107 results on '"Dianne L. Newton"'
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2. Abstract 40: NCI patient derived models repository: PDX, organoid and cell lines from the same patient - bridging the translational pipeline
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Yvonne A. Evrard, Li Chen, Sergio Alcoser, Gareth Bliss, Carrie Bonomi, Suzanne Borgel, John Carter, Ting-Chia Chang, Alice Chen, Kevin Cooper, Biswajit Das, Kelly Dougherty, Lindsay Dutko, Marion Gibson, Michelle M. Ahalt-Gottholm, Tara Grinnage-Pulley, Keegan Kalmbach, Chris Karlovich, Kimberly Klarmann, Shahanawaz Jiwani, Tiffanie Miner, Michael Mullendore, Matthew Murphy, Kevin Plater, Gloryvee Rivera, Jessica Steed, Luke Stockwin, Cindy R. Timme, Dianne L. Newton, Paul Mickey Williams, Melinda G. Hollingshead, and James H. Doroshow
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Cancer Research ,Oncology - Abstract
The National Cancer Institute’s Patient-Derived Models Repository (NCI PDMR; https://pdmr.cancer.gov) has developed a national repository of Patient-Derived Models (PDMs) comprised of patient-derived xenografts (PDXs), in vitro patient-derived tumor cell cultures (PDCs) and cancer associated fibroblasts (CAFs) as well as patient-derived organoids (PDOrg). These PDMs are clinically annotated with molecular information available in an easily accessible database for the extramural community. A key effort in developing these models is to develop matched models sets allowing for larger scale screening efforts using 2D or 3D models to prioritize selection of PDX models for preclinical translational research. To date, over 220 model sets with a PDX, PDOrg, and PDC from a single patient have been developed; 40 of these have matched CAF models allowing for exploration of research questions focused on tumor microenvironment. The largest model sets are in colorectal cancer (COADREAD, n=76), gynecologic cancers (n=33), pancreatic adenocarcinoma (PAAD, n=29), melanoma (MEL, n=19), and head and neck squamous cell carcinomas (HNSCC, n=17). Every model undergoes several quality control assessments that serve as go/no-go criteria including pathology assessment, STR validation, NGS concordance assessment and for PDXs, human:mouse DNA content assessment. It should be noted that not every model is successful in the development or QC phase so additional model sets with only one or two of the model types are also available for researcher requests, for example there are over 125 PDX/PDOrg matched model sets. The NCI is currently performing parallel preclinical screening of PDXs and PDCs or PDOrgs to determine the ability of the in vitro lines to predict in vivo activity. Genetic and histopathologic assessment of these matched model sets have demonstrated a high degree of stability by somatic mutation, copy number alteration (CNA) and gene expression data. Gene expression correlation analysis shows that mean of Spearman r between PDXs 0.89, between matched PDC/PDXs 0.79, and between matched PDOrg/PDXs 0.82. As expected, some variation at the gene expression level when comparing PDX to in vitro cultures by t-SNE can be observed, likely due to the differences in culture conditions. Funded by NCI Contract No. HHSN261200800001E Citation Format: Yvonne A. Evrard, Li Chen, Sergio Alcoser, Gareth Bliss, Carrie Bonomi, Suzanne Borgel, John Carter, Ting-Chia Chang, Alice Chen, Kevin Cooper, Biswajit Das, Kelly Dougherty, Lindsay Dutko, Marion Gibson, Michelle M. Ahalt-Gottholm, Tara Grinnage-Pulley, Keegan Kalmbach, Chris Karlovich, Kimberly Klarmann, Shahanawaz Jiwani, Tiffanie Miner, Michael Mullendore, Matthew Murphy, Kevin Plater, Gloryvee Rivera, Jessica Steed, Luke Stockwin, Cindy R. Timme, Dianne L. Newton, Paul Mickey Williams, Melinda G. Hollingshead, James H. Doroshow. NCI patient derived models repository: PDX, organoid and cell lines from the same patient - bridging the translational pipeline [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 40.
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- 2023
3. Identification of CBX3 and ABCA5 as putative biomarkers for tumor stem cells in osteosarcoma.
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Vaibhav Saini, Curtis D Hose, Anne Monks, Kunio Nagashima, Bingnan Han, Dianne L Newton, Angelena Millione, Jalpa Shah, Melinda G Hollingshead, Karen M Hite, Mark W Burkett, Rene M Delosh, Thomas E Silvers, Dominic A Scudiero, and Robert H Shoemaker
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Medicine ,Science - Abstract
Recently, there has been renewed interest in the role of tumor stem cells (TSCs) in tumorigenesis, chemoresistance, and relapse of malignant tumors including osteosarcoma. The potential exists to improve osteosarcoma treatment through characterization of TSCs and identification of therapeutic targets. Using transcriptome, proteome, immunophenotyping for cell-surface markers, and bioinformatic analyses, heterogeneous expression of previously reported TSC or osteosarcoma markers, such as CD133, nestin, POU5F1 (OCT3/4), NANOG, SOX2, and aldehyde dehydrogenase, among others, was observed in vitro. However, consistently significantly lower CD326, CD24, CD44, and higher ABCG2 expression in TSC-enriched as compared with un-enriched osteosarcoma cultures was observed. In addition, consistently higher CBX3 expression in TSC-enriched osteosarcoma cultures was identified. ABCA5 was identified as a putative biomarker of TSCs and/or osteosarcoma. Lastly, in a high-throughput screen we identified epigenetic (5-azacytidine), anti-microtubule (vincristine), and anti-telomerase (3,11-difluoro-6,8,13-trimethyl- 8H-quino [4,3,2-kl] acridinium methosulfate; RHPS4)-targeted therapeutic agents as candidates for TSC ablation in osteosarcoma.
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- 2012
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4. Abstract 3012: Patient-derived models of rare cancers in the National Cancer Institute's patient-derived models repository
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Michael Mullendore, Jenna Hull, P. Mickey Williams, Cindy R. Timme, Dianne L. Newton, Tia Shearer, Shahanawaz Jiwani, Li Chen, Chris Karlovich, Michelle Eugeni, Tara Grinnage-Polley, James H. Doroshow, Kristen Cooley, Tom Walsh, Devynn Breen, Emily Delaney, Kimberly Klarmann, Michelle M. Gottholm-Ahalt, Melinda G. Hollingshead, Jesse Stottlemyer, Chelsea McGlynn, Malorie Morris, John Mark Carter, Yvonne A. Evrard, Alice P. Chen, Ting-Chia Chang, Candace Mallow, Sergio Y. Alcoser, Shannon Uzelac, Biswajit Das, and Justine Mills
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Oncology ,Cancer Research ,medicine.medical_specialty ,business.industry ,Internal medicine ,medicine ,Cancer ,business ,medicine.disease - Abstract
There is an unmet need for preclinical models of rare cancers and rare disease sub-types. The National Cancer Institute's Patient-Derived Models Repository (NCI PDMR; https://pdmr.cancer.gov) is developing quality-controlled, early-passage, clinically-annotated patient-derived tumor xenografts (PDXs), in vitro tumor cell cultures (PDCs), cancer associated fibroblasts (CAFs), and patient-derived organoids (PDOrg) and has focused on addressing unmet needs in the preclinical model space including developing models from adult and pediatric patients with rare cancers. To date, NCI has created and molecularly characterized over 150 preclinical models of rare cancer including indications such as Hurthle cell carcinoma, osteosarcomas, Merkel cell carcinomas, salivary gland cancers, synovial sarcomas, and carcinosarcomas. Rare cancer models developed to date will be reviewed and their histopathologic and molecular characteristics compared to that reported in the clinical setting. A pipeline to identify fusion proteins in these rare cancers such as the Ewing sarcoma EWSR1-FLI1 fusion and NAB2-STAT6 fusions in solitary fibrous tumors (SFT) has been implemented. Four malignant peripheral nerve sheath tumors (MPNST) PDX models are available for researches; these models were developed from patients diagnosed between the ages of 37-68. At the time of model development, two patients were treatment naïve and two had prior radiotherapy. Two of the MPNST PDX models have NF1 oncogenic mutations, three have deep deletions in CDKN2A/B, and three have a mutation in either EED or SUZ12 consistent with the reported molecular characteristics of patients with MPNST. Also of clinical relevance, of two mesothelioma models available, one carries an NF2 driver mutation and the other BAP1 and LATS2 and a PDX model for Hurthle cell carcinoma has wide-spread loss of heterozygosity (LOH 80%). Models for other rare cancers are in development, including four cholangiocarcinoma PDXs with histopathologic confirmation that are currently being expanded for molecular characterization and distribution. Funded by NCI Contract No. HHSN261200800001E Citation Format: Cindy R. Timme, Sergio Y. Alcoser, Devynn Breen, John Carter, Ting-Chia Chang, Alice Chen, Li Chen, Kristen Cooley, Biswajit Das, Emily Delaney, Michelle A. Eugeni, Michelle M. Gottholm-Ahalt, Tara Grinnage-Polley, Jenna Hull, Chris Karlovich, Kimberly Klarmann, Shahanawaz Jiwani, Candace Mallow, Chelsea McGlynn, Justine Mills, Malorie Morris, Michael Mullendore, Dianne Newton, Tia Shearer, Jesse Stottlemyer, Shannon Uzelac, Thomas Walsh, P. Mickey Williams, Yvonne A. Evrard, Melinda G. Hollingshead, James H. Doroshow. Patient-derived models of rare cancers in the National Cancer Institute's patient-derived models repository [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3012.
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- 2021
5. Abstract 3139: Study of tumor heterogeneity and subclonality in primary pancreatic and metastatic sites from rapid autopsy patients in PDMR
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Lindsay Dutko, Shahanawaz Jiwani, Dianne L. Newton, Li Chen, Chris Karlovich, Michelle Eugeni, James H. Doroshow, Kelly Benauer, P. Mickey Williams, Nikitha Nair, Chapman Alyssa, Anna L. Fong, Biswajit Das, Luis E. Romero, Melinda G. Hollingshead, Yvonne A. Evrard, Ting-Chia Chang, Tomas Vilimas, Kelly Dougherty, Amanda Peach, and Marianne Morton
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Cancer Research ,Somatic cell ,business.industry ,Cancer ,Drug resistance ,medicine.disease ,medicine.disease_cause ,Tumor heterogeneity ,Metastasis ,Germline mutation ,Oncology ,Cancer research ,medicine ,Adenocarcinoma ,KRAS ,business - Abstract
Background: A major set of preclinical models derived from specimens acquired from rapid autopsy patients in the National Cancer Institute Patient-Derived Models Repository (NCI PDMR, https://pdmr.cancer.gov) were from pancreatic adenocarcinoma (PAAD) patients, with metastatic specimens originating from liver, colon, omentum, and lung. Genomic characterization of these preclinical models provides a unique opportunity to study tumor heterogeneity and subclonality associated with the metastatic process and potential treatment resistance. Methods: To date, 30 rapid autopsy patient-derived xenograft (PDX)/patient-derived cell (PDC)/patient-derived organoid (PDOrg ) models derived from pancreatic adenocarcinoma patients (n = 9) have been sequenced using whole-exome sequencing (WES) and RNASeq. Tumor heterogeneity between primary and metastatic sites was studied based on somatic mutation, copy number alteration (CNA) and gene expression data. A bioinformatics workflow was developed to stably infer and visualize the tumor subclonality by integrating the tools of PyClone, SCHISM, and TIMESCAPE, using somatic mutations and site-specific copy number data of multiple samples generated from PDX models in primary and metastatic sites. Results: Among 30 rapid autopsy preclinical models from primary and metastatic sites, liver is the most common metastatic site in PAAD (9/19=47%) compared to other sites. Driver mutations are conserved in all preclinical model specimens derived from a given patient. KRAS p.G12D is present in 28 PDX/PDC/PDOrg models as well as the corresponding patient specimens, and BRAF p.V600E is present in other preclinical models. The fraction of the genome affected by CNA remains stable within a PDX model across passages (n=18, mean=7.63%, sd=5.90%). However, we found that this increased when comparing PDX models derived from metastatic sites versus the primary site (n=16, mean=19.47%, sd=9.69%). This indicates the presence of tumor heterogeneity between metastatic and primary sites. Site-specific subclones were identified in PDX models from two patients (521955 and 485368) and a phylogenetic tree of primary and metastasis sites indicates that one liver metastasis had a unique seeding event compared to the other metastatic sites for both patients. Conclusion: Tumor heterogeneity and subclonality was observed in preclinical models generated from PAAD patients in the NCI PDMR. These models provide a unique resource for preclinical studies in tumor evolution, metastatic spread mediators, and drug resistance. Citation Format: Li Chen, Biswajit Das, Yvonne A. Evrard, Chris A. Karlovich, Tomas Vilimas, Amanda Peach, Chapman Alyssa, Nikitha Nair, Anna L. Fong, Luis Romero, Ting-Chia Chang, Shahanawaz Jiwani, Lindsay Dutko, Kelly Benauer, Marianne Morton, Kelly Dougherty, Michelle A. Eugeni, Dianne Newton, Melinda G. Hollingshead, P. Mickey Williams, James H. Doroshow. Study of tumor heterogeneity and subclonality in primary pancreatic and metastatic sites from rapid autopsy patients in PDMR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3139.
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- 2021
6. Abstract 3916: Patient-derived organoid and cell culture models from the NCI Patient-Derived Models Repository (NCI PDMR) preserve genomic stability and heterogeneity of patient tumor specimens
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Luis E. Romero, Kaitlyn Arthur, Robin D. Harrington, Justine N. McCutcheon, Brandie A. Fullmer, Gloryvee Rivera, Li Chen, Savanna Styers, Matthew R. Murphy, Lindsay Dutko, Rajesh Patidar, Kelly Benauer, Alyssa K. Chapman, Marion Gibson, Abigail Walke, Carrie Bonomi, Vishnuprabha R. Kannan, Luke H. Stockwin, Paul Williams, Tomas Vilimas, Kelly Dougherty, Amanda Peach, Jenna Moyer, Biswajit Das, Michelle M. Gottholm-Ahalt, Peng Wang, James H. Doroshow, Nikitha Nair, Erin Cantu, Kelsey A. Conley, Melinda G. Hollingshead, Anna Wade, Thomas Forbes, Anna J. Lee Fong, Kevin Plater, Joseph P. Geraghty, Mariah Baldwin, Dianne L. Newton, Yvonne A. Evrard, Shahanawaz Jiwani, Chris Karlovich, and Michael Mullendore
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Cancer Research ,Cancer ,Variant allele ,Early passage ,Biology ,medicine.disease ,Tumor tissue ,Tumor heterogeneity ,Genomic Stability ,Oncology ,Copy Number Alteration ,Cell culture ,medicine ,Cancer research - Abstract
Background: The National Cancer Institute (NCI) has developed a Patient-Derived Models Repository (PDMR; https://pdmr.cancer.gov) of preclinical models including patient-derived xenografts (PDX), organoids (PDOrg) and patient-derived cell cultures (PDC). Extensive clinical annotation and genomic datasets are available for these preclinical models. However, it is unclear if the molecular profiles of the corresponding patient tumors are stably propagated in these models. We have previously demonstrated that PDX models from the NCI PDMR faithfully represent the patient tumors both in terms of genomic stability and tumor heterogeneity. Here, we conduct an in-depth investigation of genomic representation of patient tumors in the PDOrgs and PDCs. Methods: PDOrgs (n=64) and PDCs (n=94) were established from tumor fragments (i.e., initiator specimens) obtained either from patient specimens or from PDX specimens of early passage. For some models (n=19), both PDOrgs and PDCs were generated from the same tumor tissue; in fewer cases (n=4), PDCs were established from organoids derived from patient specimens. Whole Exome Sequencing and RNA-Seq were performed on all PDCs and PDOrgs, and data were compared with patient specimens or early passage PDXs. Results: A majority of the PDOrgs and PDCs have stably inherited the genome of the corresponding patient specimens based on the following observations: (1) >87% of PDOrgs and PDCs maintained similar copy number alteration profiles compared with the initiator specimens of the preclinical model; (2) the variant allele frequency (VAF) of clinically relevant mutations remained consistent between the PDOrgs, PDCs, and the initiator specimens, with none of the PDCs or PDOrgs deviating by >15% VAF; and (3) clinically relevant biomarkers (e.g., MSI, LOH, mutational signatures etc.) are concordant amongst the PDOrgs, PDCs, and the initiator specimens. We observed that the majority of SNVs and indels present in the initiator specimens were also found in the PDOrgs and PDCs, suggesting almost all the tumor heterogeneity was preserved in these preclinical models. Conclusions: This large and histologically diverse set of PDOrgs and PDCs from the NCI PDMR exhibited genomic stability and faithfully represented the tumor heterogeneity observed in corresponding patient specimens. These preclinical models thus represent a valuable resource for researchers interested in pre-clinical drug or other studies. Citation Format: Biswajit Das, Yvonne A. Evrard, Li Chen, Rajesh Patidar, Tomas Vilimas, Justine N. McCutcheon, Amanda L. Peach, Nikitha V. Nair, Thomas D. Forbes, Brandie A. Fullmer, Anna J. Lee Fong, Luis E. Romero, Alyssa K. Chapman, Kelsey A. Conley, Robin D. Harrington, Shahanawaz S. Jiwani, Peng Wang, Michelle M. Gottholm-Ahalt, Erin N. Cantu, Gloryvee Rivera, Lindsay M. Dutko, Kelly M. Benauer, Vishnuprabha R. Kannan, Carrie A. Bonomi, Kelly M. Dougherty, Joseph P. Geraghty, Marion V. Gibson, Savanna S. Styers, Abigail J. Walke, Jenna E. Moyer, Anna Wade, Mariah L. Baldwin, Kaitlyn A. Arthur, Kevin J. Plater, Luke Stockwin, Matthew R. Murphy, Michael E. Mullendore, Dianne L. Newton, Melinda G. Hollingshead, Chris A. Karlovich, Paul M. Williams, James H. Doroshow. Patient-derived organoid and cell culture models from the NCI Patient-Derived Models Repository (NCI PDMR) preserve genomic stability and heterogeneity of patient tumor specimens [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3916.
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- 2020
7. Abstract 3554: Genomic landscape of acquired uniparental disomy in NCI PDMR patient derived xenograft models
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Li Chen, Yvonne A. Evrard, Michelle Eugeni, Kelly Benauer, Amanda Peach, Michelle M. Gottholm Ahalt, James H. Doroshow, Shahanawaz Jiwani, Biswajit Das, Chris Karlovich, John Carter, Candace Mallow, Tara Grinnage-Pulley, Lindsay Dutko, Tiffanie L. Miner, Sergio Y. Alcoser, Dianne L. Newton, Devynn Breen, Thomas Forbes, Emily Delaney, Alyssa K. Chapman, Marianne Radzyminski, Anna J. Lee Fong, Shannon Uzelac, Chelsea McGlynn, Tomas Vilimas, Brandie A. Fullmer, Luis E. Romero, Gloryvee Rivera, Suzanne Borgel, Robin D. Harrington, Justine N. McCutcheon, Rajesh Patidar, Jesse Stottlemyer, Vishnuprabha R. Kannan, Nikitha Nair, Erin Cantu, Peng Wang, Melinda G. Hollingshead, Kelsey A. Conley, and Paul Williams
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Cancer Research ,Haplotype ,Chromosome ,Cancer ,Biology ,medicine.disease ,Uniparental disomy ,Loss of heterozygosity ,Clear cell renal cell carcinoma ,Oncology ,Cancer research ,Carcinoma ,medicine ,Exome sequencing - Abstract
Background: Acquired Uniparental Disomy (aUPD) is relatively common in cancer. Occurrence of aUPD is more frequent in some tumor histologies (e.g., serous ovarian, colorectal) and may be relevant for choice of therapy. The Patient-Derived Models Repository (PDMR; https://pdmr.cancer.gov) developed by The National Cancer Institute (NCI) includes patient-derived xenograft (PDX) models from multiple tumor histologies with different passages and lineages. The associated clinical annotation and genomic data make it possible to assess the prevalence of aUPD in the PDMR cohort and the stability of aUPD in different passages and lineages within a PDX model. Methods: High tumor purity in the PDX specimens (after removal of mouse reads representing the stroma) enabled highly accurate assessment of loss of heterozygosity (LOH). Variants called by GATK Haplotype caller from whole exome sequencing (WES) data were used to identify segments of homozygosity using BCFtools/RoH (runs of homozygosity). The RoH segments were then intersected with the bed file for chromosome arms to get %LOH at the arm level. If %LOH on a chromosome arm was >90%, we considered the sample to have aUPD at the arm level. WES was also used to look for associations between DNA damage repair (DDR) pathway alterations and aUPD. Results: We made the following observations: a) aUPD was observed most frequently in chr18q (75/427, 17.6%) and chr3p (69/427, 16%) of PDX models; b) aUPD was observed more frequently in certain tumor histologies, e.g., clear cell renal cell carcinoma (6/8), small cell lung cancer (3/4) and non-small cell lung cancer (25/38); c) extensive aUPD was observed in 4 PDMR models (>50% of evaluated chromosome arms in these models have aUPD); d) aUPD was not observed in some tumor histologies, i.e., synovial sarcoma, uterine endometrioid carcinoma; e) in the vast majority of PDMR models (>90%), aUPD is maintained faithfully across lineages and through multiple passaging; f) subclonal aUPD events were observed in some models across different lineages; g) significant enrichment of double strand DNA break repair (DSBR) pathway alterations was observed in PDMR models without aUPD (p=0.0007, Fisher's exact test) suggesting defects in DSBR are not associated with aUPD; and h) aUPD was rarely observed in MSI-high models (1/30) suggesting mutual exclusivity of mismatch repair (MMR) pathway defects and aUPD. Conclusion: We observed a relatively high frequency of UPD in the PDMR models (at least 1 arm of a chromosome). UPD was more frequently observed in specific chromosomal arms. The frequency of aUPD was higher in some tumor histologies and absent in others. aUPD was stably maintained across passages and lineages, although some heterogeneity was observed. Our data suggest aUPD is not associated with defects in DSBR and MMR pathways. Preclinical drug studies using NCI PDMR models may suggest appropriate therapeutic options for cancers with aUPD. Citation Format: Rajesh Patidar, Li Chen, Chris A. Karlovich, Biswajit Das, Yvonne A. Evrard, Tomas Vilimas, Justine N. McCutcheon, Amanda L. Peach, Nikitha V. Nair, Thomas D. Forbes, Brandie A. Fullmer, Anna J. Lee Fong, Luis E. Romero, Alyssa K. Chapman, Kelsey A. Conley, Robin D. Harrington, Shahanawaz S. Jiwani, Peng Wang, Michelle M. Gottholm Ahalt, Erin N. Cantu, Gloryvee Rivera, Lindsay M. Dutko, Kelly M. Benauer, Vishnuprabha R. Kannan, Suzanne D. Borgel, John P. Carter, Jesse M. Stottlemyer, Tiffanie L. Miner, Devynn R. Breen, Emily T. Delaney, Chelsea A. McGlynn, Candace N. Mallow, Marianne Radzyminski, Shannon N. Uzelac, Sergio Y. Alcoser, Tara L. Grinnage-Pulley, Michelle A. Eugeni, Dianne L. Newton, Melinda G. Hollingshead, Paul M. Williams, James H. Doroshow. Genomic landscape of acquired uniparental disomy in NCI PDMR patient derived xenograft models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3554.
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- 2020
8. Abstract 3913: Evaluation of patient-derived cell lines and cancer organoids for the prediction of drug responses in patient-derived xenograft models
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Debbie Trail, Abigail Walke, Petreena Campbell, Marion Gibson, Carrie Bonomi, Kelly Dougherty, Luke H. Stockwin, Savanna Styers, Matthew R. Murphy, Yvonne A. Evrard, Annamaria Rapisarda, Jenna Moyer, Lara H. El Touny, James H. Doroshow, Tara Grinnage-Pulley, Erik Harris, Joseph P. Geraghty, John Mark Carter, Kevin Plater, Michael Mullendore, Tiffanie Chase, Nathan P. Coussens, Michelle M. Gottholm-Ahalt, John Connelly, Dianne L. Newton, Beverly A. Teicher, Ralph E. Parchment, Anna Wade, Mariah Baldwin, Curtis Hose, Kaitlyn Arthur, Melinda G. Hollingshead, and Howard Stotler
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Drug ,Cancer Research ,business.industry ,media_common.quotation_subject ,Cancer ,medicine.disease ,Oncology ,Cell culture ,Cancer research ,medicine ,Organoid ,In patient ,business ,media_common - Abstract
Cancer organoids are heterogeneous 3D cellular clusters with complexities that mimic some characteristics of tumors in situ. Thus, assays performed with cancer organoids might enable better predictions of in vivo drug responses than those performed with cell monolayers. The National Cancer Institute (NCI) is developing a national repository of Patient-Derived (PD) models comprised of clinically annotated and molecularly characterized PD xenografts (PDXs), PD tumor cell lines (PDCs), and PD cancer organoids (PDOrgs) (https://pdmr.cancer.gov/). We evaluated the therapeutic activity of a panel of FDA-approved and investigational anticancer agents, including carboplatin, gemcitabine, paclitaxel, SN38, 5-FU, adavosertib, erlotinib, trametinib, and vemurafenib, against a cohort of PDCs, PDOrgs, and PDXs from solid tumors including colon, gastroesophageal, head and neck, NSCLC, pancreatic, bladder, and uterine cancers. Our goal was to investigate whether drug sensitivities determined using PDCs and PDOrgs correlate with responses observed in the matching PDXs. Cultures were exposed to anticancer agents at concentrations ranging from 1 pM to 100 µM for periods of 4 or 6 days. The data indicated that the GI50 values for PDOrgs were in overall agreement with in vivo PDX drug responses measured as relative median to event free survival (RMEFS), where an event is the median time (days) from treatment initiation to tumor volume quadrupling, calculated as median time to tumor volume quadrupling for treated animals/median time to tumor volume quadrupling for control animals. For both paclitaxel and trametinib, responses in PDOrgs, from most sensitive to most resistant, were similar to the corresponding PDXs. Drug sensitivities determined in PDC monolayers were less clearly related to in vivo PDX responses; particularly for PDCs treated with carboplatin, gemcitabine, and SN-38. This work is part of a larger effort to provide a rigorous comparison between fully characterized and annotated PDCs-PDOrgs-PDXs to assess the value of different in vitro model systems for the prediction of PDX drug responses. This research was supported [in part] by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute. Funded by NCI Contract No. HHSN261200800001E. Citation Format: Petreena Campbell, Curtis Hose, Lara El Touny, Erik Harris, John Connelly, Carrie Bonomi, Kelly Dougherty, Savanna Styers, Abigail Walke, Jenna Moyer, Mariah Baldwin, Anna Wade, Michael Mullendore, Kaitlyn Arthur, Matthew Murphy, Kevin Plater, Marion Gibson, Joseph Geraghty, Michelle Gottholm-Ahalt, Tara Grinnage-Pulley, Tiffanie Chase, John Carter, Howard Stotler, Debbie Trail, Luke Stockwin, Dianne Newton, Yvonne Evrard, Melinda Hollingshead, Ralph E. Parchment, Nathan P. Coussens, Beverly A. Teicher, James H. Doroshow, Annamaria Rapisarda. Evaluation of patient-derived cell lines and cancer organoids for the prediction of drug responses in patient-derived xenograft models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3913.
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- 2020
9. Abstract 5056: Quality control efforts in a large-scale, preclinical trial of rare cancer PDXs by the National Cancer Institute's patient-derived models repository (NCI PDMR)
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John Carter, Dianne L. Newton, Shannon Uzelac, Gloryvee Rivera, Michael Mullendore, Malorie Morris, Abigail Walke, Lily Chen, Thomas Forbes, Kyle Georgius, Emily Delaney, Sergio . Y. Alcoser, Debbie Trail, Tom Walsh, Raymond Divelbiss, Tara Grinnage-Pulley, Shahanawaz Jiwani, Chris Karlovich, Yvonne A. Evrard, Lyndsay Dutko, Sierra Hoffman, Devynn Breen, Biswajit Das, Howard Stotler, Rajesh Patidar, Thomas Vilimas, Peng Wang, Justine Mills, Suzanne Borgel, Nicole Walters, Melinda G. Hollingshead, James Doroshow, Savanna Styers, Jesse Stottlemyer, Tiffanie Chase, Michelle M. Gottholm-Ahalt, Alice P. Chen, Kristen Cooley, and P. Mickey Williams
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Cancer Research ,medicine.medical_specialty ,Scale (ratio) ,business.industry ,media_common.quotation_subject ,Cancer ,medicine.disease ,Rare cancer ,Oncology ,Medicine ,Quality (business) ,Medical physics ,business ,media_common - Abstract
The National Cancer Institute's Patient-Derived Models Repository (NCI PDMR; https://pdmr.cancer.gov) is performing a large-scale multi-year preclinical study with 39 PDX models of rare cancers (mesothelioma, MPNST, osteosarcoma, Merkel cell carcinoma, etc) treated with 56 novel therapeutic combinations in an exploratory, n-of-4 arm, study design. Combinations that show promising responses (e.g., regression or durable inhibition of tumor growth) will be repeated along with the single agent arms to determine if the response is driven by the combination or only one of the agents. In order to do this in a timely fashion, relatively speaking, the PDX tumors are serially passaged and each passage is treated with a set of 8 combinations plus relevant vehicle control(s) while in parallel enough PDXs are retained to be expanded for the next passage and drug set. Every serial passage undergoes several quality control assessments that serve as go/no-go criteria including pathology assessment, human:mouse DNA content assessment, and low pass whole genome sequencing to determine the average fraction of genome changed compared to the original donor material. If there is a QC failure, the PDX model is restarted from early passage cryo-material (passage 1-2). An additional quality control effort is to bookend the combination studies with the first set of agents to see if tumor response is similar across passages. To date, most of the models have demonstrated a high degree of stability, though a couple of models have moved toward murine content and have been restarted from early passage material so all drug combinations can be tested. DNA and RNA are retained from all passages so a full NGS evaluation can be performed at a later date. This effort has been ongoing for over a year and the first bookend studies are beginning to be tested to determine if response at first and last passage of the study are consistent with each other, given the constraints of the inherent heterogeneity of the models themselves. Single agent studies of drug combinations that demonstrated a response in 30%-50% of the models tested are also underway to determine which combinations have a more than additive effect compared to the single agents. Promising combinations will be moved forward to early phase clinical trials for these rare cancers. Funded by NCI Contract No. HHSN261200800001E Citation Format: Yvonne A. Evrard, Biswajit Das, Sergio Y. Alcoser, Suzanne Borgel, Devynn Breen, John Carter, Tiffanie Chase, Alice Chen, Lily Chen, Kristen Cooley, Emily Delaney, Raymond Divelbiss, Lyndsay Dutko, Thomas Forbes, Kyle Georgius, Michelle Gottholm-Ahalt, Tara Grinnage-Pulley, Sierra Hoffman, Chris Karlovich, Shahanawaz Jiwani, Justine Mills, Malorie Morris, Michael Mullendore, Dianne Newton, Rajesh Patidar, Gloryvee Rivera, Howard Stotler, Jesse Stottlemyer, Savanna Styers, Debbie Trail, Shannon Uzelac, Thomas Vilimas, Abigail Walke, Thomas Walsh, Nicole Walters, Peng Wang, P. Mickey Williams, Melinda Hollingshead, James H. Doroshow. Quality control efforts in a large-scale, preclinical trial of rare cancer PDXs by the National Cancer Institute's patient-derived models repository (NCI PDMR) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5056.
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- 2020
10. Genomic characterization of preclinical models derived from primary and metastatic sites from rapid autopsy patients in PDMR
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Paul Williams, Lindsay Dutko, Marianne Radzyminski, Li Chen, Yvonne A. Evrard, Melinda G. Hollingshead, Anna Lee Fong, Luis Romero, James H. Doroshow, Tomas Vilimas, Dianne L. Newton, Rajesh Patidar, Shahanawaz Jiwani, Kelly Benauer, Chris Karlovich, Michelle Eugeni, Nikitha Nair, Biswajit Das, Kelly Dougherty, and Amanda Peach
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Oncology ,Cancer Research ,medicine.medical_specialty ,business.industry ,Internal medicine ,medicine ,Cancer ,Rapid autopsy ,medicine.disease ,business - Abstract
e13506 Background: The National Cancer Institute has developed a repository of preclinical models [Patient-Derived Models Repository (NCI PDMR, https://pdmr.cancer.gov )] including patient derived xenografts (PDXs), organoids (PDOrgs) and in vitro tumor cultures (PDCs) from patients with solid tumor cancer histologies. A subset of these preclinical models is derived from post-mortem collections from rapid autopsies representing the end point in disease progression. Clinical annotations and genomic datasets associated with these models provide a unique opportunity to study tumor evolution, mechanistic insights into the metastatic process, and treatment resistance. Methods: To date, 43 PDXs, 21 PDCs, and 23 PDOrgs using rapid autopsy specimens from 8 primary and 35 metastatic sites of 18 patients have been developed by the Biological Testing Branch (DTP, DCTD, NCI Frederick, MD) for the PDMR. Whole exome (WES) and total transcriptome (RNASeq) data were processed to generate mutation, copy number alteration (CNA) and gene expression data. Multi-model lineage trees were reconstructed based on putative somatic variants for all the models derived from all patients. The fraction of the genome affected by CNA was compared both within and across PDX models. Results: Most of the rapid autopsy PDX models (32/43) are derived from pancreatic adenocarcinoma (PAAD) patients (13/18), with metastatic specimens originating from sites including liver, colon, omentum, and lung. Driver mutations are present in all preclinical model specimens derived from the same patient. For instance, KRAS p.G12D is present in all patient-derived model specimens derived from PAAD patient 521955. The fraction of the genome affected by CNA remains stable within a PDX model across passages (n = 24, mean = 6.39%, sd = 5.90%). However, we found that this increased when comparing PDX models derived from metastatic sites versus the primary site (n = 19, mean = 16.92%, sd = 10.46%). This indicates presence of tumor heterogeneity between metastatic and primary sites. The lineage tree for models from patient 521955 indicates that one liver metastasis has a unique seeding event compared to the other 4 metastatic sites. Unsupervised clustering analysis on gene expression data also confirms the observed tumor site relationships. Conclusions: Our data demonstrate the potential use of these preclinical models available from the NCI PDMR. These models provide a unique resource for preclinical studies in tumor evolution, metastatic spread mediators, and drug resistance.
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- 2020
11. Abstract 4524: Comparison of PDX, PDC, and PDOrg models from the National Cancer Institute’s Patient-Derived Models Repository (PDMR)
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Jenna Moyer, Howard Stotler, James H. Doroshow, Tom Walsh, Lily Chen, Abigail Walke, Mike Mullendore, Matt Murphy, Tara Grinnage-Pulley, Luke H. Stockwin, Marion Gibson, Yvonne A. Evrard, John Mark Carter, Wiem Lassoued, Suzanne Borgel, Carrie Bonomi, Kelly Dougherty, Kevin Plater, Biswajit Das, Raymond Divelbiss, Joe Geraghty, Vivekananda Datta, Nikki E. Craig, Emily Delaney, Marianne Radzyminski, Alice P. Chen, Kaitlyn Arthur, Mariah Baldwin, Rajesh Patidar, Dianne L. Newton, Sergio Y. Alcoser, Debbie Trail, Kyle Georgius, Shahanawaz Jiwani, Chris Karlovich, Tiffany Chase, Candace Mallow, Mallorie Morris, Sierra Hoffman, Thomas P. Forbes, P. Mickey Williams, Nicki Scott, Savanna Styers, Anna Wade, Jesse Stottlemyer, Chelsea McGlynn, Tomas Vilimas, Melinda G. Hollingshead, Michelle M. Gottholm-Ahalt, and Kelly Hedger
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Cancer Research ,Research use ,Extramural ,Genetic stability ,Cancer ,medicine.disease ,Rare cancer ,Genealogy ,Unmet needs ,Data sequences ,Oncology ,Multiple criteria ,medicine ,Psychology - Abstract
The National Cancer Institute (NCI) has developed a Patient-Derived Models Repository (PDMR) comprised of quality-controlled, early-passage, clinically-annotated patient-derived tumor xenografts (PDXs), in vitro tumor cell cultures (PDCs), cancer associated fibroblasts (CAFs), and patient-derived organoids (PDOrg). NCI has focused on generating models to complement existing PDX collections and address unmet needs in the preclinical model space. These models are offered to the extramural community for research use (https://pdmr.cancer.gov), along with clinical annotation and molecular information (whole exome sequence, gene expression using RNASeq), via a publicly accessible database. Currently, over 200 PDX models, 50 PDC models, and 100 CAF models are available for distribution to the US research community. Approximately 50 PDOrg models will be released in early 2019. As part of its rare cancer initiative, the NCI is also targeting the collection of infrequently-observed tumor histologies to advance both biological investigations and drug development efforts for under-studied malignancies. Comparison of matched models, models where more than one model type are available (e.g., PDX and PDC), demonstrate a high degree of concordance across the model types. Genetic stability across the models is assessed using multiple criteria including genetic assessment of CNVs and presence of driver mutations. Optimal CNV assessment uses whole exome sequence data corrected for cellularity in the patient specimen using germline reads and corrected for cellularity in the PDX specimens by subtraction of the mouse reads. Histomorphologic comparison of PDXs and cell line xenografts (CLX) generated from in vitro PDCs and PDOrgs also overall show a high degree of concordance, though loss of features and dedifferentiation can be observed in some models. Overall these models demonstrate a high degree of conservation at the genetic and pathologic level when compared to the patient tumor. These models can provide researchers the ability to perform high- or mid-throughput screening in 2D or 3D culture followed by targeted selection of PDX models for in vivo studies. Funded by NCI Contract No. HHSN261200800001E Citation Format: Yvonne A. Evrard, Dianne Newton, Biswajit Das, Sergio Y. Alcoser, Kaitlyn Arthur, Mariah Baldwin, Carrie Bonomi, Suzanne Borgel, John Carter, Tiffany Chase, Alice Chen, Lily Chen, Nikki E. Craig, Vivekananda Datta, Emily Delaney, Raymond Divelbiss, Kelly Dougherty, Thomas Forbes, Kyle Georgius, Joe Geraghty, Marion Gibson, Michelle M. Gottholm-Ahalt, Tara Grinnage-Pulley, Kelly Hedger, Sierra Hoffman, Chris Karlovich, Wiem Lassoued, Shahanawaz Jiwani, Candace Mallow, Chelsea McGlynn, Mallorie Morris, Jenna Moyer, Mike Mullendore, Matt Murphy, Rajesh Patidar, Kevin Plater, Marianne Radzyminski, Nicki Scott, Luke H. Stockwin, Howard Stotler, Jesse Stottlemyer, Savanna Styers, Debbie Trail, Tomas Vilimas, Anna Wade, Abigail Walke, Thomas Walsh, P. Mickey Williams, Melinda G. Hollingshead, James H. Doroshow. Comparison of PDX, PDC, and PDOrg models from the National Cancer Institute’s Patient-Derived Models Repository (PDMR) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4524.
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- 2019
12. Oxyphenisatin acetate (NSC 59687) triggers a cell starvation response leading to autophagy, mitochondrial dysfunction, and autocrine TNFα-mediated apoptosis
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Bethanie L. Morrison, Dianne L. Newton, Melinda G. Hollingshead, Suzanne Borgel, Michael E. Mullendore, and Luke H. Stockwin
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Cancer Research ,Programmed cell death ,protein synthesis ,Drug Evaluation, Preclinical ,Mice, Nude ,Apoptosis ,Breast Neoplasms ,Biology ,Protein Serine-Threonine Kinases ,Mice ,breast cancer ,Autophagy ,TNFα ,Tumor Cells, Cultured ,Animals ,Humans ,Radiology, Nuclear Medicine and imaging ,Phosphorylation ,Autocrine signalling ,Protein kinase A ,PI3K/AKT/mTOR pathway ,Cell Proliferation ,Kinase ,Cell growth ,oxyphenisatin ,Tumor Necrosis Factor-alpha ,AMPK ,Clinical Cancer Research ,Mammary Neoplasms, Experimental ,Oxyphenisatin Acetate ,Xenograft Model Antitumor Assays ,Cell biology ,Mitochondria ,Neoplasm Proteins ,Autocrine Communication ,Oncology ,Receptors, Tumor Necrosis Factor, Type I ,Tissue Array Analysis ,Protein Biosynthesis ,Female - Abstract
Oxyphenisatin (3,3-bis(4-hydroxyphenyl)-1H-indol-2-one) and several structurally related molecules have been shown to have in vitro and in vivo antiproliferative activity. This study aims to confirm and extend mechanistic studies by focusing on oxyphenisatin acetate (OXY, NSC 59687), the pro-drug of oxyphenisatin. Results confirm that OXY inhibits the growth of the breast cancer cell lines MCF7, T47D, HS578T, and MDA-MB-468. This effect is associated with selective inhibition of translation accompanied by rapid phosphorylation of the nutrient sensing eukaryotic translation initiation factor 2α (eIF2α) kinases, GCN2 and PERK. This effect was paralleled by activation of AMP-activated protein kinase (AMPK) combined with reduced phosphorylation of the mammalian target of rapamycin (mTOR) substrates p70S6K and 4E-BP1. Microarray analysis highlighted activation of pathways involved in apoptosis induction, autophagy, RNA/protein metabolism, starvation responses, and solute transport. Pathway inhibitor combination studies suggested a role for AMPK/mTOR signaling, de novo transcription and translation, reactive oxygen species (ROS)/glutathione metabolism, calcium homeostasis and plasma membrane Na+/K+/Ca2+ transport in activity. Further examination confirmed that OXY treatment was associated with autophagy, mitochondrial dysfunction, and ROS generation. Additionally, treatment was associated with activation of both intrinsic and extrinsic apoptotic pathways. In the estrogen receptor (ER) positive MCF7 and T47D cells, OXY induced TNFα expression and TNFR1 degradation, indicating autocrine receptor-mediated apoptosis in these lines. Lastly, in an MCF7 xenograft model, OXY delivered intraperitoneally inhibited tumor growth, accompanied by phosphorylation of eIF2α and degradation of TNFR1. These data suggest that OXY induces a multifaceted cell starvation response, which ultimately induces programmed cell death. The mechanistic basis for oxyphenisatin acetate anti-cancer activity remains unresolved. This study demonstrates that exposure is associated with an acute nutrient deprivation response leading to translation inhibition, induction of autophagy, transient estrogen receptor (ER) stress and mitochondrial dysfunction. Ultimately these effects promote apoptosis induction, which in ER+ breast cancer cells is mediated by autocrine TNFα production. This is the first study implicating a nutrient deprivation response as central to the downstream effects of oxyphenisatin acetate.
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- 2013
13. The 'survivin suppressants' NSC 80467 and YM155 induce a DNA damage response
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Luke H. Stockwin, Trevor Glaros, Michael E. Mullendore, Bethanie L. Morrison, Brian J. Smith, and Dianne L. Newton
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Cancer Research ,Time Factors ,DNA damage ,Survivin ,Blotting, Western ,Repressor ,Antineoplastic Agents ,Tripartite Motif-Containing Protein 28 ,Toxicology ,Inhibitor of Apoptosis Proteins ,Histones ,chemistry.chemical_compound ,Cell Line, Tumor ,Neoplasms ,Humans ,Pharmacology (medical) ,RNA, Neoplasm ,Pharmacology ,Dose-Response Relationship, Drug ,Molecular Structure ,biology ,Chemistry ,Imidazoles ,DNA, Neoplasm ,HCT116 Cells ,Molecular biology ,Repressor Proteins ,Blot ,Histone ,Oncology ,Cell culture ,Protein Biosynthesis ,biology.protein ,Cancer research ,K562 Cells ,HT29 Cells ,DNA ,DNA Damage ,Naphthoquinones ,K562 cells - Abstract
To establish whether NSC80467, a novel fused naphthquinone imidazolium, has a similar spectrum of activity to the well-characterized "survivin suppressant" YM155 and to extend mechanistic studies for this structural class of agent.NSC80467 and YM155 were analyzed in parallel using assays measuring viability, survivin suppression, inhibition of DNA/RNA/protein synthesis and the cellular response to DNA damage.GI(50) values generated for both compounds in the NCI-60 screen yielded a correlation coefficient of 0.748, suggesting significant concordance. Both agents were also shown to inhibit protein expression of survivin [BIRC5]. COMPARE analysis identified DNA damaging agents chromomycin A3 and bisantrene HCl and one DNA-directed inhibitor of transcription, actinomycin D, as correlating with the activity of NSC80467 and YM155. Furthermore, both agents were shown to preferentially inhibit DNA, over RNA and protein synthesis. Thus, the ability of NSC80467 and YM155 to induce a DNA damage response was examined further. Treatment of PC3 cells with either agent resulted in dose-dependent induction of γH2AX and pKAP1, two markers of DNA damage. The concentrations of agent required to stimulate γH2AX were considerably lower than those required to inhibit survivin, implicating DNA damage as an initiating event. The DNA damage response was then confirmed in a panel of cell lines treated with NSC80467 or YM155, suggesting that γH2AX and pKAP1 have potential as response biomarkers.These data provide the first evidence that NSC80467 and YM155 are DNA damaging agents where suppression of survivin is a secondary event, likely a consequence of transcriptional repression.
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- 2012
14. Challenges in plasma membrane phosphoproteomics
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Benjamin C. Orsburn, Luke H. Stockwin, and Dianne L. Newton
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Phosphopeptides ,Proteomics ,Mechanism (biology) ,Phosphopeptide ,Chemistry ,Cell Membrane ,Molecular Sequence Data ,Phosphoproteomics ,Phosphoproteins ,Biochemistry ,Article ,Cell biology ,Membrane ,Solubility ,Phosphoprotein ,Animals ,Humans ,Phosphorylation ,Amino Acid Sequence ,Signal transduction ,Molecular Biology ,Integral membrane protein - Abstract
The response to extracellular stimuli often alters the phosphorylation state of plasma membrane- associated proteins. In this regard, generation of a comprehensive membrane phosphoproteome can significantly enhance signal transduction and drug mechanism studies. However, analysis of this subproteome is regarded as technically challenging, given the low abundance and insolubility of integral membrane proteins, combined with difficulties in isolating, ionizing and fragmenting phosphopeptides. In this article, we highlight recent advances in membrane and phosphoprotein enrichment techniques resulting in improved identification of these elusive peptides. We also describe the use of alternative fragmentation techniques, and assess their current and future value to the field of membrane phosphoproteomics.
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- 2011
15. A copper chelate of thiosemicarbazone NSC 689534 induces oxidative/ER stress and inhibits tumor growth in vitro and in vivo
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Jung Ho Jun, Raymond Divelbiss, Dianne L. Newton, Luke H. Stockwin, Chad N. Hancock, Melinda G. Hollingshead, Bingnan Han, and Sanjay V. Malhotra
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inorganic chemicals ,Thiosemicarbazones ,Programmed cell death ,Antioxidant ,medicine.medical_treatment ,Down-Regulation ,Mice, Nude ,Antineoplastic Agents ,HL-60 Cells ,Endoplasmic Reticulum ,medicine.disease_cause ,Biochemistry ,Article ,Mice ,chemistry.chemical_compound ,Neoplasms ,Physiology (medical) ,Organometallic Compounds ,Tumor Cells, Cultured ,medicine ,Animals ,Humans ,Buthionine sulfoximine ,reproductive and urinary physiology ,Cell Proliferation ,Chelating Agents ,Metal metabolism ,Autophagy ,Glutathione ,Oxidants ,Xenograft Model Antitumor Assays ,Molecular biology ,Up-Regulation ,nervous system diseases ,Oxidative Stress ,nervous system ,chemistry ,Unfolded Protein Response ,Unfolded protein response ,Female ,biological phenomena, cell phenomena, and immunity ,Copper ,Oxidative stress - Abstract
In this study, a Cu(2+) chelate of the novel thiosemicarbazone NSC 689534 was evaluated for in vitro and in vivo anti-cancer activity. Results demonstrated that NSC 689534 activity (low micromolar range) was enhanced four- to fivefold by copper chelation and completely attenuated by iron. Importantly, once formed, the NSC 689534/Cu(2+) complex retained activity in the presence of additional iron or iron-containing biomolecules. NSC 689534/Cu(2+) mediated its effects primarily through the induction of ROS, with depletion of cellular glutathione and protein thiols. Pretreatment of cells with the antioxidant N-acetyl-l-cysteine impaired activity, whereas NSC 689534/Cu(2+) effectively synergized with the glutathione biosynthesis inhibitor buthionine sulfoximine. Microarray analysis of NSC 689534/Cu(2+)-treated cells highlighted activation of pathways involved in oxidative and ER stress/UPR, autophagy, and metal metabolism. Further scrutiny of the role of ER stress and autophagy indicated that NSC 689534/Cu(2+)-induced cell death was ER-stress dependent and autophagy independent. Last, NSC 689534/Cu(2+) was shown to have activity in an HL60 xenograft model. These data suggest that NSC 689534/Cu(2+) is a potent oxidative stress inducer worthy of further preclinical investigation.
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- 2011
16. Abstract 986: The National Cancer Institute's patient-derived models repository (PDMR)
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Yvonne A. Evrard, Michelle M. Gottholm Ahalt, Sergio . Y. Alcoser, Kaitlyn Arthur, Mariah Baldwin, Linda L. Blumenauer, Carrie Bonomi, Suzanne Borgel, Elizabeth Bradtke, Corinne Camalier, John Carter, Tiffanie Chase, Alice Chen, Lily Chen, Donna W. Coakley, Nicole E. Craig, Biswajit Das, Vivekananda Datta, Jordyn Davidson, Margaret R. DeFreytas, Emily Delaney, Michelle A. Eugeni, Raymond Divelbiss, Palmer Fliss, Thomas Forbes, Marion Gibson, Tara Grinnage-Pulley, Sierra Hoffman, Lilia Ileva, Paula Jacobs, Franklyn Jimenez, Joseph Kalen, Catherine Karangwa, Chris Karlovich, Candace Mallow, Chelsea McGlynn, Jenna E. Moyer, Michael Mullendore, Dianne L. Newton, Nimit Patel, Rajesh Patidar, Kevin Plater, Marianne Radzyminski, Lisa Riffle, Larry Rubinstein, Luke H. Stockwin, Mickey Williams, Melinda G. Hollingshead, and James H. Doroshow
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Cancer Research ,Oncology - Abstract
The National Cancer Institute (NCI) has developed a Patient-Derived Models Repository (PDMR) comprised of quality-controlled, early-passage, clinically-annotated patient-derived xenografts (PDXs) to serve as a resource for public-private partnerships and academic drug discovery efforts. These models are offered to the extramural community for research use (https://pdmr.cancer.gov/), along with clinical annotation and molecular information (whole exome sequence, RNASeq), which is available in a publicly accessible database. The PDMR was established by NCI at the Frederick National Laboratory for Cancer Research (FNLCR) in direct response to discussions with academia and industry; the oncology community's highest priority need was preclinical models that more faithfully reflect the patient's tumor and are associated with the patient's treatment history. NCI has focused on generating models to complement existing PDX collections and address unmet needs in the preclinical model space. The PDMR generates the majority of its PDXs by subcutaneous implantation except for those histologies having better success rates in either orthotopic or alternate implant sites. All SOPs and quality-control standards developed by the PDMR as well as those shared by collaborators are posted to a public web site that houses the PDMR database. In May 2017, the public website (https://pdmr.cancer.gov/) went live with its first 100 models from histologies including pancreatic, colorectal, renal, head and neck, and lung squamous cell cancers as well as melanoma and adult soft tissue sarcomas. In early 2018, the PDMR will begin releasing models from gynecological cancers, small cell lung cancer, chondro/osteo sarcomas, lung adenocarcinoma, and squamous cell skin and Merkel cell carcinomas. In addition, wherever available germline sequence and somatic variant calls will be added to the existing molecular characterization data for each model. NCI has also increased its focus on creating PDXs from racial and ethnic minorities through several funding opportunities. The overall goal of NCI is to create a long-term home for at least 1000 models such that sufficient biological and clinical diversity is represented to allow researchers to ask questions regarding the impact of tumor heterogeneity on target qualification or clinical response, whether PDXs more faithfully represent the human tumor for pharmacodynamic assay and predictive marker development, or if adequately powered preclinical PDX clinical trials can lead to better evaluation of therapies for future clinical use. Moving forward the PDMR plans to distribute in vitro, early-passage tumor cell cultures and cancer-associated fibroblasts as well as releasing PDX drug response data for a panel of FNA-approved therapeutic agents. Funded by NCI Contract No. HHSN261200800001E Citation Format: Yvonne A. Evrard, Michelle M. Gottholm Ahalt, Sergio . Y. Alcoser, Kaitlyn Arthur, Mariah Baldwin, Linda L. Blumenauer, Carrie Bonomi, Suzanne Borgel, Elizabeth Bradtke, Corinne Camalier, John Carter, Tiffanie Chase, Alice Chen, Lily Chen, Donna W. Coakley, Nicole E. Craig, Biswajit Das, Vivekananda Datta, Jordyn Davidson, Margaret R. DeFreytas, Emily Delaney, Michelle A. Eugeni, Raymond Divelbiss, Palmer Fliss, Thomas Forbes, Marion Gibson, Tara Grinnage-Pulley, Sierra Hoffman, Lilia Ileva, Paula Jacobs, Franklyn Jimenez, Joseph Kalen, Catherine Karangwa, Chris Karlovich, Candace Mallow, Chelsea McGlynn, Jenna E. Moyer, Michael Mullendore, Dianne L. Newton, Nimit Patel, Rajesh Patidar, Kevin Plater, Marianne Radzyminski, Lisa Riffle, Larry Rubinstein, Luke H. Stockwin, Mickey Williams, Melinda G. Hollingshead, James H. Doroshow. The National Cancer Institute's patient-derived models repository (PDMR) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 986.
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- 2018
17. Sodium dichloroacetate selectively targets cells with defects in the mitochondrial ETC
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Chad N. Hancock, Luke H. Stockwin, Dianne L. Newton, Suzanne Borgel, Lawrence R. Phillips, Melinda G. Hollingshead, Sherry X. Yu, and Tracy L. Wolfe
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Proteomics ,Cancer Research ,Lung Neoplasms ,Pyruvate dehydrogenase kinase ,Cell Survival ,Apoptosis ,HL-60 Cells ,Oxidative phosphorylation ,Mitochondrion ,Biology ,Electron Transport ,Cell Line, Tumor ,Animals ,Humans ,Glycolysis ,Membrane Potential, Mitochondrial ,Dichloroacetic Acid ,Dose-Response Relationship, Drug ,Sodium Dichloroacetate ,HCT116 Cells ,Xenograft Model Antitumor Assays ,Warburg effect ,Mitochondria ,Oncology ,Biochemistry ,Anaerobic glycolysis ,Rabbits ,Pyruvate kinase - Abstract
The "Warburg effect," also termed aerobic glycolysis, describes the increased reliance of cancer cells on glycolysis for ATP production, even in the presence of oxygen. Consequently, there is continued interest in inhibitors of glycolysis as cancer therapeutics. One example is dichloroacetate (DCA), a pyruvate mimetic that stimulates oxidative phosphorylation through inhibition of pyruvate dehydrogenase kinase. In this study, the mechanistic basis for DCA anti-cancer activity was re-evaluated in vitro using biochemical, cellular and proteomic approaches. Results demonstrated that DCA is relatively inactive (IC(50) ≥ 17 mM, 48 hr), induces apoptosis only at high concentrations (≥ 25 mM, 48 hr) and is not cancer cell selective. Subsequent 2D-PAGE proteomic analysis confirmed DCA-induced growth suppression without apoptosis induction. Furthermore, DCA depolarizes mitochondria and promotes reactive oxygen species (ROS) generation in all cell types. However, DCA was found to have selective activity against rho(0) cells [mitochondrial DNA (mtDNA) deficient] and to synergize with 2-deoxyglucose in complex IV deficient HCT116 p53(-/-) cells. DCA also synergized in vitro with cisplatin and topotecan, two antineoplastic agents known to damage mitochondrial DNA. These data suggest that in cells "hardwired" to selectively utilize glycolysis for ATP generation (e.g., through mtDNA mutations), the ability of DCA to force oxidative phosphorylation confers selective toxicity. In conclusion, although we provide a mechanism distinct from that reported previously, the ability of DCA to target cell lines with defects in the electron transport chain and to synergize with existing chemotherapeutics supports further preclinical development.
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- 2010
18. Assessment of the genomic stability and molecular landscape of patient-derived xenograft (PDX) models from NCI’s Patient-Derived Models Repository (PDMR)
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John Mark Carter, Margaret R. DeFreytas, Li Chen, Michelle M. Gottholm-Ahalt, Melinda G. Hollingshead, Erin Cantu, Paul Williams, Corinne Camalier, Suzanne Borgel, Rajesh Patidar, Chris Karlovich, Vivekananda Datta, Palmer Fliss, Yvonne A. Evrard, Malorie Morris, Bishwajit Das, James H. Doroshow, Marianne Radzyminski, Gloryvee Rivera, and Dianne L. Newton
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Cancer Research ,Oncology ,business.industry ,Cancer research ,medicine ,Cancer ,Translational research ,Early passage ,medicine.disease ,business ,humanities ,Tumor xenograft ,Genomic Stability - Abstract
12023Background: Patient-derived xenografts (PDXs) are a powerful tool for cancer translational research. However, it is unclear if early passage PDXs faithfully recapitulate the molecular profiles...
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- 2018
19. Abstract A116: Temozolomide-resistant MGMT/MSI double-negative cancer cells rely on ATR signaling and homologous recombination for DNA repair and survival
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Melinda G. Hollingshead, Luke H. Stockwin, Lara H. El Touny, Curtis Hose, James H. Doroshow, Erik Harris, Anne Monks, Annamaria Rapisarda, John Connelly, Dianne L. Newton, Beverly A. Teicher, and Ralph E. Parchment
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Cancer Research ,Temozolomide ,business.industry ,DNA damage ,DNA repair ,RAD51 ,Cancer ,Microsatellite instability ,medicine.disease ,Oncology ,Cancer cell ,medicine ,Cancer research ,Null cell ,business ,medicine.drug - Abstract
Although loss of MGMT expression is used as a predictive biomarker for response to temozolomide (TMZ), some patients do not benefit from treatment, suggesting that additional criteria for patient selection remain to be identified. We used a panel of newly developed patient derived models and well-characterized cancer lines to evaluate potential determinants of TMZ response and identify strategies to overcome TMZ resistance in MGMT negative cancers. There was 100% association between microsatellite instability (MSI) and TMZ resistance in the lines tested. Moreover, only a fraction of MGMT/MSI double-negative lines were sensitive to TMZ at clinically relevant concentrations (37.6µM). TMZ-induced DNA damage and repair (DDR) were compared in a bladder line sensitive to TMZ (BLX1, IC50 11.4µM) and a resistant large neuroendocrine cancer line (LNX1, IC50>80 µM). We observed DDR activation in both models, but only BLX1 had measurable DNA damage (PARP cleavage and γH2AX accumulation, starting 24 h post treatment). An extended G2/M arrest occurred in BLX1 but not in LNX1, suggesting that LNX1 efficiently repaired TMZ-induced damage. ATR inhibition (VE821, VX970 or AZD6738), but not ATM inhibition, in LNX1 cells increased response to TMZ (IC50 27.5µM), PARP cleavage, γH2AX accumulation, and protracted G2/M arrest. BLX1 had minimal homologous recombination (HR) capacity (measured by pDR-GFP/SceI assay) compared to LNX1; however, ATR inhibition abrogated HR activity in LNX1 cells, suggesting HR importance in repair of TMZ-induced damage. To assess whether HR defects sensitize cells to TMZ, we used MGMT positive BRCA1 null cells (UWB1.289) and their BRCA1 reconstituted counterpart line. BRCA1 status did not affect TMZ response in the presence of an MGMT inhibitor (TMZ IC50>80µM); however, BRCA1 null cells were sensitive to TMZ in combination with an ATR inhibitor (VE821 0.3µM, TMZ IC50 10µM). BRCA1 reconstituted cells were sensitized to TMZ+VE821 using a Rad51 inhibitor (B02 2.5µM, TMZ IC50 22.5µM), suggesting that when HR is compromised, cells rely on ATR signaling for repair of TMZ-induced damage. Our data suggest that MGMT/MSI double-negative cells rely on ATR-dependent signaling for repair of TMZ-induced DSBs and that HR defects could be used as additional selection criteria for TMZ+ATRi treatment. Funded by NCI Contract No. HHSN261200800001E. This research was supported, in part, by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute. Citation Format: Lara H. El Touny, Erik Harris, Curtis Hose, John Connelly, Anne Monks, Dianne Newton, Luke Stockwin, Melinda Hollingshead, Ralph Parchment, James H. Doroshow, Beverly A. Teicher, Annamaria Rapisarda. Temozolomide-resistant MGMT/MSI double-negative cancer cells rely on ATR signaling and homologous recombination for DNA repair and survival [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A116.
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- 2018
20. Abstract A117: Modeling ATM loss of function via CRISPR-Cas9 as a predictive tool for therapeutic responses in cancer cells
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James H. Doroshow, Luke H. Stockwin, Beverly A. Teicher, Ralph E. Parchment, Anne Monks, Lara H. El Touny, Curtis Hose, Melinda G. Hollingshead, John Connelly, Dianne L. Newton, and Annamaria Rapisarda
- Subjects
Genome instability ,Cisplatin ,Cancer Research ,DNA damage ,Synthetic lethality ,Bleomycin ,Olaparib ,chemistry.chemical_compound ,Oncology ,chemistry ,Protein kinase domain ,Cancer cell ,Cancer research ,medicine ,medicine.drug - Abstract
ATM is a master regulator of the DNA damage response (DDR) elicited by double-strand breaks (DSB). After DNA DSB, ATM controls cell fate via cell cycle arrest, apoptosis, and DNA damage repair. Numerous tumor types have ATM gene loss-of-function mutations, resulting in genomic instability. Most knowledge on the effect of ATM loss on DDR and tumor therapeutic response is from ataxia-telangiectasia (A-T) patients and ATM knockout models. A distinct and more deleterious phenotype has been proposed for kinase-dead (KD) ATM mutants that retain ATM protein expression. Inhibition of ATM activity does not fully mimic ATM loss; therefore, KD ATM mutants may be better predictors of response to ATM inhibitors and chronic effects of ATM inhibition. Using an inducible lentiviral CRISPR/Cas9 genome-editing system, we generated in-frame ATM mutated clonal lines derived from a bladder cancer PDX model (BLX1), via targeting of ATM exon 57, which maps to the second exon in the kinase domain. CRISPR-edited clones consistently exhibited reduced phosphorylation of Kap1 (S824) but not CHK2, p53 or NBS1 when challenged with DSB-inducing agents such as ionizing radiation (IR) and etoposide. Similar results were obtained in unedited cells treated with the ATM inhibitor KU55933, confirming that changes observed are ATM-dependent and suggesting that reduced Kap1 phosphorylation is a robust readout of compromised ATM activity in response to DSB-inducing therapies. Radiosensitivity is a hallmark of the A-T syndrome and the CRISPR-edited clones had increased sensitivity to DSB-inducing agents including IR, bleomycin, etoposide, and doxorubicin but not to S-phase drugs gemcitabine and camptothecin in a 10-day colony formation assay. ATM deficiency increased the dependency of cancer cells on ATM-independent repair mechanisms. Indeed, short-term (4-day) proliferation assays demonstrated the synthetic lethality of KD ATM with ATR inhibitors (AZD6738, VE821), Chk1 inhibitors (MK8776, LY2606368), as well as high concentrations of PARP inhibitors (Olaparib, BMN673) but not the DNAPK inhibitor (NU7441). KD ATM clones did not have increased response to cisplatin as a single agent except at high concentrations (>10 µM); the combination of low-concentration cisplatin ( Citation Format: Lara H. El Touny, John Connelly, Curtis Hose, Anne Monks, Dianne Newton, Luke Stockwin, Melinda Hollingshead, Ralph Parchment, James H. Doroshow, Beverly A. Teicher, Annamaria Rapisarda. Modeling ATM loss of function via CRISPR-Cas9 as a predictive tool for therapeutic responses in cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A117.
- Published
- 2018
21. CCR4-Expressing T Cell Tumors Can Be Specifically Controlled via Delivery of Toxins to Chemokine Receptors
- Author
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Arya Biragyn, Kenya Sumitomo, Dianne L. Newton, Dolgor Baatar, and Purevdorj B. Olkhanud
- Subjects
Cytotoxicity, Immunologic ,CCR1 ,Receptors, CCR4 ,Virulence Factors ,Bacterial Toxins ,Immunology ,Exotoxins ,Antineoplastic Agents ,Eosinophil-Derived Neurotoxin ,Mice, SCID ,C-C chemokine receptor type 6 ,Biology ,Article ,Cell Line ,Mice ,Viral Proteins ,Chemokine receptor ,Mice, Inbred NOD ,Cell Line, Tumor ,Animals ,Humans ,Leukemia-Lymphoma, Adult T-Cell ,Immunology and Allergy ,CXC chemokine receptors ,CCL13 ,ADP Ribose Transferases ,Cell Death ,Immunotoxins ,Molecular biology ,Chemokines, CC ,Cancer research ,XCL2 ,Female ,Receptors, Chemokine ,Chemokine CCL17 ,Neoplasm Recurrence, Local ,CC chemokine receptors ,CCL21 - Abstract
Expression of chemokine receptors by tumors, specifically CCR4 on cutaneous T cell lymphomas, is often associated with a poor disease outcome. To test the hypothesis that chemokine receptor-expressing tumors can be successfully controlled by delivering toxins through their chemokine receptors, we have generated fusion proteins designated chemotoxins: chemokines fused with toxic moieties that are nontoxic unless delivered into the cell cytosol. We demonstrate that chemokines fused with human RNase eosinophil-derived neurotoxin or with a truncated fragment of Pseudomonas exotoxin 38 are able to specifically kill tumors in vitro upon internalization through their respective chemokine receptors. Moreover, treatment with the thymus and activation-regulated chemokine (CCL17)-expressing chemotoxin efficiently eradicated CCR4-expressing cutaneous T cell lymphoma/leukemia established in NOD-SCID mice. Taken together, this work represents a novel concept that may allow control of growth and dissemination of tumors that use chemokine receptors to metastasize and circumvent immunosurveillance.
- Published
- 2007
22. Proteomic Analysis of Plasma Membrane from Hypoxia-Adapted Malignant Melanoma
- Author
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Susanna M. Rybak, King C. Chan, David A. Lucas, Dianne L. Newton, Haleem J. Issaq, Luke H. Stockwin, Josip Blonder, Maja A. Bumke, Thomas P. Conrads, and Timothy D. Veenstra
- Subjects
Proteomics ,Vascular Endothelial Growth Factor A ,ATPase ,Molecular Sequence Data ,Cell ,Apoptosis ,Enzyme-Linked Immunosorbent Assay ,Oxygen Isotopes ,Biology ,Biochemistry ,Cell membrane ,Transcriptome ,Necrosis ,Cell Line, Tumor ,Organelle ,medicine ,Humans ,Amino Acid Sequence ,Melanoma ,Reverse Transcriptase Polymerase Chain Reaction ,Cell Membrane ,Membrane Proteins ,General Chemistry ,Chromatography, Ion Exchange ,Immunohistochemistry ,Molecular biology ,Cell Hypoxia ,Peptide Fragments ,Neoplasm Proteins ,Cell biology ,medicine.anatomical_structure ,Secretory protein ,Membrane protein ,biology.protein ,Cell Division - Abstract
Hypoxic conditions often persist within poorly vascularized tumors. At the cellular level constitutive activation of transcriptional regulators of the hypoxic response leads to the emergence of clones with aggressive phenotypes. The primary interface between the cell and the hypoxic environment is the plasma membrane. A detailed investigation of this organelle is expected to yield further targets for therapeutic perturbation of the response to hypoxia. In the present study, quantitative proteomic analysis of plasma membrane from hypoxia-adapted murine B16F10 melanoma was performed using differential 16O/18O stable isotopic labeling and multidimensional liquid chromatography-tandem mass spectrometry. The analysis resulted in the identification of 24,853 tryptic peptides, providing quantitative information for 2,433 proteins. For a subset of plasma membrane and secreted proteins, quantitative RT-PCR was used to gain further insight into the genomic regulatory events underlying the response to hypoxia. Consistent increases at the proteomic and transcriptomic levels were observed for aminopeptidase N (CD13), carbonic anhydrase IX, potassium-transporting ATPase, matrix metalloproteinase 9, and stromal cell derived factor I (SDF-1). Antibody-based analysis of a panel of human melanoma cell lines confirmed that CD13 and SDF-1 were consistently upregulated during hypoxia. This study provides the basis for the discovery of novel hypoxia-induced membrane proteins.
- Published
- 2006
23. A Dimeric Angiogenin Immunofusion Protein Mediates Selective Toxicity Toward CD22+ Tumor Cells
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Michaela A. E. Arndt, Susanna M. Rybak, Dianne L. Newton, Jürgen Krauss, and Bang K. Vu
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Cancer Research ,Angiogenin ,Recombinant Fusion Proteins ,Sialic Acid Binding Ig-like Lectin 2 ,Immunology ,Antineoplastic Agents ,Peptide ,medicine.disease_cause ,Inhibitory Concentration 50 ,Antigens, CD ,Antigens, Neoplasm ,Lectins ,Tumor Cells, Cultured ,medicine ,Protein biosynthesis ,Humans ,Immunology and Allergy ,Cytotoxicity ,Escherichia coli ,Pharmacology ,chemistry.chemical_classification ,biology ,CD22 ,Fusion protein ,Molecular biology ,Antigens, Differentiation, B-Lymphocyte ,chemistry ,Biochemistry ,Protein Biosynthesis ,biology.protein ,Antibody ,Cell Adhesion Molecules ,Dimerization - Abstract
To improve selective cytotoxicity and pharmacokinetics of an anti-CD22 antibody single chain Fv (scFv)-ribonuclease fusion protein, a dimeric derivative was generated. Human angiogenin was fused via a (G 4 S) 3 spacer peptide to the carboxy-terminal end of the stable dimeric anti-CD22 V L -V H zero-linker scFv MLT-7. The dimeric fusion protein and a monovalent counterpart were produced as soluble proteins in the periplasm of Escherichia coli. Comparative studies with homogenously purified fusion proteins revealed that both constructs specifically bound to the target antigen and retained ribonucleolytic activity. However, they exhibited a markedly different capability for killing CD22 + tumor cells. The monomeric construct inhibited protein synthesis of target cells in a dose-dependent manner, but 50% inhibition (IC 50 ) could be achieved only at the highest tested concentration (>350 nM). In contrast, the dimeric fusion protein efficiently killed CD22 + Raji and Daudi tumor cell lines with IC 50 values of 74 nM and 118 nM, respectively. These results show that the therapeutic potential of scFv-ANG fusion proteins can be markedly enhanced by engineering dimeric derivatives.
- Published
- 2005
24. Human Ribonuclease A Superfamily Members, Eosinophil-Derived Neurotoxin and Pancreatic Ribonuclease, Induce Dendritic Cell Maturation and Activation
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Barry Schweitzer, De Yang, Minjuan Wang, Joost J. Oppenheim, Helene F. Rosenberg, Stephen F. Kingsmore, O. M. Zack Howard, Qian Chen, Zhao Yuan Wang, Dianne L. Newton, Qin Fu, Velizar T. Tchernev, Susanna M. Rybak, and Dhavalkumar D. Patel
- Subjects
Chemokine ,biology ,Angiogenin ,RNase P ,medicine.medical_treatment ,Immunology ,Eosinophil-derived neurotoxin ,Dendritic cell ,Molecular biology ,Proinflammatory cytokine ,Cytokine ,Immune system ,biology.protein ,medicine ,Immunology and Allergy - Abstract
A number of mammalian antimicrobial proteins produced by neutrophils and cells of epithelial origin have chemotactic and activating effects on host cells, including cells of the immune system. Eosinophil granules contain an antimicrobial protein known as eosinophil-derived neurotoxin (EDN), which belongs to the RNase A superfamily. EDN has antiviral and chemotactic activities in vitro. In this study, we show that EDN, and to a lesser extent human pancreatic RNase (hPR), another RNase A superfamily member, activates human dendritic cells (DCs), leading to the production of a variety of inflammatory cytokines, chemokines, growth factors, and soluble receptors. Human angiogenin, a RNase evolutionarily more distant to EDN and hPR, did not display such activating effects. Additionally, EDN and hPR also induced phenotypic and functional maturation DCs. These RNases were as efficacious as TNF-α, but induced a different set of cytokine mediators. Furthermore, EDN production by human macrophages could be induced by proinflammatory stimuli. The results reveal the DC-activating activity of EDN and hPR and suggest that they are likely participants of inflammatory and immune responses. A number of endogenous mediators in addition to EDN have been reported to have both chemotactic and activating effects on APCs, and can thus amplify innate and Ag-specific immune responses to danger signals. We therefore propose these mediators be considered as endogenous multifunctional immune alarmins.
- Published
- 2004
25. Impact of antibody framework residue VH-71 on the stability of a humanised anti-MUC1 scFv and derived immunoenzyme
- Author
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Julie Richards, R Darbha, Dianne L. Newton, Bang K. Vu, Xinhua Ji, M. P. Deonarain, N S Courtenay-Luck, Juergen Krauss, Michaela A. E. Arndt, Susanna M. Rybak, Z Zhu, and V Choudhry
- Subjects
Cancer Research ,Antibody Affinity ,Immunoglobulin Variable Region ,MUC1 ,chemical and pharmacologic phenomena ,Biology ,Protein Engineering ,scFv ,law.invention ,03 medical and health sciences ,0302 clinical medicine ,Protein structure ,law ,Enzyme Stability ,Tumor Cells, Cultured ,fusion protein ,Animals ,Humans ,Computer Simulation ,Experimental Therapeutics ,Amino Acid Sequence ,Immunoglobulin Fragments ,030304 developmental biology ,0303 health sciences ,Models, Immunological ,Antibodies, Monoclonal ,Membrane Proteins ,Protein engineering ,stability ,respiratory system ,Fusion protein ,Molecular biology ,Recombinant Proteins ,Protein Structure, Tertiary ,3. Good health ,Oncology ,HMFGl ,Mutation ,Recombinant DNA ,biology.protein ,ribonuclease ,Antibody ,Immunoglobulin Heavy Chains ,Linker ,030215 immunology - Abstract
Anti-MUC1 single-chain Fv (scFv) fragments generated from the humanised antibody huHMFG1 had adequate antigen-binding properties but very poor stability irrespective of the applied linker or domain orientation. Mutagenesis of heavy-chain framework residue V(H)-71, previously described as a key residue for maintaining the CDR-H2 main-chain conformation and thus important for antigen binding, markedly stabilised the scFv while having only a minor effect on the binding affinity of the molecule. Because of its improved stability, the engineered fragment exhibited immunoreactivity with tumour cells even after 7 days of incubation in human serum at 37 degrees C. It also showed, in contrast to the wild-type scFv, a concentration-dependent binding to the target antigen when displayed on phage. When fusing the scFv to the recombinant ribonuclease rapLRI, only the fusion protein generated with the stable mutant scFv was able to kill MUC1(+) tumour cells with an IC(50) of 80 nM. We expect this novel immunoenzyme to become a promising tool for the treatment of MUC1(+) malignancies.
- Published
- 2004
26. RNA Cleavage and Inhibition of Protein Synthesis by Bleomycin
- Author
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Jih Jing Lin, Sidney M. Hecht, Anil T. Abraham, Dianne L. Newton, and Susanna M. Rybak
- Subjects
Reticulocytes ,Xenopus ,Clinical Biochemistry ,Drug Evaluation, Preclinical ,Antineoplastic Agents ,Biology ,010402 general chemistry ,Cleavage (embryo) ,Bleomycin ,01 natural sciences ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Ribonucleases ,Cell Line, Tumor ,Drug Discovery ,medicine ,Protein biosynthesis ,Animals ,Humans ,Ribonuclease ,Molecular Biology ,030304 developmental biology ,Protein Synthesis Inhibitors ,Pharmacology ,0303 health sciences ,Cell-Free System ,RNA ,General Medicine ,respiratory system ,Molecular biology ,3. Good health ,0104 chemical sciences ,Mechanism of action ,chemistry ,Protein Biosynthesis ,Transfer RNA ,Oocytes ,biology.protein ,Molecular Medicine ,Rabbits ,RNA Cleavage ,medicine.symptom ,Software - Abstract
Bleomycin is a clinically used antitumor antibiotic long thought to function therapeutically at the level of DNA cleavage. Recently, it has become clear that bleomycin can also cleave selected members of all major classes of RNA. Using the computer program COMPARE to search the database established by the Anticancer Drug Screening Program of the National Cancer Institute, a possible mechanism-based correlation was found between onconase, an antitumor ribonuclease currently being evaluated in phase III clinical trials, and the chemotherapeutic agent bleomycin. Following these observations, experimentation revealed that bleomycin caused tRNA cleavage and DNA-independent protein synthesis inhibition in rabbit reticulocyte lysate and when microinjected into Xenopus oocytes. The correlation of protein synthesis inhibition to the previously reported site-specific RNA cleavage caused by bleomycin supports the thesis that RNA cleavage may constitute an important element of the mechanism of action of bleomycin.
- Published
- 2003
27. Crystallographic and functional studies of a modified form of eosinophil-derived neurotoxin (EDN) with novel biological activities
- Author
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Alexander Wlodawer, Susanna M. Rybak, Dianne L. Newton, and Changsoo Chang
- Subjects
Models, Molecular ,Intrinsic activity ,Stereochemistry ,Fluorescent Antibody Technique ,Eosinophil-derived neurotoxin ,Eosinophil-Derived Neurotoxin ,Crystallography, X-Ray ,Protein Structure, Secondary ,Structure-Activity Relationship ,Ribonucleases ,Structural Biology ,Hydrolase ,Tumor Cells, Cultured ,Animals ,Humans ,Cytotoxic T cell ,Endothelium ,Cytotoxicity ,Sarcoma, Kaposi ,Molecular Biology ,chemistry.chemical_classification ,Binding Sites ,biology ,Active site ,Ribonuclease, Pancreatic ,N-terminus ,Crystallography ,Enzyme ,Biochemistry ,chemistry ,biology.protein ,Cattle ,Protein Processing, Post-Translational ,Protein Binding - Abstract
The crystal structure of a post-translationally modified form of eosinophil-derived neurotoxin (EDN) with four extra residues on its N terminus ((−4)EDN) has been solved and refined at atomic resolution (1 A). Two of the extra residues can be placed unambiguously, while the density corresponding to two others is poor. The modified N terminus appears to influence the position of the catalytically important His129, possibly explaining the diminished catalytic activity of this variant. However, (−4)EDN has been shown to be cytotoxic to a Kaposi’s sarcoma tumor cell line and other endothelial cell lines. Analysis of the structure and function suggests that the reason for cytotoxicity is most likely due to cellular recognition by the N-terminal extension, since the intrinsic activity of the enzyme is not sufficient for cytotoxicity and the N-terminal extension does not affect the conformation of EDN.
- Published
- 2002
28. Abstract 3840: The National Cancer Institute’s patient-derived models repository (PDMR)
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Michael E. Mullendore, Carrie Bonomi, Michelle M. Gottholm Ahalt, Michelle Eugeni, Tiffanie Chase, Dianne L. Newton, Elizabeth Bradtke, Margaret R. DeFreytas, Lilia Ileva, Sergio . Y. Alcoser, Donna W. Coakley, Suzanne Borgel, Raymond Divelbiss, Franklyn Jimenez, Biswajit Das, Alice P. Chen, Paula M. Jacobs, Nimit L. Patel, Mickey Williams, Marianne Radzyminski, James H. Doroshow, Catherine Karangwa, Tara Grinnage-Pulley, Chelsea McGlynn, Nicole E. Craig, Jordyn Davidson, Chris Karlovich, Palmer Fliss, Sierra Hoffman, Yvonne A. Evrard, Luke H. Stockwin, Emily Delaney, Thomas Forbes, Vivekananda Datta, Mariah Baldwin, Lily Chen, Lisa A. Riffle, Kaitlyn Arthur, Jenna Moyer, Joseph D. Kalen, Linda L. Blumenauer, Kevin Plater, Candace Mallow, Larry Rubinstein, Marion Gibson, Melinda G. Hollingshead, John Carter, Rajesh Patidar, and Corinne Camalier
- Subjects
0301 basic medicine ,Cancer Research ,Direct response ,Cancer ,Library science ,medicine.disease ,Tumor heterogeneity ,Clinical trial ,03 medical and health sciences ,030104 developmental biology ,Oncology ,medicine ,Subcutaneous implantation ,Sociology ,Citation ,National laboratory ,Web site - Abstract
The National Cancer Institute (NCI) has developed a Patient-Derived Models Repository (PDMR) comprised of quality-controlled, early-passage, clinically-annotated patient-derived xenografts (PDXs) to serve as a resource for public-private partnerships and academic drug discovery efforts. These models are offered to the extramural community for research use (https://pdmr.cancer.gov/), along with clinical annotation and molecular information (whole exome sequence, RNASeq), which is available in a publicly accessible database. The PDMR was established by NCI at the Frederick National Laboratory for Cancer Research (FNLCR) in direct response to discussions with academia and industry; the oncology community9s highest priority need was preclinical models that more faithfully reflect the patient9s tumor and are associated with the patient9s treatment history. NCI has focused on generating models to complement existing PDX collections and address unmet needs in the preclinical model space. The PDMR generates the majority of its PDXs by subcutaneous implantation except for those histologies having better success rates in either orthotopic or alternate implant sites. All SOPs and quality-control standards developed by the PDMR as well as those shared by collaborators are posted to a public web site that houses the PDMR database. In May 2017, the public website (https://pdmr.cancer.gov/) went live with its first 100 models from histologies including pancreatic, colorectal, renal, head and neck, and lung squamous cell cancers as well as melanoma and adult soft tissue sarcomas. In early 2018, the PDMR will begin releasing models from gynecological cancers, small cell lung cancer, chondro/osteo sarcomas, lung adenocarcinoma, and squamous cell skin and Merkel cell carcinomas. In addition, wherever available germline sequence and somatic variant calls will be added to the existing molecular characterization data for each model. NCI has also increased its focus on creating PDXs from racial and ethnic minorities through several funding opportunities. The overall goal of NCI is to create a long-term home for at least 1000 models such that sufficient biological and clinical diversity is represented to allow researchers to ask questions regarding the impact of tumor heterogeneity on target qualification or clinical response, whether PDXs more faithfully represent the human tumor for pharmacodynamic assay and predictive marker development, or if adequately powered preclinical PDX clinical trials can lead to better evaluation of therapies for future clinical use. Moving forward the PDMR plans to distribute in vitro, early-passage tumor cell cultures and cancer-associated fibroblasts as well as releasing PDX drug response data for a panel of FNA-approved therapeutic agents. Funded by NCI Contract No. HHSN261200800001E Citation Format: Yvonne A. Evrard, Michelle M. Gottholm Ahalt, Sergio . Y. Alcoser, Kaitlyn Arthur, Mariah Baldwin, Linda L. Blumenauer, Carrie Bonomi, Suzanne Borgel, Elizabeth Bradtke, Corinne Camalier, John Carter, Tiffanie Chase, Alice Chen, Lily Chen, Donna W. Coakley, Nicole E. Craig, Biswajit Das, Vivekananda Datta, Jordyn Davidson, Margaret R. DeFreytas, Emily Delaney, Michelle A. Eugeni, Raymond Divelbiss, Palmer Fliss, Thomas Forbes, Marion Gibson, Tara Grinnage-Pulley, Sierra Hoffman, Lilia Ileva, Paula Jacobs, Franklyn Jimenez, Joseph Kalen, Catherine Karangwa, Chris Karlovich, Candace Mallow, Chelsea McGlynn, Jenna E. Moyer, Michael Mullendore, Dianne L. Newton, Nimit Patel, Rajesh Patidar, Kevin Plater, Marianne Radzyminski, Lisa Riffle, Larry Rubinstein, Luke H. Stockwin, Mickey Williams, Melinda G. Hollingshead, James H. Doroshow. The National Cancer Institute9s patient-derived models repository (PDMR) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 986.
- Published
- 2017
29. Abstract 4827: Establishing a platform for the generation of organoids from diverse tumor types as part of the NCI patient-derived models (PDM) initiave
- Author
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Luke H. Stockwin, John Mark Carter, Carrie Bonomi, Vivekananda Datta, Dianne L. Newton, Jesse Stottlemeyer, Kelly Dougherty, Melinda G. Hollingshead, Lindsay Dutko, James H. Doroshow, Jenna Moyer, Anna Wade, Kaitlyn Arthur, and Michael Mullendore
- Subjects
Cancer Research ,Oncology ,Organoid ,Computational biology ,Biology - Abstract
Cancer Organoids are discrete multicellular structures that recapitulate tumor microanatomy (1). These reagents can be generated by extended culture of partially or fully dissociated tumor samples in three-dimensional matrices. By maintaining tumor and accessory cells in an appropriate context, they provide a biosimilar platform for studying disease pathogenesis and cellular pharmacology (2). Similarly, cancer organoid culture is useful for propagating slow growing tumors or those requiring heterotypic cell-cell interactions. Here, preliminary data will be presented regarding generation of organoids from diverse tumor types as part of the NCI patient-derived models (PDM) initiative. This initiative aims to develop a national repository of patient-derived cancer models (PDMs) consisting of clinically annotated patient-derived xenografts (PDXs) and patient-derived tumor cell cultures (PDCs) prepared from primary and metastatic tumors (3). A standardized panel of different organoid media formulations was constructed to optimize culture conditions for disease subsets. Using this approach, organoids were generated for colon, prostate, pancreatic, breast, melanoma, NSCLC, and bladder tumors. Although some samples were refractory to organoid generation, in several instances, samples that failed to generate 2D cultures thrived as organoids. A further finding was that direct implantation of organoid cultures was an efficient means of generating xenografts. Indeed, work will be presented detailing the exact number of organoids required to establish xenograft tumors. Protocols were developed for routine culture, passaging and long-term storage in liquid nitrogen. Similarly, organoids were amenable to characterization by FACS analysis, ICC/IHC and qRT-PCR to evaluate metrics such as tumor type, histological similarity with patient tumor, cell viability, percentage stroma and whether mouse cells persist in PDX-derived organoids. In summary, growth, expansion, analysis and storage of tumor organoids is feasible for a wide range of tumor types. Importantly, for certain samples, generation of cancer organoids appears to be a useful intermediary step for subsequent PDX model and 2D culture generation. Funded by NCI Contract No. HHSN261200800001E. References: 1. Baker LA, Tiriac H, Clevers H, Tuveson DA. Modeling pancreatic cancer with organoids. Trends Cancer. 2016;2:176-90. 2. Cantrell MA, Kuo CJ. Organoid modeling for cancer precision medicine. Genome Med. 2015;7. 3. Doroshow J, Hollingshead M, Evrard Y, Williams M, Datta V, Das B, et al. NCI patient derived models repository. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA). Mol Cancer Ther. 2015;14(12 Suppl 2). Citation Format: Luke H. Stockwin, Jenna Moyer, Anna Wade, Carrie Bonomi, Kelly Dougherty, John Carter, Jesse Stottlemeyer, Kaitlyn Arthur, Vivekananda Datta, Lindsay Dutko, Michael Mullendore, James H. Doroshow, Melinda G. Hollingshead, Dianne L. Newton. Establishing a platform for the generation of organoids from diverse tumor types as part of the NCI patient-derived models (PDM) initiave [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4827. doi:10.1158/1538-7445.AM2017-4827
- Published
- 2017
30. UNC2025, a potent and orally bioavailable MER/FLT3 dual inhibitor
- Author
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Katherine A. Minson, Michael A. Stashko, Weihe Zhang, Christopher T. Cummings, Trevor Glaros, Deborah DeRyckere, Susan Sather, Dehui Zhang, Douglas K. Graham, Stephen V. Frye, Dmitri Kireev, Xiaodong Wang, Debra Hunter, Dianne L. Newton, H. Shelton Earp, Minjung Lee, William P. Janzen, and Jing Liu
- Subjects
medicine.drug_class ,Receptor Protein-Tyrosine Kinases ,Administration, Oral ,Biological Availability ,Chemistry Techniques, Synthetic ,Mice, SCID ,C-Mer Tyrosine Kinase ,Pharmacology ,Tyrosine-kinase inhibitor ,Piperazines ,Article ,Inhibitory Concentration 50 ,Structure-Activity Relationship ,In vivo ,Cell Line, Tumor ,Proto-Oncogene Proteins ,Drug Discovery ,medicine ,Leukemia, B-Cell ,Animals ,Humans ,Kinome ,Molecular Targeted Therapy ,Protein Kinase Inhibitors ,c-Mer Tyrosine Kinase ,Chemistry ,Kinase ,Adenine ,Xenograft Model Antitumor Assays ,3. Good health ,fms-Like Tyrosine Kinase 3 ,Fms-Like Tyrosine Kinase 3 ,Molecular Medicine ,Phosphorylation - Abstract
We previously reported a potent small molecule Mer tyrosine kinase inhibitor UNC1062. However, its poor PK properties prevented further assessment in vivo. We report here the sequential modification of UNC1062 to address DMPK properties and yield a new potent and highly orally bioavailable Mer inhibitor, 11, capable of inhibiting Mer phosphorylation in vivo, following oral dosing as demonstrated by pharmaco-dynamic (PD) studies examining phospho-Mer in leukemic blasts from mouse bone marrow. Kinome profiling versus more than 300 kinases in vitro and cellular selectivity assessments demonstrate that 11 has similar subnanomolar activity against Flt3, an additional important target in acute myelogenous leukemia (AML), with pharmacologically useful selectivity versus other kinases examined.
- Published
- 2014
31. Potent and specific antitumor effects of an anti-CD22–targeted cytotoxic ribonuclease: potential for the treatment of non-Hodgkin lymphoma
- Author
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Susanna M. Rybak, David M. Goldenberg, Hans J. Hansen, Dianne L. Newton, and Stanislaw M. Mikulski
- Subjects
medicine.drug_class ,Sialic Acid Binding Ig-like Lectin 2 ,medicine.medical_treatment ,Immunology ,Drug Evaluation, Preclinical ,Antineoplastic Agents ,Mice, SCID ,Monoclonal antibody ,Biochemistry ,Mice ,Ribonucleases ,Drug Stability ,Antigens, CD ,immune system diseases ,Immunotoxin ,Lectins ,hemic and lymphatic diseases ,Tumor Cells, Cultured ,medicine ,Animals ,Humans ,Cytotoxic T cell ,Pancreas ,Mice, Inbred BALB C ,Chemotherapy ,Cell Death ,Dose-Response Relationship, Drug ,biology ,Immunotoxins ,Lymphoma, Non-Hodgkin ,CD22 ,Antibodies, Monoclonal ,Cell Biology ,Hematology ,medicine.disease ,Virology ,Lymphoma ,Antigens, Differentiation, B-Lymphocyte ,Survival Rate ,Kinetics ,Ranpirnase ,Models, Animal ,Cancer research ,biology.protein ,Female ,Antibody ,Cell Adhesion Molecules ,Neoplasm Transplantation - Abstract
LL2, an anti-CD22 monoclonal antibody against B-cell lymphoma, was covalently linked to the amphibian ribonuclease, onconase, a member of the pancreatic RNase A superfamily. LL2 increased in vitro potency (10 000-fold) and specificity against human Daudi Burkitt lymphoma cells while decreasing systemic toxicity of onconase. Monensin further increased potency of LL2-onconase on Daudi cells (IC50, 20 and 1.5 pM, absence and presence of monensin, respectively). A 1-hour exposure to LL2-onconase was sufficient to kill Daudi cells in culture. These favorable in vitro properties translated to significant antitumor activity against disseminated Daudi lymphoma in mice with severe combined immunodeficiency disease. In mice inoculated with tumor cells intraperitoneally (ip), LL2-onconase (100 μg 5 times ip every day) increased the life span of animals with minimal disease 200%. The life span of mice with advanced disseminated Daudi lymphoma (tumor cells inoculated intravenously) was increased 135%. Mice injected with LL2-onconase tolerated a dose as high as 300 mg/kg. Because both onconase and LL2 are in clinical trials as cancer therapeutics, the covalently linked agents should be considered for treatment of non-Hodgkin lymphoma.
- Published
- 2001
32. Differential Requirement for the Stress-Activated Protein Kinase/c-Jun NH2-Terminal Kinase in RNA Damage-Induced Apoptosis in Primary and in Immortalized Fibroblasts
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Susanna M. Rybak, Mihail S. Iordanov, John B. Wong, Richard A. Flavell, Dianne L. Newton, Roger J. Davis, Bruce E. Magun, and Robert K. Bright
- Subjects
Cell Survival ,Papillomavirus E7 Proteins ,p38 mitogen-activated protein kinases ,Immunoblotting ,Apoptosis ,Nerve Tissue Proteins ,Biology ,Mitogen-activated protein kinase kinase ,Transfection ,p38 Mitogen-Activated Protein Kinases ,Ligases ,Synaptotagmins ,Tumor Cells, Cultured ,Humans ,Cytotoxic T cell ,Mitogen-Activated Protein Kinase 8 ,Enzyme Inhibitors ,Phosphorylation ,Hematoxylin ,Protein kinase A ,Molecular Biology ,Membrane Glycoproteins ,MAP kinase kinase kinase ,Tumor Necrosis Factor-alpha ,Activator (genetics) ,Calcium-Binding Proteins ,NF-kappa B ,Oncogene Proteins, Viral ,Cell biology ,Enzyme Activation ,Caspases ,Mitogen-activated protein kinase ,biology.protein ,Eosine Yellowish-(YS) ,RNA ,Mitogen-Activated Protein Kinases ,HeLa Cells - Abstract
Onconase, an anticancer ribonuclease, damages cellular tRNA and causes caspase-dependent apoptosis in targeted cells (M. S. Iordanov, O. P. Ryabinina, J. Wong, T. H. Dinh, D. L. Newton, S. M. Rybak, and B. E. Magun. Cancer Res. 60, 1983–1994, 2000). The proapoptotic action of onconase depends on its RNase activity, but the molecular mechanisms leading to RNA damage-induced caspase activation are completely unknown. In this study, we have investigated whether onconase activates two signal-transduction pathways commonly stimulated by conventional chemo- and radiotherapy, namely the stress-activated protein kinase (SAPK) cascade and the pathway leading to the activation of nuclear factor-kappa B (NF-κB). We found that, in all cell types tested, onconase is a potent activator of SAPK1 (JNK1 and JNK2) and SAPK2 (p38 MAP kinase), but that it is incapable of activating NF-κB. Inhibition of p38 MAP kinase activity with a pharmacological inhibitor, SB203580, demonstrated that p38 MAP kinase is not required for onconase cytotoxicity. Using explanted fibroblasts from mice that contain targeted disruption of both jnk1 and jnk2 alleles, we found that JNKs are important mediators of onconase-induced cytotoxicity. Surprisingly, following the immortalization of these same cells with human papilloma virus (HPV16) gene products E6 and E7, additional proapoptotic pathways (exclusive of JNK) were provoked by onconase. Our results demonstrate that onconase may activate proapoptotic pathways in tumor cells that are not able to be accessed in normal cells. These results present the possibility that the cytotoxic activity of onconase in normal cells may be reduced by blocking the activity of JNKs.
- Published
- 2000
33. A gender-specific mRNA encoding a cytotoxic ribonuclease contains a 3' UTR of unusual length and structure
- Author
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Susanna M. Rybak, Shu-Yun Le, Jacob V. Maizel, Dianne L. Newton, and Shin-lin Chen
- Subjects
Models, Molecular ,Untranslated region ,DNA, Complementary ,Transcription, Genetic ,Molecular Sequence Data ,Article ,Open Reading Frames ,Ribonucleases ,Complementary DNA ,Genetics ,Protein biosynthesis ,Animals ,RNA, Messenger ,Ribonuclease ,3' Untranslated Regions ,Gene Library ,Messenger RNA ,Base Sequence ,biology ,Three prime untranslated region ,Rana pipiens ,RNA ,Molecular biology ,Open reading frame ,Liver ,Protein Biosynthesis ,Oocytes ,biology.protein ,Nucleic Acid Conformation ,Female ,Software - Abstract
A cDNA (2855 nt) encoding a putative cytotoxic ribonuclease (rapLR1) related to the antitumor protein onconase was cloned from a library derived from the liver of gravid female amphibian Rana pipiens. The cDNA was mainly comprised (83%) of 3' untranslated region (UTR). Secondary structure analysis predicted two unusual folding regions (UFRs) in the RNA 3' UTR. Two of these regions (711-1442 and 1877-2130 nt) contained remarkable, stalk-like, stem-loop structures greater than 38 and 12 standard deviations more stable than by chance, respectively. Secondary structure modeling demonstrated similar structures in the 3' UTRs of other species at low frequencies (0.01-0.3%). The size of the rapLR1 cDNA corresponded to the major hybridizing RNA cross-reactive with a genomic clone encoding onconase (3.6 kb). The transcript was found only in liver mRNA from female frogs. In contrast, immunoreactive onconase protein was detected only in oocytes. Deletion of the 3' UTR facilitated the in vitro translation of the rapLR1 cDNA. Taken together these results suggest that these unusual UFRs may affect mRNA metabolism and/or translation.
- Published
- 2000
34. Cell Cycle-Related Differences in Susceptibility of NIH/3T3 Cells to Ribonucleases
- Author
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Susanna M. Rybak, Mark R. Smith, Dianne L. Newton, and Stanley M. Mikulski
- Subjects
Microinjections ,RNase P ,Oncogene Protein p21(ras) ,Biology ,Culture Media, Serum-Free ,3T3 cells ,Mice ,Ribonucleases ,medicine ,Animals ,Cytotoxic T cell ,Annexin A5 ,Cytotoxicity ,Protein kinase A ,Interphase ,Cell Line, Transformed ,Cell Death ,Cell Cycle ,Egg Proteins ,Drug Synergism ,3T3 Cells ,Ribonuclease, Pancreatic ,Cell Biology ,Cell cycle ,Cell Transformation, Viral ,Molecular biology ,Cell biology ,medicine.anatomical_structure ,Cell culture ,Culture Media, Conditioned ,Cattle ,Signal transduction ,Extracellular Space - Abstract
Microinjection of Onconase or RNase A into NIH/3T3 cells was used to study the intracellular actions of these two proteins. Onconase preferentially killed actively growing cells in both microinjection and cell culture experiments. Moreover, agents that increased the number of cells in S phase such as serum or microinjected signal transduction mediators (Ras, protein kinase C, and mitogen-activated protein kinase) enhanced Onconase cytotoxicity. Conversely, agents that decreased these proliferative pathways (dibutyryl cAMP and protein kinase A) correspondingly diminished Onconase cytotoxicity in microinjection experiments. These results were also mimicked in cell culture experiments since log-phase v-ras-transformed NIH/3T3 cells were more sensitive to Onconase (IC50 of 7 microg/ml) than parental NIH/3T3 fibroblasts (IC50 of 40 microg/ml). Based on those data we postulated that Onconase-mediated cell death in NIH/3T3 cells was related to events occurring at two or more points in the cell cycle preferentially associated with late G1/S and S phases. In contrast, quiescent NIH/3T3 cells were more sensitive to microinjected RNase A than log phase cells and positive mediators of proliferative signal transduction did not enhance RNase A-mediated cytotoxicity. Taken together, these results demonstrate that these two RNases use different pathways and/or mechanisms to elicit cytotoxic responses in NIH/3T3 cells. Predictions formulated from these studies can be tested for relevance to RNase actions in different target tumor cells.
- Published
- 1999
35. Unique Recombinant Human Ribonuclease and Inhibition of Kaposi's Sarcoma Cell Growth
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Dianne L. Newton and Susanna M. Rybak
- Subjects
Cancer Research ,DNA, Complementary ,Blotting, Western ,Antineoplastic Agents ,Breast Neoplasms ,Eosinophil-Derived Neurotoxin ,Biology ,Polymerase Chain Reaction ,Inclusion bodies ,law.invention ,Ribonucleases ,Leucine ,law ,Genes, Synthetic ,Serine ,Tumor Cells, Cultured ,Humans ,Cytotoxic T cell ,Histidine ,Viability assay ,Cytotoxicity ,Sarcoma, Kaposi ,chemistry.chemical_classification ,Cell growth ,Proteins ,Valine ,Ribonuclease, Pancreatic ,Kidney Neoplasms ,Recombinant Proteins ,In vitro ,Amino acid ,Oncology ,Biochemistry ,chemistry ,Recombinant DNA - Abstract
BACKGROUND Preparations of human chorionic gonadotropin (hCG) have been shown to exhibit anti-Kaposi's sarcoma (KS) activity, but the identity of the responsible agent(s) remains controversial. One candidate agent is an eosinophil-derived neurotoxin (EDN)-like polypeptide that contaminates preparations of hCG. We have genetically engineered a unique form of hEDN, which is a ribonuclease, and have evaluated the cytotoxic effects of the recombinant protein on KS Y-1 cells and on cells of other cancer types. METHODS The amino-terminus of hEDN was extended by four amino acid residues, corresponding to the proximal part of the hEDN signal peptide (serine, leucine, histidine, and valine; positions -4 to -1, respectively), by use of the polymerase chain reaction and an hEDN complementary DNA. The recombinant protein was isolated from bacterial inclusion bodies. The cytotoxic activity of this hEDN variant, (-4)rhEDN, was tested on KS Y-1 cells and human glioma, melanoma, breast carcinoma, and renal carcinoma cells. RESULTS Approximately half of the anti-KS activity in a crude commercial preparation of hCG was associated with a polypeptide that reacted with anti-recombinant-hEDN (rhEDN) polyclonal antibodies. Although rhEDN protein displayed little cytotoxicity against KS Y-1 cells (IC50 [50% inhibition concentration] = >100 microg/mL), (-4)rhEDN markedly inhibited cell viability (IC50 = 6 microg/mL). Neither version of rhEDN inhibited the viability of other tested human cancer cell types. CONCLUSIONS A four amino acid extension of the amino-terminus of rhEDN confers cytotoxicity against KS Y-1 cells in vitro. Design of the (-4)rhEDN variant was based on the sequence of a natural human protein associated with hCG. Our results suggest that (-4)rhEDN is one of the agents in hCG responsible for anti-KS activity. A purified molecule is thus available for in vitro and in vivo mechanistic and, possibly, future clinical studies.
- Published
- 1998
36. Single Amino Acid Substitutions at the N-Terminus of a Recombinant Cytotoxic Ribonuclease Markedly Influence Biochemical and Biological Properties
- Author
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Lluis Boque, Dianne L. Newton, Charles Y. Huang, Alexander Wlodawer, and Susanna M. Rybak
- Subjects
Reticulocytes ,RNase P ,Antineoplastic Agents ,Protein Engineering ,medicine.disease_cause ,Biochemistry ,Substrate Specificity ,law.invention ,Structure-Activity Relationship ,Ribonucleases ,RNA, Transfer ,law ,Tumor Cells, Cultured ,medicine ,Protein biosynthesis ,Animals ,Humans ,Ribonuclease ,Escherichia coli ,Protein Synthesis Inhibitors ,chemistry.chemical_classification ,Cell-Free System ,Dose-Response Relationship, Drug ,biology ,Egg Proteins ,Molecular biology ,Recombinant Proteins ,Amino acid ,N-terminus ,Enzyme ,chemistry ,Drug Design ,Protein Biosynthesis ,Recombinant DNA ,biology.protein ,Rabbits - Abstract
Onconase is a cytotoxic ribonuclease with antitumor properties. A semisynthetic gene encoding the entire protein sequence was constructed by fusing oligonucleotides coding for the first 15 and the last 6 of the 104 amino acids to a genomic clone that encoded the remaining amino acid residues [Newton, D. L., et al. (1997) Protein Eng. 10, 463-470]. The resulting protein product expressed in Escherichia coli exhibited little enzymatic or cytotoxic activity due to the unprocessed N-terminal Met amino acid residue. In this study, we demonstrate that modification of the 5'-region of the gene to encode [Met-(-1)]Ser or [Met-(-1)]Tyr instead of the native pyroglutamate results in recombinant onconase derivatives with restored activities. [Met-(-1)]rOnc(E1S) was more active than [Met-(-1)]rOnc(E1Y) in all assays tested. Consistent with the action of native onconase, [Met-(-1)]rOnc(E1S) was a potent inhibitor of protein synthesis in the cell-free rabbit reticulocyte lysate assay, degrading tRNA at concentrations that correlated with inhibition of protein synthesis. An interesting difference between the recombinant onconase derivatives and the native protein was their susceptibility to inhibition by the major intracellular RNase inhibitor, PRI (onconase is refractory to PRI inhibition). [Met-(-1)]rOnc(E1S) and [Met-(-1)]rOnc(E1Y) inhibited protein synthesis in intact SF539 neuroblastoma cells with IC50's very similar to that of onconase (IC50 3.5, 10, and 10 microg/mL after 1 day and 0.16, 0.35, and 2.5 microg/mL after 5 days for onconase, [Met-(-1)]rOnc(E1S), and [Met-(-1)]rOnc(E1Y), respectively). Similar to that of onconase, cytotoxic activity of the recombinant derivatives was potentiated by monensin, NH4Cl, and retinoic acid. Brefeldin A completely blocked the enhancement of cytotoxicity caused by retinoic acid with all three proteins. Thus, drug-induced alterations of the intracellular trafficking of the recombinant derivatives also resembles that of onconase. Stability studies as assessed in serum-containing medium in the presence or absence of cells at 37 degreesC showed that the recombinant proteins were as stable to temperature and cell culture conditions as the native protein. Therefore, exchanging the Glu amino acid residue at the amino terminus of onconase with an amino acid residue containing a hydroxyl group produces recombinant proteins with ribonuclease and cytotoxic properties similar to native onconase.
- Published
- 1998
37. Cloning and cytotoxicity of a human pancreatic RNase immunofusion
- Author
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Stefan Dübel, Monika Zewe, Dianne L. Newton, Susanna M. Rybak, Martin Welschof, Johannes F. Coy, and Melvyn Little
- Subjects
Cytotoxicity, Immunologic ,Angiogenin ,RNase P ,Recombinant Fusion Proteins ,Immunology ,Immunoglobulin Variable Region ,Eosinophil-derived neurotoxin ,Transferrin receptor ,Bovine pancreatic ribonuclease ,Polymerase Chain Reaction ,Gene product ,Mice ,Immunotoxin ,Receptors, Transferrin ,Tumor Cells, Cultured ,Animals ,Humans ,Cloning, Molecular ,Immunoglobulin Fragments ,Protein Synthesis Inhibitors ,Cell-Free System ,biology ,Chemistry ,Immunotoxins ,Ribonuclease, Pancreatic ,Fusion protein ,Molecular biology ,Genes ,biology.protein ,Plasmids - Abstract
Background: Immunotoxins based on plant and bacterial proteins are usually very immunogenic. Human ribonucleases could provide an alternative basis for the construction of less immunogenic reagents. Two members of the human RNase family, angiogenin and eosinophil-derived neurotoxin (EDN), have been fused to a single chain antibody against the transferrin receptor, which is known to be internalised by endocytosis. The fusion proteins proved to be very efficient inhibitors of protein synthesis using various cell lines. It is not yet known whether the side effects of angiogenin and EDN will compromise their potential use as immunotoxins. Objectives: The goal of this work was to construct a human immunotoxin with no harmful side effects. Bovine pancreatic ribonuclease has been shown to be as potent as ricin at abolishing protein synthesis on injection into oocytes. We therefore decided to clone its human analogue, which is fairly ubiquitous and per se non-toxic. An immunofusion of human pancreatic RNase with a single chain antibody against the transferrin receptor was tested for its ability to inhibit protein synthesis in three different human tumor cell lines. Study design: DNA coding for the human pancreatic RNase was cloned partially from a human fetal brain cDNA library and then completed by PCR using a human placental cDNA library as a template. The RNase gene was then fused with a DNA coding for an single chain antibody against the transferrin receptor (CD71). After expressing the fusion protein in E. coli, the gene product was isolated from inclusion bodies and tested for cytotoxicity. Results: This fusion protein inhibited the protein synthesis of three human tumor cell lines derived from a melanoma, a renal carcinoma and a breast carcinoma, with IC50s of 8, 5 and 10 nM, respectively. These values were comparable with those using a similar fusion protein constructed with eosinophil derived neurotoxin (EDN) as the toxic moiety (IC50s of 8, 1.2 and 3 nM, respectively). The slightly lower activities of the human pancreatic RNase-scFv (pancRNase-scFv) with two of the cell lines suggests that fewer molecules are reaching the cytoplasmic compartment, since it was twice as active as EDN-scFv in inhibiting the protein synthesis of a rabbit reticulocyte lysate. Conclusion: These results demonstrate that the human pancreatic RNase, which is expected to have a very low immunogenic potential in humans with no inherent toxicity, may be a potent cytotoxin for tumor cells after antibody targeting.
- Published
- 1997
38. Expression and characterization of a cytotoxic human-frog chimeric ribonuclease: potential for cancer therapy
- Author
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L. Boque, A. Wlodawer, Susanna M. Rybak, Hsiang-Fu Kung, Dianne L. Newton, and Y. Xue
- Subjects
Models, Molecular ,RNase P ,Recombinant Fusion Proteins ,Molecular Sequence Data ,Neurotoxins ,Antineoplastic Agents ,Bioengineering ,Eosinophil-Derived Neurotoxin ,Biochemistry ,law.invention ,Chimera (genetics) ,Methionine ,Ribonucleases ,Protein sequencing ,law ,Aspartic acid ,Tumor Cells, Cultured ,Animals ,Humans ,Amino Acid Sequence ,Ribonuclease ,Cloning, Molecular ,Molecular Biology ,Gene ,biology ,Chemistry ,Oligonucleotide ,Egg Proteins ,Rana pipiens ,Molecular biology ,Models, Chemical ,biology.protein ,Recombinant DNA ,Electrophoresis, Polyacrylamide Gel ,Drug Screening Assays, Antitumor ,Software ,Biotechnology - Abstract
Onconase is a cytotoxic ribonuclease with antitumor properties. A semisynthetic gene encoding the entire protein sequence was constructed by fusing oligonucleotides coding for the first 15 and last six of the 104 amino acid residues to a genomic clone that encoded the remaining amino acid residues. Additionally, the 15 N-terminal amino acid residues of onconase were replaced with the first 21 amino acid residues of the homologous human RNase, eosinophil-derived neurotoxin, EDN. Two versions of the hybrid EDN-onconase protein were cloned, expressed and purified. The chimera that contained a glycine in lieu of the aspartic acid present in native onconase (position 26 in the chimera) exhibited enzymatic activity more characteristic of EDN than native onconase and was considerably more active with respect to both RNase activity and cellular cytotoxicity than recombinant onconase. In contrast to native or recombinant onconase, the EDN chimera was recognized by anti-EDN polyclonal antibodies, demonstrating that the chimera also shared structural antigenic determinants to the human enzyme. These results demonstrate that a chimeric ribonuclease has cytotoxicity comparable to onconase in two out of four cell lines tested. The implications with regard to cancer therapy are presented.
- Published
- 1997
39. Trypsin-mediated ¹⁸O/¹⁶O labeling for biomarker discovery
- Author
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Xiaoying, Ye, King C, Chan, Darue A, Prieto, Brian T, Luke, Donald J, Johann, Luke H, Stockwin, Dianne L, Newton, and Josip, Blonder
- Subjects
Proteomics ,Cell Line, Tumor ,Isotope Labeling ,Cell Membrane ,Biomarkers, Tumor ,Humans ,Proteins ,Trypsin ,Oxygen Isotopes ,Melanoma ,Cell Hypoxia ,Mass Spectrometry ,Chromatography, Liquid - Abstract
Differential (18)O/(16)O stable isotopic labeling that relies on post-digestion (18)O exchange is a simple and efficient method for the relative quantitation of proteins in complex mixtures. This method incorporates two (18)O atoms onto the C-termini of proteolytic peptides resulting in a 4 Da mass-tag difference between (18)O- and (16)O-labeled peptides. This allows for wide-range relative quantitation of proteins in complex mixtures using shotgun proteomics. Because of minimal sample consumption and unrestricted peptide tagging, the post-digestion (18)O exchange is suitable for labeling of low-abundance membrane proteins enriched from cancer cell lines or clinical specimens, including tissues and body fluids. This chapter describes a protocol that applies post-digestion (18)O labeling to elucidate putative endogenous tumor hypoxia markers in the plasma membrane fraction enriched from a hypoxia-adapted malignant melanoma cell line. Plasma membrane proteins from hypoxic and normoxic cells were differentially tagged using (18)O/(16)O stable isotopic labeling. The initial tryptic digestion and solubilization of membrane proteins were carried out in a buffer containing 60 % methanol followed by post-digestion (18)O exchange/labeling in buffered 20 % methanol. The differentially labeled peptides were mixed in a 1:1 ratio and fractionated using off-line strong cation exchange (SCX) liquid chromatography followed by on-line reversed-phase nano-flow RPLC-MS identification and quantitation of peptides/proteins in respective SCX fractions. The present protocol illustrates the utility of (18)O/(16)O stable isotope labeling in the context of quantitative shotgun proteomics that provides a basis for the discovery of hypoxia-induced membrane protein markers in malignant melanoma cell lines.
- Published
- 2013
40. Trypsin-Mediated 18O/16O Labeling for Biomarker Discovery
- Author
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DaRue A. Prieto, Luke H. Stockwin, King C. Chan, Xiaoying Ye, Dianne L. Newton, Donald J. Johann, Josip Blonder, and Brian Luke
- Subjects
chemistry.chemical_classification ,Isotopic labeling ,Chromatography ,Membrane protein ,chemistry ,Tumor hypoxia ,medicine ,Peptide ,Context (language use) ,Biomarker discovery ,Shotgun proteomics ,Trypsin ,medicine.drug - Abstract
Differential (18)O/(16)O stable isotopic labeling that relies on post-digestion (18)O exchange is a simple and efficient method for the relative quantitation of proteins in complex mixtures. This method incorporates two (18)O atoms onto the C-termini of proteolytic peptides resulting in a 4 Da mass-tag difference between (18)O- and (16)O-labeled peptides. This allows for wide-range relative quantitation of proteins in complex mixtures using shotgun proteomics. Because of minimal sample consumption and unrestricted peptide tagging, the post-digestion (18)O exchange is suitable for labeling of low-abundance membrane proteins enriched from cancer cell lines or clinical specimens, including tissues and body fluids. This chapter describes a protocol that applies post-digestion (18)O labeling to elucidate putative endogenous tumor hypoxia markers in the plasma membrane fraction enriched from a hypoxia-adapted malignant melanoma cell line. Plasma membrane proteins from hypoxic and normoxic cells were differentially tagged using (18)O/(16)O stable isotopic labeling. The initial tryptic digestion and solubilization of membrane proteins were carried out in a buffer containing 60 % methanol followed by post-digestion (18)O exchange/labeling in buffered 20 % methanol. The differentially labeled peptides were mixed in a 1:1 ratio and fractionated using off-line strong cation exchange (SCX) liquid chromatography followed by on-line reversed-phase nano-flow RPLC-MS identification and quantitation of peptides/proteins in respective SCX fractions. The present protocol illustrates the utility of (18)O/(16)O stable isotope labeling in the context of quantitative shotgun proteomics that provides a basis for the discovery of hypoxia-induced membrane protein markers in malignant melanoma cell lines.
- Published
- 2013
41. X-ray Crystallographic Structure of Recombinant Eosinophil-derived Neurotoxin at 1.83 Å Resolution
- Author
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Richard J. Youle, Steven C. Mosimann, Michael N.G. James, and Dianne L. Newton
- Subjects
Models, Molecular ,Molecular model ,Protein Conformation ,RNase P ,Stereochemistry ,Molecular Sequence Data ,Neurotoxins ,Eosinophil-Derived Neurotoxin ,Crystal structure ,Crystallography, X-Ray ,Methionine ,Ribonucleases ,Structural Biology ,Animals ,Molecule ,Molecular replacement ,Amino Acid Sequence ,Ribonuclease ,Binding site ,Molecular Biology ,Binding Sites ,biology ,Sulfates ,Chemistry ,Egg Proteins ,Active site ,Ribonuclease, Pancreatic ,Recombinant Proteins ,Crystallography ,Vertebrates ,Solvents ,biology.protein - Abstract
The X-ray crystallographic structure of recombinant eosinophil-derived neurotoxin (rEDN) has been determined by molecular replacement methods and refined at 1.83 A resolution to a conventional R-factor ( = sigma magnitute of (magnitute of F(zero)-magnitude of Fc)/ sigma magnitude of F(zero) of 0.152 with excellent stereochemistry. The molecular model of rEDN contains all 1081 non-hydrogen protein atoms, two non-covalently bound sulfate anions and 121 ordered solvent molecules. The polypeptide fold of rEDN is related to those observed in the homologous structures of RNase A, Onconase and angiogenin. rEDN is one of the largest members of the pyrimidine-specific ribonuclease superfamily of vertebrates and has small insertions in four of its seven loop structures and a large insertion from Asp115 to Tyr123. The non-covalently bound SO4(A) and SO4(B) anions occupy phosphate-binding subsites of rEDN. The active site SO4(A) anion makes contacts in rEDN that are similar to those in RNase A and involve the side-chain atoms of Gln14, His15 and His129, and the NH group of Leu130. The SO4(B) anion makes contacts with the side-chain atoms of Arg36 and Asn39 and the main-chain atoms of Asn39 and Gln40. The equivalent residues of RNase A cannot make contacts similar to those observed in rEDN. The SO4(B) binding site of rEDN likely corresponds to the P-1 subsite and may be representative of how other homologous RNases bind the P-1 phosphate.
- Published
- 1996
42. Enhancement of Vincristine Cytotoxicity in Drug-Resistant Cells by Simultaneous Treatment With Onconase, an Antitumor Ribonuclease
- Author
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Sally E. Spence, Susanna M. Rybak, Stanislaw M. Mikulski, William E. Fogler, John W. Pearson, Dianne L. Newton, Kirk Volker, Charles W. Riggs, Wojciech Ardelt, Dan L. Longo, and Hsiang-Fu Kung
- Subjects
Cancer Research ,Transplantation, Heterologous ,Drug Resistance ,Mice, Nude ,Antineoplastic Agents ,Biology ,Mice ,Ribonucleases ,In vivo ,Antineoplastic Combined Chemotherapy Protocols ,Tumor Cells, Cultured ,Animals ,Humans ,Cytotoxic T cell ,Viability assay ,Cytotoxicity ,Egg Proteins ,In vitro ,Transplantation ,Oncology ,Biochemistry ,Vincristine ,Ranpirnase ,Colonic Neoplasms ,Cancer cell ,Cancer research ,Female ,Neoplasm Transplantation - Abstract
Background : Onconase, a protein isolated from oocytes and early embryos of the frog Rana pipiens, shares extensive homology with bovine pancreatic ribonuclease (RNase A) and possesses similar enzyme activity. Onconase is cytotoxic toward cancer cells in vitro and exhibits antitumor activity in animal models. In addition, Onconase has been shown to enhance the cytotoxic activity of some chemotherapeutic agents in vitro. Purpose : We studied interactions between the cytotoxic effects of Onconase and the chemotherapeutic agent vincristine (VCR) in the treatment of drug-sensitive and multidrug-resistant human colon carcinoma cells in vitro and in mice. Methods : Transplantable human colon carcinoma cells (HT-29 par cells) were infected with a retrovirus containing human mdr1 (also known as MDRI and PGY1 complementary DNA (encoding P-glycoprotein [P-gp]), and clones that were cross-resistant to colchicine, doxorubicin, and vinblastine were selected (HT-29 mdr1 cells). Drug-resistant HT-29 mdr1 cells and drug-sensitive HT-29 par parental cells were treated with Onconase and/or VCR in vitro at varying concentrations to measure the effects on protein synthesis and cell viability. The impact of Onconase on VCR accumulation in both types of cells was determined in the presence or absence of MRK-16, an anti-P-gp monoclonal antibody capable of reversing the multidrug-resistant phenotype. The antitumor effects of Onconase and/or VCR treatment were assessed in nude mice bearing established HT-29 par or HT-29 mdr1 intraperitoneal tumors. IC 50 values (drug concentrations resulting in 50% inhibition of protein synthesis or cell viability) for Onconase and VCR were determined from semilogarithmic dose-response curves ; interactions between the cytotoxic effects of these two agents were evaluated using data from protein synthesis inhibition experiments and a two-way analysis of variance. Survival distributions from in vivo experiments were compared using Cox proportional hazards models. Results : The combination of Onconase and VCR yielded enhanced cytotoxicity in vitro that was independent of P-gp expression. Evaluation of the effects of these two compounds on protein synthesis over a wide range of drug concentrations indicated possible synergistic interactions (i.e., greater than additive effects) in both drug-resistant and drug-sensitive cells. The enhancement of VCR cytotoxicity was dependent on Onconase enzyme activity and was not associated with increased intracellular levels of VCR. Simultaneous treatment of mice bearing HT-29 par tumors with Onconase and VCR did not extend their median survival time (MST) significantly (MST with VCR = 66 days ; MST with VCR plus Onconase = 69 days ; two-tailed P =.57) ; however, the MST of mice with HT-29 mdr1 tumors was extended significantly by this treatment (MST with VCR = 44 days ; MST with VCR plus Onconase = 66 days ; two-tailed P
- Published
- 1996
43. Abstract 5178: Determinants of response to temozolomide in an exceptionally sensitive patient derived model
- Author
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John Connelly, Erik Harris, Dianne L. Newton, David Evans, Anne Monks, Luke H. Stockwin, Annamaria Rapisarda, Michael Selby, Rene Delosh, Lara H. El Touny, Beverly A. Teicher, Ralph E. Parchment, Russell Reinhart, Melinda G. Hollingshead, Chad Ogle, James H. Doroshow, Julie Laudeman, Thomas Silvers, Mark W. Burkett, and Curtis Hose
- Subjects
Trametinib ,Cancer Research ,medicine.medical_specialty ,Temozolomide ,Veliparib ,Colorectal cancer ,business.industry ,Dacarbazine ,medicine.disease ,Carboplatin ,Surgery ,chemistry.chemical_compound ,Oncology ,chemistry ,Apoptosis ,Glioma ,Cancer research ,medicine ,business ,medicine.drug - Abstract
Application of precision medicine to cancer treatment utilizes cutting-edge genomic sequencing techniques to identify specific mutations in tumors that can be matched to targeted therapies designed to treat those abnormalities. To complement NCI-MPACT, an ongoing molecular profiling-based clinical trial (NCT01827384), we used cell lines developed from several patient-derived xenograft (PDX) models to examine response to, and potential biomarkers for, the regimens of the 4-arm MPACT trial: veliparib (VLP)/temozolomide (TMZ), AZD1775/carboplatin, everolimus and trametinib. In vitro sensitivity of the PDX-derived cell lines to clinically achievable concentrations of these MPACT drugs (combinations and single agents) was examined in classic 2D cultures (monolayer on plastic) and 3D cultures (spheroids generated in ultra-low attachment culture plates). Responses in 2D and 3D cultures were similar after 7 days of drug exposure. Moreover, adding VLP (1.7μM or 5μM) in combination with TMZ did not enhance TMZ cytotoxicity. A bladder cancer cell line developed from a PDX model (BLX) showed exceptional sensitivity to TMZ (IC50 ∼ 3-5μM) compared to a lung cancer cell line (also produced from a PDX; LUX) which was insensitive (IC50 >40 μM). Loss of MGMT expression in glioma and possibly in colorectal carcinoma is considered a predictive biomarker for response to alkylating agents, such as dacarbazine and TMZ. Indeed, we could not detect MGMT protein expression in BLX, while MGMT was present at high levels in LUX. To elucidate unique determinants of BLX hypersensitivity to TMZ beyond MGMT expression, we examined DNA damage responses elicited in this model. Under both 2D and 3D conditions, exposure to TMZ (13 and 40μM) for various times (4, 8, 12, 24, 48, 72 and 96 hrs) induced γH2AX after 24 hr, while PARP1 cleavage was induced as early as 48 hrs after drug addition indicating the onset of apoptosis. TMZ activated ATR and ATM signaling pathways especially at the later time points, paralleling the pharmacodynamics of PARP cleavage. Moreover, concordant with the sensitivity profiles, signaling activation in 2D and 3D conditions was similar. In contrast, none of these pathways were activated in the TMZ non-responsive LUX model. Cell line models developed from PDXs with intermediate MGMT expression are being evaluated. A key to success of personalized medicine in oncology will be the identification of genomic determinants that predict which individual cases will show exceptional responses to particular treatments, and our data suggest that PDX cell line models may be valuable for elucidating molecular and genetic characteristics of response to specific drugs and for the identification of predictive biomarkers. Funded by NCI Contract No. HHSN261200800001E. This research was supported, in part, by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute. Citation Format: Lara H. el Touny, John Connelly, Curtis Hose, Anne Monks, Mark W. Burkett, Erik Harris, Rene’ M. Delosh, Julie Laudeman, Chad Ogle, Russell Reinhart, Michael Selby, Thomas Silvers, David Evans, Dianne Newton, Luke Stockwin, Melinda Hollingshead, Ralph Parchment, James H. Doroshow, Beverly Teicher, Annamaria Rapisarda. Determinants of response to temozolomide in an exceptionally sensitive patient derived 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 5178.
- Published
- 2016
44. Identification of CBX3 and ABCA5 as Putative Biomarkers for Tumor Stem Cells in Osteosarcoma
- Author
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Dominic A. Scudiero, Curtis Hose, Bingnan Han, Thomas Silvers, Jalpa Shah, Anne Monks, Kunio Nagashima, Angelena Millione, Rene Delosh, Robert H. Shoemaker, Vaibhav Saini, Mark W. Burkett, Dianne L. Newton, Karen M. Hite, and Melinda G. Hollingshead
- Subjects
Multidisciplinary ,business.industry ,Science ,lcsh:R ,lcsh:Medicine ,Correction ,ComputerApplications_COMPUTERSINOTHERSYSTEMS ,Computational biology ,Bioinformatics ,medicine.disease ,Tumor Stem Cells ,Medicine ,Osteosarcoma ,Table (database) ,lcsh:Q ,Identification (biology) ,lcsh:Science ,business - Abstract
During the production process, specific shading of Table 1 was lost. A correct version of Table 1 can be seen here: [^]
- Published
- 2012
45. Abstract PL08-02: NCI patient derived models repository
- Author
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P. Mickey Williams, Vivekananda Datta, Dianne L. Newton, Yvonne A. Evrard, Biswajit Das, Chih-Jian Lih, James H. Doroshow, and Melinda G. Hollingshead
- Subjects
Oncology ,Cancer Research ,medicine.medical_specialty ,Bladder cancer ,business.industry ,Cancer ,Disease ,medicine.disease ,Bioinformatics ,Clinical trial ,Prostate cancer ,Circulating tumor cell ,In vivo ,Pancreatic cancer ,Internal medicine ,medicine ,business - Abstract
The National Cancer Institute is developing a national repository of patient-derived cancer models (PDMs) comprised of[T] clinically-annotated patient-derived xenografts (PDXs); patient-derived tumor cell cultures (PDCs, including conditionally-reprogrammed tumor cell cultures) prepared from primary and metastatic tumors, circulating tumor cells (CTCs), and/or PDXs; tumor cell lysates, DNA, and RNA; and cancer-associated fibroblast cell lines (CAFs, autologous when possible) to serve as a resource for academic discovery efforts and public-private partnerships for drug discovery. NCI will provide a long-term home for >1000 PDX and PDC models, each produced from tissues and blood supplied by NCI-designated Cancer Centers and NCI-supported clinical trials networks. The effort is targeting the collection of tumors that are less prevalent in current resources, such as: small cell lung cancer, prostate cancer, bladder cancer, pancreatic cancer, head and neck cancers, as well as sarcomas and melanomas. The goals of the project are: (1) to develop a minimum of ∼50 unique patient models (both PDXs and PDCs) per disease such that the size of each molecularly-characterized subgroup is useful for subsequent validation and/or efficacy studies; (2) to perform comprehensive pre-competitive molecular characterization of patient samples and earliest passage PDXs and PDCs that includes the NCI-MPACT mutation panel, WES, RNASeq, copy number determination, histology, growth curves, and pilot proteomic/phospho-proteomic studies; and (3) to make all models and associated pre-clinical and clinical data available through a publicly available website. To date, over 1700 specimens from 1100 patients have been received for the development of PDMs; the overall ‘take' rate for PDXs originating from solid tumors is 70% with >170 assessable models and another 270 early passage tumors currently in evaluation. As expected, based on collection priorities, tumors of genitourinary, digestive, head and neck, musculoskeletal, respiratory, and skin origin are the major histological sites of origin for our PDX models. In addition, over 90 conditionally-reprogrammed cell lines have been expanded from both 18-gauge needle biopsies and surgical resections, and have passed initial quality control procedures; many of these cell cultures have a matched PDX. Over 150 CAF lines have been developed following repeated (>10) purification steps using flow cytometry and are in the process of quality control procedures that demonstrate complete lack of growth in NOD-SCID gamma IL2 receptor null (NSG) mice; of these CAFs, we have developed matched pairs of PDCs and CAFs from the same patient in 16 cases. To evaluate the potential utility of the NCI PDM Repository, we have prospectively ‘entered' 22 models in a pre-clinical trial for which eligibility (based on actionable mutations) and treatment arms are identical to those in the NCI-MPACT study (NCT01827384). Multiple objective responses (significant improvement in overall survival) have been observed in all arms of the study and in a variety of models. WES and RNASeq analysis have proven essential to explain the therapeutic responses that we have observed. We are also evaluating the relationship of in vitro and in vivo activity for the NCI-MPACT drug panel in the models where concurrent PDCs and PDXs have been produced. A web site has been developed that will provide annotated information on the models (such as DNA sequence, gene expression, prior therapy) to investigators to assist in the distribution of the contents of the repository to the research community. We expect to be able to begin distribution in late spring of 2016. Citation Format: James H. Doroshow, Melinda Hollingshead, Yvonne Evrard, P. Mickey Williams, Vivekananda Datta, Biswajit Das, Chih-Jian Lih, Dianne Newton. NCI patient derived models repository. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PL08-02.
- Published
- 2015
46. Expression and characterization of recombinant human eosinophil-derived neurotoxin and eosinophil-derived neurotoxin-anti-transferrin receptor sFv
- Author
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Richard J. Youle, Dianne L. Newton, Susanna M. Rybak, and P. J. Nicholls
- Subjects
Eosinophil-derived neurotoxin ,Transferrin receptor ,Cell Biology ,Plasma protein binding ,Biology ,Biochemistry ,Molecular biology ,Fusion protein ,Inclusion bodies ,law.invention ,Immunotoxin ,law ,Recombinant DNA ,Receptor ,Molecular Biology - Abstract
The gene for the human recombinant eosinophil-derived neurotoxin (rEDN) was synthesized and fused to the gene encoding a single chain antibody (sFv) to the human transferrin receptor (EDNsFv). Both rEDN and EDNsFv were expressed as insoluble proteins in inclusion bodies in Escherichia coli BL21(DE3). Following denaturation and renaturation, EDN and EDNsFv were partially purified by chromatography on heparin-Sepharose. Final purification of EDN was achieved by Sephadex G-100, whereas EDNsFv which contained a 6-histidyl residue carboxyl terminus was highly purified using the metal chelate resin, Ni(2+)-nitriloacetic acid. Whereas the recombinant EDN had ribonuclease activity that was similar to the native protein, the fusion protein had enzymatic activity that was 6-13% that of native EDN. The fusion protein was able to bind to the human transferrin receptor. In contrast to rEDN that had no inherent cytotoxicity to human tumor cells, the EDNsFv fusion protein was cytotoxic to human leukemia cells that express the human transferrin receptor with an IC50, 0.2-1 nM. At 1.3 nM EDNsFv, no cytotoxicity was observed on cells that lack the human transferrin receptor. Free antibody to the human transferrin receptor, E6, inhibited the cytotoxicity of the EDNsFv. Human enzymes may be engineered to acquire cytotoxic properties by fusing them to antibodies. Thus, they may be candidates for the construction of immunofusion proteins that may be less immunogenic than immunotoxins containing bacterial- or plant-derived toxin moieties.
- Published
- 1994
47. RNase inhibition of human immunodeficiency virus infection of H9 cells
- Author
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Rebecca S. Hamilton, Richard J. Youle, Kuslima Shogen, Dianne L. Newton, You-Neng Wu, Stanislaw M. Mikulski, Maneth Gravell, and Giuseppe D'Alessio
- Subjects
Male ,Time Factors ,Protein family ,Cell Survival ,RNase P ,Egg protein ,Antineoplastic Agents ,Virus Replication ,Antiviral Agents ,Cell Line ,Ribonucleases ,Semen ,Tumor Cells, Cultured ,medicine ,Homologous chromosome ,Animals ,Humans ,Ribonuclease ,Leukemia ,Multidisciplinary ,Dose-Response Relationship, Drug ,biology ,Egg Proteins ,Ribonuclease, Pancreatic ,medicine.disease ,Virology ,Kinetics ,Viral replication ,Cell culture ,HIV-1 ,biology.protein ,Cattle ,Research Article - Abstract
Onconase and bovine seminal RNase, two members of the RNase A superfamily, inhibit human immunodeficiency virus type 1 replication in H9 leukemia cells 90-99.9% over a 4-day incubation at concentrations not toxic to uninfected H9 cells. Two other members of the same protein family, bovine pancreatic RNase A and human eosinophil-derived neurotoxin, have no detectable antiviral activity, demonstrating a strikingly selective antiviral activity among homologous ribonucleases. The antiviral RNases do not appear to affect viral particles directly but inhibit replication in host cell cultures. Onconase, already in clinical trials for cancer therapy, and bovine seminal RNase have potential as antiviral therapeutics.
- Published
- 1994
48. Cytotoxic onconase and ribonuclease a chimeras: comparison andin vitrocharacterization
- Author
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Stanislaw M. Mikulski, Al Viera, Richard J. Youle, Dianne L. Newton, and Susanna M. Rybak
- Subjects
chemistry.chemical_classification ,Pharmaceutical Science ,RNA ,Transferrin receptor ,General Medicine ,Biology ,Bovine pancreatic ribonuclease ,Molecular biology ,In vitro ,Biochemistry ,chemistry ,Transferrin ,biology.protein ,Protein biosynthesis ,Pancreatic ribonuclease ,Ribonuclease - Abstract
Onconase, a protein isolated from fertilized Rana pipiens eggs, has antineoplastic properties both in vitro (Z. Darzynkiewicz, S. P. Carter, S. M. Mikulski, W. Ardelt, and K. Shogen, 1988, Cell Tissue Kinet. 21, 169–182) and in vivo (S. M. Mikulski, W. Ardelt, K. Shogen, E. H. Bernstein, and H. Menduke, 1990, J. Natl. Cancer Inst. 82, 151–153). The protein is a member of the pancreatic ribonuclease A superfamily and exhibits ribonuclease activity toward highly polymerized RNA (W. Ardelt, S. M. Mikulski, and K. Shogen, 1991, J. Biol. Chem. 266, 245–251). Previous work has demonstrated that bovine pancreatic ribonuclease A conjugated to transferrin (S. Rybak, S. Saxena, E. Ackerman, and R. Youle, 1991, J. Biol. Chem. 266, 21202–21207) or antibodies to the human transferrin receptor (D. Newton, O. Ilercil, D. Laske, E. Oldfield, S. Rybak, and R. Youle, 1992, J. Biol. Chem. 267, 169–182), acted like a class of reagents called immunotoxins to specifically inhibit protein synthesis in ligand-positive ce...
- Published
- 1993
49. Cytotoxic ribonuclease chimeras. Targeted tumoricidal activity in vitro and in vivo
- Author
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Susanna M. Rybak, Richard J. Youle, Edward H. Oldfield, O Ilercil, Douglas W. Laske, and Dianne L. Newton
- Subjects
chemistry.chemical_classification ,RNase P ,medicine.drug_class ,Transferrin receptor ,Cell Biology ,Biology ,Monoclonal antibody ,Biochemistry ,Molecular biology ,chemistry ,Cell surface receptor ,Transferrin ,biology.protein ,medicine ,Cytotoxic T cell ,Antibody ,Cytotoxicity ,Molecular Biology - Abstract
Monoclonal antibodies to the transferrin receptor or to the T cell antigen, CD5, were chemically linked to mammalian RNase A and found to specifically inhibit protein synthesis in antigen-positive cells. Antibody-mediated specificity of these cytotoxic ribonuclease chimeras (CRCs) was demonstrated in three ways. 1) Toxicity was due to the chemical linkage of RNase to antibody, as the individual components added separately or in combination did not inhibit protein synthesis; 2) the anti-transferrin receptor CRCs inhibited protein synthesis in those cells expressing the human transferrin receptor (K562, U251, Jurkat cells) but had no detectable toxicity to cells lacking the human transferrin receptor (Vero or NIH 3T3 cells); 3) free antibody to either the human transferrin receptor (454A12 or 5E-9) or to the T cell antigen, CD5 (T101), blocked the cytotoxicity of the respective CRC. Two CRC species, designated P1 and P2, that differed in size and stoichiometry of RNase A to antibody, were purified by size-exclusion high performance liquid chromatography. The higher molecular weight P1 conjugate had an IC50 of 20-30 nM, whereas the P2 conjugate had a higher IC50 of 300-500 nM. Bioactivity could be reversibly increased more than 10-fold by freezing. The cytotoxicity of the CRCs was examined in vivo in a solid tumor animal model. Intratumoral injections of an anti-transferrin receptor CRC into established U251 human glioblastoma tumors grown in the flanks of nude mice prevented tumor growth, whereas RNase A mixed with antibody was ineffective. CRCs, therefore, express cytotoxicity in vitro and in vivo. Mammalian nucleases coupled to antibodies may be utilized as cell type-selective cytotoxins and have potential as pharmacologic reagents. The systemic toxicity and immunogenicity observed with mammalian derived cytotoxins may be significantly less than that of the currently employed plant- and bacterial-derived immunotoxins.
- Published
- 1992
50. Artemisinin dimer anticancer activity correlates with heme-catalyzed reactive oxygen species generation and endoplasmic reticulum stress induction
- Author
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Luke H, Stockwin, Bingnan, Han, Sherry X, Yu, Melinda G, Hollingshead, Mahmoud A, ElSohly, Waseem, Gul, Desmond, Slade, Ahmed M, Galal, Dianne L, Newton, and Maja A, Bumke
- Subjects
Cancer Research ,Thapsigargin ,SERCA ,Blotting, Western ,Antineoplastic Agents ,Apoptosis ,Heme ,Biology ,Endoplasmic Reticulum ,Antioxidants ,Article ,Sarcoplasmic Reticulum Calcium-Transporting ATPases ,chemistry.chemical_compound ,Humans ,Enzyme Inhibitors ,Oligonucleotide Array Sequence Analysis ,Endoplasmic reticulum ,Gene Expression Profiling ,Lysine ,Cell Cycle ,Artemisinin Dimer ,Biological activity ,Artemisinins ,Acetylcysteine ,Heme oxygenase ,Oxidative Stress ,Oncology ,chemistry ,Biochemistry ,Artemisia ,Calcium ,Reactive Oxygen Species ,Dimerization ,Biomarkers ,Heme Oxygenase-1 ,Hemin - Abstract
Analogs of the malaria therapeutic, artemisinin, possess in vitro and in vivo anticancer activity. In this study, two dimeric artemisinins (NSC724910 and 735847) were studied to determine their mechanism of action. Dimers were >1,000 fold more active than monomer and treatment was associated with increased reactive oxygen species (ROS) and apoptosis induction. Dimer activity was inhibited by the antioxidant L-NAC, the iron chelator desferroxamine and exogenous hemin. Similarly, induction of heme oxygenase (HMOX) with CoPPIX inhibited activity, whereas inhibition of HMOX with SnPPIX enhanced it. These results emphasize the importance of iron, heme and ROS in activity. Microarray analysis of dimer treated cells identified DNA damage, iron/heme and cysteine/methionine metabolism, antioxidant response, and endoplasmic reticulum (ER) stress as affected pathways. Detection of an ER-stress response was relevant because in malaria, artemisinin inhibits pfATP6, the plasmodium orthologue of mammalian sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPases (SERCA). A comparative study of NSC735847 with thapsigargin, a specific SERCA inhibitor and ER-stress inducer showed similar behavior in terms of transcriptomic changes, induction of endogenous SERCA and ER calcium mobilization. However, thapsigargin had little effect on ROS production, modulated different ER-stress proteins and had greater potency against purified SERCA1. Furthermore, an inactive derivative of NSC735847 that lacked the endoperoxide had identical inhibitory activity against purified SERCA1, suggesting that direct inhibition of SERCA has little inference on overall cytotoxicity. In summary, these data implicate indirect ER-stress induction as a central mechanism of artemisinin dimer activity.
- Published
- 2009
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