Characterizing NOTCH1 PEST domain mutations in Triple Negative Breast Cancer

About 20% of newly diagnosed breast cancers are classified as triple negative breast cancer (TNBC). These tumors are very heterogenous in nature, and as the name implies, lack expression of the estrogen, progesterone and HER2 receptors. The absence of receptor expression makes currently available targeted drugs not an option for treatment. Due to the need for more personalized therapies, molecular studies have focused on the identification of commonly mutated genes, one such gene is NOTCH1 were mutations found tend to cluster in a specific region of the gene, known as the PEST domain. To better understand the potential clinical significance of these mutations, we functionally characterized NOTCH1 PEST domain mutations via gene editing targeting one allele of the NOTCH1 gene in two different nontumorigenic breast epithelial cell lines. Out of nine mutations characterized, five do not impart detectable transformative properties as measured by in vivo and in vitro assays; however, mutations A2441fs, A2441T, P2448fs and P2438_S2439 convey oncogenic properties such as increased proliferation ability, growth factor independence, anchorage independent growth, and irregular colony morphology. Additionally, they also display increase signaling thru the MAPK pathway, and higher levels of transcriptionally active form of β-catenin, that seems to be mediated in part by physical binding of mutant NOTCH1 and β-catenin. More surprisingly, in vivo, mutations A2441T and P2448fs can form palpable tumors in immunocompromised mice. Although these mutations did not impart sensitivity to Notch or MAPK targeted therapies, it did confer a strong resistance to EGFR targeted drugs. These results showcase the range of phenotypes observed in mutations within the same domain of NOTCH1. With several mutations conveying no detectable phenotype to some rendering tumorigenicity. It suggests that PEST domain mutations should not be classified under one category and therefore assumptions about their activating ability, or how they could influence clinical outcomes should be evaluated in a case by case basis