bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2021: Identification of novel brachyury target genes in lung cancer
Authors: Hu Yunping , Hsu Wesley
Published: July 2016

Lung cancer is the leading cause of cancer-related death worldwide. Previous studies have shown that the T-box transcription factor brachyury is commonly expressed in advanced lung cancer and is associated with a poor prognosis. The activation of brachyury promotes lung cancer progression, aggressiveness and resistance to conventional antitumor therapies. However, the mechanism by which brachyury drives lung cancer is unknown. Here we have sought to identify novel gene targets of brachyury to better understand the molecular pathogenesis of lung cancer. We used a bioinformatics approach and found a shortlist of putative potential brachyury target genes. We subsequently explored the modulation of candidate brachyury target genes in lung cancer. RNA interference analysis for lung cancer cell line H460 overexpressing brachyury demonstrated that fibroblast growth factor 8 (FGF8), MAS1 Proto-Oncogene G Protein-Coupled Receptor (MAS1) and Ran GTPase Activating Protein 1 (RANGAP1) played essential roles in cell growth, whereas transmembrane protein 37 (TMEM37) was involved in cell migration and invasion. ChIP-qPCR experiments confirmed the strong binding of brachyury to these functional target genes. Analysis of datasets derived from patients with lung cancer and a panel of lung cancer cell lines revealed that brachyury expression was positively correlated with FGF8, MAS1, RANGAP1 and TMEM37. In addition, the expression levels of FGF8, MAS1 and RANGAP1 were significantly associated with non-metastasis (early-stage and locally advanced lung cancer), whereas TMEM37 was associated with metastasis. Taken together, our findings suggest that brachyury may influence lung cancer progression and/or metastasis via direct regulation of FGF8, MAS1, RANGAP1 and TMEM37 expression. Identification of potential brachyury transcriptional targets provides novel insights into the molecular pathobiology of lung cancer.

Citation Format: Yunping Hu, Wesley Hsu. Identification of novel brachyury target genes in lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2021.
Abstract 2089: Therapeutic index prediction of the agonistic aglycosylated TWEAK receptor binding antibody BAY-356
Authors: Viberg Anders , Hanze Eva , Dietz Lisa , Zierz Ruprecht , Berndt Sandra , Wittemer-Rump Sabine
Published: July 2016

BAY-356 is a novel aglycosylated anti-TWEAK receptor antibody with potent agonistic activity evaluated for cancer therapy. In order to predict an efficacious therapeutic dose of BAY-356 in man, we sought to determine a therapeutic index where the exposure related to therapeutic efficacy was compared with the exposure obtained following doses tested in toxicology studies.

BAY-356 (1 mg/kg) was administered intravenously to healthy Cynomolgus monkeys and plasma concentrations measured in order to determine pharmacokinetic (PK) parameters. Using allometric scaling, the PK in humans was predicted. Efficacy data of BAY-356 in WiDr, HN10321, A253 and SCaBER xenograft models representing colorectal, head and neck squamous cell carcinoma and bladder cancer, were used to derive exposure-response models for each tumor model where the plasma exposure of BAY-356 is assumed to have an effect on the tumor size in a tumor growth model. NONMEM 7.3 was used in the estimations.

The tolerability of BAY-356 was tested in monkeys at doses of 10, 20 and 40 mg/kg by weekly intravenous injections over 4 weeks. Treatment resulted in slight to moderate toxicity in liver, kidneys and pancreas and 10 mg/kg was set to be an exposure dose well tolerated and not to be exceeded by therapeutic exposure. A dosing strategy in humans predicted to result in the same exposure as 10 mg/kg weekly in monkey is 18 mg/kg every third weeks (Q3W).

Using the estimated exposure-response in xenograft models, human tumor doubling times of 8 weeks (A253 and HN10321), 24 weeks (WiDr) and 12 and 24 weeks (SCaBER) in combination with human predicted PK parameters, tumor reduction over time in humans was predicted based on a human dosing strategy of 18 mg/kg BAY-356 Q3W. For the A253 tumor model, stable disease in humans was predicted while the results from HN10321 and SCaBER models predicted a 13-60% decrease in tumor size in humans following 12 weeks treatment. For WiDr, this dosing strategy was predicted to not be efficacious in humans.

The results based on modelling of xenograft data indicate that 18 mg/kg BAY-356 dosed Q3W is predicted to be efficacious in humans for certain tumor cells.

Citation Format: Anders Viberg, Eva Hanze, Lisa Dietz, Ruprecht Zierz, Sandra Berndt, Sabine Wittemer-Rump. Therapeutic index prediction of the agonistic aglycosylated TWEAK receptor binding antibody BAY-356. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2089.
Abstract 2093: Imaging single cell drug target engagement in vivo reveals heterogeneous fractional occupancy
Authors: Dubach Matt , Weissleder Ralph
Published: July 2016

To produce effective therapeutic treatment, drugs need to reach and engage the cognate target in the cells of interest. Incomplete target occupancy may lead to cell survival and disease progression. Therefore, for maximum efficacy, it is critical to achieve high target occupancy within each cell of a tumor. However, the tumor microenvironment likely creates a dynamic, heterogenous distribution of drug that limits target occupancy in some cells. Unfortunately, we lack the tools to determine target engagement at the cellular level, which prevents studies on heterogeneous target occupancy and contributes to the poor understanding of drug failure mechanisms. Here, we developed an approach to measure target engagement of small molecule inhibitors within single cells in mouse xenograft in vivo tumor models. Our method uses fluorescently labeled, target specific companion imaging drugs, fluorescence polarization imaging, and intravital microscopy, to measure target occupancy of unlabeled, clinical drugs in single cells. We calculated the equivalent in vitro exposure of ibrutinib, a covalent BTK inhibitor, in vivo, but found increased cellular heterogeneity, highlighting differences between in vivo activity and in vitro measurements. Additionally, following systemic delivery of the reversible PARP inhibitor olaparib, we found cells with substantial uninhibited target when average measurements indicated all target was occupied, illustrating drug activity heterogeneity that may lead to tumor survival. These results demonstrate that our technique, which is the first to measure unlabeled drug engagement with target in single cells, can quantitate cellular fractional occupancy heterogeneity during drug treatment. We expect this approach to serve as a valuable tool when measuring cellular pharmacology in vivo, providing insight on dosing, drug activity in the tumor and sources of heterogeneous drug distribution.

Citation Format: Matt Dubach, Ralph Weissleder. Imaging single cell drug target engagement in vivo reveals heterogeneous fractional occupancy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2093.
Abstract 2120: Molecular characterization of cancer stem cells derived from drug-resistant melanoma
Authors: Berlinberg Elyse J. , Crosson William , Rojas Arbis , Nazarian Ramin
Published: July 2016

BRAF kinase is a key intermediate in the MAP kinase pathway, which is associated with cell growth and proliferation. BRAFV600E activating mutation is found in 50-60% of all melanomas and 7% of all cancer malignancies. Despite an unprecedented initial response, patients treated with a highly effective BRAF inhibitor, vemurafenib (FDA approved 2011), can acquire drug resistance in as little as 6-9 months. Epigenetically driven epithelial-to-mesenchymal transition (EMT), a hallmark of cancer stem-like cells (CSCs), is a potential mechanism that melanomas employ to evade targeted therapy. We are investigating molecular alterations due to EMT in parental and vemurafenib-resistant melanoma sublines. Characterization of differential protein expression in parental and resistant cell lines through immunocytochemical, flow cytometric, and immunoblot analysis has indicated that EMT could confer drug resistance that prolongs survival of malignant melanomas. Furthermore, comparison of parental to isogenic resistant melanoma cell lines showed upregulated expression of EMT-related biomarkers that may be suggestive of the development of CSCs. These CSCs may serve as progenitor cells for therapy-resistant sublines in a heterogeneous melanoma population. Our research has identified notable EMT biomarkers in CSCs that can be used as novel targets for diagnosis and treatment of tumor resistance in melanoma patients.

Citation Format: Elyse J. Berlinberg, William Crosson, Arbis Rojas, Ramin Nazarian. Molecular characterization of cancer stem cells derived from drug-resistant melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2120.
Abstract 2148: MM-151 elicits broad and unique inhibition of cells harboring EGFR extracellular domain mutations — results of multiscale experiments with genome-edited cell lines
Authors: Wang Hongfang , Carey Shawn P. , Burenkova Olga , Tang Jian , Gerami-Moayed Nastaran , Arena Sabrina , Bardelli Alberto , Nering Rachel , Kearns Jeffrey D.
Published: July 2016

Mutations in the extracellular domain (ECD) of EGFR have been reported in colorectal (CRC) patients as a mechanism of acquired resistance to the EGFR-directed antibodies cetuximab and panitumumab. Similar to EGFR intracellular domain mutations observed in patients treated with EGFR kinase inhibitors, these EGFR-ECD mutations inhibit the binding of the antibodies while maintaining EGFR signaling activity. MM-151, an investigational agent consisting of three anti-EGFR IgG1 antibodies, has been recently reported to overcome these mutations in over-expression and patient-derived cell line models. Here, we extend these studies to characterize the mechanisms of action that enable MM-151 to provide robust inhibition of EGFR-ECD mutations beyond that achievable by other monoclonal and oligoclonal EGFR antibodies. First, utilizing a cell-free binding assay with recombinant EGFR-ECD mutants, we found that MM-151, but not the Sym004 combination of two antibodies, maintained oligoclonal binding to all mutants. Second, we performed a panel of 2D and 3D in vitro experiments with over-expression and genome-edited (CRISPR-Cas9) cell lines harboring EGFR-ECD mutations to characterize target engagement, cell signaling, proliferation, and the impact of hetero- versus homozygous expression of EGFR wild type or mutant alleles. We observed that the three mechanisms of action for MM-151- antagonism of both low- and high-affinity EGFR ligands, EGFR down-regulation, and immune-effector activity— were preserved in these models. Third, we performed xenograft experiments to evaluate the behavior of MM-151 and found that MM-151 maintained inhibitory activity in an in vivo context. The results of our multiscale experiments identified the impact of combining three antibodies to overcome the high plasticity of the EGFR extracellular domain and thus reveal the potential for clinical evaluation of MM-151 in both EGFR-naïve and EGFR-refractory CRC populations.

Citation Format: Hongfang Wang, Shawn P. Carey, Olga Burenkova, Jian Tang, Nastaran Gerami-Moayed, Sabrina Arena, Alberto Bardelli, Rachel Nering, Jeffrey D. Kearns. MM-151 elicits broad and unique inhibition of cells harboring EGFR extracellular domain mutations — results of multiscale experiments with genome-edited cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2148.
Abstract 2172: Circle Akt in: Epitope catalyzed assembly of macrocyclic therapeutics against phosphorylated Akt
Authors: Nag Arundhati
Published: July 2016

An approach to drugging ‘undruggable’ target proteins using macrocyclic, epitope targeted peptide based affinity reagents is demonstrated. An unbiased comprehensive Cu-catalyzed Azide Alkyne Cycloaddition cyclized peptide macrocyclic library is synthesized and screened against the phoshoS474 containing Hydrophobic Motif (HM) peptide epitope of Akt2. The best macrocyclic ligand, which exhibits specificity at the peptide and protein levels, is further extended through an in situ click screen to yield bivalent macrocyclic reagent with high affinity and specificity. The bivalent peptide, targeted against the phospho-S474 region of Akt, inhibits the Akt kinase in in vitro kinase assays. This ligand is being optimized, through systematic changes in its composition, to increase its cell permeability characteristics, so that its effects can be studied in Akt overexpressing carcinoma cell lines. The optimized macrocyclic peptide can eventually be used as an in vivo imaging probe and as a peptide inhibitor drug.

Citation Format: Arundhati Nag. Circle Akt in: Epitope catalyzed assembly of macrocyclic therapeutics against phosphorylated Akt. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2172.
Abstract 2181: Chemotherapeutic, chemomodulatory and vascular protective effects of naturally occurring hydroxyphenylalkanes and diarylheptanoids
Authors: Baghdadi Mohammed A. , Al-Ghamdi Eman A. , Alamoudi Abdulmohsin J. , Al-Abbasi Fahad A. , El-Bassossy Hany M. , El-Halawany Ali M. , Al-Abd Ahmed M.
Published: July 2016

Hydroxyphenylalkanes and diarylheptanoids possess potential therapeutic values in different patho-physiological conditions such as inflammation, oxidative stress, cardiovascular disorders and malignancy. Doxorubicin is widely used chemotherapeutic agent against several types of neoplastic disorders. In the current study, naturally isolated hydroxyphenylalkane and diarylheptanoid compounds were investigated for their potential chemo-modulatory effects on the top of their potential vascular protective role with doxorubicin. Shogaol and 4-methoxygingerol showed considerable cytotoxic effects against HCT116, HeLa, HepG2 and MCF7 cells with IC50 ranging from 3.1 to 18.7 μM and from 9.1 to 19.4 μM, respectively. Using DPPH free radical scavenging assay, diarylheptanoids were stronger antioxidant than hydroxyphenylalkanes with EC50 ranging from 0.5 to 5.9 μM and from 4.6 to 9.5 μM, respectively. In addition, all tested diarylheptanoids significantly ameliorated CCl4 induced disturbed intracellular GSH/GSSG balance. Gingerol moderately enhanced the cytotoxic profile of doxorubicin against HepG2 and Huh7 cells decreasing their IC50's from 0.52 to 0.37 μM and from 50 to 30 nM, respectively. Combination index of gingerol with doxorubicin (combination at equitoxic ratio of 1/100) was indicative of additive interaction (CI-value were 1.1 and 1.0, respectively). To further investigate the interactive characteristics between doxorubicin and gingerol, cell cycle distribution of HepG2 and Huh7 cells was studied using DNA cytometry after exposure to their single and equitoxic combination. Doxorubicin induced cell accumulation at S-phase and G2/M-phase while, gingerol combination with doxorubicin significantly induced cell cycle arrest at G2/M-phase. To study the potential vascular protective effect of gingerol against doxorubicin induced vascular toxicity, doxorubicin (10 μM for 1h) was incubated with or without different concentrations of 6-gingerol (3, 10 and 30 μM for 1h) on isolated aortic rings and their response to phenylephrine (PE) and acetylcholine (ACh) was examined. Co-incubation of 6-gingerol (30 μM) completely blocked the exaggerated vasoconstriction and impaired vascular relaxation induced by doxorubicin

Citation Format: Mohammed A. Baghdadi, Eman A. Al-Ghamdi, Abdulmohsin J. Alamoudi, Fahad A. Al-Abbasi, Hany M. El-Bassossy, Ali M. El-Halawany, Ahmed M. Al-Abd. Chemotherapeutic, chemomodulatory and vascular protective effects of naturally occurring hydroxyphenylalkanes and diarylheptanoids. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2181.
Abstract 2186: Incorporation of natural compounds into biomaterials to prevent breast cancer recurrence
Authors: Jordan Lauren , Walker Bailey-Jean , Moody Christopher , Booth Brian W.
Published: July 2016

Numerous studies have demonstrated that naturally occurring phytochemicals such as Tannic acid (TA) possess anti-cancer properties. Apoptotic activity is increased in breast cancer and prostate cancer cells in response to exposure to tannin extracts. Collagen type I is a biomaterial routinely used for cosmetic and reconstructive surgeries, including breast reconstruction following breast cancer mandated surgeries. TA functions as a collagen crosslinking agent through both hydrogen bonding and hydrophobic effects. Thus, if TA-crosslinked collagen type I is used for breast reconstruction procedures, the crosslinked collagen will be remodeled by growing breast epithelial cells and adipocytes where the incorporated TA will be released killing nearby residual cancer cells preventing recurrence. When adipocytes are grown on TA-crosslinked collagen beads the measureable released TA induces caspase-mediated apoptosis in ER+ and HER2+ breast cancer cells but has no effect on triple negative breast cancer cells and reduced apoptotic-inducing activity in normal breast epithelial cells. We are developing an injectable matrix comprised of TA-crosslinked collagen beads and adipocytes to serve as a tissue regeneration platform in patients post-lumpectomy. Variables such as collagen temperature, syringe pump rate, and drop distance have been studied to determine ideal method for producing beads of the desired size and shape. These promising results will lead to the construction of a matrix designed to not only promote tissue regeneration but also simultaneously help to prevent tumor recurrence. This matrix provides protection in breast cancer patients and holds promise for patients suffering from other soft tissue cancers.

Citation Format: Lauren Jordan, Bailey-Jean Walker, Christopher Moody, Brian W. Booth. Incorporation of natural compounds into biomaterials to prevent breast cancer recurrence. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2186.
Abstract 2192: Fusogenic targeted liposomes as next-generation nanomedicine for prostate cancer
Authors: Mriouah Jihane M. , Nesbitt Rae Lynn , Sosnowski Deborah , Pink Desmond , Duncan Roy , Ziljstra Andries , Lewis John D.
Published: July 2016

Metastatic or castrate-resistant is the second-leading cause of cancer mortality in males. In the past 3 years, chemotherapies have extended survival, but efficacy is limited by dose-limiting toxicities due to suboptimal biodistribution. We address the lack of specificity and accumulation in Castrate Resistant Prostate Cancer (CRPC) by developing a unique nano-carrier for drug delivery: targeted fusogenic liposomes.

We have developed a platform whereby liposomes are formulated with fusion associated small transmembrane protein p14 displaying targeting ligands. The p14 protein catalyzes mixing of the liposomal bilayer with cell membranes to deliver the cargo directly into the cytoplasm, while the targeting ligand bombesin, allows for targeting to the Gastrin-releasing Peptide (GRPR) that is overexpressed in prostate cancer.

We hypothesized that this novel targeted fusogenic liposome formulation would significantly improve the biodistribution and efficacy of chemotherapy.

A clinical liposomal doxorubicin formulation (DOXIL) was modified to incorporate targeted fusogenic p14 protein.

We then evaluated the efficacy and biodistribution of these new formulations using in vitro and in vivo models of CRPC. In PC3 cells, compared to conventional liposomes, intracellular levels of doxorubicin are increased by 15 and 25 times when p14 or p14-bombesin liposomes are used. Additionally, the IC50 is reduced from 85mM to 2mM. In mice bearing PC3 tumors treated with targeted fusogenic liposomal doxorubicin, we observed tumor growth inhibition of 57% (vs control).

This establishes a proof of concept for an innovative targeted drug delivery system that may improve the outcome of patients with CRPC by enhancing the effect of approved drugs.

Citation Format: Jihane M. Mriouah, Rae Lynn Nesbitt, Deborah Sosnowski, Desmond Pink, Roy Duncan, Andries Ziljstra, John D. Lewis. Fusogenic targeted liposomes as next-generation nanomedicine for prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2192.
Abstract 2195: Small molecule drug conjugates targeted to cholecystokinin 2 receptors
Authors: Roy Jyoti , Wayua Charity , Low Philip S.
Published: July 2016

Cholecystokinin 2 receptor (CCK2R) is over expressed on many cancers of lungs, pancreas, liver and GI tract (esophagus, colon and GIST), whereas its expression in normal tissues is limited to epithelial cells of the GI tract and brain. This restricted expression pattern renders CCK2R an excellent candidate for use in tumor-targeted drug delivery. Here we used a high affinity, low molecular weight CCK2R antagonist (CRL) to deliver imaging agents and cytotoxic drugs specifically to CCK2R positive tumors. We demonstrated that in vitro CRL conjugated to either NIR dye or radioactive imaging agent binds selectively to CCK2R positive cancer cells and exhibits low nano molar affinity for the receptor. We further showed that In vivo NIR dye and radiolabeled conjugate localizes primarily to the CCK2R positive murine tumor xenografts. Both of these conjugates also exhibited low serum protein binding and cleared rapidly from blood. Thus CRL-NIR dye conjugate have a potential application in fluorescence guided surgery where as CRL-radioactive conjugate seems promising for radioimaging in the clinic. Encouraged by the results of the imaging agents, CRL was conjugated to two potent microtubule inhibitors (tubulysin B hydrazide and desacetyl vinblastine). In vitro these two conjugates demonstrated high affinity and specificity for CCK2R. In vivo CRL conjugated to tubulysin B hydrazide was found to be more effective in eliminating tumor compared to desacetyl vinblastine conjugate. Neither of the CRL-cytotoxic drug conjugates showed any toxicity associated with the base drug nor any weight loss was observed in the mice treated with these conjugates. Results suggest that CRL has the proficiency to delivery drug specifically to tumors over expressing CCK2R receptor and lower the toxicities associated with conventional ways of treating cancer.

Citation Format: Jyoti Roy, Charity Wayua, Philip S. Low. Small molecule drug conjugates targeted to cholecystokinin 2 receptors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2195.
Abstract 2214: GITR ligand fusion protein (GITRL-Fc) induces T cell mediated anti-tumor immune response and can combine with anti-PDL1 to enhance anti-tumor immunity and long-term immune memory
Authors: Srivastava Minu K. , Yun Rui , Mayes Erin , Jie Hyun_Bae , Axelrod Fumiko , Monteon Jorge , Xie Ming-Hong , Lewicki John , Hoey Tim , Gurney Austin , Park Angie Inkyung
Published: July 2016

GITRL (Glucocorticoid-Induced Tumor Necrosis Factor Receptor Ligand, TNFSF18) is a member of the TNF superfamily and naturally exists as a membrane-anchored type II protein that self assembles as a trimer. GITRL activates the co-stimulatory receptor GITR. GITR is found primarily on activated T effector (Teff) cells and regulatory T (Treg) cells. Co-stimulation of GITR by agonist agents is hypothesized to promote anti-tumor immunity by enhancing Teff cell activity and inhibiting Treg suppression. We generated a novel single-gene GITRL trimer fused to an immunoglobulin Fc domain (GITRL-Fc). GITRL-Fc activated GITR signaling more effectively than prototype GITR agonist antibody DTA-1. GITRL-Fc promoted a robust anti-tumor immune response in multiple syngeneic mouse tumor models. GITRL-Fc enhanced tumor specific T-cell responses, particularly of the Th1 type, and also led to reduction in Treg-mediated immunesuppressive activity. GITRL-Fc displayed single agent activity in inhibiting tumor growth and promoting complete tumor rejection in the murine CT26 colon carcinoma model and combination activity with anti-PDL1 as compared to anti-PDL1 and control IgG2a alone. Mice “cured” with GITRL or GITRL/anti-PDL1 combination treatments were protected from re-challenge with tumor cells, suggesting the existence of immunologic memory. More mice were protected from tumor re-challenge with the combination of GITRL-Fc and anti-PDL1, as compared to GITRL-Fc alone. Our results demonstrate that agonist GITRL-Fc induces potent T cell responses, overcomes Treg inhibition, and promotes anti-tumor activity in preclinical models as a single agent or in combination with anti PDL1. The mechanism of tumor eradication and induction of long-term immune memory response by the combination is under investigation and will be discussed at the presentation.

Citation Format: Minu K. Srivastava, Rui Yun, Erin Mayes, Hyun_Bae Jie, Fumiko Axelrod, Jorge Monteon, Ming-Hong Xie, John Lewicki, Tim Hoey, Austin Gurney, Angie Inkyung Park. GITR ligand fusion protein (GITRL-Fc) induces T cell mediated anti-tumor immune response and can combine with anti-PDL1 to enhance anti-tumor immunity and long-term immune memory. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2214.
Abstract 2221: HHLA2, a novel member of the B7 family of inhibitory ligands, is highly expressed in osteosarcoma
Authors: Koirala Pratistha , Roth Michael , Gill Jonathan , Chinai Jordan , Ewart Michelle , Piperdi Sajida , Geller David , Hoang Bang , Fatakhova Yekaterina , Ghorpade Maya , Zang Xingxing , Gorlick Richard
Published: July 2016

Purpose: Over the past four decades there have been minimal improvements in outcomes for patients with osteosarcoma (OS). New targets and novel therapies are needed to improve outcomes for these patients. In this study, we sought to evaluate the prevalence and prognostic utility of the immune checkpoint inhibitor HHLA2 in OS.

Experimental Design: HHLA2 expression was evaluated in two cohorts of OS patients using a tumor microarray (TMA) (n = 62) and whole slides (n = 48). HHLA2 expression was assessed in primary tumor specimens and metastatic disease, and correlated with the presence of tumor infiltrating lymphocytes (TILs), and event free survival.

Results: HHLA2 was expressed in 68% of OS tumors in the TMA and in 54% in the second cohort. HHLA2 was expressed in almost all metastatic disease specimens and was more prevalent than in primary specimens without known metastases (93% vs 53%, p = 0.02). TILs were present in 75% of all osteosarcoma specimens. Patients whose tumors were ≥25% or ≥50% HHLA2 positive had significantly worse five-year event-free-survival (33% vs 64%, p = 0.03 and 14% vs 59%, p = 0.02).

Conclusions: HHLA2 is expressed in the majority of OS tumors, is associated with metastatic disease, and is associated with poorer survival. Further studies are needed assessing the effect of HHLA2 expression on the function of the immune microenvironment, as well as assessing the feasibility and utility of targeting HHLA2 in OS.

Citation Format: Pratistha Koirala, Michael Roth, Jonathan Gill, Jordan Chinai, Michelle Ewart, Sajida Piperdi, David Geller, Bang Hoang, Yekaterina Fatakhova, Maya Ghorpade, Xingxing Zang, Richard Gorlick. HHLA2, a novel member of the B7 family of inhibitory ligands, is highly expressed in osteosarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2221.
Abstract 2228: Cbl-b inhibitors as novel intra-cellular checkpoint inhibitors for cancer immunotherapy
Authors: Agarwal Saket , Wu Jian , Riling Chris , Kodrasov Matthew , Weinstock Joseph , Sokirniy Ivan , Mattern Michael , Kambayashi Taku , Kumar Suresh
Published: July 2016

The approval by the FDA and clinical use of immune checkpoint inhibitors, such as anti-PD1 and anti-CTLA4 antibodies, that activate cytotoxic T lymphocyte (CTL) mediated anti-tumor immune responses against advanced solid tumors has resulted in a paradigm shift in cancer therapy. Antitumor activity of CTLs and NK cells is tightly regulated by intracellular signaling events orchestrated by the ubiquitin signaling system. In particular, Cbl-b (Casitas B-lineage lymphoma proto-oncogene b), a RING finger E3-ubiquitin ligase primarily expressed in immune cells, acts as an intracellular checkpoint and a master negative regulator of both CTLs and NK cells. Accordingly, Cbl-b-/- mice have been observed to reject a variety of implanted metastatic and non-metastatic tumors and to delay significantly the outgrowth of spontaneous tumors; these observed antitumor effects were seen to be mediated by activated CD8+ T cells and NK cells. Based on the overwhelming evidence supporting the role of Cbl-b in immune suppression, this ubiquitin conjugating enzyme is considered a novel target for developing small molecule cancer immunotherapy agents. Using proprietary high throughput screening technologies, Progenra identified novel, selective Cbl-b inhibitors that were shown to decrease ubiquitination of substrates such as TAM receptor kinases (Tyro3) and to activate T cells (as judged by increased IL-2 production) and NK cells (as judged by increased IFNγ production and degranulation) in ex vivo functional assays. ADME/DMPK evaluation and in vivo efficacy of selected Cbl-b inhibitors will be discussed in relation to the therapeutic utility of this class of small molecule agent, which is expected to potentiate the anticancer effects of approved immunotherapy agents.

Citation Format: Saket Agarwal, Jian Wu, Chris Riling, Matthew Kodrasov, Joseph Weinstock, Ivan Sokirniy, Michael Mattern, Taku Kambayashi, Suresh Kumar. Cbl-b inhibitors as novel intra-cellular checkpoint inhibitors for cancer immunotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2228.
Abstract 2233: PI3K/Akt/mTOR signaling and plasma membrane proteins are implicated in responsiveness to adjuvant dendritic cell vaccination for metastatic colorectal cancer
Authors: Qian David , Byun Jinyoung , Xiao Xiangjun , Her Stephanie , Suriawinata Arief , Amos Christopher , Barth Richard
Published: July 2016

Purpose: We have previously demonstrated that metastatic colorectal cancer patients who exhibit immune responses to a dendritic cell (DC) vaccine have superior recurrence-free survival following surgery, compared to patients in whom immune responses do not occur. We sought to characterize the patterns of T lymphocyte infiltration and somatic mutations in metastases that are associated with and predictive of response to the DC vaccine.

Methods: Cytotoxic, memory, and regulatory T cells in resected metastases and in surrounding normal liver tissue from 22 patients (11 responders and 11 non-responders) were enumerated by immunohistochemistry prior to vaccine administration. In conjunction with tumor sequencing, the Combined Multivariate and Collapsing method was used to identify gene mutations that are associated with vaccine response. We also derived a response prediction score for each patient using his/her tumor genotype data and variant association effect sizes computed from the other 21 patients; greater weighting was placed on genes that encode plasma membrane proteins.

Results: There was no relationship between vaccine response and intra-tumor, peri-tumor, or hepatic densities of T cell subpopulations. Associated genes were found to be statistically enriched in PI3K, Akt, and mTOR signaling pathways (P < 0.001). With respect to prediction, applying a consistent score cutoff over the 22 rounds of leave-one-out cross-validation correctly inferred vaccine response in 21 of 22 patients (95%).

Conclusions: Adjuvant dendritic cell vaccination has shown promise as a form of immunotherapy for patients with metastatic colorectal cancer. Its efficacy may be influenced by somatic mutations that affect pathways involving PI3K, Akt, and mTOR, as well as neoantigens on tumor cell surfaces.

Citation Format: David Qian, Jinyoung Byun, Xiangjun Xiao, Stephanie Her, Arief Suriawinata, Christopher Amos, Richard Barth. PI3K/Akt/mTOR signaling and plasma membrane proteins are implicated in responsiveness to adjuvant dendritic cell vaccination for metastatic colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2233.
Abstract 2239: Isolation and characterization of circulating tumor cells (CTCs) in non small cell lung carcinoma
Authors: Scrima Marianna , Malanga Donatella , De Marco Carmela , Viglietto Giuseppe
Published: July 2016

Purpose: Lung cancer is the most common cause of cancer-related deaths that frequently metastasizes prior to disease diagnosis. Circulating tumor cells (CTCs) are of great clinical interest in terms of prognosis and therapy intervention and may provide important prognostic and predictive information in non-small cell lung cancer (NSCLC). The purpose of this study was to evaluate diagnostic performance of a non-invasive method based on filtration-based technology (ISET) using cytomorphologic criteria in patients undergoing surgery comparing with histologic features of corresponding primary lesions.

Materials and Methods: The CTCs analysis was performed through the following steps: isolation by ISET filtration method (ScreenCell®) from 5 ml of peripheral blood of 36 patients with NSCLC and 23 with benign lung lesions; classification of CTCs with haematoxylin and eosin (H&E) staining; identification of cell marker with co-immunostaining for epithelial and mesenchymal markers (Vimentin and Cytocheratin). CTCs were classified as tumor cells if: (i) they have a large (≥12 μm), hyperchromatic, irregular-shaped nuclei; (ii) high nuclear-cytoplasmic ratio (>0.5).

Diagnostic performance was evaluated against pathologist reported diagnoses of cancer from histological or cytological analysis.

Results: CTCs were detected in 27 of 36 (75%) NSCLC patients and in 2 of 23 (9%) negative control patients (p = 0.001). No significant differences were found for the percentage of CTCs for primary and metastatic cancer as well as for cancer stages. A test sensitivity and specificity for diagnosing lung cancer was 75% and 91% respectively. After co-immunostaining (Cytocheratin, Vimentin, CD45) CTCs showed positivity (14/15, 93%) only to vimentin (mesenchymal marker) and completely absence of cytocheratin signal (epithelial marker).

Conclusions: The performance of the tested filter-based antibody-independent technology to capture CTCs using standard cytomorphologic criteria provides the potential of a diagnostic blood test for lung cancer showed by high test sensitivity and specificity. The vimentin high expression in CTCs showed both that is a possible marker to CTCs identification and isolation and the importance of EMT mechanism transition in CTCs.

Citation Format: Marianna Scrima, Donatella Malanga, Carmela De Marco, Giuseppe Viglietto. Isolation and characterization of circulating tumor cells (CTCs) in non small cell lung carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2239.
Abstract 2267: Efficacy of pemetrexed-based chemotherapy in patients with NSCLC harboring KRAS mutations
Authors: Okumura Shunsuke , Sasaki Takaaki , Chiba Shin-ichi , Sado Masatoshi , Miyokawa Naoyuki , Funakoshi Hiroshi , Ohsaki Yoshinobu
Published: July 2016

[Background]

Biomarkers like epidermal growth factor receptor (EGFR) gene mutations or anaplastic lymphoma kinase (ALK) rearrangement have been discovered in lung cancer, leading to targeted therapies developed in non-small cell lung cancer (NSCLC). Several oncogenes have been reported to be negative biomarkers for response to tyrosine kinase inhibitors (TKIs), for example, NSCLC with KRAS mutations are resistant to EGFR-TKIs. A preclinical study showed that KRAS mutant lung cancer cells were sensitive to pemetrexed, while a retrospective study reported that pemetrexed-based chemotherapy didn't show a favorable outcomes in patients with KRAS mutant NSCLC. We, therefore, studied efficacy of pemetrexed-based chemotherapy in patients with NSCLC, and compared the efficacy between KRAS and wild-type NSCLC.

[Methods]

We retrospectively studied the medical records of patients diagnosed with NSCLC between 2013 to 2015. Genomic DNA (gDNA) was extracted from FFPE tissue samples (QIAamp DNA FFPE Tissue Kit, QIAGEN). KRAS mutations were evaluated using Ion Torrent NGS systems (Ion PGM, Thermo Fischer Scientific) or PCR-rSSO. AmpliSeq Colon and Lung Cancer Research Panel v2 was used in the present study. We used Ion Reporter software for next-generation sequencing data analysis.

[Results]

We analyzed the data of 226 patients with NSCLC and successfully extracted gDNA from 94 tissue samples. Among them, mutation analysis by Ion PGM system was performed in 48 samples and PCR-rSSO in 46 samples. We will report response rate and progression free survival in the patients treated with pemetrexed-based chemotherapy.

Citation Format: Shunsuke Okumura, Takaaki Sasaki, Shin-ichi Chiba, Masatoshi Sado, Naoyuki Miyokawa, Hiroshi Funakoshi, Yoshinobu Ohsaki. Efficacy of pemetrexed-based chemotherapy in patients with NSCLC harboring KRAS mutations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2267.
Abstract 2281: DNA methyl transferase inhibitors (DNMTis) upregulate NY-ESO expression in several human primary cells: implications for co-therapy with engineered adoptive T-cell therapies
Authors: Laugel Bruno , Sevko Alexandra , Howe Karen , Ferjentsik Zoltan , Ferronha Tiago , Maroto Miguel , Brewer Joanna , Jakobsen Bent , Binder-Scholl Gwendolyn
Published: July 2016

The DNA methyl transferase inhibitors 5-aza-2’-deoxycytidine (decitabine or DECI) and 5-azacytidine (azacitidine or AZA) have recently been approved for the treatment of a variety of hematologic malignancies. These cytidine analogues remove repressive epigenetic DNA methyl marks and induce the transcription of numerous genes normally silenced during the processes of cellular differentiation or transformation. DNMTis have a net anti-neoplastic activity through the up-regulation of tumor-suppressor genes involved in the induction of apoptosis, cellular differentiation and cell cycle arrest. In addition, DNMTis promote the expression of several cancer testis antigens (CTAs) by tumor cells, including NY-ESO and MAGE-A1/A3. These observations suggest that the combination of DNMTis with immunotherapeutic modalities may provide some clinical benefit.

Adoptive immunotherapies using autologous CD4+ and CD8+ T cells expressing an affinity optimized NY-ESO TCR are in clinical trials and show robust clinical responses in patients across several indications including synovial sarcoma, multiple myeloma or melanoma. DNMTis were previously shown to induce the expression of NY-ESO in antigen-low or -negative tumor cells and consequently enable the activation of NY-ESO-specific T-cells. While there is compelling evidence for the combination of DNMTis with T cells containing engineered affinity-enhanced TCRs recognizing NY-ESO-1, safety data supporting this approach are lacking. Initial tests into the safety of combining these treatments investigated the effects of DECI and AZA on NY-ESO and LAGE-1 expression in a panel of primary adult human cells of different tissue origins. Our data demonstrate that treatment with either compound resulted in a significant up-regulation of NY-ESO transcription in a subset of primary cells tested. The magnitude of increase in transcript expression varied between cell types and, in some cases, was sufficient to activate T cells expressing an affinity-optimized NY-ESO-specific TCR. These findings raise important safety concerns with regards to the potential combination of DNMTi compounds with NY-ESO T-cell therapy and suggest that such co-therapies may result in on-target off-tumor toxicities.

Citation Format: Bruno Laugel, Alexandra Sevko, Karen Howe, Zoltan Ferjentsik, Tiago Ferronha, Miguel Maroto, Joanna Brewer, Bent Jakobsen, Gwendolyn Binder-Scholl. DNA methyl transferase inhibitors (DNMTis) upregulate NY-ESO expression in several human primary cells: implications for co-therapy with engineered adoptive T-cell therapies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2281.
Abstract 2285: Microsomal PGE2 synthase-1 regulates melanoma cell survival and associates with melanoma disease progression
Authors: Kim Sun-Hee , Hashimoto Yuuri , Cho Sung-Nam , Roszik Jason , Milton Denái R. , Dal Fulya , Kim Sangwon F. , Menter David G. , Yang Peiying , Ekmekcioglu Suhendan , Grimm Elizabeth A.
Published: July 2016

COX-2 and its product PGE2 enhance carcinogenesis and tumor progression, which has been previously reported in melanoma. As most COX inhibitors cause much toxicity, the downstream microsomal PGE2 synthase-1 (mPGES1) is a consideration for targeting. Human melanoma TMAs were employed for testing mPGES1 protein staining intensity and percentage levels and both increased with clinical stage; employing a different Stage III TMA, mPGES1 intensity (not percentage) associated with reduced patient survival. Our results further show that iNOS was also highly expressed in melanoma tissues with high mPGES1 levels, and iNOS-mediated NO promoted mPGES1 expression and PGE2 production. An mPGES1specific inhibitor (CAY10526) as well as siRNA attenuated cell survival and increased apoptosis. CAY10526 significantly suppressed tumor growth and increased apoptosis in melanoma xenografts. Our findings support the value of a prognostic and predictive role for mPGES1, and suggest targeting this molecule in the PGE2 pathway as another avenue toward improving melanoma therapy.

Citation Format: Sun-Hee Kim, Yuuri Hashimoto, Sung-Nam Cho, Jason Roszik, Denái R. Milton, Fulya Dal, Sangwon F. Kim, David G. Menter, Peiying Yang, Suhendan Ekmekcioglu, Elizabeth A. Grimm. Microsomal PGE2 synthase-1 regulates melanoma cell survival and associates with melanoma disease progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2285.
Abstract 2286: Targeting the notch pathway: A potential therapeutic approach for desmoid tumors
Authors: Braggio Danielle , Shang Hui , Lee Ya-Jung , Al-Sannaa Ghadah A. , Creighton Chad J. , Bolshakov Svetlana , Lazar Alexander JF , Lev Dina , Pollock Raphael E.
Published: July 2016

Desmoid tumors (DTs) are rare mesenchymal lesions that can recur repeatedly. When it is feasible, DTs are surgically resected; however, this often results in high recurrence rates. Recently, treatment with PF-03084014, a potent γ-secretase inhibitor, has been shown to have antitumor activity in several tumor types by affecting the WNT/β-catenin pathway. Consequently, Notch pathway inhibition by PF-03084014 might be a promising approach for DT treatment. The expression of Notch pathway components was analyzed in DT tissues and cell strains with immunohistochemistry and Western blotting, respectively. A panel of DT cell strains was exposed to PF-03084014 and evaluated for cell proliferation. Antitumor effects were assessed via cell cycle, apoptosis, and migration and invasion analysis. Cells treated with PF-03084014 were characterized with a gene array analysis combined with Ingenuity Pathway Analysis. The results showed that Notch pathway components were expressed at different levels in DTs. Hes1 (Hes Family BHLH Transcription Factor 1) was overexpressed in DT tumors versus dermal scar tissue, and PF-03084014 caused significant decreases in Notch intracellular domain and Hes1 expression in DT cell strains. PF-03084014 decreased DT cell migration and invasion and also caused cell growth inhibition in DT cell strains, most likely through cell cycle arrest. Gene array analysis combined with Ingenuity Pathway Analysis showed that Wnt1-inducible signaling pathway protein 2 possibly regulated Notch and WNT pathways after treatment with PF-03084014 through integrin. Our findings suggest that the Notch pathway is an important DT therapeutic target. Furthermore, PF-03084014 has significant antitumor activity against DTs, and it may be an alternative strategy for DT treatment.

Citation Format: Danielle Braggio, Hui Shang, Ya-Jung Lee, Ghadah A. Al-Sannaa, Chad J. Creighton, Svetlana Bolshakov, Alexander JF Lazar, Dina Lev, Raphael E. Pollock. Targeting the notch pathway: A potential therapeutic approach for desmoid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2286.