Investigating the role of OAADPr-ACADM interaction in colorectal cancer

Project Summary/Abstract Nicotinamide Adenine Dinucleotide (NAD+) serves as a crucial redox cofactor in metabolism and acts as a substrate for poly (ADP-ribose) polymerase and sirtuins, playing roles in DNA repair, metabolism, and stress response and involving in cancer and longevity. Sirtuins consume NAD+ and produce a unique byproduct, O- acetyl-ADP-ribose (OAADPr), which holds significant potential as a signaling molecule in regulating various biological processes. Yet, the biological roles and metabolic regulations of OAADPr remain poorly elucidated. Colorectal cancer (CRC) is the third leading cause of cancer deaths in the United States. The poor outcome of CRC highlights an urgent need to identify mechanisms that regulate CRC metabolism and growth. Studies have shown elevated NAD+ levels and increased expression of NAD+ biosynthesis and salvage enzymes in CRC, suggesting a critical role of NAD+ metabolism in CRC progression. Sirtuins are involved in CRC and conflicting roles have been reported in CRC. Yet, the mechanistic links between sirtuins and cancer remain incompletely understood and the exact mechanistic links between NAD+-related metabolites and cancer remain unclear. To identify novel proteins that interact with NAD+-metabolites, I applied a proteomic thermal stability assay and discovered a previously unknown interaction between medium-chain specific acyl-CoA dehydrogenase (ACADM) and OAADPr. ACADM is a key enzyme in β-oxidation, and aberrant fat metabolism has long been recognized in CRC. I hypothesize that OAADPr may play a tumor-suppressive role in CRC through the inhibition of ACADM activity and fat utilization. I will test this hypothesis in two aims: 1) elucidate the mechanism of how OAADPr affects ACADM activity and 2) examine the impact of OAADPr-mediated inhibition on ACADM both in vitro and in vivo. In Aim 1, I will determine the kinetic and equilibrium parameters of the inhibition of OAADPr on ACADM and its mechanism. In Aim 2, I will modulate OAADPr levels in CRC cells by overexpressing (OE) SIRT3 and/or knockout (KO) MACROD1, two key enzymes that regulate OAADPr metabolism. With that, I will first assess fat oxidation in wild-type, SIRT3 OE, and/or MACROD1 KO CRC cells by measuring the rate of fatty acid oxidation, profiling acyl-carnitines, and conducting C13-palmitate tracing experiments. Then, I will assess if OAADPr modulates CRC proliferation and tumor growth by measuring cell proliferation in vitro, organoid growth ex vivo, and xenograft or spontaneous tumor growth in vivo with altered OAADPr levels. Lastly, I will investigate whether dietary supplementation of NAD+ precursors affects intratumor OAADPr levels and tumor growth in organoids and the genetically engineered mouse model. Our research is conceptually novel and will address an important gap in our understanding of the mechanism by which NAD+ metabolism affects tumor proliferation. The proposed study will also uncover new roles for OAADPr in metabolism and cancer and open a new area in NAD+ and sirtuin biology. The insight gained may open avenues for novel approaches leveraging NAD+ metabolism in cancer therapy.