br INTRODUCTION br Kinases and phosphatases control
Kinases and phosphatases control the reversible process of phosphorylation. Signaling networks involving these enzymes
compute extracellular signals into transcriptional, functional, and phenotypical responses. Deregulation of signaling networks can lead to the initiation and progression of many types of human disease, including cancer (Fleuren et al., 2016; Julien et al., 2007). Signaling network structure has been studied by mapping physical interactions of kinases and phosphatases in steady and dynamic states using biochemical approaches and reporter assays (Barrios-Rodiles et al., 2005; Breitkreutz et al., 2010; Couzens et al., 2013; Horn et al., 2011). Using in vitro kinase assays and motif-based predictions, the specificity and targets of many kinases have been revealed (Linding et al., 2007; Mok et al., 2010; Yu et al., 2009). Kinase and phosphatase perturba-tions have been applied to systematically determine network responses in yeast and human Exendin-4 (Bodenmiller et al., 2010; Ochoa et al., 2016; Sacco et al., 2012a).
Mutation-induced signaling network rewiring and modulation of signaling dynamics have also been characterized for many kinases (Creixell et al., 2015; Pawson and Warner, 2007), providing a basis for the identification of targeted therapies in cancer (Hennessy et al., 2005; Logue and Morrison, 2012). Inde-pendently of mutations, kinase overexpression drives tumori-genesis in multiple cancer types and is a critical factor in drug resistance (Eralp et al., 2008; Santarius et al., 2010; Shaffer et al., 2017). Recently, overexpression of phosphatases has been shown to mediate cancer progression and has been linked to the poor prognosis of patients (Julien et al., 2011; Liu et al., 2016; De Vriendt et al., 2013). Overexpression-induced signaling modulation remains largely uncharacterized because factors such as genetic instability induce highly heterogeneous quanti-ties of deregulated signaling proteins in cancer cells (Abbas et al., 2013), making conventional cell population-based analysis inapplicable. Only recently have technologies emerged that ac-count for such heterogeneity and that can comprehensively quantify signaling network behavior with single-cell resolution. This resolution is required to characterize abundance-related
cellular signaling states (measured as phosphorylation levels of signaling proteins) and phenotypical alterations caused by a given kinase or phosphatase of interest (Bendall et al., 2011; Lun et al., 2017). Mass cytometry allows simultaneous quantifi-cation of >40 proteins or protein modifications at single-cell res-olution, enabling the profiling of complex cellular behaviors in highly heterogeneous samples (Bendall et al., 2011; Bodenmiller et al., 2012; Chevrier et al., 2017; Levine et al., 2015). We have recently established and thoroughly validated an approach that couples transient protein overexpression with mass-cytometry-based, single-cell analysis and have revealed that protein over-expression induces complex signaling network modulations in an abundance-dependent manner (Lun et al., 2017).
Here, we applied this technique in a human kinome- and phos-phatome-wide screen to determine kinase and phosphatase abundance-dependent effects on 30 phosphorylation sites known to be involved in the regulation of growth, proliferation, survival, and stress signaling pathways. Over 10 million individ-ual cells were analyzed, covering 649 overexpression conditions with or without 10-min epidermal growth factor (EGF) stimula-tion. Assessing the effects of kinase and phosphatase on the signaling network, we expanded the functional classification of the kinome and phosphatome. Our analysis identified 1,323 pairs of overexpression-dependent signaling relationships, including 208 pairs that were previously unknown. By character-izing signaling dynamics in a follow-up EGF stimulation time course and a kinase-phosphatase combinatorial overexpression assay, we found a pro-cancer signaling response in which the overexpression of ERK-specific phosphatases sustained cell proliferative signals. Further analysis of our dataset revealed a drug-resistant mechanism through which the overexpression of tyrosine kinases, including SRC, FES, YES1, and BLK, induced MEK-independent ERK activation in melanoma A375 cells. The expression levels of these proteins could predict drug sensitivity to BRAF-MEK concurrent inhibition in patients with BRAF mutations and may be suggestive of alternative treatments.
Abundance-Dependent Effects of Human Kinases and Phosphatases on Cell Signaling
Protein abundance variance on the single-cell level is often observed in tumors as heterogeneous genomic abnormalities accumulate (Du and Elemento, 2015). Inter-tumoral heterogene-ity presumably results in highly variable signaling responses to stimuli or drug treatments. In addition, a high degree of intra-tu-moral heterogeneity further challenges cancer therapeutic inter-ventions (Patel et al., 2014; Roth et al., 2016). To understand the signaling network modulation in cells that overexpress a defined kinase or phosphatase at various levels, we applied our abun-dance-dependent signaling network assessment system (Lun et al., 2017) in a kinome- and phosphatome-wide screen.