A versatile ES cell-based melanoma mouse modeling platform

Abstract

The cumbersome and time-consuming process of generating new mouse strains and multi-allelic experimental animals often hinders the use of genetically engineered mouse models (GEMM) in cancer research. Here, we describe the development and validation of an embryonic stem cell (ESC)-GEMM platform for rapid modeling of melanoma in mice. Our platform incorporates twelve clinically relevant genotypes composed of combinations of four driver alleles (LSL-BrafV600E, LSL-NrasQ61R, PtenFlox, Cdkn2aFlox) and regulatory alleles to spatiotemporally control the perturbation of genes-of-interest. Our ESCs produce high contribution chimeras, which recapitulate the melanoma phenotypes of conventionally bred mice. Using our ESC-GEMM platform to modulate Pten expression in melanocytes in vivo, we highlight the utility and advantages of gene depletion by CRISPR-Cas9, RNAi, or conditional knockout for melanoma modeling. Moreover, we use complementary genetic methods to demonstrate the impact of Pten restoration on the prevention and maintenance of Pten-deficient melanomas. Finally, we show that chimera-derived melanoma cell lines retain regulatory allele competency and are a powerful resource to complement ESC-GEMM chimera experiments in vitro and in syngeneic grafts in vivo. Thus, when combined with sophisticated genetic tools, our ESC-GEMM platform enables rapid, high-throughput, and versatile studies aimed at addressing outstanding questions in melanoma biology.

A Series of BRAF- and NRAS-Driven Murine Melanoma Cell Lines with Inducible Gene Modulation Capabilities

“…We previously reported the derivation of 17 melanoma cell lines (Table 1) from embryonic stem cell-genetically engineered mouse model (ESC-GEMMs) chimeras (Bok et al 2019). These lines harbor allele combinations that model the most frequent genetic alterations in human melanoma (Braf V600E , Nras Q61R , Pten / , Cdkn2a / ), the Tyrosinase-CreERT2 and CAGS-LSL-rtTA3 regulatory alleles, and the collagen homing cassette (CHC) for recombination-mediated cassette exchange (RMCE) (Bok et al 2019).…”

“…Moreover, the ESCs contain alleles enabling the integration of transgenes via recombination-mediated cassette exchange and the regulation of such transgenes via the Tet-ON system. We previously derived 17 cell lines from melanomas that arose in chimeras generated from untargeted ESCs (Bok et al 2019). We here report the establishment of another 4 murine melanoma cell lines and the systematic characterization of key features for all 21 cell lines.…”

“…We recently established a speedy mouse modeling platform that relies on efficient targeting of multi-allelic embryonic stem cells (ESCs) and uses chimeras as experimental animals (Bok et al 2019). These newly derived ESCs contain driver alleles that model the most common genetic alterations in human melanoma (Braf V600E , Nras Q61R , Pten / , Cdkn2a / ).…”

Chromosome 1q amplification perturbs a ceRNA network to promote melanoma metastasis

“…ceRNA network predictions are based on miRNAs conserved among vertebrates, and the sequence of the 3'UTRs of human and mouse CEP170, NUCKS1, and ZC3H11A have approximately 80% homology, suggesting human 1q AMP ceRNAs may regulate similar networks in human and mouse cells. To test this, we assessed if overexpression of human CEP170, NUCKS1, and ZC3H11A 3'UTRs was sufficient to accelerate melanoma development using our embryonic stem cell-genetically engineered mouse model (ESC-GEMM) platform (Bok et al, 2019). 1q AMP ceRNA constructs were generated in which the CEP170, NUCKS1, or ZC3H11A 3'UTRs were linked to the GFP cDNA and expressed under the control of the Dox-inducible TRE promoter (referred to as TRE-CEP170, TRE-NUCKS1, and TRE-ZC3H11A).…”

“…TRE-GFP lacking a 3'UTR was used as control. These constructs were targeted by recombination-mediated cassette exchange to the homing cassette in BP (LSL-Braf V600E ; Pten FL/WT ) embryonic stem cells (ESCs) (Bok et al, 2019). These ESCs also harbor Tyr-CreERt2 and CAGs-LSL-rtTA3 alleles for melanocyte-specific driver allele recombination and activation of the Doxinducible transgenes, respectively (Figure S3G).…”