Facts

  • Intrinsic and extrinsic apoptosis are forms of regulated cell death (RCD) promoting the cellular demise along with the activation of proteases of the caspase family.

  • In mammalian organisms, executioner caspases are activated after cells are already committed to die.

  • Apoptosis can be manipulated by genetic or pharmacological means, and multiple genetically engineered animal models and pharmacological tools to modulate apoptosis have been developed.

  • Apoptosis is intimately involved in both (post-)embryonic development and adult tissue homeostasis.

  • Apoptosis deregulation promotes oncogenesis and contributes to the etiology of multiple human disorders, including cardiovascular, hepatic, renal, inflammatory and neurological conditions.

  • To date, venetoclax is the only apoptosis inducer that has received regulatory approval for use in humans.

Open Questions

  • Will inhibitors of apoptotic caspases with elevated target specificity become available?

  • Will agents specifically conceived to modulate apoptosis enter the clinical practice to treat solid tumors or other human disorders beyond hematological malignancies?

  • Is it conceivable to design combinatorial strategies aimed at inhibiting apoptosis while interrupting compensatory activation of other RCD signaling cascades?

  • Will it be possible to specifically inhibit apoptotic signaling without impacting on other processes dependent on apoptosis regulators such as differentiation, proliferation, and inflammatory reactions?

Introduction

The health and homeostasis of multicellular organisms depend on the tight balance between cell proliferation and cell death. In this context, a large body of experimental evidence has demonstrated the existence of a form of regulated cell death (RCD) that is executed by a genetically programmed process, and hence amenable to manipulation by genetic or pharmacological means [1]. Over the past decades, multiple variants of RCD have been characterized at the genetic, biochemical, functional, and immunological level [2,3,4,5,6,7,8]. For instance, programmed cell death (PCD) has been functionally defined as a modality of RCD activated under purely physiological conditions (i.e., in the absence of perturbations of extracellular or intracellular homeostasis) in the context of embryonic/post-embryonic development or adult tissue homeostasis [1, 9]. Conversely, pathological RCD is invariably initiated in the context of failure to adapt to shifts in extra-cellular or intra-cellular homeostasis, constituting a de facto organismal program for the elimination of excessively damaged and/or potentially harmful cells, such as cells infected with pathogens [1, 10]. From a biochemical perspective, an increasing number of RCD modalities have been defined by the Nomenclature Committee on Cell Death (NCCD) based on the mechanistic involvement of specific molecular components [1, 11]. For instance, apoptotic cell death has been defined as a form of RCD that is promoted by proteases of the caspase family, namely caspase 3 (CASP3), CASP6 and CASP7, and initiated by CASP8 and CASP9 [1, 12, 13]. However, in mammalian organisms, with the exception of CASP8, apoptotic caspases simply accelerate RCD because their activation occurs when cells are already committed to die [1, 14,15,16]. This means that contrarily to simpler organisms (e.g., Caenorhabditis elegans), in which apoptotic caspase elimination fully rescues cells from death, in mammals, apoptotic cell death can at most be retarded but not prevented by pharmacological or genetic strategies inhibiting the activity of these caspases. Mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, and autophagy-dependent cell death represent forms of RCD that involve precise molecular events and hence can also be manipulated with pharmacological or genetic interventions [1,2,3,4,5,6, 17,18,19]. Other RCD modalities have been recently identified, such as alkaliptosis [20], cuproptosis [21] and PANoptosis (involving the simultaneous activation of pyroptosis, apoptosis, and necroptosis) [22], and their signal transduction modules are under investigation. The importance of these latter forms of RCD in health and disease is not yet known.

Along with the identification of key RCD regulators and the advent of modern tools for genetic manipulation, a great experimental effort has been devoted to elucidating the role of RCD in the physiopathology of multi-cellular organisms [23]. Thus, various studies in animals (mostly rodents) genetically altered to lack or over-express components of the apoptotic apparatus (either at the whole-body level or in selected cell/tissue types) have provided formal proof of the relevance, but not always the exquisite requirement, of apoptosis for embryonic and fetal development or adult tissue homeostasis [24,25,26].

Along similar lines, pharmacological and genetic tools aimed at altering apoptotic signaling in pre-clinical disease models revealed the mechanistic contribution of apoptosis to the etiology of various conditions associated with the loss of post-mitotic or (in certain settings) non-post-mitotic cells, including a panel of neurological, cardiovascular, renal, hepatic, and inflammatory disorders [24]. Extensive studies over the last five decades highlighted the apoptotic machinery as a major target for the development of new therapeutic interventions [27], not only for the induction of cell death in the context of disrupted tissue homeostasis (e.g., for neoplastic diseases) [28], but also for the inhibition of cell death in the context of ischemic, degenerative and inflammatory conditions [29, 30]. However, while at least one drug designed to induce apoptosis is currently approved for use in humans, namely the BCL2 apoptosis regulator (BCL2) inhibitor venetoclax [31,32,33,34], which is used alone or in combinatorial regimens for the treatment of chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma and acute myeloid leukemia (AML) [31, 35,36,37,38], no other agents specifically conceived to inhibit the apoptotic apparatus have been licensed for clinical practice so far. The broad-spectrum caspase inhibitor emricasan received fast-track designation by the US Food and Drug Administration (FDA) for the treatment of non-alcoholic steatohepatitis in 2016 but demonstrated inconsistent clinical efficacy [39,40,41], and – as of now – is not approved for therapy in humans.

The lack of clinically approved, selective apoptosis inhibitors and the inconclusive performance of emricasan in recent trials reflect several aspects of (apoptotic and non-apoptotic) RCD that began to emerge only recently (Fig. 1). First, while detecting cell death as well as biomarkers of specific RCD variants in vitro is relatively straightforward [42], precise quantification of cell death in vivo in adult tissue remains challenging, at least in part because of rapid disposal of cell corpses by efferocytosis [43,44,45,46]. Thus, the actual contribution of cell death to the etiology of various human disorders is difficult to quantify by observational approaches [47, 48]. Second, while for a long-time, specific forms of RCD were considered virtually independent entities, it recently became clear that the molecular machinery for RCD is composed of highly interconnected modules characterized by substantial redundancy, backup pathways and feedback loops [10, 49, 50]. Thus, molecules that inhibit one specific form of RCD may ultimately be unable to confer actual cyto- and tissue protection instead only altering the kinetic and biochemical manifestations of death by allowing the engagement of a different RCD sub-routine. For instance, while CASP8 is a major signal transducer in death receptor (DR)-driven apoptosis (see below), it intrinsically inhibits necroptosis induced by DRs and other signaling pathways, such as Toll-like receptor (TLR) signaling [51,52,53], suggesting that caspase inhibition in the context of DR signaling may promote necroptotic cell death [54,55,56,57]. Together with a low target specificity and selectivity within the caspase family [57], this can explain the inadequate efficacy of emricasan observed in pre-clinical and clinical studies. Third, even in the hypothetical scenario of agents capable of simultaneous inhibition of all (known and unknown) RCD pathways, loss of cellular homeostasis due to failing adaptation to stress generally involve degenerative processes that at some stage cannot be reversed, such as widespread mitochondrial permeabilization and loss of RNA and protein synthesis [4, 58,59,60], i.e., even if all RCD modalities could be blocked effectively, cells might undergo uncontrolled necrotic death. In this setting, cell death may occur as a consequence of an irremediable degeneration of cellular functions that can no longer be rescued pharmacologically or even genetically [61]. Supporting these latter notions, accumulating literature indicates that, at least in mammalian systems, perhaps with the exception of CASP8, so-called apoptotic caspases mainly control the kinetics of apoptotic cell death and its immunological manifestations, but not whether cell death ultimately occurs or not [15, 16]. This points to the caspase family as a major regulator of organismal homeostasis via control of inflammatory responses [62, 63]. The simultaneous inhibition of multiple caspases, as for instance by emricasan, may thus also impact inflammation, as was demonstrated for tumor necrosis factor (TNF)-induced systemic inflammatory respiratory syndrome (SIRS) in vivo for the pan caspase-inhibitor zVAD-fmk [54, 64]. To complicate matters, multiple components of the core apoptotic machinery, including caspases and multiple members of the BCL2 family have been reported to regulate a variety of non-apoptotic functions beyond inflammation, such as mitochondrial energy production, Ca2+ signaling and terminal differentiation [65,66,67,68,69,70,71,72]. Structurally, distinguishing between apoptotic and non-apoptotic functions of caspases and the BCL2 family remains challenging. Finally, there is a hitherto unclarified heterogeneity in the regulation of RCD at distinct anatomical sites (possibly linked to micro-environmental features) at distinct stages of cellular differentiation, and in the context of diverse patho-physiological states (e.g., in young vs. adult and aged individuals).

Fig. 1: Principal causes of the therapeutic failure of intrinsic or extrinsic apoptosis inhibitors.
figure 1

The clinical development and success of agents inhibiting apoptosis is limited by multiple contributory causes, including potential non-apoptotic, accessory or even protective roles of the targeted proteins (exemplified by the involvement of certain BCL2 family members, caspases and death receptors in processes as diverse as inflammation, cell differentiation, cell proliferation and cell survival), the high interconnectivity between RCD pathway (potentially leading to the activation of compensatory RCD variants in response to the inhibition of a specific RCD type), the low specificity and selectivity of the inhibitors developed so far (exemplified by the broad-spectrum caspase inhibitors) and the difficulty to precisely determine and quantify cell death in vivo. RCD regulated cell death.

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All these issues should also be kept under consideration in the context of the present review, in which the NCCD aims at critically discussing a large amount of pre-clinical data in support of a key role for the apoptotic machinery in mammalian diseases. Specifically, the interpretation of results of genetic and pharmacological experiments presented herein should place particular attention on the aforementioned connectivity amongst different RCD variants as well as on discriminating between essential vs. accessory aspects of cell death [14]. Another issue to be considered is the fact that most conclusions are based on use of knockout/congenic mice which often present other passenger mutations potentially influencing the observed phenotype [73]. Our objective is not only to provide a critical summary of the existing literature, but also to offer an updated framework for interpretation of these findings in view of currently accepted models of RCD signaling.

Intrinsic apoptosis in disease

There are substantive supporting data from genetic studies to demonstrate that the molecular machinery for intrinsic apoptosis (described in Box 1 and Fig. 2) is involved in embryonic and fetal development as well as in adult tissue homeostasis. Numerous preclinical studies in animal models of disease demonstrate that intrinsic apoptosis contributes to etiology in various disorders involving the loss of not only post-mitotic, but also non-post-mitotic tissues, including neurological, cardiac, renal, hepatic, autoimmune/inflammatory, oncological, and infectious conditions. However, as discussed above, the interpretation of these results should be taken with caution given the high interconnectivity of RCD pathways and the crosstalk between RCD and inflammatory response. Moreover, the activation of executioner caspases occurs after cells are already committed to intrinsic apoptosis [15, 16]. Accordingly, caspase inhibition only delays the execution of cell death. In this context, the phenotypes observed under apoptotic caspase-deleted or inhibited conditions may reflect cell-extrinsic effects of caspase activity such as the release of immunomodulatory and cytotoxic signals from dying/dead cells, including damage-associated molecular patterns (DAMPs) or cytokines (this concept is extensively discussed in [14]). These phenotypes may also stem from the lack of processes independent of intrinsic (or extrinsic) apoptosis, as, for instance, the lack of CASP3-mediated cleavage of gasdermin E (GSDME) leading to impaired pyroptosis and associated inflammatory response [74, 75].

Fig. 2: Molecular machinery of the intrinsic apoptosis.
figure 2

Intrinsic apoptosis can be activated by a range of extracellular or intracellular stimuli, including, but not limited to, DNA damage, endoplasmic reticulum (ER) or oxidative stress, growth factor withdrawal or microtubular alterations. The critical step of the intrinsic apoptosis is the activation of the pro-apoptotic effectors of the BCL2 family, BAX, BAK and possibly BOK, which drives the outer membrane permeabilization (MOMP) and commits cells to death. MOMP results in the release from the mitochondrial intermembrane space into the cytosol of proapoptotic proteins, including CYCS and SMAC. CYCS assembles with APAF1, dATP and pro-CASP9 into the apoptosome, leading to the activation of CASP9, which in turn promotes the activation of the executioner caspases CASP3 and CASP7. The activation of the executioner caspases is facilitated by SMAC, which sequesters and/or degrades members of IAP family that inhibit apoptosis.

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Below, we will provide details of the pro-apoptotic BCL2 proteins, the anti-apoptotic BCL2 proteins, the components of the apoptosome—a platform for the activation of initiator caspases composed of cytochrome c, somatic (CYCS), apoptotic peptidase activating factor 1 (APAF1) and pro-CASP9—and effector caspases in disease. The instances of involvement encompass participation in the pathogenic mechanisms as well as experimental deletion or inhibition as a means of exploring potential utility as treatment targets. The effects of these regulators and effectors of the intrinsic apoptosis pathway on health are described in Box 2, Box 3 and Box 4.