In vivo genome‐editing screen identifies tumor suppressor genes that cooperate with Trp53 loss during mammary tumorigenesis

Abstract

Breast cancer is a heterogeneous disease that comprises multiple histological and molecular subtypes. To gain insight into mutations that drive breast tumorigenesis, we describe a pipeline for the identification and validation of tumor suppressor genes. Based on an in vivo genome‐wide CRISPR/Cas9 screen in Trp53 +/– heterozygous mice, we identified tumor suppressor genes that included the scaffold protein Axin1 , the protein kinase A regulatory subunit gene Prkar1a , as well as the proof‐of‐concept genes Pten , Nf1 , and Trp53 itself. Ex vivo editing of primary mammary epithelial organoids was performed to further interrogate the roles of Axin1 and Prkar1a . Increased proliferation and profound changes in mammary organoid morphology were observed for Axin1/Trp53 and Prkar1a/Trp53 double mutants compared to Pten/Trp53 double mutants. Furthermore, direct in vivo genome editing via intraductal injection of lentiviruses engineered to express dual short‐guide RNAs revealed that mutagenesis of Trp53 and either Prkar1a , Axin1 , or Pten markedly accelerated tumor development compared to Trp53 ‐only mutants. This proof‐of‐principle study highlights the application of in vivo CRISPR/Cas9 editing for uncovering cooperativity between defects in tumor suppressor genes that elicit mammary tumorigenesis.

Comprehensive review of CRISPR-based gene editing: mechanisms, challenges, and applications in cancer therapy

“…When PTEN is mutated or deleted, it leads to uncontrolled cell growth, a hallmark of cancer [ 137 ]. By using CRISPR-Cas9 to precisely target and edit the PTEN gene, researchers can potentially restore its function as a tumor suppressor, thereby inhibiting cancer cell growth and reducing tumor development [ 138 ]. The potential of CRISPR-Cas9-mediated PTEN gene editing as a treatment option varies among different cancer types [ 136 ].…”

“…Some cancers exhibit PTEN mutations as a dominant driver of tumorigenesis, making them more amenable to this approach [ 135 ]. However, the efficacy of this strategy may depend on the cancer's genetic context, as some tumors may possess alternative mechanisms to bypass PTEN function [ 138 ]. Extensive preclinical studies and clinical trials are required to determine its applicability and effectiveness across diverse cancer types [ 135 ].…”

“…Safety concerns in CRISPR-Cas9 gene editing for PTEN in cancer therapy involve potential undesirable site effects, where unintended genetic changes could occur in non-cancerous cells, leading to adverse consequences [ 136 ]. Additionally, the risk of introducing new mutations or altering other essential genes must be carefully evaluated to avoid unwanted side effects [ 138 ]. Rigorous testing in preclinical models and careful monitoring during clinical trials are crucial to ensure the safety and feasibility of this therapeutic approach [ 135 ].…”

“…One approach involves optimizing the delivery system to ensure precise targeting of cancer cells. Additionally, advancements in CRISPR-Cas9 technology, such as using base editors or prime editors, offer more precise modifications and reduced non-targeted site effects [ 138 ]. Moreover, combining CRISPR-Cas9 with other therapies, such as immunotherapies or targeted therapies, may enhance the overall therapeutic response, allowing for a more comprehensive and effective treatment strategy.…”

NEK10 tyrosine phosphorylates β-catenin to regulate its cytoplasmic turnover

“…This effect is particularly acute following DNA damage and, as a result, Nek10-deficient A549 cells exhibit heightened sensitivity to genotoxic agents. More recently, Axin1 was identified as a potential co-factor with p53 in an in vivo Crispr screen in p53+/-mice undertaken to identify genes capable of driving mammary tumorigenesis in the context of p53 heterozygosity (Heitink et al, 2022). Deletion of Axin1 in p53+/-mouse mammary organoids significantly enhanced the proliferation and altered the morphology of the organoids.…”