bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract LB-168: MicroRNA-31 initiates lung tumorigenesis and promotes mutant KRAS-driven lung cancer
Authors: Edmonds Mick D. , Boyd Kelli L. , Moyo Tamara K. , Mitra Ramkrishna , Duszynski Robert J. , Arrate Maria P. , Chen Xi , Zhao Zhongming , Blackwell Timothy S. , Eischen Christine M.
Published: July 2016

Lung cancer is the leading cause of cancer deaths and yet the genes that drive this malignancy are not entirely known. MicroRNA (miRNA) are important regulators of gene expression and aberrant miRNA expression has been linked to lung oncogenesis; however, little known about their contribution in tumor initiation. We’ve determined that miR-31 is overexpressed in human lung adenocarcinoma and this overexpression independently correlates with decreased patient survival. miR-31 promotes lung cell proliferation and adenocarcinoma growth in vivo. We also developed a transgenic mouse model that allows for lung-specific expression of miR-31 to test the oncogenic potential of miR-31 in the lung. Using this model, we observed that miR-31 induction results in lung hyperplasia, followed by adenoma formation and later adenocarcinoma development. We identified 6 negative regulators of RAS/MAPK signaling (RASA1, SPRED1, SPRED2, SPRY1, SPRY3, SPRY4) as direct targets of miR-31. Analysis of human lung adenocarcinoma revealed that high miR-31 expression correlated to decreased target mRNA expression. miR-31 overexpression in lung cells results in increased RAS signaling and pharmacologic inhibition of MEK ablates miR-31 induced lung cell proliferation. Induced expression of miR-31 in mice cooperated with mutant KRAS to accelerate lung tumorigenesis. Our study demonstrates for the first time that a miRNA can drive an epithelial cancer. Specifically we identified miR-31 as a driver of lung tumorigenesis that promotes mutant KRAS-mediated oncogenesis and demonstrate that miR-31 directly targets and reduces expression of negative regulators of RAS/MAPK signaling.

Citation Format: Mick D. Edmonds, Kelli L. Boyd, Tamara K. Moyo, Ramkrishna Mitra, Robert J. Duszynski, Maria P. Arrate, Xi Chen, Zhongming Zhao, Timothy S. Blackwell, Christine M. Eischen. MicroRNA-31 initiates lung tumorigenesis and promotes mutant KRAS-driven lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-168.
Abstract LB-169: Ovarian cancer cells selectively release exosomes containing tumor suppressor miRNAs to maintain SRC-mediated oncogenic activities
Authors: Rashed Mohammed H. , Kanlikilicer Pinar , Rodriguez Aguayo Cristian , Kabil Nashwa N. , Pichler Martin , Bayraktar Recep , Bayraktar Emine , Ivan Cristina , Mitra Rahul , Calin George A. , Sood Anil K. , Abd-Ellah Mohamed F. , Helal Gouda K. , Lopez Berestein Gabriel
Published: July 2016

Exosomes have emerged as an important mediator of diverse biological functions including tumor cell progression, invasion, immune escape, and cell-to-cell communication, through the release of molecules such as mRNAs, microRNAs, and proteins. Ovarian cancer (OC) is one of the lethal cancers with extensive local invasion, metastasis, and poor diagnosis. Thus, finding novel targets for OC therapy are urgently needed. Here, we isolated 30-140 nm exosomes from the supernatant of one normal and six different OC cells. In addition, RNAs were isolated from cells and exosomes, and profiles of two miRNA fractions were obtained using affymetrix microarray analysis. By comparing signal intensities of microarray data and validation using RT-PCR analysis, we found that miR-940 was abundant in exosomes from SKOV3IP1, HeyA8, and HeyA8-MDR. Ectopic expression of miR-940 inhibited proliferation, colony formation, invasion, migration, induced G0/G1 cell cycle arrest, and apoptosis in OC. Overexpression of miR-940 also inhibited tumor cell growth in vivo. The target gene of miRNA-940 and the downstream pathway were further investigated using several target prediction algorithms. We identified specific targeting sites for miR-940 in the 3’-untranslated region (3’-UTR) of SRC. We then experimentally validated miR-940 as a direct regulator of SRC using cell transfection and luciferase assays, and showed that miR-940 inhibited SRC expression at mRNA and protein levels. Following this reduction, the activity of proteins downstream of SRC, such as FAK, paxillin and Akt were also reduced. Collectively, our results suggest that OC cells selectively secreted the tumor suppressive miR-940 into the extracellular environment via exosomes, to maintain their invasiveness and tumorigenic phenotype.

Citation Format: Mohammed H. Rashed, Pinar Kanlikilicer, Cristian Rodriguez Aguayo, Nashwa N. Kabil, Martin Pichler, Recep Bayraktar, Emine Bayraktar, Cristina Ivan, Rahul Mitra, George A. Calin, Anil K. Sood, Mohamed F. Abd-Ellah, Gouda K. Helal, Gabriel Lopez Berestein. Ovarian cancer cells selectively release exosomes containing tumor suppressor miRNAs to maintain SRC-mediated oncogenic activities. [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 LB-169.
Abstract LB-171: Repression of miR-125b-1 by epigenetic mechanisms in breast cancer cell lines
Authors: Cisneros-Soberanis Fernanda , Andonegui Marco Alonso , Castro Clementina , Herrera Luis Alonso
Published: July 2016

miR-125b-1 downregulates targets as ERBB2, BAK1 and ETS1. These targets are involved in cell proliferation, apoptosis and cell migration, respectively. Previous studies on tumor cells reveal that downregulation of miR-125b-1 is associated with poor prognosis in breast cancer patients. DNA methylation of the miR-125b-1 promoter can repress its expression, in addition, this promoter is embedded in an intermediate CpG island thus, DNA methylation and histone modifications could also affect its transcription. Repression by DNA methylation has been well characterized, but there is no information about the role of histone modifications in the regulation of miR-125b-1 promoter. We evaluated the enrichment of two histone modifications involved in gene repression, H3K9me3 and H3K27me3, on the miR-125b-1 promoter of two breast cancer cell lines, a luminal A, MCF 7, and a triple negative, MDA-MB-231, compared with a non-transformed breast cell line, MCF 10A. We found that breast cancer cell lines are enriched with H3K27me3 and H3K9me3 in MCF 7 and MDA-MB-231, respectively. Then, we focused on reactivating miR-125b-1 in MCF 7 using an EZH2 inhibitor. After the treatment with the EZH2 inhibitor, we evaluated the transcriptional levels of the pri-miR-125b-1 and the mature miR-125b by qRT-PCR. Our results suggest that transcripts, pri-miRNA and mature miRNA, increase their expression levels after the treatment in the MCF7 cell line, but not in the MDA-MB-231 and MCF 10A cell lines. Subsequently, we evaluated the BAK1 expression and protein levels to investigate whether the miR-125b-1 reactivation could affect some targets. We observed a 60% and 70% decrease in the expression and protein levels of after treatment with the EZH2 inhibitor. To determine if the H3K9me3 is involved on miR-125b-1 silencing in MDA-MB-231, we over-expressed KDM4B/JMJD2B to reactivate this miRNA. Then, we evaluated the transcript. A three-fold increase was observed compared. We conclude that the miR-125b-1 can be repressed by different epigenetic mechanisms depending on the breast cancer subtype; the miR-125b-1 reactivation by removing the repression histone modification marks affect the expression of BAK1, a pro-apoptotic target.

Citation Format: Fernanda Cisneros-Soberanis, Marco Alonso Andonegui, Clementina Castro, Luis Alonso Herrera. Repression of miR-125b-1 by epigenetic mechanisms in breast cancer 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 LB-171.
Abstract LB-181: KLF4 suppresses T-cell acute lymphoblastic leukemia by inhibiting the stress kinase MAP2K7 pathway
Authors: Shen Ye , Park Chun Shik , Suppipat Koramit , Mistretta Toni-Ann , Puppi Monica , Horton Terzah , Rabin Karen , Lacorazza Daniel
Published: July 2016

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with the highest incidence of relapse of any pediatric ALL. A minimal two-hit model of leukemogenesis suggests that an initial genetic driver transforms hematopoietic progenitor cells into LICs, whereas a secondary genetic alteration would endow LICs with proliferative and survival advantages. Although most T-ALL patients exhibit activating mutations in NOTCH1, the cooperating genetic events required to accelerate onset of leukemia and worsen disease progression are largely unknown. Here, we show that low levels of the transcription factor KLF4 in children with T-ALL were associated with methylation of its promoter. Consistent with a tumor suppressor function, loss of KLF4 accelerated the development of NOTCH1-induced T-ALL in mice by enhancing the G1-to-S transition and promoting the expansion of leukemia-initiating cells that are responsible for chemoresistance and relapses. Mechanistically, KLF4 represses the gene encoding the kinase MAP2K7, and thus loss of KLF4 activates MAP2K7 and downstream effector JNK both in murine model of T-ALL and lymphoblasts from pediatric patients with T-ALL. Furthermore, pharmacological inhibition of JNK reduced leukemia burden in a xenograft model of human T-ALL and small molecule inhibitors exhibited anti-leukemic properties in patient-derived xenograft cells. In summary, our findings demonstrate a novel tumor suppressor function of KLF4 in a T-ALL mouse model and in pediatric T-ALL and support a model of leukemia inhibition by repression of the stress kinase MAP2K7 and its downstream targets JNK, c-JUN, and ATF2. In addition, our study provides proof-of-principle pre-clinical data supporting JNK inhibition as a potential targeted therapy for T-ALL and prompts future studies in high-risk T-ALL patients with refractory and relapsed disease.

Citation Format: Ye Shen, Chun Shik Park, Koramit Suppipat, Toni-Ann Mistretta, Monica Puppi, Terzah Horton, Karen Rabin, Daniel Lacorazza. KLF4 suppresses T-cell acute lymphoblastic leukemia by inhibiting the stress kinase MAP2K7 pathway. [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 LB-181.
Abstract LB-197: Bispecific T-cell engagers: evaluation of circulating tumor-associated antigen levels and pharmacokinetics
Authors: Gilbert Amy E. , Avilion Ariel , Doherty David , Li Shyun , Bailis Julie , Rock Dan
Published: July 2016

Immunotherapeutic approaches to activate patient T cells against cancer have emerged as a promising clinical strategy for many cancer types. One critical consideration for immune activating therapies is to establish efficacious and tolerable dosing limits. The conveyed estimate of the predicted therapeutic window could be impacted by presence of circulating tumor associated antigen. Here we evaluate a Bi-specific T cell Engager (BiTE®) directed against CD3 and CDH19, a type II cadherin overexpressed >60% of melanomas. Because, to our knowledge, the shedding of CDH19 was unknown we developed an immunoassay to detect soluble CDH19 (sCDH19) in the serum of human donors and patients with metastatic melanoma. Utilizing our immunoassay to detect sCDH19, we measured elevated levels of sCDH19 in the serum of metastatic patients relative to normal donors. Additionally, the potency of this BiTE® using T-cell dependent cellular cytotoxity assays was measured. We next incorporated the range of sCDH19 amounts in patients along with BiTE® potency and present desired pharmacokinetic profiles of this therapeutic. In sum, we report that sCDH19 is present in the circulation of patients with metastatic melanoma and evaluate the impact of sCDH19 on BiTE® pharmacokinetics and human dose projections.

Citation Format: Amy E. Gilbert, Ariel Avilion, David Doherty, Shyun Li, Julie Bailis, Dan Rock. Bispecific T-cell engagers: evaluation of circulating tumor-associated antigen levels and pharmacokinetics. [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 LB-197.
Abstract LB-203: Development of a nonclinical model for the assessment of oncology therapy-induced cardiomyopathy
Authors: Hemkens Michelle , Cardenas Samantha , Frey Jessica , Hirakawa Brad , Koza-Taylor Petra , Wang Jianying , Butler Paul
Published: July 2016

With the increasing number of people surviving cancer, patients and doctors are becoming equally focused on the quality of life in survivorship as they are on fighting cancer. Consequently, cardio-oncology has emerged as a new clinical discipline with focus on both the stratification of patients at risk and therapies that pose a cardiovascular risk, eg molecularly targeted therapies including tyrosine kinase inhibitors (TKI). Therefore, nonclinical research should strive not only to detect preclinical drug-induced changes in cardiac structure or function, but also to understand the relevance of these cardiotoxic effects to humans. As is typical for new disciplines, there is an unmet need for early and predictive tools to identify both of these factors. Currently, preclinical assessment of cardiovascular toxicity consists of repeat-dose in vivo toxicity studies focused primarily on histopathological endpoints; additional measures, such as cardiac function and biomarkers of cardiac injury, are not routinely assessed in these studies. Furthermore, analysis of these studies suggests that using young and healthy, drug-naïve animals do not adequately translate the TKI-induced cardiomyopathy phenotype; therefore, new nonclinical models are needed to address this gap. The developed model would ideally be used to assess the preclinical cardiotoxicity of proposed oncology therapies, but may also have utility in assessing cardioprotective therapies for use in cardio-oncology. This poster will propose a new model for studying cardio-oncology nonclinically that includes myocardial transcriptional signatures, strain echocardiography imaging, and pharmacologic (dobutamine) stress challenge. In general, rats with radiotelemetry were treated with vehicle or low dose doxorubicin (2 mg/kg/week IP) for 1 month. Cardiac structure and function at rest and during stress were assessed using strain echocardiography in concert with a weekly dobutamine infusion. Circulating blood biomarkers of cardiac toxicity (eg, NTproBNP, NTproANP, cTropin, and miR-208) were collected weekly and at necropsy. At the end of the study, samples of cardiac tissue were taken from the ventricular apex and were submitted for transcriptional profiling along with standard histopathology. These data as well as recent publications give high confidence in developing a rodent model predictive of drug-induced cardiomyopathy.

Citation Format: Michelle Hemkens, Samantha Cardenas, Jessica Frey, Brad Hirakawa, Petra Koza-Taylor, Jianying Wang, Paul Butler. Development of a nonclinical model for the assessment of oncology therapy-induced cardiomyopathy. [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 LB-203.
Abstract LB-204: Neoadjuvant Ezh2 inhibition amplifies an inflammatory program and sensitizes Kras-driven NSCLC to anti-NF-κB inhibition
Authors: Serresi Michela , Siteur Bjorn , Cesaroni Matteo , Proost Natalie , Hulsman Danielle , van Lohuizen Maarten , Gargiulo Gaetano
Published: July 2016

Kras mutant non-small cell lung cancer (NSCLC) is a leading cause of death but has limited therapeutic options. We investigated the impact of Polycomb repressive complex 2 (PRC2) inhibitors as single agent or as neoadjuvant treatment for subsequent anti-inflammatory therapy. Using orthotopic grafts, we show that Kras cells highly expressing the PRC2 enzymatic component Ezh2 display sensitivity to its inhibition. Ezh2 inhibition leads to transcriptional amplification of an inflammatory program involving NF-kB. Pharmacological inhibition of Ezh2 rerouted Kras tumor progression towards exploiting PRC2/NF-kB target genes as inferred by in vivo RNAi screen but also enhanced the response of Kras NSCLC to a combination of Nimesulide and Bortezomib. These data suggest that low PRC2 levels in Kras-driven NSCLC promote an epigenetic switch, providing a rationale for exploring a novel therapeutic option in which Ezh2 simultaneously acts as biomarker for therapeutic decision and as a neoadjuvant target for therapy.

Citation Format: Michela Serresi, Bjorn Siteur, Matteo Cesaroni, Natalie Proost, Danielle Hulsman, Maarten van Lohuizen, Gaetano Gargiulo. Neoadjuvant Ezh2 inhibition amplifies an inflammatory program and sensitizes Kras-driven NSCLC to anti-NF-κB inhibition. [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 LB-204.
Abstract LB-226: The effect of cabozantinib to temozolomide and bevacizumab in patients with heavily pretreated relapsed uterine leiomyosarcoma
Authors: Ikeda Sayaka , Kudoh Kazuya , Sasaki Naoki , Takano Masashi , Goto Tomoko , Kikuchi Ryoko , Kita Tsunekazu , Sakamoto Masaru , Susumu Nobuyuki , Aoki Daisuke , Kouta Hiroko , Kikuchi Yoshihiro
Published: July 2016

Background: Although uterine leiomyosarcoma (ULMS) has been treated with adriamycin, dacarbazine, ifosfamide, gemcitabine, docetaxel, et al, the effect is not satisfactory. We have reported the effect of temozolomide (T) combined with bevacizumab (B) in heavily pretreated relapsed ULMS. In this study, we evaluated the effects of addition of cabozantinib (C) to T and B.

Methods: From 2009 to 2015, total 18 patients (pts) with heavily pretreated relapsed ULMS were enrolled. They were treated with T (80mg/body/day) and B (2mg/kg; days 1, 8 and 15, q4 weeks). Since 2013, we expected better efficacy, nine pts out of 18 pts were treated by adding C (140mg/body/week) which is a c-MET inhibitor (TB, n = 9, CTB, n = 9). Treatment was continued until disease progression and/or unmanageable toxicities. The response and adverse events were evaluated using the response evaluation criteria in solid tumors (RECIST), and common terminology criteria for adverse events (CTCAE) version 3.0.

Results: As shown in Table, three (18%) of 17 pts had complete response (CR), two (12%) had partial response (PR) and eight (47%) had stable disease (SD) for at least three months. The response rate (RR; CR+PR) and clinical benefit rate (CBR; CR+PR+SD>3mo) were 29% and 76%, respectively. The median progression-free survival was 9.6 (3 - 58) months. When compared CTB with TB, CBR was better in CTB (87.5% vs 67%), but the median administration cycles and progression free interval were not improved. Two peritoneal perforation were observed in CTB.

Conclusions:

Not only TB but also CTB showed remarkable effect in heavily pretreated relapsed ULMS. These results warrant further prospective and randomized studies. nCRPRSDPDRR(%)CBR(%)TB/CTB1732842976TB921333367CTB811512587.5

Citation Format: Sayaka Ikeda, Kazuya Kudoh, Naoki Sasaki, Masashi Takano, Tomoko Goto, Ryoko Kikuchi, Tsunekazu Kita, Masaru Sakamoto, Nobuyuki Susumu, Daisuke Aoki, Hiroko Kouta, Yoshihiro Kikuchi. The effect of cabozantinib to temozolomide and bevacizumab in patients with heavily pretreated relapsed uterine leiomyosarcoma. [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 LB-226.
Abstract LB-227: Truncal activating MAPK and PI3K pathway alterations and exceptional response to dual pathway inhibition in anaplastic thyroid cancer
Authors: Gibson William J. , Ruan Daniel T. , Paulson Vera A. , Barletta Justine A. , Hanna Glenn J. , Kraft Stefan , Calles Antonio , Nehs Matthew , Moore Francis , Taylor-Weiner Amaro , Wala Jeremiah , Zack Travis , Lee Thomas , Fennessy Fiona , Alexander Erik , Thomas Tom , Jäne Pasi , Garraway Levi , Carter Scott , Beroukhim Rameen , Lorch Jochen , Van Allen Eliezer
Published: July 2016

Background: Anaplastic thyroid carcinoma (ATC) is among the most aggressive solid tumors, with a uniformly fatal prognosis. No effective systemic therapies exist. We document a case in which a patient with anaplastic thyroid carcinoma (ATC) failed to respond to either mTOR or combined RAF/MEK inhibition, but experienced a dramatic response when both drug regimens were combined.

Methods and Results: Nine biopsies of the tumor were obtained between diagnosis and autopsy. Multi-region whole-exome sequencing revealed truncal BRAF and PIK3CA mutations, which are known to activate the MAPK and PI3K/AKT pathways respectively. We performed the largest meta-analysis of ATC to date and found a 7.4% co-occurrence of MAPK and PI3K pathway alterations in ATC. We analyzed 5,255 cancer exomes in the Cancer Genome Atlas (TCGA) to determine whether similar cancer genomes occurred outside of anaplastic thyroid carcinoma. We identified 11 tumors with BRAF V600E and non-silent PIK3CA mutations. These results indicate that this genotype occurs in other cancers and may predict response to combined therapy.

Conclusions and future directions: We have identified a patient with ATC who had a dramatic response to combined inhibition of the MAPK and PI3K pathways, whereas neither MAPK inhibition nor PI3K inhibition alone was sufficient to produce tumor regression. These observations suggest that MAPK and PI3K/MTOR pathway blockade represents an effective dual therapy aimed at the founding oncogenic lesions in a subset of ATC.

Citation Format: William J. Gibson, Daniel T. Ruan, Vera A. Paulson, Justine A. Barletta, Glenn J. Hanna, Stefan Kraft, Antonio Calles, Matthew Nehs, Francis Moore, Amaro Taylor-Weiner, Jeremiah Wala, Travis Zack, Thomas Lee, Fiona Fennessy, Erik Alexander, Tom Thomas, Pasi Jäne, Levi Garraway, Scott Carter, Rameen Beroukhim, Jochen Lorch, Eliezer Van Allen. Truncal activating MAPK and PI3K pathway alterations and exceptional response to dual pathway inhibition in anaplastic thyroid 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 LB-227.
Abstract LB-235: IGF-1R/mTOR blockade and mechanisms of resistance in Ewing sarcoma
Authors: LAMHAMEDI CHERRADI Salah-Eddine , Menegaz Brian , Ramamoorthy Vandhana , Ludwig Joseph
Published: July 2016

Ewing sarcoma (ES), the second most common bone cancer in adolescents and young adults, is dependent upon the insulin-like growth factor receptor 1 (IGF-1R) and mammalian target of rapamycin (mTOR) cell-signaling axis for malignancy, survival, and metastasis. Therapies co-targeting IGF-1R and mTOR have demonstrated remarkable, although ephemeral, clinical responses in a subset of ES patients. However, the mechanisms of resistance and applicable strategies for overcoming drug resistance to IGF-1R/mTOR blockade are still undefined.

To elucidate predominant mechanism(s) of acquired drug resistance, while identifying synergistic drug combinations that improve clinical efficacy, we generated ES cell lines resistant to IGF-1R or mTOR targeted therapy. Two small-molecule inhibitors of IGF-1R were chosen, NVP-ADW-742 (IGF-1R-selective) and OSI-906 (a dual IGF-1R/insulin receptor alpha [IR-α] inhibitor). Chronic ridaforolimus exposure led to mTOR-resistant ES cell lines. Reverse phase protein lysate arrays (RPPA) revealed proteomic changes linked to IGF-1R/mTOR resistance and selected proteins were validated using in vitro cell-based assays, xenografts, and human clinical samples.

Novel mechanisms of resistance (MOR) occurred in NVP-ADW-742-, OSI-906-, and ridaforolimus-resistant cells. Of particular clinical relevance given OSI-906's ongoing phase 1b/2 clinical trials, the MOR varied depending upon whether IGF-1R or both IGF-1R and IR-α were targeted. Key proteomic alterations present in vitro also occurred in ES xenografts and human tumor specimens exposed to these agents. Novel synergistic drug combinations (e.g., inhibitors of Mnk and mTOR) were identified in vitro that are currently undergoing prospective validation.

In conclusion, we discovered new druggable targets expressed by chemo-resistant ES cells, xenografts, and relapsed human tumors. Joint suppression of these newfound targets with IGF-1R or mTOR enhanced the antineoplastic effect of IGF-1R/mTOR blockade and suggests the need for in vivo validation.

Citation Format: Salah-Eddine LAMHAMEDI CHERRADI, Brian Menegaz, Vandhana Ramamoorthy, Joseph Ludwig. IGF-1R/mTOR blockade and mechanisms of resistance in Ewing sarcoma. [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 LB-235.
Abstract LB-238: Direct targeting of oncogenic transcription factors using next-generation oligonucleotides as a novel therapeutic strategy for translocation-positive sarcomas
Authors: Slotkin Emily , Spraggon Lee , Hmeljak Julija , Martelotto Luciano , Somwar Romel , de Stanchina Elisa , Cartegni Luca , Ladanyi Marc
Published: July 2016

Many sarcoma subtypes including Ewing sarcoma (ES), alveolar rhabdomyosarcoma (ARMS), synovial sarcoma (SS), and desmoplastic small round cell tumor (DSRCT), are driven by oncogenic chimeric transcription factors. Outcomes for these diseases remain unacceptably poor despite intensification of standard therapies. Although these translocation positive sarcomas possess well-defined oncogenic drivers, as transcription factors that lack intrinsic enzymatic activity, they have been considered undruggable. We have developed a two-tiered pre-clinical therapeutic program 1) creating and characterizing faithful in vitro and in vivo models of these rare malignancies and 2) designing next generation antisense oligonucleotides (ASOs) to directly target the oncogenic chromosomal translocations underlying these multiple sarcoma subtypes. As proof of principle, we present here the program experience with DSRCT, a rare sarcoma for which there has been an overwhelming paucity of both biological tools for pre-clinical investigation, as well as clinical therapeutic options. We have extensively characterized two new DSRCT cell lines harboring the characteristic oncogenic EWSR1-WT1 t(11;22)(p13;q12) fusion, and have used these tools to develop orthotopic xenograft models. Utilizing these accurate model systems we have designed and evaluated a series of ASO compounds directly targeting the EWSR1-WT1 translocation product. Among the series of ASOs developed, we identified D2A, a selective and potent inhibitor of EWSR1-WT1. In vitro, we have shown D2A to abolish the EWSR1-WT1 fusion product with exquisite specificity in a dose-dependent fashion. D2A similarly exhibits efficacy in vivo, with anti-tumorigenic activity in our pre-clinical models resulting in an 80% reduction in xenograft tumor growth. With the aid of authentic in vitro and in vivo modeling systems, this ASO platform has wide-ranging implications for both biological as well as therapeutic advances.

Citation Format: Emily Slotkin, Lee Spraggon, Julija Hmeljak, Luciano Martelotto, Romel Somwar, Elisa de Stanchina, Luca Cartegni, Marc Ladanyi. Direct targeting of oncogenic transcription factors using next-generation oligonucleotides as a novel therapeutic strategy for translocation-positive sarcomas. [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 LB-238.
Abstract LB-239: Induction of therapeutic soluble decoy EGFR variants by antisense manipulation of RNA processing
Authors: Park Jeong Eun , Spraggon Lee , Cartegni Luca
Published: July 2016

Activation of the receptor tyrosine kinase (RTK) signaling pathways represents a key aspect of tumorigenesis in a broad range of human cancers. Thus, targeting of pathogenic RTKs, mainly with small molecule inhibitors and antibodies, has become an attractive therapeutic approach for cancer therapy. However, resistance by multiple mechanisms, including mutational re-activation of RTK signaling, activation of alternative RTK pathways, EMT transition and others, inevitably occurs over time.

We recently identified a number of novel secreted soluble decoy RTKs isoforms (sdRTKs) generated by an U1-snRNP-dependent alternative intronic polyadenylation mechanism. We therefore developed a novel antisense-based method to effectively activate the expression of these sdRTKs, both in vitro and in vivo. This leads to the generation of endogenous dominant negative variants of RTKs, which can specifically affect the signaling of pathogenic RTKs and therefore provides a novel cancer therapy approach.

Employing specific antisense compounds designed to activate intronic polyA sites, we are able to induce natural truncated EGFR variants that encode its extracellular ligand-binding domain but lack the intracellular signaling domain. These compounds thus can inhibit EGFR signaling by a three fold-mechanism: 1) removal of FL receptor, 2) sequestration of ligands and 3) non-productive dimerization with residual receptors. They are therefore designed to affect signaling both of targeted cells and of bystanders.

Here we will show that, using this approach, we can target EGFR signaling both in vitro and in tumors. Importantly, with this method we can bypass the emergence of resistance typically seen with targeted drug treatments. Further development of this methodology will therefore provide the basis for an exciting novel approach to targeting and inhibiting oncogenic EGFR signaling and other pathways in a variety of cancers.

Citation Format: Jeong Eun Park, Lee Spraggon, Luca Cartegni. Induction of therapeutic soluble decoy EGFR variants by antisense manipulation of RNA processing. [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 LB-239.
Abstract LB-242: Identification of T-cell receptors targeting KRAS-mutated human tumors
Authors: Wang Qiong J. , Yu Zhiya , Hanada Kenichi , Restifo Nicholas P. , Yang James C.
Published: July 2016

KRAS is one of the most frequently mutated proto-oncogenes in human cancers, including pancreatic and colorectal cancers. The dominant oncogenic mutations of KRAS are single amino acid substitutions at codon 12, in particular G12D and G12V. The consistency, frequency, and tumor specificity of these “neo-antigens” make them attractive therapeutic targets. Recent data associates T cells targeting mutated antigens with clinical immunotherapy responses in patients with metastatic melanoma, lung cancer or cholangiocarcinoma. We successfully generated T cells from HLA-A11*01 transgenic mice and subsequently isolated HLA-A11*01-resticted T cell receptors (TCRs) highly reactive with the mutated KRAS variants G12V and G12D. Peripheral blood lymphocytes (PBL) transduced with these TCRs can recognize multiple HLA-A*11:01+ tumor lines bearing the appropriate KRAS mutations. In a xenograft model of large established tumor, adoptive transfer of these transduced PBL reactive with a mutated pancreatic cell line could significantly reduce its growth in NSG mice. The success of adoptive transfer of TCR-engineered T cells against melanoma and other cancers support clinical trials with these T-cells recognizing mutated KRAS in patients with a variety of common cancer types. A phase I/II clinical trial targeting KRAS G12V and G12D mutations with TCR-engineered PBL in HLA-A1101+ patients will be starting soon in the Surgery Branch NCI.

Citation Format: Qiong J. Wang, Zhiya Yu, Kenichi Hanada, Nicholas P. Restifo, James C. Yang. Identification of T-cell receptors targeting KRAS-mutated human tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-242.
Abstract LB-246: Next generation sequencing (NGS) reveals high mutation rates in established and potentially novel cancer genes involved in triple-negative breast cancer
Authors: Bemanian Vahid , Sauer Torill , Touma Joel , Vetvik Katja , Chen Ying , Kristensen Vessela , Bukholm Ida , Geisler Jürgen
Published: July 2016

Introduction. Following the development of novel targeted therapies for selected breast cancer (BC) patients (like HER-2 pos. BC), the subgroup of triple-negative breast cancer (TNBC) patients is still characterized by a poor prognosis and a lack of reliable tumor markers that might guide medical therapy. Methods. Genomic DNA was extracted from paraffin embedded formalin fixed (FFPE) tissue obtained from 77 consecutive patients diagnosed with a TNBC at our hospital. The DNA samples were analyzed by next-generation sequencing (NGS) using Human Breast Cancer GeneRead DNAseq Targeted Panel V2 (Qiagen). The panel consists of a collection of PCR primers for targeted enrichment of the coding region of 44 genes commonly mutated in breast cancer. Target enrichment and library construction was performed according to the GeneReader workflow (Qiagen) and paired end sequencing was performed on a NextSeq 500 sequencer (Illumina) running 2 × 150 bp chemistry Version 2. Data analysis including alignment to the reference genome hg19 and variant calling was performed using Qiagen's online Ingenuity Variant analysis. Results. The ingenuity variant analysis classified the genetic mutations according to their clinical significance into four groups: pathogenic, likely pathogenic, benign and likely benign. We present only mutations in genes that are characterized as pathogenic or likely pathogenic, where the term “likely pathogenic” indicates greater than 90% certainty of the mutation being pathogenic (as defined by the American College of Medical Genetics and Genomics). Genetic variations were mostly observed in a subset of 44 genes included in the breast cancer panel. The tumor suppressor genes TP53 as well as BRCA1 and BRCA2 represented the highest mutation rates (>5%) among all TNBC samples. Interestingly, additional genes probably playing a pivotal role in breast cancer biology like EP300 and FGFR-1 were also found to be mutated at a high rate in TNBC. Conclusion. Next generation sequencing of samples obtained from primary TNBC patients confirmed a high level of pathogenic or likely pathogenic mutations in well-described key cancer genes like TP53, BRCA1/2, ATM, EGFR, RB1 and PIK3CA and frequently mutated additional genes (EP300, FGFR1) that will be further investigated.

Citation Format: Vahid Bemanian, Torill Sauer, Joel Touma, Katja Vetvik, Ying Chen, Vessela Kristensen, Ida Bukholm, Jürgen Geisler. Next generation sequencing (NGS) reveals high mutation rates in established and potentially novel cancer genes involved in triple-negative 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 LB-246.
Abstract LB-256: Clonogenic growth of androgen receptor-overexpressing models of castration recurrent prostate cancer cells becomes insensitive to receptor ligands when their complete cellular secretory milieu is restored
Authors: Patki Mugdha , Huang Yanfang , Ratnam Manohar
Published: July 2016

Evidence that androgen receptor (AR) overexpression causes castration recurrent prostate cancer (CRPC) cells to thrive on minimal residual post-castrate androgen is derived from studies of in vitro colony formation in the absence of cell-secreted factors or from studies demonstrating synergy between androgen and individual signaling pathways in isolation. We examined androgen-dependence using in vivo generated CRPC model cells overexpressing full length AR (C4-2 cells) or co-expressing a splice-variant (22Rv1 cells). The cells showed robust colony formation in conditioned media lacking androgenic activity derived from the same cells or from HEK293 fibroblasts. This effect was (i) not enhanced by androgen (ii) insensitive to blocking ligand-dependent transcriptional activity of AR, (iii) insensitive to mifepristone

(iv) AR-dependent, (v) associated with AR phosphorylation and (vi) similar to androgen-stimulation in sensitivity to signaling pathway inhibitors. This ligand-insensitive response of overexpressed AR to the total cellular secretory complement may limit interventions targeting hormonal factors in CRPC.

Citation Format: Mugdha Patki, Yanfang Huang, Manohar Ratnam. Clonogenic growth of androgen receptor-overexpressing models of castration recurrent prostate cancer cells becomes insensitive to receptor ligands when their complete cellular secretory milieu is restored. [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 LB-256.
Abstract LB-265: IRF8 is an essential transcriptional activator of iNOS although MDSCs up-regulate iNOS expression via an IRF8-independent mechanism
Authors: Ibrahim Mohammed M. , Paschall Amy V. , Redd Priscilla S. , Liu Kebin
Published: July 2016

Nitric Oxide Synthase (NOS) is a family of enzymes that catalyzes L-arginine metabolism to generate nitric oxide (NO). There are three main isoforms of NOS, named neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS or NOS2). These three distinct isoforms of NOS have different cellular localization, regulation, and catalytic properties. iNOS is the most extensively studied one among these three NOS isoforms. iNOS was originally identified in myeloid cells, and it is known that its expression is often induced after myeloid cell activation by endotoxins or cytokines to generate NO which then acts as a defense effector to suppress invading microorganisms or neoplastic cells. It is known that IFNγ signaling pathway and NF-κB synergistically regulate iNOS expression, and that IFN regulatory factor 8 (IRF8) enhances interaction between IFNγ signaling and NF-κB in regulation of iNOS expression. However, the molecular mechanism underlying iNOS expression regulation in myeloid cell lineage is still not fully understood. We report here that IRF8 is an essential transcription activator of iNOS in myeloid cells since IRF8-deficient myeloid cells lose iNOS expression. Furthermore, a key phenotype of IRF8 KO mice is the mass accumulation of CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs), suggesting that IRF8 is a key suppressor of MDSC differentiation under physiological conditions. On the other hand, accumulation of MDSCs is a hallmark of cancers and iNOS expression is dramatically elevated in tumor-induced MDSCs, despite that tumor-induced MDSCs exhibit diminished IRF8 expression. Furthermore, the IFNγ signaling pathway is impaired and NF-κB subunits are undetectable in tumor-induced MDSCs. Our data therefore indicate that iNOS expression is activated by an IRF8-independent mechanism that is also IFNγ and NF-κB-independent in tumor-induced MDSCs under pathological conditions. At the functional level, we determined that iNOS-KO mice exhibit lower tumor incidence as compared to WT mice. However, the sizes of established tumors are not different between iNOS-KO mice and WT mice. Taken together, our data determine that IRF8 is an essential transcriptional activator of iNOS expression in myeloid cells under physiological conditions, although iNOS expression is activated by an IFNγ-, NF-κB-, and IRF8-independent mechanism in tumor-induced MDSCs under pathological conditions. iNOS acts as a suppressor of tumor cell colonization but not tumor growth.

Citation Format: Mohammed M. Ibrahim, Amy V. Paschall, Priscilla S. Redd, Kebin Liu. IRF8 is an essential transcriptional activator of iNOS although MDSCs up-regulate iNOS expression via an IRF8-independent mechanism. [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 LB-265.
Abstract LB-269: Engineering a vascularized 3D in vitro model of cancer progression
Authors: Magdeldin Tarig , Emberton Mark , Loizidou Marilena , Cheema Umber
Published: July 2016

Microenvironmental cues regulate the neo-vascularisation and angiogenic processes within a tumour. The hallmark of cancer is the ability of cancer cells to disseminate and invade to distant sites. The presence of a ‘leaky’ vasculature in the tumour microenvironment facilitates this migration of cancer cells. Using tissue-engineering strategies, we have developed a 3D in vitro cancer model (tumouroid), where we have engineered specific compartments for both the cancer mass and the surrounding stroma. We have utilized a biomimetic matrix for the cancer mass the stroma engineered of collagen I and laminin, and used plastic compression to increase the cell and matrix density by removal of interstitial fluid in collagen hydrogels. We created artificial cancer masses (ACMs) containing colorectal cancer cells (HT29 and HCT116) and nested them within collagen/laminin stromal surrounds (SS) (both ∼10% w/v collagen) containing human dermal fibroblasts (HDFs) and endothelial cells (HUVECs). We have delineated cell specific mechanisms to describe changes in the invasion pattern of cancer cells dependent upon the stromal composition and density. HT29 cells invaded as spherical aggregates of cells while HCT116 cells invaded as contiguous cell sheets. Multicellular cultures were maintained for up to 21 days and endothelial cell morphology was investigated using immunofluorescence of CD31. In HDF-HUVEC co-cultures, HUVECs formed highly branched (29.25 ± 3.86 branches) end-to-end networks with an average length and width of 197.4μm ± 87.5μm and 19μm ± 8.1μm respectively. In the presence of HT29 cancer cells, HUVEC network formation was considerably altered with HUVECs forming much longer and wider networks (456.8μm ± 136μm and 24.2μm ± 7μm) but significantly less interconnected (13.5 ± 5.1 branches). The loss of CK20 expression by the invading cancer epithelial cell sheet was also confirmed by immunofluorescence. The loss of CK20 expression has been known to correspond with high levels of microsatellite instability and overall poor prognosis1. These findings signify the importance of a biomimetic environment in which to culture cancer cells, to mimic the native physiological response of these cells. The tumouroid model is an important tool to study the effect of microenvironmental conditions on tumor malignancy in vitro and in vivo.

1. Kim, J. H., Rhee, Y., Bae, J. M., Cho, N. & Kang, G. H. Loss of CDX2/CK20 expression is associated with poorly differentiated carcinoma, the CpG island methylator phenotype, and adverse prognosis in microsatellite-unstable colorectal cancer. Am. J. Surg. Pathol. 37, 1532-41 (2013).

Citation Format: Tarig Magdeldin, Mark Emberton, Marilena Loizidou, Umber Cheema. Engineering a vascularized 3D in vitro model of cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-269.
Abstract LB-270: Inhibition of the stromal p38MAPK pathway abrogates breast cancer metastases
Authors: Stewart Sheila A. , Murali Bhavna , Su Xinming , Flanagan Kevin , Sponagel Jasmin , Luo Xianmin , Fu Yujie , Alspach Elise , Leahy Kathleen , Monahan Joseph , Weilbeacher Katherine
Published: July 2016

Bone metastasis is a devastating and fatal complication of breast cancer for which we lack effective therapies. Thus, identifying therapies that effectively limit metastases will significantly reduce comorbidities and improve long-term survival. Recently we demonstrated that the p38MAPK pathway sustains the pro-tumorigenic senescence-associated secretory phenotype (SASP) and targeting this pathway limits the tumor-promoting capabilities of senescent cells and cancer-associated fibroblasts (CAFs). Because we found that a significant percentage of p38MAPK-dependent SASP factors are expressed in the stroma associated with breast cancer lesions, we asked whether targeting p38MAPK could limit primary and metastatic breast cancer growth. While p38MAPK inhibition modestly limited primary tumor growth, we found that this inhibition significantly reduced breast cancer bone metastases by targeting the stromal compartment. Further, p38MAPK inhibition was as effective as paclitaxel at limiting tumor growth in the bone but in contrast to paclitaxel, which failed to protect from cancer-induced bone loss, p38MAPK inhibition also protected against devastating bone loss. This contrasts our p38MAPK approach from zoledronic acid, which limits bone loss but fails to slow tumor growth in already engrafted tumors. Analysis of the mechanism(s) responsible for this reduced metastasis suggests that p38MAPK inhibition specifically targets reactive and/or senescent osteoblasts within the bones of animals harboring metastatic lesions. Because we find that senescent osteoblasts are present in human bone, we postulate that they promote metastatic outgrowth and thus p38MAPK inhibition limits the pro-metastatic activities of these cells. Finally, we will present recent data from our clinically relevant preclinical model that demonstrates that inhibition of the p38MAPK pathway can drastically reduce metastasis from the primary site. We propose that p38MAPK is an important stromal-specific therapy for breast cancer metastasis to the bone.

Citation Format: Sheila A. Stewart, Bhavna Murali, Xinming Su, Kevin Flanagan, Jasmin Sponagel, Xianmin Luo, Yujie Fu, Elise Alspach, Kathleen Leahy, Joseph Monahan, Katherine Weilbeacher. Inhibition of the stromal p38MAPK pathway abrogates breast cancer metastases. [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 LB-270.