bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4601: Dissecting the spatial heterogeneity of circulating tumor cells reveals CCL5-Treg-mediated immune evasion in hepatocellular carcinoma
Authors: Wu Liang , Jiang Miaomiao , Sun Yunfan , Yang Xinrong , Hou Yong , Fan Jia , Liu Shiping
Published: August 2018

The prognosis of hepatocellular carcinoma (HCC) is closely linked to the occurrence of recurrent, metastatic disease. Metastasis has been proposed to be initiated by circulating tumor cells (CTCs); however, the transcriptomic plasticity and adaptive mechanisms of CTCs in systemic circulation are not well defined. Here, we established single-cell RNA-sequencing profiles of 113 CTCs isolated from four key vascular sites along the dissemination route in ten HCC patients. In our study, we found that single CTCs displayed profound spatial transcriptional heterogeneity within the circulatory system, which is associated with increased transcriptional activity, cell cycle progression, and chemokine-induced immune escape programs. In particular, CTCs dynamically drove chemokine (C-C motif) ligand 5 (CCL5) expression over the course of circulation. We demonstrated that increased CCL5 expression in turn recruited regulatory T cells to facilitate immune evasion and metastatic progression, as rigorously validated by our in vitro and in vivo models. Finally, we identified an inherently dormant CTC subset with an epithelial-mesenchymal transition and immune-evasive phenotype, and demonstrated its potential prognostic implications. Collectively, our results reveal the previously unappreciated spatial heterogeneity of CTCs, and define its role in diverse regulatory programs and cellular states. Our study offers new insight into metastatic mechanisms, highlighting the potential utility of anti-CTC therapeutic strategies.

Citation Format: Liang Wu, Miaomiao Jiang, Yunfan Sun, Xinrong Yang, Yong Hou, Jia Fan, Shiping Liu. Dissecting the spatial heterogeneity of circulating tumor cells reveals CCL5-Treg-mediated immune evasion in hepatocellular 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 4601.
Abstract 4621: Risk stratification and biomarker discovery in HPV-positive oropharynx squamous cell carcinoma determined by HPV and human gene expression profile associations
Authors: Gleber-Netto Frederico O. , Rao Xiayu , Erikson Kelly , Akagi Keiko , Johnson Faye M. , Wang Jing , Califano Joseph , Gillison Maura L. , Myers Jeffrey N. , Pickering Curtis R.
Published: August 2018

Determining which HPV+ oropharyngeal squamous cell carcinoma (OPSCC) patients will respond to therapy is of utmost important for implementation of treatment de-escalation to reduce morbidity or testing of novel therapeutic approaches. Due to the lack of reliable indicators of treatment response, we explored the gene expression profile of OPSCC in order to better understand the biology of these tumors and identify novel biomarkers. In this study, we investigated human genes associated with HPV transcription in 80 OPSCC samples from The Cancer Genome Atlas (TCGA). This exploratory analysis provided a list of 582 human genes associated with HPV biology. Hierarchical clustering analysis using the 582 human genes was used to separate HPV+ OPSCC in two groups with significantly distinct survival (log-rank test p < 0.001) and differential expression of HPV genes. A refinement of this analysis generated a prognostic gene expression signature for HPV+ OPSCC containing 41 human genes. These results were validated in two independent cohorts of HPV+ OPSCC (n=83 and n=47) and one independent cohort of cervical cancer (n=83). In all three validation cohorts our 41 gene expression signature was able to identify a group of HPV+ OPSCC patients with worse survival. Further refinement and validation of the signature is ongoing. In order to understand the biology related to poor outcome in HPV+ OPSCC, a whole transcriptome analysis between the two initial HPV+ OPSCC TCGA groups was performed. Pathway analysis identified cell metabolism, cell stress, and DNA damage related genes were highly associated with poor outcome. Similar patterns of gene expression were found in vitro in a panel of 10 HPV+ cell lines, and markers of the poor outcome were found to be associated with reduced radiation sensitivity in vitro. Our study has identified prognostic biomarkers in HPV+ OPSCC with potential clinical importance. These biomarkers may be useful for selection of low risk patients for treatment de-escalation or selection of high risk patients for novel therapeutic approaches. These biomarkers are also functionally linked to radiation sensitivity and may include new therapeutic targets.

Citation Format: Frederico O. Gleber-Netto, Xiayu Rao, Kelly Erikson, Keiko Akagi, Faye M. Johnson, Jing Wang, Joseph Califano, Maura L. Gillison, Jeffrey N. Myers, Curtis R. Pickering. Risk stratification and biomarker discovery in HPV-positive oropharynx squamous cell carcinoma determined by HPV and human gene expression profile associations [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 4621.
Abstract 4630: Characterizing oncogene addiction in alveolar rhabdomyosarcoma reveals novel strategies for combination therapy
Authors: Ommer Johannes , Wachtel Marco , Schaefer Beat W.
Published: August 2018

Rhabdomyosarcoma is the most common soft tissue sarcoma in children. The aggressive alveolar subtype (aRMS) is characterized by chromosomal translocations, most often a t(2;13) resulting in the expression of the oncogenic fusion protein PAX3-FOXO1 which is critical for tumorigenesis and cell survival. Our aim here was to identify a pharmacological combination therapy approach interfering with PAX3-FOXO1 biology at different levels.

Since loss of the fusion protein results in cell death, we first aimed to pharmacologically enhance this effect. To this end, we screened aRMS tumor cells with a library of 208 drugs while simultaneously silencing PAX3-FOXO1 by shRNA. This identified the BH3-mimetic ABT-263 to sensitize aRMS cells to cell death after PAX3-FOXO1 depletion. To further characterize the cell death mechanisms we used combined shRNA and CRISPR approaches to perform a BH3 protein profiling. In accordance with identification of ABT-263 we demonstrate that aRMS cells undergo intrinsic apoptosis in a NOXA-dependent manner upon depletion of PAX3-FOXO1.

In a parallel approach, and to identify drugs altering PAX3-FOXO1 protein stability, we screened the same drug library directly measuring fusion protein levels as read-out. This revealed that inhibition of Aurora kinase A negatively affects PAX3-FOXO1 protein levels.

Finally, using both aRMS cell lines and patient-derived xenografts we demonstrate that combination treatment of Aurora kinase A inhibitors together with ABT-263 synergistically induces cell death and greatly slows tumor growth in vitro and in vivo.

Taken together, these data show a novel functional interaction of Aurora kinase A and PAX3-FOXO1 and suggest new opportunities for targeted combination treatment of aRMS.

Citation Format: Johannes Ommer, Marco Wachtel, Beat W. Schaefer. Characterizing oncogene addiction in alveolar rhabdomyosarcoma reveals novel strategies for 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 4630.
Abstract 4638: Predictive computational approaches to guide the covalent targeting of cysteine residues in drug discovery
Authors: Ward Richard A. , Lonsdale Richard
Published: August 2018

Targeted covalent inhibition is an established approach for increasing the potency and selectivity of potential drug candidates, and has also been used to identify tool compounds for target validation studies. It is evident that binding to reversible recognition elements within the pocket is essential for selective covalent inhibition, but this must also be achieved with the appropriate level of inherent reactivity of the covalent functionality and compatible geometry and reactivity of the target amino acid. We have assessed the ability of several experimental and computational approaches to predict the intrinsic reactivity of a range of cysteine targeting warheads, some of which can be used in prospective compound design. In addition, molecular dynamics (MD) simulations in combination with pKa prediction methods have been used to investigate the accessibility and potential reactivity of target cysteines towards covalent inhibition. As part of this work a number of case studies will be reported including retrospective examples from covalent drug discovery efforts along with prospective use towards novel targets. Finally, a dataset of osimertinib analogues along with other EGFR inhibitors of varying reactivity will be discussed to enable further insight into the impact of reversible and covalent interactions on inhibitor binding.

Citation Format: Richard A. Ward, Richard Lonsdale. Predictive computational approaches to guide the covalent targeting of cysteine residues in drug discovery [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 4638.
Abstract 464: Cell death and senescence: Role of protein kinase C in promoting senescence
Authors: Livneh Etta , Ari Assaf Ben , Zurgil Udi
Published: August 2018

Senescence is characterized by permanent cell cycle arrest and loss of proliferative capacity, despite continued viability and metabolic activity. Senescent cells undergo massive genome modulation including induction of the senescence-associated secretory phenotype (SAPS). Components of SASP can implement cell cycle arrest or recruit the immune system to clear senescent cells, thus contributing to tumor suppression. However, some secreted pro-inflammation molecules, act as pro-tumorigenic agents contributing to tumor progression, suggesting that elimination of senescent cells could be beneficial. Very few studies showed a role for PKC in senescence. Here we show that PKCeta, an epithelial specific and anti-apoptotic kinase (epithelia is the origin of about 90% of human tumors), promotes senescence induced by oxidative stress and DNA damage. Using PKCeta-knockdown breast adenocarcinoma MCF-7 cells we show that PKCeta promotes senescence induced by oxidative stress and DNA damage via its ability to upregulate the expression of the cell cycle inhibitors p21Cip1 and p27Kip1 and to modulate transcription of major components of SASP such as IL-6 and IL-8. Moreover, we demonstrate that PKCeta creates a positive loop for reinforcing senescence by increasing the transcription of both IL-6 and IL-6 receptor. Thus, the expression of PKCeta modulates major components of SASP. Furthermore, our studies demonstrate that PKCeta interferes with γH2AX phosphorylation, which mark DNA double-strand breaks for repair. The phosphorylation of γH2AX may reflect the extent of DNA breaks but also the cellular response to DNA damage. Our experiments suggest that the response to DNA damage (repair processes) was more efficient in PKCeta-knockdown cells. The phosphorylation on ATM and Chk2 was also lower in PKCeta expressing cells. Revealing the molecular regulators of senescence will improve our ability to develop new therapeutic strategies for clearing tumor cells. Our studies showing a role for PKCeta in senescence could identify PKCeta and/or its activated signaling cascades as targets for intervention senescence induction. Since PKCeta is highly expressed in epithelia that are the origin of about 90% of human tumors, this may provide critical inhibitors as therapeutic adjuvants for different types of carcinomas.

Citation Format: Etta Livneh, Assaf Ben Ari, Udi Zurgil. Cell death and senescence: Role of protein kinase C in promoting senescence [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 464.
Abstract 4640: Sustained co-delivery of dendritic cells and oncolytic adenovirus co-expressing IL-12 and GM-CSF using biodegradable polymer matrix for cancer immunotherapy
Authors: Oh Eonju , Oh Jung-Eun , Hong JinWoo , Chung YoonHo , Lee Yunki , Park Ki Dong , Kim Sung Wan , Yun Chae-Ok
Published: August 2018

Administration of dendritic cells (DCs) combined with oncolytic adenovirus (Ad) expressing antitumor cytokines induces a potent antitumor effect and antitumor immunity by ameliorating the immunosuppressive tumor microenvironment. However, this combination therapy has significant limitations due to rapid dissemination and inactivation of the therapeutics at the tumor site, necessitating multiple injections of both therapeutics. In this study, we investigated injectable and biodegradable gelatin-based hydrogel as a carrier for oncolytic Ad co-expressing interleukin (IL)-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (oAd) and DCs to elicit a potent antitumor effect with single administration. Gel matrix enabled sustained release of both oAd and DCs while preserving their biological activity over a considerable time period, leading to efficient retention of both therapeutics in tumor tissue. Further, tumors treated with oAd- and DC-loaded gel (oAd+DC/gel) showed a significantly greater expression level of IL-12, GM-CSF, and interferon-γ (IFN-γ) than either single treatment (oAd or DC) or oAd in combination with DC (oAd+DC), resulting in efficient activation of both endogenous and exogenous DCs, migration of DCs to draining lymph nodes, and tumor infiltration of CD4+ and CD8+ T cells. Moreover, oAd+DC/gel resulted in a significantly higher number of tumor-specific IFN-γ-secreting immune cells compared with oAd+DC. Lastly, oAd+DC/gel significantly attenuated tumor-mediated thymic atrophy, which is associated with immunosuppression in the tumor microenvironment, compared with oAd+DC. Taken together, these results demonstrate that gelatin gel-mediated co-delivery of oncolytic Ad and DCs might be a promising strategy to efficiently retain both therapeutics in tumor tissue and induce a potent antitumor immune response for an extended time period via a single administration.

Citation Format: Eonju Oh, Jung-Eun Oh, JinWoo Hong, YoonHo Chung, Yunki Lee, Ki Dong Park, Sung Wan Kim, Chae-Ok Yun. Sustained co-delivery of dendritic cells and oncolytic adenovirus co-expressing IL-12 and GM-CSF using biodegradable polymer matrix for cancer immunotherapy [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 4640.
Abstract 4649: Targeting the cancer cell lysosome with transformable self-assembling peptide nanoparticles
Authors: Baehr Christopher M. , Wang Lei , Lam Kit S.
Published: August 2018

Lysosome-mediated cell death has recently become of great interest to the cancer community as several compounds have been shown to preferentially induce cancer cell death via lysosomal membrane permeabilization (LMP) (e.g., oleocanthal, salinomycin, and TiO2 nanoparticles). Further, many cancer cell lines that are resistant to traditional cancer therapeutics are highly susceptible to lysosome-mediated cell death. Here, we develop transformable peptide nanoparticles (NP) designed to traffic to cancer cell lysosomes, form aggregates, and induce LMP. The peptide consists of three motifs: 1) a hydrophilic, cell penetrating, and pH-responsive poly-d-Arg motif, 2) a hydrogen-bonding motif derived from beta amyloid (KLVFF); and 3) hydrophobic bis(pyrene) that fluoresces upon aggregation. The peptides initially self-assemble into NPs (50nm via TEM) in PBS, but when exposed to a lysosomal pH of 4.5, form much larger aggregates. We show that these nanoaggregates colocalize with the lysosome, leading to cell death, suggesting the induction of LMP. Moreover, utilizing bis(pyrene) we show that while these aggregates form in the lung carcinoma cell line, A549, they do not form in 3T3 fibroblasts. Additionally, we demonstrate that these nanoaggregates are highly toxic to A549 cells with IC50 in the low micromolar range (~2μM). This offers a novel organelle-specific strategy to preferentially target and kill cancer cells.

Citation Format: Christopher M. Baehr, Lei Wang, Kit S. Lam. Targeting the cancer cell lysosome with transformable self-assembling peptide 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 4649.
Abstract 4687: Double positive (DP) CD4+CD8+ T cells with an exhausted phenotype in renal cell carcinoma (RCC) patients
Authors: Menard Laurence , Fischer Paul , Kakrecha Bijal , Lee Deborah , Penhallow Becky , Orduno Nataly Manjarrez , Nadler Steven
Published: August 2018

Checkpoint inhibitors target the inhibitory receptors expressed by tumor infiltrating T cells in order to reinvigorate an anti-tumor immune response. Therefore, understanding T cell composition and phenotype in human tumors is crucial. We analyzed by flow cytometry tumor infiltrating lymphocytes from two independent cohorts of patients with different cancer types, including RCC, lung and colon cancer. T cells are usually either CD4+ or CD8+ with a small percentage of CD4+ CD8+ DP cells (<5%). Compared to several other cancer types, including lung and colorectal cancers, about a third of RCC patients showed an increased proportion of DP CD4+CD8+ T cells (>5%, reaching 30-50% of T cells in some patients). These DP T cells express high level of PD1 and TIM-3 that tend to correlate with higher expression of PD1 and TIM-3 in conventional CD8 T cells. DP T cells also express markers of antigen-experienced T cells such as CD38 and HLA-DR. These results suggest that double positive T cells might be exhausted tumor specific T cells with the potential to be reactivated by checkpoint inhibitors.

Citation Format: Laurence Menard, Paul Fischer, Bijal Kakrecha, Deborah Lee, Becky Penhallow, Nataly Manjarrez Orduno, Steven Nadler. Double positive (DP) CD4+CD8+ T cells with an exhausted phenotype in renal cell carcinoma (RCC) patients [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 4687.
Abstract 470: p53 isoform delta133p53 in tumor senescence
Authors: Muhlinen Natalia Von , Beck Jessica , Harris Curtis C.
Published: August 2018

The tumor suppressor gene TP53 expresses at least 12 isoforms due to alternative splicing, alternative initiation of translation, and alternative promoter usage. These isoforms include p53β and Δ133p53, which can modulate p53 transcriptional activity and apoptosis. The p53 isoform Δ133p53 is expressed in normal tissue, but is abnormally expressed in several cancer types. However, its role in tumor progression is still unclear. Normal somatic cells undergo a limited number of cell divisions, eventually leading to an irreversible proliferative growth arrest known as replicative senescence. Replicative senescence can act as a tumor suppressor mechanism, thus constituting a critical barrier to tumor progression in vivo. Previous studies showed that, p53β and Δ133p53 are endogenous regulators of replicative senescence in normal human fibroblasts. Interestingly, expression of Δ133p53 is upregulated in several cancer types. For instance, enhanced levels of Δ133p53 are associated with inhibition of cellular senescence in colon carcinoma as well as with progression of premalignant colon adenomas to colon carcinomas. However, little is known about the role of Δ133p53 in cancer. We are studying the roles of Δ133p53 in proliferation and senescence of cancer cell lines. We hypothesized that depletion of otherwise enhanced Δ133p53 isoform induces cellular growth arrest and increases the secretion of senescence-associated secretory phenotype (SASP) pro-inflammatory cytokines such as IL-6 and IL-8 of cancer cell lines, and thus Δ133p53 may be a therapeutic target to repress tumor cell growth.

Citation Format: Natalia Von Muhlinen, Jessica Beck, Curtis C. Harris. p53 isoform delta133p53 in tumor senescence [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 470.
Abstract 4702: Upgrading cancer immunotherapy: Checkpoint blockade mAb-ODN conjugate
Authors: Benaduce Ana Paula , Gefen Tal , Rodriguez Sergio , Marples Brian , Gilboa Eli , Ishkanian Adrian
Published: August 2018

Programmed death 1 (PD-1) and its ligand PD-L1 provide inhibitory signals that affect the balance of T cell activation, playing major roles in tumor immune escape. Inhibition of negative regulators of T cell activation effectively remove signals that restrain T cell proliferation and gain of effector function. Hence, disruption of the PD-1/PD-L1 signaling pathway by the use of monoclonal antibodies (mAbs) can improve tumor control by enhancement of the endogenous antitumor immune responses. Toll-like receptors (TLR) are a specialized set of pathogen recognition receptors found in innate and adaptive immune system cells. Activation of these receptors by danger-associated molecules such as bacterial DNA or RNA, initiate signaling cascades leading to protective immune responses. Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs can mimic bacterial DNA and therefore function as ligands for intracellular TLR9, prompting consequential activation of immune responses. The purpose of this study was to assess the feasibility of using PD-1/PD-L1 mAbs conjugated to TLR9 agonists (ODNs) as a novel strategy for tumor-targeting immune checkpoint signaling and activation of immune cells. Antibodies recognizing PD-1 and PDL-1 were covalently modified with a 21mer ssDNA oligonucleotide linker sequence that served as an anchor site for the ODNs synthesized with a 3' extension. Annealing of complementary strands formed mAb-ODN conjugates. Quantification of ODNs per PD-1 or PDL-1 mAbs was verified by flow cytometry. Functionality of PD-1 and PDL-1-ODN conjugates was analyzed in vitro using splenocyte cultures and cell proliferation was evaluated by incorporation of radiolabeled H3 Thymidine. In vivo experiments were carried out using Balb/c mice bearing 4T1 subcutaneous tumors. Flow cytometry analysis confirmed each modified mAb had 5 linker sequences available to bind ODNs, and also defined the binding capability of mAb portion of the conjugate to epitope target. Splenocytes proliferation at day 5 after treatment revealed that PD-1 and PDL-1 conjugated to ODNs have significantly increased proliferation in comparison to non-conjugated mAb and ODN controls. These results indicate that conjugation of mAb-ODN is feasible and that conjugation does not affect PD-1 and PDL-1 antibody functionality. Interestingly, in vivo experiments demonstrated PDL-1-ODN but not PD1-ODN conjugate affects 4T1 tumor growth. Use of this approach to improve the therapeutic index of immunotherapeutic mAbs requires further study and validation in different murine tumor models.

Citation Format: Ana Paula Benaduce, Tal Gefen, Sergio Rodriguez, Brian Marples, Eli Gilboa, Adrian Ishkanian. Upgrading cancer immunotherapy: Checkpoint blockade mAb-ODN conjugate [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 4702.
Abstract 4712: AT1965, a novel B cell-activating immunotherapy, exerts potent anticancer activity
Authors: Roy Monideepa , Krishna Vineeth , Sengupta Aniruddha , Roy Sudip , Kulkarni Ashish , Sengupta Shiladitya
Published: August 2018

Emerging clinical evidence implicate B cells in long term survival in cancer. However, a therapeutic activation of a B cell response against cancer has not been explored. Here we describe the results with AT1965, which exerts a potent anticancer effect via the activation of B cells. AT1965 completely regressed aggressive 4T1 breast tumors established in balb/c mice. Immunophenotyping revealed that IGKC, a B cell marker, was the dominant immune biomarker that is over-expressed following AT1965 treatment. The lack of functional B cells in vivo, via the deletion of the JH gene results in the loss of heavy chain production, blocked this anti-cancer activity of AT1965. Activation of a B cell response is associated with long term memory, which should prevent relapse. To test this, tumor-bearing animals were treated with AT1965 to induce regression; Not a single animal relapsed, i.e. developed the tumor when re-challenged with the tumor thirty days after the primary tumors had regressed. T and B cells are natural partners in an immune response. We therefore tested the combination of a PD1 immune checkpoint inhibitor and AT1965 in an immunocompetent lung cancer model. Only 12.5% of the animals treated with AT1965 or the PD1-inhibitor exhibited a complete regression of lung cancer, while 37.5% exhibited delayed tumor progression. In contrast, 50% of the animals underwent complete tumor regression and 25% exhibited tumor growth inhibition when the two drugs were combined, consistent with the basic immunology premise that both B and T cells work together to mount a complete immune response. Our results indicate that B cell activation could emerge as the next paradigm in cancer immunotherapy.

Citation Format: Monideepa Roy, Vineeth Krishna, Aniruddha Sengupta, Sudip Roy, Ashish Kulkarni, Shiladitya Sengupta. AT1965, a novel B cell-activating immunotherapy, exerts potent anticancer activity [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 4712.
Abstract 4728: Iron regulation of innate immune pathogen-associated molecular pattern receptors
Authors: Stater Evan
Published: August 2018

Pathogen-associated molecular pattern (PAMP) receptors are key triggers of the pro-inflammatory immune effector functions of the cells of the innate immune system. Ectopic PAMP activation is a contributing factor to both cancer development and pathology, as well as host anti-tumor immune responses. Iron, an essential mineral in biological systems, has a similar dualistic role in cancer. We have shown that iron acts as a key modulator of intracellular signaling cascades downstream from PAMP activation. Cellular iron enrichment enhances the transcriptional activity of a pathogen-induced transcription factor downstream of PAMPs in an immortalized human macrophage cell line treated with a PAMP receptor-specific agonist ligand. Cell surface expression of the receptor was not affected by the iron enrichment; therefore, any modulation of receptor signaling by iron is likely downstream of the receptor. Furthermore, iron supplementation in vitro increases expression of immunity-related genes regulated by the pathogen-induced transcription factor; by contrast, treatment with iron-chelating reagents reduces expression of these genes. These results suggest that the iron content of the tumor microenvironment may have a regulatory role in pathological immunoregulation. In particular, the iron-retaining phenotype of many tumor cells may serve a novel pathological function, inhibiting PAMP-regulated anti-tumoral effector functions of tumor-infiltrating macrophages cells by iron deprivation.

Citation Format: Evan Stater. Iron regulation of innate immune pathogen-associated molecular pattern receptors [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 4728.
Abstract 4731: Contribution of FcγRIIB to creating the suppressive tumor microenvironment
Authors: Kasahara Yuki , Shirota Hidekazu , Ishioka Chikashi
Published: August 2018

Various immune cells are recruited in tumor microenvironment. It is well established that cellular immune responses, such as cytotoxic or suppressive activities play an important role in regulating tumor growth and metastasis. However, contribution of humoral immune responses against tumor is incompletely understood. The Fc receptors constitute critical elements for activating or down-regulating immune responses through immunoglobulin immune complex. Here, we examined the potential role of inhibitory Fc receptor, Fc gamma RIIb in tumor immunity using mice model. Current findings indicate that tumor-specific antibodies are induced and control tumor immunity. Deletion of Fc gamma RIIb receptor significantly improves both cellular and humoral anti-tumor immunity, delays tumor growth. These findings indicate that spontaneous antibody against tumor create the suppressive tumor microenvironment through Fc gamma RIIb signaling, and suggests attractive therapeutic target for cancer immunotherapy.

Citation Format: Yuki Kasahara, Hidekazu Shirota, Chikashi Ishioka. Contribution of FcγRIIB to creating the suppressive tumor microenvironment [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 4731.
Abstract 4739: Elucidation of immunosuppressive microenvironment in colorectal cancer
Authors: Sasada Tetsuro , Ohtake Junya , Satoyoshi Tetsuta , Kazama Keisuke , Wada Satoshi , Yada Erika , Shiozawa Manabu
Published: August 2018

Colorectal cancer (CRC) is one of the most increasing cancers worldwide, and novel treatment modalities remain to be developed for advanced CRC patients. In recent years, immune checkpoint inhibitors have demonstrated impressive clinical efficacy in various types of cancers, but limited clinical responses were reported in CRC, except for MSI-high or MMR-deficient tumors. It has thus been suggested that immune suppressive mechanisms other than immune checkpoints might contribute to tumor progression in CRC. In the current study, various types of immune cells were isolated from peripheral blood or resected tumor/normal tissues and regional lymph nodes in 25 primary CRC patients, and their phenotypes and functions were comprehensively assessed. Flow cytometry analysis demonstrated a significant increase of CD4+FoxP3+ T cells, which might represent regulatory T cells, in proximal lymph nodes (N1 region), compared to distal (N2 or N3 region) or unrelated lymph nodes. In addition, the functional analysis of T cells assessed by cytokine production after non-specific stimulation (PMA and A23187) showed a significant decrease in the frequencies of IFN γ-producing CD8+ T cells in N1 region. Notably, reduction of IFN γ-producing CD8+ T cells seemed to be associated with advanced stages of disease. These results indicate that the immunosuppressive microenvironment, particularly in regional lymph nodes, may be related to tumor progression in CRC. The current study suggests that monitoring of immunosuppressive microenvironment would be useful as a predictive biomarker for cancer progression and patient prognosis in CRC. Further studies remain to be performed to more thoroughly elucidate immune suppressive mechanisms, and thereby to develop a novel immunotherapeutic strategy in CRC.

Citation Format: Tetsuro Sasada, Junya Ohtake, Tetsuta Satoyoshi, Keisuke Kazama, Satoshi Wada, Erika Yada, Manabu Shiozawa. Elucidation of immunosuppressive microenvironment in colorectal 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 4739.
Abstract 4740: CLEVER-1 as a marker to identify breast cancer patients under immunosuppression
Authors: Virtakoivu Reetta E. , Boström Pia , Aaltonen Riitta , Koskivuo Ilkka , Jalkanen Sirpa , Hollmén Maija
Published: August 2018

TAMs are the main innate immune cells regulating tumor growth and metastasis. In tumors, TAMs exhibit an immunosuppressive M2 phenotype attenuating anti-tumor immune responses. CLEVER-1 is a multifunctional scavenger receptor specifically expressed on a subset of M2 macrophages and human monocytes. Interestingly, a subset of CLEVER-1+ monocytes and TAMs are increased in conditions where immunosuppressive functions prevail, like pregnancy and cancer. In addition, high CLEVER-1 mRNA levels associates with poorer overall survival of breast cancer patients. Therefore, we want to investigate can CLEVER-1 be used as a surrogate marker for identifying cancer patients under immunosuppression. Since we detected CLEVER-1+ TAMs in 40% of breast cancer patients, we will characterize whether the amount of CLEVER-1+ TAMs in tumor biopsy samples correlates with the number of CLEVER-1+ monocytes in breast cancer patient blood. We will collect blood, plasma, healthy breast tissue and tumor tissue from 200 newly diagnosed treatment naive breast cancer patients. The tissue samples and the blood from each patient are characterized by flow cytometry to identify the activation status of monocyte and macrophage populations in relation to CD4/CD8, Th1, Th2, Th17 and Treg populations. By now, we have received 25 patient samples from which 12 have been initially analyzed for CLEVER-1 expression in the peripheral blood together with other immune cell populations. Our preliminary data show that CLEVER-1 expression on peripheral blood monocytes is increased in breast cancer patients who have higher levels of Tregs. Additionally, the amount of CD4+ effector T cells (Th1 and Th17) was decreased among patients, whereas the amount of Th2 CD4+ cells was increased when comparing to the blood of healthy volunteers. The abnormal Th1/Th2 ratio is linked to cancer progression. The expected outcome is that CLEVER-1 could be used as a surrogate marker to identify patients under immune suppression. These patients could benefit from anti-CLEVER-1 therapy or other clinically available immunomodulatory drugs (e.g. anti-CTLA-4; ipilimumab, anti-PD1; nivolumab).

Citation Format: Reetta E. Virtakoivu, Pia Boström, Riitta Aaltonen, Ilkka Koskivuo, Sirpa Jalkanen, Maija Hollmén. CLEVER-1 as a marker to identify breast cancer patients under immunosuppression [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 4740.
Abstract 4749: VISTA immune checkpoint deregulation in human triple-negative breast cancer
Authors: Gruber Joshua J. , Juntilla Marisa M. , Yang Soo-Ryum , Geller Benjamin , Jager Natalie , Lin Chien-Yu , Lipchik Andrew M. , Chen Justin , Ram Ashwin , Vinayak Shaveta , Telli Melinda L. , West Robert B. , Ford James M. , Snyder Michael P.
Published: August 2018

Immunotherapy for breast cancer has lagged behind the advances in other tumor types, suggesting the need for novel immune targets. VISTA/PD-1H is a checkpoint receptor expressed on immune cells that is an effective anti-cancer target in mouse models and now in early human clinical trials. We utilized an in vitro system to examine epigenetic changes arising early after depletion of breast cancer tumor suppressor proteins. This demonstrates that epigenetic alterations occur in normal human mammary cell lines lacking BRCA2 and result in regulation of VISTA through an NF-kB-dependent pathway. VISTA is strongly induced by growth factor limitation, an effect that is blunted by BRCA2-induced NF-kB activation and binding to a kB site in the VISTA promoter. This suggests that repression of VISTA may be an early consequence of BRCA2 depletion in pre-neoplastic tissues leading to immune infiltration. In contrast, in primary, untreated human triple-negative breast cancers, VISTA is over-expressed on tumor cells, independent of its presence on immune cells. VISTA positive TNBCs (14%) have significantly increased tumor infiltrating lymphocytes (TILs) whereas VISTA strong positive TNBCs (6%) have significantly decreased TILs and tumor PD-L1 staining, suggesting a higher level of immunosuppression. Gene expression and tumor genomics in TNBCs suggests that transcriptional networks to regulate physiologic immunoreceptor levels are corrupted during malignant transformation. In summary, this work identifies a BRCA2-loss-of-function signaling pathway that regulates an important immune checkpoint molecule via NF-kB activation in pre-neoplastic tissues and triple-negative breast cancers with important consequences for the immune microenvironment. We expect these observations to enhance immunotherapeutics for breast cancer.

Citation Format: Joshua J. Gruber, Marisa M. Juntilla, Soo-Ryum Yang, Benjamin Geller, Natalie Jager, Chien-Yu Lin, Andrew M. Lipchik, Justin Chen, Ashwin Ram, Shaveta Vinayak, Melinda L. Telli, Robert B. West, James M. Ford, Michael P. Snyder. VISTA immune checkpoint deregulation in human triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4749.
Abstract 4758: Use of highly multiplexed reference materials to facilitate validation of a clinical NGS tumor fusion RNA assay
Authors: Huang Catherine , Barua Subit , Philkana Deepika , Garlick Russell , Anekella Bharathi , Fernandes Helen
Published: August 2018

Introduction: Next Generation Sequencing assays for detection of tumor RNA fusions must undergo rigorous validation before clinical implementation. Validations include assessment of the assay's accuracy, precision, reproducibility and limits of detection or reportable range. Obtaining samples with all needed variants is difficult and time consuming. When found, they are often in limited quantities such that repeated testing for precision and reproducibility studies is not possible. Sample heterogeneity or lack of characterization further complicates interpretation of results. This study demonstrates how highly characterized, uniformly manufactured reference materials can facilitate clinical NGS assay validation. Methods: An RNA panel representing decreasing levels of 16 different RNA fusions and exon skipping events compared to the total cellular RNA was generated. GM24385 cell line was engineered to contain the fusion RNAs then formalin fixed, and the total RNA was extracted using the Maxwell RSC FFPE RNA kit. The fusion RNA was serially diluted into total RNA from similarly processed non-engineered GM24385 cells to create a panel with decreasing levels of each fusion. The panel was characterized by fusion-specific, TaqMan digital PCR assays to obtain a “truth set” on which to compare NGS results. Each panel member was tested in triplicate on three separate days as part of a validation study. Results: Precision and reproducibility were assessed quantitatively through analysis of the NGS unique reads. The intra-run precision varied among fusion targets with many targets having %CV of less than 15%, while a few targets had significantly higher variability between replicates. The linearity of the assay was good, with R-squared value greater than 0.95. Lower limits of detection for each fusion target were estimated using the digital PCR data and were different for different fusion targets; for example, detection of EML4-ALK fusion was five-fold more sensitive than detection of CD74-ROS1 fusion. Conclusions: Manufactured reference materials can supplement validation studies and their uniformity and digital PCR characterization allows greater insight into assay performance such as limits of detection than use of remnant specimens alone. The sensitivity of the NGS panel used was not the same for different fusion targets and, and underscores the need for highly multiplexed reference materials and for labs to test all clinically important fusions during validation

Citation Format: Catherine Huang, Subit Barua, Deepika Philkana, Russell Garlick, Bharathi Anekella, Helen Fernandes. Use of highly multiplexed reference materials to facilitate validation of a clinical NGS tumor fusion RNA assay [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 4758.
Abstract 4759: Designing clinical trials in tumor indications with a positive signal in phase 1
Authors: Chen Cong , Anderson Keaven , Mehrotra Devan V. , Rubin Eric H. , Tse Archie
Published: August 2018

Increasingly, the conventional proof-of-concept randomized Phase 2 study is skipped in favor of directly going to Phase 3 after an experimental oncology drug has demonstrated promising anti-tumor activity in Phase 1 with a small sample size. This shift in the balance between certainty and speed is especially evident in the immune-oncology space where the tremendous success of immune checkpoint inhibitors has brought unprecedented competition in the field. The aggressive approach can be very risky no matter how promising an experimental drug appears in Phase 1. In this presentation, we introduce a novel adaptive design that mitigates the risk of late-stage programs. The proposed approach starts with a Phase 2 trial and adds an option in the design that allows the expansion of the Phase 2 trial into Phase 3 if the interim result based on the Phase 2 endpoint is promising. If the decision is to not expand, the study is kept as a Phase 2 trial and the primary analysis is conducted at the end of Phase 2. Otherwise, the study is expanded into a Phase 3 trial and the primary analysis of the study is conducted at the end of Phase 3, utilizing data from all enrolled patients including those already used for the decision making in the ongoing trial. The proposed approach is more efficient than the conventional approach that conducts and analyzes Phase 2 and Phase 3 trials sequentially, and is less risky than the contemporary approach of skipping Phase 2. Importantly, we will show that this design controls overall Type I error regardless of the expansion criterion. As a result, the study can still be considered positive even without expansion to Phase 3.

Citation Format: Cong Chen, Keaven Anderson, Devan V. Mehrotra, Eric H. Rubin, Archie Tse. Designing clinical trials in tumor indications with a positive signal in phase 1 [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 4759.
Abstract 4779: Rogaratinib, a small molecule pan-FGFR inhibitor potently inhibits FGFR4-phosphorylation and exerts anti-tumor efficacy in vivo and in vitro
Authors: Politz Oliver , Gruenewald Sylvia , Walter Alexander , Siegel Franziska , Scholz Arne , Bender Sebastian , Kneip Christoph , Ellinghaus Peter
Published: August 2018

Fibroblast growth factor signaling plays an essential role in many physiological processes, including cell proliferation, survival, differentiation, migration, and apoptosis Over-activation of the pathway can lead to the development of various cancers. There are four different FGFR subtypes with over 20 ligands providing a very complex network of interactions and signaling.

FGFR4 plays an important role in normal physiology such as myogenesis muscle regeneration and bile acid synthesis Activating mutations in the kinase domain of FGFR4 have been shown to mediate cancer development, for example, breast cancer, hepatocellular carcinoma (HCC) or rhabdomyosarcoma.

Rogaratinib is a potent inhibitor of all FGFR subtypes with low nanomolar binding kinetics towards the four kinase domains. We explored the effect of rogaratinib on cell lines with high expression levels of FGFR4 mRNA, such as breast cancer cell line MDA-MB453 and rhabdomyosarcoma cell line SH30. We could show that the proliferation of these cell lines is potently inhibited by rogaratinib and accompanied with induction of apoptosis. The Mode-of-action analysis of rogaratinib activity in these cell lines showed inhibition of FGFR4-phosphorylation and downstream signaling at clinical relevant doses.

The in vitro efficacy of rogaratinib in FGFR4 dependent cell lines was further observed as in vivo efficacy in xenograft models of rhabdomyosarcoma and HCC.

Elevation of FGFR4 expression has been associated with resistance development in several cancer types such as breast cancer.

In line with the observed anti-tumor efficacy of rogartinib in vitro and in vivo in breast cancer, sarcoma and HCC models, RNA in situ hybridization by RNAscope, revealed high FGFR4 mRNA expression in TMA's from these tumor types..)

The combination of rogaratinib with standard-of-care provides further options for addressing resistance mechanisms mediated by elevated FGFR4 expression. In addition we have profiled a number of patient derived HCC xenograft models revealing the high efficacy of rogaratinib in models with elevated FGFR expression including FGFR4.

Citation Format: Oliver Politz, Sylvia Gruenewald, Alexander Walter, Franziska Siegel, Arne Scholz, Sebastian Bender, Christoph Kneip, Peter Ellinghaus. Rogaratinib, a small molecule pan-FGFR inhibitor potently inhibits FGFR4-phosphorylation and exerts anti-tumor efficacy in vivo and in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4779.
Abstract 478: Regulation of MUC13 by microRNA in colorectal cancer
Authors: Bartu Linda , Opattova Alena , Vodicka Pavel , Vymetalkova Veronika
Published: August 2018

Colorectal cancer (CRC) represents a serious health problem with high incidence and mortality worldwide. The main reason of high mortality is the late diagnosis. For the Duke stage I, the average 5-years survival rate is 90%. In comparison to patients diagnosed at IV stage, the 5-years survival rate decreases to 3%. Mucinous CRC is generally defined as having greater than 50 % of the tumor area with a mucinous differentiation by histological examination. The increased incidence occurs in proximal colon and among younger patients, as compared to nonmucinous adenocarcinoma. Mucinous CRCs have been found to have a higher Duke stage at diagnosis, and, consequently a poorer prognosis. Mucines are surface or secreted glycoproteins with high molecular weight. Dysregulation of their expression may lead to malignant transformation. MUC13 overexpression influences colon cancer tumorigenesis and metastasis via multiple oncogenic proteins. Recently, we have observed that rs1532602 polymorphism in microRNA (miRNA) binding sites in MUC13 gene was associated with significantly decreased risk of CRC. The aim of the present study is to investigate regulation in vitro by miRNAs as a potential mechanism for increased MUC13 expression in CRC. Two different miRNAs were selected in this investigation. MiRNA-4647 is predicted to bind to the rs1532602 in MUC13. MiRNA-137 functions as a tumour suppressor in colon. Additionally, we investigated the impact of miRNAs mentioned above on regulation of MUC13 in a cohort of 113 patients with sporadic CRC. The present work provides important insights into role of miRNAs which bind to MUC13. Definition of functions, mechanisms of MUC13 regulation and its impact on CRC may contribute to better prediction and individual treatment of CRC. Acknowledgement: Grant Agency of the Ministry of Health of the Czech Republic (AZV MZ 15-26535A) and Grant Agency of the Czech Republic GACR 17-16857S.

Citation Format: Linda Bartu, Alena Opattova, Pavel Vodicka, Veronika Vymetalkova. Regulation of MUC13 by microRNA in colorectal 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 478.