Targeting the biology of aging with mTOR inhibitors

Abstract

Inhibition of the protein kinase mTOR with the FDA-approved therapeutic rapamycin promotes health and longevity in diverse model organisms. More recently, specific inhibition of mTORC1 to treat aging-related conditions has become the goal of basic and translational scientists, clinicians, and biotechnology companies. Here, we review the effects of rapamycin on the longevity and survival of both wild-type mice and mouse models of human diseases. We discuss recent clinical trials that have explored if existing mTOR inhibitors can safely prevent, delay or treat multiple diseases of aging. Finally, we discuss how new molecules may provide routes to the safer and more selective inhibition of mTORC1 in the decade ahead. We conclude by discussing what work remains to be done and the questions that will need to be addressed in order to make mTOR inhibitors part of the standard of care for diseases of aging.

Introduction

The mechanistic Target of Rapamycin (mTOR) is an evolutionarily conserved serine/threonine protein kinase found in diverse species including mice and humans. The mTOR kinase forms the catalytic core of two distinct protein complexes, mTOR Complex 1 (mTORC1) and mTORC2, each of which are composed of shared as well as unique protein subunits and phosphorylate different substrates. mTORC1 is regulated by a wide range of nutrients and hormonal cues, most notably the availability of amino acids, but also glucose, oxygen, and cholesterol ^1–3. mTORC1 activity drives a wide variety of anabolic processes through phosphorylation of substrates including ribosomal protein S6 kinase beta-1 (S6K1) and the eukaryotic translation initiation factor 4E-binding proteins (4E-BPs). mTORC1 activity also inhibits autophagy via phosphorylation of substrates including Unc-51 like autophagy activating kinase (ULK1) ⁴. In contrast to mTORC1, which is responsive to many different environmental cues, mTORC2 primarily acts as an effector of PI3K signaling by tuning the activity of substrates including AKT, serum/glucocorticoid regulated kinase (SGK), and protein kinase C alpha (PKCα).

Beginning twenty years ago, researchers discovered a role for mTORC1 signaling in the aging process. Studies in yeast, worms and flies found that genetic inhibition of mTORC1 or signaling pathways downstream of mTORC1, including S6K and translation initiation factors, extends lifespan ^5–11. mTORC1 signaling was also observed to be lower in long-lived Ames dwarf mice than in wild-type controls ¹². Studies in mice have likewise shown that genetic depletion of mTORC1 subunits, deletion of S6K1 or downstream substrates, or expression of dominant negative 4E-BP1 in specific tissues extends the lifespan and healthspan of mice ^13–16. Even partial inhibition of mTORC1 in genetic mouse models (e.g., S6K1^−/−, mTOR^+/− mLST8^+/−, mTOR^Δ/Δ, TSC1^tg) can extend lifespan and healthspan in mice ^13,17–19.

These results quite logically spurred significant interest over the possibility that a potent chemical inhibitor of mTORC1, rapamycin, could extend lifespan. This was indeed the case – and there are now numerous studies showing that rapamycin can extend the lifespan not only of model organisms including yeast, worms, and flies ^20–22, but also as we detail below, in both wild-type mice and in many disease models. In this review, we will discuss the results of these studies, as well as the possible mechanism by which reduced mTORC1 signaling via both dietary and pharmacological means may improve healthspan ^23–25.

Many of the side effects of rapamycin, which include immunosuppression, hyperlipidemia and hyperglycemia, have raised concerns about the feasibility of using rapamycin to promote healthy longevity in humans and slowed clinical evaluation of mTOR inhibitors for diseases of aging ²⁶. In this review, we will discuss data generated over the past decade suggesting that the beneficial effects of rapamycin on healthspan and lifespan are mediated by inhibition of mTORC1 whereas the negative effects of rapamycin on glucose and lipid metabolism are mediated by inhibition of mTORC2.

Importantly, these data predict that interventions that more selectively target mTORC1 – by limiting the amount of mTOR inhibitor used and the time of the exposure – will have reduced side effects as compared to chronic treatment. Here we discuss pre-clinical animal data and clinical human data exploring the use of intermittent and low dose mTOR inhibitor treatment regimens, the results of which suggest that rapalogs and other mTOR inhibitors may be able to be dosed in a way that is safer as well as geroprotective. Finally, we discuss future studies and the quest for mTORC1 selective molecules derived in part through recent discoveries about the molecular machinery through which mTORC1 senses nutrients and hormonal cues.

Extension of lifespan with the mTOR inhibitor rapamycin in mice

Rapamycin is a macrolide first discovered in soil from Easter Island almost fifty years ago ²⁷. Rapamycin is an acute inhibitor of mTORC1 but not mTORC2 ²⁸. The reasons for this difference in rapamycin sensitivity is structural; the mTOR interacting protein RICTOR, which is present in mTORC2 but not mTORC1, masks the rapamycin-interacting domain of mTOR^29–31. Although mTORC2 is not acutely inhibited by rapamycin, subsequent studies have shown that mTORC2 is inhibited in cell culture as well as in vivo in mice when exposed to high concentrations of rapamycin for a prolonged period of time, most likely due to the sequestration of free of mTOR by rapamycin so that it is unavailable for incorporation into mTORC2 ^17,32 Rapamycin and its analogs (rapalogs) inhibit T cell activation and are approved to prevent organ transplant rejection. In addition, rapalogs inhibit tumor cell growth and are approved for the treatment of a subset of tumors. Finally, rapalogs are approved for the treatment of specific genetic disorders that result in hyperactive mTOR signaling.

Multiple groups have studied the effect of rapamycin on the longevity of mice, starting with a landmark study by the National Institute on Aging Interventions Testing Program (NIA ITP) published in 2009 which demonstrated that rapamycin extended the lifespan of genetically heterogeneous mice ³³. Since that time, additional studies of rapamycin on mouse lifespan have been conducted not only by the NIA ITP, but by at least ten other groups across the globe (Table 1). A number of lessons can be drawn from this set of studies, primarily that rapamycin is an extraordinarily robust geroprotective intervention, extending lifespan in multiple wild-type mouse strains handled by multiple teams, and including inbred, outbred, and genetically heterogenous male and female mice. Every study observed an extension of lifespan in one or more dosing groups, with females typically benefiting more than males at equivalent doses of rapamycin. Importantly, unlike caloric restriction, another well validated longevity intervention, which is less efficacious when begun later in life ³⁴, rapamycin robustly extends lifespan when dosing was not initiated until late in life when the mice were 20 months of age (equivalent to about a 60 year old human) ^33,35. Dosing regimens for rapamycin are very flexible, and beneficial effects of rapamycin on longevity are observed even when rapamycin is dosed intermittently, when it is given for only a short period of time immediately postnatally, or when it is administered to older mice already past middle age ^36–43. Finally, rapamycin may be effective when combined with other geroprotectors, including metformin and acarbose ^44,45.

Table 1. The effect of rapamycin on the lifespan of wild-type mice.

The impact of rapamycin on median lifespan in mouse studies since 2009 where longevity or mortality rate was determined. Sex is listed separately for males and females where sex-specific data exists. The rapamycin dose listed for dietary administration indicates the drug concentration in the ad libitum fed diet; the dose listed for administration in water, or administered intraperitoneally (IP) or subcutaneous (SC) indicates the dose in mg per kg of body weight. Control indicates median lifespan of control group in days;

Strain	Sex	Starting age	Rapa dose	Route	Control (days)	Δ lifespan (%)	Reference
Wild-type mice – rapamycin alone via diet
UM-HET3	Male	20 months	14 ppm	Diet	--	9	Harrison et al. 2009 33
UM-HET3	Female	20 months	14 ppm	Diet	881–895	14
UM-HET3	Female	9 months	14 ppm	Diet	843–891	18	Miller et al. 2011 36
UM-HET3	Male	9 months	14 ppm	Diet	780–851	10
129/Sv	Female	2 months	1.5 mg/kg	SC 3x/week 2 weeks per 4	759	10	Anisimov et al. 2011 41
C57BL/6J.Rj	Male	4, 13, 20 months	14 ppm	Diet	~900	~10a	Neff et al. 2013 87
C57BL/6J.Nia	Male	19 months	14 ppm	Diet	954	−3.5NS	Zhang et al. 2014 194
C57BL/6J.Nia	Female	19 months	14 ppm	Diet	874	5
UM-HET3	Male	9 months	4.7 ppm	Diet	807	3NS	Miller et al. 2014 195
UM-HET3	Male	9 months	14 ppm	Diet	807	13
UM-HET3	Male	9 months	42 ppm	Diet	807	23
UM-HET3	Female	9 months	4.7 ppm	Diet	896	16
UM-HET3	Female	9 months	14 ppm	Diet	896	21
UM-HET3	Female	9 months	14 ppm	Diet	896	26
C57BL/6J.Nia	Male	4 months	14 ppm	Diet	806*	11*	Fok et al. 2014 196
C57BL/6J.Nia	Female	4 months	14 ppm	Diet	826*	16*
C57BL/6J.Nia	Male	20–21 months	126 ppm for 90 d	Diet	914	14	Bitto et al. 2016 38
C57BL/6J.Nia	Female	20–21 months	126 ppm for 90 d	Diet	960	9
UM-HET3	Male	20 months	42 ppm	Diet	772	11	Strong et al. 2020 193
UM-HET3	Male	20 months	42 ppm for 3 mo	Diet	772	11
UM-HET3	Male	20 months	42 ppm	Diet every other mo	772	9
UM-HET3	Female	20 months	42 ppm	Diet	905	15
UM-HET3	Female	20 months	42 ppm for 3 mo	Diet	905	4NS
UM-HET3	Female	20 months	42 ppm	Diet every other mo	905	8
C57BL/6 Terc+/+	Male	3 months	42 ppm	Diet	509	43	Ferrara-Romeo et al. 2020 197
C57BL/6 Terc+/+	Female	3 months	42 ppm	Diet	711	23
UM-HET3	Male	Birth (via dam)	42 ppm until 45 days	Diet	783	11.9	Shindyapina et al. 2022 42
UM-HET3	Female	Birth (via dam)	42 ppm until 45 days	Diet	822	9.1NS
Wild-type mice – rapamycin alone via I.P.
C57BL/6J.Nia	MF	22–24 months	4 mg/kg	IP every other day	~795	>14a	Chen et al. 2009 39
C57BL/6J.Nia	Female	20 months	2 mg/kg	IP 1x/5 days	897	7	Arriola Apelo et al. 2016 37
C57BL/6J.Nia	Male	20–21 months	8 mg/kg for 90 d	IP 1x/d	925	14	Bitto et al. 2016 38
C57BL/6J.Nia	Female	20–21 months	8 mg/kg for 90 d	IP 1x/d	847	0NS
Mixed	Male	600–700 d	4 mg/kg	IP every other day	911	13	Fang et al. 2018 198
Mixed	Female	600–700 d	4 mg/kg	IP every other day	896	22
CD1	Male	Day 4	10 mg/kg until 40 days	IP 1x/d	655	8.9	Aiello et al. 2022 43
CD1	Male	Day 30	10 mg/kg until 60 days	IP 1x/d	655	3.5NS
CD1	Female	Day 4	10 mg/kg until 40 days	IP 1x/d	714	8.4
CD1	Female	Day 30	10 mg/kg until 60 days	IP 1x/d	714	3.8NS
Wild-type mice – rapamycin via diet as part of a combination
UM-HET3	Male and Female	9 months	14 ppm with metformin	Diet	It’s complicated – see manuscript for details		Strong et al. 2016 44
UM-HET3	Male and Female	9 or 16 months	14 ppm with acarbose	Diet	It’s complicated – see manuscript for details		Strong et al. 2022 45

^Δlifespan is the percent change in median lifespan

^*(indicates that mean is reported instead). MF indicates that the lifespan results were not broken down by sex or that sex was not reported

^a: Lifespan study % increase was not determined

^NS: Not Statistically Significant. Control lifespan and percentage change are estimated when precise information is not available from the authors or is not listed in the referenced study. Table is adapted from Arriola Apelo et al., 2016, JGBS ¹⁹⁹ and used with permission.

Rapamycin has also been tested in a wide number of disease models, including multiple different cancers, mitochondrial disease, and progeria (Table 2). Here, the beneficial effects of rapamycin have been more varied than in wild-type mice. As might be expected, rapamycin has shown significant benefits in mouse models of mTORopathies ⁴⁶, diseases resulting from genetic activation of mTOR, including tuberous sclerosis complex and some epilepsies. Rapamycin also has substantial positive effects on the survival of mouse cancer models. Finally, rapamycin extends the lifespan of several progeroid mouse models quite effectively, suggesting that progeria mouse models, while not perfect models of aging, may have utility for rapidly assessing the potential benefits of geroprotectors.

Table 2. The effect of rapamycin on the survival of mouse disease models.

The impact of rapamycin on median lifespan in mouse studies since 2009 where longevity or mortality rate was determined. Sex is listed separately for males and females where sex-specific data exists. The rapamycin dose listed for dietary administration indicates the drug concentration in the ad libitum fed diet; the dose listed for administration in water, or administered intraperitoneally (IP) or subcutaneous (SC) indicates the dose in mg per kg of body weight. Control indicates median lifespan of control group in days;

Strain	Sex	Starting age	Rapa dose	Route	Control (days)	Δ lifespan (%)	Reference
Disease models
Autosomal dominant polycystic kidney disease
Vil-Cre;Pkd2 f3/f3	Female	10 days	50 mg/kg until 60 days	IP 1x/day	~137	~53	Li et al. 2017 200
Vil-Cre;Pkd2 f3/f3	Male	10 days	50 mg/kg until 60 days	IP 1x/day	~137	~33
Vil-Cre;Pkd2 f3/f3	MF	10 days	50 mg/kg until 110 days	IP 1x/day	~137	~75
Vil-Cre;Pkd2 f3/f3	MF	10 days	50 mg/kg until 60 days	IP 1x/day	~137	~53
Vil-Cre;Pkd2 f3/f3	MF	60 days	50 mg/kg until 110 days	IP 1x/day	~137	~53
Amyotrophic lateral sclerosis
SOD1 G93A	MF	64 days	2 mg/kg	IP 1x/d	126	−15	Zhang et al. 2011 201
SOD1 H46R/HR8Q	MF	1.5 months	14 ppm	Diet	232	NS	Bhattacharya et al. 2012 202
Cancer
Pten −/−	MF	1 month	10 mg/kg (Everolimus)	Oral	66*	>292*a	Hernando et al. 2007 203
K14Cre Pten F/F	MF	5 days	1 mg/kg	IP every other day	94	240	Squarize et al. 2008 204
ApcMin/+ C57BL/6	MF	5 weeks	40 mg/kg -not encapsulated, diet changed out frequently	Diet	156*	77	Koehl et al. 2009 205
129 Atm−/−	MF	2–3 months	15 mg/kg	IP 1x/day	~100	~32	Alexander et al. 2010 206
FVB/N HER-2/neu	Female	2 months	1.5 mg/kg	SC 3x/week 2 weeks per 4	288	13.6	Anisimov et al. 2010 40
ALB/c nu/nu + A549	Male	7 weeks	10 mg/kg (Temsirolimus)	IV 1x/5week for 5 weeks	53.5	36	Ohara et al. 2010 207
p53 −/−	Male	2 months	0.5 mg/kg	Oral 1x/day 5 d on/9 d off	161	35	Comas et al. 2012 208
Balb/c + 4T1	Female	6 weeks	5 mg/kg	IP every other day	16d	31d	Hussein et al. 2012 209
p53 +/−	Male	<5 months	1.5 mg/kg	Water	373*	28*	Komarova et al. 2012 210
p53 +/−	Male	>5 months	1.5 mg/kg	Water	373*	10*
NOD.Cg-Prkdcscid ALL (JAK2m/CRLF2R)	MF	sufficient disease burden	5 m/kg	Oral 5x/week	23	174	Maude et al. 2012 211
NOD.Cg-Prkdcscid ALL (JAK1m)	MF	sufficient disease burden	5 m/kg	Oral 5x/week	58	57
Rb1 +/−	Male	2 months	14 ppm	Diet	369	13.8	Livi et al. 2013 212
Rb1 +/−	Female	2 months	14 ppm	Diet	378	8.9
Apc Min/+	Female	8 weeks	14 ppm	Diet	174	284	Hasty et al. 2014 213
Apc Min/+	Female	8 weeks	42 ppm	Diet	174	439
Pdx1-Cre; KrasG12D/+; Ptenflox/+	MF	clinically detectable pancreatic tumors	10 mg/kg	IP 1x/day	10	460	Morran et al. 2014 214
Pdx1-Cre; KrasG12D/+; Trp53R172H/+	MF	clinically detectable pancreatic tumors	10 mg/kg	IP 1x/day	2	250
HER-2/neu	Female	2, 4, or 5 months	0.45 mg/kg	SC 3x/week 2 weeks per 4	282, 278, 289	5.7NS, 6.1, 5.5	Popovich et al. 2014 215
p53 +/+	MF	2 months	14 ppm	Diet	681	17.8	Christy et al. 2015 216
p53 +/−	MF	2 months	14 ppm	Diet	520	11.9NS
p53 −/−	MF	2 months	14 ppm	Diet	199	−3NS
C3H/HeJJcl + LM8	M	9 weeks	50 mg/kg	IV every other day	~30	~28	Ando et al. 2020 217
p53 5KR/5KR	MF	weaning	42 ppm	Diet	258	35	Kon et al. 2020 218
p53 −/−	MF	weaning	42 ppm	Diet	164	55
Apc Min/+	Male	4 weeks	42 ppm	Diet	222	348	Parihar et al. 2020 219
Apc Min/+	Female	4 weeks	42 ppm	Diet	260	156
ApcMin/+ DSS	Male	4 weeks	42 ppm	Diet	112	171	Parihar et al. 2021 220
ApcMin/+ DSS	Female	4 weeks	42 ppm	Diet	113	220
Pten +/−	Male	6 weeks	14 ppm	Diet	394	65	Tibarewal et al. 2022 221
Pten +/−	Female	6 weeks	14 ppm	Diet	239	54
Clock
Bmal1 −/−	MF	16 weeks	0.5 mg/kg	Water	~240	47	Khapre et al. 2014 222
Diabetes
C57BL/6NCr HFD	Male	12 months	1.5 mg/kg	IP 1x/week	684	b	Leontieva et al. 2014 223
C57BLKS/J leprdb/db	Male	4 months	14 ppm	Diet	349	−16	Sataranatarajan et al. 2015 224
C57BLKS/J leprdb/db	Female	4 months	14 ppm	Diet	487	−18
Epilepsy
Depdc5cc+	MF	3 weeks	6 mg/kg	IP 3x/week	126	>62c	Yuskaitis et al., 2019 225
Depdc5-Emx1-Cre	MF	13–15 days	3 mg/kg	IP 5x/week	22	>70c	Klofas et al., 2020 226
Growth hormone
ghr −/−	Male	600–700 d	4 mg/kg	IP every other day	558	−12.5	Fang et al. 2018 198
ghr −/−	Female	600–700 d	4 mg/kg	IP every other day	556	−28
Immune deficiency
Rag2 −/−	MF	3 months	14 ppm	Diet	310	121	Hurez et al. 2015 227
IFN-γ−/−	MF	5 months	14 ppm	Diet	398	34
CByB6F1 BM	Female	8–9 weeks	2 mg/kg d0–9 post LN	IP for 5–13 days	16.5 d	36 d	Feng et al. 2017 228
CByB6F1 BM	Female	8–9 weeks	2 mg/kg D0–12 and D3–12 post LN	IP for 5–13 days	16.5 d	>506c
Inflammation
nfκb1 −/−	M	4–5 months	14 ppm	Diet	598	−6NS	Correia-Melo et al. 2019 229
Lupus
MRL/l	Female	8 weeks	12.5 mg/kg	Oral 3x/week	90	72	Warner et al. 1994 230
MRL/l	Female	8 weeks	25 mg/kg	Oral 3x/week	90	163
Marfan syndrome
mgR/mgR	Female	7–8 weeks	2 mg/kg	IP 1x/d for 2 weeks	199	36	Zaradzki et al. 2022 231
mgR/mgR	Male	7–8 weeks	2 mg/kg	IP 1x/d for 2 weeks	101	45
Mitochondrial disease
Ndufs4 −/−	Female	weaning	8 mg/kg	IP every other day	~50	38	Johnson et al. 2013 132
Ndufs4 −/−	Male	weaning	8 mg/kg	IP every other day	~50	25
Ndufs4 −/−	Female	10 days	8 mg/kg	IP 1x/d	~50	~128
Ndufs4 −/−	Male	10 days	8 mg/kg	IP 1x/d	~50	~128
Ndufs4 −/−	MF	weaning	8 mg/kg	IP 1x/day	52	119	Johnson et al. 2015 232
Ndufs4 −/−	MF	weaning	42 ppm	Diet	52	29NS
Ndufs4 −/−	MF	weaning	378 ppm	Diet	52	92
Tk2 KI/KI	MF	To dams from conception	0.8 mg/kg prior to birth, 4.0 mg/kg post-birth	Water to dam	~15	~60	Siegmund et al. 2017 233
Prion
Tg(PrP-A116V)	MF	6 weeks	10 mg/kg	IP 3x/week	173*	1NS	Cortes et al 2012 234
Tg(PrP-A116V)	MF	6 weeks	20 mg/kg	IP 3x/week	173*	9
Progeria
Lmna −/−	MF	1 month	14 ppm	Diet	46	35	Ramos et al. 2012 235
Lmna −/−	MF	1 month	8 mg/kg	IP every other day	55	56
Lmna −/−	Female	4 weeks	8 mg/kg	IP every other day	59	70	Liao et al. 2016 236
Lmna −/−	Male	4 weeks	8 mg/kg	IP every other day	52	86
Lmna H222P/H222P	MF	2 months	14 ppm	Diet	309	−2NS
G2 Terc−/−	Male	3 months	42 ppm	Diet	274	−19	Ferrara-Romeo et al. 2020 197
G2 Terc−/−	Female	3 months	42 ppm	Diet	274	−12.5NS
Ercc1 Δ/−	Female	4 weeks	14 ppm	Diet	148	−7.4NS	Birkisdottir et al. 2021 237
Ercc1 Δ/−	Female	8 weeks	14 ppm	Diet	141	−6.4NS
Ercc1 Δ/−	Female	8 weeks	4.7 ppm	Diet	180	−2.2NS
Ercc1 Δ/−	Female	8 weeks	42 ppm	Diet	180	−1.6NS
Ercc1 Δ/−	Male	8 weeks	4.7 ppm	Diet	160	5NS
Ercc1 Δ/−	Male	8 weeks	42 ppm	Diet	160	−1.7NS
Tuberous sclerosis complex
GFAP-Cre Tsc1 L/L	MF	2 weeks	3 mg/kg	IP 5x/week	63	e	Zeng et al., 2008 238
GFAP-Cre Tsc1 L/L	MF	6 weeks	3 mg/kg	IP 5x/week	~74	f
CD-1 Nude with Tsc2−/−, Trp53−/− MEF tumors	MF	Tumor ≥ 40–50 mm3	8 mg/kg	IP 3x/week	24.5	88	Lee et al. 2009 239
LSL-KrasG12D Tsc1L/L	MF	13–16 weeks	6 mg/kg	IP every other day	20d	551d	Liang et al. 2010 240
LSL-KrasG12D Tsc1L/+	MF	13–16 weeks	6 mg/kg	IP every other day	48d	138d
CD-1 Nude with Tsc2−/−, Trp53−/− MEF tumors	MF	Tumor ≥ 100 mm3	8 mg/kg	IP 3x/week	31	173	Woodrum et al. 2010 241
Tsc1 L/L ; SM22cre +/−	MF	2 weeks	4 mg/kg	IP 5x/week	24	g	Malhowski et al. 2011 242
Tsc1 L/L ; SM22cre +/−	MF	2 weeks	4 mg/kg	IP 3x/week	24	g
Tsc1 L/L ; SM22cre +/−	MF	3 weeks	4 mg/kg	3x/week	24	g
Six2-Cre tg/+ ; Tsc1 L/L	MF	To dams, E12.5, E14.5. E16.5	0.2 mg/kg	3x	2	500	Nechama et al. 2020 243
Tsc2L/L, Nphs2-Cre+/− ICR	MF	4 weeks	2 mg/kg until 11 weeks	IP every other day	~53	~150	Iwata et al. 2020 244

^Δlifespan is the percent change in median lifespan

^*indicates that mean is reported instead). MF indicates that the lifespan results were not broken down by sex or that sex was not reported

^a: Lifespan study % increase was not determined

^b: 100% of rapamycin-treated mice survived to 2 years of age vs. 40% of control mice

^c: study not continued long enough to determine median lifespan

^d: survival following tumor induction, tumor implantation, or lymph node (LN) cell infusion.

^e: 91% of rapamycin-treated mice survived to 6 months of age vs. 0% of control mice.

^f100% of rapamycin-treated mice survived to 18 months of age vs. 0% of control mice.

^g: there were no deaths in the treatment cohort at 8 weeks of age vs. 100% of untreated mice.

^NS: Not Statistically Significant. ALL indicates an acute lymphoblastic leukemia xenograft model. BM indicates bone marrow failure model. DSS indicates that the mice were treated with dextran sodium sulfate to induce colon cancer. LM8 indicates a murine subcutaneous allograft tumor model. Vil-Cre refers to the expression of Cre under the control of the Vil1 promoter; K14-Cre refers to the expression of Cre under the control of the KRT14 promoter; LSL-KrasG12D refers to a the expression of a Kras mutant allele the expression of which is blocked by the a Lox-Stop-Lox cassette; SM22cre refers to Cre under the control of the Sm22α promoter; mgR refers to a hypomorphic allele of Fbn1; MEF refers to Mouse Embryonic Fibroblast; ICR is an abbreviation for Institute of Cancer Research; and Prp refers to prion protein. Control lifespan and percentage change are estimated when precise information is not available from the authors or is not listed in the referenced study. Table is adapted from Arriola Apelo et al., 2016, JGBS ¹⁹⁹ and used with permission.

While by far the most work on rapamycin and aging has been done in mice, there is significant interest in exploring the use of rapamycin in primates. A total of 66 middle-aged marmosets (Callithrix jacchus) have been fed either rapamycin or vehicle containing diets, with the lifespan and healthspan of the animals followed longitudinally. Both sexes are being examined in this study, which is still underway. A limitation of these studies is both the relatively small number of animals studied – less than 20 per sex and treatment – and the range of ages and the genetic heterogeneity of the population. Initial studies suggested that rapamycin, dosed at ~1 mg/kg of body weight via the diet, was well tolerated, with only minor effects of rapamycin on hematological parameters, and no statistically significant changes in blood glucose, cholesterol, or triglyceride levels⁴⁷. In the subset of 6 animals treated with rapamycin or vehicle for nine months, 2 rapamycin-treated animals showed an increase in fasting blood glucose, and 2 rapamycin-treated animals shown an increase in triglycerides suggesting the possibility of some inhibition of TORC2 at this dose level ⁴⁷. However, a definitive examination of the metabolic impact of rapamycin in marmosets will require studies in a larger number of animals, conducted over a longer period of time.

The second major study of rapamycin done outside of the rodent context is examining the effect of rapamycin on the aging of dogs. As companion animals, dogs share the human environment, receive regular medical care similar to humans, and develop many of the same age-related diseases that humans do. Further, many dog owners have shown interest in enrolling their dogs in studies to promote healthy aging. The short lifespan of dogs, particularly of large dog breeds, allows for studies of shorter duration compared to humans. A small pilot study found that treatment with 0.05 mg/kg or 0.1 mg/kg rapamycin three times per week did not cause statistically significant side effects but did improve measurements of cardiac function (fractional shortening and diastolic function) ⁴⁸. A larger study in which middle-aged, large-breed dogs will be treated with rapamycin or vehicle for one year will complete enrollment during 2023 ⁴⁹.

Mechanisms underlying the effects of rapamycin on healthspan and lifespan

There are many possible mechanisms underlying the geroprotective effects of rapamycin. In this section, we will discuss the effects of mTORC1 inhibition on both specific disease processes and the molecular mechanisms which may contribute to the beneficial effects of rapamycin on lifespan and healthspan.

Protein translation

mTORC1, via S6K1 and 4E-BPs, plays a central role in the regulation of translation, and defects in translation have long been theorized to contribute to aging. The error catastrophe theory suggests errors in protein translations could leads to increasingly inaccurate protein synthesis and functional decline ⁵⁰. Conceptually, slowing protein translation by treatment with rapamycin might allow mRNA to be translated into protein with higher fidelity, or to fold more accurately. In agreement with such a model, experiments in model organisms have shown that deletion or knockdown of ribosomal subunits, S6K1, or translation initiation factors results in increased lifespan ^6,10.

However, modern measurement techniques have not found evidence that protein translation errors increase with age ⁵¹. Further, neither mTOR inhibition by rapamycin or deletion of S6K1 significantly slows protein translation in mice or cells ^52–54. Studies in C. elegans have shown that the lifespan of worms lacking eukaryotic translation initiation factor 4E (eIF4E), which are long-lived and have a global reduction in protein synthesis, can be still further increased by knockdown of the gene encoding TOR ¹⁰. These results strongly suggest that rapamycin does not regulate longevity solely by downregulating protein translation.

There may be a more subtle effect of rapamycin on protein translation – namely, that rapamycin may alter the translation of specific mRNAs. Both rapamycin and complete mTOR inhibition preferentially inhibit translation of mRNAs with 5′ terminal oligopyrimidine (TOP) motifs, suggesting a potential role for these genes in longevity ^55,56. In yeast, the ability of deletion of genes encoding ribosomal subunits to extend lifespan is partially dependent upon inducing translation of the mRNA encoding the transcription factor GCN4 (General Control Nondepressible 4) ⁵⁷. Expression of the Gcn4 protein is limited by multiple upstream open reading frames (ORFs) that normally sequester ribosomes that bind to the mRNA. Under conditions of large ribosomal subunit abundance, the upstream ORFs are more frequently bypassed to initiate translation of Gcn4. It is not clear if this system is conserved in mammals because in mammals mTORC1 inhibition decreases translation of the mammalian homologue of Gcn4, ATF4 ^58,59. ATF4 instead may act as a break on mTORC1 in response to mitochondrial distress, with ATF4 inhibiting mTORC1 activity via upregulation of the mTORC1 inhibitors Sestrin2 and Redd1 ⁶⁰. Despite these differences, it is possible that changes in translation of specific mRNAs may contribute to the beneficial effects of rapamycin on healthspan and lifespan.

One potential example of this is that mice expressing a constitutively active (dephosphorylated) form of 4E-BP1 in skeletal muscle are protected from age- and diet-induced declines in insulin sensitivity and metabolic rate ¹⁵. These effects may be mediated non-cell-autonomously via increased production of Fibroblast growth factor 21 (FGF21) by skeletal muscle, activating brown adipose tissue. Interestingly, whole body overexpression of 4E-BP1 also has positive effects on healthspan, protecting male mice from diet-induced obesity ¹⁶. These mice also have increased levels of FGF21, in this case due to an upregulation of hepatic Fgf21 expression. Notably, expression of Fgf21 is regulated in part by ATF4 ^61,62. A recently described downstream effector of mTORC1 and S6K1 is glutamyl-prolyl tRNA synthetase (EPRS) which is phosphorylated by S6K1¹⁴. When phosphorylated, EPRS functions to inhibit the translation of select mRNAs by forming an interferon γ-activated inhibitor of translation (GAIT) complex which selectively inhibits mRNAs containing GAIT elements ⁶³. Mice expressing EPRS S999A, which is non phosphorylatable, have reduced body weight and adipose mass, and increased lifespan, similar to mice lacking S6K1 ¹⁴. Other aminoacyl-tRNA synthetases may similarly have non-canonical functions ⁶⁴ that may play a part in the response to rapamycin.

Autophagy

Autophagy is a process by which cells recycle their proteins and organelles, which not only allows cells to survive nutrient-limited conditions, but which is also a central mechanism by which damaged protein and subcellular organelles are removed. While autophagy thus seems to be very important from this description alone, studies in yeast, worms, and flies have shown that inactivation of autophagy shortens lifespan, while promotion of autophagy extends lifespan ^65–67. Importantly, autophagy has also been shown to be required for the extension of lifespan by reduced mTOR signaling in both yeast and worms ^65,68. In mammals, autophagy is reportedly upregulated in CR mice and in the cells of long-lived Snell dwarf mice, and is required for some of the beneficial effects of a CR diet on the heart, kidney, and liver ^69–72.

When nutrients are abundant, mTORC1 activity acts as a brake on autophagy by phosphorylating the autophagy-initiating kinase ULK1 and ATG13, as well as other components downstream of autophagy initiation and reviewed in detail elsewhere ^73–75. Conversely, when nutrients are low, autophagy is active as a result of AMP-activated protein kinase (AMPK) activation and reduced mTORC1 activity. Autophagy is normally impaired with age in multiple types of mouse cells, and genetic activation of autophagy in the aged mouse liver rejuvenates the liver histologically and improves function ^76–78. While rapamycin itself is not a strong inducer of autophagy compared to mTOR kinase inhibitors ⁵³, the effects of rapamycin on the pathology of mouse models of Alzheimer’s disease has been attributed in part to increased autophagy ^79,80.

Improvement in immune function

Premature aging of the immune system has been shown to drive aging of multiple other organ systems in mice ⁸¹. These findings suggest that therapies that improve immune aging may have more systemic healthspan and lifespan benefits. mTOR inhibition has been shown to improve the function of the aging immune system in both mice and humans. Specifically, a short 6-week course of rapamycin has been shown to rejuvenate the function of hematopoietic stem cells, increase production of naïve lymphocytes and improve the response to influenza vaccination in old mice ³⁹. Of interest, rapamycin treatment extended lifespan in this study even though it was only administered for 6 weeks when mice were already old (26 months). In elderly humans, 6 weeks of mTOR inhibitor treatment (a rapalog alone or in combination with a catalytic site mTOR inhibitor) also improved the response to influenza vaccination and was associated with a decrease in the percentage of exhausted PD1+ T cells in peripheral blood ^82,83. In addition, mTOR inhibition has been shown to upregulate antiviral immunity in older adults ^83,84. These clinical studies will be discussed in more detail below.

However, it is worth noting that rapamycin is FDA-approved as an immunosuppressant, and infections are common, particularly in humans taking high doses of rapalogs for a long period of time. In mice, rapamycin has been shown to be immunomodulatory, improving CD8⁺ T cell immunological memory while at the same time impairing defenses against acute viral and bacterial infections ⁸⁵. A recent meta-analysis suggests that mice treated with rapamycin have increased survival following an acute pathogenic challenge ⁸⁶. Thus, there may be tradeoffs, with rapalogs improving some aspects of immune function while impairing others.

Cancer

Cancer is the most common cause of death for laboratory mice, and rapamycin and its derivatives (rapalogs) inhibit the proliferation of cancer cell lines. Rapalogs are approved for use in certain oncology indications and, as shown in Table 2, rapamycin is very effective at extending the lifespan of mouse models of cancer. A study by Neff and colleagues found that rapamycin significantly reduced the proportion of 16 month old mice with cancer and/or precancerous lesions ⁸⁷, and suggested that this indicated that the effects of rapamycin on lifespan was driven by its anti-cancer effects. However, the NIA ITP has shown that the overall prevalence of cancer at death, as well as the spectrum of cancers observed, was very similar between rapamycin-treated mice and controls ³⁶. This effect on cancerous and precancerous lesions not observed in animals sacrificed at later time points, but the interpretation of these results was confounded by as cancer was not assessed in animals that died of natural causes. While cancer prevention clearly plays a role in the benefits of rapamycin in mice, cancer is itself an age-related disease, and thus prevention of cancer is an expected consequence of geroprotective therapies.

Cancer cells often express pluripotency markers, and rapamycin reduces cell size and proliferation and enhances the differentiation of mouse and human embryonic stem cells ^88–90. Rapamycin may be particularly beneficial against cancer stem cells, and has been shown to deplete leukemia-initiating cells and inhibit both self-renewal and differentiation of stem cells derived from infantile hemangioma ^91,92. Rapamycin inhibits cell proliferation, epithelial-mesenchymal transition and cancer stem cell characteristics of lung cancer stem cells and colorectal cancer stem cells ^93,94

Stem cells

In contrast to its actions of cancer stems cells, rapamycin has been shown to have beneficial effects on self-renewal and function in normal stem cells, which may contribute to the benefits of rapamycin on tissue function. Several studies have been conducted on hematopoietic stem cells (HSCs); aged mice have elevated mTORC1 activity in their HSCs, as well as functional deficits similar to those observed in a mouse model of hyperactive mTORC1 activity ³⁹. Treatment of aged mice with rapamycin restored the functional capacity of their HSCs, and boosted the immune response to influenza virus. In a separate study, rapamycin was shown to restore self-renewal capacity to a subpopulation of mouse HSCs with spontaneously high oxidative stress and reduced functional capacity ⁹⁵. Rapamycin also increases the self-renewal capacity of mouse intestinal stem cells, via a non-cell autonomous mechanism mediated by inhibition of mTORC1 in the adjacent Paneth cells ⁹⁶.

Reduction of hyperactive mTOR in aging tissues

One reason mTOR inhibition may have health benefits in older organisms is because mTOR activity may become inappropriately high with age. Higher mTORC1/S6K activity in muscle of older mice, rats and humans is associated with sarcopenia ^97–101, and in brain is associated with Alzheimer’s disease ^101–105. Altered mTORC1 signaling with age has also been reported in other tissues of mice, with most studies reporting increased mTORC1 activity with age ^{39,97,106–108}. The ability of rapamycin to promote longevity is consistent with the idea that mTOR activity is an example of antagonistic pleiotropy, with high mTOR signaling being beneficial for development and reproduction, but being harmful during a post-reproductive old age ¹⁰⁹. Under such a model, the benefits of mTOR inhibition may arise less from specific benefits on processes such as translation, and more from avoiding negative effects of hyperactive mTOR on processes such as cellular senescence. Indeed, rapamycin has been shown to inhibit the accumulation of senescent cells in mice as well as suppress the senescence associated secretory phenotype (SASP) ¹¹⁰.

Inhibition of mTORC2

In sharp contrast to mTORC1, inhibition of mTORC2 has mostly negative effects on lifespan. In worms, mTORC2 inhibition has most often been associated with reduced lifespan, while in flies increasing mTORC2 activity through overexpression of Rictor extends lifespan ^21,111–113. Whole body deficiency of mTORC2 signaling, or tissue-specific inhibition of mTORC2 signaling in the brain, liver, or adipose tissue, reduces lifespan in C57BL/6J mice ^114–117. Conversely, the lifespan of male mice is extended by acarbose and 17-α estradiol, and these compounds increased hepatic mTORC2 activity ¹¹⁸. mTORC2 activity is also elevated in long-lived Snell dwarf mice and Ghr^−/− mice ¹¹⁹.

Inhibition of mTORC2 also results in negative effects on metabolism and immunity. Genetic inhibition of mTORC2 in one or more tissues of a mouse can result in frailty, hyperphagia, insulin resistance, hyperlipidemia, hypercholesterolemia, hyperglycemia, kyphosis, and/or obesity depending upon the specific tissues in which mTORC2 is inhibited ^{114–117, 68}. Specific inhibition of mTORC1 in mice using a mTORC1-speicific inhibitor did not result in hyperglycemia, impaired glucose tolerance, hyperlipidemia or hypercholesterolemia, again demonstrating that these negative effects are mediated at least in part by inhibition of mTORC2 ¹²⁰. Finally, mTORC2 has been shown in a number of studies to play a key role in immunity and wound repair ^121–127, and in accordance with this important role for mTORC2, compounds that selectively inhibit mTORC1 have a reduced effect on the immune system compared to rapamycin ¹²⁰. Thus, lower or intermittent doses of rapamycin, or treatment with rapalogs that more specifically target mTORC1, are predicted to reduce mTORC2-associated side effects such as hyperlipidemia and hyperglycemia without impairing the lifespan-extending benefits of mTORC1 inhibition.

This is not to say that there may not be benefits to inhibiting mTORC2 in specific settings. Inhibition of mTORC2 signaling extends the lifespan of C. elegans under certain temperatures and dietary conditions^111,112, has anti-tumor effects ^128–131, is beneficial in mouse models of mitochondrial disease^132,133, and has been shown to inhibit or reverse senescence in human cells ^134,135. Certain side effects, whether mediated by mTORC1 or mTORC2, may potentially even be a consequence of “on-target” action of rapamycin and rapalogs, and avoiding these side effects may limit the benefits of such drugs for healthy aging. However, given the balance of the evidence, particularly the genetic studies showing that mTORC2 activity is associated with lifespan and that inhibition of mTORC1 alone can extend lifespan, we believe that inhibition of mTORC2 is “off-target” with respect to the beneficial effects of rapamycin and rapalogs on healthspan and longevity.

Clinical trials of mTOR inhibitors for diseases of aging

Given the extensive preclinical data confirming that mTOR inhibition extends lifespan and healthspan, there is great interest in determining whether mTOR inhibitors will have benefits for human aging. As highlighted in Figure 1, based on mouse studies rapamycin and rapalogs may have benefits in many different systems, including the brain, heart, immune system, intestine, liver, senescent skin cells, skeletal muscle and tendons. Rapalogs are approved for use in transplant and cancer patients at high doses that strongly suppress mTORC1 activity ¹³⁶and have significant side effects including mouth ulcers similar to canker sores, gastrointestinal side effects, hyperlipidemia and hyperglycemia, and impaired wound healing. The approved doses of rapalogs also inhibit immune function and therefore the FDA-approved prescribing information notes that taking rapalogs may increase the risk of infection and certain cancers associated with immunosuppression ^137,138. The risk of infectious diseases associated with higher doses of rapalogs in humans may not be adequately modeled in preclinical mouse experiments since mice are usually housed in specific pathogen free (SPF) barrier facilities.

Figure 1. Potential healthspan targets of rapamycin.

Based on studies in rodents and humans, rapamycin and rapalogs may have potential benefits for ameliorating or slowing age-related conditions associated with the brain ^{105,246–248}, heart ^25,249,250, liver ²⁵, skeletal muscle ¹⁰⁰, tendons ^25,251, the immune system ^39,82–84, the skin ¹³⁵, and the intestine ^96,252. Created with BioRender.Com.

Chronic treatment of humans with high doses of rapalogs is also associated with deleterious metabolic consequences including hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, insulin resistance and glucose intolerance ^139–143. Hyperlipidemia and hyperglycemia have also been observed in rodents treated with rapamycin or rapalogs although mice are less susceptible to developing hyperlipidemia-induced cardiovascular disease than humans. Trelinska and colleagues observed hyperlipidemia in 66% of TSC patients taking high daily doses of everolimus for 15 months; hyperglycemia was observed in 22% of subjects ¹³⁹. Another side effect of doses of rapamycin that extend lifespan in mice is testicular degeneration ^25,144, and reduced male fertility has been observed in humans treated with rapalogs ^145,146. Finally, lifespan-extending doses of rapamycin in mice also promote the formation of cataracts ²⁵, although cataracts have not been associated with rapalog treatment in humans.

Recent studies (see Table 3) have explored the safety and efficacy of lower or intermittent dosing regimens of mTOR inhibitors in older adults. Preliminary results from these studies suggest that lower or intermittent dosing regimens of mTOR inhibitors that turn down mTOR activity to “younger” levels rather than turn off mTOR activity have the potential to safely improve the function of aging organ systems or ameliorate aging-related diseases in humans. Of interest, clinical trials to date suggest that low or intermittent doses of mTOR inhibitors enhance rather than suppress immune function and decrease infection risk in older adults. Table 2 summarizes the clinical trials published to date of mTOR inhibitors for aging-related conditions; we discuss each of these briefly below.

Table 3: Completed clinical trials of mTOR inhibitors for aging-related conditions.

Information on completed clinical trials was compiled from a review of the scientific literature.

Population	mTOR Inhibitor Dosing Regimen	Duration	Primary Endpoint	Was Primary Endpoint Met?	Reference
218 adults age ≥ 65 years	Everolimus 0.5 mg daily Everolimus 5 mg weekly Everolimus 20 mg weekly Placebo	6 weeks	Improvement in influenza vaccination response	Yes	Mannick et al. 2014 82
264 adults age ≥ 65 years	Everolimus 0.1 mg daily Everolimus 0.5 mg daily BEZ235 10 mg daily BEZ235 10 mg+0.1 mg everolimus daily Placebo	6 weeks	Improvement in influenza vaccination response	Yes	Mannick et al. 2018 83
652 adults age ≥ 65 years at increased risk of respiratory tract infections	BEZ235 5 mg daily BEZ235 10 mg daily BEZ235 10 mg twice daily BEZ10 mg+ everolimus 0.1 mg daily Placebo	16 weeks	Decrease in incidence of laboratory-confirmed respiratory tract infections	Yes	Mannick et al. 2021 84
1051 adults age ≥ 65 years	BEZ235 10 mg daily Placebo	16 weeks	Decrease incidence in symptoms consistent with a respiratory tract infection	No	Mannick et al. 2021 84
25 healthy adults 70–95 years	Rapamycin 1 mg Placebo	8 weeks	Safely achieving therapeutic blood level	Yes	Kraig et al., 2018 149
36 adults with aging skin	Topical rapamycin (10 μM) Topical placebo	8 mos	Decreased in p16-positive senescent cells in skin	Yes	Chung et al. 2019 135

mTOR inhibition and the immune system

The largest clinical trials to date have investigated if mTOR inhibition can improve the function of the aging immune system. The first clinical trial was done in 218 adults age ≥ 65 years without unstable medical conditions to determine if the rapalog everolimus improved the function of the aging immune system as assessed by a response to an influenza vaccination ⁸². The rationale for the trial was a preclinical study demonstrating that 6 weeks of rapamycin treatment improved the immune response of old mice to an influenza vaccination ³⁹.

Safety was a key concern when designing this trial. Therefore, very low daily or intermittent doses of everolimus were used in the vaccination trial (1/6–1/20^th lower than the approved doses in transplant and oncology patients) that were predicted to minimize adverse events and to lower rather than completely inhibit mTORC1 activity. The clinical trial demonstrated that 6 weeks of treatment with either 0.5 mg once daily or 5 mg once weekly everolimus was well tolerated and significantly improved the response of older adults to an influenza vaccination. Immunophenotyping revealed that low or intermittent doses of everolimus decreased the percentage of PD-1+ exhausted T cells in the peripheral blood of older adults and this may have been a mechanism underlying their improved immune function. This was the first study to demonstrate that mTOR inhibition may improve the function of an aging organ system in humans⁸².

A subsequent clinical trial was done in 264 adults age ≥ 65 years to extend these findings and determine if low dose mTOR inhibitor treatment with either everolimus and/or or the catalytic site mTOR inhibitor BEZ235 improved the function of the aging immune system sufficiently to improve not only influenza vaccination response, but also to decrease total infection rates in older adults⁸³. The study confirmed that 6 weeks of treatment with a low dose of mTOR inhibitors was safe and was associated with a dose-dependent significant improvement in influenza vaccination response. In addition, low dose mTOR inhibitor treatment significantly decreased the rate of infections, the majority of which were respiratory tract infections. Exploratory transcriptional profiling found that the older adults receiving low doses of mTOR inhibitors had a significant upregulation of genes in type 1 interferon-induced antiviral pathways. This upregulation of antiviral gene expression may underlie the reduction in respiratory tract infections seen older adults treated with mTOR inhibitors since most respiratory tract infections are viral in origin ^147,148.

Large follow-up Phase 2b (n=652) and Phase 3 (n=1051) trials were done to confirm whether 16 weeks of low dose mTOR inhibitor treatment upregulated antiviral immunity and decreased the incidence of viral respiratory tract infections (RTIs) in older adults ⁸⁴. Low doses of the mTOR inhibitor BEZ235 were observed to be well-tolerated and to decrease the incidence of laboratory-confirmed RTIs (the Phase 2b primary endpoint; OR 0.601 [90% CI 0.39, 0.92]; p-value 0.025) but not the incidence of clinically symptomatic respiratory illness, which was defined as symptoms consistent with a RTI irrespective of whether an infection was laboratory-confirmed (the Phase 3 primary endpoint; OR 1.07 [90% CI 0.80, 1.42]; p-value 0.65). In both trials, significantly more interferon-induced antiviral genes were upregulated in subjects treated with BEZ235 as compared to placebo. Lessons learned from the combined Phase 2b and 3 trial results suggest that upregulation of antiviral immunity by low dose mTOR inhibitor therapy may have a greater impact on 1) severity than incidence of viral RTIs, 2) RTIs caused by coronaviruses, rhinoviruses and influenza viruses as opposed to other respiratory viruses; 3) the incidence and severity of viral RTIs in older adults ≥ 75 years of age as opposed to ≥ 65 years of age. Based on these findings, it will be important to determine in future clinical trials if mTOR inhibitors decrease the severity of specific viral respiratory tract infections in people ≥ 75 years of age.

Rapamycin in older adults

In contrast to the subjects in the above trials, who tolerated everolimus or BEZ235 quite well, a small, 8-week long randomized clinical trial of 25 older adults between 70 and 95 years of age treated with 1 mg/day of rapamycin experienced more side effects than placebo including a small increase in glycated hemoglobin (within-group p=0.03), and a 40% rise in triglyceride levels (within-group p=0.05) ¹⁴⁹. While these results tend to align with our expectation that chronic rapamycin treatment may cause hyperglycemia and hyperlipidemia, a caution in interpreting these results is the small group size and the failure of the between-group comparisons to reach statistical significance (p=0.07 and 0.12 for A1C and triglyceride changes, respectively). In addition, other parameters of glucose metabolism including insulin sensitivity and pancreatic beta cell function were not affected. It is possible that lower doses of rapamycin, intermittent treatment with rapamycin, or treatment with everolimus would be better tolerated than the daily dose of 1 mg/day examined here, but this has not been directly tested.

Rapamycin in skin

Local administration of rapamycin with negligible systemic exposure poses fewer safety risks than systemic administration. In a randomized placebo-controlled study of subjects age ≥ 40 years with signs of skin aging, 36 subjects topically applied cream containing rapamycin (10μM) to one hand and a matching placebo cream to their other hand for eight months ¹³⁵. A total of 17 subjects completed the study, 13 subjects had skin biopsies and 8 subjects had sufficient biopsy material for analysis of p16 expression levels, the primary endpoint of the study. Rapamycin-treated as compared to placebo-treated subjects were observed to have a significant decrease in senescent cells as assessed by p16 expression, increased collagen VII protein expression, and clinical and histologically assessed improvements in skin appearance. Treated subjects did not have detectable levels of rapamycin in their blood and there were no treatment-related adverse events.

Forthcoming clinical trials

A number of small clinical trials focused on geroprotective outcomes have been proposed in the United States, the United Kingdom, and New Zealand over the past two years, and many have obtained funding and/or began enrolling subjects. While the details of each study differ, most are testing low or intermittent doses of rapamycin or everolimus in subjects over the age of 50, in the range of 0.5–1 mg/day to 5–6 mg/week. These studies are testing a range of outcomes, for a range of age-related diseases, including Alzheimer’s disease, diabetes and sarcopenia; primary outcomes include disease-specific outcomes, including effects on cognitive performance, changes in insulin sensitivity, and changes in physical performance. Secondary outcomes for most planned studies include biomarkers of aging, including analysis of the DNA methylation clock; most studies are also assessing safety-related endpoints, including infections and metabolic disruption. A weakness of these planned studies is that even the largest will be studying a maximum of only 150 subjects, and the longest planned study is 18 months. Thus, while these studies are critical to gather data on the safety and effectiveness of these compounds as geroprotectors in humans, and interesting new data will likely become available over the next few years, comprehensive follow-up with well-powered double-blind placebo-controlled randomized clinical trials will still be necessary.

Novel ways to inhibit mTORC1

Given the drawbacks of rapamycin and commercially available rapalogs, most notably the side-effects and the narrowness of any potential therapeutic window, there is intense interest in identifying new ways to selectively inhibit mTORC1. In the short-term, some dietary interventions have shown potential promise. In particular, since mTORC1 activity is regulated by amino acids, while mTORC2 activity is not, reducing the content of dietary protein or specific dietary amino acids that normally stimulate mTORC1 activity has been explored as a way promote healthy aging. Several studies have shown that restriction of protein or specific restriction of either methionine or one or more of the branched-chain amino acids (BCAAs; leucine, isoleucine, and valine) reduces mTORC1 activity and extends lifespan in preclinical species ^24,150–152. Intriguingly, low blood levels of isoleucine are also associated with reduced mortality in humans ¹⁵³. Other potentially mTORC1-lowering diets include a ketogenic diet, which has been shown to extend the lifespan of mice while reducing mTORC1 activity ¹⁵⁴.

Of course, any dietary intervention will have issues with adherence, and pharmaceutical interventions can make mTOR inhibition to a much broader swath of the population. High resolution structures of mTORC1 have provided a previously unprecedented look at how mTORC1 is activated by nutrients and how it is inhibited by rapamycin ^155–157. High-resolution structures have also been generated of the complexes that regulate mTORC1 activity, including the TSC complex ¹⁵⁸, the Rag GTPases in complex with mTORC1 and Ragulator ^159–161, and the binding of leucine by Sestrin2 ¹⁶². As discussed below, this has led to potential new approaches and the development of new molecules to target mTORC1 selectively.

mTORC1 is able to phosphorylate its substrates only after allosteric interaction with Rheb-GTP, which activates mTORC1 by promoting the alignment of kinase-site residues ^163,164. In order to bring mTORC1 and Rheb-GTP together, two signaling pathways converge at the surface of the lysosome. The first of these pathways brings mTORC1 to the lysosomal surface, while a second pathway controls the GTP/GDP binding status of Rheb. The recruitment of mTORC1 to the lysosomal surface has been studied for over a decade, and has been reviewed in detail elsewhere ³; we have outlined the major regulator mechanisms in Figure 2. Briefly, mTORC1 is recruited to the lysosome by interacting with heterodimeric pairs of the Rag family of small GTPases ^165,166. In the presence of amino acids, RagA/B binds GTP and RagC/D binds GDP (e.g., RagA^GTP/RagC^GDP), permitting the Rags to interact with mTORC1 and localize it to the lysosome. The nucleotide binding status of the Rag GTPases is controlled by several different protein complexes with guanine nucleotide exchange factor (GEF) or GTPase-activating protein (GAP) activity for the Rag GTPases.

Figure 2. An overview of the mechanistic Target Of Rapamycin Complex 1 (mTORC1) signaling pathway with areas of potential pharmaceutical inhibition highlighted.

Negative regulators (CASTOR1, GATOR1, SAMTOR, Sestrin2, tuberous sclerosis complex [TSC]) and positive regulators (FLCN-FNIP2, GATOR2, KICKSTOR, LRS, RAG GTPases, RAGULATOR, Rheb, SLC38A9, v-ATPase) are shown. Potential mechanisms for the development of mTORC1 specific inhibitors include: A, B, C, D. Identifying small molecules that block the ability of amino acid sensors upstream of mTORC1 to sense the availability of leucine, arginine, or SAM; (E, F, G) developing compounds such as BC-LI-0186 that inhibit the GAP or GEF activities of FLCN-FNIP2, LRS, RAGULATOR or TARS2; (H) Inhibiting the interaction of mTORC1 and Rheb, the mechanism of action of NR1; (I) Identifying small molecules that block the ability of LYCHOS and SLC38A9 to sense the availability of cholesterol; and (J) Identifying rapamycin derivatives such as DL001 that specifically inhibit mTORC1. Select downstream substrates of mTORC1 and processes mediated by them are also show. Figure is adapted from Dumas and Lamming, 2020, JGBS ²⁴⁵ and used with permission.

One of the best described systems for regulating the nucleotide binding status of the Rag GTPases is the activity of the GATOR complexes. GATOR1 functions as a GAP for RagA and RagB, while GATOR2 acts to inhibit the activity of GATOR1 ^167,168. The GATOR complexes are regulated by levels of amino acids, cholesterol, and glycolytic intermediates, thus linking mTORC1 activity to nutrient availability. Three different amino acid sensors have been found that regulate mTORC1 activity by controlling GATOR1 or GATOR 2 activity. The Sestrin and CASTOR family of proteins link the availability of leucine and arginine, respectively, to the recruitment of mTORC1 to the lysosomal surface. Specifically, the Sestrin family of proteins binds to and inhibits the action of GATOR2 when leucine levels are low, permitting GATOR1 to inhibit the recruitment of mTORC1 to the lysosome. Binding of leucine by the Sestrins, particularly Sestrin 2, relieves this inhibition of GATOR2, resulting in the inhibition of GATOR1 GAP activity, and allowing mTORC1 to be recruited to the lysosome ^162,169.

The CASTOR proteins function similarly; when arginine levels are low, the CASTOR proteins bind to and inhibit GATOR2. When arginine levels are high, CASTOR proteins bind to arginine and release GATOR2, which allows the recruitment of mTORC1 to the lysosome^170,171. The SAMTOR protein acts as an indirect sensor of methionine levels, inhibiting GATOR1 when levels of the methionine metabolite S-adenosylmethionine (SAM) are low (128). As SAM is extremely responsive to methionine levels, SAMTOR essentially functions as a sensor of methionine levels ¹⁷². It was recently shown that cholesterol signals through a G protein-coupled receptor, LYCHOS, which links cholesterol levels to mTORC1 activity by sequestering GATOR1 in the presence of cholesterol ¹⁷³. Finally, while glucose is not directly sensed by mTORC1, the glycolytic intermediate dihydroxyacetone phosphate (DHAP) is sensed via a GATOR-dependent mechanism ².

The regulation of RagC and RagD is somewhat less well understood, but the FLCN complex has been shown to act as a GAP for these GTPases ^174–177. The leucyl-tRNA synthetase (LRS) has been shown to function as a leucine sensor for mTORC1, acting as a GAP for RagD ¹⁷⁸. The mitochondrial threonyl-tRNA synthetase TARS2 functions as a threonine sensor, interacting with GTP-RagC to promote the GTP loading of RagA, likely via the recruitment of an unidentified RagA GEF ¹⁷⁹.

At the lysosomal surface, mTORC1 activity depends upon the interaction of the mTOR protein kinase with GTP-found Rheb. Cryo-electron microscopy has revealed that Rheb-GTP binds allosterically to mTOR, resulting in a global conformational change that allosterically realigns the active-site residues to enable substrate phosphorylation ¹⁵⁷. At the lysosomal surface the Tuberous Sclerosis Complex (TSC) inhibits mTORC1 activity by acting as a GAP for Rheb. A number of different factors have been reported to control the lysosomal localization of TSC, most notably insulin/PI3K/AKT signaling, which acts to phosphorylates TSC on multiple residues, causing TSC to depart from the lysosome and permitting Rheb to be loaded by GTP ¹⁸⁰. The localization of TSC has also been reported to be sensitive to amino acids, including arginine, although the mechanism by which this takes place is unclear ¹⁸¹. The Rag GTPases have also been reported to recruit TSC to lysosomes in response to amino acid restriction, possibly via a GATOR2 or Sestrin2-dependent mechanism ¹⁸².

Researchers are capitalizing on this knowledge to develop new but still early stage mTORC1-selective drugs based on molecular and structural information about mTORC1 activation (Figure 2). One of the best examples of these is the compound is NR1, which binds the mTORC1 activator Rheb and prevents it from allosterically activating mTORC1 ¹⁶⁴. The leucyl-tRNA synthetase (LRS) has been proposed to function as a leucine sensor for mTORC1, acting as a GAP for RagD, and several recent compounds, e.g. (S)-4-isobutyloxazolidin-2-one and BC-LI-0186, have been identified that inhibit mTORC1 by interfering with LRS sensing or activity ^{178,183–186}. BC-LI-0186 inhibits mTORC1 activity in vivo in mice and slows tumor growth in a mouse model of non-small cell lung cancer ¹⁸⁷. As outlined in Figure 2, small molecules that interfere with amino acid sensors or cholesterol sensors that normally signal nutrient availability could potentially be developed as mTORC1-selective inhibitors.

Newer rapalogs have been discovered that are more selective for mTORC1 than rapamycin. One company that screened a library of modified rapalogs identified a compound, DL001, with significantly greater selectivity for mTORC1 than rapamycin ¹²⁰. As expected, mice treated with DL001 had reduced glucose intolerance, dyslipidemia, and immune disruption as compared to mice treated in parallel with rapamycin ¹²⁰. Multiple other companies are also working to bring more mTORC1-selective rapalogs to the clinic ¹⁸⁸. Pre-clinical trials of one such compound, NV-20494, has reportedly shown efficacy in a mouse model of polycystic kidney disease and in vitro in human 3D cell culture ¹⁸⁹. Finally, Rapalink-1, a compound in which a mTOR kinase inhibitor is linked to rapamycin, and delivered at low dose, has shown the ability to inhibit mTORC1 kinase activity selectively ¹⁹⁰. A potential issue with this approach for diseases of aging is that mTOR kinase inhibitors result in broader mTORC1 inhibition than rapamycin⁵³, and it remains to be determined if mTOR kinase inhibitors recapitulate the lifespan benefits of rapamycin in preclinical species.

Conclusions

There is rapidly growing interest in using mTOR inhibitors to promote healthy aging and to treat, delay or reverse numerous age-related diseases. While there is incredibly strong pre-clinical evidence in mice that rapamycin can extend lifespan and healthspan, excitement about rapamycin has outpaced rigorous evidence that rapalogs are both safe and efficacious for diseases of aging in humans. There are many unanswered questions from the trials that have been conducted thus far, but a few general lessons can be taken from the clinical trials of mTOR inhibitors that have been performed thus far. As highlighted in Figure 3, in both humans and mice, treatment with low or intermittent doses of rapamycin or everolimus, or treatment of mice with the mTORC1-selective inhibitor DL001, is much better tolerated than the high doses of mTOR inhibitors currently approved for organ transplant and oncology indications, with fewer metabolic side effects and less immunosuppression. In addition, low doses of mTOR inhibitors have been shown to have some beneficial effects on the function of aging human organ systems, in particular the immune system.

Figure 3. The known and unknown effects of rapalog dosing regimens on metabolic health, the immune system, healthspan and longevity in humans and mice.

Chronic (daily) dosing of rapamycin is associated with impaired blood glucose regulation and hyperlipidemia in humans and mice, while everolimus in mice has somewhat reduced effects on glucose homeostasis. Intermittent or low dose regimens of rapamycin or everolimus are associated with reduced side effects, while the mTORC1-selective rapalog DL001 does not impair blood glucose control or alter circulating lipid levels. Green up arrow = improvement, red down arrow = impairment; Dash indicated no change, Question mark indicates unknown.

There remains much work ahead to bring mTOR inhibitors into the clinic for age-related conditions, and as highlighted in Figure 3, many open questions remain. While the safety profile of low-dose rapamycin and rapalogs in humans appears promising, the long-term safety and efficacy of low-dose regimens remains to be determined. A much better understanding is needed of the specific dose and duration of mTOR inhibitors that both maximize efficacy and minimize risk. In humans, higher doses (e.g., 3 mg/day) of mTOR inhibitors such as everolimus inhibit T-cell function, and are therefore are used to suppress immune-mediated organ transplant rejection in patients. In contrast, a 6-fold lower dose of everolimus for 6 weeks was associated with improved immune function as assessed by vaccination response. Thus, both dose and duration may contribute to whether mTOR inhibition has positive or negative effects on healthy aging; but generally speaking, the lower the dose of a drug, the fewer expected side effects. Moreover, animal dosing regimens that extend lifespan cannot be directly translated to human doses due to differences between species in factors such as bioavailability, half-life, metabolism, plasma protein binding, and tissue distribution. These factors need to be taken into consideration when estimating the human doses that may extend healthspan or lifespan.

There is also a need to identify both the specific aging population and the specific aging-related conditions which show the greatest benefit from a safe mTOR inhibitor regimen. An important question for future research is discovering why mTOR inhibitors have sex-specific or sex-biased benefits, as pharmacological treatment with rapamycin or genetic inhibition of insulin-IGF1/PI3K/Akt/mTOR/S6K1 signaling typically (but not always) extends the lifespan of female mice a greater amount than it extends the lifespan of male mice ^191–193. We also need to define new regulatory paths for aging-related conditions such as frailty and sarcopenia, and to develop new mTOR inhibitors that improve on the safety and efficacy of currently approved mTOR inhibitors. Over the next five years, we expect results from a rapidly expanding list of human clinical trials as well as work in canines and non-human primates to shed light on the viability of mTOR inhibition as a therapy for aging-related conditions. New mTORC1-specific molecules may help widen the therapeutic window for rapalogs, limiting undesirable side effects resulting in whole or in part from inhibition of mTORC2.Collectively, we expect that researchers will soon be able to determine if clinicians can safely and effectively bring mTOR inhibitors to the geriatric bedside.