Journal articles on the topic 'CRISPR-Ca'
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Molina, Rafael, Anne Louise Grøn Jensen, Javier Marchena-Hurtado, Blanca López-Méndez, Stefano Stella, and Guillermo Montoya. "Structural basis of cyclic oligoadenylate degradation by ancillary Type III CRISPR-Cas ring nucleases." Nucleic Acids Research 49, no. 21 (November 26, 2021): 12577–90. http://dx.doi.org/10.1093/nar/gkab1130.
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Abstract Type III CRISPR-Cas effector systems detect foreign RNA triggering DNA and RNA cleavage and synthesizing cyclic oligoadenylate molecules (cA) in their Cas10 subunit. cAs act as a second messenger activating auxiliary nucleases, leading to an indiscriminate RNA degradation that can end in cell dormancy or death. Standalone ring nucleases are CRISPR ancillary proteins which downregulate the strong immune response of Type III systems by degrading cA. These enzymes contain a CRISPR-associated Rossman-fold (CARF) domain, which binds and cleaves the cA molecule. Here, we present the structures of the standalone ring nuclease from Sulfolobus islandicus (Sis) 0811 in its apo and post-catalytic states. This enzyme is composed by a N-terminal CARF and a C-terminal wHTH domain. Sis0811 presents a phosphodiester hydrolysis metal-independent mechanism, which cleaves cA4 rings to generate linear adenylate species, thus reducing the levels of the second messenger and switching off the cell antiviral state. The structural and biochemical analysis revealed the coupling of a cork-screw conformational change with the positioning of key catalytic residues to proceed with cA4 phosphodiester hydrolysis in a non-concerted manner.
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Starkuviene, Vytaute, Stefan M. Kallenberger, Nina Beil, Tautvydas Lisauskas, Bastian So-Song Schumacher, Ruben Bulkescher, Piotr Wajda, Manuel Gunkel, Jürgen Beneke, and Holger Erfle. "High-Density Cell Arrays for Genome-Scale Phenotypic Screening." SLAS DISCOVERY: Advancing the Science of Drug Discovery 24, no. 3 (January 25, 2019): 274–83. http://dx.doi.org/10.1177/2472555218818757.
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Due to high associated costs and considerable time investments of cell-based screening, there is a strong demand for new technologies that enable preclinical development and tests of diverse biologicals in a cost-saving and time-efficient manner. For those reasons we developed the high-density cell array (HD-CA) platform, which miniaturizes cell-based screening in the form of preprinted and ready-to-run screening arrays. With the HD-CA technology, up to 24,576 samples can be tested in a single experiment, thereby saving costs and time for microscopy-based screening by 75%. Experiments on the scale of the entire human genome can be addressed in a real parallel manner, with screening campaigns becoming more comfortable and devoid of robotics infrastructure on the user side. The high degree of miniaturization enables working with expensive reagents and rare and difficult-to-obtain cell lines. We have also optimized an automated imaging procedure for HD-CA and demonstrate the applicability of HD-CA to CRISPR-Cas9- and RNAi-mediated phenotypic assessment of the gene function.
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Lee, Ji Young, Mikhail Alexeyev, Natalya Kozhukhar, Viktoriya Pastukh, Roderica White, and Troy Stevens. "Carbonic anhydrase IX is a critical determinant of pulmonary microvascular endothelial cell pH regulation and angiogenesis during acidosis." American Journal of Physiology-Lung Cellular and Molecular Physiology 315, no. 1 (July 1, 2018): L41—L51. http://dx.doi.org/10.1152/ajplung.00446.2017.
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Carbonic anhydrase IX (CA IX) is highly expressed in rapidly proliferating and highly glycolytic cells, where it serves to enhance acid-regulatory capacity. Pulmonary microvascular endothelial cells (PMVECs) actively utilize aerobic glycolysis and acidify media, whereas pulmonary arterial endothelial cells (PAECs) primarily rely on oxidative phosphorylation and minimally change media pH. Therefore, we hypothesized that CA IX is critical to PMVEC angiogenesis because of its important role in regulating pH. To test this hypothesis, PMVECs and PAECs were isolated from Sprague-Dawley rats. CA IX knockout PMVECs were generated using the CRISPR-Cas9 technique. During serum-stimulated growth, mild acidosis (pH 6.8) did not affect cell counts of PMVECs, but it decreased PAEC cell number. Severe acidosis (pH 6.2) decreased cell counts of PMVECs and elicited an even more pronounced reduction of PAECs. PMVECs had a higher CA IX expression compared with PAECs. CA activity was higher in PMVECs compared with PAECs, and enzyme activity was dependent on the type IX isoform. Pharmacological inhibition and genetic ablation of CA IX caused profound dysregulation of extra- and intracellular pH in PMVECs. Matrigel assays revealed impaired angiogenesis of CA IX knockout PMVECs in acidosis. Lastly, pharmacological CA IX inhibition caused profound cell death in PMVECs, whereas genetic CA IX ablation had little effect on PMVEC cell death in acidosis. Thus CA IX controls PMVEC pH necessary for angiogenesis during acidosis. CA IX may contribute to lung vascular repair during acute lung injury that is accompanied by acidosis within the microenvironment.
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Wang, Longlong, Jianjun Liang, Yu Zhou, Tao Tian, Baoli Zhang, and Deqiang Duanmu. "Molecular Characterization of Carbonic Anhydrase Genes in Lotus japonicus and Their Potential Roles in Symbiotic Nitrogen Fixation." International Journal of Molecular Sciences 22, no. 15 (July 21, 2021): 7766. http://dx.doi.org/10.3390/ijms22157766.
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Carbonic anhydrase (CA) plays a vital role in photosynthetic tissues of higher plants, whereas its non-photosynthetic role in the symbiotic root nodule was rarely characterized. In this study, 13 CA genes were identified in the model legume Lotus japonicus by comparison with Arabidopsis CA genes. Using qPCR and promoter-reporter fusion methods, three previously identified nodule-enhanced CA genes (LjαCA2, LjαCA6, and LjβCA1) have been further characterized, which exhibit different spatiotemporal expression patterns during nodule development. LjαCA2 was expressed in the central infection zone of the mature nodule, including both infected and uninfected cells. LjαCA6 was restricted to the vascular bundle of the root and nodule. As for LjβCA1, it was expressed in most cell types of nodule primordia but only in peripheral cortical cells and uninfected cells of the mature nodule. Using CRISPR/Cas9 technology, the knockout of LjβCA1 or both LjαCA2 and its homolog, LjαCA1, did not result in abnormal symbiotic phenotype compared with the wild-type plants, suggesting that LjβCA1 or LjαCA1/2 are not essential for the nitrogen fixation under normal symbiotic conditions. Nevertheless, the nodule-enhanced expression patterns and the diverse distributions in different types of cells imply their potential functions during root nodule symbiosis, such as CO2 fixation, N assimilation, and pH regulation, which await further investigations.
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Liu, Xiaoyan, Yu Zhang, Su Wang, Guoying Liu, and Liming Ruan. "Loss of miR-143 and miR-145 in condyloma acuminatum promotes cellular proliferation and inhibits apoptosis by targeting NRAS." Royal Society Open Science 5, no. 8 (August 2018): 172376. http://dx.doi.org/10.1098/rsos.172376.
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The expression profile of miRNAs and their function in condyloma acuminatum (CA) remains unknown. In this study, we aimed to detect the effects of miR-143 and miR-145, the most downregulated in CA samples using high-throughput sequencing, on cell proliferation and apoptosis, to determine a novel therapeutic target for CA recurrence. RT-qPCR was used to validate the lower expression of miR-143 and miR-145 in a larger size of CA samples, and the expression of NRAS in CA samples was significantly higher than self-controls as determined by western blotting assay. Luciferase assay was performed to confirm that miR-143 or miR-145 targeted NRAS directly. Transduction of LV-pre-miR-143 or LV-pre-miR-145 to human papilloma virus (HPV)-infected SiHa cells led to reduced proliferation, greater apoptosis and inhibition of expression of NRAS, PI3 K p110 α and p-AKT. However, knockout of miR-143 or miR-145 in human epidermal keratinocytes by delivery of CRISPR/CAS9-gRNA for target miRNAs protected cells from apoptosis and upregulated expression of target genes as described above. MiR-143 and miR-145 sensitized cells to nutlin-3a, a p53 activator and MDM2 antagonist, while their loss protected cells from the stress of nutlin-3a. Furthermore, siRNA targeting NRAS showed similar effects on proliferation and apoptosis as miR-143 or miR-145. Taken together, our results suggest that loss of miR-143 or miR-145 in CA protects HPV-infected cells from apoptosis induced by environmental stress, in addition to promoting cellular proliferation and inhibiting apoptosis by targeting NRAS/PI3 K/ATK. Restoration of miR-143 or miR-145 might provide an applicable and novel approach to block the recurrence and progression of CA.
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Lee, Ji Young, Mher Onanyan, Ian Garrison, Roderica White, Maura Crook, Mikhail F. Alexeyev, Natalya Kozhukhar, et al. "Extrinsic acidosis suppresses glycolysis and migration while increasing network formation in pulmonary microvascular endothelial cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 317, no. 2 (August 1, 2019): L188—L201. http://dx.doi.org/10.1152/ajplung.00544.2018.
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Acidosis is common among critically ill patients, but current approaches to correct pH do not improve disease outcomes. During systemic acidosis, cells are either passively exposed to extracellular acidosis that other cells have generated (extrinsic acidosis) or they are exposed to acid that they generate and export into the extracellular space (intrinsic acidosis). Although endothelial repair following intrinsic acidosis has been studied, the impact of extrinsic acidosis on migration and angiogenesis is unclear. We hypothesized that extrinsic acidosis inhibits metabolism and migration but promotes capillary-like network formation in pulmonary microvascular endothelial cells (PMVECs). Extrinsic acidosis was modeled by titrating media pH. Two types of intrinsic acidosis were compared, including increasing cellular metabolism by chemically inhibiting carbonic anhydrases (CAs) IX and XII (SLC-0111) and with hypoxia. PMVECs maintained baseline intracellular pH for 24 h with both extrinsic and intrinsic acidosis. Whole cell CA IX protein expression was decreased by extrinsic acidosis but not affected by hypoxia. When extracellular pH was equally acidic, extrinsic acidosis suppressed glycolysis, whereas intrinsic acidosis did not. Extrinsic acidosis suppressed migration, but increased Matrigel network master junction and total segment length. CRISPR-Cas9 CA IX knockout PMVECs revealed an independent role of CA IX in promoting glycolysis, as loss of CA IX alone was accompanied by decreased hexokinase I and pyruvate dehydrogenase E1α expression and decreasing migration. 2-deoxy-d-glucose had no effect on migration but profoundly inhibited network formation and increased N-cadherin expression. Thus, we report that while extrinsic acidosis suppresses endothelial glycolysis and migration, it promotes network formation.
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Averina, Olga A., M. Y. Vysokikh, O. A. Permyakov, and P. V. Sergiev. "Simple recommendations for improving efficiency in generating genome-edited mice." Acta Naturae 12, no. 1 (April 16, 2020): 42–50. http://dx.doi.org/10.32607/actanaturae.10937.
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The generation of transgenic model organisms (primarily mice) is an integral part of modern fundamental and applied research. Simple techniques based on the biology of these laboratory rodents can often increase efficiency when generating genome-edited mouse strains. In this study, we share our three years of experience in the optimization of mouse genome editing based on microinjection of CRISPR/Cas9 components into ca. 10,000 zygotes. We tested a number of techniques meant to improve efficiency in generating knockout mice, such as optimization of the superovulation method and choosing the optimal mouse strains to be used as zygote donors and foster mothers. The presented results might be useful to laboratories aiming to quickly and efficiently create new mouse strains with tailored genome editing.
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Campbell, Hannah M., Ann P. Quick, Issam Abu-Taha, David Y. Chiang, Carlos F. Kramm, Tarah A. Word, Sören Brandenburg, et al. "Loss of SPEG Inhibitory Phosphorylation of Ryanodine Receptor Type-2 Promotes Atrial Fibrillation." Circulation 142, no. 12 (September 22, 2020): 1159–72. http://dx.doi.org/10.1161/circulationaha.120.045791.
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Background: Enhanced diastolic calcium (Ca 2+ ) release through ryanodine receptor type-2 (RyR2) has been implicated in atrial fibrillation (AF) promotion. Diastolic sarcoplasmic reticulum Ca 2+ leak is caused by increased RyR2 phosphorylation by PKA (protein kinase A) or CaMKII (Ca 2+ /calmodulin-dependent kinase-II) phosphorylation, or less dephosphorylation by protein phosphatases. However, considerable controversy remains regarding the molecular mechanisms underlying altered RyR2 function in AF. We thus aimed to determine the role of SPEG (striated muscle preferentially expressed protein kinase), a novel regulator of RyR2 phosphorylation, in AF pathogenesis. Methods: Western blotting was performed with right atrial biopsies from patients with paroxysmal AF. SPEG atrial knockout mice were generated using adeno-associated virus 9. In mice, AF inducibility was determined using intracardiac programmed electric stimulation, and diastolic Ca 2+ leak in atrial cardiomyocytes was assessed using confocal Ca 2+ imaging. Phosphoproteomics studies and Western blotting were used to measure RyR2 phosphorylation. To test the effects of RyR2-S2367 phosphorylation, knockin mice with an inactivated S2367 phosphorylation site (S2367A) and a constitutively activated S2367 residue (S2367D) were generated by using CRISPR-Cas9. Results: Western blotting revealed decreased SPEG protein levels in atrial biopsies from patients with paroxysmal AF in comparison with patients in sinus rhythm. SPEG atrial-specific knockout mice exhibited increased susceptibility to pacing-induced AF by programmed electric stimulation and enhanced Ca 2+ spark frequency in atrial cardiomyocytes with Ca 2+ imaging, establishing a causal role for decreased SPEG in AF pathogenesis. Phosphoproteomics in hearts from SPEG cardiomyocyte knockout mice identified RyR2-S2367 as a novel kinase substrate of SPEG. Western blotting demonstrated that RyR2-S2367 phosphorylation was also decreased in patients with paroxysmal AF. RyR2-S2367A mice exhibited an increased susceptibility to pacing-induced AF, and aberrant atrial sarcoplasmic reticulum Ca 2+ leak, as well. In contrast, RyR2-S2367D mice were resistant to pacing-induced AF. Conclusions: Unlike other kinases (PKA, CaMKII) that increase RyR2 activity, SPEG phosphorylation reduces RyR2-mediated sarcoplasmic reticulum Ca 2+ release. Reduced SPEG levels and RyR2-S2367 phosphorylation typified patients with paroxysmal AF. Studies in S2367 knockin mouse models showed a causal relationship between reduced S2367 phosphorylation and AF susceptibility. Thus, modulating SPEG activity and phosphorylation levels of the novel S2367 site on RyR2 may represent a novel target for AF treatment.
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Hines, Kevin M., Vishalsingh Chaudhari, Kristen N. Edgeworth, Thomas G. Owens, and Maureen R. Hanson. "Absence of carbonic anhydrase in chloroplasts affects C3 plant development but not photosynthesis." Proceedings of the National Academy of Sciences 118, no. 33 (August 11, 2021): e2107425118. http://dx.doi.org/10.1073/pnas.2107425118.
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The enzyme carbonic anhydrase (CA), which catalyzes the interconversion of bicarbonate with carbon dioxide (CO2) and water, has been hypothesized to play a role in C3 photosynthesis. We identified two tobacco stromal CAs, β-CA1 and β-CA5, and produced CRISPR/Cas9 mutants affecting their encoding genes. While single knockout lines Δβ-ca1 and Δβ-ca5 had no striking phenotypic differences compared to wild type (WT) plants, Δβ-ca1ca5 leaves developed abnormally and exhibited large necrotic lesions even when supplied with sucrose. Leaf development of Δβ-ca1ca5 plants normalized at 9,000 ppm CO2. Leaves of Δβ-ca1ca5 mutants and WT that had matured in high CO2 had identical CO2 fixation rates and photosystem II efficiency. Fatty acids, which are formed through reactions with bicarbonate substrates, exhibited abnormal profiles in the chloroplast CA-less mutant. Emerging Δβ-ca1ca5 leaves produce reactive oxygen species in chloroplasts, perhaps due to lower nonphotochemical quenching efficiency compared to WT. Δβ-ca1ca5 seedling germination and development is negatively affected at ambient CO2. Transgenes expressing full-length β-CA1 and β-CA5 proteins complemented the Δβ-ca1ca5 mutation but inactivated (ΔZn-βCA1) and cytoplasm-localized (Δ62-βCA1) forms of β-CA1 did not reverse the growth phenotype. Nevertheless, expression of the inactivated ΔZn-βCA1 protein was able to restore the hypersensitive response to tobacco mosaic virus, while Δβ-ca1 and Δβ-ca1ca5 plants failed to show a hypersensitive response. We conclude that stromal CA plays a role in plant development, likely through providing bicarbonate for biosynthetic reactions, but stromal CA is not needed for maximal rates of photosynthesis in the C3 plant tobacco.
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Lawrence, C. Martin, Alexander Charbonneau, and Colin Gauvin. "Cyclic Tetra‐Adenylate (cA 4 ) Activates CRISPR Associated Transcription Factor Csa3, Providing Feedback Activation of Protospacer Acquisition and crRNA Expression." FASEB Journal 34, S1 (April 2020): 1. http://dx.doi.org/10.1096/fasebj.2020.34.s1.05969.
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Gupta, N., K. Polkoff, L. Qiao, K. Cheng, and J. Piedrahita. "200 Developing exosomes as a mediator for CRISPR/Cas-9 delivery." Reproduction, Fertility and Development 31, no. 1 (2019): 225. http://dx.doi.org/10.1071/rdv31n1ab200.
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CRISPR/Cas systems present a powerful gene-editing tool with the potential for widespread therapeutic use; however, current methods of in vivo delivery such as adeno-associated viruses (AAV) may stimulate an immune response, creating the need for an alternative for delivery of CRISPR/Cas9. Exosomes are small vesicles that are released by cells and serve as a delivery system for RNA, proteins, and various molecules to other cells. The focus of this project was to use exosomes as a delivery system for Cas9, exploiting their high uptake by target cells and their ability to avoid the immune system in vivo. Porcine fetal fibroblasts (PFF) were grown to 80% confluency; after 48h, exosomes were isolated and concentrated from conditioned media by filtration with a 0.22-μm filter followed by 100-kDa molecular weight cutoff filter. Transmission electron microscopy, Western blotting for presence of CD81, and an uptake assay for exosomes stained with the lipophilic dye DiI (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA) were used to characterise isolated exosomes, and average particle size was evaluated by NanoSight (Salisbury, United Kingdom). After characterisation, exosomes were loaded with Cas9 (PNA Bio, Newbury Park, CA, USA) using sonication, incubation with saponin, or extrusion. For each method of loading, 1.0×1011 exosomes and 500ng of Cas9 were used. For sonication, exosomes and Cas9 were sonicated 4 times: 4s on/2s off, left on ice for 2min, and then repeated for 4 more cycles. Loaded exosomes were then incubated at 37°C for 20min. For incubation with saponin, 100μL of 0.6% saponin solution was made in PBS, mixed with exosomes and Cas9, and then incubated on a shaker at 800 rpm for 20min. For extrusion, exosomes and Cas9 were extruded (Avanti Polar Lipids, Alabaster, AL, USA) 10, 15, or 20 times through a 0.22-μm filter. To evaluate efficiency of Cas9 loading into exosomes, loaded exosome samples were split in half, with one-half receiving a proteinase K digest (100μg mL−1) to remove free Cas9 and the other receiving no treatment. Proteinase K-treated and untreated samples were then compared side by side on Western blot staining for Cas9. ImageJ software (National Institutes for Health, Bethesda, MD, USA) was used to quantify band intensity and loading efficiency. With optimal conditions, our preliminary results show loading efficiency for sonication and saponin to be 16.7 and 19.2%, respectively, whereas loading by extrusion was undetectable. For CRISPR/Cas targeting, transgenic PFF carrying one copy of H2B-GFP were used to test delivery of ribonucleotide protein complex (RNP). To verify efficiency of the guide (g)RNA targeting green fluorescent protein (GFP), cells were nucleofected with Cas9 and gRNA. The DNA was extracted, PCR amplified, and sequenced (Eton Bioscience, San Diego, CA, USA) and then evaluated for indels with TIDE, resulting in a 53.2% cleavage efficiency. Next, exosomes will be loaded with RNP to knockout GFP in H2B-GFP cells, and targeting efficiency will be evaluated by flow cytometry and TIDE. We hypothesise that based on loading efficiency and target cell uptake, exosomes will present a safe and efficient method for in vitro and in vivo delivery of Cas9. The financial support of the Comparative Medicine Institute is gratefully acknowledged.
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Zhao, Yanting, Helen Zhang, Jack M. Parent, and Lori L. Isom. "4346 Potential Sudden Unexpected Death in Epilepsy (SUDEP) Biomarkers in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes with DEPDC5 Loss-of-Function." Journal of Clinical and Translational Science 4, s1 (June 2020): 100. http://dx.doi.org/10.1017/cts.2020.311.
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OBJECTIVES/GOALS: Sudden Unexpected Death in Epilepsy (SUDEP) is a leading cause of death in epilepsy patients. This study aims to determine whether cardiac mechanisms contribute to SUDEP in epilepsy patients with variants in DEPDC5, a gene encoding a member of the mTOR GATOR complex, to identify SUDEP biomarkers. METHODS/STUDY POPULATION: SUDEP has been reported in 10% of epilepsy patients with DEPDC5 loss-of-function variants. We used human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to measure changes in cellular excitability that are known to be substrates for cardiac arrhythmias. CRISPR-derived isogenic DEPDC5 iPSC-CMs and DEPDC5 patient-derived iPSC-CMs were used in this study. Whole-cell patch-clamp was used to measure voltage-gated sodium current (INa) and calcium current (I>Ca) in single iPSC-CMs in voltage-clamp mode; and to measure action potentials (APs) in 3-dimentional iPSC-CM-derived micro-tissues in current-clamp mode. RESULTS/ANTICIPATED RESULTS: CRISPR generated heterozygous deletion of 1 base-pair in the first coding exon of DEPDC5 gene, resulting in a premature stop codon, simulated the variants identified in DEPDC5 epilepsy patients. In CRISPR generated heterozygousDEPDC5 iPSC-CMs, whole-cell voltage-clamp recordings revealed that INa was increased and ICa was reduced compared with isogenic control iPSC-CMs. Whole-cell current-clamp recordings revealed that AP duration at 80% and 90% of repolarization, APD80 and APD90, respectively, were prolonged compared to isogenic control iPSC-CMs. Similar measurements will be performed for iPSC-CMs derived from DEPDC5 patients. DISCUSSION/SIGNIFICANCE OF IMPACT: This study shows that epilepsy patients with non-ion channel gene variants in DEPDC5 have altered CM excitability, which may serve as a substrate for cardiac arrhythmias in DEPDC5 patients. Importantly, this work may allow us to identify biomarkers for SUDEP risk in these patients in the future. CONFLICT OF INTEREST DESCRIPTION: L.L.I. is the recipient of a collaborative research grant from Stoke Therapeutics.
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Saatci, Ozge, Ozge Akbulut, Metin Cetin, Vitali Sikirzhytski, and Ozgur Sahin. "Abstract P4-08-20: Inhibition of TACC3 blocks the growth of highly aggressive breast cancers with centrosome amplification." Cancer Research 83, no. 5_Supplement (March 1, 2023): P4–08–20—P4–08–20. http://dx.doi.org/10.1158/1538-7445.sabcs22-p4-08-20.
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Abstract Centrosome amplification (CA) is a hallmark of cancer that is strongly associated with highly aggressive disease and worse clinical outcome. Enhanced mitotic progression via clustering of extra centrosomes is a major coping mechanism utilized by cancer cells with CA that would otherwise undergo mitotic cell death due to formation of multipolar spindles. However, the underlying molecular mechanisms have largely been unexplored. Furthermore, mitosis-targeting inhibitors have mostly been unsuccessful in clinical settings with poor efficacy and severe side effects. Therefore, there is a dire need to uncover novel molecular mechanisms of CA-driven tumor growth and identify therapeutic targets playing key roles not only in mitosis, but also in interphase of cancer cells with CA to achieve durable anti-tumor effect with minimal toxicity. Here, we identified Transforming Acidic Coiled-Coil Containing Protein 3 (TACC3) as a novel CA-directed dependency, driving highly aggressive cell growth by forming distinct functional interactomes during cell cycle progression. We demonstrated, for the first time, that TACC3 interacts with the Kinesin Family Member C1 (KIFC1) via its TACC domain in mitotic cells with CA to promote centrosome clustering (CC) and facilitate mitotic progression. On the other hand, TACC3 interacts with the members of the nucleosome remodeling and deacetylase (NuRD) complex (HDAC2 and MBD2) in the nucleus of interphase cells with CA, thereby suppressing the transcription of key tumor suppressors to facilitate G1/S progression and cell survival. Inhibiting TACC3 in mitotic cells blocks the formation of TACC3/KIFC1 complex, leading to formation of multipolar spindles and activation of spindle assembly checkpoint (SAC)/CDK1/p-Bcl2 axis that ultimately results in mitotic cell death; whereas TACC3 inhibition in interphase cells blocks TACC3/HDAC2/MBD2 complex, leading to enhanced transcription of cyclin-dependent kinase inhibitors (e.g., p21 and p16) and apoptosis regulators (e.g., APAF1), ultimately causing p53-independent G1 arrest and strong apoptosis. Notably, inducing CA by chemical (cytochalasin D) or genomic (PLK4 overexpression or p53 loss) modulations renders cancer cells highly sensitive to TACC3 inhibition, showing the dependency of cells with CA to TACC3. Targeting TACC3 by small molecule inhibitors or CrispR-CAS9-mediated knock-out significantly reduces colony formation ability, inhibits the growth of organoids of patient-derived xenografts (PDXs) with CA, and strongly inhibits tumor growth in breast cancer cell line xenografts and PDXs with CA. Notably, we demonstrated that high CA tumors express much higher levels of TACC3, and high TACC3 expression, in association with its downstream effectors, KIFC1, HDAC2 and MBD2, leads to drastically worse clinical outcome in cancer patients with CA. Altogether, our results show, for the first time, that TACC3 is a multifunctional driver of the growth of the highly aggressive breast tumors with CA and that targeting TACC3 is a promising approach to tackle this aggressive disease. Citation Format: Ozge Saatci, Ozge Akbulut, Metin Cetin, Vitali Sikirzhytski, Ozgur Sahin. Inhibition of TACC3 blocks the growth of highly aggressive breast cancers with centrosome amplification [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-20.
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Beck, Charlotte, Tetiana Gren, Francisco Javier Ortiz-López, Tue Sparholt Jørgensen, Daniel Carretero-Molina, Jesús Martín Serrano, José R. Tormo, et al. "Activation and Identification of a Griseusin Cluster in Streptomyces sp. CA-256286 by Employing Transcriptional Regulators and Multi-Omics Methods." Molecules 26, no. 21 (October 30, 2021): 6580. http://dx.doi.org/10.3390/molecules26216580.
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Streptomyces are well-known producers of a range of different secondary metabolites, including antibiotics and other bioactive compounds. Recently, it has been demonstrated that “silent” biosynthetic gene clusters (BGCs) can be activated by heterologously expressing transcriptional regulators from other BGCs. Here, we have activated a silent BGC in Streptomyces sp. CA-256286 by overexpression of a set of SARP family transcriptional regulators. The structure of the produced compound was elucidated by NMR and found to be an N-acetyl cysteine adduct of the pyranonaphtoquinone polyketide 3′-O-α-d-forosaminyl-(+)-griseusin A. Employing a combination of multi-omics and metabolic engineering techniques, we identified the responsible BGC. These methods include genome mining, proteomics and transcriptomics analyses, in combination with CRISPR induced gene inactivations and expression of the BGC in a heterologous host strain. This work demonstrates an easy-to-implement workflow of how silent BGCs can be activated, followed by the identification and characterization of the produced compound, the responsible BGC, and hints of its biosynthetic pathway.
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Vora, Dhvani Sandip, Yugesh Verma, and Durai Sundar. "A Machine Learning Approach to Identify the Importance of Novel Features for CRISPR/Cas9 Activity Prediction." Biomolecules 12, no. 8 (August 16, 2022): 1123. http://dx.doi.org/10.3390/biom12081123.
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The reprogrammable CRISPR/Cas9 genome editing tool’s growing popularity is hindered by unwanted off-target effects. Efforts have been directed toward designing efficient guide RNAs as well as identifying potential off-target threats, yet factors that determine efficiency and off-target activity remain obscure. Based on sequence features, previous machine learning models performed poorly on new datasets, thus there is a need for the incorporation of novel features. The binding energy estimation of the gRNA-DNA hybrid as well as the Cas9-gRNA-DNA hybrid allowed generating better performing machine learning models for the prediction of Cas9 activity. The analysis of feature contribution towards the model output on a limited dataset indicated that energy features played a determining role along with the sequence features. The binding energy features proved essential for the prediction of on-target activity and off-target sites. The plateau, in the performance on unseen datasets, of current machine learning models could be overcome by incorporating novel features, such as binding energy, among others. The models are provided on GitHub (GitHub Inc., San Francisco, CA, USA).
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Mark, Zaniya A., Guoliang Li, Robert Matusik, and Zhenbang Chen. "Abstract 1464: Zaniya Mark." Cancer Research 82, no. 12_Supplement (June 15, 2022): 1464. http://dx.doi.org/10.1158/1538-7445.am2022-1464.
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Abstract Prostate cancer (PCa) is one of the most common types of cancers diagnosed in American men. Moreover, PCa malignancy disproportionally strikes more on African American (AA) men than any other ethnic groups including Caucasian American (CA) men. Forkhead Box A1 (FOXA1) is frequently mutated in hormone receptor-driven cancers including PCa. FOXA1 mutation rates in AA PCa specimens are 3-fold higher than that in CA PCa. By annotating the FOXA1 mutation landscape two hotspots were defined in AA PCa patients located in the forkhead domain: D226N and R219C. FOXA1 expression and functions are regulated by lysine [K]-specific demethylase 5B (KDM5B). KDM5B contributes to the activation of signaling pathways that promote cancer progression. Here in this project, we will focus on the mechanistic role of FOXA1 dysregulation and its interaction with KDM5B and AR on PCa progression in AA vs CA PCa cells. Therefore, we hypothesize that the dysregulation of FOXA1 plays an essential role in the progression of PCa by altering chromatin remodeling and AR interaction, and FOXA1 mutation promotes more aggressive malignancies in AA PCa cells. In order to investigate the biological function and regulation of FOXA1 in PCa cells we first need to knockout (KO) the endogenous expression using CRISPR technology and then knockin (KI) the unique mutants R219C and D226N. These in vitro studies of FOXA1 mutations found in AA PCa will be critical to determine the biological functions of these mutants for PCa progression. The findings from this study will provide valuable insights into the contributions of the FOXA1 regulation and will add to our knowledge base for the potential development of a novel therapeutic strategy to reduce PCa disparities between AA and CA populations. This project was supported, in part, by MVTCP Cancer Partnership Grant U54 CA163069. Citation Format: Zaniya A. Mark, Guoliang Li, Robert Matusik, Zhenbang Chen. Zaniya Mark [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1464.
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Komisarenko, Serhiy V., and Svitlana I. Romaniuk. "PROSPECTS FOR GENE EDITING USING CRISPR/CAS, OR HOW TO MASTER THE GENETIC SCISSORS Nobel Prize in Chemistry for 2020." Visnik Nacional'noi' academii' nauk Ukrai'ni, no. 12 (December 20, 2020): 31–49. http://dx.doi.org/10.15407/visn2020.12.031.
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The Nobel Prize in Chemistry in 2020 was awarded to two researchers in the field of molecular biology: French Emmanuelle Charpentier, who currently heads the Max Planck Unit for the Science of Pathogens (Berlin, Germany), and American Jennifer Doudna of the University of California (Berkeley, CA, USA) “for the development of a method for genome editing.” The press release of the Nobel Committee states that the winners have discovered one of the most powerful tools of genetic technology, CRISPR/Cas9, or so-called “genetic scissors.” This method has helped to obtain many important results in basic research. In particular, plant researchers have been able to create crops that are resistant to mold, pests and drought. In medicine, clinical trials of new methods of cancer treatment are underway, and the dream of curing hereditary diseases is about to become a reality. “Genetic scissors” have brought the life sciences to a new stage of development and are of great benefit to mankind.
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Kitagawa, Atsushi, Igor Kizub, Christina Jacob, Kevin Michael, Angelo D’Alessandro, Julie A. Reisz, Michael Grzybowski, et al. "CRISPR-Mediated Single Nucleotide Polymorphism Modeling in Rats Reveals Insight Into Reduced Cardiovascular Risk Associated With Mediterranean G6PD Variant." Hypertension 76, no. 2 (August 2020): 523–32. http://dx.doi.org/10.1161/hypertensionaha.120.14772.
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Epidemiological studies suggest that individuals in the Mediterranean region with a loss-of-function, nonsynonymous single nucleotide polymorphism (S188F), in glucose-6-phosphate dehydrogenase ( G6pd ) are less susceptible to vascular diseases. However, this association has not yet been experimentally proven. Here, we set out to determine whether the Mediterranean mutation confers protection from vascular diseases and to discover the underlying protective mechanism. We generated a rat model with the Mediterranean single nucleotide polymorphism (G6PD S188F ) using CRISPR-Cas9 genome editing. In rats carrying the mutation, G6PD activity, but not expression, was reduced to 20% of wild-type (WT) littermates. Additionally, unbiased metabolomics analysis revealed that the pentose phosphate pathway and other ancillary metabolic pathways connected to the pentose phosphate pathway were reduced ( P <0.05) in the arteries of G6PD S188F versus WT rats. Intriguingly, G6PD S188F mutants, as compared with WT rats, developed less large arterial stiffness and hypertension evoked by high-fat diet and nitric oxide synthase inhibition with L-N G -nitroarginine methyl ester. Intravenous injection of a voltage-gated L-type Ca 2+ channel agonist (methyl 2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-1,4-dihydropyridine-3-carboxylate; Bay K8644) acutely increased blood pressure in WT but not in G6PD S188F rats. Finally, our results suggested that (1) lower resting membrane potential of smooth muscle caused by increased expression of K + channel proteins and (2) decreased voltage-gated Ca 2+ channel activity in smooth muscle contributed to reduced hypertension and arterial stiffness evoked by L-N G -nitroarginine methyl ester and high-fat diet to G6PD S188F mutants as compared with WT rats. In summary, a mutation resulting in the replacement of a single amino acid (S188F) in G6PD, the rate-limiting enzyme in the pentose phosphate pathway, ascribed properties to the vascular smooth muscle that shields the organism from risk factors associated with vascular diseases.
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Zimmer, Alex M., Milica Mandic, Hong Meng Yew, Emma Kunert, Yihang K. Pan, Jimmy Ha, Raymond W. M. Kwong, Kathleen M. Gilmour, and Steve F. Perry. "Use of a carbonic anhydrase Ca17a knockout to investigate mechanisms of ion uptake in zebrafish (Danio rerio)." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 320, no. 1 (January 1, 2021): R55—R68. http://dx.doi.org/10.1152/ajpregu.00215.2020.
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In fishes, branchial cytosolic carbonic anhydrase (CA) plays an important role in ion and acid-base regulation. The Ca17a isoform in zebrafish ( Danio rerio) is expressed abundantly in Na+-absorbing/H+-secreting H+-ATPase-rich (HR) cells. The present study aimed to identify the role of Ca17a in ion and acid-base regulation across life stages using CRISPR/Cas9 gene editing. However, in preliminary experiments, we established that ca17a knockout is lethal with ca17a−/− mutants exhibiting a significant decrease in survival beginning at ∼12 days postfertilization (dpf) and with no individuals surviving past 19 dpf. Based on these findings, we hypothesized that ca17a−/− mutants would display alterations in ion and acid-base balance and that these physiological disturbances might underlie their early demise. Na+ uptake rates were significantly increased by up to 300% in homozygous mutants compared with wild-type individuals at 4 and 9 dpf; however, whole body Na+ content remained constant. While Cl− uptake was significantly reduced in ca17a−/− mutants, Cl− content was unaffected. Reduction of CA activity by Ca17a morpholino knockdown or ethoxzolamide treatments similarly reduced Cl− uptake, implicating Ca17a in the mechanism of Cl− uptake by larval zebrafish. H+ secretion, O2 consumption, CO2 excretion, and ammonia excretion were generally unaltered in ca17a−/− mutants. In conclusion, while the loss of Ca17a caused marked changes in ion uptake rates, providing strong evidence for a Ca17a-dependent Cl− uptake mechanism, the underlying causes of the lethality of this mutation in zebrafish remain unclear.
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Alsina, Katherina M., Mohit Hulsurkar, Sören Brandenburg, Daniel Kownatzki-Danger, Christof Lenz, Henning Urlaub, Issam Abu-Taha, et al. "Loss of Protein Phosphatase 1 Regulatory Subunit PPP1R3A Promotes Atrial Fibrillation." Circulation 140, no. 8 (August 20, 2019): 681–93. http://dx.doi.org/10.1161/circulationaha.119.039642.
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Background: Abnormal calcium (Ca 2+ ) release from the sarcoplasmic reticulum (SR) contributes to the pathogenesis of atrial fibrillation (AF). Increased phosphorylation of 2 proteins essential for normal SR-Ca 2+ cycling, the type-2 ryanodine receptor (RyR2) and phospholamban (PLN), enhances the susceptibility to AF, but the underlying mechanisms remain unclear. Protein phosphatase 1 (PP1) limits steady-state phosphorylation of both RyR2 and PLN. Proteomic analysis uncovered a novel PP1-regulatory subunit (PPP1R3A [PP1 regulatory subunit type 3A]) in the RyR2 macromolecular channel complex that has been previously shown to mediate PP1 targeting to PLN. We tested the hypothesis that reduced PPP1R3A levels contribute to AF pathogenesis by reducing PP1 binding to both RyR2 and PLN. Methods: Immunoprecipitation, mass spectrometry, and complexome profiling were performed from the atrial tissue of patients with AF and from cardiac lysates of wild-type and Pln -knockout mice. Ppp1r3a -knockout mice were generated by CRISPR-mediated deletion of exons 2 to 3. Ppp1r3a -knockout mice and wild-type littermates were subjected to in vivo programmed electrical stimulation to determine AF susceptibility. Isolated atrial cardiomyocytes were used for Stimulated Emission Depletion superresolution microscopy and confocal Ca 2+ imaging. Results: Proteomics identified the PP1-regulatory subunit PPP1R3A as a novel RyR2-binding partner, and coimmunoprecipitation confirmed PPP1R3A binding to RyR2 and PLN. Complexome profiling and Stimulated Emission Depletion imaging revealed that PLN is present in the PPP1R3A-RyR2 interaction, suggesting the existence of a previously unknown SR nanodomain composed of both RyR2 and PLN/sarco/endoplasmic reticulum calcium ATPase-2a macromolecular complexes. This novel RyR2/PLN/sarco/endoplasmic reticulum calcium ATPase-2a complex was also identified in human atria. Genetic ablation of Ppp1r3a in mice impaired binding of PP1 to both RyR2 and PLN. Reduced PP1 targeting was associated with increased phosphorylation of RyR2 and PLN, aberrant SR-Ca 2+ release in atrial cardiomyocytes, and enhanced susceptibility to pacing-induced AF. Finally, PPP1R3A was progressively downregulated in the atria of patients with paroxysmal and persistent (chronic) AF. Conclusions: PPP1R3A is a novel PP1-regulatory subunit within the RyR2 channel complex. Reduced PPP1R3A levels impair PP1 targeting and increase phosphorylation of both RyR2 and PLN. PPP1R3A deficiency promotes abnormal SR-Ca 2+ release and increases AF susceptibility in mice. Given that PPP1R3A is downregulated in patients with AF, this regulatory subunit may represent a new target for AF therapeutic strategies.
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Fischer, S., J. Trothe, Ü. Pul, D. Hartmann, and T. Ertongur-Fauth. "081 Genomic engineering using CRISPR-Cas9 in the human sweat gland cell line NCL-SG3 shows contribution of TMEM16A to Ca 2+ -activated Cl - secretion." Journal of Investigative Dermatology 136, no. 9 (September 2016): S174. http://dx.doi.org/10.1016/j.jid.2016.06.099.
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Duso, Bruno Achutti, Elena Gavilan Dorronzoro, Giulia Tini, Maria de Filippo, Marica Ippolito, Chiara Soriani, Simona Rodighiero, Stefano Santaguida, Pier Giuseppe Pelicci, and Luca Mazzarella. "Abstract 1164: Somatic NF1 loss in breast cancer leads to centrosome amplification, aneuploidy and increased sensitivity to T-DM1." Cancer Research 82, no. 12_Supplement (June 15, 2022): 1164. http://dx.doi.org/10.1158/1538-7445.am2022-1164.
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Abstract Background: Centrosome amplification (CA, the presence of >2 centrosomes) is a hallmark of several tumors. CA perturbs mitosis by generating nonphysiological pulling forces, leading to chromosome missegregation and aneuploidy. This condition may be advantageous for tumor outgrowth providing it gets corrected by clustering centrosomes into a pseudo-bipolar conformation for successful cell division. Molecular determinants of CA and therapeutic opportunities in breast cancer (BC) are still poorly understood. Preliminary data from our group show that somatic NF1 loss of function (LOF), common upon metastatic progression, is associated in vitro and in patients with selective sensitivity to maytansinoids such as T-DM1. Here, we explored the molecular basis of this increased sensitivity. Methods: Multiple CRISPR/Cas9-generated NF1KO or WT clones of HER2+ BC cell lines (BT474, SKBR3 and HCC1954) were assessed for sensitivity to T-DM1 or DM1 in BrdU-based and clonogenic assays, with RNAseq being performed. BT474 cells were furthered engineered with the FUCCI(Ca) reporter for live cell cycle imaging. Ploidy was assessed through flow cytometry. Aneuploidy in the GENIE cohort (restricted to BC patients analyzed with NF1-covering panels) was assessed by generating a segmentation score (sum of the absolute ploidy scores in segments covered by NGS panels). Centrosomes, spindle conformation and chromosome abnormalities were studied by confocal microscopy in cells synchronized both with RO3306 or thymidine block. Results: All lines showed increased sensitivity to T-DM1 in KO vs WT in BrdU-based and clonogenic assays; similar results were obtained with DM1 only, suggesting independence from HER2 targeting. RNAseq differential analysis showed significant enrichment for gene sets involved in mitotic spindle in KO but not WT cells upon T-DM1 treatment. FUCCI analysis showed significantly longer permanence in G2/M in NF1KO compared to WT cells (24.8 vs 17.9% in G2/M, p=1.81E-07). In vehicle-treated synchronized cells, significantly more KO cells showed >2 centrosomes (21.6 vs 4.7%, p<0.00001) and multiple pseudo-bipolar mitotic figures with narrow intercentriolar distances, indicative of efficient clustering. This was associated with more frequent chromosome misalignment (26.7 vs 6.7%, p=0.038). In the GENIE cohort, segmentation score was higher for patients with NF1 LOF mutations vs NF1 WT (median 46.7 vs 41.1, p=0.0023), indicative of more common aneuploidy. Upon T-DM1, KO cells exhibited significantly more non-bipolar spindles with massively wider intercentriolar distances. Conclusions: Somatic NF1 loss causes aneuploidy due to CA. This likely favors metastatization and can be exploited therapeutically. CA associated with other common oncogenic events (RAS and BRCA1/2 mutations, PTEN loss) may represent a general biomarker for drugs inhibiting centrosome clustering in BC. Citation Format: Bruno Achutti Duso, Elena Gavilan Dorronzoro, Giulia Tini, Maria de Filippo, Marica Ippolito, Chiara Soriani, Simona Rodighiero, Stefano Santaguida, Pier Giuseppe Pelicci, Luca Mazzarella. Somatic NF1 loss in breast cancer leads to centrosome amplification, aneuploidy and increased sensitivity to T-DM1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1164.
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Hambach, Julia, Kristoffer Riecken, Sophia Cichutek, Kerstin Schütze, Birte Albrecht, Katharina Petry, Jana Larissa Röckendorf, et al. "Targeting CD38-Expressing Multiple Myeloma and Burkitt Lymphoma Cells In Vitro with Nanobody-Based Chimeric Antigen Receptors (Nb-CARs)." Cells 9, no. 2 (January 29, 2020): 321. http://dx.doi.org/10.3390/cells9020321.
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The NAD-hydrolyzing ecto-enzyme CD38 is overexpressed by multiple myeloma and other hematological malignancies. We recently generated CD38-specific nanobodies, single immunoglobulin variable domains derived from heavy-chain antibodies naturally occurring in llamas. Nanobodies exhibit high solubility and stability, allowing easy reformatting into recombinant fusion proteins. Here we explore the utility of CD38-specific nanobodies as ligands for nanobody-based chimeric antigen receptors (Nb-CARs). We cloned retroviral expression vectors for CD38-specific Nb-CARs. The human natural killer cell line NK-92 was transduced to stably express these Nb-CARs. As target cells we used CD38-expressing as well as CRISPR/Cas9-generated CD38-deficient tumor cell lines (CA-46, LP-1, and Daudi) transduced with firefly luciferase. With these effector and target cells we established luminescence and flow-cytometry CAR-dependent cellular cytotoxicity assays (CARDCCs). Finally, the cytotoxic efficacy of Nb-CAR NK-92 cells was tested on primary patient-derived CD38-expressing multiple myeloma cells. NK-92 cells expressing CD38-specific Nb-CARs specifically lysed CD38-expressing but not CD38-deficient tumor cell lines. Moreover, the Nb-CAR-NK cells effectively depleted CD38-expressing multiple myeloma cells in primary human bone marrow samples. Our results demonstrate efficacy of Nb-CARs in vitro. The potential clinical efficacy of Nb-CARs in vivo remains to be evaluated.
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Matthews, Geoffrey M., Ricardo De Matos Simoes, Yiguo Hu, Michal Sheffer, Eugen Dhimolea, Paul J. Hengeveld, Megan A. Bariteau, et al. "Characterization of Lineage Vs. Context-Dependent Essential Genes in Multiple Myeloma Using Crispr/Cas9 Genome Editing." Blood 128, no. 22 (December 2, 2016): 119. http://dx.doi.org/10.1182/blood.v128.22.119.119.
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Abstract Multiple Myeloma (MM) remains an incurable malignancy in part because of an incomplete understanding of which genes are critically responsible for MM cell survival and proliferation. To address this unmet need, and building on our recent functional genomics studies with the CRISPR/Cas9 gene editing platform (ASH 2015; Int. MM Workshop, Rome 2015), we reasoned that quantification of sgRNA depletion in the absence of any treatment could identify genes essential for the survival or proliferation of MM cells and better define their role as candidate therapeutic targets. To this end, we transduced Cas9-expressing RPMI-8226 and MM.1S cells with the lentiviral genome-scale GeCKO pooled library of sgRNAs. After culture of these cell lines for 2, 6, 8 or 12 weeks without any treatment, we identified, based on next generation sequencing for the sgRNA sequences, genes with significantly depleted sgRNAs (4-6 sgRNAs/gene, >2-fold average depletion, FDR=0.05, based on MAGECK algorithm) in Cas9+ cells compared to their initial sgRNA plasmid pools, baseline cultures, or isogenic parental Cas9-negative cells. These results were confirmed for each cell line with a 2nd independent genome-wide analysis and with a focused sgRNA library containing a subset of candidates defined by the genome-wide analyses. We compared these results with data from our in-house or publicly available CRISPR/Cas9 gene editing studies, involving a total of 50 cell lines from other hematologic malignancies (leukemia, lymphoma) and from 8 different types of solid tumors. We identified 3 broad categories of essential genes in MM cells: a) core essential genes, with sgRNA depletion across the majority of MM and non-MM lines of our study, representing cellular processes critical for practically all lineages (e.g. genes involved in regulation of basic transcription factor complexes, ribosomal function, proteasome, spliceosome, structural proteins for mitochondria and other key organelles, et.c.); b) genes selectively essential for MM cell lines, but not for the overwhelming majority of leukemia, lymphoma or solid tumor cell lines; c) genes with a role in small subset(s) of cell lines, across diseases, which harbor defined genetic features correlating with this dependency. We integrated our CRISPR/Cas9-based data on MM-selective essential genes with a reanalysis of the Achilles Heel shRNA screen in MM and non-MM cell lines (10 and 493, respectively) of the Cell Line Encyclopedia Program (CCLE) program. We applied a series of statistical tests (e.g. Wilcoxon rank test or marker selection feature of GENE-E algorithm with 1000 permutation tests) to identify genes with a significantly lower rank in sgRNA or shRNA depletion in MM vs. non-MM cell lines, across different specific thresholds for fold change and statistical significance. We identified more than 50 high-value candidate target genes with preferential essentiality in MM, compared to non-MM cell lines of diverse lineages. Prominent examples of such MM-selective, essential genes included: transcription factors (e.g. IRF4, CCND2, MAF, NFKB1, NFKB2, RELA, RELB); otherNF-kB-related genes (e.g. IKBKB); PIM2 (but not PIM1 or PIM3 in this cell line panel); regulators of protein homeostasis, including diverse E2 and E3 ubiquitin ligases; and several other known or biologically-plausible dependencies which are under further evaluation. Many of these MM-selective dependencies exhibited significantly higher expression in MM, compared to non-MM cells, both in cell lines (based on the CCLE dataset) and patient-derived samples (comparison of Broad/MMRF vs. TCGA datasets, respectively). Notable observations of context-dependent essential genes include ARID1A in MM.1S cells (plausibly due to deficiency in its paralog ARID1B); and cases of both MM and non-MM cells with RAS mutations but lack of dependency on that gene. Targeting of lineage-specific dependencies (e.g. ER or AR in breast or prostate Ca, respectively) has provided major clinical benefit in some tumors; while context-specific dependencies are a cornerstone of molecularly-guided individualized treatments. Therefore, by identifying lineage- and context-dependent essential genes for MM, our integrated genome-wide CRISPR/Cas9 and shRNA analyses in molecularly annotated panels of MM vs. non-MM cell lines provide an attractive framework towards designing novel therapies for MM. Disclosures No relevant conflicts of interest to declare.
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Ercu, Maria, Michael B. Mücke, Tamara Pallien, Lajos Markó, Anastasiia Sholokh, Carolin Schächterle, Atakan Aydin, et al. "Mutant Phosphodiesterase 3A Protects From Hypertension-Induced Cardiac Damage." Circulation 146, no. 23 (December 6, 2022): 1758–78. http://dx.doi.org/10.1161/circulationaha.122.060210.
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Background: Phosphodiesterase 3A ( PDE3A ) gain-of-function mutations cause hypertension with brachydactyly (HTNB) and lead to stroke. Increased peripheral vascular resistance, rather than salt retention, is responsible. It is surprising that the few patients with HTNB examined so far did not develop cardiac hypertrophy or heart failure. We hypothesized that, in the heart, PDE3A mutations could be protective. Methods: We studied new patients. CRISPR-Cas9–engineered rat HTNB models were phenotyped by telemetric blood pressure measurements, echocardiography, microcomputed tomography, RNA-sequencing, and single nuclei RNA-sequencing. Human induced pluripotent stem cells carrying PDE3A mutations were established, differentiated to cardiomyocytes, and analyzed by Ca 2+ imaging. We used Förster resonance energy transfer and biochemical assays. Results: We identified a new PDE3A mutation in a family with HTNB. It maps to exon 13 encoding the enzyme’s catalytic domain. All hitherto identified HTNB PDE3A mutations cluster in exon 4 encoding a region N-terminally from the catalytic domain of the enzyme. The mutations were recapitulated in rat models. Both exon 4 and 13 mutations led to aberrant phosphorylation, hyperactivity, and increased PDE3A enzyme self-assembly. The left ventricles of our patients with HTNB and the rat models were normal despite preexisting hypertension. A catecholamine challenge elicited cardiac hypertrophy in HTNB rats only to the level of wild-type rats and improved the contractility of the mutant hearts, compared with wild-type rats. The β-adrenergic system, phosphodiesterase activity, and cAMP levels in the mutant hearts resembled wild-type hearts, whereas phospholamban phosphorylation was decreased in the mutants. In our induced pluripotent stem cell cardiomyocyte models, the PDE3A mutations caused adaptive changes of Ca 2+ cycling. RNA-sequencing and single nuclei RNA-sequencing identified differences in mRNA expression between wild-type and mutants, affecting, among others, metabolism and protein folding. Conclusions: Although in vascular smooth muscle, PDE3A mutations cause hypertension, they confer protection against hypertension-induced cardiac damage in hearts. Nonselective PDE3A inhibition is a final, short-term option in heart failure treatment to increase cardiac cAMP and improve contractility. Our data argue that mimicking the effect of PDE3A mutations in the heart rather than nonselective PDE3 inhibition is cardioprotective in the long term. Our findings could facilitate the search for new treatments to prevent hypertension-induced cardiac damage.
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Bulli, Lorenzo, Luis Apolonia, Juliane Kutzner, Darja Pollpeter, Caroline Goujon, Nikolas Herold, Sarah-Marie Schwarz, et al. "Complex Interplay between HIV-1 Capsid and MX2-Independent Alpha Interferon-Induced Antiviral Factors." Journal of Virology 90, no. 16 (June 8, 2016): 7469–80. http://dx.doi.org/10.1128/jvi.00458-16.
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ABSTRACTType I interferons (IFNs), including IFN-α, upregulate an array of IFN-stimulated genes (ISGs) and potently suppress Human immunodeficiency virus type 1 (HIV-1) infectivity in CD4+T cells, monocyte-derived macrophages, and dendritic cells. Recently, we and others identified ISG myxovirus resistance 2 (MX2) as an inhibitor of HIV-1 nuclear entry. However, additional antiviral blocks exist upstream of nuclear import, but the ISGs that suppress infection, e.g., prior to (or during) reverse transcription, remain to be defined. We show here that the HIV-1 CA mutations N74D and A105T, both of which allow escape from inhibition by MX2 and the truncated version of cleavage and polyadenylation specific factor 6 (CPSF6), as well as the cyclophilin A (CypA)-binding loop mutation P90A, all increase sensitivity to IFN-α-mediated inhibition. Using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology, we demonstrate that the IFN-α hypersensitivity of these mutants in THP-1 cells is independent of MX2 or CPSF6. As expected, CypA depletion had no additional effect on the behavior of the P90A mutant but modestly increased the IFN-α sensitivity of wild-type virus. Interestingly, the infectivity of wild-type or P90A virus could be rescued from the MX2-independent IFN-α-induced blocks in THP-1 cells by treatment with cyclosporine (Cs) or its nonimmunosuppressive analogue SDZ-NIM811, indicating that Cs-sensitive host cell cyclophilins other than CypA contribute to the activity of IFN-α-induced blocks. We propose that cellular interactions with incoming HIV-1 capsids help shield the virus from recognition by antiviral effector mechanisms. Thus, the CA protein is a fulcrum for the dynamic interplay between cell-encoded functions that inhibit or promote HIV-1 infection.IMPORTANCEHIV-1 is the causative agent of AIDS. During acute HIV-1 infection, numerous proinflammatory cytokines are produced, including type I interferons (IFNs). IFNs can limit HIV-1 replication by inducing the expression of a set of antiviral genes that inhibit HIV-1 at multiple steps in its life cycle, including the postentry steps of reverse transcription and nuclear import. This is observed in cultured cell systems, as well as in clinical trials in HIV-1-infected patients. The identities of the cellular antiviral factors, their viral targets, and the underpinning mechanisms are largely unknown. We show here that the HIV-1 Capsid protein plays a central role in protecting the virus from IFN-induced inhibitors that block early postentry steps of infection. We further show that host cell cyclophilins play an important role in regulating these processes, thus highlighting the complex interplay between antiviral effector mechanisms and viral survival.
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Lee, Sung Hwan, and Jiyeon Park. "Abstract 2273: Unsupervised clustering of multi-omics molecular layers reveals consensus molecular subtypes showing potential therapeutic opportunities for pancreatic cancer." Cancer Research 82, no. 12_Supplement (June 15, 2022): 2273. http://dx.doi.org/10.1158/1538-7445.am2022-2273.
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Abstract Introduction: Pancreatic cancer is a lethal disease showing dismal prognosis and therapeutic resistance. Previous molecular subtypes from genome or transcriptome did not show clinical relevance regarding precision strategy for optimal therapeutic options followed by precise patient classification. This study aims to uncover consensus molecular subtypes from cancer-specific multi-omics data showing clinically relevant therapeutic opportunities. Methods: We performed comprehensive analyses using the dataset from the cancer dependency map (DepMap) project, including cancer-specific molecular characterization with multi-omics data, genome-wide loss-of-function screening using the CRISPR-Cas9 system, and cancer drug sensitivity. The subtype-specific molecular signatures were validated in independent translational cohorts (TCGA-PAAD; n=150, ICGC-PACA-AU; n=461, ICGC-PACA-CA; n=317). Results: Integrative profiling of multi-omics molecular layers (Mutational signature, Copy number alteration, Transcriptome, MicroRNA, Chromatic profile, Proteome, and Metabolome) from pancreatic cancer cell lines (n=59) from the Cancer Cell Line Encyclopedia (CCLE) revealed a total of three cancer-specific molecular subtypes showing distinct tumor biology through all omics layer as well as clinical relevance with unique molecular dependency. Major molecular features of each subtype were reproducible in the validation cohorts. Subtype-specific molecular biomarkers, including mutational signature and metabolites, were identified. Finally, the target drug with subtype-specific genetic dependency was analyzed to provide precision strategy according to distinct subtypes’ molecular characterization. Conclusions: Integrative profiling from multi-omics molecular layers revealed precision strategies based on cancer-specific molecular subtypes of pancreatic cancer in terms of tumor classification and discriminative therapeutic opportunities. Prospective translational studies companion with clinical trials based on cancer-specific molecular subtypes is mandatory to establish the precision strategy for managing pancreatic cancer. Citation Format: Sung Hwan Lee, Jiyeon Park. Unsupervised clustering of multi-omics molecular layers reveals consensus molecular subtypes showing potential therapeutic opportunities for pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2273.
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Carnicer, Ricardo, Drew Duglan, Klemen Ziberna, Alice Recalde, Svetlana Reilly, Jillian N. Simon, Simona Mafrici, et al. "BH4 Increases nNOS Activity and Preserves Left Ventricular Function in Diabetes." Circulation Research 128, no. 5 (March 5, 2021): 585–601. http://dx.doi.org/10.1161/circresaha.120.316656.
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Rationale: In diabetic patients, heart failure with predominant left ventricular (LV) diastolic dysfunction is a common complication for which there is no effective treatment. Oxidation of the NOS (nitric oxide synthase) cofactor tetrahydrobiopterin (BH4) and dysfunctional NOS activity have been implicated in the pathogenesis of the diabetic vascular and cardiomyopathic phenotype. Objective: Using mice models and human myocardial samples, we evaluated whether and by which mechanism increasing myocardial BH4 availability prevented or reversed LV dysfunction induced by diabetes. Methods and Results: In contrast to the vascular endothelium, BH4 levels, superoxide production, and NOS activity (by liquid chromatography) did not differ in the LV myocardium of diabetic mice or in atrial tissue from diabetic patients. Nevertheless, the impairment in both cardiomyocyte relaxation and [Ca 2+ ]i (intracellular calcium) decay and in vivo LV function (echocardiography and tissue Doppler) that developed in wild-type mice 12 weeks post–diabetes induction (streptozotocin, 42–45 mg/kg) was prevented in mGCH1-Tg (mice with elevated myocardial BH4 content secondary to trangenic overexpression of GTP-cyclohydrolase 1) and reversed in wild-type mice receiving oral BH4 supplementation from the 12th to the 18th week after diabetes induction. The protective effect of BH4 was abolished by CRISPR/Cas9-mediated knockout of nNOS (the neuronal NOS isoform) in mGCH1-Tg. In HEK (human embryonic kidney) cells, S-nitrosoglutathione led to a PKG (protein kinase G)-dependent increase in plasmalemmal density of the insulin-independent glucose transporter GLUT-1 (glucose transporter-1). In cardiomyocytes, mGCH1 overexpression induced a NO/sGC (soluble guanylate cyclase)/PKG–dependent increase in glucose uptake via GLUT-1, which was instrumental in preserving mitochondrial creatine kinase activity, oxygen consumption rate, LV energetics (by 31 phosphorous magnetic resonance spectroscopy), and myocardial function. Conclusions: We uncovered a novel mechanism whereby myocardial BH4 prevents and reverses LV diastolic and systolic dysfunction associated with diabetes via an nNOS-mediated increase in insulin-independent myocardial glucose uptake and utilization. These findings highlight the potential of GCH1/BH4–based therapeutics in human diabetic cardiomyopathy. Graphic Abstract: A graphic abstract is available for this article.
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Hernandez-Cortes, Daniel, Jaime M. C. Gard, Beatrice S. Knudsen, Noel A. Warfel, and Anne E. Cress. "Abstract 3835: Kindlin-2 complexes containing α6β1 integrin are responsive to hypoxia." Cancer Research 82, no. 12_Supplement (June 15, 2022): 3835. http://dx.doi.org/10.1158/1538-7445.am2022-3835.
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Abstract The laminin-binding integrins are mechanosensory receptors critical for cell adhesion and structural organization that link the extracellular matrix (ECM) to the cytoskeleton. Integrin α6β1 is associated with prostate cancer (PCa) migration, invasion, metastasis, and decreased cancer-specific survival. Kindlin-2 (FERMT2) is a β1 integrin adaptor and mechanosensory focal adhesion (FA) protein that activates and clusters integrins in response to structural ECM alterations in the tumor microenvironment. Our goal was to determine if integrin-kindlin-2 adhesion complexes (kindlin-2:α6β1) were responsive to hypoxia, a physiologically relevant and altered microenvironment in PCa progression. Five different endpoints were tested including the biochemical analysis of kindlin-2 complexes, qRT-PCR, immunoblotting, immunocytochemistry, and electric cell impedance sensing (ECIS). Using DU145 prostate cancer cells grown under hypoxia (1% O2) for up to 16 hours, the results showed a reversible increase in kindlin-2:α6β1 complexes with maximal assembly within 4 hours and disassembly starting by 8 hours. Notably, kindlin-2:α6β1 complexes were found exclusively within membrane projections and were not observed within hypoxia-inducible paxillin (PXN)-containing FAs. The hypoxia induced kindlin-2:α6β1 complexes and classical FAs were dependent on kindlin-2 as determined by CRISPR-Cas9 heterozygous deletion of FERMT2. Protein co-localization of α6 integrin and PXN with kindlin-2 within membrane projections and FAs, respectively, was also induced under hypoxia. Further, non-invasive ECIS measurements in live cells confirmed functional cell-cell and cell-ECM dynamics driven by hypoxia and requiring kindlin-2. Our results indicate that the kindlin-2:α6β1 complexes are uniquely associated with FA-independent membrane projections induced by hypoxia, a tumor microenvironment associated with aggressive prostate cancer. The novel kindlin-2:α6β1 complexes may represent an actionable pharmacological target for blocking escape of organ confined disease and metastasis promoting steps of human prostate cancer. (Partially supported by NIH grants CA P30 23074, DOD W81XWH-19-1-0455, and NCI R01 CA242226). Citation Format: Daniel Hernandez-Cortes, Jaime M.C. Gard, Beatrice S. Knudsen, Noel A. Warfel, Anne E. Cress. Kindlin-2 complexes containing α6β1 integrin are responsive to hypoxia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3835.
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Tsai, Wen-Chin, Shuai Guo, Michael A. Olaopa, Loren J. Field, Jin Yang, Changyu Shen, Ching-Pin Chang, Peng-Sheng Chen, and Michael Rubart. "Complex Arrhythmia Syndrome in a Knock-In Mouse Model Carrier of the N98S Calm1 Mutation." Circulation 142, no. 20 (November 17, 2020): 1937–55. http://dx.doi.org/10.1161/circulationaha.120.046450.
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Background: Calmodulin mutations are associated with arrhythmia syndromes in humans. Exome sequencing previously identified a de novo mutation in CALM1 resulting in a p.N98S substitution in a patient with sinus bradycardia and stress-induced bidirectional ventricular ectopy. The objectives of the present study were to determine if mice carrying the N98S mutation knocked into Calm1 replicate the human arrhythmia phenotype and to examine arrhythmia mechanisms. Methods: Mouse lines heterozygous for the Calm1 N98S allele (Calm1 N98S/+ ) were generated using CRISPR/Cas9 technology. Adult mutant mice and their wildtype littermates (Calm1 +/+ ) underwent electrocardiographic monitoring. Ventricular de- and repolarization was assessed in isolated hearts using optical voltage mapping. Action potentials and whole-cell currents and [Ca 2+ ] i , as well, were measured in single ventricular myocytes using the patch-clamp technique and fluorescence microscopy, respectively. The microelectrode technique was used for in situ membrane voltage monitoring of ventricular conduction fibers. Results: Two biologically independent knock-in mouse lines heterozygous for the Calm1 N98S allele were generated. Calm1 N98S/+ mice of either sex and line exhibited sinus bradycardia, QT c interval prolongation, and catecholaminergic bidirectional ventricular tachycardia. Male mutant mice also showed QRS widening. Pharmacological blockade and activation of β-adrenergic receptors rescued and exacerbated, respectively, the long-QT phenotype of Calm1 N98S/+ mice. Optical and electric assessment of membrane potential in isolated hearts and single left ventricular myocytes, respectively, revealed β-adrenergically induced delay of repolarization. β-Adrenergic stimulation increased peak density, slowed inactivation, and left-shifted the activation curve of I Ca.L significantly more in Calm1 N98S/+ versus Calm1 +/+ ventricular myocytes, increasing late I Ca.L in the former. Rapidly paced Calm1 N98S/+ ventricular myocytes showed increased propensity to delayed afterdepolarization-induced triggered activity, whereas in situ His-Purkinje fibers exhibited increased susceptibility for pause-dependent early afterdepolarizations. Epicardial mapping of Calm1 N98S/+ hearts showed that both reentry and focal mechanisms contribute to arrhythmogenesis. Conclusions: Heterozygosity for the Calm1 N98S mutation is causative of an arrhythmia syndrome characterized by sinus bradycardia, QRS widening, adrenergically mediated QTc interval prolongation, and bidirectional ventricular tachycardia. β-Adrenergically induced I Ca.L dysregulation contributes to the long-QT phenotype. Pause-dependent early afterdepolarizations and tachycardia-induced delayed afterdepolarizations originating in the His-Purkinje network and ventricular myocytes, respectively, constitute potential sources of arrhythmia in Calm1 N98S/+ hearts.
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Angelotti-Mendonça, Jéssika, Alessandra Koltun, Fernanda Freitas de Oliveira, and Nathalia Volpi e Siva. "Genome editing: propelling the next generation of crop improvement." Colloquim Agrariae 17, no. 4 (August 12, 2021): 83–101. http://dx.doi.org/10.5747/ca.2021.v17.n4.a451.
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Climate change and population size records threaten food security. Therefore, the call for a more sustainable and efficient crop production has never been more urgent. Traditional plant breeding was one of the first successful approaches to expand cultivation areas and crop yield. Later, biotechnological tools and their products, such as genetically modified organisms containing exogenous DNA, further broadened the limits of agricultural results, yet bringing huge financial, bureaucratic, and public rejection hurdles. In the 90s, scientific advances brought the opportunity to drive mutations using engineered nucleases, and since 2013 CRISPR-Cas has emerged as the most practical toolkit to edit genomes. One of the most striking possibilities is to generate edited and non-transgenic plants. In this review, we present the working mechanism behind CRISPR-induced mutations and pinpoint the latest techniques developed, as well as its myriad of applications in agriculture. The enhancing scope of CRISPR ranges from introducing traits of agronomic interest – such as herbicide resistance, resistance/tolerance to biotic and abiotic stresses, and quality and durability of products – to accelerating plant breeding processes, including haploid induction, generating male-sterile lines, fixating hybrid vigor, and overcoming self-incompatibility. We also discuss regulatory issues surrounding edited plants and derived products around the world, challenges that must be overcome, and future prospects to harness all the potential of this amazing tool to guarantee the new crop production revolution.
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Noel, Eric A., Donald P. Weeks, and James L. Van Etten. "Pursuit of chlorovirus genetic transformation and CRISPR/Cas9-mediated gene editing." PLOS ONE 16, no. 10 (October 21, 2021): e0252696. http://dx.doi.org/10.1371/journal.pone.0252696.
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Genetic and molecular modifications of the large dsDNA chloroviruses, with genomes of 290 to 370 kb, would expedite studies to elucidate the functions of both identified and unidentified virus-encoded proteins. These plaque-forming viruses replicate in certain unicellular, eukaryotic chlorella-like green algae. However, to date, only a few of these algal species and virtually none of their viruses have been genetically manipulated due to lack of practical methods for genetic transformation and genome editing. Attempts at using Agrobacterium-mediated transfection of chlorovirus host Chlorella variabilis NC64A with a specially-designed binary vector resulted in successful transgenic cell selection based on expression of a hygromycin-resistance gene, initial expression of a green fluorescence gene and demonstration of integration of Agrobacterium T-DNA. However, expression of the integrated genes was soon lost. To develop gene editing tools for modifying specific chlorovirus CA-4B genes using preassembled Cas9 protein-sgRNA ribonucleoproteins (RNPs), we tested multiple methods for delivery of Cas9/sgRNA RNP complexes into infected cells including cell wall-degrading enzymes, electroporation, silicon carbide (SiC) whiskers, and cell-penetrating peptides (CPPs). In one experiment two independent virus mutants were isolated from macerozyme-treated NC64A cells incubated with Cas9/sgRNA RNPs targeting virus CA-4B-encoded gene 034r, which encodes a glycosyltransferase. Analysis of DNA sequences from the two mutant viruses showed highly targeted nucleotide sequence modifications in the 034r gene of each virus that were fully consistent with Cas9/RNP-directed gene editing. However, in ten subsequent experiments, we were unable to duplicate these results and therefore unable to achieve a reliable system to genetically edit chloroviruses. Nonetheless, these observations provide strong initial suggestions that Cas9/RNPs may function to promote editing of the chlorovirus genome, and that further experimentation is warranted and worthwhile.
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Dominici, Marco De, Johannes Menzel, and James V. DeGregori. "Abstract PR001: Dissecting the role of aging and inflammation on clonal hematopoiesis." Cancer Research 83, no. 2_Supplement_1 (January 15, 2023): PR001. http://dx.doi.org/10.1158/1538-7445.agca22-pr001.
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Abstract Aging is associated with the appearance in multiple tissues of somatic mutant clones often harboring mutations observed in cancers. In the hematopoietic system this phenomenon is named clonal hematopoiesis of indeterminant potential (CHIP) or “age-related clonal hematopoiesis”. The incidence of CHIP increases exponentially with aging, and has been associated with increased risk of myeloid malignancies, cardiovascular disease, and overall mortality. While mutations are thought to be acquired at a constant rate during life, whether aging plays a role in driving the expansion of these mutant clones is of crucial importance, as it can inform on the potential to prevent or halt the expansion of CHIP clones by intervening with the specific hallmark of aging fueling such expansion. To determine the effect of aging on the expansion of CHIP clones, we have established a method that relies on in vitro manipulation of mouse hematopoietic stem and progenitor cells (HSPC) in order to introduce by CRISPR-Cas9 selected mutations, followed by transplant into young and old recipient mice. Our method has several advantages: i) it allows rapid introduction of the desired mutations from mice of any background, including aged mice, bypassing the need to create transgenic lines; ii) by expanding the cultured cells in previously developed PVA media, it allows efficient engraftment in mildly (busulfan) conditioned recipient mice, avoiding the damaging effects on the bone marrow microenvironment that result from irradiation; iii) by manipulating HSPC with gRNAs targeting either the gene of interest (GFP tagged) or the safe harbor Rosa26 locus (dTomato tagged) prior to pooling, we can assess the competitive advantage of the mutation of interest relative to the Rosa26 control cells and determine their fitness advantage at various time points. We used this technique to generate Dnmt3a and Tet2 mutant clones and tested their expansion in young and aged recipient mice. Our data reveal that Dnmt3a exhibits a relatively mild fitness advantage and no difference in the rate of expansion between young and aged mice. By contrast Tet2-KO HSPC show a significantly faster expansion in old compared to young recipient mice, indicating a greater fitness advantage in the aged background. Tet2 mutant HSPC were previously shown to preferentially expand in inflammatory conditions such as stimulation with microbial products, IL-6 and TNFa. We have preliminary data that shows that co-housing old mice with young syngeneic mice alters their gut microbiome and results in a reduction of inflammation and restoration of more youthful gene expression profiles in stem and progenitor cells in the bone marrow. We are currently testing whether altering the gut microbiome or applying genetic models of reduced inflammation prevents the expansion of Tet2-KO cells, with associated “omics” analyses to explore underlying mechanisms. In all, these studies should contribute to our understanding of the aging-dependent occurrence of CHIP and suggest potential interventions to limit its prevalence. Citation Format: Marco De Dominici, Johannes Menzel, James V. DeGregori. Dissecting the role of aging and inflammation on clonal hematopoiesis [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr PR001.
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Gupta, Harshita, Suresh Kari, Emily Salinas, Haiyan Bai, Erica Osta, Anand Kornepati, Juan Wang, et al. "298 Tumor cell-intrinsic mTORC1 signaling through raptor makes melanoma and ovarian cancer immunotherapy resistant by regulating interferon-gamma responsiveness and promoting tumor-initiating cells." Journal for ImmunoTherapy of Cancer 9, Suppl 2 (November 2021): A321. http://dx.doi.org/10.1136/jitc-2021-sitc2021.298.
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BackgroundAlthough immunotherapy can induce durable anti-tumor response in multiple cancers, immune checkpoint blockade (ICB) therapy resistance in ovarian cancer and melanoma remains problematic. Here, we report that tumor cell-intrinsic mTORC1 regulates ICB response through mTORC1 defining subunit Raptor (Rptor) by modulating interferon-gamma (IFNg) resistance and tumor-initiating cell (TIC) virulence.MethodsWe knocked down two distinct mTORC1 signaling components: Rptor (Rptorlo, aids in mTORC1 assembly) and Lamtor1 (Ltor1lo, docks mTORC1 on lysosomes) in murine ovarian cancer ID8agg and melanoma B16 cells. PD-L1 was CRISPR knocked out in B16 and human ovarian cancer line ES2. Mice with tumors were treated with a-PD-L1± a-CD8 antibody. TICs were estimated by flow-cytometry.1ResultsRptorlo B16 and ID8agg, but not Ltor1lo B16 tumors grew slower and were a-PD-L1 responsive unlike control (ctrl) in WT mice. We noted that ctrl and Rptorlo B16 and ID8agg cells expressed similar surface PD-L1 in vitro. Thus, Rptor suppresses a-PD-L1 response in ICB-resistant tumors. Tumor immune analysis revealed increased CD8+ T cell% and a trend to increased IFNg+CD8+ T cells in a-PD-L1 treated Rptorlo, but not ctrl B16. Rptorlo a-PD-L1 efficacy was lost with a-CD8 and in IFNg knockout mice. In vitro, IFNg suppressed Rptorlo ID8agg proliferation, unlike ctrl. These data suggested that lack of Rptor makes tumors ICB responsive, possibly by making tumors IFNg-sensitive and increasing IFNg+CD8+ T cells. Further, tumor and draining lymph node (DLN) TCF1+PD-1+ T cell stem cells (critical for aPD-L1/PD-1 success2 3) were significantly higher in a-PD-L1 treated Rptorlo tumors. Thus, tumor Rptor status could regulate tumor microenvironment and distal DLN immune landscape on a-PD-L1 treatment.We previously published that mTORC1 promotes PD-L1-dependent tumor proliferation, TIC virulence1 4 PD-L1KO B16 and ES2 cells expressed similar total Rptor protein. However, lower levels of Rptor were loaded in mTOR complex in absence of PD-L1, as assessed by a-mTOR immunoprecipitation, suggesting that pro-tumorigenic Rptor functions were downstream of, and dependent on PD-L1. Successful Rptorlo aPD-L1 treatment reduced TIC in vivo, an effect reversed in absence of CD8+ T cells or host IFNg. Inhibiting ID8agg mTORC1 with rapamycin reduced stemness genes oct4, nanog expression by QPCR. Further, ID8agg Rptorlo TIC formed significantly smaller tumors versus ctrl TIC in immune-compromised NSG mice, confirming their reduced virulence. Rptor, but not Ltor1, expression inversely correlated with tumor CD8+ infiltrate in IMvigor210 trial, and strongly with TIC gene signature in ovarian cancer patients.5 6ConclusionsTumor-cell intrinsic Rptor modulates ICB resistance, IFNg responsiveness, immune microenvironment, and TIC virulence.AcknowledgementsN/ATrial RegistrationN/AReferencesGupta HB, et al. Tumor cell-intrinsic PD-L1 promotes tumor-initiating cell generation and functions in melanoma and ovarian cancer. Signal Transduct Target Ther 2016;1,Article number:16030.Im SJ, et al. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Nature 2016;537:417–421.Dammeijer F, et al. The PD-1/PD-L1-checkpoint restrains T cell immunity in tumor-draining lymph nodes. Cancer Cell 2020;38:685–700.Clark CA, et al. Tumor-intrinsic PD-L1 signals regulate cell growth, pathogenesis, and autophagy in ovarian cancer and melanoma. Cancer Res 2016;76:6964–6974.Smith BA, et al. A human adult stem cell signature marks aggressive variants across epithelial cancers. Cell Rep 2018;24:3353–3366.Hoffman-Censits JH, et al. IMvigor 210, a phase II trial of atezolizumab (MPDL3280A) in platinum-treated locally advanced or metastatic urothelial carcinoma (mUC). J Clin Oncol 2016;34, no.2_suppl:355–355.
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Duso, Bruno A., Elena Gavilán Dorronzoro, Giulia Tini, Maria R. de Filippo, Emanuele Bonetti, Maria R. Ippolito, Chiara Soriani, et al. "Abstract P5-13-04: NF1 mutations render HER2+ breast cancer highly sensitive to T-DM1 by altering microtubule dynamics." Cancer Research 82, no. 4_Supplement (February 15, 2022): P5–13–04—P5–13–04. http://dx.doi.org/10.1158/1538-7445.sabcs21-p5-13-04.
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Abstract Background: Despite major technological and conceptual advancements, treatment decisions in HER2+ metastatic breast cancer (mBC) remain largely based on clinical evidence, with no established predictive biomarkers to direct treatment for individual patients. The tumour suppressor Neurofibromatosis 1 (NF1) has been implicated in endocrine resistance but its role remains incompletely characterized in mBC. NF1 is best known as a GTPase-activating protein (GAP) that attenuates RAS signalling. However, the GAP function is likely not limited to RAS, and NF1 has been involved in other GTP-dependent processes including cytoskeletal dynamics. Data mining and analysis of public mutational registries revealed NF1 mutations as particularly enriched in HER2+ mBC compared to other molecular subtypes. Methods: To investigate the biological consequences of NF1 loss, we generated NF1 KO HER2+ mBC cell lines (BT474 and SKBR3) by CRISPR-Cas9 and both 2D and 3D proliferations assays were used for drug sensitivity profiling; live-cell imaging, high-resolution confocal microscopy and an ad-hoc computational algorithm were employed to study cell fate and microtubule conformational changes. Patient data were obtained from the Northwestern University through a prospective observational study in mBC patients. Results: Screening of several compounds approved for HER2+ mBC showed that response was generally equal or reduced in NF1 KO vs WT cells. However, response to trastuzumab-emtansine (T-DM1) was significantly increased in NF1 KO cells (IC50 ~0,3 vs 1,6 μg/mL in NF1WT). This sensitization was not observed with other antibody drug conjugates (ADCs) like DS-8201 and was reproducible with maytansine alone, suggesting a pharmacologically relevant NF1 activity on microtubules. Using the FUCCI(Ca) reporter, which tracks cell cycle progression at single-cell level, we saw a more prominent G2/M phase arrest and cell death upon T-DM1 treatment in NF1 KO compared to WT cells. Notably, NF1 KO cells exhibited a higher frequency of aberrant mitotic figures (chromosome alignment defects and multipolar spindle formation) and stronger β-galactosidase activity, an established marker of senescence. Collectively, these results suggest that NF1 KO cells become particularly subject to T-DM1-triggered mitotic catastrophe. Dephosphorylation of GTP-bound tubulin is required for appropriate microtubular dynamics; so-called “GTP islands” within the inner microtubule region are prone to rapid repolymerization and are normally kept at low levels. We hypothesize that expanded GTP-tubulin islands generated by the loss of NF1 GAP activity is a major cause of microtubular instability in NF1 KO cells. Preliminary evidence in support of this model was obtained by quantification of GTP-tubulin with a specific antibody. Finally, we assessed the predictive role of NF1 as a biomarker for T-DM1 response in a cohort of 300 mBC patients with mutational data in circulating tumour DNA (Guardant 360); we identified 13 heavily pretreated patients (>4 prior lines) who received T-DM1, of which 3 had loss-of-function NF1 mutations and 10 were NF1 WT. Median progression-free survival was higher in NF1-mutated than WT patients (334 vs 80 days); given the small sample size, these results cannot yet be considered significant (p=0.14). Conclusions: These results provide preliminary mechanistic and clinical evidence supporting the use of NF1 loss to guide treatment in HER2+ mBC. As novel HER2-specific agents are being rapidly added to the therapeutic arsenal, we propose biology-driven criteria to identify patients that may benefit specifically from T-DM1. In addition, NF1 dependence for correct microtubular dynamics may be exploited by other inhibitors of microtubular polymerization in use as ADC payloads, further extending the potential usefulness of NF1 determination. Citation Format: Bruno A Duso, Elena Gavilán Dorronzoro, Giulia Tini, Maria R de Filippo, Emanuele Bonetti, Maria R Ippolito, Chiara Soriani, Paolo D'Amico, Simona Rodighiero, Giuseppe Curigliano, Stefano Santaguida, Massimo Cristofanilli, Pier Giuseppe Pelicci, Luca Mazzarella. NF1 mutations render HER2+ breast cancer highly sensitive to T-DM1 by altering microtubule dynamics [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-04.
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Di Paola, Jorge. "Novel Congenital Platelet Disorders." Blood 128, no. 22 (December 2, 2016): SCI—39—SCI—39. http://dx.doi.org/10.1182/blood.v128.22.sci-39.sci-39.
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The processes of megakaryocyte differentiation, proplatelet formation, and the daily release of 1011 platelets into the bloodstream are tightly regulated. Genetic disturbances can lead to a cascade of downstream molecular alterations that markedly affect the function of megakaryocytes and platelets. Therefore, identifying new genes and their function in megakaryocytes and platelets is critical for understanding how these unique cells contribute to health and disease. Over the last decade advances in genomics, specifically next generation sequencing, have allowed for the discovery of several mutations and genetic variants that cause disease or influence associated hematological traits. By performing platelet RNA-Seq we were among the first to identify NBEAL2 as the causative gene for gray platelet syndrome (GPS) and showed that NBEAL2 regulates megakaryocyte development and platelet function.1-3 Mice carrying targeted Nbeal2 null alleles not only replicated the thrombocytopenia and lack of alpha granules observed in humans, but also provided new information about the role of platelets in thromboinflammation, wound healing, myelofibrosis and metastasis dissemination.4-7 More recently, we and others found that germline mutations in ETV6 lead to thrombocytopenia, red cell macrocytosis, and predisposition to lymphoblastic leukemia.8,9ETV6 encodes an ETS family transcriptional repressor, which exerts its activity by binding a consensus sequence in the promoter regions of DNA. Mice with conditional Etv6 knockout in megakaryocytic-erythroid cells are thrombocytopenic indicating the involvement of Etv6 in thrombopoiesis.10 Several of the families recently described have a missense mutation in the central domain of ETV6 (p.P214L). This mutation results in aberrant cellular localization of ETV6, decreased transcriptional repression, and impaired megakaryocyte maturation. The bone marrow of individuals affected by this mutation show hyperplasia of immature megakaryocytes suggesting a differentiation arrest. Deep sequencing of the platelet transcriptome also revealed significant differences in mRNA expression levels between patients with the ETV6 p.P214L mutation and non-affected family members, indicating that ETV6 is critically involved in defining the molecular phenotype and function of platelets. Consistent with this notion, individuals with the ETV6 p.P214L mutation experience bleeding that is disproportionate to their mild thrombocytopenia. We have also used CRISPR/Cas9 technology to generate a mouse colony where the human p.P214L ETV6 mutation was inserted into the conserved site of Etv6. Mice with this mutation (Etv6H.P214L) have reduced platelet counts. In summary, advances in human genetics that led to the discovery of novel congenital platelet disorders coupled with relevant animal models will likely contribute to our understanding of megakaryopoiesis and platelet function. References 1. Kahr WH, Hinckley J, Li L, et al. Mutations in NBEAL2, encoding a BEACH protein, cause gray platelet syndrome. Nature genetics. 2011;43(8):738-740. 2. Gunay-Aygun M, Falik-Zaccai TC, Vilboux T, et al. NBEAL2 is mutated in gray platelet syndrome and is required for biogenesis of platelet alpha-granules. Nature genetics. 2011;43(8):732-734. 3. Albers CA, Cvejic A, Favier R, et al. Exome sequencing identifies NBEAL2 as the causative gene for gray platelet syndrome. Nature genetics. 2011;43(8):735-737. 4. Deppermann C, Cherpokova D, Nurden P, et al. Gray platelet syndrome and defective thrombo-inflammation in Nbeal2-deficient mice. The Journal of clinical investigation. 2013. 5. Kahr WH, Lo RW, Li L, et al. Abnormal megakaryocyte development and platelet function in Nbeal2(-/-) mice. Blood. 2013;122(19):3349-3358. 6. Guerrero JA, Bennett C, van der Weyden L, et al. Gray platelet syndrome: proinflammatory megakaryocytes and alpha-granule loss cause myelofibrosis and confer metastasis resistance in mice. Blood.2014;124(24):3624-3635. 7. Tomberg K, Khoriaty R, Westrick RJ, et al. Spontaneous 8bp Deletion in Nbeal2 Recapitulates the Gray Platelet Syndrome in Mice. PLoS One. 2016;11(3):e0150852. 8. Noetzli L, Lo RW, Lee-Sherick AB, et al. Germline mutations in ETV6 are associated with thrombocytopenia, red cell macrocytosis and predisposition to lymphoblastic leukemia. Nature Genetics. 2015;47(5):535-538. 9. Zhang MY, Churpek JE, Keel SB, et al. Germline ETV6 mutations in familial thrombocytopenia and hematologic malignancy. Nature genetics. 2015;47(2):180-185. 10. Wang LC, Swat W, Fujiwara Y, et al. The TEL/ETV6 gene is required specifically for hematopoiesis in the bone marrow. Genes & development. 1998;12(15):2392-2402. Disclosures Di Paola: CSL BEhring: Consultancy; Biogen: Consultancy.
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Scaramellini, Natalia, Carola Arighi, Alessia Marcon, Dario Consonni, Elena Cassinerio, Giovanna Graziadei, Maria Domenica Cappellini, and Irene Motta. "Iron Chelation and Ferritin below 500 Mcg/L in Transfusion Dependent Thalassemia: Beyond the Limits of Clinical Trials." Blood 134, Supplement_1 (November 13, 2019): 3542. http://dx.doi.org/10.1182/blood-2019-130237.
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Introduction The current therapeutic management of transfusion dependent thalassemia (TDT) is based on regular blood transfusion and iron chelation therapy. Transfusion iron overload remains one of the major causes of morbidity and mortality in these patients because of the accumulation in heart, liver and endocrine glands. Three iron chelators are available in clinical practice: deferoxamine (DFO), deferiprone(DFP) and deferasirox (DFX). Guidelines clearly recommend when to start iron chelation, while discontinuation criteria are not well defined. Authorised product information state that we should consider interrupting DFX if serum ferritin (SF) falls consistently below 500mcg/L. This cut off was arbitrarily determined and there are no studies evaluating the effects of chelators in presence of SF below 500 mcg/L. In our clinical practice at Rare Diseases center of Fondazione IRCCS Ca' Granda Policlinico in Milan we do not completely interrupt iron chelation in TDT patients for SF levels below 500 mcg/L. Aims and methods Aim of our study was to evaluate the appearance of adverse events due to the assumption of iron chelation therapy in those TDT patients who had SF below 500 mcg/L. In this study we retrospectively evaluated renal and liver function from 2008 throughout December 2018 in TDT patients on DFX who presented SF below 500 mcg/L for 24 consecutive months. DFX dose are all expressed with the new tablets formulation dose. We evaluated SF, iron intake, LIC and MIC, renal and hepatic function. .A total of 5076 observations were collected, with 99.5 average per patient. We evaluated the relationships among variables with correlation models with random intercept Results One hundred ninety-two TDT patients are regularly followed at our center. They receive regular transfusion treatment and iron chelation therapy to prevent secondary iron overload. 51 out of 192 patients (32 F, 19 M, aged 44 ± 7 years) treated with DFX presented mean SF below 500 mcg/L for at least 24 consecutive months. Hematological and iron status parameters are described in Table 1. We found a strong correlation between SF and LIC (p<0.001) and for SF<500 mcg/L no hepatic iron overload was observed. Conversely we did not found a correlation between SF and MIC. For SF values below 500 mcg/L there was a minimal increase in creatinine levels, however the mean creatinine values remained within the normal range.Moreover, creatinine variation between two consecutive evaluation was below 0.3 mg/dl, cut off for acute kidney injury. Similar results were observed for liver function. Although a minimal increase of mean ALT value was observed for SF below 500 mcg/L, it remained within the normal range. None of our patient showed ALT level indicative of liver damage (ALT> 10 x upper limit of normal) We evaluated the relation between SF and DFX dose. Mean DFX dose decreases according to SF reduction. However, for SF value < 240 mcg/L, DFX dose remained stable at an average of 14 mg/kg per day. Conclusion According to our preliminary data, administration of DFX in TDT patients in presence of SF below 500 mcg/L is safe. Creatinine and ALT fluctuations, that usually remain within the range of normality, are mild, and transient and do not require specific treatment. Consistently with previously published data by Cohen et al, we show that a mean dosage of DFX of 14 mg/Kg die of film-coated tablet (20 mg/Kg of dispersable formulation) are necessary to balance an iron intake of 0.3 mg/kg die in absence of iron overload. Based on these results we suggest that in TDT patients with a continuous iron intake, iron chelation should be continued even when ferritin is below 500mcg/L. Monitoring of liver and kidney function tests are recommended in patient's follow up, as well as tailoring iron chelation. Disclosures Cappellini: Vifor Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; CRISPR Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Honoraria; Novartis: Membership on an entity's Board of Directors or advisory committees; Genzyme/Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. Motta:Sanofi-Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees.
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Mancarella, Marta, Irene Motta, Irene Rota, Margherita Migone De Amicis, Alessia Marcon, Marta Ferraresi, Marianna Giuditta, Giovanna Graziadei, Maria Domenica Cappellini, and Marco Vicenzi. "Diagnostic Work-up of Pulmonary Hypertension in Non Transfusion Dependent Thalassemia Patients: Pathophysiologichal Mechanisms and Clinical Implications." Blood 136, Supplement 1 (November 5, 2020): 21–22. http://dx.doi.org/10.1182/blood-2020-140819.
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INTRODUCTION: Pulmonary hypertension (PH) is a well documented clinical complication in Non-Transfusion Dependent Thalassemia (NTDT) patients, and it is associated with poor outcome [1]. PH prevalence is extremely variable among studied cohorts, ranging from 4.8% to 59% [2, 3]. Nevertheless, in the majority of the studies, the diagnosis is based on echocardiography, whereas the gold standard for PH definition is right heart catheterization (RHC). Thus, PH real prevalence, its pathophysiological mechanisms and risk factors still need to be elucidated. AIM:The aim of this study is to define the prevalence of PH in a cohort of NTDT patients through the application of the PH diagnostic work-up of the European Society of Cardiology Guidelines (ESC) [4]. We also aimed to investigate the involved pathophysiological mechanisms, to define risk factors, and to identify a potential role for cardiopulmonary exercise testing (CPET) in the risk stratification. MATERIALS AND METHODS: We planned a screening program for all NTDT patients referring to Rare Disease Center, in collaboration with the Dyspnea Lab of the Cardiology Unit, at Fondazione IRCCS Ca' Granda Policlinico in Milan. Following the systematic approach of ESC PH algorithm, together with an echocardiogram and cardiological evaluation, our patients underwent cardiopulmonary exercise testing (CPET) and the dosage of NT-proBNP. Patients were stratified according to PH probability (low vs intermediate-high) and further investigations (including V/Q lung scan and RHC) to confirm PH were performed in those with intermediate-high risk. RESULTS AND DISCUSSION:48 NTDT patients (18 females and 30 males) were consecutively enrolled over a period of 10 months. The mean age at enrollment was 46±12 years (median 45, range 25-72 years) and a wide spectrum of thalassemia genotypes was observed. 38 out of 48 (79%) had a low PH probability according to NYHA class and echocardiogram, thus they were addressed to regular follow-up. 10 patients presented intermediate-high PH probability and underwent further tests. So far, 6 out of 10 intermediate-high risk underwent RHC: PH was confirmed in 5 cases, allowing to identify 2 post-capillary PH, 1 pre-capillary PH (due to pulmonary embolism detected with V/Q scan) and 2 forms of PH due to high cardiac output (CO). The sixth patient showed a condition of high CO but with a mean arterial pulmonary pressure just below the value needed to diagnose PH. Thus, PH was confirmed in 5 out of 48 patients, with a prevalence of 10.4%. The four remaining patients with high PH probability are planned to be tested. Comparing those with high and low PH probability, the first were older (58.4 ±8.9 vs 42.6 ±10.7 years, p=0.0002) and presented higher NT-proBNP (450±442 vs 92±99 ng/mL, p=0.0001). Hemoglobin, erythroblasts, and platelet count were similar. Patients with high PH probability on CPET reached a lower maximal workload (87.9 W ±32.1 vs 126.9 ±41.6W) and lower O2 consumption (1207.7 ±284.5 vs 1594 ±454.8 mL/min), together with worse ventilation efficiency (VE/VCO2 slope 40.9 ±6.9 vs 29.4 ±3.85). CONCLUSIONS: In our cohort, according to these preliminary data, PH prevalence in NTDT is 10.4% and older patients seem to be at higher risk. Thus, cardiac evaluation programs are required, considering the increased life span. RHC is mandatory to confirm the diagnosis, to identify the underlying mechanisms and consequently a proper treatment. CPET may have an essential role in detecting predictive and prognostic parameters of PH and in defining different hemodynamic mechanisms (pre- or post-capillary) to select patients requiring RHC. Together with pulmonary embolism, which needs to be excluded with imaging, high CO due to chronic anemia seems to play an important role. This hypothesis could have significant treatment implications, giving more importance to increase Hb levels, either with blood transfusions or new therapies. Sleiman J, Int J Mol Sci 2018 2 Derchi G, Circulation 2014 3 Aessopos E., Blood 2001 4 Galiè N, Eur Heart J 2016 Disclosures Motta: Sanofi Genzyme:Honoraria.Cappellini:Genzyme/Sanofi:Honoraria, Membership on an entity's Board of Directors or advisory committees;CRISPR Therapeutics, Novartis, Vifor Pharma:Membership on an entity's Board of Directors or advisory committees;BMS:Honoraria.
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Li, Huiyu, Zhida Liu, Longchao Liu, Hongyi Zhang, Chuanhui Han, Luc Girard, Hyunsil Park, et al. "602 AXL targeting with bemcentinb restores PD-1 blockade sensitivity of STK11/LKB1 mutant NSCLC through innate immune cell mediated expansion of TCF1+ CD8 T cells." Journal for ImmunoTherapy of Cancer 9, Suppl 2 (November 2021): A632. http://dx.doi.org/10.1136/jitc-2021-sitc2021.602.
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BackgroundMutations in tumor suppressor STK11/LKB1 are associated with negative predictive and prognostic impact in NSCLC patients receiving immune checkpoint inhibitors (CPI) in several published cohorts, although there have been some conflicting reports on the association of such mutations with patient outcomes in this setting [1–9]. STK11/LKB1 tumors are characterized by a suppressive tumor micro-environment devoid of cytotoxic T cells, and we hypothesized that targeting the receptor tyrosine kinase AXL, a known driver of an innate immune suppressive microenvironment, would restore sensitivity to PD-1 in syngeneic pre-clinical models as well as in patients harboring STK11/LKB1 mutated NSCLC.MethodsStk11/Lkb1 (L) mutation was introduced by CRISPR technology into murine lung adenocarcinomas driven by mutant Kras and Trp53 loss (KP). Sensitivity towards anti-PD-1 was evaluated in the absence and presence of the small molecule AXL inhibitor bemcentinib in the KPL model and in a human NSCLC xenograft model carrying a STK11/LKB1 mutation. The immune tumor landscape was mapped following introduction of the Stk11/Lkb1 mutation and therapeutic intervention with anti-PD-1/pembrolizumab and bemcentinib. FFPE fine-needle aspirate biopsies of target lesions were acquired from patients at screening immediately prior to enrollment in BGBC008, a PhII single-arm, 2-stage study with bemcentinib (200mg/d) and pembrolizumab (200 mg/q3wk) for previously-treated stage IV lung adenocarcinoma patients who were CPI naïve or CPI refractory. Patients were assessed for response according to RECIST1.1 criteria at scheduled scan intervals.ResultsIntroduction of a STK11/LKB1 mutation into murine lung adenocarcinomas driven by mutant Kras and Trp53 loss resulted in a PD-1 refractory syngeneic KPL tumor. Mechanistically this occurred because KPL mutant NSCLCs lacked TCF1-expressing CD8 T cells, a phenotype that was recapitulated in human STK11/LKB1 mutant NSCLCs. Systemic inhibition of AXL with bemcentinib resulted in increased type I interferon secretion from dendritic cells resulting in expansion of tumor-associated TCF1+PD-1+CD8 T cells and restored therapeutic response to PD-1. This effect was observed in a syngeneic immunocompetent mouse model and in humanized mice bearing STK11/LKB1 mutant NSCLC human tumor xenografts.In an ongoing clinical trial (NCT03184571), 3 evaluable NSCLC patients with identified STK11/LKB1 mutations demonstrated objective clinical response/clinical benefit to the combination of AXL inhibitor bemcentinib and pembrolizumabConclusionsIn these models, AXL is a critical targetable driver of immune suppression in STK11/LKB1 mutant NSCLC contributing to CPI resistance. Our results show that inhibition of AXL rescues this deficit and represents a new clinical strategy in combination with anti-PD-1 therapy in NSCLC patients carrying a STK11/LKB1 mutationAcknowledgementsThe authors would like to thank all patients and their caretakers for participating in this trial.Trial RegistrationPatients treated with bemcentinib and pembrolizumab combination therapy were enrolled in the BGBC008 clinical trial (BerGenBio ASA and Merck & Co., Inc., Kenilworth NJ, USA, NCT03184571)ReferencesGu M, Xu T, Chang P. KRAS/LKB1 and KRAS/TP53 co-mutations create divergent immune signatures in lung adenocarcinomas. Ther Adv Med Oncol. 2021;13:17588359211006950.Cho BC, Lopes G, Kowalski DM. Relationship between STK11 and KEAP1 mutational status and efficacy in KEYNOTE-042: pembrolizumab monotherapy as first-line therapy for PD-L1 positive advanced NSCLC. Cancer Res. 2020;80(16 Supplement):CT084.Aredo JV, Padda SK, Kunder CA. Impact of KRAS mutation subtype and concurrent pathogenic mutations on non-small cell lung cancer outcomes. Lung Cancer. 2019;133:144–150.Kwack WG, Shin SY, Lee SH. Primary Resistance to Immune Checkpoint Blockade in an STK11/TP53/KRAS-Mutant Lung Adenocarcinoma with High PD-L1 Expression. Oncol Targets Ther. 2020;13:8901–8905.Shire NJ, Klein AB, Golozar A. STK11 (LKB1) mutations in metastatic NSCLC: Prognostic value in the real world. PLoS One. 2020;15(9):e0238358. 6. Skoulidis F, Goldberg ME, Greenawalt DM. STK11/LKB1 Mutations and PD-1 Inhibitor Resistance in KRAS-Mutant Lung Adenocarcinoma. Cancer Discov. 2018;8(7):822–835. 7. Wang H, Guo J, Shang X. Less immune cell infiltration and worse prognosis after immunotherapy for patients with lung adenocarcinoma who harbored STK11 mutation. Int. Immunopharmacol. 2020;84:106574. 8. Kitajima S, Ivanova E, Gou S. Suppression of STING Associated with LKB1 Loss in KRAS-Driven Lung Cancer. Cancer Discov. 2019;9(1):34–45. 9. Mograbi B, Heeke S, Hofman P. The Importance of STK11/LKB1 Assessment in Non-Small Cell Lung Carcinomas. Diagnostics (Basel). 2021;11(2):196.Ethics ApprovalThis study was approved by the following ethical committees: Use of human cord blood: UT Southwestern (UTSW) Parkland Hospital, STU 112010-047Animal studies: UTSW Medical Center, Institutional Animal Care and Use Committee, APN 2015-100921Clinical study: London Bridge Research Ethics Committee (UK): 17/LO/0418; REC-South East (Norway): 2017/473; Drug Research Ethics Committee of the University Hospital Clinic of Barcelona (Spain): BGBC008/MK-3475_PN-531; Medical College of Wisconsin Institutional Review Board #4 (USA): PRO00029453
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Al Hadidi, Samer, Ranjit Nair, Raphael E. Steiner, Sairah Ahmed, Paolo Strati, Simrit Parmar, Swami P. Iyer, et al. "Association of Epstein-Barr Virus with Advanced Stage and Survival Outcomes in Classic Hodgkin's Lymphoma." Blood 136, Supplement 1 (November 5, 2020): 37–38. http://dx.doi.org/10.1182/blood-2020-136338.
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Introduction Classic Hodgkin's lymphoma (cHL) is a highly curable lymphoid malignancy. Epstein-Barr virus (EBV) is associated with cHL, with a variable rate of detection in Hodgkin and Reed-Sternberg (HRS) cells among different histologic types and geographic areas.Although most adults worldwide are EBV seropositive, only a minority of patients infected with EBV will develop cHL. EBV is thought to be one of the causative agents for the development of cHL with an important pathobiology role. The goal of this study was to compare the presentation and the outcomes of patients with EBV+ HRS cells at the time of initial diagnosis of cHL. Methods This single-center study included patients with a diagnosis of cHL who were first seen at The University of Texas MD Anderson Cancer Center between January 1,2016 and May 28, 2020 for either newly diagnosed cHL or relapsed/refractory (R/R) cHL. Pathology was confirmed and analyzed for positivity of EBV (EBV +ve) in all patients by immunohistochemical (IHC) staining for by Epstein-Barr virus-encoded small RNA (EBER) with available paraffin blocks. The primary aims were to assess overall survival (OS), progression-free survival (PFS) and frequency of advanced disease. Descriptive statistics for categorical and continues variables were analyzed. Kaplan-Meier method was used for time-to-event analysis, including PFS and OS. Median time to event in months with 95% confidence interval (CI) was calculated. The Log-rank test was used to evaluate the difference in time-to-event endpoints between patient groups. Statistical software SAS 9.4 (SAS, Cary, NC) and S-Plus 8.2 (TIBCO Software Inc., Palo Alto, CA) were used for statistical analyses. Results Between 2016 and 2020, 644 patients met the inclusion criteria. Three hundred and fifty six patients (55%) had enough/available tissue to undergo testing for EBV at the time of initial diagnosis. The median age at diagnosis was 36 years with 51.4% males. Eighty-eight patients had positive EBV (25%) at diagnosis. The median age of +ve EBV was 37 years (Range: 18-83 years) compared to 33 years (Range: 18-85 years) for patients with -ve EBV. Mixed cellularity histology was more frequent in patients with +ve EBV when compared to the whole group of patients (32% vs. 7%; p-value: 0.03). Human immunodeficiency virus (HIV) was positive in a minority of the patients (8 patients out of 498 patients with available results) (1.6%) however 50% of the patients with HIV had +ve EBV at the time of diagnosis. Baseline demographics are summarized in Table 1. EBV was associated with the initial stage (stage II 38% in EBV +ve vs. 53% in EBV -ve, stage III 25% in EBV +ve vs. 14% in EBV -ve, stage IV 24% in EBV +ve vs. 31% in EBV -ve; p-value 0.0001). Most of the patients were treated with doxorubicin, bleomycin, vinblastine and decarbazine (ABVD) based therapy (77%). Other therapies included brentuximab vedotin (BV) (13%) and checkpoint inhibitors (CPIs) (6%). Median follow up was 1.97 years (range: 0.047- 44.09 years). PFS rate difference at 5 years was not statistically significant (64% in EBV +ve vs. 52% in EBV -ve; p-value 0.14). OS rate at 5 years was significantly lower in patients with +ve EBV (89% vs. 98%, p-value: 0.0014). OS rates at 10 years were similar (89% in EBV +ve vs. 88% in EBV -ve). Conclusions EBV at the time of diagnosis was associated with lower prevalence of localized cHL and inferior survival rates at 5 years of follow up (9% lower OS rate at 5 years). Implementation of different frontline therapy treatment algorithms specific to EBV +ve patients may help in improving the survival outcomes. Further research is needed to understand the biological significance of +ve EBV in cHL to help in developing novel agents targeting EBV positive cHL population with the ultimate goal of improving outcome. Disclosures Parmar: Cellenkos Inc.: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Iyer:Rhizen: Research Funding; CRISPR: Research Funding; Seattle Genetics, Inc.: Research Funding; Merck: Research Funding; Legend Biotech: Consultancy; Daiichi Sankyo: Consultancy; Trillium: Research Funding; Curio Biosciences: Honoraria; Target Oncology: Honoraria; Afffimed: Research Funding; Spectrum: Research Funding. Nieto:Novartis: Other: Grant Support; Astra Zeneca: Other: Grant Support; Affimed: Consultancy, Other: Grant Support; Secura Bio: Other: Grant Support. Chuang:Sage-Evidence=Based Medicine & Practice: Consultancy. Wang:Beijing Medical Award Foundation: Honoraria; Loxo Oncology: Consultancy, Research Funding; Pulse Biosciences: Consultancy; Nobel Insights: Consultancy; Verastem: Research Funding; Dava Oncology: Honoraria; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Molecular Templates: Research Funding; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; OMI: Honoraria, Other: Travel, accommodation, expenses; Targeted Oncology: Honoraria; Lu Daopei Medical Group: Honoraria; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; MoreHealth: Consultancy; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; BioInvent: Research Funding; VelosBio: Research Funding; Acerta Pharma: Research Funding; InnoCare: Consultancy; Oncternal: Consultancy, Research Funding; Juno: Consultancy, Research Funding; OncLive: Honoraria; Guidepoint Global: Consultancy. Lee:Celgene: Research Funding; Takeda: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Aptitude Health: Speakers Bureau; Seattle Genetics: Research Funding; Oncternal Therapeutics: Research Funding; Guidepoint Blogal: Consultancy.
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Mehta-Shah, Neha, Eric D. Jacobsen, Todd A. Fehniger, Brad S. Kahl, Nancy L. Bartlett, Amanda F. Cashen, Armin Ghobadi, et al. "End of Treatment Peripheral Blood TCR Evaluation for Minimal Residual Disease Evaluation in Peripheral T-Cell Lymphomas." Blood 138, Supplement 1 (November 5, 2021): 3506. http://dx.doi.org/10.1182/blood-2021-148768.
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Abstract Introduction: Peripheral T-cell lymphomas (PTCL) are a rare subset of non-Hodgkin lymphomas in which approximately 80% patients will have an overall response to CHOP based therapy but 5-year survival ranges between 20-40%. (Ellin et al Blood 2014) While response by PET/CTs have been helpful in risk stratifying patients (Mehta-Shah Blood Advances 2019), the high rate of relapse after complete response (CR) suggests that more sensitive determinants of minimal residual disease (MRD) could have prognostic and even therapeutic importance. T-cell receptor gene rearrangement sequencing (TCR) is standardly performed by next generation sequencing and is able to detect a known TCR clonotype at 10 -5. (Shah et al AMP 2017) Therefore, in a prospective multi-institutional study, we sought to evaluate the utility of TCR by next generation sequencing in quantifying MRD in PTCL. (NCT03297697). Here we report the results of the TCR evaluation at the end of CHOP-based therapy. Methods: Subjects with previously untreated PTCL (PTCL-NOS: peripheral T-cell lymphoma, NOS; angioimmunoblastic T-cell lymphoma: AITL; anaplastic large cell lymphoma: ALCL; T-follicular helper phenotype peripheral T-cell lymphoma: PTCL-TFH, monomorphic epitheliotropic intestinal T-cell lymphoma: MEITL) who were being treated with anthracycline based therapy for curative intent were eligible for the study. TCR (TRG or TRB) clonotype was established from baseline formalin fixed paraffin embedded tumor tissue or peripheral blood when tissue samples are not available. TCR clonality was identified using the LymphoTrack® TRG/TRB Assays - MiSeq® (Invivoscribe, San Diego, CA) when top %reads is ≥2.5% and is at least 2x compared to the background. Results: 43 subjects were enrolled in the study and 41 were evaluable (15 PTCL-NOS, 10 AITL, 7 ALK- ALCL, 5 ALK+ ALCL, 3 PTCL-NOS with TFH phenotype, 1 MEITL). One subject was enrolled with PTCL-NOS and later found to have T-ALL and was withdrawn from the study. One subject with ALK- ALCL withdrew consent prior to sample collection. Subjects initiated treatment with CHOEP (n=16), BV-CHP (n=11), CHOP (n=5), CEOP (n=1) CHOP+azacitidine (n=6), CHOEP+lenalidomide (n=1), EPOCH (n=1). The median age was 65 (range: 22-80). Sixteen underwent a consolidative autologous transplant. Among the 41 evaluable subjects, 73% (30/41) had tissue samples and 27% (11/41) had only PB samples for TCR clonotype assessment. TCR clonotype was identified in 78% (32/41) tissue samples and not in 23% (9/40: 1 Nodal TFH, 2 AITL, 1 ALK- ALCL, 5 ALK+ ALCL) samples. Of the 32 subjects with baseline clonotype, 2 patients (1 AITL, 1 PTCL) did not have end of treatment (EOT) samples for TCR MRD evaluation. For EOT MRD evaluation, 80% (24/30) were had detectable MRD by TCR (MRD positive) and 20% (6/30) had undetectable TCR (TCR MRD negative). For the EOT evaluation by PET/CT, 63% (19/30) had CR, 10% (3/30) had partial remission (PR) and 27% (8/30) had progressive disease (PD). Among the 6 TCR MRD negative subjects, PET/CT responses were 67% (4/6) for CR and 33% (2/6) for PR, respectively. Among the 24 TCR MRD positive subjects, PET/CT responses were 63% (15/24) for CR, 33% (1/24) for PR and 33% (8/24) for PD. Of those with positive MRD 13/24 (54%) have relapsed/progressed including 8 with primary refractory disease. At the end of CHOP-based therapy, 79% (15/19) subjects with PET CR were TCR MRD positive, and 21% (4/19) were TCR MRD negative. Among the 11 subjects with either PD or PR by PET/CT, 82% (9/11) were TCR MRD positive, and 18% (2/11) were TCR MRD negative. At a median follow up of 20.7 months, progression free survival and overall survival at 18 months did not differ between those who were TCR MRD positive or negative respectively at EOT (OS 64% vs 67% p=0.63; PFS 41% vs 50% p=0.40). We plan to present additional follow up for all patients on the study including MRD post autologous transplant at the time of the meeting. Conclusions: Measurement of peripheral blood TCR at the end of treatment is feasible in peripheral T-cell lymphomas using next generation sequencing with a known tumor clonotype. Detectable TCR at the end of treatment correlates with lack of CR but the majority of patients in complete remission by PET/CT have a detectable TCR clonotype at end of treatment. Longer follow up is required to determine if consolidative transplant alters TCR dynamics. This study was supported by the Lymphoma Research Foundation and T-cell Leukemia/Lymphoma Society. Figure 1 Figure 1. Disclosures Mehta-Shah: C4 Therapeutics: Consultancy; Ono Pharmaceuticals: Consultancy; Secura Bio: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; AstraZeneca: Research Funding; Bristol Myers Squibb: Research Funding; Celgene: Research Funding; Innate Pharmaceuticals: Research Funding; Roche/Genentech: Research Funding; Corvus Pharmaceuticals: Research Funding; Karyopharm: Consultancy; Kiowa Hakko Kirin: Consultancy; Verastem: Research Funding. Jacobsen: Takeda: Consultancy; Syros: Consultancy; Janssen: Research Funding; Novartis: Research Funding; Pharmacyclics: Research Funding; Acerta: Research Funding. Fehniger: Wugen: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties: related to memory like NK cells, Research Funding; Affimed: Research Funding; Compass Therapeutics: Research Funding; HCW Biologics: Research Funding; Kiadis: Other; ImmunityBio: Research Funding; OrcaBio: Other; Indapta: Other. Kahl: AbbVie, Acerta, ADCT, AstraZeneca, BeiGene, Genentech: Research Funding; AbbVie, Adaptive, ADCT, AstraZeneca, Bayer, BeiGene, Bristol-Myers Squibb, Celgene, Genentech, Incyte, Janssen, Karyopharm, Kite, MEI, Pharmacyclics, Roche, TG Therapeutics, and Teva: Consultancy. Bartlett: Celgene: Research Funding; Bristol Myers Squibb: Research Funding; Forty Seven: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Kite, a Gilead Company: Research Funding; Merck: Research Funding; Millennium: Research Funding; Pharmacyclics: Research Funding; Autolus: Research Funding; Seagen: Consultancy, Research Funding; Roche/Genentech: Consultancy; ADC Therapeutics: Consultancy, Research Funding. Ghobadi: Amgen: Consultancy, Research Funding; Kite, a Gilead Company: Consultancy, Honoraria, Research Funding; Atara Biotherapeutics: Consultancy; Wugen: Consultancy; Celgene: Consultancy. Moskowitz: Seattle Genetics: Consultancy, Research Funding; Imbrium Therapeutics L.P./Purdue: Consultancy; Merck: Consultancy, Research Funding; ADC Therapeutics: Research Funding; Miragen: Research Funding; Beigene: Research Funding; Bristol-Myers Squibb: Research Funding; Takeda: Consultancy; Incyte: Research Funding; Janpix Ltd.: Consultancy. Jacobsen: Invivoscribe: Current Employment. Olson: Invivoscribe: Current Employment. Vigil: Invivoscribe: Current Employment. Hill: Invivoscribe: Current Employment. Elias: Invivoscribe: Current Employment. Huang: Invivoscribe: Current Employment. Horwitz: ADC Therapeutics, Affimed, Aileron, Celgene, Daiichi Sankyo, Forty Seven, Inc., Kyowa Hakko Kirin, Millennium /Takeda, Seattle Genetics, Trillium Therapeutics, and Verastem/SecuraBio.: Consultancy, Research Funding; Affimed: Research Funding; Celgene: Research Funding; Aileron: Research Funding; Acrotech Biopharma, Affimed, ADC Therapeutics, Astex, Merck, Portola Pharma, C4 Therapeutics, Celgene, Janssen, Kura Oncology, Kyowa Hakko Kirin, Myeloid Therapeutics, ONO Pharmaceuticals, Seattle Genetics, Shoreline Biosciences, Inc, Takeda, Trillium Th: Consultancy; C4 Therapeutics: Consultancy; Crispr Therapeutics: Research Funding; Daiichi Sankyo: Research Funding; Forty Seven, Inc.: Research Funding; Kura Oncology: Consultancy; Kyowa Hakko Kirin: Consultancy, Research Funding; Millennium/Takeda: Research Funding; Myeloid Therapeutics: Consultancy; ONO Pharmaceuticals: Consultancy; Seattle Genetics: Consultancy, Research Funding; Secura Bio: Consultancy; Shoreline Biosciences, Inc.: Consultancy; Takeda: Consultancy; Trillium Therapeutics: Consultancy, Research Funding; Tubulis: Consultancy; Verastem/Securabio: Research Funding.
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Riedell, Peter A., Chase Walling, Loretta J. Nastoupil, Martina Pennisi, Richard T. Maziarz, Joseph P. McGuirk, Olalekan O. Oluwole, et al. "A Multicenter Retrospective Analysis of Clinical Outcomes, Toxicities, and Patterns of Use in Institutions Utilizing Commercial Axicabtagene Ciloleucel and Tisagenlecleucel for Relapsed/Refractory Aggressive B-Cell Lymphomas." Blood 134, Supplement_1 (November 13, 2019): 1599. http://dx.doi.org/10.1182/blood-2019-127490.
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Introduction CD19 directed CAR T cells have shown potent activity in relapsed/refractory (R/R) aggressive B-cell lymphomas (B-NHL) leading to the FDA approval of axicabtagene ciloleucel (axi-cel, Oct 2017) and tisagenlecleucel (tisa-cel, May 2018). Initial reports on commercial application of axi-cel suggest many patients (pts) would not have met eligibility criteria for the ZUMA-1 clinical trial, yet outcomes and toxicities appeared similar (Nastoupil LJ, et al. Blood 2018 132:91 and Jacobson CA, et al. Blood 2018 132:92). No data on the "real world" application of tisa-cel is available. We performed a multicenter retrospective study to include both approved commercial products, axi-cel and tisa-cel, given in centers that had the option of prescribing either product. We evaluate patterns of use, efficacy, and safety. Methods We retrospectively analyzed data from pts who underwent apheresis for commercial axi-cel or tisa-cel from 8 US academic centers. Data collection started after 5/1/2018, following FDA approval of tisa-cel when centers would have a choice to prescribe either axi-cel or tisa-cel for B-NHL. Centers were invited to participate if they were certified to administer both products. Patient and treatment characteristics were summarized descriptively. Response and toxicity were reported with 95% exact binomial CIs. Results As of 7/31/2019, 242 pts underwent apheresis for commercial CAR T-cell products. Of these, 163 (67%) underwent apheresis for axi-cel, and 79 (33%) for tisa-cel. 14 (9%) axi-cel and 3 (4%) tisa-cel pts died prior to CAR T-cell infusion from lymphoma progression, and 1 (1%) tisa-cel pt was not infused for other reasons. Detailed baseline pt characteristics were available for 180/242 pts (Table 1). Median age at apheresis was 58 years (range: 18-85) for axi-cel pts and 67 years (range: 36-88) for tisa-cel pts. ECOG PS was 0-1 in 86% of axi-cel and 94% of tisa-cel pts. By histology, 77% of axi-cel pts had DLBCL, 13% TFL, 9% HGBL and 2% PMBCL. Similarly, 81% of tisa-cel pts had DLBCL, 13% HGBL, and 6% TFL. The median number of prior therapies was 3 (range: 2-11) for axi-cel and 4 (range: 2-9) for tisa-cel pts. Prior autologous stem cell transplant was performed in 29% of axi-cel and 23% of tisa-cel pts, respectively. Bridging therapy was given in 61% of axi-cel and 72% of tisa-cel pts. Median time from apheresis to CAR T-cell infusion was 28 days for axi-cel and 44 days for tisa-cel. CAR T-cell infusion was inpatient in 100% of axi-cel and 39% of tisa-cel pts. Safety was evaluable in 213 pts. CRS was graded according to institutional practices (CARTOX (38%), Penn scale (31%), ASTCT (19%), and Lee scale (11%)). NEs were graded per CARTOX (80%), ASTCT (19%), or CTCAE V4.03 (1%). Grade ≥3 CRS and NEs occurred in 13% and 41% of axi-cel pts and 1% and 3% of tisa-cel pts. The median onset of CRS and NEs was 2 and 6 days in axi-cel, and 3 and 5 days in tisa-cel treated pts, respectively. Tocilizumab was administered in 62% of axi-cel pts with 57% receiving steroids. In tisa-cel pts, tocilizumab was administered in 13% of cases, with 7% receiving steroids. 12 deaths (8%) unrelated to lymphoma progression occurred in axi-cel pts at a median of 57 days (range: 6-373) with 5 due to infectious complications, 4 due to grade 5 NEs, 1 due to cardiac disease, 1 due to pulmonary hemorrhage, and 1 due to HLH. 4 deaths (6%) unrelated to lymphoma progression occurred in tisa-cel pts at a median of 48 days (range: 25-146) with 2 due to infectious complications, 1 due to cardiac disease, and 1 due to unknown causes. Response assessment was performed for infused pts at day 30 and/or day 90, or in those determined to have clinical progression. Of 120 axi-cel pts evaluable at day 30, the ORR was 72% with 43% achieving a CR. Of the 32 tisa-cel pts evaluable at day 30, the ORR was 59% with 44% achieving a CR. At day 90, the ORR for axi-cel was 52% with 39% achieving a CR, while for tisa-cel the ORR was 48% with 39% achieving a CR. Conclusions Efficacy outcomes in the commercial setting appear similar to responses seen in the pivotal clinical trials. Though different toxicity grading scales were employed, tisa-cel appears to be associated with less CRS and NEs. Data from a larger group of pts treated at additional centers are being gathered. Analyses of usage patterns and updated outcomes with uniform ASTCT toxicity grading will be presented in an effort to better understand therapeutic decision making. Disclosures Riedell: Novartis: Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite/Gilead: Honoraria, Research Funding, Speakers Bureau; Bayer: Honoraria, Speakers Bureau. Nastoupil:Spectrum: Honoraria; TG Therapeutics: Honoraria, Research Funding; Novartis: Honoraria; Janssen: Honoraria, Research Funding; Gilead: Honoraria; Genentech, Inc.: Honoraria, Research Funding; Bayer: Honoraria; Celgene: Honoraria, Research Funding. Maziarz:Kite: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Research Funding; Incyte: Consultancy, Honoraria; Celgene/Juno: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. McGuirk:Gamida Cell: Research Funding; Pluristem Ltd: Research Funding; Novartis: Research Funding; Fresenius Biotech: Research Funding; Astellas: Research Funding; Bellicum Pharmaceuticals: Research Funding; Kite Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Juno Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; ArticulateScience LLC: Other: Assistance with manuscript preparation. Oluwole:Pfizer: Consultancy; Spectrum: Consultancy; Gilead Sciences: Consultancy; Bayer: Consultancy. Bachanova:Novartis: Research Funding; Celgene: Research Funding; Kite: Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Gamida Cell: Research Funding; GT Biopharma: Research Funding. Hwang:Tmunity: Research Funding; Novartis: Research Funding. Schuster:Pharmacyclics: Honoraria, Research Funding; Acerta: Honoraria, Research Funding; AstraZeneca: Honoraria; Loxo Oncology: Honoraria; Nordic Nanovector: Honoraria; Pfizer: Honoraria; Novartis: Honoraria, Patents & Royalties: Combination Therapies of CAR and PD-1 Inhibitors with royalties paid to Novartis, Research Funding; Celgene: Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Merck: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Gilead: Honoraria, Research Funding. Perales:Bellicum: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; NexImmune: Membership on an entity's Board of Directors or advisory committees; MolMed: Membership on an entity's Board of Directors or advisory committees; Omeros: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Nektar Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Honoraria; Medigene: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Kyte/Gilead: Research Funding; Miltenyi: Research Funding. Bishop:Juno: Consultancy, Membership on an entity's Board of Directors or advisory committees; CRISPR Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Porter:American Board of Internal Medicine: Membership on an entity's Board of Directors or advisory committees; Immunovative: Membership on an entity's Board of Directors or advisory committees; Genentech: Employment; Wiley and Sons: Honoraria; Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Kite: Membership on an entity's Board of Directors or advisory committees; Glenmark Pharm: Membership on an entity's Board of Directors or advisory committees.
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Rahlff, Janina, Victoria Turzynski, Sarah P. Esser, Indra Monsees, Till L. V. Bornemann, Perla Abigail Figueroa-Gonzalez, Frederik Schulz, et al. "Lytic archaeal viruses infect abundant primary producers in Earth’s crust." Nature Communications 12, no. 1 (July 30, 2021). http://dx.doi.org/10.1038/s41467-021-24803-4.
Full textAbstract:
AbstractThe continental subsurface houses a major portion of life’s abundance and diversity, yet little is known about viruses infecting microbes that reside there. Here, we use a combination of metagenomics and virus-targeted direct-geneFISH (virusFISH) to show that highly abundant carbon-fixing organisms of the uncultivated genus Candidatus Altiarchaeum are frequent targets of previously unrecognized viruses in the deep subsurface. Analysis of CRISPR spacer matches display resistances of Ca. Altiarchaea against eight predicted viral clades, which show genomic relatedness across continents but little similarity to previously identified viruses. Based on metagenomic information, we tag and image a putatively viral genome rich in protospacers using fluorescence microscopy. VirusFISH reveals a lytic lifestyle of the respective virus and challenges previous predictions that lysogeny prevails as the dominant viral lifestyle in the subsurface. CRISPR development over time and imaging of 18 samples from one subsurface ecosystem suggest a sophisticated interplay of viral diversification and adapting CRISPR-mediated resistances of Ca. Altiarchaeum. We conclude that infections of primary producers with lytic viruses followed by cell lysis potentially jump-start heterotrophic carbon cycling in these subsurface ecosystems.
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Pioner, Josè Manuel, Lorenzo Santini, Chiara Palandri, Marianna Langione, Bruno Grandinetti, Silvia Querceto, Daniele Martella, et al. "Calcium handling maturation and adaptation to increased substrate stiffness in human iPSC-derived cardiomyocytes: The impact of full-length dystrophin deficiency." Frontiers in Physiology 13 (November 7, 2022). http://dx.doi.org/10.3389/fphys.2022.1030920.
Full textAbstract:
Cardiomyocytes differentiated from human induced Pluripotent Stem Cells (hiPSC- CMs) are a unique source for modelling inherited cardiomyopathies. In particular, the possibility of observing maturation processes in a simple culture dish opens novel perspectives in the study of early-disease defects caused by genetic mutations before the onset of clinical manifestations. For instance, calcium handling abnormalities are considered as a leading cause of cardiomyocyte dysfunction in several genetic-based dilated cardiomyopathies, including rare types such as Duchenne Muscular Dystrophy (DMD)-associated cardiomyopathy. To better define the maturation of calcium handling we simultaneously measured action potential and calcium transients (Ca-Ts) using fluorescent indicators at specific time points. We combined micropatterned substrates with long-term cultures to improve maturation of hiPSC-CMs (60, 75 or 90 days post-differentiation). Control-(hiPSC)-CMs displayed increased maturation over time (90 vs 60 days), with longer action potential duration (APD), increased Ca-T amplitude, faster Ca-T rise (time to peak) and Ca-T decay (RT50). The progressively increased contribution of the SR to Ca release (estimated by post-rest potentiation or Caffeine-induced Ca-Ts) appeared as the main determinant of the progressive rise of Ca-T amplitude during maturation. As an example of severe cardiomyopathy with early onset, we compared hiPSC-CMs generated from a DMD patient (DMD-ΔExon50) and a CRISPR-Cas9 genome edited cell line isogenic to the healthy control with deletion of a G base at position 263 of the DMD gene (c.263delG-CMs). In DMD-hiPSC-CMs, changes of Ca-Ts during maturation were less pronounced: indeed, DMD cells at 90 days showed reduced Ca-T amplitude and faster Ca-T rise and RT50, as compared with control hiPSC-CMs. Caffeine-Ca-T was reduced in amplitude and had a slower time course, suggesting lower SR calcium content and NCX function in DMD vs control cells. Nonetheless, the inotropic and lusitropic responses to forskolin were preserved. CRISPR-induced c.263delG-CM line recapitulated the same developmental calcium handling alterations observed in DMD-CMs. We then tested the effects of micropatterned substrates with higher stiffness. In control hiPSC-CMs, higher stiffness leads to higher amplitude of Ca-T with faster decay kinetics. In hiPSC-CMs lacking full-length dystrophin, however, stiffer substrates did not modify Ca-Ts but only led to higher SR Ca content. These findings highlighted the inability of dystrophin-deficient cardiomyocytes to adjust their calcium homeostasis in response to increases of extracellular matrix stiffness, which suggests a mechanism occurring during the physiological and pathological development (i.e. fibrosis).
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Nouzova, Marcela, Marten J. Edwards, Matthew DeGennaro, Dennys Leyva, Lilian V. Tose, Francisco Fernandez-Lima, and Fernando G. Noriega. "Genetics tools for corpora allata specific gene expression in Aedes aegypti mosquitoes." Scientific Reports 12, no. 1 (November 28, 2022). http://dx.doi.org/10.1038/s41598-022-25009-4.
Full textAbstract:
AbstractJuvenile hormone (JH) is synthesized by the corpora allata (CA) and controls development and reproduction in insects. Therefore, achieving tissue-specific expression of transgenes in the CA would be beneficial for mosquito research and control. Different CA promoters have been used to drive transgene expression in Drosophila, but mosquito CA-specific promoters have not been identified. Using the CRISPR/Cas9 system, we integrated transgenes encoding the reporter green fluorescent protein (GFP) close to the transcription start site of juvenile hormone acid methyl transferase (JHAMT), a locus encoding a JH biosynthetic enzyme, specifically and highly expressed in the CA of Aedes aegypti mosquitoes. Transgenic individuals showed specific GFP expression in the CA but failed to reproduce the full pattern of jhamt spatiotemporal expression. In addition, we created GeneSwitch driver and responder mosquito lines expressing an inducible fluorescent marker, enabling the temporal regulation of the transgene via the presence or absence of an inducer drug. The use of the GeneSwitch system has not previously been reported in mosquitoes and provides a new inducible binary system that can control transgene expression in Aedes aegypti.
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Möller, Svenning R., Christopher S. Lancefield, Nicola C. Oates, Rachael Simister, Adam Dowle, Leonardo D. Gomez, and Simon J. McQueen-Mason. "CRISPR/Cas9 suppression of OsAT10, a rice BAHD acyltransferase, reduces p-coumaric acid incorporation into arabinoxylan without increasing saccharification." Frontiers in Plant Science 13 (July 22, 2022). http://dx.doi.org/10.3389/fpls.2022.926300.
Full textAbstract:
Ester-linked hydroxycinnamic acids ferulic acid (FA) and para-coumaric acid (p-CA) play important roles in crosslinking within cell wall arabinoxylans (AX) and between AX and lignin in grass cell walls. The addition of hydroxycinnamates to AX, is mediated by the Mitchell clade of BAHD acyl-coenzyme A-utilizing transferases. Overexpression of OsAT10 (a Mitchell clade BAHD acyl transferase) in rice, has previously been shown to increase p-CA content in AX in leaves and stems, leading to increased cell wall digestibility, potentially associated with a concomitant decrease in FA content. To investigate the physiological role of OsAT10 we established CRISPR/Cas9 rice knock-out mutants devoid of OsAT10. Our analysis of hydroxycinnamic acid content in wild type plants revealed that AX associated p-CA is found almost exclusively in rice husks, with very little found in other tissues. Mutant plants were essentially devoid of ester-linked p-CA associated with AX, indicating that OsAT10 represents the major enzyme responsible for the addition of p-CA to arabinoxylan in rice plants. We found no change in the digestibility of rice husk lacking AX-associated p-CA, suggesting that the changes in digestibility seen in OsAT10 overexpressing plants were solely due to compensatory decreases in AX-associated FA.
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Perez, Maëva, Bernard Angers, C. Robert Young, and S. Kim Juniper. "Shining light on a deep-sea bacterial symbiont population structure with CRISPR." Microbial Genomics 7, no. 8 (August 27, 2021). http://dx.doi.org/10.1099/mgen.0.000625.
Full textAbstract:
Many foundation species in chemosynthesis-based ecosystems rely on environmentally acquired symbiotic bacteria for their survival. Hence, understanding the biogeographic distributions of these symbionts at regional scales is key to understanding patterns of connectivity and predicting resilience of their host populations (and thus whole communities). However, such assessments are challenging because they necessitate measuring bacterial genetic diversity at fine resolutions. For this purpose, the recently discovered clustered regularly interspaced short palindromic repeats (CRISPR) constitutes a promising new genetic marker. These DNA sequences harboured by about half of bacteria hold their viral immune memory, and as such, might allow discrimination of different lineages or strains of otherwise indistinguishable bacteria. In this study, we assessed the potential of CRISPR as a hypervariable phylogenetic marker in the context of a population genetic study of an uncultured bacterial species. We used high-throughput CRISPR-based typing along with multi-locus sequence analysis (MLSA) to characterize the regional population structure of the obligate but environmentally acquired symbiont species Candidatus Endoriftia persephone on the Juan de Fuca Ridge. Mixed symbiont populations of Ca. Endoriftia persephone were sampled across individual Ridgeia piscesae hosts from contrasting habitats in order to determine if environmental conditions rather than barriers to connectivity are more important drivers of symbiont diversity. We showed that CRISPR revealed a much higher symbiont genetic diversity than the other housekeeping genes. Several lines of evidence imply this diversity is indicative of environmental strains. Finally, we found with both CRISPR and gene markers that local symbiont populations are strongly differentiated across sites known to be isolated by deep-sea circulation patterns. This research showed the high power of CRISPR to resolve the genetic structure of uncultured bacterial populations and represents a step towards making keystone microbial species an integral part of conservation policies for upcoming mining operations on the seafloor.
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Lambert, Jonathan P., TImothy S. Luongo, Pooja Jadiya, Erhe Gao, Xueqian Zhang, Anna Maria Lucchesee, and John W. Elrod. "Abstract 432: MCUB Regulates Mitochondrial Calcium Uniporter Channel Gating and Modulates Bioenergetics and Cell Death." Circulation Research 121, suppl_1 (July 21, 2017). http://dx.doi.org/10.1161/res.121.suppl_1.432.
Full textAbstract:
The mitochondrial calcium uniporter (MCU) is a high-capacity, inward-rectifying channel in the inner mitochondrial membrane and is required for mitochondrial Ca 2+ ( m Ca 2+ ) uptake. m Ca 2+ signaling regulates bioenergetics and activates the mitochondrial permeability transition pore (MPTP) which are cellular processes implicated in cardiac pathophysiology warranting further research into the molecular regulation of the MCU. Recently, a MCU gene paralog, MCUB , was identified as a possible component of the channel. To investigate MCUB’s contribution to uniporter regulation we created a MCUB -/- HeLa cell line using CRISPR-Cas9n. Here, we report that loss of MCUB increased m Ca 2+ transient amplitude after IP3R stimulation (52% vs. con) suggesting MCUB negatively regulates m Ca 2+ uptake. Mitoplast patch-clamping confirmed that loss of MCUB increases MCU current density, suggesting MCUb modulates channel capacitance. Permeabilized MCUB -/- and WT cells exposed to various levels of Ca 2+ (0.5-20μM) revealed that MCUB -/- cells exhibited m Ca 2+ uptake at lower Ca 2+ concentrations than controls, suggesting MCUB contributes to channel gating. In m Ca 2+ retention capacity experiments MCUB -/- cells required ~30% less bath Ca 2+ to induce depolarization, suggesting a predisposition to m Ca 2+ overload. Next, we generated a cardiac-specific, tamoxifen-inducible MCUB overexpression mouse model ( MCUB -Tg). Cardiomyocytes isolated from MCUB -Tg hearts exhibited decreased m Ca 2+ uptake at low-Ca 2+ (59% vs. con) and isolated mitochondria exhibited a reduction in Ca 2+ -induced swelling (37% vs. con), suggesting a resistance to permeability transition. MCUB -Tg mice displayed a significant impairment in isoproterenol-induced contractile reserve and this correlated with a loss of isoproterenol-mediated activation of pyruvate dehydrogenase. In summary, our results suggest that MCUB limits m Ca 2+ uptake by altering channel-gating and thereby regulates bioenergetics and MPTP opening.
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Rajan, Sudarsan, Santhanam Shanmughapriya, Dhanendra Tomar, Zhiwei Dong, Joseph Y. Cheung, Peter B. Stathopulos, and Madesh Muniswamy. "Abstract 125: Perturbation of Mitochondrial Calcium Uniporter Promotes Cardiac Oxidative Stress and Autophagy During Heart Failure." Circulation Research 121, suppl_1 (July 21, 2017). http://dx.doi.org/10.1161/res.121.suppl_1.125.
Full textAbstract:
Mitochondrial calcium ([Ca 2+ ] m ) is essential for cardiomyocyte viability, and aberration of [Ca 2+ ] m is known to elicit multiple cardiac stress conditions associated with ATP depletion, reactive oxygen species, and mitochondrial permeability transition pore opening, all of which can lead to metabolic stress and the loss of dysfunctional mitochondria by aberrant autophagy. Elucidating the regulatory role of m itochondrial c alcium u niporter (MCU)-mediated [Ca 2+ ] m in modulating cardiac mitochondrial bioenergetics and autophagy has high significance and clinical impact for many pathophysiological processes. [Ca 2+ ] m is exquisitely controlled by the inner mitochondrial membrane uniporter, transporters, regulators and exchangers including MCU, MCUR1, EMRE, MICU1, MICU2 and LETM1. Our recently published findings revealed that Mitochondrial Ca 2+ Uniporter Regulator 1 (MCUR1) serves as a scaffold factor for uniporter complex assembly. We found that deletion of MCUR1 impaired [Ca 2+ ] m uptake, mitochondrial Ca 2+ current ( I MCU ) and mitochondrial bioenergetics and is associated with increased autophagy. Our new findings indicate that the impairment of [Ca 2+ ] m uptake exacerbated autophagy following ischemia-reperfusion (I/R) injury. In support of our mouse model, human failing hearts show that MCUR1 protein levels are markedly decreased and autophagy markers are increased, demonstrating a crucial link between [Ca 2+ ] m uptake and autophagy during heart failure. Additionally, our results reveal that either oxidation or disruption of human MCU Cys-97 (in mouse Cys-96; gain-of-function MCU C96A mutant) produces a conformational change within the N terminal β-grasp fold of MCU which promotes higher-order MCU complex assembly and increased I MCU activity and mitochondrial ROS levels. The results of our studies using a novel cardiac-specific MCUR1-KO model and a constitutively active global MCU C96A KI mouse model (CRISPR-Cas9 genome edited) elucidate the regulatory role of [Ca 2+ ] m in cardiac bioenergetics and autophagy during oxidative stress and myocardial infarction. Thus, targeting assembly and the activity of MCU complex will offer a new potential therapeutic target in the treatment of cardiomyopathy and heart failure.
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Chiurillo, Miguel A., Noelia Lander, Mayara S. Bertolini, Melissa Storey, Anibal E. Vercesi, and Roberto Docampo. "Different Roles of Mitochondrial Calcium Uniporter Complex Subunits in Growth and Infectivity of Trypanosoma cruzi." mBio 8, no. 3 (May 9, 2017). http://dx.doi.org/10.1128/mbio.00574-17.
Full textAbstract:
ABSTRACT Trypanosoma cruzi is the agent of Chagas disease, and the finding that this parasite possesses a mitochondrial calcium uniporter (TcMCU) with characteristics similar to that of mammalian mitochondria was fundamental for the discovery of the molecular nature of MCU in eukaryotes. We report here that ablation of TcMCU , or its paralog TcMCUb , by clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 led to a marked decrease in mitochondrial Ca 2+ uptake without affecting the membrane potential of these cells, whereas overexpression of each gene caused a significant increase in the ability of mitochondria to accumulate Ca 2+ . While TcMCU- knockout (KO) epimastigotes were viable and able to differentiate into trypomastigotes, infect host cells, and replicate normally, ablation of TcMCUb resulted in epimastigotes having an important growth defect, lower rates of respiration and metacyclogenesis, more pronounced autophagy changes under starvation, and significantly reduced infectivity. Overexpression of TcMCUb , in contrast to what was proposed for its mammalian ortholog, did not result in a dominant negative effect on TcMCU. IMPORTANCE The finding of a mitochondrial calcium uniporter (MCU) in Trypanosoma cruzi was essential for the discovery of the molecular nature of this transporter in mammals. In this work, we used the CRISPR/Cas9 technique that we recently developed for T. cruzi to knock out two components of the uniporter: MCU, the pore subunit, and MCUb, which was proposed as a negative regulator of MCU in human cells. In contrast to what occurs in human cells, MCU is not essential, while MCUb is essential for growth, differentiation, and infectivity; has a bioenergetic role; and does not act as a dominant negative subunit of MCU.