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Triple-negative breast cancer (TNBC) patients have a poor prognosis and few treatment options. Mouse models of TNBC are important for development of new therapies, however, few mouse models represent the complexity of TNBC. Here, we develop a female TNBC murine model by mimicking two common TNBC mutations with high co-occurrence: amplification of the oncogene MYC and deletion of the tumor suppressor PTEN. This Myc;Ptenfl model develops heterogeneous triple-negative mammary tumors that display histological and molecular features commonly found in human TNBC. Our research involves deep molecular and spatial analyses on Myc;Ptenfl tumors including bulk and single-cell RNA-sequencing, and multiplex tissue-imaging. Through comparison with human TNBC, we demonstrate that this genetic mouse model develops mammary tumors with differential survival and therapeutic responses that closely resemble the inter- and intra-tumoral and microenvironmental heterogeneity of human TNBC, providing a pre-clinical tool for assessing the spectrum of patient TNBC biology and drug response., (© 2023. Springer Nature Limited.)

Expression of guide RNAs in the CRISPR/Cas9 system typically requires the use of RNA polymerase III promoters, which are not cell-type specific. Flanking the gRNA with self-cleaving ribozyme motifs to create a self-cleaving gRNA overcomes this limitation. Here, we use self-cleaving gRNAs to create drug-selectable gene editing events in specific hepatocyte loci. A recombinant Adeno Associated Virus vector targeting the Albumin locus with a promoterless self-cleaving gRNA to create drug resistance is linked in cis with the therapeutic transgene. Gene expression of both are dependent on homologous recombination into the target locus. In vivo drug selection for the precisely edited hepatocytes allows >30-fold expansion of gene-edited cells and results in therapeutic levels of a human Factor 9 transgene. Importantly, self-cleaving gRNA expression is also achieved after targeting weak hepatocyte genes. We conclude that self-cleaving gRNAs are a powerful system to enable cell-type specific in vivo drug resistance for therapeutic gene editing applications., (© 2022. The Author(s).)

Intratumoral phenotypic heterogeneity has been described in many tumor types, where it can contribute to drug resistance and disease recurrence. We analyzed ductal and neuroendocrine markers in pancreatic ductal adenocarcinoma, revealing heterogeneous expression of the neuroendocrine marker Synaptophysin within ductal lesions. Higher percentages of Cytokeratin-Synaptophysin dual positive tumor cells correlate with shortened disease-free survival. We observe similar lineage marker heterogeneity in mouse models of pancreatic ductal adenocarcinoma, where lineage tracing indicates that Cytokeratin-Synaptophysin dual positive cells arise from the exocrine compartment. Mechanistically, MYC binding is enriched at neuroendocrine genes in mouse tumor cells and loss of MYC reduces ductal-neuroendocrine lineage heterogeneity, while deregulated MYC expression in KRAS mutant mice increases this phenotype. Neuroendocrine marker expression is associated with chemoresistance and reducing MYC levels decreases gemcitabine-induced neuroendocrine marker expression and increases chemosensitivity. Altogether, we demonstrate that MYC facilitates ductal-neuroendocrine lineage plasticity in pancreatic ductal adenocarcinoma, contributing to poor survival and chemoresistance.