bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3130: Dependency on c-MYC in multiple myeloma.
Authors: Holien Toril , Våtsveen Thea K. , Hella Hanne , Waage Anders , Sundan Anders
Published: August 2014

The oncogene c-MYC is deregulated in multiple myeloma. Importantly, the activity of c-MYC is elevated in plasma cells in patients with myeloma compared to the premalignant state monoclonal gammopathy of undetermined significance, indicating a prominent role for c-MYC in myeloma pathogenesis. We earlier found the bone morphogenetic proteins induce apoptosis in myeloma cell lines and primary cells by SMAD-dependent downregulation of c-MYC. Repression of c-MYC by short hairpin RNA in myeloma cell lines has by others also been shown to induce apoptosis. We wanted to go one step further and study the role of c-MYC in primary myeloma cells. Transcriptional regulation by c-MYC requires heterodimerization of c-MYC and its obligate partner MAX. The small molecular inhibitor 10058-F4 disrupts the formation of such heterodimers and was used in vitro to test the dependency on c-MYC activity in primary myeloma cells and myeloma cell lines. The inhibitor induced apoptosis in a dose-dependent manner in the 22 primary samples tested. Apoptosis was induced in five of six cell lines tested. The sixth cell line, U266, does not express c-MYC, and was not sensitive to the inhibitor. Using the INA-6 myeloma cell line, we found that apoptosis induced by 10058-F4 correlated with downregulation of the anti-apoptotic protein Bcl-xL. We also found that apoptosis was induced by the inhibitor even in the presence of bone marrow stromal cells derived from myeloma patients. Our results further support that c-MYC is a potential target for therapy in multiple myeloma.

Citation Format: Toril Holien, Thea K. Våtsveen, Hanne Hella, Anders Waage, Anders Sundan. Dependency on c-MYC in multiple myeloma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3130. doi:10.1158/1538-7445.AM2013-3130
Abstract 3131: Androgen receptor promotes ligand-independent prostate cancer progression through c-Myc upregulation.
Authors: Gao Lina , Schwartzman Jacob , Gibbs Angela , Kleinschmidt Richard , Wilmot Beth , Bottomly Daniel , Coleman Ilsa , Nelson Peter , McWeeney Shannon , Alumkal Joshi J.
Published: August 2014

Prostate cancer is the most common cancer in men in the United States and the second leading cause of cancer death. The androgen receptor (AR) is the central signaling pathway in prostate cancer. Because of this, androgen deprivation therapy (ADT), which involves reducing levels of androgen ligands or interfering with binding of ligands to AR, has been the major therapeutic focus for the past 70 years. However, in many patients there is no benefit from ADT, and all prostate cancers eventually progress. At progression on ADT, the AR is ubiquitously expressed. For this reason, we contend that AR-dependent mechanisms are critical for prostate cancer progression after ADT. Indeed, recent work demonstrates that the AR can function independently of ligands to promote prostate cancer cell survival. However, specific ligand-independent AR target genes that account for this effect were not well-characterized. We show here that c-Myc, which is a key mediator of ligand-independent prostate cancer progression, is a key ligand-independent AR target gene.

Using microarray analysis, we found that c-Myc and AR expression levels strongly correlated with each other in tumors from patients with castration-resistant prostate cancer (CRPC) progressing despite ADT. We confirmed that AR directly regulates c-Myc transcription in a ligand-independent manner, that AR and c-Myc suppression reduces ligand-independent prostate cancer cell growth, and that ectopic expression of c-Myc attenuates the anti-tumor activity of AR suppression. Importantly, treatment with the bromodomain inhibitor JQ1 suppressed c-Myc function and suppressed ligand-independent prostate cancer cell survival. Our results define a new link between two critical proteins in prostate cancer - AR and c-Myc - and demonstrate the potential of AR and c-Myc-directed therapies to improve prostate cancer control.

Citation Format: Lina Gao, Jacob Schwartzman, Angela Gibbs, Richard Kleinschmidt, Beth Wilmot, Daniel Bottomly, Ilsa Coleman, Peter Nelson, Shannon McWeeney, Joshi J. Alumkal. Androgen receptor promotes ligand-independent prostate cancer progression through c-Myc upregulation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3131. doi:10.1158/1538-7445.AM2013-3131
Abstract 3151: High-throughput RNAi screening identifies a novel metastatic melanoma drug target in the context of PTEN mutation.
Authors: Yin Holly , Chow Donald , Sereduk Chris , Aziz Meraj , Kim Seungchan , Brown Kevin M. , Trent Jeffrey M.
Published: August 2014

It has become increasingly important to identify targeted therapy in a defined molecular context. Here we will present our data which suggests TERT, a telomerase reverse transcriptase, serves as a novel drug target in melanoma in the context of PTEN mutation. PTEN is mutated in ∼15% of melanoma patients. Genomic RNA-interference (RNAi) technology was applied for synthetic lethality screens in an effort to identify the targets which upon knock-down significantly decrease cell viability specifically in the context of somatic mutations of the PTEN gene. We carried out such screen in a panel of 15 melanoma cell lines, including four PTEN mutated cell lines, against an siRNA library covering the kinome (∼700 genes with 4 siRNA per target) using CellTiter Glo as the endpoint to measure cell viability. Lethal genes, which decreased cell viability significantly upon knockdown, were initially identified for each cell line. Then the hit lists were compared between wild-type PTEN and mutant PTEN using a chi-square test, a statistical analysis. 29 gene candidates were selected for hit confirmation using the same set of siRNA, among which 24 genes were confirmed as potential PTEN synthetic lethal candidates, this results in a high confirmation rate of 83%. Based upon its association with melanoma risk (Law et al, J Invest Dermatol, 2012), We further validated TERT using a commercially available small molecule inhibitor. This inhibitor has significantly higherpotency in the PTEN mutant context (average IC50 of 0.34uM) compared to PTEN wild-type background (average IC50 of 0.75uM). This result both validates our RNAi screening results, but more importantly, this compound can serve as an initial structure scaffold for further optimization towards development as a novel drug for metastatic melanoma patients who carry PTEN mutations. Evaluation of more TERT inhibitors in a diverse panel of melanoma cell lines is presently underway.

Citation Format: Holly Yin, Donald Chow, Chris Sereduk, Meraj Aziz, Seungchan Kim, Kevin M. Brown, Jeffrey M. Trent. High-throughput RNAi screening identifies a novel metastatic melanoma drug target in the context of PTEN mutation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3151. doi:10.1158/1538-7445.AM2013-3151
Abstract 3159: Modulating therapeutic targets in B-cell lymphoma: Loss-of-function RNAi screen for CD20 regulators.
Authors: Slabicki Mikolaj , Sellner Leopold , Jethwa Alexander , Boettcher Michael , Zenz Thorsten
Published: August 2014

The surface glycoprotein CD20 is expressed on healthy and malignant B-cells and can be therapeutically exploited by monoclonal antibodies (e.g. Rituximab, Ofatumumab, GA-101). Stem cells and pro-B-cells are lacking CD20 surface protein, which allows the reestablishment of B-cell populations after treatment. Other cell lineages do not express CD20. Rituximab has revolutionized clinical practice in the treatment of malignant B-cell lymphoma. However, refractory lymphoma cells can lose the expression of CD20 after Rituximab exposure and B-cell lymphoma with low CD20 surface expression may be less responsive to treatment. Despite the widespread use of CD20 antibodies across lymphoma subtypes, little is known about the endogenous regulation of CD20. It was shown that several chemical compounds may modulate the surface density of CD20 (e.g. histone deacetylase inhibitors), but the regulatory network controlling CD20 levels is not understood. We aim to dissect the CD20 regulatome and to exploit the findings for novel therapeutic strategies.

The CD20 regulatome is studied in a Burkitt lymphoma cell line (Raji) characterized by medium expression of CD20 in comparison to other lymphoma models. As a proof of principle, we were able to down-regulate the surface expression of CD20 assayed by FACS with four independent silencing triggers targeting CD20 mRNA. To discover novel regulators of CD20 expression, we performed an unbiased genome-wide RNAi screen. The screen hits are involved in diverse molecular mechanisms such as signaling, trafficking, chromatin remodeling and membrane composition.

The group of genes decreasing the expression of CD20 expression can provide an insight into the physiological and pathological machinery involved in CD20 trafficking and processing. The strongest hit in this group, next to CD20 knockdown, is potentially involved in the direct transport of CD20 to the cell surface. Clinically most relevant is the group of genes where silencing increases the expression of CD20. The two strongest hits in this category are a transcription factor and a gene involved in signaling. Detailed molecular characterization of several chosen hits is currently ongoing. Their inhibition may lead to increased sensitivity to anti-CD20 antibodies and thus provide starting points for rational drug design.

Citation Format: Mikolaj Slabicki, Leopold Sellner, Alexander Jethwa, Michael Boettcher, Thorsten Zenz. Modulating therapeutic targets in B-cell lymphoma: Loss-of-function RNAi screen for CD20 regulators. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3159. doi:10.1158/1538-7445.AM2013-3159
Abstract 3174: A real-time qPCR approach to detect fusions between the KIF5B and RET genes in non-small cell lung cancer.
Authors: Bell Katherine , Gaffney Dana , Martinez Gabriela , Platero Suso , Karkera Jayaprakash
Published: August 2014

Lung cancer is the leading cause of death in cancer worldwide. There are two major forms of lung cancer, including small cell lung cancer (SCLC) which accounts for approximately 20% of all lung cancers and non-small cell lung cancer (NSCLC) which accounts for approximately 80% of lung cancers. Around 25% of these lung cancer patients are never smokers and these cancers tend to be the result of single somatic mutation events. Several somatic events have been reported in NSCLC, including mutations in EGFR and KRAS along with an EML4-ALK fusion gene, however more than 40% of these cancers are the result of unknown genetic events. Recently several papers have reported a novel fusion gene resulting from a 10.6 Mb inversion on chromosome 10 which leads to a fusion between the KIF5B and RET genes. The RET gene is a well-known tyrosine-kinase proto-oncogene which has been linked to papillary thyroid carcinomas and its expression is generally very low in lung. RET tyrosine kinase stimulates autophosphorylation of the tyrosine kinase unit which activates several pathways including STAT3, RAS/ERK, MAPK, PI3K/AKT and SRC. In the KIF5B-RET fusion KIF5B retains its coiled-coil domain necessary for homodimerization and the RET retains its kinase function leading to aberrant activation of several kinase pathways. Several fusion genes between the exons of KIF5B and RET have been previously reported including KIF5B15:RET12, KIF5B16:RET12, KIF5B22:RET12, KIF5B23:RET12, KIF5B15:RET11, KIF5B24:RET8 and KIF5B24:RET11. In this study we developed a TaqMan qRT-PCR-based approach to evaluate the expression of these seven (7) KIF5B-RET fusion transcripts in 64 NSCLC fresh frozen biopsies, ranging from stage I to stage III, including 25 adenocarcinoma and 37 squamous cell carcinoma samples, respectively. Our findings confirm the presence of the fusion between KIF5B15 (exon 15) and RET12 (exon 12) at a frequency of 1.56% in all subtypes. The clinicopathological background of the KIF5B/RET fusion-positive patient agrees with previously reported trends for this fusion event consisting of a caucasian female, non smoker, with adenocarcinoma subtype. Although this percentage is relatively small, it still represents around 12,000 individuals worldwide that express this fusion transcript, presenting a promising biomarker for targeted therapeutics in the treatment of NSCLC disease.

Citation Format: Katherine Bell, Dana Gaffney, Gabriela Martinez, Suso Platero, Jayaprakash Karkera. A real-time qPCR approach to detect fusions between the KIF5B and RET genes in non-small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3174. doi:10.1158/1538-7445.AM2013-3174
Abstract 3185: The eSNV-Detect: a computational system to identify expressed single nucleotide variants from tumor transcriptomes.
Authors: Tang Xiaojia , Baheti Saurabh , Shameer Khader , Thompson Kevin J. , Kocher Jean-Pierre , Perez Edith A. , Thompson Aubrey E. , Kalari Krishna R.
Published: August 2014

MOTIVATION: Transcriptome sequencing (RNA-seq) is the most economical and information-rich next generation sequencing technology to study genomic medicine. Genomic features such as gene expression, transcript expression, fusion transcripts, long non-coding RNAs and expressed single nucleotide variants (eSNVs) can be obtained from RNA-seq data. There are several computational workflows to obtain gene counts, transcript counts and fusion gene candidates, but not a comprehensive system to identify expressed SNVs from RNA-sequencing data.

METHODS: We have developed a novel computational system (The eSNV-Detect) with parallel alignment strategies to identify expressed SNVs accurately from tumor transcriptome sequencing data. Sequence-based features like reads supporting reference allele, alternate allele, ratio of alternate reads to total read depth, base qualities, mapping qualities, forward strand and reverse strand supporting reads etc. along with a variety of databases such as dbSNP, 1000 genome, 5400 exome, refGene, avsift, ljb_phylop from UCSC were used to call SNVs from RNA-sequencing data. For the expressed SNVs detected in tumors, we can also identify the amino acid change and classify the protein domains using our method.

RESULTS: We have applied the eSNV-Detect method to call novel expressed SNVs in two tumors (one HER2 positive and one ER positive breast tumor). BWA and Bowtie were used to align the two breast tumors. Several filtering criteria's were applied to the tumors to remove any known (germ line) or low confidence SNVs. We randomly selected 83 and 31 eSNVs for the HER2 and ER positive tumors respectively and performed Sanger sequencing validation in tumor and matched normal tissues of the tumor. We were able to validate 81 out of 83 eSNVs in the HER2 tumor sample, and 29 out of 31 eSNVs in the ER positive tumor sample. Of the eSNVs that we validated 39/81 and 10/31 were localized to functional protein domains in HER2 and ER positive tumors respectively. We are also in the process of validating eSNVs detected from RNA-seq with the tumor and blood exome sequencing data we have for the same patients.

Citation Format: Xiaojia Tang, Saurabh Baheti, Khader Shameer, Kevin J. Thompson, Jean-Pierre Kocher, Edith A. Perez, Aubrey E. Thompson, Krishna R. Kalari. The eSNV-Detect: a computational system to identify expressed single nucleotide variants from tumor transcriptomes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3185. doi:10.1158/1538-7445.AM2013-3185
Abstract 3194: Tristetraprolin controls the stability of uPA/uPAR mRNA through binding to the 3’UTR of uPA/uPAR mRNA in U87MG glioma cells.
Authors: Ryu Jinhyun , Choi Jungil , Lee Dong Hoon , Roh Gu Seob , Kim Hyun Joon , Cho Gyeong Jae , Choi Wan Sung , Park Jae-Yong , Park Jeong Woo , Kang Sang Soo
Published: August 2014

Malignant gliomas are one of the most common primary intrinsic brain tumors. In particular, glioblastoma multiforme (GBM), WHO grade IV, is highly invasive. Because of its high invasiveness, patients with unresectable GBM have a poor prognosis and have median survival of only a few months. The invasiveness of glioma depends on proteolysis of the extracellular matrix, a complex event occurring through the surrounding tissue during tumor cell invasion. Urokinase plasminogen activator (uPA) and its specific receptor (uPAR) play a major role in the infiltrative growth of glioblastoma. Tristetraprolin (TTP or ZFP36) is a tandem CCCH zinc-finger RNA-binding protein that regulates the stability of certain AU-rich element (ARE) mRNAs. Recent work reports that TTP is deficient in many cancer cells when compared with normal cell types. Moreover, the lack of TTP is associated with a variety of cancer-related processes. In the present study, we investigated the post-transcriptional regulation of uPA/uPAR expression by the tristetraprolin (TTP) in U87MG human glioma cells. Here, we showed that the overexpression of TTP decreased the stability of uPA/uPAR mRNA and the expression level of uPA/uPAR protein. uPA/uPAR mRNA include AU-rich elements (AREs) within the 3’-untranslated region, and TTP destabilized a luciferase mRNA containing uPA/uPAR AREs. In addition, TTP inhibits cell invasion in U87MG glioma cells. Present study demonstrates that TTP suppress the progression of glioma through the regulation of uPA/uPAR mRNA stability.

Citation Format: Jinhyun Ryu, Jungil Choi, Dong Hoon Lee, Gu Seob Roh, Hyun Joon Kim, Gyeong Jae Cho, Wan Sung Choi, Jae-Yong Park, Jeong Woo Park, Sang Soo Kang. Tristetraprolin controls the stability of uPA/uPAR mRNA through binding to the 3’UTR of uPA/uPAR mRNA in U87MG glioma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3194. doi:10.1158/1538-7445.AM2013-3194
Abstract 3196: Androgen receptor 3’ untranslated region (UTR): a conundrum and a significant therapeutic opportunity.
Authors: Shukla Girish C. , Sikand Kavleen , Ebron Jey S.
Published: August 2014

Master transcriptional regulator Androgen Receptor (AR) remained a fascinating and an enigmatic molecule, particularly in Prostate cancer biology. Major impetus in PCa research is currently focused on the development of new generation of therapeutics to target AR expression, especially in castration-resistant stage of the disease. 920 amino acid coding AR gene encompasses over 180 kilobase (kb) of X-chromosome containing eight exons that produces a nearly 10.7 kb AR mRNA. The fully processed mature AR mRNA account for only 26% of total coding capacity and most of the transcript codes for noncoding regions that includes 5’ and 3’ UTRs. However, functional roles of UTRs in AR gene expression, principally its regulatory involvement in development of CRPC remained indefinable. Our research is focusing on understanding the functional roles of 6.8 kb long AR 3’ UTR in AR gene expression and especially the molecular mechanism that revitalizes AR expression in CRPC. We have shown that a number of regulatory noncoding miRNAs play a critical 3’ UTR targeting role in AR gene expression and has potential therapeutic implications. Here we show a synergistic approach of targeting AR expression mediated via its 3’ UTR, using a miRNA cocktail combinatorial approach. In addition, we are investigating the potential functional association of 3’ UTR in AR gene expression and implication in PCa.

Citation Format: Girish C. Shukla, Kavleen Sikand, Jey S. Ebron. Androgen receptor 3’ untranslated region (UTR): a conundrum and a significant therapeutic opportunity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3196. doi:10.1158/1538-7445.AM2013-3196

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 3199: Ceramide restores the expression of wild-type p53 tumor suppressor in mRNA splicing through PP1 and ASF/SF2.
Authors: Liu Yong-Yu , Patwardhan Gauri A.
Published: August 2014

p53 mutants that have been detected in approximately 50% of tumors loss the activity in stabilizing the genome and tumor suppression, and further exhibit oncogenic function in cancer cells. Most tumors that exhibit disrupted p53 signaling pathways remain addicted to p53 mutants, and p53 mutants have emerged as perhaps the most important target to improve cancer treatment. Most works in restoring p53 activity focus on either altering protein conformations of the mutants or introducing foreign p53 gene into cells. Using gene silencing strategy and mass spectrometry, here we demonstrated that ceramide modulates the RNA splicing to restore the expression of wild-type p53 in p53 deletion-mutant ovarian cancer cells. Enhanced endogenous long-carbon chain ceramide species (C16- to C24-ceramides) and exogenous C6-ceramide, but not other sphingolipids, restored wild-type p53 mRNA (intact exon-5), phosphorylated p53 protein (Ser15 in exon-5) and p53-responsive proteins (p21, Bax) in cells that dominantly expressed a deleted p53-mRNA and protein in exon-5. Thus, the restored p53 sensitized the cells to induced apoptosis. Furthermore, ceramide activated protein phosphatase 1 (PP1), rather than PP2a, increased the translocation of alternative splicing factor ASF/SF2 from cytoplasm to nucleus and thus determined the pre-mRNA splicing for wild-type p53. Alternative pre-mRNA splicing not only generates more than one protein isoform from single gene, it also results in gene mutants in cancer and other diseases. Our study elucidates a previous unrecognized mechanism in regulating the expression of wild-type versus mutant protein of

p53 and indicates mutants can be restored in posttranscriptional levels by using an epigenetic approach.

Citation Format: Yong-Yu Liu, Gauri A. Patwardhan. Ceramide restores the expression of wild-type p53 tumor suppressor in mRNA splicing through PP1 and ASF/SF2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3199. doi:10.1158/1538-7445.AM2013-3199
Abstract 32: Single and multiple dose-ranging evaluation of safety, pharmacokinetics, and pharmacodynamics of GS-9973, a novel pSYK inhibitor.
Authors: Ramanathan Srini , DiPaolo Julie A. , Doan Tom , Burge Dan
Published: August 2014

Background: GS-9973 is a selective, reversible, ATP-competitive small molecule spleen tyrosine kinase (Syk) inhibitor that blocks BCR-mediated activation/ proliferation, Fc receptor signaling/ function, and inhibits immune complex-mediated inflammatory cytokine production, including TNFα. The safety, pharmacokinetics (PK), and pharmacodynamics (PD) of GS-9973 were evaluated in a double-blind, single/multiple ascending dose study.

Methods: In sequential cohorts (n=8 active, 2 placebo), subjects (fasted) received GS-9973 (25 mg to 1200 mg) as a single dose (Part A) or multiple twice-daily doses for 7 days (Part B). Along with PK, PD sampling Parts A and B assessed functional inhibition of ex vivo αIgE-stimulated CD63 expression on basophils. Safety and tolerability were assessed throughout the study, with dose-escalation gated by pre-specified stopping criteria. PK-PD was evaluated using GS-9973 concentrations vs CD63 inhibition.

Results: Across dose cohorts, subjects were ≥ 60% white, ≥ 50% male, with mean age of 28 to 37 years. Adverse events (AEs) were generally mild to moderate, with no AE-driven study drug discontinuations noted. GS-9973 exposures (Table 1) reached a plateau at ≥ 600 mg BID (due to solubility-limited absorption), provided > 90% CD63 inhibition at peak levels, and > 60% inhibition over the dosing interval, due to 10-14 hr plasma half-life. PK-PD analyses indicated a sigmoidal exposure-response relationship, with EC50 of ∼ 400-450 ng/ml and hill slope > 1.

Single and Multiple dose PK of GS-9973 Part A Single Dose (mg) AUCinf (ng.h/ml) Cmax (ng/ml) 25 231 (80) 42.3 (76) 75 413 (98) 67.8 (84) 200 4250 (64) 534 (56) 600 5960 (90) 779 (70) 1200 9740 (111) 1060 (101) Part B Multiple Dose (mg) AUCtau Cmax Ctrough T1/2 (hr) 25 356 (65) 57.8 (68) 11.9 (65) 8.7 (7.7, 9.5) 75 1880 (48) 273 (52) 79.2 (64) 8.9 (6.9, 10.8) 200 7650 (57) 1050 (59) 380 (63) 10.0 (8.4, 14.9) 600 21500 (64) 2960 (61) 1240 (66) 15.4 (12.8, 19.7) 900 23900 (62) 2810 (58) 1470 (67) 13.5 (11.7, 16.9) 1200 26800 (77) 3430 (77) 1470 (80) 13.9 (9.8, 16.4) Values presented as mean (%CV) except for T1/2 (median (Q1,Q3)); Part B: Cmax, Ctrough units ng/ml; AUCtau: ng.h/ml

Conclusions: GS-9973 was generally well tolerated over a 48-fold dose range. GS-9973 exposures plateaued ≥ 600 mg and provided robust exposure-dependent PD activity over the dosing interval. The overall safety, PK, and PD profile of GS-9973 support further clinical evaluation of doses with strong PD activity.

Citation Format: Srini Ramanathan, Julie A. DiPaolo, Tom Doan, Dan Burge. Single and multiple dose-ranging evaluation of safety, pharmacokinetics, and pharmacodynamics of GS-9973, a novel pSYK inhibitor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 32. doi:10.1158/1538-7445.AM2013-32
Abstract 3203: The tri-snRNP spliceosome complex is required for MYC-dependent cancer growth.
Authors: Adler Adam , Jiang Zhaoshi , McCleland Mark , Blackwood Elizabeth , Yee Sharon , Haley Benjamin , Stephan Jean-Philippe , Hussain Sofia , Chopra Vivek , Firestein Ron
Published: August 2014

Spliceosome coordinated RNA splicing is an essential cellular process that can generate an immensely diverse repertoire of RNA. While cancer cells have been known to hijack this process to generate splice forms with oncogenic function, the specific role of spliceosome components in this process is not well understood. In this study, we use an integrative genomic approach to identify PRPF6, a member of the tri-snRNP spliceosome complex as essential for colon cancer growth. We show that, in addition to PRPF6, other tri-snRNP components are coordinately overexpressed or amplified in cancer cells. Inhibition of the tri-snRNP complex, but not other spliceosome components, abrogated cancer cell growth only in dependent cancer cell lines in vitro and in vivo. High resolution transcriptome analysis reveals that the tri-snRNP complex binds and regulates the splicing of a relatively small number of genes, many of which are transcriptional targets of the c-MYC oncogene. Intriguingly, many components of the tri-snRNP complex are mutated in a genetic form of Retinitis Pigmentosa. This genetic corollary suggests that the tri-snRNP complex is necessary in specific cellular contexts that may depend on the proper transcription and splicing of a tri-snRNP regulated set of genes.

Citation Format: Adam Adler, Zhaoshi Jiang, Mark McCleland, Elizabeth Blackwood, Sharon Yee, Benjamin Haley, Jean-Philippe Stephan, Sofia Hussain, Vivek Chopra, Ron Firestein. The tri-snRNP spliceosome complex is required for MYC-dependent cancer growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3203. doi:10.1158/1538-7445.AM2013-3203

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 321: Sunitinib alternated with everolimus in renal cell carcinoma: a sequential combination strategy for antitumor efficacy in mice clinical trial.
Authors: Dos Santos Célia , Tijeras-Raballand Annemilaï , Serova Maria , Dreyer Chantal , Slimane Khemaies , de Gramont Armand , Faivre Sandrine , Raymond Eric
Published: August 2014

Background: Sunitinib (VEGFR/PDGFR inhibitor) and everolimus (mTOR inhibitor) are approved for advanced renal cell carcinoma (RCC), as first-line and second-line therapy, respectively. No data have been reported on programmed sequential strategies combining alternative use of sunitinib and everolimus prior to progression. Such strategy is expected to delay the emergence of first-line acquired resistance and improve tumor control. The aim of our study was to assess the effects of three sequences administration of sunitinib and everolimus.

Methods: Nude mice (n=5/group) were engrafted with human RCC cells (CAKI-1) then randomized to vehicle, everolimus, sunitinib, sunitinib alternated with everolimus every week (S1), every two weeks (S2), and every three weeks (S3). Median time to tumor progression (mTTP) was determined in each group. Angiogenesis and necrosis were evaluated using CD31 and HE staining. Inhibition of mTOR pathway was evaluated using phospho-S6 (pS6) and e-cadherin/vimentin expressions were analyzed by immunofluorescence staining.

Results: The S1, S2 and S3 regimens increased mTTP by 30 to 80% relative to sunitinib or everolimus alone. The vascular surface areas (vessels size and number) in tumors were reduced in all treated groups relative to control, especially S1 and S2. All three sequential therapies induced larger necrosis areas in tumors as compared to control. In each group, tumor response seemed associated with a decrease in pS6 expression and changes in epithelial-mesenchymal phenotype with a decrease in vimentin and an increase in e-cadherin expression

Conclusion: Sunitinib and everolimus programmed sequential regimens prior to progression yielded longer mTTP than sunitinib and everolimus monotherapies. The sequential combinations of these two agents in a RCC mouse clinical trial, increased antiangiogenic effects, leading potentially to tumor necrosis, especially with the S1 and S2 regimens.

mTTP for each group and effects of sequential treatments in tumors angiogenesis and necrosis Treatment groups mTTP (in days) Vascular Surface Area per field (% vs control) Number of vessels per field % of necrosis surface per field Control 24 100 22 23 Sunitinib 36 30 5 47 Everolimus 50 28 7 27 S1 64 15 5 37 S2 59 20 4 51 S3 59 42 5 37

Citation Format: Célia Dos Santos, Annemilaï Tijeras-Raballand, Maria Serova, Chantal Dreyer, Khemaies Slimane, Armand de Gramont, Sandrine Faivre, Eric Raymond. Sunitinib alternated with everolimus in renal cell carcinoma: a sequential combination strategy for antitumor efficacy in mice clinical trial. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 321. doi:10.1158/1538-7445.AM2013-321
Abstract 3210: Proteomic study of 5-fluorouracil resistance in colon cancer cell lines.
Authors: Guerrero-Valero Marta , Marin-Vicente Consuelo , Zubarev Roman A. , Visa Neus
Published: August 2014

The anticancer drug 5-fluorouracil (5-FU) is a chemotherapy agent that has been in use against colon cancer for about 40 years. However, inherent and acquired drug resistance phenomena often limit the effectiveness of 5-FU chemotherapy, which makes it necessary to explore the underlying resistance mechanisms. In addition, it is important to identify useful markers to predict the sensitivities of different tumors to 5-FU therapy.

In order to study the mechanism of 5-FU resistance, we used three 5-FU-resistant cell lines (HCT116/5-FU, H630/5-FU and RKO/5-FU) and their parental cell lines (HCT116, H630 and RKO). The proteomes of the untreated cells and of cells treated with 5-FU for 24 h were analyzed by label-free mass spectrometry using a nano-LC-ESI-LTQ Orbitrap Velos. When we compared the untreated proteomes, we could detect significant differences among resistant and parental cell lines, and we identified 21 proteins overexpressed and 12 underexpressed in the three resistant cell lines. Many of these proteins were associated with the mitochondria or were involved in carboxylic acid metabolism, in response to chemical stimuli, in regulation of programmed cell death, in oxidoreductase ativity or in DNA repair/cell cycle control pathways.

The observed differences among the 5-FU resistant and the parental proteomes were even more pronounced in cells treated with 5-FU for 24 h. The numbers of proteins overexpressed and underexpressed in the resistant cell lines rose to 25 and 17, respectively. Moreover, many of the differentially expressed proteins were associated to the ribosome and were involved in translation and RNA metabolism.

The mass spectrometric results were validated by semi-quantitative Western blotting and complemented with cell proliferation assays and with pathway analysis. Altogether, our results not only led to the identification of potential protein markers of 5-FU resistance, but also provided information about the underlying resistance mechanisms.

Citation Format: Marta Guerrero-Valero, Consuelo Marin-Vicente, Roman A. Zubarev, Neus Visa. Proteomic study of 5-fluorouracil resistance in colon cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3210. doi:10.1158/1538-7445.AM2013-3210
Abstract 3211: Proteomic analysis of nuclei dissected from FFPE tissue using expression microdissection.
Authors: Blackler Adele R. , Tangrea Michael A. , Pohida Tom , Morgan Nicole , Bonner Robert , Mora Keshia , Kakareka John , Markey Sanford , Emmert-Buck Michael R.
Published: August 2014

Currently, proteomic analysis of formalin-fixed, paraffin-embedded (FFPE) tissue (the most common method of storing tissue specimens) are limited by the overwhelming complexity of the cellular proteome, which makes disease related changes in protein expression difficult to detect. Expression Microdissection (xMD) allows for automated, operator-independent dissection of specific cells or subcellular organelles in FFPE tissue, resulting in the enrichment of specific, selectable protein populations. In xMD, tissue slices that have been stained for specific cellular markers using immunohistochemical (HC) methods are placed under a layer of ethylene vinyl acetate (EVA) polymer film. When the stack is uniformly illuminated (using a rastering laser or a light pulse), the EVA melts and makes contact only over the stained tissue, enabling specific capture and transfer of these areas for further biochemical analysis, while leaving unstained tissue behind.

As a proof of principle, we are using the xMD technique to enrich for nuclei in FFPE rat brain tissue sections followed by proteomic analysis. Shotgun proteomic analysis of these samples show a significant enrichment in nuclear localized proteins, with an average 18% of recovered proteins localized to the nucleus-versus 11% of recovered proteins in whole tissue scrapes. Targeted mass spectrometry, using Multiple Reaction Monitoring (MRM), also shows an enrichment of nuclear markers such as histones and DNA-binding proteins, and a de-enrichment of non-nuclear proteins. The enrichment for nuclei allows for the identification of methylated lysines on histones that are not detected in whole tissue scrapes, most likely due to their low abundance.

The evolving methodology may allow researchers to profile the sub-cellular proteome of patient samples to identify potential new disease biomarkers and drug targets. This novel technology could also be used to advance our understanding of the biomolecular changes responsible for a specific disease state.

Citation Format: Adele R. Blackler, Michael A. Tangrea, Tom Pohida, Nicole Morgan, Robert Bonner, Keshia Mora, John Kakareka, Sanford Markey, Michael R. Emmert-Buck. Proteomic analysis of nuclei dissected from FFPE tissue using expression microdissection. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3211. doi:10.1158/1538-7445.AM2013-3211
Abstract 3235: Characterization of tumor explant models using next-generation sequencing.
Authors: Lai Zhongwu , Bradford James , Zhang Qi , Dempster Sara , Dougherty Brian , D'Cruz Celina , Dry Jonathan
Published: August 2014

Tumor explant models (human tumor tissue directly transplanted into mice) are believed to conserve original tumor characteristics, including molecular heterogeneity and tumor architecture. Thus, they better represent patient population and can provide more relevant predictive insight into clinical outcome. They potentially offer a clinically relevant model in which to compare drug response to genomic and molecular profiles. So called next generation sequencing (NGS) technologies (Exome sequencing and RNA-seq) are becoming the standard choices for genetic profiling due to the rich information they can provide. The sequence based nature of these technologies offers the potential to differentiate results by species and remove mouse derived stromal ‘contamination’ when assessing tumor cells directly. This also presents a unique opportunity to differentiate and assess molecular interaction between the tumor and the stromal tissue.

To this aim, we have developed algorithms to differentiate NGS sequences between human and mouse. Using mixtures of human and mouse derived cell lines we demonstrated that the algorithm achieved high sensitivity (99.8%) and specificity (99.5%). More than 90% of sequence reads mapped to both human and mouse can be effectively differentiated. We applied the approach to differentiate human and mouse sequences for several explant models derived from both exome sequencing and RNA-seq. We demonstrated that the process resulted in more accurate expression quantification, and reduction of false positives in variance calling. In addition, we compared the variance calling between paired exome and RNA-seq and found that the two methods are complimentary to each other where genes were expressed. We also highlighted a number of interesting features and anomalies between RNAseq and exome data.

In summary, we demonstrated the feasibility of using exome and RNA-seq to effectively differentiate human and mouse sequences in tumor explants models. We highlight the value of this approach to rationally guide their application in drug discovery.

Citation Format: Zhongwu Lai, James Bradford, Qi Zhang, Sara Dempster, Brian Dougherty, Celina D'Cruz, Jonathan Dry. Characterization of tumor explant models using next-generation sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3235. doi:10.1158/1538-7445.AM2013-3235
Abstract 3254: Discovery of S3I-1757, a STAT3-STAT3 dimerization disruptor that inhibits STAT3 activity, tumor survival, migration and invasion.
Authors: Zhang Xiaolei , Sun Ying , Pireddu Roberta , Yang Hua , Urlam Murali K. , Lawrence Harshani R. , Guida Wayne C. , Lawrence Nicholas J. , Sebti Said M.
Published: August 2014

The ability of STAT3 to translocate to the nucleus, bind DNA and transcriptionally regulate STAT3 target genes depends on tyrosine phosphorylation STAT3-STAT3 dimerization. Our chemistry efforts led to the discovery of S3I-1757 that disrupts STAT3-STAT3 dimerization. Fluorescence polarization assays and molecular modeling show that S3I-1757 interacts with Y-705 binding site in the STAT3-SH2 domain and displaces fluorescein-labelled GpYLPQTV phosphotyrosine peptide from binding to STAT3. Furthermore, using co-immunoprecipitation and co-localization studies we show that S3I-1757 inhibits STAT3 dimerization and STAT3-EGF receptor binding in intact cells. Treatment of human cancer cells with S3I-1757 (but not a closely related analogue, S3I-1756, that does not inhibit STAT3 dimerization), inhibits selectively the phosphorylation of STAT3 over Akt and Erk1/2, nuclear accumulation of P-Y705-STAT3, STAT3-DNA binding and transcriptional activation and suppresses the expression levels of STAT3 target genes such as Bcl-xL, survivin, cyclin D1 and MMP9. Furthermore, S3I-1757 but not S3I-1756 inhibits anchorage-dependent and -independent growth, migration and invasion of human cancer cells which depend on STAT3 selectively over those that do not. Finally, STAT3-C, a genetically engineered mutant of STAT3 that forms a constitutively dimerized STAT3, rescues from S3I-1757 effects. Our results warrant further development of S3I-1757 as a STAT3-STAT3 dimerization inhibitor capable of blocking hyper activated STAT3 and suppressing malignant transformation of STAT3-dependent human tumors.

Citation Format: Xiaolei Zhang, Ying Sun, Roberta Pireddu, Hua Yang, Murali K. Urlam, Harshani R. Lawrence, Wayne C. Guida, Nicholas J. Lawrence, Said M. Sebti. Discovery of S3I-1757, a STAT3-STAT3 dimerization disruptor that inhibits STAT3 activity, tumor survival, migration and invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3254. doi:10.1158/1538-7445.AM2013-3254
Abstract 3257: Targeting small cell lung cancer harboring PIK3CA mutation with a selective oral PI3K inhibitor, PF-4989216.
Authors: Walls Marlena , Baxi Sangita M. , Mehta Pramod P. , Epps Elizabeth , Estrella Heather , Liu Kevin KC , Zhu JinJiang , Li Chunze , Smeal Tod , Yin Min-Jean
Published: August 2014

Aberrant PI3K/AKT signaling occurs commonly in cancer. Gene mutation, amplification, and copy number gains in the catalytic p110α of PI3K have been shown in a variety of human cancers. Tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) dephosphorylates the 3-phosphoinositides and is frequently mutated, deleted, or down-regulated in many human cancers to constitutively activate the PI3K pathway. Small cell lung cancer (SCLC) patients have poor prognosis; response to second-line chemotherapy for patients with refractory disease is less than 10%, and survival is 3-4 months. Multiple phase III trials have been conducted, however the survival of SCLC patients has not significantly improved over the years. In this study, we characterized a selective PI3K inhibitor, PF-4989216, in preclinical SCLC models to investigate whether targeting the PI3K pathway may be a potential therapy for SCLC. PF-4989216 inhibits phosphorylation of PI3K downstream molecules and subsequently leads to induction in apoptosis and cell cycle block, as well as inhibition of cell proliferation, transformation, and xenograft tumor growth in SCLCs harboring a PIK3CA mutation. Moreover, our results suggest that there may be different mechanisms of tumorigenesis between PIK3CA mutation and PTEN loss in SCLCs, and indicate that PF-4989216 is a potential cancer drug candidate for small-cell lung cancer patients harboring a PIK3CA mutation.

Citation Format: Marlena Walls, Sangita M. Baxi, Pramod P. Mehta, Elizabeth Epps, Heather Estrella, Kevin KC Liu, JinJiang Zhu, Chunze Li, Tod Smeal, Min-Jean Yin. Targeting small cell lung cancer harboring PIK3CA mutation with a selective oral PI3K inhibitor, PF-4989216. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3257. doi:10.1158/1538-7445.AM2013-3257
Abstract 326: Overexpression of the carboxyl-terminus of MUC16 can accelerate the occurrence of phenotypes in p53 heterozygous mouse model.
Authors: Tian Huasong , Zhou Qin , Iasonos Alexia , David Spriggs
Published: August 2014

Ovarian cancer is the fifth leading cause of cancer death in women. Cancer antigen 125 (CA125), also known as MUC16, is a high molecular weight glycoprotein overexpressed in sera of women with epithelial ovarian cancer, but not in normal epithelial ovarian tissue. As a biomarker, CA125 is widely used to screen, diagnose and monitor the epithelial ovarian cancer. To further understand the roles of MUC16 in tumorigenesis, recently, some mouse models related Muc16 and MUC16 were created. There is no phenotype for the Muc16 knockout mice (Cheon et al., PLoS ONE, 2009). We built a MUC16 carboxyl-terminus transgenic mouse (MUC16ΔC354) and found out that the transgenic mice appear to be viable, fertile, and develop normally through the more than two years of life. In order to investigate the relationship of p53 mutation and MUC16 overexpression in tumorigensis, we generated mice in which MUC16ΔC354 can be overxepressed with p53 heterozygote. Like the MUC16ΔC354 transgenic mice, the MUC16ΔC354; p53+/- mice develop normally. Both mouse strains are susceptible to spontaneous tumors, including sarcoma and lymphoma. However, comparing p53+/- mice, overexpression of MUC16ΔC354 significantly accelerates the occurrence of phenotypes in MUC16ΔC354; p53+/- mice. Out of a study population of 94 MUC16ΔC354; p53+/- mice, 22 (23.4%) developed obvious neoplasms by 18 months of age, 13 (11.9%) out of 109 for p53+/- mice (ρ <0.014). Tumor tissues and tumor cells carrying MUC16ΔC354 and p53+/- are transplantable. MAPK/ERK signaling pathway is upregulated in these tumors. Our findings demonstrate our hypothesis that MUC16 may play a role in tumorigensis.

Citation Format: Huasong Tian, Qin Zhou, Alexia Iasonos, Spriggs David. Overexpression of the carboxyl-terminus of MUC16 can accelerate the occurrence of phenotypes in p53 heterozygous mouse model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 326. doi:10.1158/1538-7445.AM2013-326