bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3552: Regulation of immune homeostasis by direct activator BH3-only proteins
Authors: Ludwig Lindsey M. , Roach Lauren E. , Fisher Jill K. , Walensky Loren D. , LaBelle James L.
Published: July 2016

The BH3-only proteins BIM, BID, and PUMA have emerged as both inhibitors of anti-apoptotic BCL-2 family proteins and direct activators of the pro-apoptotic executioner proteins BAX and BAK, and have been implicated in regulating immune cell homeostasis. For example, BIM is a master regulator of B and T cells and its deletion in mice causes splenomegaly and lymphocytosis. Although deletion of Bid or Puma do not grossly impact murine hematopoietic organs, loss of each can protect lymphocytes from certain forms of cell death. Combined deletion of Puma and Bim causes increased lymphocytosis and spontaneous development of B-cell lymphoma, while loss of BID and BIM leads to delayed T cell contraction following antigen-mediated proliferation. To investigate the role of direct activator BH3-only proteins in regulating the adaptive immune response over time, we performed peripheral lymphocyte analyses on single and combinatorial knock-outs of Bim, Puma, and Bid. Select genotypes, such as Bim−/−, Bim−/−Puma−/−, and Bim−/−Puma−/−Bid−/− mice, show lymphocytosis early in life but, unexpectedly, counts were observed to gradually normalize by 40 weeks of age. Given the dominant role of BIM in immune cell homeostasis, we investigated whether this phenotype was dictated by global deletion of Bim or intrinsic to T or B cell-specific Bim deletion. In generating and analyzing Lck-cre and CD19-cre Bim floxed animals, we found that T cell-specific Bim deletion recapitulated early lymphocyte expansion followed by contraction, whereas the lymphocytosis of mice with B cell-specific Bim deletion showed no age-related changes. Further characterization of aged Lck-cre Bim floxed mice revealed spleen weights similar to wild type mice, and thymocytes with increased sensitivity to a range of apoptotic stimuli; these mice also have an increased percentage of regulatory T cells compared to similarly aged wild-type mice, as has been reported for globally-deleted Bim−/− animals. To determine the mechanism underlying the observed lymphocyte contraction, we performed qRT-PCR analysis for a panel of BCL-2 family proteins. We find that aged Lck-cre Bim floxed mice manifest upregulation of a series of BH3-only transcripts, including Puma and Bid, reflective of an intriguing compensatory mechanism to remedy, over time, the lymphocytosis that derives from Bim deletion in T cells. These studies provide new insight into the complexity and dynamism underlying BH3-only regulation of immune cell homeostasis during health, disease, and aging.

Citation Format: Lindsey M. Ludwig, Lauren E. Roach, Jill K. Fisher, Loren D. Walensky, James L. LaBelle. Regulation of immune homeostasis by direct activator BH3-only proteins. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3552.
Abstract 3565: Dinaciclib, a CDK inhibitor promotes proteasomal degradation of Mcl-1 and enhances ABT-737 mediated cell death in malignant human glioma cell lines
Authors: Jane Esther P. , Premkumar Daniel , Cavaleri Jonathon , Rajasekar Thatchana , Sutera Philip , Pollack Ian
Published: July 2016

The prognosis of malignant glioma, the most common brain tumor, is still poor, thus underscoring the need to develop novel treatment strategies. Because glioma cells commonly exhibit genomic alterations involving genes that regulate cell cycle control, there is a strong rationale to examine the potential efficacy of strategies to counteract this process. In this study, we examined the antiproliferative effects of the cyclin-dependent kinase inhibitor dinaciclib in malignant human glioma cell lines, with intact or deleted or mutated p53 or PTEN or p14ARF, or EGFR amplification status. Dinaciclib was found to inhibit cell proliferation and induce cell cycle arrest at the G2/M checkpoint, independent of p53 mutational status. In a standard 72 h MTS assay, at clinically relevant concentrations, dose-dependent antiproliferative effects were observed but cell death was not induced. Moreover, the combination of conventional chemotherapeutic agents and various growth signaling inhibitors with dinaciclib did not yield synergistic cytotoxicity. In contrast, combination of the Bcl-2/Bcl-xL inhibitor ABT-263 or ABT-737 with dinaciclib potentiated the apoptotic response induced by each single drug. The synergistic killing by ABT-737 with dinaciclib led to cell death accompanied by the hallmarks of apoptosis, including an early loss of the mitochondrial transmembrane potential, the release of cytochrome c, smac/DIABLO and apoptosis-inducing factor (AIF), phosphatidylserine exposure on the plasma membrane surface and activation of caspases and PARP. Mechanistic studies revealed that dinaciclib promoted proteasomal degradation of Mcl-1.

Citation Format: Esther P. Jane, Daniel Premkumar, Jonathon Cavaleri, Thatchana Rajasekar, Philip Sutera, Ian Pollack. Dinaciclib, a CDK inhibitor promotes proteasomal degradation of Mcl-1 and enhances ABT-737 mediated cell death in malignant human glioma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3565.
Abstract 363: Establishment of multicellular tumor spheroids-based assay for screening of novel therapeutics
Authors: Song Yeonhwa , Seo Haengran
Published: July 2016

3D cell culture system is highlighted as new method to screen for new cancer therapies. The multicellular tumor spheroid cultures are closely similar to pathophysiological gradients of in vivo tumors and subsequently an ideal screening model for new drugs. Hepatocellular carcinomas (HCC) is resistant to conventional chemotherapeutic agents and remains an unmet medical need. Hence, we focused in developing tumor spheroids-based screening assay to identify new drugs to treat HCC.

Tumor spheroid cultures were formed in hydrogel coated multi-well plates within 3 days followed by 3 days treatment with reference compounds (Doxorubicin, Etoposide, Methotrexate, Taxol, etc.). Bright images of tumor spheroids were visualized by Operetta imaging system in the presence and absence of reference compounds. Cell viability of spheroids were measured by resazurin assay.

In the results, assay was developed and validated using multicellular tumor spheroids (MCTS) cultures composed of Huh7 (HCC cells), WI38 (fibroblasts), LX2 (hepatic stellate cells) and HUVEC (endothelial cells) to recapitulate HCC tumor microenvironments. We also addressed the effect of sorafenib on the growth of spheroids composed of Huh7 alone or MCTS. Results from this study indicate that sorafenib was less efficacious (IC50 = 8.0μM) in the MCTS compared to its effect on Huh7 spheroid (IC50 = 3.8 μM).

In summary, we have established and validated a highly reproducible MCTS-based assay. Based on this results, MCTS based-HTS system can offer a new model for drug screening and significantly improve the efficiency of identifying new drugs for liver cancer therapy.

Citation Format: Yeonhwa Song, Haengran Seo. Establishment of multicellular tumor spheroids-based assay for screening of novel therapeutics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 363.
Abstract 3641: ReCapSeg: Validation of somatic copy number alterations for CLIA whole exome sequencing
Authors: Lichtenstein Lee , Woolf Betty , MacBeth Alyssa , Birsoy Ozge , Lennon Niall
Published: July 2016

The presence of specific somatic copy number alterations (SCNAs) in tumor genomes can be used to predict sensitivity to specific treatments and to predict outcomes. CapSeg Revised (ReCapSeg) detects SCNAs from tumor sequencing data. The Broad Institute is integrating ReCapSeg into the Clinical Research Sequencing Platform (CRSP), a CLIA-certified platform, to enable physicians to use detected SCNAs in treatment decisions. By using sequencing data CRSP can produce SCNA reports using the same data being generated for other reports (e.g. somatic single nucleotide variants). ReCapSeg produces copy ratio estimates for regions of the genome and includes a caller that labels regions as amplified, deleted, or neutral. ReCapSeg leverages a panel of normals (PoN), which obviates the need for the matched normal for a given tumor sample.

Validation for ReCapSeg, in CRSP, is repeatable and standardized with a process for creating a PoN and gathering performance metrics from case samples. Additional QC metrics and criteria on input sequencing data were identified and included, so that reports can indicate when reduced performance can be expected.

Citation Format: Lee Lichtenstein, Betty Woolf, Alyssa MacBeth, Ozge Birsoy, Niall Lennon. ReCapSeg: Validation of somatic copy number alterations for CLIA whole exome sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3641.
Abstract 3654: In vivo CRISPR screen identidied Nf1 as a new tumor suppressor recurrently inactivated in liver cancer
Authors: Song Chunqing
Published: July 2016

Our CRISPR screen identified NF1, encoding a RasGAP, as a new tumor-suppressor gene. CRISPR mediated loss-of-function mutations in NF1 led to increase RAS activation and increased tumor formation in liver cells. NF1 expression was reduced in 60% (225/373) of human HCC and was associated with reduced patient survival. Point mutations of NF1 was found in 3% of HCC. These findings identify NF1 inactivation as a new tumor suppressor in the liver and highlight the importance of RasGAPs in liver cancer.

Citation Format: Chunqing Song. In vivo CRISPR screen identidied Nf1 as a new tumor suppressor recurrently inactivated in liver cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3654.
Abstract 3676: Prognostic significance of PTEN alteration in Middle Eastern colorectal carcinoma
Authors: Siraj Abdul K. , Beg Shaham , Bu Rong , Al-Rasheed Maha , Al-Sanea Nasser , Al-Dayel Fouad , Al-Kuraya Khawla
Published: July 2016

Phosphatase and tensin homolog (PTEN) detected on chromosome 10 is a tumour suppressor commonly reported to be inactivated in colorectal cancers, but the prognostic significance of PTEN remains controversial. Here, we investigated the clinical associations and the significant prognostic value of combined PTEN mutation, genomic deletion and loss of expression in a large cohort of Middle Eastern colorectal cancers. We examined PTEN protein expression using immunohistochemistry in 400 sporadic colorectal cancers with full clinical investigations and follow up data. PTEN mutations were studied using capture sequencing and Sanger sequencing validation. PTEN copy number alteration was carried out using Fluorescence In-Situ Hybridization (FISH). Of the all CRC tumours analysed 83/347 (24%) cases showed absence of PTEN staining, 189/347 (54.5%%) cases showed weak staining and remaining 75/347 (21.5%) cases showed staining similar to normal & internal positive control. 17 mutations in coding region of PTEN were identified in 13 (3.2%) CRC cases. Further, PTEN deletion was identified in 24/338 (7.1%) of CRC cases. No amplification of PTEN gene was detected. Interestingly when combining all PTEN alterations (protein expression loss, PTEN mutation and PTEN genomic deletion), it accounted for 97 (29.7%) CRC cases. Significant association of combined PTEN alterations was seen disease free survival (p = 0.0367). On multivariate analysis, this stands true (p = 0.0227) when adjusted for Age, Sex, Site, Grade and Stage. Thus, PTEN alteration is an independent marker of poor prognosis in CRC. In conclusion, PTEN gene alteration plays an important role in Middle Eastern colorectal cancer pathogenesis and it can help in identifying a subgroup of CRC with particular poor outcome.

Citation Format: Abdul K. Siraj, Shaham Beg, Rong Bu, Maha Al-Rasheed, Nasser Al-Sanea, Fouad Al-Dayel, Khawla Al-Kuraya. Prognostic significance of PTEN alteration in Middle Eastern colorectal carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3676.
Abstract 3690: Metabolic regulation of mutant p53 stability by the mevalonate pathway
Authors: Iwakuma Tomoo , Ranjan Atul , Iyer Swathi V. , Padhye Subhash , Weir Scott , Roy Anuradha
Published: July 2016

Introduction: Mutations in the p53 gene are mostly missense mutations and result in accumulation of dysfunctional p53 protein in tumors with oncogenic gain-of-function activities. Increasing evidence indicates that stabilization of mutant p53 (mutp53) in tumors is crucial for its oncogenic activities including tumor progression and drug resistance, while downregulation of mutp53 reduces oncogenicity of cancer cells. These observations suggest that malignant properties of cancer cells are dependent on the presence of mutp53, thus providing a rationale to identify compounds that deplete mutant p53 with little impact on wild-type p53.

Experimental procedures: Toward this goal, we performed high throughput screens of chemical libraries (∼9,000 compounds) with Saos2 (p53 null) cells expressing a fusion protein of p53R175H and luciferase, using luciferase as a reporter.

Summary of data: This screening led us to identify “statins”, a class of cholesterol-lowering medications, as compounds that induced degradation of p53R175H. We found that other inhibitors of the mevalonate pathway, such as 6-fluoromevalonate and zoledronic acid, failed to induce p53R175H degradation, while statin-mediated inhibition of HMG-CoA reductase and subsequent reduction in mevalonte-5-phosphate triggered p53R175H degradation. These results suggest that statin's effect on p53R175H is specific and independent of protein prenylation/lipidation or cholesterol synthesis. Moreover, nuclear export of p53R175H was required for the statin-mediated degradation, which was mediated through an E3 ubiquitin ligase CHIP, but not MDM2. Interestingly, statins induced degradation of mainly conformational p53 mutants with minimal effects on the levels of wild-type p53 and DNA contact mutants.

Conclusions: This is the first study demonstrating that mutp53 stability is regulated through a specific process of the mevalonate pathway, thereby providing a novel regulatory mechanism of mutp53 degradation. Our findings suggest that p53 mutation status in tumors may have an impact on efficacy of statins in cancer therapy.

Citation Format: Tomoo Iwakuma, Atul Ranjan, Swathi V. Iyer, Subhash Padhye, Scott Weir, Anuradha Roy. Metabolic regulation of mutant p53 stability by the mevalonate pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3690.
Abstract 3698: The effect of epigenetic silencing and p53 mutation on DLL4 expression in human cancer stem cell disorder: The Li-Fraumeni syndrome
Authors: Yao Zhixing , Sherif Zaki A.
Published: July 2016

Tumorigenesis results from an accumulation of mutational and epigenetic changes that alter normal cell growth and survival pathways. Li-Fraumeni syndrome (LFS) is a clinically and genetically heterogeneous inherited cancer syndrome that can provide powerful insights into our understanding of the somatic mutations present in sporadic cancers. LFS is currently linked to heterozygous mutation in the tumor suppressor gene p53 (TP53), which encodes a transcription factor that responds to diverse cellular stresses through the regulation of target genes that induce apoptosis, cell cycle arrest, DNA repair, and senescence. We previously showed a balanced reciprocal chromosomal translocation t(11;15)(q23;q15) in the non-cancerous skin fibroblasts of a bilateral breast cancer patient in LFS family. This prompted us to investigate the breakpoint region of the translocation, which uncovered a gene that encodes DLL4, (Delta-like ligand 4; locus at 15q15.1), a Notch ligand that is a key target in tumor vasculature. DLL4 regulates T-cell lineage thymopoiesis and further affects cancer immunosurveillance. In the breakpoint region of chromosome 15q15, we examined THBS1, DNAJC17, Rad51, DLL4, CHAC1 and INO80 gene expression levels. We found that specifically DLL4 is abrogated in all the LFS cell lines as well as MDA231 breast cancer cell line, and drastically down-regulated in MCF7 breast and IMR32 brain cancer cell lines and several human organ-specific tumor samples. Furthermore, DNA methylation studies revealed that DLL4 promoter is silenced only in MCF7 and MDA231 but not in LFS cell lines. ChIP and siRNA knockdown assays demonstrated that p53 binds to DLL4 promoter through its association with CTCF, a chromosomal networking protein CCCTC binding factor. These results imply a possible karyotype-phenotype correlation with respect to DLL4 in LFS and breast cancer initiation and progression. The drastic reduction or absence in the expression of DLL4 in normal skin fibroblasts of LFS patients as well as breast and brain cancer cells is significant and supports the concept that this ligand may also play a role in cancer immune-surveillance; and its resuscitation in the tumor microenvironment may stimulate T-cell immunity and suppress tumor growth.

Citation Format: Zhixing Yao, Zaki A. Sherif. The effect of epigenetic silencing and p53 mutation on DLL4 expression in human cancer stem cell disorder: The Li-Fraumeni syndrome. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3698.
Abstract 3702: Src phosphorylated Mdm2 requires MdmX to act as a neddylating ligase
Authors: Hauck Paula M. , Mayo Lindsey D.
Published: July 2016

Mdm2 is an oncoprotein and, along with its family member MdmX, have been shown to be elevated in human cancers. Like Mdm2, MdmX, is a RING containing protein, yet it lacks intrinsic E3 ligase function. Mdm2 has been shown to facilitate ubiquitination as a monomer, a homodimer, and as a heterodimer with MdmX depending on the cellular stress. Our recent report establishes that under growth conditions that activate Src, Mdm2 functions as a neddylating enzyme. Whether MdmX is necessary for Mdm2 to neddylate p53 has yet to be shown. Here we demonstrate that Src phosphorylation results in increased levels of MdmX, increased binding between Mdm2 and MdmX, and increased neddylation of MdmX and p53. Interestingly, the lack of MdmX (in transient assays or in shRNA cell lines) results in decreased neddylation of p53. These data support a critical role for MdmX as part of the Mdm2 neddylating complex.

Citation Format: Paula M. Hauck, Lindsey D. Mayo. Src phosphorylated Mdm2 requires MdmX to act as a neddylating ligase. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3702.
Abstract 3703: MDM2 regulation of MYC and MYCN in pediatric neural cancers
Authors: Tran Hung N. , Qi Donglai , Cobrinik David
Published: July 2016

Background

MYC and MYCN have critical roles in a wide range of cancers and can be deregulated through diverse transcriptional and post-transcriptional mechanisms. Recent studies have shown that the high-level MYCN expression needed to drive retinoblastoma and MYCN-amplified neuroblastoma proliferation depends upon efficient MYCN translation mediated by highly expressed MDM2 and acting via a p53-independent mechanism. Here, we assessed MDM2-mediated regulation of MYC as well as MYCN in other pediatric neural tumors, specifically in Group 3 medulloblastoma and in MYC-overexpressing neuroblastoma. We also assessed MDM2-mediated regulation of MYCN in small cell lung cancer (SCLC), which has genetic and morphologic features in common with retinoblastoma.

Methods

MDM2, MYCN, and MYC expression were defined in Group 3 medulloblastoma lines D283, D425, D341; in MYC-overexpressing neuroblastoma lines CHLA 255 and SY-5Y; and in p53-wild type and p53-mutant SCLC lines H69 and H526 by western blotting. Cells were infected with shMDM2 and scrambled control lentivirus and effects on MDM2, MYC, and MYCN protein expression defined using western blot and effects on cell growth defined using Cell Titer-Glo. In neuroblastoma cells, MDM2 knockdown effects were defined with or without p53 co-knockdown.

Results

Group 3 medulloblastoma lines D283, D341, D425 highly expressed MYC but not MYCN. MDM2 knockdown did not decrease MYC expression in these cells. MDM2 knockdown decreased MYC expression In CHLA 255 and SY5Y neuroblastoma cells, along with p53 induction and cell death, and MYC down-regulation and cell death were mitigated by co-knockdown of p53. MDM2 knockdown decreased MYCN expression and impaired viability in p53-wild type H69 SCLC cells and in p53-mutant H526 SCLC cells.

Conclusions

MDM2 is not needed to sustain high-level MYC expression in three Group 3 medulloblastoma cell lines. MDM2 sustains high-level MYC in two MYC-overexpressing neuroblastoma cell lines via a p53-dependent mechanism distinct from the p53-independent MYCN regulation in MYCN-amplified neuroblastomas. In SCLC, MDM2 sustains high-level MYCN expression and viability in a p53-independent manner, similar to MDM2 effects in retinoblastoma and in MYCN-amplified neuroblastoma. These findings suggest that MDM2 selectively sustains MYCN but not MYC expression through a p53-independent mechanism and raise the possibility that cis-acting elements specific to MYCN-RNA mediate MDM2 regulation.

Citation Format: Hung N. Tran, Donglai Qi, David Cobrinik. MDM2 regulation of MYC and MYCN in pediatric neural cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3703.
Abstract 3707: Semiconductor-based next-generation sequencing analysis of 409 cancer-related genes for mutations and copy-number variations in oral squamous cell carcinoma
Authors: Nakagaki Takafumi , Sasaki Yasushi , Idogawa Masashi , Koyama Ryota , Kobashi Kenta , Tamura Miyuki , Ohashi Tomoko , Ogi Kazuhiro , Hiratsuka Hiroyoshi , Tokino Takashi
Published: July 2016

Somatic mutation analysis is standard of practice for human cancers in order to identify therapeutic sensitizing and resistance mutations. To better understand the molecular pathogenesis of oral squamous cell carcinoma (OSCC) patients, we performed comprehensive genomic analyses that use PCR target enrichment and next-generation sequencing on the Ion Torrent semiconductor sequencers (PGM and Proton). DNA (40 ng) was extracted from 45 human OSCC specimens and their corresponding non-cancerous tissues including FFPE samples. Using the Ion Ampliseq Comprehensive Cancer Panel, we sequenced 15992 loci from 409 tumor suppressor genes and oncogenes frequently cited and frequently mutated in human cancers (covered regions = 95.4% of total, 1.5 megabases of target sequence). We also detected copy number variations (CNVs) in which segments of the genome can be duplicated or deleted from sequencing data.

Each sample underwent on average 5.9 million sequencing reads after quality filtering. The mean read depths were 367.8 x, and >95% of targeted bases were represented .The number of somatic mutations (SNVs and indels) in 45 patients with OSCC ranged from 1 to 36 with a median of 7.33 (6.40/Mb). The most frequent mutations were detected on TP53 (28 of 45; 62.2%). Many of the mutations on TP53 were detected in the DNA-binding domain (23 of 28; 82.1%). NOTCH1 mutations were identified in 10 cases (8 missense, 1 coding frameshift, and 1 essential splice site mutations). CDKN2A mutations were observed in 8 cases; and PIK3CA were mutated 3 cases. Although the most common mutations in OSCC were C/G>T/A transitions, which are consistent with previous reports on head and neck, lung and oesophageal SCCs, the second most frequent mutations were C/G>A/T transversions. We also identified a median of 100 significant CNVs (range of 0-481) per sample. Pathway assessment has shown that somatic aberrations within OSCC genomes are mainly involved in several important pathways, including cell cycle regulation (p53 pathway, 84.4%) and RTK-MAPK-PI3K (62.2%). This targeted next generation sequencing using low amounts of FFPE DNA is a valuable tool for high-throughput genetic testing in research and clinical settings.

Citation Format: Takafumi Nakagaki, Yasushi Sasaki, Masashi Idogawa, Ryota Koyama, Kenta Kobashi, Miyuki Tamura, Tomoko Ohashi, Kazuhiro Ogi, Hiroyoshi Hiratsuka, Takashi Tokino. Semiconductor-based next-generation sequencing analysis of 409 cancer-related genes for mutations and copy-number variations in oral squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3707.
Abstract 3714: Optimisation of quinazolinedione sulphonamides as novel inhibitors of poly(ADP Ribose) glycohydrolase (PARG)
Authors: Smith Kate , Acton Ben , James Dominic , Jones Cliff , Jones Stuart , Jordan Allan , Hamilton Nicola , McGonagle Alison , Mould Daniel , Small Helen , Stowell Alex , Tucker Julie , Waddell Ian , Waszkowycz Bohdan , Ogilvie Donald
Published: July 2016

The macrodomain protein poly(ADP ribose) glycohydrolase (PARG) has been shown to be a critical component in the repair of single stand DNA breaks and counteracts the function of the ARTD family of poly(ADP ribose) polymerases, commonly known as the PARPs. As PARG exists as a single protein, it presents an attractive target for therapeutic intervention in cancer cells with enhanced dependence upon DNA repair.

Inhibitors of this enzyme have proved difficult to discover and develop. Moreover, intact cell-active tool compounds which have the propensity to be used as robust chemical probes to understand PARG pharmacology, are absent from the literature.

This poster will describe our work in this emerging area, optimising a series of drug-like quinazolinedione derivatives to deliver molecules with the correct physicochemical and biochemical properties to function as in vitro cell probe compounds. These unprecedented agents display potent on-target biochemical (5 nM) and cell (10 nM) activity with a significant window to acute 3-day cytotoxicity. Moreover, these agents are selective against PARP family members and the close glycohydrolase homologue ARH3. The medicinal chemistry optimisation of the scaffold will be described, alongside the outline pharmacology demonstrating on-target, selective inhibition of PARG in cells. Such tool compounds will be of value in revealing the detailed mechanisms of action of PARG in DNA repair and other PAR chain-mediated cellular processes, with the ultimate goal of delivering novel and clinically relevant therapeutic agents.

Citation Format: Kate Smith, Ben Acton, Dominic James, Cliff Jones, Stuart Jones, Allan Jordan, Nicola Hamilton, Alison McGonagle, Daniel Mould, Helen Small, Alex Stowell, Julie Tucker, Ian Waddell, Bohdan Waszkowycz, Donald Ogilvie. Optimisation of quinazolinedione sulphonamides as novel inhibitors of poly(ADP Ribose) glycohydrolase (PARG). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3714.
Abstract 373: Discovery of selective PI3Kb inhibitors for PTEN-deficient tumors and other indications
Authors: Chowdhury Arnab R. , Athisayamani Jeyaraj D. , Mitra Sayan , Maddi Srinivas , Adluri Ram Sudheer , Bethi Sridhar R. , Joshi Hemant , Tirunagaru Vijaya G. , Gupta Jang B.
Published: July 2016

PTEN somatic mutations occur in a large percentage of human cancers, from breast to prostate. Deletions of PTEN e.g. in prostate cancers are associated with tumor aggression and poor outcome, and up to 70 percent of prostate cancer patients have lost one copy of the PTEN gene at the time of diagnosis. Recent pharmacologic and genetic studies have identified the β isoform of PI3K to be required for growth of PTEN-deficient tumors in vivo and highly potent compounds are currently being explored in the clinic. These clinical candidates, however, are either limited by poor bioavailability or moderate selectivity against the delta isoform, thus warranting more selective compounds with better exposure. We have undertaken a comprehensive knowledge-based discovery of selective inhibitors of PI3Kβ with admissible pharmacokinetic and drug like properties. We have successfully identified a chemical series with potency ranging as low as 2.5 nM for PI3Kβ with >100 - fold selectivity for PI3Kδ. The α and γ isoform selectivity was around 1000 and 500 fold respectively. Compounds did not decrease insulin sensitivity and T cell proliferation thus mitigating the PI3Kα and PI3Kδ mediated side effects of nonselective inhibitors. In vitro biochemical potency translated into cell survival assay and pAKT target engagement in recombinant and wild type mammalian cell lines. In vivo tumor regression was observed in xenograft models of prostate cancer. The series did not show any observable hERG or CYP liabilities. The considerable potency, high selectivity and good bioavailability of this novel class of compounds not only makes them attractive for prostate cancer, but also for rare disease indications like hamartoma and other drug resistant cancers that are dependent on PTEN deletions.

Citation Format: Arnab R. Chowdhury, Jeyaraj D. Athisayamani, Sayan Mitra, Srinivas Maddi, Ram Sudheer Adluri, Sridhar R. Bethi, Hemant Joshi, Vijaya G. Tirunagaru, Jang B. Gupta. Discovery of selective PI3Kb inhibitors for PTEN-deficient tumors and other indications. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 373.
Abstract 3731: TSC deficiency unmasks a novel protective role of RIP1/RIP3 signaling against mitochondrial and oxidative stress-induced cell death
Authors: Filipczak Piotr T. , Thomas Cindy , Chen Wenshu , Salzman Andrew , McDonald Jacob D. , Lin Yong , Belinsky Steven A.
Published: July 2016

Tuberous sclerosis complex (TSC) is a genetic multi-organ disorder characterized by the development of neoplastic lesions in kidney, lung, brain, heart and skin. It is caused by an inactivating mutation in tumor suppressor genes coding the TSC1/TSC2 complex, resulting in hyperactivation of mTOR- and Raf/MEK/MAPK-dependent signaling that stimulates tumor cell proliferation and metastasis. Despite its oncogenic effect, cells with TSC deficiency were more sensitive to oxidative stress and dependent on mitochondrial metabolism, providing a rationale for a new therapeutic approach. The presented study shows that simultaneous inhibition of two major pathways regulating redox homeostasis using L-Buthionine-sulfoximine (BSO, glutathione synthesis inhibitor) and auranofin (thioredoxin reductase inhibitor) induces oxidative burst, mitochondrial damage and necrotic cell death in TSC2 deficient cells in a highly synergistic and cell context-specific manner. Furthermore, blocking RIP1/RIP3-dependent signaling using chemical inhibitors necrostatin-1 and necrosulfonamide (NSA), reported by others to protect from some cytotoxic stimuli, synergizes with BSO and auranofin in killing TSC2 deficient cells. Expression analysis demonstrated that RIP1 and RIP3 protein levels are elevated in cells with TSC2 deficiency, and their inactivation enhances mitochondrial dysfunction. Finally, supplementation with the mitochondrial metabolite alpha-ketoglutarate, whose synthesis is regulated by RIP1/RIP3, rescues cells from the sensitizing effect of necrostatin-1 and NSA. Together, this study identifies a novel TSC context-dependent role of RIP1/RIP3 signaling in regulating mitochondrial and oxidative homeostasis, and substantiates glutathione, thioredoxin and RIP1/RIP3 pathways targeting as a promising therapeutic approach against TSC-associated tumors.

Citation Format: Piotr T. Filipczak, Cindy Thomas, Wenshu Chen, Andrew Salzman, Jacob D. McDonald, Yong Lin, Steven A. Belinsky. TSC deficiency unmasks a novel protective role of RIP1/RIP3 signaling against mitochondrial and oxidative stress-induced cell death. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3731.
Abstract 3741: Antimetastatic activity of ISTH0047, a potent and selective TGF-beta 2 antisense oligonucleotide, in syngeneic lung metastatic model of mouse 4T1 mammary carcinoma
Authors: Wosikowski Katja , Hasenbach Kathy , Petschenka Jutta , Barea Roldan Diana , Kreiter Sebastian , Sahin Ugur , Serin Guillaume , Redon Julie-Orlane , Hillairet de Boisferon Marc , Bichat Francis , Kohonen Hanna , Janicot Michel
Published: July 2016

Transforming growth factor beta (TGF-β) isoforms are the primary mediators for TGF-β signaling via TGF-β receptors and downstream phosphorylation/dephosphorylation cascade. TGF-β is associated with a wide range of biological processes in oncology, including tumor cell invasion, migration, angiogenesis, immunosuppression, as well as regulation of tumor stem cell properties. Mouse 4T1 mammary carcinoma cell line is a transplantable tumor cell line that is highly tumorigenic and invasive and, unlike most tumor models, can spontaneously metastasize from the primary tumor in the mammary gland to multiple distant sites including lymph nodes, blood, liver, lung, brain, and bone. Considering rather challenging preclinical evaluation of antitumor activity in tumor models, mouse 4T1 mammary carcinoma model has been widely used in the literature for evaluation of TGF-β antagonists. In this report, we describe the efficacy of ISTH0047 - a potent and selective TGF-β2 antisense oligonucleotide - in murine 4T1 primary tumors and lung metastasis following tumor cell injection into the mammary fat pad (orthotopic tumor model) of syngeneic Balb/c mice. Consistent with literature data generated with other classes of TGF-β antagonists (e.g., small-molecule kinase inhibitors, antibodies or TGF-β trap agents), although limited antitumor activity was demonstrated on primary tumor growth, marked and statistically significant decrease of lung metastasis number was observed upon subcutaneous administrations of ISTH0047. In addition, side by side comparison with murine surrogates of CTLA-4 or PD-1 antibodies indicated similar efficacy of all test items on lung metastasis in this model. Taken together, these encouraging results pave the way for in-depth preclinical evaluation of both ‘seed and soil’ theory and efficacy of combination regimen (immunomodulation) for better clinical outcome.

Citation Format: Katja Wosikowski, Kathy Hasenbach, Jutta Petschenka, Diana Barea Roldan, Sebastian Kreiter, Ugur Sahin, Guillaume Serin, Julie-Orlane Redon, Marc Hillairet de Boisferon, Francis Bichat, Hanna Kohonen, Michel Janicot. Antimetastatic activity of ISTH0047, a potent and selective TGF-beta 2 antisense oligonucleotide, in syngeneic lung metastatic model of mouse 4T1 mammary carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3741.
Abstract 3771: Discovery of a covalent inhibitor of ERK docking-interactions that inhibits A375 melanoma cells proliferation
Authors: Kaoud Tamer S. , Johnson William H. , Ebelt Nancy D. , Piserchio Andrea , Warthaka Mangalika , Cano Micael , Sammons Rachel , Wang Qiantao , Ren Pengyu , Ghose Ranajeet , Dalby Kevin N.
Published: July 2016

Acquired drug resistance, especially mechanisms associated with the reactivation of the MAPK (RAF/MEK/ERK) pathway represent a major challenge to current treatments of melanoma. Recently, targeting ERK has evolved as a potentially attractive strategy to overcome this resistance. Several ERK inhibitors have already entered clinical trials.

Most of the available ERK inhibitors are reversible inhibitors that either act through an allosteric mechanism, or by targeting the ATP binding site. Taking advantage of our understanding of ERK-docking interactions we tried to discover an irreversible substrate-selective inhibitor that targets the protein-binding site of ERK. Here, we report the discovery of a covalent inhibitor of ERK that targets its protein-docking site. Protein NMR, Mass spectroscopy, mutagenesis and molecular docking studies indicate a covalent interaction of the inhibitor with a conserved cysteine residue, Cys-159. Extensive biochemical studies provide an estimate of its kinetic parameters and its kinase-selectivity profile. The new ERK inhibitor inhibits ERK activation, as well as its ability to phosphorylate downstream substrates (e.g. p90RSK and Elk-1) in HEK293T and A375 melanoma cells. The targeting of ERK in HEK293T cells was confirmed using a chemical-genetic approach where the ERK2 C159A mutant was used to rescue the effects of this compound on ERK2 signaling and cell proliferation. Currently, we are testing the effect of the compound on tumor growth inhibition in an A375 melanoma cancer xenografts model. This covalent inhibitor represents a potentially valuable lead molecule whose development may result in a novel class of pharmacologically useful ERK inhibitors for targeting resistant forms of melanoma.

Citation Format: Tamer S. Kaoud, William H. Johnson, Nancy D. Ebelt, Andrea Piserchio, Mangalika Warthaka, Micael Cano, Rachel Sammons, Qiantao Wang, Pengyu Ren, Ranajeet Ghose, Kevin N. Dalby. Discovery of a covalent inhibitor of ERK docking-interactions that inhibits A375 melanoma cells proliferation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3771.
Abstract 3784: The role of the taurine transporter SLC6A6 in promoting prosurvival activity and multidrug resistance of colorectal cancer
Authors: Yasunaga Masahiro , Matsumura Yasuhiro
Published: July 2016

The identification of a novel therapeutic target is desired to improve the treatment of colorectal cancer (CRC). In a comprehensive gene expression analysis, we found that the taurine transporter SLC6A6 was highly expressed in CRC. In the functional analysis, SLC6A6-knocked down (KD) CRC cells showed attenuated cell-survival activity accompanying enhanced sensitivity of the drugs (5-FU, DOX and SN-38). Moreover, the number of side population (SP) cells and their cancer stem cell (CSC)-like properties such as tumor-initiation were abrogated by SLC6A6-KD. Conversely, increased the cell-survival activity, the fraction of SP cells and enhancement of multidrug resistance (MDR) were observed in SLC6A6-overexpressed cells. Furthermore, SLC6A6-siRNA treatment enhanced the cytotoxic effect of all 3 drugs as distinct from ABCG2-siRNA treatment, the efficacy of which was limited to its 2 substrate-drug, DOX and SN-38. Finally, we clarified the prosurvival and anti-apoptotic effects of the taurine transporter SLC6A6 in CRC cells. Moreover, we found that SLC6A6 plays an important role in the maintenance of CSC characteristics promoting cell survival signal and MDR. Our findings indicate that the SLC6A6 may be a novel therapeutic target for refractory CRC.

Citation Format: Masahiro Yasunaga, Yasuhiro Matsumura. The role of the taurine transporter SLC6A6 in promoting prosurvival activity and multidrug resistance of colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3784.
Abstract 3802: Evaluation of microfilament-directed cytochalasins as novel antineoplastic agents
Authors: Trendowski Matthew , Christen Timothy D. , Acquafondata Christopher , Fondy Thomas P.
Published: July 2016

Despite inherent differences between the cytoskeletal networks of malignant and normal cells, and the clinical antineoplastic activity of microtubule-directed agents, there has yet to be a microfilament-directed agent approved for clinical use. Cytochalasins are mycotoxins known to potently inhibit the polymerization of filamentous (F)-actin, and have long been used in vitro to examine the importance of microfilaments in fundamental cellular processes. It has previously been demonstrated that cytochalasins preferentially multinucleate malignant cells, suggesting that the congeners may exert novel antineoplastic activity. We have taken these findings, and further expanded upon their potential importance. We have shown in vitro efficacy with multiple congeners, including cytochalasin B, which potentiates substantial sensitivity to clinically approved antineoplastic agents, X-radiation, and low frequency ultrasound. Further, we have shown that cytochalasin B is more cytotoxic against neoplastic cells selected for high metastatic propensity than are their less invasive counterparts. Interestingly, we have also demonstrated that nine cytochalasin congeners may be substantially less affected by drug efflux due to overexpression of ATP-binding cassette (ABC) transporters than are doxorubicin, paclitaxel, or vinblastine, as demonstrated with SK human ovarian carcinoma cell lines of varying levels of drug resistance. Finally, we have shown that cytochalasins B and D elicit substantial antitumor and antimetastatic activity in numerous preclinical mammalian models of malignancy, suggesting that the novel mechanisms by which these congeners exert antineoplastic activity is worth further examination.

Citation Format: Matthew Trendowski, Timothy D. Christen, Christopher Acquafondata, Thomas P. Fondy. Evaluation of microfilament-directed cytochalasins as novel antineoplastic agents. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3802.
Abstract 3836: Novel conditionally active biologic anti-Axl antibody-drug conjugate demonstrates anti-tumor efficacy and improved safety profile
Authors: Chang Cathy , Frey Gerhard , Boyle William J. , Sharp Leslie L. , Short Jay M.
Published: July 2016

Axl is a TAM family receptor tyrosine kinase that has been implicated in the pathogenesis of many cancer types. The high level of expression on the cancer cell surface has made it an attractive target for antibody therapeutics. However, Axl is expressed on many normal tissues and has been implicated in wide ranging requisite biological processes including response of endothelial cells to vascular injury, hematopoiesis, and regulation of immune responses. This normal tissue expression may limit Axl as a target for antibody-drug-conjugates (ADC). Conditionally Active Biologics (CAB) technology is a proprietary platform that selects antibodies that bind to target antigen in the context of diseased tissues, but not normal tissues, by taking advantage of the unique cancer microenvironment that is produced largely as a result of the Warburg effect. Using our CAB technology, we have identified anti-Axl Abs that reversibly bind to recombinant Axl and Axl expressing cells under conditions that are present in the tumor microenvironment but not in normal tissues.

CAB-Axl antibodies were then conjugated to a model toxin payload to generate CAB-Axl-ADCs. The CAB-Axl-ADCs were active against Axl positive human tumor xenografts with tumor stasis observed at 1mg/kg weekly and tumor regressions observed at 1 mg/kg twice a week dose levels. A non-specific IgG-ADC showed minimal efficacy at the same dose levels. Single dose studies in cynomolgus macaques have demonstrated that CAB-Axl-ADC has reduced liver toxicity and immune system effects compared to Axl-ADCs that bind to Axl under normal conditions.

In conclusion, our data is consistent with our work on CAB-EGFR antibodies, and suggests that ADCs generated using the CAB technology provides biologics with increased therapeutic index. Specifically, the CAB-Axl-ADC is an excellent candidate for evaluation as a treatment for human cancers that are Axl positive.

Citation Format: Cathy Chang, Gerhard Frey, William J. Boyle, Leslie L. Sharp, Jay M. Short. Novel conditionally active biologic anti-Axl antibody-drug conjugate demonstrates anti-tumor efficacy and improved safety profile. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3836.
Abstract 3902: Novel selective estrogen receptor downregulators developed using endocrine-independent breast cancer cell lines
Authors: Xiong Rui , Zhao Jiong , Gutgesell Lauren , Tonetti Debra , Thatcher Gregory
Published: July 2016

Approximately 70% of breast cancer patients are estrogen receptor positive (ER+). Aromatase inhibitors and the selective estrogen receptor modulator (SERM), tamoxifen, are the first line treatments for these patients; however, almost 50% of patients either do not respond or acquire resistance. Multiple mechanisms, including mutations of the ESR1 gene, contribute to resistance via ligand-independent constitutive activation of ER. Selective estrogen down-regulators (SERDs) that block ligand-dependent and independent ER signaling by ablation of ER, offer a therapeutic approach to treatment-resistant, advanced stage and early stage ER+ breast cancer. Therapeutic use of the first generation SERD, fulvestrant (Faslodex), has largely remained 2nd and 3rd line, because of poor physiochemical/pharmacokinetic properties. Novel benzothiophene based SERDs were designed, synthesized, and optimized and assayed in three tamoxifen-resistant (TR), endocrine-independent ER+ MCF-7 and T-47D cell lines. Cell viability, ERE-luciferase response, and ER degradation was measured and compared to parent endocrine-dependent MCF-7 and T-47D cell lines in 2D and/or 3D spheroid cell cultures and compared to SERDs, fulvestrant and GDC-0810. Pharmacokinetic analysis was used to select novel SERDs for xenograft studies.

Citation Format: Rui Xiong, Jiong Zhao, Lauren Gutgesell, Debra Tonetti, Gregory Thatcher. Novel selective estrogen receptor downregulators developed using endocrine-independent breast cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3902.