Structure-guided design of small molecules to modulate the secreted phosphoproteome in breast cancer

Joshua Mayfield
Appointment Period: 2017-2019, Grant Year: [32, 33]

Signaling by reversible phosphorylation is often disrupted in disease states, such as cancer, and inhibitors targeting intracellular kinases and phosphatases are effective research and therapeutic agents. Despite the extensive research around intracellular kinases, phosphatases, and their substrates – little is known about phosphorylation events occurring extracellularly or on secreted proteins. When Vincent Tagliabracci was a post-doctoral fellow in the lab funded by this training grant, he identified Fam20C as the kinase responsible for phosphorylating the majority of the secreted phosphoproteome, primarily within S-x-E motifs. Fam20C is up-regulated in some forms of invasive breast cancer, and increased phosphorylation of several secreted proteins within S-x-E motifs have been correlated with metastatic breast cancer in humans. Indeed, genetic depletion of Fam20C in breast cancer cells alters their ability to adhere, migrate, and invade in cell-based assays and in xenograft animal models. These observations suggest that Fam20C is important to the progression of breast cancer via its influence on substrate proteins and ultimately the extracellular environment.

There are currently no known high affinity inhibitors of Fam20C. Small molecule inhibitors would provide for inhibition of Fam20C with spatial and temporal resolution and be applicable across a wide variety of cell-based and animal models of cancer. Thus in collaboration with Novartis, high throughput screening of the GNF compound library has been performed and revealed multiple Fam20C inhibitors with nano-molar affinity. Crystals of Fam20C have been previously obtained in the Dixon lab. Cocomplex x-ray crystal structures of Fam20C and these lead compounds will be obtained to determine chemical characteristics that define effective inhibitors. Once identified, these characteristics can direct the synthesis of novel compounds that are potent and specific with desirable pharmacological properties. These optimized inhibitors will be utilized in a series of biological experiments to determine the importance of extracellular protein phosphorylation to the tumor microenvironment, cancer cell migration, and the disease process.

We predict that these flexible tools will help determine the impact of secreted protein phosphorylation during distinct stages of breast cancer cell progression including tumor growth and metastasis. Furthermore, Fam20C inhibition may represent viable treatment strategy to alter the tumor microenvironment and modulate cancer cell migration in patients with breast cancer. The Fam20C project has been funded on the Growth, Regulation, & Oncogenesis Training Grant since its beginning and has resulted in high profile publications in Science, Cell, Elife, and PNAS. Extensive progress has been made on this emerging family of proteins in a very short time, and I will bring expertise in structural biology to this exciting project.

PUBLICATIONS (resulting from this training): 

Mayfield JE, Irani S, Mehaffey MR, Walker L, Robinson MR, Burkholder NT, Prescott NA, Kathuria K, Brodbelt JS, Zhang YJ. Tyr1 phosphorylation of RNA polymerase II CTD by c-Abl directs Ser2 phosphorylation by P-TEFb. In preparation.

Banerjee S, Ji C, Mayfield JE, Goel A, Xiao J, Dixon JE, Guo X. Ancient drug curcumin impedes 26S proteasome activity by direct inhibition of dual-specificity tyrosine- regulated kinase 2. Proc Natl Acad Sci U S A. In review.