Citation: D’Souza, A.M.; Gnanamony, M.; Thomas, M.; Hanley, P.; Kanabar, D.; de Alarcon, P.; Muth, A.; Timchenko, N. Second Generation Small Molecule Inhibitors of Gankyrin for the Treatment of Pediatric Liver Cancer. Cancers 2022, 14, 3068. https://doi.org/10.3390/ cancers14133068 Academic Editor: Jennifer S. Carew Received: 29 April 2022 Accepted: 14 June 2022 Published: 22 June 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). cancers Article Second Generation Small Molecule Inhibitors of Gankyrin for the Treatment of Pediatric Liver Cancer Amber M. D’Souza 1, * , Manu Gnanamony 1 , Maria Thomas 1 , Peter Hanley 1 , Dipti Kanabar 2 , Pedro de Alarcon 1 , Aaron Muth 2 and Nikolai Timchenko 3 1 Department of Pediatrics, University of Illinois College of Medicine Peoria, 1 Illini Drive, Peoria, IL 61605, USA; manug@uic.edu (M.G.); tmaria@uic.edu (M.T.); pjhanle2@uic.edu (P.H.); pdealarc@uic.edu (P.d.A.) 2 Department of Pharmaceutical Sciences, St. John’s University, 8000 Utopia Pkwy, Jamaica, NY 11439, USA; dipti.kanabar17@my.stjohns.edu (D.K.); mutha@stjohns.edu (A.M.) 3 Division of Pediatric Surgery, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229, USA; nikolai.timchenko@cchmc.org * Correspondence: dsom6v@uic.edu; Tel.: +1-309-624-4945 Simple Summary: New therapy options are needed for children with liver cancer. The goal of this study was to evaluate the role of three new compounds in the treatment of liver cancer cells. These compounds all inhibit a protein called Gankyrin, which is known to promote liver cancer by destroying tumor suppressor proteins. We demonstrated that liver cancer cells have significantly reduced proliferation when treated with these compounds by preventing the degradation of tumor suppressor proteins. We also discovered that these compounds enhance the effects of doxorubicin, which is a chemotherapy drug commonly used in liver cancer. These results support continued efforts in Gankyrin-based therapy for the treatment of pediatric liver cancer. Abstract: Background: Gankyrin, a member of the 26S proteasome, is an overexpressed oncoprotein in hepatoblastoma (HBL) and hepatocellular carcinoma (HCC). Cjoc42 was the first small molecule inhibitor of Gankyrin developed; however, the IC 50 values of >50 μM made them unattractive for clinical use. Second-generation inhibitors demonstrate a stronger affinity toward Gankyrin and increased cytotoxicity. The aim of this study was to characterize the in vitro effects of three cjoc42 derivatives. Methods: Experiments were performed on the HepG2 (HBL) and Hep3B (pediatric HCC) cell lines. We evaluated the expression of TSPs, cell cycle markers, and stem cell markers by Western blotting and/or real-time quantitative reverse transcription PCR. We also performed apoptotic, synergy, and methylation assays. Results: The treatment with cjoc42 derivatives led to an increase in TSPs and a dose-dependent decrease in the stem cell phenotype in both cell lines. An increase in apoptosis was only seen with AFM-1 and -2 in Hep3B cells. Drug synergy was seen with doxorubicin, and antagonism was seen with cisplatin. In the presence of cjoc42 derivatives, the 20S subunit of the 26S proteasome was more available to transport doxorubicin to the nucleus, leading to synergy. Conclusion: Small-molecule inhibitors for Gankyrin are a promising therapeutic strategy, especially in combination with doxorubicin. Keywords: Gankyrin; cjoc42 derivative; hepatoblastoma; pediatric hepatocellular carcinoma; therapy; drug synergy 1. Introduction Pediatric liver cancer represents 2% of childhood malignancies, with the two most common types being hepatoblastoma (HBL) and hepatocellular carcinoma (HCC) [1]. Chemotherapy remains a cornerstone of therapy; however, chemoresistance remains an issue [2,3]. About 30% of patients with HBL will eventually relapse or die of chemoresistant Cancers 2022, 14, 3068. https://doi.org/10.3390/cancers14133068 https://www.mdpi.com/journal/cancers