Abstract
Despite its central role in human cancer, MYC deregulation is insufficient by itself to transform cells. Because inherent mechanisms of neoplastic control prevent precancerous lesions from becoming fully malignant, identifying transforming alleles of MYC that bypass such controls may provide fundamental insights into tumorigenesis. To date, the only activated allele of MYC known is T58A, the study of which led to identification of the tumor suppressor FBXW7 and its regulator USP28 as a novel therapeutic target. In this study, we screened a panel of MYC phosphorylation mutants for their ability to promote anchorage-independent colony growth of human MCF10A mammary epithelial cells, identifying S71A/S81A and T343A/S344A/S347A/S348A as more potent oncogenic mutants compared with wild-type (WT) MYC. The increased cell-transforming activity of these mutants was confirmed in SH-EP neuroblastoma cells and in three-dimensional MCF10A acini. Mechanistic investigations initiated by a genome-wide mRNA expression analysis of MCF10A acini identified 158 genes regulated by the mutant MYC alleles, compared with only 112 genes regulated by both WT and mutant alleles. Transcriptional gain-of-function was a common feature of the mutant alleles, with many additional genes uniquely dysregulated by individual mutant. Our work identifies novel sites of negative regulation in MYC and thus new sites for its therapeutic attack.
Highlights
The c-Myc (MYC) oncoprotein is a prominent contributor to tumorigenesis
Given the requirement of phosphorylation at serine 62 (S62) for the subsequent phosphorylation of threonine 58 (T58) and the knowledge that certain kinases can phosphorylate more than one residue within the N-terminus of MYC, we generated a panel of T58, S62, S71, and S81 mutants that contained every possible combination of these four N-terminal phosphorylation sites mutated to alanine [15, 18]
We evaluated our mutant panel by stable overexpression in nontransformed MCF10A mammary epithelial cells, as we have shown that expression of WT MYC alone is sufficient to promote anchorage-independent colony growth, which is further potentiated by the T58A point mutant [13]
Summary
The c-Myc (MYC) oncoprotein is a prominent contributor to tumorigenesis. Understanding pathways that both regulate and cooperate with MYC has the potential to identify novel therapeutic strategies to inhibit this oncogene [1]. The powerful antitumor effect of both direct (e.g., Omomyc) and indirect (e.g., JQ1) MYC inhibition has been experimentally reinforced by important recent publications, underscoring the need to better understand the mechanisms both regulating and contributing to MYC-induced transformation [2,3,4,5]. Phosphorylation has been shown to be an important mechanism, regulating MYC activity. Two phosphorylation sites within MYC homology box I (MBI), threonine 58 (T58), and serine 62 (S62) undergo hierarchical phosphorylation. S62 can be phosphorylated by a number of kinases, including extracellular signal–regulated kinase (ERK), c-jun–NH2–kinase
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