bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3636: Improved clinical variant calling and HLA genotyping with GRCh38
Authors: Chapman Brad , Meynert Alison , Church Deanna , Johnson Justin , Hofmann Oliver
Published: July 2016

The Genome Reference Consortium released human genome build 38 (hg38, GRCh38) two years ago and it offers major improvements over the six year old previous build, 37 (hg19, GRCh37). The new build reflects our increased understanding of the heterogeneity within human sub-populations, contains a large number of alternative genomic loci that better capture our knowledge of genome structure, and includes highly diverse human leukocyte antigen (HLA) regions enabling HLA typing.

To help with the migration of clinical sequencing workflows we provide support for variant calling on build 38, validate methods using truth sets from the Genome in a Bottle consortium and Illumina's Platinum Genome and show that feature projection approaches are a viable intermediate approach to port existing annotation resources. We show how the refined genomic representation improves mapping and variant calling by reducing the number of false positive SNP calls and improving InDel detection, and how post-processing of sequence reads mapping to HLA regions enables accurate HLA genotyping from targeted panels, exome- and WGS data. Using Omixon HLA validation data, integrated OptiType HLA calling achieved 100% accuracy on 8 targeted panels and better than 90% accuracy on 1000 genome exome samples.

This work provides a freely available, validated, ready to run pipeline making use of the improved heterogeneity representation in build 38 and demonstrating the value of moving towards a more accurate, graph-based representation of human genomes.

Citation Format: Brad Chapman, Alison Meynert, Deanna Church, Justin Johnson, Oliver Hofmann. Improved clinical variant calling and HLA genotyping with GRCh38. [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 3636.
Abstract 3644: Optimization of library construction for massively parallel sequencing using low-input, FFPE-derived DNA without additional PCR amplification
Authors: Thorner Aaron R. , Ziaugra Liuda , Ducar Matthew D. , Lin Ling , Laing Angelica , Coleman Haley A. , McShane Suzanne R. , Clapp Andrea , Paquette Rachel R. , Wollison Bruce M. , Hoeftberger Johann , Patel Neil A. , Hunter Samuel S. , Manam Monica D. , MacConaill Laura E. , Hahn William C. , Meyerson Matthew L. , van Hummelen. Paul
Published: July 2016

Massively parallel sequencing (MPS) is increasingly used in the laboratory and clinic to identify genomic factors contributing to tumorigenesis, such as somatic mutations, DNA insertions and deletions, transcriptome and epigenetic changes, and chromosomal abnormalities. Because tumor specimens are often quite limited or are from rare and precious samples, it is necessary to prepare sequencing libraries from minimal amounts of DNA. Indeed, it can be challenging to extract sufficient amounts of starting material from fresh-frozen paraffin embedded (FFPE) samples for library construction, and frequently samples are not processed because they do not reach minimum requirements. Therefore, it is of great importance to improve currently available protocols to process low yield FFPE-derived DNA for MPS. This will allow for the inclusion of more tumor samples to be sequenced per cohort, resulting in increased ability to identify genomic factors involved in tumorigenesis.

Here, we optimized the KAPA Library Preparation Kit (Kapa Biosystems, Wilmington, MA) to successfully perform library construction from low-yield FFPE and fresh frozen samples. The kit normally requires an input of at least 100 ng of DNA. However, we were able to modify the protocol to prepare libraries from as little as 10 ng of starting material by optimizing the ratio of adapter:input DNA and SPRI-clean-up, performing low-volume reactions, and using IDT universal blockers during hybrid capture. Additional library enrichment PCR was not required. Library yields were sufficient for downstream hybrid capture and sequencing, and sequencing metrics were comparable to samples that were prepped using the manufacturer's recommendations. Experiments are underway to demonstrate that library complexity remains unchanged.

Citation Format: Aaron R. Thorner, Liuda Ziaugra, Matthew D. Ducar, Ling Lin, Angelica Laing, Haley A. Coleman, Suzanne R. McShane, Andrea Clapp, Rachel R. Paquette, Bruce M. Wollison, Johann Hoeftberger, Neil A. Patel, Samuel S. Hunter, Monica D. Manam, Laura E. MacConaill, William C. Hahn, Matthew L. Meyerson, Paul van Hummelen. Optimization of library construction for massively parallel sequencing using low-input, FFPE-derived DNA without additional PCR amplification. [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 3644.
Abstract 3645: Low level somatic variant detection by Sanger sequencing of formalin-fixed paraffin-embedded (FFPE) samples
Authors: Gerstner Arpad , Schreiber Edgar , Jackson Stephen , Varma Kamini
Published: July 2016

Deleterious sequence variants play an important role in the initiation and progression of many different cancer types. The detection of germline variants by the gold standard Sanger sequencing has been well established, however, the detection of somatic mutations, especially in heterogeneous tumor samples where variants may be present at a lower level, has been more challenging.

To facilitate analysis of somatic mutations in tumor samples, we have developed Sanger sequencing panels that cover the entire coding regions of specific genes implicated in tumorigenesis (e.g. TP53, KRAS and NRAS). We have also developed companion software, Minor Variant Finder (MVF), that facilitates detection of low levels of somatic mutations in Sanger sequencing studies. To demonstrate the workflow of these panels with MVF, we analyzed DNA from lung cancer FFPE samples. We initially determined variants of TP53 and KRAS in these samples using Ion Torrent™ Personal Genome Machine (PGM™) next generation sequencing (NGS). We confirmed the identity and variant allele frequency of these variants by Sanger sequencing coupled with MVF. Furthermore, we were able to confirm these results in 1 ng, 0.5 ng or 0.1 ng of DNA from these samples. Finally, we made serial dilutions of one of these samples to establish limit of detection (LOD). We show that this workflow can detect as little as 3% of a minor variant in an FFPE sample.

Sanger sequencing is the gold standard for confirmation of minor variants detected by NGS. In this study, we show that Sanger sequencing of limited number of targets, in conjunction with the MVF software, can also be an ideal first line screening choice for tumor FFPE samples where limited amount of DNA is available.

For Research Use only - Not for use in diagnostic procedures.

Citation Format: Arpad Gerstner, Edgar Schreiber, Stephen Jackson, Kamini Varma. Low level somatic variant detection by Sanger sequencing of formalin-fixed paraffin-embedded (FFPE) samples. [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 3645.
Abstract 3646: Highly sensitive and cost-effective detection of somatic cancer variants using single-molecule, real-time sequencing
Authors: Kujawa Steve , Sethuraman Anand , Eng Kevin , Baybayan Primo , Heyrman Lien , Del Favero Jurgen
Published: July 2016

Next-Generation Sequencing (NGS) technologies allow for molecular profiling of cancer samples with high sensitivity and speed at reduced cost. For efficient profiling of cancer samples, it is important that the NGS methods used are not only robust but also capable of accurately detecting low-frequency somatic mutations. Single Molecule, Real-Time (SMRT®) Sequencing offers several advantages, including the ability to sequence single molecules with very high accuracy (>QV40) using the circular consensus sequencing (CCS) approach. The availability of genetically defined, human genomic reference standards provides an industry standard for the development and quality control of molecular assays for studying cancer variants. Here we characterize SMRT Sequencing for the detection of low-frequency somatic variants using the Quantitative Multiplex DNA Reference Standards from Horizon Diagnostics, combined with amplification of the variants using the Multiplicom Tumor Hotspot MASTR Plus assay.

First, we sequenced a reference standard containing precise allelic frequencies from 1% to 24.5% for major oncology targets verified using digital PCR. This reference material recapitulates the complexity of tumor composition and serves as a well-characterized control. The control sample was amplified using the Multiplicom Tumor Hotspot MASTR Plus assay that targets 252 amplicons (121-254 bp) from 26 relevant cancer genes, which includes all 11 variants in the control sample. We also sequenced a second sample containing a series of mixes, each with known mutations, at levels below 10% and down to 0.01%. PCR-amplified targets were sequenced and analyzed using SMRT Sequencing to identify the variants and determine the observed frequency. The random error profile and high-accuracy CCS reads make it possible to accurately detect low-frequency somatic variants.

Citation Format: Steve Kujawa, Anand Sethuraman, Kevin Eng, Primo Baybayan, Lien Heyrman, Jurgen Del Favero. Highly sensitive and cost-effective detection of somatic cancer variants using single-molecule, real-time 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 3646.
Abstract 3656: Cooperation of Rab25 loss with oncogenes in breast cancer
Authors: Rao Krishna A. , Joshi Pooja S.
Published: July 2016

Breast cancer is the most common type of cancer in females worldwide. A number of genes such as BRCA1, BRCA-2, Hras, and p53 undergo mutations and contribute to tumorigenesis of the mammary tissue. The RAB guanosine triphosphates (RAS-related in brain) belong to the Ras superfamily of GTPases. The human RAS family consists of 3 proto oncogenes - H-ras, K-ras, N-ras. Rab 25 belongs to the Rab11 subfamily and plays an important role in vesicular trafficking. The Rab proteins are ubiquitously expressed. Loss of Rab 25 expression has been reported in a number of breast cancer cases and in transformed mammary cell lines containing H-ras point mutations.

Human Mammary Epithelial Cells (HMEC) were obtained from healthy individuals undergoing reduction mammoplasty. The cells were immortalized by transducing with LXSN CDK4 R24C. This was followed by transduction with hTERT, catalytic subunit of the telomerase enzyme.

In this study, we are trying to understand if loss of Rab25 expression in HMEC co-operates with wild and mutant H-ras as well as other oncogenes and contributes to tumorigenesis. HMEC lines in which Rab25 has been knocked down were transduced with various oncogenes, including wild type and mutant H-ras, Her-2, and IGFR1. We examined the co-operativity between loss of Rab25 and these oncogenes in migration, anchorage independent growth, stem cell marker expression, and also in vivo tumorigenesis and metastasis in nude mice.

Citation Format: Krishna A. Rao, Pooja S. Joshi. Cooperation of Rab25 loss with oncogenes in breast 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 3656.
Abstract 3662: Analysis of the tumor suppressor function of B55alpha, the PP2A regulatory subunit encoded by PPP2R2A, in prostate cancer cells
Authors: Zhao Ziran , kurimchak Alison , Adeyemi Mary , Woodruff Patrick , Kolenko Vladimir , Graña Xavier
Published: July 2016

Genome wide DNA copy number studies in prostate cancer (PCa) have identified deletions on chromosome 8p21.2 affecting the PPP2R2A gene, which encodes B55α, a B regulatory subunit of Protein Phosphatase 2A (PP2A). The frequency of PPP2R2A hemizygous and homozygous deletions in prostate tumors is 38-58% and 2-7%, respectively. Moreover, PPP2R2A hemizygous loss correlates with reduced mRNA expression. In contrast, no significant correlation is observed for NKX3.1, a tumor suppressor also located at 8p21.2. Bioinformatics analysis indicates that PPP2R2A may belong to the class of “mutator alterations” occurring early in tumorigenesis that promote driver gene mutations. Indeed, PCa patients with these alterations exhibit reduced disease free progression. PP2A consists of a collection of trimeric holoenzymes whose substrate specificity and function is determined by the regulatory B subunit. There are 14 B subunit genes. While it is well accepted that PP2A plays a key tumor suppressor function in human cells, the particular holoenzyme(s) implicated in different tumor types is unclear. We have found, that limited ectopic expression of B55α in PCa cell lines that naturally express low levels of B55α results in dramatic toxicity. We have linked this to G2/M arrest, without concomitant loss in DNA replication, which leads to euploidy, nuclear enlargement and cell death. Thus, early hemizygous loss of PPP2R2A may facilitate effective mitotic progression. We have also seen that increased expression of B55α in these cells dramatically reduces their tumorigenic potential in SCID mice. Moreover, we observe inhibition in the phosphorylation of PP2A/B55α substrates when B55α activity is reconstituted, including AKT and pRB, suggesting alterations in multiple cancer pathways when B55α is downregulated in tumor cells. Interestingly, treatment of PC3 cells with PP2A activating drugs mimic cell cycle effects seen with B55α reconstitution.

Citation Format: Ziran Zhao, Alison kurimchak, Mary Adeyemi, Patrick Woodruff, Vladimir Kolenko, Xavier Graña. Analysis of the tumor suppressor function of B55alpha, the PP2A regulatory subunit encoded by PPP2R2A, in prostate cancer cells. [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 3662.
Abstract 3668: Investigating a tumor suppressor role for Parkinson's susceptibility gene LRRK2 in lung cancer
Authors: Lebovitz Chandra , Chow Norman , Lam Wan , Gorski Sharon
Published: July 2016

In this study we investigate a novel and previously untested research question: does a known disease susceptibility gene in Parkinson's disease (PD) also act as a tumor suppressor gene in lung cancer? Our screen of publicly available cancer patient sequence data for genome and transcriptome alterations of genes that modulate autophagy, an important tumor cell survival process, revealed a dramatic loss of gene expression of Leucine-rich repeat kinase 2 (LRRK2) in non-small cell lung tumors compared to matched normal tissue. We further confirmed reduced or absent LRRK2 protein expression in a panel of lung cancer cell lines. Activating kinase-domain mutations in LRRK2 and their effect on autophagy in neuronal cell types is under study in PD; however, the pathological role of altered LRRK2 in cancer and its effect on autophagy status and tumorigenicity is completely unexplored. Pharmacological inhibition of the LRRK2 kinase domain was recently shown to stimulate autophagy in PD cell lines, while many tumors are thought to exploit elevated autophagy to survive metabolic stress and chemotherapy. Ongoing clinical trials are testing combination autophagy inhibition with current standard of care treatments in multiple cancers. Therefore, a better understanding of pathogenic LRRK2-mediated autophagy in lung cancer could form the basis of a new approach to help identify lung cancer patients that may benefit from treatment strategies employing autophagy inhibitors. Our preliminary data indicates that LRRK2 knockdown in lung cancer cell lines leads to altered cellular phenotypes and increased protein levels of a known lung cancer gene. We are further testing whether genetic and/or pharmacological inhibition of LRRK2 facilitates tumorigenesis in standard murine models of lung cancer. Our investigation of the biological relevance and therapeutic potential of a surprising discovery linking neurodegenerative disease-associated LRRK2 and lung cancer may uncover a new tumor suppressor gene and provide a novel marker (i.e. LRRK2 loss) to aid in lung cancer detection.

Citation Format: Chandra Lebovitz, Norman Chow, Wan Lam, Sharon Gorski. Investigating a tumor suppressor role for Parkinson's susceptibility gene LRRK2 in lung 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 3668.
Abstract 3675: Loss of Pkhd1 promotes intestinal tumorigenesis in Apc mice
Authors: Hu Bo , Li Wei , Qiu Qingchao , He Zhengxi , He Xiusheng , Wu Guanqing
Published: July 2016

Objectives: PKHD1 (Polycystic Kidney and Hepatic Disease 1), a causal gene for ARPKD (Autosomal Recessive Polycystic Kidney Disease), has been proposed to play a role in the colorectal carcinogenesis. This study is to explore the role of PKHD1 in colorectal carcinogenesis.

Methods: Mouse model with Pkhd1 mutation (Pkhd1-/-) was used to mate with ApcMin/+ mouse in the same genetic background. The resulting ApcMin/+ mice with and without Pkhd1-/- alleles were randomly sorted into two experimental groups (i.e. Pkhd1-/-;ApcMin/+ n = 36; ApcMin/+ n = 23). Tumorigenic susceptibility of Pkhd1-/-;ApcMin/+ and ApcMin/+ mice was compared. The number, size, pathology of intestinal tumors in the mice were analyzed at the age of 1-, 3-, and 6-month. The survival rates were investigated in the two groups of mice.

Results: There was no statistical difference in tumor number and size between 1-month-old ApcMin/+ and Pkhd1-/-;ApcMin/+ mice. However, the number and size of the intestinal tumors in 3- and 6-month-old Pkhd1-/-;ApcMin/+ mice were significantly increased compared with those of ApcMin/+ mice alone (P = 0.017,P = 0.022). In addition, the pathology of tumors was also analyzed between the two groups. There was no pathological difference between 1-month-old ApcMin/+ and Pkhd1-/-;ApcMin/+ mice. However, at 3-month-old, Pkhd1-/-;ApcMin/+ mice exhibited severer atypical hyperplasia in the intestinal tumor than those of ApcMin/+ mice; while at 6-month-old, focal necrosis were often seen in intestinal tumors of Pkhd1-/-;ApcMin/+ mice, but none from ApcMin/+ mice. Strikingly, tumor infiltration into the muscular mucosa was also seen in a 6-month-old Pkhd1-/-;ApcMin/+ mice. There was no statistically survival difference between ApcMin/+ and Pkhd1-/-;ApcMin/+ mice.

Conclution: Loss of Pkhd1 promotes the intestinal tumorigenesis and induces tumor malignant transformation in ApcMin/+ mice.

Citation Format: Bo Hu, Wei Li, Qingchao Qiu, Zhengxi He, Xiusheng He, Guanqing Wu. Loss of Pkhd1 promotes intestinal tumorigenesis in Apc mice. [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 3675.
Abstract 3679: Klotho suppresses colon cancer through modulation of the Wnt pathway and unfolded protein response
Authors: Rubinek Tami , Rubinstein Tammi , Shahmoon Shiri , Varol Chen , Zigmond Ehud , Rosin-Absesfelf Rina , Barshack Iris , Wolf Ido
Published: July 2016

Aberrant activation of the canonical Wnt pathway is implicated in colorectal cancer (CC) development and progression. β-catenin, the key effector of this pathway, functions with T-cell factor/lymphoid-enhancer-factor (TCF/LEF) to activate expression of Wnt target genes. Klotho is a transmembrane protein, which can be shed and act as a circulating hormone. Klotho-deficient mice manifest a syndrome resembling accelerated aging, while klotho overexpression extends life span. We previously identified klotho as a breast and pancreatic tumor suppressor (TS). Recent data indicate klotho as a TS in an array of malignancies, including CC. Among other activities, klotho inhibits the insulin-like growth factor (IGF)-1 and Wnt pathways. We aimed to decipher the role of klotho as a TS in CC.

We first noted that klotho overexpression inhibited CC cell colony formation. Next, we examined in vivo effects using the azoxymethane mouse model, and found that klotho inhibited colon polyp formation. We then used an orthotopic model by injecting MC38 cells to mice colons, and found that klotho inhibited colonic obstruction and tumor formation.

While we did not see an effect of klotho on the IGF-1 pathway, klotho did affect the Wnt pathway. Klotho reduced Wnt3A and β-catenin levels as measured by Western blotting, and inhibited TFC/LEF transcriptional activation in luciferase assay. As transcriptional inhibition was abrogated by a constitutively active β-catenin, we suspected that klotho inhibited the pathway upstream of β-catenin. Indeed, co-immunoprecipitation analyses indicated direct interaction between klotho and Wnt3A. As the inhibitory effect of klotho on CC cell colony formation was only partially rescued by a constitutively active β-catenin, additional mechanisms may be involved. Thus, we conducted a cDNA expression microarray and involvement of klotho in endoplasmic reticulum (ER) stress and unfolded protein response (UPR) was observed. Further studies showed that klotho induced elevation in XBP1 RNA, GRP78 protein levels, and eIF2α phosphorylation, all indicative of a role klotho plays in UPR regulation.

Our data indicate klotho as a potent TS in CC, and suggest its role at early stages of tumor formation. The Wnt pathway partially mediates klotho effects in CC, however additional mechanisms, including ER stress response regulation, are involved.

Citation Format: Tami Rubinek, Tammi Rubinstein, Shiri Shahmoon, Chen Varol, Ehud Zigmond, Rina Rosin-Absesfelf, Iris Barshack, Ido Wolf. Klotho suppresses colon cancer through modulation of the Wnt pathway and unfolded protein response. [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 3679.
Abstract 3680: Loss of ARID1A expression leads to sensitivity to ROS-inducing agents in ovarian cancer cells
Authors: Kwan Suet-Yan , Wong Kwong-Kwok
Published: July 2016

AT-rich interactive domain-containing protein 1A (ARID1A) is frequently inactivated by mutations in a broad spectrum of cancer types, including ovarian and endometrial cancers. However, therapeutic strategies targeting ARID1A-mutant cancers remain limited. In this study, we aimed to identify drugs that selectively target ARID1A-mutant ovarian cancer cells. To this end, we compared the drug sensitivities of ARID1A-mutant and ARID1A-wildtype cancer cell lines using the Genomics of Drug Sensitivity in Cancer database. We found that ARID1A-mutant cancer cell lines were more sensitive to treatment with the reactive oxygen species (ROS)-inducing agent elesclomol compared to ARID1A-wildtype cancer cell lines. We validated this finding in a panel of ovarian and endometrial cancer cell lines, observing that treatment with elesclomol inhibited growth and induced apoptosis more potently in ARID1A-mutant cells than in ARID1A-wildtype cells. Knockdown of ARID1A expression in ARID1A-wildtype cells resulted in increased sensitivity to ROS-inducing agents, whereas re-expression of ARID1A in ARID1A-mutant cells resulted in decreased sensitivity. Finally, we found that knockdown of ARID1A expression resulted in increased intracellular ROS levels and subsequently promoted cell growth. In summary, we identified a novel role for ARID1A in protecting cells from oxidative stress and that ROS-inducing agents may be used to target ARID1A-mutant ovarian cancer cells.

Citation Format: Suet-Yan Kwan, Kwong-Kwok Wong. Loss of ARID1A expression leads to sensitivity to ROS-inducing agents in ovarian cancer cells. [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 3680.
Abstract 3685: Redox regulation of p53 stability is a function of inhibition of PP2A-mediated dephosphorylation at threonine 55
Authors: Chong Stephen , Pervaiz Shazib
Published: July 2016

Protein Phosphatase 2A (PP2A) is a heterotrimeric serine/threonine phosphatase that has critical roles in numerous cellular pathways. The role of PP2A as an essential tumor suppressor in numerous death signaling pathways is well documented. In this regard, recent work from our laboratory has highlighted a novel mechanism of apoptosis inhibition via post-translational redox-modification of PP2A. Increase in intracellular superoxide, either by pharmacological inhibition or siRNA-mediated silencing of superoxide dismutase 1 (SOD1), results in the dissociation of PP2A-AC catalytic core from its B regulatory subunit. Dissociation of the PP2A heterotrimer is a result of the selective nitration [by peroxynitrite derived from the reaction of superoxide with nitric oxide] of a conserved tyrosine residue on B56δ which we identified as Y289 (B56δY289). Importantly, we showed in lymphoma cells that PP2A-AC catalytic core is typically targeted to Bcl-2 by its B56δ regulatory subunit. When O2- is elevated, it signals for the sustained phosphorylation of Bcl-2 at S70 by inducing the release of PP2A-AC catalytic core from B56δ-bound Bcl-2. This results in the sustained phospho-activation of anti-apoptotic Bcl-2 and resistance to apoptotic stimuli. Notably, Bcl-2 is not the only substrate of PP2A, nor is B56δ the only PP2A regulatory subunit susceptible to redox modification. Thus, implications from redox-modification of PP2A may extend beyond the phospho-activation of Bcl-2. In the context of carcinogenesis, another notable substrate of PP2A is the tumor suppressor, p53. p53 stability is negatively influenced by its phosphorylation status at threonine-55 (T55). Phosphorylation of T55, on the other hand, is countered by PP2A harboring the B56γ3 subunit. Here we provide evidence that the B56γ3 sub-unit of PP2A complexes with p53 and this complex is sustained upon an increase in intracellular superoxide. Importantly, redox-mediated inhibition of B56γ3-containing PP2A (PP2AB56γ3) prevents the dephosphorylation of p53 at T55, thereby negatively impacting the stability of p53 in tumor cells with elevated superoxide level. This, we hypothesize, may be a novel death inhibitory mechanism that renders tumor cells resistant to conventional DNA-damaging therapeutic agents that rely heavily on the activation of p53 for death induction.

Citation Format: Stephen Chong, Shazib Pervaiz. Redox regulation of p53 stability is a function of inhibition of PP2A-mediated dephosphorylation at threonine 55. [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 3685.
Abstract 3704: TRISENOX disrupts MDM2-DAXX-HAUSP interaction and increased promyelocytes formation in murine model of APL
Authors: Kumar Sanjay , Tchounwou Paul
Published: July 2016

Trisenox (ATO) is widely used in the treatment of all age group of acute promyelocytic leukemia (APL) patients in both induction and consolidation therapy. In vitro studies are shown that it works through oxidative stress, cell cycle regulation and apoptosis to kill APL cells having PML-RAR α oncogene. Some ATO resistant case of APL patients are reported with different oncogenes combination. However, in vivo anti-leukemic mechanism of action of ATO in APL patients poorly known. We have used transgenic murine model to elucidate the anti-cancer properties of ATO through new target. We hypothesized that ATO disrupts MDM2-DAXX-HAUSP interaction and also increased more number of promyelocytes formation inside bone marrow cells of APL mice. To test the hypothesis, we used different groups of younger (4-5weeks old) transgenic APL mice and standardized different dose of ATO (1.25, 2.5, 5.0 and 7.5 mg/kg body wt) after 21days continuous treatment in PBS. We utilized western blotting, immunocytochemistry, confocal imaging and other molecular techniques to identify molecular mechanisms of ATO action in APL mice hepatocytes as well as bone marrow cells. We found that it increases number of promyelocyte formation in bone marrow cells and also degradation of PML-RARα dose dependent fashion. ATO nicely activated p53 expression in both liver tissue as well as bone marrow cells of APL mice through degradation of MDM2. It was also disrupted both expression and association of MDM2-DAXX-HAUSP complex molecules dose dependent manner in mice liver tissues. On the basis of these findings, we conclude that ATO disrupts MDM2-DAXX-HAUSP interaction, increased promyelocytes formation, MDM2 degradation by accumulating P53 in APL mice liver tissues. It is novel target for treatment of APL patients through designing of new anti-leukemic drugs.

Keywords: Trisenox, P53, MDM2-DAXX-HAUSP complex molecules APL Mice hepatocytes and bone marrow cells.

Acknowledgements: This research was financially supported by National Institutes of Health NCRR Grant No. 5G12RR013459 and MIMHD Grant No. 8G12MD007581, through the RCMI-Center for Environmental Health at Jackson State University.

Citation Format: Sanjay Kumar, Paul Tchounwou. TRISENOX disrupts MDM2-DAXX-HAUSP interaction and increased promyelocytes formation in murine model of APL. [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 3704.
Abstract 3713: Targeting alternative DNA repair pathways for cancer cell killing with heavy ion particles
Authors: Wang Huichen , Saganti Premkuma
Published: July 2016

Heavy ion therapy is a promising approach for cancer treatment, particularly for locally advanced solid tumors. High linear energy transfer (LET) radiation induces greater lesions density in close proximity (clusters) compared that in low-LET radiation. Such clustered DNA damage is thought to be more difficult to repair than isolated DNA damage produced by equivalent doses of low-LET radiation and persist longer in irradiated cells. It has been known that homologous combination contributes to the repair of clustered DNA damage other than classic non-homologous end-joining (NHEJ). DNA double-strand breaks are also repaired by an alternative NHEJ pathway with PARP1, DNA ligase III and Mre11. This alternative pathway is error prone and contributes to genomic instability, such as chromosomal translocation and telomere fusion. Genetic analysis of tumors in TCGA database revealed that these genes are amplified in human tumors and their overexpression is correlated with poor overall survival of cancer patients. However, influence of alternative NHEJ pathways on the relative biological effectiveness (RBE) of heavy ions remains unclear. Here we investigated the effect of PARP-1 and DNA ligase III on RBE of heavy ion particles on cell survival and chromosomal aberration in breast cancer cells and pancreatic cancer cells. Cells were irradiated with X-ray, Carbon (290 MeV/n, 13 KeV/μM) and Iron (1 GeV/n, 150 KeV/μM). We found that overexpression of PARP-1 and DNA ligase III increased RBE of carbon and iron. PARP inhibitor reduced chromosomal aberration induced by heavy ions. This study may provide predictor of radiation responsive of tumor cells from cancer patients that would lead to patient stratification for heavy ion therapy selection

Citation Format: Huichen Wang, Premkuma Saganti. Targeting alternative DNA repair pathways for cancer cell killing with heavy ion particles. [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 3713.
Abstract 3733: Augmentation of response to therapeutic agents by (-)-gallocatechin gallate through inhibition of RAD51 nuclear translocation
Authors: Wu Ming-Hsi , Lin Lie-Chwen , Lee Te-Chang
Published: July 2016

Lung cancer is the most common cancer worldwide. Platinum-based combination chemotherapy is one of first line treatments for patients with lung cancer. Most of the cytotoxic drugs are targeting DNA, interfere with DNA synthesis, and triggering DNA damage response (DDR). Since DDR usually activates important defensive pathways to repair damaged DNA, DDR is known as a key factor determining the outcome of chemotherapy. Among them, RAD51, playing a central role in homologous recombination (HR) DNA repair, has been shown to interfere with the prognosis of patients treated with chemoradiotherapy. Since we have demonstrated that inhibition of RAD51 nuclear focus formation synergistically enhances the anticancer activity of cisplatin, we further screened purified natural products in order to find products with activity to inhibit RAD51 nuclear focus formation. In the present study, we found that a green tea catechin derivative, the (-)-Gallocatechin gallate (GCG) has potent inhibitory activity against RAD51 nuclear focus formation in cisplatin-treated H460 cells. The in vitro cytotoxicity assay showed that the combination of cisplatin or irinotecan with GCG significantly increases the sensitivity of H460 cells to these DNA damage agents. We also evaluated the anti-tumor growth activity of cisplatin or irinotecan in combination with GCG using H460 xenografts. Our results showed that cisplatin and irinotecan suppresses tumor growth by 17.6% and 33.8%, while in combination with GCG suppresses tumor growth by 52.2% and 68.9%, respectively. We further demonstrated that GCG is able to inhibit cisplatin-induced ATM and Chk2 phosphorylation. Together, our results suggest that GCG may interfere with ATM-Chk2 pathway and hence suppress RAD51 nuclear focus formation and DNA repair activities.

Citation Format: Ming-Hsi Wu, Lie-Chwen Lin, Te-Chang Lee. Augmentation of response to therapeutic agents by (-)-gallocatechin gallate through inhibition of RAD51 nuclear translocation. [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 3733.
Abstract 3737: Inhibition of O-GlcNAc transferase in tamoxifen resistant breast cancer cells
Authors: Barkovskaya Anna , Prasmickaite Lina , Mills Ian G. , Mælandsmo Gunhild M. , Moestue Siver A. , Itkonen Harri M.
Published: July 2016

O-linked N-acetyl-glucosamine transferase (OGT) is an enzyme that catalyzes addition of the O-GlcNAc modification to a wide range of intracellular proteins. The O-GlcNAc modification is a product of the hexosamine biosynthetic pathway, which requires glucose and glutamine as substrates. Uptake of both of these nutrients is often up-regulated in cancer, which in turn leads to an increase in the total protein O-GlcNAcylation. Increased OGT expression has also been reported in most cancer types, including the most frequently diagnosed cancer in women, breast cancer. Many of the breast cancers rely on estrogen receptor alpha (ERα) for proliferation and have shown a strong response to the ERα inhibition, most commonly achieved by treatment with tamoxifen. However, while efficient, prolonged exposure to tamoxifen commonly causes resistance and relapse of the disease. It is therefore vital to uncover mechanisms which contribute to the resistance in order to develop adequate treatment strategy for these patients.

Here, we have investigated the effect of targeting OGT in an isogenic pair of ERα-positive tamoxifen-sensitive MCF7 and tamoxifen-resistant TAMR breast cancer cell lines. OGT inhibition decreased viability and triggered cell death in both cell lines. These responses were associated with over 50% reduction in ERα expression in both MCF7 and TAMR cells. Reduced O-GlcNAcylation has previously been reported to induce endoplasmic reticulum stress and activation of transcription factor C/EBP homologous protein (CHOP), which promotes cell death. Targeting OGT resulted in a strong increase of CHOP expression, which appeared more prominent in the TAMR cells. Finally, targeting OGT induced a very pronounced cell cycle arrest in the G2/M phase in the TAMR cells, while the MCF7 cell lined showed a very modest response.

Taken together, these results indicate that targeting OGT leads to a differential response in the tamoxifen-sensitive and resistant breast cancer cells. Currently, we are using an expanded panel of tamoxifen-resistant cell lines to perform expression microarrays, metabolic flux assays and DNA damage response analysis in order to uncover the cause of the differential response to OGT targeting. This may help us identify potential therapeutic combinations that might be suitable for treatment of tamoxifen-resistant cancers.

Citation Format: Anna Barkovskaya, Lina Prasmickaite, Ian G. Mills, Gunhild M. Mælandsmo, Siver A. Moestue, Harri M. Itkonen. Inhibition of O-GlcNAc transferase in tamoxifen resistant breast cancer cells. [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 3737.
Abstract 3750: CD133-targeted oncolytic adenovirus shows therapeutic effect by attacking colon cancer stem cells
Authors: Sato Mizuho , Miura Yoshiaki , Yamamoto Masato
Published: July 2016

Colorectal cancer is the third most common cancer in the world and about 50% of patients relapse after treatment. Cancer stem cells (CSCs) have contribution to recurrence, metastasis and and chemotherapy resistant of colorectal cancers. CD133 (Prominin-1), a member of the transmembrane glycoprotein family, is a marker of CSCs in several cancers and is also generally accepted as a colorectal cancer stem cell marker. CD133 expression was correlated with recurrence, metastases and chemotherapy resistance, as well as poor prognosis in colorectal cancer. It is therefore reasonable to develop CSCs-directed therapeutic strategies by employing CD133 as a target molecule. Recently, we have established a method for isolating transductionally-targeted adenovirus by high-throughput screening. In this study, using this adenovirus library screening system, we isolated the CD133-specific Oncolytic Adenovirus (OAd) and tested the oncolytic activity of CD133-targeted Ad in both in vitro and in vivo. CD133-targeted OAd (AdML-TYML) showed strong binding to CD133-positive cells (293-CD133 and LoVo (CD133 (+) human colon carcinoma)), not to CD133-negative cells (parental 293 and LS174T (CD133 (-) human colon carcinoma)) and this virus also showed strong oncolysis selectively in LoVo cells, whereas there was no effect on LS174T cells.

In order to assess the effect of AdML-TYML on stemness, tumor establishment assay was performed. AdML-TYML treatment leads to inhibition of tumor establishment of CD133-positive colorectal cancer cell lines in nude mice. 100% of the mice inoculated with non-infected LoVo cells had developed tumors, while 0% of the mice inoculated with AdML-TYML infected LoVo cells had done so. In addition, when the anti-tumor effect of the CD133-targeted OAd was analyzed in established tumors of CD133(+) colorectal cancer subcutaneous xenografts, intra-tumor (i.t.) administration of AdML-TYML exhibited significantly stronger antitumor effect compared to its equivalent Ad5-WT (wild type Ad5 fiber).

We focused on the CD133 as a target molecule of CSCs in colon cancer and successfully identified potent CD133-targeting OAd using high-throughput screening system. The CD133-targeted OAd exhibited selective infectivity and oncolysis to CD133-positive cells and anti-tumor effect against established colon cancer cells. Our results suggest that CD133-targeted oncolytic adenovirus selectively eliminate CD133 (+) colorectal cancer stem cells from the tumor and also this virus could be a key tool for the prevention of metastases and relapses in a variety of cancers.

Citation Format: Mizuho Sato, Yoshiaki Miura, Masato Yamamoto. CD133-targeted oncolytic adenovirus shows therapeutic effect by attacking colon cancer stem cells. [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 3750.
Abstract 3754: Pre-clinical studies of a highly potent folate receptor targeted DNA crosslinking agent
Authors: Reddy Joseph A. , Nelson Melissa , Johnson Theresa , Dirksen Christina , Vetzel Marilynn , Hahn Spencer , Qi LongWu , Vlahov Iontcho , Leamon Christopher
Published: July 2016

Folate receptor (FR) targeted small molecule drug conjugates (SMDC) have shown promising results in early stage clinical trials with Vintafolide and EC1456. In our effort to develop FR targeted SMDCs with varying mechanisms of action, we have now built a folate conjugate of a DNA crosslinking agent based on a novel DNA-alkylating moiety. This agent was found to be extremely potent with an in vitro IC50 ∼ 100 x lower than any other folate SMDC we have created to date. Treatment of nude mice bearing FR positive human xenografts led to cures in 100% of the mice at very low doses (> 500 nmol/kg) and a convenient once a week schedule. The observed activity was not accompanied by any noticeable weight loss (up to 20 weeks post end of dosing) or major organ tissue degeneration. In contrast, no significant anti-tumor activity (0% CR’s) was observed in treated animals that were co-dosed with an excess of a benign folate ligand, thus demonstrating its target-specific activity. Complete responses were also observed in other FR positive drug resistant (paclitaxel and cisplatin) models. Taken together, these studies demonstrated that this SMDC with a distinct DNA reacting mechanism has significant anti-tumor growth activity and tolerability, thus lending support to future clinical development of this novel FR-targeted agent.

Citation Format: Joseph A. Reddy, Melissa Nelson, Theresa Johnson, Christina Dirksen, Marilynn Vetzel, Spencer Hahn, LongWu Qi, Iontcho Vlahov, Christopher Leamon. Pre-clinical studies of a highly potent folate receptor targeted DNA crosslinking agent. [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 3754.
Abstract 3759: Inhibition of heat shock protein 27 (Hsp27) expression by apatorsen as a therapeutic strategy in breast cancer
Authors: Komulainen Emilia , Shia Alice , Bansal Jasmin , Cavicchioli Francessa , O’Leary Karen , Foster John , Schmid Peter
Published: July 2016

Background: Heat shock protein 27 (Hsp27) is over-expressed in a number of cancers, including breast cancer, and interacts with apoptotic proteins inhibiting the apoptotic process and promoting tumourgenesis. Clinically, Hsp27 expression has been associated with a more aggressive phenotype and resistance to chemotherapy. Apatorsen (OGX-427) is a therapeutic antisense oligonucleotide designed to specifically target Hsp27. Apatorsen has shown single agent clinical activity, and randomised trials in combination with chemotherapy are underway. Here we present pre-clinical validation of inhibition of Hsp27 expression by apatorsen as a novel therapeutic strategy in breast cancer.

Results: Basal expression analysis of Hsp27 by qPCR and western blot in a panel of >20 breast cancer cell lines demonstrated a wide variation with up to 3-log difference in expression between cell lines and good correlation of RNA and protein expression levels. Hsp27 expression increased after treatment with chemotherapy agents. Treatment with apatorsen caused rapid reduction of cellular Hsp27 levels within 24-48 hours; effects on Hsp27 levels were sustained for 5 and 7 days. Knockdown of Hsp27 was associated with significant reduction of cell survival with 38-93% loss of cell viability across the cell line panel (compared to mismatched oligonucleotide). There was no association between response to apatorsen and basal Hsp27 expression or molecular breast cancer subtype. Using whole genome mRNA (Illumina Human HT-12 v4 Expression BeadChip Kit) and methylation analysis (Illumina Human 450K Methylation BeadChip), we were unable to define a predictive signature of response to apatorsen. Co-treatment of breast cancer cell lines with apatorsen and Doxorubicin, Paclitaxel and Vinorelbine chemotherapy showed an additive cytotoxic effect. In vivo validation of the effects of apatorsen is ongoing.

Conclusions: Therapeutic silencing of Hsp27 expression in breast cancer cell lines with apatorsen presents a novel strategy for the treatment of disease. Here we present data to support the use of apatorsen in combination with conventional chemotherapy for the treatment of breast cancer.

Citation Format: Emilia Komulainen, Alice Shia, Jasmin Bansal, Francessa Cavicchioli, Karen O’Leary, John Foster, Peter Schmid. Inhibition of heat shock protein 27 (Hsp27) expression by apatorsen as a therapeutic strategy in breast 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 3759.