31 results on '"Michael C. Nicastri"'
Search Results
2. PPT1 inhibition enhances the antitumor activity of anti–PD-1 antibody in melanoma
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Gaurav Sharma, Rani Ojha, Estela Noguera-Ortega, Vito W. Rebecca, John Attanasio, Shujing Liu, Shengfu Piao, Jennifer J. Lee, Michael C. Nicastri, Sandra L. Harper, Amruta Ronghe, Vaibhav Jain, Jeffrey D. Winkler, David W. Speicher, Jerome Mastio, Phyllis A. Gimotty, Xiaowei Xu, E. John Wherry, Dmitry I. Gabrilovich, and Ravi K. Amaravadi
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Oncology ,Therapeutics ,Medicine - Abstract
New strategies are needed to enhance the efficacy of anti–programmed cell death protein antibody (anti–PD-1 Ab) in cancer. Here, we report that inhibiting palmitoyl-protein thioesterase 1 (PPT1), a target of chloroquine derivatives like hydroxychloroquine (HCQ), enhances the antitumor efficacy of anti–PD-1 Ab in melanoma. The combination resulted in tumor growth impairment and improved survival in mouse models. Genetic suppression of core autophagy genes, but not Ppt1, in cancer cells reduced priming and cytotoxic capacity of primed T cells. Exposure of antigen-primed T cells to macrophage-conditioned medium derived from macrophages treated with PPT1 inhibitors enhanced melanoma-specific killing. Genetic or chemical Ppt1 inhibition resulted in M2 to M1 phenotype switching in macrophages. The combination was associated with a reduction in myeloid-derived suppressor cells in the tumor. Ppt1 inhibition by HCQ, or DC661, induced cyclic GMP-AMP synthase/stimulator of interferon genes/TANK binding kinase 1 pathway activation and the secretion of interferon-β in macrophages, the latter being a key component for augmented T cell–mediated cytotoxicity. Genetic Ppt1 inhibition produced similar findings. These data provide the rationale for this combination in melanoma clinical trials and further investigation in other cancers.
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- 2020
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3. Dimeric quinacrines as chemical tools to identify PPT1, a new regulator of autophagy in cancer cells
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Michael C. Nicastri, Vito W. Rebecca, Ravi K. Amaravadi, and Jeffrey D. Winkler
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ppt1 ,mtor ,autophagy ,lysosome ,macropinocytosis ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
DQ661 is a novel dimeric quinacrine that affects multiple lysosomal functions (autophagy and macropinocytosis) and mTORC1 (mechanistic target of rapamycin) activity by specifically targeting protein-palmitoyl thioesterase 1 (PPT1). DQ661 has in vivo activity in immunocompetent mouse models of cancer, and constitutes a new tool compound for the study of lysosomal function in cancer and therapeutic resistance.
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- 2018
- Full Text
- View/download PDF
4. Chemical Methods from A Unified Approach to Targeting the Lysosome's Degradative and Growth Signaling Roles
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Ravi K. Amaravadi, Jeffrey D. Winkler, David W. Speicher, Donita C. Brady, Roberto Zoncu, Meenhard Herlyn, Xiaowei Xu, Shujing Liu, Qin Liu, Ronen Marmorstein, Joshua Rabinowitz, Yiling Lu, Gordon B. Mills, Maureen E. Murphy, Julie S. Barber-Rotenberg, Zhi Wei, Samuel M. Levi, Rani Ojha, Sengottuvelan Murugan, Shengfu Piao, Gretchen M. Alicea, Gao Zhang, Cynthia I. Chude, Eric Witze, Chun-Yan Lim, Michel Nofal, Amruta Ronghe, Quentin McAfee, Colin Fennelly, Noel McLaughlin, Michael C. Nicastri, and Vito W. Rebecca
- Abstract
Supplemental Chemical Methods
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- 2023
5. Supplemental Figures S1-S7 and Supplemental Table S1 from A Unified Approach to Targeting the Lysosome's Degradative and Growth Signaling Roles
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Ravi K. Amaravadi, Jeffrey D. Winkler, David W. Speicher, Donita C. Brady, Roberto Zoncu, Meenhard Herlyn, Xiaowei Xu, Shujing Liu, Qin Liu, Ronen Marmorstein, Joshua Rabinowitz, Yiling Lu, Gordon B. Mills, Maureen E. Murphy, Julie S. Barber-Rotenberg, Zhi Wei, Samuel M. Levi, Rani Ojha, Sengottuvelan Murugan, Shengfu Piao, Gretchen M. Alicea, Gao Zhang, Cynthia I. Chude, Eric Witze, Chun-Yan Lim, Michel Nofal, Amruta Ronghe, Quentin McAfee, Colin Fennelly, Noel McLaughlin, Michael C. Nicastri, and Vito W. Rebecca
- Abstract
Supplemental Figures and Table
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- 2023
6. Supplementary Figures S1-S4 from PPT1 Promotes Tumor Growth and Is the Molecular Target of Chloroquine Derivatives in Cancer
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Ravi K. Amaravadi, Jeffrey D. Winkler, David W. Speicher, Phyllis A. Gimotty, Ronen Marmorstein, Meenhard Herlyn, Xiaowei Xu, Lynn M. Schuchter, Jennifer J. Lee, Gretchen M. Alicea, Alessandra Martorella, Estela Noguera-Ortega, Shengfu Piao, Rani Ojha, Aaron R. Goldman, Gao Zhang, Noel P. McLaughlin, Quentin McAfee, Amruta Ronghe, Julie S. Barber-Rotenberg, Cynthia I. Chude, Colin Fennelly, Michael C. Nicastri, and Vito W. Rebecca
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Supplementary Figures S1-S4
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- 2023
7. Chemical Methods from PPT1 Promotes Tumor Growth and Is the Molecular Target of Chloroquine Derivatives in Cancer
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Ravi K. Amaravadi, Jeffrey D. Winkler, David W. Speicher, Phyllis A. Gimotty, Ronen Marmorstein, Meenhard Herlyn, Xiaowei Xu, Lynn M. Schuchter, Jennifer J. Lee, Gretchen M. Alicea, Alessandra Martorella, Estela Noguera-Ortega, Shengfu Piao, Rani Ojha, Aaron R. Goldman, Gao Zhang, Noel P. McLaughlin, Quentin McAfee, Amruta Ronghe, Julie S. Barber-Rotenberg, Cynthia I. Chude, Colin Fennelly, Michael C. Nicastri, and Vito W. Rebecca
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Chemical Methods
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- 2023
8. Data from PPT1 Promotes Tumor Growth and Is the Molecular Target of Chloroquine Derivatives in Cancer
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Ravi K. Amaravadi, Jeffrey D. Winkler, David W. Speicher, Phyllis A. Gimotty, Ronen Marmorstein, Meenhard Herlyn, Xiaowei Xu, Lynn M. Schuchter, Jennifer J. Lee, Gretchen M. Alicea, Alessandra Martorella, Estela Noguera-Ortega, Shengfu Piao, Rani Ojha, Aaron R. Goldman, Gao Zhang, Noel P. McLaughlin, Quentin McAfee, Amruta Ronghe, Julie S. Barber-Rotenberg, Cynthia I. Chude, Colin Fennelly, Michael C. Nicastri, and Vito W. Rebecca
- Abstract
Clinical trials repurposing lysosomotropic chloroquine (CQ) derivatives as autophagy inhibitors in cancer demonstrate encouraging results, but the underlying mechanism of action remains unknown. Here, we report a novel dimeric CQ (DC661) capable of deacidifying the lysosome and inhibiting autophagy significantly better than hydroxychloroquine (HCQ). Using an in situ photoaffinity pulldown strategy, we identified palmitoyl-protein thioesterase 1 (PPT1) as a molecular target shared across monomeric and dimeric CQ derivatives. HCQ and Lys05 also bound to and inhibited PPT1 activity, but only DC661 maintained activity in acidic media. Knockout of PPT1 in cancer cells using CRISPR/Cas9 editing abrogates autophagy modulation and cytotoxicity of CQ derivatives, and results in significant impairment of tumor growth similar to that observed with DC661. Elevated expression of PPT1 in tumors correlates with poor survival in patients in a variety of cancers. Thus, PPT1 represents a new target in cancer that can be inhibited with CQ derivatives.Significance:This study identifies PPT1 as the previously unknown lysosomal molecular target of monomeric and dimeric CQ derivatives. Genetic suppression of PPT1 impairs tumor growth, and PPT1 levels are elevated in cancer and associated with poor survival. These findings provide a strong rationale for targeting PPT1 in cancer.This article is highlighted in the In This Issue feature, p. 151
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- 2023
9. Figure S1-4, TABLES1, S2 from ATG5 Mediates a Positive Feedback Loop between Wnt Signaling and Autophagy in Melanoma
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Ashani T. Weeraratna, Maureen E. Murphy, Ravi K. Amaravadi, Maria S. Soengas, Jeffrey D. Winkler, Michael C. Nicastri, Meenhard Herlyn, Hong Wu, Xiaowei Xu, Gordon B. Mills, Michael A. Davies, Y.N. Vashisht Gopal, Qin Liu, Patricia A. Brafford, Ling Li, Vito W. Rebecca, Reeti Behera, Amanpreet Kaur, Marie R. Webster, Curtis H. Kugel, Anna Budina-Kolomets, and Abibatou Ndoye
- Abstract
Figure S1: Wnt5A expression correlates with high autophagy in melanoma. Figure S2: Wnt5A increases autophagy in melanoma Figure S3: ATG5 affects Wnt signaling in melanoma Figure S4: β-catenin increases the sensitivity of melanoma cells to autophagy inhibition Supplemental Figure Legends SUPPLEMENTAL TABLE 1: DENSITOMETRY FOR ALL WESTERNS Supplemental Table 2. Tissue microarrays (TMA) H scores
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- 2023
10. Data from ATG5 Mediates a Positive Feedback Loop between Wnt Signaling and Autophagy in Melanoma
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Ashani T. Weeraratna, Maureen E. Murphy, Ravi K. Amaravadi, Maria S. Soengas, Jeffrey D. Winkler, Michael C. Nicastri, Meenhard Herlyn, Hong Wu, Xiaowei Xu, Gordon B. Mills, Michael A. Davies, Y.N. Vashisht Gopal, Qin Liu, Patricia A. Brafford, Ling Li, Vito W. Rebecca, Reeti Behera, Amanpreet Kaur, Marie R. Webster, Curtis H. Kugel, Anna Budina-Kolomets, and Abibatou Ndoye
- Abstract
Autophagy mediates resistance to various anticancer agents. In melanoma, resistance to targeted therapy has been linked to expression of Wnt5A, an intrinsic inhibitor of β-catenin, which also promotes invasion. In this study, we assessed the interplay between Wnt5A and autophagy by combining expression studies in human clinical biopsies with functional analyses in cell lines and mouse models. Melanoma cells with high Wnt5A and low β-catenin displayed increased basal autophagy. Genetic blockade of autophagy revealed an unexpected feedback loop whereby knocking down the autophagy factor ATG5 in Wnt5Ahigh cells decreased Wnt5A and increased β-catenin. To define the physiologic relevance of this loop, melanoma cells with different Wnt status were treated in vitro and in vivo with the potent lysosomotropic compound Lys05. Wnt5Ahigh cells were less sensitive to Lys05 and could be reverted by inducing β-catenin activity. Our results suggest the efficacy of autophagy inhibitors might be improved by taking the Wnt signature of melanoma cells into account. Cancer Res; 77(21); 5873–85. ©2017 AACR.
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- 2023
11. SIRT1 is downregulated by autophagy in senescence and ageing
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Parinaz Fozouni, Vemika Chandra, Congcong Lu, Lu Wang, Jing Jiang, Caiyue Xu, Corey Bretz, Melanie Ott, Terje Johansen, Peter D. Adams, Jeffrey D. Winkler, Ravi K. Amaravadi, Shelley L. Berger, Gry Evjen, Michael C. Nicastri, Zhixun Dou, Wei Tong, and Benjamin A. Garcia
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Male ,Senescence ,Cell Survival ,T-Lymphocytes ,Inbred C57BL ,Medical and Health Sciences ,Article ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,Sirtuin 1 ,Downregulation and upregulation ,Autophagy ,Animals ,Humans ,Progenitor cell ,Cellular Senescence ,Cell Proliferation ,030304 developmental biology ,Cell Nucleus ,0303 health sciences ,Chemistry ,Stem Cells ,Autophagosomes ,Cell Biology ,Middle Aged ,Biological Sciences ,Cell biology ,Mice, Inbred C57BL ,enzymes and coenzymes (carbohydrates) ,Haematopoiesis ,Ageing ,030220 oncology & carcinogenesis ,Female ,NAD+ kinase ,biological phenomena, cell phenomena, and immunity ,Lysosomes ,Microtubule-Associated Proteins ,hormones, hormone substitutes, and hormone antagonists ,Developmental Biology - Abstract
SIRT1 (Sir2) is an NAD+-dependent deacetylase that plays critical roles in a broad range of biological events, including metabolism, the immune response and ageing1,2,3,4,5. Although there is strong interest in stimulating SIRT1 catalytic activity, the homeostasis of SIRT1 at the protein level is poorly understood. Here we report that macroautophagy (hereafter referred to as autophagy), a catabolic membrane trafficking pathway that degrades cellular components through autophagosomes and lysosomes, mediates the downregulation of mammalian SIRT1 protein during senescence and in vivo ageing. In senescence, nuclear SIRT1 is recognized as an autophagy substrate and is subjected to cytoplasmic autophagosome–lysosome degradation, via the autophagy protein LC3. Importantly, the autophagy–lysosome pathway contributes to the loss of SIRT1 during ageing of several tissues related to the immune and haematopoietic system in mice, including the spleen, thymus, and haematopoietic stem and progenitor cells, as well as in CD8+CD28- T cells from aged human donors. Our study reveals a mechanism in the regulation of the protein homeostasis of SIRT1 and suggests a potential strategy to stabilize SIRT1 to promote productive ageing.
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- 2020
12. PPT1 Promotes Tumor Growth and Is the Molecular Target of Chloroquine Derivatives in Cancer
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Noel P. McLaughlin, Ronen Marmorstein, Julie S. Barber-Rotenberg, Aaron R. Goldman, Estela Noguera-Ortega, Gao Zhang, Michael C. Nicastri, Cynthia I. Chude, Quentin McAfee, David W. Speicher, Shengfu Piao, Vito W. Rebecca, Jeffrey D. Winkler, Rani Ojha, Lynn M. Schuchter, Alessandra Martorella, Phyllis A. Gimotty, Meenhard Herlyn, Xiaowei Xu, Gretchen M. Alicea, Jennifer J. Lee, Colin Fennelly, Amruta Ronghe, and Ravi K. Amaravadi
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0301 basic medicine ,Apoptosis ,Article ,Antimalarials ,03 medical and health sciences ,0302 clinical medicine ,Chloroquine ,Neoplasms ,Lysosome ,Biomarkers, Tumor ,Polyamines ,Tumor Cells, Cultured ,medicine ,Humans ,Cytotoxicity ,Cell Proliferation ,Cell growth ,Chemistry ,Autophagy ,Membrane Proteins ,Cancer ,Prognosis ,medicine.disease ,Xenograft Model Antitumor Assays ,Survival Rate ,030104 developmental biology ,medicine.anatomical_structure ,Oncology ,Mechanism of action ,030220 oncology & carcinogenesis ,Cancer cell ,Aminoquinolines ,Cancer research ,Thiolester Hydrolases ,medicine.symptom ,medicine.drug - Abstract
Clinical trials repurposing lysosomotropic chloroquine (CQ) derivatives as autophagy inhibitors in cancer demonstrate encouraging results, but the underlying mechanism of action remains unknown. Here, we report a novel dimeric CQ (DC661) capable of deacidifying the lysosome and inhibiting autophagy significantly better than hydroxychloroquine (HCQ). Using an in situ photoaffinity pulldown strategy, we identified palmitoyl-protein thioesterase 1 (PPT1) as a molecular target shared across monomeric and dimeric CQ derivatives. HCQ and Lys05 also bound to and inhibited PPT1 activity, but only DC661 maintained activity in acidic media. Knockout of PPT1 in cancer cells using CRISPR/Cas9 editing abrogates autophagy modulation and cytotoxicity of CQ derivatives, and results in significant impairment of tumor growth similar to that observed with DC661. Elevated expression of PPT1 in tumors correlates with poor survival in patients in a variety of cancers. Thus, PPT1 represents a new target in cancer that can be inhibited with CQ derivatives. Significance: This study identifies PPT1 as the previously unknown lysosomal molecular target of monomeric and dimeric CQ derivatives. Genetic suppression of PPT1 impairs tumor growth, and PPT1 levels are elevated in cancer and associated with poor survival. These findings provide a strong rationale for targeting PPT1 in cancer. This article is highlighted in the In This Issue feature, p. 151
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- 2019
13. Synthesis of Sterically Hindered Primary Amines by Concurrent Tandem Photoredox Catalysis
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Tomislav Rovis, Daniel A. DiRocco, Dan Lehnherr, Michael C. Nicastri, and Yu-hong Lam
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Steric effects ,Active ingredient ,Primary (chemistry) ,Tandem ,Chemistry ,Ligand ,Photoredox catalysis ,General Chemistry ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Combinatorial chemistry ,Catalysis ,0104 chemical sciences ,Colloid and Surface Chemistry ,Structural motif - Abstract
Primary amines are an important structural motif in active pharmaceutical ingredients (APIs) and intermediates thereof, as well as members of ligand libraries for either biological or catalytic applications. Many chemical methodologies exist for amine synthesis, but the direct synthesis of primary amines with a fully substituted α carbon center is an underdeveloped area. We report a method which utilizes photoredox catalysis to couple readily available
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- 2020
14. Targeting quiescent leukemic stem cells using second generation autophagy inhibitors
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Orianne Olivares, Arunima Mukhopadhyay, Elodie M. Kuntz, Tessa L. Holyoake, Leon Murphy, Ravi K. Amaravadi, Eyal Gottlieb, Mary T. Scott, Amy Dawson, Jeffrey D. Winkler, Erin P. Keaney, Karen Dunn, Rebecca Mitchell, G. Vignir Helgason, Pablo Baquero, Michael C. Nicastri, Kevin M. Ryan, Christina Halsey, Angela Ianniciello, and Alison M. Michie
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0301 basic medicine ,Cancer Research ,Myeloid ,medicine.drug_class ,Fusion Proteins, bcr-abl ,Apoptosis ,Article ,Tyrosine-kinase inhibitor ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Leukemia, Myelogenous, Chronic, BCR-ABL Positive ,hemic and lymphatic diseases ,Autophagy ,Polyamines ,Tumor Cells, Cultured ,medicine ,Animals ,Humans ,Protein Kinase Inhibitors ,Cell Proliferation ,business.industry ,Cell growth ,Myeloid leukemia ,Hematology ,medicine.disease ,3. Good health ,Mice, Inbred C57BL ,Leukemia ,Haematopoiesis ,030104 developmental biology ,medicine.anatomical_structure ,Oncology ,Drug Resistance, Neoplasm ,030220 oncology & carcinogenesis ,Aminoquinolines ,Neoplastic Stem Cells ,Cancer research ,Stem cell ,business - Abstract
In chronic myeloid leukemia (CML), tyrosine kinase inhibitor (TKI) treatment induces autophagy that promotes survival and TKI-resistance in leukemic stem cells (LSCs). In clinical studies hydroxychloroquine (HCQ), the only clinically approved autophagy inhibitor, does not consistently inhibit autophagy in cancer patients, so more potent autophagy inhibitors are needed. We generated a murine model of CML in which autophagic flux can be measured in bone marrow-located LSCs. In parallel, we use cell division tracing, phenotyping of primary CML cells, and a robust xenotransplantation model of human CML, to investigate the effect of Lys05, a highly potent lysosomotropic agent, and PIK-III, a selective inhibitor of VPS34, on the survival and function of LSCs. We demonstrate that long-term haematopoietic stem cells (LT-HSCs: Lin-Sca-1+c-kit+CD48-CD150+) isolated from leukemic mice have higher basal autophagy levels compared with non-leukemic LT-HSCs and more mature leukemic cells. Additionally, we present that while HCQ is ineffective, Lys05-mediated autophagy inhibition reduces LSCs quiescence and drives myeloid cell expansion. Furthermore, Lys05 and PIK-III reduced the number of primary CML LSCs and target xenografted LSCs when used in combination with TKI treatment, providing a strong rationale for clinical use of second generation autophagy inhibitors as a novel treatment for CML patients with LSC persistence.
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- 2018
15. Electrochemical Synthesis of Hindered Primary and Secondary Amines via Proton-Coupled Electron Transfer
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Erik L. Regalado, Jinchu Liu, Tomislav Rovis, Daniel A. DiRocco, Michael C. Nicastri, Justin A. Newman, Yu-hong Lam, and Dan Lehnherr
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chemistry.chemical_classification ,Trifluoromethyl ,Pyrazine ,Iminium ,Alkyne ,Ether ,General Chemistry ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Combinatorial chemistry ,Catalysis ,0104 chemical sciences ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,chemistry ,Amide ,Pyridine ,Proton-coupled electron transfer - Abstract
Accessing hindered amines, particularly primary amines α to a fully substituted carbon center, is synthetically challenging. We report an electrochemical method to access such hindered amines starting from benchtop-stable iminium salts and cyanoheteroarenes. A wide variety of substituted heterocycles (pyridine, pyrimidine, pyrazine, purine, azaindole) can be utilized in the cross-coupling reaction, including those substituted with a halide, trifluoromethyl, ester, amide, or ether group, a heterocycle, or an unprotected alcohol or alkyne. Mechanistic insight based on DFT data, as well as cyclic voltammetry and NMR spectroscopy, suggests that a proton-coupled electron-transfer mechanism is operational as part of a hetero-biradical cross-coupling of α-amino radicals and radicals derived from cyanoheteroarenes.
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- 2019
16. A Unified Approach to Targeting the Lysosome's Degradative and Growth Signaling Roles
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Zhi Wei, Gao Zhang, Noel P. McLaughlin, Joshua D. Rabinowitz, Xiaowei Xu, David W. Speicher, Amruta Ronghe, Jeffrey D. Winkler, Gordon B. Mills, Michael C. Nicastri, Quentin McAfee, Ravi K. Amaravadi, Julie S. Barber-Rotenberg, Sengottuvelan Murugan, Qin Liu, Cynthia I. Chude, Donita C. Brady, Meenhard Herlyn, Michel Nofal, Roberto Zoncu, Yiling Lu, Rani Ojha, Gretchen M. Alicea, Vito W. Rebecca, Shengfu Piao, Ronen Marmorstein, Chun Yan Lim, Eric S. Witze, Maureen E. Murphy, Samuel M. Levi, Colin Fennelly, and Shujing Liu
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0301 basic medicine ,Catabolism ,Pinocytosis ,Autophagy ,Cancer ,PPT1 ,mTORC1 ,Biology ,medicine.disease ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,medicine.anatomical_structure ,Oncology ,Lysosome ,medicine ,PI3K/AKT/mTOR pathway - Abstract
Lysosomes serve dual roles in cancer metabolism, executing catabolic programs (i.e., autophagy and macropinocytosis) while promoting mTORC1-dependent anabolism. Antimalarial compounds such as chloroquine or quinacrine have been used as lysosomal inhibitors, but fail to inhibit mTOR signaling. Further, the molecular target of these agents has not been identified. We report a screen of novel dimeric antimalarials that identifies dimeric quinacrines (DQ) as potent anticancer compounds, which concurrently inhibit mTOR and autophagy. Central nitrogen methylation of the DQ linker enhances lysosomal localization and potency. An in situ photoaffinity pulldown identified palmitoyl-protein thioesterase 1 (PPT1) as the molecular target of DQ661. PPT1 inhibition concurrently impairs mTOR and lysosomal catabolism through the rapid accumulation of palmitoylated proteins. DQ661 inhibits the in vivo tumor growth of melanoma, pancreatic cancer, and colorectal cancer mouse models and can be safely combined with chemotherapy. Thus, lysosome-directed PPT1 inhibitors represent a new approach to concurrently targeting mTORC1 and lysosomal catabolism in cancer. Significance: This study identifies chemical features of dimeric compounds that increase their lysosomal specificity, and a new molecular target for these compounds, reclassifying these compounds as targeted therapies. Targeting PPT1 blocks mTOR signaling in a manner distinct from catalytic inhibitors, while concurrently inhibiting autophagy, thereby providing a new strategy for cancer therapy. Cancer Discov; 7(11); 1266–83. ©2017 AACR. See related commentary by Towers and Thorburn, p. 1218. This article is highlighted in the In This Issue feature, p. 1201
- Published
- 2017
17. PPT1 inhibition enhances the antitumor activity of anti-PD-1 antibody in melanoma
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David W. Speicher, Jeffrey D. Winkler, Rani Ojha, Dmitry I. Gabrilovich, Michael C. Nicastri, Vaibhav Jain, Shengfu Piao, Shujing Liu, John Attanasio, Xiaowei Xu, Phyllis A. Gimotty, Ravi K. Amaravadi, Estela Noguera-Ortega, Jennifer J. Lee, Amruta Ronghe, Vito W. Rebecca, Jérôme Mastio, E. John Wherry, Gaurav Sharma, and Sandra L. Harper
- Subjects
0301 basic medicine ,Programmed cell death ,T-Lymphocytes ,Programmed Cell Death 1 Receptor ,Priming (immunology) ,Therapeutics ,Antibodies ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Antineoplastic Combined Chemotherapy Protocols ,medicine ,Autophagy ,Tumor Cells, Cultured ,Cytotoxic T cell ,Humans ,Animals ,Enzyme Inhibitors ,Cytotoxicity ,Immune Checkpoint Inhibitors ,Melanoma ,Chemistry ,Macrophages ,Membrane Proteins ,General Medicine ,Interferon-beta ,medicine.disease ,Nucleotidyltransferases ,Mice, Inbred C57BL ,030104 developmental biology ,RAW 264.7 Cells ,Oncology ,030220 oncology & carcinogenesis ,Stimulator of interferon genes ,Cancer cell ,Cancer research ,Medicine ,Thiolester Hydrolases ,Lysosomes ,Research Article ,Hydroxychloroquine - Abstract
New strategies are needed to enhance the efficacy of anti–programmed cell death protein antibody (anti–PD-1 Ab) in cancer. Here, we report that inhibiting palmitoyl-protein thioesterase 1 (PPT1), a target of chloroquine derivatives like hydroxychloroquine (HCQ), enhances the antitumor efficacy of anti–PD-1 Ab in melanoma. The combination resulted in tumor growth impairment and improved survival in mouse models. Genetic suppression of core autophagy genes, but not Ppt1, in cancer cells reduced priming and cytotoxic capacity of primed T cells. Exposure of antigen-primed T cells to macrophage-conditioned medium derived from macrophages treated with PPT1 inhibitors enhanced melanoma-specific killing. Genetic or chemical Ppt1 inhibition resulted in M2 to M1 phenotype switching in macrophages. The combination was associated with a reduction in myeloid-derived suppressor cells in the tumor. Ppt1 inhibition by HCQ, or DC661, induced cyclic GMP-AMP synthase/stimulator of interferon genes/TANK binding kinase 1 pathway activation and the secretion of interferon-β in macrophages, the latter being a key component for augmented T cell–mediated cytotoxicity. Genetic Ppt1 inhibition produced similar findings. These data provide the rationale for this combination in melanoma clinical trials and further investigation in other cancers., Inhibiting palmitoyl-protein thioesterase 1 (PPT1), a target of CQ derivatives like hydroxychloroquine (HCQ), enhances the antitumor efficacy of anti-PD-1 Ab in murine melanoma models.
- Published
- 2019
18. ALDH1A1 and HLTF modulate the activity of lysosomal autophagy inhibitors in cancer cells
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Michael C. Nicastri, Phyllis A. Gimotty, Jeffrey D. Winkler, Shengfu Piao, Meghan Buckley, Arabinda Samanta, Xiao Hong Ma, Rani Ojha, Quentin McAfee, Eric J. Brown, Vito W. Rebecca, and Ravi K. Amaravadi
- Subjects
0301 basic medicine ,Colorectal cancer ,DNA damage ,Mice, Nude ,Biology ,Aldehyde Dehydrogenase 1 Family ,Epigenesis, Genetic ,03 medical and health sciences ,Helicase-Like Transcription Factor ,Lysosome ,Cell Line, Tumor ,medicine ,Autophagy ,Animals ,Humans ,HLTF ,Molecular Biology ,Reproducibility of Results ,Retinal Dehydrogenase ,Chloroquine ,Cell Biology ,Aldehyde Dehydrogenase ,medicine.disease ,Molecular biology ,ALDH1A1 ,DNA-Binding Proteins ,Gene Expression Regulation, Neoplastic ,030104 developmental biology ,medicine.anatomical_structure ,Drug Resistance, Neoplasm ,Research Paper-Basic Science ,Cancer cell ,Cancer research ,biology.protein ,Lysosomes ,Reactive Oxygen Species ,DNA Damage ,Hydroxychloroquine ,Transcription Factors - Abstract
Lysosomal autophagy inhibitors (LAI) such as hydroxychloroquine (HCQ) have significant activity in a subset of cancer cell lines. LAIs are being evaluated in cancer clinical trials, but genetic determinants of sensitivity to LAIs are unknown, making it difficult to predict which tumors would be most susceptible. Here we characterize differentially expressed genes in HCQ-sensitive (-S) and -resistant (-R) cancer cells. Notably, expression of canonical macroautophagy/autophagy genes was not associated with sensitivity to HCQ. Expression patterns of ALDH1A1 (aldehyde dehydrogenase 1 family member A1) and HLTF (helicase like transcription factor) identified HCQ-S (ALDH1A1high HLTFlow; ALDH1A1low HLTFlow) and HCQ-R (ALDH1A1low HLTFhigh) cells. ALDH1A1 overexpression was found to enhance LAI cell entry and cytotoxicity without directly affecting lysosome function or autophagic flux. Expression of HLTF allows repair of DNA damage caused by LAI-induced reactive oxygen species, leading to HCQ resistance. Sensitivity to HCQ is increased in cells where HLTF is silenced by promoter methylation. HLTF overexpression blunted the antitumor efficacy of chloroquine derivatives in vitro and in vivo. Analysis of tumor RNA sequencing data from >700 patients in the Cancer Genome Atlas identified cancers including colon cancer, renal cell carcinoma, and gastric cancers, that were enriched for the HCQ-S or HCQ-R signature. These results provide mechanistic insights into LAI efficacy, and guidance for LAI clinical development.
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- 2017
19. ATG5 mediates a positive feedback loop between Wnt signaling and autophagy in melanoma
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Hong Wu, Y.N. Vashisht Gopal, Curtis H. Kugel, Qin Liu, Reeti Behera, Xiaowei Xu, Maureen E. Murphy, Ashani T. Weeraratna, Michael A. Davies, Gordon B. Mills, Amanpreet Kaur, Ravi K. Amaravadi, Vito W. Rebecca, Maria S. Soengas, Jeffrey D. Winkle, Meenhard Herlyn, Patricia Brafford, Ling Li, Anna Budina-Kolomets, Marie R. Webster, Michael C. Nicastri, and Abibatou Ndoye
- Subjects
0301 basic medicine ,Cancer Research ,Beta-catenin ,medicine.medical_treatment ,ATG5 ,Blotting, Western ,Article ,Wnt-5a Protein ,Targeted therapy ,Autophagy-Related Protein 5 ,03 medical and health sciences ,Mice ,Cell Line, Tumor ,medicine ,Autophagy ,Polyamines ,Animals ,Humans ,Melanoma ,Wnt Signaling Pathway ,beta Catenin ,Feedback, Physiological ,biology ,Reverse Transcriptase Polymerase Chain Reaction ,Wnt signaling pathway ,medicine.disease ,Cell biology ,WNT5A ,Gene Expression Regulation, Neoplastic ,030104 developmental biology ,Oncology ,Cell culture ,biology.protein ,Aminoquinolines ,RNA Interference - Abstract
Autophagy mediates resistance to various anticancer agents. In melanoma, resistance to targeted therapy has been linked to expression of Wnt5A, an intrinsic inhibitor of β-catenin, which also promotes invasion. In this study, we assessed the interplay between Wnt5A and autophagy by combining expression studies in human clinical biopsies with functional analyses in cell lines and mouse models. Melanoma cells with high Wnt5A and low β-catenin displayed increased basal autophagy. Genetic blockade of autophagy revealed an unexpected feedback loop whereby knocking down the autophagy factor ATG5 in Wnt5Ahigh cells decreased Wnt5A and increased β-catenin. To define the physiologic relevance of this loop, melanoma cells with different Wnt status were treated in vitro and in vivo with the potent lysosomotropic compound Lys05. Wnt5Ahigh cells were less sensitive to Lys05 and could be reverted by inducing β-catenin activity. Our results suggest the efficacy of autophagy inhibitors might be improved by taking the Wnt signature of melanoma cells into account. Cancer Res; 77(21); 5873–85. ©2017 AACR.
- Published
- 2017
20. Internal Disulfide Bond Acts as a Switch for Intein Activity
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Lingyun Li, Jian Xie, Kristina Xega, Robert J. Linhardt, Julie N. Reitter, Kenneth V. Mills, Chunyu Wang, and Michael C. Nicastri
- Subjects
Protein subunit ,DNA polymerase II ,Cell ,food and beverages ,DNA Polymerase II ,Protein tag ,Biology ,Biochemistry ,Article ,Inteins ,medicine.anatomical_structure ,Tandem Mass Spectrometry ,Protein splicing ,Exteins ,biology.protein ,medicine ,Protein Splicing ,Electrophoresis, Polyacrylamide Gel ,Cysteine ,Disulfides ,Intein ,Oxidation-Reduction ,Polyacrylamide gel electrophoresis - Abstract
Inteins are intervening polypeptides that catalyze their own removal from flanking exteins, concomitant to the ligation of the exteins. The intein that interrupts the DP2 (large) subunit of DNA polymerase II from Methanoculleus marisnigri (Mma) can promote protein splicing. However, protein splicing can be prevented or reduced by overexpression under nonreducing conditions because of the formation of a disulfide bond between two internal intein Cys residues. This redox sensitivity leads to differential activity in different strains of E. coli as well as in different cell compartments. The redox-dependent control of in vivo protein splicing in an intein derived from an anaerobe that can occupy multiple environments hints at a possible physiological role for protein splicing.
- Published
- 2013
21. An Investigation of Salt‐Dependent Protein Splicing
- Author
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Mario S. Jaramillo, Kenneth V. Mills, Julie N. Reitter, Michael C. Nicastri, and Christopher E. Cousin
- Subjects
chemistry.chemical_classification ,Biochemistry ,Chemistry ,Protein splicing ,Genetics ,Salt (chemistry) ,Molecular Biology ,Biotechnology - Published
- 2016
22. Salt-Dependent Conditional Protein Splicing of an Intein from Halobacterium salinarum
- Author
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Kenneth V. Mills, Mario V. Jaramillo, Michael C. Nicastri, Julie N. Reitter, and Christopher E. Cousin
- Subjects
0301 basic medicine ,Halobacterium salinarum ,biology ,Disulfide bond ,Active site ,Salt Tolerance ,Cleavage (embryo) ,medicine.disease_cause ,biology.organism_classification ,Biochemistry ,In vitro ,Inteins ,03 medical and health sciences ,030104 developmental biology ,Bacterial Proteins ,Protein splicing ,biology.protein ,medicine ,Protein Splicing ,Intein ,Escherichia coli - Abstract
An intein from Halobacterium salinarum can be isolated as an unspliced precursor protein with exogenous exteins after Escherichia coli overexpression. The intein promotes protein splicing and uncoupled N-terminal cleavage in vitro, conditional on incubation with NaCl or KCl at concentrations of >1.5 M. The protein splicing reaction also is conditional on reduction of a disulfide bond between two active site cysteines. Conditional protein splicing under these relatively mild conditions may lead to advances in intein-based biotechnology applications and hints at the possibility that this H. salinarum intein could serve as a switch to control extein activity under physiologically relevant conditions.
- Published
- 2016
23. Dimeric quinacrines as chemical tools to identify PPT1, a new regulator of autophagy in cancer cells
- Author
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Jeffrey D. Winkler, Vito W. Rebecca, Ravi K. Amaravadi, and Michael C. Nicastri
- Subjects
0301 basic medicine ,autophagy ,Cancer Research ,macropinocytosis ,Regulator ,mTORC1 ,PPT1 ,03 medical and health sciences ,Lysosome ,medicine ,Mechanistic target of rapamycin ,biology ,Chemistry ,Autophagy ,Cancer ,medicine.disease ,3. Good health ,Cell biology ,Author's Views ,030104 developmental biology ,medicine.anatomical_structure ,Cancer cell ,mTOR ,lysosome ,biology.protein ,Molecular Medicine - Abstract
DQ661 is a novel dimeric quinacrine that affects multiple lysosomal functions (autophagy and macropinocytosis) and mTORC1 (mechanistic target of rapamycin) activity by specifically targeting protein-palmitoyl thioesterase 1 (PPT1). DQ661 has in vivo activity in immunocompetent mouse models of cancer, and constitutes a new tool compound for the study of lysosomal function in cancer and therapeutic resistance.
- Published
- 2017
24. Protein splicing of an intein from the extreme halophile Halobacterium salinarum (768.9)
- Author
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Michael C. Nicastri, Kenneth V. Mills, Julie N. Reitter, and Mario V. Jaramillo
- Subjects
biology ,Biochemistry ,Chemistry ,Protein splicing ,Genetics ,Halobacterium salinarum ,biology.organism_classification ,Intein ,Molecular Biology ,Halophile ,Biotechnology - Published
- 2014
25. The stops and starts of protein splicing: regulation and mechanism of non‐canonical inteins (584.1)
- Author
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Julie N. Reitter, Kenneth V. Mills, Jennie E. Williams, Michael C. Nicastri, and Kathryn M. Colelli
- Subjects
Genetics ,Non canonical ,Protein splicing ,Mechanism (biology) ,Computational biology ,Biology ,Molecular Biology ,Biochemistry ,Biotechnology - Published
- 2014
26. Conditional protein splicing via disulfide bond formation
- Author
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Kristina Xega, Michael C. Nicastri, Kenneth V. Mills, and Julie N. Reitter
- Subjects
Chemistry ,Stereochemistry ,Protein splicing ,Genetics ,Disulfide bond ,Molecular Biology ,Biochemistry ,Biotechnology - Published
- 2013
27. The structure, regulation and activity of non‐canonical inteins
- Author
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Kathryn M. Colelli, Jennie E. Williams, Michelle D. Marieni, Katherine R. Connor, Kenneth V. Mills, Julie N. Reitter, and Michael C. Nicastri
- Subjects
Physics ,Pure mathematics ,Non canonical ,Genetics ,Structure (category theory) ,Molecular Biology ,Biochemistry ,Biotechnology - Published
- 2013
28. Abstract 1018: Structural features of novel dimeric quinacrines that have single-agent antitumor activity determine the mechanism of action: destabilization of mTORC1/lysosomal interaction versus DNA damage
- Author
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Sengottuvelan Murugan, Shengfu Piao, Gao Zhang, Colin Fennelly, Jeffrey D. Winkler, Meenhard Herlyn, Michael C. Nicastri, Gretchen M. Alicea, Vito W. Rebecca, Noel McGlaughlin, Zhi Wei, Yiling Lu, Quentin McAfee, Gordon B. Mills, and Ravi K. Amaravadi
- Subjects
Cancer Research ,DNA damage ,Chemistry ,Cancer ,mTORC1 ,medicine.disease ,Molecular biology ,medicine.anatomical_structure ,Oncology ,Biochemistry ,Mechanism of action ,Lysosome ,medicine ,Viability assay ,medicine.symptom ,IC50 ,Linker - Abstract
The safety and preliminary activity of hydroxychloroquine in phase I cancer clinical trials have established the feasibility and rationale for targeting the lysosome in cancer. We previously reported a more potent lysosomal inhibitor Lys05, which is a dimeric chloroquine (CQ) linked with a triamine linker. Here we report that high throughput screening of >100 Lys05 derivatives (72-hour viability assay) revealed extending linker length between the CQ motifs markedly enhanced anti-proliferative potency (IC50 Citation Format: Vito W. Rebecca, Michael Nicastri, Noel McGlaughlin, Quentin McAfee, Gao Zhang, Gretchen M. Alicea, Shengfu Piao, Colin Fennelly, Sengottuvelan Murugan, Zhi Wei, Gordon B. Mills, Yiling Lu, Meenhard Herlyn, Jeffrey D. Winkler, Ravi K. Amaravadi. Structural features of novel dimeric quinacrines that have single-agent antitumor activity determine the mechanism of action: destabilization of mTORC1/lysosomal interaction versus DNA damage. [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 1018.
- Published
- 2016
29. Conditional protein splicing of inteins with a non‐canonical C‐terminal glutamine
- Author
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Kristina Xega, Kenneth V. Mills, Michael C. Nicastri, and Julie N. Reitter
- Subjects
Glutamine ,Non canonical ,Terminal (electronics) ,Protein splicing ,Chemistry ,Genetics ,Molecular Biology ,Biochemistry ,Biotechnology ,Cell biology - Published
- 2012
30. Non‐canonical inteins: Protein splicing by alternate mechanisms
- Author
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Kathryn M. Colelli, George Savidis, Julie N. Reitter, Michelle D. Marieni, Michael C. Nicastri, Jennie E. Williams, Laura M. Urbanski, Katherine R. Connor, and Kenneth V. Mills
- Subjects
Non canonical ,Chemistry ,Protein splicing ,Genetics ,Computational biology ,Molecular Biology ,Biochemistry ,Biotechnology - Published
- 2012
31. Protein Splicing: Regulation by Temperature and Oxidation State of Non-Canonical Inteins
- Author
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Julie N. Reitter, Kathryn Colleli, Jennie E. Williams, Kenneth V. Mills, and Michael C. Nicastri
- Subjects
0303 health sciences ,Chemistry ,Stereochemistry ,Thermophile ,Kinetics ,Biophysics ,010402 general chemistry ,01 natural sciences ,Solution structure ,0104 chemical sciences ,03 medical and health sciences ,Non canonical ,Oxidation state ,Protein splicing ,parasitic diseases ,RNA splicing ,Intein ,030304 developmental biology - Abstract
Protein splicing is the post-translational process by which an intein, or intervening protein, catalyzes its own excision from the flanking polypeptides, or exteins, concomitant with extein ligation. We have shown that protein splicing can be regulated by the oxidation state of a disulfide bond between two Cys residues involved in the splicing reaction, and that this link of oxidation state to activity can serve as a biosensor. The unusual stability of a pair of related inteins from thermophilic archaebacteria has allowed for structural study and analysis of the kinetics of each step of splicing in vitro. An NMR solution structure of a thermophilic intein reveals a particularly rigid structure, a disordered loop that influences activity and is absent in a highly similar intein, and a β-hairpin specific to inteins from archaebacteria.This material is based upon work supported by the National Science Foundation under grant MCB-1244089 and by the Camille and Henry Dreyfus Foundation.
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