Abstract

Background: Immune checkpoint therapies combination with anti-VEGF is the standard-of-care in first-line of hepatocellular carcinoma (HCC). However, only 30 % of patients present a response to first line therapy. Autophagy inhibitors were recently spotted out as a potential robust strategy to promote antigen presentation and therefore reinforce immune checkpoint inhibitors (ICIs) potency conducting to strong anti-tumoral response. Hereafter, we aimed to assess the efficacy of GNS561 (Ezurpimtrostat), an autophagy inhibitor targeting palmitoyl-protein thioesterase-1 (PPT-1) lysosomal enzyme, in combination with atezolizumab/bevacizumab in HCC in a chicken embryos model.

Methods: Chorioallantoic membrane (CAM assay) model was used, inoculated with liver adenocarcinoma Hep3B cell line at day 9 (D9). Between D9 and D18, eggs were treated every two day with GNS561 alone (0.92 mg/kg), or in combination with atezolizumab/bevacizumab (at 1 mg/kg/1mg/kg (R1), or 2 mg/kg/2mg/kg (R2)). At D18, the upper portion of the CAM containing tumor is removed. The tumors are cut away from normal CAM tissue and weighed, and the number of dead embryos is counted. A one-way analysis of variance with post-tests is done. No cytotoxicity was reported.

Results: All regimens induced a significant tumor growth regression compared to the control group, GNS561 alone having a more potent anti-tumoral effect (p<0.0004) compared to both atezolizumab/bevacizumab regimens (R1, p<0.0147; R2, p<0.0071). Interestingly, atezolizumab/bevacizumab combination regimens exhibited no significant difference between the two tested dose-regimens. A triple combination regimen led to significantly improved anti-tumoral efficacy compared to the two atezolizumab/bevacizumab groups, for comparable dosing (R1, p<0.0076, R2, p<0.0001), spotlighting triple combination superior benefit. Results further pointed out GNS561 combination with atezolizumab/bevacizumab significant superior anti-tumor effect at the highest dose in comparison with GNS561 alone, reinforcing the interest of triple therapy (p<0.0030).

Conclusion: This study reports that GNS561 potentiates the anti-tumor effect of atezolizumab/bevacizumab combination emphasizing PPT1 inhibitors’ therapeutic interest in combination with immunotherapy in HCC. This triple therapy is currently tested in the randomized phase 2 clinical trial ABE-LIVER, in first line setting in advanced HCC (NCT05448677).