Carla Tangermann

Although the functional effects of many recurrent cancer mutations have been characterized, The Cancer Genome Atlas contains more than 10 million functionally uncharacterized, nonrecurrent events. It is proposed that the context-specific activity of transcription factors assessed through the expression of their transcriptional targets serves as a sensitive and accurate reporter assay for evaluating the functional roles of oncogene mutations. Analysis of transcription factor activity in samples with mutations of unknown significance, compared to established gain-of-function (hypermorph) or loss-of-function (hypomorph) mutations in the same gene, enabled functional characterization of 583,089 individual mutational events across TCGA. This approach facilitated the identification of neomorphic mutations (gain of new function) or mutations that phenocopy mutations in other genes (mutational mimicry). Validation using exogenous mutation expression assays confirmed the majority of predicted loss-of-function, gain-of-function, neomorphic and neutral (no predicted functional effect) mutations in PIK3CA and FGFR2. These findings may inform targeted therapy decisions for patients with mutations of unknown significance in established oncogenes.

Variants of uncertain significance represent the biggest challenge for genomics-based precision oncology. Activated fibroblast growth factor receptors (FGFRs) frequently drive tumorigenesis by different genetic aberrations. However, it remains unknown which of the many point mutations affecting FGFR1 , FGFR2 , FGFR3 or FGFR4 in cancer are druggable, that is, activating signaling while not mediating FGFR inhibitor resistance. Here we implemented a saturation mutational scanning platform to screen all 11,520 possible point mutations covering the kinase domains of FGFR1 – 4 . Pooled positive selection screens identified 474 activating and 738 mutations mediating resistance to the FGFR inhibitors pemigatinib and futibatinib, together revealing 301 druggable FGFR mutations analogous to a strong PS3/BS3 evidence level. The screens also identified loss-of-function mutations and an association of gain-of-function mutations with hydrophobic changes. The functional screens identified 97% of acquired resistance mutations in clinical trials. Our comprehensive catalog of every druggable mutation in the FGFR kinase domains is readily available for clinical decision support.

Nonstop extension mutations, a.k.a. stop-lost or stop-loss mutations, convert a stop codon into a sense codon resulting in translation into the 3’ untranslated region until the next in-frame stop codon, thereby extending the C-terminus of a protein. In cancer, only nonstop mutations in SMAD4 have been functionally characterized, while the impact of other nonstop mutations remain unknown. Here, we exploit our pan-cancer NonStopDB dataset and test all 2335 C-terminal extensions arising from somatic nonstop mutations in cancer for their impact on protein expression. In a high-throughput screen, 56.1% of the extensions effectively reduce protein abundance. Extensions of multiple tumor suppressor genes like PTEN, APC, B2M, CASP8, CDKN1B and MLH1 are effective and validated for their suppressive impact. Importantly, the effective extensions possess a higher hydrophobicity than the neutral extensions linking C-terminal hydrophobicity with protein destabilization. Analyzing the proteomes of eleven different species reveals conserved patterns of amino acid distribution in the C-terminal regions of all proteins compared to the proteomes like an enrichment of lysine and arginine and a depletion of glycine, leucine, valine and isoleucine across species and kingdoms. These evolutionary selection patterns are disrupted in the cancer-derived effective nonstop extensions.

Recent studies have demonstrated that not only genes but also entire chromosomes can be engineered using clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPER-associated protein 9 (Cas9)^1,2,3,4,5. A major objective of applying chromosome restructuring in plant breeding is the manipulation of genetic exchange⁶. Here we show that meiotic recombination can be suppressed in nearly the entire chromosome using chromosome restructuring. We were able to induce a heritable inversion of a >17 Mb-long chromosome fragment that contained the centromere and covered most of chromosome 2 of the Arabidopsis ecotype Col-0. Only the 2 and 0.5 Mb-long telomeric ends remained in their original orientation. In single-nucleotide polymorphism marker analysis of the offspring of crosses with the ecotype Ler-1, we detected a massive reduction of crossovers within the inverted chromosome region, coupled with a shift of crossovers to the telomeric ends. The few genetic exchanges detected within the inversion all originated from double crossovers. This not only indicates that heritable genetic exchange can occur by interstitial chromosome pairing, but also that it is restricted to the production of viable progeny.

CRISPR/Cas has been mainly used for mutagenesis through the induction of double strand breaks (DSBs) within unique protein-coding genes. Using the SaCas9 nuclease to induce multiple DSBs in functional repetitive DNA of Arabidopsis thaliana, we can now show that cell death can be induced in a controlled way. This approach, named CRISPR-Kill, can be used as tool for tissue engineering. By simply exchanging the constitutive promoter of SaCas9 with cell type-specific promoters, it is possible to block organogenesis in Arabidopsis. By AP1-specific expression of CRISPR-Kill, we are able to restore the apetala1 phenotype and to specifically eliminate petals. In addition, by expressing CRISPR-Kill in root-specific pericycle cells, we are able to dramatically reduce the number and the length of lateral roots. In the future, the application of CRISPR-Kill may not only help to control development but could also be used to change the biochemical properties of plants.

Recent studies have demonstrated that not only genes, but also entire chromosomes can be engineered using CRISPR-Cas9 1–5. A major objective of applying chromosome restructuring (CR) in plant breeding is the manipulation of genetic exchange 6. Here we show that meiotic recombination can be suppressed in nearly the entire chromosome using CR. We were able to induce a heritable inversion of a more than 17 Mb-long chromosome fragment, which contained the centromere and covered most of chromosome 2 of the Arabidopsis ecotype Col-0. Only the 2 and 0.5 Mb-long telomeric ends remained in their original orientation. In SNP marker analysis with the offspring of crosses with the ecotype Ler-1, we detected a massive reduction of crossovers (CO) within the inverted chromosome region, coupled with a shift of CO to the telomeric ends. Interestingly, the few genetic exchanges detected within the inversion all originated from double CO. This not only indicates that heritable genetic exchange can occur by interstitial chromosome pairing, but also that it is restricted to the production of viable progeny.