Dynamic investigation of hypoxia-induced L-lactylation

The recently identified histone modification lysine lactylation can be stimulated by L-lactate and glycolysis. Although the chemical group added upon lysine lactylation was originally proposed to be the L-enantiomer of lactate (KL-la), two isomeric modifications, lysine D-lactylation (KD-la), and N-ε-(carboxyethyl) lysine (Kce), also exist in cells, with their precursors being metabolites of glycolysis. The dynamic regulation and differences among these three modifications in response to hypoxia have not been investigated previously. In this study, we demonstrate that intracellular KL-la, but not KD-la or Kce, is upregulated in response to hypoxia. Depletion of glyoxalase enzymes, GLO1 and GLO2, had minimal impact on KD_x001E_la, Kce, or hypoxia-induced KL-la. Conversely, blocking glycolytic flux to L-lactate under hypoxic conditions by knocking out LDHA/B completely abolished the induction of KL-la, but increased KD-la and Kce. We further observed a correlation between the level of KL-la and HIF-1α expression under hypoxic conditions and when small molecules were used to stabilize HIF-1α in the normoxia condition. Our result demonstrated that there is a strong correlation between HIF-1α and KL-la in lung cancer tissues, and that patient samples with higher grade tend to have higher KL-la levels. Using a proteomics approach, we quantified 66 KL-la sites that were upregulated by hypoxia and demonstrated that p300/CBP contributes to hypoxia-induced KL-la. Collectively, our study demonstrates that KL-la, rather than KD-la or Kce, is the prevailing lysine lactylation in response to hypoxia. Our results therefore demonstrate a link between KL-la and the hypoxia-induced adaptation of tumor cells.