Generation of precision preclinical cancer models using regulated in vivo base editing.

Although single-nucleotide variants (SNVs) make up the majority of cancer-associated genetic changes and have been comprehensively catalogued, little is known about their impact on tumor initiation and progression. To enable the functional interrogation of cancer-associated SNVs, we developed a mouse system for temporal and regulatable in vivo base editing. The inducible base editing (iBE) mouse carries a single expression-optimized cytosine base editor transgene under the control of a tetracycline response element and enables robust, doxycycline-dependent expression across a broad range of tissues in vivo. Combined with plasmid-based or synthetic guide RNAs, iBE drives efficient engineering of individual or multiple SNVs in intestinal, lung and pancreatic organoids. Temporal regulation of base editor activity allows controlled sequential genome editing ex vivo and in vivo, and delivery of sgRNAs directly to target tissues facilitates generation of in situ preclinical cancer models.

CRISPR in cancer biology and therapy.

Over the past decade, CRISPR has become as much a verb as it is an acronym, transforming biomedical research and providing entirely new approaches for dissecting all facets of cell biology. In cancer research, CRISPR and related tools have offered a window into previously intractable problems in our understanding of cancer genetics, the noncoding genome and tumour heterogeneity, and provided new insights into therapeutic vulnerabilities. Here, we review the progress made in the development of CRISPR systems as a tool to study cancer, and the emerging adaptation of these technologies to improve diagnosis and treatment.

Base editing sensor libraries for high-throughput engineering and functional analysis of cancer-associated single nucleotide variants.

Base editing can be applied to characterize single nucleotide variants of unknown function, yet defining effective combinations of single guide RNAs (sgRNAs) and base editors remains challenging. Here, we describe modular base-editing-activity 'sensors' that link sgRNAs and cognate target sites in cis and use them to systematically measure the editing efficiency and precision of thousands of sgRNAs paired with functionally distinct base editors. By quantifying sensor editing across >200,000 editor-sgRNA combinations, we provide a comprehensive resource of sgRNAs for introducing and interrogating cancer-associated single nucleotide variants in multiple model systems. We demonstrate that sensor-validated tools streamline production of in vivo cancer models and that integrating sensor modules in pooled sgRNA libraries can aid interpretation of high-throughput base editing screens. Using this approach, we identify several previously uncharacterized mutant TP53 alleles as drivers of cancer cell proliferation and in vivo tumor development. We anticipate that the framework described here will facilitate the functional interrogation of cancer variants in cell and animal models.

Dietary fructose improves intestinal cell survival and nutrient absorption.

Fructose consumption is linked to the rising incidence of obesity and cancer, which are two of the leading causes of morbidity and mortality globally1,2. Dietary fructose metabolism begins at the epithelium of the small intestine, where fructose is transported by glucose transporter type 5 (GLUT5; encoded by SLC2A5) and phosphorylated by ketohexokinase to form fructose 1-phosphate, which accumulates to high levels in the cell3,4. Although this pathway has been implicated in obesity and tumour promotion, the exact mechanism that drives these pathologies in the intestine remains unclear. Here we show that dietary fructose improves the survival of intestinal cells and increases intestinal villus length in several mouse models. The increase in villus length expands the surface area of the gut and increases nutrient absorption and adiposity in mice that are fed a high-fat diet. In hypoxic intestinal cells, fructose 1-phosphate inhibits the M2 isoform of pyruvate kinase to promote cell survival5-7. Genetic ablation of ketohexokinase or stimulation of pyruvate kinase prevents villus elongation and abolishes the nutrient absorption and tumour growth that are induced by feeding mice with high-fructose corn syrup. The ability of fructose to promote cell survival through an allosteric metabolite thus provides additional insights into the excess adiposity generated by a Western diet, and a compelling explanation for the promotion of tumour growth by high-fructose corn syrup.

Functional Common and Rare ERBB2 Germline Variants Cooperate in Familial and Sporadic Cancer Susceptibility.

We investigated a Spanish and Catalan family in which multiple cancer types tracked across three generations, but for which no genetic etiology had been identified. Whole-exome sequencing of germline DNA from multiple affected family members was performed to identify candidate variants to explain this occurrence of familial cancer. We discovered in all cancer-affected family members a single rare heterozygous germline variant (I654V, rs1801201) in ERBB2/HER2, which is located in a transmembrane glycine zipper motif critical for ERBB2-mediated signaling and in complete linkage disequilibrium (D' = 1) with a common polymorphism (I655V, rs1136201) previously reported in some populations as associated with cancer risk. Because multiple cancer types occurred in this family, we tested both the I654V and the I655V variants for association with cancer across multiple tumor types in 6,371 cases of Northern European ancestry drawn from The Cancer Genome Atlas and 6,647 controls, and found that the rare variant (I654V) was significantly associated with an increased risk for cancer (OR = 1.40; P = 0.021; 95% confidence interval (CI), 1.05-1.89). Functional assays performed in HEK 293T cells revealed that both the I655V single mutant (SM) and the I654V;I655V double mutant (DM) stabilized ERBB2 protein and activated ERBB2 signaling, with the DM activating ERBB2 significantly more than the SM alone. Thus, our results suggest a model whereby heritable genetic variation in the transmembrane domain activating ERBB2 signaling is associated with both sporadic and familial cancer risk, with increased ERBB2 stabilization and activation associated with increased cancer risk. PREVENTION RELEVANCE: By performing whole-exome sequencing on germline DNA from multiple cancer-affected individuals belonging to a family in which multiple cancer types track across three generations, we identified and then characterized functional common and rare variation in ERBB2 associated with both sporadic and familial cancer. Our results suggest that heritable variation activating ERBB2 signaling is associated with risk for multiple cancer types, with increases in signaling correlated with increases in risk, and modified by ancestry or family history.

An In Vivo Kras Allelic Series Reveals Distinct Phenotypes of Common Oncogenic Variants.

KRAS is the most frequently mutated oncogene in cancer, yet there is little understanding of how specific KRAS amino acid changes affect tumor initiation, progression, or therapy response. Using high-fidelity CRISPR-based engineering, we created an allelic series of new LSL-Kras mutant mice, reflecting codon 12 and 13 mutations that are highly prevalent in lung (KRASG12C), pancreas (KRASG12R), and colon (KRASG13D) cancers. Induction of each allele in either the murine colon or pancreas revealed striking quantitative and qualitative differences between KRAS mutants in driving the early stages of transformation. Furthermore, using pancreatic organoid models, we show that KRASG13D mutants are sensitive to EGFR inhibition, whereas KRASG12C-mutant organoids are selectively responsive to covalent G12C inhibitors only when EGFR is suppressed. Together, these new mouse strains provide an ideal platform for investigating KRAS biology in vivo and for developing preclinical precision oncology models of KRAS-mutant pancreas, colon, and lung cancers. SIGNIFICANCE: KRAS is the most frequently mutated oncogene. Here, we describe new preclinical models that mimic tissue-selective KRAS mutations and show that each mutation has distinct cellular consequences in vivo and carries differential sensitivity to targeted therapeutic agents.See related commentary by Kostyrko and Sweet-Cordero, p. 1626.This article is highlighted in the In This Issue feature, p. 1611.

GO: a functional reporter system to identify and enrich base editing activity.

Base editing (BE) is a powerful tool for engineering single nucleotide variants (SNVs) and has been used to create targeted mutations in cell lines, organoids and animal models. Recent development of new BE enzymes has provided an extensive toolkit for genome modification; however, identifying and isolating edited cells for analysis has proven challenging. Here we report a 'Gene On' (GO) reporter system that indicates precise cytosine or adenine base editing in situ with high sensitivity and specificity. We test GO using an activatable GFP and use it to measure the kinetics, efficiency and PAM specificity of a range of new BE variants. Further, GO is flexible and can be easily adapted to induce expression of numerous genetically encoded markers, antibiotic resistance genes or enzymes, such as Cre recombinase. With these tools, GO can be exploited to functionally link BE events at endogenous genomic loci to cellular enzymatic activities in human and mouse cell lines and organoids. Thus, GO provides a powerful approach to increase the practicality and feasibility of implementing CRISPR BE in biomedical research.

Distinct Colorectal Cancer-Associated APC Mutations Dictate Response to Tankyrase Inhibition.

The majority of colorectal cancers show hyperactivated WNT signaling due to inactivating mutations in the adenomatous polyposis coli (APC) tumor suppressor. Genetically restoring APC suppresses WNT and induces rapid and sustained tumor regression, implying that reengaging this endogenous tumor-suppressive mechanism may be an effective therapeutic strategy. Here, using new animal models, human cell lines, and ex vivo organoid cultures, we show that tankyrase (TNKS) inhibition can control WNT hyperactivation and provide long-term tumor control in vivo, but that effective responses are critically dependent on how APC is disrupted. Mutant APC proteins truncated within the mutation cluster region physically engage the destruction complex and suppress the WNT transcriptional program, while APC variants with early truncations (e.g., Apc Min) show limited interaction with AXIN1 and ß-catenin, and do not respond to TNKS blockade. Together, this work shows that TNKS inhibition, like APC restoration, can reestablish endogenous control of WNT/ß-catenin signaling, but that APC genotype is a crucial determinant of this response. SIGNIFICANCE: This study reveals how subtle changes to the mutations in a critical colorectal tumor suppressor, APC, influence the cellular response to a targeted therapy. It underscores how investigating the specific genetic alterations that occur in human cancer can identify important biological mechanisms of drug response and resistance.This article is highlighted in the In This Issue feature, p. 1325.

Optimized base editors enable efficient editing in cells, organoids and mice.

CRISPR base editing enables the creation of targeted single-base conversions without generating double-stranded breaks. However, the efficiency of current base editors is very low in many cell types. We reengineered the sequences of BE3, BE4Gam, and xBE3 by codon optimization and incorporation of additional nuclear-localization sequences. Our collection of optimized constitutive and inducible base-editing vector systems dramatically improves the efficiency by which single-nucleotide variants can be created. The reengineered base editors enable target modification in a wide range of mouse and human cell lines, and intestinal organoids. We also show that the optimized base editors mediate efficient in vivo somatic editing in the liver in adult mice.