Combination therapy for HCC: from CRISPR screening to the design of clinical therapies

Abstract

CRISPR screening has been broadly used to discover new therapy targets, but these finding’ successful clinical transition is still limited. In a recent study published in Nature, Jin et al. adopted CRISPR screening to dissect the resistance of hepatocellular carcinoma to lenvatinib, discovering the synergistic effect of EGFR inhibitor (gefitinib) and lenvatinib (multi-tyrosine kinase inhibitor, muti-TKI) combination therapy with encouraging clinical benefits. Liver cancer is still a major threat to global health, ranking as the fourth most common cause of cancer-related death and the second most lethal tumor, with a 5-year survival rate of 18%. Unfortunately, the global incidence of liver cancer is still growing, with estimated cases of >1 million by 2025. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. Despite the large armamentarium of therapies that have been used to treat HCC, including surgery, ablation therapy, embolization, chemotherapy, and radiation therapy, the majority of late-stage HCC patients succumb to the disease. In advanced stages, only systemic therapy is applicable. For decades, sorafenib, an oral multi-TKI, was the only approved systemic therapy for HCC. Recently, TKIs and immunecheckpoint inhibitors (ICIs) have changed conventional therapies for HCC. Among them, lenvatinib showed high efficacy (median overall survival of 13.6 months for lenvatinib vs 12.3 months for sorafenib) in a global open-label randomized phase III study and was approved as the new first-line for advanced-stage HCC. This result puts lenvatinib as one of the best single therapies for HCC. However, the overall response rate for lenvatinib is only 24.1%, highlighting the need for developing combination therapies. The scenario was supported by promising results observed in the combination of ICIs and TKIs or anti-angiogenic therapies. For example, the combination of PD-L1 antibody (atezolizumab) and VEGF inhibitor (bevacizumab) has demonstrated striking superiority in first-line treatment, opening a new standard of care for HCC. However, the overall survival and response rate for patients with advanced HCC is still very low, reflecting the need to explore new combination therapies. A recent milestone study by Jin et al. reports the use of synthetic lethal CRISPR screen in discovering the synergistic effects of EGFR inhibitor and lenvatinib combination in HCC, achieving an approximately 50% of clinical responses in patients with advanced HCC who were unresponsive to lenvatinib. In this study, the authors used SNU499, a lenvatinib-resistant HCC cell line, to carry out synthetic lethal CRISPR screens, and discovered the knockout of EGFR under lenvatinib treatment is lethal to EGFR-expressing HCC cells. Considering the fact that most liver cancers express high levels of EGFR, they explored the therapeutic potentials of combinating EGFR targeting gefitinib and lenvatinib in HCC. According to the in vitro and in vivo data, the combination of lenvatinib and gefitinib had potent inhibition of tumor growth and their synergistic effect seems only applied to EGFR cells. Nevertherless, another seemingly similar EGFR inhibitor plus multi-TKI combination pair, erlotinib and sorafenib, failed to yield significant clinical benefit. Exploring the underlying reasons, they noticed although both sorafenib and lenvatinib are multi-TKIs, only lenvatinib inhibits FGFR signaling which is essential for the combination therapy, and the combination of FGFR inhibitor and EGFR inhibitor had similar synergistic effects against liver cancer. Further mechanistic studies guided by the CRISPR screening data revealed that the feedback activation of the EGFR-PAK2-ERK5 pathway after lenvatinib treatment contributes to the resistance of HCC to lenvatinib. Therefore, the addition of the EGFR inhibitor, gefinitib, abolishes or at least alleviates the resistance of HCC to lenvatinib by suppressing this pathway. Based on the above mentioned results, they initiated a clinical trial to evaluate the therapeutic potentials of lenvatinib and gefitinib combination in patients with unresectable, lenvatinib-resistant, EGFR HCC. Although still early, this combination therapy showed encouraging results in patients with advanced HCC who are not responsive to the treatments of levatinib or lenvatinib plus PD-1 antibody. According to the mRECIST criteria, 4 of 12 cases showed confirmed partial response, 4 showed stable disease, and 4 showed progression disease. Notably, according to their MRI data, the responders already showed sharp tumor burden reduction and serum AFP decrease within 4 weeks of the combination therapy. Even promising, it might still be early to draw firm conclusions, and we are eager to see the long-term survival outcome. It has to be mentioned that the vast majority of patients with advanced HCC still had a very poor prognosis due to primary or acquired resistance to systemic therapies. This seminal study has demonstrated that, by designing appropriate CRISPR screens, we can systematically dissect the pathways and mechanisms of the drug resistance and the identified key regulators especially the synergistic targets that may hold great potentials for new combination therapies (Fig. 1). Notably, this is not their first effort to identify potential synergistic targets for combination therapy with CRISPR screening. At 2019, Prof. Wenxin Qin’s group and Prof. Rene Bernards’s group already performed CRISPR screening on HCC vulnerabilities and identified the combined inhibition of CDC7 and mTOR as a “one-two punch” therapy for liver cancer with TP53 mutation, reducing tumor relapse by alleviating both the cell-autonomous and non-cell-autonomous attributes of