Inhibition of Kynurenine Metabolism and Its Effect in Mitochondrial Function in Hepatocellular Carcinoma

Abstract Hepatocellular carcinoma (HCC) is the most prevalent cancer in the liver. The majority of ingested tryptophan is processed in the liver through the kynurenine pathway, the endpoint of which is de novo NAD+ biosynthesis. Dysregulation of tryptophan-kynurenine metabolism and NAD+ synthesis may promote mitochondrial malfunction, tumor reprogramming, and carcinogenesis. Using a publicly available gene expression dataset from liver hepatocellular carcinoma (LIHC) samples available through The Cancer Genome Atlas (TCGA; n = 371), we employed Principal Component Analysis (PCA), hierarchical clustering, gene-pattern expression profiling, and survival analysis to cluster patients and determine overall survival. Our analysis of genes encoding kynurenine pathway enzymes determined that patients with high QPRT expression had a poor prognosis with decreased median survival, with no effect on the maximum survival. There is a significant difference in the survival between patients with high QPRT expression relative to patients with high HAAO/AFMID expression (HR = 1.2, [95% CI 0.5-1.8] P = 0.0181, Gehan-Breslow-Wilcoxon Test). Patients with high QPRT expression have higher survival rates compared with low QPRT expression (HR = 1.4, [95% CI 0.9-2.2] P = 0.0344, Gehan-Breslow-Wilcoxon Test). To test the consequences of kynurenine-pathway inhibition in mitochondrial function and morphology we use 4-Cl-3HAA, an irreversible HAAO inhibitor, and observed a small increase in mitochondrial fragmentation in HepG2 cells after 24 hours of treatment. We conclude that kynurenine metabolism may be useful as a biomarker to predict patient prognosis among HCC patients. In ongoing work, we are testing QPRT inhibitors in cell culture as a potential adjuvant for chemotherapies.

analysis showed that GW treatment reduced BV/TV in old mice but had no effect in young mice.FACS analysis data showed that GW treatment significantly increased lymphoid (CD3) and decreased myeloid (CD11b) lineage cells only in young mice.GW treatment significantly decreased CD4 and CD8 T cell subpopulations in young mice, but had no effect in old mice.Interestingly, Nanostring analysis of inflammation genes revealed an opposite effects of GW treatment in young vs. old mice, i.e., genes whose expression were downregulated by GW in bone cells of the young mice were upregulated in the old mice.These results suggested that systemic inhibition of PPARg, while enhancing CD3 and reducing CD11b cell populations in the bone marrow of young mice, may have a negative effect on bone architecture in old mice.

GREATER SKELETAL MUSCLE OXIDATIVE CAPACITY IS ASSOCIATED WITH HIGHER RESTING METABOLIC RATE: RESULTS FROM THE BLSA
Marta Zampino, 1 Richard Semba, 2 Fatemeh Adelnia, 3 Jennifer Schrack, 4 Richard Spencer, 1 Kenneth Fishbein, 1 Eleanor Simonsick, 1 and Luigi Ferrucci, 1 1.National Institute on Aging,Bethesda,Maryland,United States,2. Johns Hopkins University,Baltimore,Maryland,United States,3. Vanderbilt University Medical Center,Nashville,Tennessee,United States,4. Johns Hopkins Bloomberg School of Public Health,Baltimore,Maryland,United States Resting metabolic rate (RMR) tends to decline with aging.The age-trajectory of decline in RMR is similar to changes that occur in muscle mass, muscle strength and fitness.However, while the decline in these phenotypes have been related to changes of mitochondrial function and oxidative capacity, whether lower RMR is associated with poorer mitochondrial oxidative capacity is unknown.In 619 participants of the Baltimore Longitudinal Study of Aging, we analyzed the cross-sectional association between RMR (kcal/day), assessed by indirect calorimetry, and skeletal muscle maximal oxidative phosphorylation capacity, assessed as post-exercise phosphocreatine recovery time constant (tau-PCr), by phosphorous magnetic resonance spectroscopy.Linear regression models were used to evaluate the relationship between tau-PCr and RMR, adjusting for potential confounders.We found that independent of age, sex, lean body mass, muscle density and fat mass, higher RMR was significantly associated with shorter tau-PCr, indicating greater mitochondrial oxidative capacity.In conclusion, higher RMR appears to be associated with a higher mitochondrial oxidative capacity in skeletal muscle.This association may reflect a relationship between better muscle quality and greater mitochondrial health.

INHIBITION OF KYNURENINE METABOLISM AND ITS EFFECT IN MITOCHONDRIAL FUNCTION IN HEPATOCELLULAR CARCINOMA
Raul Castro-Portuguez, 1 Samuel Freitas, 2 and George Sutphin, 1 1.University of Arizona, Tucson, Arizona, United States, 2. University of Arizona, Tucson, United States Hepatocellular carcinoma (HCC) is the most prevalent cancer in the liver.The majority of ingested tryptophan is processed in the liver through the kynurenine pathway, the endpoint of which is de novo NAD+ biosynthesis.
Dysregulation of tryptophan-kynurenine metabolism and NAD+ synthesis may promote mitochondrial malfunction, tumor reprogramming, and carcinogenesis.Using a publicly available gene expression dataset from liver hepatocellular carcinoma (LIHC) samples available through The Cancer Genome Atlas (TCGA; n = 371), we employed Principal Component Analysis (PCA), hierarchical clustering, genepattern expression profiling, and survival analysis to cluster patients and determine overall survival.Our analysis of genes encoding kynurenine pathway enzymes determined that patients with high QPRT expression had a poor prognosis with decreased median survival, with no effect on the maximum survival.There is a significant difference in the survival between patients with high QPRT expression relative to patients with high HAAO/AFMID expression (HR = 1.2, [95% CI 0.5-1.8]P = 0.0181, Gehan-Breslow-Wilcoxon Test).Patients with high QPRT expression have higher survival rates compared with low QPRT expression (HR = 1.4,[95% CI 0.9-2.2]P = 0.0344, Gehan-Breslow-Wilcoxon Test).To test the consequences of kynurenine-pathway inhibition in mitochondrial function and morphology we use 4-Cl-3HAA, an irreversible HAAO inhibitor, and observed a small increase in mitochondrial fragmentation in HepG2 cells after 24 hours of treatment.We conclude that kynurenine metabolism may be useful as a biomarker to predict patient prognosis among HCC patients.In ongoing work, we are testing QPRT inhibitors in cell culture as a potential adjuvant for chemotherapies.

LATE-ONSET INTERMITTENT FASTING DECREASES AGING-RELATED FRAILTY
Yoko Henderson, 1 Nazmin Bithi, 2 Christopher Link, 3 Jie Yang, 1 and Christopher Hine, 1 1.Cleveland Clinic Lerner Research Institute, Cleveland, Ohio, United States,2. Lerner Research Institute,Cleveland Clinic,Cleveland,Ohio,United States,3. Cleveland Clinic,Cleveland,Ohio,United States Global average life expectancy continues to rise.As aging increases likelihoods of exhibiting geriatric syndromes (a.k.a.frailty), there is a need for effective anti-aging treatments.Multiple studies have shown the positive effects of dietary restriction (DR) on lifespan in various model organisms.However, DR is not widely implemented in older adults due to issues with patient compliance and the overall lack of understanding on the effects of DR initiated later in life.Thus, the present study tested whether late-life DR, specifically Every-Other-Day (EOD) fasting, attenuates agingrelated frailty using a modified and simplified frailty index in mice.Briefly, 20-month old male and female C57BL/6 mice (human equivalent of 65 years) that had been on a control chow diet ad libitum during adulthood were placed on EOD fasting or ad libitum feeding for 2.5 months.Their frailty index was identified using an indirect calorimeter, glucose tolerance test, novel object place recognition test, forelimb grip strength meter, and rotarod.We found that late-life EOD fasting decreased overall caloric intake in males but not in females.In addition, EOD fasting significantly improved metabolic, musculoskeletal, and cognitive endpoints in male mice, but enhanced only some of these in female mice.Furthermore, EOD fasting improved hydrogen sulfide (H2S) production capacity and its associated sulfhydration signaling in tissues, which positively correlated with improvements in frailty measures.We conclude that EOD fasting implemented late in life can have therapeutic potential in the clinic.We are currently investigating the necessity of H2S production for DR mediated benefits and longevity.

LATE-ONSET PHARMACOLOGICAL OR DIETARY INTERVENTIONS IMPROVE HEALTHSPAN AND LIFESPAN IN MALE AND FEMALE MICE
Sarah Mitchell, 1 Michael MacArthur, 1 Alice Kane, 2 Margaret Torrence, 3 Huseyin Mehmet, 4 James Vath, 5 Brendan Manning, 1 and James Mitchell, 6 1. Harvard T.H. Chan School of Public Health,Boston,Massachusetts,United States,2. Harvard Medical School Center for Animal Resources School of Public Health,Boston,Maine,United States,3. Harvard TH Chan School of Public Health,Boston,Massachusetts,United States,4. Zafgen,Inc.,Boston,Massachusetts,United States,5. Zafgen,Inc,Boston,Massachusetts,United States,6. ETH Zurich,Zurich,Massachusetts,Switzerland While late-onset dietary or pharmacological interventions can extend longevity in rodents, whether or not they can be used to reverse or forestall onset of aging-related symptoms (i.e.frailty) remains untested.Here, we employed three interventions to test this hypothesis.Male and female C57BL/6 mice were randomized to one of four groups: control, 15% calorie restriction (15CR), 0.1% Methionine Restriction (MR)or ZGN1062 (1.5mg/kg, drug in feed) starting at 21mo.Healthspan measurements (mouse clinical frailty index (FI), blood collection, and hematology) were performed every three months, and survival was assessed for all mice.At baseline there were no significant differences in frailty index.After 6mo FI increased consistent with reduced healthspan in control males (0.23□0.01 to 0.34□0.01A.U, p<0.0001) and females at this age (0.19□0.03 to 0.24□0.01,p<0.0001).Male 15CR, MR and ZGN1062 mice had significantly lower FI scores at 27mo age (15CR: 0.32□0.01,p=0.02;MR: 0.31□0.01,p=0.0009;ZGN1062: 0.30□0.01,p<0.0001).Female mice were less frail than males at 27mo, suggesting sexual dimorphism in the timing of frailty onset in mice.ZGN1062 significantly extended lifespan in males (HR=0.56,p=0.007) and females (HR=0.46,p=0.001).There was a sexual dimorphism in the ability of 15CR and MR to extend lifespan, and a trend towards increased lifespan in males (HR=0.69,p=0.057 and HR=0.71, p=0.09) but not in females.Histological analysis for cause-of-death is ongoing.Taken together these data suggest that a pharmacological intervention associated with weight loss, which may be a more practical therapeutic strategy towards mitigation of age-related healthspan decline than dietary restriction-based interventions.