bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5487: Targeting glycolysis for the treatment of cancer
Authors: Chesney Jason , Mojesky Aubrey , Imbert-Fernandez Yoannis , Trent John , Telang Sucheta
Published: August 2018

Tumors exhibit markedly increased glucose uptake and glycolysis and this metabolic phenotype serves to satisfy their increased requirement for energy and biosynthetic intermediates. Tumor glucose metabolism is stimulated in part by fructose-2,6-bisphosphate (F26BP), which is the product of a family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) and allosterically activates a key glycolytic enzyme, 6-phosphofructo-1-kinase (PFK-1). Previous studies have found the PFKFB3 family member to be a dominant source of F26BP in cancer cells due to a high kinase activity, expression in multiple tumor types and the inhibition of tumor growth by specific PFKFB3 inhibitors including a compound, PFK158, that is currently under evaluation in a phase I trial. Recent data, however, indicate that the PFKFB4 enzyme plays a significant role in regulating glucose metabolism in neoplastic cells. We have found that PFKFB4 is highly expressed in multiple cancers, strongly induced by HIF-1α, and that its suppression markedly reduced F26BP and glycolytic flux, increased apoptosis and inhibited growth of xenograft tumors in vivo, thus providing strong rationale for the development of antineoplastic agents selectively targeting PFKFB4. Using virtual screening, we have discovered a first-in-class small molecule inhibitor of PFKFB4, termed 5MPN, that selectively inhibits recombinant PFKFB4 activity with a resultant decrease in F26BP, glycolytic flux and cancer cell growth in vitro and markedly reduces the growth of established tumors without systemic toxicity. During our studies, we unexpectedly observed that PFKFB4 expression is increased by PFKFB3 inhibition, suggesting that PFKFB4 may compensate for decreased PFKFB3 expression and activity. We examined the effects of simultaneous silencing of PFKFB4 and PFKFB3 on cancer cell lines and found that co-knockdown led to a significant decrease in cell viability and glycolysis and to a near-complete abrogation of anchorage-independent growth in vitro. We additionally examined the effect of simultaneous inhibition of PFKFB4 and PFKFB3 using 5MPN and several PFKFB3 inhibitors in vitro and found a synergistic increase in cell death. Taken together, our data indicate that targeting PFKFB4 may be a viable therapeutic option against cancer and strongly support the further development of PFKFB4 small-molecule inhibitors as well as the co-targeting of PFKFB4 and PFKFB3 as potential strategies for the treatment of cancer.

Citation Format: Jason Chesney, Aubrey Mojesky, Yoannis Imbert-Fernandez, John Trent, Sucheta Telang. Targeting glycolysis for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5487.
Abstract 5493: Targeted inhibition of glutaminase in GRM1-expressing melanoma cells inhibits cell proliferation by reducing glutamate bioavailability
Authors: Shah Raj , Zhu Mengying , Boreland Andrew , Filipp Fabian , Chen Suzie
Published: August 2018

Aberrant glutamatergic signaling has been implicated in many cancer types. Our laboratory has previously illustrated the role of metabotropic glutamate receptor 1 (GRM1) in neoplastic transformation of melanocytes in vitro and spontaneous metastatic melanoma in vivo. Glutamate, the natural ligand of GRM1 is also the predominant excitatory neurotransmitter in the central nervous system. We have demonstrated significant upregulation of glutaminase (GLS) expression in GRM1 expressing melanoma cells, resulting in excess glutamate production and the establishment of autocrine loops in vitro. GLS catalyzes the first step in glutamine metabolism–the conversion of glutamine to glutamate. Comparison of glutamate levels in circulating blood plasma between melanoma prone and wild type mice showed elevated glutamate levels in melanoma prone GRM1-transgenic mice, suggesting that aberrant GRM1 expression also promotes an increase in circulating glutamate, to ensure constitutive activation of the GRM1 receptor and its downstream signaling affecters in vivo. Inclusion of CB-839, a potent, selective, and orally bioavailable inhibitor of GLS, resulted in significant inhibition of GRM1+ melanoma cell proliferation compared to GRM1- ones. We also demonstrated that treatment with Riluzole, an inhibitor of glutamatergic signaling by decreasing the available ligand, glutamate, led to significantly reduced melanoma cell proliferation in vitro and tumor progression in vivo. We found that simultaneous targeting of glutamate production (CB-839) and release (Riluzole) in GRM1+ melanoma cells led to synergistic suppression of cell proliferation in vitro. Furthermore, we found that CB-839 altered the function not the expression of GLS in melanoma cells. Additionally, we also discovered that manipulation of GRM1 expression by genetic means led to robust parallel changes in gene expression levels and intracellular glutamate concentrations by mass spectrometry analysis, as well as the endogenous levels of GLS in melanoma cells, suggesting association between GRM1 and GLS. The sensitivity of GRM1+ melanoma cells to modulation of GLS point to the dependency of these cells to glutamate amounts via GLS activity. We are continuing to investigate the mechanisms and regulation of GLS in our experimental system, with the goal of developing a rational approach for the treatment of GRM1 expressing tumors.

Citation Format: Raj Shah, Mengying Zhu, Andrew Boreland, Fabian Filipp, Suzie Chen. Targeted inhibition of glutaminase in GRM1-expressing melanoma cells inhibits cell proliferation by reducing glutamate bioavailability [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 5493.
Abstract 5501: The role of G-protein coupled receptor 64 (GPR64) as a tumor suppressor in endometrial cancer
Authors: Yoo Jung-Yoon , Ahn Jong Il , Kim Tae Hoon , Broaddus Russell R. , Lim Jeong Mook , Yoon Ho-Geun , Jeong Jae-Wook
Published: August 2018

Endometrial cancer is the most common gynecologic cancer. G-protein coupled receptor 64 (GPR64) belongs to a family of adhesion GPCRs and plays an important role for male fertility. However, GPR64 function has not been reported in endometrial cancer. Our objective is to investigate the role of GPR64 in endometrial cancer. We examined the levels of GPR64 in human endometrial cancer tissue by immunohistochemistry analysis. GPR64 levels were significantly lower in 10 of 21 (47.62%) of endometrial carcinoma samples compared to control. To test whether GPR64 has a role of tumor suppressor in endometrial cancer, we used an siRNA loss-of-function approach in human endometrial adenocarcinoma cell lines. Depletion of GPR64 by siRNA transfection in Ishikawa and HEC1A cells showed an increase of colony formation ability and cell proliferation. Depletion of GPR64 also significantly increased the migration and invasion activity of Ishikawa and HEC1A cells. Furthermore, the phosphorylation of AMP-activated protein kinase (AMPK) and expression of Connexin 43 (Cx43), a member of the large family of gap junction proteins, were regulated in GPR64-deficient Ishikawa cells. These results suggest that GPR64 plays an important role for cell proliferation, migration, and invasion as a tumor suppressor in endometrial cancer.

Citation Format: Jung-Yoon Yoo, Jong Il Ahn, Tae Hoon Kim, Russell R. Broaddus, Jeong Mook Lim, Ho-Geun Yoon, Jae-Wook Jeong. The role of G-protein coupled receptor 64 (GPR64) as a tumor suppressor in endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5501.
Abstract 5504: TRAF3 acts as a tumor suppressor through modulation of TP53 and RB in human papillomavirus associated head and neck squamous cell carcinomas
Authors: Chen Tony Wayne , Zhang Jialing , Yang Xinping , Chen Zhong , Waes Carter Van
Published: August 2018

The number of human papillomavirus-associated (HPV+) head and neck squamous cell carcinoma (HNSCC) cases has increased sharply in recent years, especially among tonsillar and oropharyngeal cancers. The Cancer Genome Atlas (TCGA) network identified novel loss-of-function genomic alterations of TNF receptor-associated factor 3 (TRAF3) in a subset of HPV+ HNSCC specimens. Previously, most studies of TRAF3 function are from the immune system and lymphoid malignancies, where TRAF3 has been implicated as a ring-finger E3 ubiquitin ligase that promotes anti-viral immunity, while inhibiting alternative NF-κB signaling by which DNA viruses can transform lymphoid cells. However, the function of TRAF3 and the mechanisms by which TRAF3 deficiency contributes to tumorigenesis are largely unknown. To explore the potential mechanisms of TRAF3 defects observed in the tumor subset identified by TCGA, we surveyed a panel of HPV+ HNSCC cell lines, and identified lines with lower steady-state protein levels of TRAF3 than normal oral keratinocytes. Functional studies show that TRAF3 expression leads to suppression of oncogenic phenotypes, including decreased alternative NF-κB signaling, cell proliferation, colony formation, and cell migration. Additionally, restoring TRAF3 increased anti-viral cytokine interferon alpha (IFNA) expression, and sensitivity to chemotherapy agent cisplatin. The anti-proliferative activity of TRAF3 was partially mediated by restoration of expression of classical tumor suppressor genes, TP53 and RB, which are degraded in HPV+ cancers. TRAF3 re-expression increased the steady-state protein levels of TP53 and gene expression of TP53AIP, which mediates cell cycle arrest and apoptosis. Knockdown of TP53 in TRAF3 transfected cell lines partially reversed the slowed cell growth rate, supporting the contribution of TP53 to TRAF3 mediated growth inhibition. TRAF3 expression also increased RB protein expression and decreased RB regulated transcription factor E2F1, which regulates cellular genes involved in cell proliferation and DNA synthesis. In conclusion, we have revealed a novel role whereby TRAF3 loss leads to enhanced HPV mediated repression of the TP53 and RB tumor suppressors that control cell growth and cell death in HPV+ HNSCCs.

(Supported by NIDCD intramural project ZIA-DC-000016, 73 and 74)

Citation Format: Tony Wayne Chen, Jialing Zhang, Xinping Yang, Zhong Chen, Carter Van Waes. TRAF3 acts as a tumor suppressor through modulation of TP53 and RB in human papillomavirus associated head and neck squamous cell carcinomas [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 5504.
Abstract 5505: GAN gene exon 8 SNP is related to gigaxonin expression and increased expression of e-cadherin in head and neck cancer
Authors: Ko Albert , Venkatesan Natarajan , Chatoff Jenna , Morselli Marco , Fishbein Gregory , Bomont Pascale , Pellegrini Matteo , Wang Marilene , Srivatsan Eri
Published: August 2018

Gigaxonin is the protein product of Giant Axonal Neuropathy (GAN) gene and is involved in the processing of intermediate filaments of neural cells and vimentin fibers in fibroblasts. We have shown earlier that gigaxonin is associated with p16 in NFkB ubiquitination of cisplatin sensitive tumor cells. Our studies have shown that there is a direct relationship between the presence of exon 8 SNP (C>T) and expression of giagxonin in cancer cell lines. We have also seen an inverse relationship between exon 8 SNP and tumor recurrence and metastasis. RNA-seq studies of cancer cell lines and primary tumors have shown a direct relationship in the expression of gigaxonin to the expression of e-cadherin and inverse relationship to the expression of Vimentin and Zeb1. Immunohistochemical (IHC) studies of primary head and neck cancers have confirmed the inverse relationship between e-cadherin and vimentin. We hypothesize therefore that gigaxonin is involved in the ubiquitination of vimentin and cancer stem cell markers such as zeb1 resulting in the chemo sensitization of human tumor cells.

Citation Format: Albert Ko, Natarajan Venkatesan, Jenna Chatoff, Marco Morselli, Gregory Fishbein, Pascale Bomont, Matteo Pellegrini, Marilene Wang, Eri Srivatsan. GAN gene exon 8 SNP is related to gigaxonin expression and increased expression of e-cadherin in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5505.
Abstract 552: PEPD is an essential regulator of p53 tumor suppressor
Authors: Yang Lu , Li Yun , Bhattacharya Arup , Zhang Yuesheng
Published: August 2018

Peptidase D (PEPD), also known as prolidase among other names, is an enzyme that hydrolyzes dipeptides with proline or hydroxyproline at the carboxy terminus and is believed to be important for collagen metabolism, as proline and hydroxyproline are very abundant in collagen. Interestingly, we recently found that eliminating cellular PEPD causes cell death and tumor regression due to p53 activation. Here, we show that PEPD binds to and suppresses over half of nuclear and cytoplasmic p53 under normal conditions, independent of its enzymatic activity. PEPD binds to the proline-rich domain in p53, which inhibits phosphorylation of nuclear p53 and MDM2-mediated mitochondrial translocation of nuclear and cytoplasmic p53. Indeed, PEPD competes with MDM2 for p53 binding. However, the PEPD-p53 complex is critical for p53 response to stress, as stress signals doxorubicin (DOX) and hydrogen peroxide each must free p53 from PEPD in order to achieve robust p53 activation, which is mediated by reactive oxygen species. Thus, PEPD stores p53 for stress response, but this also renders cells dependent on PEPD for survival as it suppresses p53. Our results reveal a major p53 regulatory mechanism and a critical physiological function of PEPD. The p53-PEPD system likely operates in most if not all cells, since both p53 and PEPD are expressed ubiquitously. Disrupting PEPD suppression of p53 may be an important therapeutic strategy in cancer, as our data show that PEPD knockdown by RNA interference in tumors in mice causes p53 activation in the tumor

tissues and tumor regression. Our study also reveals a previously unrecognized anticancer mechanism of DOX. We show that the key step in DOX-induced p53 activation is the disruption of p53 association with PEPD via reactive oxygen species. This finding also raises the intriguing question of whether other stress-inducing anticancer agents also disrupt the PEPD-p53 complex for p53 activation and suggests that antioxidants may inhibit the anticancer activity of DOX and other agents by inhibiting p53 separation from PEPD. This work is supported by NCI grants and Roswell Park Alliance Foundation Grants.

Citation Format: Lu Yang, Yun Li, Arup Bhattacharya, Yuesheng Zhang. PEPD is an essential regulator of p53 tumor suppressor [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 552.
Abstract 5526: Mapping signaling networks in Nf1 mutant tumors
Authors: Pucciarelli Daniela , Krishnamurthi Ganesh , Braunstein Steve , Nakamura Jean L.
Published: August 2018

Introduction: The tumor suppressor gene Neurofibromatosis type 1 (NF1) encodes the Ras-GTPase activating protein neurofibromin, which negatively regulates Ras signaling. Loss of NF1 increases signaling through Raf/MEK/ERK and PI3K/AKT/mTOR. Inhibition of each of these pathways can produce variable responses, suggesting compensatory mechanisms and heterogeneity in signaling mechanisms in cancers. To study the biochemical sources of signaling we sought to functionally characterize Nf1-mutant tumors based on their variable drug sensitivities and development of drug resistance.

Methods: We previously established Nf1 null tumor cell lines from malignancies arising in irradiated Nf1+/- mice. We functionally profiled this panel of tumor cell lines using a custom 94 compound drug library targeting core signaling pathways commonly activated in cancers. Phosphorylated and total ERK, AKT, and S6 were assessed by western blotting. Cell lines resistant to treatment with MAPK and mTOR inhibitors were generated from parental cell lines. Parental and resistant cell lines were compared using MTS, exome and transcriptome sequencing, western blotting and kinome analysis.

Results: Nf1 mutant tumor cell lines demonstrated heterogeneous drug responses and, based on this observation, they could be organized into 3 functional sub-classes sharing similar drug sensitivities. All 12 Nf1 null cell lines demonstrated heterogeneous basal Ras pathway activation, as indicated by phosphorylated ERK, AKT, and S6. This variability was associated with variable sensitivity to treatment with MAPK and mTOR inhibitors. Acquired resistance to MAPK or mTOR inhibition (MEKi and mTORi) revealed differences in Ras activation that distinguished resistant from parental lines. For example, in contrast to parental lines, Ras-GTP levels in mTORi resistant cell lines demonstrated stability after serum stimulation compared to the parental counterpart, suggesting a reprogrammed role for Ras activation when mTORi resistance is acquired. These data highlight the plasticity of Ras-driven signaling in Nf1 tumors, which likely must be addressed to optimize therapy and counter the emergence of resistant clones.

Conclusion: Our analysis indicates significant variability in Ras pathway signaling in the context of Nf1 loss, which has implications for the choice of specific kinase inhibitors to use therapeutically. Cancers, including Nf1 null tumors, develop resistance to therapy and our data points to specific reprogramming of signaling pathways that distinguish resistance mechanisms.

Citation Format: Daniela Pucciarelli, Ganesh Krishnamurthi, Steve Braunstein, Jean L. Nakamura. Mapping signaling networks in Nf1 mutant tumors [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 5526.
Abstract 553: p53 target BRMS1A is a metastatic suppressor
Authors: Tokino Takashi , Koyama Ryota , Idogawa Masashi , Sasaki Yasushi
Published: August 2018

Tumor suppressor p53 plays an important role in the cell fate determination in response to diverse upstream signals. As a transcription factor, p53 regulates a number of genes that are involved in cell-cycle arrest, apoptosis, senescence, and maintenance of genomic stability. Recent studies revealed that p53 is critical for the regulation of cell invasion and migration. Microarray analysis showed that breast cancer metastasis-suppressor 1-like (BRMS1L) is upregulated by p53. We identified two responsive sequences of p53 in the first intron and upstream of the BRMS1L gene. These two response elements are well conserved among mammals. Functional analysis showed that ectopic expression of BRMS1L inhibited cancer cell invasion and migration. In contrast, knockdown of BRMS1L by siRNA induced the opposite effect. Notably, clinical databases revealed that reduced BRMS1L expression correlated with poor prognosis in patients with breast and brain cancer. Altogether, these results strongly indicate that BRMS1L is one of the mediators downstream of the p53 pathway, and inhibits cancer cell invasion and migration, which are essential steps in cancer metastasis. Collectively, our results demonstrate that BRMS1L is involved in cancer cell invasion and migration, and may be a therapeutic target for cancer.

Citation Format: Takashi Tokino, Ryota Koyama, Masashi Idogawa, Yasushi Sasaki. p53 target BRMS1A is a metastatic suppressor [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 553.
Abstract 5556: Combining adenosine receptor inhibition with AB928 and chemotherapy results in greater immune activation and tumor control
Authors: Walters Matt J. , Piovesan Dana , Tan Joanne , DiRenzo Daniel , Yin Fangfang , Miles Dillon , Leleti Manmohan R. , Park Tim , Soriano Ferdie , Sharif Eshan , Schindler Ulrike , Powers Jay P.
Published: August 2018

INTRODUCTION: Extracellular adenosine triphosphate (ATP) is efficiently hydrolyzed to adenosine by ecto-nucleotidases CD39 and CD73, which converts adenosine-monophosphate (AMP) into adenosine (ADO). ADO suppresses immune responses including those of T cells, natural killer (NK) cells, and dendritic cells (DC) through activation of A2aR and A2bR receptors. Treatment of cancer cells with platinum-based and anthracycline chemotherapy has been shown to induce immunogenic cell death (ICD), characterized by increased extracellular ATP levels, and upregulation of CD39 and CD73, leading to the enhanced generation of adenosine.

METHODS: The ability of AB928 to inhibit adenosine-mediated suppression of immune cell function was assessed using human CD4 and CD8 cells. BALB/c mice were inoculated with AT-3-OVA tumors, which express the model antigen ovalbumin, and subsequently treated with chemotherapy or AB928 alone, or a combination of both. The growth rate and immune composition of the tumors were assessed (flow cytometry).

RESULTS: Consistent with the ability of adenosine to suppress immune function, AB928 inhibited the ability of adenosine to suppress CD4 or CD8 T cell activation. Tumor-bearing mice that have been treated with either doxorubicin (DOX, an anthracycline) or oxaliplatin (OX) exhibit increased expression of CD39 and CD73. Established AT-3-OVA tumors treated with AB928 alone had a small but significant decrease in their growth rate; similarly, tumors treated with chemotherapy exhibited a reduction in growth rate. Consistent with the ICD hypothesis, treatment of AT-3-OVA-bearing mice with DOX or OX resulted in an increase in OVA-specific CD8 T cells in the tumor. Concurrent treatment with AB928 and chemotherapy resulted in significantly reduced tumor growth rates, when compared to chemotherapy alone. Analysis of the tumor-infiltrating cell populations showed a significant increase in OVA-specific CD8 T cells, relative to those treated with chemotherapy alone.

CONCLUSIONS: Treatment of tumor-bearing mice with a combination of AB928, a dual A2aR and A2bR antagonist, and ICD-inducing chemotherapy results in increased tumor antigen-specific CD8 T cell responses and significantly reduced tumor growth. AB928 is currently undergoing clinical evaluation.

Citation Format: Matt J. Walters, Dana Piovesan, Joanne Tan, Daniel DiRenzo, Fangfang Yin, Dillon Miles, Manmohan R. Leleti, Tim Park, Ferdie Soriano, Eshan Sharif, Ulrike Schindler, Jay P. Powers. Combining adenosine receptor inhibition with AB928 and chemotherapy results in greater immune activation and tumor control [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 5556.
Abstract 5573: Highly multiplexed diagnostic test for the detection of extended EGFR del19 variants in patients with NSCLC
Authors: Mellert Hestia S. , Jackson Leisa , Alexander Kristin , Weaver Amanda , Maar Dianna , Shelton Dawne , Pestano Gary
Published: August 2018

The detection of nucleic acids in circulation has become an increasingly important part of the clinical workup and testing for cancer patients, particularly those with non-small cell lung cancer (NSCLC). The utilization of blood-based PCR platforms for detection of somatic variant mutations addresses several limitations of previously established molecular testing approaches such as an inability to access tissue, insufficient tissue quantity or quality, and slow turnaround time for results. However, the number of variants detected may be limited by use of some rapid PCR technologies. We have focused in this study on the analytic and clinical development of a highly multiplexed assay for the detection of EGFR del19 while maintaining a rapid turn-around time. The test system includes the prospective collection of whole blood into a cfDNA BCT and the reproducible detection of del19 variants out of plasma using ddPCR-based technology. Analytic specificity and sensitivity was examined using in vitro DNA designed to mimic the EGFR del19 variants in the assay. Analytic sensitivity was performed and demonstrates that the theoretical limit of detection of the test was below 0.02% minor variant frequency when using high concentrations of synthetic standards. Precision studies were conducted with both analytic and clinical samples at high, medium and low mutation frequencies, over three consecutive days, three runs in one day and with two operators. All test parameters were successfully met. Normal healthy donor (n = 11) and reference ddPCR positive and negative NSCLC donor (n = 80) samples were tested. EGFR del19 variants were not identified in the plasma from the normal healthy donors or the EGFR del19 reference negative NSCLC donors. All reference positive cases were accurately detected above our required threshold of 2 variant copies with detection ranging from 14 to 1584. For all donor specimens the wild-type copies were well above the predefined clinical QC threshold of 300, ranging from 380 to 760 copies. Of note, no negative reference samples contained more than two mutant copies. We conclude that the developed test is highly suited for the sensitive and reproducible detection of multiple EGFR del19 variants from blood specimens in the clinical laboratory.

Citation Format: Hestia S. Mellert, Leisa Jackson, Kristin Alexander, Amanda Weaver, Dianna Maar, Dawne Shelton, Gary Pestano. Highly multiplexed diagnostic test for the detection of extended EGFR del19 variants in patients with NSCLC [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 5573.
Abstract 5587: Sample quality control of cell-free DNA
Authors: Liu Melissa H. , Graf Eva
Published: August 2018

Sequencing of cell-free DNA (cfDNA) extracted from blood specimens or other body fluids is possible due to the establishment of low input library protocols for next-generation sequencing workflows. Accurate quantification of cfDNA samples is essential to determine suitable input amounts for cfDNA library preparation prior to sequencing. The main component of cfDNA samples is the mononucleosome with a size around 170 bp, sometimes with additional species representing nucleosome multimers. Further, cfDNA samples may contain larger DNA fragments dependent on the donor's health status, preanalytical sample treatment, or extraction method. High molecular weight material can negatively influence library preparation and subsequently result in lower sequencing depth. Therefore, reliable quantification of cfDNA requires a method that separates DNA fragments by size, such as electrophoresis. This poster shows the use of a new cfDNA assay for the 2100 Bioanalyzer system as an add-on for the High Sensitivity DNA assay. The assay enables automated cfDNA quantification with pre-defined regions. Moreover, the results include sample purity as a score to qualify cfDNA samples according to their contamination level with high molecular weight material. The features of the assay are described with examples of typical sample patterns.

Citation Format: Melissa H. Liu, Eva Graf. Sample quality control of cell-free DNA [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 5587.
Abstract 5588: Uneven distribution of CTCs in magnetic fields following binding with iron nanoparticles
Authors: Xue Chengsen , Swenson Christina D. , Closkey Thomas W. Mc
Published: August 2018

Circulating tumor cell (CTC) detection and characterization have recently become an integral part of precision medicine. Several large, multicenter clinical studies confirmed that the prevalence of CTC is a useful predictor of treatment efficacy in metastatic breast cancer. The number of CTCs is strongly correlated with clinical progression. Oncology clinics widely use this biomarker to provide prognostic information. There are several platforms to isolate and measure CTCs. The only FDA-approved method is the CellSearch system developed by Veridex. The CellSearch System uses anti-EpCAM antibodies and polymer-coated iron nanoparticles in order to isolate CTC with the use of a magnetic field. The enriched CTC fraction is then placed in a MagNest chamber and the transparent slide is read by the automatic fluorescent CellTracks Analyzer. By using prelabeled breast cancer cells as quality control (QC) samples, we semiquantitated the distribution of CTC cells on slides in eleven QC samples. We found that CTC cells distribute unevenly on the slides. In order to count all CTCs, the whole area of the slide is divided into 175 small regions by the manufacturer. To better quantitate the CTC number, it is important to have all cells evenly spread over the slide. Our results showed that 41 out of 175 regions contained higher QC CTC counts. The heat map indicates that the magnetic field lines may have an effect on CTC localization. The CTC platforms are susceptible for underestimating actual CTC counts. Our findings indicate that cell concentration in certain regions caused by a magnetic field effect could be one of several contributing factors.

Citation Format: Chengsen Xue, Christina D. Swenson, Thomas W. Mc Closkey. Uneven distribution of CTCs in magnetic fields following binding with iron nanoparticles [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 5588.
Abstract 5589: Detection of epidermal growth factor receptor mutations from circulating tumor DNA versus archival tissue of lung cancer
Authors: Oh In-Jae , Seo Hyeong-Won , Cho Hyun-Ju , Park Ha-Young , Kho Bo-Gun , Chang Jin-Sun , Kim Tae-Ok , Shin Hong-Jun , Park Cheol Kyu , Lim Jung-Hwan , Kwon Yong-Soo , Kim Yu-Il , Lim Sung-Chul , Kim Young-Chul , Choi Yoo-Duk
Published: August 2018

Background: Epidermal growth factor receptor (EGFR) mutations are well known predictive marker of targeted therapy. We compared the sensitivity of EGFR mutation detection techniques between matched achival lung cancer tissue and peripheral blood sample.

Methods: We collected the paired samples from plasma and paraffin-embedded tumor tissue obtained before EGFR-tyrosine kinase inhibitor (EGFR-TKI). DNA extraction was performed using the QIAamp MinElute virus spin kit and EGFR mutation analysis was done by two detection methods. The current standard test is the PNAClampTM (Clamp) which is the PNA-based PCR clamping that selectively amplifies only the mutated target DNA sequence. The new technique is the PANAMutyperTM EGFR test (Mutyper), which use PNA clamping-assisted fluorescence melting curve analysis.

Results: A total of 295 (188 male and 107 female) patients were analyzed in this study. The histologic types were composed of 258 adenocarcinoma, 22 squamous cell carcinoma and 15 others. Most were clinical stage IV (137, 46.4%) and the EGFR mutation rate of tissue sample by standard Clamp test was 25.7%. Plasma sensitivity was significantly higher in Mutyper than Clamp (71.2% vs. 30.0%, p<0.001) with tissue as reference. The overall concordance and degree of agreement between two samples were better in Mutyper (91.9%, k=0.762, p<0.001) than Clamp (81.7%, k=0.344, p<0.001). In tissue sample, the median progression-free survival (PFS) of EGFR sensitive group was significantly longer than negative group regardless of the two methods. In plasma sample, the median PFS was significantly longer only by Mutyper (9.9 vs. 2.2 months, p=0.001), not by Clamp (9.8 vs. 7.6 months, p=0.968).

Conclusions: Mutyper was useful liquid biopsy technique with better sensitivity than standard Clamp test.

Citation Format: In-Jae Oh, Hyeong-Won Seo, Hyun-Ju Cho, Ha-Young Park, Bo-Gun Kho, Jin-Sun Chang, Tae-Ok Kim, Hong-Jun Shin, Cheol Kyu Park, Jung-Hwan Lim, Yong-Soo Kwon, Yu-Il Kim, Sung-Chul Lim, Young-Chul Kim, Yoo-Duk Choi. Detection of epidermal growth factor receptor mutations from circulating tumor DNA versus archival tissue of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5589.
Abstract 5613: Generation of a therapeutic monoclonal antibody for acute myeloid leukemia by employing the U937 cell line as a surrogate antigen
Authors: Chen Bethany , Kandov Evelyn , Ghebrehiwet Berhane
Published: August 2018

The prognosis of acute myeloid leukemia (AML) has improved for younger patients, but it still remains grim for patients over 65 years old with a likelihood of death within a year of diagnosis. Here, we evaluated the effects of novel anti-gC1qR monoclonal antibodies (mAbs) that were generated against intact U937 cells, which are used here as a model for AML. The U937 cell line is known to overexpress gC1qR, which in turn serves not only as a proliferative molecular signal but also provides a platform for the generation of angiogenic and metastatic factors. Cell viability results reveal that all of the mAbs significantly decrease (p<0.05) the percentage of viable AML cells although the mixture of those mAbs was not significant when compared to the individual mAbs themselves. The anti-U937 antibodies, monoclonal and polyclonal, showed a significant difference (p<0.05) against the anti-purified recombinant gC1qR antibodies. The anti-U937 mAb methodology of immunizing with the mammalian cell instead of the purified protein allowed the mAbs to better target the native endogenous protein present on human cells. Our study not only provided novel and potent mAbs against AML, but also implicated a potentially more efficient technique of creating therapeutic mAbs against cancer cells.

Citation Format: Bethany Chen, Evelyn Kandov, Berhane Ghebrehiwet. Generation of a therapeutic monoclonal antibody for acute myeloid leukemia by employing the U937 cell line as a surrogate antigen [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 5613.
Abstract 5637: Combination of oncolytic vaccinia virus and immune checkpoint blockade overcomes resistance to immunotherapy in renal cell carcinoma
Authors: Chon Hongjae , Kim Chan
Published: August 2018

Renal cell carcinoma (RCC) is a hypervascular tumor with variable immunogenic responses, and VEGF-targeted anti-angiogenic agent has become a standard treatment during the past decade. Recently, immune checkpoint inhibitor (ICI) has merged as another standard of care in RCC, but response is limited as a monotherapy. To determine the optimal combination strategy for immunotherapy in RCC, we employed JX594 (Pexa-Vec), an oncolytic vaccinia virus currently in phase 3 clinical trial, as a combination partner for ICI in RCC. Intratumoral injection of JX594 remodeled the tumor microenvironment of Renca tumor model through dynamic changes in immune system, leading to the conversion of an immunuosuppressive, non-inflamed tumor into an inflamed tumor. The JX594 virotherapy increased the intratumoral infiltration of CD8+ T cell by 16-fold, resulting in systemic and tumor-specific anti-cancer immune response against RCC. Moreover, the expression of immune checkpoint molecules such as PD-1 (4.0-fold), CTLA-4 (2.3-fold), and LAG-3 (3.1-fold) were increased. Depletion experiment showed that the therapeutic effect of JX594 is largely dependent on the cooperation of CD8+ and CD4+ T cells. In addition, GM-CSF has been shown to potentiate the immunotherapeutic efficacy of JX594. Combination of JX594 and ICI targeting PD-1 or CTLA-4 further increased the intratumoral infiltration of activated CD8+ T cells and effectively suppressed the growth of RCC. Furthermore, the optimal schedule was rationalized by

combination treatment of various administration methods. Finally, based on these combination results, triple combination of JX594, anti-PD-1, and anti-CTLA-4 maximized the anti-cancer immune responses and induced durable responses with improved overall survival. Collectively, we demonstrate that intratumoral injection of JX594 sensitizes non-inflamed RCC to ICI treatment through CD8+ T cell infiltration in tumor and induces anti-cancer immune responses that can overcome immunotherapy resistance.

Citation Format: Hongjae Chon, Chan Kim. Combination of oncolytic vaccinia virus and immune checkpoint blockade overcomes resistance to immunotherapy in renal cell carcinoma [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 5637.
Abstract 5648: Proteomics analysis of oral cancer cell-derived extracellular vesicles
Authors: Oliveira Ana K. , Busso-Lopes Ariane F. , Sá Jamile Oliveira , Rivera César , Leme Adriana F. Paes
Published: August 2018

More than 300,000 new cases of head and neck cancer occur each year around the world, and although it is not one of the most common tumors in the world, has an elevated morbidity rate (37.8%) after five years of diagnosis. The development and progression of the Oral Squamous Cell Carcinoma (OSCC) involve complex cellular mechanisms which contribute to a tumor progression. Recently extracellular vesicles (EVs) have taken a significant physiological and pathological role in cell-cell communication through interactions mediated by receptors or by transfer of bioactive molecules such as receptors, proteins, lipids, mRNA, microRNA, and DNA, by changing the phenotype of the recipient cell. The tumor cells can transfer information to different cell types using EVs as a vehicle, modifying the characteristics of the tumor microenvironment, and contributing to cancer sustaining. The detection of alterations in the EVs proteome may, therefore, contribute to the understanding the cellular mechanisms modulated in OSCC and help to improve the clinical prognosis and potential therapeutic targets in oral cancer. Thus, this study analyzed the cargo proteins of different EVs from less and high aggressive oral cancer cells to understand their possible role in microenvironment modulation and oral cancer progression. Our results indicate that EVs of aggressive cells have exclusive proteins that may contribute to lymph node metastasis, which is the worse prognostic factor in OSCC and can be considered potential targets in OSCC.FAPESP: 2016/19337-3

Citation Format: Ana K. Oliveira, Ariane F. Busso-Lopes, Jamile Oliveira Sá, César Rivera, Adriana F. Paes Leme. Proteomics analysis of oral cancer cell-derived extracellular vesicles [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 5648.
Abstract 5685: Nanocage-therapeutics prevailing phagocytosis and immunogenic cell death awakens immunity against cancer
Authors: Nam Gihoon , Kim Gi Beom , Kim In-San
Published: August 2018

A growing appreciation of the relationship between the immune system and tumorigenesis has led to the development of strategies aimed at ‘re-editing' the immune system to kill tumors. Here, we report a novel tactic for overcoming the activation-energy threshold of the immunosuppressive tumor microenvironment and mediating the delivery and presentation of tumor neoantigens to the host's immune system. This nature-derived nanocage not only efficiently presents ligands that enhance cancer cell phagocytosis, it also delivers drugs that induce immunogenic cancer cell death. The designed nanocage-therapeutics induces the release of neoantigens and danger signals in dying tumor cells, and leads to enhancement of tumor cell phagocytosis and cross-priming of tumor specific T cells by neoantigen peptide-loaded antigen-presenting cells. We observed potent inhibition of tumor growth and complete eradication of tumor through systemic tumor-specific T-cell responses in tumor draining lymph node and spleen and further, infiltration of CD8+ T cells into the tumor site. Remarkably, after removal of the primary tumor, all mice treated with this nanocage-therapeutics are protected against subsequent challenge with the same tumor cells, suggesting development of lasting, tumor-specific responses. This designed nanocage-therapeutics ‘awakens' the host's immune system and provokes a durable systemic immune response against cancer.

Citation Format: Gihoon Nam, Gi Beom Kim, In-San Kim. Nanocage-therapeutics prevailing phagocytosis and immunogenic cell death awakens immunity against cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5685.
Abstract 5712: Identification of a highly suppressive Treg subset associated with immunotherapy response
Authors: Troiani Teresa , Romano Simona , D'Arrigo Paolo , Rea Anna , Tufano Martina , Giunta Emilio F. , Matarrese Giuseppe , Procaccini Claudio , Novizio Nunzia , Vigorito Vincenza , Faicchia Deriggio , Argenziano Giuseppe , Ciardiello Fortunato , Romano Maria F.
Published: August 2018

Melanoma often exploits Treg to avoid immune attack. Treg is a heterogeneous population with respect to immunosuppressive capability. Lymphocytes are particularly rich in FKBP51 (encoded by FKBP5 gene), known as the receptor for FK506. Melanoma aberrantly expresses this protein, which sustains resistance and invasion. Melanoma/immune cell interaction, through PD-L1/PD1, bidirectionally generates FKBP5 splicing inducing a lower molecular weight form termed FKBP51s. In 64 advanced melanoma patient PBMCs, we found that FKBP51s marked a Treg subset that correlated to anti-CTLA4 response. More precisely, a Treg FKBP51s+ count <1% was associated with unresponsiveness (normal donor-range 0.1-0.7%). Aim of the present study was to assess the role of Treg FKBP51s+ as potential biomarker of response in a different cohort of patients treated with anti-PD1. In addition, the suppressive potential of Treg FKBP51s+ in comparison with that of Treg FKBP51s- is investigated. To date, we have outcomes of 11 patients. For each patient, we have collected up to 10 blood samples, at T0 and before each treatment, to monitor Tregs. In 5 responder patients, Treg FKBP51s+ was >1.2 and <4.8; in 5 non responder patients, the count was >0.04 and <0.8 After a transient increase following the first administration, the count decreased to 0.3+0.2% in responder patients. Interestingly, a patient with count =0.7% developed autoimmune side effects that led to therapy discontinuation. Resolution of side effects was accompanied by a value increase to 9.9%; anti-PD1 re-administration was then successful. In vitro iTreg generation showed that FKBP51s was upregulated in Treg CD25high, Ki67high and p70S6khigh, corresponding to a highly metabolically active profile with strong suppressive capability. In conclusion, melanoma patients that benefit from immune checkpoint targeted therapy are recognizable by an expansion of a Treg subset which is marked by FKBP51s, a splicing protein isoform generated by triggering of surface antigens (PD-L1, PD1), abundantly expressed on highly suppressive Tregs.

Citation Format: Teresa Troiani, Simona Romano, Paolo D'Arrigo, Anna Rea, Martina Tufano, Emilio F. Giunta, Giuseppe Matarrese, Claudio Procaccini, Nunzia Novizio, Vincenza Vigorito, Deriggio Faicchia, Giuseppe Argenziano, Fortunato Ciardiello, Maria F. Romano. Identification of a highly suppressive Treg subset associated with immunotherapy response [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 5712.
Abstract 5719: RNA-based tumor neoantigens from intron retention
Authors: Margolis Claire A. , Smart Alicia C. , Adeegbe Dennis , Miao Diana , He Meng Xiao , Pimentel Harold , Fugmann Tim , Wong Kwok-Kin , Allen Eliezer M. Van
Published: August 2018

Background: Tumor neoantigens contribute to cancer immunity and may influence selective immune checkpoint inhibitor response. Translation of nonsynonymous somatic mutations in tumor cells generates mutant peptides that can be recognized as foreign by the host immune system. Alterations in tumor RNA, rather than DNA, may also contribute to the tumor neoantigen landscape. Intron retention, a widespread feature of cancer transcriptomes, represents a novel source of tumor neoantigens.

Methods: We developed an in silico pipeline leveraging existing tools and novel methods to computationally identify retained-intron neoantigens (RI-neoantigens) from transcriptome sequencing data. We applied our computational pipeline to three cohorts of melanoma patients (n = 89) treated with immune checkpoint blockade.

Results: This approach identified a mean RI-neoantigen burden of 3,643 across cohorts, which augments the mean DNA-derived somatic neoantigen burden by roughly 4.5-fold. 3,253 RI-neoantigens were exclusively present in two or more patients who responded to immunotherapy. A subset of responder-exclusive RI-neoantigens were validated by peptide restimulation assay. A candidate RI-neoantigen from a cancer cell line was identified in complex with MHC I by mass spectrometry, supporting the hypothesis that aberrant splicing results in intron retention, which generates abnormal transcripts that are translated into immunogenic peptides and presented to the immune system.

Conclusions: Our results demonstrate the contribution of transcriptional dysregulation to the overall burden of tumor neoantigens. Further study of RI-neoantigens may expand our understanding of tumor immunity and identify immunogenic peptide sequences for personalized cancer vaccines.

Citation Format: Claire A. Margolis, Alicia C. Smart, Dennis Adeegbe, Diana Miao, Meng Xiao He, Harold Pimentel, Tim Fugmann, Kwok-Kin Wong, Eliezer M. Van Allen. RNA-based tumor neoantigens from intron retention [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 5719.