SUMMARY: The long-term goal of Project 2 is to understand the molecular mechanisms responsible for longevity and cancer resistance of long-lived mammalian species and then translate these strategies to humans. The naked mole rat (NMR) is the longest-lived rodent, with a maximum lifespan of 32 years. Moreover, NMRs are highly resistant to cancer and other age-related diseases. Since NMRs are related to the short-lived laboratory rodents, they represent an ideal model for studies of longevity. We identified a novel mechanism of cancer- resistance in the NMR which is mediated by abundant high molecular weight hyaluronan (HMW-HA) in naked mole rat tissues. HA is a polymer glycosaminoglycan, and a component of the extracellular matrix produced by hyaluronan synthase 2 gene (HAS2). Depleting HMW-HA from NMR cells make them prone to form tumors. In the current funding cycle, we generated transgenic mice expressing naked mole rat HAS2 (nmrHAS2 mice). Our preliminary results suggest that nmrHAS2 mice have a longer lifespan and improved fitness. These mice are resistant to osteoarthritis and have improved hematopoietic stem cell maintenance. Importantly, we found that HMW-HA levels were only modestly elevated in nmrHAS2 mice due to very high hyaluronidase (HA-degrading) activity in the mouse tissues compared to the naked mole rat. Human tissues have similarly high hyaluronidase activity. We therefore propose that downregulating hyaluronidases using small molecules is a promising strategy to increase endogenous HMW-HA levels and alleviate age-related diseases, which has translational potential. We designed a strategy for a high throughput screen for hyaluronidase inhibitors and identified the first compounds that upregulate HMW-HA levels. In search for additional mechanisms that promote NMR health and longevity, we found that NMRs have more stable epigenomes characterized by resistance to iPSC reprogramming and specific differences in histone modifications. Based on these findings, our future objectives are focused on two longevity mechanisms we identified in the NMR, namely HMW-HA and a stable epigenome. Our aims are: (1) Complete the lifespan study and characterize cancer susceptibility, tissue pathology, genome stability (with Project 3), metabolism, and methylation age (with Project 4) of nmrHAS2 mice. (2) Perform a high throughput screen for hyaluronidase inhibitors and test the effect of these compounds on health, fitness and age- related pathologies in mice. (3) Determine molecular mechanisms responsible for NMR epigenome stability. We will identify specific histone modifying enzymes that are differentially regulated in the NMR, and manipulate them to stabilize the mouse epigenome. We will collaborate with Project 1 to determine the role of SIRT6 in NMR epigenome stability, and with Project 3 to investigate genome and methylome stability in the NMR and mouse cells after epigenetic interventions. The proposed research will identify strategies for exporting the longevity mechanisms found in the naked mole rat to other species.