Exploiting signaling signatures in triple-negative breast cancer to identify targetable vulnerabilities

Abstract

Triple-negative breast cancer (TNBC) is an aggressive and heterogeneous disease with the worst five-year survival rate of all breast cancer subtypes. The standard of care for TNBC is chemotherapy, but not all patients respond to chemotherapy, and responders often recur with chemotherapy-resistant disease. There are limited targeted therapy options for TNBC, in part, because tumor heterogeneity has precluded the identification of druggable drivers of disease. However, TNBC has several common genetic alterations, including hyperactivation of the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway, epidermal growth factor receptor (EGFR) overexpression, and protein tyrosine phosphatase (PTP) loss. In this thesis, we exploit the signaling signatures of TNBC, using two distinct approaches, to identify targetable vulnerabilities.

In the first approach, we performed a genome-wide negative selection CRISPR/Cas9 screen with PI3K/AKT inhibitors in TNBC cells and identified synthetic lethality between PI3K/AKT pathway inhibition and disruption of cholesterol homeostasis genes. AKT inhibitors synergized with statins, cholesterol biosynthesis inhibitors, to induce apoptotic cell death in TNBC cells but not in estrogen receptor (ER)- positive breast cancer cells or non-tumorigenic breast epithelial cells. We showed that TNBC cells are hypersensitive to statins due to dysregulated cholesterol homeostasis, which prevents statin-induced upregulation of HMG-CoA reductase (HMGCR), a rate-limiting enzyme in cholesterol biosynthesis. This work proposes a novel combination treatment strategy for TNBC, combining a potent statin, pitavastatin, with AKT inhibition.

In the second approach, we characterized the metabolic tyrosine phosphoproteome in TNBC, driven by EGFR overexpression and PTP loss. We performed parallel phosphoproteomics and metabolomics in triple-negative, non-tumorigenic mammary epithelial cells, followed by an integrated analysis of the two datasets. This analysis identified both previously characterized tyrosine phosphorylation (pTyr) sites and uncharacterized pTyr sites on metabolic enzymes. We developed a CRISPR interference (CRISPRi) system to further define the functional consequences of the uncharacterized pTyr sites. By combining metabolomics with phosphoproteomics, we propose a method to globally characterize the functional significance of the metabolic tyrosine phosphoproteome, which can be used to identify functionally relevant targets in TNBC. Altogether, this thesis identifies a novel targeted therapy combination for TNBC and proposes multiple methods by which to functionally characterize TNBC and identify druggable targets.