bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3378: Variance in the distance between chromosome territories 9 and 22 in lymphocytes from peripheral blood healthy subjects: A proof of concept for a possible risk factor in chronic myeloid leukemia
Authors: Vallejo David G. , Fabian Eunice , Herrera Luis A.
Published: August 2018

The objective of our study was to assess the variations in the 3D organization of Chromosome Territories (CT) of chromosomes 9 and 22 in lymphocytes from healthy subjects. The differences in the distances of CTs 9 and 22 between subjects may provide a new perspective in the predisposition for specific chromosome translocations. We quantified the intra- and inter-subject variation in the distance of CT 9 and 22 as a proof of concept for new questions on the physiological and pathological effects of these differences. Methods: Blood samples from healthy subjects were processed for the isolation of lymphocytes with Ficoll as the gradient media. After the isolation, we fixed the cells directly onto coverslips for a 3D Fish protocol with 9 and 22 whole chromosome probes. Our protocol has been standardized to maintain the integrity of the nucleus with only minor changes in its volume. After the hybridization, we used the Elyra System of Zeiss to obtain super resolution images in Z stack. The images were processed with the Imaris software for its 3D reconstruction, measurement (distances, volumes and colocalization) and analysis. Results: The CTs 9 and 22 behave differently in peripheral blood lymphocytes. The CT 22 has a more internal position (closer to the center of the nucleus) in comparison with the CT 9, which can be seen most of the times in contact with the border of the nucleus. The CT 22 occupies a smaller volume than CT 9, which has almost a twice as larger volume. The CTs 9 have a polarized distribution inside the nucleus, therefore the distance between the CT 9-9 was larger (~7.3 μm ± 0.9) than the distance between CT 22-22. Although the position of the CTs was highly variable in cells from the same individual, we found a statistically significant difference in the inter-individual distance between CT 9-9 and the minimal distance between CT 9-22. Conclusion: Our study suggests that there are differences in the distribution of CT in lymphocytes isolated from peripheral blood between our subjects. These differences could prove to be a new individual characteristic with important implications in the predisposition for specific diseases, mainly to those related with chromosomal rearrangements.

Citation Format: David G. Vallejo, Eunice Fabian, Luis A. Herrera. Variance in the distance between chromosome territories 9 and 22 in lymphocytes from peripheral blood healthy subjects: A proof of concept for a possible risk factor in chronic myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3378.
Abstract 3447: IL-6 promotes tumor metastasis by promoting anoikis resistance and stemness phenotype in cancer cells via the activation of FAK and nanog stabilization
Authors: Yadav Arti , Kumar Bhavna , Kumar Pawan
Published: August 2018

The process of tumor metastasis is highly complex and involves dissemination of tumor cells from the primary tumor through the vascular system. However, most of the cancer cells, particularly epithelial cancer cells, have very low survival rates in circulation and undergo rapid anoikis. Tumor cells that acquire malignant potential have developed mechanisms to resist anoikis and thereby survive while travelling though the lymphatic or circulatory systems. Recently, we have shown that IL-6 promotes epithelial-mesenchymal transition (EMT) and tumor metastasis. A number of studies have shown that FAK signaling pathways play an important role in inducing anoikis resistance in tumor cells. In this study, we examined if IL-6 promoted tumor metastasis by enhancing stemness and anoikis resistance in cancer cells via the activation of FAK signaling. Our results demonstrate that IL-6 significantly enhanced stemness phenotype in cancer cells as measured by tumorsphere formation and ALDH positive cells by upregulating nanog protein levels. Nanog knockdown significantly reversed IL-6-mediated cancer stem cell (CSC) phenotype. IL-6 treatment of cancer cells induced FAK phosphorylation in a time dependent manner. Interestingly, FAK knockdown did not significantly decrease nanog mRNA levels but markedly decreased nanog protein levels. FAK depletion completely blocked IL-6-mediated nanog phosphorylation and enhanced nanog protein ubiquitination and degradation. These results suggest that FAK may be modulating nanog protein levels by stabilization nanog protein through phosphorylation. Furthermore, FAK knockdown significantly decreased IL-6-mediated tumorsphere formation, anoikis resistance and tumor metastasis. Taken together, our results show that IL-6 promotes tumor metastasis by enhancing stem cell phenotype and anoikis resistance in head and neck cancer cells via FAK activation and nanog protein stabilization.

Citation Format: Arti Yadav, Bhavna Kumar, Pawan Kumar. IL-6 promotes tumor metastasis by promoting anoikis resistance and stemness phenotype in cancer cells via the activation of FAK and nanog stabilization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3447.
Abstract 3498: Regulation of AR through an FGF5 driven mechanism
Authors: McLean Dalton
Published: August 2018

Purpose

To investigate the role of increasing FGF5 expression in the progression of prostate cancer and emergence of castration resistant prostate cancer.

Methods

Utilizing a cell line model representing different stages of prostate cancer progression, we assessed FGF5 expression and function in vitro and in vivo. We analyzed both an outcomes and progression tissue microarray (TMA) containing ~400 prostate cancer patient cores to assess FGF5 expression in a clinical setting.

Results

There was a significant increase in the both RNA and protein expression of FGF5 in tumorigenic cell lines compared to nontumorigenic. In TMAs that were analyzed, there was a significant increase of FGF5-positive cells across progression of prostate cancer from benign to metastatic, with the highest percentage occurring in cases of cancer.

When performing gain of function experiments with recombinant FGF5 (rFGF5) or overexpression plasmids, we found that FGF5 significantly increased the proliferation of AR-positive cell lines. Conversely, in AR-negative cell lines, FGF5 inhibited proliferation. Because FGF5 is associated with stem cells and differentiation events, we next asked whether the population of FGF5 cells that increases through cancer progression could lead to molecular changes in the total population. In addition to inhibiting proliferation of AR-negative nontumorigenic BPH1 cells, rFGF5 resulted in a robust AR expression. In LNCaP, C4-2, and C4-2B rFGF5 resulted in an increase in AR expression. In C4-2 and C4-2B cells an increase in AR protein coincided with an increase in nuclear YAP1, a potent transcriptional regulator.

We hypothesized that androgens could be regulating FGF5 expression in the prostate. When androgen dependent LNCaP cells are subjected to an androgen-depleted environment, FGF5 protein and RNA significantly increased within 24hrs. Additionally, when AR is stimulated using dihyrotestosterone, FGF5 protein and RNA levels significantly decreased. These effects are not observed in cell lines that do not express AR.

Conclusions

These results suggest that FGF5 is increased in prostate cancer progression cell lines as well as patient samples, with highest expression in aggressive cancer cores. Data also suggests that AR plays a role in the regulation of FGF5 and vice versa. These data suggest that FGF5 driven AR expression could be driven by YAP1 nuclear localization. This mechanism will be further interrogated.

Citation Format: Dalton McLean. Regulation of AR through an FGF5 driven mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3498.
Abstract 3505: Tumor radiosensitization by suppression of hexosamine biosynthetic pathway
Authors: Efimova Elena V. , Appelbe Oliver K. , Lee Steve Seung-Young , Ricco Natalia , Ramamarthy Aishwarya , Rymut Nicolas , Collins Tamica , Wolfgeher Donald J. , Warrington Sara , Kron Stephen J.
Published: August 2018

The elevation of glucose and glutamine metabolism characteristic of cancer has been ascribed to increased demand for metabolic intermediates that provide building blocks for rapid cell growth. However, recent discoveries have highlighted complementary effects mediated by metabolites serving as cofactors for chromatin modifying enzymes and providing metabolic intermediates for proteins posttranslational modification. Along with epigenetic regulation of gene expression, multiple chromatin modifiers modulate DNA damage repair, raising the question of whether metabolic reprogramming may thereby promote genomic instability and cell immortality. In our prior work, we uncovered a role for the glycolysis hexosamine biosynthetic pathway (HBP) in DNA repair, ionizing radiation induced foci (IRIF) resolution, and prevention of radiation induced cancer cell senescence. Here we report that the targeting of key enzymes in the HBP pathway, O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) or O-GlcNAcase (OGA/ MGEA5), have contrary effects on the DNA damage response in tumors. shRNA knockdown of OGA resulted in increased GlcNAc protein modification, protection of cancer cells from irradiation through improved double strand break (DSB) repair and IRIF resolution in tumor cells, increased proliferation, and prevented radiation-induced senescence. In turn, silencing of OGT diminished O-GlcNAc modification, prevented DSB repair, increased IRIF persistence, reduced proliferation, and promoted radiation-induced senescence in tumor xenografts. These findings reveal an important relationship between cancer cell metabolism, DSB repair, and senescence and provide a rationale for

targeting metabolic reprogramming, specifically O-GlcNAc modification, to block cancer cell immortality and overcome resistance to genotoxic stress.

Citation Format: Elena V. Efimova, Oliver K. Appelbe, Steve Seung-Young Lee, Natalia Ricco, Aishwarya Ramamarthy, Nicolas Rymut, Tamica Collins, Donald J. Wolfgeher, Sara Warrington, Stephen J. Kron. Tumor radiosensitization by suppression of hexosamine biosynthetic pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3505.
Abstract 3509: The glutaminase inhibitor CB-839 synergizes with CDK4/6 and PARP inhibitors in pre-clinical tumor models
Authors: Emberley Ethan D. , Bennett Mark , Chen Jason , Gross Matthew , Huang Tony , Makkouk Amani , Marguier Gisele , Pan Alison , Spurlock Sandra M. , Steggerda Susanne , Parlati Francesco
Published: August 2018

Many tumor cells utilize the amino acid glutamine to meet the elevated bioenergetic and biosynthetic demands of rapid cell growth. The enzyme glutaminase converts glutamine to glutamate, which is used to fuel the TCA cycle, synthesize amino acids and nucleotides, and balance cellular oxidative stress. We developed CB-839, a potent and orally bioavailable small molecule inhibitor of glutaminase, that blocks production of glutamate and generation of downstream metabolites glutathione, malate and aspartate. Mice treated with CB-839 had decreased levels of nucleotides in their tumors, likely due to glutamine-derived aspartate being required for nucleotide biosynthesis. Consistent with our finding that CB-839 decreases nucleotide pools, CB-839 treatment delayed cancer cells from either entry into S-phase or progression through S-phase. Based on this observation, the ability of CB-839 to synergize with therapies that block cell cycle progression was tested. CB-839 synergized with the CDK4/6 inhibitor palbociclib in colorectal carcinoma (CRC), triple negative breast cancer (TNBC) and ER+ breast cancer cell lines resulting in anti-proliferative activity. The combination of CB-839 with palbociclib also led to decreased cell cycle progression through S-phase and caused an accumulation of cells in G1 as measured by an EdU DNA incorporation assay. In vivo, the combination of CB-839 with palbociclib resulted in enhanced anti-tumor activity in both an ER+ breast cancer and CRC xenograft tumor model. We next investigated whether CB-839 treatment would enhance the anti-tumor effects of DNA repair inhibitors, given the ability of CB-839 treatment to decrease nucleotide pools. CB-839 treatment in combination with the PARP inhibitors niraparib and talazoparib led to synergistic anti-proliferative activity in TNBC, CRC, non-small cell lung carcinoma, ovarian and prostate cancer cells. In vivo, the combination of CB-839 with PARP inhibitors showed enhanced anti-tumor activity compared to single agent treatment in a CRC tumor xenograft model. CB-839 is currently undergoing evaluation for efficacy in the treatment of cancer in several phase I/II clinical trials. These encouraging pre-clinical results support the testing of CB-839 with CDK4/6 or PARP inhibitors in cancer patients.

Citation Format: Ethan D. Emberley, Mark Bennett, Jason Chen, Matthew Gross, Tony Huang, Amani Makkouk, Gisele Marguier, Alison Pan, Sandra M. Spurlock, Susanne Steggerda, Francesco Parlati. The glutaminase inhibitor CB-839 synergizes with CDK4/6 and PARP inhibitors in pre-clinical tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3509.
Abstract 3511: The mechanism of antitumor effect of metformin forgemcitabine-resistant pancreatic adenocarcinoma
Authors: Suzuki Keiichi , Takeuchi Osamu , Suzuki Yukio
Published: August 2018

Metformin (MET) is the first-line treatment for type 2 diabetes mellitus. Several epidemiological studies have reported anti-cancer effects of MET, including against pancreatic ductal adenocarcinoma (PDAC), which mainly acts through induction of AMP activated protein kinase (AMPK). Gemcitabine (GEM) has become the standard chemotherapy for PDAC but tolerance to GEM has become a burdensome issue. We evaluated the anti-tumor effects of MET for GEM-resistant PDAC in a xenograft mouse model. For this in vivo study, BxG30 (the cell line for GEM-resistant PDCA) was implanted into both flanks of female BALB/c nude mice. Mice were divided into four groups: (i) control (no treatment); (ii) the GEM-treated group (100 mg/kg); (iii) the MET-treated group (600 mg/kg); and (iv) the combined treatment group (G+M). Mice were fed for 4 weeks. Estimated tumor volumes and body weights were measured each week. Treatments were initiated 2 weeks after implantation. MET was administrated orally once per day. GEM was given by intraperitoneal injection once per week. Compared with the control, the final tumor volumes were decreased significantly only in the G+M group. [SCW1] The treated control ratio (T/C%) was calculated: GEM, 80.2%; MET, 54.0%; G+M, 47.2%. The anti-tumor effect of GEM for BxG30 was clearly limited[SCW2] . MET group showed satisfactory anti-tumor effects, but T/C% was <50% only in the G+M group. This result revealed that combination therapy had excellent anti-tumor effects even for GEM-resistant PDAC. The phosphorylation of ribosomal proteins S6 and 4E-BP, important targets of the mammalian target of rapamycin (mTOR) signaling pathway, was surveyed by western blot analysis. Western blot analysis showed inhibition of S6 and 4E-BP phosphorylation by co-incubation with MET, but not with GEM. Hypoxia-inducible factor 1 (HIF-1) is the one of the most important target of mTOR signaling pathway influencing tumor cell to progress, thus the expression level of HIF-1 was evaluated by western blot analysis as well. The results showed significant inhibition of HIF-1 expression by MET treatment, but not by GEM incubated under hypoxia (95%N2, 5%O2). Then the production of VEGF was evaluated by ELISA under hypoxia. The result showed suppression of VEGF production by MET treatment, but not by GEM. Our data showed that MET develops a different anti-tumor effect from GEM by suppression of mTOR-HIF-1 signaling. These results are of great clinical interest and reveal the potential of another anti-tumor agent for treatment of PDAC.

Citation Format: Keiichi Suzuki, Osamu Takeuchi, Yukio Suzuki. The mechanism of antitumor effect of metformin forgemcitabine-resistant pancreatic adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3511.
Abstract 3513: Curcumin induces a fatal energetic impairment by inhibiting ATP-synthase activity and decreasing ATP generation and oxygen consumption in in vitro and in vivo tumor models
Authors: Pfeffer Ulrich , Bianchi Giovanna , Ravera Silvia , Traverso Chiara , Amaro Adriana , Piaggio Francesca , Emionite Laura , Bacchetti Tiziana , Raffaghello Lizzia
Published: August 2018

Curcumin has been reported to inhibit inflammation, tumor growth, angiogenesis and metastasis by decreasing cell growth and by inducing apoptosis mainly through the inhibition of nuclear factor kappa-B (NFkB), a master regulator of inflammation. Recent reports also indicate potential metabolic effects of the polyphenol and we therefore analyzed whether and how it affects the energy metabolism of tumor cells. We show that curcumin inhibits the activity of ATP-synthase in isolated mitochondrial membranes leading to a dramatic drop of ATP and a reduction of oxygen consumption in in vitro in several murine tumor cell lines (CT26 colon cancer, B16 melanoma, L1210 lymphocytic leukemia, 4T1 breast cancer) and in vivo in syngeneic tumor models. The effects of curcumin on ATP-synthase are independent of the inhibition of nuclear factor kB (NFkB) since the IkB Kinase inhibitor, SC-514, inhibits the expression of the NFkB target gene, BCL2, but does not affect the activity of the ATP-synthase. The activities of the glucose metabolism enzymes hexokinase, phosphofructokinase, pyruvate kinase and lactate dehydrogenase are only slightly affected in a cell type specific manner. The energy impairment translates into decreased tumor cell viability. Apoptosis is induced by promoting the generation of reactive oxygen species and malondialdehyde (MDA), a marker of lipid oxidation. Tumor autophagy is induced by curcumin at least in part due to the activation of the AMP-activated protein kinase (AMPK). These activities translate into a significant delay of in vivo tumor growth likely due to a reduction of tumor angiogenesis since we observe reduced number and size of tumor vessels in vivo. These results establish the ATP-synthase, a central enzyme of the cellular energy metabolism, as a target of the anti-tumoral polyphenol leading to inhibition of cancer cell growth and a general reprogramming of tumor metabolism.

Citation Format: Ulrich Pfeffer, Giovanna Bianchi, Silvia Ravera, Chiara Traverso, Adriana Amaro, Francesca Piaggio, Laura Emionite, Tiziana Bacchetti, Lizzia Raffaghello. Curcumin induces a fatal energetic impairment by inhibiting ATP-synthase activity and decreasing ATP generation and oxygen consumption in in vitro and in vivo tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3513.
Abstract 3534: Protein acylation mediates encapsulation of Src kinase into exosomes
Authors: Wen Ru , Li Qianjin , Kim Sungjin , Ma Yongjie , Xie Jin , Cai Houjian
Published: August 2018

Exosomes are nanosized vesicles (30-150 nm) secreted from almost all cell types (1). Exosomes play important roles in cell-cell communication through the transfer of exosomal proteins, mRNA, and microRNAs, mediating immune escape and promoting tumor progression (2,3). However, it remains unclear how oncogenic proteins are selectively disseminated through exosomes. Myristoylation is lipid modification of protein involved in the attachment of N-terminal glycine of proteins with a myristoyl group. It is critical for various biologic functions including cell signaling, protein localization, and cell-cell communication (4). In this study, we evaluated the role of protein acylation for the encapsulation of Src family kinases in exosomes. Myristoylation promotes the enrichment of Src kinase in exosomes whereas palmitoylation inhibits the encapsulation process. Additionally, phosphorylated Src protein was more favored to be capsulated in exosomes. Mechanistically, knockdown of TSG101 decreased expression levels of Src kinase in exosomes, indicating that Src encapsulation into exosomes is associated with endosomal sorting complexes required for transport (ESCRT). Finally, Src kinase can be translocated to normal cells via exosomes cargo to induce pathologic signaling. These results provide new insights of Src kinase encapsulation mechanism into exosomes, and therapeutic approaches for inhibiting the transfer of oncogenic proteins through targeting protein myristoylation process.

References:

1. Simons M, Raposo G. Exosomes–vesicular carriers for intercellular communication. Curr Opin Cell Biol 2009;21(4):575-81.

2. Skog J et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nature Cell Biol 2008;10(12):1470-6.

3. Abusamra AJ et al. Tumor exosomes expressing Fas ligand mediate CD8+ T-cell apoptosis. Blood Cells Mol Dis 2005;35(2):169-73.

4. Casey PJ. Protein lipidation in cell signaling. Science 1995;268(5208):221.

Citation Format: Ru Wen, Qianjin Li, Sungjin Kim, Yongjie Ma, Jin Xie, Houjian Cai. Protein acylation mediates encapsulation of Src kinase into exosomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3534.
Abstract 3537: FBXW7-mediated extracellular signal-regulated kinase 3 stability in cancer development
Authors: Kim Seung-Min , Lee Cheol-Jung , Yoo Sun-Mi , Cho Seon-Yeon , Park Juhee , Cho Yong-Yeon
Published: August 2018

Extracellular signal-regulated kinase (ERK) is an atypical member of the mitogen-activated protein kinase (MAPK) family. Initial studies have demonstrated that ERK3 was highly expressed in the central nervous system, skeletal muscle, lung, thymus, and testis. Interestingly, unlikely with classical MAPKs including ERK1/2, JNKs and p38s, ERK3 is known to have narrow substrate specificity. Although the function of ERK3 has studied for last three decades, some information for the role of ERK3 is currently available. Recent studies indicate that ERK3 is upregulated in diverse cancer tissues including lung cancer and colon cancer and ERK2 promotes cancer cell migration and invasion in triple-negative breast cancer. Interestingly, ERK3 showed short protein half-life about 30-60 min and ubiquitin-specific protease 20, a deubiquitinase, induced the ERK3 protein level. Thus, identification of E3 ubiquitin ligase is important to understand ERK3 function in cell transformation and cancer development. In this study, we identified that FBXW7, a member of F-box protein complex, bind with ERK3. Moreover, we found that ectopic expression of FBXW7 decreased ERK3 protein level in a dose dependent manner. By proteome analysis, we found that ERK3 contained FBXW7 specific degron motif in C-terminal variable region. Importantly, FBXW7 depletion in HCT-116 restored ERK3 protein level. Taken together, we concluded that FBXW7 is a specific binding partner of ERK3 and may regulate ERK3 protein stability.

Key words: MAPK, ERK3, FBXW7, ubiquitination, protein stability

Citation Format: Seung-Min Kim, Cheol-Jung Lee, Sun-Mi Yoo, Seon-Yeon Cho, Juhee Park, Yong-Yeon Cho. FBXW7-mediated extracellular signal-regulated kinase 3 stability in cancer development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3537.
Abstract 3568: Phosphatidylserine targeting antibody enhances antitumor activity of CAR T cells in mouse melanoma
Authors: Schad Sara , Hirschhorn-Cymerman Daniel , Budhu Sadna , Zhong Hong , Yang Xia , Shan Joseph , King Steven , Merghoub Taha , Wolchok Jedd
Published: August 2018

A viable strategy to treat advanced cancers includes transferring of tumor-specific T cells. T cells that recognize tumor antigens can be expanded and reinvigorated ex-vivo. Furthermore, autologous T cells can be genetically modified to express antitumor T cell receptors or chimeric antigen receptors (CARs). Although the potency and specificity of tumor-specific T cells can be manipulated ex vivo, once reinfused into patients, the T cells are subjected to immunosuppressive mechanisms established by the tumor. An important immune checkpoint regulator within tumors is phosphatidylserine (PS), a phospholipid that is exposed on apoptotic cells, tumor cells and tumor endothelium. Innate immune cells exposed to PS secrete suppressive cytokines and chemokines that can significantly impair the function and activation of antitumor T cells. Antibodies that target PS have been shown to reactivate antitumor immunity by polarizing tumor-associated macrophages into a proinflammatory M1 phenotype, reducing the number of MDSCs in tumors and promoting the maturation of dendritic cells into functional APCs. Our lab has previously shown that a PS targeting monoclonal antibody (mch1N11), in combination with transgenic CD4+ T cells that recognize the melanoma antigen Trp1, can regress very advanced melanomas in all treated mice. Here, we further those studies with data showing that a 2nd-generation CAR T cell that binds Trp1 on the surface of B16 melanoma, in combination with mch1N11 can improve antitumor activity and overall survival in B16 tumor-bearing mice. Additionally, in vitro killing assays with antigen-specific T cells sorted from the tumor reveal that mch1N11 enhances the cytolytic function of these T cells against B16 melanoma. Flow cytometry analysis of local immune responses in the tumors of animals treated with tumor-specific T cells and mch1N11 showed a decrease in anti-inflammatory (M2) macrophages and FoxP3+ regulatory T cells. These findings highlight that diminishing suppressive mechanisms locally with mch1N11 can enhance the efficacy of transgenic TCR and CAR T cells to improve the outcome in patients with advanced-stage melanoma. Our studies may inform the design of clinical trials combining PS targeting antibodies with CAR T cell therapy in solid tumors.

Citation Format: Sara Schad, Daniel Hirschhorn-Cymerman, Sadna Budhu, Hong Zhong, Xia Yang, Joseph Shan, Steven King, Taha Merghoub, Jedd Wolchok. Phosphatidylserine targeting antibody enhances antitumor activity of CAR T cells in mouse melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3568.
Abstract 3570: Adoptive transfer of tumor antigen-specific CTLs requires anti-CTLA-4 and anti-PD-1 to drive tumor eradication
Authors: Shi Lewis Zhichang , Gao Jianjun , Vence Luis , Brando Jorge , Allison James , Sharma Padmanee
Published: August 2018

Adoptive transfer of tumor-reactive T cells (ACT) has led to significant clinical responses in hematologic malignancies. However, its efficacy in solid tumors has been dismal, likely due to the immunosuppression imposed by the tumor microenvironment (TME) of solid tumors. A proven effective strategy to revert the immunosuppressive TME is immune checkpoint blockades (ICBs), including anti-CTLA-4 and anti-PD-1/PD-L1, which have been recently approved by FDA for cancer therapy. Here, using the orthotopic B16-F10 melanoma model, we combine ACT of PMEL-1 Tg+ CTLs that recognize the endogenous melanoma antigen gp100 with dual blockade of CTLA-4 and PD-1, the highly pursued effective combinatorial ICB, to treat established tumors. Although ACT alone transiently suppresses tumor growth, complete tumor regression requires additional dual anti-CTLA-4 and anti-PD-1 blockade. Mechanistically, we demonstrate that the ICOS-Eomes-IFN-γαμμα axis, but not the ICOS-T-bet-IFN-γαμμα axis, in CTLs mediates the therapeutic effects of ACT and ICBs. Our data provide direct evidence that combining ACT of CTLs with ICBs confers long-term protection to tumor challenges, which largely depends on CTL-intrinsic expression of ICOS.

Citation Format: Lewis Zhichang Shi, Jianjun Gao, Luis Vence, Jorge Brando, James Allison, Padmanee Sharma. Adoptive transfer of tumor antigen-specific CTLs requires anti-CTLA-4 and anti-PD-1 to drive tumor eradication [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3570.
Abstract 3677: Scalable assembly of individual patient profiles for clinical trials accrual and research
Authors: Duren Ryan , Smith Ryan , Tackes Nick , Neeley Shane , Welsh James , Li Xuan Shirley
Published: August 2018

As clinical data are digitized in electronic medical records (EMR), the amount of historical data becomes a challenge for utilization and outcomes studies. An automated approach for structuring clinical data into machine-readable format is essential due to scale. We previously developed MMPower, an ElasticSearch-based technology platform for named entity extraction defined by SNOMED conditions, >2,000 Entrez Genes, >900,000 cancer alterations, and ~7,000 FDA-approved and experimental therapeutics/progression that enables clinical trials search. In a pilot study, MMPower was applied to characterize and index oncology EMR for elucidating clinical histories and identifying clinical trial participants. 52,509 individual EMR were collated by Medical Record Number (MRN) across facets including Demographics, Providers/Sites, Diagnosis, Medication, Imaging, Lab results, Pathology, Performance status, and Encounter with Physician Notes. Applying MMPower yielded a total cohort of 43,987 (83.8% of EMR received) EMR with searchable, structured metadata profiles. We found that 39,317 (89.4%) of the total cohort had a cancer diagnosis as defined by SNOMED cancer types. We also extracted an Entrez gene from 36,152 (82.2%) EMR, while cancer alterations or stratification biomarkers such as ER/PR status, ALK fusions, IGH-BCL1, EGFR exon 19 deletion, CDKN2A loss, etc., were identified in 25,208 (57.3%) EMR. Furthermore, we found that the majority of EMR (69.7%) with identifiable genes also contained a cancer alteration. Interestingly, although most EMR (39,615 or 90% of total cohort) held identifiable cancer therapeutics, only 1.7% of the total cohort were flagged as cases of progression. We then determined the feasibility of matching EMR to clinical trials and found that, strikingly, 16.7% of the total cohort were potentially eligible for institution-specific clinical trials. Finally, we sought to enrich individual EMR with molecular alterations data from external lab reports, so we developed a PDF to JSON transform that enables mapping of report results by MRN to our cancer alterations model, yielding a single searchable record from multiple data sources. These results suggest that the ability to structure machine-readable clinical and molecular data for individuals from EMR at scale would accelerate translational research by promoting more efficient clinical trials accrual and could be extended to prevalence calculations, evidence-backed treatment guidance through molecular assertions, biomarkers discovery, and outcomes studies of therapeutic efficacy.

Citation Format: Ryan Duren, Ryan Smith, Nick Tackes, Shane Neeley, James Welsh, Xuan Shirley Li. Scalable assembly of individual patient profiles for clinical trials accrual and research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3677.
Abstract 3684: HDAC inhibitors restore BRAF inhibitor sensitivity by altering PI3K and survival signaling in melanoma
Authors: Hersey Peter , Gallagher Stuart J.
Published: August 2018

Mutations in BRAF activate oncogenic MAPK signaling in almost half of cutaneous melanomas. Inhibitors of BRAF (BRAFi) and its target MEK are widely used to treat melanoma patients with BRAF mutations but unfortunately acquired resistance occurs in the majority of patients. Resistance results from mutations or non-genomic changes that either reactivate MAPK signalling or activate other pathways that provide alternate survival and growth signaling.Here we show the histone deacetylase inhibitor (HDACi) panobinostat overcomes BRAFi resistance in melanoma, but this is dependent on the resistant cells showing a partial response to BRAFi treatment. Using patient- and in vivo- derived melanoma cell lines with acquired BRAFi resistance, we show that combined treatment with the BRAFi encorafenib and HDACi panobinostat synergistically induced caspase-dependent apoptotic cell death. HDAC inhibition decreased PI3K pathway activity associated with a reduction in the protein level of a number of receptor tyrosine kinases including AXL, YAP, ERBB2 and ERBB3 but not EGFR or ERBB4. Both NOXA and BIM proteins were implicated as causing cell death, but in a cell line specific manner. Independent of these changes, panobinostat reduced c-Myc and pre-treatment of cells with siRNA against c-Myc reduced BRAFi/HDACi drug-induced cell death. These results suggest that a combination of HDAC and MAPK inhibitors may play a role in treatment of melanoma where the resistance is due to activation of MAPK-independent pathways. This work identifies patients that may respond well to this drug combination and demonstrates the necessity of disrupting multiple cell survival pathways in melanoma.

Citation Format: Peter Hersey, Stuart J. Gallagher. HDAC inhibitors restore BRAF inhibitor sensitivity by altering PI3K and survival signaling in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3684.
Abstract 3707: PD-L1 antibody drug conjugate for cancer immune-chemo combination therapy
Authors: Sau Samaresh , Petrovici Alex , Alsaab Hashem , Iyer Arun K.
Published: August 2018

Antibody drug conjugates (ADCs) are a new form of targeted therapy to cancer, consisting of an antibody, a conditionally stable linker, and a cytotoxic drug. Immune checkpoint molecule, such as programmed death ligand-1 (PD-L1) overexpressed in various solid tumors including triple negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC) and it has a crucial role in tumor immune evasion. Blocking the interaction between PD-L1 and PD-1 (programmed death-1 receptor) resurrects tumor immune surveillance, resulting CD8+ cytotoxic T cells activation to attack the tumor cells. This is an emerging anticancer mechanism and more than four antibody inhibitors have been recently FDA approved against PD-1 or PD-L1 biomarker. Targeting PD-L1 in TNBC and NSCLC is an excellent approach for tumor immunotherapy. However, the major challenges of anti-PD-L1 therapy (Atezolizumab) are (i) the transient expression of PD-L1 in tumor microenvironment, (ii) development of anti-PD-L1 resistance, (iii) difficulty of tumor stroma penetration that retard its clinical outcome. To overcome these challenges, we chemically conjugated Atezolizumab (ATZ) with potent chemotherapeutic payload, such as Doxorubicin (Dox) through polyethylene glycol (PEG) linkers. The role of Dox is to break the tumor stroma so that PD-L1 antibody can penetrate to the core of the tumor. Introduction of PEG-linker with high molecular weight ATZ (155 kD) has drastically improved the solubility of ADC and successful chemical conjugation of between ATZ and PEG-Dox was evaluated by MALDI-TOF analysis. Our data has revealed significantly tumor cell killing effect and induction of apoptosis in PD-L1-Dox treated MDA-MB-231 cells. The near infrared imaging of PD-L1 dye conjugate in TNBC and NSCLC patient derived xenograft (PDx) supports the selective tumor targetability. Based on the synergistic anticancer effect and selective tumor targetability demonstrated in this study build a strong rational of using PD-L1 ADC approach for cancer immune-chemo therapy and diagnosis.

Citation Format: Samaresh Sau, Alex Petrovici, Hashem Alsaab, Arun K. Iyer. PD-L1 antibody drug conjugate for cancer immune-chemo combination therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3707.
Abstract 3730: Structure determination, mode of action and safe delivery of an improved K6L9-type peptide
Authors: Shrestha Tej B. , Yu Jing , Bossmann Stefan , Troyer Deryl
Published: August 2018

There is a significantly increased interest in using antimicrobial peptides for anticancer therapy. D-K6L9, LKLLKKLLKKLLKLL is an antimicrobial peptide that has been explored. It kills cancer cells by depolarizing their cell membranes, thus inducing apoptosis. Further modification of the peptide for increased toxicity and safe packaging into a delivery vehicle for targeted delivery is highly desired for the in vivo applications. Here, the peptide was modified with two extra amino acids (serine and alanine) at both C and N terminals, resulting in SA-LKLLKKLLKKLLKLL-AS. The sequence and structure of the modified peptide were determined by means of 2D 1H-1H -COSY, NOESY, and TOCSY-NMR spectroscopy. The 3D structure of the peptide in the solution phase was generated by CNS software using a distance constraint generated by NOE spectroscopy. The 3-D structure of SA-D-K6L9-AS is maintained, as well as the amphiphilicity required for insertion into cell membranes. This peptide was tested on the following mouse cancer cell lines: GL 26 (glioma), B16-F10 (melanoma lung metastases) and 4T1 (metastasizing breast cancer). The LC50 values of the modified peptide were found to be 0.707 µM for B16-F10, 6.237 µM for 4T1 and 1.707 µM for GL 26, which is 5- 10 fold more active than the original D-K6L9. To explore the findings using the modified peptide, SA-D-K6L9-AS tagged with a rhodamine dye was incubated with GL 26 cancer cells. Sequential confocal imaging (every 30 seconds) revealed that the peptide gets internalized into the cytoplasm in less than 5 min and then localizes in the mitochondria. This peptide is found to be toxic to neuronal stem cells and monocytes as well. To avoid the non-specific toxicity of the peptide for in vivo applications, highly mesoporous silica nanoparticles (MSN) were synthesized. The peptide was loaded into the MSN. MSN were further coated with a polysilazane as a “gift wrap” after loading the peptide. This gift wrap can hold the contents inside the MSN, showing no toxicity at 24 hours and subsequent slow release of the peptide payload into the cytoplasm within 72 hours. This gift wrapping of the highly toxic peptide into the MSN could be very useful for in vivo cancer therapy by means of targeted delivery to the cancer site with appropriate surface modification of MSN.

Citation Format: Tej B. Shrestha, Jing Yu, Stefan Bossmann, Deryl Troyer. Structure determination, mode of action and safe delivery of an improved K6L9-type peptide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3730.
Abstract 3757: Targeting the IDO/TDO pathway through degradation of the immunosuppressive metabolite kynurenine
Authors: Coma Silvia , Cavanaugh Jill , Nolan James , Tchaicha Jeremy , McGovern Karen , Stone Everett , Lamb Candice , Karamitros Christos , Blazek John , Garrison Kendra , Georgiou George , Manfredi Mark , Zhang Xiaoyan Michelle
Published: August 2018

The tryptophan/kynurenine pathway has been clinically validated in several tumor types with small-molecule IDO1 inhibitors in combination with checkpoint inhibition. Indoleamine-pyrrole 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase 2 (TDO2) are upregulated in a number of tumor types, metabolizing tryptophan to form immunosuppressive kynurenine. We are developing Kynureninase (Kynase), a kynurenine depleting enzyme, to treat IDO1 and TDO2 positive tumors. The human Kynase has been successfully engineered to vastly increase catalytic activity and stability toward kynurenine over the wild type enzyme. In mice, Kynase achieved prolonged Kynurenine degradation (≥5 days) in plasma and tumor draining lymph node (TDLN), leading to anti-tumor activity as a single agent and in combination with check point inhibitors in mouse syngeneic tumor models. Kynase demonstrated superior tumor growth inhibition and survival benefit relative to a leading IDO1 inhibitor epacadostat in these models. The effects of Kynase on a number of immune cell types, both in vitro and in vivo, are being investigated. Human Kynase has also shown a favorable PK profile and kynurenine degradation in non-human primates, and Kynase variants are now moving toward development candidate selection for treatment of cancers where both IDO/TDO pathways play a significant immunosuppressive role.

Citation Format: Silvia Coma, Jill Cavanaugh, James Nolan, Jeremy Tchaicha, Karen McGovern, Everett Stone, Candice Lamb, Christos Karamitros, John Blazek, Kendra Garrison, George Georgiou, Mark Manfredi, Xiaoyan Michelle Zhang. Targeting the IDO/TDO pathway through degradation of the immunosuppressive metabolite kynurenine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3757.
Abstract 3825: INCAGN02390, a novel antagonist antibody that targets the co-inhibitory receptor TIM-3
Authors: Waight Jeremy , Iyer Priyadarshini , Breous-Nystrom Ekaterina , Riordan Christina , Findeis Mark , Underwood Dennis , Connolly Joseph , Sanicola-Nadel Michele , Nastri Horacio , Scherle Peggy , Hollis Gregory , Huber Reid , Stein Robert , Dijk Mark van , Wilson Nicholas S.
Published: August 2018

Unprecedented rates of durable clinical responses have been observed for antibody-based therapeutics targeting immune checkpoint proteins such as cytotoxic T lymphocyte antigen-4 (CTLA-4) or programmed death receptor-1 (PD-1). Nonetheless, a significant number of patients experience de novo resistance or relapse due to adaptive resistance mechanisms. T-cell immunoglobulin and mucin domain containing-3 (TIM-3) is an inhibitory receptor involved in immune tolerance often co-opted by tumors to prevent successful antitumor responses. Accordingly, TIM-3 is frequently expressed on myeloid and so-called exhausted T and NK cells within the tumor microenvironment. Targeting the TIM-3 pathway in preclinical models has provided additional rationale for pharmacologic modulation of this axis in cancer patients. INCAGN02390 is a novel and fully human Fc-engineered IgG1κ antibody developed to antagonize the TIM-3 pathway for the treatment of human malignancies. INCAGN02390 forms a high-affinity interaction with TIM-3, occluding access to the CC'-FG binding cleft and blocking phosphatidylserine binding. In addition, INCAGN02390 elicits rapid receptor internalization, potentially obviating interactions with other described or undescribed ligands. INCAGN02390 also enhances IFN-γ production from T cells undergoing tonic TCR stimulation when combined with PD-1 blockade. Finally, to demonstrate combinatorial potential, we show potent antitumor activity of an anti-mouse TIM-3 antibody in concert with other checkpoint antibodies in vivo. In summary, these data support assessment of INCAGN02390 in patients with advanced or metastatic solid tumors.

Citation Format: Jeremy Waight, Priyadarshini Iyer, Ekaterina Breous-Nystrom, Christina Riordan, Mark Findeis, Dennis Underwood, Joseph Connolly, Michele Sanicola-Nadel, Horacio Nastri, Peggy Scherle, Gregory Hollis, Reid Huber, Robert Stein, Mark van Dijk, Nicholas S. Wilson. INCAGN02390, a novel antagonist antibody that targets the co-inhibitory receptor TIM-3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3825.
Abstract 3834: Antibodies targeting Resokine, a soluble immune modulator, inhibit tumor growth in syngeneic mouse models
Authors: Ogilvie Kathy , Ng Cherie , Nangle Leslie , Ampudia Jeanette , Chang Joon , Cubitt Andrea , King David J. , Mendlein John
Published: August 2018

A number of non-canonical functions have been established for proteins generated from the tRNA synthetase gene family. One of these, termed Resokine, is derived from histidyl tRNA synthetase and plays an important role in controlling immune cell activation. Circulating levels are sufficient to down-regulate the extent of T cell activation that can be achieved in vitro. A panel of specific monoclonal antibodies have been generated and tested for their anti-tumor activity in mouse syngeneic tumor models. Antibodies to Resokine demonstrated anti-tumor activity across three different tumor models. Treatment of subcutaneous CT26 tumors resulted in improved efficacy compared to treatment with antibodies that block the PD-1/PD-L1 interaction. Significant efficacy was also observed in the difficult to treat subcutaneous B16F10 melanoma and 4T1 breast tumor models. In addition anti-Resokine demonstrated significant activity in a tumor seeding model using B16F10 melanoma which resulted in inhibition of tumor nodules in the lung, and was more efficacious than a combination of antibodies to PD-L1 and CTLA-4. Combinations of anti-Resokine antibody with either anti-PD-1 or anti-PD-L1 demonstrated at least additive, and potentially synergistic activity in these models. Animals with long-term tumor regressions were re-implanted with viable tumor cells, and demonstrated long-term immune memory with rejection of the newly implanted tumors. To understand the mechanism of anti-Resokine antibody therapy, cell depletion studies were carried out in the B16F10 tumor model. In these experiments activity of anti-Resokine antibodies was demonstrated to be dependent upon the presence of CD8 T cells and also NK cells, but independent of CD4 T cells. The immune based mechanism of antibodies to Resokine was further demonstrated by re-challenge of mice that had regressed tumors upon treatment. Tumor re-growth was not observed even in the absence of further treatment whereas control mice grew tumors at the normal rate, suggesting that immune memory had been induced. Antibodies to Resokine offer an exciting new potential option for immunotherapy of cancer, which has significant activity as monotherapy and is compatible with more established modalities. Anti-Resokine antibodies are currently being developed to initiate clinical evaluation.

Citation Format: Kathy Ogilvie, Cherie Ng, Leslie Nangle, Jeanette Ampudia, Joon Chang, Andrea Cubitt, David J. King, John Mendlein. Antibodies targeting Resokine, a soluble immune modulator, inhibit tumor growth in syngeneic mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3834.