Abstract B061: Discovering co-driver genes and pathways of mutant <i>TP53</i> in breast cancer by CRISPR screening and multi-omics approaches
Authors: Lilian Nwekwo, Anasuya Pal, Yining Zheng, Lydia Sakala, Laura Gonzalez-Malerva, Dustin Grief, Dustin Lord, Jin Park, Joshua LaBaer
Journal: Cancer Research (2024)

Abstract Triple-Negative Breast Cancer (TNBC) is the most aggressive and highly heterogeneous molecular subtype of breast cancer lacking the progesterone, HER2 and estrogen receptors. The heterogeneity of these receptors makes it difficult to target and treat. The TP53 gene which encodes the p53 protein is mutated in 80% of TNBC cases. We hypothesized that, in addition to the neo-morphic activities of different p53 mutants themselves, the heterogeneity in clinical phenotypes observed in TNBC is due to functional interactions of the p53 driver mutants with other functionally important co-existing mutations called ‘co-drivers’. To identify co-driver mutations, our lab performed a genome-wide screening by using lentiviral CRISPR libraries on a cell line over-expressing p53-R273C (a prevalent mutant form of p53), and MYC (an oncogene). Upon injection of the library-transduced cells into mice, large tumor was formed, which was extracted and subjected to targeted PCR and whole exome sequencing. This identified truncation mutations in the EVC2 (EvC Ciliary Complex Subunit 2) and the NR1D1 (Nuclear Receptor 1D1) genes. This suggested that they are tumor suppressors in their wild type functions, and loss of this native function would co-drive the formation of tumors implicated with mutant p53-R273C to promote tumor formation in vivo. EVC2 positively modulates the Hedgehog signaling pathway. Interestingly, a majority of mutations in NR1D1 were an in-frame deletion of a tyrosine residue, which may be a critical regulatory residue of NR1D1. It is known to modulate the JAK/STAT3 pathway and, just like p53, NR1D1 can inhibit the circadian clock genes. We are testing whether deleting EVC2 and NR1D1 genes by CRISPR or expressing the mutant forms in the presence of p53-R273C increase invasion and/or other cancer hallmark phenotypes and also whether the amino acid, tyrosine is critical to the regulation of NR1D1, with the goal of understanding their contributions to the development of various hallmarks of cancer and the underlying mechanisms which will identify how these mutations contribute to the progression of TNBC and could potentially provide molecularly targeted therapies for TNBC. In parallel, our lab previously carried out RNA-Seq on cell lines expressing 10 different mutant p53 proteins, where pathway analysis identified the Hippo pathway, a tumor suppressor pathway, highly associated with aggressive cellular phenotypes in the mutant p53 contexts. In cancer, it is known that, upon suppression of the upstream Hippo pathway, unphosphorylated TAZ (or WWTR1) localizes in the nucleus binds to TEAD, leading to the activation of a series of genes to promote cancer hallmark phenotypes. We are investigating the molecular mechanisms of actions through which Hippo pathway drives the progression of TNBC and assess the pathway as the target for potential therapy for TNBC with specific TP53 mutations. This study will reveal how different mutant p53 proteins regulate Hippo pathway, TAZ activation/localization, and TEAD-mediated transcription to promote invasiveness. Citation Format: Lilian Nwekwo, Anasuya Pal, Yining Zheng, Lydia Sakala, Laura Gonzalez-Malerva, Dustin Grief, Dustin Lord, Jin Park, Joshua LaBaer. Discovering co-driver genes and pathways of mutant TP53 in breast cancer by CRISPR screening and multi-omics approaches [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B061.