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FBXW7 is an E3 ubiquitin ligase that targets proteins for proteasome-mediated degradation and is mutated in various cancer types. Here, we use CRISPR base editors to introduce different FBXW7 hotspot mutations in human colon organoids. Functionally, FBXW7 mutation reduces EGF dependency of organoid growth by ~10,000-fold. Combined transcriptomic and proteomic analyses revealed increased EGFR protein stability in FBXW7 mutants. Two distinct phosphodegron motifs reside in the cytoplasmic tail of EGFR. Mutations in these phosphodegron motifs occur in human cancer. CRISPR-mediated disruption of the phosphodegron motif at T693 reduced EGFR degradation and EGF growth factor dependency. FBXW7 mutant organoids showed reduced sensitivity to EGFR-MAPK inhibitors. These observations were further strengthened in CRC-derived organoid lines and validated in a cohort of patients treated with panitumumab. Our data imply that FBXW7 mutations reduce EGF dependency by disabling EGFR turnover., Competing Interests: Competing interests statement:H.C. is inventor of several patents related to organoid technology, is cofounder of Xilis Inc. and is currently employee of Roche, Basel (CH) Switzerland. His full disclosure is given at https://www.uu.nl/staff/JCClevers/.

Extending the success of cellular immunotherapies against blood cancers to the realm of solid tumors will require improved in vitro models that reveal therapeutic modes of action at the molecular level. Here we describe a system, called BEHAV3D, developed to study the dynamic interactions of immune cells and patient cancer organoids by means of imaging and transcriptomics. We apply BEHAV3D to live-track >150,000 engineered T cells cultured with patient-derived, solid-tumor organoids, identifying a 'super engager' behavioral cluster comprising T cells with potent serial killing capacity. Among other T cell concepts we also study cancer metabolome-sensing engineered T cells (TEGs) and detect behavior-specific gene signatures that include a group of 27 genes with no previously described T cell function that are expressed by super engager killer TEGs. We further show that type I interferon can prime resistant organoids for TEG-mediated killing. BEHAV3D is a promising tool for the characterization of behavioral-phenotypic heterogeneity of cellular immunotherapies and may support the optimization of personalized solid-tumor-targeting cell therapies., (© 2022. The Author(s).)

Breast cancer is characterized by histological and functional heterogeneity, posing a clinical challenge for patient treatment. Emerging evidence suggests that the distinct subtypes reflect the repertoire of genetic alterations and the target cell. However, the precise initiating events that predispose normal epithelium to neoplasia are poorly understood. Here, we demonstrate that breast epithelial organoids can be generated from human reduction mammoplasties (12 out of 12 donors), thus creating a tool to study the clonal evolution of breast cancer. To recapitulate de novo oncogenesis, we exploited clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 for targeted knockout of four breast cancer-associated tumor suppressor genes (P53, PTEN, RB1, NF1) in mammary progenitor cells from six donors. Mutant organoids gained long-term culturing capacity and formed estrogen-receptor positive luminal tumors on transplantation into mice for one out of six P53/PTEN/RB1-mutated and three out of six P53/PTEN/RB1/NF1-mutated lines. These organoids responded to endocrine therapy or chemotherapy, supporting the potential utility of this model to enhance our understanding of the molecular events that culminate in specific subtypes of breast cancer. [ABSTRACT FROM AUTHOR]