Abstract Hepatocellular carcinoma (HCC), a major type of liver cancer, is a leading cause of cancer deaths worldwide. HCC remains refractory to most treatment options; hence, new therapeutic modalities are urgently needed. Our long-term goal is to develop novel HCC therapeutics based on a deep understanding of its pathogenesis. The specific goal of this application is to define a novel tumor suppressive role for the alkaline ceramidase 2 (ACER2) pathway in HCC development and progression. ACER2 is a member in the alkaline ceramidase family that we identified initially from the yeast Saccharomyces cerevisiae and then from mammals. ACER2 catalyzes the hydrolysis of ceramides to generate sphingosine (SPH), a bioactive lipid implicated in programmed cell death (PCD) and autophagy. We identify ACER2 as a novel transcriptional target of p53 and demonstrate that the ACER2/SPH pathway is a novel signaling axis that operates downstream of p53 to mediate PCD in response to DNA damage. According to the TCGA database, ACER2 is mutated or deleted suppressed in several cancers. A previous study finds that ACER2 is epigenetically repressed in HCC and our preliminary results reveal that ACER2 is downregulated in liver tumors compared to patient-matched adjacent non-tumor liver tissues. Remarkably, we find that mice deficient in the alkaline ceramidase 2 (Acer2) gene are more susceptible to age- related development of various spontaneous tumor types including liver tumors, suggesting that ACER2 is a novel tumor suppressor. According to these exciting findings, we hypothesize that ACER2 is a novel tumor suppressor whose suppression promotes HCC development, progression, and resistance to DNA-damaging chemotherapeutics. As a further corollary, we hypothesize that rectifying the ACER2/SPH pathway will inhibit HCC and overcome the resistance of HCC to chemotherapeutics. These hypotheses will be tested in three interrelated specific aims: Aim 1 Establish that ACER2 plays a key tumor suppressive role in HCC using a liver carcinogenesis mouse model and liver organoid model, Aim 2 Define the cellular and molecular mechanisms by which the repression of the ACER2/SPH pathway promotes liver tumorigenesis, and Aim 3 Establish the role of the ACER2 pathway in improving the radio/chemotherapy of HCC. Successful completion of these aims will 1) validate the tumor suppressive role of the newly-identified tumor suppressor ACER2; 2) offer novel insights into the mechanisms by which the newly identified p53/ACER2/SPH signaling axis mediates the DNA damage response and tumor suppression; and 3) provide a proof of concept to develop the ACER2/SPH pathway into a novel therapeutic target for HCC. Given the poor clinical outcome of patients with HCC, these studies may have widespread impact on the clinical management of these patients.