109 results on '"Kost-Alimova M"'
Search Results
2. The position of t(11;22)(q23;q11) constitutional translocation breakpoint is conserved among its carriers
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Tapia-Páez, I., Kost-Alimova, M., Hu, P., Roe, B., Blennow, E., Fedorova, L., Imreh, S., and Dumanski, J.
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- 2001
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3. A novel gene containing LIM domains (LIMD1) is located within the common eliminated region 1 (C3CER1) in 3p21.3
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Kiss, H., Kedra, D., Yang, Y., Kost-Alimova, M., Kiss, C., O'Brien, K.P., Fransson, I., Klein, G., Imreh, S., and Dumanski, J.P.
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- 1999
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4. Microdissection molecular copy-number counting (μMCC)—unlocking cancer archives with digital PCR#
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McCaughan, F, Darai-Ramqvist, E, Bankier, A T, Konfortov, B A, Foster, N, George, P J, Rabbitts, T H, Kost-Alimova, M, Rabbitts, P H, and Dear, P H
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- 2008
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5. A microcell hybrid based approach to identify chromosome 3p genes that antagonize tumor growth
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Kholodnyuk, I.D., Szeles, A., Kost-Alimova, M., Kashuba, V., Yang, Y., Klein, G., and Imreh, S.
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Genetic research -- Analysis ,Human genetics -- Research ,Tumors -- Genetic aspects ,Biological sciences - Published
- 2000
6. Assignment1 of inosine ′-monophosphate dehydrogenase type 2 (IMPDH2) to human chromosome band 3p21.2 by in situ hybridization
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Kost-Alimova, M. V., Glesne, D. A., Huberman, E., and Zelenin, A. V.
- Abstract
n/a
- Published
- 1998
7. Horizontal transfer of tumor DNA to endothelial cells in vivo
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Ehnfors, J, primary, Kost-Alimova, M, additional, Persson, N Luna, additional, Bergsmedh, A, additional, Castro, J, additional, Levchenko-Tegnebratt, T, additional, Yang, L, additional, Panaretakis, T, additional, and Holmgren, L, additional
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- 2009
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8. Segmental duplications and evolutionary plasticity at tumor chromosome break-prone regions
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Darai-Ramqvist, E., primary, Sandlund, A., additional, Muller, S., additional, Klein, G., additional, Imreh, S., additional, and Kost-Alimova, M., additional
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- 2008
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9. Evolutionarily plastic regions at human 3p21.3 coincide with tumor breakpoints identified by the “elimination test”
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Darai, E., primary, Kost-Alimova, M., additional, Kiss, H., additional, Kansoul, H., additional, Klein, G., additional, and Imreh, S., additional
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- 2005
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10. Coincidence of synteny breakpoints with malignancy-related deletions on human chromosome 3
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Kost-Alimova, M., primary, Kiss, H., additional, Fedorova, L., additional, Yang, Y., additional, Dumanski, J. P., additional, Klein, G., additional, and Imreh, S., additional
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- 2003
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11. A microcell hybrid based approach identifies human chromosome 3p genes that are silenced following tumor growth, at four distinct regions
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Kholodnyuk, I.D., primary, Kost-Alimova, M., additional, Szeles, A., additional, Kashuba, V., additional, Yang, Y., additional, Klein, G., additional, and Imreh, S., additional
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- 2001
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12. Similar regions of human chromosome 3 are eliminated from or retained in human/human and human/mouse microcell hybrids during tumor growth in severe combined immunodeficient (SCID) mice
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Yang, Y., primary, Kost-Alimova, M., additional, Ingvarsson, S., additional, Qianhui, Q., additional, Kiss, H., additional, Szeles, A., additional, Kholodnyuk, I., additional, Cuthbert, A., additional, Klein, G., additional, and Imreh, S., additional
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- 2001
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13. Analysis of NotI linking clones isolated from human chromosome 3 specific libraries
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Kashuba, V.I, primary, Gizatullin, R.Z, additional, Protopopov, A.I, additional, Li, J, additional, Vorobieva, N.V, additional, Fedorova, L, additional, Zabarovska, V.I, additional, Muravenko, O.V, additional, Kost-Alimova, M, additional, Domninsky, D.A, additional, Kiss, C, additional, Allikmets, R, additional, Zakharyev, V.M, additional, Braga, E.A, additional, Sumegi, J, additional, Lerman, M, additional, Wahlestedt, C, additional, Zelenin, A.V, additional, Sheer, D, additional, Winberg, G, additional, Grafodatsky, A, additional, Kisselev, L.L, additional, Klein, G, additional, and Zabarovsky, E.R, additional
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- 1999
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14. Assignment and Ordering of Twenty-Three Unique NotI-Linking Clones Containing Expressed Genes Including the Guanosine 5'-Monophosphate Synthetase Gene to Human Chromosome 3
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Fedorova, L., primary, Kost-Alimova, M., additional, Gizatullin, R.Z., additional, Alimov, A., additional, Zabarovska, V.I., additional, Szeles, A., additional, Protopopov, A.I., additional, Vorobieva, N.V., additional, Kashuba, VI., additional, Klein, G., additional, Zelenin, A.V., additional, Sheer, D., additional, and Zabarovsky, E.R., additional
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- 1997
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15. Assignment and ordering of twenty-three unique NotI-Linking clones containing expressed genes including the guanosine 5'-monophosphate synthetase gene to human chromosome 3
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Fedorova, L., Kost-Alimova, M., Gizatullin, R. Z., Andrei Alimov, Zabarovska, V. I., Szeles, A., Protopopov, A. I., Vorobieva, N. V., Kashuba, V. I., Klein, G., Zelenin, A. V., Sheer, D., and Zabarovsky, E. R.
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Genetics ,Genetics (clinical)
16. Assignment and ordering of twenty-three unique NotI-linking clones containing expressed genes including the guanosine 5'-monophosphate synthetase gene to human chromosome 3
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Fedorova L, Kost-Alimova M, Rz, Gizatullin, Alimov A, Vi, Zabarovska, Szeles A, Ai, Protopopov, Nv, Vorobieva, Vladimir Kashuba, Klein G, Av, Zelenin, Sheer D, and Er, Zabarovsky
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Base Sequence ,Molecular Sequence Data ,Restriction Mapping ,Chromosome Mapping ,Gene Expression ,Ligases ,Humans ,Carbon-Nitrogen Ligases ,CpG Islands ,Chromosomes, Human, Pair 3 ,Cloning, Molecular ,Deoxyribonucleases, Type II Site-Specific ,Conserved Sequence ,In Situ Hybridization, Fluorescence - Abstract
Twenty-three unique NotI-linking clones, mainly isolated from the NRL1 library, were mapped and ordered by fluorescence in situ hybridization to human chromosome 3. All these clones were partially sequenced around the NotI sites and thus represent sequence-tagged sites. The EMBL nucleotide database was then searched with sequences from the NotI-linking clones using the FASTA program. This search revealed that the NRL-090 clone (at 3q24) contains the gene encoding human guanosine 5'-monophosphate synthetase (GMPS-PEN). To our knowledge, this is the first localization of this gene. Clone NL1-320 (at 3p21.3) contains a gene encoding arginine tRNA (97.3% identity in 73 bp), while clones NRL-063, NRL-097 and NRL-143 contain expressed sequences with unknown functions. Other clones displayed 60-85% similarities to cDNAs, CpG islands and other genes.
17. Mandatory chromosomal segment balance in aneuploid tumor cells
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Li Lung Maria, Cheng Yue, Kholodnyuk Irina, Yang Ying, Fedorova Ludmila, Sandlund Agneta, Yau Wing, Darai-Ramqvist Eva, Kost-Alimova Maria, Stanbridge Eric, Klein George, and Imreh Stefan
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Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
Abstract Background Euploid chromosome balance is vitally important for normal development, but is profoundly changed in many tumors. Is each tumor dependent on its own structurally and numerically changed chromosome complement that has evolved during its development and progression? We have previously shown that normal chromosome 3 transfer into the KH39 renal cell carcinoma line and into the Hone1 nasopharyngeal carcinoma line inhibited their tumorigenicity. The aim of the present study was to distinguish between a qualitative and a quantitative model of this suppression. According to the former, a damaged or deleted tumor suppressor gene would be restored by the transfer of a normal chromosome. If so, suppression would be released only when the corresponding sequences of the exogenous normal chromosome are lost or inactivated. According to the alternative quantitative model, the tumor cell would not tolerate an increased dosage of the relevant gene or segment. If so, either a normal cell derived, or, a tumor derived endogenous segment could be lost. Methods Fluorescence in Situ Hybridization based methods, as well as analysis of polymorphic microsatellite markers were used to follow chromosome 3 constitution changes in monochromosomal hybrids. Results In both tumor lines with introduced supernumerary chromosomes 3, the copy number of 3p21 or the entire 3p tended to fall back to the original level during both in vitro and in vivo growth. An exogenous, normal cell derived, or an endogenous, tumor derived, chromosome segment was lost with similar probability. Identification of the lost versus retained segments showed that the intolerance for increased copy number was particularly strong for 3p14-p21, and weaker for other 3p regions. Gains in copy number were, on the other hand, well tolerated in the long arm and particularly the 3q26-q27 region. Conclusion The inability of the cell to tolerate an experimentally imposed gain in 3p14-p21 in contrast to the well tolerated gain in 3q26-q27 is consistent with the fact that the former is often deleted in human tumors, whereas the latter is frequently amplified. The findings emphasize the importance of even minor changes in copy number in seemingly unbalanced aneuploid tumors.
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- 2007
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18. Array-CGH and multipoint FISH to decode complex chromosomal rearrangements
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Dumanski Jan, Klein George, Mantripragada Kiran, Sandlund Agneta, de Ståhl Teresita, Darai-Ramqvist Eva, Imreh Stefan, and Kost-Alimova Maria
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Biotechnology ,TP248.13-248.65 ,Genetics ,QH426-470 - Abstract
Abstract Background Recently, several high-resolution methods of chromosome analysis have been developed. It is important to compare these methods and to select reliable combinations of techniques to analyze complex chromosomal rearrangements in tumours. In this study we have compared array-CGH (comparative genomic hybridization) and multipoint FISH (mpFISH) for their ability to characterize complex rearrangements on human chromosome 3 (chr3) in tumour cell lines. We have used 179 BAC/PAC clones covering chr3 with an approximately 1 Mb resolution to analyze nine carcinoma lines. Chr3 was chosen for analysis, because of its frequent rearrangements in human solid tumours. Results The ploidy of the tumour cell lines ranged from near-diploid to near-pentaploid. Chr3 locus copy number was assessed by interphase and metaphase mpFISH. Totally 53 chr3 fragments were identified having copy numbers from 0 to 14. MpFISH results from the BAC/PAC clones and array-CGH gave mainly corresponding results. Each copy number change on the array profile could be related to a specific chromosome aberration detected by metaphase mpFISH. The analysis of the correlation between real copy number from mpFISH and the average normalized inter-locus fluorescence ratio (ANILFR) value detected by array-CGH demonstrated that copy number is a linear function of parameters that include the variable, ANILFR, and two constants, ploidy and background normalized fluorescence ratio. Conclusion In most cases, the changes in copy number seen on array-CGH profiles reflected cumulative chromosome rearrangements. Most of them stemmed from unbalanced translocations. Although our chr3 BAC/PAC array could identify single copy number changes even in pentaploid cells, mpFISH provided a more accurate analysis in the dissection of complex karyotypes at high ploidy levels.
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- 2006
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19. Assignment<FOOTREF>[sup 1] </FOOTREF> of inosine ′-monophosphate dehydrogenase type 2 (IMPDH2) to human chromosome band 3p21.2 by in situ hybridization.
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Kost-Alimova, M. V., Glesne, D. A., Huberman, E., and Zelenin, A. V.
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- *
IMP dehydrogenase , *HUMAN genetics , *HUMAN chromosomes , *HUMAN gene mapping , *ONCOLOGY , *IN situ hybridization , *NUCLEOSIDES , *DEHYDROGENASES - Abstract
This article describes an experiment on mapping of inosine 5-monophosphate dehydrogenase type 2 (IMPDH2) to human chromosome band 3p21.2 by in situ hybridization. Inosine 5'-monophosphate dehydrogenase (IMPDH, E.C. 1.1.1.205) is the rate limiting enzyme in de novo guanine nucleotide biosynthesis, and is encoded by two genes termed types I and II. Increased IMPDH activity has been found in an assortment of tumors, while decreased IMPDH activity has been observed during differentiation of various cell types.
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- 1998
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20. Telomere dysfunction induces metabolic and mitochondrial compromise
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Simona Colla, Darrell N. Kotton, Y. Alan Wang, Sachet A. Shukla, Elena Ivanova, Marcus P. Cooper, Ronald A. DePinho, Giovanni Tonon, Eric S. Martin, Lynda Chin, John E. Mahoney, Javid Moslehi, Richard S. Maser, Mariela Jaskelioff, Ronglih Liao, Maria Kost-Alimova, Mira Guo, Timothy P. Heffernan, Alexei Protopopov, Florian L. Muller, Samuel R. Perry, Robert Xiong, Richard C. Mulligan, Friedrich Foerster, Ergiin Sahin, Carl Walkey, Marc Liesa, Roderick T. Bronson, Orian S. Shirihai, Attila J. Fabian, Sahin, E, Colla, S, Liesa, M, Moslehi, J, Müller, Fl, Guo, M, Cooper, M, Kotton, D, Fabian, Aj, Walkey, C, Maser, R, Tonon, G, Foerster, F, Xiong, R, Wang, Ya, Shukla, Sa, Jaskelioff, M, Martin, E, Heffernan, Tp, Protopopov, A, Ivanova, E, Mahoney, Je, Kost-Alimova, M, Perry, Sr, Bronson, R, Liao, R, Mulligan, R, Shirihai, O, Chin, L, and Depinho, Ra.
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Senescence ,Telomerase RNA component ,Telomerase ,Multidisciplinary ,Mitochondrial biogenesis ,Telomerase reverse transcriptase ,PPARGC1A ,Mitochondrion ,Biology ,Molecular biology ,Cell biology ,Telomere - Abstract
Telomere dysfunction activates p53-mediated cellular growth arrest, senescence and apoptosis to drive progressive atrophy and functional decline in high-turnover tissues. The broader adverse impact of telomere dysfunction across many tissues including more quiescent systems prompted transcriptomic network analyses to identify common mechanisms operative in haematopoietic stem cells, heart and liver. These unbiased studies revealed profound repression of peroxisome proliferator-activated receptor gamma, coactivator 1 alpha and beta (PGC-1α and PGC-1β, also known as Ppargc1a and Ppargc1b, respectively) and the downstream network in mice null for either telomerase reverse transcriptase (Tert) or telomerase RNA component (Terc) genes. Consistent with PGCs as master regulators of mitochondrial physiology and metabolism, telomere dysfunction is associated with impaired mitochondrial biogenesis and function, decreased gluconeogenesis, cardiomyopathy, and increased reactive oxygen species. In the setting of telomere dysfunction, enforced Tert or PGC-1α expression or germline deletion of p53 (also known as Trp53) substantially restores PGC network expression, mitochondrial respiration, cardiac function and gluconeogenesis. We demonstrate that telomere dysfunction activates p53 which in turn binds and represses PGC-1α and PGC-1β promoters, thereby forging a direct link between telomere and mitochondrial biology. We propose that this telomere-p53-PGC axis contributes to organ and metabolic failure and to diminishing organismal fitness in the setting of telomere dysfunction.
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- 2011
21. Three million images and morphological profiles of cells treated with matched chemical and genetic perturbations.
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Chandrasekaran SN, Cimini BA, Goodale A, Miller L, Kost-Alimova M, Jamali N, Doench JG, Fritchman B, Skepner A, Melanson M, Kalinin AA, Arevalo J, Haghighi M, Caicedo JC, Kuhn D, Hernandez D, Berstler J, Shafqat-Abbasi H, Root DE, Swalley SE, Garg S, Singh S, and Carpenter AE
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- Humans, Microscopy methods, Image Processing, Computer-Assisted methods
- Abstract
The identification of genetic and chemical perturbations with similar impacts on cell morphology can elucidate compounds' mechanisms of action or novel regulators of genetic pathways. Research on methods for identifying such similarities has lagged due to a lack of carefully designed and well-annotated image sets of cells treated with chemical and genetic perturbations. Here we create such a Resource dataset, CPJUMP1, in which each perturbed gene's product is a known target of at least two chemical compounds in the dataset. We systematically explore the directionality of correlations among perturbations that target the same protein encoded by a given gene, and we find that identifying matches between chemical and genetic perturbations is a challenging task. Our dataset and baseline analyses provide a benchmark for evaluating methods that measure perturbation similarities and impact, and more generally, learn effective representations of cellular state from microscopy images. Such advancements would accelerate the applications of image-based profiling of cellular states, such as uncovering drug mode of action or probing functional genomics., (© 2024. The Author(s).)
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- 2024
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22. Optimizing the Cell Painting assay for image-based profiling.
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Cimini BA, Chandrasekaran SN, Kost-Alimova M, Miller L, Goodale A, Fritchman B, Byrne P, Garg S, Jamali N, Logan DJ, Concannon JB, Lardeau CH, Mouchet E, Singh S, Shafqat Abbasi H, Aspesi P Jr, Boyd JD, Gilbert T, Gnutt D, Hariharan S, Hernandez D, Hormel G, Juhani K, Melanson M, Mervin LH, Monteverde T, Pilling JE, Skepner A, Swalley SE, Vrcic A, Weisbart E, Williams G, Yu S, Zapiec B, and Carpenter AE
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- Microscopy, Fluorescence, Mitochondria, Software, Image Processing, Computer-Assisted methods, Cell Culture Techniques
- Abstract
In image-based profiling, software extracts thousands of morphological features of cells from multi-channel fluorescence microscopy images, yielding single-cell profiles that can be used for basic research and drug discovery. Powerful applications have been proven, including clustering chemical and genetic perturbations on the basis of their similar morphological impact, identifying disease phenotypes by observing differences in profiles between healthy and diseased cells and predicting assay outcomes by using machine learning, among many others. Here, we provide an updated protocol for the most popular assay for image-based profiling, Cell Painting. Introduced in 2013, it uses six stains imaged in five channels and labels eight diverse components of the cell: DNA, cytoplasmic RNA, nucleoli, actin, Golgi apparatus, plasma membrane, endoplasmic reticulum and mitochondria. The original protocol was updated in 2016 on the basis of several years' experience running it at two sites, after optimizing it by visual stain quality. Here, we describe the work of the Joint Undertaking for Morphological Profiling Cell Painting Consortium, to improve upon the assay via quantitative optimization by measuring the assay's ability to detect morphological phenotypes and group similar perturbations together. The assay gives very robust outputs despite various changes to the protocol, and two vendors' dyes work equivalently well. We present Cell Painting version 3, in which some steps are simplified and several stain concentrations can be reduced, saving costs. Cell culture and image acquisition take 1-2 weeks for typically sized batches of ≤20 plates; feature extraction and data analysis take an additional 1-2 weeks.This protocol is an update to Nat. Protoc. 11, 1757-1774 (2016): https://doi.org/10.1038/nprot.2016.105., (© 2023. Springer Nature Limited.)
- Published
- 2023
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23. nELISA: A high-throughput, high-plex platform enables quantitative profiling of the secretome.
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Dagher M, Ongo G, Robichaud N, Kong J, Rho W, Teahulos I, Tavakoli A, Bovaird S, Merjaneh S, Tan A, Edwardson K, Scheepers C, Ng A, Hajjar A, Sow B, Vrouvides M, Lee A, DeCorwin-Martin P, Rasool S, Huang J, Han Y, Erps T, Coffin S, Chandrasekaran SN, Miller L, Kost-Alimova M, Skepner A, Singh S, Carpenter AE, Munzar J, and Juncker D
- Abstract
We present the nELISA, a high-throughput, high-fidelity, and high-plex protein profiling platform. DNA oligonucleotides are used to pre-assemble antibody pairs on spectrally encoded microparticles and perform displacement-mediated detection. Spatial separation between non-cognate antibodies prevents the rise of reagent-driven cross-reactivity, while read-out is performed cost-efficiently and at high-throughput using flow cytometry. We assembled an inflammatory panel of 191 targets that were multiplexed without cross-reactivity or impact on performance vs 1-plex signals, with sensitivities as low as 0.1pg/mL and measurements spanning 7 orders of magnitude. We then performed a large-scale secretome perturbation screen of peripheral blood mononuclear cells (PBMCs), with cytokines as both perturbagens and read-outs, measuring 7,392 samples and generating ~1.5M protein datapoints in under a week, a significant advance in throughput compared to other highly multiplexed immunoassays. We uncovered 447 significant cytokine responses, including multiple putatively novel ones, that were conserved across donors and stimulation conditions. We also validated the nELISA's use in phenotypic screening, and propose its application to drug discovery.
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- 2023
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24. Discovering cellular programs of intrinsic and extrinsic drivers of metabolic traits using LipocyteProfiler.
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Laber S, Strobel S, Mercader JM, Dashti H, Dos Santos FRC, Kubitz P, Jackson M, Ainbinder A, Honecker J, Agrawal S, Garborcauskas G, Stirling DR, Leong A, Figueroa K, Sinnott-Armstrong N, Kost-Alimova M, Deodato G, Harney A, Way GP, Saadat A, Harken S, Reibe-Pal S, Ebert H, Zhang Y, Calabuig-Navarro V, McGonagle E, Stefek A, Dupuis J, Cimini BA, Hauner H, Udler MS, Carpenter AE, Florez JC, Lindgren C, Jacobs SBR, and Claussnitzer M
- Abstract
A primary obstacle in translating genetic associations with disease into therapeutic strategies is elucidating the cellular programs affected by genetic risk variants and effector genes. Here, we introduce LipocyteProfiler, a cardiometabolic-disease-oriented high-content image-based profiling tool that enables evaluation of thousands of morphological and cellular profiles that can be systematically linked to genes and genetic variants relevant to cardiometabolic disease. We show that LipocyteProfiler allows surveillance of diverse cellular programs by generating rich context- and process-specific cellular profiles across hepatocyte and adipocyte cell-state transitions. We use LipocyteProfiler to identify known and novel cellular mechanisms altered by polygenic risk of metabolic disease, including insulin resistance, fat distribution, and the polygenic contribution to lipodystrophy. LipocyteProfiler paves the way for large-scale forward and reverse deep phenotypic profiling in lipocytes and provides a framework for the unbiased identification of causal relationships between genetic variants and cellular programs relevant to human disease., Competing Interests: J.C.F. has received consulting honoraria from Goldfinch Bio and Astra Zeneca and speaking honoraria from Novo Nordisk, Astra Zeneca, and Merck for research presentations over which he had full control of content. M.C. holds equity in Waypoint Bio, serves as a consultant for Pfizer, and is a member of the Nestle Scientific Advisory Board. The authors have filed a provisional patent application (63/218,656)., (© 2023.)
- Published
- 2023
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25. FALCON systematically interrogates free fatty acid biology and identifies a novel mediator of lipotoxicity.
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Wieder N, Fried JC, Kim C, Sidhom EH, Brown MR, Marshall JL, Arevalo C, Dvela-Levitt M, Kost-Alimova M, Sieber J, Gabriel KR, Pacheco J, Clish C, Abbasi HS, Singh S, Rutter JC, Therrien M, Yoon H, Lai ZW, Baublis A, Subramanian R, Devkota R, Small J, Sreekanth V, Han M, Lim D, Carpenter AE, Flannick J, Finucane H, Haigis MC, Claussnitzer M, Sheu E, Stevens B, Wagner BK, Choudhary A, Shaw JL, Pablo JL, and Greka A
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- Humans, Fatty Acids, Signal Transduction, Biology, Fatty Acids, Nonesterified metabolism, Diabetes Mellitus, Type 2
- Abstract
Cellular exposure to free fatty acids (FFAs) is implicated in the pathogenesis of obesity-associated diseases. However, there are no scalable approaches to comprehensively assess the diverse FFAs circulating in human plasma. Furthermore, assessing how FFA-mediated processes interact with genetic risk for disease remains elusive. Here, we report the design and implementation of fatty acid library for comprehensive ontologies (FALCON), an unbiased, scalable, and multimodal interrogation of 61 structurally diverse FFAs. We identified a subset of lipotoxic monounsaturated fatty acids associated with decreased membrane fluidity. Furthermore, we prioritized genes that reflect the combined effects of harmful FFA exposure and genetic risk for type 2 diabetes (T2D). We found that c-MAF-inducing protein (CMIP) protects cells from FFA exposure by modulating Akt signaling. In sum, FALCON empowers the study of fundamental FFA biology and offers an integrative approach to identify much needed targets for diverse diseases associated with disordered FFA metabolism., Competing Interests: Declaration of interests N.W., J.C.F., and A.G. are co-inventors of a patent on the composition, method, and use for FFA screening, application no: 52199-550P01US. A.G. serves as a founding advisor to a new company launched by Atlas Ventures, an agreement reviewed and managed by Brigham and Women’s Hospital, Mass General Brigham, and the Broad Institute of MIT and Harvard in accordance with their conflict of interest policies., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
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26. Morphology and gene expression profiling provide complementary information for mapping cell state.
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Way GP, Natoli T, Adeboye A, Litichevskiy L, Yang A, Lu X, Caicedo JC, Cimini BA, Karhohs K, Logan DJ, Rohban MH, Kost-Alimova M, Hartland K, Bornholdt M, Chandrasekaran SN, Haghighi M, Weisbart E, Singh S, Subramanian A, and Carpenter AE
- Subjects
- Humans, Phenotype, Gene Expression Profiling methods
- Abstract
Morphological and gene expression profiling can cost-effectively capture thousands of features in thousands of samples across perturbations by disease, mutation, or drug treatments, but it is unclear to what extent the two modalities capture overlapping versus complementary information. Here, using both the L1000 and Cell Painting assays to profile gene expression and cell morphology, respectively, we perturb human A549 lung cancer cells with 1,327 small molecules from the Drug Repurposing Hub across six doses, providing a data resource including dose-response data from both assays. The two assays capture both shared and complementary information for mapping cell state. Cell Painting profiles from compound perturbations are more reproducible and show more diversity but measure fewer distinct groups of features. Applying unsupervised and supervised methods to predict compound mechanisms of action (MOAs) and gene targets, we find that the two assays not only provide a partially shared but also a complementary view of drug mechanisms. Given the numerous applications of profiling in biology, our analyses provide guidance for planning experiments that profile cells for detecting distinct cell types, disease phenotypes, and response to chemical or genetic perturbations., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)
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- 2022
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27. Single-nucleus profiling of human dilated and hypertrophic cardiomyopathy.
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Chaffin M, Papangeli I, Simonson B, Akkad AD, Hill MC, Arduini A, Fleming SJ, Melanson M, Hayat S, Kost-Alimova M, Atwa O, Ye J, Bedi KC Jr, Nahrendorf M, Kaushik VK, Stegmann CM, Margulies KB, Tucker NR, and Ellinor PT
- Subjects
- CRISPR-Cas Systems, Case-Control Studies, Cells, Cultured, Gene Knockout Techniques, Heart Ventricles metabolism, Heart Ventricles pathology, Humans, Myocardium metabolism, Myocardium pathology, Myofibroblasts metabolism, Myofibroblasts pathology, RNA-Seq, Transcription, Genetic, Transforming Growth Factor beta1, Cardiomyopathy, Dilated genetics, Cardiomyopathy, Dilated pathology, Cardiomyopathy, Hypertrophic genetics, Cardiomyopathy, Hypertrophic pathology, Cell Nucleus genetics, Gene Expression Profiling, Heart Failure genetics, Heart Failure pathology, Single-Cell Analysis
- Abstract
Heart failure encompasses a heterogeneous set of clinical features that converge on impaired cardiac contractile function
1,2 and presents a growing public health concern. Previous work has highlighted changes in both transcription and protein expression in failing hearts3,4 , but may overlook molecular changes in less prevalent cell types. Here we identify extensive molecular alterations in failing hearts at single-cell resolution by performing single-nucleus RNA sequencing of nearly 600,000 nuclei in left ventricle samples from 11 hearts with dilated cardiomyopathy and 15 hearts with hypertrophic cardiomyopathy as well as 16 non-failing hearts. The transcriptional profiles of dilated or hypertrophic cardiomyopathy hearts broadly converged at the tissue and cell-type level. Further, a subset of hearts from patients with cardiomyopathy harbour a unique population of activated fibroblasts that is almost entirely absent from non-failing samples. We performed a CRISPR-knockout screen in primary human cardiac fibroblasts to evaluate this fibrotic cell state transition; knockout of genes associated with fibroblast transition resulted in a reduction of myofibroblast cell-state transition upon TGFβ1 stimulation for a subset of genes. Our results provide insights into the transcriptional diversity of the human heart in health and disease as well as new potential therapeutic targets and biomarkers for heart failure., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)- Published
- 2022
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28. Phenotypic Screening for Small Molecules that Protect β-Cells from Glucolipotoxicity.
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Small JC, Joblin-Mills A, Carbone K, Kost-Alimova M, Ayukawa K, Khodier C, Dancik V, Clemons PA, Munkacsi AB, and Wagner BK
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- Animals, Apoptosis, Glucose metabolism, Humans, Palmitates metabolism, Phosphatidylinositol 3-Kinases metabolism, Rats, Diabetes Mellitus, Type 2 drug therapy, Diabetes Mellitus, Type 2 metabolism, Insulin-Secreting Cells metabolism
- Abstract
Type 2 diabetes is marked by progressive β-cell failure, leading to loss of β-cell mass. Increased levels of circulating glucose and free fatty acids associated with obesity lead to β-cell glucolipotoxicity. There are currently no therapeutic options to address this facet of β-cell loss in obese type 2 diabetes patients. To identify small molecules capable of protecting β-cells, we performed a high-throughput screen of 20,876 compounds in the rat insulinoma cell line INS-1E in the presence of elevated glucose and palmitate. We found 312 glucolipotoxicity-protective small molecules (1.49% hit rate) capable of restoring INS-1E viability, and we focused on 17 with known biological targets. 16 of the 17 compounds were kinase inhibitors with activity against specific families including but not limited to cyclin-dependent kinases (CDK), PI-3 kinase (PI3K), Janus kinase (JAK), and Rho-associated kinase 2 (ROCK2). 7 of the 16 kinase inhibitors were PI3K inhibitors. Validation studies in dissociated human islets identified 10 of the 17 compounds, namely, KD025, ETP-45658, BMS-536924, AT-9283, PF-03814735, torin-2, AZD5438, CP-640186, ETP-46464, and GSK2126458 that reduced glucolipotoxicity-induced β-cell death. These 10 compounds decreased markers of glucolipotoxicity including caspase activation, mitochondrial depolarization, and increased calcium flux. Together, these results provide a path forward toward identifying novel treatments to preserve β-cell viability in the face of glucolipotoxicity.
- Published
- 2022
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29. High-Throughput Assay to Screen Small Molecules for Their Ability to Prevent Sickling of Red Blood Cells.
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Nakagawa A, Cooper MK, Kost-Alimova M, Berstler J, Yu B, Berra L, Klings ES, Huang MS, Heeney MM, Bloch DB, and Zapol WM
- Abstract
Sickle cell disease (SCD) is an inherited disorder of hemoglobin (Hb); approximately 300,000 babies are born worldwide with SCD each year. In SCD, fibers of polymerized sickle Hb (HbS) form in red blood cells (RBCs), which cause RBCs to develop their characteristic "sickled" shape, resulting in hemolytic anemia and numerous vascular complications including vaso-occlusive crises. The development of novel antisickling compounds will provide new therapeutic options for patients with SCD. We developed a high-throughput "sickling assay" that is based on an automated high-content imaging system to quantify the effects of hypoxia on the shape and size of RBCs from HbSS SCD patients (SS RBCs). We used this assay to screen thousands of compounds for their ability to inhibit sickling. In the assay, voxelotor (an FDA-approved medication used to treat SCD) prevented sickling with a z '-factor > 0.4, suggesting that the assay is capable of identifying compounds that inhibit sickling. We screened the Broad Repurposing Library of 5393 compounds for their ability to prevent sickling in 4% oxygen/96% nitrogen. We identified two compounds, SNS-314 mesylate and voxelotor itself, that successfully prevented sickling. SNS-314 mesylate prevented sickling in the absence of oxygen, while voxelotor did not, suggesting that SNS-314 mesylate acts by a mechanism that is different from that of voxelotor. The sickling assay described in this study will permit the identification of additional, novel antisickling compounds, which will potentially expand the therapeutic options for SCD., Competing Interests: The authors declare the following competing financial interest(s): Massachusetts General Hospital has filed a patent related to SNS-314 mesylate., (© 2022 The Authors. Published by American Chemical Society.)
- Published
- 2022
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30. Control of osteocyte dendrite formation by Sp7 and its target gene osteocrin.
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Wang JS, Kamath T, Mazur CM, Mirzamohammadi F, Rotter D, Hojo H, Castro CD, Tokavanich N, Patel R, Govea N, Enishi T, Wu Y, da Silva Martins J, Bruce M, Brooks DJ, Bouxsein ML, Tokarz D, Lin CP, Abdul A, Macosko EZ, Fiscaletti M, Munns CF, Ryder P, Kost-Alimova M, Byrne P, Cimini B, Fujiwara M, Kronenberg HM, and Wein MN
- Subjects
- Adolescent, Animals, Bone Diseases genetics, Bone Diseases physiopathology, Female, Gene Expression Regulation, Humans, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Muscle Proteins genetics, Mutation, Sp7 Transcription Factor genetics, Transcription Factors genetics, Bone Diseases metabolism, Dendrites metabolism, Muscle Proteins metabolism, Osteocytes metabolism, Sp7 Transcription Factor metabolism, Transcription Factors metabolism
- Abstract
Some osteoblasts embed within bone matrix, change shape, and become dendrite-bearing osteocytes. The circuitry that drives dendrite formation during "osteocytogenesis" is poorly understood. Here we show that deletion of Sp7 in osteoblasts and osteocytes causes defects in osteocyte dendrites. Profiling of Sp7 target genes and binding sites reveals unexpected repurposing of this transcription factor to drive dendrite formation. Osteocrin is a Sp7 target gene that promotes osteocyte dendrite formation and rescues defects in Sp7-deficient mice. Single-cell RNA-sequencing demonstrates defects in osteocyte maturation in the absence of Sp7. Sp7-dependent osteocyte gene networks are associated with human skeletal diseases. Moreover, humans with a SP7
R316C mutation show defective osteocyte morphology. Sp7-dependent genes that mark osteocytes are enriched in neurons, highlighting shared features between osteocytic and neuronal connectivity. These findings reveal a role for Sp7 and its target gene Osteocrin in osteocytogenesis, revealing that pathways that control osteocyte development influence human bone diseases., (© 2021. The Author(s).)- Published
- 2021
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31. Predicting cell health phenotypes using image-based morphology profiling.
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Way GP, Kost-Alimova M, Shibue T, Harrington WF, Gill S, Piccioni F, Becker T, Shafqat-Abbasi H, Hahn WC, Carpenter AE, Vazquez F, and Singh S
- Subjects
- Algorithms, Biological Assay, Cell Line, Humans, Machine Learning, Microscopy, Phenotype, Cells cytology, Forecasting methods, Image Processing, Computer-Assisted methods
- Abstract
Genetic and chemical perturbations impact diverse cellular phenotypes, including multiple indicators of cell health. These readouts reveal toxicity and antitumorigenic effects relevant to drug discovery and personalized medicine. We developed two customized microscopy assays, one using four targeted reagents and the other three targeted reagents, to collectively measure 70 specific cell health phenotypes including proliferation, apoptosis, reactive oxygen species, DNA damage, and cell cycle stage. We then tested an approach to predict multiple cell health phenotypes using Cell Painting, an inexpensive and scalable image-based morphology assay. In matched CRISPR perturbations of three cancer cell lines, we collected both Cell Painting and cell health data. We found that simple machine learning algorithms can predict many cell health readouts directly from Cell Painting images, at less than half the cost. We hypothesized that these models can be applied to accurately predict cell health assay outcomes for any future or existing Cell Painting dataset. For Cell Painting images from a set of 1500+ compound perturbations across multiple doses, we validated predictions by orthogonal assay readouts. We provide a web app to browse predictions: http://broad.io/cell-health-app. Our approach can be used to add cell health annotations to Cell Painting datasets.
- Published
- 2021
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32. Targeting a Braf/Mapk pathway rescues podocyte lipid peroxidation in CoQ-deficiency kidney disease.
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Sidhom EH, Kim C, Kost-Alimova M, Ting MT, Keller K, Avila-Pacheco J, Watts AJ, Vernon KA, Marshall JL, Reyes-Bricio E, Racette M, Wieder N, Kleiner G, Grinkevich EJ, Chen F, Weins A, Clish CB, Shaw JL, Quinzii CM, and Greka A
- Subjects
- Alkyl and Aryl Transferases genetics, Alkyl and Aryl Transferases metabolism, Animals, Ataxia drug therapy, Ataxia genetics, Ataxia pathology, Drug Delivery Systems, HEK293 Cells, Humans, Kidney Diseases drug therapy, Kidney Diseases genetics, Kidney Diseases pathology, Lipid Peroxidation genetics, MAP Kinase Signaling System genetics, Mice, Mitochondrial Diseases drug therapy, Mitochondrial Diseases genetics, Mitochondrial Diseases pathology, Muscle Weakness drug therapy, Muscle Weakness genetics, Muscle Weakness pathology, Podocytes pathology, Proto-Oncogene Proteins B-raf genetics, RNA-Seq, Ubiquinone genetics, Ubiquinone metabolism, Ataxia metabolism, Indenes pharmacology, Kidney Diseases metabolism, Lipid Peroxidation drug effects, MAP Kinase Signaling System drug effects, Mitochondrial Diseases metabolism, Muscle Weakness metabolism, Podocytes metabolism, Proto-Oncogene Proteins B-raf metabolism, Pyrazoles pharmacology, Ubiquinone deficiency
- Abstract
Mutations affecting mitochondrial coenzyme Q (CoQ) biosynthesis lead to kidney failure due to selective loss of podocytes, essential cells of the kidney filter. Curiously, neighboring tubular epithelial cells are spared early in disease despite higher mitochondrial content. We sought to illuminate noncanonical, cell-specific roles for CoQ, independently of the electron transport chain (ETC). Here, we demonstrate that CoQ depletion caused by Pdss2 enzyme deficiency in podocytes results in perturbations in polyunsaturated fatty acid (PUFA) metabolism and the Braf/Mapk pathway rather than ETC dysfunction. Single-nucleus RNA-Seq from kidneys of Pdss2kd/kd mice with nephrotic syndrome and global CoQ deficiency identified a podocyte-specific perturbation of the Braf/Mapk pathway. Treatment with GDC-0879, a Braf/Mapk-targeting compound, ameliorated kidney disease in Pdss2kd/kd mice. Mechanistic studies in Pdss2-depleted podocytes revealed a previously unknown perturbation in PUFA metabolism that was confirmed in vivo. Gpx4, an enzyme that protects against PUFA-mediated lipid peroxidation, was elevated in disease and restored after GDC-0879 treatment. We demonstrate broader human disease relevance by uncovering patterns of GPX4 and Braf/Mapk pathway gene expression in tissue from patients with kidney diseases. Our studies reveal ETC-independent roles for CoQ in podocytes and point to Braf/Mapk as a candidate pathway for the treatment of kidney diseases.
- Published
- 2021
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33. TFEB Transcriptional Responses Reveal Negative Feedback by BHLHE40 and BHLHE41.
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Carey KL, Paulus GLC, Wang L, Balce DR, Luo JW, Bergman P, Ferder IC, Kong L, Renaud N, Singh S, Kost-Alimova M, Nyfeler B, Lassen KG, Virgin HW, and Xavier RJ
- Subjects
- Basic Helix-Loop-Helix Leucine Zipper Transcription Factors genetics, Basic Helix-Loop-Helix Transcription Factors genetics, Cell Nucleus genetics, Cell Nucleus metabolism, Gene Knockout Techniques, HeLa Cells, Homeodomain Proteins genetics, Humans, Lysosomes metabolism, Mitochondria metabolism, Transcription, Genetic, Basic Helix-Loop-Helix Leucine Zipper Transcription Factors metabolism, Basic Helix-Loop-Helix Transcription Factors metabolism, Homeodomain Proteins metabolism
- Abstract
Transcription factor EB (TFEB) activates lysosomal biogenesis genes in response to environmental cues. Given implications of impaired TFEB signaling and lysosomal dysfunction in metabolic, neurological, and infectious diseases, we aim to systematically identify TFEB-directed circuits by examining transcriptional responses to TFEB subcellular localization and stimulation. We reveal that steady-state nuclear TFEB is sufficient to activate transcription of lysosomal, autophagy, and innate immunity genes, whereas other targets require higher thresholds of stimulation. Furthermore, we identify shared and distinct transcriptional signatures between mTOR inhibition and bacterial autophagy. Using a genome-wide CRISPR library, we find TFEB targets that protect cells from or sensitize cells to lysosomal cell death. BHLHE40 and BHLHE41, genes responsive to high, sustained levels of nuclear TFEB, act in opposition to TFEB upon lysosomal cell death induction. Further investigation identifies genes counter-regulated by TFEB and BHLHE40/41, adding this negative feedback to the current understanding of TFEB regulatory mechanisms., Competing Interests: Declaration of Interests P.B., N.R., and B.N. are employees of Novartis. D.R.B. and H.W.V. are employees of Vir Biotechnology. R.J.X. is a consultant to Novartis and a cofounder of Jnana Therapeutics and Celsius Therapeutics. H.W.V. is a founder of Casma Therapeutics and PierianDx. These organizations did not participate in funding this work., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2020
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34. A High-Content Screen for Mucin-1-Reducing Compounds Identifies Fostamatinib as a Candidate for Rapid Repurposing for Acute Lung Injury.
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Kost-Alimova M, Sidhom EH, Satyam A, Chamberlain BT, Dvela-Levitt M, Melanson M, Alper SL, Santos J, Gutierrez J, Subramanian A, Byrne PJ, Grinkevich E, Reyes-Bricio E, Kim C, Clark AR, Watts AJB, Thompson R, Marshall J, Pablo JL, Coraor J, Roignot J, Vernon KA, Keller K, Campbell A, Emani M, Racette M, Bazua-Valenti S, Padovano V, Weins A, McAdoo SP, Tam FWK, Ronco L, Wagner F, Tsokos GC, Shaw JL, and Greka A
- Abstract
Drug repurposing has the advantage of identifying potential treatments on a shortened timescale. In response to the pandemic spread of SARS-CoV-2, we took advantage of a high-content screen of 3,713 compounds at different stages of clinical development to identify FDA-approved compounds that reduce mucin-1 (MUC1) protein abundance. Elevated MUC1 levels predict the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) and correlate with poor clinical outcomes. Our screen identifies fostamatinib (R788), an inhibitor of spleen tyrosine kinase (SYK) approved for the treatment of chronic immune thrombocytopenia, as a repurposing candidate for the treatment of ALI. In vivo , fostamatinib reduces MUC1 abundance in lung epithelial cells in a mouse model of ALI. In vitro , SYK inhibition by the active metabolite R406 promotes MUC1 removal from the cell surface. Our work suggests fostamatinib as a repurposing drug candidate for ALI., Competing Interests: F.W.K.T. has received research project grants from and has consultancy agreements with Rigel Pharmaceuticals, and is the chief investigator of an international clinical trial of a SYK inhibitor in IgA nephropathy (ClinicalTrials.gov NCT02112838), funded by Rigel Pharmaceuticals., (© 2020 The Author(s).)
- Published
- 2020
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35. Single cell census of human kidney organoids shows reproducibility and diminished off-target cells after transplantation.
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Subramanian A, Sidhom EH, Emani M, Vernon K, Sahakian N, Zhou Y, Kost-Alimova M, Slyper M, Waldman J, Dionne D, Nguyen LT, Weins A, Marshall JL, Rosenblatt-Rosen O, Regev A, and Greka A
- Subjects
- Animals, Cell Differentiation, Cell Line, Gene Expression Regulation, Developmental, Humans, Induced Pluripotent Stem Cells metabolism, Induced Pluripotent Stem Cells transplantation, Kidney metabolism, Kidney Transplantation, Mice, Organoids metabolism, Organoids transplantation, Reproducibility of Results, Sequence Analysis, RNA, Single-Cell Analysis, Transplantation, Heterologous, Induced Pluripotent Stem Cells cytology, Kidney cytology, Organoids cytology
- Abstract
Human iPSC-derived kidney organoids have the potential to revolutionize discovery, but assessing their consistency and reproducibility across iPSC lines, and reducing the generation of off-target cells remain an open challenge. Here, we profile four human iPSC lines for a total of 450,118 single cells to show how organoid composition and development are comparable to human fetal and adult kidneys. Although cell classes are largely reproducible across time points, protocols, and replicates, we detect variability in cell proportions between different iPSC lines, largely due to off-target cells. To address this, we analyze organoids transplanted under the mouse kidney capsule and find diminished off-target cells. Our work shows how single cell RNA-seq (scRNA-seq) can score organoids for reproducibility, faithfulness and quality, that kidney organoids derived from different iPSC lines are comparable surrogates for human kidney, and that transplantation enhances their formation by diminishing off-target cells.
- Published
- 2019
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36. Small Molecule Targets TMED9 and Promotes Lysosomal Degradation to Reverse Proteinopathy.
- Author
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Dvela-Levitt M, Kost-Alimova M, Emani M, Kohnert E, Thompson R, Sidhom EH, Rivadeneira A, Sahakian N, Roignot J, Papagregoriou G, Montesinos MS, Clark AR, McKinney D, Gutierrez J, Roth M, Ronco L, Elonga E, Carter TA, Gnirke A, Melanson M, Hartland K, Wieder N, Hsu JC, Deltas C, Hughey R, Bleyer AJ, Kmoch S, Živná M, Barešova V, Kota S, Schlondorff J, Heiman M, Alper SL, Wagner F, Weins A, Golub TR, Lander ES, and Greka A
- Subjects
- Activating Transcription Factor 6 metabolism, Animals, Benzamides chemistry, Benzamides pharmacology, Bridged Bicyclo Compounds therapeutic use, Epithelial Cells cytology, Epithelial Cells metabolism, Female, Frameshift Mutation, Heptanes therapeutic use, Humans, Imidazoline Receptors antagonists & inhibitors, Imidazoline Receptors genetics, Imidazoline Receptors metabolism, Induced Pluripotent Stem Cells cytology, Induced Pluripotent Stem Cells metabolism, Kidney cytology, Kidney metabolism, Kidney pathology, Kidney Diseases metabolism, Kidney Diseases pathology, Lysosomes metabolism, Male, Mice, Mice, Transgenic, Mucin-1 chemistry, Mucin-1 genetics, Mucin-1 metabolism, RNA Interference, RNA, Small Interfering metabolism, Unfolded Protein Response drug effects, Vesicular Transport Proteins chemistry, Benzamides metabolism, Bridged Bicyclo Compounds pharmacology, Heptanes pharmacology, Lysosomes drug effects, Vesicular Transport Proteins metabolism
- Abstract
Intracellular accumulation of misfolded proteins causes toxic proteinopathies, diseases without targeted therapies. Mucin 1 kidney disease (MKD) results from a frameshift mutation in the MUC1 gene (MUC1-fs). Here, we show that MKD is a toxic proteinopathy. Intracellular MUC1-fs accumulation activated the ATF6 unfolded protein response (UPR) branch. We identified BRD4780, a small molecule that clears MUC1-fs from patient cells, from kidneys of knockin mice and from patient kidney organoids. MUC1-fs is trapped in TMED9 cargo receptor-containing vesicles of the early secretory pathway. BRD4780 binds TMED9, releases MUC1-fs, and re-routes it for lysosomal degradation, an effect phenocopied by TMED9 deletion. Our findings reveal BRD4780 as a promising lead for the treatment of MKD and other toxic proteinopathies. Generally, we elucidate a novel mechanism for the entrapment of misfolded proteins by cargo receptors and a strategy for their release and anterograde trafficking to the lysosome., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2019
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37. Discovering metabolic disease gene interactions by correlated effects on cellular morphology.
- Author
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Jiao Y, Ahmed U, Sim MFM, Bejar A, Zhang X, Talukder MMU, Rice R, Flannick J, Podgornaia AI, Reilly DF, Engreitz JM, Kost-Alimova M, Hartland K, Mercader JM, Georges S, Wagh V, Tadin-Strapps M, Doench JG, Edwardson JM, Rochford JJ, Rosen ED, and Majithia AR
- Subjects
- Acyltransferases genetics, Acyltransferases metabolism, Adipocytes metabolism, Adipocytes pathology, CCAAT-Enhancer-Binding Proteins genetics, CCAAT-Enhancer-Binding Proteins metabolism, Cells, Cultured, Diabetes Mellitus pathology, GTP-Binding Protein gamma Subunits genetics, GTP-Binding Protein gamma Subunits metabolism, HEK293 Cells, Humans, Insulin Resistance, Perilipin-1 genetics, Perilipin-1 metabolism, Phenotype, Transcriptome, Adipocytes cytology, Adipogenesis, Diabetes Mellitus genetics, Gene Regulatory Networks, Protein Interaction Maps
- Abstract
Objective: Impaired expansion of peripheral fat contributes to the pathogenesis of insulin resistance and Type 2 Diabetes (T2D). We aimed to identify novel disease-gene interactions during adipocyte differentiation., Methods: Genes in disease-associated loci for T2D, adiposity and insulin resistance were ranked according to expression in human adipocytes. The top 125 genes were ablated in human pre-adipocytes via CRISPR/CAS9 and the resulting cellular phenotypes quantified during adipocyte differentiation with high-content microscopy and automated image analysis. Morphometric measurements were extracted from all images and used to construct morphologic profiles for each gene., Results: Over 10
7 morphometric measurements were obtained. Clustering of the morphologic profiles accross all genes revealed a group of 14 genes characterized by decreased lipid accumulation, and enriched for known lipodystrophy genes. For two lipodystrophy genes, BSCL2 and AGPAT2, sub-clusters with PLIN1 and CEBPA identifed by morphological similarity were validated by independent experiments as novel protein-protein and gene regulatory interactions., Conclusions: A morphometric approach in adipocytes can resolve multiple cellular mechanisms for metabolic disease loci; this approach enables mechanistic interrogation of the hundreds of metabolic disease loci whose function still remains unknown., (Copyright © 2019 The Authors. Published by Elsevier GmbH.. All rights reserved.)- Published
- 2019
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38. Genome-Wide Interrogation of Human Cancers Identifies EGLN1 Dependency in Clear Cell Ovarian Cancers.
- Author
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Price C, Gill S, Ho ZV, Davidson SM, Merkel E, McFarland JM, Leung L, Tang A, Kost-Alimova M, Tsherniak A, Jonas O, Vazquez F, and Hahn WC
- Subjects
- CRISPR-Cas Systems, Cell Line, Tumor, Female, Humans, Ovarian Neoplasms pathology, RNA Interference, Genome-Wide Association Study, Hypoxia-Inducible Factor-Proline Dioxygenases genetics, Ovarian Neoplasms genetics
- Abstract
We hypothesized that candidate dependencies for which there are small molecules that are either approved or in advanced development for a nononcology indication may represent potential therapeutic targets. To test this hypothesis, we performed genome-scale loss-of-function screens in hundreds of cancer cell lines. We found that knockout of EGLN1 , which encodes prolyl hydroxylase domain-containing protein 2 (PHD2), reduced the proliferation of a subset of clear cell ovarian cancer cell lines in vitro . EGLN1-dependent cells exhibited sensitivity to the pan-EGLN inhibitor FG-4592. The response to FG-4592 was reversed by deletion of HIF1A, demonstrating that EGLN1 dependency was related to negative regulation of HIF1A. We also found that ovarian clear cell tumors susceptible to both genetic and pharmacologic inhibition of EGLN1 required intact HIF1A. Collectively, these observations identify EGLN1 as a cancer target with therapeutic potential. SIGNIFICANCE: These findings reveal a differential dependency of clear cell ovarian cancers on EGLN1, thus identifying EGLN1 as a potential therapeutic target in clear cell ovarian cancer patients., (©2019 American Association for Cancer Research.)
- Published
- 2019
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39. A GPX4-dependent cancer cell state underlies the clear-cell morphology and confers sensitivity to ferroptosis.
- Author
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Zou Y, Palte MJ, Deik AA, Li H, Eaton JK, Wang W, Tseng YY, Deasy R, Kost-Alimova M, Dančík V, Leshchiner ES, Viswanathan VS, Signoretti S, Choueiri TK, Boehm JS, Wagner BK, Doench JG, Clish CB, Clemons PA, and Schreiber SL
- Subjects
- Aged, Animals, Apoptosis genetics, Basic Helix-Loop-Helix Transcription Factors genetics, CRISPR-Cas Systems genetics, Carcinoma, Renal Cell genetics, Cell Line, Tumor, Female, Gene Expression Profiling, Gene Expression Regulation, Neoplastic, Gene Knockout Techniques, Glutathione Peroxidase genetics, HEK293 Cells, Humans, Iron metabolism, Kidney Neoplasms genetics, Lipid Peroxidation genetics, Male, Mice, Nude, Middle Aged, Neoplasm Proteins genetics, Phospholipid Hydroperoxide Glutathione Peroxidase, RNA Interference, Xenograft Model Antitumor Assays, Basic Helix-Loop-Helix Transcription Factors metabolism, Carcinoma, Renal Cell pathology, Glutathione Peroxidase metabolism, Kidney Neoplasms pathology, Neoplasm Proteins metabolism
- Abstract
Clear-cell carcinomas (CCCs) are a histological group of highly aggressive malignancies commonly originating in the kidney and ovary. CCCs are distinguished by aberrant lipid and glycogen accumulation and are refractory to a broad range of anti-cancer therapies. Here we identify an intrinsic vulnerability to ferroptosis associated with the unique metabolic state in CCCs. This vulnerability transcends lineage and genetic landscape, and can be exploited by inhibiting glutathione peroxidase 4 (GPX4) with small-molecules. Using CRISPR screening and lipidomic profiling, we identify the hypoxia-inducible factor (HIF) pathway as a driver of this vulnerability. In renal CCCs, HIF-2α selectively enriches polyunsaturated lipids, the rate-limiting substrates for lipid peroxidation, by activating the expression of hypoxia-inducible, lipid droplet-associated protein (HILPDA). Our study suggests targeting GPX4 as a therapeutic opportunity in CCCs, and highlights that therapeutic approaches can be identified on the basis of cell states manifested by morphological and metabolic features in hard-to-treat cancers.
- Published
- 2019
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40. Cell Painting, a high-content image-based assay for morphological profiling using multiplexed fluorescent dyes.
- Author
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Bray MA, Singh S, Han H, Davis CT, Borgeson B, Hartland C, Kost-Alimova M, Gustafsdottir SM, Gibson CC, and Carpenter AE
- Subjects
- Cell Line, Tumor, Cell Shape, Cell Size, Humans, Image Processing, Computer-Assisted, Fluorescent Dyes metabolism, Molecular Imaging methods, Staining and Labeling methods
- Abstract
In morphological profiling, quantitative data are extracted from microscopy images of cells to identify biologically relevant similarities and differences among samples based on these profiles. This protocol describes the design and execution of experiments using Cell Painting, which is a morphological profiling assay that multiplexes six fluorescent dyes, imaged in five channels, to reveal eight broadly relevant cellular components or organelles. Cells are plated in multiwell plates, perturbed with the treatments to be tested, stained, fixed, and imaged on a high-throughput microscope. Next, an automated image analysis software identifies individual cells and measures ∼1,500 morphological features (various measures of size, shape, texture, intensity, and so on) to produce a rich profile that is suitable for the detection of subtle phenotypes. Profiles of cell populations treated with different experimental perturbations can be compared to suit many goals, such as identifying the phenotypic impact of chemical or genetic perturbations, grouping compounds and/or genes into functional pathways, and identifying signatures of disease. Cell culture and image acquisition takes 2 weeks; feature extraction and data analysis take an additional 1-2 weeks., Competing Interests: The authors declare competing financial interests. Recursion Pharmaceuticals is a biotechnology company in which CCG, BB, CTD, HH, and AEC have real or optional ownership interest (see the HTML version of this article for details).
- Published
- 2016
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41. Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting.
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Aguirre AJ, Meyers RM, Weir BA, Vazquez F, Zhang CZ, Ben-David U, Cook A, Ha G, Harrington WF, Doshi MB, Kost-Alimova M, Gill S, Xu H, Ali LD, Jiang G, Pantel S, Lee Y, Goodale A, Cherniack AD, Oh C, Kryukov G, Cowley GS, Garraway LA, Stegmaier K, Roberts CW, Golub TR, Meyerson M, Root DE, Tsherniak A, and Hahn WC
- Subjects
- Cell Line, Tumor, DNA Cleavage, DNA Copy Number Variations, DNA Damage, G2 Phase Cell Cycle Checkpoints, Gene Amplification, Gene Editing, Gene Expression, Gene Knockout Techniques, Genes, Essential, High-Throughput Screening Assays, Humans, RNA, Guide, CRISPR-Cas Systems, CRISPR-Cas Systems, Clustered Regularly Interspaced Short Palindromic Repeats, Gene Dosage, Gene Targeting methods, Genomics methods
- Abstract
Unlabelled: The CRISPR/Cas9 system enables genome editing and somatic cell genetic screens in mammalian cells. We performed genome-scale loss-of-function screens in 33 cancer cell lines to identify genes essential for proliferation/survival and found a strong correlation between increased gene copy number and decreased cell viability after genome editing. Within regions of copy-number gain, CRISPR/Cas9 targeting of both expressed and unexpressed genes, as well as intergenic loci, led to significantly decreased cell proliferation through induction of a G2 cell-cycle arrest. By examining single-guide RNAs that map to multiple genomic sites, we found that this cell response to CRISPR/Cas9 editing correlated strongly with the number of target loci. These observations indicate that genome targeting by CRISPR/Cas9 elicits a gene-independent antiproliferative cell response. This effect has important practical implications for the interpretation of CRISPR/Cas9 screening data and confounds the use of this technology for the identification of essential genes in amplified regions., Significance: We found that the number of CRISPR/Cas9-induced DNA breaks dictates a gene-independent antiproliferative response in cells. These observations have practical implications for using CRISPR/Cas9 to interrogate cancer gene function and illustrate that cancer cells are highly sensitive to site-specific DNA damage, which may provide a path to novel therapeutic strategies. Cancer Discov; 6(8); 914-29. ©2016 AACR.See related commentary by Sheel and Xue, p. 824See related article by Munoz et al., p. 900This article is highlighted in the In This Issue feature, p. 803., (2016 American Association for Cancer Research.)
- Published
- 2016
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42. Structure-Guided Design of IACS-9571, a Selective High-Affinity Dual TRIM24-BRPF1 Bromodomain Inhibitor.
- Author
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Palmer WS, Poncet-Montange G, Liu G, Petrocchi A, Reyna N, Subramanian G, Theroff J, Yau A, Kost-Alimova M, Bardenhagen JP, Leo E, Shepard HE, Tieu TN, Shi X, Zhan Y, Zhao S, Barton MC, Draetta G, Toniatti C, Jones P, Geck Do M, and Andersen JN
- Subjects
- Adaptor Proteins, Signal Transducing chemistry, Animals, Benzimidazoles pharmacokinetics, Carrier Proteins chemistry, DNA-Binding Proteins, Female, Humans, Methylation, Mice, Molecular Docking Simulation, Nuclear Proteins chemistry, Protein Binding, Adaptor Proteins, Signal Transducing antagonists & inhibitors, Adaptor Proteins, Signal Transducing metabolism, Benzimidazoles chemistry, Benzimidazoles pharmacology, Carrier Proteins antagonists & inhibitors, Carrier Proteins metabolism, Drug Design, Nuclear Proteins antagonists & inhibitors, Nuclear Proteins metabolism
- Abstract
The bromodomain containing proteins TRIM24 (tripartite motif containing protein 24) and BRPF1 (bromodomain and PHD finger containing protein 1) are involved in the epigenetic regulation of gene expression and have been implicated in human cancer. Overexpression of TRIM24 correlates with poor patient prognosis, and BRPF1 is a scaffolding protein required for the assembly of histone acetyltransferase complexes, where the gene of MOZ (monocytic leukemia zinc finger protein) was first identified as a recurrent fusion partner in leukemia patients (8p11 chromosomal rearrangements). Here, we present the structure guided development of a series of N,N-dimethylbenzimidazolone bromodomain inhibitors through the iterative use of X-ray cocrystal structures. A unique binding mode enabled the design of a potent and selective inhibitor 8i (IACS-9571) with low nanomolar affinities for TRIM24 and BRPF1 (ITC Kd = 31 nM and ITC Kd = 14 nM, respectively). With its excellent cellular potency (EC50 = 50 nM) and favorable pharmacokinetic properties (F = 29%), 8i is a high-quality chemical probe for the evaluation of TRIM24 and/or BRPF1 bromodomain function in vitro and in vivo.
- Published
- 2016
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43. Development of novel cellular histone-binding and chromatin-displacement assays for bromodomain drug discovery.
- Author
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Zhan Y, Kost-Alimova M, Shi X, Leo E, Bardenhagen JP, Shepard HE, Appikonda S, Vangamudi B, Zhao S, Tieu TN, Jiang S, Heffernan TP, Marszalek JR, Toniatti C, Draetta G, Tyler J, Barton M, Jones P, Palmer WS, Geck Do MK, and Andersen JN
- Abstract
Background: Proteins that 'read' the histone code are central elements in epigenetic control and bromodomains, which bind acetyl-lysine motifs, are increasingly recognized as potential mediators of disease states. Notably, the first BET bromodomain-based therapies have entered clinical trials and there is a broad interest in dissecting the therapeutic relevance of other bromodomain-containing proteins in human disease. Typically, drug development is facilitated and expedited by high-throughput screening, where assays need to be sensitive, robust, cost-effective and scalable. However, for bromodomains, which lack catalytic activity that otherwise can be monitored (using classical enzymology), the development of cell-based, drug-target engagement assays has been challenging. Consequently, cell biochemical assays have lagged behind compared to other protein families (e.g., histone deacetylases and methyltransferases)., Results: Here, we present a suite of novel chromatin and histone-binding assays using AlphaLISA, in situ cell extraction and fluorescence-based, high-content imaging. First, using TRIM24 as an example, the homogenous, bead-based AlphaScreen technology was modified from a biochemical peptide-competition assay to measure binding of the TRIM24 bromodomain to endogenous histone H3 in cells (AlphaLISA). Second, a target agnostic, high-throughput imaging platform was developed to quantify the ability of chemical probes to dissociate endogenous proteins from chromatin/nuclear structures. While overall nuclear morphology is maintained, the procedure extracts soluble, non-chromatin-bound proteins from cells with drug-target displacement visualized by immunofluorescence (IF) or microscopy of fluorescent proteins. Pharmacological evaluation of these assays cross-validated their utility, sensitivity and robustness. Finally, using genetic and pharmacological approaches, we dissect domain contribution of TRIM24, BRD4, ATAD2 and SMARCA2 to chromatin binding illustrating the versatility/utility of the in situ cell extraction platform., Conclusions: In summary, we have developed two novel complementary and cell-based drug-target engagement assays, expanding the repertoire of pharmacodynamic assays for bromodomain tool compound development. These assays have been validated through a successful TRIM24 bromodomain inhibitor program, where a micromolar lead molecule (IACS-6558) was optimized using cell-based assays to yield the first single-digit nanomolar TRIM24 inhibitor (IACS-9571). Altogether, the assay platforms described herein are poised to accelerate the discovery and development of novel chemical probes to deliver on the promise of epigenetic-based therapies.
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- 2015
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44. The SMARCA2/4 ATPase Domain Surpasses the Bromodomain as a Drug Target in SWI/SNF-Mutant Cancers: Insights from cDNA Rescue and PFI-3 Inhibitor Studies.
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Vangamudi B, Paul TA, Shah PK, Kost-Alimova M, Nottebaum L, Shi X, Zhan Y, Leo E, Mahadeshwar HS, Protopopov A, Futreal A, Tieu TN, Peoples M, Heffernan TP, Marszalek JR, Toniatti C, Petrocchi A, Verhelle D, Owen DR, Draetta G, Jones P, Palmer WS, Sharma S, and Andersen JN
- Subjects
- Binding, Competitive, Catalysis, Cell Line, Tumor, Chromatin metabolism, Chromosomal Proteins, Non-Histone genetics, DNA Helicases chemistry, DNA Helicases deficiency, DNA, Complementary genetics, Gene Knockout Techniques, Genetic Complementation Test, Humans, Lung Neoplasms pathology, Microarray Analysis, Neoplasms genetics, Nuclear Proteins chemistry, Nuclear Proteins deficiency, Protein Structure, Tertiary, RNA Interference, RNA, Small Interfering pharmacology, Rhabdoid Tumor genetics, Rhabdoid Tumor pathology, Sarcoma, Synovial genetics, Sarcoma, Synovial pathology, Transcription Factors chemistry, Transcription Factors genetics, Azabicyclo Compounds pharmacology, Chromatin Assembly and Disassembly drug effects, Chromosomal Proteins, Non-Histone deficiency, DNA Helicases antagonists & inhibitors, Molecular Targeted Therapy, Neoplasm Proteins antagonists & inhibitors, Neoplasms drug therapy, Nuclear Proteins antagonists & inhibitors, Pyridines pharmacology, Transcription Factors antagonists & inhibitors, Transcription Factors deficiency
- Abstract
The SWI/SNF multisubunit complex modulates chromatin structure through the activity of two mutually exclusive catalytic subunits, SMARCA2 and SMARCA4, which both contain a bromodomain and an ATPase domain. Using RNAi, cancer-specific vulnerabilities have been identified in SWI/SNF-mutant tumors, including SMARCA4-deficient lung cancer; however, the contribution of conserved, druggable protein domains to this anticancer phenotype is unknown. Here, we functionally deconstruct the SMARCA2/4 paralog dependence of cancer cells using bioinformatics, genetic, and pharmacologic tools. We evaluate a selective SMARCA2/4 bromodomain inhibitor (PFI-3) and characterize its activity in chromatin-binding and cell-functional assays focusing on cells with altered SWI/SNF complex (e.g., lung, synovial sarcoma, leukemia, and rhabdoid tumors). We demonstrate that PFI-3 is a potent, cell-permeable probe capable of displacing ectopically expressed, GFP-tagged SMARCA2-bromodomain from chromatin, yet contrary to target knockdown, the inhibitor fails to display an antiproliferative phenotype. Mechanistically, the lack of pharmacologic efficacy is reconciled by the failure of bromodomain inhibition to displace endogenous, full-length SMARCA2 from chromatin as determined by in situ cell extraction, chromatin immunoprecipitation, and target gene expression studies. Furthermore, using inducible RNAi and cDNA complementation (bromodomain- and ATPase-dead constructs), we unequivocally identify the ATPase domain, and not the bromodomain of SMARCA2, as the relevant therapeutic target with the catalytic activity suppressing defined transcriptional programs. Taken together, our complementary genetic and pharmacologic studies exemplify a general strategy for multidomain protein drug-target validation and in case of SMARCA2/4 highlight the potential for drugging the more challenging helicase/ATPase domain to deliver on the promise of synthetic-lethality therapy., (©2015 American Association for Cancer Research.)
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- 2015
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45. Utilizing murine inducible telomerase alleles in the studies of tissue degeneration/regeneration and cancer.
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Shingu T, Jaskelioff M, Yuan L, Ding Z, Protopopov A, Kost-Alimova M, and Hu J
- Subjects
- Animals, Cells, Cultured, Female, Gene Knock-In Techniques methods, In Situ Hybridization, Fluorescence, Male, Mice, Mice, Inbred C57BL, Neoplasms pathology, Neural Stem Cells cytology, Neural Stem Cells enzymology, Telomere metabolism, Alleles, Neoplasms genetics, Regeneration genetics, Telomerase genetics
- Abstract
Telomere dysfunction-induced loss of genome integrity and its associated DNA damage signaling and checkpoint responses are well-established drivers that cause tissue degeneration during ageing. Cancer, with incidence rates greatly increasing with age, is characterized by short telomere lengths and high telomerase activity. To study the roles of telomere dysfunction and telomerase reactivation in ageing and cancer, the protocol shows how to generate two murine inducible telomerase knock-in alleles 4-Hydroxytamoxifen (4-OHT)-inducible TERT-Estrogen Receptor (mTERT-ER) and Lox-Stopper-LoxTERT (LSL-mTERT). The protocol describes the procedures to induce telomere dysfunction and reactivate telomerase activity in mTERT-ER and LSL-mTERT mice in vivo. The representative data show that reactivation of telomerase activity can ameliorate the tissue degenerative phenotypes induced by telomere dysfunction. In order to determine the impact of telomerase reactivation on tumorigenesis, we generated prostate tumor model G4 PB-Cre4 Pten(L/L) p53(L/L) LSL-mTERT(L/L) and thymic T-cell lymphoma model G4 Atm(-/-) mTERT(ER/ER). The representative data show that telomerase reactivation in the backdrop of genomic instability induced by telomere dysfunction can greatly enhance tumorigenesis. The protocol also describes the procedures used to isolate neural stem cells (NSCs) from mTERT-ER and LSL-mTERT mice and reactivate telomerase activity in NSCs in vitro. The representative data show that reactivation of telomerase can enhance the self-renewal capability and neurogenesis in vitro. Finally, the protocol describes the procedures for performing telomere FISH (Fluorescence In Situ Hybridization) on both mouse FFPE (Formalin Fixed and Paraffin Embedded) brain tissues and metaphase chromosomes of cultured cells.
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- 2015
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46. Oncogene ablation-resistant pancreatic cancer cells depend on mitochondrial function.
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Viale A, Pettazzoni P, Lyssiotis CA, Ying H, Sánchez N, Marchesini M, Carugo A, Green T, Seth S, Giuliani V, Kost-Alimova M, Muller F, Colla S, Nezi L, Genovese G, Deem AK, Kapoor A, Yao W, Brunetto E, Kang Y, Yuan M, Asara JM, Wang YA, Heffernan TP, Kimmelman AC, Wang H, Fleming JB, Cantley LC, DePinho RA, and Draetta GF
- Subjects
- Animals, Autophagy, Carcinoma, Pancreatic Ductal drug therapy, Carcinoma, Pancreatic Ductal genetics, Cell Respiration drug effects, Cell Survival drug effects, Disease Models, Animal, Female, Gene Expression Regulation, Neoplastic, Genes, p53 genetics, Glycolysis, Lysosomes metabolism, Mice, Mitochondria drug effects, Mutation genetics, Neoplasm Recurrence, Local prevention & control, Neoplastic Stem Cells drug effects, Neoplastic Stem Cells metabolism, Neoplastic Stem Cells pathology, Oxidative Phosphorylation drug effects, Pancreatic Neoplasms drug therapy, Pancreatic Neoplasms genetics, Proto-Oncogene Proteins p21(ras) metabolism, Recurrence, Signal Transduction, Pancreatic Neoplasms, Carcinoma, Pancreatic Ductal metabolism, Carcinoma, Pancreatic Ductal pathology, Mitochondria metabolism, Pancreatic Neoplasms metabolism, Pancreatic Neoplasms pathology, Proto-Oncogene Proteins p21(ras) genetics
- Abstract
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers in western countries, with a median survival of 6 months and an extremely low percentage of long-term surviving patients. KRAS mutations are known to be a driver event of PDAC, but targeting mutant KRAS has proved challenging. Targeting oncogene-driven signalling pathways is a clinically validated approach for several devastating diseases. Still, despite marked tumour shrinkage, the frequency of relapse indicates that a fraction of tumour cells survives shut down of oncogenic signalling. Here we explore the role of mutant KRAS in PDAC maintenance using a recently developed inducible mouse model of mutated Kras (Kras(G12D), herein KRas) in a p53(LoxP/WT) background. We demonstrate that a subpopulation of dormant tumour cells surviving oncogene ablation (surviving cells) and responsible for tumour relapse has features of cancer stem cells and relies on oxidative phosphorylation for survival. Transcriptomic and metabolic analyses of surviving cells reveal prominent expression of genes governing mitochondrial function, autophagy and lysosome activity, as well as a strong reliance on mitochondrial respiration and a decreased dependence on glycolysis for cellular energetics. Accordingly, surviving cells show high sensitivity to oxidative phosphorylation inhibitors, which can inhibit tumour recurrence. Our integrated analyses illuminate a therapeutic strategy of combined targeting of the KRAS pathway and mitochondrial respiration to manage pancreatic cancer.
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- 2014
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47. Telomere dysfunction suppresses multiple endocrine neoplasia in mice.
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Lee JH, Anver M, Kost-Alimova M, Protopopov A, DePinho RA, and Rane SG
- Abstract
Multiple endocrine neoplasia (MEN) syndrome is typified by the occurrence of tumors in two or more hormonal tissues. Whereas the genetics of MEN syndrome is relatively well understood, the tumorigenic mechanisms for these cancers remain relatively obscure. The Cdk4 (R24C) mouse model develops highly penetrant pituitary tumors and endocrine pancreas adenomas, and, as such, this model is appropriate to gain insight into mechanisms underlying MEN. Using this model, here we provide evidence supporting an important role for telomerase in the pathogenesis of MEN. We observed increased aneuploidy in Cdk4 (R/R) fibroblasts along with significantly elevated telomerase activity and telomere length in Cdk4 (R/R) islets and embryonic fibroblasts. To better understand the role of telomerase, we generated Cdk4 (R24C) mice with inactivation of the mTERC locus, which codes for the essential RNA component of the enzyme telomerase (mTERC (-/-) Cdk4 (R/R) mice). Embryonic fibroblasts and islets derived from mTERC (-/-) Cdk4 (R/R) mice exhibit reduced telomere length and proliferative capacity. Further, mTERC (-/-) Cdk4 (R/R) fibroblasts display reduced transformation potential. Importantly, mTERC (-/-) Cdk4 (R/R) mice display significantly reduced spontaneous tumorigenesis. Strikingly, we observed dramatic suppression of pituitary tumors and endocrine pancreas adenomas in mTERC (-/-) Cdk4 (R/R) mice. Telomere dysfunction suppressed tumor initiation and increased latency of tumor development while not affecting the progression of established tumors. In summary, these results are suggestive of an important role for telomerase in tumor development in the Cdk4 (R24C) mouse model, specifically in the genesis of tumors in the pituitary and the endocrine pancreas.
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- 2014
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48. Role of telomere dysfunction in cardiac failure in Duchenne muscular dystrophy.
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Mourkioti F, Kustan J, Kraft P, Day JW, Zhao MM, Kost-Alimova M, Protopopov A, DePinho RA, Bernstein D, Meeker AK, and Blau HM
- Subjects
- Animals, Cell Size, Disease Models, Animal, Dystrophin genetics, Humans, Mice, Mice, Inbred mdx, Myocytes, Cardiac pathology, Heart Failure etiology, Muscular Dystrophy, Duchenne complications, Muscular Dystrophy, Duchenne genetics, Telomere pathology
- Abstract
Duchenne muscular dystrophy (DMD), the most common inherited muscular dystrophy of childhood, leads to death due to cardiorespiratory failure. Paradoxically, mdx mice with the same genetic deficiency of dystrophin exhibit minimal cardiac dysfunction, impeding the development of therapies. We postulated that the difference between mdx and DMD might result from differences in telomere lengths in mice and humans. We show here that, like DMD patients, mice that lack dystrophin and have shortened telomeres (mdx/mTR(KO)) develop severe functional cardiac deficits including ventricular dilation, contractile and conductance dysfunction, and accelerated mortality. These cardiac defects are accompanied by telomere erosion, mitochondrial fragmentation and increased oxidative stress. Treatment with antioxidants significantly retards the onset of cardiac dysfunction and death of mdx/mTR(KO) mice. In corroboration, all four of the DMD patients analysed had 45% shorter telomeres in their cardiomyocytes relative to age- and sex-matched controls. We propose that the demands of contraction in the absence of dystrophin coupled with increased oxidative stress conspire to accelerate telomere erosion culminating in cardiac failure and death. These findings provide strong support for a link between telomere length and dystrophin deficiency in the etiology of dilated cardiomyopathy in DMD and suggest preventive interventions.
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- 2013
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49. Telomerase reactivation following telomere dysfunction yields murine prostate tumors with bone metastases.
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Ding Z, Wu CJ, Jaskelioff M, Ivanova E, Kost-Alimova M, Protopopov A, Chu GC, Wang G, Lu X, Labrot ES, Hu J, Wang W, Xiao Y, Zhang H, Zhang J, Zhang J, Gan B, Perry SR, Jiang S, Li L, Horner JW, Wang YA, Chin L, and DePinho RA
- Subjects
- Animals, Bone Neoplasms secondary, Cell Line, Tumor, Crosses, Genetic, DNA Copy Number Variations, Disease Models, Animal, Female, Genomic Instability, Humans, Male, Mice, Tumor Suppressor Protein p53 metabolism, Prostatic Neoplasms genetics, Prostatic Neoplasms pathology, Telomerase metabolism, Telomere metabolism
- Abstract
To determine the role of telomere dysfunction and telomerase reactivation in generating pro-oncogenic genomic events and in carcinoma progression, an inducible telomerase reverse transcriptase (mTert) allele was crossed onto a prostate cancer-prone mouse model null for Pten and p53 tumor suppressors. Constitutive telomerase deficiency and associated telomere dysfunction constrained cancer progression. In contrast, telomerase reactivation in the setting of telomere dysfunction alleviated intratumoral DNA-damage signaling and generated aggressive cancers with rearranged genomes and new tumor biological properties (bone metastases). Comparative oncogenomic analysis revealed numerous recurrent amplifications and deletions of relevance to human prostate cancer. Murine tumors show enrichment of the TGF-β/SMAD4 network, and genetic validation studies confirmed the cooperative roles of Pten, p53, and Smad4 deficiencies in prostate cancer progression, including skeletal metastases. Thus, telomerase reactivation in tumor cells experiencing telomere dysfunction enables full malignant progression and provides a mechanism for acquisition of cancer-relevant genomic events endowing new tumor biological capabilities., (Copyright © 2012 Elsevier Inc. All rights reserved.)
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- 2012
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50. Antitelomerase therapy provokes ALT and mitochondrial adaptive mechanisms in cancer.
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Hu J, Hwang SS, Liesa M, Gan B, Sahin E, Jaskelioff M, Ding Z, Ying H, Boutin AT, Zhang H, Johnson S, Ivanova E, Kost-Alimova M, Protopopov A, Wang YA, Shirihai OS, Chin L, and DePinho RA
- Subjects
- Animals, Ataxia Telangiectasia Mutated Proteins, Cell Cycle Proteins genetics, DNA-Binding Proteins genetics, Gene Knockdown Techniques, Genes, cdc, Humans, Lymphoma, T-Cell genetics, Lymphoma, T-Cell metabolism, Lymphoma, T-Cell pathology, Mice, Neoplasm Invasiveness pathology, Neoplasms genetics, Neoplasms metabolism, Protein Serine-Threonine Kinases genetics, Reactive Oxygen Species metabolism, Receptors, Estrogen metabolism, Superoxide Dismutase genetics, Superoxide Dismutase metabolism, Telomerase genetics, Telomerase metabolism, Transcription Factors genetics, Transcription Factors metabolism, Tumor Suppressor Proteins genetics, Mitochondria metabolism, Telomerase antagonists & inhibitors, Telomere Homeostasis
- Abstract
To assess telomerase as a cancer therapeutic target and determine adaptive mechanisms to telomerase inhibition, we modeled telomerase reactivation and subsequent extinction in T cell lymphomas arising in Atm(-/-) mice engineered with an inducible telomerase reverse transcriptase allele. Telomerase reactivation in the setting of telomere dysfunction enabled full malignant progression with alleviation of telomere dysfunction-induced checkpoints. These cancers possessed copy number alterations targeting key loci in human T cell lymphomagenesis. Upon telomerase extinction, tumor growth eventually slowed with reinstatement of telomere dysfunction-induced checkpoints, yet growth subsequently resumed as tumors acquired alternative lengthening of telomeres (ALT) and aberrant transcriptional networks centering on mitochondrial biology and oxidative defense. ALT+ tumors acquired amplification/overexpression of PGC-1β, a master regulator of mitochondrial biogenesis and function, and they showed marked sensitivity to PGC-1β or SOD2 knockdown. Genetic modeling of telomerase extinction reveals vulnerabilities that motivate coincidental inhibition of mitochondrial maintenance and oxidative defense mechanisms to enhance antitelomerase cancer therapy., (Copyright © 2012 Elsevier Inc. All rights reserved.)
- Published
- 2012
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