bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 847: Optimization of radiation regimen in a carcinogen-induced mouse model for lung cancer
Authors: Bhayana Sagar , Liu Joseph , Chakravarti Arnab , Jacob Naduparambil K.
Published: August 2018

Purpose: Lung cancer remains the most common cause of cancer-related mortality in United States and worldwide. The major cause of lung cancer is attributed to carcinogens present in tobacco smoke, air pollution, diet and food supplements, occupational and other environmental exposures. Lung Cancer Mutation Consortium has identified many recognized “driver mutations” in nearly two-thirds of the patients who developed advanced form of lung cancer. Our primary goal is to understand the mechanism underlying the variations in radiation sensitivity with driver mutations and optimize radiation dose and treatment regimen. A better understanding of the most frequent driving mutations is required for progression towards personalized therapy. Methods: Urethane, a carcinogen injected via intraperitoneal route into A/J mice induced multiple lung tumors by week 25 that resembled non-small cell lung carcinoma. These lung tumors were assessed via MRI and each tumor was longitudinally tracked via surface rendered 3D imaging following X-ray exposure using small animal radiation research platform (SARRP). We employed acute high single dose (12 Gy) and clinically relevant fractionated dose (3 X 5 Gy). Individual lung tumor was also dissected from non-irradiated and irradiated mice followed by collagenase digestion and then cultured in a specific media for in-vitro mutational analysis. Results: Urethane induced multiple lung tumors (20-25 tumors with 1mg/kg urethane) of varying size (0.5 - 2mm) were detectable by MRI at 25 week following injection. Longitudinal MRI performed on irradiated tumor bearing mice showed significant regression after radiation with commitment survival benefit. Mice performed better when exposed to fractionated regimen than high single dose with relatively low level radiation toxicity. Primary lung cancer cells were prepared from the explanted tumors for prospectively examining relevant driver mutations and studying the genetic determinants of resistance and to identify novel targets. We also have collected body fluids for investigating changes in molecular profile of exosomes at time points after irradiation. Conclusions: Our study thus far set up a platform for longitudinal 3D tracking of individual lung tumors following treatment and recurrence. Tumor regression was evident after a week of radiation exposure. Primary lung tumor cell cultures were successfully established for molecular analysis and mechanistic studies

Citation Format: Sagar Bhayana, Joseph Liu, Arnab Chakravarti, Naduparambil K. Jacob. Optimization of radiation regimen in a carcinogen-induced mouse model for 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 847.
Abstract 865: Small molecule-mediated modulation of Ras elicits inhibition of phospho ERK signaling through negative feedback on SOS1
Authors: Howes Jennifer E. , Akan Denis T. , Burns Michael C. , Sun Qi , Little Andrew J. , Camper DeMarco V. , Abbott Jason R. , Phan Jason , Lee Taekyu , Rossanese Olivia W. , Waterson Alex G. , Fesik Stephen W.
Published: August 2018

Oncogenic mutation of Ras is responsible for more than 30% of all human tumors. Therefore, pharmacological modulation of Ras has attracted great interest as a therapeutic strategy. Our laboratory has recently discovered small molecules that activate Son of Sevenless (SOS)-catalyzed nucleotide exchange on Ras and paradoxically inhibit downstream signaling. Here we describe how pharmacologically targeting SOS1 induces biphasic modulation of Ras-GTP and downstream ERK levels. We consistently observed this phenotype in a variety of cell lines expressing different Ras mutant isoforms, and using multiple chemotypes of compound. In elucidating the molecular mechanism, we observed that compound treatment caused an increase in phosphorylation at ERK consensus motifs on SOS1 that was not observed with the expression of a non-phosphorylatable S1178A SOS1 mutant, or after pre-treatment with an ERK inhibitor. Phosphorylation at S1178 on SOS1 is known to inhibit the association between SOS1 and GRB2 and disrupt SOS1 membrane localization. Consistent with this, we show that wild-type SOS1 and GRB2 dissociated in a time dependent fashion in response to compound treatment, and conversely, this interaction was enhanced with the expression of a S1178A SOS1 mutant. Pre-treatment with an ERK inhibitor prevented compound-induced inhibition of the association between SOS1 and GRB2. Furthermore, in cells expressing either S1178A SOS1 or a constitutively membrane-bound CAAX box tagged SOS1 mutant, we observed elevated Ras-GTP levels over time in response to compound, as compared to the biphasic changes in Ras-GTP exhibited in cells expressing wild-type SOS1. These results show that small molecule agonists of SOS1 cause paradoxical inhibition of Ras-GTP and MAPK signaling in Ras mutant cancer cells, through negative feedback on SOS1. Overall, these compounds provide us with the unique opportunity to better understand the biological functions of SOS and Ras in cancer cells, and may aid in the discovery of small molecules to treat Ras-driven tumors.

Citation Format: Jennifer E. Howes, Denis T. Akan, Michael C. Burns, Qi Sun, Andrew J. Little, DeMarco V. Camper, Jason R. Abbott, Jason Phan, Taekyu Lee, Olivia W. Rossanese, Alex G. Waterson, Stephen W. Fesik. Small molecule-mediated modulation of Ras elicits inhibition of phospho ERK signaling through negative feedback on SOS1 [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 865.
Abstract 867: Blocking breast cancer metastasis by targeting HuR-FOXQ1 signaling axis
Authors: Wu Xiaoqing , Gardashova Gulhumay , Lan Lan , Zhan Yu , Liu Jiajun , Dixon Dan A. , Aubé Jeffrey , Welch Danny R. , Xu Liang
Published: August 2018

As most of treatment options do not work very well for metastatic cancer. Patients with metastatic cancer have a greatly lower survival rate compared with patients with local cancer. Metastasis remains a life-threat to cancer patients and an unmet medical need. The RBP Hu antigen R (HuR) is overexpressed in virtually all malignancies tested, including breast cancer. Cytoplasmic HuR accumulation correlates with high-grade malignancy, poor distant disease-free survival and serves as a prognostic factor for poor clinical outcome in breast cancer. HuR promotes tumorigenesis by promoting mRNA stability and translation of proteins implicated in proliferation, survival, angiogenesis, invasion, and metastasis. We found that silencing of HuR inhibited cell invasion in vitro in breast cancer. Using RIP-seq (ribonucleoprotein immunoprecipitation-sequencing), a transcription factor FOXQ1, which is recently revealed to implicate in breast cancer invasion and metastasis processes, is found to be a direct HuR target. Furthermore, exogenous introduction of FOXQ1 can rescue cell invasive ability inhibited by HuR knockout. Taken together, HuR-FOXQ1 signaling axis is a potential target for blocking breast cancer metastasis. RNA-binding proteins had previously been considered “undruggable” due to lack of a well-defined binding pocket for target RNAs. Using high throughput screening followed by structure-based rational design and lead optimization, we have identified small molecules that inhibit HuR-mRNA interaction at nM to sub-µM potency. Our lead compound, KH-3, potently inhibits breast cancer cell growth and decreases cell invasion in vitro similar to HuR knockout, as well as increasing the expression of epithelial marker E-cadherin. FOXQ1 overexpression abolishes the effect of KH-3 on blocking metastasis in breast cancer cells, demonstrating that the HuR inhibitor KH-3 inhibits cell metastasis by blocking FOXQ1 function. Moreover, KH-3 treatment disrupts HuR-FOXQ1 interaction in RNP-IP and FOXQ1 3′-UTR luciferase reporter assays. In vivo efficacy studies show that KH-3 not only exhibits potent antitumor efficacy in orthotopic xenograft models of breast cancer, but also efficiently blocks lung metastasis in experimental metastatic cancer model. In conclusion, we identified a potent and specific small molecule disrupter of HuR-FOXQ1 interaction for potential novel anti-metastatic therapy of breast cancer with HuR overexpression.

Citation Format: Xiaoqing Wu, Gulhumay Gardashova, Lan Lan, Yu Zhan, Jiajun Liu, Dan A. Dixon, Jeffrey Aubé, Danny R. Welch, Liang Xu. Blocking breast cancer metastasis by targeting HuR-FOXQ1 signaling axis [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 867.
Abstract 872: IDH1-mutated cancer cells are sensitive to cisplatin and an IDH1-mutant inhibitor counteracts this sensitivity
Authors: Khurshed Mohammed , Molenaar Remco J. , Wilmink Johanna W. , Laarhoven Hanneke W. van , Noorden Cornelis J. van
Published: August 2018

Isocitrate dehydrogenase 1 (IDH1) is mutated in various types of human cancer and predicts improved response to treatment with irradiation or chemotherapy. Mutated IDH1 enzymes catalyze neomorphic conversion of α-ketoglutarate (α-KG) to the oncometabolite D-2-hydroxyglutarate (D-2HG) with concomitant consumption of NADPH, resulting in decreased reducing power needed for detoxification of e.g. reactive oxygen species (ROS). We report that a small-molecule inhibitor of IDH1-mutation (IDH1MUT) that is being investigated for cancer therapy may limit efficacy of treatment when co-administered with cisplatin.

In the present study, we investigated whether the efficacy of treatment with cisplatin, which is a widely-used chemotherapeutic agent, induces DNA strand breaks and oxidative damage in IDH1MUT cancer cells. We found that exposure to cisplatin induced higher levels of ROS, DNA double-strand breaks, and cell death in IDH1MUT cancer cells as compared to IDH1 wild-type (IDH1WT) cancer cells. Besides these cytotoxic effects, mechanistic investigations revealed that cisplatin treatment causes dose-dependent reduction of oxygen consumption and thus the oxidative respiration in lDH1MUT cells and not in lDH1WT cells, which was accompanied by disturbed mitochondrial proteostasis and impaired mitochondrial activity. These effects were abolished by the IDH1MUT inhibitor AGI-5198 and were recapitulated by treatment with D-2HG. Thus, our study shows that altered oxidative stress responses due to a vulnerable oxidative metabolism underlie the sensitivity of IDH1MUT cancer cells to cisplatin. Furthermore, our data offer an explanation for the relatively longer survival of patients with IDH1-mutated tumors, and imply that administration of IDH1MUT inhibitors in these patients limit efficacy of cisplatin treatment.

Citation Format: Mohammed Khurshed, Remco J. Molenaar, Johanna W. Wilmink, Hanneke W. van Laarhoven, Cornelis J. van Noorden. IDH1-mutated cancer cells are sensitive to cisplatin and an IDH1-mutant inhibitor counteracts this sensitivity [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 872.
Abstract 881: Identifying the context of vulnerability to MLN4924 in glioblastoma (GBM)
Authors: Chaudhuri Leena , Bybee Rita L. , Hartman Lauren K. , Peng Sen , Finlay Darren , Vuori Kristiina , Berens Michael E. , Dhruv Harshil D.
Published: August 2018

Neddylation is a post-translational mechanism that marks proteins for degradation through activity of NEDD8 Activating Enzyme (NAE). NAE activates cullin-RING ligases (CRL), which ubiquitylate selected substrates and mark them for proteosomal degradation. MLN4924, or Pevonedistat, targets NAE and inhibits Neddylation and induces apoptosis in sensitive cells. To assess the preferential sensitivity of cell lines to MLN4924, we performed a 10-point drug dose response (DDR) assay on long-term established GBM cell lines. Efficacy of MLN4924 in glioma cell lines was evaluated by measuring cell viability (CellTiterGlo®) and cell cycle progression (flow cytometry with propidium iodide staining). To identify mechanism of differential response to MLN4924 treatment, cell cycle regulatory pathway and DNA damage were also examined by Western blotting. GB1 (IC50 = 0.28 μM) & LN18 (IC50 = 0.19 μM) were established as sensitive and M059K (IC50 = 5.5 μM) & SNU1105 (IC50 = 20.9 μM) as non-sensitive cell lines based on the IC50 values. Flow cytometry analysis of DNA content revealed significant arrest of cells in G2/M even at low doses of 100 nM of MLN4924 preferentially in GB1 and LN18. This was consistent with an increase in CRL substrates p21, p27 and WEE1 in GB1 and LN18 possibly contributing to the G2/M arrest. While CDT1 accumulation was observed starting at 2h post MLN4924 treatment in sensitive cell lines, it took upto 8h for CDT1 accumulation in the non-sensitive cell lines. Increases in CDT1 induced re-replication causing massive arrest in G2/M phase lead to increased DNA damage, validated by higher expression of γH2AX in the sensitive cell lines. Additionally, we also investigated the efficacy of MLN4924 against orthotopic glioma PDX models in vitro and in vivo to validate our findings. In a cohort of glioblastoma PDX models we discovered that GBM PDX models with lower Neddylation gene set enrichment score (GBM116, GBM59, SF7300) were markedly more vulnerable to MLN4924 than GBM PDX models with higher Neddylation gene set enrichment score (GBM91 and GBM102) in vitro and in vivo. Orthotopic PDX models of selected GBM revealed survival prolongation of GBM116, but minimal survival benefit to GBM102 tumors. Validation of the predictive markers of vulnerability to MLN4924 in additional PDX models will set the stage for prospective clinical trials of MLN4924 in glioblastoma patients.

Citation Format: Leena Chaudhuri, Rita L. Bybee, Lauren K. Hartman, Sen Peng, Darren Finlay, Kristiina Vuori, Michael E. Berens, Harshil D. Dhruv. Identifying the context of vulnerability to MLN4924 in glioblastoma (GBM) [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 881.
Abstract 894: Overexpression and extended degradation rate of Axl in acquired gefitinib-resistant non-small cell lung cancer cells
Authors: Kim Donghwa , Bach Duc-Hiep , Hong Ji-Young , Park Hyen Joo , Lee Sang Kook
Published: August 2018

Non-small cell lung cancer (NSCLC) is a major type of lung cancer which accounts for approximately 80-85% of all lung cancers. Epidermal growth factor receptor (EGFR)- targeted tyrosine kinase inhibitors (TKIs), such as gefitinib have improved the survival of NSCLC patients with activating mutations in EGFR. However, the drug responses are only transient because tumors acquire resistance via secondary mutations in EGFR (T790M) or amplification of other receptor tyrosine kinases (RTKs) including MET or AXL. Therefore, the precise mechanisms of chemoresistance and finding the novel ways of targeting cancer cells are needed to be identified. AXL, a TAM (Tyro3, AXL, Mer) family member, has been studied as a tumorigenesis, chemoresistance, and EMT marker. Also, we found that AXL is overexpressed in EGFR mutated gefitinib-resistant non-small lung cancer cells (PC9-gef) compared to its parent cells (PC9). Moreover, the AXL degradation rate was found to be suppressed. These findings suggest that the modulation of AXL turnover can be a therapeutic target to overcome gefitinib-resistance. In this study, we confirmed the possibilities of overcoming gefitinib-resistance through the down-regulation and turnover acceleration of AXL by employing yuanhadine, an anti-tumor agent derived from Daphne Genkwa. Yuanhuadine was also effectively suppressed the tumor growth in nude mouse xenograft models implanted with PC9 or PC9-gef cells. Therefore, regulation of AXL expression by modulating its degradation rate can be a therapeutic strategy to overcome acquired gefitinib-resistance in NSCLC.

Citation Format: Donghwa Kim, Duc-Hiep Bach, Ji-Young Hong, Hyen Joo Park, Sang Kook Lee. Overexpression and extended degradation rate of Axl in acquired gefitinib-resistant non-small cell lung 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 894.
Abstract 897: A novel fusion gene responsible for colon cancer drug resistance
Authors: Xie Jingwu , Gu Dongsheng , Song Ruolan
Published: August 2018

Colorectal cancer is a leading cause of cancer-related mortality worldwide. Patients with advanced colorectal cancer eventually succumb to the disease, despite the fact that some patients respond initially to therapies (chemotherapy and targeted therapy). Thus, identifying molecular mechanisms responsible for drug resistance will help design novel strategies to treat colorectal cancer. In this study, we identified a novel mechanism by which colorectal cancer cells acquired 5-FU resistance. Through exome sequencing, we discovered a novel fusion gene common in 5-FU-resistant colorectal cancer cells. This fusion gene, once ectopically expressed in the sensitive cancer cells, promotes drug resistance to 5-FU treatment. Further analyses have revealed a signaling network linking this fusion gene to drug resistance. Taken together, we have revealed a novel mechanism for drug resistance in colorectal cancer, which may be a novel target to overcome drug resistance in colorectal cancer.

Citation Format: Jingwu Xie, Dongsheng Gu, Ruolan Song. A novel fusion gene responsible for colon cancer drug resistance [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 897.
Abstract 910: Mechanisms of Ewing sarcoma resistance to LSD1 inhibition
Authors: Pishas Kathleen I. , Sharma Sunil , Lessnick Stephen L.
Published: August 2018

Given the modest efficacy of current sarcoma therapeutic modalities, there is a major unmet clinical need for the translation of novel agents to tackle the burden of Ewing sarcoma (pediatric and young adolescent bone cancer). We have recently shown that therapeutic targeting of LSD1, a highly expressed lysine specific demethylase, with the reversible small molecule inhibitor SP-2509, comprehensively disrupts the global transcriptional function of EWS-FLI, and significantly impairs the growth of Ewing sarcoma cell lines both in vitro and in vivo. Prior to clinical trial evaluation, the goal of this study was to elucidate mechanisms of Ewing sarcoma SP-2509 drug resistance.

In order to investigate SP-2509 resistance, A673 Ewing sarcoma cells were cultured in escalating concentrations of SP-2509 for 8 months. Cell Titer Glo analysis revealed that these SP-2509 drug resistant (DR) cells were 54.9 fold more resistant to SP-2509 compared to parental A673 cells (72hr IC50 0.138μM versus 7.586μM). SP-2509 DR cells also exhibited a significant decrease in their ability to form anchorage-independent colonies in soft agar. Whole exome (250X coverage) and RNA seq analysis of parental and SP-2509 DR A673 cells revealed 25 drug induced mutations, with 88% of the whole cell population harboring a Glu53* stop-gain mutation in MRPL45 (mitochondrial ribosomal protein L45). In addition, SP-2509 DR cells displayed a distinct transcriptomic profile, with 3250/2376 genes significantly up/down regulated (≥1.5 fold) in SP-2509 DR cells compared to parental controls. KEGG Pathway analysis revealed that the genes were highly enriched for hepatic stellate cell activation/interferon signaling and cholesterol biosynthesis respectively. Lastly, SP-2509 DR cells were shown to be equally sensitive to etoposide, resistant to vincristine/doxorubicin and surprisingly more sensitive to the HDAC inhibitors vorinostat and entinostat compared to parental A673 cells.

Together, our preliminary findings provide key insights into the mechanisms of SP-2509 drug resistance, information of which can be used to find combinatorial agents that can potentially be used to circumvent resistant clones to achieve maximal therapeutic effect.

Citation Format: Kathleen I. Pishas, Sunil Sharma, Stephen L. Lessnick. Mechanisms of Ewing sarcoma resistance to LSD1 inhibition [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 910.
Abstract 918: A secondary RET mutation allosterically conferring resistance to vandetanib
Authors: Nakaoku Takashi , Kohno Takashi , Araki Mitsugu , Nino Seiji , Chauhan Rakhee , Knowles Phillip P. , Tsuchihara Katsuya , Matsumoto Shingo , Shimada Yoko , Mimaki Sachiyo , Ishii Genichiro , Ichikawa Hitoshi , Okuno Yasushi , Yoh Kiyotaka , McDonald Neil Q. , Goto Koichi
Published: August 2018

Targeted therapy against oncogenic ALK and ROS1 fusion using type I tyrosine kinase inhibitors (TKIs), which bind to the ATP-binding cleft of kinases, is highly effective in lung adenocarcinoma (LADC); however, such cancers inevitably acquire resistance, which severely limits the efficacy of cancer treatments. The identification of these resistance mutations led to the development of novel TKIs to overcome acquired resistance. Here we report resistance to vandetanib, a type I RET kinase inhibitor, developed in a patient with metastatic LADC harboring a CCDC6-RET fusion that initially exhibited a response to treatment. The resistant tumor acquired a secondary mutation resulting in substitution of an amino acid located outside the nucleotide binding cleft. The mutation conferred resistance to vandetanib by increasing the ATP affinity and auto-phosphorylation activity of RET kinase. A crystal structure of the mutant revealed a small hydrophobic core around the mutated amino acid likely to enhance basal kinase activity by stabilizing an active conformer. In addition, a thermal shift assay suggested that the mutation was less able to bind vandetanib. Molecular dynamics simulation supported the mutant underwent conformational changes decreasing the binding affinity of vandetanib. Our findings indicate that the missense mutation is able to increase kinase activity and confers drug resistance through allosteric effects.

Citation Format: Takashi Nakaoku, Takashi Kohno, Mitsugu Araki, Seiji Nino, Rakhee Chauhan, Phillip P. Knowles, Katsuya Tsuchihara, Shingo Matsumoto, Yoko Shimada, Sachiyo Mimaki, Genichiro Ishii, Hitoshi Ichikawa, Yasushi Okuno, Kiyotaka Yoh, Neil Q. McDonald, Koichi Goto. A secondary RET mutation allosterically conferring resistance to vandetanib [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 918.
Abstract 920: COP1 E3 ligase regulates response to oncogenic MAPK pathway inhibition
Authors: Mayekar Manasi K. , Lin Luping , Bivona Trever G.
Published: August 2018

Oncogenically activated RAS-MAPK pathway is the driver of several cancers including the majority of non-small cell lung adenocarcinomas (NSCLC). RAS-MAPK pathway is activated in NSCLC tumors via diverse genetic alterations in upstream receptor tyrosine kinases such as EGFR and ALK as well as in RAS, BRAF, MEK and RAS GTPase activating protein (GAP) and tumor suppressor, NF1. Molecular targeting of players of RAS-MAPK pathway can elicit an initial tumor response in many patients. However, most patients show an incomplete response and some fail to respond despite the presence of RAS-MAPK pathway activating genetic lesion in the tumor. Hence, it is important to understand the molecular basis of response and resistance to targeted therapies inhibiting the RAS-MAPK pathway in lung adenocarcinomas in order to improve patient survival. We conducted a genetic screen to identify regulators of response to MAPK pathway inhibition in lung adenocarcinomas. Our genetic screen uncovered the E3 ubiquitin ligase COP1/RFWD2 as a novel modulator of response to inhibition of RAS-MAPK pathway. We found that depletion of COP1 and members of its complex, as well as proteasomal subunits, confers resistance to RAS-MAPK pathway inhibition in human lung adenocarcinoma cells with oncogenically activated RAS-MAPK pathway. Interestingly, oncogenic targets of COP1 include MAPK pathway effectors, presence of which has been shown to support the survival of cancer cells with oncogenically activated MAPK upon inhibition of the RAS-MAPK pathway. Hence, we tested if depletion of COP1 alters the levels of those oncogenic substrates. Excitingly, we observed a substantial impact of COP1 depletion on the levels of certain effectors in the presence of RAS-MAPK inhibitors in genetically diverse NSCLC cells and also in oncogenic-BRAF driven melanoma cells. Our studies suggests that depletion of COP1 confers resistance to MAPK pathway inhibition in RAS-MAPK pathway driven cancers through accumulation of specific MAPK pathway effectors. This work has improved our understanding of the molecular basis of tumor cell resilience during initial treatment as well as of primary treatment resistance. Additionally, we are examining if levels of COP1 could be a biomarker for predicting response to RAS-MAPK pathway inhibitor therapy. We will also determine if the resistance-conferring COP1 substrate could be cotargeted along with RAS-MAPK pathway to improve the patient response in RAS-MAPK pathway driven lung adenocarcinomas and other cancers with low levels of COP1.

Citation Format: Manasi K. Mayekar, Luping Lin, Trever G. Bivona. COP1 E3 ligase regulates response to oncogenic MAPK pathway inhibition [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 920.
Abstract 928: Regulatory heterogeneity in glioblastoma multiforme informs novel drug target discovery
Authors: Liu Yunpeng , He Shan , Hemann Michael T. , Regev Aviv
Published: August 2018

Glioblastoma multiforme (GBM) is one of the most malignant forms of cancer. Its heterogeneity has been assessed at multiple levels, including bulk and single-cell transcriptome profiling. The disease has been stratified into four molecular subtypes - Classical, Neural, Proneural and Mesenchymal according to their gene expression profiles, and each of these subtypes display distinct mutational signatures and histological features reflective of their nomenclature. However, the underlying regulatory circuitry that gave rise to such heterogeneity and its implications for specialized therapeutic options are unclear. To dissect subtype-specific regulatory programs, we have developed a nonlinear regression model to unveil key transcriptional rewiring across the four GBM subtypes. Using a backbone network of potential transcription factor (TF) - target gene pairs, we applied nonlinear regression using expression profiles of each subtype to derive subtype-specific regulatory parameters for each TF-gene pair. Coupled with a novel analysis pipeline, the model revealed a set of transcription regulators whose regulatory capacities for target genes are unique to each subtype. The target genes of these signature TFs are enriched for expression signature genes for the corresponding subtype, implying that subtype-specific rewiring of TFs play a significant role in shaping differential transcription landscapes. The model was also able to capture known physical and functional interactions between TFs, as well as identify potential new co-regulatory relationships. Detailed examination of both expression and co-regulatory profiles of signature TFs showed that altered regulatory behavior could be a result of differential expression of the TF itself, differential functional partnering of the TF with other TFs, or a combination of both mechanisms. Importantly, our model also demonstrates that differential regulatory programs could provide new options for targeted therapy in GBM. We simulated the effects of knockdown/inhibition of these co-regulatory partners by propagating changes in expression to the corresponding target genes and then to the protein signaling layer using a novel random walk-based algorithm. Using the apoptosis pathway as readout, we show that targeting the retinoic acid receptor RXRA may be specifically beneficial for killing tumor populations of the Neural subtype, whereas the HNF4A gene is a potentially beneficial drug target for cells of the Mesenchymal subtype.

Citation Format: Yunpeng Liu, Shan He, Michael T. Hemann, Aviv Regev. Regulatory heterogeneity in glioblastoma multiforme informs novel drug target 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 928.
Abstract 94: DDX31 cooperates with mutant p53 and EGFR to promote the multistep progression of invasive bladder cancer
Authors: Katagiri Toyomasa , Daizumoto Kei , Yoshimaru Tetsuro , Matsushita Yosuke , Fukawa Tomoya , Ono Masaya , Kanayama Hiro-omi
Published: August 2018

Bladder cancer is the most common malignancy of the urinary tract worldwide. Approximately 70% of cases are diagnosed as non-muscle-invasive bladder cancer (NMIBC), while the remaining 30% of cases are classified as muscle-invasive bladder cancer (MIBC). Evidence based on molecular biology has highlighted that the aggressiveness of MIBC advances through a multistep mechanism due to many genomic alterations. Notably, alterations of TP53 and EGFR pathways frequently occur in bladder cancers and are associated with poor prognosis, respectively. However, the connection between the overexpression of EGFR and the p53 mutation in multistep carcinogenesis and the progression of MIBC remains unknown. Here we report the distinct critical roles of DEAD box polypeptide 31 (DDX31) in the multistep progression of muscle invasive bladder cancer (MIBC) through its sequential interaction with mutant p53 (mutp53) and EGFR. In early MIBC cells, nuclear DDX31 acts as a transcriptional co-activator that binds to mutp53/SP1 and enhances mutp53 transcriptional activation, thereby upregulating the EPB41L4B gene, which plays a critical role in metastatic behavior and promotes the invasion and migration of MIBC. Notably, in advanced MIBC, cytoplasmic DDX31, which is transported from the nucleus, functions as an adaptor scaffold protein that forms a complex with EGFR via its interaction with phosho-nucleolin (NCL), leading to constitutive activation of EGFR-Akt signaling. Significantly, high expression of both cytoplasmic DDX31 and p53 proteins is correlated with a poor prognosis in MIBC patients. More importantly, blocking the DDX31-NCL interaction via a dominant-negative peptide led to downregulation of EGFR-Akt signaling, resulting in a significant anti-tumoral effect of bladder cancer in vivo. Our findings reveal that DDX31 cooperates with mutp53 and EGFR to promote the multistep progression of MIBC and that inhibition of DDX31-NCL formation may lead to potential treatment strategies for advanced MIBC.

Citation Format: Toyomasa Katagiri, Kei Daizumoto, Tetsuro Yoshimaru, Yosuke Matsushita, Tomoya Fukawa, Masaya Ono, Hiro-omi Kanayama. DDX31 cooperates with mutant p53 and EGFR to promote the multistep progression of invasive bladder 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 94.
Abstract 948: Preventing estrogen receptor alpha-positive breast cancer outgrowth with the use of hormone replacement therapy
Authors: Dembo Anna G. , Aledort Emily S. , Greene Geoffrey L.
Published: August 2018

Menopause occurs in all women, usually between the ages of 45 and 55. As a result of menopause, women often experience undesirable vasomotor symptoms, which can be alleviated by hormone replacement therapy (HRT). The Women's Health Initiative (WHI) trial concluded that PremPro, a HRT formulation that combines conjugated equine estrogens (CE) with medroxyprogesterone acetate, increases the risk of breast cancer. Over the years, the number of women taking HRT has dramatically decreased due to the perceived risk based largely on the results of the WHI trial. Follow-up sudies suggest that breast cancer cases from PremPro treatment were primarily due to the outgrowth of occult tumors, not the formation of new disease. Duavee, a more recent form of HRT that combines CE and bazedoxifene (BZA), a selective estrogen receptor modulator (SERM) and degrader (SERD), has been approved by the FDA for treatment of moderate to severe hot flashes and to reduce the risk of osteoporosis. More importantly, this CE+BZA mixture not only relieves symptoms associated with menopause, but it also does not stimulate the breast or uterus. Several preclinical studies suggest that CE+BZA might be protective in the breast, however the mechanism of action of this new combination therapy is not known. Our goal, therefore, is to elucidate the underlying molecular mechanisms by which CE+BZA differentially affects estrogen receptor alpha (ERα) action in the mammary gland, using transcriptome and whole genome occupancy analysis in breast cancer cell lines. RNA-Seq and ChIP-Seq studies suggest that CE+BZA decreases pathways related to S-phase entry, cell cycle regulation and BRCA1. We are also studying the effects of CE+BZA on early mammary cancer progression in the estrogen-sensitive polyoma middle T antigen (PyMT) transgenic mouse model and have observed that treatment with CE+BZA delayed the onset of tumors and decreased their rate of growth. Mice treated with CE+BZA also survived longer. In addition, CE+BZA was able to decrease the rate of tumor growth in an ERα-positive patient-derived xenograft (PDX) mouse model. An improved understanding of the molecular mechanisms of CE+BZA action in hormone sensitive breast cancer cell and animal models should have important implications for women considering HRT.

Citation Format: Anna G. Dembo, Emily S. Aledort, Geoffrey L. Greene. Preventing estrogen receptor alpha-positive breast cancer outgrowth with the use of hormone replacement therapy [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 948.
Abstract 972: Defining the molecular context of MYC and WDR5 at chromatin
Authors: Guarnaccia Alissa D. , Tansey William P.
Published: August 2018

MYC is an oncoprotein transcription factor that is overexpressed in the majority of malignancies and contributes to the deaths of ~100,000 Americans each year. The tumorigenic functions of MYC stem from its influence on the transcriptional program of a cell, promoting the expression of thousands of genes involved in cell cycle progression, growth, and metabolism. In order to understand how MYC promotes cancer, we must understand the mechanisms by which MYC selects its target genes. Epigenetic signatures, DNA sequence motifs, and protein-protein interactions have all been implicated to play a role in MYC target gene selection, but a deep molecular understanding of the determinants of target gene recognition by MYC is missing. Our laboratory discovered that the chromatin-associated scaffolding protein WDR5 directly interacts with MYC and co-localizes on chromatin at a majority of loci (Thomas et al., Mol. Cell, 58: 440-452, 2015). The MYC-WDR5 interaction is disrupted by point mutation of three charged residues in MYC. While this MYC mutant retains the ability to bind to naked DNA, the mutant does not bind target loci in the context of chromatin and is unable to form tumors in mice. From these findings we propose that WDR5 is a crucial factor for enabling MYC activity in cancer. Both MYC and WDR5 are known to be highly networked proteins with multiple simultaneous interactions. Our aim is to tease apart this complicated network and define the molecular context specifically for the MYC-WDR5 interaction. To do this, we are employing our WDR5-binding deficient MYC mutant as a tool to genetically distinguish MYC interactions that are dependent upon WDR5. Our approach is to use Stable Isotope Labeling of Amino acids in Cell Culture (SILAC) because it enables us to quantitatively compare wild-type and mutant MYC and reduces background noise common to non-quantitative MYC-centered proteomics. Once identified by SILAC, interacting proteins are validated with biochemical methods and by proximity ligation assay in cells. This dual validation confirms the interaction in two ways, and provides information on the sub-cellular localization of the interaction. Thus far we have identified a preliminary set of ~25 interacting proteins that are currently undergoing validation. Characterization of these new factors by genetic, genomic, and biochemical approaches will expand our understanding of MYC biology, and potentially identify new factors that can be therapeutically targeted in cancer.

Citation Format: Alissa D. Guarnaccia, William P. Tansey. Defining the molecular context of MYC and WDR5 at chromatin [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 972.
Abstract 973: MEKK3 sustains EMT and stemness in pancreatic cancer by regulating YAP and TAZ transcriptional activity
Authors: Piro Geny , Santoro Raffaela , Zanotto Marco , Carbone Carmine , Tortora Giampaolo , Melisi Davide
Published: August 2018

Background: Pancreatic cancer (PC) is one of the most threatening and poorly understood human malignancies. MEKK3 (MAP3K3) is an intracellular serine/threonine kinase activated by different signaling pathways, including IL1, TNFα and TLR8, which regulates in turn NF-κB, JNK and p38 activation.The transcriptional regulators Yes Associated Protein (YAP) and transcriptional coactivator with a PDZ binding domain (TAZ) are central determinants of malignancy and have been identified as a critical oncogenic effectors of KRAS in pancreatic cancer. We hypothesized that MEKK3 could sustain pancreatic cancer by inducing YAP/TAZ oncogenic activities. Methods: MEKK3 was knocked-out (KO) in AsPC1, Panc1, and MDA-Panc28 PC cell lines by CRISPR/Cas9. The expression of MEKK3, E-cadherin, YAP/TAZ and their target genes was measured by both Western blot and qPCR. Cell migration was assessed by transwell and wound healing assays. Stemness was measured by spheroid formation and percentage of CD44+/CD24+/EpCAM+ cancer stem cells (CSC). Effects of MEKK3 KO was evaluated in vivo in an orthotopic nude mouse model with GFP-expressing Panc1 PC cells. Expression of YAP/TAZ and their target genes in tumor samples was measured by IHC. Results: MEKK3 KO resulted in the reversion of both Epithelial-to-Mesenchymal Transition (EMT) and cell migration, as well as in the impairment of cell proliferation. More interestingly, the size of 3D colonies arising from MEKK3 KO cell lines was smaller than the ones formed by their respective controls, due to a reduced percentage of CD44+/CD24+/EpCAM+ CSC. In MEKK3 KO cells, the recruitment of YAP/TAZ onto the promoters of their target genes was significantly impaired and this paired with a reduction of the expression of the same genes. In nude mice, MEKK3 KO significantly reduced tumor growth and prolonged overall survival. Conclusions: Our study is the first to demonstrate that genetic silencing of MEKK3 is a valid approach to revert in vivo the aggressiveness of PC by modulating YAP and TAZ transcriptional activities. Inhibition of MEKK3 could represent an exciting therapeutic strategy for the treatment of PC, which should be warranted further development.

Citation Format: Geny Piro, Raffaela Santoro, Marco Zanotto, Carmine Carbone, Giampaolo Tortora, Davide Melisi. MEKK3 sustains EMT and stemness in pancreatic cancer by regulating YAP and TAZ transcriptional activity [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 973.
Abstract 987: Organoid-based characterization of patient tumors and microenvironments at single cell resolution
Authors: Salahudeen Ameen A. , Zhu Junjie , Ju Jihang , Batish Arpit , Sutha Ken , Neal James T. , Giangarra Valeria , Montesclaros Luz , Sapida Jerald , Sharifi Osman , Lee Josephine , Zheng Grace X. , Wagh Dhananjay A. , Coller John A. , Neal Joel W. , Padda Sukhmani K. , Sabatti Chiara , Kuo Calvin J.
Published: August 2018

The advent of microenvironment directed cancer treatments such as antiangiogenic agents and recent immunotherapies have produced a pressing need for robust and systematic characterization of patient tumors and corresponding stromal populations. However, in vitro cultures of tumor biopsies do not typically preserve both the tumor epithelium and tumor microenvironment as an intact syngeneic unit. To address these limitations, we developed organoid cultures of surgically resected patient cancer biopsies that intrinsically retain diverse tumor microenvironmental cellular components without requiring reconstitution. We compared transcriptome profiles of fresh tumor cell populations to organoid cultures by droplet based single cell 5' RNA sequencing (scRNA-seq). Analysis of >50k single cell transcriptome profiles revealed tumor and stromal cell populations including tumor epithelia, fibroblasts and immune cells in fresh tumors. scRNA-seq of organoid cultures revealed a similar composition of native cancer cells and stromal components in both immune and non immune populations. In particular, 5' scRNA-seq enabled simultaneous characterization of paired T cell receptor alpha and beta chains as well as generalized gene expression in individual cells. We observed clonotype expansion of cytotoxic T cells in both fresh tumors and organoid cultures. The faithful recapitulation of tumor microenvironmental diversity within human organoid cultures should facilitate the in vitro exploration of immunotherapeutic agents and modeling of associated patient-specific responses.

Citation Format: Ameen A. Salahudeen, Junjie Zhu, Jihang Ju, Arpit Batish, Ken Sutha, James T. Neal, Valeria Giangarra, Luz Montesclaros, Jerald Sapida, Osman Sharifi, Josephine Lee, Grace X. Zheng, Dhananjay A. Wagh, John A. Coller, Joel W. Neal, Sukhmani K. Padda, Chiara Sabatti, Calvin J. Kuo. Organoid-based characterization of patient tumors and microenvironments at single cell resolution [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 987.
Abstract 99: An oncogenic role of Deleted in Liver Cancer 1 (DLC1) in metastatic melanoma
Authors: Yang Xintao , Cheung May , Lung Hong-Lok , Cheung Martin
Published: August 2018

Melanoma is a type of skin malignancy originated from neural crest-derived melanocytes. Although the mortality rate has been significantly reduced due to early cancer diagnosis and treatment, distant metastasis still exhibits much inferior survival rates compared to the early stage of this disorder. Therefore, developing new target for therapy is of particular significance in combating metastatic melanoma. DLC1 is well known for its tumor suppressor role through activating small Rho-GTPases' (RHOA and CDC42) intrinsic capability to convert an active GTP to an inactive GDP-bound state, leading to inhibition of tumor growth and metastasis. So far, downregulation of DLC1 expression has been widely reported to associate with increased risk of various malignancies whereas elevation of its expression prevents tumor progression. In contrast to these studies, our pilot findings reveal that multiple melanoma cell lines exhibit higher levels of DLC1 than benign melanocytes and other cancer types, suggesting its unique expression profile in melanoma. Furthermore, in vitro functional assays demonstrate that silenced DLC1 inhibits proliferative, clonogenic, migratory and invasive capabilities of melanoma cells, whereas restoration of DLC1 rescue the oncogenic properties. More importantly, in lung metastasis model using nude mice, DLC1 knockdown suppresses melanoma cells colonizing to the lungs whereas their pulmonary metastases can be recovered with the reconstitution of DLC1. In addition, the manipulation of DLC1 expression does not alter the RHO-ROCK-MRLC signalling, indicating that its oncogenic effect is mediated through a RHO-independent manner. Thus, our results unravel an unprecedented paradigm-shift of DLC1 functioning as an oncogene instead of a tumor suppressor in metastatic melanoma, which may shed new light on disease pathogenesis and uncover novel therapeutic targets to treat this deadly cancer.

Citation Format: Xintao Yang, May Cheung, Hong-Lok Lung, Martin Cheung. An oncogenic role of Deleted in Liver Cancer 1 (DLC1) in metastatic melanoma [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 99.
Abstract CT138: Open-label personalized targeted intervention to maximize TTFields intensity in recurrent GBM (OptimalTTF Trial)
Authors: Mikic Nikola , Korshøj Anders Rosendal
Published: August 2018

Introduction Tumor Treating Fields (TTFields) is approved for the treatment of recurrent glioblastoma (rGBM). Skull remodeling surgery involves formation of strategically placed craniectomies, burr holes or skull thinning, in order to facilitate electric current flow into the tumor region. Preclinical studies have indicated that these procedures provide a marked and focal enhancement (~100%) of TTFields intensity without significantly compromising patient safety. In this open-label phase 1 investigator-initiated interventional trial, we test the safety, feasibility and efficacy of TTfields plus personalized targeted skull remodeling surgery and best choice chemotherapy. We believe this study has high-impact potential at a low clinical risk and may advance current state-of-the-art therapy of GBM therapy.

Methods The trial will include fifteen patients. Major eligibility criteria include age > 18 years, first recurrence of supratentorial GBM, Karnofsky performance score (KPS) > 60, focal tumor < 2 cm to cortical surface, and lack of uncontrollable epilepsy or significant co-morbidity. Upon inclusion, personalized field calculations will be performed to validate TTFields enhancement > 25% due by skull remodeling surgery. The primary endpoint is toxicity assessed by CTCAEv4.0. Secondary endpoints include overall survival, progression free survival (PFS), PFS at six months (PFS6), objective response rate, quality of life (EORTC QLQ-C30 and BN20), KPS, and steroid dose. Follow-up is 18 months and includes regular toxicity assessment (6-week intervals) as well as quality of life and response assessments (3-month intervals). Endpoint data will be collected at the end of scheduled follow-up, occurrence of suspected unexpected serious adverse reactions (SUSARs) or unacceptable serious adverse events (SAEs), withdrawal of consent, or loss to follow-up. Interim analysis will be performed upon endpoint registration for the first five-10 patients.

Citation Format: Nikola Mikic, Anders Rosendal Korshøj. Open-label personalized targeted intervention to maximize TTFields intensity in recurrent GBM (OptimalTTF Trial) [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 CT138.