A new CRISPR mediated intestinal tumor mouse model targeting four canonical tumor suppressor genes.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e16064-e16064
Author(s):  
Hajime Kashima ◽  
Daniel Veronese-Paniagua ◽  
Anthony Fischer ◽  
Blair Madison ◽  
Deborah Rubin
Keyword(s):  
Tumor Suppressor ◽  
Mouse Model ◽  
Tumor Suppressor Genes ◽  
Rapid Development ◽  
Tumor Development ◽  
Intestinal Tumor ◽  
Intestinal Tumors ◽  
Suppressor Genes ◽  
Intestinal Tumorigenesis ◽  
Guide Rnas

e16064 Background: Mouse models of intestinal tumorigenesis have been developed and many of them involve mutations in the Apc gene. However, human intestinal tumors contain multiple additional sporadic mutations in tumor suppressor genes (TSGs). Our goal is to develop a novel mouse model of intestinal tumorigenesis that can recapitulate the natural history of mutations in diverse stages of tumor development. Methods: We used multiple guide RNAs to achieve random mutations in the canonical TSGs, Apc, Pten, Smad4, and Tp53. We generated transgenic (PPAS) mice that constitutively express the appropriate guide RNAs. Moreover, we achieved inducible Cas9 expression in icCas9N mice intestine using the Villin promoter to drive both a doxycycline-dependent activator and a doxycycline-inactivated repressor. We fed the doxycycline chow to PPAS:icCas9 double transgenic mice from the age of 6 to 8 weeks, and harvested intestine at 12 weeks. Results: We examined seven PPAS;icCas9 mice, and detected intestinal tumors in all the mice. Two mice had small intestinal tumor, three mice had colonic tumor, and two mice had tumors in both small and large intestine. The average number of tumors were 0.86, 1.57, 2.43 in small intestine, colon, and both respectively. We analyzed mutations in 11 tumors in 6 mice. The mutation patterns of Apc, Pten, Smad4 and Tp53 in tumors shared three distinct patterns. One was characterized by mutations in all four TSGs (n = 9). The second showed mutation in APC and Smad4 and Pten (n = 1). The third showed mutation only in Tp53 (n = 1). Normal intestine and colon in PPAS:icCas9 mice had no mutations. Conclusions: This model provides a powerful platform for modeling intestinal tumorigenesis driven by the canonical signaling pathway which are commonly dysregulated in colon cancer. This model provides a means for rapid development of intestinal tumors in mice, enabling an investigation of the relationship between novel candidate regulators of tumorigenesis and the canonical signaling pathways regulated by these four common TSGs. [Table: see text]

scholarly journals TMOD-18. TARGETING THE PI3K/AKT PATHWAY IN MYCN AMPLIFIED HIGH GRADE GLIOMAS

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii231-ii232
Author(s):  
Katharine Halligan ◽  
Ann-Catherine Stanton ◽  
Matthew Halbert ◽  
Brian Golbourn ◽  
Stephen Mack ◽  
...  
Keyword(s):  
Stem Cells ◽  
Tumor Suppressor ◽  
Mouse Model ◽  
Protein Expression ◽  
Neural Stem Cells ◽  
Tumor Suppressor Genes ◽  
Akt Pathway ◽  
High Grade ◽  
Suppressor Genes ◽  
High Grade Gliomas

Abstract Pediatric glioblastoma (pGBM) are incurable brain tumors with overall poor prognosis and response to treatments due to molecular and epigenetic heterogeneity. In particular, the MYCN subtype of pGBM are a highly aggressive form of GBM with a dismal median survival of only 14 months. Furthermore, this subtype is enriched with loss of the tumor suppressor genes TP53 and PTEN, leading to aberrantly active PI3K-AKT signaling pathway and DNA-checkpoint abnormalities. Here, we report the generation of a novel syngeneic mouse model that recapitulates the features of the MYCN subtype of pGBM. We isolated Sox2-Cre neural stem cells from C57BL/6 mice and transduced inverted retroviral-cassettes of the murine Mycn oncogene simultaneously with shRNA targeting tumor suppressor genes p53 and Pten. Retroviral-cassettes are flanked by tandem LoxP sites arranged so that Cre recombinase expression inverts the cassettes in frame allowing for MYCN protein expression and loss of the P53/PTEN proteins. Transgene activation is accompanied with selectable cell surface markers and fluorescent tags enabling for fluorescent activated cell sorting (FACS) of the desired cell populations. Neural stem cells with MYCN protein expression and concurrent silencing of P53 and PTEN protein (NPP cells) result in significantly increased proliferation and activation of PI3K-AKT pathway as compared to control neural stem cells and have. Injection of NPP cells into the forebrain of immune competent C57BL/6 mice result in the formation of invasive high-grade gliomas with a lethal phenotype at ~50 days post injection. Using several next generation brain penetrant small molecule inhibitors of the PI3K-AKT pathway, we show inhibition of tumorigenesis in vitro. Moreover, we have identified several novel mechanisms of PI3KAKT treatment resistance and are currently identifying therapies that may overcome this resistance through RNA seq analysis. In summary, well defined genetic drivers of GBM can lead to informed mouse model generation to test promising therapies.

scholarly journals Simultaneous haploinsufficiency of Pten and Trp53 tumor suppressor genes accelerates tumorigenesis in a mouse model of prostate cancer

2009 ◽  
Vol 77 (1) ◽  
pp. 103-111 ◽  
Author(s):  
Suzana S. Couto ◽  
Mei Cao ◽  
Paulo C. Duarte ◽  
Whitney Banach-Petrosky ◽  
Shunyou Wang ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Tumor Suppressor ◽  
Mouse Model ◽  
Tumor Suppressor Genes ◽  
Suppressor Genes

scholarly journals Genotyping of Adrenocortical Tumors: Very Frequent Deletions of the MEN1 Locus in 11q13 and of a 1-Centimorgan Region in 2p161

1999 ◽  
Vol 84 (2) ◽  
pp. 730-735
Author(s):  
Magnus Kjellman ◽  
Leyla Roshani ◽  
Bin Tean Teh ◽  
Olli-Pekka Kallioniemi ◽  
Anders Höög ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Loss Of Heterozygosity ◽  
Tumor Suppressor Genes ◽  
Familial Cancer ◽  
Genetic Material ◽  
Tumor Development ◽  
Carney Complex ◽  
Chromosome 11 ◽  
Suppressor Genes ◽  
Men1 Gene

To identify chromosomal regions that may contain loci for tumor suppressor genes involved in adrenocortical tumor development, a panel of 60 tumors (39 carcinomas and 21 adenomas) were screened for loss of heterozygosity. Although the vast majority of loss of heterozygosity (LOH) were detected in the carcinomas and involved chromosomes 2, 4, 11, and 18, only few were found in the adenomas. Therefore, 2 loci that harbor the familial cancer syndromes Carney complex in 2p16 and the multiple endocrine neoplasia type 1 gene in 11q13 were further studied in 27 (13 carcinomas and 14 adenomas) of the 60 tumors. Detailed analysis of the 2p16 region mapped a minimal area of overlapping deletions to a 1-centimorgan region, which is separate from the Carney complex locus. LOH for a microsatellite marker (PYGM), very close to the MEN1 gene, was detected in all 8 informative carcinomas (100%) and in 2 of 14 adenomas. Of the 27 cases analyzed in detail, 13 cases (11 carcinomas and 2 adenomas) showed LOH on chromosome 11 and was therefore selected for MEN1 gene mutation analysis. In 6 cases a common polymorphism (Asp418Asp) was found, but no mutation was detected. In conclusion, our data indicate the existence of tumor suppressor genes at multiple chromosomal locations, whose inactivations are involved in the development of adrenocortical carcinomas. Loss of genetic material from 2p16 was strongly associated with the malignant phenotype, as it was seen in almost all carcinomas but not in any of the adenomas. LOH in 11q13 also occurred frequently in the carcinomas, but was not associated with a MEN1 mutation, suggesting the involvement of a different tumor suppressor gene on this chromosome.

scholarly journals Promoter Hypermethylation of Tumor Suppressor Genes Located on Short Arm of the Chromosome 3 as Potential Biomarker for the Diagnosis of Nasopharyngeal Carcinoma

Diagnostics ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1404
Author(s):  
Thuan Duc Lao ◽  
Toan Ngoc Nguyen ◽  
Thuy Ai Huyen Le
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Tumor Suppressor ◽  
Tumor Suppressor Genes ◽  
Target Genes ◽  
Tumor Development ◽  
Promoter Hypermethylation ◽  
Chromosome 3 ◽  
Suppressor Genes ◽  
Chromosome 3P ◽  
Potential Biomarker

DNA methylation, the most common epigenetic alteration, has been proven to play important roles in nasopharyngeal carcinoma (NPC). Numerous tumor suppressor genes located on the chromosome 3p, particularly in the region of 3p21, are frequently methylated in NPC, thus suggesting great potential for diagnosis of NPC. In this review, we summarize recent findings of tumor suppressor genes on chromosome 3 that likely drive nasopharyngeal tumor development and progression, based on previous studies related to the hypermethylation of these target genes. Better understanding will allow us to design further experiments to establish a potential test for diagnosis of NPC, as well as bring about methylated therapies to improve the treatment of NPC.

Molecular Pathogenesis of Oral Squamous Cell Carcinoma: Implications for Therapy

2008 ◽  
Vol 87 (1) ◽  
pp. 14-32 ◽  
Author(s):  
S. Choi ◽  
J.N. Myers
Keyword(s):  
Squamous Cell Carcinoma ◽  
Tumor Suppressor ◽  
Oral Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Tumor Suppressor Genes ◽  
Tumor Development ◽  
Genetic Alterations ◽  
Suppressor Genes ◽  
Promote Tumor Development

The development of oral squamous cell carcinoma (OSCC) is a multistep process requiring the accumulation of multiple genetic alterations, influenced by a patient’s genetic predisposition as well as by environmental influences, including tobacco, alcohol, chronic inflammation, and viral infection. Tumorigenic genetic alterations consist of two major types: tumor suppressor genes, which promote tumor development when inactivated; and oncogenes, which promote tumor development when activated. Tumor suppressor genes can be inactivated through genetic events such as mutation, loss of heterozygosity, or deletion, or by epigenetic modifications such as DNA methylation or chromatin remodeling. Oncogenes can be activated through overexpression due to gene amplification, increased transcription, or changes in structure due to mutations that lead to increased transforming activity. This review focuses on the molecular mechanisms of oral carcinogenesis and the use of biologic therapy to specifically target molecules altered in OSCC. The rapid progress that has been made in our understanding of the molecular alterations contributing to the development of OSCC is leading to improvements in the early diagnosis of tumors and the refinement of biologic treatments individualized to the specific characteristics of a patient’s tumor.

A Mouse Model of Alkylator-Induced Myelodysplastic Syndrome.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 368-368 ◽  
Author(s):  
Deepa Edwin ◽  
Christine McMahon ◽  
Friederike Kreisel ◽  
Molly Bogue ◽  
Timothy S. Fenske ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Mouse Model ◽  
Tumor Suppressor Genes ◽  
Bone Marrow Cells ◽  
Point Mutations ◽  
Comparative Genomic ◽  
Suppressor Genes ◽  
Hematopoietic Stem ◽  
Molecular Abnormalities ◽  
Key Features

Abstract The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis and cellular dysplasia. Peripheral blood cytopenias and progression to AML are important clinical sequelae of MDS. 10–20% of MDS cases are a consequence of prior treatment with alkylators. The molecular basis of therapy-related MDS (t-MDS) is poorly understood. Point mutations of RAS family members and inactivation of the p53 and p15 tumor suppressor genes by mutation or hypermethylation represent the most frequently reported molecular abnormalities in MDS. Clonal cytogenetic changes, usually involving loss of material from chromosomes 5 and/or 7 are present in >90% of t-MDS cases. These recurring deletions suggest that myeloid tumor suppressor genes may be present in these regions, although their identify has not yet been established. Progress in understanding the genetic basis of human MDS has been hampered by a lack of suitable animal models. To develop a mouse model of t-MDS, we screened 32 inbred strains for susceptibility to t-MDS after treatment with the prototypical alkylating agent, N-nitroso-N-ethylurea (ENU). Mice (n=12 per strain) received two doses of ENU (100mg/kg, IP) or no treatment (n=12 per strain) at 9 and 10 weeks of age. Among the strains tested, SWR/J mice were found to be highly susceptible to myeloid malignancies (MDS/AML). We confirmed this in a second cohort. 10 of 33 (30%) ENU-treated SWR/J mice developed key features of MDS, including anemia (mean Hb=10.9 ±1.1 g/dL, compared to mean Hb=14.0 ±0.3 g/dL in 32 untreated age and sex-matched SWR/J mice, p=0.0006) and erythroid dysplasia (megaloblastic maturation, nuclear budding and blebbing in normoblasts) with a latency of approximately 30 weeks after ENU exposure. There was also evidence of dysplasia in the megakaryocytic lineage, manifested by numerous micromegakaryocytes with unilobar nuclei. Mild dysplastic features were detected rarely in untreated controls from this strain. The t-MDS mice developed significant splenomegaly (mean=0.49 ±0.19 g compared to control mean=0.15 ±0.01 g, p=0.004) with histologic evidence of increased extramedullary hematopoiesis. No significant immunophenotypic differences were detected in bone marrow cells from the t-MDS cases compared to controls. Iron stores were normal with no evidence of ringed sideroblasts. In 2 of the 10 affected mice, MDS evolved to AML, manifested by rapid breathing, circulating myeloid blasts, and leukocytosis (21-621,000 cells/μL). An additional 3 ENU-treated SWR/J mice developed AML without evidence of a preceding MDS phase. All AML cases had a Kit+Gr1+CD34- phenotype with no expression of lymphoid markers. The blasts were myeloperoxidase negative. This mouse model recapitulates many key features of human alkylator-associated t-MDS/AML and should be useful for discovery of mutations involved in the pathogenesis of this syndrome. We are employing array-based comparative genomic hybridization and candidate gene resequencing as tools for mutation discovery in this model. Because of their unique sensitivity to t-MDS, the SWR/J strain will also be particularly useful for identification of germline polymorphisms that affect susceptibility to alkylator-associated t-MDS/AML.

scholarly journals Ribosomal Proteins Rps19 and Rps14 Cooperate As Tumor Suppressor Genes with p53

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2943-2943
Author(s):  
Maria Virgilio ◽  
Grzegorz Pietka ◽  
Elspeth M Payne
Keyword(s):  
Tumor Suppressor ◽  
Tumor Suppressor Genes ◽  
Tumor Development ◽  
Tumor Formation ◽  
Transcription Activator ◽  
Suppressor Genes ◽  
Exon 2 ◽  
Peripheral Nerve Sheath Tumors ◽  
Exon 1 ◽  
Mutant Lines

Abstract Diamond-Blackfan Anemia (DBA) is a congenital bone marrow failure syndrome that manifests as a profound macrocytic anemia and classically presents within the first year of life. Heterozygous mutations in, or genomic loss of one of several Ribosomal Protein (RP) genes have been identified in over 50% of DBA patients, most commonly RPS19, accounting for 25% of all cases. DBA shares a similar erythroid phenotype to the 5q- subtype of myelodysplastic syndrome in which anemia is thought to arise from heterozygous loss of RPS14. Anemia in these conditions is at least partially due to p53-mediated apoptosis and cell cycle arrest of erythroid progenitors. To further study the role of p53 in the pathogenesis of DBA and 5q- syndrome, we employed genome editing tools to generate stable Rps14 and Rps19 knockout zebrafish lines. We generated Transcription Activator-Like Effector Nucleases (TALENs) targeting exon 1 of rps19 and exon 1 of rps14 as well as Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR) single guide RNAs (sgRNA) targeting exon 2 of rps19. TALENs or CRISPRs were injected into p53m214k/m214k zebrafish embryos at the single-cell stage. This zebrafish line carries a mutated p53 that is insensitive to DNA damage and hence prone to tumor formation. rps19 CRISPR sgRNAs were injected with mRNAs encoding Cas9, Cas9D10A nickase, and a ssDNA guide with a human DBA mutation. For each cohort of embryos injected, genomic DNA analysis from 20 phenotypically normal embryos from each clutch was screened to determine the efficacy of cleavage by TALEN and CRISPR using MiSeq. Mutations were identified in 30% (rps14 TALEN) 29% (rps19 TALEN), 27% (rps19 Crispr Cas9) and 12% (rps19 Crispr Cas9D10A) of reads. None of the rps19Crispr Cas9D10A carried the ssDNA guide mutation, rather single nucleotide variants and indels similar to those observed with Cas9. The remaining embryos from each F0 clutch were raised in order to generate stable mutant lines in the F1 generation; however, early, overt tumor growth was noted in all RP injected lines. Tumors were observed from 4 months post fertilization compared with 9 months for uninjected controls. F0 RP mosaic fish continued to develop tumors earlier than uninjected counterparts. At 10 months of age tumor development was statistically significantly higher in rps19 and rps14 TALEN and rps19 Cas9D10A and trended towards significance in rps19 Cas9 injected fish. Overall survival was significantly reduced in each of the cohorts compared to p53m214k/m214k uninjected controls (p<0.0001). Preliminary histology of grown tumors has shown melanomas and malignant peripheral nerve sheath tumors. Zebrafish injected with an unrelated TALEN targeting a zinc transporter (SLC30A10), into p53m214k mutant embryos do not show any increase in tumor formation compared to uninjected controls. Notably several RP’s have been shown to be haploinsufficient tumor suppressor genes in their own right in zebrafish and drosophila models. To determine if the early tumor development in p53m214k zebrafish was simply additive to a potential tumor suppressor effect of Rps14 or Rps19 alone, we injected WT embryos with the rps14 and rps19 TALENS. High mortality in rps14 and rps19 TALEN injected WT embryos impeded this analysis; however recent published reports on stable Rps19 mutant zebrafish do not report an increase in tumor incidence. Interestingly, embryo survival was not affected when TALENs were injected into p53m214/+. Analysis of these zebrafish is ongoing. Our results show that loss of Rps14 or Rps19 accelerates the development of tumors in the p53m214k/m214k mutant line. This effect is independent of the RP or the method of mutation (TALEN vs CRISPR), indicating that off target effects are unlikely to be responsible for this observation. As these are mosaic F0 fish, it is possible that tumors may arise from cells with homozygous RP mutations. Further molecular analysis will reveal this. We have now identified 2 stable Rps19 mutant lines, and tumor analysis of F1 fish from these lines is ongoing. In conclusion, we have shown that loss of Rps14 or Rps19 cooperates with a loss of function p53 mutation to accelerate tumor formation and death. Our results highlight the importance of caution in using p53 suppressors as a therapeutic option in RP deficient patients. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document