TALENs and CRISPR/Cas9 fuel genetically engineered clinically relevant <i>Xenopus tropicalis</i> tumor models

Naert, Thomas; Nieuwenhuysen, Tom Van; Vleminckx, Kris

doi:10.1002/dvg.23005

Cited by 27 publications

(25 citation statements)

References 117 publications

(156 reference statements)

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“…We believe the latter to be attributable to the life-span of X. tropicalis that is substantially longer (>3x) than mice and may also underlie interspecies physiological differences. Interestingly, our In the comparison to established mammalian cancer models, we believe that Xenopus holds unique experimental advantages such as extremely straightforward tissue-restrictive CRISPR/Cas9 delivery and multiplexing in externally developing embryos 18 . Additionally, the X. tropicalis SC-PaNEC, glioblastoma and choroid plexus cancer models we report here are competitive with or outperform the existing Rb1/Tp53-inactivated mice models in terms of latency and penetrance of tumor development (Table S4).…”

Section: Discussionmentioning

confidence: 97%

RBL1 (p107) functions as tumor suppressor in glioblastoma and small-cell pancreatic neuroendocrine carcinoma

Naert¹,

Dimitrakopoulou²,

Tulkens³

et al. 2019

Preprint

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27Alterations of the retinoblastoma and/or the p53 signaling network are associated with specific cancers 28 such as high-grade astrocytoma/glioblastoma, small cell lung cancer (SCLC), choroid plexus tumors and 29 small-cell pancreatic neuroendocrine carcinoma (SC-PaNEC). However, the intricate functional 30 compensation between RB1 and the related pocket proteins RBL1/p107 and RBL2/p130 in suppressing 31 tumorigenesis remains poorly understood. Here we performed lineage-restricted parallel inactivation of 32 rb1 and rbl1 by multiplex CRISPR/Cas9 genome editing in the true diploid Xenopus tropicalis to gain 33 insight into these in vivo compensatory mechanisms. We show that while rb1 inactivation is sufficient to 34 induce choroid plexus papilloma, combined rb1 and rbl1 inactivation is required and sufficient to drive 35 SC-PaNEC, retinoblastoma and astrocytoma. Further, using a novel Li-Fraumeni syndrome-mimicking 36 tp53 mutant X. tropicalis line, we demonstrate increased malignancy of retinoblastoma-mutant neural 37 malignancies upon concomitant inactivation of tp53. Interestingly, although clinical SC-PaNEC samples 38 are characterized by abnormal p53 expression or localization, in the current experimental models, the 39 tp53 status had little effect on the establishment and growth of SC-PaNEC, but may rather be essential 40 for maintaining chromosomal stability. SCLC was only rarely observed in our experimental set-up, 41 indicating requirement of additional or alternative oncogenic insults. In conclusion, we used CRISPR/Cas9 42 to delineate the tumor suppressor properties of Rbl1 and generate new insights in functional 43 compensation within the retinoblastoma protein family in suppressing pancreatic and specific neural 44 cancers. 45 Keywords 46 p107, p53, CRISPR/Cas9, Pancreatic neuroendocrine carcinoma, choroid plexus, retinoblastoma, Xenopus 47 tropicalis 48 49 50The interplay of the signaling networks controlling cell cycle (e.g. Retinoblastoma (RB)) and cell death 51 (e.g. p53) in suppressing the development of cancers including, amongst others, glioblastoma, choroid 52 plexus carcinoma, pancreatic neuroendocrine carcinoma and small-cell lung cancer was previously 53 demonstrated by clinical and animal modeling studies 1-6 . Unsatisfactory, the median survival prospects 54 for patients diagnosed with p53 and RB1 deficient cancers are extremely dismal, e.g. for small cell lung 55 cancer (SCLC) (stage IV: 8-10 months 7 ), small cell pancreatic neuroendocrine carcinoma (SC-PaNEC) (11 56 months 8 ), choroid plexus tumors (tp53 mutated WHO grade III: 2-4 months 9 ) and glioblastoma (12-18 57 months 10 ). As such, continued generation of novel and short latency preclinical models for these highly 58 aggressive cancers remains necessary to fuel rational design of molecular targeted drug therapies. 59 Intriguingly, the RB signaling network entails a family of three pocket proteins (RB1, RBL1, RBL2), 60 which in union tightly regulate G1/S cell cycle progression, and whose differential expression may 61 underl...

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Section: Discussionmentioning

confidence: 97%

RBL1 (p107) functions as tumor suppressor in glioblastoma and small-cell pancreatic neuroendocrine carcinoma

Naert¹,

Dimitrakopoulou²,

Tulkens³

et al. 2019

Preprint

Self Cite

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“…As in X. laevis , tracer fluorescent mRNAs can be injected into specific X. tropicalis blastomeres to act as reporters in a whole‐embryo or tissue‐specific manner; and morpholinos or mRNAs can be injected for knock down or overexpression of gene products. In the same way, TALEN and CRISPR reagents can be injected to generate loss‐of‐function mutations . Both F 0 mosaic genome editing and stable edited lines have proliferated in recent years, and community resources, notably at the national Xenopus Resource Center, have prioritized generation of hundreds of mutant X. tropicalis lines for genes valuable to the greater Xenopus and medical genetics communities.…”

Section: Genetic Engineering Toolsmentioning

confidence: 99%

“…In the same way, TALEN and CRISPR reagents can be injected to generate loss-of-function mutations. 11 Both F 0 mosaic genome editing and stable edited lines have proliferated in recent years, and community resources, notably at the national Xenopus Resource Center, 12 have prioritized generation of hundreds of mutant X. tropicalis lines for genes valuable to the greater Xenopus and medical genetics communities.…”

Section: Introductionmentioning

confidence: 99%

Advancing genetic and genomic technologies deepen the pool for discovery in Xenopus tropicalis

Kakebeen

Wills

2019

Developmental Dynamics

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Xenopus laevis and Xenopus tropicalis have long been used to drive discovery in developmental, cell, and molecular biology. These dual frog species boast experimental strengths for embryology including large egg sizes that develop externally, well‐defined fate maps, and cell‐intrinsic sources of nutrients that allow explanted tissues to grow in culture. Development of the Xenopus cell extract system has been used to study cell cycle and DNA replication. Xenopus tadpole tail and limb regeneration have provided fundamental insights into the underlying mechanisms of this processes, and the loss of regenerative competency in adults adds a complexity to the system that can be more directly compared to humans. Moreover, Xenopus genetics and especially disease‐causing mutations are highly conserved with humans, making them a tractable system to model human disease. In the last several years, genome editing, expanding genomic resources, and intersectional approaches leveraging the distinct characteristics of each species have generated new frontiers in cell biology. While Xenopus have enduringly represented a leading embryological model, new technologies are generating exciting diversity in the range of discoveries being made in areas from genomics and proteomics to regenerative biology, neurobiology, cell scaling, and human disease modeling.

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“…We previously demonstrated penetrant induction of desmoid tumors (DT) by tissue-restricted TALENsmediated genome engineering of apc in the anterior endoderm 8 . In the meanwhile, the advent of multiplex CRISPR/Cas9 for parallel gene inactivation pruned us to investigate whether DTs could similarly be induced by CRISPR/Cas9, in order to facilitate further downstream multiplexed gene inactivation studies 12 . We performed CRISPR/Cas9-mediated genome engineering of apc, in transgenic 8-cell embryos carrying a Wnt/β-catenin reporter construct 13 (Table S1a).…”

Section: Apc Tumor Suppressor Gene Inactivation Initiates Desmoid Tummentioning

confidence: 99%

CRISPR-SID: identifying EZH2 as a druggable target for desmoid tumors viain vivodependency mapping

Naert

Tulkens

Nieuwenhuysen

et al. 2019

Preprint

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Identification of true dependencies in cancer is pivotal to the elucidation of novel therapeutic strategies to increase prospects for cancer patients. Unfortunately, in vivo identification of genetic dependencies has long relied on expensive and time-consuming breeding of genetically engineered animal models.Recently, in vitro CRISPR/Cas9 screens provided a new method for rapid and genome-wide identification of genetic dependencies. Nevertheless, genetic dependencies would ideally be identified using in vivo cancer models initiated by clinically relevant oncogenic driver or tumor suppressor insults. Here, we report a new methodology called CRISPR/Cas9-mediated Negative Selection Identification of genetic Dependencies (CRISPR-NSID) that allows in vivo elucidation of cancer cell vulnerabilities in genetic cancer models. The methodology hinges on the fact that for a genetic dependency there is an incapability for recovering tumors carrying biallelic frameshift mutations in this gene. We demonstrate how integrating experimentally determined, or in silico predicted, probabilities of frameshift editing for any given gRNA can be employed to ascertain negative selection pressure on inactivation of a genetic dependency during tumorigenesis. As a proof-of-principle, we use CRISPR-NSID to identify ezh2 and creb3l1 as genetic dependencies in desmoid tumors (desmoid-type fibromatosis) occurring in a Xenopus tropicalis cancer model driven by apc mutations. Bridging CRISPR-NSID to a clinically unmet need, we further demonstrate the promise for EZH2 inhibition as a new therapeutic strategy for desmoid tumors.This study establishes a new methodology for rapid identification of genetic dependencies in monoclonal disorders with wide adaptability to other model systems and organisms.

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TALENs and CRISPR/Cas9 fuel genetically engineered clinically relevant Xenopus tropicalis tumor models

Cited by 27 publications

References 117 publications

RBL1 (p107) functions as tumor suppressor in glioblastoma and small-cell pancreatic neuroendocrine carcinoma

RBL1 (p107) functions as tumor suppressor in glioblastoma and small-cell pancreatic neuroendocrine carcinoma

Advancing genetic and genomic technologies deepen the pool for discovery in Xenopus tropicalis

CRISPR-SID: identifying EZH2 as a druggable target for desmoid tumors viain vivodependency mapping

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