bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5717: Mass spectrometry as a tool for MHC class I and II neoantigen discovery
Authors: Ford Michael J. , Jones Richard , Allen David , Amunugama Ravi , Pisano Michael , Mobley James , Domanski Paul , Ho Bill , Bochar Daniel , Holt Melissa
Published: August 2018

A neoantigen is a patient-specific tumor antigen resulting from mutations during oncogenesis (1). Advances in genome sequencing of cancer tumor tissues combined with bioinformatics have enabled the identification of tumor specific mutations and the prediction of the peptides that will be presented by the MHC complex. The presented peptides are recognized as foreign by the immune system and can be used to discriminate cancerous from normal cells. Neoantigen prediction by gene sequencing and in silico approaches can be strengthened and further supported by mass spectrometry. The molecular level characterization of peptides associated with molecules of the major histocompatibility complex requires a targeted protein complex enrichment, an unbiased peptide elution, and finally a peptide analysis method. We use immunoaffinity to isolate the target complex, elute the peptides under conditions minimizing protein contamination and finally analyze the peptides by mass spectrometry. Here we present a case studies of our recent work applying workflows for the analysis of peptides associated with both Class I and Class II MHC molecules. Combining state-of-the-art mass spectrometry and bioinformatics, we demonstrate the utility of this approach for neoantigen identification and quantitation.

Reference: 1. Sun et al. Cancer Lett 2017;392:17-25.

Citation Format: Michael J. Ford, Richard Jones, David Allen, Ravi Amunugama, Michael Pisano, James Mobley, Paul Domanski, Bill Ho, Daniel Bochar, Melissa Holt. Mass spectrometry as a tool for MHC class I and II neoantigen discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5717.
Abstract 5732: Zinc transporter, ZIP14, as a mediator of systemic muscle wasting in metastatic cancers
Authors: Biswas Anup , Wang Gang , Ma Wanchao , Coker Courtney , Tanji Kurenai , Kandpal Manoj , Davuluri Ramana , Acharyya Swarnali
Published: August 2018

Metastasis contributes to the vast majority of cancer-related mortalities. Metastatic tumors secrete factors that systemically affect various organs leading to dysfunction and accelerated death. More than 80% of metastatic cancer patients experience a progressive and debilitating loss of muscle mass and function by a process known as cachexia. Cachectic patients suffer deterioration of diaphragm and cardiac muscles and often die prematurely due to respiratory and cardiac failure. The prognosis for these patients is further diminished by the fact that they are often too weak to tolerate standard doses of anti-cancer treatments. Cachexia is therefore an important determinant of therapeutic response, outcome and patient survival in advanced cancer patients. Although systemic metabolic derangements and chronic inflammation predominate in cachexia, the underlying molecular mechanisms driving its development are not well understood. Therefore, insights into the specific interventions that could either reverse or prevent cachexia are expected to improve treatment outcome, survival and quality of life in cancer patients. In this study, we identified a metal ion transporter, ZIP14 that was upregulated in cachectic muscles from five independent metastatic models, as well as in metastatic cancer patients. We find that TNF-alpha and TGF-beta cytokines upregulate ZIP14 in muscles, which in turn results in the accumulation of intracellular zinc. Increased zinc influx in muscle cells is associated with the degradation of myofibrillar proteins that contributes to muscle atrophy and muscle mass loss. Importantly, germline ablation or muscle-specific depletion of Zip14 markedly inhibits cancer-induced muscle wasting. Our study demonstrates a novel function of ZIP14 in muscles as a mediator of cachexia in advanced cancers. Insights from this study can be used to develop therapeutic strategies to prevent cachexia, and improve the survival and quality of life in metastatic cancer patients.

Citation Format: Anup Biswas, Gang Wang, Wanchao Ma, Courtney Coker, Kurenai Tanji, Manoj Kandpal, Ramana Davuluri, Swarnali Acharyya. Zinc transporter, ZIP14, as a mediator of systemic muscle wasting in metastatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5732.
Abstract 5745: Effect of activator of G-protein signaling 3 (AGS3) on prostate tumorigenesis
Authors: Adekoya Timothy O. , Smith Nikia , Mowatt Tonelia , Aladeniyi Temilade , Richardson Ricardo M.
Published: August 2018

Prostate cancer (PCa) is a leading cause of cancer death among men, with greater prevalence of the disease among the African American population in the US. Activator of G-protein Signaling 3 (AGS3/GPSM1), a receptor-independent activator of G-protein signaling has been shown to affect different cellular processes and cell cycle activity as well as tumor growth and development. AGS3 contains seven tetratricopeptide (TPR) repeats in its N-terminal half and four G-protein regulatory (GPR) motifs in its C-terminal half. The aim of this study is to assess the role of AGS3 in prostate cancer development and metastasis as well as to understand the molecular dynamics involved. To that end, the metastatic PCa cell lines LNCaP, PC3, MDA PCa 2b and DU-145 were analysed for AGS3 expression relative to RWPE-1, a non-metastatic Pca cell line. Quantitative RT-PCR and western blot analysis have shown that AGS3 expression varies in the PCa cell lines relative to control RWPE-1. Overexpression of AGS3 in PC3 and LNCaP cells significantly enhanced tumor progression in nude mice xenografts. Interestingly, expression of the TPR, not the GPR, repeats in LNCaP promoted tumor growth as well as the full length AGS3. Preliminary studies with a Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice deficient in AGS3 expression (TRAMP+/AGS3-/-) displayed decrease prostate tumorigenesis. Taken together, these results indicate that AGS3 expression promotes prostate tumor growth. The data also suggest that the effect of AGS3 in prostate tumorigenesis is mediated via its TPR, not GPR motif.

Citation Format: Timothy O. Adekoya, Nikia Smith, Tonelia Mowatt, Temilade Aladeniyi, Ricardo M. Richardson. Effect of activator of G-protein signaling 3 (AGS3) on prostate tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5745.
Abstract 5752: Protumoral and pro-metastatic effects of TLR7 in lung cancer
Authors: Dajon Marion , Iribarren Kristina , Alifano Marco , Damotte Diane , Cremer Isabelle
Published: August 2018

TLR7 agonists are currently under investigations for their ability to enhance anti-tumor immune responses. However, in some tumor models, these agonists also stimulate tumor cells, which can express high levels of TLR7, a receptor for single-stranded RNA [1]. We have demonstrated that stimulation of lung tumor cell lines expressing TLR7 with synthetic TLR7 agonists led to upregulation of the antiapoptotic protein Bcl-2, tumor cell survival and chemoresistance [2]. In Non-Small-Cell Lung Carcinoma (NSCLC) cohorts of patients, we have observed high expression of TLR7 on tumor cells of 70% of patients, which conferred poor clinical outcome and was strongly associated with resistance to chemotherapy [3]. This pro-tumoral effect of TLR7 has been validated in murine models in which the injection of TLR7 agonists in NOD/SCID mice, in C57BL/6 wild-type or in TLR7-deficient mice grafted with lung adenocarcinoma tumor cells led to increased tumor progression, increased lung metastasis, and resistance to chemotherapy. On the contrary, we demonstrated that TLR7 antagonist injection led to antitumoral effect. Additionally, TLR7 stimulation resulted in a significant increase of Myeloid-Derived Suppressor Cells (MDSC) in the tumor microenvironment. Depletion experiments of MDSC indicated that these cells are involved in the pro-tumoral effect induced upon TLR7 stimulation. Finally, we have demonstrated that the pro-tumoral effect of TLR7 stimulation, mediated by the MDSC recruitment, was independent of TLR7 stimulation on immune cells. Our results reveal the mechanism by which TLR7 stimulation induce the pro-tumoral effect in mice, and open the way to the development of novel cancer therapeutics including TLR7 inhibitors, for NSCLC patients. This results demonstrate the important role of TLR7 in lung tumor progression. Knowing that natural ligands of TLR7 are ssRNA, we suppose that this effect could be linked to viral infections or RNA released in the tumor microenvironment. [1] Dajon, et al. Immunobiologie. 2016 [2] Cherfils-Vicini J, et al. J Clin Invest. 2010 [3] Chatterjee J, et al. Cancer Res. 2014

Citation Format: Marion Dajon, Kristina Iribarren, Marco Alifano, Diane Damotte, Isabelle Cremer. Protumoral and pro-metastatic effects of TLR7 in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5752.
Abstract 5762: Fast screening of CD40 agonistic antibodies for cancer immunotherapy
Authors: Jin James , Guo Yanan , Shen Yuelei , Yang Benny
Published: August 2018

CD40, Cluster of Differentiation 40, is a member of the TNF-receptor superfamily. CD40 is a co-stimulatory protein found primarily on antigen presenting cells, including B cells, macrophages and dendritic cells. Its ligand, CD40L (CD154) is predominantly expressed on T helper cells, whose engagement activates antigen presenting cells and induces a variety of downstream effects. Whereas most immune checkpoint receptors are expressed on the surface of T cells, CD40 stands out as it is expressed by antigen presenting cells and controls the proper activation of these cells. In turn, CD40 activation exerts a significant impact on T cell-mediated activity against cancer cells. Several pharmaceutical companies are pursuinganti-CD40 therapiesfor treating hematological and solid cancers. Having an access to Biocytogen's antibody discovery and in vivo efficacy screening platform for immune checkpoints, we asked whether we could find suitable anti-CD40 reagents which can successfullycombat developed tumors in preclinical settings. We designed a powerful screening strategy using Biocytogen's humanized CD40 mouse (B-hCD40). Candidate antibodies were generated by immunizing wild-type mice with recombinant hCD40 protein. Then the hybridomas were screened by high-throughput flow cytometry. Instead of examining the immune modulating activity in cell culture, we applied these antibodies to B-hCD40 mice bearing MC38 tumors. Through this straightforwardscreening, several clones stood out by their unparallelingbenefits. These clones were further selected for humanization. Again, the cohort of recombinant humanized CD40 antibodies was screened in B-hCD40 mice. In the end, we discovered the leads with the most potent anti-tumor activity. In conclusion, our work successfully identified several CD40 antibodies that are highly effective for cancer treatment in the preclinical setting.

Citation Format: James Jin, Yanan Guo, Yuelei Shen, Benny Yang. Fast screening of CD40 agonistic antibodies for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5762.
Abstract 5774: Novel cell-based high-throughput hybridoma screening method using the Celigo image cytometer for antibody discovery
Authors: Chan Leo L. , Zhang Haohai , Rice William , Kassam Nasim , Longhi Maria S. , Zhao Haitao , Robson Simon C. , Gao Wenda , Wu Yan
Published: August 2018

Hybridoma screening is a highly important process for antibody discovery, which can identify potential clones from hundreds to thousands of hybridoma cultures against the desired target antigen. Traditional screening methods using ELISA and flow cytometry presented technical issues such as limited accuracy, reduced high-throughput capability, increased time and cost. Conventional ELISA screening with coated antigen can also be impractical for difficult to express hydrophobic membrane antigens or multi-chain protein complexes. Here, we demonstrate novel cell-based high-throughput screening methodology using plate-based Celigo Image Cytometer, which avoids nonspecific signals by contrasting antibody binding signals directly on living cells, with and without recombinant antigen expression. The image cytometry-based high-throughput screening method was optimized by detecting the binding of hybridoma supernatants to the recombinant antigen CD39 expressed on Chinese hamster ovary (CHO) cells. Both target CHO and CFSE-stained wild type were co-cultured to simultaneously detect target antibodies (Alexa Fluor 594) and nonspecific binding, which can eliminate the need to produce a separate control sample for each hybridoma supernatant. Next, the image cytometer was used to screen 672 unique hybridoma supernatants to develop the high-throughput screening workflow. The Celigo was used to quickly image and analyze antibody binding of 672 samples, using Hoechst, Alexa Fluor 594 and CFSE fluorescence to identify high, medium, and low binding hits. Finally, the sensitivity of the image cytometer was demonstrated by serial dilution of purified CD39 antibody. Celigo was used to measure antibody affinities of commercial and in-house antibodies to membrane-bound CD39. This cell-based screening procedure can be completely accomplished within one day, significantly improving throughput and efficiency of hybridoma screening. Furthermore, measuring direct antibody binding to living cells eliminated both false positive and false negative hits. The image cytometry method was highly sensitive and versatile, and could detect positive antibody in supernatants at concentrations as low as ~5 ng/mL, with concurrent Kd binding affinity coefficient determination. We propose that this screening method will greatly improve screening technologies and facilitate more efficient antibody discovery.

Citation Format: Leo L. Chan, Haohai Zhang, William Rice, Nasim Kassam, Maria S. Longhi, Haitao Zhao, Simon C. Robson, Wenda Gao, Yan Wu. Novel cell-based high-throughput hybridoma screening method using the Celigo image cytometer for antibody discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5774.
Abstract 5776: Pharmacologic and PK/PD study of D-0502: An orally bioavailable SERD with potent antitumor activity in ER-positive breast cancer cell lines and xenograft models
Authors: Wang Yaolin , Shi Zhe , Jiang Yueheng , Dai Xing
Published: August 2018

Breast cancer is the most common form of cancer in women in the developed world, with estrogen receptor-positive (ER+) breast cancer accounting for 70-80% of all breast cancers. Endocrine therapy such as selective estrogen receptor degrader (SERD) fulvestrant has been used effectively to extend the life of ER+ breast cancer patients, either alone or in combination with CDK4/6 inhibitor (palbociclib or abemaciclib). However, due to poor pharmacokinetic property, fulvestrant has to be given via intramuscular injection. In addition, patients relapsed after prolonged endocrine therapy with aromatase inhibitors such as letrozole and anastrozole due to emergence of ER mutations. Here, we report the discovery of D-0502, an orally bioavailable SERD with potent activity in various ER+ breast cancer cell lines and xenograft models. In addition, combination of our SERD candidates and palbociclib in both MCF-7 xenograft model and ESR-1 mutated (Y537S) patient-derived breast cancer xenograft model resulted in further tumor growth inhibition or regression. Comparison of various SERD molecules showed our candidate has more potent antitumor activity in MCF-7 xenograft model than GDC-0810, AZD9496 and fulvestrant. More importantly, drug metabolism and pharmacokinetic (PK) studies both in vitro and in vivo demonstrated that our SERD molecule D-0502 exhibits superior PK profiles suitable for clinical development.

Citation Format: Yaolin Wang, Zhe Shi, Yueheng Jiang, Xing Dai. Pharmacologic and PK/PD study of D-0502: An orally bioavailable SERD with potent antitumor activity in ER-positive breast cancer cell lines and xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5776.
Abstract 5800: Vorinostat and indole-3-carbinol modulate microRNAs and histone deacetylase activity in subtypes of triple-negative breast cancer
Authors: Lyn-Cook Beverly , NouriEmamzaden Fatemeh , Hawkins Anfernee , Hammons George , Word Beverly
Published: August 2018

Triple-negative breast cancer is a highly heterogeneous and aggressive subtype of breast cancer. Epigenetic drugs are continuing to emerge as promising therapies for triple-negative breast cancer (TNBC). These drugs, including the one used in this study, vorinostat, a histone deacetylase inhibitor (HDAC), are capable of multiple actions and are considered multifunctional drugs. Epigenetic mechanisms have been shown to play a role in the etiology of this cancer, through modulation of DNA methylation, histone modification or microRNAs. We have shown in this study that vorinostat and indole-3-carbinol (I3C) modulated the expression of microRNAs 221 and 222 differently in basal-like-2 (BL2), MSL, basal-like1 (BL1) and M subtypes of TNBC cells. Additional studies demonstrated that combination of tamoxifen and vorinostat significantly decreased expression of miRNA 221 in TNBC cell lines. MiRNAs are short noncoding RNAs that regulate the function of target genes post-transcriptionally. In addition, breast cancer microarrays further showed modulation of a number of other miRNAs that play a role in cell proliferation, drug resistance, cell invasion and metastasis. MicroRNAs are epigenetic targets for drug development. Using a global HDAC activity assay, it was also shown that subtypes of TNBC cells respond differently to vorinostat and I3C treatment, depending on their HDAC activity. HCC70, an African American cell line and a BL2 subtype, had the highest HDAC activity and its activity was significantly decreased by vorinostat treatment compared to BT549 (M), HCC 1806 (BL2), MCF7 (ER positive) and a basal nonmalignant cell line, MCF10A. Both vorinostat and I3C show great promise alone and in combination as effective agents in triple-negative breast cancer. Histone deacetylase inhibitors and dietary agents are emerging as therapy for triple-negative breast cancer and are currently in clinical trials.

Citation Format: Beverly Lyn-Cook, Fatemeh NouriEmamzaden, Anfernee Hawkins, George Hammons, Beverly Word. Vorinostat and indole-3-carbinol modulate microRNAs and histone deacetylase activity in subtypes of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5800.
Abstract 5822: Secretory acetylated APE1/Ref-1 requirement for suppression of tumor growth in triple-negative breast cancer in vivo: Extracellular apoptotic initiator
Authors: Lee Yu Ran , Park Myoung Soo , Joo Hee Kyoung , Kim Ki Mo , Kim Jeryong , Jeon Byeong Hwa , Choi Sung
Published: August 2018

Triple-negative breast cancer (TNBC) represents a relatively small proportion of all BCs but a relatively large proportion of BC-related death. Thus more effective therapeutic strategies are needed for the management of TNBC. We previously demonstrated that the stimulation of apoptosis by the binding of secreted acetylated-apurinic apyrimidinic endonuclease 1/redox factor-1 (Ac-APE1/Ref-1) to the receptor for advanced glycation end products (RAGE) was essential for the TNBC cell death in response to hyperacetylation. The aim of the present study was to assess the potential therapeutic efficacy of secretory Ac-APE1/Ref-1 in orthotopic TNBC xenografts in vivo. We found that hyperacetylation in xenografts caused secretion of Ac-APE1/Ref-1 into the blood, where the factor bound directly to RAGE in hyperacetylated tumor tissues. Hyperacetylation in the TNBC xenografts induced strong inhibition of tumor growth and development. Hyperacetylation also caused cell death in tumors, accompanied by increased RAGE expression and generation of reactive oxygen species, which induced apoptosis. Tissues exhibited markedly higher counts of apoptotic bodies, a reduced proliferation index, and reduced neovascularization compared with control tumors. Ac-APE1/Ref-1-stimulated apoptosis was markedly reduced in RAGE-knockdown tumors compared with RAGE-overexpressing tumors, even in the presence of hyperacetylation. The function of secreted Ac-APE1/Ref-1 was confirmed in other hyperacetylated TNBCs xenografts using BT-459 and MDA-MB-468 cells, demonstrating its relevance as an anticancer molecule. Our findings suggest that the Ac-APE1/Ref-1 possesses potent chemotherapeutic efficacy against hyperacetylated TNBCs based on stimulation of cell death by RAGE-dependent triggering of Ac-APE1/Ref-1.

Citation Format: Yu Ran Lee, Myoung Soo Park, Hee Kyoung Joo, Ki Mo Kim, Jeryong Kim, Byeong Hwa Jeon, Sung Choi. Secretory acetylated APE1/Ref-1 requirement for suppression of tumor growth in triple-negative breast cancer in vivo: Extracellular apoptotic initiator [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5822.
Abstract 5849: Valproic acid reverses trastuzumab resistance in erbB2-positive breast cancer cells via downregulation of ErbB2/ErbB3 signaling through induction of ErbB family members-targeting miRNAs
Authors: Lin Tingting , Ren Qun , Zuo Weimin , Lin Rong , Zhao Hu , Wang Ping , Tan Jianming , Lyu Hui , Liu Bolin , Wang Shuiliang
Published: August 2018

Amplification and/or overexpression of erbB2 occur in approximately 25% of invasive breast cancer and are significantly associated with a worse prognosis for breast cancer patients. Trastuzumab, a humanized monoclonal antibody against erbB2 receptor, has been successfully used in early-stage and metastatic breast cancer therapy of patients with erbB2-overexpressing tumors as monotherapy and in combination with other agents; however, both primary and acquired resistances to trastuzumab are common and currently represent a significant clinical problem. Thus, seeking of novel therapeutic strategies/agents to overcome trastuzumab resistance is vital to improve the survival of breast cancer patients whose tumors overexpress erbB2. In our current study, we explored whether valproic acid (VPA), a clinically used anticonvulsant drug with reported HDACi activity, reverses trastuzumab resistance in erbB2-positive breast cancer cells. We have found that VPA inhibits proliferation of both trastuzumab-sensitive SKBR3 and BT474 cell lines as well as their corresponding trastuzumab-resistant SKBR3-pool2 and BT474-HR20 cell lines in a dose- and time-dependent manner, which is accompanied by significant reduction of cyclin D1 as well as induction of P21. Meanwhile, VPA also induces caspase-dependent apoptosis in all aforementioned four cell lines. More interestingly, VPA not only significantly enhances the anti-proliferative effects of trastuzumab, but also potentiates trastuzumab-induced apoptosis in trastuzumab-sensitive and resistant cells. Our further mechanistic studies revealed that VPA treatment results in significant inactivation of PI3K/Akt signaling via simultaneous down-regulation of ErbB2 and ErbB3 in erbB2-positive breast cancer cancer cells, an effect which could be mainly arrtibuted to induction of ErbB family members-targeting microRNAs including miR-125a, miR-125b and miR-205 by VPA. Taken together, we presented here that VPA reverses trastuzumab resistance in erbB2-positive breast cance cells via downregulation of ErbB2/ErbB3 signaling in a miRNAs-inducing-dependent manner. Our data suggested that further investigation regarding the in vivo therapeutic effect of VAP against trastuzumab-resistant breast cancer should be of especial interest.

Citation Format: Tingting Lin, Qun Ren, Weimin Zuo, Rong Lin, Hu Zhao, Ping Wang, Jianming Tan, Hui Lyu, Bolin Liu, Shuiliang Wang. Valproic acid reverses trastuzumab resistance in erbB2-positive breast cancer cells via downregulation of ErbB2/ErbB3 signaling through induction of ErbB family members-targeting miRNAs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5849.
Abstract 5861: RX-3117 promotes epigenetic effects in cancer cells through enhanced degradation of DNMT1
Authors: Sarkisjan Dzjemma , Pronk Ferry , Kuin Rosan , Hassouni Btissame el , Lee Young B. , Kim Deog J. , Dijk Marc , Geerke Daan P. , Peters G. J.
Published: August 2018

RX-3117 is a novel, investigational, oral, small molecule nucleoside compound that shows promising antitumor activity in xenografts including patient-derived xenografts resistant to gemcitabine. RX-3117 is currently being evaluated in a Phase IIa multi-center, open-label clinical study in patients with relapsed or refractory pancreatic cancer and advanced bladder cancer. RX-3117 can downregulate DNMT1 and activate genes controlled by methylation, such as E-cadherin and p16. We aimed to elucidate the mechanism of RX-3117 mediated downregulation of DNMT1.

Expression of DNMT1 was evaluated by western blotting. Binding of DNMT1 to DNA was measured after DNA isolation, DNAse treatment and gel electrophoresis. DNMT1 trapping in the nucleus was also studied with ImageStream FACS analysis. DNMT1 ubiquitination was measured by immunoprecipitation and quantification of the ubiquitin tag K48. Translocation of DNMT1 was studied by confocal microscopy. Modeling studies were performed with Quantum Mechanics (QM) and Molecular Dynamics (MD).

DNMT1 acts on the DNA during the so-called base flipping necessary for methyl transfer. To determine whether RX-3117 treatment would lead to trapping of DNMT1, NSCLC A549 and PDAC SUIT-028 cells were treated for 24 hr with 1 µM RX-3117. Immunostaining of DNMT1-DNA and ImageStream FACS analysis demonstrated binding of DNMT1 to DNA. Treatment with RX-3117 also resulted in DNMT1 ubiquitination, as shown by an increase in the ubiquitin tag K48, reaching the highest level after 48 hr. Incubation of A549 and SUIT-028 cells with 80 nM of the specific proteasome inhibitor bortezomib prevented RX-3117 mediated degradation of DNMT1 (at 24 and 24 hr exposure to 1 µM RX-3117). This underlines the role of the proteasome in DNMT1 degradation. With confocal microscopy it was demonstrated that DNMT1 and the proteasome co-localized and translocated to the cytosol of A549 and SUIT-028 cells treated for 48 hours with 1 µM RX-3117. With molecular modelling using QM and MD, changes in nucleophilicity of C4-C5 double bound were observed indicating that DNMT1 would initiate methyl transfer but cannot proceed.

In summary we conclude that RX-3117 induces epigenetic changes in cancer cells by trapping DNMT1 in the nucleus followed by translocation of the protein to the proteasome for degradation.

Citation Format: Dzjemma Sarkisjan, Ferry Pronk, Rosan Kuin, Btissame el Hassouni, Young B. Lee, Deog J. Kim, Marc Dijk, Daan P. Geerke, G. J. Peters. RX-3117 promotes epigenetic effects in cancer cells through enhanced degradation of DNMT1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5861.
Abstract 5874: Targeting DDX3 in medulloblastoma by RK-33
Authors: Tantravedi Saritha , Vesuna Farhad , Martin Allison , Lim Michael , Eberhart Charles G. Eberhart G. , Diest Paul J. van , Raman Venu
Published: August 2018

Introduction: Medulloblastoma is the most common form of pediatric brain cancer. Current treatments of surgical resection, craniospinal irradiation, and chemotherapy increase survival but around 40% of patients experience disease recurrence. Classically, four molecular subgroups of medulloblastoma have been identified: Wnt; Sonic hedgehog (SHH); Group 3; and Group 4. More recent work has subtyped up to seven molecular subgroups. The RNA helicase DDX3 is involved in many biological activities such as transcription, mRNA translation, splicing, and nuclear export. About 8% of medulloblastoma cases involve mutations in DDX3, of which 11% involve the pediatric Wnt and SHH subtypes. In addition, coinciding DDX3 and CTNNB1 mutations associated with dysregulated Wnt /β-catenin signaling in Wnt-type medulloblastomas have also been reported.

Aims and Methods: The goal of this work was to study whether RK-33, a small molecule inhibitor of DDX3, would be effective against medulloblastoma as a single agent and in combination with radiotherapy. We studied DDX3 expression in medulloblastoma cell lines DAOY and UW228 and human tissue microarrays by immunohistochemistry, western blotting, and qRT-PCR. We targeted DDX3 by siRNA and by RK-33 and observed proliferation and viability changes by MTS assays. Flow cytometric analysis was used to determine effect of RK-33 on cell cycle and apoptosis. TOP/FOP promoter assay was utilized to study effect on TCF promoter activity. We used colony formation assays to study effect of combining radiation and RK-33 treatment on cells. DAOY tumors were generated in nude mice and treated with combination therapy to study effects in vivo.

Results: High DDX3 expression was observed in 55% of pediatric and 66% of adult medulloblastomas. DDX3 knockdown resulted in reduced cellular proliferation. Both the medulloblastoma cell lines were sensitive to RK-33, with IC50 values of 2.5 µM and 3.5 µM, respectively and resulted in decreased levels of DDX3 expression, with no observable toxicity. RK-33 resulted in a G1 phase cell cycle arrest before the onset of apoptosis. DDX3 knockdown resulted in a significant decrease of TCF activity and reduced transcription of Axin2, CCND1, MYC, and Survivin. A combination of RK-33 and radiation resulted in a significantly higher inhibitory effect than using single treatment of either RK-33 or radiation. We conclude that inhibiting DDX3 via the small molecule inhibitor RK-33 is effective in reducing medulloblastoma tumors and that the effect is synergistic with radiation treatment.

Citation Format: Saritha Tantravedi, Farhad Vesuna, Allison Martin, Michael Lim, Charles G. Eberhart G. Eberhart, Paul J. van Diest, Venu Raman. Targeting DDX3 in medulloblastoma by RK-33 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5874.
Abstract 5881: Novel inhibitors of the epigenetic reader protein MBD2
Authors: Giovinazzo Hugh , Reichert Zachary R. , Bergman Andries , Lin Xiaohui , Wyhs Nicolas , Esopi David , Vaghasia Ajay , Liu Jianyong , Jain Yash , Bhamidipati Akshay , Steinberg Ruchama , Speed Traci , Vaughn Matthew , Zhang Yonggang , Brennen Nate , Deweese Theodore , Yegnasubramanian Srinivasan , Nelson William G.
Published: August 2018

DNA hypermethylation can trigger silencing of tumor suppressor genes during cancer development and progression, partly through binding by methylated-DNA binding proteins (MBD), such as MBD2, that function as “epigenetic readers” and recruit co-repressor complexes to promote gene repression. Inhibiting MBD2-mediated repression represents an attractive cancer therapeutic strategy. Here, we used a cell-based screen to identify small molecules capable of reactivating hypermethylated promoter sequences. We used biochemical, molecular biologic, and pharmacologic approaches to characterize mechanism of action of identified MBD2 inhibitors. A subset of these compounds represent a new class of inhibitors capable of selectively antagonizing interactions between MBD2 and methylated DNA, leading to reactivation of the hypermethylated gene GSTP1 and the epigenetically silenced retinoic acid signaling pathway. Combinations of one of the newly identified MBD2 inhibitors, KCC-08, with the retinoic acid receptor agonist, isotretinoin, significantly reduced cancer cell growth/survival in vitro and in vivo. These novel MBD2 inhibitors are thus positioned for further pharmacologic lead development for use as probes to interrogate epigenetic gene silencing mechanisms and as cancer therapeutics.

Citation Format: Hugh Giovinazzo, Zachary R. Reichert, Andries Bergman, Xiaohui Lin, Nicolas Wyhs, David Esopi, Ajay Vaghasia, Jianyong Liu, Yash Jain, Akshay Bhamidipati, Ruchama Steinberg, Traci Speed, Matthew Vaughn, Yonggang Zhang, Nate Brennen, Theodore Deweese, Srinivasan Yegnasubramanian, William G. Nelson. Novel inhibitors of the epigenetic reader protein MBD2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5881.
Abstract 5888: Disruption of FOXO3a-miRNAs mediated feedback inhibition of IGF2-IRS1 signaling contributes to Herceptin resistance in HER2 positive breast cancer cells
Authors: He Zhimin
Published: August 2018

Resistance to HER2-target therapy remains a clinical challenge for HER2+ breast cancer subtype. The molecular mechanisms for Herceptin resistance remain elusive. Here, we demonstrated that the IGF2 and IRS1 protein levels were increased in Herceptin resistant breast cancer cells and that the IGF2/IGF1R/IRS1/Akt/mTOR signaling was involved in Herceptin resistance. In Herceptin sensitive HER2+ breast cancer cells, IRS1 protein level resulted from mTOR2 mediated FOXO3a status determined the feedback regulation of IGF2. Upon high dose rhIGF2 treatment, the mTOR1 translationally upregulated PPP3CB protein level that recovered the function of FOXO3a inactivated by mTOR2. FOXO3a positively transcriptionally regulated miR-128-3p and miR-30-5p expression that targeted IRS1 mRNA to control the feedback regulation of IGF2 signaling. In agreement, in Herceptin resistant cells, the decrease of miR-128-3p and miR-30-5p resulted from constitutively inactivation of FOXO3a by mTOR2, led to increase of IRS1 protein level, even in response to high endogenous IGF2 protein level in culture medium. High IGF2 protein level was resulted from decrease of miR-193a-5p that was also regulated by FOXO3a. Unexpectedly, in Herceptin resistant cells, Src/p-STAT5 signaling cooperating with HDAC1 transcriptionally repressed PPP3CB expression resulting into FOXO3a inactivation. Moreover, we confirmed that IRS1 knockdown reversed the Herceptin resistance in vivo using a xenograft mouse model. Serums form patients with poor response to Herceptin, had higher IGF2 protein level. Tissues from patients with poor response had higher IRS1, p-Akts473, p-FOXO3a and p-Src levels, but had lower PPP3CB, miR-128-3p, miR-30-5p and miR-193a-5p levels. In conclusions, disruption of PPP3CB-FOXO3a-miRNAs mediated feedback inhibition of IGF2-IRS1 signaling plays pivotal role in Herceptin resistance of HER2+ breast cancer. Related molecules in this signaling may represent potential biomarkers and therapeutic targets for HER2+ breast cancer treatment.

Keywords: Breast cancer, Herceptin resistance, IGF2, IRS1, FOXO3a

This study was supported by grants from the National Natural Science Foundation of China: No.81672616, No.81402196 and No.81272450, supported by grants from Guangdong Natural Science Funds for Distinguished Young Scholars No.2016A030306003 (G Zheng).

Citation Format: Zhimin He. Disruption of FOXO3a-miRNAs mediated feedback inhibition of IGF2-IRS1 signaling contributes to Herceptin resistance in HER2 positive breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5888.
Abstract 589: A systematic review of randomized clinical trials for therapeutic outcomes of anti-EGFR agents used in combination with other targeted therapies
Authors: Kurup Sonali , Liskova Pavlina , Jungsuwadee Paiboon , Sakharkar Prashant , Tambe Soniya
Published: August 2018

Introduction: The epidermal growth factor receptor (EGFR) plays a prominent role in cancer cell proliferation and metastasis in non-small-cell lung cancer (NSCLC). Targeted cancer therapies include agents that block tumor-specific molecular targets and thereby interfere with tumor growth, progression and metastasis. This study focuses on a systematic review of outcomes from phase II and III clinical trials for standard anti-EGFR therapy plus a targeted therapy to EGFR monotherapy.

Methods: Studies were identified through a thorough literature search using, EMBASE, Pubmed, Scifinder Scholar, clinicaltrials.gov and the Cochrane library using the following terms: “epidermal growth factor receptor kinase”, “combination therapy”, “non small cell lung cancer”. Eligible studies included patients of any stage and mutation status. Data abstraction included patient demographic characteristics; sample size, patient inclusion and exclusion criteria, overall response rate (ORR), median progression free survival (PFS), median overall survival (OS), and adverse effects (AE).

Results: Literature search resulted in the identification of 1543 studies of which 153 were found to be relevant for this review and 31 fit the inclusion criteria. Of the 31 studies, 28 included erlotinib and 3 studies included gefitinib as the control. Twenty-five of the studies focused on kinase inhibitors targeting VEGFR, EGFR, MET, IGF-1R, AKT, mTOR and HER3 as the combination agent. The remaining six studies focused on alternate pathways of targeted inhibition including hormonal agents and histone deacetylase inhibitors. The identified studies included patients (N=7552) of mean age (63 ± 2), female (22-80%), smokers in combination therapy (12-96%) versus monotherapy (8-95%). Median OS (months) for patients on the EGFR-based combination regimen (range 5.7-24.7) vs monotherapy (range 4.5-21.8). Median PFS (months) for patients on the EGFR-based combination regimen (range 1.3-16.0) vs monotherapy (1.5-9.7). A greater incidence of AE was observed for EGFR-based combination therapy compared to EGFR monotherapy.

Conclusions: Several different targeted therapies have been evaluated as part of clinical trials for NSCLC in combination with EGFR-based therapies, and compared to EGFR-based monotherapy. For patients with NSCLC, a higher range for median OS and median PFS was observed for EGFR-based combination regimens compared to EGFR monotherapy.

Citation Format: Sonali Kurup, Pavlina Liskova, Paiboon Jungsuwadee, Prashant Sakharkar, Soniya Tambe. A systematic review of randomized clinical trials for therapeutic outcomes of anti-EGFR agents used in combination with other targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 589.
Abstract 5893: Expression and role of autophagy-associated p62 (SQSTM1) in multidrug-resistant ovarian cancer
Authors: Wang Jinglu , Garbutt Cassandra , Hornicek Francis J , Duan Zhenfeng
Published: August 2018

Multidrug resistance is the major cause of treatment failure in ovarian cancer. p62 (SQSTM1) is a multifunctional protein involved in multiple cellular processes including proliferation, drug sensitivity and autophagy-associated cancer cell growth. p62 is a critical indicator of autophagic flux, which is inversely associated with autophagy activity. However, the role of p62 remains controversial in drug resistance in human ovarian cancer. In this study, we examined p62 expression by immunohistochemistry in a unique ovarian cancer tissue microarray (TMA), which was constructed with paired primary, metastatic, and recurrent tumor tissues from 26 individual patients. Results showed that both the metastatic and recurrent tumor tissues expressed less p62 than the patient-matched primary tumor. A significant inverse correlation has been found between p62 expression and both the disease free survival and overall survival. In addition, multidrug resistant cancer cell lines expressed lower levels of p62 as compared with their parental drug sensitive cell lines. Importantly, cell viabilities determined by MTT assay after exposure to different concentrations of paclitaxel showed inhibition of autophagy or accumulation of p62 enhances paclitaxel sensitivity in ovarian cancer drug resistant cells. Furthermore, the wound healing assay exhibited that inhibition of autophagy significantly decreased multidrug resistant ovarian cancer cell migration in vitro. Collectively, these data highlight that autophagy pathway may be a promising therapeutic target to prevent metastasis, recurrence and to reverse drug resistance in ovarian cancer.

Citation Format: Jinglu Wang, Cassandra Garbutt, Francis J Hornicek, Zhenfeng Duan. Expression and role of autophagy-associated p62 (SQSTM1) in multidrug-resistant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5893.
Abstract 5902: RNA sequencing analysis of taxane-resistant prostate cancer cells reveals potential candidate genes for therapeutic targeting
Authors: Ross Christina K. Cajigas-Du , Woods-Burnham Leanne , Ramirez Joshua , Chen Xin , Wang Charles , Casiano Carlos A.
Published: August 2018

There is no cure for metastatic prostate cancer (PCa), the most commonly diagnosed cancer and third leading cause of cancer mortality among U.S. men. Patients with advanced PCa often develop resistance to conventional therapies resulting in metastatic castration-resistant PCa (mCRPC). Docetaxel (DTX) chemotherapy is the standard-of-care for mCRPC, followed by secondary chemotherapy with cabazitaxel (CBZ), yet chemoresistance and death inevitably occurs. While pharmacological agents targeting single survival pathways have shown promise at the bench side, clinical trials have failed. This is mostly due to the presence of redundant pathways utilized by PCa tumor cells to maintain chemoresistance. Therefore, we are interested in further defining molecular pathways associated with DTX-resistance in order to identify novel therapeutic targets to overcome this resistance. We hypothesized that RNA sequencing (RNAseq) analysis comparing the transcriptomes of chemosensitive and chemoresistant mCRPC cells will reveal potential targets for combinatorial therapies. RNAseq analysis of DTX-sensitive and -resistant PC3 and DU145 cells revealed a number of differentially regulated genes that were either unique to each cell line or common to both. We found that dipeptidyl peptidase 4 (DPP4), fatty acid binding protein 5 (FABP5), nestin (NES), and tetraspanin 8 (TSPAN8) were among the top most robustly and significantly upregulated genes in both DTX-resistant PC3 and DU145 cell lines. In-house qPCR studies validated the RNAseq results for these genes, and Western blotting analysis showed increased expression of these proteins in DTX-resistant cells, which correlated with increased mRNA expression. Interestingly, NES, TSPAN8, DPP4 are genes associated with development of the cancer stem-cell (CSC) phenotype. Other genes associated with CSCs were also upregulated in the DTX-resistant cells, suggesting that in vitro selection of mCRPC cells for DTX resistance may enrich for CSCs. This novel finding is consistent with recent studies implicating CSCs in PCa chemoresistance. Further analysis and validation of our RNAseq data will increase our understanding of DTX-resistance mechanisms in PCa cells, including the yet undefined functional role of CSC-related genes. These studies also reveal potential therapeutic targets to circumvent DTX resistance in PCa.

Citation Format: Christina K. Cajigas-Du Ross, Leanne Woods-Burnham, Joshua Ramirez, Xin Chen, Charles Wang, Carlos A. Casiano. RNA sequencing analysis of taxane-resistant prostate cancer cells reveals potential candidate genes for therapeutic targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5902.
Abstract 5905: Sp1 acetylation associates with stemness characteristics in temozolomide-resistant glioblastoma
Authors: Chuang Jian-Ying , Hsu Che-Chia , Chang Kwang-Yu , Chang Wen-Chang
Published: August 2018

The prognosis of glioblastoma (GBM) is usually poor even following treatment with the first-line chemotherapeutic agent temozolomide (TMZ). One most known resistant mechanism is the presence of DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). However, compared with MGMT-mediated innate TMZ resistance, the development of acquired resistance is considered more complex with multi-factorial involvement such as the presence of cancer stem cells (CSCs). In this study, we treated the MGMT-negative GBM cells with TMZ to investigate the acquired resistance, and found that both histone deacetylases (HDACs) and Sp1 are key factors protecting GBM against TMZ. These results include the following: (1) Stemness markers were highly increased in TMZ-resistant GBMs; (2) The activity of HDACs affected the stem-like characteristics and cell survival of GBM stem cells (GSCs); (3) An HDAC1/2/6-selective inhibitor MPT0B291 increased TMZ-sensitivity and induced senescence in TMZ-resistant cells; (4) MPT0B291 suppressed anti-senescence genes (hTERT and BMI1) expression via inhibition Sp1 transactivation; (5) Both HDACs and Sp1 were elevated and interacted with each other in GSCs and resistant GBM cells; (6) TMZ treatment induced Sp1 deacetylation, but MPT0B291 attenuated that. In summary, we verified that HDACs increases Sp1 activation via protein deacetylation and causes Sp1-downstream target upregulation, which may enrich stemness properties and protect GBM against chemotherapeutic drugs.

Citation Format: Jian-Ying Chuang, Che-Chia Hsu, Kwang-Yu Chang, Wen-Chang Chang. Sp1 acetylation associates with stemness characteristics in temozolomide-resistant glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5905.