Targeting NAD⁺ metabolism of hepatocellular carcinoma cells by lenvatinib promotes M2 macrophages reverse polarization, suppressing the HCC progression

Background: Lowered nicotinamide adenine dinucleotide (NAD⁺) levels in tumor cells drive tumor hyperprogression during immunotherapy, and its restoration activates immune cells. However, the effect of lenvatinib, a first-line treatment for unresectable hepatocellular carcinoma (HCC), on NAD⁺ metabolism in HCC cells, and the metabolite crosstalk between HCC and immune cells after targeting NAD⁺ metabolism of HCC cells remain unelucidated.

Methods: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and ultra-high-performance liquid chromatography multiple reaction monitoring-mass spectrometry (UHPLC-MRM-MS) were used to detect and validate differential metabolites. RNA sequencing was used to explore mRNA expression in macrophages and HCC cells. HCC mouse models were used to validate the effects of lenvatinib on immune cells and NAD⁺ metabolism. The macrophage properties were elucidated using cell proliferation, apoptosis, and co-culture assays. In silico structural analysis and interaction assays were used to determine whether lenvatinib targets tet methylcytosine dioxygenase 2 (TET2). Flow cytometry was performed to assess changes in immune cells.

Results: Lenvatinib targeted TET2 to synthesize and increase NAD⁺ levels, thereby inhibiting decomposition in HCC cells. NAD⁺ salvage increased lenvatinib-induced apoptosis of HCC cells. Lenvatinib also induced CD8⁺ T cells and M1 macrophages infiltration in vivo. And lenvatinib suppressed niacinamide, 5-Hydroxy-L-tryptophan and quinoline secretion of HCC cells, and increased hypoxanthine secretion, which contributed to proliferation, migration and polarization function of macrophages. Consequently, lenvatinib targeted NAD⁺ metabolism and elevated HCC-derived hypoxanthine to enhance the macrophages polarization from M2 to M1. Glycosaminoglycan binding disorder and positive regulation of cytosolic calcium ion concentration were characteristic features of the reverse polarization.

Conclusions: Targeting HCC cells NAD⁺ metabolism by lenvatinib-TET2 pathway drives metabolite crosstalk, leading to M2 macrophages reverse polarization, thereby suppressing HCC progression. Collectively, these novel insights highlight the role of lenvatinib or its combination therapies as promising therapeutic alternatives for HCC patients with low NAD⁺ levels or high TET2 levels.