Technical Advance
Abstract
To date, inheritance of a mutant BRCA1 or BRCA2 gene is the best-established indicator of an increased risk of developing breast cancer. Sequence analysis of these genes is being used to identify BRCA1/2 mutation carriers, though these efforts are hampered by the high frequency of variants of unknown clinical significance (VUSs). Functional evaluation of such variants has been restricted due to lack of a physiologically relevant assay. In this study we developed a functional assay using mouse ES cells to study variants of BRCA1. We introduced BAC clones with human wild-type BRCA1 or variants into Brca1-null ES cells and confirmed that only wild-type and a known neutral variant rescued cell lethality. The same neutral variant was also able to rescue embryogenesis in Brca1-null mice. A test of several BRCT domain mutants revealed all to be deleterious, including a VUS. Furthermore, we used this assay to determine the effects of BRCA1 variants on cell cycle regulation, differentiation, and genomic stability. Importantly, we discovered that ES cells rescued by S1497A BRCA1 exhibited significant hypersensitivity after γ-irradiation. Our results demonstrate that this ES cell–based assay is a powerful and reliable method for analyzing the functional impact of BRCA1 variants, which we believe could be used to determine which patients may require preventative treatments.
Authors
Suhwan Chang, Kajal Biswas, Betty K. Martin, Stacey Stauffer, Shyam K. Sharan
Abstract
The presence of circulating tumor cells (CTCs) in the peripheral blood is associated with short survival, making the detection of CTCs clinically useful as a prognostic factor of disease outcome and/or a surrogate marker of treatment response. Recent technical advances in immunocytometric analysis and quantitative real-time PCR have made it possible to detect a few CTCs in the blood; however, there is no sensitive assay to specifically detect viable CTCs. Here, we report what we believe to be a new approach to visually detect live human CTCs among millions of peripheral blood leukocytes, using a telomerase-specific replication-selective adenovirus expressing GFP. First, we constructed a GFP-expressing attenuated adenovirus, in which the telomerase promoter regulates viral replication (OBP-401; TelomeScan). We then used OBP-401 to establish a simple ex vivo method that was able to detect viable human CTCs in the peripheral blood. The detection method involved a 3-step procedure, including the lysis of rbc, the subsequent addition of OBP-401 to the cell pellets, and an automated scan using fluorescence microscopy. OBP-401 infection increased the signal-to-background ratio as a tumor-specific probe, because the fluorescent signal was amplified only in viable, infected human tumor cells, by viral replication. This GFP-expressing virus-based method is remarkably simple and allows precise enumeration of CTCs.
Authors
Toru Kojima, Yuuri Hashimoto, Yuichi Watanabe, Shunsuke Kagawa, Futoshi Uno, Shinji Kuroda, Hiroshi Tazawa, Satoru Kyo, Hiroyuki Mizuguchi, Yasuo Urata, Noriaki Tanaka, Toshiyoshi Fujiwara
Abstract
Basic research into human mature myelomonocytic cell function, myeloid lineage diversification and leukemic transformation, and assessment of myelotoxicity in preclinical drug development requires a constant supply of donor blood or bone marrow samples and laborious purification of mature myeloid cells or progenitors, which are present in very small quantities. To overcome these limitations, we have developed a protocol for efficient generation of neutrophils, eosinophils, macrophages, osteoclasts, DCs, and Langerhans cells from human embryonic stem cells (hESCs). As a first step, we generated lin–CD34+CD43+CD45+ hematopoietic cells highly enriched in myeloid progenitors through coculture of hESCs with OP9 feeder cells. After expansion in the presence of GM-CSF, these cells were directly differentiated with specific cytokine combinations toward mature cells of particular types. Morphologic, phenotypic, molecular, and functional analyses revealed that hESC-derived myelomonocytic cells were comparable to their corresponding somatic counterparts. In addition, we demonstrated that a similar protocol could be used to generate myelomonocytic cells from induced pluripotent stem cells (iPSCs). This technology offers an opportunity to generate large numbers of patient-specific myelomonocytic cells for in vitro studies of human disease mechanisms as well as for drug screening.
Authors
Kyung-Dal Choi, Maxim A. Vodyanik, Igor I. Slukvin
Abstract
The in vivo application of cytolytic peptides for cancer therapeutics is hampered by toxicity, nonspecificity, and degradation. We previously developed a specific strategy to synthesize a nanoscale delivery vehicle for cytolytic peptides by incorporating the nonspecific amphipathic cytolytic peptide melittin into the outer lipid monolayer of a perfluorocarbon nanoparticle. Here, we have demonstrated that the favorable pharmacokinetics of this nanocarrier allows accumulation of melittin in murine tumors in vivo and a dramatic reduction in tumor growth without any apparent signs of toxicity. Furthermore, direct assays demonstrated that molecularly targeted nanocarriers selectively delivered melittin to multiple tumor targets, including endothelial and cancer cells, through a hemifusion mechanism. In cells, this hemifusion and transfer process did not disrupt the surface membrane but did trigger apoptosis and in animals caused regression of precancerous dysplastic lesions. Collectively, these data suggest that the ability to restrain the wide-spectrum lytic potential of a potent cytolytic peptide in a nanovehicle, combined with the flexibility of passive or active molecular targeting, represents an innovative molecular design for chemotherapy with broad-spectrum cytolytic peptides for the treatment of cancer at multiple stages.
Authors
Neelesh R. Soman, Steven L. Baldwin, Grace Hu, Jon N. Marsh, Gregory M. Lanza, John E. Heuser, Jeffrey M. Arbeit, Samuel A. Wickline, Paul H. Schlesinger
Abstract
Dorsal root ganglion (DRG) neuron dysfunction occurs in a variety of sensory neuronopathies for which there are currently no satisfactory treatments. Here we describe the development of a strategy to target therapeutic genes to DRG neurons for the treatment of these disorders. We genetically modified an adenovirus (Ad) to generate a helper virus (HV) that was detargeted for native adenoviral tropism and contained DRG homing peptides in the adenoviral capsid fiber protein; we used this HV to generate DRG-targeted helper-dependent Ad (HDAd). In mice, intrathecal injection of this HDAd produced a 100-fold higher transduction of DRG neurons and a markedly attenuated inflammatory response compared with unmodified HDAd. We also injected HDAd encoding the β subunit of β-hexosaminidase (Hexb) into Hexb-deficient mice, a model of the neuronopathy Sandhoff disease. Delivery of the DRG-targeted HDAd reinstated neuron-specific Hexb production, reversed gangliosidosis, and ameliorated peripheral sensory dysfunction. The development of DRG neuron–targeted HDAd with proven efficacy in a preclinical model may have implications for the treatment of sensory neuronopathies of diverse etiologies.
Authors
Tomoya Terashima, Kazuhiro Oka, Angelika B. Kritz, Hideto Kojima, Andrew H. Baker, Lawrence Chan
Abstract
Liver sinusoidal endothelial cells are a major endogenous source of Factor VIII (FVIII), lack of which causes the human congenital bleeding disorder hemophilia A. Despite extensive efforts, gene therapy using viral vectors has shown little success in clinical hemophilia trials. Here we achieved cell type–specific gene targeting using hyaluronan- and asialoorosomucoid-coated nanocapsules, generated using dispersion atomization, to direct genes to liver sinusoidal endothelial cells and hepatocytes, respectively. To highlight the therapeutic potential of this approach, we encapsulated Sleeping Beauty transposon expressing the B domain–deleted canine FVIII in cis with Sleeping Beauty transposase in hyaluronan nanocapsules and injected them intravenously into hemophilia A mice. The treated mice exhibited activated partial thromboplastin times that were comparable to those of wild-type mice at 5 and 50 weeks and substantially shorter than those of untreated controls at the same time points. Further, plasma FVIII activity in the treated hemophilia A mice was nearly identical to that in wild-type mice through 50 weeks, while untreated hemophilia A mice exhibited no detectable FVIII activity. Thus, Sleeping Beauty transposon targeted to liver sinusoidal endothelial cells provided long-term expression of FVIII, without apparent antibody formation, and improved the phenotype of hemophilia A mice.
Authors
Betsy T. Kren, Gretchen M. Unger, Lucas Sjeklocha, Alycia A. Trossen, Vicci Korman, Brenda M. Diethelm-Okita, Mark T. Reding, Clifford J. Steer
Abstract
Acute promyelocytic leukemia (APL) is characterized by the t(15;17) chromosomal translocation, which results in fusion of the retinoic acid receptor α (RARA) gene to another gene, most commonly promyelocytic leukemia (PML). The resulting fusion protein, PML-RARA, initiates APL, which is a subtype (M3) of acute myeloid leukemia (AML). In this report, we identify a gene expression signature that is specific to M3 samples; it was not found in other AML subtypes and did not simply represent the normal gene expression pattern of primary promyelocytes. To validate this signature for a large number of genes, we tested a recently developed high throughput digital technology (NanoString nCounter). Nearly all of the genes tested demonstrated highly significant concordance with our microarray data (P < 0.05). The validated gene signature reliably identified M3 samples in 2 other AML datasets, and the validated genes were substantially enriched in our mouse model of APL, but not in a cell line that inducibly expressed PML-RARA. These results demonstrate that nCounter is a highly reproducible, customizable system for mRNA quantification using limited amounts of clinical material, which provides a valuable tool for biomarker measurement in low-abundance patient samples.
Authors
Jacqueline E. Payton, Nicole R. Grieselhuber, Li-Wei Chang, Mark Murakami, Gary K. Geiss, Daniel C. Link, Rakesh Nagarajan, Mark A. Watson, Timothy J. Ley
Abstract
Somatic genetic alterations in cancers have been linked with response to targeted therapeutics by creation of specific dependency on activated oncogenic signaling pathways. However, no tools currently exist to systematically connect such genetic lesions to therapeutic vulnerability. We have therefore developed a genomics approach to identify lesions associated with therapeutically relevant oncogene dependency. Using integrated genomic profiling, we have demonstrated that the genomes of a large panel of human non–small cell lung cancer (NSCLC) cell lines are highly representative of those of primary NSCLC tumors. Using cell-based compound screening coupled with diverse computational approaches to integrate orthogonal genomic and biochemical data sets, we identified molecular and genomic predictors of therapeutic response to clinically relevant compounds. Using this approach, we showed that v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations confer enhanced Hsp90 dependency and validated this finding in mice with KRAS-driven lung adenocarcinoma, as these mice exhibited dramatic tumor regression when treated with an Hsp90 inhibitor. In addition, we found that cells with copy number enhancement of v-abl Abelson murine leukemia viral oncogene homolog 2 (ABL2) and ephrin receptor kinase and v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) (SRC) kinase family genes were exquisitely sensitive to treatment with the SRC/ABL inhibitor dasatinib, both in vitro and when it xenografted into mice. Thus, genomically annotated cell-line collections may help translate cancer genomics information into clinical practice by defining critical pathway dependencies amenable to therapeutic inhibition.
Authors
Martin L. Sos, Kathrin Michel, Thomas Zander, Jonathan Weiss, Peter Frommolt, Martin Peifer, Danan Li, Roland Ullrich, Mirjam Koker, Florian Fischer, Takeshi Shimamura, Daniel Rauh, Craig Mermel, Stefanie Fischer, Isabel Stückrath, Stefanie Heynck, Rameen Beroukhim, William Lin, Wendy Winckler, Kinjal Shah, Thomas LaFramboise, Whei F. Moriarty, Megan Hanna, Laura Tolosi, Jörg Rahnenführer, Roel Verhaak, Derek Chiang, Gad Getz, Martin Hellmich, Jürgen Wolf, Luc Girard, Michael Peyton, Barbara A. Weir, Tzu-Hsiu Chen, Heidi Greulich, Jordi Barretina, Geoffrey I. Shapiro, Levi A. Garraway, Adi F. Gazdar, John D. Minna, Matthew Meyerson, Kwok-Kin Wong, Roman K. Thomas
Abstract
The uptake of lipoproteins by macrophages is a critical step in the development of atherosclerotic lesions. Cultured monocyte-derived macrophages take up large amounts of native LDL by receptor-independent fluid-phase pinocytosis, either constitutively or in response to specific activating stimuli, depending on the macrophage phenotype. We therefore sought to determine whether fluid-phase pinocytosis occurs in vivo in macrophages in atherosclerotic lesions. We demonstrated that fluorescent pegylated nanoparticles similar in size to LDL (specifically nontargeted Qtracker quantum dot and AngioSPARK nanoparticles) can serve as models of LDL uptake by fluid-phase pinocytosis in cultured human monocyte–derived macrophages and mouse bone marrow–derived macrophages. Using fluorescence microscopy, we showed that atherosclerosis-prone Apoe-knockout mice injected with these nanoparticles displayed massive accumulation of the nanoparticles within CD68+ macrophages, including lipid-containing foam cells, in atherosclerotic lesions in the aortic arch. Similar results were obtained when atherosclerotic mouse aortas were cultured with nanoparticles in vitro. These results show that macrophages within atherosclerotic lesions can take up LDL-sized nanoparticles by fluid-phase pinocytosis and indicate that fluid-phase pinocytosis of LDL is a mechanism for macrophage foam cell formation in vivo.
Authors
Chiara Buono, Joshua J. Anzinger, Marcelo Amar, Howard S. Kruth
Abstract
Our aging society is confronted with a dramatic increase of patients suffering from tauopathies, which include Alzheimer disease and certain frontotemporal dementias. These disorders are characterized by typical neuropathological lesions including hyperphosphorylation and subsequent aggregation of TAU protein and neuronal cell death. Currently, no mechanism-based cures are available. We generated fluorescently labeled TAU transgenic zebrafish, which rapidly recapitulated key pathological features of tauopathies, including phosphorylation and conformational changes of human TAU protein, tangle formation, neuronal and behavioral disturbances, and cell death. Due to their optical transparency and small size, zebrafish larvae are well suited for both in vivo imaging and drug development. TAU-induced neuronal cell death was imaged by time-lapse microscopy in vivo. Furthermore, we used this zebrafish model to identify compounds targeting the TAU kinase glycogen synthase kinase 3β (GSK3β). We identified a newly developed highly active GSK3β inhibitor, AR-534, by rational drug design. AR-534 reduced TAU phosphorylation in TAU transgenic zebrafish. This transgenic zebrafish model may become a valuable tool for further studies of the neuropathology of dementia.
Authors
Dominik Paquet, Ratan Bhat, Astrid Sydow, Eva-Maria Mandelkow, Stefan Berg, Sven Hellberg, Johanna Fälting, Martin Distel, Reinhard W. Köster, Bettina Schmid, Christian Haass
Copyright © 2025 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)