Efficient genetic editing of human intestinal organoids using ribonucleoprotein-based CRISPR.

Organoids, combined with genetic editing strategies, have the potential to offer rapid and efficient investigation of gene function in many models of human disease. However, to date, the editing efficiency of organoids with the use of non-viral electroporation methods has only been up to 30%, with implications for the subsequent need for selection, including turnaround time and exhaustion or adaptation of the organoid population. Here, we describe an efficient method for intestinal organoid editing using a ribonucleoprotein-based CRISPR approach. Editing efficiencies of up to 98% in target genes were robustly achieved across different gut anatomical locations and developmental timepoints from multiple patient samples with no observed off-target editing. The method allowed us to study the effect of loss of the tumour suppressor gene PTEN in normal human intestinal cells. Analysis of PTEN-deficient organoids defined phenotypes that likely relate to its tumour suppressive function in vivo, such as a proliferative advantage and increased organoid budding. Transcriptional profiling revealed differential expression of genes in pathways commonly known to be associated with PTEN loss, including mTORC1 activation.

Metabolic Imaging Detects Resistance to PI3Kα Inhibition Mediated by Persistent FOXM1 Expression in ER+ Breast Cancer.

PIK3CA, encoding the PI3Kα isoform, is the most frequently mutated oncogene in estrogen receptor (ER)-positive breast cancer. Isoform-selective PI3K inhibitors are used clinically but intrinsic and acquired resistance limits their utility. Improved selection of patients that will benefit from these drugs requires predictive biomarkers. We show here that persistent FOXM1 expression following drug treatment is a biomarker of resistance to PI3Kα inhibition in ER+ breast cancer. FOXM1 drives expression of lactate dehydrogenase (LDH) but not hexokinase 2 (HK-II). The downstream metabolic changes can therefore be detected using MRI of LDH-catalyzed hyperpolarized 13C label exchange between pyruvate and lactate but not by positron emission tomography measurements of HK-II-mediated trapping of the glucose analog 2-deoxy-2-[18F]fluorodeoxyglucose. Rapid assessment of treatment response in breast cancer using this imaging method could help identify patients that benefit from PI3Kα inhibition and design drug combinations to counteract the emergence of resistance.

NOTCH-mediated non-cell autonomous regulation of chromatin structure during senescence.

Senescent cells interact with the surrounding microenvironment achieving diverse functional outcomes. We have recently identified that NOTCH1 can drive 'lateral induction' of a unique senescence phenotype in adjacent cells by specifically upregulating the NOTCH ligand JAG1. Here we show that NOTCH signalling can modulate chromatin structure autonomously and non-autonomously. In addition to senescence-associated heterochromatic foci (SAHF), oncogenic RAS-induced senescent (RIS) cells exhibit a massive increase in chromatin accessibility. NOTCH signalling suppresses SAHF and increased chromatin accessibility in this context. Strikingly, NOTCH-induced senescent cells, or cancer cells with high JAG1 expression, drive similar chromatin architectural changes in adjacent cells through cell-cell contact. Mechanistically, we show that NOTCH signalling represses the chromatin architectural protein HMGA1, an association found in multiple human cancers. Thus, HMGA1 is involved not only in SAHFs but also in RIS-driven chromatin accessibility. In conclusion, this study identifies that the JAG1-NOTCH-HMGA1 axis mediates the juxtacrine regulation of chromatin architecture.

Versatile and enhanced tumour modelling in mice via somatic cell transduction.

Genetically engineered mouse (GEM) models of cancer currently comprise the most accurate way to experimentally recapitulate the human disease in the laboratory. Given recent advances in genomics and genetic screens, however, as well as an increasing urgency for the translation of effective preclinical treatments into the clinic, there is a pressing need to make these models easier and more efficient to work with. Accordingly, we have developed a versatile lentivirus-based approach to induce tumours from somatic cells of GEMs, add or subtract gene expression and render the tumours imageable from a simple breeding stock. The vectors deliver a tamoxifen-inducible and self-inactivating Cre recombinase, conditional bioluminescent and fluorescent proteins and an shRNA component. Following the transduction of somatic cells, tumours are initiated by Cre-mediated recombination of the inherited floxed alleles. Self-inactivation of Cre expression switches on the expression of luciferase, thereby rendering the recombined cells and resulting tumours bioluminescent. We demonstrate proof of concept of this approach by inducing bioluminescent lung tumours in conditional Kras and p53 mice. We also show that a variant vector expressing shRNA alters tumour growth dynamics and the histological grade associated with the inherited genotype. This approach comprises a versatile means to induce imageable and spontaneous tumour burden in mice. The vectors can be readily customized at the bench to modify reporter readout or tumour phenotype without additional transgenic strain development or breeding. They should also be useful for inducing imageable tumours in organs other than the lung, provided that the inherited conditional genotype is sufficiently penetrant.