bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5173: Discovery of a series of benzimidazoles as potent Wnt/β-catenin signaling inhibitors in colorectal cancer cells
Authors: Li Yonghe , Oliver Patsy G. , Lu Wenyan , Pathak Vibha , Augelli-Szafran Corinne E. , Buchsbaum Donald J. , Suto Mark J.
Published: July 2017

Aberrant activation of Wnt/β-catenin signaling is a necessary initiating event in the genesis of most colorectal cancers. Loss-of-function mutations in the tumor suppressor gene adenomatous polyposis coli (APC) are present in about 80% of all colorectal cancers, and gain-of-function mutations in the oncogene CTNNB1 (β-catenin encoding gene) exist in approximately 10% of colorectal cancers. Both APC and CTNNB1 mutations lead to cytosolic accumulation and nuclear translocation of β-catenin, and therefore constitutive activation of β-catenin/TCF4-mediated gene transcription. As such, the Wnt/β-catenin pathway has emerged as one of the most promising targets for colorectal cancer treatment. Despite tremendous efforts in the past decade, there are no small molecule Wnt inhibitors approved by the FDA for cancer treatment. Recent work in our laboratories has identified a series of benzimidazoles as potent Wnt/β-catenin inhibitors. Here, we show that several benzimidazoles displayed strong activities against Wnt/β-catenin signaling in colorectal cancer cells harboring mutations in CTNNB1 or APC. In particular, SRI36324, the lead compound in this series, inhibited Wnt/β-catenin signaling in colorectal cancer HCT116 and DLD-1 cells with IC50 values of 4.1 and 3.7 nM, respectively. Moreover, the benzimidazole compounds exhibited potent activities against colorectal cancer cell proliferation under both standard cell culture conditions (adherent cells in complete medium containing 10% FBS) and conditions designed to enrich for cancer initiating cells. In addition, the benzimidazole compounds had no off-target effects on other pathways (e.g., STAT3 and mTORC1 signaling) in colorectal cancer cells, and were less antiproliferative to non-cancerous cells. Together, our findings indicate that the benzimidazole compounds are promising candidates for development as novel therapeutic agents for colorectal cancer.

Note: This abstract was not presented at the meeting.

Citation Format: Yonghe Li, Patsy G. Oliver, Wenyan Lu, Vibha Pathak, Corinne E. Augelli-Szafran, Donald J. Buchsbaum, Mark J. Suto. Discovery of a series of benzimidazoles as potent Wnt/β-catenin signaling inhibitors in colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5173. doi:10.1158/1538-7445.AM2017-5173
Abstract 5174: Phosphodiesterase 10A inhibition as a novel approach to suppress β-catenin signaling in ovarian cancer cells
Authors: Silva Luciana Madeira Da , Gavin Elaine , Lee Kevin J. , Ramírez-Alcántara Veronica , Berry Kristy L. , Taylor Holly T. , Musiyenko Alla , Aragon Ileana V. , Keeton Adam B. , Scalici Jennifer , Rocconi Rodney P. , Piazza Gary A.
Published: July 2017

Canonical Wnt/β-catenin signaling is known to be associated with platinum resistance in ovarian cancer in which inhibitors hold promise for the treatment of refractory disease. Phosphodiesterase 10A (PDE10A) is a dual cyclic AMP and cyclic GMP phosphodiesterase isozyme recently implicated in colon cancer. PDE10A inhibition in colon cancer cells by siRNA or small molecule inhibitors increased cGMP levels and activated PKG to inhibit β-catenin signaling. A novel PDE10 inhibitor, ADT-061, was identified by screening a library of indene derivatives, and showed strong antineoplastic activity in the Apc+/min-FCCC mouse (Lee K et al., unpublished data). Cyclic GMP and phosphodiesterases participate in the ovarian follicular development, although little is known about PDE10A expression in ovaries, especially with regard to a potential role in ovarian tumorigenesis. PDE10A protein was found to be expressed in various established ovarian cancer cell lines at higher levels than immortalized or primary ovarian surface epithelial cell lines. Pf-2545920, a known PDE10A inhibitor, and ADT-061 inhibited the growth of multiple ovarian tumor cell lines with IC50s around 20µM and 0.5µM, respectively. Both compounds induced apoptosis after 24h treatment, as measured by PI/Annexin-V staining and PARP cleavage. Pf-2545920 and ADT-061 induced phosphorylation of VASP at Ser157 and Ser239 in various ovarian cancer cell lines, indicating activation of cyclic AMP and cyclic GMP signaling, respectively. Treatment also decreased levels of β-catenin and downstream targets of TCF-dependent transcription, including c-MYC, survivin and cyclin-D1. Homozygous knockout PDE10A clones of OV-90 ovarian cancer cells obtained using CRISPR/Cas9 showed decreased clonogenic potential, decreased Pf-2545920-mediated VASP phosphorylation and β-catenin, c-MYC and survivin expression. Ongoing efforts are focused on the development of more potent ADT-061 analogs. These observations support further study of a role of PDE10 in ovarian tumorigenesis and the development of ADT-061 or analogs for the treatment of refractory ovarian cancer as well as the prevention of malignant recurrence.

Citation Format: Luciana Madeira Da Silva, Elaine Gavin, Kevin J. Lee, Veronica Ramírez-Alcántara, Kristy L. Berry, Holly T. Taylor, Alla Musiyenko, Ileana V. Aragon, Adam B. Keeton, Jennifer Scalici, Rodney P. Rocconi, Gary A. Piazza. Phosphodiesterase 10A inhibition as a novel approach to suppress β-catenin signaling in ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5174. doi:10.1158/1538-7445.AM2017-5174
Abstract 5181: Targeted knockdown of MYC in AML cells using G-quadruplex interacting small molecules
Authors: Turnidge Megan A. , Patel Apurvi , Montoya Justin J. , Lee David W. , Wai Daniel H. , Gokhale Vijay , Hurley Laurence , Arceci Robert J. , Azorsa David O.
Published: July 2017

Acute Myeloid Leukemia (AML) is a disease that occurs when genomic changes alter expression of key genes, causing cells to resume an undifferentiated state, proliferate, and maintain tumor growth throughout the body. Recently, there has been an increase in efforts toward developing therapies that specifically target the protein products of these aberrantly expressed genes. However, many of the proteins are difficult to target because of structural challenges, protein overexpression that requires high drug doses, or mutations that confer resistance to therapy. One potential type of targeted therapy that circumvents these issues is the use of small molecules that stabilize DNA secondary structures called G-quadruplexes. G-quadruplexes are present in the promoters of many potential oncogenes, and have regulatory roles in their transcription. This study analyzes the therapeutic potential of compounds that target the regulatory G-quadruplex in the MYC oncogene, which is commonly misregulated in AML. Treatment of the MYC expressing AML cell lines KG-1 and UT-7epo with the compound GQC-05 resulted in decreased expression of MYC mRNA, as determined by qPCR. Moreover, GQC-05 treatment induced a more significant decrease in c-Myc protein expression than similar treatment with JQ-1(+), as shown by western blot analysis. Treatment of AML cells with GQC-05 also decreased cell viability and increased apoptosis. Concurrent treatment of AML cells with GQC-05 and JQ-1(+) showed an antagonistic effect, indicating potential competition in the silencing of MYC. These results demonstrate that targeting the MYC promoter G-quadruplex in AML cells leads to knockdown of MYC expression and induces apoptosis. These results further support the development of a novel mechanism for targeting key genetic drivers in AML, and lay the groundwork for advances in treatment of other cancers driven by G-quadruplex regulated oncogenes.

Citation Format: Megan A. Turnidge, Apurvi Patel, Justin J. Montoya, David W. Lee, Daniel H. Wai, Vijay Gokhale, Laurence Hurley, Robert J. Arceci, David O. Azorsa. Targeted knockdown of MYC in AML cells using G-quadruplex interacting small molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5181. doi:10.1158/1538-7445.AM2017-5181
Abstract 5182: Targeting abnormal metabolism downstream of MYC in atypical teratoid/rhabdoid tumors
Authors: Wang Sabrina , Rubens Jeffrey , Allen Sariah , Orr Brent , Eberhart Charles , Raabe Eric
Published: July 2017

Atypical teratoid rhabdoid tumors (AT/RT) are deadly infantile brain tumors in dire need of new, targeted therapies. Recent molecular analysis revealed considerable tumor heterogeneity subdividing AT/RT into 3 distinct groups. The MYC subgroup has a dismal 5 year survival of 18.5%. MYC is known to drive reliance on glutamine for cellular metabolism suggesting that high-MYC expressing neoplasms may be sensitive to glutamine metabolic inhibitors. 6-Diazo-5-oxo-L-norleucine (DON) is a glutamine antagonist that decreases the tumorigenicity of high MYC expressing models of medulloblastoma and neuroblastoma . We hypothesize that DON will reduce the tumorigenicity of the MYC subgroup of AT/RT. High levels of c-MYC protein are expressed in about 1/3 of human AT/RT (Immunohistochemistry for c-MYC on 22 human AT/RT, H-score > 1 standard deviation over the median considered high c-MYC expression). Similarly two of six of AT/RT cell lines expressed high levels of c-MYC protein by western blot. DON treatment slowed cell growth of high c-MYC expressing AT/RT cell lines BT12 and CHLA06 (MTS assay p<0.005 10uM DON vs DMSO control; t-test), whereas cell lines BT37 and CHLA05, which had low c-MYC expression were not affected. Cell cycle analysis demonstrated that DON caused cell cycle arrest in the G2/M phase for the high c-MYC expressing lines BT12 and CHLA06 (31.2% of BT12 DMSO controls in G2/M phase vs 54.5% after 10uM DON treatment and 27.4% vs 48.9% in CHLA06). In contrast the cell cycle was not affected by DON treatment in the cell lines with low c-MYC expression (21.3% DMSO vs 20.2% after 10uM DON in BT37). DON also increased apoptosis in cell lines with high c-MYC and induced DNA strand breaks (Western blot for c-PARP and p-H2A.X respectively). DON significantly improved survival in BT-12 high c-MYC expressing orthotopic mouse xenografts (median survival increased from 21 to 36 days, p<0.005; log-rank test). These data develop the framework for future clinical trials using glutamine antagonists to target the MYC subgroup of AT/RT.

Citation Format: Sabrina Wang, Jeffrey Rubens, Sariah Allen, Brent Orr, Charles Eberhart, Eric Raabe. Targeting abnormal metabolism downstream of MYC in atypical teratoid/rhabdoid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5182. doi:10.1158/1538-7445.AM2017-5182
Abstract 5183: The radioenhancer NBTXR3 brings anticancer efficacy to the cisplatin-based chemoradiation in vitro and in vivo
Authors: Pottier Agnès , Vivet Sonia , Paris Sébastien , Lu Bo
Published: July 2017

NBTXR3 is a radioenhancer composed of functionalized hafnium oxide nanoparticles, designed to enhance the radiation dose deposit within the cancer cells when activated by ionizing radiations. NBTXR3 is intended for single intratumor administration and is currently evaluated in cancer clinical trials including soft tissue sarcoma, head and neck (H&N), prostate, liver and rectum.

Cisplatin (CDDP) is a cytotoxic agent considered to be a radiosensitizer and inhibits the repair of sublethal damage from irradiation. Concurrent chemoradiation using CDDP is the mainstream treatment for high risk H&N, cervix and non-small cell lung cancer patients. We hypothesized that adding NBTXR3 to radiation treatment (RT) may improve significantly anticancer effect of the chemoradiation combination

In vitro, no specific clonogenic toxicity was observed for the cells exposed only to NBTXR3. For the combined treatment CDDP was used at its IC50 concentrations. A marked and enhanced cell destruction (DEF) was observed with the CDDP combined treatment (added 6h or 16h prior to RT) and NBTXR3 (800µM, added 16h prior to RT) at ≥2Gy when compared to the single agent.

TreatmentCell line (DEF*)CDDPNBTXR3IrradiationFaDuNCI-H460-Luc26h-2 Gy1.0±0.061.2±0.06-16h1.4±0.151.3±0.256h16h1.4±0.141.6±0.1216h-1.0±0.121.9±0.32-16h1.2±0.131.5±0.1716h16h1.8±0.232.6±0.456h-4 Gy1.3±0.082.2±0.16-16h2.2±0.241.9±0.416h16h3.1±0.643.8±0.4816h-1.2±0.192.9±0.52-16h2.1±0.312.8±0.3916h16h5.3±0.977.6±2.76

*For a given radiation dose, the DEF value was obtained by taking the ratio of the survival fraction in the control (radiotherapy alone) and the survival fraction of the treatment.

In vivo, NBTXR3 enhanced the fractionated radiation treatment (5x2Gy) when injected intratumorally at 25% of tumor volume. NBTXR3 activated with ionizing radiation in combination with low dose of CDDP (1mg/kg administered 5 consecutive days, 2 hours prior to RT) delayed tumor growth when compared to single agent CDDP in combination with RT.

These results strongly support the concurrent chemoradiotherapy regimen with NBTXR3 nanoparticles as an effective radioenhancer to increase the anticancer effect. A new phase I/II trial with H&N cancers patient receiving radiotherapy plus CDDP has been started evaluating the optimal dose, safety and preliminary efficacy of NBTXR3.

Citation Format: Agnès Pottier, Sonia Vivet, Sébastien Paris, Bo Lu. The radioenhancer NBTXR3 brings anticancer efficacy to the cisplatin-based chemoradiation in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5183. doi:10.1158/1538-7445.AM2017-5183
Abstract 5191: A combination of Stat3 inhibitor and radiation for the treatment of malignant pleural mesothelioma
Authors: Matsumoto Seiji , Doi Hiroshi , Fukuda Akihiro , Nakamichi Tohoru , Kuroda Ayumi , Hashimoto Masaki , Takuwa Teruhisa , Kondo Nobuyuki , Hasegawa Seiki
Published: July 2017

Background: Trimodality therapy, including surgery, is the treatment for patients with malignant pleural mesothelioma (MPM). However, the 5-year survival rate remains less than 40%. To improve the survival rate, we investigated Stat3 as a potential therapeutic target for MPM.

Methods:Cell viability was assessed using Cell Counting Kit-8 (CCK-8: WST-8 Dojindo). MPM cells (NCI-H28, NCI-H226, NCI-H2052, NCI-H2452, MSTO-211H) were seeded into 96-well plates. After treatment with a Stat3 inhibitor (BBI-608 : napabucasin), Cell Counting Kit-8 solution was added to each well and absorbance was measured using a microplate reader. The level of phosphorylated Stat3 (p-Stat3) in cell lysates was measured by ELISA. ROS level in cells was measured by FACS analysis. In the in vivo study, H226 cells were injected subcutaneously in the flank region of nude mice randomly assigned to four treatment groups (5 mice/group)—(1) treatment with vehicle control, (2) treatment with Stat3 inhibitor, (3) radiation therapy (RT), and (4) Stat3 inhibitor and RT. Tumor size was measured twice/week.

Results: Napabucasin decreased the viability of MPM cells (epithelioid, sarcoma, biphasic) in a dose-dependent manner. p-Stat3 levels decreased by 90% after treatment with 10 μM napabucasin in H226 cells. Napabucasin treatment induced more ROS production in MPM cells than did Pemetrexed. Moreover, combinatorial treatment with napabucasin and radiation (2 Gy, 5 Gy) induced more ROS production than did RT alone. In the mouse model, treatment with napabucasin alone significantly suppressed tumor growth (p < 0.05), but napabucasin and RT completely suppressed tumor growth.

Conclusion:We showed that napabucasin inhibited proliferation of the MPM cells (epithelial, biphasic, sarcomatoid) and demonstrated that combination therapy with napabucasin and radiation completely suppressed tumor growth in a mouse model. Treatment with the napabucasin alone and the combination of napabucasin and RT induced high ROS production in MPM cells. Collectively, the combination of Stat3 inhibitor with Radiation Therapy (START) is a promising approach for the treatment of MPM.TheA combination of of Stat3 inhibitor and radiation with for the treatment of malignant pleural mesothelioma

Citation Format: Seiji Matsumoto, Hiroshi Doi, Akihiro Fukuda, Tohoru Nakamichi, Ayumi Kuroda, Masaki Hashimoto, Teruhisa Takuwa, Nobuyuki Kondo, Seiki Hasegawa. A combination of Stat3 inhibitor and radiation for the treatment of malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5191. doi:10.1158/1538-7445.AM2017-5191
Abstract 5192: Selection of radiosensitizers based on HRAS mutation in head and neck cancer
Authors: Fisher Michael M. , Swick Adam D. , Nickel Kwangok P. , Kimple Randall J.
Published: July 2017

Purpose/Objective(s): The ability of activated HRAS to promote radiation resistance and predict for sensitivity to potential radiosensitizers was tested in a head and neck cancer model system.

Materials/Methods: Mutations in HRAS, KRAS, and NRAS were analyzed using cBioPortal in a head and neck cancer cohort. The Illumina TruSeq Amplicon Cancer panel was used to screen a panel of head and neck cancer cell lines for mutations in RAS-family genes. Cell growth and radiation survival was assessed by clonogenic survival assay. Immunoblots were used to confirm target activation/knockdown in overexpression and knockdown studies. A tumor control dose 50% (TCD50) assay was employed to investigate radiosensitization of a mutated-HRAS head and neck cancer cell line in a flank xenograft model in nude mice.

Results: Activating mutations in one of the three RAS genes are seen in 5-10% of head and neck cancer patients. Mutations in HRAS represent over 50% of these. Screening our panel of head and neck cancer cell lines identified a canonical activating mutation in HRAS (i.e. G12V) in SCC22B. Consistent with known roles for activated HRASG12V, SCC22B is relatively insensitive to both cetuximab and radiation. Using both in vitro and in vivo studies, cetuximab exhibited no ability to radiosensitize SCC22B. Cetuximab treatment decreased AKT, but not ERK activation. Direct inhibition pathways downstream of HRAS by selumetinib (MEK/ERK) or BEZ235 (PI3K/MTOR) decreased target protein activation and resulted in significant growth inhibition compared to control (p<0.05). Treatment with either selumetinib or BEZ235 radiosensitized HRASG12V expressing cells (SER 1.3-1.8) but had more modest effects on cells with wildtype HRAS. siRNA knockdown of HRAS radiosensitized SCC22B, but not SCC1 or SCC6 cells relative to non-targeting control. Overexpression of HRASG12V conferred relative radioresistance in wildtype cell lines. In vivo assessment of the radiosensitizing effects of these compounds (TCD50) is pending.

Conclusion: More than 5% of head and neck cancers harbor activating mutations in one of the RAS family of genes. These mutations lead to resistance to cetuximab, either as a single agent, or as a radiosensitizer. Inhibition of downstream targets such as the MEK/ERK and PI3K/MTOR pathways can radiosensitize tumors harboring activating mutations in HRAS. Identification of additional mutation/drug combinations that result in radiosensitization may be valuable to advance the design of personalized radiation therapy.

Citation Format: Michael M. Fisher, Adam D. Swick, Kwangok P. Nickel, Randall J. Kimple. Selection of radiosensitizers based on HRAS mutation in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5192. doi:10.1158/1538-7445.AM2017-5192
Abstract 5215: Time dependent blood-brain barrier modulation by vascular endothelial growth factor to improve CNS drug delivery
Authors: McCord Matthew
Published: July 2017

Introduction:

Efforts to transiently increase blood brain barrier permeability would facilitate improved CNS delivery of chemotherapy agents. Previous studies demonstrated that vascular endothelial factor (VEGF) increases permeability in traumatic brain injury and ischemic brain disease. Exogenously administered VEGF increases barrier permeability in the non-diseased CNS. In our study, we investigated the role for exogenous VEGF therapy to modulate blood-brain barrier permeability

Methods:

For in vitro studies, confluent mouse brain endothelial cells (BEND.3) were treated with VEGF165. Trans-endothelial electrical resistance measured cell impedance changes and immunofluorescence was used to assess qualitative expression of tight and adherens junction proteins at key time points. Western blotting and qPCR were done to evaluate the effect of VEGF on expression of protein and RNA associated with tight and adherens junctions.

Results:

Confluent endothelial cells treated with rVEGF165 (200ng/mL) evaluated by TEER show a significant drop in cell impedance approximately 6 hours post treatment, compared to control (more than 10-fold) reflecting increased permeability of the monolayer. Treatment with progressively higher doses of VEGF (200, 400, and 600ng/mL) showed a progressively greater drop in cell impedance. Immunofluorescence of endothelial cells treated with rVEGF165 (200ng/mL) shows disassembly of both tight junctions and adherens junctions between 4 and 8 hours post-treatment compared to controls. Western blots and qPCR demonstrate that rVEGF165 treatment induces down-regulation of genes associated with tight junctions, such as Claudin5, beginning as early as 4 hours post treatment and continuing up to 24 hours post treatment.

Conclusion:

Our results suggest that VEGF is capable of inducing transient breakdown of the blood-brain barrier, which may facilitate improved CNS delivery of chemotherapeutic agents. Our in vitro data warrants further investigations of the applicability of VEGF therapy to induce endothelial cell permeability in animal tumor models, potentially improving delivery of chemotherapeutic drugs.

Citation Format: Matthew McCord. Time dependent blood-brain barrier modulation by vascular endothelial growth factor to improve CNS drug delivery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5215. doi:10.1158/1538-7445.AM2017-5215
Abstract 522: Expression and functional analysis of signal peptidase complex 18 in bladder cancer
Authors: Shigematsu Yoshinori , Sekino Yohei , Sakamoto Naoya , Sentani Kazuhiro , Oue Naohide , Hayashi Tetsutaro , Teishima Jun , Matsubara Akio , Yasui Wataru
Published: July 2017

Urothelial carcinoma of the bladder is a common malignancy that causes approximately 150,000 deaths per year worldwide. So far, no molecularly targeted agents have been approved for treatment of the disease. In the present study, we built an in-house oligonucleotide array, on which 394 genes were selected based on our SAGE data and previously reported array data, in order to identify the genes of most relevance to gastric carcinogenesis. Among these genes, we focused on SEC11A, because it is frequently overexpressed in gastric cancer (GC). SEC11A encodes the SPC18 protein, which is one of the subunits of the signal peptidase complex (SPC). Most secretory proteins contain amino terminal- or internal signal peptides that direct their sorting to the endoplasmic reticulum (ER). From the ER, proteins are transported to either the extracellular space or the plasma membrane through the ER-Golgi secretory pathway. The ER signal peptides are then cleaved by the SPC. In GC, SPC18 contributes to progression via EGFR pathway associated with TGF-a secretion. However, the expression and function of SPC18 have not been investigated in bladder cancer (BC). In this study, we analyzed the expression and distribution of SPC18 in human BC. Expression of SPC18 was observed in 44 (54.3%) out of 81 BC cases that was associated with T category (p=0.0002), Grade (p=0.0425), vascular invasion (p=0.0112) and CK5/6 positive/ CK20 negative basal type BC (p=0.0004). The univariate analysis indicated that expression of SPC18 (HR, 3.14; 95% CI, 1.47-7.47; P=0.0027) was associated with overall survival. In the multivariate model, SPC18 expression was an independent prognostic indicator (HR, 3.46; 95% CI, 1.35-10.00; P=0.0087). BC cells KMBC2 transfected with SPC18 expression vector significantly induced the cell growth (p<0.01) and invasion (p<0.01) activity. Western blot showed the overexpression of SPC18 induced the phosphorylation of EGFR, Akt and Erk. Real-time reverse transcription PCR analysis revealed the SPC18 forced expression cells induced mesenchymal character. These results suggest that SPC18 might contribute to the progression of BC.

Citation Format: Yoshinori Shigematsu, Yohei Sekino, Naoya Sakamoto, Kazuhiro Sentani, Naohide Oue, Tetsutaro Hayashi, Jun Teishima, Akio Matsubara, Wataru Yasui. Expression and functional analysis of signal peptidase complex 18 in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 522. doi:10.1158/1538-7445.AM2017-522
Abstract 5224: Identification of drug-target interactions through rapid selection of drug resistant protein variants generated by CRISPR/Cas9-induced NHEJ
Authors: Neggers Jasper E. , Kwanten Bert , Dierckx Tim , Daelemans Dirk
Published: July 2017

Identification and validation of the cellular target of bioactive hits identified in a phenotypic screen is crucial for their further development into a drug. The gold standard proof for identification of a small molecule's target is the discovery of mutations that confer resistance. However, selection of drug resistance and subsequent deconvolution of the relevant mutations that confer drug resistance remains time consuming. Therefore, a methodology that can accelerate the drug resistance selection process and that can simplify identification of relevant drug resistance mutations would benefit the drug discovery and development process greatly.

Here we report a simple method to rapidly identify the mechanism of action of small molecules with anti-cancer activity. The method utilizes the positive selection of in-frame drug resistance mutations in the target protein of a small molecule derived from the localized genetic variation created by non-homologous end joining (NHEJ) repair of CRISPR/Cas9-induced double strand breaks. In brief, we introduced double strand breaks in the target proteins of selinexor, ispinesib and triptolide and treated these cells with the respective drug. Resistance was obtained rapidly and sequencing of resistant colonies revealed known as well as many new drug resistant protein variants. Next, we investigated whether this approach can be applied as a screening strategy on a subset of genes to identify the direct cellular target protein of a small molecule. We therefore, as proof of concept, designed a pooled sgRNA tiling library targeting all PAM sites of multiple genes and applied this to ispinesib. Drug resistant colonies formed rapidly and were enriched with sgRNAs targeting the ispinesib binding site in KIF11, ispinesib’s target protein. Further sequencing of the sgRNA target gene locus revealed drug resistance mutations in KIF11, validating the feasibility of the methodology to uncover the target of a small molecule from a small pool of candidate genes. To conclude, we provide a new method for identification of the cellular target protein and binding site of a small molecule based on positive selection of drug resistant protein variants generated by targeted CRISPR/Cas-induced NHEJ repair.

Citation Format: Jasper E. Neggers, Bert Kwanten, Tim Dierckx, Dirk Daelemans. Identification of drug-target interactions through rapid selection of drug resistant protein variants generated by CRISPR/Cas9-induced NHEJ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5224. doi:10.1158/1538-7445.AM2017-5224
Abstract 5230: Targeting two unique end-insertion G-quadruplexes formed in the 3′-end of the PDGFR-β core promoter nuclease hypersensitive element with ellipticine analog
Authors: Onel Buket , Agrawal Prashansa , Carver Megan , Hurley Laurence H. , Yang Danzhou
Published: July 2017

Aberrant expression of PDGFR-β promotes multiple hallmarks of cancer, and its transcriptional regulation provides an attractive means to inhibit the PDGFR-β signaling pathway. The nuclease hypersensitive element (NHE) region of the human PDGFR-β core promoter has been shown to be responsible in regulating approximately 60% of the PDGFR-β basal promoter activity. Multiple overlapping G-quadruplexes (G4s) have been shown to form in this NHE region. We previously determined the most stable G4 structure formed in the 5’-mid of PDGFR-β NHE region. Significantly, an ellipticine analog, GSA1129, has been shown to selectively bind to the G4 formed in the 3’-end NHE region, thereby shifting the dynamic equilibrium toward the 3’-end G4. This selective interaction was shown to result in suppression of PDGFR-β activity in both cancer cell lines and a preclinical animal model. Thus, characterization of 3’-end G4 provides an important basis for understanding the molecular recognition of this G4 and for rational drug design. In this study, we used NMR spectroscopy to show that the PDGFR-β NHE 3′-end sequence forms two novel end-insertion intramolecular G4s which exist in a dynamic equilibrium under physiological salt condition. One is the 3′-insertion-G4 with a 3′ non-adjacent flanking guanine inserted into the 3′-external tetrad. The other is a 5′-insertion-G4 with a 5′ non-adjacent flanking guanine inserted into the 5′-external tetrad. We further confirmed that these novel end-insertion G4s can readily form in the 3’-end NHE sequence under physiologically relevant ionic conditions using dimethyl sulfate (DMS) footprinting experiments. To elucidate the molecular interactions of ellipticine analog with the 3’-end G4s, we studied the binding of GSA1129 to both of the end-insertion G4s. Significantly, our CD melting studies showed that GSA1129 markedly increased the stability of both end-insertion G4s by about 25°C. Furthermore, 1H NMR titration experiments have shown that GSA1129 selectively binds to the G4 formed in the 3’-end NHE region. The novel end-insertion structures and the dynamic nature of the PDGFR-β NHE 3′-end G4 may make it an effective target for GSA1129 binding and regulation. Our result highlights the dynamic nature of the PDGFR-β NHE 3′-end sequence and the importance of identifying the exact sequence for the formation of the naturally occurring G4 structure.

Citation Format: Buket Onel, Prashansa Agrawal, Megan Carver, Laurence H. Hurley, Danzhou Yang. Targeting two unique end-insertion G-quadruplexes formed in the 3′-end of the PDGFR-β core promoter nuclease hypersensitive element with ellipticine analog [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5230. doi:10.1158/1538-7445.AM2017-5230
Abstract 5232: The molecular basis for specific recognition of the biologically relevant hybrid-2 type human telomeric G-quadruplex by epiberberine
Authors: Lin Clement , Wu Guanhui , Shao Yong , Yang Danzhou
Published: July 2017

G-quadruplex structures have been shown to form in human telomeres, and the formation of G-quadruplexes can inhibit telomerase, which plays a key role in cancers by stabilizing telomere length and integrity thereby granting limitless replicative potential. The human telomeric G-quadruplex is thus considered to be a potential target for cancer therapeutics. In physiologically relevant potassium solution, human telomeric DNA sequences form two equilibrating hybrid-type G-quadruplex structures, with the hybrid-2 structure being the predominant form in extended sequences. We discovered that epiberberine (EPI), a naturally occurring protoberberine alkaloid, can specifically bind the hybrid-2 human telomeric G-quadruplex and can induce the conversion to the hybrid-2 structure. Using nuclear magnetic resonance (NMR) spectroscopy, we determined the solution structure of the 1:1 complex of EPI and hybrid-2 human telomeric G-quadruplex. Our NMR structure shows EPI bound at the 5’ end of the hybrid-2 telomeric quadruplex with an unexpectedly large drug-induced conformational change in the flanking and loop regions, creating a very well-defined drug binding pocket with extensive capping structures. The EPI molecule and 5’ flanking adenine form an induced quasi-triad plane which is intercalated between the external 5’ tetrad and capping structures. Two layers of new capping structures are formed with the 5’ flanking segment and the second TTA lateral loop to cover the “intercalated quasi-triad”. Notably, the well-defined EPI-induced multi-layer binding-site arrangement is only possible in the hybrid-2 folding topology of telomeric DNA. Our results provide important insights into specific targeting of the physiologically relevant hybrid-2 human telomeric G-quadruplex by a small molecule compound; EPI’s asymmetric crescent-shape and the positioning of its dioxolane moiety, as well as the hybrid-2 folding and the loop and flanking bases in the human telomeric DNA sequence, all contribute toward the specific recognition of EPI. In addition, we have conducted polymerase stop assays, which confirmed that EPI can stabilize the human telomeric G-quadruplex to inhibit polymerase activity.

Citation Format: Clement Lin, Guanhui Wu, Yong Shao, Danzhou Yang. The molecular basis for specific recognition of the biologically relevant hybrid-2 type human telomeric G-quadruplex by epiberberine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5232. doi:10.1158/1538-7445.AM2017-5232
Abstract 5234: Expression of cytochrome P450 in head and neck cancer cell lines
Authors: Presa Daniela F. , Morais Goreti Ribeiro , Sutherland Mark , McCaul Jim , Robert Jacques , Patterson Laurence , Pors Klaus
Published: July 2017

Background: The high expression of cytochrome P450 (CYP) enzymes including isoforms CYP1A1, 1B1 and 2W1 in some tumor types compared to normal tissue make these potential targets for targeted drug discovery. Our efforts in this space have resulted in the discovery of duocarmycin bioprecursors that are bioactivated by CYP1A1 and CYP2W1 expressed in malignant tissues1-2. Epidemiological studies have shown that head and neck cancer (HNC) is a complex multistage process that in part involves exposure to a combination of carcinogens and the capacity of certain drug-metabolising enzymes including CYPs to detoxify or activate such carcinogens. Here, we report on the expression of CYP1A1, CYP1B1 and CYP2W1 in HNC cell lines and human primary tissues.

Materials and methods: A panel of HNC cell lines (FaDu, Detroit-562, A253, OSC19) was characterized for CYP1A1, CYP1B1 and CYP2W1 gene expression using real time PCR. Furthermore, immunofluorescence (IF) was used to detect protein expression in HNC cell lines and immunohistochemistry (IHC) in xenografts, paraffin-embedded and snap-frozen human HNC tissue. Anti-proliferative activity was measured using the MTT assay and DNA damage was measured using the comet assay.

Results: In the HNC cell lines significant expression (5-10 fold) was shown at the gene level for CYP1A1 and CYP1B1 whereas CYP2W1 was hardly detected. However, CYP2W1 was expressed in FaDu and Detroit xenografts and in a small cohort of 10 human HNC samples as measured by IF and IHC, respectively. In agreement with CYP1 expression, the cytotoxicity results of ICT2700 and ICT2706 revealed FaDu and Detroit-562 as the most sensitive HNC cell lines suggesting bioactivation of these two lead duocarmycin bioprecursors.

Conclusion: Accordingly, from these data we can conclude that CYP1A1, CYP1B1 and CYP2W1 are expressed in selected HNC cell lines and tumor tissue. In addition, early evidence suggests that CYP2W1 expression is stimulated by the presence of an in vivo microenvironment. However a larger cohort (≥ 60) of clinical HNC samples will be used to confirm this observation and will be reported at the annual AACR meeting. [1] Sheldrake et al.Reengineering of the duocarmycin structural architecture enables bioprecursors development targeting CYP1A1 and CYP2W1 for biological activity. J Med Chem. 2013, 56, 62737. [2] Travica et al. Colon cancer-specific cytochrome P450 2W1 converts duocarmycin analogues into potent tumor cytotoxins. Clin Cancer Res. 2013, 19, 295261.

Citation Format: Daniela F. Presa, Goreti Ribeiro Morais, Mark Sutherland, Jim McCaul, Jacques Robert, Laurence Patterson, Klaus Pors. Expression of cytochrome P450 in head and neck cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5234. doi:10.1158/1538-7445.AM2017-5234
Abstract 5236: The dynamic interplay between the cancer genome mutating enzyme APOBEC3B and DNA substrates
Authors: Demir Ozlem , Wagner Jeffrey R. , Shi Ke , Carpenter Michael A. , Banerjee Surajit , Shaban Nadine M. , Kurahashi Kayo , Salamango Daniel J. , McCann Jennifer L. , Starrett Gabriel J. , Duffy Justin V. , Harki Daniel , Aihara Hideki , Amaro Rommie E. , Harris Reuben S.
Published: July 2017

The APOBEC3 (A3) family of enzymes catalyze deamination of cytosines to uracils (C-to-U) in single-stranded (ss)DNA, and is an important part of innate immune responses against viruses and transposons. Recent data also indicate that APOBEC3B (A3B) is a major source of genomic mutations in multiple cancers and a contributor to the evolution of drug resistance. A3B is therefore a promising anti-cancer target. Using existing structural information and structural modeling, we generated a model for wild-type A3B in complex with ssDNA and used molecular dynamics to monitor the dynamics of the native interactions of wild-type A3B with ssDNA. The details of A3B-ssDNA interaction and its implications for potential inhibitor design will be presented.

Citation Format: Ozlem Demir, Jeffrey R. Wagner, Ke Shi, Michael A. Carpenter, Surajit Banerjee, Nadine M. Shaban, Kayo Kurahashi, Daniel J. Salamango, Jennifer L. McCann, Gabriel J. Starrett, Justin V. Duffy, Daniel Harki, Hideki Aihara, Rommie E. Amaro, Reuben S. Harris. The dynamic interplay between the cancer genome mutating enzyme APOBEC3B and DNA substrates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5236. doi:10.1158/1538-7445.AM2017-5236
Abstract 5238: Structural and biochemical characterization of membrane bound KRAS
Authors: Stephen Andrew G. , Van Que , Tonelli Marco , Gillette William , Esposito Dominic , Niu Ben , Gross Michael , Heinrich Frank , Ramanathan Arvind , Stanley Christopher
Published: July 2017

KRAS4b functions as a molecular switch cycling between an active GTP bound state and inactive GDP state. Active KRAS4b binds tightly to RAF1 via its Ras binding domain (RBD), however activation of RAF1 competent for MAPK signal transduction requires membrane bound KRAS4b. Using recombinant farnesylated and methylated KRAS4b (KRAS4b-FMe) we have used a variety of biophysical methodologies including paramagnetic relaxation enhancement NMR, neutron reflectivity, small angle neutron and x-ray scattering and protein footprinting to gain insights into the orientation and structure of KRAS-FMe bound to a membrane. In these experiments we have used Nanodiscs or tethered lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline or -serine (70% POPC 30% POPS). Our data indicate that KRAS-FMe is oriented perpendicular to the membrane with minimal direct contacts to the membrane. The nucleotide bound state does not significantly change the orientation relative to the membrane. We will also present data on the effect of the RBD domain of RAF1 binding to KRAS-FMe at the membrane.

Citation Format: Andrew G. Stephen, Que Van, Marco Tonelli, William Gillette, Dominic Esposito, Ben Niu, Michael Gross, Frank Heinrich, Arvind Ramanathan, Christopher Stanley. Structural and biochemical characterization of membrane bound KRAS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5238. doi:10.1158/1538-7445.AM2017-5238
Abstract 5244: Partial knockdown of RLIP76 prevents cancer susceptibility of p53 null mice
Authors: Singhal Sharad S. , Singhal Jyotsana , Tompkins Joshua , Horne David , Wu Xiwei , Riggs Arthur , Awasthi Sanjay
Published: July 2017

P53 is a stress-responsive, genome protective, tumor suppressor whose functions are lost or altered in nearly all neoplasia. P53 homozygous knockout mice (p53-/-) develop spontaneous lymphoma and other malignancies in nearly 100% of cases by 6 months of life. In stark contrast, homozygous gene knockout of the stress-responsive, anti-apoptotic, mercapturic acid pathway transporter protein, RLIP76, results in marked protection from chemical carcinogenesis. P53 expression and activation is regulated by intermediary metabolites of this pathway, and p53 regulates the expression of key enzymes of this pathway. P53 can directly bind to RLIP76 through a CDK-interaction domain, and inhibit its transport activity as well as endocytosis stimulatory activity. These results indicate that RLIP76 is an effector of p53 in a manner analogous to its known function as the first known effector of the Ral pathways that regulate membrane plasticity, motility and invasion. This translates into reduced kinase signaling down-stream of membrane-receptor/ligand interactions. An inverse relationship between RLIP76 and p53 protein and mRNA expression has been observed in-vitro, and is confirmed by analysis of human cancer genomic analyses in the COH database. RLIP76 depletion causes apoptosis in cancer cells independent of p53 status, strengthening the view that RLIP76 is key down-stream effector of p53. We reasoned that if RLIP76 is necessary for cancer to develop in p53-deficient animals, its suppression will reduce the incidence of malignancy in 12-week old p53-/- mice treated with COHSA007 phosphorothioated antisense, 200 μg i.p.q.2wk till age 8 months. Results of our studies confirmed these predictions, since 4/4 scrambled antisense treated p53-/- mice developed T-cell lymphoma whereas 8/8 COHSA007 treated p53-/- mice survived to the end of the experiment without development of any malignancy. These findings demonstrate that RLIP76 is required for malignancy in the p53-/- mice. Taken in context of our previous studies showing that RLIP76-/- mice are highly resistant to inflammation and carcinogenesis in chemical carcinogenesis models of the skin and lung, the present findings strongly support the idea of an existential role of RLIP76 in the malignant phenotype, and strengthen the rationale for using targeted systemic depletion of RLIP76 for treatment of human neoplasia (Supported in part by the Department of Defense grant (W81XWH-16-1-0641) and USPHS grant CA 77495).

Citation Format: Sharad S. Singhal, Jyotsana Singhal, Joshua Tompkins, David Horne, Xiwei Wu, Arthur Riggs, Sanjay Awasthi. Partial knockdown of RLIP76 prevents cancer susceptibility of p53 null mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5244. doi:10.1158/1538-7445.AM2017-5244
Abstract 5247: δ-Tocopherol inhibits the development of prostate adenocarcinoma in prostate specific Pten-/- mice
Authors: Yang Xu , Hong Wang , Wang Guocan , Liu Anna , Bosland Maarten c. , Yang Chung s.
Published: July 2017

PTEN/PI3K/AKT axis plays a critical role in regulating cell growth, metabolism, migration, differentiation, and survival. Activation of this signal pathway is frequently found in human cancers. Data from our previous studies demonstrated that δ-tocopherol (δ-T) attenuates the activation of AKT by growth factor in prostate cancer cell lines, leading to inhibiting proliferation and inducing apoptosis. Herein, we investigated whether δ-T is able to inhibit the development of prostate adenocarcinoma in prostate-specific Pten-/- (Ptenp-/-) mice, in which the activation of AKT resulted from loss of Pten is the driver. By feeding Ptenp-/- mice with the AIN93M or 0.2% δ-T supplemented diet starting at the age of 6 or 12 weeks, we found that, at the age of 40 weeks, δ-T treatment reduced the number of invasive prostate adenocarcinoma by 53.3% (p<0.005) and 42.7% (p<0.001), respectively. By immunohistochemical analysis, the activation of AKT via phosphorylation of AKT(T308) was found reduced in the prostate of the mice on δ-T diet. Consistently, proliferation was reduced and apoptosis was increased in prostate of the mice on δ-T diet. We also determined oxidative stress by immunohistochemical staining of 8-OH-dG and nitro-tyrosine, and found that these markers were not altered during prostate tumorigenesis, nor they were affected by δ-T. A 0.2% α-tocopherol diet was also used to feed Ptenp-/- mice, but did not inhibit the development of prostate adenocarcinoma, consistent with previous data demonstrating that α-tocopherol did not inhibit the activation of AKT. Together, these results support that δ-T is effective in supressing the development of prostate adenocarcinoma in Ptenp-/- mice through inhibition of AKT activation.

Citation Format: Xu Yang, Wang Hong, Guocan Wang, Anna Liu, Maarten c. Bosland, Chung s. Yang. δ-Tocopherol inhibits the development of prostate adenocarcinoma in prostate specific Pten-/- mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5247. doi:10.1158/1538-7445.AM2017-5247