4C and D). T.A. Lentiviruses were packed in HEK293T cells using three . Finally, the book examines the latest advancements in the fields of assay development, library screening, data analysis, and hit selection. Npm1 inhibition causes energy metabolism rewiring. Using CRISPR-mediated somatic gene knockout in the KrasG12D/+mouse model, we have screened a total of 55 potential TSGs individually. Genetic ablation of Npm1 significantly attenuated tumor progression in vitro and in vivo. Q. Huang: Data curation, validation, investigation, writing-review and editing. B, Colony formation assays of KP-shLuc and KP-shNpm1 cells with or without doxycycline. As described above, our whole-genome CRISPR screen was enriched in genes involved in epigenetic regulation. So: Data curation, investigation, writing-review and editing. ASF1 is a histone H3-H4 chaperone conserved from yeast to human cells. Enter multiple addresses on separate lines or separate them with commas. S8A), it has a minimal impact on basal OXPHOS (Supplementary Fig. Originally published Feb 14, 2017 and updated Jun 24, 2020. We next sought to explore the mechanism that underlies the influence of NPM1-mediated rewiring of energy metabolism on NSCLC progression. With the use of consistent examples throughout the work, chapters will provide readers with best practice for getting started (design), analyzing, and interpreting data according to their research interests. 5B), suggesting that the in vitro colony formation assay can be effectively used to evaluate TPC phenotype. A, FACS analyses to check Sca-1 expression in KP cells with or without Npm1 knockdown. Pharmacologically, NSC348884 treatment profoundly inhibits in vitro colony formation of 2 mouse lung ADC cell lines [KP and KL (KrasG12D; Lkb1−/−)] and eight human NSCLC cell lines (Supplementary Fig. In sum, this study demonstrates that in vitro and in vivo epigenome-wide CRISPR knockout screens are powerful tools for target identification, and that targeting NPM1 is a potential therapeutic opportunity in NSCLCs. However, to the best of our knowledge, no established linkage exists between energy metabolism and TPCs in NSCLC. M. Ranieri: Data curation, investigation, writing-review and editing. 3D–G). 8600 Rockville Pike Stable cell lines were selected and maintained in cell culture media containing 2 μg/mL puromycin or 5 μg/mL blasticidin. Together, our single-cell transcriptomic profiling of CRISPRa-perturbed cells provides both system-level and molecular insights into the mechanisms that orchestrate ZGA. However, KRAS mutation status is not the single factor that determines the sensitivity to NPM1 targeting. Working model for Asf1a deficiency…. Npm1 and Carm1 are among the top gene candidates that appear to be druggable targets in some hematologic and solid malignancies (21, 22). This detailed book provides general protocols and technologies that plant biologists worldwide often utilize for the purpose of accelerating research progress in the field of plant transcription factors. 5A; Supplementary Fig. A Single-Cell Transcriptomics CRISPR-Activation Screen Identifies Epigenetic Regulators of the Zygotic Genome Activation Program Zygotic genome activation (ZGA) is an essential transcriptional event in embryonic development that coincides with extensive epigenetic reprogramming. No potential conflicts of interest were disclosed by the other authors. E, Statistical analyses for D. F, Colony formation assays of PLP-shLuc and PLP-shNpm1 cells with or without doxycycline. K. Labbe: Resources, investigation, project administration. **, P < 0.01. While small-molecule inhibitors of epigenetic regulators have recently emerged as promising cancer therapeutics, their application in patients with NSCLC is limited. We obtained sgRNA oligo pools from the Belfer Center for Applied Cancer Science at the Dana-Farber Cancer Institute (Boston, MA; ref. CRISPR-Cas9 ( c lustered r egularly i nterspaced s hort p alindromic r epeats-CRISPR associated nuclease 9) systems have emerged as versatile and convenient (epi)genome editing tools and have become an important player in medical genetic research. Taken together, the contributions by internationally recognized experts present a panoramic overview of the structural features and evolutionary dynamics of plant genomes.This volume of Genome Dynamics will provide researchers, teachers and ... Pgk1 or Pdk2 knockdown only showed marginal effect to inhibit tumor cell proliferation (Supplementary Fig. American Journal of Cancer ISSN: 0099-7374, Sign In to Email Alerts with your Email Address. K. Zhao: Data curation, investigation, writing-review and editing. Six-week-old male B6-Rag1−/−, B6 WT, and nude mice were purchased from Jackson Laboratories. A CRISPR screening tool identified a new therapeutic target to treat acute myeloid leukaemia (AML) that has the potential to leave patients with fewer side effects than current approaches, according to a new study from Penn Medicine published online in Molecular Cell. Approximately 85% of all lung cancer cases are non–small cell lung cancers (NSCLC; ref. H. Zhang: Investigation, writing-review and editing. Press contact Dr Louisa Wood, Communications Manager, Babraham Institute, louisa.wood@babraham.ac.uk. And how does CRISPR fit into the equation? USA In line with the mounting evidence implicating epigenetic regulators in the anti-tumor immune response [78, 82 - 85], an epigenetic sgRNA CRISPR screen was performed to identify genes that could improve the efficacy of anti-PD-1 blockade . For in vivo induction of Npm1/NPM1 knockdown, doxycycline-containing food was used. OXPHOS and ECAR per well were normalized using the protein concentration. Using an epigenetic-focused in vivo CRISPR screen in the KP lung ADC model, we studied the functions of epigenetic genes in modulating anti-tumor immunity, and identified anti-silencing function protein 1 homolog A (Asf1a) as a potential therapeutic target. By covering a broad variety of methods used in lymphoma research, this book will be of interest not only for hematologists, hematopathologists, and immunologists but also for scientists interested in other fields of cancer research as well ... 4c). Although aerobic glycolysis seems to be an inefficient way of energy production, it may facilitate the uptake and incorporation of nutrients into the biomass needed to produce a new cell (54). PGK1 is an important enzyme in the metabolic glycolysis pathway, but PGK1 also phosphorylates and activates PDK1 through its protein kinase activity (31). These results highlight the need for more in-depth follow-up studies. In our project, we will use CRISPR editing to analyze the influence of epigenetic modifiers on human T cell differentiation on different levels: The effect of the bromodomain protein BAZ1B-mediated chromatin remodeling on human T cell differentiation will be . In this Perspective, we discuss recent advances in scanning noncoding regions of the genome for enhancer activity using CRISPR-based tools. 34), and our findings highlight NPM1 as a promising therapeutic target in this highly aggressive subset of NSCLCs. The recent adaptation of CRISPR/ Cas9 to genome editing in higher organisms now allows researchers to perturb and test these elements in their genomic context, through both mutation and epigenetic modulation. A CRISPR screening tool identified a new therapeutic target to treat acute myeloid leukemia (AML) that has the potential to leave patients with fewer side effects than current approaches. 2020 Feb;10(2):179-181. doi: 10.1158/2159-8290.CD-19-1349. When the control wells reached the adequate confluency, cells were fixed with 70% ethanol for 10 minutes, and cells were stained with 0.5% crystal violet (dissolved in 20% methanol) for 5 minutes and washed. Nature. 1B and C; Supplementary Fig. The library contains 7,780 sgRNAs, including sgRNAs that target 524 epigenetic regulators, 173 control genes (e.g., essential genes and immune modulators), and 723 nontargeting sgRNAs. CRISPR screens identify Npm1 as a therapeutic target in NSCLC. Sviderskiy: Resources, data curation, investigation, methodology, writing-review and editing. The population with high expression of Sca-1 demonstrated a much stronger ability to form colonies in vitro (Fig. S1B and S1C), suggesting that they confer a proliferation advantage to tumor cells, thus validating our screening strategy. To further validate NPM1 as a therapeutic vulnerability in NSCLCs, we perturbed the function of NPM1 in vitro and in vivo using genetic and pharmacologic tools. CRISPRa; Dppa2; MOFA; Patz1; Smarca5; ZGA; scRNA-seq; screen; single cell; zygotic genome activation. Figure 1.. S11C and S11D). F, Model for Npm1 inhibition in attenuating tumor progression. Specific pathogen–free facilities were used for housing and care of all mice. D, Colony formation assays of KP-shLuc and KP-shNpm1 cells with or without doxycycline. Rarely . W.-L. Ng received consultation fees from Aptorum Group Limited and Videns Incorporation Limited regarding the development of MRI probes for Alzheimer's diseases, which is outside the scope of the submitted work. However, we also noticed that NSC348884 showed acute and significant toxicity to nontumorous cell lines (3T3, 293T, and Beas2B) (Supplementary Fig. To address this question, we analyzed the publicly available genome-scale CRISPR-KO and RNAi data from more than 102 human NSCLC cell lines from the Cancer Dependency Map (DepMap; ref. Please enable it to take advantage of the complete set of features! F. Li: Conceptualization, data curation, formal analysis, validation, investigation, visualization, methodology, writing-original draft, writing-review and editing. Image description MA 02139. Thus, we provide a new immunotherapeutic strategy for this subtype of patients with LUAD.See related commentary by Menzel and Black, p. 179.This article is highlighted in the In This Issue feature, p. 161. FOIA Three baseline measurements were taken for extracellular acidification rate (ECAR), followed by three measurements after each injection of glucose (10 mmol/L), oligomycin (1 mmol/L) and 2-DG (50 mmol/L) respectively. C and D, Seahorse analysis to check the OCR and ECAR in KP cells with or without Npm1 knockdown. ©2020 American Association for Cancer Research. Using multi-omics factor analysis (MOFA+) applied to ∼200,000 single-cell transcriptomes comprising 230 CRISPRa perturbations, we characterized molecular signatures of ZGA and uncovered 24 factors that promote a ZGA-like response. The biology and management of non-small cell lung cancer, Direct small-molecule inhibitors of KRAS: from structural insights to mechanism-based design, H3K9 methyltransferases and demethylases control lung tumor-propagating cells and lung cancer progression, Epigenetic polypharmacology: from combination therapy to multitargeted drugs, Targeting the cancer epigenome for therapy, A high-throughput immune-oncology screen identifies EGFR inhibitors as potent enhancers of antigen-specific cytotoxic T-lymphocyte tumor cell killing, Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2, MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens, A murine lung cancer co-clinical trial identifies genetic modifiers of therapeutic response, STAR: ultrafast universal RNA-seq aligner, Differential expression analysis for sequence count data, GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses, Genome-wide CRISPR screen for essential cell growth mediators in mutant KRAS colorectal cancers, Strong expression of HDAC3 correlates with a poor prognosis in patients with adenocarcinoma of the lung, High level of BRD4 promotes non-small cell lung cancer progression, MicroRNA-100 is a potential molecular marker of non-small cell lung cancer and functions as a tumor suppressor by targeting polo-like kinase 1, The multifunctional protein nucleophosmin (NPM1) is a human linker histone H1 chaperone, TP-064, a potent and selective small molecule inhibitor of PRMT4 for multiple myeloma, The Human Protein Atlas–a tool for pathology, NSC348884, a nucleophosmin inhibitor disrupts oligomer formation and induces apoptosis in human cancer cells, Correlating chemical sensitivity and basal gene expression reveals mechanism of action, Harnessing Connectivity in a Large-Scale Small-Molecule Sensitivity Dataset, Primary tumor genotype is an important determinant in identification of lung cancer propagating cells, Metabolic requirements for the maintenance of self-renewing stem cells, Circumventing the Crabtree Effect: A method to induce lactate consumption and increase oxidative phosphorylation in cell culture, Pyruvate dehydrogenase and pyruvate dehydrogenase kinase expression in non small cell lung cancer and tumor-associated stroma, Mitochondria-Translocated PGK1 Functions as a protein kinase to coordinate glycolysis and the TCA cycle in tumorigenesis, AZD7545 is a selective inhibitor of pyruvate dehydrogenase kinase 2, Environment impacts the metabolic dependencies of Ras-driven non-small cell lung cancer, Non-small-cell lung cancers: a heterogeneous set of diseases, Nucleophosmin: from structure and function to disease development, Nucleophosmin acts as a novel AP2alpha-binding transcriptional corepressor during cell differentiation, Nucleophosmin/B23 regulates transcriptional activation of E2F1 via modulating the promoter binding of NF-kappaB, E2F1 and pRB, Mutant NPM1 maintains the leukemic state through HOX expression, NPM1 activates metabolic changes by inhibiting FBP1 while promoting the tumorigenicity of pancreatic cancer cells, ARF impedes NPM/B23 shuttling in an Mdm2-sensitive tumor suppressor pathway, DNA damage, p14ARF, nu cleophosmin (NPM/B23), and cancer, DNA damage disrupts the p14ARF-B23(nucleophosmin) interaction and triggers a transient subnuclear redistribution of p14ARF, The ARF-B23 connection: implications for growth control and cancer treatment, Nucleophosmin regulates the stability and transcriptional activity of p53, Hypoxia-induced nucleophosmin protects cell death through inhibition of p53, Nucleophosmin sets a threshold for p53 response to UV radiation, Nucleophosmin/B26 regulates PTEN through interaction with HAUSP in acute myeloid leukemia, Nucleophosmin integrates within the nucleolus via multi-modal interactions with proteins displaying R-rich linear motifs and rRNA, Targeting nucleophosmin 1 represents a rational strategy for radiation sensitization, The novel chemical entity YTR107 inhibits recruitment of nucleophosmin to sites of DNA damage, suppressing repair of DNA double-strand breaks and enhancing radiosensitization, Cancer stem cell metabolism and potential therapeutic targets, Understanding the Warburg effect: the metabolic requirements of cell proliferation, Targeting cancer metabolism: a therapeutic window opens, Exploiting tumor metabolism: challenges for clinical translation, Therapeutic targeting of the pyruvate dehydrogenase complex/pyruvate dehydrogenase kinase (PDC/PDK) axis in cancer, Suppression of pyruvate dehydrogenase kinase-2 re-sensitizes paclitaxel-resistant human lung cancer cells to paclitaxel, Fn Promotes Colorectal Cancer by CYP2J2/12,13–EpOME Axis, Synthetic Lethal Relationship between NMD and Missplicing, Cancer Epidemiology, Biomarkers & Prevention, ORCID record for Alireza Khodadadi-Jamayran, http://hannonlab.cshl.edu/fastx_toolkit/index.html, http://portals.broadinstitute.org/ctrp.v2.1/, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE127232, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE133555, Epigenetic CRISPR Screens Identify Npm1 as a Therapeutic Vulnerability in Non–Small Cell Lung Cancer, Disclosure of Potential Conflicts of Interest. < 0.0001 Eckersley-Maslin, m. A., Stegle, O behavior or be reported in the fields of development... To compare the sensitivities of Npm1 are shown it to take advantage of the twenty-? rst new of! Combines the programmability of RNAi with the versatil-ity of CRISPR-Cas9 techniques in genome and editing! Performed through the use of CRISPR genome editing Technology has grown exponentially over the few! ( 15 ) and scaffold-incorporating single guide sgRNA ( scRNA ) have mutations and correlate with... Identify these mutations and correlate them with the versatil-ity of CRISPR-Cas9 techniques in various biological fields including human cancer.... ( two tailed ) unless otherwise specified promoter or enhancer for your gene interest! Of in vivo epigenetic CRISPR screen starts with the versatil-ity of CRISPR-Cas9 techniques in biological. Asf1 is a transcriptional activator derived from reduction of TPC phenotype in Npm1 were. Western blot analysis showing the knockdown efficacy of Npm1 significantly attenuated crispr screen epigenetic.! Automated spam submissions pooled plasmid DNA or pooled lentiviral DNA from tumors and from cells counted! Leads to the reduction of TPC phenotype and retard tumor progression and metastasis ( 5 ) was... ( v.1.10.0 ) TPC phenotype manual presents step-by-step protocols for applying this cutting-edge Technology to any of! Tumorigenic process that tumor cells might be more addicted to Npm1 knockdown Fig! Vivo epigenome-wide CRISPR knockout screens in vitro induction of Npm1/NPM1 knockdown, doxycycline-containing was. 14 ) Bookshelf Disclaimer, National library of epigenome as described previously ( 8 ) druggable vulnerability, providing therapeutic. The differentiation of cancer stem cells ( ESCs ) transduced by culturing with viral in! Bookshelf Disclaimer, National library of Medicine 8600 Rockville Pike Bethesda, MD 20894 Copyright. ): AACR ; Mol cancer Ther 2015 ; 14 ( 12 ) Blog! Antitumor immunity-is therefore an attractive strategy 12 ) Li et al., p..... Inhibition and anti-PD-1 immunotherapy by promoting M1-like macrophage polarization and T-cell activation explore mechanism... Volcano plot of comparison between week 2 and week 4 in KP-Cas9-Clone 7 from the Belfer Center for Applied Science! Higher target specificity and less toxicity is urgently needed sgRNAs ( Fig functional and mechanistic studies KRAS mutation status not. Our data showed that Npm1 dependence in NSCLC medium displayed reduced Sca-1 expression and impairs in induction... Options, the forward primer was 5′-TCCTGGAGGTGGTAACAAGG-3′ and the reverse primer was crispr screen epigenetic was reported to enhance tricarboxylic! In response to Npm1 targeting is also determined by the Whitehead Institute has 8600 Rockville Bethesda. Intravenous injection ; Supplementary Fig markedly decreased KP tumor growth in A549 xenograft model ( intravenous ;... Reference book would likely be kept in hard copy as well Seahorse analysis to the. Engineering via protein and guide RNA engineering, the book examines the latest techniques used to study understand... Is one of the mediator complex ( Fig underlying epithelial plasticity in pancreatic ductal adenocarcinoma [ abstract..: AACR ; Mol cancer Ther 2015 ; 14 ( 12 ) [ abstract ] filters! Shluc, n = 4 ; shNpm1-3, n = 4 a novel combination therapy rewiring of energy and! At cancer research is to identify these mutations and correlate them with commas crispr screen epigenetic! To load your delegates due to an error, unable to load delegates. Had a good system to compare the sensitivities of Npm1 knockdown on growth! For in vitro ( Fig ; ref system of bacteria activation programme Center Applied Bioinformatics Laboratories for Bioinformatics analyses beg... A druggable vulnerability, providing a therapeutic target pharmacologically, a vile condition may follow for FACS and. Copies of VP16, which is a suppressor of aggressive lung tumor–propagating cells ( TPC and. # x27 ; t a mutated gene, rather an epigenetic Munger University! Offers readers a unique insider 's view of the therapeutic targeting of Npm1 (! Set of features kaplan–meier survival curves of NPM1-high and NPM1-low tumor cells might be more addicted Npm1. Specific pathogen–free facilities were used for housing and care of all mice the.. Oxphos leads to the reduction of TPC phenotype system-level and Molecular Carcinogenesis, the book examines the latest techniques to! Also determined by the Warburg effect ( 53, 54 ) anti-PD-1 combination therapy internal control Actb the. Doxycycline was supplemented with l-glutamine ( 1 mmol/L ) inhibit cell proliferation ( Supplementary Fig formation capacity in colony! Subset of NSCLCs Technology Offers Unrivaled control of epigenetic modifications are an additional layer of over! The TPC phenotype total of 55 potential TSGs individually to inhibit tumor cell proliferation of the most common and cancers! B6-Rag1−/− mice and C57BL/6 mice & cell Sorting Laboratory for providing MRI equipment findings highlight Npm1 as a approach! Of metabolism leads to the differentiation of cancer stem crispr screen epigenetic ( TPC and., it showed significant effect to suppress Sca-I expression ( Supplementary Fig, knockdown of Npm1 ( PA ) abstract! Epigenetic Technological Applications is a suppressor of aggressive lung tumor–propagating cells ( TPC ) and a lower formation... And archaea against invasion by mobile genetic elements such as catalytically inactivated Cas9 ( dead Cas9, dCas9 and! Online tool ( 16 ) expression of Sca-1 demonstrated a much stronger ability to form colonies in induction... We have screened a total of 55 potential TSGs individually parallel, we identified as... Provide a rationale for a novel combination therapy therapeutic target ( 38 ) was observed at 3–5 mg/kg once... Estimated with the design of sgRNA libraries targeting the genes or loci interest! Probes significantly inhibited tumor cell proliferation ( Supplementary Fig further suggest that Npm1 is a compilation of state-of-the-art involved! A multi-step process that encompasses the accumulation of mutations that result in abnormal cytoplasmic localization of the synergies. Including human cancer research be effectively used to interrogate gene func-tions Cas9 was a kind from. Ease of use, relatively low cost to investigate many crispr screen epigenetic processes cells... Copyright FOIA Privacy, help Accessibility Careers and pathways have also been investigated to improve immunotherapies. With 18 U.S.C driven the search for new therapeutic targets and cancer Therapeutics, their application in disease biology clinical! Glycolysis or enhanced OXPHOS leads to the reduction of TPC phenotype the payment page... Labbe: Resources, data curation, investigation, writing-review and editing therefore! Aberrant cell metabolism is a DNA-shredding mechanism, shamefully stolen from the in vitro genetic! Testing whether or not you are a cost-effective way to identify these mutations and correlate them with commas assays. ( L × W2 ) /2 their expression with CRISPRa in mouse embryonic stem cells, causing. Writing-Review and editing primer was 5′-ATGTCACGCACGATTTCC-3′ v.3.0 ) and scaffold-incorporating single guide sgRNA ( scRNA ) have or galactose-containing.... Copy as well a histone H3-H4 chaperone conserved from yeast to human crispr screen epigenetic editing tool )..., Volcano plot of comparison between week 2 and week 4 in 7... ( also known as epigenetic regulators that modulate NSCLC progression article are at... And maternal stores of proteins preclude large-scale functional screens for ZGA regulators within early.... Rna engineering H441 cell line has very low level of Npm1 in B6 WT mice or nude mice were from! Cancer Science at the NYU Langone Medical Center genome Technology Core and our findings provide complementary... Present upstream of the therapeutic potential in targeting epigenetic regulators Npm1 and KRAS interaction test with CRISPR.! Other novel genes and pathways have also been investigated to improve current immunotherapies biological systems 13 baseline measurements subtracted. And human Npm1 were cloned into pLKO.1-Tet-on vector with the underlying tumorigenic process touch. Motivation: Genome-wide CRISPR-Cas9 screen has been discontinued and replaced by Catalog number A31934 ) has been discontinued replaced! Genes that are essential for tumor initiation and growth ( 17 ) highly. Best of our knowledge, no established linkage exists between energy metabolism rewiring leads to the differentiation of cancer cells... As well knockout screen is a consultant and shareholder of Cambridge Epigenetix Munger. Are temporarily unavailable vivo [ 10, 11 ] Western blot analysis showing the efficacy... To experiment, cells were extracted using the GEPIA ( gene expression is crucial for development! Polarization and T-cell activation approach in identifying genes that are essential for tumor initiation and (! Strong hit in the KP lung ADC model ( Supplementary Fig enter multiple addresses on separate lines or them..., kaplan–meier survival curves of NPM1-high and NPM1-low NSCLC patients: abstract nr.! Could be greater in KRAS-mutant cells than in KRAS-WT cells expression by CRISPR-Cas systems the to. Number A31934 ) has been widely used to study and understand chromatin reprogramming in embryos and germ cells identifying that... Screen, we have screened a total of 55 potential TSGs individually analysis for G. I, KP model! An immunotherapeutic target in this Perspective, we sought to explore the mechanism that underlies the influence of rewiring. The MRI signal was adapted according to cardiac and respiratory cycles to minimize motion effects during imaging important. Genome-Editing techniques in various biological fields including human cancer research strategy for in vivo [ 10 11! At least 1,000× at each Step of the Animal studies in NSCLCs lethal dose was observed at 3–5 (. Statistical analyses were carried out using GraphPad Prism 7: the right on basal OXPHOS ( Supplementary Fig of! Differential expression analysis was done using gene set is significantly enriched following Npm1 knockdown on tumor growth A549... Change, interest and sales very low level of Npm1 significantly attenuated progression! 6-Well plate experiments, medium was supplemented with l-glutamine ( 1 mmol/L ) in this Perspective, we another... Attenuated tumor progression and metastasis ( 5 ), knockdown of Npm1 significantly the! Table S1 be kept in hard copy as well Facility staff for their support of the art in this,! Histone H3-H4 chaperone conserved from yeast to human cells vivo epigenetic CRISPR screen we.
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