid autopsy program. We used a multiplex-immunofluorescent technique that allows staining of a single slide with up to 35 antigens and characterized the presence, position and functional statues of cells that comprise the pancreatic TME. These included: tumor cells, vasculature, fibroblasts, macrophages, mast cells, basophils, eosinophils, B cells, T cells, and NK cells, along with multiple subtypes of these. Based on our preliminary data from this, we hypothesize that the organ determines the features of the TME. Analyzing primary tumors and metastases revealed that both the composition and spatial distribution of different cell types are distinct between the primary tumor and liver metastases. PAM clustering of the cellular featured grouped primary tumor with primary and metastasis with metastatis 87% of the time. As the TME has been shown to impact chemotherapeutic response, we further examined chemotherapeutic effect between sites. Comparing gemcitabine (standard of care) and untreated patients, we observed distinct site-based responses. In the primary tumors with gemcitabine treatment there was a significant increase in inflammatory cells (neutrophils and macrophages) in comparison to untreated patients, while in the liver gemcitabine induced an increase in desmoplasia (SMA). Combined this data indicates that the distinct TME’s exist within the same patient in an organ specific manner which can impact chemotherapeutic response. Understand these differences is therapeutically critical to patients whose therapeutic strategies involve targeting the TME to improve drug delivery, modulate immune suppression or otherwise modulate the microenvironment.

Citation Format: Krysten E. Vance, Micheal A. Hollingsworth, Paul Grandgenett. Identifying site-specific disparities in the tumor microenvironment of primary and metastatic disease [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 3760.
Abstract 3763: Effect of cancer-associated fibroblasts on lymphatic vessel formation in malignant melanoma
Authors: Maeda Shusaku , Yashiro Masakazu , Motomura Hisashi , Hatano Takaharu , Fujikawa Heishiro
Published: August 2019

Introduction: It has been reported that cancer-associated fibroblasts (CAFs) in tumor microenvironment may play an important role for the progression and metastasis of cancer cells. Some reports suggested that CAFs may promote tumor lymphangiogenesis. In malignant melanoma, lymph node metastasis is one of important prognostic factors. Tumor lymphangiogenesis may be controlled not only by tumor cells but also tumor microenvironment such as CAFs, however no study about the interaction between CAFs and human lymphatic endothelial cells (HLECs) was found. Then, we evaluated the effect of CAFs on lymphatic vessel formation in malignant melanoma.

Materials and methods: We used CAFs, normal tissue associated fibroblasts (NAFs) established from a patient of malignant melanoma, and HLECs. Serum-free conditioned medium was obtained from NAFs (NAF-CM) or CAFs (CAFs-CM). CAFs and NAFs were cultured with or without supernatant from malignant melanoma cells. HLECs were cultured by EBM with 50% of NAF-CM or CAFs-CM. Control group was cultured with 50% EBM and 50% of serum-free medium. The effect of each CM on the proliferation of HLECs was examined by CCK assay and tube formation assay. Tube formation was assessed at 12 hours of incubation by fluorescence microscope.

Result: CAFs-CM significantly decreased the proliferation of HLECs in comparison with NAFs-CM after 48, 72 and 96 hours of incubation by CCK assay. In tube formation assay, tube length and number of tubes and junctions were also significantly shorter and smaller in CAFs group, in compared with the control and NAFs group.

Conclusion: CAFs might have a suppressive effect on HLECs, resulting in inhibition of lymphangiogenesis.

Citation Format: Shusaku Maeda, Masakazu Yashiro, Hisashi Motomura, Takaharu Hatano, Heishiro Fujikawa. Effect of cancer-associated fibroblasts on lymphatic vessel formation in malignant melanoma [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 3763.
Abstract 3777: Exploring phenotypic mosaicism in a mouse model of glioma towards understanding intratumoral heterogeneity
Authors: Hara Toshiro
Published: August 2019

The lethality of glioblastoma multiforme (GBM) and the failure of treatment is largely attributable to the heterogeneous properties of this cancer. We aim specifically to characterize the driving event for GBMs to acquire a heterogeneous phenotype during progression. Central to achieving this goal is the ability to interrogate possible sources of heterogeneity that guide cellular states that are similar to those identified in human patients. We therefore employ a mouse model of glioma that recapitulates the pathophysiology and gene expression signatures of human GBM. While initiated with identical oncogenic drivers, this mouse model unexpectedly displays heterogeneous phenotypes between animals. Our preliminary data using time-series single-cell transcriptomics approach shows that transformed cells, even without acquiring additional genetic alterations, switch cellular states at the beginning of tumor formation and one population commit themselves to expand to establish gliomas that are mainly comprised of one cell type. We further observe the strong association of microenvironmental cues with glioma cell populations and their roles in the state transitioning of glioma cells. Through the characterization of mechanisms that drive phenotypic mosaicism, we hope to uncover general principles that govern tumor progression and heterogeneity, and then ultimately provide novel therapeutic strategies to cure GBM.

Citation Format: Toshiro Hara. Exploring phenotypic mosaicism in a mouse model of glioma towards understanding intratumoral heterogeneity [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 3777.
Abstract 3784: Role of HPV-human fusion transcripts in oropharyngeal cancer development
Authors: Pinatti Lisa , Walline Heather , Carey Thomas
Published: August 2019

Human papillomavirus (HPV) genomic integration is frequently seen in oropharyngeal squamous cell carcinoma (OPSCC). Integration into cellular genes can cause the generation of HPV-host fusion transcripts, which have been associated with worse survival in OPSCC patients, but their functional consequences are unknown, as HPV alone is capable of transformation. The forced expression of fusion transcripts versus HPV transcripts in spontaneously immortalized human oral keratinocytes (NOKSI) could demonstrate their effects on cell behavior. Our research focuses on characterizing HPV integration sites and fusion transcripts in cell lines and tumors, followed by in vitro analysis of fusion transcript function. By Detection of Integrated Papillomavirus Sequences (DIPS-PCR) and RT-PCR, we have identified integration events and fusion transcripts in a subset of samples in both intergenic and genic regions of the genome. We began our analysis with a fusion transcript reading from HPV16 E6-E7-E1 into the tumor suppressor gene TP63 from UM-SCC-47. The full fusion transcript and the HPV portion only were cloned into lentiviral vectors, and stable NOKSI populations expressing these transcripts were selected. These cultures, as well as control cultures, were subjected to proliferation, migration and invasion assays. Subsequent in-depth molecular characterization and in vivo studies will help clarify their effects on cancer formation and metastasis. These studies will advance our understanding of how fusion transcripts lead to worse patient survival and help develop new therapies for these patients.

Citation Format: Lisa Pinatti, Heather Walline, Thomas Carey. Role of HPV-human fusion transcripts in oropharyngeal cancer development [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 3784.
Abstract 3793: Understanding the function of LMP1 CTAR3 in EBV-associated lymphomas
Authors: Ross Tabithia , Bentz Gretchen L.
Published: August 2019

Background: Our present study aims to determine the function of the repetitive elements in the biology of Epstein-Barr virus (EBV) Latent Membrane Protein-1 (LMP1). Latent EBV infection is associated with distinct lymphoid and epithelial malignancies, and LMP1 is the principal oncoprotein of the virus. LMP1 has three C-terminal activating regions (CTARs) that induce multiple signal transduction pathways resulting in sustained cellular proliferation, increased cellular survival, and its oncogenic nature. CTAR1 and CTAR2 have been extensively characterized, but CTAR3 is much less studied. CTAR3 contains the LMP1 repetitive elements, which consist of 11-amino acid repeats and PxxPxP/Jak-interacting motifs. Analysis of over 200 clinical isolates revealed that there is significant variability in the number of 11-aaR (1.5-7.5 repeats) and PxxPxP-motives (1-2 repeats), but the exact function of these elements in oncogenesis is unknown. The findings of the study will greatly add to the understanding of the oncogenic nature of LMP1 and help to elucidate a specific purpose for the repetitive elements found within CTAR3.

Methods: Using EBV B95.8 as our wild type strain, we constructed GFP-tagged LMP1 expression constructs that contain (1) wild type CTAR3; (2) PxxPxP only (no 11-aaR); (3) 11-aaR only (no PxxPxP); and (4) ΔCTAR3 (no 11-aaR or PxxPxP). These expression constructs were transfected into HEK 293 cells to assess the effect of the repeats on the trafficking and oncogenic potential of LMP1.

Results: Findings suggest that the 11-aaR are necessary for correct intracellular and extracellular trafficking of LMP1. Loss of the 11-aaRs results in decreased plasma membrane-expressed LMP1 and the accumulation of LMP1 in intracellular membranes. Further, loss of the 11-aaR inhibits LMP1-induced cell migration, which suggests that the 11-aaR contribute to the oncogenic nature of LMP1.

Conclusions: The number of 11-aaR found in LMP1 CTAR3 is variable in nature. Here we show that loss of the 11-aaR inhibits the normal biology of the oncoprotein, in that LMP1 trafficking is altered and the ability of the oncoprotein in promote cell migration is abrogated. Therefore, we propose that the CTAR3 11-aaR are essential to the oncogenic nature of LMP1. The role of these repeats during the transformation process and during tumorigenesis will be determined. Ultimately, further appreciation of the role of LMP1 CTAR3 in the biology of the virus may help identify potential targets for therapeutic interventions for EBV-associated malignancies.

Citation Format: Tabithia Ross, Gretchen L. Bentz. Understanding the function of LMP1 CTAR3 in EBV-associated lymphomas [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 3793.
Abstract 3795: Antibiotics induce mitochondrial dysfunction, DNA damage and aerobic glycolysis of human mammary epithelia and fibroblast
Authors: Elliott Robert L. , Jiang Xianpeng
Published: August 2019

Mitochondria evolved from free-living bacteria after being endocytosed by eukaryotic host cells millions of year ago. We hypothesized that antibiotics could cause mammalian mitochondrial damage while causing bacterial lethality. Mitochondrial toxicity of three antibiotics, azithromycin, doxycycline and ciprofloxacin, were tested in human mammary epithelia MCF-12A and fibroblast by fluorescent and transmission electron microscopy. Gene expression and DNA damage were tested by real time polymerase chain reaction (qPCR) and ELISA. We found antibiotics suppressed the mitochondrial membrane potential gradient of MCF-12A and fibroblast. Ultrastructural studies showed that antibiotics caused vacuolated swollen mitochondria with disrupted cristae in MCF-12A and fibroblast, compared to the ultrastructure of mitochondria in the cells without antibiotic treatment. Fluorescent microscopy revealed antibiotics induce mitochondrial reactive oxygen species (ROS), superoxide, after 3 hours of culture. The DNA oxidative damage product, 8-OHdG, was significantly increased in the media after MCF-12A and fibroblast were cultured in antibiotic media for 24 hours. Antibiotics upregulated gene expression of hypoxia inducible factor 1 alpha (HIF1a), glycolytic enzymes including hexokinase 2(HK2), phosphofructokinase 1 (PFKM) and pyruvate kinase muscle isozyme M2 (PKM2), lactate dehydrogenase A (LDHA), and glucose transporters 1 (SLC2A1) and 3 (SLC2A3) in MCF-12A and fibroblast. Lactate production was also increased in the culture media of healthy cells MCF-12A and fibroblast cells, confirming aerobic glycolysis, or “the Warburg effect”, the method to generate energy after cells are treated with antibiotics. In summary, antibiotics caused mitochondrial toxicity, ROS overproduction, DNA oxidant damage, upregulation of HIF1a gene and aerobic glycolysis in healthy mammalian cells. Over-usage of antibiotics could contribute to tumorigenesis and neurodegeneration and aggravate existing mitochondria-associated diseases.

Citation Format: Robert L. Elliott, Xianpeng Jiang. Antibiotics induce mitochondrial dysfunction, DNA damage and aerobic glycolysis of human mammary epithelia and fibroblast [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 3795.
Abstract 3796: Selonsertib, an ASK1 inhibitor, antagonizes ABCB1- and ABCG2-mediated chemotherapeutic drug resistance
Authors: Ji Ning , Yang Yuqi , Cai Chao-Yun , Lei Zi-Ning , Wang Jing-Quan , Gupta Pranav , Shukla Suneet , Ambudkar Suresh V. , Kong Dexin , Chen Zhe-Sheng
Published: August 2019

The overexpression of ATP-binding cassette (ABC) transporters has known to be one of the most important mechanisms responsible for the development of multidrug resistance (MDR). Selonsertib, an apoptosis signal-regulating kinase 1 (ASK1) inhibitor, is in phase III clinical trial for the treatment of non-alcoholic steatohepatitis (NASH). In this study, we investigated whether selonsertib could antagonize MDR mediated by ABC transporters, involving in ABCB1, ABCG2, ABCC1 and ABCC10. The results showed that selonsertib significantly reversed ABCB1- and ABCG2-mediated MDR, but not MDR-mediated by ABCC1 or ABCC10. Mechanistically, our studies indicated that the reversal effect of selonsertib was related to the attenuation of the efflux function of ABCB1 and ABCG2 transporters, consequently enhancing intracellular accumulation of substrate drugs. Meanwhile, selonsertib, at reversal concentration, affected neither the expression level of ABCB1 and ABCG2 nor the localization of corresponding proteins in subcellular level. Selonsertib stimulated the ATPase activity of both ABCB1 and ABCG2 in a concentration-dependent manner. Our in silico docking study showed that selonsertib could interact with the substrate-binding sites of both ABCB1 and ABCG2. This study provides a clue into a novel treatment strategy, which includes a combination of selonsertib with antineoplastic drugs to circumvent ABCB1- or ABCG2- mediated MDR.

Citation Format: Ning Ji, Yuqi Yang, Chao-Yun Cai, Zi-Ning Lei, Jing-Quan Wang, Pranav Gupta, Suneet Shukla, Suresh V. Ambudkar, Dexin Kong, Zhe-Sheng Chen. Selonsertib, an ASK1 inhibitor, antagonizes ABCB1- and ABCG2-mediated chemotherapeutic drug resistance [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 3796.
Abstract 3798: Acetate supplementation eliminates hypoxia mediated resistance to differentiation therapy in neuroblastoma cells
Authors: Li Yang , Zhou Yiren , Maris John M. , Giaccia Amato J. , Ye Jiangbin
Published: August 2019

Neuroblastoma is the most common extracranial solid tumor in childhood. Retinoic acid (RA) leads to cell cycle arrest and induces cell differentiation in neuroblastoma cells, thus it has been used to treat neuroblastoma as differentiating agent for decades. However, therapy resistance often occurs and the overall survival remains low. Hypoxia, a common metabolic stress existing in tumor microenvironment, increases tumor resistance to RA by promoting dedifferentiation of neuroblastoma cells. The aim of this study was to elucidate the mechanisms underlying hypoxia-mediated therapy resistance and develop therapeutic approach to improve the response to differentiation therapy. By RNA-Seq analysis, we identified a group of neuron-specific differentiation markers that induced by RA treatment, while the induction was diminished under hypoxia. Associated with the silencing of differentiation markers expression, a significant loss of histone acetylation of H3K9, H3K27 and total H3 was observed under hypoxia. Metabolomics analysis indicated hypoxia inhibited mitochondrial oxidative phosphorylation to decrease intracellular acetyl-CoA and citrate, which provides acetyl-CoA for histone acetyltransferase. Importantly, acetate supplementation restored cellular acetyl-CoA level, histone acetylation, and the expression of differentiation markers under hypoxia. Differentiation morphology was also restored by acetate supplementation under hypoxia. In conclusion, our finding suggested hypoxia contributed to the resistance to RA-induced differentiation by reprogramming cellular metabolism and inducing histone hypoacetylation. Acetate supplementation is an efficacious therapeutic approach to improve the sensitivity of hypoxic tumor to RA-based differentiation therapy.

Citation Format: Yang Li, Yiren Zhou, John M. Maris, Amato J. Giaccia, Jiangbin Ye. Acetate supplementation eliminates hypoxia mediated resistance to differentiation therapy in neuroblastoma cells [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 3798.
Abstract 3805: Artesunate reduces tumor growth in sunitinb-resistant renal cell carcinoma cells
Authors: Juengel Eva , Markowitsch Sascha , Erb Holger H. , Efferth Thomas , Haferkamp Axel
Published: August 2019

Introduction: Renal cell carcinoma (RCC) is the most common cancer of the kidney. Due to therapy resistance effect of the approved compounds, like the tyrosine kinase inhibitor (TKI) sunitinib, is limited. Thus, new treatment approaches are urgently needed. Artesunate (ART) is derived from Traditional Chinese Medicine (TCM), showing anti-tumor activity, also in urologic tumors. However, little is known about the efficiency of ART on therapy-resistant RCC. Therefore, we investigated the effect of ART on sunitinib-resistant RCC cell lines.

Material and Methods: Parental (=sensitive) and sunitinib-resistant (1 µM) RCC cell lines, KTC-26, A498, and Caki-1, were exposed to ART (1-100 µM) for 24, 48 and 72 hours. ART untreated cells served as controls. Tumor cell growth, proliferation, cell cycling and the expression of cell cycle regulating proteins were then evaluated. Aside, the appearance of apoptotic and necrotic effects was determined.

Results: Exposure to different concentrations of ART significantly reduced the tumor cell growth and proliferation of parental and sunitinib-resistant cells in a time- and dose-dependent manner, compared to the controls. At the same time, exposure to ART resulted in a cell cycle arrest in the G0/G1 phase. Notably, this effect was more prominent in the sunitinib-resistant cells. G0/G1 phase elevation was associated with a decrease in the percentage of S and G2/M phase cells. The observed changes in cell growth and cell cycle phases were accompanied by distinct modulations of the cell cycle regulating proteins, indicating for an inhibitory regulation of ART through cell cycle arrest. Indeed, neither necrotic nor apoptotic effects events were detectable after ART treatment, corroborating this assumption.

Conclusion: Due to our data we conclude that ART has anti-tumor effects not only in parental but also in sunitinib-resistant RCC cells. Thus, adding ART to the standard therapies might be a promising treatment strategy for patients with advanced RCC. Further investigations are necessary to verify our findings.

Citation Format: Eva Juengel, Sascha Markowitsch, Holger H. Erb, Thomas Efferth, Axel Haferkamp. Artesunate reduces tumor growth in sunitinb-resistant renal cell carcinoma cells [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 3805.
Abstract 3813: PHB2 inactivation by AKAP-BIG3 is required for progression of HER2-overexpressing breast cancer
Authors: Yoshimaru Tetsuro , Matsushita Yosuke , Sasa Mitsunori , Miyoshi Yasuo , Katagiri Toyomasa
Published: August 2019

Overexpression of the human epidermal growth factor receptor 2 (HER2) in breast cancer is linked to aggressive tumor behavior and a poorer prognosis. Anti-HER2 monoclonal antibody, trastuzumab extends the overall survival of patients with HER2-overexpressing breast cancer, but resistance to trastuzumab and/or other HER2-targeted therapies remains a serious clinical problem. Our previous studies demonstrated that Brefeldin A-Inhibited Guanine nucleotide-exchange protein 3 (BIG3), which is exclusively overexpressed in the majority of breast cancers, functions as a cancer specific A-kinase anchoring protein that binds protein kinase A (PKA) and protein phosphatase 1 (PP1Cα), thereby inactivating prohibitin2 (PHB2) suppressive activity through its dephosphorylation on Ser39. We further developed dominant-negative stapling peptide (stERAP), which is designed to specifically disrupt binding of BIG3-PHB2 complex and improve duration of their anti-tumor effects. Here we newly report that stERAP has significant suppressive effects on the HER2 signaling network by activating the tumor suppressive ability of PHB2. stERAP resulted in intrinsic PHB2 release from BIG3, followed by rapid phosphorylation on Ser39, Thr42 and Thr169 via PKCα, TTK and MK5, respectively. Importantly, PHB2 released from BIG3 by stERAP treatment translocated into nucleus, followed by acting as a transcriptional co-repressor by recruiting HDAC1 and NcoR through its Ser39-phosphorylation. More importantly, we observed that serine/threonine phosphorylations of PHB2 are necessary for disruption of HER2-HER3 hetero-dimerization and NF-κB signalling activation which are associated with acquired resistance to trastuzumab. We demonstrated that weekly treatment of stERAP completely suppressed the proliferation of trastuzumab-resistant HER2-positive breast cancer cells in vitro and in vivo. We are currently examining the association of both BIG3 overexpression and PHB2 dephosphorylation with poor prognosis of patients with HER2-positive breast cancers by immunohistochemical staining, and will report these results at this meeting. We demonstrated for the first time that PHB2 inactivation by the AKAP-BIG3 (BIG3/PKA/PP1Cα tri-complex) is required for oncogenic HER2 signalling activation in breast cancer cells, and stERAP may be a promising anti-cancer drug for trastuzumab-resistant breast cancer.

Citation Format: Tetsuro Yoshimaru, Yosuke Matsushita, Mitsunori Sasa, Yasuo Miyoshi, Toyomasa Katagiri. PHB2 inactivation by AKAP-BIG3 is required for progression of HER2-overexpressing breast cancer [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 3813.
Abstract 3824: Increased production of endogenous hydrogen sulfide (H2S) induced resistance to 5-FU and Oxaliplatin in colon cancer cells
Authors: Chen Shanwen , Zuo Shuai
Published: August 2019

The combination of 5-FU and Oxaliplatin has been widely adopted as one of the first line chemotherapeutic regimens for colon cancer patients. However, the development of chemoresistance can lead to early recurrence and metastasis to distant organs, thus remaining a leading cause of death from colon cancer. In the present study, the increased production of hydrogen sulfide (H2S) in colon cancer tissues was validated and the effect of inhibition of production of endogenous H2S on the responses of colon cancer cells to 5-FU and Oxaliplatin was further analyzed. The results suggested that inhibition of the enzymatic activity of cystathionine-β-synthase (CBS), a major producer of H2S in colon cancer cells, significantly decreased the IC50s of both 5-FU and Oxaliplatin. Inhibition of CBS leads to decreased PI3K-Akt signaling and consequently decreased expression of thymidylate synthetase (TYMS), which is the target of 5-FU. Inhibition of CBS utilizing aminooxyacetic acid (AOAA) leads to increased production of reactive oxygen species (ROS) and exaggerated mitochondrial damages in colon cancer cells. Analysis performed on CompuSyn confirmed the synergetic effect of AOAA with both 5-FU and Oxaliplatin. In vivo imaging of tumorigenesis in nude mice further confirmed the effect of inhibition of CBS on the therapeutic effect of both 5-FU and Oxaliplatin. Together, these results suggested that inhibition of production of endogenous H2S might be a promising target for chemoresistance in colon cancer.

Citation Format: Shanwen Chen, Shuai Zuo. Increased production of endogenous hydrogen sulfide (H2S) induced resistance to 5-FU and Oxaliplatin in colon cancer cells [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 3824.
Abstract 3828: Differential transcriptional response of MYC to bromodomain inhibition in HPV positive head and neck squamous cell carcinoma
Authors: Rengifo Hassler , Wang Albert R. , Titi Mohammad , Nickel Kwang , Kimple Randy , Iyer Gopal
Published: August 2019

Differential transcriptional response of MYC to bromodomain inhibition in HPV positive head and neck squamous cell carcinoma

Targeting of bromodomain (BRD) and extra-terminal domain (BET) family protein activity has the potential to reduce proliferation in many cancers. We hypothesize that targeting BRDs using its inhibitors will lead to reduced proliferation in human papilloma virus (HPV) infected head and neck cancer squamous cell carcinoma (HNSCC). HPV infected HNSCC is predicted to surpass the incidence of cervical cancer by 2020. Amongst various HPV serotypes, HPV16 is classified as oncogenic for a various number of cancer sites including head and neck squamous cell carcinoma (HNSCC). Integration of HPV into host genomes is not part of its life cycle; however, the exception is HNSCC wherein E6 and E7 viral proteins predominantly drive its tumor progression. HPV integrated HNSCC cells will be utilized to unravel the fundamental question of epigenetic regulation of viral-host interaction using the HPV-positive HNSCC model. Results from our lab indicate that despite high nanomolar IC50 in cell proliferation assays, downregulation of MYC expression was not observed using BRD inhibitor in all MYC expressing HNSCC cell lines. Further interrogation revealed differential phosphorylation of tumor suppressors - p53 and pRb might influence the sensitivity to BRD inhibition, which in turn could explain the contrasting regulation of MYC RNA levels. Intriguingly, combining radiation with bromodomain inhibitors led to increased radio-sensitization suggesting that BRDs have a role to play in DNA damage repair pathway and altered cell cycle regulation. Taken together, we will present MYC dependent and independent mechanism following BRD inhibition in HPV positive HNSCC.

Note: This abstract was not presented at the meeting.

Citation Format: Hassler Rengifo, Albert R. Wang, Mohammad Titi, Kwang Nickel, Randy Kimple, Gopal Iyer. Differential transcriptional response of MYC to bromodomain inhibition in HPV positive head and neck squamous cell carcinoma [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 3828.
Abstract 3830: Novel bromodomains and extra-terminal motif (BET) inhibitors developed against estrogen receptor positive, fulvestrant- and CDK4/6-resistant breast cancer models
Authors: Xiong Rui , Li Yangfeng , Zhao Jiong , Tonetti Debra , Thatcher Gregory
Published: August 2019

Approximately 70% of breast cancer patients have estrogen receptor positive (ER+) tumors. Fulvestrant (FUL), a selective estrogen receptor degrader (SERD) able to ablate ER, was first introduced to clinical practice in 2002 as the second and third line treatment for ER+ metastatic breast cancer. In August 2017, FUL was added to first-line therapy in postmenopausal women by demonstrating superiority compared to first-line AI therapy, anastrozole, with improved progression-free-survival (PFS) and a 20% reduction in the risk of disease progression or death. However, acquisition of FUL-resistance in both first-line setting or as combination therapy with CDK4/6 inhibitor has been observed clinically, leaving patients and oncologists with chemotherapy as the only backup therapy. Little is known about resistance mechanisms to FUL alone or in combination with CDK4/6 inhibitors. Here, we showed that targeting epigenetic vulnerabilities in breast cancer using novel bromodomain and extra-terminal (BET) inhibitors can effectively inhibit growth of tamoxifen-, FUL- and CDK4/6- resistant breast cancer cells. We designed and developed a novel series of pyridinone-based BET inhibitors, optimized by growth inhibition in tamoxifen- and FUL- resistant breast cancer cell lines (MCF7:CFR) in both 2D and 3D cultures. Optimized compounds showed superior in vitro activity to BET inhibitors in clinical trials. Selected compounds were further evaluated for pharmacokinetics and shown efficacy in endocrine-resistant xenograft models.

Citation Format: Rui Xiong, Yangfeng Li, Jiong Zhao, Debra Tonetti, Gregory Thatcher. Novel bromodomains and extra-terminal motif (BET) inhibitors developed against estrogen receptor positive, fulvestrant- and CDK4/6-resistant breast cancer models [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 3830.
Abstract 3849: Structure-based PROTAC design demonstrates BAF complex ATPase vulnerabilities in cancer
Authors: Koegl Manfred , Farnaby William , Whitworth Claire , Roy Michael , Diers Emelyne , Trainor Nicole , Steurer Steffen , Riedmueller Carina , Gmaschitz Teresa , Wachter Johannes , Dank Christian , Gallant Michael , Sharps Bernadette , Rumpel Klaus , Traxler Elisabeth , Lorenz Romario , Petermann Oliver , Greb Peter , Weinstabl Harald , Bader Gerd , Zoephel Andreas , Weiss-Puxbaum Alexander , Rinnenthal Joerg , Arnnhof Heribert , Wiechens Nicola , Wu Meng-Ying , Owen-Hughes Tom , Ettmayer Peter , Pearson Mark , McConnell Darryl B. , Ciulli Alessio
Published: August 2019

Targeting subunits of BAF/PBAF complexes has been proposed as an approach to exploit cancer vulnerabilities, yet small molecules developed to date have remained largely ineffectual. Here we develop PROTAC degraders of the BAF ATPase subunits SMARCA2 and SMARCA4 using a bromodomain ligand and recruitment of the E3 ubiquitin ligase VHL. High-resolution ternary complex crystal structures and biophysical investigation guided the rational optimization of ACBI1, a potent cooperative SMARCA2/4 degrader. ACBI1 induced antiproliferative effects and cell death caused by SMARCA2 depletion in SMARCA4 mutant cancer cells, and in acute myeloid leukemia cells dependent on SMARCA4 ATPase activity. These findings exemplify a successful biophysics and structure driven campaign to degrade targets previously considered undruggable, and pave the way towards new therapeutics for the treatment of tumors sensitive to the loss of BAF complex ATPases.

Citation Format: Manfred Koegl, William Farnaby, Claire Whitworth, Michael Roy, Emelyne Diers, Nicole Trainor, Steffen Steurer, Carina Riedmueller, Teresa Gmaschitz, Johannes Wachter, Christian Dank, Michael Gallant, Bernadette Sharps, Klaus Rumpel, Elisabeth Traxler, Romario Lorenz, Oliver Petermann, Peter Greb, Harald Weinstabl, Gerd Bader, Andreas Zoephel, Alexander Weiss-Puxbaum, Joerg Rinnenthal, Heribert Arnnhof, Nicola Wiechens, Meng-Ying Wu, Tom Owen-Hughes, Peter Ettmayer, Mark Pearson, Darryl B. McConnell, Alessio Ciulli. Structure-based PROTAC design demonstrates BAF complex ATPase vulnerabilities in cancer [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 3849.