Citations

The microtubule-associated protein, tau, is the principal component of paired helical filaments (PHFs) in Alzheimer's disease. PHF-tau is highly phosphorylated and a total of 25 sites of phosphorylation have so far been identified. Many of these sites are serine or threonine residues that are immediately followed in the sequence by proline residues, and hence are candidate phosphorylation sites for proline-directed kinases. In vitro, glycogen synthase kinase-3 (GSK-3), extracellular signal-related kinase-1 and -2, and mitogen-activated protein kinases, p38 kinase and c-jun N-terminal kinase, all phosphorylate many of these sites, although with different efficiencies for particular sites. Phosphorylation studies in transfected cells and neurons show that GSK-3 phosphorylates tau more extensively than do these other proline-directed kinases. Mutations in tau have been shown to affect in vitro phosphorylation of tau by GSK-3. The Arg406-->Trp (R406W) tau mutation also affects tau phosphorylation in cells.

Abstract Patient derived xenografts (PDX) mouse models for many types of cancer have demonstrated therapeutic responses similar to those seen in patients. Thus, resected patient tumor tissue directly implanted into immune compromised mice followed by therapy and tumor growth analysis is thought to be the closest preclinical model to predict patient therapeutic responses. To date, few models are available for the different histotypes of thyroid cancer derived from follicular thyrocytes; these include papillary, follicular, Hurthle cell, squamous and anaplastic thyroid carcinoma. We have developed eight PDX models in athymic nude mice representing all of these subtypes. The models have been extensively characterized for mutational status (i.e. BRAF, telomerase, RAS, PTEN, TP53, RB, etc) as well as validation by short tandem repeat (STR) analysis to match that of the originating patient tumor tissue. Squamous and anaplastic thyroid cancers are rare tumor types with no FDA approved therapies. Each model demonstrated their own unique responses to radiation, cytotoxic or molecular targeted therapies such as doxorubicin, sorafenib (tyrosine kinase inhibitor), cisplatin, paclitaxel, sunitinib (tyrosine kinase inhibitor), erlotinib (EGFR inhibitor), trametinib (MEK inhibitor) and vemurafenib (BRAF inhibitor). We expect that these models may provide useful in vivo models for thyroid cancer research as well as models for therapeutic guidance based upon histotype, mutational status and response to therapies. Citation Format: Laura A. Marlow, Adam C. Mathias, Louis K. Dawson, William F. Durham, Kenneth A. Meshaw, Robert J. Mullin, Daniel L. Small, Aidan J. Synnott, Dragana Milosevic, Brian C. Netzel, Stefan K. Grebe, Kevin Wu, Robert C. Smallridge, John A. Copland. Characterization of novel thyroid PDX models and their response to combination therapies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1458. doi:10.1158/1538-7445.AM2015-1458

Abstract Methyl-lysine (Kme) recognition domains play a central role in epigenetic regulation during cellular differentiation, development, and gene transcription with more than 200 known “reader” domains in the human proteome. We describe our target-class approach to ligand design and discovery for three cancer relevant members of this large family: L3MBTL3 (a Malignant Brain Tumor (MBT) domain containing reader), 53BP1 (a tandem Tudor domain) and CBX7 (a chromo domain). The advantages of a small molecule driven approach to modulating chromatin biology are numerous: temporal resolution; mechanistic flexibility (targeting a specific activity of a protein as opposed to ablating them all with transgenic knock-outs or RNA-interference techniques); ease of delivery; and most significantly, when warranted, a small molecule tool has the potential to provide an immediate transition to a drug discovery effort, potentially cutting years off the time between target validation and therapeutic intervention. UNC1215 binds the MBT domains of L3MBTL3 with a Kd of 120 nM, competitively displacing mono- or dimethyl-lysine containing peptides. This probe is greater than 50-fold selective versus other members of the human MBT family and also demonstrates selectivity against more than 200 other Kme reader domains examined. UNC1215 increases the cellular mobility of GFP-L3MBTL3 fusion proteins and point mutants that disrupt the Kme binding function of GFP-L3MBTL3 phenocopy the effects of UNC1215. The potency, specificity, and cellular effects of UNC1215 establish it as the first cell-active antagonist of a Kme reader domain and a useful chemical probe for biological studies of the function of L3MBTL3 (James, L. I. et al. Nat Chem Biol 2013, 9, 184-191). 53BP1 is a Kme binding protein that plays a central biological role in DNA Damage Repair (DDR) pathway via its recruitment to sites of DNA double strand breaks (DSB). BRCA1 is a checkpoint and DNA damage repair gene that is required for maintenance of genomic integrity, and the inheritance of mutated BRCA1 is a major risk factor for breast and ovarian cancer. The BRCA1 knockout murine model is embryonic lethal and a conditional knockout in mammary glands results in low frequency and long latency of mammary tumor formation. It was recently reported that the BRCA1-null developmental phenotype is rescued when placed on a 53BP1-null background. Adult mice from this model, that are null for both the 53BP1 and BRCA1 genes, age normally and display a very low incidence of tumor formation. Genomic instability can be rescued in 53BP1 knockouts because the homologous recombination (HR) pathway is largely restored in cells lacking both BRCA1 and 53BP1 (Cao, L. et al. Molecular cell 2009, 35 (4), 534-41). Based on this data, small molecule antagonists of 53BP1's biological function could act as viable therapeutics to restore HR in the DDR response in humans with BRAC1 mutations. We will report the first small molecule, fragment-like ligands with selectivity for binding to 53BP1. CBX7 is a chromo domain containing Kme reader that, along with other CBX domains, recruits the PRC1 complex to histone 3, lysine 27 trimethylation sites (H3K27me3) and enables transcriptional silencing downstream of the PRC2 complex which ‘writes’ the H3K27me3 mark via the catalytic activity of EZH2. As EZH2 is a well validated drug target with inhibitors now entering the clinic for the treatment of various cancers, we hypothesize that antagonism of the PRC1 complex recognition of H3K27me3 will phenocopy and perhaps synergize with the activity of EZH2 inhibitors (Konze, K. D. et al. ACS Chemical Biology 2013, 8, 1324-1334). Progress towards a chemical probe to validate CBX7 as a therapeutic target will be presented. The pursuit of high quality chemical probes for KMe reader proteins represents an emerging area that will open new avenues of research in chromatin biology and may in time, translate to new therapeutic approaches. Antagonists of the readers of acetylated lysine (BRD4 bromo domains) derived from pioneering chemical probe efforts (Filippakopoulos, P. et al. Nature 468, 1067-73 (2010)) have recently entered the clinic for the treatment of various malignancies and we hope that our chemical probe work will help to validate Kme readers as novel and significant anti-tumor targets. Citation Format: Lindsey I. James, Jake I. Stuckey, Brandi M. Baughman, Michael T. Perfetti, Samantha G. Pattenden, Brad M. Dickson, Jacqueline L. Norris, Gaofeng Cui, Pavel Mader, Aiping Dong, Yunxiang Mu, Scott B. Rothbart, Brian D. Strahl, Jinrong Min, Peter J. Brown, Dmitri B. Kireev, William P. Janzen, Kevin M. McBride, Mark T. Bedford, Georges Mer, Cheryl H. Arrowsmith, Stephen V. Frye. Targeting chromatin regulation for cancer therapy: progress towards chemical probes for methyl-lysine readers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr SY08-03. doi:10.1158/1538-7445.AM2015-SY08-03

Abstract Glioblastoma (GBM), a highly invasive primary brain tumour, remains an incurable disease (1). Angiogenesis, the formation of new blood vessels, is a defining feature of GBM (2). Targeting GBM angiogenesis with Bevacizumab (Bev) is associated with improved progression free survival, but may also significantly enhance local tumour invasion (3). RhoGTPases and their activators, guanine nucleotide exchange factors (GEFs), play central roles in the invasive process (4). Herein, we sought to identify and target GEFs of importance in meditating GBM invasion with a view to improving Bev response. We report a novel strategy by which GBM tumours invade and proliferate via overexpression of the GEF beta-PIX which was shown to be increased at the invasive edge in 74% of GBM tumours assessed (n = 19) compared with tumour core (5). We have further shown that siRNA-mediated knockdown of beta-PIX in GBM patient-derived xenograft cell cultures and cell lines resulted in decreased cell invasion in 3D, cell proliferation and survival assays in vitro. Furthermore, we have uncovered a role for beta-PIX expression in endothelial cell function, as knockdown of beta-PIX inhibits HUVEC cell migration in vitro. To interrogate in vivo effects, we established an imagable orthotopic model of invasive GBM in mice, by stereotactically implanting GBM cells stably overexpressing firefly luciferase (U87R-GFP-luc2). beta-PIX-(human and mouse) specific siRNA or scrambled control siRNA (2 × 10 μg i.c.) were delivered to tumours using an alphaVbeta3 integrin targeting-(RGD peptide conjugated)-nanoparticle (InVivoPlex Aparna Bio Corp, Rockville, MD). Animals were treated with- or without- Bev (6 × 10mg/kg i.p. over 12 days). Initial data has shown that animals treated with beta-PIX siRNA nanoparticles in combination with Bev had an improved median survival of 100 days compared with Bev- alone treated mice (median: 88 days). Our data may support the future clinical use of a beta-PIX-targeting nanoparticle therapeutic, to improve Bev outcomes in GBM. 1. Kohler BA et al J Natl Cancer Inst. 2011; 103, 714-736 2. Jain RK et al Nat Rev Neurosci. 2007; 8(8):610-22 3. Norden AD et al Neurology 2008; 70: 779-787 4. Murray D et al Brit J Can 2014; 110(5):1307-15 5. Hoelzinger DB et al Neoplasia 2005; 7(1): 7-16 Citation Format: David W. Murray, Philip O'Halloran, Monika Jarzabek, Brian MacCarthy, Jann N. Sarkaria, Raymond M. Schiffelers, Marc Symons, Annette T. Byrne. Pre-clinical assessment of a novel anti-invasion nanoparticle therapeutic in combination with bevacizumab for the treatment of glioblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5093. doi:10.1158/1538-7445.AM2015-5093

Abstract Genome-wide association studies (GWAS) of multiple myeloma (MM) in Northern Europeans have identified seven novel risk loci (2p23.3, 3p22.1, 3q26.2, 6p21.33, 7p15.3, 17p11.2, 22q13.1). We performed a multiethnic meta-analysis of these regions in 1,274 MM patients and 1,486 controls of European ancestry (EA) and 1,049 MM patients and 7,080 controls of African ancestry (AA), leveraging the differential linkage-disequilibrium of these populations in order to better localize the putative functional variants. We observed directionally consistent effects for all seven index SNPs in both populations, with four significantly associated (p<0.05) with risk in EAs (3p22.1, 7p15.3, 17p11.2, 22q13.1), and two significantly associated with risk in AAs (7p15.3 and 22q13.1). In a fixed effects meta-analysis of six regions (excluding the HLA region on chromosome 6), variation in five of the regions (2p33.3, 3p22.1, 7p15.3, 17p11.2, 22q13.1) had statistically significant associations with risk (Table 1). In one region, the index variant had the strongest association [rs4487645 at 7p15.3, (OR = 1.30, p = 8.7×10−8)]. Five of the six most significantly associated variants identified in the multiethnic analyses overlapped with biologically relevant features indicating regulatory activity based on CD20+ (B lymphocyte) cells, showing evidence of potential function; those included a missense variant in (17p11.2, rs34562254, Pro251Leu) in TNFRSF13B, which encodes a lymphocyte-specific protein in the tumor necrosis factor receptor family that interacts with the NF-kb pathway. Our study shows that these regions are important in MM risk across ethnicities and further supports the use of multiple ethnic groups in genetic studies to enhance identification of risk variants. Table 1.Most significant associations for each region in the multiethnic meta-analysis.Individuals of European AncestryIndividuals of African AncestryMulitethnic Metar2 with IndexcCHRSNPRAaFreqbORP-valueFreqbORP-valueORP-valueP-het2rs732075G0.591.222.0×10−30.621.122.0×10−21.162.6×10−40.280.09/0.283rs73069394A0.191.243.0×10−30.621.181.5×10−21.201.3×10−50.550.77/0.963rs12637184G0.761.136.0×10−20.921.192.7×10−11.151.0×10−20.640.94/1.007rs4487645C0.671.237.0×10−40.891.485.5×10−51.308.7×10−80.07-d17rs34562254A0.121.452.4×10−50.131.212.2×10−31.312.5×10−60.120.33/0.9022rs139400T0.491.224.0×10−40.531.172.1×10−31.191.2×10−60.630.63/0.96aRisk allelebFrequency of the risk allele in European and African ancestry studiescr2 metrics based on 1000 Genomes Project AFR/EUR populationsdIndex SNP Citation Format: Kristin A. Rand, Chi Song, Eric Dean, Daniel Serie, Karen Curtin, Dennis Hazelett, Amie E. Hwang, Xin Sheng, Alex Stram, David J. Van Den Berg, Carol Ann Huff, Leon Bernal-Mizrachi, Michael H. Tomasson, Sikander Ailawadhi, Anneclaire De Roos, Seema Singhal, Karen Pawlish, Edward Peters, Catherine Bock, David V. Conti, Graham Colditz, Todd Zimmerman, Scott Huntsman, John Graff, African Ancestry Prostate Cancer GWAS Consortium,African Ancestry Breast Cancer GWAS Consortium, Stephen J. Chanock, Michael Lieber, Jayesh Mehta, Eric A. Klein, Nalini Janakiraman, Richard K. Severson, Angela R. Brooks-Wilson, Vincent Rajkumar, Elizabeth E. Brown, Laurence Kolonel, Susan Slager, Brian E. Henderson, Graham G. Giles, John J. Spinelli, Brian Chiu, Kenneth C. Anderson, Jeffrey Zonder, Robert Z. Orlowski, Sagar Lonial, Nicola Camp, Celine Vachon, Elad Ziv, Dan O. Stram, Christopher A. Haiman, Wendy Cozen. Multiple myeloma susceptibility loci examined in African and European ancestry populations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4629. doi:10.1158/1538-7445.AM2015-4629

Abstract The success of clinical trials for patients with malignant melanoma (MM) designed from the “promising” results using pre-clinical models is minimal. With its increase in incidence, there is a dire need to develop both pre-clinical models comparable to clinical practice that have the capability to translate from bench-to-bedside and novel therapeutic agents for this life-threatening form of skin cancer. To address this concern, we have developed a panel of pre-clinical MM models including patient matched cell lines, patient cell-line derived xenograft models and patient tumor tissue-derived xenograft models. Patient derived xenograft (PDX) models are purported to most closely replicate a patient's response to therapy. Since the development of patient-derived cell lines and tumors grown in mice were more efficacious, their genetic, phenotypic, and therapeutic responses were compared to those of their corresponding patient-matched PDX model. Each model was molecularly characterized to confirm a match to that of the originating patient tumor tissue via protein immunohistochemistry (IHC), mRNA quantitative polymerase chain reaction (qPCR), DNA short tandem repeat (STR) analysis, and Sanger sequencing. Each pre-clinical model provided a genetic match to their respective patient tumor sample. All pre-clinical models were positive for the melanoma-specific markers (S-100 beta, HMB-45, and Melan-A) confirming a melanoma phenotype. Soft agar studies and tumor growth of our PDX and patient cell-line derived xenograft models confirmed tumorigenicity within our models providing the foundation to perform therapeutic studies. Mutation status data (i.e. BRAF, RAS), patient therapeutic clinical history and oncogene overexpression analysis (pERK, pAKT, PI3K, pmTOR, etc.) provide the platform and rationale behind the use of specific therapeutic agents per model. BRAF, NRAS and KRAS mutations were identified along with elevated pERK, pAKT and pmTOR in the models. Targeted therapies (trametinib/MEK inhibitor; vemurafenib/BRAF inhibitor; MK-2206/pAKT inhibitor; and everolimus/mTOR inhibitor) demonstrated dose dependent inhibition of cell proliferation in each of the models based upon oncogenic expression or mutations. In vivo experiments are in progress, which will allow comparison of the three models. The next steps include combination therapy to identify antitumor synergy targeting oncogenic signaling pathways. Citation Format: Antoneicka L. Harris, Laura Marlow, Adam Mathias, Louis Dawson, William Durham, Kenneth Meshaw, Robert Mullin, Daniel Small, Aidan Synnott, Kevin Wu, Dragana Milosevic, Brian Netzel, Stefan Grebe, Svetomir Markovic, John Copland. Characterization of novel patient derived melanoma xenografts and cell lines in response to targeted therapies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2417. doi:10.1158/1538-7445.AM2015-2417