bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2137: Discovery of dual MNK 1 and 2 and BCR-ABL kinase inhibitors for the treatment of blast crisis chronic myeloid leukemia
Authors: Cherian Joseph , Nacro Kassoum , Poh Zhi Ying , Guo Samantha , Ho Melvyn Wai Tuck , Yang Haiyan , Lim Sharon , Choong Meng Ling , Joy Joma Kanikadu , Zekui Perlyn Z. , Liu Boping , Ong Esther Hongqian , Pendharkar Vishal , Ding Lijun , Poulsen Anders , Lee May Ann , Sangthongpitag Kanda , Chuah Charles , Ong Tiong S. , Hill Jeffrey , Keller Thomas H. , Matter Alex
Published: July 2016

Chronic Phase Chronic Myelogenous Leukemia (CP-CML) is well treated with tyrosine kinase inhibitors (TKI) through BCR-ABL kinase modulation. However, TKI are ineffective for late-stage or blast crisis (BC) CML in which phase, granulocyte macrophage progenitors (GMPs) have acquired the ability to function as leukemic stem cells (LSCs), and are thought to act as a reservoir for TKI resistance with poor prognosis. An effective BC-CML therapy will likely have to target the BC-LSC population to ensure long-term disease control.

Recent studies have shown the importance of the MAP kinase interacting serine/threonine kinase (MNK1/2)-eukaryotic translation initiation factor 4E (eIF4E) axis in sustaining BC-LSC self-renewal. Thus, a single agent which inhibits both BCR-ABL and MNK1/2 kinase simultaneously represents a rational approach to target BC-LSCs. Such an agent should be able to distinguish between normal hematopoietic stem cells (HSC) and LSCs.

Here, we report the discovery of a potential BC-CML therapy that relies on the inhibition of MNK1/2 and BCR-ABL kinases using a dual specific MNK/ABL inhibitor. We will also report the in vitro and in vivo biological data, pharmacokinetic properties, and biochemical characteristics of such compounds. These compounds are able to prevent eIF4E phosphorylation, thus selectively inhibiting the MNK-eIF4E-dependent self-renewal function of BC-LSCs as a single agent while leaving normal HSCs unscathed.

Citation Format: Joseph Cherian, Kassoum Nacro, Zhi Ying Poh, Samantha Guo, Melvyn Wai Tuck Ho, Haiyan Yang, Sharon Lim, Meng Ling Choong, Joma Kanikadu Joy, Perlyn Z. Zekui, Boping Liu, Esther Hongqian Ong, Vishal Pendharkar, Lijun Ding, Anders Poulsen, May Ann Lee, Kanda Sangthongpitag, Charles Chuah, Tiong S. Ong, Jeffrey Hill, Thomas H. Keller, Alex Matter. Discovery of dual MNK 1 and 2 and BCR-ABL kinase inhibitors for the treatment of blast crisis chronic myeloid leukemia. [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 2137.
Abstract 2146: YAP is an important pathway downstream of EGFR that is a potential target for EGFR-TKI-resistance lung adenocarcinomas
Authors: Lee Ting-Fang , Tseng Yu-Chi , Liu Ying-Pu , Kao Yu-Rung , Wu Cheng-Wen
Published: July 2016

Epidermal growth factor receptor (EGFR) mutations are found in lung adenocarcinomas leading to tumor cells proliferation and survival. EGFR tyrosine kinase inhibitors (TKIs) that block EGFR activity are effective therapeutics for EGFR-mutant lung adenocarcinoma patients, but TKI-resistance inevitably occurs. The YES-associated protein (YAP) transcription coactivator has been implicated as an oncogene and is amplified in human cancers and provides tumor cells strong proliferation and survival cues. This study investigated the roles of YAP in lung adenocarcinoma by exploring its regulation and functions mediated by EGFR signaling. Using lung adenocarcinoma cell lines, we detected enhanced YAP expression and activity mediated by EGFR signaling due to enhanced protein stability. The Src family protein YES was involved in EGFR-regulated YAP expression. The YAP/YES pathway was crucial for proliferation in EGFR-dependent cells. Small molecules that reduced YAP levels by mechanisms bypassing EGFR were effective in killing EGFR-dependent cells including EGFR T790M, the major cause of TKI-resistance. These observations unveiled the significance of YAP in EGFR mutant lung adenocarcinomas and identified YAP as an promising therapeutic target for EGFR-dependent lung adenocarcinoma patients, including those with EGFR T790M-caused TKI resistance.

Citation Format: Ting-Fang Lee, Yu-Chi Tseng, Ying-Pu Liu, Yu-Rung Kao, Cheng-Wen Wu. YAP is an important pathway downstream of EGFR that is a potential target for EGFR-TKI-resistance lung adenocarcinomas. [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 2146.
Abstract 2150: Alternative treatment strategies to overcome MAPK inhibitor resistance in melanoma
Authors: Goetz Eva , Rabasha Bokang , Garraway Levi
Published: July 2016

Inhibitors of the BRAF/MEK/ERK pathway have promise in the treatment of BRAF mutant melanoma and other MAPK activated cancers. Treatment of metastatic melanoma patients with the combination of RAF + MEK inhibitor has extended progression free survival over single agent therapy. However, in a large subset of patients, resistance invariably develops. Most clinical models show reactivation of the MAPK pathway in resistant samples, suggesting further downregulation of MAPK signaling might be beneficial. Therefore, we took BRAF mutant melanoma grown to resistance by continuous culture in dabrafenib plus trametinib (RAF + MEK inhibitor) or SCH772984 (ERK inhibitor), and treated them with a triple combination of RAF+MEK+ERK inhibitors. We found cells resistant to RAF+MEK inhibitors were cross-resistant to ERK inhibitor, but sensitive to RAF+MEK+ERK inhibitors. Conversely, cells resistant to the ERK inhibitor were cross-resistant to RAF+MEK inhibitor, but sensitive to the triple combination. However, given the possible deleterious side effects of a triple combination in patients, we explored whether alternative dosing and timing strategies could delay development of resistance. We compared chronic dosing of RAF+MEK or ERK inhibitors to weekly exposures alternating between RAF+MEK and ERK inhibitors. This alternative dosing strategy delayed development of resistance in vitro over chronic dosing, while also resulting in a slower proliferation rate in resistant cells. In addition, cells exposed to a higher dose of inhibitor for a shorter period of time was equivalent to chronic exposure. These studies suggest that alternative dosing strategies (other than chronic exposure) may prevent development of resistance, and allow for treatment of patients with higher order combinations and less side effects.

Citation Format: Eva Goetz, Bokang Rabasha, Levi Garraway. Alternative treatment strategies to overcome MAPK inhibitor resistance in 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 2150.
Abstract 2164: Tenascin-C binding peptides for cancer targeting
Authors: Lingasamy Prakash , Tobi Allan , Hunt Hedi , Säälik Pille , Salumäe Markko , Teesalu Tambet , Teesalu Tambet
Published: July 2016

Extracellular matrix protein tenascin C (TNC) is strongly overexpressed in the stroma of many solid tumors. TNC expression is upregulated at the invasive front of tumors while being nearly undetectable in normal adult tissues. TNC-targeted humanized antibodies are undergoing clinical development for targeting compounds to solid tumors. Compared to antibodies, homing peptides may have more favorable extravasation and tumor penetration properties.

We used in vitro T7 peptide display to identify peptides that bind to the alternatively spliced domain C of the large isoform of Tenascin-C (TNC FnIIIC). The selected phage pool showed 500-fold increase in binding to immobilized TNC FnIIIC. Candidate TNC FnIIIC-binding peptides (designated PL1- 3) were found to bind to TNC FnIII C protein, to the extracellular matrix extracted from PC3 prostate tumors and to cultured tumor cells known to overexpress TNC. To evaluate candidate peptides in vivo, we used mice bearing patient-derived xenografts of P3 glioma (R. Bjerkvig, Bergen, Norway), 4T1 breast tumors, and PC-3prostate carcinoma. A pool of the phages displaying TNC FnIIIC binding peptides, and a panel of phages displaying published tumor homing and control peptides was injected intravenously in tumor-bearing mice, and relative tumor homing and background in control tissues was determined for each phage using high-throughput sequencing (HTS) of phage genomic DNA. These studies demonstrated that among TNC FnIIIC targeting peptides, the PL3 peptide showed the best tumor homing and specificity profile. To evaluate the potential of the synthetic PL3 peptide for tumor targeting of nanoparticles, we coupled PL3 peptide to iron oxide nanoworms (IONW) - paramagnetic nanoparticles that are PEGylated to extend blood half-life, and have because of their elongated shape more effective targeting properties than spherical nanoparticles. 5 hours after intravenous administration of 7.5mg/kg of IONW in mice bearing PC3 prostate cancer xenografts, PL3-IONW but not control IONW showed a robust accumulation in tumor vasculature, and some extravasation into extravascular tumor tissue. Our data suggests that the newly identified TNC FnIIIC targeting peptide, PL3, may be used for payload delivery into tumor stroma. Currently, we are evaluating PL3-targeted IONWs as a MRI contrast agent and as carriers for cytotoxic compounds to TNC expressing tumors.

Citation Format: Prakash Lingasamy, Allan Tobi, Hedi Hunt, Pille Säälik, Markko Salumäe, Tambet Teesalu, Tambet Teesalu. Tenascin-C binding peptides for cancer targeting. [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 2164.
Abstract 2170: Molecular modeling of novel peptide-receptor interaction for targeted drug delivery of prostate cancer
Authors: Salam Ahmad , Hembrick Vincent , Jaynes Jesse , Turner Timothy , Abdalla Mohamed
Published: July 2016

Prostate Cancer (PCa) is the second leading cause of cancer-related deaths among men in the United States. Due to the harsh side effects of conventional chemo- and radiotherapies, targeted drug delivery is now gaining the focus of cancer researchers. The key for success of any newly developed targeted drug delivery systems is the novel design of targeting peptides which have high binding affinities to differentially over-expressed receptors on the surface of PCa cells. Two over-expressed such receptors, which can be targeted to the cell surface of PCa cells are: Gonadotropin Releasing Hormone Receptor (GnRH-R), and Prostate Specific Membrane Antigen (PSMA). The analysis done from molecular dynamics simulation data gave an important information about each interaction; such as RMSD, The number of contacts between receptor and peptide within a cutoff distance of 5 Å and the binding free energies, ΔGGBSA of the interactions. In all systems, the back bone RMSD deviation of the receptor was calculated using the last 10 ns of the sampling process and averaged over all trajectories. The RMSD data deviated between 1.22 - 2.51 Å. The free binding energies or ΔGGBSA was calculated from the last 10 ns of the sampling process. The QH1 system has the more negative ΔGGBSA -36.15 kcal mol-1 and the QH4 system has the least negative ΔGGBSA of -18.4 kcal mol-1 among the eight systems of GnRH-R. QH2 system exhibits a highly interactive hydrogen bond (bond distance is 2.65 Å) between 134th position of the receptor amino acid sequence GLU and 5th position of the qh2 peptide's TYR. The ΔGGBSA is for the qh2 system is – 34.92 kcal mol-1. In the PSMA and its targeting peptide interactions, T2IA has the least negative ΔGGBSA of -47.37 kcal mol-1. A salt bridge was observed between the 10th position of the T2IA peptide sequence ARG and the 328 position of the 3RBU receptor's GLU. Based on our molecular modeling studies, we have determined the best targeting peptides among all the newly designed peptides. The binding efficiency of the targeting peptides with the lowest binding energies will be further investigated surface plasmon resonance (SPR) in near future.

Citation Format: Ahmad Salam, Vincent Hembrick, Jesse Jaynes, Timothy Turner, Mohamed Abdalla. Molecular modeling of novel peptide-receptor interaction for targeted drug delivery of 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 2170.
Abstract 222: Inhibitors of mitochondrial transcription (IMT) specifically affect cancer cell proliferation
Authors: Bergbrede Tim , Choidas Axel , Di Lucrezia Raffaela , Menninger Sascha , Unger Anke , Uwe Koch , Nussbaumer Peter , Bonekamp Nina , Hoberg Emily , Posse Victor , Felser Andrea , Falkenberg Maria , Larsson Nils Göran , Gustafsson Claes M. , Klebl Bert M.
Published: July 2016

Mitochondria contain a unique DNA genome (mtDNA) that encodes a number of key components of the respiratory chain. Levels of mtDNA gene expression correlate with the metabolic requirements of mammalian cells. Recently, the role of mitochondrial function for the development and progression of cancer has been highlighted. Oncogenes have been identified to redirect mitochondrial functions to increase cancer cell survival. Increased levels of oxidative phosphorylation have also been implicated in cancer cell resistance towards drugs. In agreement with this notion, compounds that inhibit respiration may specifically target such resistant cells. Finally, it was demonstrated that inhibition of mitochondrial gene expression leads to apoptosis in rapidly dividing cells. Based on these observations, we present a completely novel principle different from known electron transfer chain blockers to treat cancer by inhibiting mtDNA transcription. To this end a study to identify and characterize inhibitors of mitochondrial transcription (IMTs) with lead-like properties was initiated. A high throughput biochemical screening campaign was performed identifying small molecular weight inhibitors of PolRMT, the mitochondrial RNA polymerase, which controls mtDNA transcription. Upon hit optimization, potent compounds with biochemical and cellular IC50s in the low nanomolar range have been identified. IMTs displayed good physicochemical and pharmacological properties. They have been shown to be inactive on other RNA polymerases proving their excellent specificity. Testing IMTs in assays for cancer cell proliferation revealed potent inhibition of sensitive cell lines (double digit nanomolar range). Interestingly, none of the tested primary non-cancer cell lines was affected by the IMTs, indicating a good therapeutic window. Testing selected IMTs in pharmacokinetic studies in vivo revealed high exposures and no detectable adverse effects (single and repeated dosing). Medicinal chemistry optimization will help to identify candidate compounds for efficacy testing in xenograft models.

Citation Format: Tim Bergbrede, Axel Choidas, Raffaela Di Lucrezia, Sascha Menninger, Anke Unger, Koch Uwe, Peter Nussbaumer, Nina Bonekamp, Emily Hoberg, Victor Posse, Andrea Felser, Maria Falkenberg, Nils Göran Larsson, Claes M. Gustafsson, Bert M. Klebl. Inhibitors of mitochondrial transcription (IMT) specifically affect cancer cell 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 222.
Abstract 2222: Dual inhibition of cancer stemness and immune checkpoint genes by targeting Stat3
Authors: Gao Yuan , Keates Sarah , Hsu Eric , Huang Janet , Brooks Emily , Hitron Matt , Li Youzhi , Rogoff Harry A. , Li Chiang
Published: July 2016

Highly malignant stemness-high cancer cells, also termed cancer stem cells, have been found to hijack stemness genes important for embryonic stem cells to acquire a state of high-stemness. Cancer stemness has been associated with enhanced tumorigenicity and resistance to chemotherapies. Moreover, stemness-high cancer cells have been isolated from a variety of human cancer types. Therefore, targeting cancer stemness holds significant promise for advancing cancer treatment.

Recent clinical success with antibodies targeting PD1 and PDL1 has validated that cancer cells can hijack immune checkpoint genes to subvert endogenous anticancer surveillance by the immune system. It is, therefore, highly desirable to develop therapeutics that simultaneously target both cancer cell stemness and immune checkpoints.

The transcription factor Signal Transducer and Activator of Transcription 3 (Stat3) is activated by a multitude of upstream oncogenic pathways and cytokine receptors. Aberrant and constitutive activation of Stat3 has been found in a wide variety of human cancers, and has also been implicated in cancer cell proliferation, survival, and immune evasion.

By using aiRNA (asymmetric RNA duplexes) we have achieved potent Stat3 silencing with precision in stemness-high cancer cells. We observed that Stat3 silencing leads to simultaneous down regulation of cancer cell stemness and immune checkpoint gene expression. Treatment of stemness-high cancer cells with BB608, a small molecule inhibitor of Stat3, led to simultaneous inhibition of stemness-high cancer cell survival and self-renewal, as well as downregulation of immune checkpoint genes, including IDO1 in vitro and in vivo. Furthermore, while expression of stemness genes and immune checkpoint genes in tumor tissues are known to predict poor patient survival, patients with higher expression levels of stemness genes and immune checkpoint genes showed prolonged overall survival after treatment with BB608 in clinical trials. Targeting Stat3 is, therefore, a promising strategy to achieve dual inhibition of cancer cell stemness and immune evasion.

Citation Format: Yuan Gao, Sarah Keates, Eric Hsu, Janet Huang, Emily Brooks, Matt Hitron, Youzhi Li, Harry A. Rogoff, Chiang Li. Dual inhibition of cancer stemness and immune checkpoint genes by targeting Stat3. [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 2222.
Abstract 2268: Detection of T790M mutation in EGFR gene, an EGFR-TKI resistant mutation, in tumor samples unexposed to EGFR TKIs
Authors: Koba Hayato , Kimura Hideharu , Nishikawa Shingo , Yoneda Taro , Sone Takashi , Kasahara Kazuo
Published: July 2016

Background: T790M mutation in EGFR gene is the commonest mechanism of resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) patients with EGFR mutation. The third-generation EGFR-TKIs are expected to overcome the resistance caused by T790M existence. On the other hand, several reports using highly sensitive detection assays showed a minute amount of T790M mutation alleles was detected in tumor tissues obtained before EGFR-TKI treatment.

Aim: To clear whether T790M dominancy in tumor cells before EGFR-TKI treatment reflects between about the EGFR-mutated lung cancer which was not until exposed EGFR TKI therapy and actual resistance mechanism. Methods: We assessed T790M dominancy in tumor cells from 29 NSCLC patients with EGFR mutations, who had not received EGFR-TKIs. T790M mutation and common mutations, such as deletional mutations in exon 19 and L858R, were detected by droplet digital PCR (ddPCR) separately. T790M dominancy was calculated from each level of mutation-alleles about common mutation and T790M mutation.

Results: We can detect the T790M mutation in initial biopsy tissues from 21 cases (72%). The mean value of T790M mutation dominancy ratio before EGFR-TKI treatment was 0.48% (0.00-69.09%) and it is not difference between in patients with T790M-positive at resistance and in that with negative (p = 0.87). Conclusions: T790M mutation was detected in most of the tumor tissues unexposed to EGFR TKIs. T790M mutation dominancy calculated by our assays in tumor tissues before EGFR-TKI treatment may not be related to induce T790M resistance to EGFR-TKIs.

Citation Format: Hayato Koba, Hideharu Kimura, Shingo Nishikawa, Taro Yoneda, Takashi Sone, Kazuo Kasahara. Detection of T790M mutation in EGFR gene, an EGFR-TKI resistant mutation, in tumor samples unexposed to EGFR TKIs. [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 2268.
Abstract 2277: Multiplexed gene expression profiling of resected intrahepatic cholangiocarcinoma: Implication of FGFR4-FGF19 as a novel treatment target
Authors: Yoo Changhoon , Kim Kyu-pyo , Ryoo Baek-Yeol , Hong Seung-Mo , Hwang Jung Jin , Jeong Seong-Yun , Hoeflich Klaus , Schmidt-Kittler Oleg , Miller Stephen , Choi Eun Kyung
Published: July 2016

Background: The fibroblast growth factor receptor 4 (FGFR4) pathway is an essential regulatory component of bile acid synthesis and its relationship with hepatocellular carcinoma (HCC) has been reported. We investigated the gene expression and clinical significance of FGFR4 and related pathways in intrahepatic cholangiocarcinoma (IH-CCa).

Methods: We assessed the expression of 98 genes in formalin-fixed paraffin embedded tumor tissue specimens from 46 patients with IH-CCa surgically resected using the NanoString platform. This included 10 FGF pathway genes (e.g. FGFR1-4, KLB, FGF3, 4, 19, 21, 23), 19 distal marker genes (e.g. CYP7A1, CYP17A1) and 31 genes relevant to HCC and CCa (e.g. AFP, TS) and 18 CNV matched genes and 20 control genes. Log-transformation of the gene expression was done for normalization and statistical analysis. Overall survival of 44/46 patients was correlated with gene expression (< median vs ≥ median) using a log-rank test. Two patients were excluded from analysis, due to death from postoperative complications and metastasectomy respectively.

Results: The median age of our patient cohort was 56 years (range 30-78) and 34 patients (74%) were male. All but one tumor specimen (98%) were from the primary tumor resection and microscopic complete resection (R0) was achieved in 37 patients (80%). Six patients (13%) had hepatitis B virus infection with or without liver cirrhosis. FGFR4 was expressed 100 times over background in IH-CCa, the expression of FGF19, FGF21, CYP7A1, CYP17A1, AFP and KLB was lower. Overall survival was significantly stratified according to the expression of FGFR4 (p = 0.004), FGF19 (p = 0.046), FGF21 (p = 0.04), KLB (p = 0.03), CYP8B1 (p = 0.02), ADH1B (p = 0.045), CDKN1B (p = 0.005), NCOR1 (p = 0.03), NF1 (p = 0.006), NROB2 (p = 0.001), MAP2K3 (p = 0.009), MYC (p = 0.04), CYP2E1 (p<0.001), and LGR5 (p = 0.03).

Conclusions: Our results indicate that FGFR4 might have a crucial role in IH-CCa, and its overexpression was associated with better prognosis, supporting that FGFR4 may define a subset of IH-CCa and be used as a promising target for therapeutic interventions.

Citation Format: Changhoon Yoo, Kyu-pyo Kim, Baek-Yeol Ryoo, Seung-Mo Hong, Jung Jin Hwang, Seong-Yun Jeong, Klaus Hoeflich, Oleg Schmidt-Kittler, Stephen Miller, Eun Kyung Choi. Multiplexed gene expression profiling of resected intrahepatic cholangiocarcinoma: Implication of FGFR4-FGF19 as a novel treatment target. [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 2277.
Abstract 2295: GDNF family receptor alpha 4 (GFRa4)-targeted adoptive T-cell immunotherapy for medullary thyroid carcinoma
Authors: Bhoj Vijay G. , Nunez-Cruz Selene , Zhou Kenneth , Arhontoulis Dimitrios , Feldman Michael , Mansfield Keith , Peng Haiyong , Rader Christoph , Siegel Don L. , Milone Michael C.
Published: July 2016

Metastatic medullary thyroid cancer (MTC) is a rare, but often aggressive, thyroid malignancy with a 5-year survival rate of 28% and few effective therapeutic options. Adoptive T-cell immunotherapy using chimeric antigen receptor (CAR)-modified T cells (CARTs) is showing encouraging results in the treatment of cancer, but development is challenged by the availability of suitable target antigens. We identified glial-derived neurotrophic factor (GDNF) family receptor alpha 4 (GFRα4), which associates with the RET receptor tyrosine kinase, as a putative antigenic target for CAR-based therapy of MTC. Using RNA in situ hybridization (RNAscope) and quantitative RT-PCR, we show that GFRα4 is highly expressed in MTC. Normal tissue expression of GFRα4 mRNA is restricted to parafollicular cells (C-cells) within the thyroid, the normal cell type from which MTC originates, and normal thymus.Based upon the highly restricted expression, we generated two high affinity single chain variable fragments (scFvs) targeting GFRα4 isoforms a and b by selecting a naïve rabbit antibody library by phage display. Second generation CARs bearing the CD137 costimulatory domain were constructed using these GFRα4-specific scFvs. GFRα4-specific CARTs trigger antigen-dependent cytotoxicity and cytokine production in vitro, and they are able to control pre-established TT cell tumors in an immunodeficient mouse xenograft model of MTC. These data demonstrate the feasibility of targeting GFRα4 by CARTs, and support this molecule as a promising target for adoptive T cell immunotherapy and other antibody-based therapy of MTC.

Citation Format: Vijay G. Bhoj, Selene Nunez-Cruz, Kenneth Zhou, Dimitrios Arhontoulis, Michael Feldman, Keith Mansfield, Haiyong Peng, Christoph Rader, Don L. Siegel, Michael C. Milone. GDNF family receptor alpha 4 (GFRa4)-targeted adoptive T-cell immunotherapy for medullary thyroid 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 2295.
Abstract 23: Caloric restriction slows tumor growth and metastases in both hormone-sensitive and hormone-resistant prostate cancers
Authors: Gitman Robert S. , LaRose Meredith , Trabulsi Edouard J. , Palagani Ajay , DeAngelis Tiziana , Kelly William K. , Gomella Leonard G. , Simone Nicole L.
Published: July 2016

Introduction: The purpose of this study is to assess the effect of caloric restriction (CR) on prostate cancer (PCa) tumor burden, proliferation, and overall survival with and without radiation treatment (RT). Additionally, we determined if the beneficial effect of CR noted are due to a reduction in pro-inflammatory markers hypothesized to enhance PCa tumorgenesis.

Methods: To assess the effect of CR in vivo, 40 male 12 week-old NCRNu-M mice were injected with LNCaP or PC3 tumor cells. Once tumors were palpable, mice were randomly assigned to one of four treatment groups in cohorts of 10: ad libitum (AL) diet, 6.5Gy of radiation (RT), 30% reduction in caloric intake (CR), or CR+RT. Tumor growth and proliferation rate were measured 3 times per week via calipers and live bioluminescent imaging. Additionally, PCa tumor tissue were analyzed to determine if CR can exert its effect on tumor growth and metastases via IGF-1R signaling pathway.

Results: Adding CR to RT decreased tumor progression. In the PC3 murine model, when compared to AL, RT reduced tumor size by 22%, 77% with CR, and 80% with CR+RT. In the LNCaP murine model, compared with AL, CR reduced tumor size by 49% and a 55% reduction with CR+RT. Primary tumor formation began 9 weeks post tumor injection in the radiated cohort and was delayed to 15 weeks post tumor injection in the CR and CT+ RT cohorts. Additionally, time to metastasis was delayed with CR (86 days to metastasis in AL, 93 with RT, and 108 when combining CR+RT). Molecularly, CR decreased multiple members of the IGF-1R signaling pathway. The total IGF-1 and IGF-1R levels exhibited 50% reduction with CR and CR+RT but total INSR levels. CR also induced a significant decrease of pGSK3β and pAKT levels.

Conclusions: For the first time, we have shown that decreasing calories by 30% in both hormone-sensitive and hormone-refractory prostate cancer models, enhance the efficacy of radiation. Our experiments show that even CR alone and in combination with RT can improve PCa tumor proliferation rate, tumor burden, time to metastasis, and overall survival. We hypothesize that these changes are in part, due to the decrease of the IGF-1R signaling pathway. We propose that CR may be used as a novel therapeutic intervention to enhance outcomes of radiation treatment by altering the molecular profile of prostate tumors.

Citation Format: Robert S. Gitman, Meredith LaRose, Edouard J. Trabulsi, Ajay Palagani, Tiziana DeAngelis, William K. Kelly, Leonard G. Gomella, Nicole L. Simone. Caloric restriction slows tumor growth and metastases in both hormone-sensitive and hormone-resistant prostate cancers. [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 23.
Abstract 2307: Improved CAR safety by a non-lethal switch to control CAR activity at the T-cell surface membrane
Authors: Juillerat Alexandre , Marechal Alan , Filhol Jean-Marie , Duchateau Philippe , Poirot Laurent
Published: July 2016

Adoptive immunotherapy using engineered T-cells has emerged as a powerful approach to treat cancer. The potential of this approach relies on the ability to redirect the specificity of T cells through genetic engineering. Novel specificities in T cells have been typically implemented through the genetic transfer of the so-called chimeric antigen receptors (CARs). CARs are synthetic receptors that associated an extracellular targeting moiety with one or more intracytoplasmic signaling domain derived from lymphocyte activation receptors. Present CAR architectures are designed to combine all relevant domains within a single polypeptide, thereby; they combines advantages of MHC unrestricted target recognition to the potent native effector mechanisms of the T cell. Although adoptive transfer of CAR T cells is proven to be an effective strategy to cancer therapy, potential adverse effects such as Cytokines Release Syndrome (CRS) and/or the risk of on-target off-tumor targeting are still a major concern. To date only suicide mechanisms that can eradicate the engineered T-Cell “at will” or mRNA CAR transfection approaches have been proposed to addresses this safety issue.

Here, we describe the development of a small molecule based switch-on technology to control the surface presentation of a chimeric antigen receptor in T-cells. By grafting protein domains that can interact upon addition of a small molecule drug in the hinge domain of the CAR architecture, we are able to turn a T-cell endowed with an engineered CAR from an off-state to an on-state, in term surface presentation as well as for its specific cytolytic properties. This system offers the advantage of a “transient CAR T-cell” for safety while letting open the possibility of multiple cytotoxicity cycles using a small molecule drug. This non-lethal control system of CAR engineered T-cells represents an important advance for the safety of this technology.

Citation Format: Alexandre Juillerat, Alan Marechal, Jean-Marie Filhol, Philippe Duchateau, Laurent Poirot. Improved CAR safety by a non-lethal switch to control CAR activity at the T-cell surface membrane. [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 2307.
Abstract 2315: Dynamic SPECT imaging of PSMA-specific CAR T cells in mice bearing prostate cancer
Authors: Emami-Shahri Nia , Foster Julie , Sosabowski Jane , Maher John , Papa Sophie
Published: July 2016

Immunotherapy using CAR-T-cells is acquiring an expanding role in the treatment of malignant disease. However, efficacy of solid tumour CAR-T-immunotherapy has proven inconsistent. Limitations include poor T-cell trafficking to tumour deposits, insufficient T-cell longevity in-vivo and the occurrence of both predicted and unanticipated on-target and off-target toxicity. To refine this therapeutic approach, it is desirable to develop systems that allow the monitoring of T-cell location and persistence in-vivo.

A retroviral vector named PiN-4 was constructed, which co-expresses: (i) an interleukin (IL)-4-responsive chimeric cytokine receptor (4αβ); (ii) a prostate-specific membrane antigen (PSMA)-targeted CAR (P28z) and (iii) hNIS, which promotes the uptake of technetium-99m pertechnetate (99mTcO4−) in viable cells. IL-4 enriched human 4P28zN+ T-cells were administered intravenously to Nod Scid Gamma (NSG) mice bearing prostate tumour xenografts.

Measurement of the tumour xenografts by bioluminescent imaging (BLI) found that only PSMA-expressing tumours responded to 4P28zN+ T-cell treatment. Longitudinal SPECT-CT imaging further confirmed this with the preferential accumulation of 4P28zN+ T-cells in PSMA-expressing tumours compared to PSMA-negative tumours.

Use of NIS as a T-cell imaging reporter brings several potential advantages. It promotes receptor-mediated uptake of the inexpensive, low toxicity and clinically useful SPECT tracer, technetium-99m pertechnetate (99mTcO4−). In addition, the ectopic expression of NIS is well tolerated by host cells and hNIS functions only in viable cells. These data demonstrate proof of concept for the utility of hNIS as an imaging reporter in genetically engineered T-cells.

Citation Format: Nia Emami-Shahri, Julie Foster, Jane Sosabowski, John Maher, Sophie Papa. Dynamic SPECT imaging of PSMA-specific CAR T cells in mice bearing 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 2315.
Abstract 2344: Discovery and characterization of new original blocking antibodies targeting the CD73 immune checkpoint for cancer immunotherapy
Authors: Giraudon Paoli Marc , Augier Severine , Royannez Blemont Marilyne , Rodriguez Céline , Rispaud Blanc Hélène , Chanteux Stéphanie , Gourdin Nicolas , Gauthier Laurent , Ménétrier Caux Christine , Morel Yannis , Caux Christophe , Paturel Carine , Perrot Ivan
Published: July 2016

CD73 (NT5E) is a cell membrane ectoenzyme of the NTPDase family that plays a major role in the conversion of AMP into Adenosine (Ado). Within the tumor microenvironment, accumulation of Ado causes immune suppression and dysregulation of immune cell infiltrates resulting in tumor spreading. CD73 expression in the tumor environment has been associated with poor disease outcome and/or with a pro-metastatic phenotype. Thus, targeting CD73 may promote anti-tumor immunity by reducing Ado accumulation and may block tumor cell metastasis by inhibiting CD73 on tumor cells. Here, we describe the generation and characterization of novel anti-human CD73 antibodies, intended for the treatment of a wide range of cancers. The research leading to these results has received funding from the European Community's Seventh Framework Program (FP7/2007-2013) under grant agreement n°602200. Antibodies were discovered that inhibited CD73 function by different mechanisms, including the direct blocking of CD73 enzymatic activity or the down-modulation of membrane CD73 expression. Epitope mapping revealed that antibodies acting by these different modes of action bound to distinct sites on CD73. All selected antibodies cross-react with cynomolgus CD73 protein and have strong avidity and affinity for membrane or recombinant CD73, by flow cytometry and Surface Plasmon Resonance, respectively. Antibodies that inhibit CD73 enzymatic activity strongly reduce AMP catabolism by both recombinant and cellular CD73 with IC50 in the nanomolar range. They also efficiently reverse ATP- and AMP-mediated T cell suppression in in vitro assays in presence of both CD39+ and CD73+ cells. The antibodies that induce down-modulation of cellular CD73 expression do not block recombinant CD73 enzyme activity and partially inhibit cellular CD73 activity; they reverse ATP- but not AMP-dependent T cell suppression. The antibodies displaying the most interesting features were humanized. Evaluation of their activity in animal models is ongoing.

Citation Format: Marc Giraudon Paoli, Severine Augier, Marilyne Royannez Blemont, Céline Rodriguez, Hélène Rispaud Blanc, Stéphanie Chanteux, Nicolas Gourdin, Laurent Gauthier, Christine Ménétrier Caux, Yannis Morel, Christophe Caux, Carine Paturel, Ivan Perrot. Discovery and characterization of new original blocking antibodies targeting the CD73 immune checkpoint 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 2344.
Abstract 2354: Cancer vaccine development for hepatocellular carcinoma - HEPAVAC
Authors: Kutscher Sarah , Accolla Roberto , Ma Yuk T. , Heidenreich Regina , Izzo Francesco , Koenigsrainer Alfred , Loeffler Markus , Mueller Phillip , Mayer Andrea , Rammensee Hans-Georg , Sangro Bruno , Francque Sven , Valmori Danila , Weinschenk Toni , Singh-Jasuja Harpreet , Buonaguro Luigi
Published: July 2016

The HEPAVAC Consortium aims to develop a highly innovative, novel cancer vaccine approach for hepatocellular carcinoma (HCC). The international project consortium consists of 9 European Partners from academia and the biotech industry with complementary and substantial expertise in developing immunotherapeutic strategies to treat cancer. The project has started in September 2013 and is supported by the European Commission's 7th Framework Program (www.hepavac.eu).

HCC/normal adjacent tissue matched samples have been collected for HLA immunopeptidome analysis. 17 HCC samples from HLA-A*02+ patients and 15 samples from HLA-A*24+ patients have been analysed by mass spectrometry (LC-MS/MS). RNA-expression profiles have been established for 12 HCC samples. HLA-presentation/expression of peptides and mRNA on primary HCC samples are compared to >140 normal tissue samples from relevant organs (including heart, brain, lung, kidney, liver, nerve, skin etc.) from Immatics’ database.

A total of 9051 HLA-A*02-restricted different tumor-associated peptides (TUMAPs) have been identified from HLA-A*02+ samples, while a total of 3286 different HLA-A*24-restricted TUMAPs have been identified from HLA-A*24+ samples. Of these, 33 HLA-A*02+ TUMAPS and 33 HLA-A*24+ TUMAPs have been validated, including peptide synthesis, immunogenicity testing and pharmaceutical evaluation. Most promising TUMAP candidates show selective expression only in HCC samples and no expression in normal tissues. In parallel, more than 6600 HLA-DR TUMAPs have been identified in Hep3B cells transfected with CIITA as well as an average of 1500 HLA-DR TUMAPs have been identified in HCC samples.

A total of 16 newly identified HCC-specific epitopes have been selected for the HEPAVAC vaccine cocktail and are currently synthesized according to GMP standard. Of these, 7 are restricted to HLA- class I A*02; 5 HLA-A*24 and 4 HLA class II. In parallel, preclinical studies assessing the formulation and combination of the immunological RNA-based adjuvant (RNAdjuvant®) with peptide cocktails are underway. A multi Center phase I/II clinical trial in early HCC patients is predicted to start in July 2016.

Citation Format: Sarah Kutscher, Roberto Accolla, Yuk T. Ma, Regina Heidenreich, Francesco Izzo, Alfred Koenigsrainer, Markus Loeffler, Phillip Mueller, Andrea Mayer, Hans-Georg Rammensee, Bruno Sangro, Sven Francque, Danila Valmori, Toni Weinschenk, Harpreet Singh-Jasuja, Luigi Buonaguro. Cancer vaccine development for hepatocellular carcinoma - HEPAVAC. [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 2354.
Abstract 2401: RD3-loss orchestrates GC-signaling independent evolution of progressive neuroblastoma
Authors: Meghna Singh , Rachel Wolansky , Herman Terence S. , Aravindan Natarajan
Published: July 2016

Recently we recognized the transcriptional/translational loss of Retinal degeneration Protein 3 (RD3) in high-risk progressive neuroblastoma (HR-NB) and defined its novel tumor evolution stabilization function. Studies have shown that RD3 competitively binds to guanylate cyclases (GCs) and maintains GC expression, stability, as well as restores GC localization. Accordingly, we investigated the role of GCs, if any, in RD3-loss associated evolution of HR-NB. Utilizing an in vivo mouse model of spontaneous HR-NB coupled with ex vivo approaches using metastatic site derived aggressive cells (MSDACs), we examined the transcriptional and translational expression of GCs and validated with high-content confocal immunofluorescence. Further, utilizing a cGMP competitive enzyme immunoassay, we investigated both intracellular and soluble GC activity. Whole genome microarray analysis revealed a differential activation of GC signaling transcriptional machinery in three different clones of MSDACs (compared to parental SH-SY5Y cells) ex vivo. Immunoblotting identified a consistent translation of GCs (GC-1 and GC-2) in a manifold of metastatic tumors (compared to non-metastatic primary xenografts) in vivo and in MSDACs ex vivo. Confocal immunofluorescence analysis validated the near-identical expression of GC1 and GC2 both in SH-SY5Y cells and MSDACs. cGMP immunoassay recognized indistinguishable intracellular and soluble GC activity in clones of differentiated/undifferentiated MSDACs as opposed to the clones of SH-SY5Y. For the first time, the results of this study define the transcription and translation blueprint of GCs in HR-NB and, further imply that the RD3-loss may dictate GCs-signaling independent alternate molecular flow-through in the evolution of HR-NB.

Acknowledgements: Stephenson Cancer Center, NIH COBRE 1P20GM103639-01, Broken Arrow Lady Elks Auxiliary, and Oklahoma TSET.

Citation Format: Singh Meghna, Wolansky Rachel, Terence S. Herman, Natarajan Aravindan. RD3-loss orchestrates GC-signaling independent evolution of progressive neuroblastoma. [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 2401.
Abstract 2436: Exploring genomic alterations in pediatric cancer using ProteinPaint
Authors: Zhou Xin , Edmonson Michael N. , Wilkinson Mark R. , Patel Aman , Wu Gang , Liu Yu , Li Yongjin , Zhang Zhaojie , Rusch Michael , Parker Matthew , Becksfort Jared , Downing James R. , Zhang Jinghui
Published: July 2016

Current cancer genome data portals have focused primarily on presenting data generated from adult cancer studies. These portals typically lack features for exploring pathogenic germline mutations, somatic gene fusions, and gene expression profiling, all of which are important biomarkers for risk stratification of pediatric cancer. We have developed ProteinPaint (https://pecan.stjude.org/proteinpaint/), a web service hosting 30,000+ validated somatic SNV/indels and fusion transcripts detected in 1,654 pediatric tumor samples from 17 subtypes, 252 pathogenic or loss-of-function germline lesions detected in >1000 pediatric cancer patients of 21 subtypes, and gene expression profiles derived from RNA-Seq of 928 pediatric tumors. Cancer genomic alterations are shown on novel “disc-on-stem” skewer graphs which were designed to depict the diverse prevalence, complex allelic alteration, and temporal origin of sequence mutations and gene fusions. Adult somatic cancer mutation data from the COSMIC database can be displayed in parallel with pediatric cancer data sets for cross-study comparison. We will demonstrate examples of how ProteinPaint's integrative view of genomic alteration, gene expression and pediatric-adult data comparison has facilitated the evaluation of somatic and germline mutation pathogenicity in a clinical setting. Custom data including sequence mutations in the MAF format used by the Cancer Genome Atlas (TCGA) project, copy number alterations, and structural variations can all be imported and visualized alongside published pediatric and adult cancer data sets. Furthermore, ProteinPaint supports curation and annotation of fusion transcripts predicted from RNASeq data and analysis of tumor clonal evolution with a 2-D plot of mutation frequency of paired diagnosis and relapse samples. ProteinPaint delivers a premium user experience with animation and interactive features for visualizing large cancer mutation datasets, and can serve as a workbench to import, explore and interpret user data. Its framework continues to expand as its intuitive visualization has enabled non-bioinformatics scientists and clinicians to access and manipulate genomic data for discovery and clinical reporting.

Citation Format: Xin Zhou, Michael N. Edmonson, Mark R. Wilkinson, Aman Patel, Gang Wu, Yu Liu, Yongjin Li, Zhaojie Zhang, Michael Rusch, Matthew Parker, Jared Becksfort, James R. Downing, Jinghui Zhang. Exploring genomic alterations in pediatric cancer using ProteinPaint. [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 2436.