bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4989: Cannabinoid receptor 2 and C-X-C chemokine receptor 4 interact to abrogate CXCL12-mediated cellular response
Authors: Coke Christopher , Johnson Ahriea , Jones Kia , Hinton Cimona
Published: August 2015

The expression of C-X-C Chemokine Receptor 4 (CXCR4) has been correlated with increased metastatic potential of cancer cells. CXCR4 increases tumor malignancy by encouraging tumors cells to migrate to distal organs expressing its cognate ligand, CXCL12, facilitating metastasis. Thus, targeting the CXCR4/CXCL12 signaling axis provides a good strategy to inhibit the metastatic spread of tumor cells and slow cancer progression. Recent studies suggest that cannabis may have anti-proliferative as well as anti-metastatic properties, though a biochemical mechanism describing how this occurs has yet to be discovered. Our lab has confirmed that an interaction between agonist-bound CXCR4 and agonist-bound Cannabinoid Receptor 2 (CB2) can decrease cancer cell migration. Simultaneous treatment of the breast cancer cell line, MDA-MB-231 with CXCL12 and AM1241, a synthetic ligand for CB2, desensitized the intrinsic cellular response to migrate toward areas of high CXCL12 concentration. Furthermore, through co-immunoprecipitation, we determined that there was increased interaction between the two receptors with co-stimulation. When CXCR4 and CB2 were activated simultaneously with various agonists, decreases in migration were observed, confirming that the regulatory activity was receptor-based, not agonist-based. Moreover, when cells were treated with the CB2 antagonist (AM630) and CXCL12 simultaneously, the decrease in migration was no longer seen, as CXCL12 was able to induce cell movement. In order to determine whether receptors were activated in response to simultaneous stimulation, calcium mobilization assays were performed and results showed that transiently activated calcium levels were significantly lower in response to simultaneous treated cells when compared to cells treat with their individual ligands. Therefore, we propose that the interaction of CB2 with CXCR4 may play a role in inhibiting the cells response to CXCL12, leading to a loss in metastatic potential and presenting a new mechanism for regulating oncogenic signaling.

Citation Format: Christopher Coke, Ahriea Johnson, Kia Jones, Cimona Hinton. Cannabinoid receptor 2 and C-X-C chemokine receptor 4 interact to abrogate CXCL12-mediated cellular response. [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 4989. doi:10.1158/1538-7445.AM2015-4989
Abstract 499: Characterization of the cell death mechanism after silencing of PAX3-FOXO1 in alveolar rhabdomyosarcoma using a CRISPR-Cas mediated knockout approach
Authors: Wachtel Marco , Niggli Felix , Schäfer Beat
Published: August 2015

Chromosomal translocations leading to the generation of PAX-FOXO1 fusion oncogenes are found in about 80 percent of all alveolar Rhabdomyosarcoma (aRMS). Recent whole genome sequencing studies revealed that apart from these translocations mutation frequency is generally very low in aRMS, with no other genetic changes detected in some cases. aRMS tumor cells depend on the fusion protein and exhibit cell cycle arrest or undergo cell death upon blocking of its activity. Taken together this strongly suggests that the PAX-FOXO1 fusion represents the tumor initiating event and that it remains the most important tumorigenic factor for aRMS maintenance at later tumor stages.

However, the molecular mechanisms involved in tumorigenesis downstream of PAX3-FOXO1 are only partially understood yet, including the ones involved in oncogene addiction. We therefore aimed to further characterize the molecular events leading to cell death after silencing of PAX3-FOXO1. Towards this aim, we used an inducible shRNA system to silence PAX3-FOXO1 in different aRMS cell lines. Reduction of PAX3-FOXO1 below a threshold of about 20 percent of its basal level efficiently induced Caspase-dependent cell death. CRISPR-Cas mediated knock-out of individual members of the apoptotic cascade was then used to further characterize the involved apoptotic mechanism. BH3-only proteins including Bim were found as relevant sensors detecting cellular stress after silencing of PAX3-FOXO1.

This data suggests that combined interference with PAX-FOXO1 activity and anti-apoptotic Bcl-2 family members might accelerate tumor cell death.

Citation Format: Marco Wachtel, Felix Niggli, Beat Schäfer. Characterization of the cell death mechanism after silencing of PAX3-FOXO1 in alveolar rhabdomyosarcoma using a CRISPR-Cas mediated knockout approach. [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 499. doi:10.1158/1538-7445.AM2015-499
Abstract 5: Mcl-1-mediated resistance to ABT-263 is combated by mTOR inhibition in luminal breast cancers
Authors: Williams Michelle M. , Lee Linus , Sanchez Violeta , Morrison Meghan M. , Hicks Donna , Cook Rebecca S.
Published: August 2015

We found that 8.1% of luminal breast cancer cases curated by The Cancer Genome Atlas (TCGA), harbor gene amplification of anti-apoptotic Bcl-2 family members, warranting further study of anti-apoptotic Bcl-2 family proteins (A1, Bcl-2, Bcl-xL, Bcl-w and Mcl-1) in luminal breast cancers. A complex balance of pro- and anti-apoptotic Bcl-2 proteins guides activation versus repression of the intrinsic apoptotic pathway, with overexpression of anti-apoptotic factors tipping the balance towards cell survival. This mechanism is commonly used by cancers to evade apoptosis.

We used the BH3-mimetic ABT-263 to inhibit Bcl-2, Bcl-xL and Bcl-w in a panel of human luminal breast cancer lines, causing only modest apoptosis in monolayer and growth inhibition in three-dimensional (3D) matrix. Expression and activity of Mcl-1, another anti-apoptotic Bcl-2 family member, was rapidly upregulated in response to ABT-263 through increased cap-dependent MCL1 translation. Mcl-1 knock-down decreased tumor cell growth and improved ABT-263-mediated tumor cell killing. Inhibition of cap-dependent translation using the mTOR inhibitor RAD001/everolimus or the EIF4 complex inhibitor 4E1RCat blocked Mcl-1 induction in ABT-263 treated cells, enhancing apoptosis and growth inhibition. In vivo, single agent ABT-263 was ineffective at reducing tumor growth in xenografted human breast cancers and in transgenic WAP-Myc luminal mammary tumors. However, RAD001 decreased Mcl-1 levels and decreased WAP-Myc tumor growth by 40%. These results demonstrate that heightened cap-dependent translation of Mcl-1 prevents therapeutically induced tumor cell killing in ABT-263 treated luminal tumors, highlighting the importance of Mcl-1 regulation in therapeutic resistance, and identifying Mcl-1 as a potential therapeutic target in luminal breast cancers. These studies suggest that targeted inhibition of Mcl-1 alone or in combination with other Bcl-2 family inhibitors will enhance tumor cell killing in luminal breast cancers.

Citation Format: Michelle M. Williams, Linus Lee, Violeta Sanchez, Meghan M. Morrison, Donna Hicks, Rebecca S. Cook. Mcl-1-mediated resistance to ABT-263 is combated by mTOR inhibition in luminal breast cancers. [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 5. doi:10.1158/1538-7445.AM2015-5
Abstract 5002: Abstract Submission
Authors: Acharya Swati , Petersen Kira Y.D , Golubkov Vladislav , Kwong Mandy , Adams Christopher M. , Jackson Peter K. , Lewis David B.
Published: August 2015

Protein tyrosine kinase 7 (PTK7), a catalytically inactive receptor tyrosine kinase (RTK) that is highly expressed by T-lineage cells during intrathymic development, is a novel marker for human CD4+ recent thymic emigrants (RTEs), and is also highly expressed on some T-lineage thymomas, e.g., Jurkat cells as well as inprimary T-Acute Lymphoblastic leukemia. The function of PTK7 in normal human T-cell development and in oncogenesis remains unclear. Here, using RNAi-mediated gene silencing in T-lineage tumor cells, primary human peripheral T-cells, and thymocytes, we found that targeting PTK7 consistently decreased cell survival by augmenting caspase-3 activation of apoptosis. The PTK7 knockdown also decreased AKT phosphorylation and PI3 kinase activity, suggesting an essential role for PTK7 in survival of RTEs and developing thymocytes involving the PI3K/AKT pathway. Using mass spectrometry we identified insulin-like growth factor-1 (IGF-1) receptor as an active kinase partner of PTK7. This interaction was biologically relevant in that PTK7 downregulation also reduced IGF-1R-dependent survival signals in T-lineage cells. As enhanced IGF-1-dependent signaling is a frequent event in oncogenesis, the intersection of PTK7 with the IGF-1 signaling pathway suggests the potential of PTK7-directed therapy of T-lineage tumors.

Citation Format: Swati Acharya, Kira Y.D Petersen, Vladislav Golubkov, Mandy Kwong, Christopher M. Adams, Peter K. Jackson, David B. Lewis. Abstract Submission. [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 5002. doi:10.1158/1538-7445.AM2015-5002
Abstract 5004: Expression profiling of receptor tyrosine kinases in high-grade neuroendocrine carcinoma of the lung: A comparative analysis with adenocarcinoma and squamous cell carcinoma
Authors: Matsumura Yuki , Umemura Shigeki , Ishii Genichiro , Tsuta Koji , Mastsumoto Shingo , Aokage Keiju , Hishida Tomoyuki , Yoshida Junji , Ohe Yuichiro , Suzuki Hiroyuki , Ochiai Atsushi , Goto Koichi , Nagai Kanji , Tsuchihara Katsuya
Published: August 2015

Background:

As the comprehensive genomic analysis of small cell lung cancer (SCLC) progresses, novel treatments for this disease need to be explored. With attention to the direct connection between the receptor tyrosine kinases (RTKs) of tumor cells and the pharmacological effects of specific inhibitors, we systematically assessed the RTK expressions of high-grade neuroendocrine carcinomas of the lung (HGNEC), including SCLC and large cell neuroendocrine carcinoma (LCNEC).

Patients and Methods:

Fifty-one LCNEC and 61 SCLC patients who underwent surgical resection were enrolled in this research. As a control group, 202 patients with adenocarcinomas (ADCs) and 122 patients with squamous cell carcinomas (SQCCs) were also analyzed. Using immunohistochemical staining (IHC), we stained all tumors with antibodies for 10 RTKs; c-Kit, EGFR, IGF1R, KDR, ERBB2, FGFR1, c-Met, ALK, RET and ROS1. We scored the expression of these RTKs for all enrolled tumors, and compared them according to the histological types.

Results:

The LCNEC and SCLC patients exhibited similar clinicopathological characteristics. The IHC scores for each RTK were almost equivalent between the LCNEC and SCLC groups, but they were significantly different from those of the ADC or SQCC groups. Especially, c-Kit was the only RTK that was remarkably expressed in both LCNECs and SCLCs. On the other hand, about 20% of the HGNEC tumors exhibited strongly positive RTK expression, and this rate was similar to those for the ADC and SQCC tumors. Intriguingly, strongly positive RTKs were almost mutually exclusive in individual tumors.

Conclusions:

Compared with ADC or SQCC, LCNEC and SCLC had similar expression profiles for the major RTKs. The exclusive c-Kit positivity observed among HGNECs suggested that c-Kit might be a distinctive RTK in HGNEC. These HGNECs which have strongly positive RTKs could be the targets of personalized therapies.

Citation Format: Yuki Matsumura, Shigeki Umemura, Genichiro Ishii, Koji Tsuta, Shingo Mastsumoto, Keiju Aokage, Tomoyuki Hishida, Junji Yoshida, Yuichiro Ohe, Hiroyuki Suzuki, Atsushi Ochiai, Koichi Goto, Kanji Nagai, Katsuya Tsuchihara. Expression profiling of receptor tyrosine kinases in high-grade neuroendocrine carcinoma of the lung: A comparative analysis with adenocarcinoma and squamous cell carcinoma. [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 5004. doi:10.1158/1538-7445.AM2015-5004
Abstract 5005: Androgen-mediated genomic and non-genomic regulation of TRPM8 in prostate cancer
Authors: Asuthkar Swapna , Zakharian Eleonora
Published: August 2015

Transient receptor potential melastatin 8 (TRPM8) is a cold-sensing and Ca2+ permeable channel protein. The TRPM8 mRNA is overexpressed in early prostate tumors with high androgen levels, while anti-androgen therapy greatly reduces its expression. Thus, androgens, which are at the crossroads of several signaling pathways, appear to be associated with TRPM8-mediated Ca2+ -dependent signaling. Genomic regulation; From the chromatin-immunoprecipitation (ChIP) analysis, we observed that an androgen response element (ARE) mediates androgen regulation of trpm8. Although TRPM8 mRNA is expressed at high levels in prostate cancer (PC) cells, we found that the TRPM8 protein undergoes UBA1-mediated ubiquitination and degradation. PYR-41, a potent inhibitor of initial enzyme in the ubiquitination cascade, UBA1 increased TRPM8 activity on the plasma membrane of LNCaP cells. Further, PYR-41-mediated PMTRPM8 activity was accompanied by enhanced activation of p53 and Caspase-9. In addition, we found that the promoter region of trpm8 possesses putative binding sites for p53 and that the overexpression of p53 increased the TRPM8 mRNA levels. Our findings support previous studies that suggest the fine balance between androgen receptor (AR) and p53 expression that regulates androgen-dependent growth of PC. Non-genomic regulation; Furthermore, using immunofluorescence, calcium-imaging and planar lipid bilayers, we identified that TRPM8 channel is functionally associated with androgens in the prostate. Most importantly, we report that testosterone can directly activate TRPM8 channel at low picomolar concentrations and resulted in full openings of the purified TRPM8 channel in planar lipid bilayers. Our data demonstrated that testosterone is an endogenous and highly potent agonist of TRPM8 channel. In light of these observations our data of non-genomic effect of testosterone on PMTRPM8 may support a strategy for targeting the androgen-TRPM8-AR interaction or rescuing PMTRPM8 expression as a new therapeutic application for PC.

Note: This abstract was not presented at the meeting.

Citation Format: Swapna Asuthkar, Eleonora Zakharian. Androgen-mediated genomic and non-genomic regulation of TRPM8 in 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 5005. doi:10.1158/1538-7445.AM2015-5005
Abstract 5012: Establishment of a transplantable, NY-BR-1 expressing breast cancer model in HLA-transgenic mice
Authors: Das Krishna , Gardyan Adriane , Vormehr Mathias , Mueller-Decker Karin , Zörnig Inka , Jäger Dirk , Osen Wolfram , Eichmüller Stefan B.
Published: August 2015

Introduction: Breast cancer is one of the leading causes of cancer related deaths in women worldwide and current standard therapies show limited efficacy. However, immunotherapeutic approaches like adoptive T cell transfer might represent an attractive option for the treatment of breast carcinoma. The differentiation antigen NY-BR-1 appears as a suitable target for T cell immunotherapy against breast cancer as it is overexpressed in 60% of breast carcinomas compared to healthy breast.

Objectives: The aim of this project is to establish a NY-BR-1-expressing, transplantable tumor model in HLA transgenic mice that would allow to investigate the functional role of NY-BR-1-specific HLA-restricted CD4+ T cells in vivo with respect to their capacity to sustain cytotoxic T lymphocytes (CTL)-mediated tumor attack. Furthermore, the capability of NY-BR-1-specific CD4+ T cells to interact with tumor-associated macrophages (TAMs) thereby potentially promoting differentiation of TAMs into immunostimulatory type-1 macrophages (M1) will be investigated.

Materials and methods: Stable transfectant clones of the C57BL/6 derived lymphoma cell line EL4 and of the mammary adenocarcinoma cell line EO771 expressing NY-BR-1 were established. Furthermore, an H2Db-restricted, NY-BR-1-specific CTL epitope could be identified which upon peptide immunization of C57BL/6 mice resulted in the generation of NY-BR-1 specific, H2Db-restricted CTL lines, similarly to immunization with a rec. NY-BR-1 encoding adenovirus as observed in parallel experiments.

Results and conclusions: Transplanted EL4 and EO771 derived transfectant clones stably expressing NY-BR-1 gave rise to subcutaneous tumors in H2-compatible, HLA-DR4 transgenic mice. In addition, a NY-BR-1-specific CTL line recognizing EL-4/NY-BR-1 expressing transfectants in vitro was successfully generated upon peptide immunization as described above. Studies to investigate the cooperative effect between the aforementioned CTL line and HLA-DR3- and HLA-DR4-restricted NY-BR-1-specifc CD4+ T cell lines, upon joint adoptive transfer into tumor bearing HLA-transgenic mice are currently ongoing. The results presented here demonstrate the first NY-BR-1 expressing mouse tumor model, allowing the investigation of NY-BR-1-specific immune responses in vivo.

Citation Format: Krishna Das, Adriane Gardyan, Mathias Vormehr, Karin Mueller-Decker, Inka Zörnig, Dirk Jäger, Wolfram Osen, Stefan B. Eichmüller. Establishment of a transplantable, NY-BR-1 expressing breast cancer model in HLA-transgenic mice. [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 5012. doi:10.1158/1538-7445.AM2015-5012
Abstract 5017: The discovery of novel therapeutics that restore antigen processing pathways in immune-edited metastatic cancers
Authors: Nohara Lilian , Gabathuler Reinhard , Ahn Paul , Cheng Ping , Williams David , Andersen Raymond , Jefferies Wilfred A.
Published: August 2015

Cancer immune-editing resulting in immune escape where Major Histocompatibility Complex I (MHC I) molecules and the endogenous Antigen Processing Pathway (APP) leading to MHC I expression are down regulated, are prevalent in metastatic forms of cancer. In the absence of MHC I, these tumors subvert host immune surveillance mechanisms and are thus resistant to many of the immunotherapies approaches that evoke adaptive immunity to eradicate tumors in humans.

Previously, we showed that by restoring TAP-1 expression in metastatic disease it is possible to restore the APP and the CTL recognition of tumor specific antigen loaded MHC-I molecules in carcinomas. We continued to investigate this mechanism of TAP deficiencies leading to APP defects and were the first to discover that this phenotype is not regulated by defects or mutations in the TAP-1 gene, but it is epigenetically regulated and can be restored by treatment with Histone Deacetylase Inhibitors (HDACi).

Here, we describe a novel mechanism of action of compounds in promoting immune responses against tumors. We have developed a high-throughput screening assay to identify compounds that induce antigen presentation in metastatic prostate and lung carcinomas. We have used this system to screen a pharmacological library made from deep-sea sponge extracts, as it has been found that marine invertebrates are a diverse source of pharmaceutical leads and also of chemical diversity. Our results exploiting this system indicate several extracts isolated from amongst 500 sea sponges result in an increase a significant increase in surface MHC I expression, while at the same time exhibiting low cytotoxicity. The chemical structures of several of the active components of these extracts have been determined and one particularly promising candidate has been synthesized and produced in sufficient quantities to commence animal testing. Initial studies indicate the compound is well tolerated and there is no toxicity in animals at the doses that were studied. Results will be presented regarding the activity of the compound against subcutaneously implanted metastatic tumors.

The work described herein will provide new therapeutic candidates for harnessing the power of the immune system to recognize and destroy metastatic cancers.

Citation Format: Lilian Nohara, Reinhard Gabathuler, Paul Ahn, Ping Cheng, David Williams, Raymond Andersen, Wilfred A. Jefferies. The discovery of novel therapeutics that restore antigen processing pathways in immune-edited metastatic cancers. [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 5017. doi:10.1158/1538-7445.AM2015-5017
Abstract 5019: Characterization of sentinel node-derived antibodies from breast cancer patients
Authors: Shukla Girja S. , Pero Stephanie C. , Sun Yu-Jing , Carman Chelsea L. , Krag David N.
Published: August 2015

Lymph nodes are one of the important sites in the body where immune responses to antigens are initiated. The tumor-draining lymph node or sentinel node is the first site where cancer cells and cancer-related antigens are most likely to spread. In response to antigen exposure and immune activation, the lymph node B cells undergo clonal expansion and somatic hypermutation, leading to the affinity-matured populations of effector B cells secreting antibodies that bind to the tumor. The cancer-draining node is therefore an ideal source of B cells that produce anticancer antibodies. In the present investigation, we have characterized the antibodies derived from immortalization of sentinel node B cells from 29 breast cancer patients. The antibodies were screened for their isotypes and for binding to breast cancers classified as luminal A, luminal B, HER2, and basal/normal subtypes, based on the expressions of estrogen, progesterone and HER2 receptors. The isotype analysis showed a higher percentages of antibodies belong to IgG (48%) and IgM (34%) isotypes along with a smaller share of IgA (18%). The cell-binding studies were conducted using ELISA, immunofluorescence microscopy, and flow cytometry techniques. Of the studied antibodies, about 28% showed binding to the tumor cells. The comparative studies of antibody-binding to breast tumor cells and non-cancer breast cells identified several antibodies showing differential binding profile. The binding analyses also demonstrated that all of the tumor-binding antibodies belonged to IgM isotype. It is evident from the results that the tumor-reactive antibodies are generated in the sentinel nodes of breast cancer patients; however, the absence of class-switched anti-tumor antibodies in the repertoire raises a possibility of tumor-induced node suppression leading to an inefficient humoral immune response towards tumor antigens. The results are important in assessing the tumor immune response/lymph node suppression, and the possible role of the autoantibodies in diagnosis and therapy of breast cancer.

Citation Format: Girja S. Shukla, Stephanie C. Pero, Yu-Jing Sun, Chelsea L. Carman, David N. Krag. Characterization of sentinel node-derived antibodies from breast cancer patients. [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 5019. doi:10.1158/1538-7445.AM2015-5019
Abstract 5024: Implications of MAPK pathway inhibition on monocytes and tumor-associated macrophages in melanoma
Authors: Gremel Gabriela , Girotti Maria Romina , Viros Amaya , Ashton Garry , Bradley Helen , Galvani Elena , Lee Rebecca , Fusi Alberto , Lorigan Paul , Marais Richard
Published: August 2015

Plasticity in T-cell populations during response and onset of resistance to targeted therapy has been well documented. More recently, macrophage-derived tumor necrosis factor has been described to drive resistance to MAPK pathway inhibition, yet little is known on the adaptation of tumor-associated macrophage populations to treatment and disease progression in human melanoma.

To test for alterations in macrophage abundance following resistance to MAPK pathway inhibition, we applied a fluorescence-based co-staining strategy on melanoma specimens, representing tissue collected from patients before treatment and at relapse. In addition, we established in vitro co-culture systems to study the paracrine effects of melanoma cells on various aspects of monocyte differentiation and macrophage activation in the background of MAPK inhibition and resistance.

Resistance to targeted therapy was found to be associated with an increase in the number of tumor-resident macrophages. In vitro co-culture of melanoma cell lines with monocytes isolated from the peripheral blood of healthy donors or with CSF1-differentiated macrophages in the presence or absence of MAPK inhibition led to profound changes in monocyte/macrophage marker expression, indicating a direct interplay between the two cell populations upon MAPK pathway inhibition. Our data show that macrophages play an integral role in the changing immune-microenvironment during the course of MAPK pathway inhibition. Elucidation of mechanisms driving these processes will result in strategies to optimize treatment combinations and consecutive treatment outcomes for melanoma patients.

Note: This abstract was not presented at the meeting.

Citation Format: Gabriela Gremel, Maria Romina Girotti, Amaya Viros, Garry Ashton, Helen Bradley, Elena Galvani, Rebecca Lee, Alberto Fusi, Paul Lorigan, Richard Marais. Implications of MAPK pathway inhibition on monocytes and tumor-associated macrophages in 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 5024. doi:10.1158/1538-7445.AM2015-5024
Abstract 5025: Immune expression profiling of MAPK inhibitor resistant tumors based upon mechanisms of resistance
Authors: Wilmott James S. , Kakavand Hojabr , Menzies Alexander M. , Vilain Ricardo , Pupo Gulietta M. , Thompson John F. , Kefford Richard F. , Carlino Matteo S. , Rizos Helen , Long Georgina V. , Scolyer Richard A.
Published: August 2015

Purpose: To evaluate the association between the immunoreactivity of the immune inhibitory ligand PD-L1 with tumour infiltrating lymphocytes (TILs) and known mechanisms of MAPK inhibitor (MAPKi) resistance in tumor biopsies resected at the time of MAPKi disease progression.

Experimental Design: 23 patients with disease progression on MAPKi (vemurafenib, dabrafenib, and combination dabrafenib and trametinib) each had a progressing tumor biospied. All tumors underwent MAPKi resistance screening including RNA expression profiling. Morphological features, PD-L1 immunohistochemical immunoreactivity, and RNA expression profile were correlated with mechanisms of resistance (including mutations in NRAS and MEK, and amplification and aberrant splicing of BRAF).

Results: PD-L1 immunoreactivity was significantly higher in tumors that harbored an aberrant BRAF splicing variant (n = 8) or MEK mutations (n = 5) than those with BRAF amplification (n = 4), NRAS mutations (n = 2) or those with unknown resistance mechanisms (n = 4) (mean 27% vs 1%, p = 0.049). Additionally, the PD-L1 immunoreactivity in tumors with known MAPK-reactivating mechanisms (n = 19) tended to be higher than those with unknown resistance mechanisms (n = 4) (mean 17% vs 0%, p = 0.283). The immunoreactivity of PDL1 in progressed lesions was independent of BRAFV600 genotype, RECIST response and type of MAPK inhibitor. Correlations with TIL subsets, RNA expression, and response to subsequent anti-PD-1 therapy are underway.

Conclusions: The induction of PD-L1 immunoreactivity following MAPKi treatment in human melanoma is variable, however specific mechanisms of MAPKi resistance may confer increased expression of the inhibitory ligand. Further study is underway examining the association of the above with TIL infiltration, the RNA expression profile, and the response to subsequent anti-PD1 therapy.

Citation Format: James S. Wilmott, Hojabr Kakavand, Alexander M. Menzies, Ricardo Vilain, Gulietta M. Pupo, John F. Thompson, Richard F. Kefford, Matteo S. Carlino, Helen Rizos, Georgina V. Long, Richard A. Scolyer. Immune expression profiling of MAPK inhibitor resistant tumors based upon mechanisms of resistance. [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 5025. doi:10.1158/1538-7445.AM2015-5025
Abstract 5028: CXCL10 within the tumor microenvironment induces gemcitabine resistance in pancreatic cancer cells
Authors: Delitto Daniel , Perez Chelsey , Black Brian S. , Sorenson Heather L. , Knowlton Andrea E. , Han Song , Zhang Dongyu , Sarosi George A. , Moldawer Lyle L. , Behrns Kevin E. , Liu Chen , George Thomas J. , Thomas Ryan M. , Trevino Jose G. , Wallet Shannon M. , Hughes Steven J.
Published: August 2015

Background: The systemic treatment of pancreatic cancer (PC) is hindered by the rapid development of chemoresistance to current cytotoxic therapies. Mechanisms governing the development of chemoresistance remain poorly characterized, particularly with respect to contributions from the tumor microenvironment. Thus, the goal of this study was to identify novel mechanisms acting within the tumor microenvironment which lead to PC chemoresistance.

Methods: Intratumoral soluble mediator concentrations from resected PC specimens (n = 26) as well as supernatants from co-cultures of primary tumor-associated pancreatic stellate cells (PSCs) and PC cells (n = 12) were evaluated using a panel of 41 growth factors, chemokines and cytokines. The effect of CXCL10, a highly expressed soluble mediator during co-culture, on viability, proliferation, and apoptosis of PC cells was evaluated with and without gemcitabine treatment. In addition, the contribution of CXCL10 on migration patterns of peripheral blood mononuclear cells (PBMCs) was assessed.

Results: Co-culture of tumor-associated PSCs with PC cells revealed increased CXCL10 levels compared to either cell type cultured alone. In addition, high intratumoral CXCL10 concentrations correlated with reduced overall survival (HR 6.9; P = .006). While CXCL10 treatment had a small effect on the viability of PC cells, it led to significantly increased PC cell viability in the presence of gemcitabine. Further, gemcitabine treatment induced the expression of the CXCL10 receptor, CXCR3, and this induction of CXCR3 was associated with the absence of apoptotic markers in PC cells. Finally, constitutive expression of CXCL10 by PC cells preferentially led to the migration of regulatory immune cell subsets.

Conclusion: Paracrine CXCL10 signaling between stromal, PC and immune cells may be responsible not only for chemoresistance to gemcitabine, but also the recruitment and potential polarization of regulatory immune cell subsets in the pancreatic cancer microenvironment.

Citation Format: Daniel Delitto, Chelsey Perez, Brian S. Black, Heather L. Sorenson, Andrea E. Knowlton, Song Han, Dongyu Zhang, George A. Sarosi, Lyle L. Moldawer, Kevin E. Behrns, Chen Liu, Thomas J. George, Ryan M. Thomas, Jose G. Trevino, Shannon M. Wallet, Steven J. Hughes. CXCL10 within the tumor microenvironment induces gemcitabine resistance in pancreatic cancer 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 5028. doi:10.1158/1538-7445.AM2015-5028
Abstract 5030: Polarizing tumor associated macrophages (TAMs) towards an anti-tumor phenotype with a novel compound reveals a new subset of TAMs within breast tumors which facilitate tumor regression
Authors: Sotayo Alaba O. , Guerriero Jennnifer L. , Ponichtera Holly E. , Letai Anthony G.
Published: August 2015

Manipulation of the innate immune system is a relatively understudied strategy for anti-cancer immunotherapy. The current focus on immunotherapy is centered on the adaptive immune system. However, there is growing evidence that manipulation of the innate immune system, including macrophages, is also a promising method to combat cancer.

Tumor associated macrophages (TAMs) are one of the major infiltrating leukocyte populations associated with solid tumors. There are two major types of macrophages: classically activated macrophages, which can kill bacteria, pathogens, and similarly tumor cells; and alternatively activated macrophages which facilitate wound repair and are generally found in the tumor microenvironment. TAMs are generally alternatively activated cells with immunosuppressive properties that have been shown to enhance tumorogenesis by facilitating metastasis, angiogenesis, and inhibiting a protecting adaptive immune response. The presence of macrophages in the tumor microenvironment correlates with poor prognosis. Here we describe a novel, first in class compound that activates macrophages to an anti-tumor phenotype. While this compound has no direct cytotoxic activity, we show in the PyMT mouse model of breast cancer that breast tumors regress in response to therapy in a manner dependent on myeloid cells. Through the use of flow cytometry, we have been able to identify different subpopulations of TAMs using the markers CD45, MHCII, and CD11b. Correlating with tumor regression, we see an increase in PARP and cleaved caspase 3, indicating apoptotic cell death. Here we show that a novel compound effectively polarize macrophages to an anti-tumor phenotype to induce tumor regression. This strategy may have great therapeutic promise.

Citation Format: Alaba O. Sotayo, Jennnifer L. Guerriero, Holly E. Ponichtera, Anthony G. Letai. Polarizing tumor associated macrophages (TAMs) towards an anti-tumor phenotype with a novel compound reveals a new subset of TAMs within breast tumors which facilitate tumor regression. [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 5030. doi:10.1158/1538-7445.AM2015-5030
Abstract 5037: Immunotherapy meets epigenetics: GP96-Ig cellular vaccine genetic signature is modulated by the inhibition of histone modifications
Authors: Fromm George J. , Schilling Neal , Schreiber Taylor H.
Published: August 2015

A number of novel cancer treatment strategies have been identified and developed over the last decade with hopes of eradicating a diverse range of cancers, however the molecular mechanisms behind patient immune responses and tumor microenvironment signaling are complex, and have often hindered the translation of these ideas into successful patient treatments. Two modalities at the forefront of these potential blockbuster strategies are combinatorial immunotherapy and epigenetic manipulation. These approaches are supported by compelling clinical data, but have been encumbered by a number of factors including: adverse event profiles, complicated manufacturing and regulatory issues, toxicity due to non-specific small molecule interactions, limited overlap in patient/therapy antigen presentation, and exorbitant cost of biological treatments. To circumvent a number of these limitations, we have taken a novel approach by engineering allogeneic cellular vaccines tailored to specific cancers, that secrete heat-shock protein gp96-Ig and achieve high-frequency polyclonal CD8+ T cell responses to femto-molar concentrations of tumor antigens through antigen cross-priming in vivo. Using genomics (RNA-sequencing) we identified differential mRNA expression levels genome-wide between normal bladder tissue, our proprietary bladder cancer cellular vaccine Vesigenurtacel-L or HS410, and patient bladder tumor biopsy samples, to generate a genomic signature of Bladder Cancer Altered Genes, or BCAGs. Next, we examined a number of small molecule inhibitors of epigenetic modifications (i.e. histone deacetylases or HDACs), and determined their effect on BCAG expression and antigen secretion in HS410 cells. We find that the inhibition of certain HDACs results in decreased proliferation, dampened secretion of gp96-Ig, and attenuated expression of tumor specific genes. Together our work has defined the pattern of gene dysregulation genome-wide in bladder cancer and shown that epigenetic modifiers are capable of modulating the expression of many of these genes, which may be important for the efficacy of our cancer vaccine strategy. Therefore, epigenetic manipulation may present an attractive means to fine-tune gene expression programs and maximize antigen secretion thereby increasing vaccine potency and the ability of a single vaccine to target a variety of cancer types.

Citation Format: George J. Fromm, Neal Schilling, Taylor H. Schreiber. Immunotherapy meets epigenetics: GP96-Ig cellular vaccine genetic signature is modulated by the inhibition of histone modifications. [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 5037. doi:10.1158/1538-7445.AM2015-5037
Abstract 504: Glutamine metabolic inhibitors suppress growth and tumorigenicity in MYC and MYCN-driven pediatric malignancies
Authors: Hanaford Allison , Guerra Catherine , Eberhart Charles , Raabe Eric , Price Antoinette
Published: August 2015

The MYC family of oncogenes is associated with aggressive pediatric cancers such as medulloblastoma and neuroblastoma. These transcription factors promote oncogenesis in part by altering cellular metabolism. In multiple types of cancer, high-level MYC expression drives increased glutamine metabolism. We hypothesized that MYC-driven medulloblastoma and MYCN amplified neuroblastoma would be sensitive to the glutamine metabolic inhibitors 6-diazo-5-oxo-l-norleucine (DON) and Acivicin. These drugs have previously be used in pediatric clinical trials, but never extensively clinically tested in MYC-driven malignancies. The high MYC medulloblastoma cell lines D425Med and D283Med experienced an 82% decrease in growth as measured by MTS assay when treated with Acivicin or DON (p<0.004). DON and Acivicin treatment also caused a 400% increase in apoptosis as measured by cleaved caspase-3 immunofluorescence (p<0.03). In human neural stem cells transformed with MYC, Acivicin treatment decreased proliferation by 50% as measured by BrdU incorporation (p<0.003) and increased apoptosis by over 100% as measured by cleaved caspase-3 immunofluoresence (p<0.0001). Human neural stem cells immortalized with SV40 were unaffected by Acivicin and DON treatment (p = 0.1). In mice with D283Med flank xenografts, Acivicin decreased the size of tumors by 70%. DON treatment decreased the volume of flank tumors by 94% in animals with D425Med xenografts (p = 0.001). In mice with D425Med cerebellar xenografts (p = 0.0018), DON tripled median survival from 26 to 90 days in animals with. The MYCN amplified neuroblastoma cell lines LAN5 and IMR32 were also sensitive to treatment with DON and Acivicin. DON and acivicin treatment significantly decreased proliferation as measured by BrdU incorporation (p<0.03) and significantly increased apoptosis as measured by cleaved caspase-3 immunofluroescence (p<0.003). The non-MYCN amplified neuroblastoma cell line SKNAS was resistant to treatment with both DON and Acivicin, suggesting that expression confers sensitivity to glutamine analogs. These data suggest glutamine metabolism is a therapeutic target in medulloblastoma and neuroblastoma with elevated MYC or MYCN levels, and potentially other pediatric cancer types associated with MYC oncogenes.

Citation Format: Allison Hanaford, Catherine Guerra, Charles Eberhart, Eric Raabe, Antoinette Price. Glutamine metabolic inhibitors suppress growth and tumorigenicity in MYC and MYCN-driven pediatric malignancies. [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 504. doi:10.1158/1538-7445.AM2015-504
Abstract 5044: Reducing breast cancer risk with hormone replacement therapy
Authors: Dembo Anna , Greene Geoffrey
Published: August 2015

Menopause occurs in women between the ages of 45 and 55 and often results in symptoms that can be alleviated with hormone replacement therapy (HRT). HRT improves the symptoms associated with menopause that negatively affect quality of life, such as hot flashes, difficulty sleeping, fatigue, and vaginal atrophy. HRT also prevents osteoporosis. A current HRT formulation, which includes conjugated equine estrogens (CE) and medroxyprogesterone acetate, unfortunately increases the risk of breast cancer. Over the years, the number of women taking HRT has decreased due to this perceived increased risk. A new form of HRT, which contains CE and bazedoxifene (BZA), a selective estrogen receptor modulator, has recently been approved by the FDA for treatment of moderate to severe hot flashes and for the reduction of osteoporosis development. The CE/BZA mixture has been shown to not only relieve symptoms associated with menopause, but also does not stimulate the breast or uterus. Various studies show that CE/BZA may be protective in the breast. Currently, the mechanism of action of this new combination therapy is not known. Our goal is to identify the molecular mechanism of action of CE/BZA through transcriptome and whole genome occupancy analysis in breast cancer cell lines. In addition, we are determining the effects of the combination therapy on breast cancer progression in a transgenic mouse model. The overall hypothesis of our project is that the ability of CE/BZA to delay the onset of mammary tumors and decrease the number of lesions in the polyoma middle T antigen transgenic mouse model is due to its action as an estrogen receptor α antagonist in the mammary gland. We have determined that CE stimulates gene expression in MCF7 cells at a similar rate to 17β-estradiol, and that BZA is able to decrease this stimulation. Similarly, we have found that CE increases ERα occupancy at promoters of some estrogen target genes, while a combination of CE and BZA decreases this occupancy. Through understanding the molecular mechanism of CE/BZA and by determining whether it reduces the incidence of invasive breast cancer in a transgenic mouse model, we will be able to improve quality of life for many women by relieving menopausal symptoms in a safe manner.

Citation Format: Anna Dembo, Geoffrey Greene. Reducing breast cancer risk with hormone replacement 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 5044. doi:10.1158/1538-7445.AM2015-5044
Abstract 5045: Characterization of estrogen receptor alpha phosphorylation sites as a marker for tamoxifen resistant breast cancer
Authors: Speckmann Wayne A. , Brockett Robert , Yang Chun , Mohan Chandra , Long Kevin , Hatler Michele , Xu Xiaoyi
Published: August 2015

A major focus of breast cancer research is to understand the mechanisms responsible for disease progression and drug resistance. Most treatment options target endocrine therapy, however, the processes that modulate these signaling pathways are poorly understood. Here, we seek to gain additional understanding about the phosphorylation status of the estrogen receptor as it relates to tamoxifin drug treatment. Tamoxifen, a selective ER inhibitor, was the first targeted breast cancer therapy developed. Unfortunately, tamoxifen therapy was shown to be ineffective for certain kinds of cancers and even in affected tumors an acquired resistance was eventually seen. Previous research has shown that the estrogen receptor protein is phosphorylated at more than 20 different sites and some of these sites are linked with poor prognosis in tamoxifen treated patients. For example, phosphorylation at the S305 site may lead to an activation of ERα and to transcription of ERα-responsive gene after tamoxifen treatment, leading to tamoxifen resistance. In addition, other phosphorylation sites have also been found to correlate with poor prognosis of tamoxifen treated patients. Thus, differential phosphorylation of ERα may serve as a biomarker to identify patients unlikely to respond to tamoxifen treatment. Here, utilizing a newly developed medium throughput IHC test system we have interrogated multiple sites of ERα for phosphorylation status by screening an extensive panel of different breast cancer patient samples and other non-breast cancer tissue microarray (TMA) samples. These patient samples were classified according to their clinical grade (ER positive or negative, Her2, progesterone positive or negative, and Ki67 index) and tamoxifen treatment and then screened for their ERα phosphorylation status. The results show unique staining patterns depending upon the tumor classification and conclusions about the utilization of this information as a potential for diagnostic applications will be discussed.

Citation Format: Wayne A. Speckmann, Robert Brockett, Chun Yang, Chandra Mohan, Kevin Long, Michele Hatler, Xiaoyi Xu. Characterization of estrogen receptor alpha phosphorylation sites as a marker for tamoxifen resistant breast 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 5045. doi:10.1158/1538-7445.AM2015-5045