Abstract 1419: Small molecule inhibitor of NRF2 selectively overcomes sorafenib resistance in KEAP1-deficient hepatocellular carcinoma
Authors: Chen Jiang , Jiang Shi , Li Bixia , Ji Tong , Daniel Staiculescu , He Jiayan , Xu Junjie , Cai Xiujun 
Published: July 2021

Background and Aims: Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world. A recent randomized phase III trial of combined immunotherapy (atezolizumab) with antiangiogenesis (bevacizumab) versus the standard sorafenib monotherapy reported an unprecedented response rate of 27%, leading to FDA approval of this combination as the new standard treatment in May 2020. Yet, despite this undisputable progress, the majority of the HCC cases are likely to recur or develop resistance to both first line therapy. In this study, we aimed to identify novel, druggable candidate genes for patients with sorafenib resistant HCC.

Method: CRISPR/Cas9 genome (GeCKO v2) library screening was used to identify loss of function mutations conferring sorafenib resistance upon HCC cells. CCK-8 and clonogenic assays were used to measure cell viability and cytotoxicity. Flow cytometry was performed to analyze cellular reactive oxidative species (ROS) and apoptosis level. Multiple animal models were used to study the synergistic efficacy of the combination of nuclear factor erythroid 2- related factor 2 (NRF2) inhibition and sorafenib.

Results: In our study, we explored the mechanisms underlying sensitivity of HCC cells to sorafenib using genome-wide CRISPR-Cas9 screening. Kelch-like ECH- associated protein-1(KAEP1) was discovered as a previously unknown factor for determining the sensitivity of HCC cells to sorafenib. Using established sorafenib resistant HCC cell lines and the parental cell, we validated that KEAP1 mediates NRF2 degradation through the ubiquitin-proteasome pathway, thereby conferring sensitivity to sorafenib. Conversely, the loss of function of KEAP1 reduced degradation of NRF2 and conferred increased sorafenib resistance. Analysis of 55 patients with HCC, who had received hepatic resection followed by postoperative standard sorafenib treatment, revealed that high NRF2 expression shortened both overall survival (P&lt;0.05) and disease free survival (P&lt;0.01). CCK-8 and clonogenic assays were used in combination with sorafenib revealed that NRF2 inhibitor substantially enhances cytotoxicity and leads to robust proliferation inhibition of sorafenib resistant HCC cells, as compared with single agents. In both cell derived xenograft (CDX) and patient-derived xenograft (PDX) HCC models with preserved NRF2 function, NRF2 inhibitor, in combination with sorafenib, showed significant antitumor activity (P&lt;0.05). Furthermore, the combination of NRF2 inhibitor and sorafenib shows striking synergy by increasing ROS and inducing apoptosis in HCC cells.

Conclusion: Our results uncovered the potential of NRF2 as a drug target for patients with sorafenib resistant HCC. The impressive efficacy of NRF2 inhibitor and sorafenib suggests a potential combination therapy to supplement the existing standard of treatment for sorafenib resistant HCC.

Citation Format: Jiang Chen, Shi Jiang, Bixia Li, Tong Ji, Staiculescu Daniel, Jiayan He, Junjie Xu, Xiujun Cai. Small molecule inhibitor of NRF2 selectively overcomes sorafenib resistance in KEAP1-deficient hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1419.