bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract LB-043: Improving rectal cancer therapy using a patient-derived tumor xenograft model
Authors: Camp Ernest R. , Wang Cindy , Hurst Katie , Nogueira Lourdes M. , Findlay Victoria J.
Published: July 2016

Mechanisms of locally advanced rectal cancer (LARC) neoadjuvant chemoradiation resistance are poorly understood. Current research models to study this issue are incomplete and restricted by the limited supply of untreated tissue samples. Patient-derived xenografts (PDX) are commonly developed from surgical specimens and can serve as effective tools to investigate therapy. We previously demonstrated that the SNAI2/ miR-145 signaling axis enhanced the tumor-initiating cell (TIC) and chemotherapy resistance. We developed a unique PDX model from pre-neoadjuvant therapy LARC biopsy samples to study chemoradiation resistance. Further, we hypothesized that TICs isolated from LARC patient-derived xenografts would demonstrated increased SNAI2/ miR-145 signaling.

Methods: Fresh LARC endoscopic biopsy samples were digested using Liberase DH to yield a dissociated single-cell solution. All in vivo experiments consisted of injecting cells with Matrigel subcutaneously into NSG NODSCID/IL2Rgnull immunocompromised mice. Xenograft (Passage 1, P1) single cells were resuspended in ALDEFLUOR assay buffer. ALDEFLUOR and EPCAM positive cells (ALDH+/EPCAM+) were then detected using a flow cytometer to identify the TICs. Using the negative control ALDH inhibitor, DEAB (1.5 μM), identify the ALDH+ cells compared with background. For limited dilution (LD) in vitro spheroid assay, 5000 EPCAM+/ALDH+ and ALDH- cells were cultured in ultra-low attachment plates in cancer stem-cell medium (DMEM-F12; 1X B-27, βEGF, βFGF, 1% penicillin/streptomycin, BSA, heparin, β-mercaptoethanol). For LD in vivo study, EPCAM+ ALDH sorted cells were implanted subcutaneously with Matrigel and closely observed. Droplet Digital PCR was conducted using the QX200 Droplet Digital PCR System. miR-145 PCR was performed using TaqMan MicroRNA Assays (Applied Biosystems) and normalized to RNU6B.

Results: Our LARC PDX model had high outgrowth and engraftment rates of 70 and 85% respectively and maintained critical histologic features. Both LD in vitro and in vivo experiments demonstrated that EPCAM+/ALDH+ cells were highly tumorigenic suggestive of TICs (p<0.05). The ALDH+ population enriched for SNAI2/ miR-145 signaling. miR-145 was inhibited by at least 8.8 fold in the TIC population.

Conclusions: The lack of novel strategies plays a central role in the inability to overcome therapeutic resistance in LARC. Our results demonstrate a novel model to investigate LARC and suggest that a novel miR-145 therapeutic strategy may be able to target the TIC in LARC patients.

Citation Format: Ernest R. Camp, Cindy Wang, Katie Hurst, Lourdes M. Nogueira, Victoria J. Findlay. Improving rectal cancer therapy using a patient-derived tumor xenograft model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-043.
Abstract LB-067: Proteomic analysis identifies multi-dimensional deregulated signaling pathways in SCLC lung cancer
Authors: Rikova Klarisa , Hall Ben , Levy Tyler , Possemato Anthony , Aguiar Mike , Beausoleil Sean , Min Ren Jian , Lee Kimberly Lee , Lonning Scott , Comb Michael
Published: July 2016

Lung cancer is the most common type of cancer in the U.S., with 5 yr. survival rates of 18% and 6% for non-small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC) patients, respectively. In spite of success with targeted therapies for 15-20% of patients with NSCLCs, the success is generally short-lived as the tumors become resistant to treatment. In contrast to NSCLC, no successful targeted therapies have been identified in SCLC. Further understanding of the regulatory cell signaling networks for SCLCs, may enable the discovery of new actionable lung cancer drivers.

Posttranslational modifications (PTMs), including phosphorylation, acetylation and methylation, act alone and in combination to regulate protein function, cellular behavior and epigenetics. All three types of PTMs are deregulated in various cancers. In this study we developed a highly quantitative MS/MS approach combining TMT labeling with a number of motif and site-specific antibodies to identify deregulated signaling pathways between tumor and normal specimens. PTM-specific antibodies were used to enrich modified peptides followed by 6-plex TMT mass-spec analyses to quantitatively measure over 15,300 differentially phosphorylated, acetylated, and methylated sites on over 4,600 proteins from 17 SCLC patient tissues and 5 para-normals.

Understanding cell-signaling networks at the intersection of multiple pathways and protein modification systems in SCLC lung cancers leads to the identification of new druggable disease drivers.

Citation Format: Klarisa Rikova, Ben Hall, Tyler Levy, Anthony Possemato, Mike Aguiar, Sean Beausoleil, Jian Min Ren, Kimberly Lee Lee, Scott Lonning, Michael Comb. Proteomic analysis identifies multi-dimensional deregulated signaling pathways in SCLC 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-067.
Abstract LB-082: Endoplasmic reticulum stress induces secretion of high mobility group (HMG) proteins and is associated with tumor-infiltrating lymphocytes in triple-negative breast cancer
Authors: Heo Sun-Hee , Park In Ah , Lee Hee Jin , Kim Young-Ae , Gong Gyungyub
Published: July 2016

Purpose: Even though prognostic and predictive significance of tumor-infiltrating lymphocytes (TILs) in triple-negative breast cancer (TNBC) have been demonstrated widely, the cause of TIL influx is not clear. High mobility group (HMG) N1, one of the three subtypes of HMGs which are nucleosome-binding proteins is recently known as a potent alarmin that binds to TLR4 and induces Th1 immune responses. The mechanisms which involve transportation of HMGN1 are not identified. We, in this study, suggest that extracellular secretion of HMGN1 might be associated with influx of TILs and subsequent formation of TLSs, as well as dendritic cell maturation and activation in human TNBC.

Experimental Design: We reviewed the hematoxylin and eosin (H&E)-stained slides of 767 patients with TNBC and evaluated the degree of TILs. We also measured the degree of expression of HMGs (HMGB1 and HMGN1) and several endoplasmic reticulum stress (ERS)-associated molecules (PERK, p-eIF2a, and XBP1) by immunohistochemical (IHC) staining in tissue microarrays of the patients. The hypothesis that HMGN1 migrates from nucleus to cytoplasm and extracellular space as the cell gets variable ER stresses was confirmed by Western blot analyses using human TNBC cell lines and pharmacological ER stress inducers. Results: On IHC staining, high cytoplasmic expression of HMGB1, HMGN1, PERK, p-eIF2a, and XBP1 were identified in 43.1%, 25.7%, 45.9%, 46.9%, and 41.8% of TNBCs, respectively. Cytoplasmic expression of HMGB1 was significantly correlated with cytoplasmic expression of HMGN1, amount of TILs, expression of PERK and XBP1 (p<0.001∼0.039). Cytoplasmic expression of HMGN1 was also significantly correlated with amount of TILs, expression of p-eIF2a and XBP1 (p<0.001∼0.004). Higher nuclear expression of both HMGB1 (p<0.001) and HMGN1 (p<0.001) showed significant association with ER stress. On Western blot analyses using TBNC cell lines, treatment of a ERS inducer resulted in the secretion of HMG proteins to the extracellular space. Conclusions: To the best of our knowledge, it is the first study that elucidated the associations among ERS, extracellular secretion of HMGs, and degree of TILs in TNBCs. Understanding the mechanisms of TIL influx will help to develop effective immunotherapeutic agents for treatment of TNBC.

Citation Format: Sun-Hee Heo, In Ah Park, Hee Jin Lee, Young-Ae Kim, Gyungyub Gong. Endoplasmic reticulum stress induces secretion of high mobility group (HMG) proteins and is associated with tumor-infiltrating lymphocytes 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-082.
Abstract LB-101: The expression of death receptor 4 is positively regulated by MEK/ERK signaling and suppressed upon MEK inhibition
Authors: Oh You-Take , Yao Weilong , Deng Jiusheng , Yue Ping , Sun Shi-Yong
Published: July 2016

Death receptor 4 (DR4) is a cell surface receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and triggers apoptosis upon ligation with TRAIL or aggregation. MEK/ERK signaling is a well-known and the best-studied effector pathway downstream of Ras and Raf. This study focuses on determining the impact of pharmacological MEK inhibition on DR4 expression and elucidating the underlying mechanism.We found that several MEK inhibitors, including MEK162, AZD6244 and PD0325901, effectively decreased the levels of DR4 protein including cell surface DR4 in different cancer cell lines while suppressing ERK1/2 phosphorylation. Accordingly, pre-treatment of TRAIL-sensitive cancer cell lines with a MEK inhibitor attenuated TRAIL-induced apoptosis. These results indicate that MEK inhibition negatively regulates DR4 expression and cell response to TRAIL-induced apoptosis. MEK inhibitors did not alter DR4 protein stability, rather decreased its mRNA levels, suggesting a transcriptional regulation of DR4 expression. In contrast, enforced activation of MEK/ERK signaling by expressing ectopic B-Raf (V600E) or constitutively activated MEK1 (MEK1-CA) or MEK2 (MEK2-CA) activated ERK1/2 and increased DR4 expression; these effects were inhibited when a MEK inhibitor was present. Moreover, enforced expression of MEK1-CA enhanced DR4promoter activity; this effect could be abolished by the presence of a MEK inhibitor. Hence it is clear that activation of the MEK/ERK kinase cascade positively regulates DR4 transcription, resulting in increased DR4 expression. Detailed promoter analysis through deletion and mutation identified the AP-1 binding site as an essential response element for MEK enhancement of DR4 transactivation. In addition, inhibition of AP-1 by knocking down c-Jun abrogated the ability of MEK1-CA to increase DR4 promoter activity and DR4 expression. These results suggest an essential role of AP-1 in mediating MEK/ERK activation-induced DR4 expression. Our findings together highlight a previously undiscovered mechanism that positively regulates DR4 expression through activation of the MEK/ERK/AP-1 signaling pathway. (WY and YTO contribute equally to this work. This study was supported by Emory Winship Cancer Institute Robbins Scholar awards to YTO and to JD and Halpern Research Scholar award to SYS. SYS is a Georgia Research Alliance Distinguished Cancer Scientist and Halpern Research Scholar)

Citation Format: You-Take Oh, Weilong Yao, Jiusheng Deng, Ping Yue, Shi-Yong Sun. The expression of death receptor 4 is positively regulated by MEK/ERK signaling and suppressed upon MEK 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-101.
Abstract LB-109: Identification of anaplastic lymphoma kinase fusions in clear cell renal cell carcinoma
Authors: Wei Huafeng , Chen Yang
Published: July 2016

As the most common type of renal cancer, clear cell renal cell carcinoma (ccRCC) with advanced stages constitutes a continued major challenge for uro-oncologists where identification of novel driver mutations and development of novel targeted therapies against them remain an unmet need. Aberrations of the anaplastic lymphoma kinase (ALK) gene have been implicated in the pathogenesis of multiple human cancers where ALK represents a rational therapeutic target as demonstrated in lung cancer with ALK rearrangement. In this study, we performed the screening of ALK expression in 87 pathologically defined ccRCC by immunohistochemistry (IHC) using a newly developed rabbit anti-human ALK monoclonal antibody (clones D5F3). Four patients were positive for ALK expression as determined by IHC among which 2 cases were further confirmed by fluorescence in situ hybridization (FISH) assay using the Vysis LSI ALK dual color break-apart probe; furthermore, we detected the presence of EML4-ALK (E13:A20, variant 1) fusion gene in tumors from these two patients by using RACE-coupled PCR sequencing and RT-PCR. Notably, we first showed that enforced EML4-ALK expression significantly promoted the in vitro proliferation, clonogenic colony formation and apoptosis resistance in immortalized normal renal tubal epithelial cell HK2 and their in vivo outgrowth when injected into immunocompromised nude mice, and importantly, this pro-tumorigenic effect could be completely abolished by ALK-specific inhibitor Crizotinib, indicating the potential effectiveness of ALK-specific inhibitors in treating ALK-positive ccRCC patients. Our data show that ALK fusions exist in adult ccRCC, which provides the rationale for ALK inhibitor therapy in selected patients of ccRCC.

Citation Format: Huafeng Wei, Yang Chen. Identification of anaplastic lymphoma kinase fusions in clear cell renal cell 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 LB-109.
Abstract LB-123: STAT3 mediates APC-driven chemoresistance in breast cancer
Authors: VanKlompenberg Monica K. , Leyden Emily A. , Zhang Jian-Ting , Prosperi Jenifer R.
Published: July 2016

Breast tumors frequently develop resistance to treatment with a variety of standard (i.e. cisplatin or doxorubicin) or targeted chemotherapeutic agents, resulting in tumor recurrence and increased patient mortality. The Adenomatous Polyposis Coli (APC) tumor suppressor is lost by hypermethylation or mutation in up to 70% of sporadic breast cancers; however, the downstream effects of APC loss have not been well explored in breast cancer. Heterozygous Apc mutation using the ApcMin/+ mouse model enhanced tumorigenesis in the mouse mammary tumor virus - Polyoma Middle T transgenic model (MMTV-PyMT). Cells isolated from MMTV-PyMT;ApcMin/+ mice are resistant to chemotherapy, express higher levels of multidrug resistance protein 1 (MDR1), and have a greater population of tumor initiating cells (TICs) compared to controls. Here we demonstrate that APC loss-of-function cells have increased activation of signal transducer and activator of transcription 3 (STAT3) and the anti-apoptotic protein Mcl-1. STAT3 over-expression is common in breast cancer, leads to poor prognosis, and up-regulates MDR1 expression leading to chemotherapeutic resistance. To explore the functional role of STAT3 in mediating therapeutic resistance in APC loss-of-function models, we examined the impact of STAT3 inhibition on MDR1 and the TIC population to cause therapeutic resistance in the MMTV-PyMT;ApcMin/+ vs MMTV-PyMT;Apc+/+ murine breast cancer cells. We have made the novel observation that chemotherapy treatment has a time-dependent effect on the activation of STAT3 and expression of Mcl-1 specific to Apc-mutant cells. Finally, we have assessed the use of STAT3 inhibitors in combination with standard chemotherapy to alleviate APC-mediated resistance. Combined these data suggest that loss of APC activates STAT3-driven pathways resulting in the development of chemotherapeutic resistance.

Citation Format: Monica K. VanKlompenberg, Emily A. Leyden, Jian-Ting Zhang, Jenifer R. Prosperi. STAT3 mediates APC-driven chemoresistance in 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-123.
Abstract LB-151: Positively selected enhancer elements endow tumor cells with metastatic competence
Authors: Morrow James J. , Miller Tyler E. , Saiakhova Alina , Lizardo Michael M. , Bartels Cynthia F. , Bayles Ian , Hung Stevephen , Mendoza Arnulfo , Myers Jay T. , Allen Frederick , DiFeo Analisa , Rubin Brian P. , Huang Alex Y. , Meltzer Paul S. , Helman Lee J. , Khanna Chand , Scacheri Peter C.
Published: July 2016

Metastasis results from a complex set of traits acquired by tumor cells, distinct from those necessary for tumorigenesis. Here, we investigate the contribution of enhancer elements to the metastatic phenotype of osteosarcoma. Through epigenomic profiling, we identify substantial differences in signature enhancer-histone marks between near-isogenic pairs of high and low lung-metastatic osteosarcoma cells. We term these regions Metastatic Variant Enhancer Loci (Met-VELs). Met-VELs drive coordinated waves of gene expression during metastatic colonization of the lung. Met-VELs cluster non-randomly, indicating that activity of these enhancers and their associated gene targets is positively selected. Osteosarcoma lung metastasis is inhibited by global interruptions of Met-VEL associated gene expression via pharmacologic BET inhibition, by knockdown of AP-1 transcription factors whose motifs are enriched in Met-VELs, and by knockdown of individual genes activated by Met-VELs. These observations have implications for the discovery and development of targeted anti-metastatic therapies.

Citation Format: James J. Morrow, Tyler E. Miller, Alina Saiakhova, Michael M. Lizardo, Cynthia F. Bartels, Ian Bayles, Stevephen Hung, Arnulfo Mendoza, Jay T. Myers, Frederick Allen, Analisa DiFeo, Brian P. Rubin, Alex Y. Huang, Paul S. Meltzer, Lee J. Helman, Chand Khanna, Peter C. Scacheri. Positively selected enhancer elements endow tumor cells with metastatic competence. [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-151.
Abstract LB-154: Epigenetic silencing of triple-negative breast cancer hallmarks by Withaferin A
Authors: Vanden Berghe Wim , Szarc vel Szic Katarzyna , Declerck Ken , Crans René A.J. , Diddens Jolien , Gerhäuser Clarissa
Published: July 2016

Background: Triple negative breast cancer (TNBC) is characterized by poor prognosis and a DNA hypomethylation profile. We found that the steroidal lactone Withaferin A (WA), isolated from the plant Withania somnifera, triggers active chromatin remodeling and elicits promising chemosensitizing effects in triple negative breast cancer (BC) cells. Here, we have further characterized potential involvement of epigenetic reprogramming by Withaferin A in TNBC treatment.

Materials and methods: By Infinium HumanMethylation450 arrays, Epityper Mass array and CpG pyrosequencing, we determined genome-wide DNA methylation changes in weakly-metastatic and aggressive, metastatic BC cell lines treated with a pharmacologically relevant concentration of 700 nM WA. In parallel, we performed ChIP analysis of histone mark H3K4Me3 to evaluate histone demethylation by JARID1B/KDM5B. Furthermore, epigenetic changes in response to WA were crosscompared with genomewide methylation changes and clinical breast cancer patient characteristics in The Cancer Genome Atlas database.

Results: In contrast to the DNA hypomethylating agent 5-aza-2’-deoxycytidine (DAC), WA treatment of TNBC cells silences an epigenetic cancer network through gene-specific hypermethylation of tumor promoting genes including ADAM metallopeptidase domain 8 (ADAM8), urokinase-type plasminogen activator (PLAU), tumor necrosis factor (ligand) superfamily, member 12 (TNFSF12), and genes related to detoxification (glutathione S-transferase mu 1, GSTM1), or mitochondrial metabolism (malic enzyme 3, ME3). Remarkably, DNA hypermethylation of corresponding CpG sites in these genes correlates with receptor tyrosine-protein kinase erbB-2 amplification (HER2)/estrogen receptor (ER)/progesterone receptor (PR) positive status in primary BC tumors. Increased expression levels of JARID1B, promoter-specific loss of active H3K4me3 chromatin marks and gain of DNA methylation suggest that WA-specific gene silencing involves epigenetic reprogramming of HER2/ER/PR-dependent gene expression programs to suppress aggressive TNBC characteristics in favor of luminal BC hallmarks with an improved therapeutic sensitivity.

Conclusions: WA may represent a novel and attractive phytopharmaceutical to overcome therapy resistance in TNBC.

Citation Format: Wim Vanden Berghe, Katarzyna Szarc vel Szic, Ken Declerck, René A.J. Crans, Jolien Diddens, Clarissa Gerhäuser. Epigenetic silencing of triple-negative breast cancer hallmarks by Withaferin A. [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-154.
Abstract LB-158: Simultaneous targeting of the cell cycle and transcription through irreversible inhibition of CDK7: a novel, new therapeutic approach for high-grade glioma
Authors: Greenall Sameer , Johns Terrance G.
Published: July 2016

High Grade Glioma (HGG), an incurable aggressive neoplasm of the brain, is characterised by a diverse array of genetic aberrations including those associated with receptor tyrosine kinase (RTK) signalling and cell cycle regulation pathways. Furthermore, HGG possess a highly heterogeneous intratumoral transcriptional profile, both at the global and subclonal levels, that allows for rapid resistance to a wide spectrum of therapeutics. Cyclin dependent kinase 7 (CDK7) presents an opportunity to target proliferation and global transcription simultaneously as it is the CDK activating kinase that controls multiple checkpoint progressions through the cell cycle as well as stabilising the RNA polymerase II-based transcriptional apparatus. Using a new irreversible inhibitor of CDK7, THZ1, we demonstrate that a panel of 10 patient-derived primary HGG cells lines, representing a diverse array of genotypes and phenotypes, were all exquisitely sensitive to THZ1 treatment at low nanomolar doses. THZ1 induced global transcription downregulation as measured by microarray analysis and resulted in potent caspase 3/7 activation, DNA damage and apoptosis. Cell cycle in all lines was arrested at the G2/M phase regardless of p53, CDKN2A/B or RB status, with substantial aneuploidy observed. Additionally, THZ1 treatment results in the dramatic loss of several RTKs critical in driving HGG including EGFR, EGFRvIII, PDGFR-α, PDGFR-β, MET, FGFR-3 and AXL, resulting in reduced PI3K/AKT, MAPK/ERK and STAT3 activation. Efficacy in ex vivo tumor slices and in vivo orthotopic experiments will be presented as well as insights into possible mechanisms of resistance. Our work suggest that irreversible inhibition of CDK7 represents a novel and effective treatment for HGG.

Citation Format: Sameer Greenall, Terrance G. Johns. Simultaneous targeting of the cell cycle and transcription through irreversible inhibition of CDK7: a novel, new therapeutic approach for high-grade glioma. [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-158.
Abstract LB-176: The human androgen receptor regulates DNA damage response (DDR) in prostate cancer
Authors: Asim Mohammad , Zecchini Heather , Tarish Firas , Massie Charlie , Baridi Ajoeb , Warren Anne , Zhao Wanfeng , McDuffus Leigh-Anne , Mascalchi Patrice , Shaw Greg , Dev Harveer , Wadhwa Karan , Wijnhoven Paul , Forment Josep , Lyons Scott , Lynch Andy , O’Neill Cormac , Zecchini Vincent , Rennie Paul , Baniahmad Aria , Tavare Simon , Mills Ian , Schulz Niklas , Galanty Yaron , Helleday Thomas , Neal David
Published: July 2016

Clinical studies have shown the improved benefit of combining androgen deprivation therapy (ADT) with radiotherapy, indicating a potential interaction between the androgen signalling and DNA damage response (DDR) pathways in PCa. We have explored the underlying basis of this interaction by interrogating the mechanism of androgen regulation of the DDR in men with PCa, both untreated and treated with an androgen inhibitor. We show that androgen receptor (AR) signalling regulates the DDR and promotes DDR foci formation leading to ATM activation in PCa. Our data showing that AR regulates DDR provides an explanation for the improved outcome of combined ADT and radiotherapy. Thus, our work identifies the rationale for targeting both the AR and DDR pathways simultaneously to improve therapy outcome in advanced or high risk prostate cancer.

Citation Format: Mohammad Asim, Heather Zecchini, Firas Tarish, Charlie Massie, Ajoeb Baridi, Anne Warren, Wanfeng Zhao, Leigh-Anne McDuffus, Patrice Mascalchi, Greg Shaw, Harveer Dev, Karan Wadhwa, Paul Wijnhoven, Josep Forment, Scott Lyons, Andy Lynch, Cormac O’Neill, Vincent Zecchini, Paul Rennie, Aria Baniahmad, Simon Tavare, Ian Mills, Niklas Schulz, Yaron Galanty, Thomas Helleday, David Neal. The human androgen receptor regulates DNA damage response (DDR) in 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 LB-176.
Abstract LB-183: The impact of protein concentration on the distribution of 111In-antibody conjugate for imaging of programmed death-ligand 1 (PD-L1)
Authors: Nedrow Jessie , Josefsson Anders , Park Sunju , Ranka Sagar , Sgouros George
Published: July 2016

Background: Programmed cell Death Ligand 1 (PD-L1) is part of an immune checkpoint system that is essential for preventing autoimmunity. Tumor cells have developed the ability to co-opt these immune checkpoints to suppress anti-tumor immunity. Recent approaches in immunotherapy targeting immune checkpoints have shown great promise in a variety of cancers. PD-L1 overexpression is associated with a poorer prognosis in a variety of cancers, but typically have a stronger response to anti-PD-L1 therapy. PD-L1 is a dynamic biomarker and the ability to image its dynamic nature could provide key information in identifying patients who will best respond to anti-PD-L1 treatment with limited adverse effects. Here we evaluated an 111In-antibody conjugate targeted to PD-L1 for SPECT imaging in an aggressive mouse melanoma model. In addition, we investigated the relationship between the spleen, which we have found to be a sink for anti-PD-L1 antibodies, and specific activity (activity/protein concentration) of the 111In-antibody conjugate.

Methods: An anti-PD-L1 antibody was conjugated to DTPA for 111In-labeling. The biodistribution of the resulting conjugate, 111In-DTPA-anti-PD-L1, was performed over a range of specific activities in B16F10 tumor bearing mice. SPECT imaging was also performed with the 111In tracer.

Results: 111In-DTPA-anti-PD-L1 (Kd = 0.6±0.1 nM) having a specific activity (SA) of 0.57 Ci/μmol demonstrated uptake in the B16F10 tumor (6.1±2.8%ID/g) at 24 hours, as well as high uptake in the spleen (32.8±5.1%ID/g). Decreasing the SA to 0.01 Ci/μmol blocked PD-L1-postive cells in the spleen (13.4±4.8%ID/g) forcing more of the tracer to remain in circulation, allowing for increase accumulation in the B16F10 tumor (16.8±6.2%ID/g) at 24 hours. The lower SA also demonstrated a reduction in tumor to blood ratios compared to the higher SA (2.2 vs. 4.1), but an increase was shown in the tumor to muscle ratio (27 vs. 4.6).

Conclusion: 111In-DTPA-anti-PD-L1 is capable of identifying tumors that overexpresses PD-L1, and has the potential to help select patients that will best respond to anti-PD-L1 therapy. In addition, the further understanding of the spleen/tumor relationship could be utilized to tailor individual doses for anti-PD-L1 therapy and minimize adverse effects.

Citation Format: Jessie Nedrow, Anders Josefsson, Sunju Park, Sagar Ranka, George Sgouros. The impact of protein concentration on the distribution of 111In-antibody conjugate for imaging of programmed death-ligand 1 (PD-L1). [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-183.
Abstract LB-213: NEDD8 pathway inhibition is effective in preclinical models of poorly differentiated, clinically aggressive colorectal cancer
Authors: Picco Gabriele , Sassi Francesco , Isella Claudio , Di Nicolantonio Federica , Sapino Anna , Bardelli Alberto , Trusolino Livio , Bertotti Andrea , Medico Enzo
Published: July 2016

The NEDD8 conjugation pathway modulates the ubiquitination, stability and activity of a wide range of intracellular proteins, and its blockade by pevonedistat is emerging as a promising therapeutic approach in various cancer settings. However, systematic characterization of pevonedistat efficacy in specific tumor types and definition of molecular predictors of response are still missing. We investigated the in vitro sensitivity to pevonedistat in a large panel of molecularly and functionally annotated colorectal cancer (CRC) cell lines, and exploited global gene expression profiles to define a transcriptional predictor. Out of 122 tested cell lines, 16 (13%) displayed a marked response to pevonedistat, featuring DNA re-replication, proliferative block and apoptosis. Pevonedistat sensitivity did not significantly correlate with microsatellite instability or mutations in KRAS or BRAF, but was functionally associated with resistance to EGFR pathway blockade. A 184-gene transcriptional predictor generated in cell lines was applied to 87 metastatic CRC clinical samples for which patient-derived xenografts (PDX) were available. For in vivo tests, pevonedistat was provided by Millennium Pharmaceuticals, Inc., a subsidiary of Takeda Pharmaceutical Company Ltd. In vivo administration of pevonedistat did not affect the growth of any predicted resistant PDX; whereas, it significantly impaired growth of five out of six predicted sensitive models. In human CRC gene expression datasets, transcriptional prediction of pevonedistat sensitivity was associated with poor prognosis after surgery and early progression under cetuximab treatment. Histological and immunohistochemical analyses revealed that pevonedistat sensitivity signature captures transcriptional traits of poor differentiation and high-grade mucinous adenocarcinoma. These results highlight NEDD8-pathway inhibition by pevonedistat as a potentially effective strategy to treat poorly differentiated, clinically aggressive CRC and identify transcriptional and histological response predictors with possible clinical applicability.

Citation Format: Gabriele Picco, Francesco Sassi, Claudio Isella, Federica Di Nicolantonio, Anna Sapino, Alberto Bardelli, Livio Trusolino, Andrea Bertotti, Enzo Medico. NEDD8 pathway inhibition is effective in preclinical models of poorly differentiated, clinically aggressive colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-213.
Abstract LB-222: Optimization and preclinical characterization of highly potent and stable anti-CD70 ADCs for the potential treatment of CD70 positive cancers
Authors: Skidmore Lillian , Cox Kari , Kirtley Jessica , Le Tiep , Firdos Juhi , Manibusan Anthony , Fang Shiao-Yan , Beck Andy , Bhowmik Shiva , Knudsen Nick , Wang Jianing , Sun Ying , Ayala Armando , Powell Alyssa , Wahl Alan , Tian Feng , Humphreys Robin
Published: July 2016

Antibody drug conjugates (ADCs) require coordinated optimization of antibody, linker, and payload to generate potent, specific, and stable cancer therapeutics. CD70 is an attractive ADC tumor target due to its limited expression in normal tissues and over-expression in many solid tumors and hematologic malignancies. Utilizing Ambrx's site-specific EuCODE technology, we have generated and evaluated multiple ADCs with different combinations of linkers and payloads for treatment of CD70 positive cancers. In vitro activity identified specific CD70 ADCs with picomolar potency/activity against CD70 positive cancer cell lines. Conjugation of drug-linkers at different sites in the antibody and modification of linker design improved in vitro activity and in vivo stability of lead CD70 ADCs. Optimized anti-CD70 ADCs were evaluated for pharmacokinetic profile and demonstrated to be efficacious in reducing tumor burden and significantly prolonging survival in an in vivo orthotopic xenograft model of multiple myeloma. In summary, we have generated encouraging preclinical in vitro and in vivo data with stable, site-specific, DAR = 2 anti-CD70 ADCs for the potential treatment of CD70 positive cancers such as multiple myeloma.

Citation Format: Lillian Skidmore, Kari Cox, Jessica Kirtley, Tiep Le, Juhi Firdos, Anthony Manibusan, Shiao-Yan Fang, Andy Beck, Shiva Bhowmik, Nick Knudsen, Jianing Wang, Ying Sun, Armando Ayala, Alyssa Powell, Alan Wahl, Feng Tian, Robin Humphreys. Optimization and preclinical characterization of highly potent and stable anti-CD70 ADCs for the potential treatment of CD70 positive 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 LB-222.
Abstract LB-232: Nano-CaCO3 as a novel pH-sensitive nanoparticle platform for cancer therapy
Authors: Som Avik , Raliya Ramesh , Akers Walter , Ippolito Joseph , Singamaneni Srikanth , Biswas Pratim , Achilefu Samuel
Published: July 2016

Introduction: The acidic extracellular environment of tumors potentiates their aggressiveness and metastasis, but few methods exist to selectively modulate the extracellular pH (pHe) environment of tumors. Transient flushing of biological systems with alkaline fluids or proton pump inhibitors is impractical and nonselective. Here we report a nanoparticles-based strategy to intentionally modulate the pHe in tumors and restricts it growth.

Methods: We developed two independent facile methods to synthesize monodisperse non-doped vaterite nano-CaCO3 with distinct size range between 20 and 300 nm. Biochemical simulations were used to simulate pH changes in vivo using the particle. In vitro and in vivo assays were used to determine pH changes. IV injections of the nanoparticles were implemented in a xenograft model of fibrosarcoma to determine growth changes.

Results: Biochemical simulations indicate that the dissolution of calcium carbonate nanoparticles (nano-CaCO3) dissolution in vivo increases pH asymptotically to 7.4. Using murine models of cancer, we demonstrate that the selective accumulation of nano-CaCO3 in tumors increases tumor pHe over time. The persistent neutralization of tumor pHe from nano-CaCO3 induces tumor growth stasis.

Conclusions: We have been able to create a novel nanoparticle based on widely existing in vivo components (Calcium and CO3) that safely equilibrates acidic pH of tumors up to normal physiological pH. As expected, this modulation restricts growth of implanted tumors in vivo. Future studies will study the effect of these particles on metastasis, and synergy with other therapeutic modalities.

Citation Format: Avik Som, Ramesh Raliya, Walter Akers, Joseph Ippolito, Srikanth Singamaneni, Pratim Biswas, Samuel Achilefu. Nano-CaCO3 as a novel pH-sensitive nanoparticle platform for cancer therapy. [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-232.
Abstract LB-234: A persistently activated signaling network regulates the DNA damage secretory program to promote cancer resistance driven by the therapeutically damaged tumor microenvironment
Authors: Sun Yu , Zhang Boyi , Chen Fei , Chiao Paul
Published: July 2016

In cancer clinics, an in vivo form of the senescence-associated secretory phenotype (SASP) is driven by a DNA damage secretory program (DDSP) provoked by side effects of genotoxic therapeutics including chemotherapy and radiation which fuel advanced pathologies particularly cancer resistance. However, the complete mechanism underlying DDSP development in the tumor microenvironment (TME) remains unclear. Here we report that genotoxic agents frequently used in clinical oncology induce a gene expression pattern distinct from the one that is shaped by non-genotoxic drugs. Specifically, the zinc finger and scan domain containing 4 (Zscan4) is upregulated upon DNA damage in stromal cells and engaged in DNA damage response (DDR) in genotoxic settings. Apart from the established functions in telomere elongation and genomic integrity of stem cells, Zscan4 is central for the Rap1/TRF2 assembly and orchestrates the long term DDR signaling in differentiated stromal cells. In response to DNA damage, the mammalian target of rapamycin (mTOR) is phosphorylated in the cytoplasm of stromal cells and relays DDR signals, a phenomenon different from human cancer cells. There are physical interactions between the mTOR and the IKK complexes, events functionally implicating the NF-κB machinery that is regulated by both the IL-1α/IRAK1 and mTOR/Raptor axes. We determined that mTORC1 is a critical mediator of DDSP programmatic implementation by conducting signals from upstream molecules including p38 and PI3K/Akt, molecules activated in DNA damage events. We revealed that the ligand IL-1α signals through IL-1R and TAK1, the latter a member of the mitogen-activated kinase kinase kinase (MAPKKK) family that induces a proinflammatory cascade by activating JNK/p38 and NF-κB. Further, targeting p38, mTOR or TAK1 with small molecule inhibitors in vivo significantly abrogated cancer resistance acquired from the TME in chemotherapeutic cycles. Overall, our study substantiates p38, mTOR and TAK1 as a group of new candidates for future TME-oriented intervention to minimize resistance provoked by anticancer regimes, and suggests that anti-p38/mTOR/TAK1 agents may be repurposed to sensitize tumors by effectively targeting the TME. Our data disclose a novel mechanism of SASP (or DDSP) signaling in classical therapeutic settings, thus opening new avenues to improve clinical outcomes in the current era of precision medicine.

Citation Format: Yu Sun, Boyi Zhang, Fei Chen, Paul Chiao. A persistently activated signaling network regulates the DNA damage secretory program to promote cancer resistance driven by the therapeutically damaged tumor microenvironment. [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-234.
Abstract LB-284: Multiscale systems pharmacology model for time sequence combination chemotherapy in breast cancer
Authors: Ait-Oudhia Sihem
Published: July 2016

Background: HER2-positive (HER2+) breast cancer (BC) is more quickly-growing and aggressive than other types of BC. Trastuzumab (TZM) is a humanized monoclonal antibody directed against HER2 receptor. It is clinically used for BC in combination with paclitaxel (PAC) in a 24 h sequence treatment during the first week of therapy followed by a simultaneous administration during the subsequent weeks. To our knowledge there is no scientific rationale for such therapeutic regimes. We propose to develop and optimize a sequential combination of PAC+TZM to optimize their anti-tumoral activity, and hence, HER2+ BC patients’ outcome.

Method: Six therapeutic regimens were investigated in vivo on Balbc nude mice xenografted with BT474 cells, a human breast cancer cell line that overexpresses HER2 receptor, including 1) a tumor priming regimen (TPR) with PAC administered 24 h prior to TZM, 2) reverse-TPR with TZM administration first then PAC 24 h later, 3) simultaneous administration of both drugs, 4) PAC alone, 5) TZM alone, 6) vehicle as a control. The doses were 10 and 3 mg/kg for PAC and TZM. The anti-tumoral response in all treatment arms was monitored over time through the measurement of the reduction in tumor volumes as well as animals survival.

Results: In vivo studies showed a greater tumor volume shrinkage response in the animals from the TPR treatment arm compared to the other therapeutic schemes with: 1) an enhanced in vivo antitumor response, and 2) a longer animal survival. These results support our in vitro findings of drug synergy and apoptosis.

Conclusion: TPR anti-tumor results are promising. The proposed research will develop and employ a systems pharmacology model based on our results to hypothesize optimal regimens for the association (PAC+TZM) breast cancer killing, which will be tested in vivo.

Citation Format: Sihem Ait-Oudhia. Multiscale systems pharmacology model for time sequence combination chemotherapy in 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-284.
Abstract LB-286: Dynamic alternative splicing correlates with drug synergy and induces novel gene regulatory networks in MCF7
Authors: Chen Xintong , Bashir Ali , Stolovitzky Gustavo , Losic Bojan
Published: July 2016

Drug combination therapies in the cancer setting often succeed where mono-therapies fail, facilitating durable and robust responses that may curtail metastases and even be accompanied by milder side-effects. Here we incorporate transcriptional splicing dynamics with a gene level expression analysis to assess the transcriptional landscape of MCF-7 (ER+) cell-lines across mono or combination therapies in a time course short-read RNA-seq experiment. We leverage the most comprehensive known splicing database of MCF7 to assess the sensitivity and specificity our short-read splicing inference by interrogating the long-read PacBio isoform sequencing (IsoSeq) MCF7 database. We infer robust isoforms which are strongly associated with phenotypic synergy and demonstrate their novel functional role by computing gene-gene coexpression networks which are sensitive to their expression.

Briefly, we use RNA-seq to study the transcriptional response over time (0, 3, 6, 9, 12, and 24 h) for three drugs (A, B and C) and their combinations (AB, AC and BC) in MCF-7 (ER+) breast cancer cells lines. Cell viability measurements show that one of the combinations (AB) is strongly synergistic, whereas the other two (AC and BC) are merely additive. We show that in addition to a novel transcriptional signature driven by differential expression, the combination AB transcriptional landscape is characterized by persistent alternative splicing signatures mostly comprised of genes which are not differentially expressed with respect to A or B, but whose functional role has been dramatically changed by the addition (deletion) of a key regulatory protein domain encoded by the extra(missing) exon. We integrate this synergistic splicing signal into a gene regulatory network (GRN) via adaptive network refinement to show that it strongly contributes to the emergence of extensive transcriptional cascades by creating and removing key gene-gene correlations and altering the modular structure of the network. Using this approach we show that a key (heretofor poorly understood) isoform of Protein beta-arrestin-1 (ARBB1), which participates in the desensitization of G-protein coupled receptors through intracellular response to Estrogen, becomes enriched in the calcium ion-dependent exocytosis coexpression module for synergistic therapies only, despite being absent in the baseline MCF-7 long-read PacBio IsoSeq data.

Our results directly show that alternative splicing is crucial in any molecular characterization of phenotypic drug synergy in drug-tumor interactions.

Citation Format: Xintong Chen, Ali Bashir, Gustavo Stolovitzky, Bojan Losic. Dynamic alternative splicing correlates with drug synergy and induces novel gene regulatory networks in MCF7. [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-286.
Abstract LB-291: Environment or accident? A more stringent bound on environmental contributions to cancerogenesis
Authors: Bilke Sven , Meltzer Paul S.
Published: July 2016

Finding the right balance between cancer prevention and early detection is essential for an efficient anti-cancer strategy. Yet, the underlying biological question “how much cancer risk results from chance, how much from environmental factors’’ continues to be controversial. Recently, the “bad luck’’ hypothesis was strengthened based on observing a tight correlation between a tissues cancer risk and its number of stem cell divisions [1]. This interpretation was later challenged by pointing out that certain even drastic changes of environmental risk factors would not affect theat correlation [2]. Based on this argument they derived an estimate of the upper bound of environmental contributions to canceriogenesis. Here we argue that the argument made in [2] did not fully appreciate that typically several mutations are requited before cancer initiation occurs [3]. We show that by incorporating this aspect into the analysis the correlation between the number of stem cell divisions and cancer risk would respond to changes of environmental factors. We re-analyze the data in [1] to derive a slightly more stringent upper bound of the environmental risk contributions for a number of cancers than the ones obtained in [2]. [1] C. Tomasetti, B. Vogelstein, Science 347(6217), 78-81 (2015) [2] S. Wu, S.Powers et al., Nature 529, 43-47 (2015) [3] C. Nordling, British Journal of Cancer 7(1), 68-72 (1953)

Citation Format: Sven Bilke, Paul S. Meltzer. Environment or accident? A more stringent bound on environmental contributions to cancerogenesis. [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-291.