Identification and Characterization of CLK3 in the Colonic Epithelium as a Regulator of the Wnt Pathway

Abstract

Colorectal cancer (CRC) is the third most prevalent cancer in the world for both men and women. Overall, rates of CRC are on a steady decline, yet rates in young adults have seen a steady incline. New information about signaling pathways involved in the progression of CRC may uncover a mechanism for targeted therapy. The Wnt signaling pathway is a major contributor to the homeostasis of the gut epithelium. Wnt signaling is tightly controlled to regulate patterning and organization, and dysregulation of this pathway is often seen in cancer. Recently, new data has suggested that splicing kinase CLK3 can regulate the Wnt signaling pathway. Little is known about the role of CLK3 in the colonic epithelium, but it has been shown that CLK3 is upregulated in several cancers, including CRC. Additionally, it regulates development and cell fate through the phosphorylation of proteins that impact the alternative splicing of developmental genes. Our work begins to uncover the role of CLK3 in colonic cell signaling. Using human colonic epithelial cells, we show that CLK3 regulates the Wnt pathway at the Wnt enhanceosome complex where Wnt target gene transcription occurs. Using RNA-sequencing, we define sets of genes regulated by CLK3 with or without the stimulation of the Wnt pathway. Further, using phosphoproteomic data, we predict the proteins are being phosphorylated by CLK3 and begin to expose a signaling pathway between CLK3 and the Wnt pathway. To evaluate CLK3 in a more relevant model system, we turned to colon organoids to recapitulate the human tissue in vitro. With our 2D monolayer system, we found CLK3 to be localized in the stem cell niche of the colonic tissue, where most of the Wnt signaling is occurring. These data suggest that CLK3 is regulating Wnt signaling in the crypts of the colon, and high expression of CLK3 may lead to overactivation of Wnt target genes that promote proliferation and cell survival. Thus, we developed Wnt assays to screen for inhibitor compounds against the CLK family, and see great potential for CLK3 as a future target for colon cancer therapeutics. ---- The sudden rise of the SARS-CoV-2 virus and the delay in the development of effective therapeutics to mitigate it made evident an urgent need for ways to screen for compounds that can block infection and prevent further pathogenesis and spread. Yet, identifying effective drugs efficacious against viral infection and replication with minimal toxicity for the patient can be difficult. Monoclonal antibodies were shown to be effective, yet as the SARS-CoV-2 mutated, these antibodies became ineffective. Small molecule antivirals were identified using pseudovirus constructs to recapitulate infection in non-human cells, such as Vero E6 cells. However, the impact was limited due to poor translation of these compounds in the clinical setting. This is in part due to the lack of similarity of screening platforms to the in vivo physiology of the patient and in part because drugs effective in vitro showed dose-limiting toxicities. In this study, we performed two high-throughput screens in human lung adenocarcinoma cells with authentic SARS-CoV-2 virus to identify both monoclonal antibodies that neutralize the virus and clinically useful kinase inhibitors to block the virus and prioritize minimal host toxicity. Using high-content imaging combined with single-cell and multidimensional analysis, we identified antibodies and kinase inhibitors that reduce virus infection without affecting the host. Our screening technique uncovered novel antibodies and overlooked kinase inhibitors that could be effective against SARS-CoV-2 virus. Further characterization of these molecules will streamline the repurposing of compounds for the treatment of future pandemics and uncover novel mechanisms viruses use to hijack and infect host cells.