Senolytics: The Path to Translation James L. Kirkland, M.D., Ph.D. - - PowerPoint PPT Presentation

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- Galactosidase γH2A.X 25th passage human abdominal subcutaneous preadipocytes

Senescent Preadipocytes Accumulate in Human Adipose Tissue with Aging

4 younger (31 ± 5 y) and 4 older (71 ± 2 y) healthy male volunteers. * P < 0.05 Young Old

Senescent Cells Accumulate in Human Fat in Obesity

BMI = 32.8; 30 years old SA-βGal quantification : 2.18 OD/ g Abdominal subcutaneous fat BMI = 23.53; 37 years old SA-βGal quantification : 0.221 OD/ g 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

Sen enesc escen ence- ac act ivat at ed prom

• m ot
• t er

ATTA TTAC

AP20187

p1 p16 I nk

nk4A or p5

p53-related senescence-activated

promoter ATTAC Accelerate accumulation of senescent cells Healthspan, not maximum lifespan, as key outcome

Original idea and experimental strategy: 2006-7; JK, TT

GFP FP

Nature 479:232, 2011

FKBP Caspase 8- Flag

I NK-ATTA TTAC;BubR1 H/

H/ H

Nature 479:232, 2011

I nk nk promoter ATTA TTAC GFP FP

• Cross with BubR

bR1 H/

H/ H

• Transgenic as opposed to knock-in

Further experimental strategy: JvD after 6/ 2008

I RES ES

AP20187

• 2617

FKBP Caspase 8- Flag

Activating ATTA

TTAC Eliminates

Senescent Cells

SA-β-galactosidase activity

Untreated Treated with AP20187 every 3 days

Untreated Treated with AP20187 GFP in I AT

Nature 479:232, 2011

Activated ATTA

TTAC kills senescent cells in v vit ro and in

mice, so senescent cells are susceptible to the caspase apoptosis execution cascade downstream of caspase 8

Experimental procedures: TT, NKL, TP, RM, DJB, MW, BGC after 6/ 2008

Wild-type INK-ATTAC+/- SAGB DAPI

AP20187 Reduces Senescent Cell Burden in 18 Month Old I NK

NK-ATTA TTAC Mice

eLife Dec., 2015

60 80 100 120

Baseline 3 weeks Fat mass%

*

1 2 3 4

Relative mRNA level

WT INK-ATTAC

* * * * * *

20 40 60 80 100 120 140 160

Serum Activin A %

*

Baseline 3 weeks 60 80 100 120

Baseline 3 weeks Lean mass%

60 80 100 120

Baseline 3 weeks Body weight%

WT INK-ATTAC

5 10 15 20 25

SABG+ cells%

WT INK-ATTAC

*

PPARγ C/EBPα aP2 LIPIN-1 AdipoQ Activin A IL6 p16 p21

Genetic Clearance of Senescent Cells Blunts Fat Loss, I ncreases Adipogenic Markers, and Reduces Activin A in Fat From 18-Month-Old I NK

NK-ATTA TTAC Mice

eLife Dec., 2015

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

Anti-Apoptosis Negative Regulation of Apoptosis GSEA Gene Sets

Aging Cell March, 2015

siRNA’s Against Anti-Apoptotic Regulators Selectively Decrease Senescent Cell Viability

Radiation-induced senescent cells Day 0 vs

• vs. 4

Selected from 39 pro-survival transcripts targeted by siRNA, 17 of which affected senescent cell viability

Aging Cell March, 2015

Networks of Anti-Apoptotic Regulators Conferring Resistance to Apoptosis in Senescent Cells

Aging Cell March, 2015

Pathways: Ephrins/ dependence receptors, PI 3K/ Akt, Bcl-2, p53/ p21, serpine, HI F-1

D Acts on Senescent Human Preadipocytes, Q on Senescent HUVECs

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

Aging Cell March, 2015

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

• N. LaRusso lab

24 month mice, single dose of D+ Q, livers analyzed after 5 days N= 8 animals/ group. * P< 0.05

p16 FI SH

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

L Niedernhofer Y I keno N= 7-8 mice/ group; * P< 0.05; * * P< 0.01

Aging Cell March, 2015

Senolytics Delay Neurologic Dysfunction in Progeroid Mice

Vehicle Senolytics

L Niedernhofer

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 2nd 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 COPD/ tobacco Glaucoma

• J. Clin. I nvest. 123:966-972, 2013
• J. Gerontol. 48:1-5, 2013
• 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

Acknowledgements

T Tchkonia T Pirtskhalava N Giorgadze T Thomou Y Zhu M Xu M Stout R Kandhaya Pillai A Palmer M Mahlman I Karagiannides E Lubbers S Brozovich E Trushina J van Deursen D Baker N LeBrasseur T White S Khosla H Fuhrmann-Stroissnigg L Niedernhofer P Robbins J Armstrong K Ness T von Zglinicki J Miller J Wren J Campisi A Bouloumie C Pothoulakis M Jensen A Teferi C Conover A Bartke M Masternak Y I keno J Kopchick D Berryman E List M Adamo M Lenburg J Hubbard R Miller C Boney W Guo A Oberg A Terzic N Barzilai S Austad

AG13925, AG31736, AG41122, AG44396 The Connor Group, Noaber Foundation, Ted Nash Foundation, Ellison Foundation, Glenn Foundation, AFAR