Abstract LB-093: Refolding & restoring function to mutant p53: HDAC inhibitor MS275 partially restores activity to R175H mutant p53
Authors: Baatz Margaux , Flores Brianna , Moore Jacob , Leyva Kathryn J. , Hull Elizabeth E. 
Published: August 2019

Introduction: Most p53 mutations occur in the DNA binding domain, resulting not only in a loss of WT p53 function but also in the gain of additional onocogenic properties such as increased cellular proliferation and metastasis. Two newly developed pharmaceuticals, NSC 319726 and SCH 529074, refold mutant p53 to a WT conformation, reduce cell proliferation, decrease aggregate formation, and represent a novel therapeutic approach. Another approach to regulating p53 activity is through acetylation. This post-translational modification leads to an activating conformational change in WT p53, but it is unclear how acetylation affects mutant p53. Two histone deacetylase inhibitors (HDACi), MS275 (Entinostat) and FK228 (Romidepsin), are promising chemotherapeutics that increase acetylation of many proteins. In this study, we ask if treatment with these HDACi leads to refolding of mutant p53, reduction of proliferation, and p53 transcription factor activity.

Methods: R175H, H193Y, R248Q, and R273H constructs were transfected into a p53-null cell line. Inducible p53 expression in stable subclones was quantitated by immunofluorescence, western blot, and ELISA. Proliferation was measured by incorporation of EdU after treatment with FK228, MS275, NSC 319726, and SCH 529074. Refolding of p53 was determined before and after treatment using the conformational antibodies pAb240 and pAb1620, which bind to unfolded and folded p53, respectively. Functionality of the R175H mutant p53 was assessed by measuring the ability of p53 to bind its response element.

Results: We focused on the R175H conformational p53 mutation as had a higher rate of cellular proliferation measured by EdU incorporation compared to the other mutants (p&lt; 0.05). As expected, NSC 319726 treatment reduces R175H proliferation (p&lt; 0.0001) and promotes refolding into the WT p53 conformation (p&lt; 0.05) while no difference was detected with the broad spectrum mutant p53 reactivator SCH 529074. The HDAC inhibitors MS275 and FK228 reduce R175H proliferation (p&lt; 0.0001) and MS275 appears to promote refolding into the WT p53 conformation while FK228 does not. When compared to control, transcription factor activity increased 3.8-fold with NSC 319726 treatment (p &lt; 0.05), 6.0-fold with MS275 treatment (p&lt; 0.0001), and 4.6-fold with FK228 treatment (p&lt;0.01). Results suggest that HDACi may activate mutant p53 and that MS275 may do so by restoring WT conformation.

Conclusion: The HDAC inhibitor MS275 reactivates R175H mutant p53 as measured by increased transcription factor activity apparently through refolding, as determined by pAb1620 binding following treatment. These results imply a novel mode of action for HDACi. Clinically this suggests that MS275 may be able to restore WT p53 tumor suppressor activity.

Citation Format: Margaux Baatz, Brianna Flores, Jacob Moore, Kathryn J. Leyva, Elizabeth E. Hull. Refolding & restoring function to mutant p53: HDAC inhibitor MS275 partially restores activity to R175H mutant p53 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-093.