bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2925: Anti-tumor activity of a MYC-targeting dicer substrate siRNA in combination with BRD4/CDK7 inhibitors
Authors: Chipumuro Edmond , Siddiquee Zakir , Ganesh Shanthi , Shui Serena , Shah Anee , Kim Boyoung , Chen Dongyu , Pandya Purva , Storr Rachel , Wang Weimin , Dudek Hank , Lai Cheng , Abrams Marc , Brown Bob
Published: July 2016

MYC is a well-characterized driver of numerous tumor types. Since the protein encoded by this gene is challenging to target via conventional modalities, progress in new therapeutic agents has been slow despite decades of research. RNA interference technology has enabled the inhibition of previously-undruggable genetic targets at the mRNA level, and has advanced to clinical development for several indications. DCR-MYC, a lipid nanoparticle (LNP)-formulated Dicer substrate siRNA (DsiRNA) targeting MYC mRNA, is currently in Phase Ib/II clinical trials and showing promising results. In this study we used an improved EnCore LNP and MYC DsiRNA, which demonstrated MYC mRNA silencing activity and efficacy in mouse models of human hepatocellular carcinoma (HCC). Small molecule inhibitors that target BRD4; JQ1 and CDK7; THZ1 has previously reported anti-proliferative effects in various cancer types and efficacy in several tumor mouse models including HCC. Treatment with both JQ1 and THZ1 induced cell cycle arrest and apoptosis in various cancer cells by repressing MYC expression. Here we observed striking anti-proliferative effects in vitro when MYC-DsiRNA was combined with THZ1 in cancer cells. In addition, when mice bearing HCC tumors were treated with MYC-DsiRNA combined with either THZ1 or JQ1, the antitumor efficacy was additive or synergistic relative to either single agent alone. We observed significantly more MYC mRNA knockdown in the tumors that had the combination treatment compared to the tumors that received either of the single agent treatment. These preclinical data suggests the possibility of a significant and practical benefit of combining MYC-DsiRNA and small molecule inhibitors that target an epigenetic regulator BRD4 (JQ1) and a global gene regulator CDK7 (THZ1).

Citation Format: Edmond Chipumuro, Zakir Siddiquee, Shanthi Ganesh, Serena Shui, Anee Shah, Boyoung Kim, Dongyu Chen, Purva Pandya, Rachel Storr, Weimin Wang, Hank Dudek, Cheng Lai, Marc Abrams, Bob Brown. Anti-tumor activity of a MYC-targeting dicer substrate siRNA in combination with BRD4/CDK7 inhibitors. [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 2925.
Abstract 2965: In vitro and in vivo activity of a site-specific SeriMab antibody-drug conjugate (ADC) using an indolino-benzodiazepine DNA-alkylating agent
Authors: Vitharana Dilrukshi , Wilhelm Alan , Harris Luke , Archer Katie , Shizuka Manami , Maloney Erin , Ab Olga , Laleau Rassol , Sun Xiuxia , Pinkas Jan , Miller Michael , Chari Ravi , Keating Thomas , Fishkin Nathan
Published: July 2016

Previously we have described the characterization of a proprietary class of indolino-benzodiazepine dimers, IGNs, with high potency against many cancer lines. Antibody-drug conjugates (ADCs) made with mono-imine containing IGNs were designed to only alkylate DNA and not cause DNA crosslinking. ADCs with the IGN linked via lysine residues of the antibody were shown to be highly potent and antigen specific. Here we apply our SeriMab site-specific technology platform, which employs N-terminal conjugatable aldehydes derived from oxidation of a serine residue, to link an IGN molecule to the antibody using a peptide linker. This ADC (SeriMab-IGN-P1) has exactly 2 IGN molecules per antibody conjugated to the N-termini of the heavy chains, as determined by MS analysis.

Serimab-IGN-P1 was found to maintain binding to the target antigen with comparable affinity to the unconjugated antibody by FACS analysis (Kd = 300 pM). The conjugate was highly potent (IC50 = 4 pM) against a cancer cell line with high target expression (3 × 106 antibodies bound per cell) as well as a cell line with much lower target expression (2 × 104 antibodies bound per cell, IC50 = 22 pM). SeriMab-IGN-P1 also demonstrated strong bystander killing activity against proximal target-negative cancer cells, likely due to a cell permeable metabolite identified in cells expressing the target antigen.

The unique oxime linkage in SeriMab-IGN-P1 was found to be stable in circulation in mice for 3 days, as determined by affinity capture LC-MS; the conjugate species with 2 IGNs per antibody remained predominant over this period with an estimated t1/2 of >10 days for payload release.

In vivo, SeriMab-IGN-P1 was found to be effective against human lung cancer xenografts (NCI-H2110), with anti-tumor activity observed at single doses as low as 2 μg/kg (payload dose), and complete responses observed at 5 μg/kg. This conjugate was also well tolerated in CD-1 mice at doses significantly higher than the minimum effective dose that produced tumor regression.

These data are highly encouraging for new ADC therapies using the SeriMab site-specific conjugation platform with potent DNA-acting payloads.

Citation Format: Dilrukshi Vitharana, Alan Wilhelm, Luke Harris, Katie Archer, Manami Shizuka, Erin Maloney, Olga Ab, Rassol Laleau, Xiuxia Sun, Jan Pinkas, Michael Miller, Ravi Chari, Thomas Keating, Nathan Fishkin. In vitro and in vivo activity of a site-specific SeriMab antibody-drug conjugate (ADC) using an indolino-benzodiazepine DNA-alkylating 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 2965.
Abstract 3024: Targeting TMEPAI/PMEPA1 inhibits triple negative breast cancer cell growth and metastasis by enhancing growth suppressive TGF-β signaling
Authors: Singha Prajjal Kanti , Pandeswara Srilakshmi , Venkatachalam Manjeri A. , Saikumar Pothana
Published: July 2016

Triple negative breast cancers (TNBC) that lack estrogen receptor (ER), progesterone receptor (PR) and hormone epidermal growth factor receptor 2 (HER-2/neu) are aggressive and cause high mortality among breast cancer patients. Transforming growth factor beta (TGF-β) dependency for their aggressive behavior (growth and metastasis) has been well established in many of these tumors. Although targeting TGF-β signaling has major potential to treat TNBCs, however it carries the risk of disturbing the tumor suppressive effects of TGF-β in early tumors and its homeostatic control of normal tissues. Hence there is a need to identify novel targets to impede tumor progression without compromising the beneficial effects of TGF-β signaling. To satisfy this unmet need, earlier we identified that transmembrane prostate androgen induced (TMEPAI/PMEPA1), a direct target gene of TGF-β could act as a “molecular switch” that converts TGF-β from a tumor suppressor to promoter in TNBC. Thus, we undertook the present study that identified a novel compound that blocked TMEPAI expression.

We identified terpenoid derivatives (TD) which inhibited the expression of TMEPAI, enhanced TGF-β signaling and blocked proliferation, migration and invasion of several triple negative breast cancer cells in vitro. Our results showed that TD increased phosphorylation of Smad2/3 and increased PTEN, p21 and p27 proteins that cause growth suppression. Concomitantly, TD decreased Akt phosphorylation and reduced Snail and Slug that are required for cell growth and metastasis. Interestingly, TD did not affect the growth of normal human mammary epithelial cells and failed to cause associated molecular changes that can result in growth suppression. Notably, using mice models (nude mice and syngeneic BALB/c mice), we showed that TD reduced tumor burden significantly with little or no toxicity. Consistent with its ability to inhibit Akt phosphorylation and induction of Snail and Slug proteins, TD suppressed lung metastasis of TNBC in mice model. Importantly, reduced tumors derived from TD treated mice exhibited increased expression of pSmad2/3, PTEN, p21, p27 and reduced expression of pAkt compared to tumors obtained from vehicle treated mice. These results suggest that drugs that target TMEPAI will restore homeostatic functions of TGF-β to inhibit growth and metastasis of TNBC and thus prevent new tumor development.

Citation Format: Prajjal Kanti Singha, Srilakshmi Pandeswara, Manjeri A. Venkatachalam, Pothana Saikumar. Targeting TMEPAI/PMEPA1 inhibits triple negative breast cancer cell growth and metastasis by enhancing growth suppressive TGF-β signaling. [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 3024.
Abstract 3068: Anti-tumor effect of DHP107, a mucoadhesive lipid formulae of oral paclitaxel, in bladder cancer model
Authors: Kim Se Hyun , Kim Jung Min , Kim Tae Soo , Lee Won Suk , Kwon Woo Sun , Lim Seung Tak , Ahn Joong Bae , Kim Se-Kyu , Jo Yeong Woo , Rha Sun Young
Published: July 2016

Purpose: Paclitaxel is an effective anti-neoplastic agent in many solid tumors including bladder cancer. With the development of new drug delivery strategies, novel formulations of Cremophor EL-free paclitaxel have been developed. Here, we investigated the in vitro and in vivo anti-tumor effect of a mucoadhesive oral formulation of paclitaxel (DHP107) in comparison with iv paclitaxel in a human bladder cancer model.

Methods: Four human bladder cancer cell lines (RT4, T24, J82, HT1376) and one murine bladder cancer cell line (MBT-2) were used in this study. In vitro antitumor activity and underlying mechanisms were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, cell-cycle analysis and apoptosis assay. In the in vivo study, RT4 cell line was implanted subcutaneously in the flank of nu/nu mice, and treated with iv paclitaxel and 2 doses of DHP107. The effects were assessed by the tumor growth, body weight and Ki-67 staining in tumor tissues.

Results: The IC50 values of DHP107 ranged from 4.15 to 34.72 μg/ml with MTT assay. In RT4 cell line, both drugs induced similar G2-M arrest and apoptosis. The xenograft experiments showed that both DHP107 (per os, 50 mg/kg, equivalent of paclitaxel 10 mg/kg) and iv paclitaxel (intraperitoneal, 10 mg/kg) attenuated the growth of RT4 tumors in mice, equivalently. The higher dose (per os, 100 mg/kg) of DHP107 resulted in increased efficacy compared with the lower dose (50 mg/kg) without weight loss in mice. However, significant weight loss was observed in iv paclitaxel (-20% at Day 11) treated group. We observed significantly decreased proliferation with Ki-67 staining in DHP107 (100 mg/kg) treated group than in DHP107 (50 mg/kg) or iv paclitaxel (10 mg/kg) treated groups.

Conclusion: Our findings provide the evidence that DHP107 showed comparable antitumor effect with iv paclitaxel and is a promising anti-tumor agent in bladder cancer. Clinical studies in patients with bladder cancer warrant further investigation.

Citation Format: Se Hyun Kim, Jung Min Kim, Tae Soo Kim, Won Suk Lee, Woo Sun Kwon, Seung Tak Lim, Joong Bae Ahn, Se-Kyu Kim, Yeong Woo Jo, Sun Young Rha. Anti-tumor effect of DHP107, a mucoadhesive lipid formulae of oral paclitaxel, in bladder cancer model. [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 3068.
Abstract 3080: OPB-51602: a novel STAT3 inhibitor that targets mitochondrial respiratory chain and triggers STAT3 dependent ROS production
Authors: Hirpara Jayshree L. , Higuchi Kumi , Tsunoda Takeshi , Goh Boon Cher , Pervaiz Shazib
Published: July 2016

Signal transducer and activator of Transcription (STAT) proteins are activated upon extracellular signals resulting in their phosphorylation and nuclear translocation. Once in the nucleus, STAT proteins, in specific STAT3 activates gene transcription of factors involved in a myriad of cellular processes, including proliferation and survival. By implication, increased expression and activity of STAT3 are associated with human cancers, which argue strongly for the development of STAT inhibitors as a promising chemotherapeutic strategy. We are working with a novel STAT3 inhibitor, OPB-51602, that not only block STAT3 phosphorylation but also inhibits growth and colony formation in a variety of human cancer cell lines. Of note, in addition to targeting STAT3, the compound inhibits mitochondrial respiratory chain and triggers a significant increase in mitochondrial superoxide (O2-) production (within 1 hour of exposure) in NSCLC cell line H1975. Using functional mutants of pY705 and pS727 STAT3, the growth inhibitory activity of the compound could be compromised, however, only S727 mutant rescued the mitochondrial effects. In addition, exposure of cells to OPB-51602 resulted in extracellular acidification (ECAR) together with a complete shutdown of mitochondrial ATP production. Interestingly, these effects could be rescued by 2-deoxyglucose. These data provide evidence that the pharmacological inhibition of STAT3 not only blocks cell proliferation but also induces mitochondrial redox catastrophe and bio-energetic crisis, which could have potential therapeutic implications against cancers that are rendered drug resistant due to the activation of surrogate STAT3 signaling axis.

Citation Format: Jayshree L. Hirpara, Kumi Higuchi, Takeshi Tsunoda, Boon Cher Goh, Shazib Pervaiz. OPB-51602: a novel STAT3 inhibitor that targets mitochondrial respiratory chain and triggers STAT3 dependent ROS production. [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 3080.
Abstract 3237: Assessment and targeting of M2-tumor associated macrophages (M2-TAMs) in prostate cancer
Authors: Zarif Jelani C. , Henandez James R. , Pienta Kenneth J.
Published: July 2016

Prostate cancer is a leading cause of cancer-related deaths of men in the U.S., and in the past year, over 30,000 men died from this disease. While localized prostate cancer is highly treatable by surgical resection and radiation, cancer that has metastasized remains incurable. Alternatively activated macrophages, also known as M2-macrophages, primarily scavenge debris and in the process, promote angiogenesis and wound repair. M2-macrophages are phenotypically similar to M2 tumor-associated macrophages (M2-TAMs) have been reported to associate with solid tumors such as prostate cancer to facilitate epithelial to mesenchymal transition (EMT), tumor invasiveness, metastasis, and resistance to therapy. As an invasive species within the tumor microenvironment, this makes M2-TAMs an ideal therapeutic target in prostate cancer. The purpose of this project is to develop novel therapeutics that will directly target M2-TAMs for destruction and subsequently attenuate prostate tumor growth, progression and metastasis. The central hypothesis of this study is to determine if targeting of M2-TAMs using specific surface antigens will be an effective therapy for lethal prostate cancer and potentially, other cancers. Our study elucidated M2-Macrophage biology including creating homogenous populations of both M1 and M2 macrophages using a new strategy as to allow for identification of novel surface antigen markers. While using our new method for polarization of human monocyte polarization into macrophages, we found that M2 macrophages polarization was stymied in vitro after glutamine deprivation. We then assessed novel markers of macrophage populations, cytokine and chemokine production, T-Cell inactivation and we also assessed whether these surface antigens that are expressed on M2-macrophages are were found in metastatic human prostate cancers indicative of myeloid infiltration. Validation studies using metastatic prostate cancer tissues from rapid autopsies and surgical specimens supplied by the Department of Urology at Johns Hopkins University School of Medicine were also be performed. Complementary studies will be executed to target these markers for anti-tumor cell efficacy using these relevant model systems. By identifying and targeting specific markers on M2-TAMs, we predict that this targeting will provide a better prognosis for patients who have been diagnosed with lethal prostate cancer.

Citation Format: Jelani C. Zarif, James R. Henandez, Kenneth J. Pienta. Assessment and targeting of M2-tumor associated macrophages (M2-TAMs) in prostate 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 3237.
Abstract 3437: Cancer and all-cause mortality among white and blue collar workers in middle-aged and elderly chinese women in a prospective cohort study
Authors: Ji. Bu-Tian
Published: July 2016

Inequalities in health and mortality between white and blue collar workers have been well documented in western countries, particularly among men. Studies regarding occupation related mortality disparities in Asian populations including Chinese are limited. We included 74,941 40-70 years old Chinese women from the Shanghai Women's Health Study (SWHS) in an analysis of occupation and mortality. An in-person interview was conducted at enrolment to obtain lifetime job history as well as lifestyle and other potential risk factors for each participant. Each job title was coded in three digits according to the Chinese National Standard Occupation and Industry Codes Manual, and the categories of white and blue collar work were defined based on this coding. The information on mortality was provided by annual linkage to the Shanghai Vital Statistics Registry. Cox regression models were applied to evaluate the hazard ratios (HRs) and 95% confidence intervals (CIs) of all-cause and cause-specific mortality. In comparison with lifetime white collar workers, lifetime blue collar workers had 18% excess in all cause-mortality (HR = 1.18, 95% CI = 1.04-1.34) and 12% excess in cancer deaths (HR = 1.12, 95% CI = 0.93-1.35) after adjustment for lifestyle and other potential risk factors. A significantly increased risk was also associated with mortality from diabetes (HR = 1.89, 95% CI = 1.06-3.37). There were no statistically significant differences in mortality between white and blue collar workers for other causes of death including circulatory, respiratory, gastrointestinal or other diseases among this population. Our study provides suggestive evidence that blue collar workers may have a higher risk of total cancer mortality compared to white collar workers among women in Shanghai.

Citation Format: Bu-Tian Ji. Cancer and all-cause mortality among white and blue collar workers in middle-aged and elderly chinese women in a prospective cohort study. [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 3437.
Abstract 3521: The effect of MazF, Escherichia coli ribonuclease, on gastric adenocarcinoma (AGS cells)
Authors: Saffarian Maryam , Tzeng Jeremy
Published: July 2016

Apoptosis is a gene-directed program that could be inhibited by several factors such as the disruption of the balance between pro-apoptotic and anti-apoptotic proteins, the impaired death receptor signalling or the reduced caspase functions. Several studies have shown that ribonucleases or antibiotics can induce apoptosis in mammalian cells through the shutoff of the protein synthesises. MazF produced by E. coli is a ribonuclease protein that cleaves mRNAs at ACA sequence sites and inhibits their translation. Thus, we hypothesized that the overexpression of MazF proteins in cancer cells could lead to the induction of apoptosis. In the present study, the ACA-less mazF gene was inserted into pSF-T7-EMCV T7 IRES expression plasmid. The mRNAs of mazF gene were synthesized in in vitro conditions, and one μg/ml of the mRNA was transferred into AGS (Gastric adenocarcinoma) cell line. The incidence of apoptosis was detected by conducting caspase 3/7 and Annexin-V assays. The results showed that 99.83% of cells were detached after 18 hours, 0.0086% of cells remained attached without apparent apoptosis, but 0.101% of attached cells were under apoptotic conditions. The results suggest that MazF protein has an ability to induce apoptosis in AGS cells. Since this protein can cleave mRNAs at ACA sites, it could inhibit protein synthesis, reduce the growth rate, and induce apoptosis in cancer cells. This finding is the first report of the application of MazF as an anti-cancer agent for the induction of apoptosis in AGS cell line.

Citation Format: Maryam Saffarian, Jeremy Tzeng. The effect of MazF, Escherichia coli ribonuclease, on gastric adenocarcinoma (AGS 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 3521.
Abstract 3572: AZ5576, a novel potent and selective CDK9 inhibitor, induces rapid cell death and achieves efficacy in multiple preclinical hematological models
Authors: Cidado Justin , Shen Minhui , Grondine Michael , Boiko Scott , Wang Haiyun , Borodovsky Alexandra , Mazzola Anne Marie , Wu Alan , Lawson Deborah , Ferguson Douglas , Gao Beirong , Cui Andy , D’Cruz Celina , Drew Lisa
Published: July 2016

Cyclin-dependent kinase 9 (Cdk9) is a serine/threonine kinase that regulates elongation of transcription through phosphorylation of RNA polymerase II at serine 2

(pSer2-RNAPII). Mcl1, an anti-apoptotic protein that has been linked to increased survival and chemotherapy resistance in various cancers, can be indirectly modulated through transient inhibition of Cdk9 due to it having a short-lived transcript and being a labile protein. Transient inhibition of Cdk9, therefore, represents a potential therapeutic opportunity in tumors dependent on Mcl1 for survival, including various hematological malignancies. AZ5576 is a potent, highly selective, and orally bioavailable inhibitor of Cdk9 that inhibits Cdk9 enzyme activity with an IC50 <5nM and decreases phosphorlyation of Ser2-RNAPII in cells with an IC50 of 96nM. In the MLL-fusion containing acute myeloid leukemia (AML) line, MV411, short-term treatment with AZ5576 led to a rapid dose-dependent decrease in pSer2-RNAPII with concomitant loss of Mcl1 mRNA and protein, resulting in the cleavage and activation of caspase 3 and loss of cell viability. However, protein expression of other Bcl2 family members (e.g. Bcl2, BclxL, Bim) remained unchanged. Further, AZ5576 induced rapid caspase activation and loss of viability in a diverse set of hematological cancer cell lines, including those from acute myeloid leukemia (16/20), multiple myeloma (10/19), and diffuse large B-cell lymphoma (8/13). In vivo efficacy with intermittent dosing of AZ5576 was also demonstrated in multiple xenograft models from each hematological tumor type. Finally, AZ5576 was still able to induce loss of viability in various multiple myeloma lines cultured in the presence of cytokines or bone marrow stromal cells that represent potential protective effects of the tumor microenvironment. Together, these results highlight the therapeutic potential for selective CDK9 inhibition in the treatment of hematological malignancies.

Citation Format: Justin Cidado, Minhui Shen, Michael Grondine, Scott Boiko, Haiyun Wang, Alexandra Borodovsky, Anne Marie Mazzola, Alan Wu, Deborah Lawson, Douglas Ferguson, Beirong Gao, Andy Cui, Celina D’Cruz, Lisa Drew. AZ5576, a novel potent and selective CDK9 inhibitor, induces rapid cell death and achieves efficacy in multiple preclinical hematological models. [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 3572.
Abstract 3622: Complete workflow for detection of low frequency somatic mutations from cell-free DNA using Ion Torrent™ platforms
Authors: Gu Jian , Brinza Dumitru , Mongan Ann , Chien Richard , Dhingra Dalia , Hyland Fiona , Bramlett Kelli
Published: July 2016

Research detecting of somatic mutations in circulating cell-free DNA (cfDNA) using research blood samples from subjects previously diagnosed with cancer provides a potential non-invasive approach to monitor cancer status and evaluate cancer evolution in the future. However, most of the existing mutation detection methods show insufficient sensitivity to detect cfDNA mutations since only small amount of mutant gene fragments, derived from tumor cells, is present in a large amount of normal circulating DNA background.

We demonstrated a complete workflow that includes blood collection, cfDNA isolation, library preparation, sequencing, and data analysis to enable detection of rare DNA variants in blood plasma samples. Blood samples were collected using Streck™ DNA tubes followed by plasma preparation and cfDNA isolation using MagMAX™ Cell-Free DNA Isolation Kit. Library preparation was performed using Oncomine™ lung cfDNA kit. Barcoded libraries were pooled and sequenced on Ion Torrent™ Next Generation Sequencing Platforms. Sequencing data was analyzed in Torrent Suite™ using variantCaller-cfDNA plugin. ∼150 biomarkers relevant to non-small cell lung cancer were interrogated in one sequencing run.

We demonstrated detection sensitivity at 0.1% frequency using engineered mutants that were spiked into control DNA samples. The workflow was tested on a set of research samples from matched tumor FFPE and blood plasma collected from research subjects with non-small cell lung cancer (NSCLC). About 1 mL of plasma was processed using the workflow described above. RecoverAll™ Multi-Sample RNA/DNA Isolation Workflow was used to isolate DNA from FFPE samples, followed by library preparation, sequencing and data analysis using the same workflow described above. Summary of variant calls from matched cfDNA and FFPE tumor samples are presented here. Results indicate high sensitivity of the workflow and expected levels of concordance between variants detected in the two types of research samples.

In this study, we developed a highly sensitive and reliable research workflow to detect rare somatic mutations in circulating cfDNA samples. Significant overlapping of mutations discovered in FFPE tumor and cfDNA samples suggests that this workflow may be used to monitor tumor dynamics in NSCLC and potentially other tumors in the future.

Disclaimer: For research use only. Not for use in diagnostic procedures.

Citation Format: Jian Gu, Dumitru Brinza, Ann Mongan, Richard Chien, Dalia Dhingra, Fiona Hyland, Kelli Bramlett. Complete workflow for detection of low frequency somatic mutations from cell-free DNA using Ion Torrent™ platforms. [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 3622.
Abstract 3643: Flow cytometry multiplexing used to analyze metabolic activity and assess pharmaceutical compound toxicity as a high throughput screening tool
Authors: OGrady Mike , Montoya Leticia A. , Yan Michelle , Clarke Scott T. , Low Quentin , DeMarco Carolyn , Anderson April , Kihn Kathy , Finnessy Carmen , Wickett Marcy , Calderon Veronica
Published: July 2016

High throughput screening (HTS) is an extremely effective method for allowing researchers to identify putative active compounds for therapeutics. Recently, flow cytometry has emerged as a powerful HTS tool with the added benefit of cell-by-cell analysis. Flow cytometry allows a researcher to study protein-protein interactions, metabolic activities, as well as DNA content in a single or multiplexed assay format. For this study, we screened the Tocriscreen Total compound library and compared HTS using a plate reader with flow cytometer multiplexing.

Peripheral blood mononuclear (PBMCs) and Jurkat cells were used as cell models. Cells were cultured under standard conditions and either at normoxic (5%) or hyperoxic (19%) oxygen levels. We also varied length of time the cells were treated with compound library (24, 48, or 72 hrs). Membrane integrity and metabolic activity were measured as an output for evaluating cellular viability. To further assess cellular activity, post-screening analysis was implemented to establish EC50s of “hits” from compound library.

Screening analysis identified target drugs that were further analyzed using cell health readouts with different mechanisms of action to assess compound toxicity. Results indicate that compound “hits” and potency differed in the screen depending on cell type, the mechanistic readout, length of compound exposure, and mode of readout used to perform the HTS experiments.

Citation Format: Mike OGrady, Leticia A. Montoya, Michelle Yan, Scott T. Clarke, Quentin Low, Carolyn DeMarco, April Anderson, Kathy Kihn, Carmen Finnessy, Marcy Wickett, Veronica Calderon. Flow cytometry multiplexing used to analyze metabolic activity and assess pharmaceutical compound toxicity as a high throughput screening tool. [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 3643.
Abstract 3764: Trabectedin activity in patient-derived mesothelioma xenografts
Authors: Licandro Simonetta Andrea , Frapolli Roberta , Bello Ezia , Libener Roberta , Orecchia Sara , Grosso Federica , D’Incalci Maurizio
Published: July 2016

Malignant pleural mesothelioma (MPM) incidence is increased over the past two decades and it is predicted to increase further in the next 20 years especially in developing countries where asbestos has not yet been banned. Surgery is not an option for the majority of patients due to the diffuse growth of MPM, making chemotherapy the treatment of choice, but prognosis remains very poor with a median survival time of 10-17 months. Trabectedin (ET-743, Yondelis) is a marine alkaloid used for the 2nd line therapy of ovarian cancer (combined with pegylated doxorubicin) and soft tissue sarcomas. It binds the DNA minor groove modulating transcription of cancer cells and has an anti-inflammatory effect modifying tumor microenvironment and reducing tumor associated macrophages.

The aim was to test antitumor activity of trabectedin in newly established patient derived xenografts of MPM.

MPM473, MPM484 and MPM487 xenografts were obtained in athymic nude mice by s.c. injection of 107 cells isolated from the pleural effusion of MPM patients and maintained by serial passages in mice. H&E staining and immunohistochemistry were performed. Mice were randomized when tumor reached about 200 mg to receive trabectedin, cisplatin, pemetrexed, gemcitabine, and imatinib. Antitumor activity was expressed as T/C% were T and C are the mean tumor weight of treated and control mice, respectively.

The three xenografts reproduced the morphology and the histochemical profile of the human tumors. As reported in the table trabectedin was able to induce a 50% reduction in tumor growth in MPM473 and MPM 487 xenografts and only a marginal effect in MPM484 xenograft. TreatmentDose (mg/kg)ScheduleBest T/C% (Day)MPM473MPM484MPM487Cisplatin5i.v. q7dx361.9 (69)66.1 (69)67.3 (39)Gemcitabine100i.p. q4dx583.2 (41)67.7 (48)49.7 (56)Imatinib200p.o. qdx1568.9 (76)89.4 (55)87.1 (39)Pemetrexed250i.p. q4dx477.4 (52)76.8 (49)84.3 (39)Trabectedin0.15i.v. q7dx351.0 (73)63.6 (69)51.3 (56)

In conclusion we report three new MPM xenografts able to mimic the pathological features of human MPM. Although trabectedin showed only a moderate antitumor activity in our experimental models, its efficacy is comparable or better than that of the other drugs currently used for this disease. Studies are in progress in syngenic models that are more appropriate to test also the ability of trabectedin to modulate the immune system.

Citation Format: Simonetta Andrea Licandro, Roberta Frapolli, Ezia Bello, Roberta Libener, Sara Orecchia, Federica Grosso, Maurizio D’Incalci. Trabectedin activity in patient-derived mesothelioma xenografts. [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 3764.
Abstract 3785: Targeting PDLIM5 for lung cancer
Authors: Park Deborah , Cheng Han , Chen Tianji , Tor Merve , Khatib Nour , Huang Jason , Zhou Qiyuan , Rong Lijun , Zhou Guofei
Published: July 2016

Rationale: Lung cancer is the most common cause of cancer-related death in the United States and worldwide. Despite decades of efforts on research and smoking cessation, the 5-year survival rate of lung cancer patients remains poor at 15%, and the population of adenocarcinoma cases in nonsmokers is rising. PDLIM5, a member of the Enigma subfamily of PDZ and LIM domain protein family, contains an N-terminal PDZ domain and 3 LIM domains at its C-terminus. We have previously shown that PDLIM5 regulates TGF-β/Smad family, which are known to participate in the pathogenesis of lung cancer. In this study, we aim to investigate whether PDLIM5 plays a role in lung cancer and establish a high throughput screening (HTS) platform for PDLIM5-targeted drug discovery.

Methods: We searched the Oncomine data base for PDLIM5 gene expression. We suppressed PDLIM5 in A549 cells and determined the expression levels of epithelial-mesenchymal transition markers. We exposed A549 cells to hypoxia or treated them with TGF-β1 and measured the expression levels of PDLIM5. We generated a stable mink lung epithelial cell line (MLEC) containing a TGFβ/Smad luciferase reporter with lentivirus-mediated suppression of PDLIM5 (MLEC-shPDLIM5) and measured levels of Smad2/3 and pSmad2/3. We used MLEC-shPDLIM5 and a control cell line (MLEC-shCTL) to screen the Prestwick library (1,200 compounds). We treated MLEC and A549 cells with paclitaxel and determined levels of Smad2/3 and pSmad2/3.

Results: We found that PDLIM5 was overexpressed in non-small-cell lung carcinoma (NSCLC) patients and that the expression levels of PDLIM5 inversely correlated with the survival rate of lung cancer patients. Hypoxia but not TGF-β1 induced PDLIM5 expression levels. Suppression of PDLIM5 induced E-cadherin levels while decreased levels of vimentin and α-SMA. In MLEC, suppression of PDLIM5 decreased Smad-dependent luciferase activity, smad3, and pSmad2. More importantly, we identified and validated that paclitaxel as a PDLIM5 inhibitor in MLEC. Furthermore, we showed that paclitaxel inhibited Smad2 expression and Smad3 phosphorylation in A549 cells.

Conclusions: We have shown that PDLIM5 is overexpressed in lung cancer cells and its upregulation is hypoxia-dependent but TGF-β independent. Loss of PDLIM5 inhibits EMT. We have identified paclitaxel as a PDLIM5 inhibitor. This system is robust and suitable for PDLIM5 targeted drug discovery. Support: a UIC UICentre Arm1 Inception Grant, a Gilead Sciences Research Scholars Program in Pulmonary Arterial Hypertension award and R01 HL123804 (G. Zhou).

Citation Format: Deborah Park, Han Cheng, Tianji Chen, Merve Tor, Nour Khatib, Jason Huang, Qiyuan Zhou, Lijun Rong, Guofei Zhou. Targeting PDLIM5 for 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 3785.
Abstract 3961: Methodological considerations in the preparation of biomimetic reference materials for ctDNA assays
Authors: Harkins Seth B. , Tomson Farol L. , Anekella Bharathi , Garlick Russell
Published: July 2016

Clinical utilization of cell-free DNA (cfDNA) has become commonplace for NIPS testing and has recently been more widely deployed to quantitate cell-free circulating tumor DNA (ctDNA) for use in cancer patient care longitudinally across the treatment spectrum from subclinical, to diagnosis and through treatment. The scarcity and unstable nature of the cfDNA analyte in human plasma is problematic and thus the lack of robust detection methods has limited clinical validation. In this work we sought to avoid the inherent limitations of plasma-based patient reference materials and developed a strategy to prepare a biomimetic reference material which is sufficiently equivalent to the cfDNA found in patients with malignant cancers. Thus far the scarcity and physical form of ctDNA in human plasma has attenuated the validation of assays for clinical utility. To this end, we have generated a biosynthetic construct with short DNA fragments containing relevant cancer mutation sequences, including single-nucleotide variations, insertions and deletions in BRAF, EGFR, KRAS, NRAS, PIK3CA or HER/ERBB2. The mutant fragments were titrated into normal human reference DNA, GM24385, and ultrasonically sheared to ∼160 bp. Precise allele frequency blends were verified by digital PCR (dPCR) prior to encapsulation to enhance stability and compatibility with commutable plasma. The reference material is formulated to 20 ng/mL of extractable DNA in modified Seracare Matribase™ to confer commutability in pre-analytic processes. These materials have been quantitated by dPCR and/or Next Generation Sequencing (NGS). A dilution panel was prepared at 10%, 5%, 2.5%, 1.25%, 0.1% allele frequency (mutant:normal DNA). Linearity (R2 = 0.9997) was observed by dPCR (measured vs. expected) and the NGS detected, Swift Accel-Amplicon 56G Oncology Panel (R2 = 0.997). Due to the low abundance of ctDNA, a characterized reference set is critical to validate assays that are developed with extremely low limit of detection. Data will be presented to show the breadth of this methodology and its utility in validating NGS and dPCR assays.

Citation Format: Seth B. Harkins, Farol L. Tomson, Bharathi Anekella, Russell Garlick. Methodological considerations in the preparation of biomimetic reference materials for ctDNA assays. [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 3961.
Abstract 4026: Epigenetic changes in T cells in response to immune checkpoint blockade
Authors: Goswami Sangeeta , Chen Jianfeng , Zhao Hao , Zhang Xuejun , Sharma Padmanee
Published: July 2016

Immune checkpoint blockade therapy has led to clinical success and have established immunotherapy as one of the primary modality of cancer treatment. It leads to a durable response in around 20% of the patients (responders). Success of immune checkpoint blockade therapy relies on the increase of effector T cells and decrease of regulatory T cells in the tumor microenvironment. However, the underlying mechanism that govern the ratio of effector and regulatory T cells between responder and non-responder to checkpoint blockade is not understood. Understanding the process could reveal novel mechanisms to boost efficacy of immune checkpoint blockade. Differentiation of naïve T cells into effector and regulatory phenotype requires lineage-specifying transcription factors and epigenetic modifications that allow appropriate repression or activation of gene transcription. One key epigenetic modification for T cell differentiation is trimethylation of lysine 27 on histone H3 (H3K27me3). Loss of H3K27me3 results in increased Th-1 plasticity whereas presence of H3K27me3 on Foxp3 locus is required to maintain the function of T-regulatory cells. We hypothesize that immune checkpoint blockade changes the epigenetic landscape in tumor infiltrating T cells that results in an effector phenotype and epigenetic modification of H3K27me3 could promote an anti-tumor immune microenvironment in tumor bearing mice.We showed that in-vivo inhibition of H3K27me3 in combination with anti-CTLA4 results in reduction of tumor size and also reduction in regulatory T cells in a B16-F10 melanoma mice model compared to anti-CTLA4 treatment alone, suggesting H3K27me3 inhibition could increase the efficacy of checkpoint blockade therapy. Future studies will aim to elucidate epigenetic changes in H3K27me3 and define an epigenetic signature as a result of immune checkpoint therapy by ChIP-sequencing. Data generated in this project will greatly enhance our understanding of epigenetic regulation of T cell differentiation is response to immune checkpoint blockade and aid identifying key changes that could be further modified to overcome resistance to immune checkpoint blockade in non-responders.

Citation Format: Sangeeta Goswami, Jianfeng Chen, Hao Zhao, Xuejun Zhang, Padmanee Sharma. Epigenetic changes in T cells in response to immune checkpoint blockade. [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 4026.
Abstract 4260: 3D colorectal cancer organoids as preclinical models for assessment of activity of RAF inhibitor BGB-283
Authors: Wu Xiaoran , Fu Chunyan , Yuan Xi , Zhang Lianhai , Wang Xiaohong , Ji Jiafu , Luo Lusong
Published: July 2016

Tumor organoids are three-dimensional (3D) cultures of cancer cells. They can be derived from the tumor cell lines, patient-derived xenografts and tumor tissues from cancer patients, thereby providing an attractive in vitro assay to optimize anticancer treatment. Oncogenic B-RAF, which drives cell transformation and proliferation, has been detected in 5-15% of colorectal cancers (CRC). Despite the remarkable clinical activities achieved by vemurafenib in treating B-RAF V600E metastatic melanoma, their clinical efficacy in B-RAF V600E CRC is far less impressive. BGB-283, currently under clinical development for RAF and RAS mutated solid tumors, is a novel inhibitor of the RAF dimer with inhibitory activities against RAF family kinases. In this study, we evaluated the anti-proliferative effect of BGB-283 and compared it to BRAF inhibitor vemurafenib in CRC patient tumor derived organoids models. BGB-283 exhibited therapeutic potentials in tumors with mutations in the MAPK pathway. Herein, we reported the establishment of tumor organoid models from 19 CRC patients’ tumor tissues with a success rate of 83%. Organoids maintained many characteristics of their original tumors based on their histology. Our studies using primary CRC tumor organoids showed that in contrast to vemurafenib, which only inhibited B-RAF V600E CRC tumors, BGB-283 had inhibition effect on both B-RAF V600E and KRAS mutated tumors. These findings support BGB-283 as a promising clinical stage antitumor agent in treating CRC harboring B-RAF V600E and KRAS mutations. In addition, we showed that patient-derived organoids can be a valuable tool to test drug sensitivity in a personalized treatment and help to fill the gap between tumor genetics and patient trials.

Citation Format: Xiaoran Wu, Chunyan Fu, Xi Yuan, Lianhai Zhang, Xiaohong Wang, Jiafu Ji, Lusong Luo. 3D colorectal cancer organoids as preclinical models for assessment of activity of RAF inhibitor BGB-283. [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 4260.
Abstract 4293: Combination of hepatocyte growth factor, genistein and daidzein concentrations as a biomarker for gastric cancer risk: A nested case-control study
Authors: Jang Jieun , Hwang Yunji , Ahn Choonghyun , Ko Kwang-Pil , Yoo Keun-Young , Park Sue K.
Published: July 2016

Backgrounds: Hepatocyte growth factor (HGF) induces ERK pathway which is associated with carcinogenesis by promoting cell proliferation. Inhibition of ERK signal transduction pathways by genistein, one of the isoflavones, has been reported by several in vitro studies. Therefore, gastric cancer risk by HGF and isoflavone concentrations was evaluated in this study.

Methods: Nested case-control study with 93 gastric cancer cases and 1:1 matched controls was conducted within the Korean Multi-center Cancer Cohort (KMCC). HGF and ISF (genistein, daidzein, equol) concentrations in plasma were measured with enzyme-linked immunosorbent assay and time-resolved fluoroimmunoassay, respectively. Odds ratios (ORs) and 95% confidence intervals (95% CIs) on gastric cancer risk according to HGF and ISF level was calculated using conditional logistic regression models.

Results: Compared to the group with low level of HGF (<315pg/mL) and high level of genistein (≥212.5nmol/L), the group having high level of HGF and lower level of genistein was associated with increased gastric cancer risk (OR = 5.08, 95% CI = 1.39-18.57). Elevated gastric cancer risk in the subjects having high HGF level and low daidzein level than subjects with low HGF and high daidzein levels was also shown (OR = 3.67, 95% CI = 1.18-11.45). There was significant interaction between HGF and genistein levels on gastric cancer was found (p for interaction<0.01). Continual increase in gastric cancer risk according to the counts of risky status by HGF, genistein, and daidzein levels was found (p for trend = 0.03).

Conclusion: Our study suggests that combination of HGF, genistein and daidzein concentrations has potential as a biomarker for gastric cancer risk.

Citation Format: Jieun Jang, Yunji Hwang, Choonghyun Ahn, Kwang-Pil Ko, Keun-Young Yoo, Sue K. Park. Combination of hepatocyte growth factor, genistein and daidzein concentrations as a biomarker for gastric cancer risk: A nested case-control study. [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 4293.
Abstract 4313: Periodic fasting mimicking diet delays cancer development and progression
Authors: Brandhorst Sebastian , Navarrete Gerardo , Wei Min , Morgan Todd E. , Longo Valter D.
Published: July 2016

Dietary intake and composition can have profound effects on aging and age-related pathologies including cancer. As such, studies of fasting (the complete withdrawal of food) and dietary restriction (DR, generally described as a 20- 40% reduction in calories) have contributed to the understanding of the relationship between nutrients, cellular aging, cancer development and cancer cell survival. DR may only be feasible in delaying tumor development but might not present an effective treatment modality due to the time required to achieve beneficial effects. Further, chronic DR may also have negative impacts on wound healing, which could be detrimental in patients undergoing surgery. Our findings in yeast indicated that starvation decreases age-dependent mutations whereas in mouse xenograft tumor models fasting alone can be as effective as chemotherapy drugs in delaying the progression of certain tumor types, in part through the reduction of extracellular glucose and IGF-1 concentration/signaling. Fasting, although results indicate efficacy as a treatment option, on the other hand may not be feasible for patients at risk for undernutrition or cachexia.

To address the need for a dietary based intervention that both delays cancer development and progression, we developed a fasting-mimicking diet (FMD) to ensure micronutrient supply and tested its effectiveness in rodent models. Periodic bi-weekly FMD cycles had a significant impact on the development and progression of multiple cancer models while allowing the preservation of lean body mass even at old age and extending longevity in healthspan study. As a treatment modality, FMD cycles alone significantly reduced tumor progression and sensitized various cancer models to chemotherapeutic drugs.

Citation Format: Sebastian Brandhorst, Gerardo Navarrete, Min Wei, Todd E. Morgan, Valter D. Longo. Periodic fasting mimicking diet delays cancer development and progression. [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 4313.