An Oxanthroquinone Derivative Disrupts Ras Plasma Membrane Localization and Function By Inhibition of Acylpeptide Hydrolase and Perturbation of Sphingomyelin Metabolism

<p>Oncogenic RAS proteins are commonly expressed in human cancer. To be functional, RAS proteins must undergo post-translational modification and localize to the plasma membrane (PM). Therefore, compounds that prevent RAS PM targeting have potential as putative RAS inhibitors. Here we examined the mechanism of action of oxanthroquinone G01 (G01), a recently described inhibitor of KRAS PM localization. We show that G01 mislocalized HRAS and KRAS from the PM with similar potency and disrupted the spatial organization of RAS proteins remaining on the PM. G01 also inhibited recycling of epidermal growth factor receptor and transferrin receptor, but did not impair internalization of cholera toxin, indicating suppression of recycling endosome function. In searching for the mechanism of impaired endosomal recycling we observed that G01 also enhanced cellular sphingomyelin (SM) and ceramide levels and disrupted the localization of several lipid and cholesterol reporters, suggesting that the G01 molecular target may involve SM metabolism. Indeed, G01 exhibited potent synergy with other compounds that target SM metabolism in KRAS localization assays.</p> <p>When attached to a biotin moiety, G01 bound acylpeptide hydrolase (APEH) and blocked its enzymatic activity. Inhibition of APEH by ebelactone, an esterase inhibitor, or the knockdown of APEH mislocalized KRASG12V as well as phosphatidylserine from the PM. Together, these results suggest that G01 mislocalizes KRASG12V from the PM by binding to and inhibiting APEH function.</p> <p>Furthermore, G01 significantly abrogated RAS-RAF-MAPK signaling in MDCK cells expressing constitutively activated, oncogenic mutant RASG12V. G01 also inhibited the proliferation of RAS-less mouse embryonic fibroblasts (MEFs) expressing oncogenic mutant KRASG12V or KRASG12D but not RAS-less MEFs expressing oncogenic mutant BRAFV600E. Consistent with these effects, G01 inhibited the proliferation of KRAS-transformed pancreatic, colon, and endometrial cancer cells. Taken together, these results suggest that G01 should undergo further evaluation as a potential anti-RAS therapeutic.</p> <p>To enhance the potency of G01 for KRAS inhibition, we synthesized D01 and D02, two derivatives of G01 containing an additional geranyl or farnesyl group, respectively. D01 or D02 mislocalized KRAS more effectively than G01, and disrupted the clustering of KRAS at a much lower concentration than G01. As a consequence, D01 or D02 inhibited MAPK signaling downstream of oncogenic KRAS, as well as the proliferation of KRAS-dependent pancreatic cancer cells.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation (PhD)

Year dc:date.available

2019

Authors dc:creator

• Tan, Lingxiao
• <p>0000-0001-9758-891X</p>

Contributors dc:contributor

• John F. Hancock
• Swathi Arur
• Carmen W. Dessauer

• RAS
• KRAS
• oxanthroquinone
• plasma membrane
• cancer
• sphingomyelin
• signaling
• Biochemistry
• Cancer Biology
• Cell Biology

Repository record dc:identifier

https://digitalcommons.library.tmc.edu/utgsbs_dissertations/925

OAI identifier oai:identifier

oai:digitalcommons.library.tmc.edu:utgsbs_dissertations-1972