bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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&lt;0.005) and 42.7% (p&lt;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