The potential of Senolytics in transplantation

Abstract

Older organs provide a substantial unrealized potential with the capacity to close the gap between demand and supply in organ transplantation.

The potential of senolytics in improving age-related conditions has been shown in various experimental studies and early clinical trials. Those encouraging data may also be of relevance for transplantation.

As age-differences between donor and recipients are not uncommon, aging may be accelerated in recipients when transplanting older organs; young organs may, at least in theory, have the potential to ‘rejuvenate’ old recipients.

Here, we review the relevance of senescent cells and the effects of senolytics on organ quality, alloimmune responses and outcomes in solid organ transplantation.

Senescent cells accumulate in aging and impact organ quality and -function

Senescent cells are characterized by the expression of p21^CIP1 and/or p16^INK4a, DNA segments with chromatin alterations reinforcing senescence (Rodier et al., 2011), senescence-associated heterochromatin foci (Narita et al., 2003), senescence-associated β-galactosidase (Lee et al., 2006), short telomeres (Bernadotte et al., 2016) and the production of a senescence-associated secretory phenotype (SASP). From a physiological perspective, senescence represents a cell-autonomous mechanism, preventing the transformation into malignant cells (Campisi, 2001). Indeed, approaches aimed to inhibit cellular senescence by targeting p16^INK4a, Rb, p53 or p21^CIP1 have been shown to promote cancer development (Larsen, 2004; Takeuchi et al., 2010).

Characteristically, senescent cells accumulate in various tissues with aging (Dimri et al., 1995; Korolchuk et al., 2017) and have a compromised mitochondrial capacity with a limited tissue regeneration (Jurk et al., 2014). Senescent cells also have the ability to induce senescence in surrounding, non-senescent cells through the production of SASP consisting of pro-inflammatory cytokines, chemokines, proteases, and other factors, (Coppe et al., 2008; Xu et al., 2015b). SASP constitutes a fundamental feature of senescent cells disrupting tissue homeostasis and impeding neighboring cellular functions resulting into age-related tissue dysfunction, chronic age-related diseases, and biological aging (Kirkland and Tchkonia, 2017). Consequently, senescent cell accumulation promotes a variety of age-related diseases. For instance, senescent cells accumulate in adipose tissue in individuals with diabetes, contributing to age-related metabolic dysfunction (Minamino et al., 2009; Tchkonia et al., 2010; Xu et al., 2015a), in the aorta during atherosclerosis (Roos et al., 2016), and in the lungs as observed in idiopathic pulmonary fibrosis (Schafer et al., 2017).

Multimorbidity, defined asthe coexistence of multiple chronic diseases a clinical condition that is particularly common in the elderly. Metabolic syndrome and cardiovascular disease both display established causes of aging. These conditions are associated with changes that are also observed in aging, such as increased intensity of intracellular processes that generate free radicals and cause chronic low-grade inflammation, a phenomenon also called ‘Inflamm-aging’ (Edrey and Salmon, 2014; Franceschi et al., 2007). Inflamm-aging itself is a risk factor not only for cardiovascular disease, cancer and chronic kidney disease, but also an indicator of poor health status lined to multimorbidity, sarcopenia, frailty and premature death (Ferrucci and Fabbri, 2018). Based on these findings, an interactive concept is suggested: while the accumulation of senescent cells is the cause of age-related diseases, the presence of multiple chronic diseases may, in turn, accelerate aging. (Hodes et al., 2016) Since, both transplant recipients and donors have frequently an extensive past-medical history it is also possible that accelerated biological aging due to senescence derived multimorbidity may affect transplant organ function and outcome.

Senescent cell accumulation and Immunogenicity

Senescent cells secrete various inflammatory proteins most of which are transcriptionally regulated by nuclear factor κB and CCAAT/Enhancer-binding transcribed by protein β (Kuilman et al., 2008). Epigenetic factors such as G9a/GLP, SIRT1, MLL1, BRD4, and HMGB2 have been shown to drive the expression of SASP factors including IL-6, IFN-γ, and TNF-α (Freund et al., 2010; Loo et al., 2020; Takahashi et al., 2012) that contribute to the sterile, low grade, chronic inflammation in aging (Franceschi et al., 2000; Tchkonia et al., 2013).

Ischemia-reperfusion injury (IRI) is an inevitable component of transplantation occurring during organ procurement, transport and, finally, while reconstructing blood flow. Oxygen demand increases rapidly in ischemic tissues after organ reperfusion. IRI induces oxidative stress, mitochondrial damage, electrolyte imbalance resulting into local inflammation, the release of ROS (Fisher et al., 1991), pro-inflammatory cytokines, especially TNF-α, IL-1, IL-6, IL-8 (Krishnadasan et al., 2003; Naidu et al., 2003) in addition to various proteases (Yano et al., 2001). All of those factors have been implicated in accelerated senescence via SASP activation, mitochondrial dysfunction, and activation of the p53 and RAS pathways. Recent studies in experimental models confirm that IRI induces senescence in both cardiomyocytes and interstitial cells, both within and around the infarcted left ventricular myocardium (Dookun et al., 2020). Senomorphic drugs that inhibit SASP including ATM kinase inhibitors and Janus kinase inhibitors may ameliorate the consequences of SASP factors released upon IRI in transplanted aged organs (Birch and Gil, 2020).

IRI also activates the recipient’s innate immunity through the release of damage associated molecular patterns (DAMPs) consisting of intracellular contents (e.g., mt-DNA, HMGB1) or membrane components (e.g., hyaluron, syndecan). Donor and recipient derived antigen presenting cells recognize and activated DAMPs through pattern recognition receptors (PRR) that initiate adaptive immune responses targeting the allograft through MHC-class-II mediated antigen-presentation (Yi et al., 2012).

Old donor organs contain augmented frequencies of senescent cells as a key source of cell-free-mt-DNA. IRI, in turn, leads to an increased release of cf-mt-DNA, particularly in old organs promoting an amplified dendritic cell mediated Th1/Th17 alloimmune response leading to an inferior graft survival of older organs (Iske et al., 2020; Oberhuber et al., 2015). Clinically, increased levels of mt-DNA have also been linked to higher rejection rates and delayed graft function following kidney transplantation (Kim et al., 2019).

These findings are consistent with preclinical and clinical studies showing that donor age poses a significant risk for adverse outcomes, an augmented immunogenicity and more frequent acute rejections (Oberhuber et al., 2015; Tullius and Milford, 2011).

Improving outcomes of older organs for transplantation

Although organ transplantation represents the treatment of choice for end-stage organ failure, the approach cannot achieve its potential as there is a dramatic gap between demand and supply. While the number of organ transplants has increased over the last years, more than 100,000 patients are currently waiting for an organ transplant in the United States (Hart et al., 2021). In contrast, it is estimated that approximately 24.000 organs from older donors are currently not utilized or discarded (Klassen et al., 2016). The age of organ donors is the most prominent reason for organ discard. (Messina et al., 2017) Utilizing available organs for transplantation successfully has therefore the potential to substantially reduce wait-times, morbidity and mortality for patients on transplant waitlists.

Senolytics have the capacity to selectively clear senescent cells. In preclinical models, senolytics delay, prevent or alleviate age-related dysfunction and diseases in multiple organs. Several clinical studies testing the potential of senolytics are currently underway (Kirkland and Tchkonia, 2020). As senolytics effectively reduce the burden of senescent cells and alleviate age-related dysfunction and inflammation, these agents may ultimately be effective when utilizing organs from older donors. We have been able to show that Dasatinib plus Quercetin (D & Q) applied prior to experimental IRI and transplantation significantly reduced the burden of senescent cells while alleviating systemic inflammation and decreasing levels of cf-mt-DNA, Th17 and IFNγ⁺ T cells. Notably, pretreatment of old donor mice with senolytics prolonged the survival of older to that of young organs in a mouse cardiac transplant model (Iske et al., 2020).

In the heart, for example, senescence accumulate with aging, resulting in augmented amounts of senescent cardiomyocytes, endothelial cells, smooth muscle cells, cardiac fibroblasts and cardiac progenitor cells (Anderson et al., 2019; Walaszczyk et al., 2019). Preclinical models of biological aging have demonstrated that senescence contributes to the pathophysiology of age-related remodeling and cardiac dysfunction indicating a role for senolytics (Chimenti et al., 2003). Navitoclax has been shown to reduce cardiomyocyte senescence and SASP of cardiomyocytes attenuating cardiac hypertrophy reducing the size of the left ventricle (Anderson et al., 2019; Owens et al., 2021; Walaszczyk et al., 2019).

During and following the transplant procedure, several factors including ischemia reperfusion injury, early allo-immune responses in addition to immunosuppressive treatment mediate graft damage and impede tissue regeneration. Moreover, tissue senescence with aberrant p21 expression has been identified to further compromise tissue generation following partial hepatectomy (Ritschka et al., 2020). Accumulating senescent cells have also been shown to impair tissue regeneration following cardiac infarction (Lewis-McDougall et al., 2019). Notably, ABT-737, a senolytic drug inhibiting Bcl-2 and Bcl-x, has been shown to disrupt aberrant p21 expression, thus restoring liver generation in mice (Ritschka et al., 2020) while global senescent cell elimination in the INK-ATTAK system increased the number of proliferating cardiomyocytes in old hearts (Lewis-McDougall et al., 2019).

There are several time points during which an organ for transplant could be treated with senolytics: Starting prior to transplantation with a treatment of the donor (Fig. 1A), during organ preservation with a treatment of the graft itself (Fig. 1 B), and finally, following transplantation with a treatment of the recipient (Fig. 1C).

Figure 1. Potential Opportunities to Apply Senolytics in Organ Transplantation.

(A) Donor: Senolytics may be administered prior to organ donation. When applied in living donors, a regimen with minimal to no side effects is desirable.

(B) Graft: Treatment of the graft after organ procurement and prior to transplantation represents an attractive therapeutic intervention. Ex-vivo perfusion systems allowing for prolonged organ evaluation may not only support the treatment with senolytics but may also aide in monitoring treatment effects.

(C) Recipient: Recipients can be treated with senolytics at the time of transplantation or in the immediate post-transplant period; interaction between senolytics and immunosuppressants need to be considered.

Administering senolytics to the donor may ameliorate the consequences of IRI in older organs. Senolytics that target only a single senescent cell anti-apoptotic pathways (SCAP), such as navitoclax, are likely to have significant off-target apoptotic effects on non-aging cell types including platelets and immune cells while eliminating a limited range of senescent cells (Zhu et al., 2016). Moreover, some senescent cells have a beneficial role in tissue repair. (Demaria et al., 2014) Intermittent administration, combined with the short elimination half-life of senolytics may thus be most effective, minimizing drug toxicity and off-target effects (Christopher et al., 2008; Graefe et al., 2001). Notably, only a brief exposure to senolytics is required for senescent cells to initiate apoptosis thus avoiding the risks of long-term exposure and side-effects (Palmer et al., 2021). Indeed, the tolerability of dasatinib in patients with chronic myeloid leukemia has improved with intermittent dosing schedules (La Rosee et al., 2013).

Treatment of the allograft itself following organ procurement and prior to transplantation, may provide another opportunity for intervention. Adding senolytics to organ preservation solutions represents a unique opportunity for targeted delivery. New technologies including ex-vivo hypo- or normothermic organ perfusion do not only facilitate the assessment of organ function prior to transplantation but may also provide a platform for an isolated treatment of donor organs (Chandak et al., 2019; Nasralla et al., 2018; Reddy et al., 2009; Van Raemdonck et al., 2015). Overall, we submit that that senolytics may help to significantly expand the donor pool and improve outcomes of transplants derived from older donors. With an increased utilization of older organs, organ repair ‘centers’, an increased utilization of ex vivo organ perfusion systems and an adjusted allocation may be become necessary. The cost of machine perfusion can be an obstacle to their wider use. However, the cost of performing normothermic machine perfusion on grafts that have already been discarded was estimated to be only slightly more than the estimated monthly Medicare expenditure for the care of a MELD 30 patient, in addition to preventing deaths on the waiting list (Raigani et al., 2020). Moreover, economic analysis of kidney transplantation demonstrated that transplantation of low-quality kidneys is more cost-effective that waiting and receiving dialysis (Senanayake et al., 2021).

Biological age affecting transplant outcome

While the effects of donor age on transplant outcomes are clearly recognized (Tullius and Milford, 2011), it has become clear that biological rather than chronological donor age determines organ quality and long-term function after transplantation. Indeed, cyclin-dependent kinase inhibitor 2A (CDKN2A) expression in zero-hour biopsies in kidney transplantation was associated with donor age and graft function. In addition, linear regression analysis showed that CDKN2A was the best single predictor, followed by donor age and telomere length (Koppelstaetter et al., 2008). The concept that biological organ age impacting outcomes after transplantation is also supported by findings that delayed graft function, acute rejection, and chronic allograft dysfunction, are associated with telomere shortening (Domanski et al., 2015). At the same time, improved graft survival of renal transplants from p16 ^INK4a−/− mice compared to wild-type mice (Braun et al., 2012) underscore the concept of a cellular based biological age affecting graft outcomes. Thus, assessing chronological donor age utilizing markers of biological organ age, may provide additional valuable information.

Interference of senolytics with immunosuppressants

When administering senolytics to transplant recipient both, the pharmacological interaction as well as the mechanistic interaction between senolytics and immunosuppressive drugs require consideration. Notably, a broad range of senolytics has been shown to exert immunosuppressive effects on various immune cells which may augment the effects of established immunosuppressants.

Dasatinib is a Tyrosine kinase inhibitor (TKI) targeting the Src family (O’Hare et al., 2005) that also interferes with T cell receptor signaling (Smith-Garvin et al., 2009). Phosphorylation of Lck and Fyn, both members of the Src family, for instance, represent an early step within the T cell receptor activation cascade. Indeed, depleting Lcy and Fyn simultaneously has been shown to impair T cell function in mice (Denny et al., 2000; Lovatt et al., 2006; Tsun et al., 2011). SRC family proteins also display relevant targets of glucocorticoids mediated T cell suppression (Giese et al., 2004; Moes et al., 2014). Co-administration of Dasatinib and glucocorticoids may therefore affect similar pathways with potential additive or synergistic effects on T-cell suppression. Indeed, Dasatinib has been shown to exert synergistic effects with glucocorticoids in a murine model of acute T-cell leukemia (Serafin et al., 2017).

Calcineurin inhibitors (CNI) including tacrolimus (FK506) and cyclosporine (CsA) are widely administered as maintenance immunosuppressants in solid organ transplantation (Matas et al., 2015). Calcineurin dephosphorylates the cytoplasmic nuclear factor of activated T cells (NFAT), facilitating the migration into the nucleus, thus preventing the transcription of critical cytokines including IL-2 (Giese et al., 2004; Moes et al., 2014). Notably, the histone deacetylase inhibitor and senolytic drug Panobinostat have also been shown to induce calcineurin degradation through inhibiting the chaperone function of heat shock protein 90 (Imai et al., 2016) suggesting synergistic effects with tacrolimus or CsA.

Further evidence for synergistic effects of senolytics drugs with immunosuppressants derives from clinical studies assessing the impact of combinatorial treatment on hematopoietic tumors. Of relevance, synergistic effects of the bcl-family inhibitor navitoclax and rituximab treating B cell lymphoma have been reported (Ackler et al., 2012).

Senolytics may also interfere with the immunosuppressive effects of mTOR inhibitors such as rapamycin. In addition to their senolytic effects, the flavonoids quercetin and fisetin have been identified as inhibitors of the mTOR pathway which may result in synergistic effects on inhibiting T cell alloimmunity. In support, quercetin has been shown to inhibit the induction and effector function of cytotoxic T cells in mixed leucocyte reactions (Schwartz et al., 1982) while fisetin compromised Th1 and Th2 cytokine production, and proliferation of murine T cells in vitro (Song et al., 2013).

Interestingly, rapamycin reduces cellular senescence and systemic inflammation in the elderly while improving physical performance (Kim and Guan, 2015; Singh et al., 2016) suggesting a potential interference of immunosuppressants with senolytics. Mechanistically, mTOR inhibition has been shown to impede on protein translation and augmentation of autophagy expanded lifespan in flies and worms (Bjedov et al., 2010; Hansen et al., 2008; Hansen et al., 2007; Toth et al., 2008) (Figure 2).

Figure 2. Interaction of Senolytic and Immunosuppressive drugs.

Members of the Src family including Lck and Fyn are involved in early TCR signal transduction. Both, Dasatinib and Glucocorticoids target Src family proteins.

IP3 is the trigger for Ca²⁺ release from the endoplasmic reticulum (ER) promoting the entry of extracellular Ca²⁺ into the cell via calcium release-activated Ca²⁺ channels. Calmodulin bound to calcium activates calcineurin while promoting the transcription of IL-2 through the NFAT transcription factor. CNI inhibits calcineurin, thus suppressing dephosphorylations of NFAT. Panobinostat induces calcineurin degradation through inhibiting the chaperone function of heat shock protein 90.

CD28 co-stimulation of the TCR phosphorylates and activates PI3K, thereby activating the PI3K/mTOR pathway. Rapamycin inhibits T cell proliferation by inhibiting the formation of mTORC1.

Transfer of senescent cells with organ transplantation: A theoretical concept with relevance for senolytics

Old organs represent an underutilized source for transplantation (Saidi and Hejazii Kenari, 2014; Tullius and Rabb, 2018). Transplanting older organs, in turn, may lead to the transfer of senescent cells that may accumulate in recipients, potentially accelerating aging.

Intraperitoneal transplantation of relatively small numbers of senescent cells transferred into young mice accelerated aging of visceral adipose tissue and has been accompanied by a decline in physical performance (Xu et al., 2018). In addition, autologous transplantation of senescent cells into healthy knee joints of young mice promoted the development of an osteoarthritis-like condition (Xu et al., 2017). Moreover, transplanting senescent cells into the skeletal muscle of immunocompromised NOD SCID gamma mice revealed an increase in the number of senescent cells with an enhanced expression of SASP markers including IL-1α, IL-1β, IL-6, and TNF-α (da Silva et al., 2019). Most recently it has been shown that the transfer of splenocytes augmented SASP proteins systemically in recipient mice resulting into accelerated aging (Yousefzadeh et al., 2021). These observations are consistent with our preliminary findings showing that young mice transplanted with old hearts accumulated senescent cells in various tissues with an inferior physical performance that improved if grafts had been treated with senolytics.

Thus, removing senescent cells from donor organs may not only reduce immunogenicity and improve organ function but also inhibit the transfer of senescent cells in organ transplantation. The finding that D&Q slowed aging and the associated decline in physical function in mice injected intraperitoneally with senescent cells supports this concept (Xu et al., 2018).

At least in theory, it is also possible that young donor organs could exert a rejuvenating effect when transplanted into older recipients. Experiments with parabiosis models have shown the potential to rejuvenate brain, heart muscle, pancreas, bone, and skeletal muscle of old mice. (Baht et al., 2015; Conboy et al., 2005; Katsimpardi et al., 2014; Villeda et al., 2011; Villeda et al., 2014). It has also been shown that the transfer of plasma from young into old mice augmented the plasticity of hippocampal neurons and improved cognitive function (Villeda et al., 2014). Subsequent studies have indicated that several blood soluble factors may mediate rejuvenation. Moreover, extracellular vesicles derived from young mesenchymal stromal cells rejuvenated old endothelial progenitor cells in vitro (Kang and Yang, 2020; Rybtsova et al., 2020; Wang et al., 2020). Although representing a theoretical possibility, the rejuvenation potential when transplanting a young organ remains speculative currently.

Conclusion

Reducing the burden of senescent cells to improve organ quality while ameliorating alloimmune responses represents a novel concept. Diminishing the amounts of senescent cells in old organs with senolytics may thus have the potential of increasing organ availability and improving transplant outcomes. Confirmatory clinical trials will be necessary.

With many open questions remaining, optimal treatment time points, regimens and detailed mechanisms including an interfere with immunosuppressants will require more detailed investigations.

Transferring aging with the transplantation of older organs will be of clinical relevance if confirmed. Mechanisms linking the transplantation of old organs with an acceleration of senescence in recipients need to be delineated in detail.

Glossary & Abbreviations

Akt

Protein kinase B

ATM

ataxia telangiectasia mutated

Bcl-2 /

Bcl-x B-cell lymphoma 2 / B-cell lymphoma x

BRD4

Bromodomain-containing protein 4

CaA

cyclosporine

CCAAT

cytosine-cytosine-adenosine-adenosine-thymidine

CDKN2A

cyclin-dependent kinase inhibitor 2A

CNI

Calcineurin inhibitors

DAMPs

damage associated molecular patterns

D & Q

Dasatinib plus Quercetin

ER

endoplasmic reticulum

FK506

Tacrolimus

GLP

G9a-like-protein

HMGB2

high-mobility group box 2

IFN

Interferon

IL

interleukin

INK-ATTAK-mouse

INK-linked apoptosis through targeted activation of caspase), which selectively expresses a FK506-binding protein (FKBP)–CASP8 fusion protein in p16INK4A-postive senescent cells and triggers apoptosis following administration of AP20187, a molecule that dimerizes the FKBP–CASP8 fusion protein

IP3

inositol trisphosphate

IRI

Ischemia-reperfusion injury

Lat

linker for activation of T cells

Lck

lymphocyte protein tyrosine kinase

MLL1

Mixed lineage leukemia protein-1

mTOR

mammalian/mechanistic target of rapamycin

mTORC1

mTOR complex 1

NFAT

nuclear factor of activated T cells

NMP

normothermic mechanical perfusion

NOD

nucleotide-binding oligomerization domain-containing protein

PI3K

Phosphoinositide 3-kinase

PIP2

phosphatidylinositol 4,5-bisphosphate

PIP3

Phosphatidylinositol 3,4,5-trisphosphate

PLCγ1

Phosphorylation of phospholipase C γ1

PRR

pattern recognition receptors

RAS

Rat sarcoma

ROS

reactive oxygen species

SASP

senescence-associated secretory phenotype

SCAP

single senescent cell anti-apoptotic pathways

SCID

severe combined immunodeficiency

SIRT1

stimulator of interferon genes 1

TCR

T cell antigen receptor

TKI

Tyrosine kinase inhibitor

TNF

tumor necrosis factor

Zap70

Zeta-chain associated protein kinase 70