Hepatocellular carcinoma, the most common form of liver cancer, is notorious for its silent progression. Global cancer statistics from 2018 ranked liver cancer as the sixth most common malignancy and the fourth leading cause of cancer death worldwide. Because most patients are diagnosed only after the disease has reached intermediate or advanced stages, surgical resection is often no longer an option. For these individuals, treatment relies on non-curative approaches such as chemotherapy, targeted drug therapy, and immunotherapy, making any improvement in the effectiveness of these tools a matter of life and death.

Tyrosine kinase inhibitors, or TKIs, form the backbone of pharmacological treatment for advanced liver cancer. First-line drugs such as sorafenib and lenvatinib block the signaling pathways that tumor cells use to proliferate and survive, but many patients eventually progress or develop resistance. Regorafenib, approved by the U.S. Food and Drug Administration in 2017 for patients whose disease advances on sorafenib, targets a broad panel of receptors including VEGFR1-3, PDGFR-beta, KIT, RET, and RAF, and uniquely also hits fibroblast growth factor receptors and the angiopoietin 1 receptor known as TIE2. Preclinical work has suggested regorafenib is pharmacologically more potent than sorafenib, capable of blocking angiogenesis, tumorigenesis, and metastasis while also modulating tumor immunity. Earlier studies showed it can drive liver cancer cells into apoptosis by directly activating the phosphatase SHP-1, which suppresses phosphorylated STAT3, an effect independent of its anti-angiogenic activity.

On the immunotherapy side, the team turned to BMS-1, a small molecule first described in a 2015 patent filed by Bristol-Myers Squibb as part of a series of compounds that disrupt the interaction between the immune checkpoint proteins PD-1 and PD-L1. Antibody drugs such as atezolizumab and avelumab have shown promise against several malignancies, but antibodies suffer from drawbacks including immunogenicity and poor penetration of tumor tissue, which contribute to low response rates in some patients. Small-molecule checkpoint inhibitors could, in principle, sidestep these limitations, and combining them with other agents has become an active research frontier.

To test whether BMS-1 truly engages its intended target, the researchers first ran molecular docking simulations using AutoDock Vina software. The predicted binding energy of the BMS-1-PD-L1 complex was a favorable minus 11.633 kilocalories per mole, and the structural model placed BMS-1 wedged between PD-L1 dimers, consistent with the idea that the compound prevents the ligand from engaging PD-1 on T cells. The team then measured how effectively each drug killed three human hepatocellular carcinoma cell lines, SMMC-7721, Hep3B, and SK-Hep1, using CCK-8 viability assays. The half-maximal inhibitory concentrations came out at roughly 13.35, 13.32, and 9.50 micromolar for regorafenib, and 74.11, 73.08, and 53.76 micromolar for BMS-1 across the three lines, establishing dose ranges for combination testing.

When the drugs were combined at concentrations below each one’s IC50, the results were striking. Using CompuSyn software to calculate combination indices, the team found values below 1, the mathematical signature of synergy rather than simple additivity, in SMMC-7721 cells across the tested combinations, and in Hep3B and SK-Hep1 cells for most combinations. The pairing of 6 micromolar regorafenib with 30 micromolar BMS-1 showed synergy in all three cell lines and was selected for deeper analysis. Transwell assays added another dimension: regorafenib alone significantly curbed the migration and invasion of the cancer cells, BMS-1 alone had little effect, and the combination suppressed these malignant behaviors even more strongly than regorafenib by itself.

Digging into the mechanism, the researchers tracked how the drug pair altered cell cycle machinery. EdU incorporation assays revealed a sharp drop in the fraction of cells in S phase after combination treatment, and western blotting showed that levels of Cyclin A2, Cyclin B1, Cyclin D1, and Cyclin D3, the proteins that drive cells through the G1/S and G2/M checkpoints, were downregulated, with Cyclin A2 and Cyclin B1 significantly lower in the combination group than with either drug alone. Apoptosis assays told a parallel story. Flow cytometry with Annexin V and propidium iodide staining, backed up by TUNEL staining, showed that the combination produced the highest rate of late apoptosis in all three cell lines, while western blots revealed a marked increase in cleaved PARP, a canonical executioner of programmed cell death, and a decrease in the proliferation marker PCNA.

One of the most intriguing findings emerged when the team introduced immune cells into the picture. Hepatocellular carcinoma cells barely expressed PD-L1 when grown alone, but when the researchers co-cultured them with peripheral blood mononuclear cells isolated from healthy volunteers, PD-L1 expression surged, mimicking the immune pressure of a real tumor microenvironment. Under those conditions, regorafenib pushed PD-L1 levels down, BMS-1 had a modest effect, and the combination drove PD-L1 expression down significantly. The authors speculate that regorafenib’s shared target FGFR4, which lenvatinib uses to promote proteasomal degradation of PD-L1 through the FGFR4-glycogen synthase kinase 3-beta pathway, may underlie this effect, though they caution that the exact mechanism remains unproven.

The combination also held up in living animals. In nude mice bearing subcutaneous tumors derived from SMMC-7721 cells, four weeks of treatment with intraperitoneal BMS-1 at 20 milligrams per kilogram twice weekly plus oral regorafenib at 5 milligrams per kilogram five times weekly shrank tumor volume and weight more dramatically than either drug alone. The authors are careful to note the caveats: the xenograft model lacks a fully functional immune system, so the immunological component of the synergy could not be fully assessed, and systemic tolerability has yet to be evaluated prospectively. They also point to related work suggesting that BMS-series compounds and other biphenyl derivatives can trigger cancer cell death through non-immune pathways, such as inhibition of AKT phosphorylation or modulation of TGF-beta/Smad signaling, hinting that BMS-1 may be doing double duty.

Even so, the study adds momentum to one of the hottest ideas in oncology: that molecularly targeted drugs can remodel the tumor microenvironment in ways that make immune checkpoint blockade work better. Prior research has shown that regorafenib can boost CD8-positive T cell activation by blocking interferon-gamma-induced PD-L1 expression in melanoma, and that combining kinase inhibitors with checkpoint inhibitors can allow cytotoxic T lymphocytes to infiltrate tumors more effectively. With single-agent checkpoint inhibitors such as nivolumab and pembrolizumab delivering median overall survival of only 15.6 and 12.9 months in liver cancer trials, respectively, the need for better combinations is acute. If future studies in immunocompetent models and eventually patients confirm what the cell cultures and mouse xenografts suggest, the regorafenib-BMS-1 pairing could provide a theoretical foundation for a new generation of chemo-immunotherapy against hepatocellular carcinoma, one in which a kinase inhibitor and a checkpoint blocker amplify each other’s strengths while keeping doses, and side effects, in check.

Subject of Research: Combination therapy with regorafenib and the small-molecule PD-1/PD-L1 inhibitor BMS-1 for hepatocellular carcinoma

Article Title: BMS-1 synergistically promotes the anti-tumor effect of regorafenib on hepatocellular carcinoma

Article References: Yu, G., Li, J., Wang, K., Fu, F., Lin, J., Xu, W., Liu, S., & Yang, H. (2026). BMS-1 synergistically promotes the anti-tumor effect of regorafenib on hepatocellular carcinoma. Heliyon, 12(15), Article e45420. https://doi.org/10.1016/j.heliyon.2026.e45420

Image Credits: AI Generated

DOI: 10.1016/j.heliyon.2026.e45420

Keywords: hepatocellular carcinoma, regorafenib, BMS-1, PD-1/PD-L1 inhibitors, tyrosine kinase inhibitors, immunotherapy, combination therapy, apoptosis, cell cycle, tumor microenvironment, xenograft model, synergy