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.

In situ CRISPR-Cas9 base editing for the development of genetically engineered mouse models of breast cancer.

Genetically engineered mouse models (GEMMs) of cancer have proven to be of great value for basic and translational research. Although CRISPR-based gene disruption offers a fast-track approach for perturbing gene function and circumvents certain limitations of standard GEMM development, it does not provide a flexible platform for recapitulating clinically relevant missense mutations in vivo. To this end, we generated knock-in mice with Cre-conditional expression of a cytidine base editor and tested their utility for precise somatic engineering of missense mutations in key cancer drivers. Upon intraductal delivery of sgRNA-encoding vectors, we could install point mutations with high efficiency in one or multiple endogenous genes in situ and assess the effect of defined allelic variants on mammary tumorigenesis. While the system also produces bystander insertions and deletions that can stochastically be selected for when targeting a tumor suppressor gene, we could effectively recapitulate oncogenic nonsense mutations. We successfully applied this system in a model of triple-negative breast cancer, providing the proof of concept for extending this flexible somatic base editing platform to other tissues and tumor types.

Base editing the mammalian genome.

Base editing is a powerful technology that enables programmable conversion of single nucleotides in the mammalian genome. Base editors consist of a partially active Cas9 nuclease (Cas9D10A) tethered to a natural or synthetic DNA modifying enzyme. Though only recently described, BE has already shown enormous potential for basic and translational research, allowing the creation or repair of disease alleles in a variety of cell types and model organisms. In the past 2 years, a vast array of new and modified base editor variants have been described, expanding the flexibility and usefulness of the approach. Though simple in concept, effective implementation of base editing requires an understanding of the advantages and limitations of each of these tools. Here, we provide an overview of the concepts of DNA base editing, and discuss the recent progress toward the development of optimized base editing systems for mammalian cells. In addition, we highlight key technical aspects of designing and executing BE experiments, and provide detailed experimental examples of successful base editing in cell lines and organoids to help guide the effective use of these tools for genome modification.

Endogenous spacing enables co-processing of microRNAs and efficient combinatorial RNAi.

We present Multi-miR, a microRNA-embedded shRNA system modeled after endogenous microRNA clusters that enables simultaneous expression of up to three or four short hairpin RNAs (shRNAs) from a single promoter without loss of activity, enabling robust combinatorial RNA interference (RNAi). We further developed complementary all-in-one vectors that are over one log-scale more sensitive to doxycycline-mediated activation in vitro than previous methods and resistant to shRNA inactivation in vivo. We demonstrate the utility of this system for intracranial expression of shRNAs in a glioblastoma model. Additionally, we leverage this platform to target the redundant RAF signaling node in a mouse model of KRAS-mutant cancer and show that robust combinatorial synthetic lethality efficiently abolishes tumor growth.

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.

Detection of Marker-Free Precision Genome Editing and Genetic Variation through the Capture of Genomic Signatures.

Genome editing technologies have transformed our ability to engineer desired genomic changes within living systems. However, detecting precise genomic modifications often requires sophisticated, expensive, and time-consuming experimental approaches. Here, we describe DTECT (Dinucleotide signaTurE CapTure), a rapid and versatile detection method that relies on the capture of targeted dinucleotide signatures resulting from the digestion of genomic DNA amplicons by the type IIS restriction enzyme AcuI. DTECT enables the accurate quantification of marker-free precision genome editing events introduced by CRISPR-dependent homology-directed repair, base editing, or prime editing in various biological systems, such as mammalian cell lines, organoids, and tissues. Furthermore, DTECT allows the identification of oncogenic mutations in cancer mouse models, patient-derived xenografts, and human cancer patient samples. The ease, speed, and cost efficiency by which DTECT identifies genomic signatures should facilitate the generation of marker-free cellular and animal models of human disease and expedite the detection of human pathogenic variants.

Adenine base editing in an adult mouse model of tyrosinaemia.

In contrast to traditional CRISPR-Cas9 homology-directed repair, base editing can correct point mutations without supplying a DNA-repair template. Here we show in a mouse model of tyrosinaemia that hydrodynamic tail-vein injection of plasmid DNA encoding the adenine base editor (ABE) and a single-guide RNA (sgRNA) can correct an A>G splice-site mutation. ABE treatment partially restored splicing, generated fumarylacetoacetate hydrolase (FAH)-positive hepatocytes in the liver, and rescued weight loss in mice. We also generated FAH+ hepatocytes in the liver via lipid-nanoparticle-mediated delivery of a chemically modified sgRNA and an mRNA of a codon-optimized base editor that displayed higher base-editing efficiency than the standard ABEs. Our findings suggest that adenine base editing can be used for the correction of genetic diseases in adult animals.

Exploiting the Therapeutic Interaction of WNT Pathway Activation and Asparaginase for Colorectal Cancer Therapy.

Colorectal cancer is driven by mutations that activate canonical WNT/ß-catenin signaling, but inhibiting WNT has significant on-target toxicity, and there are no approved therapies targeting dominant oncogenic drivers. We recently found that activating a ß-catenin-independent branch of WNT signaling that inhibits GSK3-dependent protein degradation induces asparaginase sensitivity in drug-resistant leukemias. To test predictions from our model, we turned to colorectal cancer because these cancers can have WNT-activating mutations that function either upstream (i.e., R-spondin fusions) or downstream (APC or ß-catenin mutations) of GSK3, thus allowing WNT/ß-catenin and WNT-induced asparaginase sensitivity to be unlinked genetically. We found that asparaginase had little efficacy in APC or ß-catenin-mutant colorectal cancer, but was profoundly toxic in the setting of R-spondin fusions. Pharmacologic GSK3α inhibition was sufficient for asparaginase sensitization in APC or ß-catenin-mutant colorectal cancer, but not in normal intestinal progenitors. Our findings demonstrate that WNT-induced therapeutic vulnerabilities can be exploited for colorectal cancer therapy. SIGNIFICANCE: Solid tumors are thought to be asparaginase-resistant via de novo asparagine synthesis. In leukemia, GSK3α-dependent protein degradation, a catabolic amino acid source, mediates asparaginase resistance. We found that asparaginase is profoundly toxic to colorectal cancers with WNT-activating mutations that inhibit GSK3. Aberrant WNT activation can provide a therapeutic vulnerability in colorectal cancer.See related commentary by Davidsen and Sullivan, p. 1632.This article is highlighted in the In This Issue feature, p. 1611.

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.

Tankyrase inhibition sensitizes cells to CDK4 blockade.

Tankyrase (TNKS) 1/2 are positive regulators of WNT signaling by controlling the activity of the ß-catenin destruction complex. TNKS inhibitors provide an opportunity to suppress hyperactive WNT signaling in tumors, however, they have shown limited anti-proliferative activity as a monotherapy in human cancer cell lines. Here we perform a kinome-focused CRISPR screen to identify potential effective drug combinations with TNKS inhibition. We show that the loss of CDK4, but not CDK6, synergizes with TNKS1/2 blockade to drive G1 cell cycle arrest and senescence. Through precise modelling of cancer-associated mutations using cytidine base editors, we show that this therapeutic approach is absolutely dependent on suppression of canonical WNT signaling by TNKS inhibitors and is effective in cells from multiple epithelial cancer types. Together, our results suggest that combined WNT and CDK4 inhibition might provide a potential therapeutic strategy for difficult-to-treat epithelial tumors.