bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 268: Combined inhibition of cyclooxygenase with docosahexaenoic acid augments apoptosis in KRAS mutant non-small cell lung cancer cells
Authors: Amissah Felix , Jones Kylee J. , Poku Rosemary A.
Published: August 2018

Cyclooxygenase (COX) inhibitors are widely used in the treatment of various inflammatory disorders. Their antiproliferative effects have also been widely reported. This health benefit of COX inhibitors is limited by some potential adverse effects, including gastrointestinal irritation. Docosahexaenoic (DHA), commonly derived from fish oils, is also known to possess anticancer effects. We examined whether COX inhibitors (aspirin and diclofenac) combined with DHA would synergize their antiproliferative activity. This would allow for a decrease in the amounts of each compound used for chemoprevention, which may likely decrease the risk of adverse effects. While treatment with DHA resulted in concentration-dependent cell death in K-Ras mutant (A549 and NCI-H1573) lung cancer cells (EC50 of 5.5 µM and 9.5 µM), combination with diclofenac resulted in relatively more significant reduction in cell viability (EC50 of 3.7 µM and 3.2 µM) and cell proliferation (p<0.0001). In addition, the combination treatment was more effective in inhibiting clonogenic cell growth and inducing apoptosis with a significant increase in proapoptotic proteins (p<0.001). Furthermore, the combination treatment yielded more marked inhibition of K-Ras expression as well as altered localization in the cell membrane. These data point to the promising chemopreventive potential of combining low doses of COX inhibitors with DHA with a possible decrease in their adverse effects.

Citation Format: Felix Amissah, Kylee J. Jones, Rosemary A. Poku. Combined inhibition of cyclooxygenase with docosahexaenoic acid augments apoptosis in KRAS mutant non-small cell lung cancer cells [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 268.
Abstract 2692: Elucidation of the pre-B-cell receptor signaling network in acute lymphocytic leukemia cells
Authors: Walter Roland , Kouidri Khouloud , Peter Jessica , Pape Martine , Merschen Laura , Haeupl Bjoern , Doebele Carmen , Serve Hubert , Yi Yanlong , Urlaub Henning , Oellerich Thomas
Published: August 2018

Antigen receptors transduce complex signals and thereby control important cell fate decisions of the B-lineage including differentiation, proliferation and B-cell survival. The initial steps of B-cell development take place in the bone marrow, and the first dependency of a developing B-cell on antigen receptor signaling occurs at the pre-B-cell stage where expression of the pre-BCR is essential for cell survival. In contrast to B-cell-receptor (BCR) signaling, the nature of pre-BCR signals is far less characterized. It is believed that pre-BCRs transduce a constitutive baseline signal that is essential for survival and differentiation of pre-B-cells into immature B-cells. Since pre-BCRs are crucial regulators of pre-B-cell survival and differentiation, it is not surprising that dysregulation of pre-BCR signaling is observed in acute lymphocytic leukemia (ALL) cells where it contributes to survival of the malignant cell clones. To identify drug targets in the pre-BCR signaling network, we have systematically characterized pre-BCR signaling in two pre-BCR-dependent ALL cell lines by quantitative phosphoproteomics. Because pre-BCRs transduce a constitutive baseline signal, we interfered with pre-BCR signaling by either Syk inhibitor treatment or by an inducible shRNA-mediated knock-down of the pre-BCR component Ig-α to monitor pre-BCR-dependent signals. We identified and quantified more than 15,000 phospho-sites. Among these sites, more than 500 were regulated upon interference with pre-BCR signaling. Although the upstream components are shared between pre-BCRs and BCRs, the wiring of their downstream signals occurred to be different which is reflected by our finding that the majority of the identified pre-BCR signaling effectors have not been described in the context of BCR signaling in mature B-cells or lymphomas. Finally, we have identified vulnerabilities in the pre-BCR signaling network by intersecting our phosphoproteomic data with data from loss-of-function CRISPR-Cas9 screens for essential genes in ALL cell lines. Taken together, this study provides a comprehensive dataset for pre-BCR signaling and identifies effector proteins that are relevant for the regulation of ALL cell survival and proliferation.

Citation Format: Roland Walter, Khouloud Kouidri, Jessica Peter, Martine Pape, Laura Merschen, Bjoern Haeupl, Carmen Doebele, Hubert Serve, Yanlong Yi, Henning Urlaub, Thomas Oellerich. Elucidation of the pre-B-cell receptor signaling network in acute lymphocytic leukemia cells [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 2692.
Abstract 2695: Quantitative phosphoproteome analysis identifies novel functional pathways of tumor suppressor DLC1 in estrogen receptor-positive breast cancer
Authors: Gökmen-Polar Yesim , True Jason D. , Vieth Edyta , Gu Yuan , Gu Xiaoping , Qi Guihong D. , Mosley Amber L. , Badve Sunil
Published: August 2018

Deleted in Liver Cancer-1 (DLC1), a member of the RhoGAP family of proteins, functions as a tumor suppressor in several cancers including breast cancer. However, its clinical relevance is unclear in breast cancer. In this study, expression of DLC1 was correlated with prognosis using publicly available breast cancer gene expression datasets and quantitative polymerase chain reaction (qPCR) in cohorts of Estrogen Receptor-positive (ER+) breast cancer. Mutation and methylation status of DLC1 were assessed in these datasets including The Cancer Genome Atlas (TCGA). To seek further insights in understanding the role of DLC1 in ER+ breast cancer, we developed a knock-in model of DLC1 in T47D breast cancer cells. Label-free global proteomic and TiO2 phosphopeptide enrichment assays were performed and validated using Western blotting. Data are available via ProteomeXchange with identifier PXD008220. Here, we report that low expression of DLC1 correlates with poor prognosis in patients with ER+ breast cancer with further decrease in metastatic lesions. Analysis of the TCGA data showed that down regulation of DLC1 is not due to methylation or mutations. Stable knock-in of DLC1-full-length inhibits cell colony formation significantly in vitro compared to its control counterpart. 6726 phosphopeptides were quantified by phosphoproteomics analysis in both conditons, whereas 205 and 122 were unique to DLC1 knock-in and T47D-control cells, respectively. Pathway analysis using DAVID showed the top three significant clusters of differentially identified phosphopeptides involving cell-cell adhesion, mRNA processing and splicing, and transcription regulation. Decreased phosphorylation of epithelial cell transforming sequence 2 (ECT2) at the residue T359, critical for its active conformational change, was validated. In conclusion, this data suggests that high expression of DLC1 reduces cell growth and is associated with favorable prognosis. Our results document an inverse relation between ECT2 phosphorylation and DLC1 expression, promising a novel strategy for treating ER+ breast cancer.

Citation Format: Yesim Gökmen-Polar, Jason D. True, Edyta Vieth, Yuan Gu, Xiaoping Gu, Guihong D. Qi, Amber L. Mosley, Sunil Badve. Quantitative phosphoproteome analysis identifies novel functional pathways of tumor suppressor DLC1 in estrogen receptor-positive breast cancer [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 2695.
Abstract 27: Regulating Rac1 signaling proteins influences migratory and protrusive behaviors of metastatic breast cancer cells in aligned, 3D collagen and collagen-like matrices
Authors: Doorn Rachel Van , Szulczewski Joseph , Ponik Suzanne
Published: August 2018

Breast cancer is the most commonly diagnosed cancer in women, with one out of every eight women being diagnosed in their lifetime. Our lab has previously shown that aligned collagen promotes tumor progression to metastatic disease, and predicts poor prognosis for breast cancer patients. Aligned collagen increases the persistence of cellular migration by limiting the number of protrusions from the cell body. The regulation of Rac1 and Rho proteins are important for cell motility through formation of lamelliepodia. Deregulation of these proteins and those upstream of Rac1, can result in cancer cell migration, invasion and metastases. Understanding how these proteins are regulated in different 3D collagen tumor microenvironments is important for determining phenotypes of invasive breast cancers. Inhibition of cell motility through the Rac1 pathway may limit cancer cell migration and prevent metastatic disease. While many studies have investigated cell migration in 2D, little is known regarding the cellular mechanisms that regulate both breast cancer cell migration and protrusive events in 3D aligned collagen. In vitro models will be used to recapitulate 3D aligned, and random collagen environments and assess the mechanisms of cell migration and protrusion dynamics by live-cell, time-lapse microscopy. Previous work from our group determined that MDA-MB-231 cells seeded into collagen environments had fewer protrusions, which localized in the directions of collagen alignment, when migrating along aligned fibers compared to MDA-MB-231 cells in the random collagen matrices. Preliminary data demonstrates that signaling proteins upstream of Rac1 such as, small GTPases, DOCK180 and FilGAP, dictate cellular protrusions and migratory persistence. MDA-MB-231 cell lines with doxycycline-induced knock-down Rac1 GTPase activating protein (GAP), FilGAP, and guanine nucleotide exchange factor (GEF), DOCK180, demonstrate that the regulation of Rac1 activity through these specific proteins plays a role in breast cancer cell migration and protrusion dynamics in an aligned collagen environment. Understanding the regulation of breast cancer cell migration in aligned and random 3D collagen environments will enhance the current understanding of progression to metastatic disease on a cellular level.

Citation Format: Rachel Van Doorn, Joseph Szulczewski, Suzanne Ponik. Regulating Rac1 signaling proteins influences migratory and protrusive behaviors of metastatic breast cancer cells in aligned, 3D collagen and collagen-like matrices [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 27.
Abstract 2731: Checkpoint inhibitor therapy in combination with the implantation of agarose encapsulated cancer cells inhibits tumor growth in a mouse model of osteosarcoma
Authors: Dumpala Pradeep R. , Martis Prithy C. , Bemrose Melissa A. , Dudley Atira , Smith Barry H. , Gazda Lawrence S.
Published: August 2018

We have previously shown that murine renal adenocarcinoma (RENCA) cells encapsulated in agarose macrobeads (MB) secrete factors that inhibit the proliferation of freely growing tumor cells outside the MB, both in vitro and in vivo. This effect is being investigated in ongoing clinical trials with colorectal patients who underwent laparoscopic intraperitoneal implantations of RENCA MBs (NCT01053013, NCT02046174). In the current study, we report the effect of immune checkpoint inhibitors (anti-PD-1 and anti-PD-L1 monoclonal antibodies) in combination with RENCA MBs on tumor burden in BALB/cJ mice induced with K7M2 murine osteosarcoma tumors. Mice that developed tumors (2-5 mm) on subcutaneous injection of K7M2 cells were randomly enrolled in the following study groups: sham, RENCA MBs only, anti-PD-1 only, RENCA MBs + anti-PD-1, anti-PD-L1 only, RENCA MBs + anti-PD-L1. Anti-PD-1 and anti-PD-L1 antibodies were administered ip bi-weekly for 3-6 weeks. RENCA MBs were implanted in the peritoneal cavity on the day of enrollment. Mean tumor volumes (mm3) throughout days 0-24 were: RENCA MB (199.17 ± 125.57), anti-PD-1 only (278.87 ± 180.34), anti-PD-L1 only (332.46 ± 185.13) and sham treated mice (512.61 ± 230.12). RENCA MB and anti-PD-L1 combination therapy did not inhibit tumor growth (375.00 ± 511.61). PD-L1 expression was absent in primary tumors as observed by immunostaining. Treatment with RENCA MBs plus anti-PD-1 treatment demonstrated an additive effect (116.94 ± 83.77) compared to RENCA MB only or anti-PD-1 alone. Moreover, tumors in 4 out of 16 mice from the RENCA MB plus anti-PD-1 group receded and were undetectable (3 in less than 1 week and 1 at 39 days) following treatment. Tumors also regressed and were not palpable in 1 mouse each in the anti-PD-1 and RENCA MB plus anti-PD-L1 groups at day 14 and 9, respectively. Discontinuation of anti-PD-1 and anti-PD-L1 treatment after 3 weeks of treatment resulted in loss of tumor growth regulation. Preliminary data suggests that continued treatment with these immune checkpoint inhibitors beyond 3 weeks prolongs the regulation of tumor growth in this model. These data support the use of anti-PD-1 in combination with the implantation of RENCA macrobeads in this mouse model of osteosarcoma, and the possibility of similar approaches in the clinic.

Citation Format: Pradeep R. Dumpala, Prithy C. Martis, Melissa A. Bemrose, Atira Dudley, Barry H. Smith, Lawrence S. Gazda. Checkpoint inhibitor therapy in combination with the implantation of agarose encapsulated cancer cells inhibits tumor growth in a mouse model of osteosarcoma [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 2731.
Abstract 2736: Preclinical pharmacologic and pharmacodynamic studies of a novel and potent IDO1 inhibitor D-0751
Authors: Wang Yaolin , Shi Zhe , Han Zixing , Wang Zhenwu , Jiang Yueheng , Dai Xing
Published: August 2018

Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-containing enzyme that catalyzes the degradation of the essential amino acid L-tryptophan to kynurenine. It plays an important role in the initial and rate-limiting step in the breakdown of tryptophan. It has been reported that IDO1, an enzyme induced by IFN-gamma, is one of the central regulators of immune responses in various physiologic and pathologic settings and the enzyme is frequently expressed in cancers. Kynurenine produced by IDO1 suppresses T-cell function in the tumor microenvironment, resulting in tumor cell evasion from immune cells. IDO1 inhibitors, such as epacadostat and BMS-986205, have shown preliminary clinical therapeutic response in melanoma and other solid tumors when combined with anti-PD-1 antibody. Here, we report the discovery of a novel and selective IDO1 inhibitor with potent activity in various cell-based assays. Our lead molecule D-0751 inhibits IDO1 activity in HeLa cell and human whole blood assay with IC50 of 0.6 nM and 13 nM, respectively. It activates the human T-cell activity in a HeLa co-culture study with EC50 at 9 nM. D-0751 is optimized to have good PK profile in rodent, dog and monkey as well as slow clearance when incubated with human hepatocyte. Pharmacodynamic (PD) effect has been observed in mouse models (blood and tumor samples) as well as in dog (blood samples) with efficient kynurenine reduction observed at optimal dose level. Taken together, these results support the advancement of D-0751 as a clinical candidate in combination with anti-PD-1/L1 antibody in various solid tumor indications.

Citation Format: Yaolin Wang, Zhe Shi, Zixing Han, Zhenwu Wang, Yueheng Jiang, Xing Dai. Preclinical pharmacologic and pharmacodynamic studies of a novel and potent IDO1 inhibitor D-0751 [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 2736.
Abstract 2739: The ketone body β-hydroxybutyrate alters expression of PD-L1 on malignant glioma cells but does not directly affect T cells in vitro
Authors: Woolf Eric C. , Manhas Kavita R. , Healey Deborah R. , Blattman Joseph N. , Scheck Adrienne C.
Published: August 2018

Patients with newly diagnosed glioblastoma multiforme (GBM), the most aggressive grade of glioma, have an average life expectancy of ~15 months, and less than 6% survive five years, despite the current standard of care, which consists of aggressive surgery followed by radiation and chemotherapy. Advances in the survival of GBM patients require the design of new therapeutic approaches. One novel strategy being explored in our laboratory and others involves using ketogenic therapy (KT). This approach involves shifting the body's metabolic physiology by increasing the production of ketone bodies such as β-hydroxybutyrate (BHB) and decreasing blood glucose. We have demonstrated that nutritionally-induced KT enhances survival and potentiates standard therapy in a mouse model of malignant glioma. Our previous study shows that KT works in part as an immune adjuvant, boosting tumor-reactive immune responses in the microenvironment by alleviating immune suppression in vivo. In particular we found that KT reduced expression of programmed cell death-1 (PD-1) on tumor-infiltrating T cells and its ligand PD-L1 on tumor cells. The PD-1/PD-L1 axis represents a crucial mechanism of glioma-mediated immunosuppression and is a major focus of immunotherapy in GBM and other cancers. The current study aims to determine the role of BHB in the anti-tumor and immune-enhancing effects of KT. We show that in vitro treatment with BHB induces a variety of molecular changes in glioma cells including reduced protein expression of PD-L1. Given that our in vivo data suggests that KT enhances T cell function against tumors we also sought to determine the impact of BHB on T cell function in vitro. Activated CD8+ and CD4+ T cells were cultured with varying doses of BHB prior to flow cytometric analysis. There were no significant changes in expression of immune checkpoint receptors (PD-1, CTLA-4 and LAG-3), production of anti-tumor promoting cytokines (IFN-y, TNF, IL-2) or viability in either T cell population following treatment with any dose of BHB. This suggests that BHB and KT may indirectly impact T cell function by influencing the way tumor cells manipulate the microenvironment. Taken together this data helps us better understand the anti-tumor mechanisms underlying KT and warrants further exploration of combinational treatment strategies using this approach and immunotherapies for GBM.

Citation Format: Eric C. Woolf, Kavita R. Manhas, Deborah R. Healey, Joseph N. Blattman, Adrienne C. Scheck. The ketone body β-hydroxybutyrate alters expression of PD-L1 on malignant glioma cells but does not directly affect T cells in vitro [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 2739.
Abstract 2746: Synergistic immunotherapeutic effects of TLR7/8 agonist on low-dose cyclophosphamide-treated C26 model
Authors: Yang Jing , Li Patrick Z. , Yin Xiangyun , Nie Xiaohua , Wang Jingshu , Lau Walter , Zhang Liguo , Li Lixin
Published: August 2018

In recent years, low dose cyclophosphamide has been used clinically either as a single agent or as combination therapy to treat lymphomas, and breast and ovarian cancers. The efficacy of low dose cyclophosphamide is primarily due to its ability to immune-modulate in addition to its direct anti-tumor effects. This ability highlights the potential for synergism between conventional chemotherapy and novel immunotherapy. Toll-like receptors (TLRs) are a crucial part of the innate immunity and present the first line of defense against pathogens. Resiquimod is the ligand for TLR7 and 8 and directly activate innate immune cells, including myeloid dendritic cells, plasmacytoid dendritic cells, and monocytes/macrophages. This activation may result in activation of co-stimulatory molecules, production of antiviral cytokines, and stimulation of cell-mediated NK and T cell immune responses. The aim of this study was to analyze whether immunopotentiating TLR7/8 agonist might synergize with low doses of cyclophosphamide in the treatment of colorectal carcinoma. The effect of combining a weekly dosing of cyclophosphamide intraperitoneally with systemic administration of Resiquimod was evaluated in an in vivo murine C26 synergistic model. Systemic administration of Resiquimod in mice induced significant systemic immune responses as evidenced by upregulating IFNα inducible gene clusters. The combined therapy with Resiquimod and cyclophosphamide was superior to each single treatment and increased efficacy of tumor inhibition and prolonged their survival significantly. Importantly, the combined treatment generated a significant numbers of tumor-specific T cell infiltration in tumor microenvironment. Our data suggest that systemic administration of TLR7/8 agonist enhances anti-tumor effect of low dose of cyclophosphamide. The data supports the use of a combination of TLR7/8 agonist and cyclophosphamide as a novel therapeutic strategy against colorectal carcinoma.

Citation Format: Jing Yang, Patrick Z. Li, Xiangyun Yin, Xiaohua Nie, Jingshu Wang, Walter Lau, Liguo Zhang, Lixin Li. Synergistic immunotherapeutic effects of TLR7/8 agonist on low-dose cyclophosphamide-treated C26 model [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 2746.
Abstract 275: Flavokawain B, a kava chalcone, acts synergistically with proteasome inhibitors (i.e., bortezomib and MG132) to reduce the growth of prostate cancer cells via downregulation of Skp2
Authors: Li Xuesen , Pham Victor , Tippin Matthew , Xu Shan , Fu Dongjun , Song Liankun , Hoang Bang H. , Zi Xiaolin
Published: August 2018

Flavokawain B, a chalcone isolated from kava root extracts, has been shown to inhibit the in vitro and in vivo growth of various cancer cell lines via induction of apoptosis. However, the mechanism of flavokawain B's action remains largely unknown. Here we have demonstrated that flavokawain B caused Skp2 degradation in an ubiquitin and proteasome dependent manner. In addition, flavokawain B inhibited NEDD8 conjugations to both Cullin1 and Ubc12 in PC3 cells and Ubc12 NEDDylation in an in vitro assay. Overexpression of dominant-negative cullin1 (1-452) or K720R mutant cullin1 rescued the effect of flavokawain B on Skp2 degradation, but further increased the expression of p27. However, siRNA knock-down of Skp1, CSN5, and UBC12 expression resulted in down-regulation of Skp2 expression and further enhanced the effect of flavokawain B on Skp2 degradation. siRNA knock-down of Cdh1, a known E3 ligase of Skp2 for targeted degradation, also didn't rescue the effect of flavokawain B on Skp2 degradation, but overexpression of F-box deleted Skp2 can reverse the effect of flavokawain B on Skp2 degradation. These results suggest that degradation of Skp2 by flavokawain B is involved in Cullin1. Furthermore, flavokawain B selectively inhibited the growth of pRb deficient cell lines and PC3 cells with Skp2 overexpression. Flavokawain B also acts synergistically with Bortezomib and MG132 to inhibit the growth of prostate cancer cell lines via down-regulation of Skp2 and up-regulation of p27 and p21 expression. These finding provide rationale for combination of flavokawain B and Bortezomib in treatment of prostate cancer.

Citation Format: Xuesen Li, Victor Pham, Matthew Tippin, Shan Xu, Dongjun Fu, Liankun Song, Bang H. Hoang, Xiaolin Zi. Flavokawain B, a kava chalcone, acts synergistically with proteasome inhibitors (i.e., bortezomib and MG132) to reduce the growth of prostate cancer cells via downregulation of Skp2 [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 275.
Abstract 276: Targeting cancer stem cells with chemopreventive agents for breast cancer prevention
Authors: Shan Naing Lin , Yang Jeffrey , Bak Min Ji , Wahler Joseph , Suh Nanjoo
Published: August 2018

Breast cancer accounts for one of the most common causes of cancer-related deaths in women. Despite the available treatment modalities to cure breast cancer, therapeutic resistance and recurrence remain major challenges, owing to the culminating evidence of the presence of cancer stem cell subpopulations in breast tumors. The purpose of the study is to identify chemopreventive and dietary compounds that effectively target cancer stem cells to inhibit breast cancer development and progression. The mammosphere culture system has been proven to enrich cancer stem-like cells, and cells from mammospheres are shown to be more tumorigenic in vivo. By culturing mammospheres from breast cancer cell lines, we have shown that 1,25 vitamin D3, a vitamin D3 analog, BXL0124, pterostilbene (trans-3,5-dimethoxy-4-hydroxystilbene), tocopherols and triterpenoids (CDDO-Im) decreased the mammosphere forming efficiency (MFE) in a dose-dependent manner. Vitamin D compounds (1,25D3 and BXL0124) decreased the MFE in SUM159, MCF10DCIS and MCF-7 breast cancer cell lines (dose range 1-100 nM). Pterostilbene, a bioactive component of blueberries, effectively decreased the MFE in the MCF-7 cell line. The size of the mammospheres was found to be smaller with increasing concentrations (10-100 μM). Tocopherols significantly decreased estrogen-stimulated MFE in MCF-7 cells (1-10 μM). CDDO-Im, a synthetic triterpenoid derived from natural oleanolic acid, has also been shown to decrease MFE and sphere size in MCF-7 cells (50-100 nM). The effect on the shape of the spheres was also observed, suggesting the differentiation-inducing properties of these compounds. Taken together, we demonstrate that naturally occurring compounds as well as synthetic analogs derived from natural and dietary compounds significantly inhibit sphere forming efficiency and cancer stemness in breast cancer cells. Our study suggests that chemopreventive compounds, 1,25D3, BXL0124, pterostilbene, tocopherols, and CDDO-Im, are agents with the potential to target cancer stem-like cells to prevent breast cancer.

Citation Format: Naing Lin Shan, Jeffrey Yang, Min Ji Bak, Joseph Wahler, Nanjoo Suh. Targeting cancer stem cells with chemopreventive agents for breast cancer prevention [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 276.
Abstract 278: Inhibition of skin tumor promotion by TPA using a combination of topically applied ursolic acid and curcumin
Authors: Tremmel Lisa , Rho Okkyung , DiGiovanni John
Published: August 2018

Despite the many advances that have been made in understanding cancer development and the discovery of new therapies, cancer still remains the second leading cause of death in the US. In addition, approximately two out of five people are expected to be diagnosed with an invasive cancer in their lifetime. Prevention remains an important strategy to reduce the burden of cancer. One approach to prevent cancer is the use of phytochemicals in various combinations as safe and effective cancer preventive agents. The purpose of this study was to examine the effects of the combination of ursolic acid (UA) and curcumin (Curc) for potential synergistic chemopreventive activity to inhibit tumor promotion using the mouse two-stage skin carcinogenesis model. UA and Curc alone have been shown to inhibit tumorigenesis in several mouse models, and both have been shown to inhibit mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). The anti-cancer activity of these phytochemicals is due primarily to their anti-inflammatory, anti-oxidant, and anti-proliferative effects. These effects are accomplished by acting on a broad spectrum of signaling pathways. In initial short-term experiments (mice treated twice weekly for two weeks with TPA), the combination of UA and Curc pretreated topically inhibited TPA-induced activation of epidermal NF-κB, Cox-2, epidermal growth factor receptor (EGFR) and Stat3 to a greater extent than either compound alone. Additionally, these compounds increased levels of programmed cell death protein 4 (Pdcd4). The alterations in these signaling pathways by the combination of UA and Curc were associated with decreased epidermal proliferation and skin inflammation as assessed by measuring BrdU incorporation and inflammatory gene expression, respectively. Furthermore, results from skin tumor experiments (mice initiated with DMBA and promoted twice weekly with TPA) indicate that the combination of UA and Curc given topically significantly inhibited mouse skin tumor promotion by TPA to a greater extent than the individual compounds given alone. The greatest effects were seen on tumor free survival and on tumor size. These results suggests the potential for the combination of UA and Curc to be used topically for chemopreventive strategies in non-melanoma skin cancer.

Citation Format: Lisa Tremmel, Okkyung Rho, John DiGiovanni. Inhibition of skin tumor promotion by TPA using a combination of topically applied ursolic acid and curcumin [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 278.
Abstract 2791: Physiologically based pharmacokinetic modeling and simulations to estimate the efficacious dose of the CEACAM6 function-blocking antibody BAY 1834942
Authors: Wittemer-Rump Sabine , Niederalt Christoph , Willuda Joerg , Trautwein Mark , Luetke-Eversloh Merlin , Doecke Wolf-Dietrich , Guenther Clemens , Scheerans Christian
Published: August 2018

BAY 1834942 is an immunostimulatory function-blocking (fb) antibody (Ab) against the target carcinoembryonic antigen related cell adhesion molecule 6 (CEACAM6) expressed on tumor cells in multiple cancer indications. The suggested mode of action of BAY 1834942 is the blockade of the immunosuppressive effect of CEACAM6 on activated T cells which restores the immune response against cancer cells.

Available preclinical pharmacokinetic (PK) and in vitro pharmacodynamic (PD) data, target receptor density information and tumor (patho-)physiology were used to create a model framework taking into account the three most essential ‘pillars' (target exposure, target binding and drug activity) to estimate the human efficacious dosing of BAY 1834942. For this purpose, a physiologically-based pharmacokinetic (PBPK) model considering target binding of BAY 1834942 in tumor tissue and on blood granulocytes has been developed. The aim of the PBPK model was to estimate the dose range and regimen for humans leading to exposure level at the tumor site that allows sufficient target binding. The PBPK simulations were based on 1) an analysis of PD in vitro data in order to estimate the degree of saturation needed for maximum drug activity, 2) the assessment of CEACAM6 receptor numbers on tumor cells and blood granulocytes and 3) in vivo PK data in order to develop and evaluate the PBPK model. For the latter, plasma PK data of BAY 1834942 in monkeys were used as well as known tumor concentration-time profiles of the antibodies MOPC21 (non-targeting Ab) and ZCE025 (anti-CEA Ab) in mice and humans. Uncertainty of parameters which are relevant for CEACAM6 target saturation was considered by stochastic in silico simulations to estimate the CEACAM6 saturation at the tumor vs. dosing. This analysis revealed that the predicted human efficacious dose strongly depends on CEACAM6 density. Thus, a low CEACAM6 density scenario (25,000 CEACAM6/tumor cell) and a high CEACAM6 density scenario (250,000 CEACAM6/tumor cell) were simulated and used to support dose selection for the first-in-man (FIM) study of BAY 1834942. The FIM study is currently under preparation.

Citation Format: Sabine Wittemer-Rump, Christoph Niederalt, Joerg Willuda, Mark Trautwein, Merlin Luetke-Eversloh, Wolf-Dietrich Doecke, Clemens Guenther, Christian Scheerans. Physiologically based pharmacokinetic modeling and simulations to estimate the efficacious dose of the CEACAM6 function-blocking antibody BAY 1834942 [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 2791.
Abstract 2795: Screening and characterization of drugs that inhibit the ubiquitin-proteasome system
Authors: Selvaraju Karthik , Mofers Arjan , Pellegrini Paola , Hillert Ellin-Kristina , D'Arcy Padraig , Linder Stig
Published: August 2018

Pharmacological inhibition of proteasomal cysteine deubiquitinase (DUB) activity (i.e., USP14 and UCHL5 DUBs) has been shown to be particularly cytotoxic to cancer cells and to result in inhibition of tumor growth in several in vivo models. Most proteasome DUB inhibitors contain α, β-unsaturated ketones with the potential to covalently bind DUB active site cysteines. We here performed a screen of 5000 compounds containing α, β-unsaturated ketones in order to identify novel proteasome DUB inhibitors and to examine the question of the specificity of such compounds. Surprisingly, only ~3% of 5000 compounds containing α, β-unsaturated ketones showed anti proliferative activity at 5 uM. Almost one third of these cytotoxic compounds were found to block proteasome processing. Treatment of cells with the hit compounds induced the accumulation of proteasome bound high molecular weight polyubiquitin conjugates, induction of the chaperone HSP70B' and the oxidative stress marker heme oxygenase-1 (HO-1) and to induce an apoptotic response. A subset of 10 compounds were studied in detail and found to inhibit proteasome DUB activity in vitro. Importantly, hit compounds did not show inhibitory activity on a panel of recombinant non-proteasome DUBs. Inhibition of proteasome processing by hit compounds was not due to inhibition of 20S proteasome enzymatic activity. Exposure of multiple myeloma cells to the hit compounds resulted in thermostabilization of USP14 at the relevant concentrations using CETSA. These results suggest that α, β-carbonyls-containing compounds are promising candidates for development of drugs targeting proteasome DUBs and that drugs showing unexpected selectivity can be identified.

Citation Format: Karthik Selvaraju, Arjan Mofers, Paola Pellegrini, Ellin-Kristina Hillert, Padraig D'Arcy, Stig Linder. Screening and characterization of drugs that inhibit the ubiquitin-proteasome system [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 2795.
Abstract 2801: Novel selenodiazoles as antitumor and radical scavenging agents
Authors: Ruberte Ana Carolina , Plano Daniel , Encio Ignacio , Sharma Arun K. , Sanmartin Carmen
Published: August 2018

Heterocyclic compounds with selenium atom in endo position, such as ebselen (EBS), have achieved very promising activity against cancer and hold an excellent pharmacological profile. In fact, EBS is currently under Phase II clinical trial for the evaluation of its therapeutic safety and efficacy. The purpose of this study was to make structural modifications over this scaffold to improve its anticancer and antioxidant characteristics.

Twenty-eight novel benzo[c][1,2,5]selenodiazole-5-carboxamide derivatives were designed and synthesized. Cytotoxicity of these structures, using the MTT assay, was tested in vitro against a panel of five human cancer cell lines, including prostate (PC-3), colon (HT-29), leukemia (CCRF-CEM), lung (HTB-54) and breast (MCF-7). The selectivity of all the compounds was further examined in two non-malignant cell lines derived from breast (184B5) and lung (BEAS-2B). For MCF-7 cells, the apoptotic status and cell cycle analysis of the cells were based on the TUNEL technique. Likewise, their radical scavenging activity was determined using the DPPH assay.

Interestingly, three compounds exhibited higher cytotoxic activity than EBS in solid tumors, along with higher selectivity indexes. The cytotoxic activity of lead compound 8 was remarkable in MCF-7 cells. The antioxidant capacity of four compounds was greater than EBS.

The toxicity of derivative 8 in the non-tumoral cell line 184B5 was low (lethal dose (LD50) > 100 µM) and it exhibited promising antiproliferative values in MCF-7 cells with a growth inhibition of 50% (GI50) of 3.7 µM. The radical scavenging capacity of four compounds was greater than EBS.

Given the low toxicity and moderate antioxidant activity, these compounds can serve as an excellent scaffolds to achieve new and potent antioxidant compounds useful for several diseases such as cancer, neurodegenerative and heart diseases.

Citation Format: Ana Carolina Ruberte, Daniel Plano, Ignacio Encio, Arun K. Sharma, Carmen Sanmartin. Novel selenodiazoles as antitumor and radical scavenging agents [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 2801.
Abstract 2804: Identifying an optimal site for linker conjugation of the taccalonolides, novel microtubule stabilizing drugs
Authors: Risinger April L. , Du Lin , Ola Antonius , Cichewicz Robert H. , Mooberry Susan L.
Published: August 2018

The taccalonolides are a structurally distinct class of microtubule stabilizers purified from the roots and rhizomes of plants of the genus Tacca. The covalent binding of taccalonolides bearing a C-22,23 epoxide to β-tubulin generates a distinct profile of microtubule stabilization that results in a high level of cellular persistence and the ability to circumvent multiple mechanisms of clinically relevant drug resistance, including expression of P-glycoprotein. The biological effects of 121 structurally distinct taccalonolides, including 23 isolated natural products and 98 semi-synthetic derivatives have been evaluated. These compounds range in potency from 0.6 nM to over 50 µM, allowing for detailed structure activity relationship studies.

Some of the most potent taccalonolide derivatives include semi-synthetic modifications at C-6, C-7, or C-15 in combination with C-22,23 epoxidation. While these compounds did not have an acceptable therapeutic window for in vivo antitumor activity by systemic administration, they demonstrated highly effective and exquisitely persistent antitumor activity in a drug-resistant tumor model when delivered directly into the tumor. Additional studies were undertaken to see whether large chemical moieties could be added at these sites to facilitate targeted delivery of these compounds. In contrast to C-7 and C-15 derivatives, which are cleaved, C-6 modifications are stable and retain microtubule stabilizing and cytotoxic activities. In particular, fluorescent C-6 taccalonolide conjugates allowed, for the first time, direct visualization of these compounds in live cells, which co-localized with bundled microtubules. Further probing of the functionality at taccalonolide C-6 will allow for more detailed mechanistic studies of drug transport and distribution as well as optimization of a linker that could be used for antibody-based drug delivery.

Citation Format: April L. Risinger, Lin Du, Antonius Ola, Robert H. Cichewicz, Susan L. Mooberry. Identifying an optimal site for linker conjugation of the taccalonolides, novel microtubule stabilizing drugs [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 2804.
Abstract 2809: Novel 2,5,8-trihydro-6,7-disubstituted-4-(3'-alkylsulfonamidoanilino) quinazoline derivatives as RAF inhibitor
Authors: Yen Shih Chieh , Peng Shao-Zheng , Huang Hung-Jyun , Yang Ju-Ying , Liu Yen-Hsi , Ciou Sian-Yi , Kuo Mann-Yan , Liao Chu-Bin
Published: August 2018

The oncogenic mutations of BRAF which occur most frequently in V600E, leading to constitutive activation of the MAPK signaling pathway, are common in a variety of human cancers. Recently, drugs targeting RAF kinase have been approved as an effective treatment for human malignancies that rely on this target for their growth. Thus, suppressing RAF kinase activity is a clinically meaningful approach to treat cancer patients harboring RAF driven oncogene. In this study, a series of 2,5,8-trihydro-6,7-disubstituted-4-(3'-alkylsulfonamidoanilino)quinazoline was synthesized and evaluated for their RAF kinases inhibition activity. Several potent compounds displayed double-digit nanomolar IC50 values for RAF kinases including CRAF, BRAF, and BRAFV600E. Preliminary profiling of one of the most active compounds in a panel of protein kinases revealed its selectivity for RAF kinases. In cells, this compound exhibited selective cytotoxicity against cancer cells harboring BRAF V600E mutation and dose-dependent inhibition of phosphorylation of RAF downstream effectors MEK and ERK. Moreover, the compound was oral active in mice bearing A375 BRAFV600E-mutant melanoma xenograft. Therefore, the novel quinazoline derivative proved to be active as RAF inhibitor.

Citation Format: Shih Chieh Yen, Shao-Zheng Peng, Hung-Jyun Huang, Ju-Ying Yang, Yen-Hsi Liu, Sian-Yi Ciou, Mann-Yan Kuo, Chu-Bin Liao. Novel 2,5,8-trihydro-6,7-disubstituted-4-(3'-alkylsulfonamidoanilino) quinazoline derivatives as RAF inhibitor [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 2809.
Abstract 2828: MMR status affects efficiency of homologous recombination repair of F10-induced DNA DSBs
Authors: Gmeiner William H. , Mani Chinnadurai , Palle Komaraiah
Published: August 2018

Background: DNA mismatch repair (MMR) is an important stratification factor in colorectal cancer (CRC). 10-20% of sporadic CRC cases display high microsatellite instability (MSI-H) due to DNA mismatch repair (MMR) deficiency, and these are refractory to current fluoropyrimidine (FP) chemotherapy. Our studies with DNA-directed FP polymers, such as F10, indicate they are highly effective regardless of MMR-deficiency consistent with inducing MMR-independent cell death. We investigate the role of MMR-status on potency of F10 and induction of DNA damage and activation of the DNA damage response as part of an overall effort to determine if fluoropyrimidine polymers can be used effectively in the treatment of MMR-deficient CRC.

Methods: Studies were conducted using MMR-deficient HCT-116 CRC cells and HCT-116 3-6 cells. Potency was assessed by cell-titer glo and clonogenic assays. Replication stress was evaluated by DNA fiber combing. DNA damage and activation of the DNA damage response was evaluated by Western blots for gH2AX, Chk1 activation, and proteins important for homologous recombination repair.

Results: F10 is highly potent to CRC cells regardless of MMR status and is ~1,000-fold more potent than 5-FU to MMR-deficient HCT-116 cells. The increased potency of F10 parallels increased efficiency at causing replication fork slowing as assessed by DNA fiber analysis. F10 causes DNA double strand breaks (DSBs) and activates Chk1 consistent with causing replication fork collapse. DNA DSBs, Chk1 activation, and upregulation of FANCD2, which mediates HR-repair of DNA DSBs all show a marked MMR-dependence consistent with hMLH1 being a factor in HR-mediated repair of F10-induced DNA DSBs.

Conclusions: F10 is highly potent to CRC cells regardless of MMR status via a mechanism involving replication fork slowing and increased replication stress. MMR status affects basal levels of proteins involved in HR and the upregulation of these proteins in response to F10 treatment. Our studies indicate FP polymers are likely to be generally effective in CRC regardless of MMR status however MMR status may be an important factor in a personalized medicine approach to their use as combination with Chk1 inhibitors and other drugs modulating the DNA damage response may depend on MMR status.

Citation Format: William H. Gmeiner, Chinnadurai Mani, Komaraiah Palle. MMR status affects efficiency of homologous recombination repair of F10-induced DNA DSBs [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 2828.
Abstract 2830: Understanding chemotherapy-induced replicative stress to identify rational combination therapies
Authors: Alkan Ozan , Schoeberl Birgit , Shah Millie , Koshkaryev Alexander , Heinemann Tim , Drummond Daryl , Yaffe Michael B. , Raue Andreas
Published: August 2018

Cells respond to DNA damage by activating complex signaling networks that decide cell fate, promoting not only DNA damage repair and survival but also cell death. We have developed a multi-scale computational model using the U2OS osteosarcoma cancer cell line that quantitatively links chemotherapy-induced DNA damage response signaling to cell fate. The computational model was trained and calibrated based on an extensive data set that comprises cell cycle distribution of the initial cell population, signaling data measured by western blot, and cell fate data in response to chemotherapy treatment measured by time-lapse microscopy. The resulting mechanistic model can predict the cellular responses to chemotherapy alone and in combination with targeted inhibitors of the DNA damage response pathway, which we were able to confirm experimentally. Computational models, like the one presented here, can be used to understand the molecular basis that defines the complex interplay between cell survival and cell death, as well as to rationally identify chemotherapy-potentiating drug combinations.

Citation Format: Ozan Alkan, Birgit Schoeberl, Millie Shah, Alexander Koshkaryev, Tim Heinemann, Daryl Drummond, Michael B. Yaffe, Andreas Raue. Understanding chemotherapy-induced replicative stress to identify rational combination therapies [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 2830.