Ghena Krdi
Ghena Krdi
Ghena Krdi
Helios Scholar
School: Arizona State University
Hometown: Phoenix, Arizona
Mentor: David Azorsa, Ph.D.
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The Effect of CDK Inhibitors on ATF6α Activity in Osteosarcoma Cells

Osteosarcoma (OS) is the most common type of bone cancer that mainly targets children and teenagers. Localized OS has a survival rate of 60% - 75%, which drops to less than 30% for metastatic, recurrent or refractory disease. Recent studies have shown an association of drug resistant OS cells with high expression of ATF6α. ATF6α is a sensor that the endoplasmic reticulum (ER) releases when it is stressed. Upon its release as a stress sensor, ATF6α is transferred from the endoplasmic reticulum to the Golgi then it is cleaved to the nucleus where it increases the transcription of specific genes including BiP and HERPUD1. 

Previous work in our laboratory used drug library screening to identify inhibitors of ATF6α activity.  Results showed that inhibitors of cyclin-dependent kinase (CDK) were able to decrease ATF6α activity in a reporter assay. For this project, our objective was to study the effect of CDK inhibitors on ATF6α activity in OS cells and their effects on OS cell growth. We performed drug dose response (DDR) assays on four OS cells lines, U2OS, Sa-OS2, MG-63 and 143b as well as control cell lines HS-5 and HEK-293 (non-cancer cells lines). The cells were treated for 72 hours with varying doses of the CDK inhibitors Flavopiridol and Dinaciclib. Our results show that OS cells responded in a similar manner to the drugs with similar EC50 values indicating that OS cells were not more sensitive to treatment with CDK inhibitors compared to control cells. We further tested the effect of the CDK inhibitor Dinaciclib on ATF6α activity induced by the ER stressor Tunicamycin. U2OS cells were treated with Tunicamycin for 18 hours to induce ER stress in the presence of 1, 10 or 100 nM Dinaciclib. Both protein lysates and RNA were isolated and analyzed by western blot or RT-qPCR respectively. Our results show that in cells treated with 100 nM Dinaciclib, ATF6α protein expression is lost upon ER stress induction with Tunicamycin. Gene expression studies of U2OS cells treated with Tunicamycin showed increased expression of the ATF6 targets BiP and HERPUD1. Furthermore, the addition of Dinacicilib was able to decrease the Tunicamycin-induced gene expression in a dose dependent manner. Our results suggest that CDK inhibitors such as Dinaciclib could limit the activity of ATF6α and could be considered a treatment for drug resistant OS cells possibly as part of a combination therapy approach.