bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3746: Targeting adrenal androgen to treat prostate cancer
Authors: Wu Yue , Wang Jianmin , Tang Li , Pop Elena , Stocking John J. , Attwood Kristopher M. , Smith Gary J.
Published: August 2018

Background: Adrenal androgens are the most abundant androgens in humans, with dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEA-S) being the major adrenal androgens in circulation. Significantly, adrenal androgens may serve as substrates for intratumoral (intracrine) production of testosterone (T) and/or dihydrotestosterone (DHT), the ligands for androgen receptor (AR). However, adrenal androgens also have functions that are independent of AR. The present study addressed the capability of prostate cancer tissue to use adrenal androgens for T/DHT production, delineated signaling pathways activated by adrenal androgens, and determined whether adrenal androgens sustained tumor growth after androgen-deprivation therapy (ADT).

Experimental Procedures: Metabolism of adrenal androgen was evaluated ex vivo using fresh clinical prostate tissue specimens. The production of DHT or DHEA was determined using ELISA. Human prostate cancer cells lines were used for in vitro experiments. The effects of adrenal androgens on tumor growth were evaluated in vivo using xenografts of the human prostate cancer cell line VCaP in castrated or intact mice.

Results: 1) Human prostate tissue was able to produce DHT using DHEA-S at a physiologic concentration (3.5 μM). The production of DHT from DHEA-S was absolutely dependent on the activity of steroid sulfatase (STS). DHEA at a physiologic concentration (10 nM) was not a suitable substrate for DHT production, but was used by prostate tissue when available at a supra-physiologic concentration (3.5 μM) to produce DHT. 2) DHEA-S was able to stimulate growth of prostate cancer cells in vitro. The growth stimulatory effect of DHEA-S was mediated by AR-dependent and AR-independent signaling pathways. A subset of the genes modulated specifically by DHEA-S were distinctive from the T-modulated genes. 3) DHEA sustained tumor growth after castration. DHEA-stimulated growth was attributed to activation of the insulin-like growth factor 1 receptor (IGF1R) signaling pathway.

Conclusions: Prostate tissue can use adrenal androgens for DHT production. Adrenal androgens may stimulate prostate cancer growth using AR-dependent pathways that are mediated via intratumoral T/DHT production, or using AR-independent signaling pathways such as the IGF1R signaling pathway. Identification of targets and therapeutics to block the adrenal androgen-stimulated pathways may help develop novel strategies to treat prostate cancer.

Citation Format: Yue Wu, Jianmin Wang, Li Tang, Elena Pop, John J. Stocking, Kristopher M. Attwood, Gary J. Smith. Targeting adrenal androgen to treat prostate 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 3746.
Abstract 3761: Histone deacetylase inhibitor, Entinostat enhances the tumor specific immune response by generating T-cell central memory in the TME
Authors: Gaur Pankaj , Verma Vivek , Nandre Rahul , Vir Pooja , Wang Hua , Kang Baolin , Ordentlich Peter , Wang Lei , Gupta Seema , Khleif Samir N.
Published: August 2018

The epigenetic deregulation of T-cells and enhanced numbers of immunosuppressive cells in the tumor microenvironment (TME) are associated with decreased anti-tumor effects. Hence, targeting the epigenetic modifications using modulators such as histone deacetylase inhibitors (HDACi) provides the basis for a potential role for these agents in cancer-immunotherapy. In this context, Entinostat, an oral HDACi has been shown to reprogram the TME by impacting the numbers and activity of the immunosuppressive cell populations resulting in enhanced anti-tumor activity when combined with immune checkpoint blockade. However, the effect of this inhibitor on anti-tumor activity of effector T-cells is not well understood. Here, using animal tumor-models that require antigen-vaccine for T cell priming, we showed that Entinostat significantly synergized with tumor antigen-specific vaccine in delaying the tumor growth and prolonging mice survival. Further, we found that administering Entinostat in combination with antigen-priming resulted in sustainable long-term anti-tumor effects. Immunologically, Entinostat significantly enhanced the total CD8+, antigen-specific CD8+, granzyme B+ (GB+) CD8+, and antigen-specific GB+CD8+ T-cells in the TME when combined with the vaccine. Interestingly, we found that Entinostat led to a significant increase in the number of central memory T-cells (TCM; CD62L+CD44+) and decrease in the effector memory cells (TEM; CD62L-CD44+) in the TME. Notably, we did not find significant immune modulation in the periphery following the combination treatment indicating that the immune modulatory effects of Entinostat were localized to TME and hence may be associated with less systemic toxicity. Taken together these results highlight the ability of Entinostat to significantly enhance anti-tumor responses partly through the generation of central-memory which may translate to durable responses. These data provide further support on the potential of utilizing Entinostat in combinatorial immunotherapeutic strategies.

Citation Format: Pankaj Gaur, Vivek Verma, Rahul Nandre, Pooja Vir, Hua Wang, Baolin Kang, Peter Ordentlich, Lei Wang, Seema Gupta, Samir N. Khleif. Histone deacetylase inhibitor, Entinostat enhances the tumor specific immune response by generating T-cell central memory in the TME [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 3761.
Abstract 3762: Novel RORγt agonist induces antitumor immune effect through enhancement of tumor antigen-specific CD8+ T-cell infiltration into the TME
Authors: Gaur Pankaj , Verma Vivek , Nandre Rahul , Vir Pooja , Wang Hua , Kang Baolin , Carter Laura , Hu Xiao , Liu Xikui , Gupta Seema , Khleif Samir
Published: August 2018

RORγt is a master transcription factor, which regulates the proliferation and functionality of Type 17 T-cells (TH17 and Tc17). Recent nonclinical studies have shown that in addition to promoting effector differentiation, stemness and plasticity, RORγt also inhibits Treg differentiation, an important component of suppressive tumor microenvironment (TME). Therefore, stimulation of RORγt by synthetic, small-molecule agonists holds promise as an immunotherapy. We tested the effects of a RORγ agonist (LYC-54143; 100 mg/kg BID given continuously till the end of study) on the tumor growth and survival in mice in combination with a tumor antigen-specific vaccine (3 doses, one week apart), which is required for proper priming in this model. We found that the RORγ agonist resulted in a highly significant enhancement of the antitumor effect delaying the tumor growth (p≤0.001 at day 21 compared to vaccine alone treatment) and prolonging survival. At day 36 after tumor implantation, 80% of mice survived in the RORγ agonist + vaccine treatment group compared to 0% survival following RORγ agonist or vaccine alone treatments. Immunologically, we found that RORγ agonist treatment led to a significant decrease in the numbers of Tregs and a significant increase in IL-17+ IFNγ+ T cells in the TME. Moreover, RORγ agonist treatment led to a significant increase in the numbers of T cells including increased antigen-specific CD8+ T-cells in the TME. Interestingly, scheduling RORγ agonist before or with the vaccine demonstrated comparable antitumor activity, suggesting that the scheduling of RORγ agonist treatment does not affect the therapeutic outcomes. These results show that an RORγ agonist can enhance the antitumor immune response by enhancing effector functions and by decreasing immune suppression in the TME by increasing cytokine production and switching the differentiation of immune-suppressive Tregs to effector Th17 cells and support that RORγ agonists are promising immune-modulatory agents in cancer.

Citation Format: Pankaj Gaur, Vivek Verma, Rahul Nandre, Pooja Vir, Hua Wang, Baolin Kang, Laura Carter, Xiao Hu, Xikui Liu, Seema Gupta, Samir Khleif. Novel RORγt agonist induces antitumor immune effect through enhancement of tumor antigen-specific CD8+ T-cell infiltration into the TME [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 3762.
Abstract 3765: An anti-CD137 antibody can show greater efficacy in syngeneic mouse tumor models when combined with other immuno-oncology (IO) therapies
Authors: Robinson Matthew J. , Osma-Garcia Ines , Coates-Ulrichisen Jane , Watkins Amanda , Mosely Suzanne , Lloyd Chrisopher , Williams Geoff , Morrow Michelle , Dovedi Simon , Herbst Ronald , Wilkinson Robert
Published: August 2018

CD137 is a costimulatory molecule associated with tumour infiltrating lymphocytes and cytotoxic T lymphocytes in particular. Murine anti-CD137 monoclonal antibodies (mAbs) have shown potent anti-tumour effects in syngeneic tumour models, which can be further enhanced by combination with checkpoint inhibitor (CPI) mAbs to PD-1, or CTLA-4 and OX40. Two anti-CD137 monoclonal antibodies (mAbs), Urelumab and Utomilumab, are in development for both haematological and solid cancers, and studies include combinations with other IO therapies such as anti-PD-1, anti-PD-L1 and anti-OX40 mabs. We studied the impact of combining anti-CD137 with CPIs anti-PD-L1, anti-CTLA4, and IO agonist GITRL fusion protein (GITRL-FP) and anti-OX40 on pharmacodynamic responses, tumour growth and survival in mouse syngeneic models. In a responsive model, CT26, combining anti-CD137 with anti-PD-L1 had a greater impact on tumour growth inhibition, which correlated with an enhanced proliferation of CD8 T cells, as determined Ki67 expression using flow cytometry. In MC-38, a model that is less sensitive to both monotherapies, the combination was synergistic and resulted in enhanced tumour growth inhibition. Next, we compared the anti-CTLA4, anti-OX40 and GITRL-FP as monotherapies and in combination with anti-CD137 in the CT26 model. As before, anti-CD137 alone induced CD8 T cell proliferation, whereas anti-OX40 and GITRL-FP stimulated expansion of CD8 and CD4 T cells. Interestingly, in all combinations anti-CD137 appeared to enhance both CD8 and CD4 T cell proliferation. Anti-CTLA4 and GITR-FP had a greater impact on tumour growth inhibition when combined with anti-CD137. In summary, in vivo models indicate that anti-CD137 mAbs are a promising therapy in cancer, but the most benefit may be obtained in combination with other IO therapies.

Citation Format: Matthew J. Robinson, Ines Osma-Garcia, Jane Coates-Ulrichisen, Amanda Watkins, Suzanne Mosely, Chrisopher Lloyd, Geoff Williams, Michelle Morrow, Simon Dovedi, Ronald Herbst, Robert Wilkinson. An anti-CD137 antibody can show greater efficacy in syngeneic mouse tumor models when combined with other immuno-oncology (IO) therapies [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 3765.
Abstract 3785: Loss of vacuolar ATPase in hematopoietic stem cells promotes breast tumor progression and pathogenesis
Authors: Sahoo Manoranjan , Katara Gajendra K. , Bilal Mahamood Y. , Fleetwood Sara , Kulshrestha Arpita , Ibrahim Safaa A. , Beaman Kenneth D.
Published: August 2018

Vacuolar ATPase (V-ATPase) is expressed intracellularly on vesicular membranes, and controls intracellular pH and acidification of various intracellular vesicles. Most cancer cells express V-ATPase on their cell surface where they appear to acidify the extracellular environment and help cancer cells to metastasize. We have previously demonstrated that the a2 isoform of V-ATPase (a2V) of mammary epithelial cells plays a key role during breast tumor progression and metastasis. However, the role of a2V of hematopoietic stem cells during breast tumor pathogenesis is not known. Therefore, to understand the role of a2V in hematopoietic stem cells during breast tumor progression, we conditionally knocked out a2V (cKO) from the hematopoietic stem cells of mice. We observed that deletion of a2V leads to a significant reduction of CD4+ and CD8+ T cells in the periphery and spleen of the a2V cKO mice compared to control mice. We hypothesized that reduction of helper CD4+ cells, or cytotoxic CD8+ T cells would result in larger and faster-growing tumors in the a2V-cKO mice. After implantation of a syngeneic tumor cell line (E0771) in the breast tissue of mice, we observed that deletion of a2V led to larger and faster-growing breast tumors compared to control mice. Further investigation of the tumor microenvironment revealed that there was a significant reduction in percentage of CD4+ and CD8+ T cells in the a2V-cKO mice compared to control mice. Targeted RNAseq of tumor microenvironment demonstrated that out of 499 genes that were analyzed, only 3 genes were significantly upregulated while 144 genes were significantly downregulated in the a2V cKO mice. The 144 downregulated genes included genes for pro-inflammatory cytokines, T cell specific genes, death receptors and proapoptotic genes. Collectively, these results demonstrate that deletion of a2V from the hematopoietic stem cells leads to a decrease in CD4+ and CD8+ T cells in the periphery, which results in a faster-growing and larger breast tumor. Further study is needed to understand the relative contribution of CD4+ or CD8+ T cells during breast tumor pathogenesis.

Citation Format: Manoranjan Sahoo, Gajendra K. Katara, Mahamood Y. Bilal, Sara Fleetwood, Arpita Kulshrestha, Safaa A. Ibrahim, Kenneth D. Beaman. Loss of vacuolar ATPase in hematopoietic stem cells promotes breast tumor progression and pathogenesis [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 3785.
Abstract 3791: Triggering receptor expressed in myeloid cells 1 (TREM1) is a predictive marker for breast cancer therapeutics
Authors: Pullikuth Ashok K. , Chifman Julia , Routh Eric D. , Chou Jeff , Jin Guangxu , Black Michael A. , Miller Lance D.
Published: August 2018

Triggering receptor expressed on myeloid cells-1 (TREM1) is a cell surface immunoglobulin that amplifies pro-inflammatory responses in myeloid cell populations and has been implicated in carcinogen-induced liver cancer. However, its role in breast cancer is unknown. In a recent screen to identify gene expression patterns associated with breast cancer outcomes, we identified a significant and reproducible positive association between TREM1 expression and breast cancer recurrence in two large breast cancer cohorts comprising 1,984 and 616 patients (p<0.001 and p=0.003, respectively). TREM1 expression was significantly associated with recurrence in both basal-like (p<0.001) and luminal tumor subtypes (Luminal A, p=0.047; Luminal B, p=0.022) and remained significant (p<0.001) in a multivariate Cox regression model, independent of histologic grade, tumor size, nodal status, patient age, treatment and molecular subtype. In pre-treatment biopsies of patients treated with neoadjuvant chemotherapy, high TREM1 expression was associated with a significant decrease in pathologic complete response (pCR) rate (p<0.001), especially in those tumors estimated to have high levels of lymphocytic infiltrate that otherwise associated with an increased pCR rate, consistent with an immune regulatory function for TREM1. Bioinformatics analysis of genes reproducibly correlated with TREM1 expression in breast tumors revealed significant enrichment in myeloid immune cell populations (p<0.001) in general, and neutrophils (p<0.001), M0 macrophages (p<0.001) and M2 macrophages (p<0.001), in particular. Immunohistochemical analysis of breast tumor tissues confirmed colocalization of TREM1 protein expression with the pan-macrophage marker, CD68. These findings, together with the established role for TREM1 in promoting inflammation, fit a model where tumor-infiltrating TREM1-positive myeloid cells impact breast cancer outcomes by promoting immune evasion.

Citation Format: Ashok K. Pullikuth, Julia Chifman, Eric D. Routh, Jeff Chou, Guangxu Jin, Michael A. Black, Lance D. Miller. Triggering receptor expressed in myeloid cells 1 (TREM1) is a predictive marker for breast cancer therapeutics [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 3791.
Abstract 3798: Gut microbiota modulates adoptive cell therapy via CD8α dendritic cells
Authors: Uribe-Herranz Mireia , Bittinger Kyle , Rafail Stavros , Guedan Sonia , Pierini Stefano , Tanes Ceylan , Gill Saar A. , Tany Janos , Morgan Mark A. , Bushman Frederic D. , June Carl H. , Facciabene Andrea
Published: August 2018

Adoptive T-Cell therapy (ACT) is a promising new modality for malignancies. Here, we report that adoptive T-cell efficacy in tumor-bearing mice is significantly affected by differences in the native composition of the gut microbiome, treatment with antibiotics or by heterologous fecal transfer. Depletion of bacteria with vancomycin, decreased the rate of tumor growth in mice receiving ACT, whereas treatment with neomycin and metronidazole had no effect, indicating the role of specific bacteria in host response. Vancomycin treatment induced an increase in systemic CD8α+ dendritic cells, which mediated a sustained systemic expansion of adoptively transferred antitumor T cells in an IL-12-dependent manner. In subjects undergoing allogeneic hematopoietic cell transplantation, we found that oral vancomycin also increased IL-12 levels. Collectively, our findings demonstrate an important role played by the gut microbiota in the antitumor effectiveness of ACT and suggest new avenues to improve response to ACT by altering the gut microbiota.

Citation Format: Mireia Uribe-Herranz, Kyle Bittinger, Stavros Rafail, Sonia Guedan, Stefano Pierini, Ceylan Tanes, Saar A. Gill, Janos Tany, Mark A. Morgan, Frederic D. Bushman, Carl H. June, Andrea Facciabene. Gut microbiota modulates adoptive cell therapy via CD8α dendritic cells [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 3798.
Abstract 3802: Human NKG2D ligand regulation of Natural Killer cell function and its implications in cancer and inflammation
Authors: Dhar Payal , Wu Jennifer
Published: August 2018

The significance of ligand-induced activation of Natural Killer cell activating receptor NKG2D in controlling tumor growth has been well established in various experimental animal models. Amongst different types of NKG2D ligands, MHC I chain related molecule MICA/B is expressed most prevalently in various human malignancies, but has not been found in rodent family. Proteolytic-mediated shed-form of soluble MICA/B (sMIC) has been shown to suppress the immune system and correlate with advanced disease stages in cancer patients. Previous studies in our lab using two humanized bi-transgenic mice, one expressing native ligand (TRAMP/MICB) and alternatively expressing the shedding-resistant mutant (TRAMP/MICB.A2) revealed opposite roles of membrane-bound and soluble form of MIC in regulating tumor immunity. Mice with high levels of sMIC exhibited increased tumor progression, whereas mice expressing membrane-bound MICB.A2 in tumors enjoyed tumor-free survival. These findings raised the intriguing question of why do these structurally similar forms of MIC have vastly different effects on tumor immunity. To address this question, we investigated the signaling events and functional outcomes in NK cells upon stimulation by the two forms of ligands. In vitro co-culture studies of NK cells with tumor cell lines expressing sMICB and MICB.A2 revealed elevated pro-inflammatory cytokine production by NK cells upon stimulation with sMICB. In contrast, NK cells stimulated with MICB.A2 displayed enhanced expression of cytotoxicity mediators and signaling molecules of cytotoxicity pathway. This suggests that signaling through sMICB may polarize the NKG2D signaling pathways with preferential activation of inflammatory cytokine pathways. Our data has uncovered a new potential mechanism whereby sMIC promotes tumor progression and supports the notion that sMIC is a viable target for cancer immunotherapy.

Citation Format: Payal Dhar, Jennifer Wu. Human NKG2D ligand regulation of Natural Killer cell function and its implications in cancer and inflammation [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 3802.
Abstract 381: Uptake of biguanides in mitochondria appears to be related to increased glucose uptake and medium acidification but not necessarily related to inhibition of growth of bladder and colon cancer cells
Authors: Lea Michael A. , Kim Haneulsol , desBordes Charles
Published: August 2018

Biguanides including metformin have been used in the treatment of Type 2 diabetes and they are under investigation as anti-cancer agents. Investigation on the mechanism of action of biguanides has largely focused on inhibition of complex 1 in the mitochondrial electron transport chain with subsequent activation of AMP-dependent protein kinase. Studies by Bridges et al. (BMC Biology 14:65, 2016) indicate that inhibitory effects on complex 1 in cells require uptake of biguanides into the mitochondria. In the present work the action of two biguanides that enter the mitochondria (buformin and phenformin) were compared with the action of two biguanides with poor uptake (phenyl biguanide and proguanil). Effects on growth, glucose uptake and medium acidification were studied with two human colon cancer cells (Caco-2 and HT29) and seven bladder cancer cell lines (5637, HT1197, HT1376, RT4, T24, TCCSUP and UM-UC-3). After incubation for 72 hours with 25 µM and 50 µM concentrations of the biguanides, growth inhibition was greatest with proguanil followed by phenformin and with little or no growth inhibition at these concentrations with buformin and phenylbiguanide. In contrast, increased glucose uptake and acidification of the medium was observed with buformin and phenformin with no change or decreased medium acidification with phenyl biguanide and proguanil. Studies with buformin over a concentration range from 10 µM to 500 µM indicated that there was generally growth inhibition at concentrations greater than 50 µM. Studies on medium acidification and glucose uptake revealed biphasic responses with increases at lower concentrations that were reversed at higher concentrations of buformin where growth inhibition was observed. The data suggested that the effect of biguanides on glucose metabolism requires mitochondrial uptake while the mechanism for growth inhibition by biguanides remains to be established.

Citation Format: Michael A. Lea, Haneulsol Kim, Charles desBordes. Uptake of biguanides in mitochondria appears to be related to increased glucose uptake and medium acidification but not necessarily related to inhibition of growth of bladder and colon cancer cells [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 381.
Abstract 3815: Decomposition of parameters contributing to the improved therapeutic index of ADCs obtained by GlycoConnect™ and HydraSpace™ Technologies
Authors: Delft Floris van , Janssen Brian , Geel Remon van , Wijdeven Marloes , Verkade Jorge , Berkel Sander van
Published: August 2018

We have found that conjugation of toxic payloads to the native glycan of a monoclonal antibody by chemoenzymatic remodeling (GlycoConnect™ technology) consistently provides antibody-drug conjugates (ADCs) with enhanced therapeutic index (TI) increases versus those of ADCs prepared by mainstream clinical technologies. For example, head-to-head comparison of GlycoConnect™ ADCs -based on the same antibody and payload components- with the marketed drugs Adcetris and Kadcyla revealed an improvement in both efficacy and safety, which is further enhanced by use of a polar HydraSpace™ technology. Importantly, a similar improvement in TI was noted for comparison of a GlycoConnect™ ADC versus or a site-specific ADC derived from a cysteine-engineered antibody, the most important emerging technology in the clinic. With the aim to better understand the superiority of ADCs based on combined GlycoConnect™ and HydraSpace™ technologies, we have performed in-depth in vitro and in vivo investigation into factors contributing to the overall performance of ADCs prepared by glycan conjugation with a polar spacer. For example, it is found that stability is significantly improved versus mainstream technologies in terms of aggregation and linker stability. Rapid aggregation was observed for both Kadcyla and Adcetris, as well as ADC decomposition due to retro-Michael reaction resulting in free payload (for Adcetris) or albumin-conjugated DM1 (for Kadcyla), as indicated by LC-MS and immunoassay analysis. The latter image is confirmed by in vivo pharmacokinetic analysis, showing a dramatic difference in total antibody versus total conjugated antibody by premature release of payload. Finally, analysis with a solid 3D tumoroid model indicates enhanced penetration of site-specific versus randomly conjugated ADC, which may also contribute to better efficacy in solid tumors. The data generated further underline the importance of conjugation and linker technology as critical quality attributes for next-generation ADCs with higher therapeutic index.

Citation Format: Floris van Delft, Brian Janssen, Remon van Geel, Marloes Wijdeven, Jorge Verkade, Sander van Berkel. Decomposition of parameters contributing to the improved therapeutic index of ADCs obtained by GlycoConnect™ and HydraSpace™ Technologies [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 3815.
Abstract 3820: Novel PD-1/GITRL bispecific fusion protein delivering concurrent T cell co-stimulation and checkpoint pathway inhibition
Authors: Dovedi Simon J. , Seaman Jonathan , Osma-Garcia Ines , Deschler Katharina , Poon Edmund , Herbst Ronald , Wilkinson Robert
Published: August 2018

Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) is a co-stimulatory receptor, and programmed cell death protein 1 (PD-1) is an important co-inhibitory receptor involved in controlling T-cell activation and function. Preclinical studies using murine cross-reactive molecules demonstrate that the combination of an αPD-1 monoclonal antibody (mAb) with a GITR-ligand (GITRL) fusion protein can elicit synergistic antitumor activity and long-term survival benefit in syngeneic mouse tumor models. Here we describe the activity of two bispecific fusion proteins, each comprising two GITR ligand trimers fused to the Fc region of an αPD1 mAb, in an IgG1 (MEDI5771) or IgG4P (MEDI3387) format. We show that these bispecific molecules are able to concurrently engage both targets and are effective in cell-based PD-1 and GITR reporter assays, and in a range of human primary T-cell assays. Furthermore, the PD-1/GITRL FP bispecific constructs elicit T-cell responses in vitro that are at least equivalent to a combination of a) the parental molecules and b) MEDI1873 (a GITR agonist currently undergoing clinical evaluation) and an αPD-1 mAb. Moreover, using surrogate molecules we demonstrate that IV administration leads to dose-dependent pharmacodynamic responses on CD4+ and CD8+ T cells for both isotypes of the bispecific molecule. These data demonstrate that the antitumor activity for these novel molecules is equivalent to the combination of GITRL-FP and PD-1 mAb in the B16 model. In summary, the simultaneous dual targeting of PD-1 and GITR pathways mediated by these two novel bispecific molecules induces robust T-cell activity that is at least equivalent to a combination of molecules targeting these pathways, thereby demonstrating strong potential as a novel anticancer therapy.

Citation Format: Simon J. Dovedi, Jonathan Seaman, Ines Osma-Garcia, Katharina Deschler, Edmund Poon, Ronald Herbst, Robert Wilkinson. Novel PD-1/GITRL bispecific fusion protein delivering concurrent T cell co-stimulation and checkpoint pathway inhibition [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 3820.
Abstract 3838: Inhibition of the spectraplakin protein MACF1 sensitizes glioblastomas to BCNU
Authors: Borlay Danielle , Bonner Kala , Kutten Orica , Atkinson Katelyn , Quick Quincy A.
Published: August 2018

Glioblastoma heterogeneity and diversity is recognized as a major contributing factor to the resistance of these cancers to clinical chemo- and radiotherapy treatment regimens. This diversity is attributed in part to a plethora of novel unevaluated proteins whose functions participate in glioblastoma etiology, progression, and recurrence. One such protein is the cytoskeletal cross-linker, Microtubule Actin Crosslinking Factor 1 (MACF1), which we have previously shown was expressed at high levels in glioblastoma tissue as compared to its absence in normal brain tissue. In support of these findings immunoblotting expression analysis in this study showed that MACF1 protein expression was upregulated in response to BCNU exposure in glioblastoma cells. Subsequently, experiments were conducted to assess the combinatorial effect of MACF1 inhibition in conjunction with BCNU treatment. A CRISPR-Cas9 genome editing approach was used to inhibit MACF1 function prior to drug exposure. Cell proliferation analysis revealed a cooperative synergistic effect of MACF1 inhibition and BCNU treatment on the reduction of glioblastoma cells as compared to cells treated with either BCNU or CRISPR inhibition of MACF1 alone. In conclusion, upregulation of MACF1 in response to BCNU suggests that this spectraplakin protein may play a role in the mechanistic resistance of glioblastomas to BCNU and the therapeutic prognosis of glioblastomas treated with this drug. Additionally, this study demonstrated that MACF1 inhibition is a novel neoadjuvant approach that enhances the therapeutic efficacy of BCNU.

Citation Format: Danielle Borlay, Kala Bonner, Orica Kutten, Katelyn Atkinson, Quincy A. Quick. Inhibition of the spectraplakin protein MACF1 sensitizes glioblastomas to BCNU [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 3838.
Abstract 3843: The potential of heat shock proteins 70/90 as molecular therapeutic targets in breast cancer
Authors: Kim Ho Young , Kim Sanghwa , Kim Lee Su
Published: August 2018

Purpose: Heat shock proteins (hsps) are molecular chaperones that are synthesized by cells in response to various stress conditions. The expression of hsps have been shown to be associated with carcinogenesis and the expression of hsps have been implicated in the biological behavior of tumors. Recently, hsps have emerged as novel molecular targets in anticancer protocols. The objectives of this study were to investigate the significance of hsp 70/90 in breast carcinogenesis and effect of geldanamycin (a blocker of hsp 90) and quercetin (a blocker of hsp 70) on growth inhibition in different breast cancer cell lines. Methods: Breast tissues from 82 patients were obtained between January 2010 and December 2010 at the Division of Breast & Endocrine Surgery, Hallym University Sacred Heart Hospital. Expression of hsp 70/90 was studied by immunohistochemistry (IHC) on tissue sections from 63 breast carcinomas and 19 benign breast tissues. Both cytoplasmic and nuclear expression was measured. Expression of hsp 70/90 was also analyzed by use of a Western blot with the breast cancer cell lines. We next investigated the effects of blockers of hsp 70/90 on cell growth of the human breast cancer cell lines. Results: More prominent hsp 90 expression was observed in malignant tissue than in benign tissue by both cytoplasmic and nuclear IHC staining (p<0.001, p<0.001). Nuclear hsp 90 expression was associated with a positive lymph node status (p=0.003) and the presence of poorly differentiated tumors (p=0.028). Expression of hsp 70 was not different in malignant and benign tissues as determined by both cytoplasmic and nuclear IHC staining. The breast cancer cell lines all expressed hsp 70/90. Geldanamycin markedly inhibited the cell growth of these breast cancer cell lines in a dose dependent manner and induced apoptosis in the cell lines. Quercetin inhibited cell growth of the cell lines less efficiently. Conclusion: The expression of hsp 90 was associated with breast carcinogenesis and the presence of more aggressive tumors. Geldanamycin inhibited cell growth of hsp 90 expressing breast cancer cell lines. We suggest that Hsp 90 may be a possible molecular target against breast cancer.

Citation Format: Ho Young Kim, Sanghwa Kim, Lee Su Kim. The potential of heat shock proteins 70/90 as molecular therapeutic targets in 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 3843.
Abstract 3847: Inhibition of GCN2 sensitizes ASNS-downregulated cancer cells to asparaginase by disrupting amino acid response
Authors: Nakamura Akito , Nambu Tadahiro , Ebara Shunsuke , Hasegawa Yuka , Toyoshima Kosei , Tsuchiya Yasuko , Tomita Daisuke , Fujimoto Jun , Kurasawa Osamu , Takahara Chisato , Kawamura Ayumi , Nishigaki Ryuichi , Satomi Yoshinori , Hata Akito , Hara Takahito
Published: August 2018

General control nonderepressible 2 (GCN2) plays a major role in cellular response to amino acid limitation. Although maintenance of amino acid homeostasis is critical for tumor growth, little is known concerning the contribution of GCN2 to cancer cell proliferation and its potential as a therapeutic target. In this study, we demonstrate that inhibition of GCN2 with small-molecule compounds sensitizes cancer cells to anti-leukemic agent asparaginase (ASNase). We first tested acute lymphoblastic leukemia (ALL) cells and showed that treatment with GCN2 inhibitors rendered ALL cells sensitive to ASNase by preventing the induction of asparagine synthetase (ASNS). The prevention of ASNS expression suppresses asparagine synthesis and thus inhibits protein translation. In addition, comprehensive gene expression profiling revealed that the combined treatment induced stress-activated MAPK pathway and thereby triggering apoptosis. Using cell-panel analyses, we also identified a clear synergy between GCN2 inhibitors and ASNase in acute myelogenous leukaemia (AML) and pancreatic cancer cells besides ALL. Notably, basal ASNS expression levels were significantly correlated with sensitivity to combinatorial treatment. Furthermore, the combination displayed robust antitumor activity in mouse xenograft models of ALL, AML, and pancreatic cancer. These results demonstrate a novel mechanistic insight into a role of GCN2 in amino acid response and provide a rationale for further investigating GCN2 inhibitors for the treatment of cancer.

Citation Format: Akito Nakamura, Tadahiro Nambu, Shunsuke Ebara, Yuka Hasegawa, Kosei Toyoshima, Yasuko Tsuchiya, Daisuke Tomita, Jun Fujimoto, Osamu Kurasawa, Chisato Takahara, Ayumi Kawamura, Ryuichi Nishigaki, Yoshinori Satomi, Akito Hata, Takahito Hara. Inhibition of GCN2 sensitizes ASNS-downregulated cancer cells to asparaginase by disrupting amino acid 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 3847.
Abstract 3849: Identification and optimization of chemical compounds as potent agonists of human STING with anticancer activity in mice
Authors: Wu Jian Hui
Published: August 2018

The host STING pathway plays a critical role in innate immune sensing of cancer, that drives type-I interferons (IFNs) production and promotes aggressive antitumor responses. Compound DMXAA is an agonist of mouse STING (mSTING) and demonstrated potent antitumor activities in several tumor models, including melanoma. However, DMXAA cannot activate human STING (hSTING), which provides a possible rationale for its failure in recent clinical trial. In this project, we aim to identify novel chemical compounds as potent STING agonists. By combining structure-based drug design and in vitro assays, we have discovered two initial hits as STING agonists that belong to two different chemical scaffolds. Direct binding of our compounds with hSTING was confirmed by Surface plasmon resonance (SPR) analysis. Chemical optimization of our initial hits has led to compound #150 and #171. We demonstrated that #150 and #171 at 10 uM have substantially activated STING pathway in HEK293 cells that were transiently transfected with plasmid expressing hSTING. Compound #171 at 20 uM potently activated STING signaling in human THP-1 cells that harbor hSTINGHAQ as well as PBMC cells from a panel of human donors. Importantly, with DMXAA as the control, we demonstrated that #171 at 20 mg/kg via i.p. has significant antitumor effect against TRAMP-c1 tumor in C57/BL6 mice.

Citation Format: Jian Hui Wu. Identification and optimization of chemical compounds as potent agonists of human STING with anticancer activity in mice [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 3849.
Abstract 3861: 3D ex vivo PDX cell model screening to better predict in vivo outcome
Authors: Xu Xiaoxi , Li Zhongliang , Liu Yan , Meng Fanxiu , Lu Yu , Zhang Songling , Dong Chunlan , Xing Frank , Shi Qian
Published: August 2018

Patient-derived xenograft (PDX) models have been highly demanded in preclinical drug discovery and development due to several advantages over conventional 2D cell culture system: 1) reflection of the heterogeneity, molecular and histopathologic signatures of the original tumor than cell lines or genetically engineered mouse models, and 2) certain degree of correlation of their drug-response profiles with clinical response. Certain limitations of using PDX models, however, do exist, including low throughput in candidate drug screening, lack of dose-response curves, high cost and time-consuming studies, and progressive loss of human-derived stromal elements over passages. To overcome these disadvantages, we sought to develop ex vivo 3D assay format on cells isolated from early passage PDX models, and provide genetic mutation information to better interpret results. Moreover, we attempted to incorporate immunotherapy strategy into the 3D system as well. We have recently built up an ex vivo cell bank containing more than 300 frozen ex vivo tumor cells dissociated from freshly isolated PDX tumor tissues. We have collected five panels of such frozen ex vivo cells, each panel representing more than 50 models in a specific tumor type. SOC compounds corresponding to each unique tumor panel were tested, and will be discussed. Moreover, we have established a 3D immune cell and ex vivo somatic tumor cell co-culture system to evaluate drug efficacy of immune checkpoint inhibitors aPD1, aPDL1 and CTLA4 antibodies either as single agent or in combination with small-molecule inhibitors. The data will be presented. In summary, we have developed a robust and reproducible 3D ex vivo assay platform for medium-throughput compound screening with bioinformatics information for data interpretation.

Citation Format: Xiaoxi Xu, Zhongliang Li, Yan Liu, Fanxiu Meng, Yu Lu, Songling Zhang, Chunlan Dong, Frank Xing, Qian Shi. 3D ex vivo PDX cell model screening to better predict in vivo outcome [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 3861.
Abstract 3865: ActivSignal platform technology for direct multiplex analysis of signaling pathway activation profiles
Authors: Alexandrov Ilya , Brandina Irina , Rahimi Nader , Sherman Michael , Yaglom Julia , Meyer Rosana , Ali Marwa , Ersoy Esma
Published: August 2018

The multiplex IPAD assay technology from ActivSignal monitors activity of 26 major signaling pathways by examining phosphorylation events or expression of more than 60 most relevant human and mouse protein targets. The technology uses antibodies against established and well known targets that reflect activities of canonical pathways. We utilize paired antibodies for each protein target for high specificity, and these pairs are carefully selected and tested. Only if both antibodies in a pair bind to their specific target molecule will the detection take place during the analysis phase - avoiding false hits. The IPAD assay can be used to profile samples made from cell culture, tissues, blood, etc. The IPAD assay requires only minute amounts of sample, which can be easily taken from your precious sample where available amounts may be limited. Applications include:

Direct snapshot of signaling pathway activity rather than downstream effects.

Understanding the physiological effects of a drug.

What pathways are regulated in response to lead candidate drug?

Does the drug of interest cause biologically significant secondary and off target effects?

Evaluation of signaling pathway activities in response to shRNA, CRISPR, hormones, stresses and other treatments.

Elucidating the signaling proteome in a medium-throughput format (96-well format).

Profiling of tissue samples from patients and animals.

Signaling profiling of tumor biopsies.

In one application, our collaborators used the IPAD assay to understand signaling effects of a novel protein, TMIGD1, whose expression is markedly downregulated in renal cancer. The Activsignal platform analysis showed changes in activities of several major pathways, e.g. inhibition of cell cycle and cell proliferation after reintroduction of TMIGD1. In another application of our platform our collaborators studied the mechanism of action of a small molecule inhibitor that targets Hsp70. They used ActivSignal platform technology to profile signaling pathways in response to the inhibitor. They identified several pathways whose activities were affected by the inhibitor. ActivSignal is a breakthrough, low-cost tool for the analysis of signaling pathway activation profiles. It enables researchers to rapidly and efficiently gain insight into function of their favorite drug molecules, genes etc.

Citation Format: Ilya Alexandrov, Irina Brandina, Nader Rahimi, Michael Sherman, Julia Yaglom, Rosana Meyer, Marwa Ali, Esma Ersoy. ActivSignal platform technology for direct multiplex analysis of signaling pathway activation profiles [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 3865.
Abstract 3866: A novel assay for rapid identification and isolation of single intact circulating tumor cells
Authors: Huang Lili , Shen Le , Yu Min , Liu Paul
Published: August 2018

We present a magnetic bead assay for rapid identification and isolation of single intact circulating tumor cells (CTCs). Cell viability is essential for cell culture and downstream molecular analysis. Many CTC isolation technologies, including CellSearch, uses fixation and fluorescent imaging for CTC detection that the captured CTCs are no longer viable. This novel assay utilize magnetic sensing for CTC identification, and therefore preserve CTC integrity and viability. The assay is performed on a microelectronic chip which also generates spatially-patterned microscopic magnetic field to sort single CTCs. We evaluated the performance metrics of the assay with EGFP-expression breast cancer patient derived CTC cells mixed with peripheral blood mononuclear cells (PBMCs) in vitro. Cell suspensions were mixed with magnetic microbeads coated with anti-EpCAM antibodies and then isolated with a magnet. Different amount (20, 50, 100, 500) of CTCs from serial dilution were spiked into 5x10^6 human PBMCs labelled with Alexa Flour 488 anti-human CD45 antibodies. After the assay, no PBMCs are visible which shows high purity in recovered CTCs. Recovery rate, defined by the ratio of the mean of the recovered CTC count to the mean of the spiked CTC count, is in the range of 87% to 92% for the various CTC spiked-in amount. In each spiking experiment, triplicate measurement and controls are used. Total assay time is less than 80 minutes. Conclusion: A rapid assay for isolation and detection of CTCs from PBMCs is demonstrated which shows high recovery rate and viability. This novel assay has great potential for single intact rare cell isolation.

Citation Format: Lili Huang, Le Shen, Min Yu, Paul Liu. A novel assay for rapid identification and isolation of single intact circulating tumor cells [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 3866.