bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2845: The mechanisms of resistance to temozolomide in glioma cells
Authors: Ohba Shigeo , Hirose Yuichi
Published: August 2018

Glioblastoma is one of the most aggressive tumor in the central nervous system tumors, with 5-year survival rates of less than 10%. The standard therapy for glioblastomas is maximal safe resection, followed by radiation therapy and chemotherapy with temozolomide (TMZ). One of the reasons of the worse prognosis of the tumor is the acquisition of resistance to TMZ in glioma cells. TMZ is a DNA-methylating agent. TMZ-induced O6-methylguanine adducts, in the absence of repair by O6-methylguanine DNA methyltransferase (MGMT), mispair with thymine during the next DNA replication cycle, leading to futile DNA mismatch repair (MMR) by the MMR system, formation of double strand breaks (DSBs) and eventual cell death of glioma cells. Several mechanisms have been proposed about the acquisition of resistance to TMZ in glioblastomas, such as changes of expression of MGMT, MMR, nucleotide excision repair and homologous recombination (HR). To clarify the mechanisms of resistance to TMZ and to find the way to overcome the resistance to TMZ, several clones of U251 cells resistant to TMZ were obtained and analyzed. FACS analyses showed several patterns of cell cycle distributions of the cells treated with TMZ. #3 resistant clone showed G2 arrest at day3 after TMZ exposure and this arrest was abrogated sooner compared to the G2 arrest of parental U251. TMZ did not induce G2 arrest in #8 resistant clone. Consistent with the results, TMZ-induced DSBs in #3 resistant cells were rapidly disappeared compared to those in U251 cells, and the DSBs were least induced by TMZ in #8 resistant cells. The expression of MGMT was not found in U251 parental cells, #3 cells nor #8 cells, which mean MGMT was not associated with the resistance to TMZ in those cells. The protein levels of MSH6, which was associated with MMR, was reduced in #8 cells. The inhibitor of PLK1 or PARP reduced the cell growth independent of MMR. The ability of HR was increased in #3 resistant clone. By suppression of Rad51, which was major factor associated with HR, the resistant #3 clone was re-sensitized to TMZ, however #8 was not. These results suggested that inhibition of HR may be a viable means to resensitize some kinds of temozolomide-resistant gliomas to TMZ.

Citation Format: Shigeo Ohba, Yuichi Hirose. The mechanisms of resistance to temozolomide in glioma 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 2845.
Abstract 2848: Radiographic and genomic evolution of individual metastases during HER2 blockade in colorectal cancer
Authors: Siravegna Giulia , Lazzari Luca , Sartore-Bianchi Andrea , Crisafulli Giovanni , Mussolin Benedetta , Cassingena Andrea , Martino Cosimo , Lanman Richard , Nagy Rebecca , Corti Giorgio , Bartolini Alice , Arcella Pamela , Montone Monica , Lodi Francesca , Vanzati Alice , Valtorta Emanuele , Cappello Giovanni , Bertotti Andrea , Lonardi Sara , Zagonel Vittorina , Leone Francesco , Russo Mariangela , Balsamo Antonella , Truini Mauro , Nicolantonio Federica Di , Amatu Alessio , Bonazzina Erica , Ghezzi Silvia , Regge Daniele , Vanzulli Angelo , Trusolino Livio , Siena Salvatore , Marsoni Silvia , Bardelli Alberto
Published: August 2018

Targeting HER2 with trastuzumab and lapatinib is effective in ERBB2 amplified metastatic colorectal cancer (mCRC). Although at least 30% of the patients initially respond, secondary resistance occurs in most of the cases.

Since the drivers of secondary resistance to trastuzumab and lapatinib in ERBB2 amplified mCRC are unknown, we exploited longitudinal plasma collections and patient-derived cell models to define the molecular bases of resistance to HER2 blockade. Levels of ERBB2 amplification in plasma circulating tumor DNA (ctDNA) paralleled response and relapse. The emergence of EGFR, ERBB2, RAS, BRAF and PIK3CA variants in ctDNA was associated with resistance. Radiographic measurements of individual metastases coupled with longitudinal liquid biopsies unveiled lesion-specific patterns of heterogeneous response in several patients. Phylogenetic tracking and functional analyses on tissue samples and patient-derived cell models established from eight metastases of a single case revealed new druggable oncogenic dependencies and genomic evolution associated with resistance. These data highlight the relevance of coupling imaging and liquid biopsies analyses in precision oncology and provide the rationale for additional lines of therapies in HER2 positive mCRC relapsing upon HER2 blockade.

Citation Format: Giulia Siravegna, Luca Lazzari, Andrea Sartore-Bianchi, Giovanni Crisafulli, Benedetta Mussolin, Andrea Cassingena, Cosimo Martino, Richard Lanman, Rebecca Nagy, Giorgio Corti, Alice Bartolini, Pamela Arcella, Monica Montone, Francesca Lodi, Alice Vanzati, Emanuele Valtorta, Giovanni Cappello, Andrea Bertotti, Sara Lonardi, Vittorina Zagonel, Francesco Leone, Mariangela Russo, Antonella Balsamo, Mauro Truini, Federica Di Nicolantonio, Alessio Amatu, Erica Bonazzina, Silvia Ghezzi, Daniele Regge, Angelo Vanzulli, Livio Trusolino, Salvatore Siena, Silvia Marsoni, Alberto Bardelli. Radiographic and genomic evolution of individual metastases during HER2 blockade in colorectal 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 2848.
Abstract 2849: Biodynamic imaging predicts response of breast cancer patients to neoadjuvant chemotherapy
Authors: Turek John J. , Nolte David D. , An Ran
Published: August 2018

The goal of this research was to determine if biodynamic imaging (BDI) and frozen breast cancer tumors from tissue banks could be used to retrospectively assess response to chemotherapy and identify the more chemoresistant patients. Neoadjuvant chemotherapy (NAC) is a common protocol for breast cancer patients prior to surgery. Many patients will respond to initial chemotherapy, but there is a significant percentage of patients that do not receive a benefit from NAC due to chemoresistance. Accurate prediction of chemoresistance prior to therapy, could enhance the effectiveness of NAC by allowing for better drug selection. BDI uses Doppler detection of light scattered by intracellular motion in living tissue, and several xenograft, and clinical studies have shown that this phenotypic profiling method is a highly accurate predictor of resistance to chemotherapy. Frozen, clinically annotated breast cancer samples were obtained from several tissue banks. All of the patients received adriamycin-cyclophosphamide and paclitaxel (AC-T) neoadjuvant chemotherapy prior to surgery. The samples were rapidly thawed and 1mm3 pieces were placed in tissue culture medium in a 96 well plate, sealed with a gas permeable membrane, and 6 replicates for each drug treatment assayed over an 18 hour period. Drugs tested were the AC combination, and also the individual drugs (adriamycin, 4-hydroxycyclophosphamide, and paclitaxel). A negative drug vehicle control was 0.1% DMSO in growth medium, and vincristine was used as a positive control. For the analysis, patients were classified as sensitive or resistant based upon the clinically annotated pathology reports. The BDI spectral data for each drug treatment was analyzed using a machine learning algorithm to identify patterns in the frequency vector biomarkers for each patient using a combination of a similarity matrix and a linear separability analysis using a neural net perceptron. The neural net perceptron and hold-out cross validation of BDI spectral data correctly identified all patients as sensitive or resistant. Frozen samples from tissue banks contain enough viable cells to evaluate drug response using BDI. This makes it possible to conduct retrospective studies on chemoresistance using frozen banked tissue samples and also makes it possible to study the relationship between genotype to chemo-phenotype. Supported by Animated Dynamics, Inc.

Citation Format: John J. Turek, David D. Nolte, Ran An. Biodynamic imaging predicts response of breast cancer patients to neoadjuvant chemotherapy [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 2849.
Abstract 2851: The evolution of tumor cells under AsiDNA treatment results in “autosensitization”
Authors: Kozlac Maria , Jdey Wael , Girard Pierre-Marie , Bono Françoise , Dutreix Marie
Published: August 2018

Purpose: The Achilles' heel of all conventional and targeted anticancer treatments is intrinsic or acquired resistance. The era of precision medicine where tumors are selected for specific treatments based on their genetic alterations is revolutionary, but unfortunately prolonged responses are rarely observed due to rapid emergence of resistant clones. We recently developed a new concept of DNA repair inhibitor (Dbait) acting by activating enzymes involved in DNA damage signaling. We tested how such agonist activity would be prone to induce resistance to Dbait treatment.

Experimental design: We performed repeated cycles of treatment with various targeted therapy agents (Imatinib, Olaparib, 6-thioguanine and the clinical form of Dbait, AsiDNATM). We analyzed the specific sensitivity of independent cultures after each treatment cycle. The study was performed in different tumor cell lines ((MDAMB231, MDAMB468, HCC1143, THP1, U937, KBM7) and in two non-malignant breast cell lines (MCF10A, MCF12A). Extensive transcriptome and genome studies were performed on MDAMB231 breast cancer cell lines.

Results: Resistant clones appeared at a frequency of 1.45% for Olaparib, 0.62% for imatinib, and 1.66% for 6-thioguanine. Similar protocol did not select for resistance to AsiDNA. Unexpectendly, tumor cells became more sensitive to AsiDNA after each cycle of treatment. This behavior was specific of AsiDNA and was not observed with other treatments. The six tumor cell lines tested developed AsiDNA autosensitisation and no resistance. Non tumoral cells were not affected by repeated treatments. The acquired sensitivity of the treated tumor populations was conserved for months after end of treatment Transcriptional and genetic analysis of independently treated MDAMB-231 populations reveals that all evolved similarly. They display a few conserved genome modifications and a large deregulation of 1160 genes resulting in an overexpression of luminal associated genes and a decrease in mesenchymal associated genes.

Conclusion: Our results indicate a phenomenon of “autosensitisation”, along treatment which has never been described for anticancer treatment and could prevent development of resistance during treatment.

Citation Format: Maria Kozlac, Wael Jdey, Pierre-Marie Girard, Françoise Bono, Marie Dutreix. The evolution of tumor cells under AsiDNA treatment results in “autosensitization” [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 2851.
Abstract 288: Genomic profiling of metastatic gastric adenocarcinoma
Authors: Harada Kazuto , Song Shumei , Xu Yan , Bagewell Brain D. , Pizzi Melissa P. , Jin Jiankang , Wang Ying , Scott Ailing W. , Ma Lang , Amlashi Fatemeh G. , Kobayashi Makoto , Vykoukal Jody V. , Ivan Cristina , Estrella Jeannelyn S. , Chowdhuri Sinchita R. , Calin George A. , Hanash Samir , Lee Ju-Seog , Liu Bin , Ajani Jaffer A.
Published: August 2018

Objectives: Metastatic gastric adenocarcinoma (mGAC) is highly resistant to therapy and is incurable. Particularly, peritoneal carcinomatosis leads to severe morbidities and short survival. The genome wide approaches have been conducted in primary GAC and have uncovered useful information. However, detailed understanding of mGAC is limited. Thus, we aimed to characterize the genomic landscape of mGAC.

Methods: Thirty-four malignant ascites specimens were prospectively collected from at The University of Texas MD Anderson Cancer Center (Houston, USA) from February 2016. 25 specimens had whole exome sequencing and 30 specimens had RNA sequencing.

Results: The top highly mutated genes in mGAC were TP53, ATM, SYNE2, TTN, CDH11, FAT4/3, KMT2C, FGF23, NCOR1, RP1, CYP2A, and ADAMTS8/9. Compared with mutations in primary GAC (TCGA and TMUCIH), mutations in mGAC shared only 13.7% with TCGA and 10.9% with TMUCIH data. The commonly shared mutations in all three sets were TP53, ATM, FAT4, CDH1, SYNE1, PLEC, PRKDC, and ZFHX3, while unique mutations in mGAC were TTN, SYNE2, ZNF503, PUS7L, PPP2R5C, NBPF12, MROH1, IFNL1, and COL18A1. Furthermore, RNA- sequencing revealed two frequent fusions; 36.7% of samples with C15orf57-CBX3 (t(15;7)) fusion and 10% with SLC35A1-TSHZ2 (t(6;20)). We explored gene signatures in C15orf57-CBX3 fusion subgroup and C15orf57-CBX3 non-fusion subgroup. Gene set enrichment analysis revealed that C15orf57-CBX3 fusion group had enrichment of immune-response related genes, while non-fusion subgroup had enrichment of genes related to the segregation of chromatid, DNA replication, and histone exchange. The clustering analysis of RNA expression using the top 2000 genes with most median absolute deviation identified two subgroups that correlated either with C15orf57-CBX3 fusion or mutations.

Conclusions: We have identified a novel genomic landscape of mGAC, such as unique mutation and fusions. Further studies are needed to gain insights into the mechanism of progression and resistance phenotype of mGAC.

Citation Format: Kazuto Harada, Shumei Song, Yan Xu, Brain D. Bagewell, Melissa P. Pizzi, Jiankang Jin, Ying Wang, Ailing W. Scott, Lang Ma, Fatemeh G. Amlashi, Makoto Kobayashi, Jody V. Vykoukal, Cristina Ivan, Jeannelyn S. Estrella, Sinchita R. Chowdhuri, George A. Calin, Samir Hanash, Ju-Seog Lee, Bin Liu, Jaffer A. Ajani. Genomic profiling of metastatic gastric adenocarcinoma [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 288.
Abstract 2883: Repurposing atovaquone, an antiprotozoal drug for management of breast cancer by inhibiting HER2/β-catenin signaling
Authors: Gupta Nehal , Srivastava Sanjay
Published: August 2018

Breast cancer is the second highest cause of cancer-related mortality in women. HER2 is an oncogene overexpressed in about 30% of breast cancer patients and β-catenin, a proto-oncogene plays a pivotal role in breast cancer metastasis and drug resistance leading to poor prognosis. In the current study, we evaluated the anti-cancer effects of an anti-protozoal drug, atovaquone against several breast cancer cell lines such as MCF-7, 4T1, HCC1806, CI66, SKBR3 and T47D. Our results showed that atovaquone treatment induced apoptosis as evaluated by PI/Annexin assay and cleavage of caspase-3 and PARP, and inhibited the growth of all the breast cancer cell lines tested. Similar observations were made in patient-derived cancer cell lines. In addition, atovaquone treatment significantly reduced the expression of HER2, β-catenin and its downstream molecules such as pGSK-3β, TCF-4, cyclin D1 and c-Myc in these cell lines. Treatment of HCC1806 cells with 5 µM atovaquone resulted in significant reduction in mammosphere forming efficiency (MFE) of breast cancer cells. Mammosphere-forming cells are known to exhibit cancer stem cell (CSC)-like properties together with drug resistance. Efficacy of atovaquone was evaluated in the in-vivo tumor model by orthotopic implantation of two highly aggressive 4T1 and CI66, triple negative breast cancer cells in the mammary fats of female mice. Our results demonstrate that oral administration of atovaquone suppressed the growth of CI66 and 4T1 tumors by 70% and 60% respectively. Atovaquone treated tumors exhibited reduced HER2, β-catenin and increased apoptosis when analyzed by western blot. We also developed resistance in 4T1 breast cancer cells towards paclitaxel, a first line chemotherapeutic agent for treatment of metastatic breast cancer. 4T1 paclitaxel resistant cells were orthotopically implanted in female balb/c mice and mice were treated with atovaquone for 20 days. Our results showed that atovaquone was able to suppress the growth of paclitaxel resistant tumors by 42%. Conclusively, our results indicate that atovaquone effectively reduces the growth of primary breast tumors as well as paclitaxel resistant tumors by inhibiting HER2/β-catenin signaling. Most importantly, atovaquone is already in clinical use with an established safety record therefore, any positive findings from our studies will prompt further clinical investigation into repurposing atovaquone for the treatment of advanced breast cancer patients. [Supported in part by R01 grant CA 129038 awarded by National Cancer Institute, NIH].

Citation Format: Nehal Gupta, Sanjay Srivastava. Repurposing atovaquone, an antiprotozoal drug for management of breast cancer by inhibiting HER2/β-catenin signaling [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 2883.
Abstract 2890: Trifluridine efficacy in cancer stem cell
Authors: Tsunekuni Kenta , Konno Masamitsu , Koseki Jun , Asai Ayumu , Doki Yuichiro , Mori Masaki , Ishii Hideshi
Published: August 2018

Background: Cancer stem cells (CSC) have been involved in disease recurrence, metastasis, and therapeutic resistance, but effective anti-cancer agent for these cells is not still currently available. Trifluridine (FTD) is a key component of the novel oral antitumor drug trifluridine/tipiracil, which was approved for the treatment of patients with metastatic colorectal cancer who have been previously treated with fluoropyrimidine, oxaliplatin and irinotecan based chemotherapy etc. and improved overall survival. Here we showed the efficacy of FTD in CD44 and CD133 high expressing (CD44high/CD133high) cancer cells which has CSC like property.

Method: CD44high /CD133high and CD44low /CD133low DLD-1 cells were separated by Fluorescence-Activated Cell Sorting (FACS) and subjected to sphere formation assay. Anti-proliferative and anti-sphere forming effects of FTD in CD44high /CD133high cells were measured. Then, 150 days FTD long exposed colorectal cell line's CSC markers CD44 and CD133 were measured by flow cytometry and its tumor initiating property in in vitro and in vivo were evaluated.

Results: CD44 high /CD133 high DLD-1 cells formed much more sphere than CD44low /CD133low cells significantly. Compared to unsorted cells, CD44high /CD133high cells displayed 5-FU resistance (IC50 value of 5-FU for unsorted and CD44 high /CD133 high cells were 2.5 μM and 5.5 μM respectively) but not FTD resistance (IC50 value of FTD for unsorted and CD44 high /CD133 high cells were 8.9 μM and 10.7 μM, respectively) in in vitro proliferation assay. In addition, FTD treatment in a subtoxic concentration 1 μM for DLD-1 cells disrupted sphere formation. FTD-long exposed DLD-1 cell lines showed decreased CD44high /CD133high population compared to its parental cell line from 11.3% to 0.7%, and showed decreased sphere-forming efficacy. In addition, FTD-long exposed DLD-1 cell lines decreased tumor forming potential in in vivo. These results suggest that FTD is effective for CSC like cells and that long exposure to FTD might lead CSC depletion.

Conclusion: FTD was proved to be effective for CSC like CD44high /CD133high cells. It suggests that FTD might be useful for CSC targeted chemotherapy in case of CD44 and CD133 are highly expressed subtype of tumors.

Citation Format: Kenta Tsunekuni, Masamitsu Konno, Jun Koseki, Ayumu Asai, Yuichiro Doki, Masaki Mori, Hideshi Ishii. Trifluridine efficacy in cancer stem cell [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 2890.
Abstract 2893: Balancing the efficacy and toxicity of anti-CD47 antibodies by direct screening in humanized mouse models
Authors: Guo Yanan , Guo Chaoshe , Shen Yuelei , Yang Benny
Published: August 2018

CD47 is a transmembrane protein found on the surface of many cells in the body. Its expression is often up-regulated in many cancer cells. The receptor of CD47 was identified as SIRPa, which is expressed on phagocytic cells. Engagement of SIRPa by CD47 serves as “do not eat me” signal, thus inhibiting the phagocytic activity of macrophages. Cancer cells often hijack this pathway by boosting CD47 expression on their surface, in order to prevent innate cell-mediated phagocytosis. Molecules blocking the CD47-SIRPa interaction will unleash such inhibition and promote tumor destruction. Accordingly, anti-CD47 antibodies offer new hope to successful cancer treatment. Despite people are enthusiastic about CD47 as a potential target, the side effects associated with CD47 blockade have emerged as a major concern. For example, treatment with anti-CD47 antibody greatly reduced the number of circulating red blood cells and platelets that also express CD47. Thus, the class of CD47 antibodies that stimulate tumor cell killing while sparing normal cells in vivois desirable for the cancer patients. We are interested in understanding whether humanized mouse models could aid the evaluation of a variety of monoclonal CD47 antibodies we generated. We first screened them using the CDX platformestablished at Biocytogen. Indeed, most antibodies were able to clear human tumor cells in B-NDG mice. However, when we screened the toxicity of these antibodies using humanized CD47 mice (B-hCD47), many antibodies caused rapid death of animals upon the first administration. Interestingly, we found two clonal antibodies bearing the least toxicity since the treated animals exhibited minimal weight loss. Thus, we concluded that humanized mice could expedite the development of safer CD47 antibodies that can be advanced to human clinical trials.

Citation Format: Yanan Guo, Chaoshe Guo, Yuelei Shen, Benny Yang. Balancing the efficacy and toxicity of anti-CD47 antibodies by direct screening in humanized mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2893.
Abstract 2902: Targeting the tight junction protein claudin-1 in colorectal cancer
Authors: Cherradi Sara , Ayrolles-Torro Adeline , Vezzo-Vié Nadia , Combes Eve , Canterel-Thouennon Lucile , Gava Fabien , Lobjois Valérie , Ducommun Bernard , Gongora Celine , Rio Maguy Del
Published: August 2018

Colorectal cancer (CRC) is one of the major causes of cancer-related deaths in the Western world. When localized, CRC is often curable by surgery, but the prognosis of patients with metastatic disease remains very poor. The current treatment of metastatic CRC (mCRC) relies on therapy combining chemotherapy and targeted therapies. However, relapses are observed in most cases due to the occurrence of drug resistance. Therefore, more therapeutic options are required particularly by identifying new molecular targets that can be reached by antibodies.

We previously showed that Claudin-1 (CLDN1), a major constituent of tight junctions, is overexpressed at the membrane of CRC cells which makes it a good target for antibodies. Besides, CLDN1 is differentially expressed in the new CRC molecular subtypes. We have then developed a monoclonal antibody (mAb) against CLDN1, called 6F6, which targets the extracellular part of CLDN1. Results showed that the 6F6 mAb significantly decreased cell growth, survival, and migration in vitro as well as in vivo. Furthermore, we demonstrated the cytostatic effect of the 6F6 mAb through proliferation assays.

We are now deciphering the role of CLDN1 on 3D spheroids, a model that reflects the physiopathology. Using immunofluorescence staining on frozen sections we are exploring the impact of CLDN1 on spheroids proliferation. We are also investigating on the distribution of tight junction proteins implicated on cell proliferation in the presence or absence of CLDN1.

CLDN1 is a new promising therapeutic target in CRC, it is now interesting to elucidate its role in colorectal cancer cell proliferation.

Keywords: Colorectal, Cancer, Claudin-1, monoclonal antibodies, spheroids, proliferation

Citation Format: Sara Cherradi, Adeline Ayrolles-Torro, Nadia Vezzo-Vié, Eve Combes, Lucile Canterel-Thouennon, Fabien Gava, Valérie Lobjois, Bernard Ducommun, Celine Gongora, Maguy Del Rio. Targeting the tight junction protein claudin-1 in colorectal 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 2902.
Abstract 2904: Awakening metastatic breast cancer cells to chemotherapy
Authors: Lourenco Ana Rita , Lopez Jay , Crowley Michael , Mittal Vivek , Gao Dingcheng
Published: August 2018

Breast cancer is the most common cancer in women with more than 316,000 new cases and expected 40,610 deaths in the US in 2017. Despite vast improvement in cancer management and treatment in the past years, not all subtypes of breast cancer have benefited equally. This includes the most aggressive form of invasive breast cancer, triple negative breast cancer (TNBC) subtype (ER-, PR- and HER2-), which accounts for about 20-25% of all breast cancer cases and presents a higher breast cancer-related mortality compared to other breast cancer subtypes. The poor survival rates attributed to TNBC is due to the lack of available biological targets and high rates of metastatic recurrence. Therefore, chemotherapy is the only therapeutic option for patients with metastatic TNBC. However, development of chemotherapy resistance is observed in the great majority of the cases and patients relapse within 5 years of a diagnosis. Given the unmet clinical needs, the discovery of novel therapeutic options that overcome chemoresistance of metastatic breast cancer is required. We have identified a group of genes which expression was enriched in chemotherapy resistant tumors cells, suggesting that these are potential therapeutic candidates to overcome chemotherapy resistance. Of these, CDKN1A (p21) was included. Furthermore, Immunostaining of metastatic lung tissue confirmed that surviving tumor cells under CTX treatment highly express p21. The enrichment of p21 in surviving tumor cells under chemotherapy, together with its well documented functions in cell-cycle arrest and regulation of apoptosis, provides a rationale to explore the underlying mechanisms that govern p21-induced chemoresistance and whether targeting p21 will re-sensitize the chemoresistant tumor cells in vivo.

Citation Format: Ana Rita Lourenco, Jay Lopez, Michael Crowley, Vivek Mittal, Dingcheng Gao. Awakening metastatic breast cancer cells to chemotherapy [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 2904.
Abstract 2907: Targeting the epigenetic regulator KAT2a in cancer
Authors: Gunnell Andrea , Downs Jessica , Pennicott Lewis , Osborn Kay , Grand Darren Le , Duffell Katie , Patel Hitesh , Hudson Jessica , Booth Jessica R. , Ward Simon
Published: August 2018

Gene expression frequently requires the dynamic remodelling of the chromatin architecture to orchestrate the assembly of transcriptional protein complexes at the promoter sites of DNA. Chromatin remodelling complexes are multicomponent proteins that facilitate reorganisation of chromatin structure using the energy derived from ATP hydrolysis, and as a result play a pivotal role in controlling gene expression. Mammalian SWI/SNF complexes are examples of chromatin remodelling complexes with multiple roles in maintaining genome stability. It is estimated that 19.6% of all human tumours have mutations in SWI/SNF complexes, leading to the deregulation of gene expression as an early driver of carcinogenesis. Our interest in developing novel treatments for renal cancer focused on the role of the chromatin remodelling complex, PBAF. The BAF180 component of PBAF is mutated in 40 % of clear cell renal carcinomas and its loss is an early event in renal cell tumorigenesis. We performed an siRNA based high throughput screen to identify genes associated with genome stability that were synthetically lethal with the loss of BAF180 and identified KAT2a as a potential druggable target. KAT2a is a protein that also functions in the regulation of gene expression as a component of the SAGA histone modifying complex. It is a multifunctional protein that can both ‘read' and ‘write' acetylation marks on histone proteins through its bromodomain and a histone acetyltransferase domain respectively. We will describe our efforts to establish suitable assays to support the discovery of selective inhibitors of the bromodomain of KAT2a. The work will include the establishment of a high throughput assay to measure compound binding, crystallographic characterisation of small molecule inhibitors of the bromodomain, and our initial studies towards the target validation of the bromodomain to recapitulate the synthetic lethality observed with depletion of BAF180.

Citation Format: Andrea Gunnell, Jessica Downs, Lewis Pennicott, Kay Osborn, Darren Le Grand, Katie Duffell, Hitesh Patel, Jessica Hudson, Jessica R. Booth, Simon Ward. Targeting the epigenetic regulator KAT2a in 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 2907.
Abstract 2912: CRISPR-Cas9: A tool for rapid target discovery and validation
Authors: Wiggins Ceri M. , Walter David , Russell Paul , Sheridan Clare , Hudson Chantelle , Yarker Joanne , Sage Carlos le , McCarthy Nicola , Moore Jonathan D.
Published: August 2018

Genetic perturbations mediated by CRISPR-Cas9 are transforming the drug discovery process. In the hunt for novel drug targets, researchers finally have a screening tool with sufficient penetrance and power to identify new interactions, followed by a rapid gene editing technology to streamline validation approaches. At Horizon Discovery, we have now initiated more than 250 CRISPR-Cas9 knockout (KO) screens, including a significant effort to identify novel synthetic lethal interactions in colon and lung cancer genotypes. These screens have identified a reassuring number of known essential interactions, such as MDM2 and PPMID in TP53 wild type cancer cell lines. These results provide confidence in the ability of these screens to also discover novel targets that should make it through the validation process. Indeed, we have discovered novel targets for selective ablation of TP53 mutant colon cancers, several of which are strongly supported by the literature. In FBXW7 mutant colon cancers, CRISPR-Cas9 KO screens identified a set of potential novel druggable targets in addition to MCL1 - a known substrate of FBXW7, which showed a strong dependency in FBXW7 mutant lines. An important step in triaging primary hits ahead of extensive validation work has been secondary screens that target functional domains with high density sgRNA tiling. They allow more targets to be examined than would be feasible by any arrayed approach. Data from such screens, which test tens to hundreds of guides in unison, not only increases hit calling power, but can also yield information regarding important domains and mechanism of action. This was evident in the FBXW7 secondary screen data, where guides targeting the key functional BH domains of MCL1 proved to be the most effective. This information can be used to prioritise individual targets for further validation, using techniques that include an inducible CRISPR-Cas9 system to assess the effect of individual guides in an arrayed format on cell proliferation and survival, and CRISPRi. These data exemplify how CRISPR-Cas9 can enable the discovery of a new, more robust generation of drug and antibody targets.

Citation Format: Ceri M. Wiggins, David Walter, Paul Russell, Clare Sheridan, Chantelle Hudson, Joanne Yarker, Carlos le Sage, Nicola McCarthy, Jonathan D. Moore. CRISPR-Cas9: A tool for rapid target discovery and validation [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 2912.
Abstract 2924: Preclinical analysis of synergistic antitumor effect of eribulin and HDAC inhibitor for triple-negative breast cancer cells
Authors: Oba Takaaki , Izumi Hiroto , Ito Ken-ichi
Published: August 2018

Eribulin (ERI), an inhibitor of microtubule dynamics, is a key drug for metastatic breast cancer. While HDAC inhibitors have been shown to presents antitumor effect for various malignancies. Among HDAC family, HDAC6 is known as a deacetylase of tubulin. To develop a novel therapeutic strategy for triple negative breast cancer (TNBC), we tested the combinational effect of ERI and HDAC inhibitor for TNBC cells in vitro. Two TNBC cell lines (MDA-MB-231, Hs578T) and their ERI-resistant cells (MDA-MB-231/E, Hs578T/E) established in our laboratory were used in the study. Vorinostat (VOR), a pan-HDAC inhibitor, and ricolinostat (RICO), a selective HDAC6 inhibitor were used. The sensitivity to VOR or RICO was not different between the parental and the ERI-resistant cells. In the protein expression analysis, HDAC6 expression was not altered between parental and ERI-resistant cells. Prior treatment with VOR or RICO increased the expression of acetylatedα-tubulin in both parental and ERI-resistant TNBC cell lines. When low concentration of VOR (0.5 μM) or RICO (0.5 μM) was added simultaneously with ERI, the sensitivity to ERI was enhanced significantly in both parental MDA-MB-231 and Hs578T cell lines. Furthermore, when both parental cell lines were treated with VOR (5 μM) or RICO (5 μM) for 48 hrs prior to addition of ERI, the sensitivity to ERI was significantly enhanced. The enhancement of sensitivity to ERI both by the simultaneous addition with low concentration of VOR or RICO and by the prior treatment with VOR or RICO was observed in the ERI-resistant cells as well. Moreover, inhibition of HDAC6 by small interfering RNA partially enhanced the sensitivity to ERI in the both parental cell lines. Our study demonstrated the possibility that the combination of ERI with HDAC inhibitor might enhance the antitumor effect of ERI and overcome ERI resistance through acetylation of α-tubulin by HDAC6 inhibition in TNBC cells.

Citation Format: Takaaki Oba, Hiroto Izumi, Ken-ichi Ito. Preclinical analysis of synergistic antitumor effect of eribulin and HDAC inhibitor for triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2924.
Abstract 2926: The glycogen synthase kinase-3 inhibitor, 9-ING-41, synergizes with chemotherapy to inhibit pancreatic tumor growth in vivo
Authors: Ding Li , Zhang Jin-san , Dube John R. , Madamsetty Vijay S. , Schmitt Daniel M. , Mukhopadhyay Debabrata , Billadeau Daniel D.
Published: August 2018

Glycogen synthase kinase-3 (GSK3) is a ubiquitously expressed serine-threonine protein kinase involved in multiple cellular functions ranging from the control of glycogen metabolism to transcriptional regulation. We have previously demonstrated that GSK-3β is overexpressed in human pancreatic ductal adenocarcinoma (PDAC) and aberrant nuclear accumulation of GSK-3β serves as a hallmark of poorly differentiated PDAC. Moreover, genetic ablation of GSK-3β or GSK-3 inhibition led to PDAC tumor cell killing, identifying GSK-3β as a therapeutic target in PDAC. Herein, we describe a novel GSK-3 inhibitor, 9-ING-41. The effect of 9-ING-41 on tumor cell growth was tested using several PDAC and patient-derived xenograft (PDX) cell lines in vitro and in vivo using an orthotopic pancreatic cancer animal model. Interestingly, 9-ING-41 treatment resulted in the stabilization of cyclin B levels as well as enhanced serine-10 phosphorylated histone H3 (pS10HH3) and consequently M phase arrest and blocked mitotic exit. The prolonged M phase arrest induced mitotic catastrophe in pancreatic cancer cell lines and inhibited in vitro colony formation. An in vivo orthotopic pancreatic cancer animal model study using PDX-derived cell lines demonstrated that 9-ING-41 hindered tumor growth in combination with several standard chemotherapeutic agents. Significantly, we found that 9-ING-41 treatment impaired the DNA damage response pathway activated by Gemcitabine in pancreatic cancer cell lines. In conclusion, our study indicates that GSK-3 plays an important role in pancreatic cancer progression and 9-ING-41 in combination with existing gemcitabine or irinotecan could be a new strategy for treating patients with PDAC.

Citation Format: Li Ding, Jin-san Zhang, John R. Dube, Vijay S. Madamsetty, Daniel M. Schmitt, Debabrata Mukhopadhyay, Daniel D. Billadeau. The glycogen synthase kinase-3 inhibitor, 9-ING-41, synergizes with chemotherapy to inhibit pancreatic tumor growth in vivo [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 2926.
Abstract 293: Heterogeneous nucleocytoplasmic regulation of an incompletely penetrant ErbB2 onco-phenotype
Authors: Wang Lixin , Janes Kevin A.
Published: August 2018

Perturbation of cancer cells often leads to heterogeneous outcomes, in that most cells exhibit a dominant phenotype, but the rest appear resistant or hypersensitive to the perturbation. If the penetrance of such a phenotype is heritably incomplete, then it becomes extremely difficult to decipher the upstream molecular events that heterogenize the population and cause response variability. By combining quantitative measurements with dynamical models, systems approaches should be useful if provided with a core network of important biomolecules. The daunting hurdle lies in identifying phenotype-relevant regulatory heterogeneities that define the network for penetrance at the single-cell level. Here, we exploit a new approach, called stochastic frequency matching (SFM), which elaborates the molecular networks upstream of incompletely penetrant phenotypes. SFM identifies and parameterizes single-cell heterogeneities—which emerge after a uniform perturbation but before the appearance of a variable phenotype—to hone in on regulatory states corresponding to future penetrance. For a multi-acinar onco-phenotype incompletely triggered by ErbB receptor tyrosine kinase signaling in 3D cultured breast epithelia, we implemented SFM using microarrays to uncover a network of critical nucleocytoplasmic regulators, including the inner-ring nucleoporin NUP37 and the exportin CSE1L. Gain- and loss-of-function perturbations of NUP37 and CSE1L alter the frequency of ErbB2-triggered multiacinus formation, supporting that SFM can identify mechanisms of incompletely penetrant phenotypes. In addition, population-level transcriptomics revealed other ErbB2-induced changes in nucleocytoplasmic shuttling proteins: KPNA2, RANBP1, and XPO1. The net result of these alterations on shuttling is complicated, and the specific cargo relocalized by ErbB2 signaling is not known. We hypothesize that ErbB signaling heterogeneously reconfigures the nucleocytoplasmic shuttling state of cells to determine incomplete penetrance of the onco-phenotype. Using proximity labeling, we seek to identify novel ErbB2-induced NUP37 and CSE1L interactors that may serve as effectors for the multiacinar phenotype. Systems models of the nucleocytoplasmic shuttling network will evaluate the impact of observed abundance changes and identify characteristics of cargo predicted to be most sensitive to network reconfiguration. The importance of key effectors will ultimately be tested in vivo with murine cells engineered for spontaneous Erbb2 amplification and in patient samples with HER2 amplification.

Citation Format: Lixin Wang, Kevin A. Janes. Heterogeneous nucleocytoplasmic regulation of an incompletely penetrant ErbB2 onco-phenotype [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 293.
Abstract 2933: Synergistic antitumor effect of ONC201 in combination with radiation therapy
Authors: Zhou Lanlan , Wagner Jessica , El-Deiry Wafik S.
Published: August 2018

Radiation therapy is used as the main treatment when surgery is not feasible, before/after surgery, and to palliate symptoms in advanced stage. About 50% of cancer patients receive radiation therapy. 40% of the cure is achieved by radiation therapy. First-in-class small-molecule imipridone ONC201 can cause dual inactivation of ERK and AKT and induce an integrated stress response, proapoptotic TRAIL receptor DR5 activation, cancer stem cell depletion, and cell cycle arrest. ONC201 could synergize with radiation therapy because of its proapoptotic and antimetastasis effects. We tested this hypothesis in colorectal cancer, non-small cell lung cancer and glioblastoma cell lines (HCT116, RKO, LS513, H460, A549 and SNB19). Firstly, CellTiter-Glo® luminescent cell viability assays were performed with single treatment or combination of radiation therapy (0 to 4 Gy) and ONC201 (0 to 20 μM). For combination treatment, cancer cells were pretreated with ONC201 for 24 hours followed by radiation therapy. Next, colony formation assays with single treatment or combination were used to evaluate the effective dose range from cell viability assays. Flow cytometry and Western blot analysis were used for determination of apoptosis. We observed synergism between ONC201 and radiation therapy in several colorectal cancer, non-small cell lung cancer and glioblastoma cell lines. Combination indices are 0.53, 0.59 and 0.68 when non-small cell lung cancer cell line H460 was treated with ONC201 at 5 μM and radiation therapy at 1, 2 and 4 Gy. We are currently pursuing this promising combinational therapy in vivo, using the H460 xenograft model and potential syngeneic lung tumor models with wild-type p53. We plan to pursue the mechanism of this combination from in vivo samples. Our results indicate that the combination of radiation therapy and ONC201 could be an important approach in the application of ONC201 in clinical practice.

Citation Format: Lanlan Zhou, Jessica Wagner, Wafik S. El-Deiry. Synergistic antitumor effect of ONC201 in combination with radiation 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 2933.
Abstract 2942: Enhanced cytotoxic activity of BRAF, MEK and PI3K inhibition with combination CDK4/6 inhibition in melanoma cells
Authors: Zhang Yanping , Peng Guangyong , Hsueh Eddy C.
Published: August 2018

Introduction: We have previously reported that CDK4/6 inhibition enhanced the anti-proliferation and anti-migration effects of BRAF, MEK and PI3K inhibitors in melanoma cells. (Zhang Y et al, AACR 2017) In this study, we investigated the effect of combination CDK4/6 inhibition and BRAF, MEK and PI3K inhibition on induction of cell cycle arrest and apoptosis in melanoma.

Methods: Five human melanoma cell lines were used. CDK4/6 inhibitor (PD0332991; PD), BRAF inhibitor (PLX4032; PLX), MEK inhibitor (AZD6244; AZD), and PI3K/AKT inhibitor (XL765; XL) were used alone or in combination. Cell apoptosis analysis was performed using the FITC Annexin V Apoptosis Detection Kit. Autophagy was evaluated by immunofluorescence using anti-LC3A/B and NECN-1 antibodies. Lysosomal morphology was evaluated using lysosomal associated membrane protein 1-green fluorescent protein fusion construct. Western blotting was performed for apoptosis markers (PARP and caspase-9) and autophagy markers (LC3A/B and Beclin1) using GAPDH as internal control. Data were presented as means ± SD for triplicate experiments. For comparison between groups, the student's t test was used and p< 0.05 was considered to be statically significant.

Results: Apoptosis was evaluated by Annexin V assay. Various levels of apoptosis were observed with single agent PD, PLX, XL and AZD in all 5 melanoma cell lines. Combination of PD enhanced the apoptosis effects of PLX, XL, and AZD in all 5 melanoma cell lines. Increased LC3 and BECN1 detection and increased density of autophagosomes on immunofluorescence was observed after combining CDK4 inhibition with BRAF, MEK, and PI3K/AKT inhibition. Inhibition of PARP and Beclin-1 expression and increase in caspase-9 expression were observed with combination of PD compared with single agent PLX, XL, and AZD.

Conclusion: CDK4 inhibition enhanced anti-melanoma activity of BRAF , PI3K/AKT and MEK inhibitions through increased induction of early autophagy and caspase-mediated apoptosis. This suggests that inhibition of CDK4/6 plus BRAF , MEK and PI3K/AKT inhibition may help understanding the interplay between autophagy and apoptosis and inform the development of future targeted agents.

Citation Format: Yanping Zhang, Guangyong Peng, Eddy C. Hsueh. Enhanced cytotoxic activity of BRAF, MEK and PI3K inhibition with combination CDK4/6 inhibition in melanoma 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 2942.
Abstract 2949: Synergistic effects of tyrosine kinase inhibitors and conventional chemotherapeutics on canine and human osteosarcoma cells
Authors: Yang Ya-Ting , Yuzbasiyan-Gurkan Vilma
Published: August 2018

Osteosarcoma (OS) is the most common bone tumor in both humans and dogs, and has a nearly ten fold higher incidence in dogs than humans. Despite advances in treatment of other cancers, the overall survival rates for OS have stagnated for the past decades. Therefore, there is a great need to identify novel and effective treatments. In the past few years, several classes of novel small molecules with anti-tumor activities have become available. Among them, we selected tyrosine kinase inhibitors (TKIs) with potential anti-tumor medications to evaluate the effects of treating OS. In our previous studies, two TKIs (sunitinib and sorafenib) showed good growth inhibition effects at low concentrations on three canine OS cell lines (D17, Abrams, and Gracie) and two human OS cell lines (SAOS2 and U2OS) in a series of drug screening assay. In addition to viability, migration, and invasion ability of OS cells were decreased by these two TKIs. Furthermore, we investigated the effect of combing one of the TKIs with current chemotherapeutics for OS. We determined the combination index to assess whether sorafenib and sunitinib have synergistic effects with conventional chemotherapeutics including cisplatin, carboplatin, or doxorubicin. Data from our cell lines point to synergistic effects of sorafenib combined with doxorubicin, but not when combined with cisplatin or carboplatin in both human and canine OS. Combination studies with sunitinib are ongoing. Based on current findings, clinical trials using combination of doxorubicin and sorafenib in proof of concept studies in dogs are warranted. These studies can be carried out relatively quickly in dogs where case load is high, and in turn, provide useful data for initiation of clinical trials in humans.

Citation Format: Ya-Ting Yang, Vilma Yuzbasiyan-Gurkan. Synergistic effects of tyrosine kinase inhibitors and conventional chemotherapeutics on canine and human osteosarcoma 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 2949.