Lysine lactylation (Kla) is a recently discovered histone mark derived from metabolic lactate. The NAD + -dependent deacetylase SIRT3, which can also catalyze removal of the lactyl moiety from lysine, is expressed at low levels in hepatocellular carcinoma (HCC) and has been suggested to be an HCC tumor suppressor. Here we report that SIRT3 can delactylate non-histone proteins and suppress HCC development. Using SILAC-based quantitative proteomics, we identify cyclin E2 (CCNE2) as one of the lactylated substrates of SIRT3 in HCC cells. Furthermore, our crystallographic study elucidates the mechanism of CCNE2 K348la delactylation by SIRT3. Our results further suggest that lactylated CCNE2 promotes HCC cell growth, while SIRT3 activation by Honokiol induces HCC cell apoptosis and prevents HCC outgrowth in vivo by regulating Kla levels of CCNE2. Together, our results establish a physiological function of SIRT3 as a delactylase that is important for suppressing HCC, and our structural data could be useful for the future design of activators.

Relapse is a leading cause of death after allogeneic hematopoietic stem cell transplantation (allo-HSCT) for acute myeloid leukemia (AML). However, the underlying mechanisms remain poorly understood. Natural killer (NK) cells play a crucial role in tumor surveillance and cancer immunotherapy, and NK cell dysfunction has been observed in various tumors. Here, we performed ex vivo experiments to systematically characterize the mechanisms underlying the dysfunction of bone marrow-derived NK (BMNK) cells isolated from AML patients experiencing early relapse after allo-HSCT. We demonstrated that higher levels of active transforming growth factor-β1 (TGF-β1) were associated with impaired effector function of BMNK cells in these AML patients. TGF-β1 activation was induced by the overexpression of glycoprotein A repetitions predominant (GARP) on the surface of CD4+ T cells. Active TGF-β1 significantly suppressed mTORC1 activity, mitochondrial oxidative phosphorylation, the proliferation, and cytotoxicity of BMNK cells. Furthermore, pretreatment with the clinical stage TGF-β1 pathway inhibitor, galunisertib, significantly restored mTORC1 activity, mitochondrial homeostasis, and cytotoxicity. Importantly, the blockade of the TGF-β1 signaling improved the anti-tumor activity of NK cells in a leukemia xenograft mouse model. Thus, our findings reveal a mechanism explaining BMNK cell dysfunction and suggest that targeted inhibition of TGF-β1 signaling may represent a potential therapeutic intervention to improve outcomes in AML patients undergoing allo-HSCT or NK cell-based immunotherapy.