bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A120: The effect of targeted nanoemulsion on pancreatic cancer cells
Authors: Fei QinQin , O'Barr Sophia , Lambros Maria P.
Published: August 2018

Purpose: Pancreatic cancer is characterized by its extreme aggressiveness and limited treatment options. This work evaluates the targeting of a nanoemulsion containing doxorubicin (DOX) to different pancreatic cancer cell lines and its mechanism of uptake. Methods: Three human pancreatic cancer cell lines, PANC-1, BxPC-3, and MIA PACA-2, were seeded separately into 12-well plates (1.0 x 105 cells/well). Then the cells were treated with DOX alone or DOX in targeted or nontargeted nanoemulsions. Cells treated with culture medium only were used as negative controls. The cellular uptake of the different DOX formulations by the different pancreatic cancer cell lines was evaluated using a fluorescence microscope (EVOS) and a flow cytometer (Accuri). The cytotoxicity of different formulations was evaluated using the SRB assay. The uptake mechanism of the DOX in targeted and nontargeted nanoemulsions was evaluated by comparing the uptake when cells were incubated at 40C and 370C. The uptake mechanism was further evaluated by pretreatment of the cells with inhibitors: cytochalasin D (inhibits caveolin-mediated endocytosis), chlorpromazine (inhibits clathrin mediated endocytosis), amiloride (inhibits micropinocytosis), and nystatin (depletes cholesterol and affects rafts). Results: The uptake of DOX in targeted nanoemulsion by PANC-1, BxPC-3, and MIA PACA-2 cell lines was 1.4-fold, 1.2-fold, and 1.2-fold higher, respectively, compared to that of DOX in the nontargeted nanoemulsion. The IC50 value for DOX in targeted nanoemulsion was 0.3 uM, for DOX in nontargeted nanoemulsion was 0.6 uM, and for DOX only was 1.3 uM. The uptake of DOX in targeted nanoemulsion and nontargeted nanoemulsion was higher at 370C compared to that at 40C. None of the inhibitors used had a significant effect on the uptake of DOX in the targeted or nontargeted nanoemulsions. Conclusion: The results show that DOX in the targeted nanoemulsion has significantly higher cellular uptake and cytotoxicity compared to that of the nontargeted nanoemulsion. The uptake of DOX in targeted and nontargeted nanoemulsion is energy dependent. However, none of the inhibitors used has a significant effect on the uptake of DOX in the targeted nanoemulsion, which requires further studies.

Citation Format: QinQin Fei, Sophia O'Barr, Maria P. Lambros. The effect of targeted nanoemulsion on pancreatic cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A120.
Abstract A129: A second-site mutation in BRAF confers resistance to RAF inhibition in a BRAF V600E-mutant brain tumor
Authors: Wang Jiawan , Yao Zhan , Jonsson Philip , Allen Amy , Qin Alice Can Ran , Pisapia David , Rosen Neal , Taylor Barry S. , Pratilas Christine A.
Published: August 2018

BRAF V600E drives tumors by hyperactivating ERK signaling, is activated in a RAS-independent manner, and signals as a monomer. Selective inhibitors of RAF potently inhibit BRAF V600E and downstream ERK signaling, and have been associated with impressive clinical responses in patients whose tumors harbor V600E mutations in BRAF. Almost all patients, however, ultimately develop resistance. To date, acquired resistance mechanisms validated in patient samples have included splice forms of BRAF, mutations in NRAS, and BRAF amplification, all of which promote formation of RAF dimers, and mutations in MEK. Second-site point mutations in BRAF that promote dimer formation and are associated with acquired resistance have not previously been identified in RAF inhibitor-resistant tumors.

Here, we report a complete response followed by clinical progression of a BRAF V600E-mutant pediatric brain tumor treated with the RAF inhibitor dabrafenib. We identified a second-site mutation in BRAF at the time of progression that was not present in the pretreatment tumor. Further study indicated the acquired mutation is in cis with V600E. We demonstrate that the acquired resistance mutation induces RAF dimer formation, and is sufficient to confer resistance to dabrafenib. Moreover, we showed that ERK signaling activated by the double mutation is sensitive to MEK inhibition or to the RAF plus and MEK inhibitor combination. We also find that two novel RAF dimer inhibitors may overcome resistance mediated by the novel mutation in BRAF.

This is the first study, to our knowledge, that demonstrates the emergence of a confirmed second-site mutation in BRAF V600E and validates that this mutation is responsible for acquired resistance in an initially responding patient treated with dabrafenib. The mechanism of acquired resistance can be abrogated by the novel RAF dimer inhibitors. Our data confirm that a novel class of RAF dimer inhibitors are active against an acquired resistance mutation, suggesting an improved personalized treatment option for patients who harbor similar second-site mutations in BRAF.

Citation Format: Jiawan Wang, Zhan Yao, Philip Jonsson, Amy Allen, Alice Can Ran Qin, David Pisapia, Neal Rosen, Barry S. Taylor, Christine A. Pratilas. A second-site mutation in BRAF confers resistance to RAF inhibition in a BRAF V600E-mutant brain tumor [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A129.
Abstract A130: ANXA1 is involved in resistance to 5-FU in colon cancer cells
Authors: Saito Motonobu , Onozawa Hisashi , Sakuyama Misato , Ashizawa Mai , Kikuchi Tomohiro , Okayama Hirokazu , Endo Hisahito , Fujita Shotaro , Sakamoto Wataru , Saze Zenichiro , Momma Tomoyuki , Ohki Shinji , Kono Koji
Published: August 2018

Annexin A1 (ANXA1), which belongs to the annexin superfamily, is a 37 kDa calcium-dependent phospholipid-linked protein that is associated with regulation of cellular differentiation, proliferation, and apoptosis, anti-inflammatory effects, and drug resistance. Through these functions, ANXA1 is considered to be associated with cancer development and metastasis in various malignant tumors. We previously reported that a case of positive ANXA1 expression by immunohistochemical (IHC) staining in colorectal cancer was associated with venous invasion (P=0.023) as well as lymph node metastasis (P=0.042). We therefore performed further investigation of the role of ANXA1 in colorectal cancer metastasis and treatment. In vitro cell experiments revealed that ANXA1 expression was significantly induced in the colon cancer cells resistant to 5-FU compared to the control cells. We then revealed that overexpression of ANXA1 induced a significant increase of cell viability to 5-FU, whereas ANXA1 knockdown induced a significant decrease of cell viability to 5-FU. Further experiments revealed that ANXA1 expression was induced by hypoxia in colon cancer cells, inducing cellar invasion. These results suggest that ANXA1 expression may be induced by hypoxia during colorectal cancer progression and metastasis. Our results provide a possible strategy to overcome 5-FU resistance by modulating ANXA1 expression.

Citation Format: Motonobu Saito, Hisashi Onozawa, Misato Sakuyama, Mai Ashizawa, Tomohiro Kikuchi, Hirokazu Okayama, Hisahito Endo, Shotaro Fujita, Wataru Sakamoto, Zenichiro Saze, Tomoyuki Momma, Shinji Ohki, Koji Kono. ANXA1 is involved in resistance to 5-FU in colon cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A130.
Abstract A138: Loss of the lysosomal transporter SLC46A3 is a mechanism for innate and acquired resistance to non-cleavable maytansine and pyrrolobenzodiazepine-based antibody-drug conjugates
Authors: Kinneer Krista , Meekin John , Phipps Sandrina , Bezabeh Binyam , Fleming Ryan , Kiefer Christine , Rebelatto Marlon , Dimasi Nazzareno , Wick Michael J. , Moriarty Alyssa , Groves Megan , Herbst Ronald , Tiberghien Arnaud , Masterson Luke , Howard Philip , Tice David
Published: August 2018

Antibody-drug conjugates (ADCs) combine a monoclonal antibody with a potent cytotoxic drug to preferentially eliminate antigen-positive cells for the treatment of cancer. ADCs bearing non-cleavable warheads such as the pyrrolobenzodiazepine (PBD) SG3376 rely upon lysosomal degradation of the antibody for release of the cytotoxic warhead. Here we sought to identify the mechanism of lysosomal membrane transport following release of these membrane-insoluble warheads. We used a multiplexed RT-PCR screen to compare the expression of 43 lysosomal membrane-specific genes in cell lines with inherent sensitivity or resistance to various SG3376-based ADCs. We discovered that only the expression of SLC46A3 correlated with sensitivity to the ADCs. Functional validation through gene knockout or forced gene expression in vitro revealed that loss or gain of SLC46A3 altered the potency of SG3376- and DM1-ADCs but had no effect on ADCs carrying the non-cleavable warhead MMAF or the cleavable PBD payload SG3249. To explore a role in acquired resistance, we interrogated trastuzumab-DM1 (T-DM1) sensitive and resistant cell line pairs and found that resistant lines had lost expression of SLC46A3. Sensitivity to both SG3376- and DM1-ADCs was restored upon reintroduction of SLC46A3. Furthermore, we examined a patient-derived xenograft (PDX) breast cancer model of acquired T-DM1 resistance and found that expression of SLC46A3 was decreased by 92% compared to the parental, T-DM1 sensitive model. Taken together, our results establish SLC46A3 as a transporter of both SG3376 and DM1 catabolites from the lysosome, and suggest that SLC46A3 may be a predictive response marker for ADCs bearing these non-cleavable warheads.

Citation Format: Krista Kinneer, John Meekin, Sandrina Phipps, Binyam Bezabeh, Ryan Fleming, Christine Kiefer, Marlon Rebelatto, Nazzareno Dimasi, Michael J. Wick, Alyssa Moriarty, Megan Groves, Ronald Herbst, Arnaud Tiberghien, Luke Masterson, Philip Howard, David Tice. Loss of the lysosomal transporter SLC46A3 is a mechanism for innate and acquired resistance to non-cleavable maytansine and pyrrolobenzodiazepine-based antibody-drug conjugates [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A138.
Abstract A141: Preclinical evaluation of a new ROS1 inhibitor to overcome resistance to EGFR therapy in glioblastoma
Authors: Aljohani Hashim M. , Aittaleb Mohamed , Park Byung Sun , Lee So Ha , Bahassi El Mustapha
Published: August 2018

Glioblastoma (GBM) is the most aggressive brain tumor in adults and remains incurable despite multimodal intensive treatment regimens. The majority of GBM tumors show a mutated or overexpressed epidermal growth factor receptor (EGFR); however, tumors treated with EGFR inhibitors such as gefitinib or erlotinib inevitably recur. This recurrence highlights the need to identify signaling pathways involved in GBM resistance to EGFR therapy that may serve as targets for intervention. In our recently published work, we found that activation of the ROS1 pathway is a primary mechanism by which glioma cells become resistant to EGFR-targeted therapy. ROS1 is a proto-oncogene receptor tyrosine kinase activated by chromosomal rearrangement in several human cancers, including non-small cell lung cancer (NSCLC), cholangiocarcinoma, gastric cancer, ovarian cancer, and glioblastoma. We therefore designed, synthesized, and screened a series of new ROS1 inhibitors. The lead compound, ROS-Ic, is very potent (IC50 = 24nM) and highly specific to ROS1 protein. Treatment of gefitinib-resistant GBM cells with ROS1-lc sensitized the resistant cells to low concentrations of gefitinib, resulting in apoptosis following a prolonged S-phase cell cycle arrest. When compared to crizotinib, an FDA-approved small-molecule tyrosine kinase inhibitor currently used to treat ALK- and ROS1+ NSCLC patients, ROS-Ic showed superior cytotoxic activity in vitro towards ROS1-positive GBM cells. Crizotinib resistance is thought to be due to abolished crizotinib binding by ROS1 mutations in crizotinib binding pocket. Interestingly, the binding of our leading compound ROS1-Ic was not affected by these mutations and crizotinib-resistant cells harboring these mutations were still sensitive to ROS1-Ic, highlighting the potential therapeutic value of this compound.

Citation Format: Hashim M. Aljohani, Mohamed Aittaleb, Byung Sun Park, So Ha Lee, El Mustapha Bahassi. Preclinical evaluation of a new ROS1 inhibitor to overcome resistance to EGFR therapy in glioblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A141.
Abstract A150: Complex 3D cultures for combination compound screens reveal new insights
Authors: Evans David M. , Michael Selby H. , Bowles Lori , Delosh Rene M. , Julie Laudeman , Ogle Chad C. , Russell Reinhart , Thomas Silvers , Parchment Ralph , Doroshow James , Teicher Beverly
Published: August 2018

3D tumor cell cultures more closely mimic the tumor environment in drug screening assays. The tumor environment includes endothelial cells, infiltrating cells, and stromal cells and the extracellular matrix excreted by these cells. It is likely that the tumor microenvironment affects the response of tumor cells to therapeutic agents. We have used a complex spheroid model consisting of tumor cells, endothelial cells (HUVEC), and mesenchymal stem cells (MSCs) to explore response to selected therapeutic agents. Using 9-10 tumor lines of each of NSCLC, melanoma, and pancreatic cancer, we tested combinations of FDA-approved agents and investigational agents on viability. All agents were studied at multiple concentrations at their reported clinical Cmax values and below in a 6 by 5 concentration matrix. Bliss independence calculations identified drug combinations that were additive, synergistic, or antagonistic. Gemcitabine plus the Chk1/2 inhibitor AZD 7762 showed strong synergy in PANC-1 cells and weaker synergy in MiaPAca-2 complex spheroids. Under the same conditions, gemcitabine and AZD7762 were additive to antagonistic in cell killing and strong antagonism was observed in CFPAC-1 cells. A combination of gemcitabine with the BET inhibitor (GSK525762) was extremely cytotoxic even at low concentrations (Gem, 0.2uM; GSK525762, 10uM)–demonstrating synergy between the Bromodomain (BET) inhibitor GSK525762 and gemcitabine in PANC-1 cells. In one NSCLC cell line (NCI-H23) the combination of GSK525762 (concentrations >1uM) was synergistic with carboplatin; however, in 5 NSCLC PDX lines, the combination had no synergy. In the LOX-IMVI melanoma line, GSK525762 was synergistic with cobimetinib. Cobimetinib and rabusertib were synergistic in the MDA-MB-435 line with >99% cell killing at concentrations < clinical Cmax. The results demonstrate that complex multicellular spheroids can be utilized in prolonged compound exposure screens (7 days) with robust and statistically relevant results using cell viability as an initial measure of cytotoxicity and can inform selection of combinations for progression. The observed additivity, synergy, or antagonism was cell line and tumor type dependent.

Citation Format: David M. Evans, Selby H. Michael, Lori Bowles, Rene M. Delosh, Laudeman Julie, Chad C. Ogle, Reinhart Russell, Silvers Thomas, Ralph Parchment, James Doroshow, Beverly Teicher. Complex 3D cultures for combination compound screens reveal new insights [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A150.
Abstract A178: Targeting the ATM-mediated DNA damage response pathway for cancer treatment
Authors: Grindrod Scott , Velena Alfredo , Jung Mira
Published: August 2018

Epigenetic modulators have emerged as potent therapeutic agents for treatment of patients with cancers. We have developed dual-function small molecules targeting activation of ATM, the DNA damage response transducer, ataxia telangiectasia mutated gene product, and inhibition of histone deacetylases (HDACi). The goal is to develop a single-molecule therapeutic that optimizes the protective effects of ATM activation in normal tissue while targeting HDAC inhibition to sensitize cancerous tissue to radiation. The candidate compound SP-1-161 is a hydroxamic acid that was rationally designed by optimizing ATM activation in the cap domain. In vitro assays against a panel of Class I and Class II HDAC enzymes identified SP-1-161 as a pan-HDAC inhibitor with nanomolar potency (IC50 = 8 nM). Western analysis confirms that SP-1-161 increases acetylated histone H3/H4 and α-tubulin and activates ATM (p-ATM/S1987) in MCF7 cells. The cytotoxicity values (IC50) were in low micro-molar ranges in normal breast epithelial (184A1) and breast cancer (MCF7) cell lines. Radiation clonogenic survival assays demonstrated that the compound protected nonmalignant 184A1 cells (from D0 = 1 Gy to D0 =1.4 Gy) while increasing sensitivity of malignant MCF7 cells to IR (from D0=1.6 Gy to D0=1.12 Gy). Furthermore, in vivo plasma pharmacokinetics following oral administration revealed the area under the curve of 415.79 ng/ml × h with the half-life 1.94 h. Together, our results demonstrate that SP-1-161 is an unprecedented radiation chemotherapeutic agent for treating cancers and protecting normal cells, potentially by targeting the DDR signaling pathways.

Citation Format: Scott Grindrod, Alfredo Velena, Mira Jung. Targeting the ATM-mediated DNA damage response pathway for cancer treatment [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A178.
Abstract B018: Breaking down cancer: IDH and its impact on metabolism and DNA damage and repair
Authors: Neville Kathryn , Arcand Elizabeth , Chiaramida Gabriela , O'Connell Kaylie , Tipping Marla
Published: August 2018

Metabolic reprogramming has been detected in many diseases, including cancer. Recently, a small number of metabolic enzymes have been identified as driver mutations for various types of cancer. To better understand how these genes lead to cancer, and to identify molecular targets for treatment, we are investigating the isocitrate dehydrogenase (IDH) mutation implicated in human glioma and acute myeloid leukemia. We are currently using Drosophila melanogaster as a model to study both the metabolic changes that occur when IDH is mutated, and the potential impact on DNA damage repair pathways. To analyze metabolic changes in the brain of IDH mutant flies, we have optimized a protocol for measuring the metabolism of whole brains using the XFe96 metabolic analyzer. We have also detected the expression of D2-HG, a metabolite produced by mutant IDH in human cells, in our fly IDH model. Generation of D2-HG in human cells has been reported to correlate with insufficient DNA repair. To study this further we are testing for increased DNA damage and lack of DNA repair initiation. In particular, we are investigating a hypothesized connection between the Fanconi anemia DNA repair pathway and IDH mutations.

Citation Format: Kathryn Neville, Elizabeth Arcand, Gabriela Chiaramida, Kaylie O'Connell, Marla Tipping. Breaking down cancer: IDH and its impact on metabolism and DNA damage and repair [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B018.
Abstract B031: Semaphorin 3E promotes pancreatic cancer metastasis through activating stromal cell
Authors: Liang Zhengdong , Yong Lin-Kin , Van Buren George , Fisher William , Hwang Rosa , Chen Changyi , Yao Qizhi Cathy
Published: August 2018

Background: Better understanding of the molecular mechanisms of pancreatic adenocarcinoma (PDAC) metastasis is important for the development of effective treatments for this devastating disease. We have previously shown that overexpression of Semaphorin 3E (Sema3E) enhances PDAC cell growth and is associated with poor patient survival. Here, we further determined the effect of Sema3E on stromal cells and the molecular mechanisms contributing to PDAC metastasis. Materials and Methods: Panels of stable PDAC cell lines were generated using either lenti-Sema3E for overexpression or the Crispr/Cas9 system to knock out (KO) Sema3E in both panc28 and panc48 cell lines. The effects of Sema3E overexpression or KO were measured in orthotopically implanted xenograft tumor mouse models in respect to tumor size, metastasis, and survival. After coculture with pancreatic stellate cells (PSCs), the effects of Sema3E on PSC activation were evaluated by GFAP and αSMA staining. Mixtures of PDAC cell lines with PSCs in vivo tumor growth were also evaluated. Result: After orthotopically implanting a panel of Sema3E-overexpressing or knockout panc28 cell lines, we found that the survival span of the sema3E-overexpression cell implant group was significantly shorter than the vector control group (31±9 days vs. 51±15 days) (p<0.05). In addition, more lung metastases were found in the Panc28-Sema3E overexpressing group. Compared to the primary tumor, the lung metastases had increased sema3E expression. Furthermore, Sema3E overexpression promoted PSC activation with a greater ratio of αSMA to GFAP staining when cocultured with PSC cells. Cotransplant of Sema3E-overexpression cells and PSC cells promoted tumor growth and metastasis when compare to Sema3E-overexpressed cells alone. Conclusion: Sema3E overexpression in pancreatic cancer cells promotes tumor growth, lung metastasis, and poor survival. The interaction between Sema3E and PSC cells may play an important role in contributing to tumor growth and metastasis.

Citation Format: Zhengdong Liang, Lin-Kin Yong, George Van Buren, William Fisher, Rosa Hwang, Changyi Chen, Qizhi Cathy Yao. Semaphorin 3E promotes pancreatic cancer metastasis through activating stromal cell [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B031.
Abstract B032: The study of the role of Romo1 for angiogenesis/lymphangiogenesis in colorectal cancer
Authors: Kim Hong Jun , Lee Dae-Hee , Jo Min Jee , Oh Sang Cheul
Published: August 2018

Reactive oxygen species modulator-1 (Romo1) is a novel protein that has been reported to be crucial for cancer cell proliferation and invasion. In a previous work, for the first time we reported that Romo1 correlated with poor survival of patients with colorectal cancer (CRC). In the current study, we investigated the role of Romo1 for angiogenesis/lymphangiogenesis in CRC. Romo1 overexpression in clinical tumor samples was associated with a high lymph node ratio between the metastatic and examined lymph nodes (p=0.025). IHC staining was used to demonstrate the Romo1 expression in clinical tissues. In the Matrigel invasion assay, cell invasiveness decreased in the Romo1 knockdown CRC cells compared with the controlled cells; this tendency was shown to be associated with epithelial-mesenchymal transition (EMT) induced by Romo1. We identified EMT markers at the protein levels in the Romo1-overexpressing cells. Epithelial marker E-cadherin was decreased while mesenchymal markers N-cadherin and Snail were increased in the Romo1-overexpressing cells compared with controlled cells. We also evaluated the mechanism by which Romo1 affects vascular endothelial growth factors (VEGF) transcription and promotes angiogenesis/lymphangiogenesis. The expression levels of VEGF were detected by real-time PCR and Western blot. We found that VEGF expression levels were upregulated in Romo1 overexpressed cells. Also in clinical tumor samples, Romo1 expression level determined by IHC staining was positively related with VEGF. Based on these results, we used Romo1 overexpressed cell’s conditional medium (CM) to culture the human umbilical vein endothelial cells (HUVEC), and detected the tube formation. We found that Romo1 overexpressing cell CM enhanced the tube formation compared with the control CM. In conclusion, lymphatic metastasis induced by Romo1 was a key reason for the poor survival associated with highly expressed Romo1 in CRC. We have shown that Romo1 not only promotes cancer invasion via EMT, but also has a direct role in lymphangiogenesis and/or angiogenesis.

Citation Format: Hong Jun Kim, Dae-Hee Lee, Min Jee Jo, Sang Cheul Oh. The study of the role of Romo1 for angiogenesis/lymphangiogenesis in colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B032.
Abstract B033: Transcriptional dissection and therapeutic modulation of metastasis development in pancreatic cancer
Authors: Lopez-Casas Pedro P. , Dimitrov Spas , Perales-Patón Javier , Baños Natalia , Muñoz Manuel , Dopazo Ana , Al-Shahrour Fátima , Hidalgo Manuel
Published: August 2018

Pancreatic cancer (PDAC) is a highly metastatic disease, and up to 70% of patients who are diagnosed with early-stage disease recur with metastatic disease despite treatment with chemotherapy. The development of metastasis remains the leading cause of cancer-related mortality in this disease, and little is known about the molecular mechanisms that drive the spreading of the primary tumor cells to the metastatic niche. Therefore, there is an urgent need to discover druggable antimetastatic targets. To generate a robust model of PDAC metastasis we implanted PDAC patient-derived samples orthotopically in NOD scid gamma (NSG) mice. We observed recurrent dissemination to distant organs, liver, and lung that resulted in animal death with a median of 50 days. We obtained human cancer single-cell suspensions at the primary location (graft), the mouse blood, and the metastatic lesions by using magnetic methods and an anti-HLA-ABC antibody, which recognized the human cells. Next, we captured single-cells by utilizing the Fluidigm technology and performed RNA-seq on each isolated cell. Using bioinformatic tools, we analyzed the transcriptional landscape of the cells isolated from the three compartments to search for potential druggable metastatic drivers. The data show that PDAC circulating tumor cells (CTCs) keep high tumorigenic and metastatic capability and that CTC-derived tumors preserve the morphologic characteristics of parental tumor. The metastatic disease of pancreatic cancer is a well-organized evolutionary process, in which the primary tumor cells, metastatic cells, and CTCs are transcriptionally divergent. The transcriptional heterogeneity between these three biological emerges from three biologic hallmarks including extracellular matrix interaction and focal adhesion, cell cycle, and ribosome biogenesis. The analysis of transcriptionally overexpressed genes identified BIRC5 (Survivin), a key regulator of mitosis and apoptosis, as one of the highest expressed genes in CTCs as well as the most promising target. Intriguingly, in vivo targeting of BIRC5 with YM155, a small molecule described as transcriptional repressor of BIRC5, aborted the metastatic process and significantly increased the survival of treated mice compared to control.

Citation Format: Pedro P. Lopez-Casas, Spas Dimitrov, Javier Perales-Patón, Natalia Baños, Manuel Muñoz, Ana Dopazo, Fátima Al-Shahrour, Manuel Hidalgo. Transcriptional dissection and therapeutic modulation of metastasis development in pancreatic cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B033.
Abstract B039: KDM4B inhibition confers suseptibility to PI3K inhibitor in PTEN-deficient TNBC through overactivating UPR pathway
Authors: Wang Wenyu , Oguz Gokce , Lee Puay Leng , Yatim Siti Maryam , Yu Qiang
Published: August 2018

Triple-negative breast cancer (TNBC) is among the most lethal cancers in females. Up to 35% of TNBC show PTEN mutation or PTEN loss (PTEN deficiency). Although PTEN deficiency leads to aberrant activation of PI3K/AKT signaling, therapeutic targeting of PI3K/AKT pathway has yielded very limited clinical benefit. The effective therapeutic targets for PTEN-deficient cancers are in urgent need of identifying common vulnerabilities of this important pathway. To identify additional chemical drugs that target PTEN-deficient TNBC, we performed a drug screening together with a series of in vitro and in vivo validation. Here, we show that PTEN deficiency confers vulnerability to inhibition of histone demethylase KDM4B, resulting from perturbation of endoplasmic reticulum (ER) homeostasis through activating eIF2α/ATF4 unfolded protein response (UPR) pathway. Mechanistically, KDM4B suppresses UPR pathway by interacting with and inhibiting eIF2α activation, and this function requires its demethylase activity but is independent of histone modification. Consistent with the sensitivity, PTEN-deficient cells and samples from PTEN-deficient patients show constitutively activation of UPR, which usually confers survival advantage but induces cell death once it is further activated and prolonged by KDM4B inhibition. More importantly, targeting KDM4B can robustly synergize with PI3K inhibitor to induce cell death in PTEN-deficient TNBC. This synergistic killing is caused by "overactivating" the UPR pathway resulting from transcriptional activation of UPR genes by ATF4 and FoxO1. Collectively, our study thus identifies a novel cytoplasmic function of KDM4B in regulating cellular stress and provides a new therapeutic approach to treat PTEN-deficient TNBC.

Citation Format: Wenyu Wang, Gokce Oguz, Puay Leng Lee, Siti Maryam Yatim, Qiang Yu. KDM4B inhibition confers suseptibility to PI3K inhibitor in PTEN-deficient TNBC through overactivating UPR pathway [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B039.
Abstract B040: MERTK activates Akt by antagonizing SAV1 binding
Authors: Liu Pengda , Jiang Yao
Published: August 2018

Hyperactivation of the PI3K-Akt pathway is observed in virtually all solid tumors. Multiple genetic events such as PTEN loss and PIK3CA amplification partially account for the increase in the pathway activation. In addition to genetic changes, dysregulation of post-translational modifications of Akt, including phosphorylation, acetylation, hydroxylation, methylation, and others that are necessary for Akt activation is also frequently observed in various types of cancers. However, whether and how Akt is regulated by its binding partners is minimally understood. To this end, through a proteomic approach, we have identified the tumor suppressor SAV1 as a novel Akt-interacting partner to suppress Akt activation. Furthermore, we identified MERTK as a major upstream kinase phosphorylating Akt1-Y26 to disrupt SAV1 binding and subsequently activate Akt. Mechanistically, MERTK-mediated Akt1-Y26 phosphorylation facilitates Akt plasma membrane recruitment by PI(3,4,5)P3 and promotes PDK1 binding to promote Akt activation. Importantly, cancer patient-derived SAV1 mutants and Akt1 mutants were identified to exert elevated oncogenicity by bypassing SAV1 binding and suppression, further supporting a pathophysiologic role of the identified MERTK/Akt/SAV1 signaling in tumorigenesis.

Citation Format: Pengda Liu, Yao Jiang. MERTK activates Akt by antagonizing SAV1 binding [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B040.
Abstract B042: Anticancer effects of processed garlic constituent diallyl trisulfide on head and neck squamous cell carcinoma
Authors: Mathan Sivapar , Singh Shivendra , Singh Rana P.
Published: August 2018

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Despite significant advances in therapeutic approaches, survival rate is less than 50%. Early detection and treatment will help in prevention of spreading of invasive cancer and thereby improve the survival rates. Studies showed that dietary intake of Allium vegetables lowered the risk of various cancers. Studies reported DATS as a promising chemopreventive constituent of processed garlic that induced cell cycle arrest and apoptosis in prostate and breast cancer. However, it has not been investigated for its efficacy and molecular mechanisms of chemopreventive effects of DATS against HNSCC. Herein, we investigated the effect of DATS on HNSCC FaDu, D562, UMSCC-22A, UMSCC-22B, and Cal33 cells in culture. DATS (10-100 µM) treatment significantly reduced the growth, proliferation, and survival of HNSCC cells. We observed that DATS treatments significantly induced G2/M cell cycle arrest. DATS treatment resulted in the accumulation of cyclin B1 and induced the levels of cip1/p21. DATS-induced mitotic arrest was attenuated by NAC pretreatment, which suggested that DATS mediates growth-suppressive effects by induction of ROS. DATS induced cell death through apoptosis, which was associated with ROS production. Further, DATS increased the ratio of Bax/Bcl2 and cleavage of caspase-3 and PARP and decreased the levels of XIAP. Exposure of HNSCC cells to pharmacologic concentrations of DATS (1-10 μM) resulted in inhibition of cancer stem cells (CSC) fraction as evidenced by CD133high/CD44high fraction of cells, aldehyde dehydrogenase 1 (ALDH1) activity, and spheroid formation. DATS-treated tumor xenograft also revealed its in vivo inhibitory effect on CSCs. We observed for the first time that DATS treatment decreased tumor incidence and inhibited tumor promotion and progression in 4-Nitroquinoline 1-oxide (4-NQO)-induced mouse oral carcinogenesis model. Taken together, DATS strongly inhibited cell proliferation, induced cell cycle arrest, cell death via apoptosis, mitochondrial dysfunction and ROS generation and reduced CSC fraction, and decreased tumor incidence, promotion, and progression in 4-NQO model.

Citation Format: Sivapar Mathan, Shivendra Singh, Rana P. Singh. Anticancer effects of processed garlic constituent diallyl trisulfide on head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B042.
Abstract B044: Novel compounds suppressing p53-dependent growth of tumor cells
Authors: Tatsuda Daisuke , Momose Isao , Watanabe Takumi , Kunisada Takao , Kawada Manabu , Shibasaki Masakatsu
Published: August 2018

Background: p53, a tumor suppressor gene, is required for cell cycle checkpoint signaling pathways and induction of apoptosis. In cell growth conditions, Mdm2, a ubiquitin E3 ligase, binds and ubiquitinates p53 and induces p53 degradation by 26S proteasomes. Suppression of p53 degradation, however, can activate p53 and induce cell cycle arrest and apoptosis. Because p53 regulates tumor cell survival, activation of p53 is an attractive target for antitumor drugs. We established a cell-based assay to screen natural products and searched for a new p53 activator. As a result of screening, we identified quinofuracins A-E. Materials and Methods: To search for a new activator of the p53-dependent signaling pathway, we screened microbial metabolites using human glioblastoma LNZTA3 cells in which p53 expression can be regulated by tetracycline. Human glioblastoma LNZTA3 cells (tetracycline regulated p53 expression) and human gastric cancer MKN45 cells (p53 wild-type) and MKN7 cells (p53 mutant-type) were incubated with the compounds for 3 days, and cell viability was measured by MTT assay. Results: As a result of screening, we identified quinofuracins A-E, novel anthraquinone derivatives containing ß-D-galactofuranose, from Staphylotrichumboninense PF1444. Quinofuracins induced p53-dependent cell death; upregulated the expression of p53, p21, and PUMA; and stimulated PARP degradation in p53-expressing LNZTA3 cells. Quinofuracins also induced p53-dependent cell death in colon and stomach cancer cells. Conclusions: These findings suggest that quinofuracins induce the apoptotic pathway in vitro by activating p53, and thus may suppress tumor growth in vivo.

Citation Format: Daisuke Tatsuda, Isao Momose, Takumi Watanabe, Takao Kunisada, Manabu Kawada, Masakatsu Shibasaki. Novel compounds suppressing p53-dependent growth of tumor cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B044.
Abstract B054: Impairing the reseeding of circulating tumor cells decelerates metastatic progression
Authors: Qian Chen , Worrede-Mahdi Asurayya , Shen Fei , Salvino Joseph , Meucci Olimpia , Fatatis Alessandro
Published: August 2018

This study aims to understand the mechanisms and dynamics by which cancer cells, departing from existing metastases as circulating cancer cells (CTCs), further seed and colonize skeleton and soft tissues, expanding metastatic spreading and precipitating the clinical progression to terminal disease. We have previously shown that the chemokine receptor CX3CR1 is implicated in the metastatic seeding of breast cancer cells and that FX-68, a novel small-molecule antagonist for this receptor, effectively contains both number of lesions and total tumor burden in animal models of metastatic disease. Here we established that FX-68 impairs the reseeding of skeleton and soft-tissue and that the CTCs unable to reseed and forced to remain in the blood eventually succumb to apoptotic death. The metastatic potential of CTCs was confirmed by reinoculation experiments as well as by forcefully mobilizing cancer cells from metastases back in the blood and assessing the number of additional lesions generated, in the presence and absence of FX-68. Finally, we hypothesized that prolonging the permanence of CTCs in the blood could improve their exposure to chemotherapy drugs. This paradigm was tested by combining FX-68 with doxorubicin or docetaxel and assessing drug bioavailability and antimetastatic effects. In summary, our results strongly support developing CX3CR1 antagonists and promoting their clinical use, as this approach will provide novel and effective tools to contain the progression of metastatic disease in patients.

Citation Format: Chen Qian, Asurayya Worrede-Mahdi, Fei Shen, Joseph Salvino, Olimpia Meucci, Alessandro Fatatis. Impairing the reseeding of circulating tumor cells decelerates metastatic progression [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B054.
Abstract B070: Deciphering the ubiquitin code with polyubiquitin chain selective affinity matrices
Authors: Singh Rajesh , Than Don Bosco , Julius Sarah , Lu Xiaolong , Foote Peter , Song Chengcheng
Published: August 2018

Post-translational modification of proteins by ubiquitin (Ub) is a versatile process, highly dynamic, and involved in nearly all aspects of biologic functions in eukaryotes. Dysregulation of ubiquitylation has been implicated in a plethora of pathologic conditions ranging from developmental abnormalities and autoimmune to neurodegenerative diseases and a variety of cancers. Ub is attached, via isopeptide bonds, to lysine residues in the target protein through a cascade of enzymatic reactions. These Ub-moieties can then serve as substrates for the conjugation of additional Ubs, forming poly-ubiquitin chains, again through the formation of isopeptide bonds between the C-terminus of one Ub and any of seven lysine, or N-terminal methionine in the target Ub. The reversibility, heterogeneity, and diversity of these modifications combined with the lack of suitable tools have made it difficult to properly isolate and characterize poly-ubiquitylated cellular proteins. This chain complexity known as the “Ub code” requires diverse technical approaches to unravel its complexity. Recent publications have demonstrated that antibodies against Ub display linkage bias or are of poor quality for experiments requiring precise measurements. However, in 2009 a novel technology called TUBEs (Tandem-repeated Ub-Binding Entities) was developed. TUBEs have revolutionized the Ub field by allowing poly-ubiquitylated proteins to be purified from cellular extracts. Development of TUBEs that selectively bind K48, K63 poly-ubiquitylated proteins has helped understand the role of modified proteins in cell physiology. In addition, derivatives of chain selective TUBEs have been used to develop novel assays for DUBs and ubiquitin ligases. This presentation will describe variants of TUBEs and their applications, as well as the new iterations of TUBEs aimed at identifying Ub-linkage specific functions and their biologic relevance.

Citation Format: Rajesh Singh, Don Bosco Than, Sarah Julius, Xiaolong Lu, Peter Foote, Chengcheng Song. Deciphering the ubiquitin code with polyubiquitin chain selective affinity matrices [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B070.
Abstract B073: PD-L1 expression in urothelial cancer: first results from RUSSCO-RSP assay comparision study
Authors: Zavalishina Larisa , Povilaitite Patritsiya , Andreeva Yulia , Petrov A. , Pugach Inna , Kharitonova Ekaterina , Rumyantsev Alexey , Raskin Grigory , Frank Georgy , Tjulandin Sergei , Tsimafeyeu Ilya
Published: August 2018

Background: To compare similarities and differences between PD-L1 IHC assays in patients with urothelial cancer (UC), collaborative Russian Society of Clinical Oncology (RUSSCO) and Russian Society of Pathology (RSP) study was conducted. The first results, presented here, describe a rate of PD-L1 IHC expression assessed by each test and evaluate concordance between 3 assays. Methods: 100 UC tumors were stained with PD-L1 IHC assays (clones SP142, 22C3 and SP263) as used in the clinical trials of second-line therapy of atezolizumab (Ventana SP142), pembrolizumab (Dako 22C3), and durvalumab (Ventana SP263). Four trained pathologists certified by Ventana/Roche and Dako/Agilent in interpreting respective assays independently evaluated the percentages of tumor (TC) and tumor-infiltrating immune cells (IC) staining positive at any intensity. Single clinical cut-off for inclusion in this comparative analysis was used: IC ≥5% for SP142, TC+IC/TC ≥10% for 22C3, and TC or IC ≥25% for SP263. Results: Patients were predominantly male (89%), 47/100 (47%) had grade 3 tumors, 45/100 (45%) and 45/100 (45%) had T1 and T2 stages, respectively. 300 IHC slides were scored. The percent of immune cell staining across 3 assays appears to be higher than for tumor cell staining (45% vs. 8% for SP142; 55% vs. 24% for 22C3; 72% vs. 27% for SP263). There were variabilities in immune cell staining for all assays and in tumor cell staining across SP142 and other assays. The percentage of PD-L1-positive patients was comparable with SP142 (9%), 22C3 (11%) and SP263 (7%). Table indicates the number of cases that were concordant with the index assay scoring algorithm, when an alternative cut-off was used to determine the allocation of cases to clinical groups above and below the cut point.

Table Assay clone used for slide stainingScoring AlgorithmSP142IC ≥5%22C3TC+IC / TC ≥10%SP263TC or IC ≥25%SP142100/100 (100%)92/100 (92%)95/100 (95%)22C395/100 (95%)100/100 (100%)95/100 (95%)SP26396/100 (96%)95/100 (95%)100/100 (100%)

Conclusions: First comparison showed that concordance was more than 90% between SP142, 22C3, and SP263 assays used for evaluation of PD-L1 expression in patients with urothelial carcinoma. Further analysis is required to draw a final conclusion.

Citation Format: Larisa Zavalishina, Patritsiya Povilaitite, Yulia Andreeva, A. Petrov, Inna Pugach, Ekaterina Kharitonova, Alexey Rumyantsev, Grigory Raskin, Georgy Frank, Sergei Tjulandin, Ilya Tsimafeyeu. PD-L1 expression in urothelial cancer: first results from RUSSCO-RSP assay comparision study [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B073.
Abstract B074: Ubiquitin proteomics: profiling the landscape of ubiquitin modification by ubisite-omics
Authors: Song Chengcheng , Akimov Vyacheslav , Foote Peter , Lu Xiaolong , Blagoev Blagoy , Singh Rajesh
Published: August 2018

The ubiquitin pathway has emerged as a promising druggable target for cancer. Ubiquitylation regulates various cellular functions including cell cycle, differentiation, apoptosis, and stress response. Ubiquitylation of proteins is encrypted bya variety of chain-selective K48, K63, K6, K11 poly-ubiquitins as well as mono-ubiquitin attached to target proteins. Proteomics provides an effective approach for decoding the complex ubiquitin signals. Ubiquitylation is a very dynamic process and ubiquitylated proteins are low in abundance and stoichiometry. Enrichment of ubiquitylated proteins by affinity metrics TUBE (Tandem Ubiquitin Binding Entities) is essential to improve quantitative, reproducible, and sensitive proteomics analysis. The most common approach is to use antibody that pulls down DiGlyGly-lysine (C-terminal di-Gly of ubiquitin attached to epsilon amino group of lysine), the so-called “ubiquitin remnant.” However, ubiquitylation from ubiquitin-like proteins (UBLs) such as SUMO, NEDD8, and ISG15 that are undistinguishable by DiGlyGly-lysine antibody makes it impossible to assign a role for ubiquitin or UBLs. Here we demonstrate a novel ubiquitylation-specific proteomics approach. This method utilizes UbiSite antibody that recognizes the ubiquitin C-terminus (13 a.a.), which creates the specificity for ubiquitin remnant. HepG2 and Jurkat cells were chosen as model systems. The alteration in cellular protein ubiquitylation were promoted by treatment with the inhibitor of a proteasome-associated DUB, USP14, a USP7 DUB inhibitor that is an immune oncology target or bortezomib, a 26S proteasome inhibitor. Cell lysates were digested with Lys C and immuneprecipitated with UbiSite antibody. Enriched ubiquitylated peptides were further digested with trypsin and analyzed by LC-MS/MS. The total proteome and ubiquitome were compared among treated and untreated samples. In addition, the ubiquitome of proteins, the site-specific ubiquitylation levels, were also compared with other published studies. We have identified a number of novel ubiquitylated proteins using the UbiSite proteomics approach. We believe this approach will facilitate discovery of pharmacologic markers for drugs, uncover new substrates for DUBs and ubiquitin ligases, and open doors for cell biologists to specifically identify genome-wide patterns of poly-ubiquitylated proteins.

Citation Format: Chengcheng Song, Vyacheslav Akimov, Peter Foote, Xiaolong Lu, Blagoy Blagoev, Rajesh Singh. Ubiquitin proteomics: profiling the landscape of ubiquitin modification by ubisite-omics [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B074.