bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 270: Preclinical assessment of combination therapy with selumetinib and CTLA-4 for cancer
Authors: Mullins Stefanie R. , Poon Edmund , Watkins Amanda , Smith Paul D. , Leishman Andrew , Stewart Ross , Wilkinson Robert
Published: August 2015

Immune mediated therapies for Cancer (IMT-C), such as anti-CTLA-4 and anti-PD-1/PD-L1 monoclonal antibodies (mAb), are showing promise in the treatment of solid tumors. However, although these treatments demonstrate clinical activity in a range of tumors, the durable benefit of these agents appears to be limited to a subset of patients. One hypothesis is that activity in these patients is limited by a lack of immune priming or by additional immunosuppressive pathways. Combination with molecular targeted therapies offers the potential to explore additional treatment strategies for patients. However, in order to select the best combination partners, a greater understanding is needed as to how each therapy affects the immune system both directly, through effects on

leukocytes, and indirectly, through effects on tumor immunogenicity and the microenvironment. Selumetinib is a potent inhibitor of the mitogen-activated protein kinase (MAPK) kinase 1/2 (MEK1/2), involved in the activation of signaling pathways which regulates cellular growth, proliferation and survival. Given that these pathways are often found aberrantly activated in human tumors, and also play key roles in the regulation of immunological processes, it is difficult to predict the combinatorial effect of MEK1/2 inhibition and IMT in cancer treatment. To assess the effect of selumetinib in a tumor model with an intact immune system, we have characterized the impact of dosing selumetinib with or without anti-CTLA-4 mAb on the immune system in the CT26 syngeneic tumor model. Flow cytometric analysis showed that this combination reduced T-cell proliferation and memory T-cell generation, but increased the frequency of splenic T cells. Selumetinib treatment decreased PD-L1 expression and increased MHC class II expression on the tumor cells, whilst decreasing myeloid-derived suppressor cell (MDSC)-like cells in the tumor. Despite the apparent ability of selumetinib to have both positive and negative effects on immune responses in vivo, the combination of selumetinib and anti-CTLA4 mAb was found to show anti-tumor activity in the CT26 colorectal tumor mouse model. These studies highlight the potential for enhancing anti-tumor immune response through combinations of molecular targeted therapies and IMT-Cs.

Citation Format: Stefanie R. Mullins, Edmund Poon, Amanda Watkins, Paul D. Smith, Andrew Leishman, Ross Stewart, Robert Wilkinson. Preclinical assessment of combination therapy with selumetinib and CTLA-4 for cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 270. doi:10.1158/1538-7445.AM2015-270
Abstract 2707: Downregulation of cyclin D1 sensitizes cancer cells to MDM2 antagonist Nutlin-3
Authors: Ou Wenbin , Chen Weicai , Zhu Jiaqing , Eilers Grant , Li Xuhui , Yang Peipei , Li Hailong , Meng Fanguo , Fletcher Jonathan
Published: August 2015

The MDM2-p53 pathway plays a prominent role in various cancer pathogenesis. Nutlin-3, a small-molecule antagonist of MDM2, inhibits proliferation in cancer cells with wild-type p53 by blocking the interaction of MDM2 and p53. However, different cancer cells differ greatly sensitivity of Nutlin-3 because of MDM2 amplification and/or p53 mutation status. Herein, we evaluate the sensitivity of Nutlin-3 in different cancer cell lines including four mesothelioma cell lines, one breast cancer line, and one leiomyosarcoma cell line with nonmutant p53 expression, two liposarcoma cell lines harboring MDM2 amplification and wild-type p53, and one control mesothelioma cell line JMN1B harboring p53 point mutation. Unexpectedly, Nutlin-3 treatment induced cyclin D1 expression accompanied with inhibition of MDM2-p53 interaction in nonmutant cancer cell lines. Additive effects were obtained through a coordinated attack on MDM2-p53 interaction and cyclin D1, as demonstrated by immunoblots, cell viability and cell cycle analyses, showing that lentiviral mediated cyclin D1 knockdown or drug inhibition, and block of MDM2-p53 interaction, in cancer cell lines containing wild-type p53, induced greater anti-proliferative effects, compared to either intervention alone. Further results demonstrated that upregulation of cyclin D1 after Nutlin-3 treatment to some extent was dependent on expression and ubiquitin E3-ligase activity of MDM2. These compelling anti-proliferative responses indicate that combination inhibition of cyclin D1 and MDM2-p53 interaction warrants clinical evaluation as a novel therapeutic strategy in cancer cells harboring wild-type p53.

Citation Format: Wenbin Ou, Weicai Chen, Jiaqing Zhu, Grant Eilers, Xuhui Li, Peipei Yang, Hailong Li, Fanguo Meng, Jonathan Fletcher. Downregulation of cyclin D1 sensitizes cancer cells to MDM2 antagonist Nutlin-3. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2707. doi:10.1158/1538-7445.AM2015-2707
Abstract 2730: Additional mutations in genes relevant to the Ras signaling pathway promote the malignant characteristics of NF1-associated malignant peripheral nerve sheath tumor (NF1-MPNST) cells
Authors: Arima Yoshimi , Kosaki Kenjiro , Hirose Chikako , Saya Hideyuki
Published: August 2015

Malignant peripheral nerve sheath tumors (MPNST) are soft tissue sarcomas surrounding the peripheral nerve and occur sporadically or more often in association with neurofibromatosis type 1 (NF1, von Recklinghausen disease), which is an inherited disease caused by NF1 gene germline mutations. The NF1 tumor suppressor gene encodes neurofibromin and is a functional Ras GTPase-activating protein (RasGAP) that regulates the Ras signal negatively by accelerating the conversion of activated Ras-GTP to inactive Ras-GDP.

We hypothesized that additional genetic alterations promote malignancy of NF1-associated tumors. Hence, we inoculated a GFP-labeled human NF1-MPNST cell line, sNF96.2-GFP, which has a frame-shift mutation (c.3683delC, p.Asn1229MetfsTer11) in the NF1 gene, into the renal sub-capsules of immunodeficient mice. A subclonal cell line, the A-1 cell, was established from the developed tumor. We found that A-1 cells show much higher tumorigenic activity and Akt activation than parental sNF96.2-GFP cells.

We analyzed the genomic DNA of both the sNF96.2 and the A-1 cells by using the next-generation sequencing and the medical exome panel including 4813 genes, which are known to be responsible for most human genetic disorders. We identified 18 heterozygous variants in 17 genes that were present in the A-1 cells, but not in the original sNF96.2 cells. Two of these genes are associated with the regulation of the Ras signaling pathway.

Our data suggests that the additional gene mutations that regulate Ras pathways do in fact promote the malignant characteristics of NF1-associated tumors through the Akt activation.

Citation Format: Yoshimi Arima, Kenjiro Kosaki, Chikako Hirose, Hideyuki Saya. Additional mutations in genes relevant to the Ras signaling pathway promote the malignant characteristics of NF1-associated malignant peripheral nerve sheath tumor (NF1-MPNST) cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2730. doi:10.1158/1538-7445.AM2015-2730
Abstract 275: ER-886046, an antagonist of PGE2 receptor type-4, induces an effective antitumor immune response in mice by attenuating intratumoral MDSCs and TAMs
Authors: Albu Diana I. , Wang Zichun , Wu Jiayi , Huang Kuan-chun , Li Wei , Liu Diana , Kuznetsov Galina , Chen Qian , Bao Xingfeng , Woodall-Jappe Mary
Published: August 2015

One of the hallmarks of an immunosuppressive tumor microenvironment is the presence of myeloid-derived suppressor cells (MDSCs) and type 2 tumor-associated macrophages (TAMs). These myeloid cells derive from immature monocytes and their differentiation is highly regulated by the engagement of prostaglandin E2 (PGE2) receptor type 4 (EP4) with PGE2 in the tumor microenvironment. To understand the importance of EP4 for tumor support, we implanted mouse tumors in EP4 inducible knockout mice and found that these tumors grew significantly more slowly than in wild type mice, indicating an important role of host cell EP4 signaling for tumor progression. Here we report the development and evaluation of ER-886046, a novel and specific EP4 antagonist, for cancer treatment. Daily oral administration of ER-886046 inhibited the growth of multiple mouse syngeneic tumor models with an inhibitory activity up to 100%. The anti-tumor activity of ER-886046 was T cell-dependent since it was not observed in T cell deficient mice. Furthermore, we found in vitro and in vivo mechanistic evidence that ER-886046 interferes with tumor-induced monocyte differentiation into immunosuppressive type 2 macrophages and MDSCs, instead supporting monocyte differentiation into APCs and facilitating intratumoral T cell accumulation. Importantly, ER-886046 has a desirable pharmacokinetic and metabolism profile in mice, rats and dogs which qualifies it as a good candidate for clinical studies in humans. Thus, the preclinical data support further investigation of targeting EP4 signaling by ER-886046 as a novel immune therapy for cancer treatment in clinical setting.

Citation Format: Diana I. Albu, Zichun Wang, Jiayi Wu, Kuan-chun Huang, Wei Li, Diana Liu, Galina Kuznetsov, Qian Chen, Xingfeng Bao, Mary Woodall-Jappe. ER-886046, an antagonist of PGE2 receptor type-4, induces an effective antitumor immune response in mice by attenuating intratumoral MDSCs and TAMs. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 275. doi:10.1158/1538-7445.AM2015-275
Abstract 2753: The germline TP53 p.R337H mutation: a putative selective advantage
Authors: Andrade Kelvin César , Leite Ana Carolina , Nobrega Amanda , Savage Sharon , Achatz Maria Isabel
Published: August 2015

Li-Fraumeni syndrome is a hereditary cancer predisposition disorder related to germline TP53 mutations. Although it is considered to be a rare condition worldwide, a high occurrence of a founder mutation, the p.R337H TP53, is present in 0.3% of Southern Brazilian population. In a subgroup of p.R337H carriers, specific characteristics associated with late aging were clinically observed, such as a lower frequency of metabolic alterations and diseases typical of aging. However, despite of these particular features no study has clinically characterized these individuals until the moment. Therefore, our aim was to evaluate clinical features of p.R337H carriers and compare them with a control group. We used a clinical form composed by information on physical characteristics, personal habits, clinical history of diseases and physical activity throughout life. After IRB approval, we applied it in 48 p.R337H carriers (mean age: 46,8; range: 16-100) and in 38 carrier's relatives who were mutation-negative, as controls, (mean age 52,7; range: 18-90). Fisher's exact test was performed for statistical analysis. Regarding personal characteristics and habits, no difference was found between groups neither among p.R337H asymptomatic and affected carriers. However, some diseases discrepancies were found: In individuals older than 55 years of age, prevalence of osteoporosis was significantly lower in the p.R337H group when compared to controls (12% vs 59%; p<0,05). Anemia was also less frequent in p.R337H carriers (0% vs 13%; p<0,05) when all ages are considered. Although non-statistically significant, lower frequencies of neurodegenerative disorders, gastritis and thyroid dysfunction were also observed in the p.R337H group. These findings provide further information about a better overall health in p.R337H carriers, suggesting a possible selective advantage. This could be a plausible explanation for why this deleterious mutation has persisted throughout successive generations. More detailed clinical characterization studies are under way as well as molecular approaches looking for interactions between the p.R337H mutation and cellular senescence mechanisms.

Citation Format: Kelvin César Andrade, Ana Carolina Leite, Amanda Nobrega, Sharon Savage, Maria Isabel Achatz. The germline TP53 p.R337H mutation: a putative selective advantage. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2753. doi:10.1158/1538-7445.AM2015-2753
Abstract 2798: Quercetin inhibits Cr(VI)-induced malignant transformation of human lung epithelial cells by targeting miR-21-Pdcd4 signaling pathway
Authors: Poyil Pratheeshkumar
Published: August 2015

Hexavalent chromium [Cr(VI)] is an important human carcinogen associated with pulmonary diseases and lung cancer. Cancer prevention using natural products has become an integral part of cancer control. Quercetin is one of the most abundant dietary flavonoids widely present in many fruits and vegetables, possesses potent antioxidant activity capable of protecting normal cells from various stimuli-induced oxidative stress and cell death. MicroRNA-21 (miR-21) is a key regulator of oncogenic processes. It is significantly elevated in the majority of human tumors and functionally linked to cellular proliferation, invasion and migration. Studies have shown that miR-21 exerts its oncogenic activity by targeting the tumor suppressor gene programmed cell death 4 (Pdcd4). The present study examined the effect of quercetin on the inhibition of Cr(VI) induced carcinogenesis and the role of miR-21-Pdcd4 signaling involved. Our results showed that quercetin decreased ROS generation induced by Cr(VI) exposure in Beas-2B cells. Chronic Cr(VI) exposure induced malignant cell transformation, increased miR-21 expression and caused inhibition of Pdcd4, which were significantly inhibited by the treatment of quercetin in a dose dependent manner. Stable knockdown of miR-21 or overexpression of pdcd4 in Beas-2B cells significantly reduced the Cr(VI) induced cell transformation. Furthermore, Quercetin also inhibited the Cr(VI) induced beta-catenin/TCF-dependent transcription. Taken together, these results demonstrate that quercetin is able to protect Beas-2B cells from Cr(VI)-induced carcinogenesis by targeting miR-21-Pdcd4 signaling.

Keywords: Hexavalent chromium, Quercetin, miR-21-Pdcd4 signaling

Citation Format: Pratheeshkumar Poyil. Quercetin inhibits Cr(VI)-induced malignant transformation of human lung epithelial cells by targeting miR-21-Pdcd4 signaling pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2798. doi:10.1158/1538-7445.AM2015-2798
Abstract 2808: Luteolin inhibits cell proliferation and induces cell apoptosis via down-regulation of mitochondrial membrane potential in esophageal carcinoma cells EC1 and KYSE450
Authors: Chen Ping , Hu Tao , Ma Yane , Chen Xiaoyu , Dai Liping , Lei Ningjing , Dong Ziming , Li Pei
Published: August 2015

Esophageal squamous carcinoma (ESCC) may be developed through a progressive sequence from mild to severe dysplasia, carcinoma in situ, and finally, invasive carcinoma. Chemoprevention can block or weaken the influence of development. Recent study has shown luteolin, a bioflavonoid, possesses anti-inflammatory, antioxidant, and anti-proliferative effects, and it might have a preventive effect in this progress. In this study, we focused on the effect of luteolin on cell cycle regulation in human Esophageal Squamous Carcinoma Cell Line EC1 and KYSE450 Cells in vitro and its potential mechanisms. Observations by flow cytometer showed that luteolin inhibited cell cycle progression at G2/M phase in a dose- and time-dependent manner. We also found that luteolin could induce cell apoptosis via decreasing activation of caspase-3 and down-regulation of mitochondrial membrane potential. Western blot results showed the protein expression of cycle related protein CyclinD1 and apoptosis related proteins caspase-3, caspase-9, and Bak were also significantly decreased in luteolin treated cells compared with the non-luteolin treated cells. Our results suggest that the proper use of luteolin might be a practical approach to the prevention of esophageal carcinoma via the inhibition of cell proliferation and other potential high risk regions for this disease.

Citation Format: Ping Chen, Tao Hu, Yane Ma, Xiaoyu Chen, Liping Dai, Ningjing Lei, Ziming Dong, Pei Li. Luteolin inhibits cell proliferation and induces cell apoptosis via down-regulation of mitochondrial membrane potential in esophageal carcinoma cells EC1 and KYSE450. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2808. doi:10.1158/1538-7445.AM2015-2808
Abstract 2812: The chemo-prevention effect of statin on hepatocellular carcinoma cell lines
Authors: Ghalali Aram , Martin-Renedo Javier , Högberg Johan , Stenius Ulla
Published: August 2015

Hepatocellular carcinoma (HCC) is rated as the fifth most common malignancy and third in cancer related deaths worldwide. Statins, HMG-CoA reductase inhibitors, are potent cholesterol lowering drugs. Recent epidemiology suggests that statins prevent aggressive HCC development (J Clin. Oncol. 2012; 30:623-630). In previous cell experiments we have shown that statins down-regulate nuclear Akt responses to insulin (J Biol Chem 2010; 285, 27900). We showed that atorvastatin decreases nuclear phosphorylated Akt (pAkt) levels in pancreatic and lung cancer cell lines within minutes. This rapid effect was apparently not mediated by HMG-CoA reductase inhibition; instead there were indications that it was mediated by the P2×7 receptor. Akt has oncogenic activity in HCC and many other cancer types, and we have now tested the possibility that the P2×7-Akt pathway is of importance for the anticipated anticancer effects of statins in HCC. We performed western blotting, immunocytochemistry and transfections assays and found that statins decrease insulin induced pAkt levels in the nucleus. Further, statin decrease the proliferation of liver cells significantly. Hepatocytes lacking P2×7 receptor was insensitive to these effects of statins. Overexpression of P2×7 restituted the statin effect on pAkt levels. Overexpression of Hepatit B X protein vector has been shown to increase pAkt levels (FEBS J 279, 1220) and we found that statin counteract the Hepatit B vector-induced pAkt. These data provide evidence for a signaling pathway by which statins decrease pAkt and its downstream effect on pGSK3β Ser9 in hepatocytes via the P2×7 receptor. As the Akt pathway is deregulated and may act as a driver in HCC development this signaling pathway may have a role in the indicated chemo-preventive effect of statins.

Citation Format: Aram Ghalali, Javier Martin-Renedo, Johan Högberg, Ulla Stenius. The chemo-prevention effect of statin on hepatocellular carcinoma cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2812. doi:10.1158/1538-7445.AM2015-2812
Abstract 2826: Equimolar vs. pharmacokinetics-guided dosing in anti-cancer efficacy assessment of precursor-product pairs: Example with pyranocoumarins from Korean Angelica
Authors: Wu Wei , Puppala Manohar , Tang Suni , Zhang Jinhui , Xing Chengguo , Jiang Cheng , Lu Junxuan
Published: August 2015

Prostate cancer is a malignant disease quite unique to men and has the second highest mortality rate next to lung cancer in the United States, with an estimated 233,000 new cases to be diagnosed in 2014. Angelica gigas Nakai (AGN) is a traditional medicinal herb in Korea and China. Ethanol extract of dried AGN root has been shown by us and others to exert anti-cancer activity in several allograft and xenograft models. In recently completed studies in primary carcinogenesis model (TRAMP mice), our group has demonstrated that AGN ethanol extract can significantly suppress the growth of neuroendocrine (NE)-lineage cancers and inhibit prostate epithelial lineage lesions. These studies also found that major pyranocoumarin isomer compounds decursin (D) and decursinol angelate (DA) from AGN when provided at equimolar intake to that provided by AGN accounted for the epithelial lineage suppression efficacy, but was less active than AGN extract against NE-Ca growth. Because rodent models and humans convert D/DA rapidly and extensively (>95%) into decusinol (DOH), we hypothesize that DOH (and/or its metabolites) may be the true “active” chemical for suppressing the epithelial cancer. To test this, we compared oral gavage of DOH at equimolar dose as D/DA in SCID mice carrying human LNCaP/AR-Luc cells inoculated subcutaneously. Whereas DOH treated mice showed greater suppression of tumor growth than D/DA, measurement of plasma DOH levels showed 1.7 fold higher in the former at 3 h post-dose. Our earlier work has shown in a rat model that gavage of DOH led to 2.4 fold area under curve (AUC) over that achieved by equimolar dosing of D/DA, suggesting differences for absorption, distribution, metabolism, and excretion (ADME) of DOH vs. D/DA in other species as well. We are currently conducting PK studies of DOH and D/DA in mice to establish their PK parameters in order to achieve bio-equivalent dosing to critically test DOH efficacy and our hypothesis. In summary, PK-evaluation of precursor-product pairs might be important to determine bio-equivalent dosing before costly anti-cancer efficacy tests are conducted to ensure valid efficacy comparison.

Citation Format: Wei Wu, Manohar Puppala, Suni Tang, Jinhui Zhang, Chengguo Xing, Cheng Jiang, Junxuan Lu. Equimolar vs. pharmacokinetics-guided dosing in anti-cancer efficacy assessment of precursor-product pairs: Example with pyranocoumarins from Korean Angelica. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2826. doi:10.1158/1538-7445.AM2015-2826
Abstract 2832: Targeting and depletion of circulating cancer cells by lipophilic antibody-modified erythrocytes
Authors: Simberg Dmitri
Published: August 2015

There is a great interest in targeting and selective ablation of populations of circulating cells for research or therapeutic purposes. Red blood cells (RBCs) are readily available and fully biocompatible long-circulating intravascular carriers (natural life is 120 days) that are amenable to chemical modifications, drug loading and reinjection. Here we demonstrate that using our previously described lipophilic ligand painting strategy, red blood cells (RBCs) could be in one step converted into targeted entities that selectively seek and bind various cells in vitro and in vivo. In vitro, RBCs modified with lipophilic anti-EpCAM or anti-CD45 antibodies efficiently bound to cancer cells and leukocytes, forming characteristic rosettes. In vivo, intravenously injected RBCs painted with anti-CD45 antibody immediately associated with CD45 positive cells in blood, forming RBC-leukocyte rosettes. Moreover, anti-CD45-modified RBCs, but not the same amount of anti-CD45 antibody or anti-CD45-lipid conjugate (1-2 microgram per mouse), depleted over 50% of CD45+ leukocytes from circulation, with main clearance organs of leukocytes being liver and spleen with no visible deposition in kidneys and lungs. Anti-CD20 (rituximab)-painted RBCs efficiently (over 90%) depleted CD19+/CD20+/CD45+ human lymphoma cells in mantle cell lymphoma (MCL) JeKo-1 model, while the same amount of rituximab-lipid (2 microgram per mouse) was much less efficient in lymphoma cell depletion. Treatment of MCL mice with rituximab-modified RBCs carrying only 2 μg of the antibody resulted in a significant prolongation of survival as compared to the same amount of antibody control. Lipophilic ligand-painted RBCs is a novel tool that can be utilized for targeting blood borne cells for experimental immunology and cancer therapy applications.

Citation Format: Dmitri Simberg. Targeting and depletion of circulating cancer cells by lipophilic antibody-modified erythrocytes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2832. doi:10.1158/1538-7445.AM2015-2832
Abstract 2847: High complexity barcoding to study clonal dynamics in response to cancer therapy
Authors: Bhang Hyo-eun C. , Ruddy David A. , Krishnamurthy Radhakrishna Viveksagar , Zhao Rui , Kao Iris , Rakiec Daniel , Shaw Pamela , Balak Marissa , Caushi Justina X. , Ackley Elizabeth , Keen Nicholas , Schlabach Michael R. , Palmer Michael , Sellers William R. , Michor Franziska , Cooke Vesselina G. , Korn Joshua M. , Stegmeier Frank
Published: August 2015

Targeted therapies, such as erlotinib and imatinib, lead to dramatic clinical responses, but the emergence of resistance presents a significant challenge. Recent studies have revealed intratumoral heterogeneity as a potential source for the emergence of therapeutic resistance. However, it is still unclear if relapse/resistance is driven predominantly by pre-existing or de novo acquired alterations. To address this question, we developed a high-complexity barcode library, ClonTracer, which contains over 27 million unique DNA barcodes and thus enables the high resolution tracking of cancer cells under drug treatment. Using this library in two clinically relevant resistance models, we demonstrate that the majority of resistant clones pre-exist as rare subpopulations that become selected in response to therapeutic challenge. Furthermore, our data provide direct evidence that both genetic and non-genetic resistance mechanisms pre-exist in cancer cell populations. The ClonTracer barcoding strategy, together with mathematical modeling, enabled us to quantitatively dissect the frequency of drug-resistant subpopulations and evaluate the impact of combination treatments on the clonal complexity of these cancer models. Hence, monitoring of clonal diversity in drug-resistant cell populations by the ClonTracer barcoding strategy described here may provide a valuable tool to optimize therapeutic regimens towards the goal of curative cancer therapies.

Citation Format: Hyo-eun C. Bhang, David A. Ruddy, Viveksagar Krishnamurthy Radhakrishna, Rui Zhao, Iris Kao, Daniel Rakiec, Pamela Shaw, Marissa Balak, Justina X. Caushi, Elizabeth Ackley, Nicholas Keen, Michael R. Schlabach, Michael Palmer, William R. Sellers, Franziska Michor, Vesselina G. Cooke, Joshua M. Korn, Frank Stegmeier. High complexity barcoding to study clonal dynamics in response to cancer therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2847. doi:10.1158/1538-7445.AM2015-2847
Abstract 2853: Investigating DNAPK as a biomarker and a novel therapeutic target in aggressive prostate cancer
Authors: Kothari Vishal , Goodwin Jonathan F. , Zhao Shuang , Davicioni Elai , Karnes Jeffrey R. , Den Robert B. , Mehra Rohit , Knudsen Karen E. , Feng Felix Y.
Published: August 2015

Background/purpose: Despite recent advances in the development of androgen deprivation therapies (ADT), prostate cancer (PCa) remains the second leading cause of cancer death in American men. Thus, there is a critical need to improve treatment outcomes for men with aggressive PCa. The purpose of this study was to identify and functionally characterize kinases that can serve as both prognostic biomarkers and therapeutic targets in PCa.

Methods: To identify kinases associated with metastatic progression of PCa, we utilized high-density oligonucleotide arrays to interrogate the expression of all kinases in 545 prostatectomy samples, obtained from high-risk patients with long-term clinical follow-up (>10 years). We ranked all kinases by their fold change in expression between PCas that subsequently metastasized versus those that did not. We then characterized the top ranked kinase in preclinical PCa models with in vitro mechanistic experiments and in vivo therapeutic studies.

Results: We nominated DNA-dependent protein kinase (DNAPK) as the top kinase associated with metastatic PCa progression. Interrogation of pathways associated with DNAPK knockdown in PC3, DU145, VCaP and C4-2B, identified canonical Wnt signaling as the top DNAPK-activated pathway. In PCa cells (PC3, LNCaP C.S. {LNCaP grown under charcoal-stripped serum condition}, C4-2B and LNCaP-AR) DNAPK inhibition significantly abrogates Wnt signaling and abolishes cell proliferation, migration and invasion. Interestingly, we found that DNAPK directly modulates the Wnt signaling independent of the androgen receptor (AR), highlighting the therapeutic potential of DNAPK inhibition in cancers resistant to ADT. Indeed, in VCaP xenograft models, pharmacologic inhibition of DNAPK with NU7441 demonstrated 3.4 fold (p<0.01) reduction of tumor growth at non-toxic doses. Using an independent prostate cancer tissue cohort, we validate DNAPK expression as a biomarker for metastatic disease progression (p = 6.4 e−06, hazard ratio = 2).

Conclusion: In summary, we nominate DNAPK as a potential biomarker of disease progression and as a novel therapeutic target in aggressive prostate cancer. Our data suggests that DNAPK mediates PCa progression by upregulating Wnt signaling, and demonstrates that DNAPK inhibition results in significant tumor responses in PCa xenografts.

Citation Format: Vishal Kothari, Jonathan F. Goodwin, Shuang Zhao, Elai Davicioni, Jeffrey R. Karnes, Robert B. Den, Rohit Mehra, Karen E. Knudsen, Felix Y. Feng. Investigating DNAPK as a biomarker and a novel therapeutic target in aggressive prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2853. doi:10.1158/1538-7445.AM2015-2853
Abstract 289: The combination of CD40 agonism and PD-L1 blockade enhances anti-tumor immunity in a mouse syngeneic orthotopic pancreatic tumor model
Authors: Luheshi Nadia , Coates-Ulrichsen Jane , Harper James , Davies Gareth , Legg James , Wilkinson Robert
Published: August 2015

Infiltration of immunosuppressive myeloid cells into tumors correlates with poorer prognosis in many cancer types. The immunosuppressive effects of these cells help to create a microenvironment that may inhibit responses to T cell-directed immunotherapies. Anti-CD40 agonistic antibodies (αCD40) promote myeloid cell maturation, license cross-presentation of antigens to cytotoxic T cells, and enhance direct macrophage tumoricidal activity. We tested the hypothesis that αCD40 can improve responses to PD-L1 blockade via enhancing anti-tumor immunity in a syngeneic orthotopic mouse model of pancreatic cancer (Pan02). Whilst PD-L1 blockade alone had limited effect on tumor growth, we demonstrated that αCD40 drove antigen presenting cell maturation and systemic immune activation, inhibiting tumor growth and improving overall survival. αCD40 also induced upregulation of PD-L1 both systemically and within the tumor microenvironment. Furthermore, the combination of αCD40 and PD-L1 blockade led to enhanced tumor growth inhibition and improved anti-tumor immunity compared to either monotherapy alone. These data provide further support for the potential benefits of re-educating tumor myeloid cells.

Citation Format: Nadia Luheshi, Jane Coates-Ulrichsen, James Harper, Gareth Davies, James Legg, Robert Wilkinson. The combination of CD40 agonism and PD-L1 blockade enhances anti-tumor immunity in a mouse syngeneic orthotopic pancreatic tumor model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 289. doi:10.1158/1538-7445.AM2015-289
Abstract 293: Therapeutic effects of the bromodomain inhibitors JQ1 and I-BET 762 on pancreatic cancer
Authors: Leal Ana S. , Williams Charlotte R. , Sporn Michael B. , Liby Karen T.
Published: August 2015

Pancreatic cancer is expected to be the second most deadly cancer by 2030 with very few effective therapeutic options to improve patient survival. Bromodomain inhibitors (JQ1 and I-BET 762) are a new generation of chemotherapeutic drugs that target BET proteins, impairing their ability to bind to acetyl modified lysines and therefore interfering with transcriptional initiation and elongation. BET proteins regulate several genes responsible for cell cycle, apoptosis and inflammation. I-BET 762 is currently in clinical trials for cancer and has the advantage of oral administration.

Kras is one of the most common mutated genes in pancreatic cancer (90%); however its protein is a difficult drug target. p-Erk, responsible for several pathways regulating cell survival, is a downstream effector of Kras that has been shown to be essential for the progression and maintenance of pancreatic cancer. LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) and LSL-KrasG12D/+;Pdx-1-Cre (KC) mouse models are commonly used to study pancreatic cancer. KPC mice develop the full spectrum of pancreatic cancer by an average age of 20 weeks. The KC mouse model is commonly used to study pancreatic cancer progression after stimulation with a pancreatic inflammatory agent, such as cerulein.

JQ1 and I-BET 762 reduce the protein levels of p-ERK 1/2 in cells lines derived from a pancreatic tumor or ascites of KPC animals. JQ1 and I-BET 762 are also effective at inhibiting cell growth in human pancreatic cells harboring Kras mutations. When KC mice are injected with LPS to induce inflammation and pancreatitis, p-ERK 1/2 protein levels are significantly increased. This model is being used to test the effects of JQ1 and I-BET 762 after a single LPS injection, in KC mice.

Pancreatic cancer is driven by several cytokines and chemokines, including CCL2 and IL-6, which recruit and regulate the inflammatory cells (macrophages, T cells and MDSC (Myeloid derived suppressor cells) cells) in the tumor. Levels of IL-6 and CCL2 in cell lines developed from KPC mice are reduced when treated with JQ1 and I-BET 762. KC mice stimulated with LPS also have higher levels of CCL2 and IL-6 than mice stimulated with saline.

In conclusion this study showed that the bromodomain inhibitors JQ1 and I-BET 762 had positive effects in suppressing targets of inflammation in pancreatic cancer.

We thank James Bradner for the generous gift of JQ1.

Citation Format: Ana S. Leal, Charlotte R. Williams, Michael B. Sporn, Karen T. Liby. Therapeutic effects of the bromodomain inhibitors JQ1 and I-BET 762 on pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 293. doi:10.1158/1538-7445.AM2015-293
Abstract 2933: The apoptotic mechanism of action of SKI-178, a novel Sphingosine kinase 1 selective inhibitor
Authors: Dick Taryn E. , Hengst Jeremy , Kale Vijay , Colledge Ashley , Yun Jong K.
Published: August 2015

We previously developed SKI-178 as a novel Sphingosine kinase 1 (SphK1) selective inhibitor that is cytotoxic toward a broad panel of cancer types. Herein, we sought to determine the mechanism-of-action of SKI-178-induced cell death. We present evidence that SKI-178 induces prolonged mitosis followed by apoptotic cell death through the intrinsic apoptotic cascade. Further examination of the mechanism-of-action (MOA) of SKI-178 implicated c-Jun NH2-terminal kinase (JNK) and cyclin-dependent protein kinase 1 (CDK1) as critical factors required for SKI-178-induced apoptosis. In cell cycle synchronized human AML cell lines, we demonstrate that entry into mitosis is required for apoptotic induction by SKI-178 and that CDK1, not JNK, is required for SKI-178-induced apoptosis. We further demonstrate that the sustained activation of CDK1 during prolonged mitosis, mediated by SKI-178, leads to the simultaneous phosphorylation of the pro-survival Bcl-2 family members, Bcl-2 and Bcl-xl, as well as the phosphorylation and subsequent degradation of Mcl-1. Moreover, multi-drug resistance mediated by MDR-1 and/or pro-survival Bcl-2 family member over-expression did not affect the sensitivity of AML cells to SKI-178. In addition to AML, we extend the MOA of SKI-178 to include various solid tumor cell lines, including pancreatic cancer and glioblastoma. Taken together, we provide evidence that SKI-178 induces apoptosis in a CDK1-dependent manner and is not a substrate for MDR1, making it a promising chemotherapeutic candidate for the treatment of various cancer types, including those known to be drug resistant.

Citation Format: Taryn E. Dick, Jeremy Hengst, Vijay Kale, Ashley Colledge, Jong K. Yun. The apoptotic mechanism of action of SKI-178, a novel Sphingosine kinase 1 selective inhibitor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2933. doi:10.1158/1538-7445.AM2015-2933
Abstract 2943: Novel combination therapy of DNMT inhibitor SGI-110 and PARP inhibitor BMN-673 (talazoparib) for BRCA-proficient ovarian cancer
Authors: Pulliam Nicholas , Taverna Pietro , Lyons John , Nephew Kenneth P.
Published: August 2015

Ovarian cancer (OC) is initially chemoresponsive but the majority of patients relapse after first line platinum-, taxane-based chemotherapy. Recurrence has been shown to be associated with increased DNA damage response (DDR) mediated by poly-(ADP)-ribose polymerase 1/2 (PARP1/2), which can be therapeutically targeted by PARP inhibitors (PARPi). Although PARPi are indicated for platinum-responsive, BRCA-mutated OC, most OC patients have BRCA-proficient disease. Based on our previous studies supporting a role for DNA methylation in chemoresistant OC, mediated by the enzyme DNA methyltransferase 1 (DNMT1), and reports on a functional role for DNMT1 in DNA double strand break repair mediated by BRCA1/2, we hypothesized that combining DNMTi and PARPi will impair BRCA-mediated DDR, resulting in cytotoxicity in OC cells. A panel of OC cell lines, including A2780 (platinum sensitive, BRCA-wild type (wt)), A2780-cp and HeyC2 (platinum resistant, BRCA-wt) and Kuramochi (platinum resistant, BRCA2 mutant) were examined for cell growth using colony formation assays after treatment with DNMTi SGI-110 (5, 20, or 100nM) and PARPi talazoparib (1 or 10nM), alone or in combination. Combination treatment schemas consisted of: 1) “priming”: SGI-110 for three days, 24 hour recovery, then talazoparib treatment on Day 5 or 2) “co-administration”: talazoparib administration on day 1 and SGI-110 treatment on days 1-3. Colony formation was inhibited by talazoparib alone in all cell lines but in a dose-dependent manner. Decreased colony formation after SGI-110 alone (20 or 100 nM) was observed only in Kuramochi cells. Combination drug treatments resulted in significant reductions in colony formation. For example, regardless of BRCA and platinum sensitivity status, co-administration of SGI-110 and talazoparib reduced (P<0.05) cell survival, albeit%survival was dependent on drug dose and OC cell line. In general, priming with SGI-110 prior to treatment with talazoparib reduced (P<0.05) survival of A2780, Kuramochi and HeyC2 (majority of combinations tested); however, decreased (P<0.05) survival of A2780-cp was observed only after priming with 100 nM SGI-110. In summary, talazoparib combined with SGI-110 displayed effective cytotoxicity in OC cell lines harboring either wt- or mutant-BRCA, indicating that this DNMTi-PARPi drug combination impairs BRCA-mediated DDR and may represent an effective treatment regimen for OC.

Citation Format: Nicholas Pulliam, Pietro Taverna, John Lyons, Kenneth P. Nephew. Novel combination therapy of DNMT inhibitor SGI-110 and PARP inhibitor BMN-673 (talazoparib) for BRCA-proficient ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2943. doi:10.1158/1538-7445.AM2015-2943
Abstract 2972: Genomic classification of cutaneous melanoma
Authors: Watson Ian R. , Wu Chang-Jiun , Zou Lihua , Gershenwald Jeffrey E. , Chin Lynda ,   on behalf of the Melanoma Analysis Working Group and The Cancer Genome Analysis Research Network
Published: August 2015

We describe here the landscape of genomic alterations in cutaneous melanomas through DNA, RNA, and protein-based analysis of 333 resected primary and/or metastatic melanomas from 331 patients. We establish a framework for genomic classification based on the pattern of the most prevalent significantly mutated genes into one of four subtypes: mutant BRAF, RAS, NF1, and Triple-WT (wild-type). Integrative analysis reveals enrichment of mutations in KIT, focal amplifications and complex structural rearrangements as a feature of the Triple-WT subtype. We find no significant outcome correlation with genomic classification, but samples assigned to a transcriptomic subclass enriched for immune gene expression are significantly associated with improved patient survival. That favorable prognosis of the ‘immune’ transcriptomic subclass is associated with high LCK protein expression, a T-cell marker, and presence of lymphocyte infiltrate on pathology review. This clinicopathological and multi-dimensional analysis suggests prognosis of melanoma patients with nodal metastases is influenced by immunobiology of the tumor stroma, offering insights to further personalize therapeutic decision-making.

Citation Format: Ian R. Watson, Chang-Jiun Wu, Lihua Zou, Jeffrey E. Gershenwald, Lynda Chin, on behalf of the Melanoma Analysis Working Group and The Cancer Genome Analysis Research Network. Genomic classification of cutaneous melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2972. doi:10.1158/1538-7445.AM2015-2972
Abstract 2974: Comprehensive and integrative genomic characterization of diffuse lower grade gliomas
Authors: Brat Daniel J. ,   The Cancer Genome Atlas (TCGA) Research Network
Published: August 2015

Background: Diffuse low and intermediate grade gliomas (LGG; WHO grades II and III) have highly variable clinical behaviors that are not optimally predicted by histology; some are indolent, while others quickly progress to glioblastoma. Uncertainty is compounded by inter-observer variability in diagnosis, in part due to modest correlation between histology and biologic classes. Mutations in isocitrate dehydrogenase (IDH), TP53, ATRX, and co-deletion of chromosomes 1p and 19q are implicated as clinically relevant LGG markers.

Methods: We performed a comprehensive genome-wide analysis of 293 LGG, incorporating exome sequence, DNA copy number, DNA methylation, messenger RNA and microRNA expression and targeted protein expression. Molecular data was integrated with clinical outcomes to identify biologically robust LGG subtypes.

Results: We uncovered 19 mutated genes, including recognized drivers and novel mutations. Unsupervised clustering of genome-wide data from RNA-, DNA- and DNA methylation platforms uncovered concordant classification of three robust, non-overlapping LGG subtypes that were captured with highest correlation by IDH and 1p/19q status. The large majority of IDH wild-type LGG showed strikingly similar genomic aberrations and clinical behavior to primary glioblastoma. Nearly all IDH-mutant LGG lacking 1p/19q co-deletion had mutations in TP53 (94%) and ATRX inactivation (86%). IDH-mutant, 1p/19q-co-deleted LGG had most favorable clinical outcomes and harbored mutations in CIC, FUBP1, NOTCH1 and the TERT promoter.

Conclusions: Integration of genome-wide data from multiple platforms cleanly delineates three LGG molecular subtypes concordant with IDH and 1p/19q status: IDH-mutant LGG are either 1p/19 co-deleted or TP53 mutant, whereas most IDH wild-type LGG are molecularly and clinically similar to glioblastoma. Overall, results support a molecular-based, clinically relevant classification for implementation into routine clinical practice.

Citation Format: Daniel J. Brat, The Cancer Genome Atlas (TCGA) Research Network. Comprehensive and integrative genomic characterization of diffuse lower grade gliomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2974. doi:10.1158/1538-7445.AM2015-2974