bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4828: Enhanced cytotoxicity following sequential treatment with filanesib and melphalan in human myeloma cell lines
Authors: Destephanis Darla , Norris Eric J. , Turnquist Brian , Usmani Saad , Ganapathi Mahrukh , Ganapathi Ram
Published: July 2016

As a single agent, melphalan is used as a preparative treatment prior to autologous stem cell transplant (ASCT) in patients with multiple myeloma (MM) given its myelosuppressive and anti-myeloma effects. While most patients achieve complete remission following ASCT, a majority will develop recurrent disease. Therefore, novel combinations that improve the anti-myeloma activity of melphalan may improve post-transplant outcomes. The first-in-class agent Filanesib (Fil) is a kinesin spindle protein inhibitor which induces mitotic arrest/apoptosis in proliferating cells and has demonstrated anti-myeloma activity in vitro and in vivo. In this study, we tested the effects of combination treatment with filanesib and melphalan in the human myeloma cell lines (HMCLs), U266, MM1S and NCI-H929. As single agents, melphalan and filanesib both inhibit myeloma cell proliferation as assessed by the MTS assay. However, when HMCLs were treated simultaneously with both drugs an antagonistic interaction was observed. This antagonism was in part due to cell cycle arrest in the S-phase, which prevented filanesib from downregulating the anti-apoptotic proteins, BCL-2 and MCL-1 and exerting its cytotoxic effect in the G2/M-phase.. To elucidate whether sequential treatment could enhance cytotoxicity, HMCLs were treated with either melphalan (1 hour) followed by filanesib (MelFil) for 48 hours or filanesib for 48 hours followed by 1 hour melphalan treatment (FilMel). Compared to the apoptosis observed with individual drugs, the MelFil sequence led to significantly lower apoptosis, whereas the the FilMel treatment led to enhanced apoptosis following 48 hours after drug washout. Furthermore examination of recovery of cell proliferation following seven days of recovery after drug removal revealed an increase in cell proliferation for the MelFil treatment compared to a decline in the number of viable cells for FilMel treatment. Taken together, our findings suggest that filanesib pretreatment prior to melphalan may be a clinically relevant therapeutic combination strategy. Moreover, our data underscore the importance of testing sequencing strategies for optimizing therapeutic efficacy of combination treatment.

Citation Format: Darla Destephanis, Eric J. Norris, Brian Turnquist, Saad Usmani, Mahrukh Ganapathi, Ram Ganapathi. Enhanced cytotoxicity following sequential treatment with filanesib and melphalan in human myeloma 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 4828.
Abstract 4830: Preclinical anti-tumor activity of nanoliposomal irinotecan (Nal-IRI, MM-398) + 5-FU + oxaliplatin in pancreatic cancer
Authors: Gaddy Daniel F. , Lee Helen , Paz Nancy , Leonard Shannon C. , Kalra Ashish , Straubinger Ninfa L. , Straubinger Robert M. , Gillard Bryan M. , Moser Michael T. , Drummond Daryl C. , Klinz Stephan G. , Hendriks Bart S. , Fitzgerald Jonathan B.
Published: July 2016

Nanoliposomal irinotecan (Nal-IRI, MM-398) recently gained approval in combination with 5-fluorouracil/leucovorin (5-FU/LV) in post-gemcitabine metastatic pancreatic cancer based on results of the Phase 3 NAPOLI-1 trial. Nal-IRI, in combination with 5-FU/LV, improved overall survival in gemcitabine-refractory metastatic PDAC relative to 5-FU/LV alone with a well-defined and manageable toxicity profile in pretreated patients. FOLFIRINOX (5-FU/LV, irinotecan and oxaliplatin) is a chemotherapy regimen active in first-line metastatic PDAC. Herein, we evaluate the preclinical anti-tumor activity of a nal-IRI + 5-FU + oxaliplatin regimen relative to the FOLFIRINOX regimen. Using pancreatic cancer cell lines, we demonstrate enhanced cell death when nal-IRI treatment is simulated using prolonged exposure of SN-38 (the active metabolite of irinotecan) in combination with 5-FU and oxaliplatin. In cell line-derived and patient-derived xenograft models of pancreatic cancer we demonstrate improved anti-tumor activity of nal-IRI relative to exposure-matched doses of unencapsulated irinotecan. Further, nal-IRI consistently improved tumor growth inhibition and survival relative to unencapsulated irinotecan in preclinical models, both as a monotherapy and in combination with 5-FU and oxaliplatin. The addition of nal-IRI to 5-FU and/or oxaliplatin did not exacerbate the baseline toxicities of these agents, including weight loss and neutropenia, and tolerability could be further improved by delaying the administration of oxaliplatin to 1 day post-MM-398. These findings illustrate the therapeutic potential of nal-IRI in combination with 5-FU/LV and oxaliplatin and support an ongoing Phase 2 trial (NCT02551991) of this triplet regimen in first-line PDAC.

Citation Format: Daniel F. Gaddy, Helen Lee, Nancy Paz, Shannon C. Leonard, Ashish Kalra, Ninfa L. Straubinger, Robert M. Straubinger, Bryan M. Gillard, Michael T. Moser, Daryl C. Drummond, Stephan G. Klinz, Bart S. Hendriks, Jonathan B. Fitzgerald. Preclinical anti-tumor activity of nanoliposomal irinotecan (Nal-IRI, MM-398) + 5-FU + oxaliplatin in pancreatic 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 4830.
Abstract 4832: Overcoming breast cancer chemoresistance and blocking metastasis by targeting RNA-binding protein HuR
Authors: Wu Xiaoqing , Marquez Rebecca T. , Han Shuang , Li Ke , Lan Lan , Guo Yuxiao , Dixon Dan A. , Aubé Jeffrey , Welch Danny R. , Xu Liang
Published: July 2016

Patients with metastatic breast cancer have a dismal 5-year survival rate of only 24%. Chemoresistance is another contributor to the high mortality of advanced breast cancer. The RNA-binding protein, Hu antigen R (HuR), is overexpressed at all stages of breast cancer development. Cytoplasmic HuR accumulation correlates with high-grade malignancy, poor distant disease-free survival and serves as a prognostic factor for poor clinical outcome in breast cancer. HuR promotes tumorigenesis by promoting mRNA stability and translation of proteins implicated in proliferation, survival, angiogenesis, invasion, and metastasis. HuR also modulates sensitivity of breast cancer cells to chemotherapy. Taken together, HuR is an emerging target for breast cancer therapy, especially metastatic breast cancer. Using shRNA and CRISPR technologies to modulate HuR expression in breast cancer cells, we found that cells with HuR KD/KO were less invasive and more sensitive to chemotherapy. In an effort to discover small molecules that disrupt the HuR-mRNA interaction and block HuR functions in breast cancer progression, metastasis and chemoresistance, high throughput screening (HTS) was carried out using several chemical libraries (∼23,000 compounds) using fluorescence polarization (FP) assay and identified several initial hits with sub-micromolar inhibitory constants (Ki). Those potential disruptors were then validated by ALPHA assay (Amplified Luminescent Proximity Homogeneous Assay), confirmed by Surface Plasmon Resonance (SPR). In cell-based assays, top hit KH-3 and its optimized analogs, specifically shortened HuR target mRNA half-lifes and decreased the level of the encoded proteins. Moreover, those compounds inhibited invasion and restored chemosensitivity. qPCR arrays focusing on specific pathways revealed that HuR inhibitors potently upregulated metastatic suppressors and downregulated genes frequently overexpressed in lung metastases. In animal studies, KH-3 not only powerfully exhibited antitumor efficacy in orthotopic xenografts, but also efficiently blocked experimental metastasis. In conclusion, we identified potent and specific small molecule disrupters of HuR-mRNA interaction for potential anti-metastatic therapy for breast cancer with HuR overexpression.

Citation Format: Xiaoqing Wu, Rebecca T. Marquez, Shuang Han, Ke Li, Lan Lan, Yuxiao Guo, Dan A. Dixon, Jeffrey Aubé, Danny R. Welch, Liang Xu. Overcoming breast cancer chemoresistance and blocking metastasis by targeting RNA-binding protein HuR. [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 4832.
Abstract 4848: Discovery of ABI-231 analogs targeting the colchicine site in tubulin for advanced melanoma
Authors: Qinghui Wang , Kinsie Arnst E. , Miller Duane D. , Li Wei
Published: July 2016

We previously discovered a novel 2-aryl-4-benzoyl-imidazole (ABI) scaffold exerts its potent anti-proliferative effects through interacting with the colchicine binding site in tubulin. Extensive structure-activity relationship (SAR) studies of this novel scaffold showed that the best analog in this series, ABI-231, has an average IC50 = 5.2 nM against panels of melanoma and prostate cancer cell lines, is orally bioavailable, and strongly suppress melanoma tumors in vivo. Analyses of the potential binding pose of ABI-231 at the colchicine binding pocket suggested small substitutions to the indole moiety in ABI-231 could further increase its interactions to tubulin.

Based this hypothesis, we have established an efficient synthetic method for the synthesis of ABI-231 analogs by either rotating the indole ring or introducing substituents on the indole of ABI-231. Using this method, we synthesized and tested 27 analogs of ABI-231. Several of the compounds show excellent potency against a panel of melanoma cancer cell lines with a diverse genomic background (mutations in BRAF, TP53, CDKN2A and PTEN) that are frequently encountered in clinical metastatic melanoma tumors. Additionally, they are not substrates for the P-glycoprotein (Pgp) efflux pump and therefore strongly inhibited the multidrug resistant cell lines. The potency of the compounds was further verified by their potent growth inhibition of cancer cell colonies. Results from cell cycle analysis, apoptosis evaluation and tubulin polymerization inhibition are consistent with their expected mechanisms of action. Potential off-target analyses performed by Eurofins Cerep-Panlabs SafetyScreen 44 indicated that they are clean and does not inihibt any of the tested off-targets at 100 fold of its IC50 value in melanoma cells, suggesting the good safety profile for this compound and the probably these scaffold in general. We are currently performing in vivo efficacy studies for the most potent analogs. These compounds hold great promise as a new generation of orally available tubulin inhibitors.

Citation Format: Wang Qinghui, Arnst E. Kinsie, Duane D. Miller, Wei Li. Discovery of ABI-231 analogs targeting the colchicine site in tubulin for advanced 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 4848.
Abstract 485: Blood-based profiling of patients with NSCLC using CLIA certified cell-free DNA tests for EGFR, KRAS and BRAF point mutations and test use in clinical practice
Authors: Mellert Hestia , Thurston Scott , Foreman Trudi , Hahn Westen , Dupuis Nicholas , Pestano Gary
Published: July 2016

Nearly 80% of patients will not have mutation results available at their initial oncology consult and as many as 1 in 4 patients will begin treatment in advance of receiving their tumor mutation results. These factors hinder diagnosis at the molecular level, which is an important component in the decision to commence treatment with specific targeted therapies. The goal of this study was to measure the utility of a CLIA-certified Laboratory Developed Test (LDT) in the absence of a tissue diagnosis. EGFR, KRAS and BRAF point mutations within the cell-free DNA (cfDNA) isolated from the plasma of patients with advanced stages of cancer were analyzed using Droplet Digital™ PCR (ddPCR) technology. The specific tests detected the EGFR sensitizing mutations L858R and exon 19 deletion (E746 - A750) and EGFR resistance mutation, T790M, the KRAS mutations G12C, G12V and G12D, as well as the BRAF V600E mutation. The tests were comprised of three components: (i) a whole-blood collection kit that ships at ambient temperature, (ii) cfDNA isolated from plasma for analysis using ddPCR, and (iii) a secure laboratory information management system (LIMS) for sample accessioning and report generation. Test mutation status results were reported within 72 hours of blood shipment from the physicians’ office. To date we have processed greater than 2000 individual variant tests for patients with NSCLC. The predominant tumor type for EGFR sensitizing mutations requested was NSCLC (95%). Interestingly, even BRAF test orders were > 90% from NSCLC patients. Additionally, the percentage of tests requested that were positive for each mutation category were 12% for EGFR sensitizing, 20% for EGFR resistance, 14% for KRAS and 1.8% for BRAF. Significantly, T790M test orders were observed to have increased five-fold over the last three months of testing. We have developed highly sensitive, 72 hour test to results blood-based assays as a part of the GeneStratTM panel, that expand the utility of laboratory testing for patients previously diagnosed with cancer.

Citation Format: Hestia Mellert, Scott Thurston, Trudi Foreman, Westen Hahn, Nicholas Dupuis, Gary Pestano. Blood-based profiling of patients with NSCLC using CLIA certified cell-free DNA tests for EGFR, KRAS and BRAF point mutations and test use in clinical practice. [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 485.
Abstract 4852: Multivalent peptoid conjugates suppress enzalutamide-resistant prostate cancer cellular proliferation
Authors: Wang Yu , Levine Paul , Dehigaspitiya Dilani , Profit Adam , Logan Susan , Imberg-Kazdan Keren , Kirshenbaum Kent , Garabedian Michael
Published: July 2016

Development of resistance to latest anti-androgens treatment for advanced prostate cancer is a growing concern. New strategies to block androgen receptor (AR) function in castration resistant prostate cancer are therefore required. Here we report the characterization of a multivalent conjugate presenting two spatially defined bioactive ethisterone ligands as spatially defined pendant groups on a peptoid oligomer. The conjugate, termed Multivalent Peptoid Conjugate 6 (MPC6), suppressed the proliferation of multiple AR-expressing prostate cancer cell lines including those that failed to respond to enzalutamide and ARN509. The structure-activity relationships of MPC6 variants were evaluated, revealing that increased spacing between ethisterone moieties and changes in peptoid topology eliminated its anti-proliferative effect. Mechanistically, MPC6 blocked AR coactivator-peptide interaction, and decreased AR protein half-life. Pharmacological studies revealed that MPC6 was metabolically stable and displayed a low plasma clearance rate. Importantly, MPC6 treatment reduced tumor growth and decreased Ki67 and AR expression in mouse xenograft models of enzalutamide-resistant LNCaP-abl cells. Thus, MPC6 represents a new class of compounds with the potential to combat treatment-resistant prostate cancer.

Citation Format: Yu Wang, Paul Levine, Dilani Dehigaspitiya, Adam Profit, Susan Logan, Keren Imberg-Kazdan, Kent Kirshenbaum, Michael Garabedian. Multivalent peptoid conjugates suppress enzalutamide-resistant prostate cancer cellular proliferation. [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 4852.
Abstract 4856: Selective human estrogen receptor partial agonist (ShERPAs) for treatment of tamoxifen-resistant ER+ breast cancer
Authors: Delgado-Rivera Loruhama M.
Published: July 2016

Estrogen receptor positive (ER+) breast cancer represents a significant cause of death among women in the US, which is exacerbated by resistance to 1st line endocrine therapies, notably tamoxifen, which occurs in 50% of patients. Estrogen fuels growth of endocrine-dependent cancers and yet estradiol (E2) and ERá agonist, diethylstilbestrol, were used successfully in the clinic in greats cancer therapy in the 20th century. The side effects associated with these treatments are considered unacceptable, spurring the pursuit of safer, alternative therapeutic agents and the model systems to develop and optimize these agents. Three breast cancer cell lines that demonstrate resistance to tamoxifen and endocrine independence in 2D (MCF-7:5C), or 3D (T-47D:PKCá and T-47D:TAM1) cell cultures; and comparison to the parent endocrine-dependent cell lines provides a set of model systems able to test the hypothesis that a selective estrogen mimic (SEM) will provide a safe, therapeutic approach to ER+ endocrine-independent breast cancer overexpressing the PKCá biomarker. A partial agonist at ERá was selected as such a therapeutic approach: The use of partial agonists will allow us to retain the beneficial properties of E2, while attenuating the unwanted side effects. Selective human Estrogen Receptor Partial Agonists (ShERPAs) were designed, synthesized, and validated, using a combination of time-resolved FRET (TR-FRET) on ERá/SRC3 recruitment, isolated ERá affinity, and cell-based assays. ShERPAs were identified containing novel structures derived from the selective estrogen receptor modulators (SERMs) bazedoxifene and raloxifene, and their scaffolds, 3-methylindole and benzothiophene, respectively. Examples with favorable pharmacokinetic properties were validated in xenograft models of tamoxifen-resistant breast cancer.

Citation Format: Loruhama M. Delgado-Rivera. Selective human estrogen receptor partial agonist (ShERPAs) for treatment of tamoxifen-resistant ER+ breast 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 4856.
Abstract 4859: Identifying high quality, potent and selective inhibitors of ATM kinase: Discovery of AZD0156
Authors: Pike Kurt G.
Published: July 2016

Ataxia telangiectasia mutant (ATM) is a serine/threonine protein kinase from the phosphatidylinositol 3-kinase-related kinase (PIKK) family of protein kinases (also comprising ATR, DNA-PKcs, mTOR etc.) and plays a crucial role in the cellular DNA damage response signalling activated by DNA double strand breaks (DSB). Activated ATM promotes DNA repair and S/G1-cell cycle checkpoints to prevent premature mitosis, maintain genomic integrity and promote appropriate cell survival or death pathways. DSBs arise intrinsically through the collapse of stalled replication forks, which are induced by a wide range of chemotherapies, or extrinsically through exposure to ionizing radiation. Therefore, ATM inhibition represents an exciting clinical opportunity as a target to hyper-sensitize tumors to chemo/radiotherapy.

Herein, we describe our efforts to identify multiple, novel series of ATM inhibitors. We will describe our optimization efforts with particular attention given to improving the potency of the compounds in cellular systems and the selectivity of the compounds over other closely related proteins (e.g. ATR, mTOR etc.). We will also describe our efforts to optimize both the physicochemical properties of the molecules as well as the pharmacokinetic profile to enable low dose, oral administration in the clinic. These efforts culminated in the discovery of the clinical candidate AZD0156, a first in class orally available ATM inhibitor. AZD0156 shows sub-nanomolar potency in cell based assays of ATM inhibition with selectivities of greater than 1000 fold over other members of the PIKK family of enzymes. AZD0156 is a permeable, highly soluble compound with excellent preclinical pharmacokinetic properties including oral bioavailability. AZD0156 shows robust efficacy in mouse xenograft models after oral administration when combined with DSB inducing agents. AZD0156 is currently undergoing early clinical assessment.

Citation Format: Kurt G. Pike. Identifying high quality, potent and selective inhibitors of ATM kinase: Discovery of AZD0156. [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 4859.
Abstract 4862: AGI-134: a fully synthetic alpha-Gal glycolipid that prevents the development of distal lesions and is synergistic with an anti-PD-1 antibody in a mouse melanoma model
Authors: Shaw Stephen , Kristian Sascha , Wigglesworth Kim , Middleton Jenny , Glossop Mel , Whalen Giles , Old Robert , Westby Mike , Pickford Chris
Published: July 2016

Background: AGI-134 is a fully synthetic glycolipid, composed of an alpha-Gal (Galá1-3Galâ1-4GlcNAc-R) sugar epitope attached via a linker to a lipid tail. Natural antibodies to the alpha-Gal epitope are responsible for the hyperacute rejection of xenografts in humans. It is proposed that intratumorally administered AGI-134 will incorporate into the cell membranes of the tumor cells, presenting the alpha-Gal epitope for binding of anti-Gal antibodies to the tumor cells. This will initiate an immune response that attacks the injected tumor and, through uptake of immune-complexed tumor antigens by antigen presenting cells, will create a patient-specific, systemic anti-tumor response against distant metastases.

Results: We demonstrate that AGI-134 incorporates into tumor cell membranes in vitro and that the exposed alpha-Gal epitope binds anti-Gal IgG and IgM antibodies from human serum to the tumor cell surface. Using flow cytometry and a complement-dependent cytotoxicity assay we show that tumor cell opsonization with anti-Gal antibodies leads to deposition of complement proteins C3b and C5b-9, which ultimately leads to tumor cell lysis. Furthermore, we demonstrate that AGI-134-labeled tumor cells opsonized with human serum proteins are phagocytosed by professional APCs.

Using the B16-F10 melanoma model in anti-Gal producing á1,3-galactosyltransferase knockout (GT KO) mice we present data to demonstrate that AGI-134 injection into a primary tumor provides significant dose-dependent protection from the development of established distant lesions. Using GT KO mouse serum we demonstrate in vitro that deposition of complement on AGI-134-labeled mouse tumor cells is both alpha-Gal and anti-Gal dependent. In vivo, we demonstrate that the effect of AGI-134 is due to the alpha-Gal moiety by replacing it with human blood group antigens. The protection from secondary lesions conferred by AGI-134 is long lasting in the GT KO mouse melanoma model (monitored up to 90 days). Importantly, when sub-optimal concentrations of AGI-134 were tested in vivo in combination with an anti-PD-1 antibody (RMP1-14), a significant enhancement in efficacy over either of the agents administered alone was observed.

Citation Format: Stephen Shaw, Sascha Kristian, Kim Wigglesworth, Jenny Middleton, Mel Glossop, Giles Whalen, Robert Old, Mike Westby, Chris Pickford. AGI-134: a fully synthetic alpha-Gal glycolipid that prevents the development of distal lesions and is synergistic with an anti-PD-1 antibody in a mouse melanoma model. [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 4862.
Abstract 4867: The CD40 agonistic antibody APX005M ‘licenses’ antigen presenting cells to promote tumor-specific T-cell responses
Authors: Filbert Erin L. , Björck Pia , Yang Xiaodong , Trifan Ovidiu C.
Published: July 2016

Modulation of co-stimulatory and co-inhibitory molecules on immune cells has become a promising approach for cancer immunotherapy. CD40 engagement on antigen presenting cells (APCs) by its ligand CD154 leads to maturation and expression of co-stimulatory molecules such as CD80, CD86, OX-40L, and 4-1BBL that are requisite for optimal antigen-specific T-cell activation, an essential component of the anti-tumor immune response. To evaluate the therapeutic potential of targeting the CD40-CD154 pathway, Apexigen has developed APX005M - a humanized IgG1 CD40 agonistic antibody that binds with high affinity to human CD40. APX005M mimics CD154, has potent CD40 agonistic activity and promotes activation of APCs. Monocyte-derived dendritic cells treated with APX005M display a mature phenotype characterized by upregulation of CD80, CD86 and HLA-DR and increased secretion of IL-12. In mixed lymphocyte reaction and viral antigen recall assays, APX005M significantly enhances proliferation of and IFN-ã secretion from both naïve and memory T cells. Since CD40-CD154 signaling is involved in the priming phase of T cell activation and acts upstream of many critical costimulatory pathways, CD40 agonistic antibodies may represent an essential component of combination immunotherapy. In support of this, we show that APX005M synergizes with checkpoint inhibitors to promote antigen-specific T-cell responses. Importantly, in an ex vivo tumor assay APX005M induces T-cell proliferation and cytokine secretion. These data demonstrate that APX005M binds to APCs and induces their activation and maturation to ultimately drive a potent tumor-specific T-cell response. T cell activation in cancer patients receiving APX005M will be assessed in current and future clinical studies.

Citation Format: Erin L. Filbert, Pia Björck, Xiaodong Yang, Ovidiu C. Trifan. The CD40 agonistic antibody APX005M ‘licenses’ antigen presenting cells to promote tumor-specific T-cell responses. [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 4867.
Abstract 487: Usefulness of exosome quantification by cholesteryl ester transfer protein activity as a biomarker for human esophageal squamous cell carcinoma (ESCC)
Authors: Matsumoto Yasunori , Kano Masayuki , Akutsu Yasunori , Hanari Naoyuki , Hoshino Isamu , Murakami Kentaro , Usui Akihiro , Suito Hiroshi , Takahashi Masahiko , Otsuka Ryota , Matsubara Hisahiro
Published: July 2016

Exosomes are small membrane vesicles (30-100 nm) and are thought to play an important role in tumor progression, chemoresistance, dormancy etc. However, the exosome dynamics and transportation between cancer cells and the other cells are still unclear. In addition, although it is common method to quantify exosomes using standard protein quantification methods, it is actually incorrect.

In this study, we assessed the exosome dynamics with mouse model and quantify plasma exosome by cholesteryl ester transfer protein activity, which is known to be enriched within exosomes. We analyzed the relationship between exosome quantification and clinical characters in ESCC patients.

In order to image and analyze the movement of cancer cell-derived exosomes, green fluorescent protein (GFP)-tagged CD63, which is a general marker of exosomes, was expressed in human esophageal squamous cancer cell line TE2. Mouse model of human ESCC were then established by s.c. injection of TE2-CD63-GFP or TE2 cell lines. Eight weeks after injection, the tumor, blood and other organs were harvested and imaged with fluorescence. Our data indicates tumor derived exosome can emitted from the tumor and circulate in the blood flow.

Quantification of plasma exosome in ESCC patients (n = 64) and no malignant patient (n = 10) were performed by measurement of cholesteryl ester protein activity, and then analyzed the relationship between the exosome number and the patients’ clinical characteristics, such as age, sex, TNM classification, prognosis, tumor marker. Our data indicates the exosome number was higher in ESCC patients than no malignant patients (P = 0.0014). Althogh there were no difference in tumor depth, lymph node status, distant metastasis, the exosome number can be a prognostic marker for ESCC (P = 0.04).

Citation Format: Yasunori Matsumoto, Masayuki Kano, Yasunori Akutsu, Naoyuki Hanari, Isamu Hoshino, Kentaro Murakami, Akihiro Usui, Hiroshi Suito, Masahiko Takahashi, Ryota Otsuka, Hisahiro Matsubara. Usefulness of exosome quantification by cholesteryl ester transfer protein activity as a biomarker for human esophageal squamous cell carcinoma (ESCC). [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 487.
Abstract 4873: ImmTACs re-direct the immune system efficiently to eradicate cancer
Authors: Bossi Giovanna , Kenefeck Rupert , Caroline Boudousquie Caroline , Harper Jane , Dukes Joseph , Liddy Nathaniel , Paston Samantha , Mahon Tara , Molloy Peter , Sami Malkit , Baston Emma , Cameron Brian , Vuidepot Annelise , Hassan Namir , Jakobsen Bent K.
Published: July 2016

Immunotherapy strategies that are able to induce T cell infiltration into tumors and activate a cytotoxic T cell response have the potential to destroy the tumor. Although T cells can mediate clearance of a tumor, thymic selection and the suppressive microenvironment limit their effectiveness. To overcome poor tumor immunogenicity, we have developed a unique platform that enables the generation of ImmTACs (Immune-mobilising monoclonal TCRs Against Cancer); these are comprised of an affinity enhanced TCR specific for a cancer antigen fused to an anti-CD3 scFv. The TCR end targets and binds MHC class I/peptide complex displayed on cancerous cells while the anti-CD3 scFv end engages polyclonal T cells to mediate a potent anti-tumor response.

The most advanced ImmTAC, IMCgp100, targets the HLA-A2/gp100280-288 epitope presented by melanoma cells. IMCgp100 is currently in a Phase I/IIa clinical trial for advance malignant melanoma and is showing promising clinical efficacy in some patients. Here we report a series of in vitro experiments evaluating IMCgp100 mechanism of action. IMCgp100 is able to redirect T cells from healthy donors or from melanoma patients to destroy cancer cells and secrete a range of inflammatory cytokines and chemokines associated with T cell trafficking into tumors; some of these cytokines also induce upregulation of inhibitory pathway molecules. We show that effector memory cells in the CD8+ and CD4+ T cell compartments are very efficient in eliminating melanoma cells and in expanding upon IMCgp100 engagement. The effects of combining IMCgp100 with agents that relieve the suppression imposed by immune check-point molecules have also been investigated.

Citation Format: Giovanna Bossi, Rupert Kenefeck, Caroline Caroline Boudousquie, Jane Harper, Joseph Dukes, Nathaniel Liddy, Samantha Paston, Tara Mahon, Peter Molloy, Malkit Sami, Emma Baston, Brian Cameron, Annelise Vuidepot, Namir Hassan, Bent K. Jakobsen. ImmTACs re-direct the immune system efficiently to eradicate 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 4873.
Abstract 4877: Discovery of novel and potent inhibitors of indoleamine-2,3-dioxygenase (IDO1) for cancer immunotherapy
Authors: Liu Shengyang , Tao Taoliangshan , Liang Yue , Guo Hongli , Ye Xiaogang , Wu Zhiheng , Bao Fang , Yang Heping , Cai Yaxian , Yang Gangjing , Wang Longsheng , Liu Fang , Fu Guangliang , Liu Xiaolei , Du Jiangang , Qin Ping , Ma Yuanfei , Zhu Panhu , Yu Yajun , Ma Chen , Wang Pei , Zhao Liping , Wang Yuxun , Gao Daxin , Li Qun
Published: July 2016

Indoleamine-2,3-dioxygenase (IDO1) is an immune regulatory enzyme that oxidizes tryptophan to kynurenine. IDO1 is overexpressed in numerous tumor cells that block T-cell activation, induce T-cell apoptosis and increase regulatory T cells, which create an environment in which tumor-specific cytotoxic T lymphocytes are no longer able to attack a patient's cancer cells. IDO1 inhibition reverses the immune suppression at the tumor site and allows the generation of an effective anticancer immune response. Preclinical and clinical studies have shown that IDO1 inhibitors increase the anticancer immune response and dramatically increase the efficacy of various therapeutic agents especially the immune checkpoint antibodies targeting PD-1, PD-L1 and CTLA-4.

Via structure-based design, we have discovered a series of novel and potent IDO1 inhibitors that demonstrate strong IDO1 on-target activity with IC50 values ranging from 10 nM to 50 nM against human IDO1 enzyme. These compounds potently inhibit IDO1 in tumor-bearing mice as well as in Hela cells with lower single-digit to lower double digit nM IC50. The lead compound selected for clinical development shows good ADME profile, low serum binding, and good efficacy in various murine animal models when dosed orally and subcutaneously alone or in combination with immune checkpoint inhibitors. Preclinical evaluation of the compound will be presented in details.

Citation Format: Shengyang Liu, Taoliangshan Tao, Yue Liang, Hongli Guo, Xiaogang Ye, Zhiheng Wu, Fang Bao, Heping Yang, Yaxian Cai, Gangjing Yang, Longsheng Wang, Fang Liu, Guangliang Fu, Xiaolei Liu, Jiangang Du, Ping Qin, Yuanfei Ma, Panhu Zhu, Yajun Yu, Chen Ma, Pei Wang, Liping Zhao, Yuxun Wang, Daxin Gao, Qun Li. Discovery of novel and potent inhibitors of indoleamine-2,3-dioxygenase (IDO1) 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 4877.
Abstract 4879: Cancer cell-mediated signaling of TLR 2, 4, and 9 causes activation of PI3K/Akt/mTOR pathway and induces tumor cell proliferation in pancreatic cancer
Authors: Kreckel Jennifer , Grimmig Tanja , Moench Romana , Germer Christoph T. , Gasser Martin , Waaga-Gasser Ana Maria
Published: July 2016

Background: Toll like receptor (TLR) ligands are in clinical use for the immunotherapy of different cancers. They are supposed to induce an inflammatory immune response against the tumor. Interestingly, several studies showed that TLRs were expressed by cancer cells in different tumor entities and that their activation can contribute to an inflammatory microenvironment. Yet, for pancreatic cancer, TLR expression and its impact in tumor cells is only poorly understood. Therefore this study analyzed the influence of TLR2, 4, and 9 expression and activation on tumor cell signaling and proliferation in pancreatic cancer.

Methods: The expression of TLR 2, 4, and 9 was analyzed in vitro in several established as well as primary human pancreatic cancer cell lines by qRT-PCR and Western Blot. TLR stimulation was then performed in BxPC-3, MIA Paca2, and PacaDD135 cells using the TLR ligands oligodeoxyribonucleotide2006 (ODN2006), lipoteichonic acid of Staphylococcus aureus (LTA-SA) and High-Mobility Group Box 1 (HMGB1). Expression of MyD88 and pAkt was then analyzed by Western Blot. Functional analysis on proliferation (ATP assay) and cytokine expression (Luminex) was additionally performed.

Results: Expression of TLR2, 4 and/or 9 was demonstrated in all investigated human pancreatic cancer cell lines. Receptor activation by single or combined use of TLR ligands resulted in increased MyD88 and pAkt (Ser473) expression. Additionally, up-regulated anti-apoptotic protein Bcl-2 expression was found in stimulated cells, but not in the unstimulated cells. Furthermore, TLR activation resulted in the production of the interleukin(IL)-6, IL-8 and tumor necrosis factor á (TNF-á). Interestingly, tumor cell proliferation was increased within 24 and 48 hours of stimulation.

Conclusion: Our results demonstrate TLR2, 4 and/or 9 expression in all human pancreatic cancer cell lines. Additionally, our findings on TLR activation suggest chronic inflammation-mediated TLR signaling to negatively influence tumor cell apoptosis and to shift the cytokine release in pancreatic cancer towards an inflammatory microenvironment. These findings emphasize the particular role of TLR2, 4 and 9 and their activation in pancreatic cancer, outlining their relevance as potential targets for cancer therapy.

Citation Format: Jennifer Kreckel, Tanja Grimmig, Romana Moench, Christoph T. Germer, Martin Gasser, Ana Maria Waaga-Gasser. Cancer cell-mediated signaling of TLR 2, 4, and 9 causes activation of PI3K/Akt/mTOR pathway and induces tumor cell proliferation in pancreatic 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 4879.
Abstract 4880: A high-throughput siRNA screen identifies Nucleoside Diphosphate Kinase (NME3) as a novel host regulator of NF-êB signaling in response to Salmonella-induced activation of TLR-5
Authors: Gonzalez Caleb , Flentie Kelly , Kocher Brandon , Jayne Marasa Jayne , Piwnica-Worms David
Published: July 2016

Bacterial flagellin is a potent activator of NF-êB signaling, inflammation and host innate immunity. In comparison to TNFá, perhaps the most studied NF-êB-inducing ligand, flagellin elicits an overlapping, yet unique downstream transcriptional response by host cells. In contrast to the pro-oncogenic functions of TNFá, recent data indicate that flagellin may represent a novel anti-tumor ligand that acts through TLR5 and the NF-êB pathway to induce host immunity and to aid in the clearance of tumor xenografts. To begin to characterize the molecular basis for the anti-tumor effect of flagellin and to identify downstream innate signaling components of NF-êB that are responsible for the pro-inflammatory program necessary for these anti-tumor effects, we employed a high throughput screen. Utilizing HCT116 colon carcinoma cells expressing a dynamic NF-êB bioluminescent reporter and Salmonella typhimurium expressing flagellin, we performed a live cell screen of a siRNA library targeting 691 known and predicted human kinases to identify novel modulators of flagellin-induced NF-êB activation. This screen uncovered nucleoside diphosphate kinase (NME3) as a previously unrecognized, positive regulator of NF-êB signaling in response to flagellin. Targeted knockdown and overexpression assays confirmed the regulatory contribution of NME3. Furthermore, Kaplan-Meier survival analysis for breast, lung and ovarian cancer patients showed that high level expression of NME3 correlated with increased overall survival. Together, these data identify a novel pro-inflammatory role for NME3 in NF-êB signaling that may potentially be exploited to optimize cancer immunotherapy.

Citation Format: Caleb Gonzalez, Kelly Flentie, Brandon Kocher, Jayne Jayne Marasa, David Piwnica-Worms. A high-throughput siRNA screen identifies Nucleoside Diphosphate Kinase (NME3) as a novel host regulator of NF-êB signaling in response to Salmonella-induced activation of TLR-5. [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 4880.
Abstract 4899: The regulation by kinases of the expression of human major histocompatibility class I molecules
Authors: Brea Elliott J. , Oh Claire , Manchado Eusebio , Gejman Ron , Mo George , Mondello Patrizia , Garippa Ralph , Rosen Neal , Scheinberg David A.
Published: July 2016

The major histocompatability complex (MHC) is a central receptor in the adaptive immune response and is the underlying target of several effective therapies for cancer. Druggable kinases may provide the opportunity to modulate the immune response toward MHC. However, the regulation of MHC-I by kinases is largely unstudied, even though many patients with cancer are receiving therapeutic kinase inhibitors. The entire human kinome was screened using a pooled shRNA interference-based approach in a human mesothelioma cell line to uncover kinase regulators of MHC-I. Negative and positive regulators of cell surface HLA levels were discovered. A subset of highly scoring positive and negative kinase hits were subsequently validated by additional RNAi, and pharmacologic inhibitors when available. MAP2K1 (MEK), EGFR, and RET were validated as negative regulators of HLA expression in multiple cancer types. We mapped the pathways responsible for increased HLA upon kinase inhibition. Interestingly, inhibition of the MAP Kinase pathway broadly influenced expression of other components of the antigen presentation machinery. Moreover, DDR2 and MINK1 were shown to positively regulate HLA-A*02:01. This had therapeutic relevance, as shown with a therapeutic TCR mimic antibody to a MHC/peptide complex. Druggable kinases may thus serve as immediately applicable targets for modulating immunotherapy for many diseases.

Citation Format: Elliott J. Brea, Claire Oh, Eusebio Manchado, Ron Gejman, George Mo, Patrizia Mondello, Ralph Garippa, Neal Rosen, David A. Scheinberg. The regulation by kinases of the expression of human major histocompatibility class I molecules. [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 4899.
Abstract 490: High-throughput mutational analysis in cell-free DNA by targeted next-generation sequencing
Authors: Tam Nga wan Rachel , Shames David , Darbonne Walter
Published: July 2016

Circulating cell-free DNA (cfDNA) in plasma offers a non-invasive approach to monitor tumor molecular profiling in real-time at multiple time-points, detection of emerging genomic alterations associated with drug resistance and clarifying cancer prognosis and diagnosis of cancer recurrence or progression. We developed a robust, multiplexed and PCR-based targeted next-generation sequencing approach to detect actionable mutations in cfDNA with low DNA input (1-5 ng). We employed the latest Ion TorrentTM S5/XL bench-top sequencer with Ion ChefTM Automation System and successfully automated the entire 2-day workflow from libraries preparation to semi-conducting chip sequencing. The hands-on time has been reduced to only 30 min.

We will present data showing validation of this NGS platform using commercially available DNA control, archival FFPE tissue and cfDNA matched samples and demonstrate robust sensitivity and specificity by using the off-the-shelf Ion AmpliSeq Cancer Hotspot Panel v2 that covers 50 oncogenes. We also compared this approach to other orthogonal technologies, including quantitative PCR (qPCR) and droplet digital PCR (ddPCR) and achieved 100% concordance across these platforms. NGS can detect mutations down to 1% allelic frequency with 100% sensitivity and specificity.

This robust and automated NGS platform is being routinely used in our lab to analyze cell-free DNA samples from multiple clinical trials. These efforts enable the development of a non-invasive method to overcome existing challenges to provide molecular understanding of patient's tumor evolution in real time, and aid in the development of personalized therapies for cancer patients.

Citation Format: Nga wan Rachel Tam, David Shames, Walter Darbonne. High-throughput mutational analysis in cell-free DNA by targeted next-generation sequencing. [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 490.
Abstract 491: Salvage MET amplification detection and therapy through cell-free DNA NGS in a progressing lung cancer patient
Authors: Peled Nir , Belilovski Anna , Soussan-Gutman Lior , Lanman Richard B. , Talasaz AmirAli
Published: July 2016

Background: Mutational analysis has become standard of care in lung cancer. However, many patients do not have tissue available due to lack of tissue, or more commonly due to tissue exhaustion by previous tissue analysis. Next generation sequencing of circulating cell-free DNA (cfDNA) provides a non-invasive alternative. EGFR point mutations and indels are well reported in this manner, however gene copy number amplification is more challenging. Here we present a cases where MET amplification has been detected and guided highly efficient therapy in advanced lung cancer patient.

Methods: Guardant360TM is a targeted cfDNA NGS panel using hybrid capture and complete exon sequencing exons for single nucleotide variant detection in 68 genes, copy number amplifications (CNA) in 16 genes, and selected indels and fusions. CNA has been validated against cell lines with known amplifications.

Results: A 70 y old former light smoker (15 PY) with pulmonary fibrosis and moderate pulmonary hypertension was diagnosed with a 30 mm RMB lung adenocarcinoma that was treated by stereotactic beam radiotherapy due to poor pulmonary reserve. After 5 months, mediastinal, liver and multiple bone metastases were diagnosed. Genomic analysis of the primary tissue biopsy yielded a rare EGFR mutation (I744F) and afatinib therapy was started to cover uncommon EGFR mutations. However, 2 months later, a significant progression has occurred and the patient had severe local progression with respiratory near-failure. The patient was not a candidate for chemotherapy and there was no further tissue available for molecular testing. Therefore cfDNA testing was performed finding no residual EGFR mutation in the blood but a MET amplification reported above the 90th percentile for the Guardant Health laboratory with copy number of 53.6 in the blood.

Upon this result, crizotinib 250 mg BID was started with immediate clinical improvement, followed with a dramatic imaging response on CT/PET scans. Currently, the patient is in PS-0 and free of any symptoms, 3 months since treatment has been started.

Conclusion: cfDNA detection of MET amplification as a key resistance mechanism after EGFR TKI therapy is feasible in patients where tissue is not accessible or was undergenotyped and may be accompanied by a fast and dramatic clinical improvement.

Citation Format: Nir Peled, Anna Belilovski, Lior Soussan-Gutman, Richard B. Lanman, AmirAli Talasaz. Salvage MET amplification detection and therapy through cell-free DNA NGS in a progressing lung cancer patient. [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 491.