Biology subjects

Weiss, T. J.

Publications and source records attributed to Weiss, T. J..

3 recordsLinked to original sources

bioRxiv2021

Functional differences drive the selection of NRAS mutants in melanoma

Distinct NRAS mutants are enriched in various tumor types. Here, we generated a suite of fully congenic, conditional, Nras knock-in mouse models (LSL-Nras Q61R, -K, -L, -H, -P, -Q; G12D and G13D, -R) to test the hypothesis that melanocyte transformation requires functions specific to the NRAS mutants enriched in human melanoma (Q61R and Q61K). Consistent with the rarity of NRAS codon 12 and 13 mutants in human melanoma, spontaneous melanomas were rare or absent in mice expressing NRAS G12D, G13D or G13R. Mice expressing less common codon 61 alleles (Q61H, Q61P) also developed few or no tumors. NRAS Q61R, Q61K, or Q61L expression, by contrast, induced rapid melanoma onset with high penetrance. Cohorts of heterozygous mice containing one LSL-Nras Q61R and one LSL-Nras Q61K, -L, -H, -P, or -Q allele were generated to assess potential interactions between NRAS mutants. The ability of each Nras variant to substitute for an Nras Q61R allele was consistent with its own ability to drive spontaneous melanoma formation. However, LSL-Nras Q61Q/Q61R mice rarely developed tumors. In vitro experiments in mouse embryonic fibroblasts (MEFs) highlighted activation of the MAPK pathway as a defining difference between tumorigenic and non-tumorigenic NRAS mutants. Enhanced MAPK activation was associated with the promotion of BRAF-BRAF and BRAF-CRAF dimers. These results support the development of cancer preventative strategies specific to the properties of the commonly observed RAS mutants in each tumor type.

cancer biology↗

bioRxiv2019

UVA and UVB elicit distinct mutational signatures in melanoma

BRAF-mutant melanomas are more likely than NRAS-mutant melanomas to arise in anatomical locations protected from chronic sun damage. We hypothesized that this discrepancy in tumor location is a consequence of the differential sensitivity of BRAF and NRAS-mutant melanocytes to ultraviolet light (UV)-mediated carcinogenesis. We tested this hypothesis by comparing the mutagenic consequences of a single, narrow band ultraviolet-A (UVA; 320-400nm) or ultraviolet-B (UVB; 280-320nm) exposure in mouse models predisposed to Braf- or Nras-mutant melanoma. Exposures approximated the amount of UVA or UVB energy contained in ~40 minutes of summer sunlight. Tumor onset was accelerated in all UVB-, but only half of UVA- irradiated mice as compared to unirradiated controls. Melanomas from both mouse models, harbored recurrent mutations affecting the RING domain of MAP3K1 and Actin-binding domain of Filamin A irrespective of UV status. Melanomas from UVB-irradiated, Braf-mutant mice averaged twice as many SNVs (1,025 vs. 435) and five times as many dipyrimidine variants (33.3 vs. 5.7) than tumors from similarly irradiated Nras-mutant mice. We identified a mutational signature enriched in UVB-accelerated tumors which mirrored COSMIC signatures associated with human skin cancer. Notably, this signature was enriched to a greater extent in Braf- than Nras-mutant murine melanomas. These data suggest that oncogenic BRAF may enhance UVB carcinogenesis to promote melanoma formation at anatomic sites with low or intermittent sun exposure.

cancer biology↗

bioRxiv2018

GeneWeld: a method for efficient targeted integration directed by short homology

Choices for genome engineering and integration involve high efficiency with little or no target specificity or high specificity with low activity. Here, we describe a targeted integration strategy, called GeneWeld, and a vector series for gene tagging, pGTag (plasmids for Gene Tagging), which promote highly efficient and precise targeted integration in zebrafish embryos, pig fibroblasts, and human cells utilizing the CRISPR/Cas9 system. Our work demonstrates that in vivo targeting of a genomic locus of interest with CRISPR/Cas9 and a donor vector containing as little as 24 to 48 base pairs of homology directs precise and efficient knock-in when the homology arms are exposed with a double strand break in vivo. Our results suggest that the length of homology is not important in the design of knock-in vectors but rather how the homology is presented to a double strand break in the genome. Given our results targeting multiple loci in different species, we expect the accompanying protocols, vectors, and web interface for homology arm design to help streamline gene targeting and applications in CRISPR and TALEN compatible systems.

genetics↗