Senolytics: The Path to Translation James L. Kirkland, M.D., Ph.D. Director, Mayo Clinic Kogod Center on Aging Claude D. Pepper Older Americans I ndependence Center Annual Meeting Arlington April 19, 2016
Aging is at the Nexus of Chronic Disease
Fundamental Aging Mechanisms Shared by Chronological Aging and Age-Related Chronic Diseases • I nflammation (chronic, low-grade, sterile) • Cellular Senescence • Macromolecular Dysfunction (DNA, protein aggregation, autophagy, AGE’s, lipotoxicity) • Stem Cell and Progenitor Dysfunction
Cellular Senescence Senescence Associated β - γ H2A.X Galactosidase 25 th passage human abdominal subcutaneous preadipocytes
Senescent Preadipocytes Accumulate in Human Adipose Tissue with Aging Young Old 4 younger (31 ± 5 y) and 4 older (71 ± 2 y) healthy male volunteers. * P < 0.05
Senescent Cells Accumulate in Human Fat in Obesity BMI = 23.53; 37 years old BMI = 32.8; 30 years old SA- β Gal quantification : 0.221 OD/ g SA- β Gal quantification : 2.18 OD/ g Abdominal subcutaneous fat Abdominal subcutaneous fat A. Bouloumie
Senescent Human Preadipocytes Develop a SASP Control (CON), irradiation-induced (I RA), and serial passage-induced (SP) senescent human preadipocytes. * P < 0.05 compared with non-senescent controls; n = 5 PNAS, 2015
Targeting Senescent Cells I n n Vivo vo AP20187 Sen enesc escen ence- ac act ivat at ed prom om ot ot er ATTA TTAC GFP FP FKBP Caspase 8- Flag p16 I nk nk4A or p5 p53 -related senescence-activated p1 promoter ATTAC Original idea and experimental strategy: Accelerate accumulation of senescent cells 2006-7; JK, TT Healthspan, not maximum lifespan, as key outcome Nature 479:232, 2011
TTAC;BubR1 H/ H/ H I NK-ATTA AP20187 nk promoter I nk ATTA TTAC I RES ES GFP FP -2617 FKBP Caspase 8- Flag • Cross with BubR bR1 H/ H/ H Further experimental • Transgenic as opposed to knock-in strategy: JvD after 6/ 2008 Nature 479:232, 2011
Activating ATTA TTAC Eliminates Senescent Cells Untreated Untreated Treated with AP20187 Treated with AP20187 every 3 days SA- β -galactosidase activity Experimental GFP in I AT procedures: TT, NKL, Activated ATTA TTAC kills senescent cells in v vit ro and in TP, RM, DJB, MW, BGC mice, so senescent cells are susceptible to the caspase after 6/ 2008 apoptosis execution cascade downstream of caspase 8 Nature 479:232, 2011
AP20187 Reduces Senescent Cell Burden in 18 Month Old I NK TTAC Mice NK-ATTA Wild-type INK-ATTAC +/- SAGB DAPI eLife Dec., 2015
Genetic Clearance of Senescent Cells Blunts Fat Loss, I ncreases Adipogenic Markers, and Reduces Activin A in Fat From 18-Month-Old I NK TTAC Mice NK-ATTA 25 120 120 120 WT WT INK-ATTAC INK-ATTAC 20 Body weight% SABG+ cells% Lean mass% Fat mass% 100 100 100 * * 15 10 80 80 80 5 60 60 60 0 Baseline 3 weeks Baseline 3 weeks Baseline 3 weeks 160 4 WT * * 140 Relative mRNA level INK-ATTAC Serum Activin A % 120 3 100 2 80 * * 60 eLife 1 * 40 * * Dec., 20 2015 0 0 PPAR γ C/EBP α aP2 LIPIN-1 AdipoQ Activin A IL6 p16 p21 Baseline 3 weeks
Hypothesis-Driven Senolytic Drug Development 1) Senescent cells can resist apoptotic stimuli, implying increased pro-survival and anti-apoptotic defenses 2) I n some respects, senescent cells are like cancer cells that do not divide 2013
Anti-Apoptotic Gene Networks Are Active in Senescent Cells Negative Regulation of Anti-Apoptosis Apoptosis GSEA Gene Sets Aging Cell March, 2015
siRNA’s Against Anti-Apoptotic Regulators Selectively Decrease Senescent Cell Viability Selected from 39 pro-survival transcripts targeted by siRNA, Radiation-induced senescent cells 17 of which affected Day 0 vs vs . 4 senescent cell viability Aging Cell March, 2015
Networks of Anti-Apoptotic Regulators Conferring Resistance to Apoptosis in Senescent Cells Pathways: Ephrins/ dependence receptors, PI 3K/ Akt, Bcl-2, p53/ p21, serpine, HI F-1 Aging Cell March, 2015
D Acts on Senescent Human Preadipocytes, Q on Senescent HUVECs Aging Cell March, 2015 ATP Lite; validated by crystal violet; abdominal subcutaneous preadipocytes from 4 healthy kidney transplant donors; for HUVEC’s N= 5 replicates. Day 0 vs vs . 3
D+ Q Target Senescent, But Not Proliferating or Differentiated Cells For Apoptosis Aging Cell March, 2015
Navitoclax, A Bcl-2 Family I nhibitor, I s Senolytic I n Some, But Not All Cell Types Aging Cell Dec., 2015
D+ Q Reduce p16 + , SA β -Gal + , and TAF + Cells I n n Vivo vo SA β -Gal p16 24 month old mouse inguinal fat; 5 days after single dose Also: mouse quadriceps muscle p16 mRNA and inguinal fat SA β -Gal + cells after leg radiation; mouse adipose tissue p16 mRNA and SA β - Gal + cells after high fat diet; TAF’s in mouse tissues with aging and after high fat diet; monkey skin p16 mRNA Aging Cell March, 2015
D+ Q Are Synergistic in Reducing p16 + Cholangiocytes From Old Mice p16 FI SH 24 month mice, single dose of D+ Q, livers analyzed after 5 days N= 8 animals/ group. * P < 0.05 N. LaRusso lab Aging Cell March, 2015
A Single Dose of Senolytics Alleviates Radiation-I nduced Gait Disturbance for 7 Months N= 6-9 mice/ group; * P < 0.05; * * P < 0.001 Aging Cell March, 2015
Senolytics Delay Frailty in Progeroid Mice N= 7-8 mice/ group; * P < 0.05; * * P < 0.01 L Niedernhofer Y I keno Aging Cell March, 2015
Senolytics Delay Neurologic Dysfunction in Progeroid Mice Vehicle L Niedernhofer Senolytics Aging Cell March, 2015
Senolytics Delay Osteoporosis in Progeroid Mice S Khosla Aging Cell March, 2015
Senolytics Enhance Cardiac and Vascular Function in Old Mice 24 month old mice J Miller Aging Cell March, 2015, Feb., 2016
Senolytics Reduce I ntimal Plaque Calcification in ApoE -/ - Atherosclerotic Mice Aging Cell, 2016
D+ Q Phenocopies Effects of Genetic Clearance on Glucose Tolerance in Diet-I nduced Obese Mice Genetic Targeting Senolytic A. Palmer (with J Campisi)
I ntermittent Treatment • Single or intermittent doses of senolytics appear to alleviate at least some age- or senescence-related conditions • This suggests that intermittent treatment may eventually be feasible in humans, perhaps given during periods of good health • I f so, this would reduce side effects • Senescent cells do not divide, so drug resistance as with antibiotics or anti-cancer drugs is unlikely
Emerging Evidence for Effects of Senescent Cells or Their Removal On: Diabetes/ Obesity Age-Related Lipodystrophy Cardiac Dysfunction Vascular Hyporeactivity Aortic Lipid Deposits Frailty/ Sarcopenia Response to Chemotherapy Response to Radiation Cancer Cognition/ Alzheimer’s/ Parkinson’s/ ALS Renal Dysfunction Osteoporosis/ Osteoarthritis COPD/ I diopathic Pulmonary Fibrosis/ Tobacco Primary Biliary Cirrhosis Progerias Cataracts/ Macular Degeneration/ Glaucoma HI V Prostatic Hypertrophy Skin Disorders
Clinical Scenarios for Testing Agents That Target Aging Processes ( e.g .g ., Cellular Senescence or the SASP) Simultaneous Alleviation of Co-Morbidities 3 or more of: diabetes, atherosclerosis, hypertension, MCI , sarcopenia, osteoarthritis, etc. Delay in 2 nd or later co-morbidities (TAME ) “Accelerated Aging” Conditions Childhood cancer survivors Bone marrow transplant survivors Progeroid syndromes Diabetes due to obesity HI V (dementia, frailty) Conditions with Localized Cellular Senescence Osteoarthritis Fracture non-union Atherosclerotic plaques Radiation I diopathic pulmonary fibrosis J. Clin. I nvest. 123:966-972, 2013 COPD/ tobacco J. Gerontol. 48:1-5, 2013 Glaucoma Exp. Gerontol. 2014
Clinical Scenarios for Testing Agents That Target Aging Processes ( e.g .g ., Cellular Senescence or the SASP) Otherwise Fatal Conditions I diopathic pulmonary fibrosis Primary biliary cirrhosis Cancers HI V dementia Resilience/ Clinical Stresses in Pre-frail Subjects Chemotherapy Radiation Elective surgery Bone marrow transplantation Rehabilitation after MI I mmunization Recovery after pneumonia Frailty Slow gait/ decreased strength/ sarcopenia Loss of independence in moderately frail subjects J. Clin. I nvest. 123:966-972, 2013 J. Gerontol. 48:1-5, 2013 Exp. Gerontol. 2014
Conclusions • Clearing senescent cells improves function in chronologically-aged mice • Senolytic agents may delay, prevent, or alleviate multiple senescence- and age-related conditions and enhance healthspan • I ntermittent treatment may be effective • Senolytics may be translated into clinical interventions in humans – if pre-clinical studies demonstrate effectiveness and low toxicity and if the right clinical study approaches can be devised
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