Chapter 15: Apoptosis Mechanisms: Relevance to the Hematopoietic System

It is now well established that defects in the normal mechanisms that control programmed cell death (PCD) occur commonly in human diseases. Cell numbers in the body are governed not only by cell division, which determines the rate of cell production, but also by cell death, which dictates the rate of cell loss. In the course of a typical day, an average adult human produces, and in parallel eradicates, approximately 50 to 70 billion cells, representing approximately 1 million cells per second. Normally, these two processes of cell division and cell death are tightly coupled so that no net increase in cell numbers occurs, or so thatsuch increases represent only temporary responses to environmental stimuli. However, alternations in the expression or function of the genes that control PCD can upset this delicate balance, contributing to or causing disease.

In most cases, PCD occurs by apoptosis. Apoptosis is defined by its morphologic features. As viewed with the assistance of the light- (or, preferably, electron-) microscope, the characteristics of the apoptotic cell include chromatin condensation and nuclear fragmentation (pyknosis), plasma membrane blebbing, and cell shrinkage. Eventually, the cell breaks into small membrane-surrounded fragments (apoptotic bodies), which are cleared by phagocytosis, without inciting an inflammatory response. The release of apoptotic bodies is what inspired the term “apoptosis” from the Greek, meaning “to fall away from” and conjuring notions of the falling of leaves in the autumn from deciduous trees.¹

In recent years, the molecular machinery responsible for apoptosis has been elucidated, revealing a family of intracellular proteases, caspases (cysteine aspartyl proteases), which are responsible directly or indirectly for most of the morphologic and biochemical changes that characterize the phenomenon of apoptosis.^2,3 Diverse regulators of the caspases have also been discovered, including activators and inhibitors of these cell death proteases. Inputs from signal transduction pathways into the core of the cell death machinery have also been identified, demonstrating ways of linking environmental stimuli to cell death responses or cell survival maintenance. Knowledge of the molecular mechanisms of apoptosis is providing insights into the pathogenesis of many diseases, revealing strategies for possible novel treatments.

Intracellular proteases called caspases are responsible for most of the morphologic changes that we recognize as “apoptosis,” as well as many of the biochemical changes often associated with this route of cell demise. Specifically, activation of a family of intracellular cysteine proteases that cleave their substrates at aspartic acid residues, known as “caspases” for cysteine aspartyl-specific proteases.⁴ These proteases are present as inactive zymogens in essentially all animal cells, but can be triggered to assume active states, generally involving their proteolytic processing at conserved aspartic acid (Asp) residues. During activation, the zymogen proproteins are cleaved to generate the large (~20 kDa) and small (~10 kDa) subunits of the active enzymes, typically liberating an N-terminal prodomain from the processed polypeptide chain. The active enzymes consist of heterotetramers composed of two large and two small subunits, generally with two active sites per molecule.^2,3

The observation that caspases cleave their substrates at Asp residues and are also activated by proteolytic processing at Asp residues makes evident that these proteases collaborate in proteolytic cascades, where caspases activate themselves and each other. Humans contain 11 caspases. They can be subgrouped according to either their amino-acid sequence similarities or their protease specificities.

From a functional perceptive, it is useful to view the caspases as either upstream “initiator” caspases or downstream “effector” caspases.⁵ The zymogen forms of upstream initiator caspases possess large N-terminal prodomains, which function as protein interaction modules, allowing them to interact with various proteins that trigger caspase activation. In contrast, the proforms of downstream effector caspases contain only short N-terminal prodomains, serving no apparent function. Downstream caspases are largely dependent on upstream caspases for their proteolytic processing and activation. The substrates of effector caspases are myriad, as revealed in recent years by unbiased proteomics approaches. Substrates include cytoskeletal and nuclear matrix proteins, chromatin-modifying (e.g., poly-ADP ribosyl polymerase [PARP]) and DNA repair proteins, inhibitory subunits of endonucleases (CIDE-family proteins), protein kinases (often separating the autorepressing regulatory domains from catalytic domains) and other signal transduction proteins.

Several pathways for activating caspases have been delineated (Fig. 15–1). The simplest is exploited by cytolytic T lymphocytes (CTLs) and natural killer (NK) cells, which introduce apoptosis-inducing proteases, particularly granzyme B (a serine protease), into effective intracellular compartments of target cells via perforin-dependent mechanisms.⁶ Unlike the caspases, granzyme B is a serine protease. However, similar to the caspases, granzyme B specifically cleaves its substrates at Asp residues. Granzyme B is capable of cleaving and activating multiple caspases and some caspase substrates.⁷ Endogenous and viral inhibitors of granzyme B have been identified, accounting for resistance to this apoptotic inducer.^8,9,10

Another caspase-activation pathway is represented by tumor necrosis factor (TNF) family receptors. Eight of the approximately 30 known members of the TNF family in humans contain a so-called death domain (DD) in their cytosolic tails.¹¹ Several of these DD-containing TNF family receptors use caspase activation as a signaling mechanism, including TNF receptor-1 (TNFR1)/CD120a; Fas/APO1/CD95; death receptor (DR)-3 (DR3)/Apo2/Weasle; DR4/ tumor necrosis factor–related apoptosis-inducing ligand receptor 1 (TRAILR1); DR5/TRAILR2; and DR6. Ligation of these receptors at the cell surface results in receptor clustering and recruitment of several intracellular proteins, including certain procaspases, to the cytosolic domains of these receptors, forming a “death-inducing signaling complex” that triggers caspase activation and leads to apoptosis.^12,13

The specific caspases summoned to the DISC are caspase-8 and, in some cases, caspase-10. These caspases contain so-called death effector domains (DEDs) in their N-terminal prodomains that bind to a corresponding DED in the Fas-associated death domain (FADD), a bipartite adapter protein containing a DD and a DED. FADD functions as a molecular bridge between the DD and DED domain families, and is, in fact, the only protein in the human genome with this dual domain structure. Consequently, cells from mice in which the fadd gene has been knocked out are resistant to apoptosis induction by TNF family cytokines and their receptors. Cells derived from caspase-8 knockout mice also fail to undergo apoptosis in response to ligands or antibodies that activate TNF family DRs, demonstrating an essential role for this caspase in this pathway.¹⁴ However, mice lack the highly homologous protease, caspase-10, which is found in humans, having arisen from an apparent gene duplication on chromosome 2.¹⁵ Thus, caspases 8 and 10 may play redundant roles in human cells.

Mitochondria also play important roles in apoptosis, releasing cytochrome c (Cyt-c) into the cytosol, which then causes assembly of a multiprotein caspase-activating complex, referred to as the “apoptosome.”^16,17 The central component of the apoptosome is Apaf1, a caspase-activating protein that oligomerizes upon binding Cyt-c and which specifically binds procaspase-9. Apaf1 and procaspase-9 interact with each other via their caspase recruitment domains (CARDs). Such CARD–CARD interactions play important roles in many steps in apoptosis pathways. In addition to Cyt-c, mitochondria also release several other proteins of relevance to apoptosis, including endonuclease G, AIF (an activator of nuclear endonucleases), and SMAC (Diablo) and Omi (HtrA), antagonists of a family of caspase-inhibitory proteins known as the IAPs (inhibitors of apoptosis) (see section “Inhibitors of Apoptosis” below).

The central importance of the Cyt-c–dependent pathway for apoptosis is underscored by the observation that cells derived from mice in which either the apaf1 or procaspase-9 genes have been ablated are incapable of undergoing apoptosis in response to agents that trigger Cyt-c release from mitochondria.¹⁸ Nevertheless, such cells can die by nonapoptotic routes,¹⁹ demonstrating that mitochondria control both caspase-dependent and caspase-independent cell death pathways. Moreover, distinguishing mitochondria-driven apoptotic from nonapoptotic cell death can be challenging in many contexts because of the similar morphologic features caused probably by some of the proteins released from these organelle such as endonuclease G and AIF, which promote chromatin condensation and DNA fragmentation. The mitochondrial mechanisms for apoptotic and nonapoptotic cell death are activated by myriad stimuli, including growth factor deprivation, oxidants, Ca²⁺ overload, DNA-damaging agents, microtubule-modifying drugs, and much more.^17,20 In this sense, mitochondria are sometimes viewed as central integrators of cell stress signals that dictate ultimately cell life and death decisions.

Mitochondria can also participate in cell death pathways induced via TNF family DRs, through crosstalk mechanisms involving proteins such as Bid, BAR, and Bap31.^21,22,23,24 However, mitochondrial (“intrinsic”) and DR (“extrinsic”) pathways for caspase activation are fully capable of independent operation in most types of cells.²⁵

Cell death mechanisms are also linked to endoplasmic reticulum (ER). In most cases, however, these ER-initiated signals ultimately seem to impinge on mitochondria as the downstream effectors of the cell death pathway. In this regard, the ER is a central regulator of intracellular Ca²⁺, and ER membranes form close contacts with mitochondria to create structures where Ca²⁺ effluxes from ER into mitochondria, thereby impacting mitochondrial function in profound ways that can either promote cell life or cause death. Too much Ca²⁺ entry into mitochondria, for instance, triggers a phenomenon called mitochondrial permeability transition (MPT) in which the organelles swell and eventually rupture.

However, in addition to the role of ER Ca²⁺ and mitochondria-driven cell death, another pathway for apoptosis has been linked to accumulation of unfolded proteins in the ER. Specifically, ER stress induces expression of the proapoptotic transcription factor CHOP, which, in turn, stimulates expression of DR5 (TRAILR2), causing caspase-8-dependent apoptosis.²⁶ Additionally, CHOP has been reported to directly stimulate transcription of the gene encoding Bim, a proapoptotic member of the Bcl-2 family (see section “Suppressors of Apoptosis” below) that stimulates Cyt-c release from mitochondria. Thus, ER stress has multiple potential routes of stimulating cell death pathways, with the predominant pathway probably varying among cell types and pathophysiologic contexts.

Although diverse mechanisms exist for activating initiator Caspases, as outlined above, in most instances, the biochemical mechanisms appear to be remarkably similar. Much of caspase activation and can be explained by the “induced proximity model,”²⁷ in which forcing dimerization of procaspases results in conformational states that promote protease activation, typically resulting in cleavage events that lock the proteases into their fully active state. This mechanism is clearly operative in the caspase-activation pathways induced by TNF family receptors (extrinsic pathway) and Cyt-c/mitochondria (“intrinsic pathway”).

Given the critical importance of making the correct choices about cell life–death decisions in complex multicellular organisms, it is not surprising that the pathways governing caspase activation are under exquisite control by networks of proteins that directly or indirectly communicate with these proteases. A delicate balance between proapoptotic and antiapoptotic regulators of apoptosis pathways is at play on a continual basis, ensuring the survival of long-lived cells and the proper turnover of short-lived cells in a variety of tissues, including the marrow, thymus, and peripheral lymphoid tissues. The antiapoptotic proteins responsible for creating roadblocks to cell death have been mapped to specific caspase-activation pathways.

BCL-2 FAMILY

The Bcl-2 family represents a large group of proteins (number >26 in humans) that control mitochondria-dependent steps in cell-death pathways, including dictating whether Cyt-c is or is not released from these organelles (Fig. 15–2). Both proapoptotic and antiapoptotic Bcl-2 family proteins have been delineated.²⁸ These proteins are best known for their roles in controlling the intrinsic (mitochondrial) cell-death pathway,²⁰ although effects on the ER-pathway for cell death have also been documented.²⁹ Even though the human genome encodes at least 26 Bcl-2 family proteins, only six of these are antiapoptotic (in humans, Bcl-2, Bcl-X_L [BCL2L1], Bcl-W [BCL2L2], Mcl-1 [BCL2L3], Bfl-1 [BCL2L5], and Bcl-B [BCL2L10]). Several types of animal viruses also harbor Bcl-2 family genes within their genomes, including herpes viruses implicated in cancer such as the Epstein-Barr virus (EBV) and Kaposi sarcoma virus (KSV). The relative ratios of anti- and proapoptotic Bcl-2 family proteins dictate the ultimate sensitivity or resistance of cells to various apoptotic stimuli, including growth factor deprivation, hypoxia, radiation, anticancer drugs, oxidants, and Ca²⁺ overload.

Various Bcl-2 family members play important roles in controlling the life spans of hematopoietic cells, as evidenced by phenotypes generated in genetically engineered mice (gene knockouts and transgenics) and also (in some cases) by human clinical experiences with experimental therapeutics targeting some of these proteins. For example, antiapoptotic protein Bcl-2 is required for survival of mature T cells and B cells, with deficiency of Bcl-2 causing lymphopenia. Conversely, the proapoptotic protein Bim is necessary for limiting expansion of T and B lymphocytes, with deficiency of Bim causing lymphocytosis. Bim also plays important role in eradicating autoreactive T cells in the thymus (“negative selection”), having important implications for mechanisms of autoimmune diseases.³⁰ Bcl-X_L is required for platelet homeostasis, such that either genetic or pharmacologically induced Bcl-X_L deficiency causes thrombocytopenia. Antiapoptotic protein Mcl-1 is particularly important for survival of the myeloid lineage in mice, as well as contributing to lymphocyte survival. Conversely, antiapoptotic protein Bcl-W is not required for hematopoiesis in mice, despite being widely expressed in myeloid lineage cells. It should be noted that direct comparisons of gene manipulations in mice with the human circumstance are not always possible because of genomic differences in the Bcl-2 family genes of mice versus humans (e.g., human Bfl-1 versus murine A1; human Bcl-B versus murine Boo/Diva).

Many members of the Bcl-2 family have a hydrophobic stretch of amino acids near their carboxyl-terminus that anchors them in the outer mitochondrial membrane.¹⁷ In contrast, other Bcl-2 family members such as Bid, Bim, and Bad, lack these membrane-anchoring domains, but dynamically target mitochondria in response to specific stimuli. Still others have the membrane-anchoring domain but keep it latched against the body of the protein until stimulated to expose it (e.g., Bax).³¹

Based on their predicted (or experimentally determined) three-dimensional structures, Bcl-2 family proteins can be broadly divided into two groups. One subset of these proteins is probably similar in structure to the pore-forming domains of bacterial toxins, such as the colicins and diphtheria toxin.^32,33,34,35 These α-helical pore-like proteins include both antiapoptotic proteins (Bcl-2, Bcl-X_L, Mcl-1, Bfl-1, Bcl-W, Bcl-B) and proapoptotic proteins (Bax, Bak, Bok, and Bid). Most of the proteins in this subcategory can be recognized by conserved stretches of amino acid sequence homology, including the presence of Bcl-2 homology (BH) domains, BH1, BH2, BH3, and sometimes BH4. However, this is not uniformly the case, as the Bid protein contains only a BH3 domain but has been determined to share the same overall protein-fold with Bcl-X_L, Bcl-2, and Bax.^33,34 Where tested to date, these proteins have all been shown to form ion-conducting channels in synthetic membranes in vitro, including Bcl-2, Bcl-X_L, Bax, and Bid,^{36,37,38,39,40} but the significance of this pore activity remains unclear.

The other subset of Bcl-2 family proteins appears to have in common only the presence of the BH3 domain, including Bad, Bik, Bim, Hrk, Bcl-G_S, p193, APR (Noxa), and PUMA. These “BH3-only” proteins are uniformly proapoptotic. Their cell-death–inducing activity depends, in most cases, on their ability to dimerize with antiapoptotic Bcl-2 family members, functioning as trans-dominant inhibitors of proteins such as Bcl-2 and Bcl-X_L.^41,42 However, some of these proteins (e.g., Bid, PUMA, Bim) can also interact with proapoptotic proteins (e.g., Bax, Bak), functioning as agonists of the killers, in addition to dimerizing with antiapoptotics (e.g., Bcl-2; Bcl-X_L) to function as antagonists of these cell-survival proteins (see Fig. 15–2).^28,43 Binding of Bid to Bax or Bak promotes insertion of these proteins into membranes where they oligomerize, apparently forming large pores through which molecules such as Cyt-c, SMAC, and Omi can escape from mitochondria or causing an increase in the permeability of the outer membrane of mitochondria through more complex mechanisms.^44,45 Bax and Bak thus induce mitochondrial outer membrane permeabilization (MOMP), which is a critical event that not only causes release of death-inducing mitochondrial proteins but also secondarily causes necrosis by uncoupling of oxidative phosphorylation (when Cyt-c becomes limiting) and diversion of electrons from the respiratory chain into production of toxic free radicals.^46,47

The BH3 domain mediates dimerization among Bcl-2 family proteins. This domain consists of an amphipathic α-helix of approximately 16 amino acids that inserts into a hydrophobic crevice on the surface of antiapoptotic proteins such as Bcl-2 and Bcl-X_L.⁴⁸ The BH3-only proteins link a wide variety of environmental stimuli to the mitochondrial pathway for apoptosis, with some examples outlined below.

In addition to mitochondria, mechanistic links for Bcl-2 family proteins to ER stress and autophagy have also been delineated. For example, Bax and Bak can bind the ER stress signaling protein, IRE-1, thereby stimulating its intrinsic autokinase activity and its endoribonuclease activity.⁴⁹ Proapoptotic (Bak/Bax) and antiapoptotic (Bcl-2/Bcl-X_L) family members also have opposing effects on basal ER Ca²⁺ levels, probably via effects on Ca²⁺ channel proteins in ER membranes (e.g., IP3Rs, BI-1, and TmBim3). Autophagy protein Beclin contains a BH3-like domain that mediates interactions with antiapoptotic Bcl-2 family proteins, which sequester Beclin and thereby reduce autophagic flux.⁵⁰ Autophagy, which is a lysosome-dependent catabolic pathway, can either promote cell survival by providing access to nutrients during times of nutrient insufficiency and hypoxia, or it can cause cell death when stimulated to an extreme.⁵¹

FLIP

The c-FLIP proteins are another type of apoptosis suppressor that operates via directly binding to certain caspases and their upstream activator, FADD. The c-FLIP gene of humans resides in a tandem gene cluster on chromosome 2, which contains the genes encoding procaspases 8 and 10, suggestive of gene duplication events. Two isoforms of c-FLIP are produced from a single gene, including the long form, which is highly similar in overall sequence to procaspases 8 and 10, containing tandem copies of DEDs, followed by a pseudocaspase domain that lacks enzymatic activity. The shorter isoform consists only of the DED domains, thus resembling analogous proteins encoded in the genomes of some mammalian viruses.⁵² FLIP-S is exclusively antoptotic whereas FLIP-L can be either pro- or antiapoptotic, depending on its levels of expression relative to procaspases 8 and 10.⁵³ In general, FLIP proteins form complexes with procaspases 8 and 10, preventing their dimerization and activation, as well as competing for binding to adapter protein FADD, which is required for caspase recruitment to DR complexes.^54,55 Thus, in most circumstances, FLIP proteins create blockades in the extrinsic pathway for apoptosis.

Additionally, a role in suppressing nonapoptotic cell death (necroptosis) has been described for FLIP in partnership with caspase-8.⁵⁶ In this regard, TNFR1 signaling has been shown to stimulate caspase-independent cell death in some circumstances (commonly called “necroptosis”) via a mechanism that is suppressed by FLIP and caspase-8 but that is dependent on the protein kinase Rip3 (see section “Inhibitors of Apoptosis” below). It is thought that dimers consisting of the longer isoform of c-FLIP plus caspase-8 direct the proteolytic activity of caspase-8 to substrates that promote cell survival rather than cell death.⁵⁷ Among the relevant substrates is the kinase Rip1, an upstream activator of Rip3. Thus, FLIP proteins play complex roles in cell death regulation mediated by various members of the TNF Receptor family.

INHIBITORS OF APOPTOSIS

The IAP proteins (n = 8 in humans) suppress apoptosis via a diversity of mechanisms, including directly binding to and inhibiting certain caspases.^58,59 IAPs are characterized by the presence of protein interaction domains called BIRs (baculovirus internal repeats), numbering between 1 and 3 per protein. Most IAPs also carry RING domains that endow them with E3 ligase activity through interactions with ubiquitin conjugating enzymes (UBCs). Some of the apoptogenic proteins released from mitochondria, notably SMAC and HtrA2, bind certain BIRs and thereby compete for protein interactions on the surface of IAPs. Some examples of IAP mechanisms are provided here.

XIAP (so-called because its encoding gene resides on the X-chromosome) contains 3 BIR domains. BIR2 of XIAP binds downstream effector proteases, caspases-3 and -7, to suppress apoptosis at a distal point. BIR3 of XIAP binds upstream initiator protease, caspase-9, to suppress an apical step in the mitochondrial pathway for apoptosis.

The c-IAP1 (BIRC2) and c-IAP2 (BIRC3) proteins are also capable of binding to caspases 3, 7, and 9, although they are less potent by far as direct enzymatic inhibitors and may rely on their E3 ligase activity for controlling caspase degradation. However, these IAP family members also participate in other cell death-relevant mechanisms by impacting signal transduction by TNF family receptors. Binding of TNF to one of its principal cellular receptors expressed widely on cells, TNFR1, is capable of triggering at least three different signaling pathways, each involving overlapping but distinct protein complexes that are assembled at the receptor (Fig. 15–3). One of these TNFR1-initiated pathways causes caspase activation and apoptosis by DISC assembly (described in the section “Caspase Activation Pathways,” and Fig. 15-1 above). Another pathway causes activation of the kinase Rip3, usually via the upstream kinase Rip1, which associates with TNFR1 complexes.⁶⁰ The Rip3-dependent cell death pathway is caspase-independent, leading to nonapoptotic cell death (“necroptosis”) through a process involving reactive oxygen species (ROS) generated by mitochondria. Another serine/threonine kinase, mixed-lineage kinase domain-like (MLKL) protein, appears to be a critical downstream mediator of Rip3-induced necroptosis. This Rip3-dependent pathway for necroptosis is suppressed by c-IAP1 and c-IAP2, probably via their roles as E3 ligases and possibly involving ubiquitin/proteasome-mediated reductions in Rip3 protein levels.⁶¹ Finally, TNFR1 stimulates a cell survival pathway in which c-IAP1 and c-IAP2 participate. In this TNFR1-mediated survival pathway, the kinase Rip1 comes together with the E3 ligases c-IAP1, c-IAP2, and tumor receptor-associated factor 2 (TRAF2) to stimulate noncanonical (lysine 63, rather than lysine 48) ubiquitination of Rip1, initiating a signal transduction pathway that causes activation of transcription factor nuclear factor (NF)-κB. NF-κB influences the expression of many target genes involved in host defenses and immune regulation, among which are several genes that suppress apoptosis. As a result, this NF-κB pathway nullifies the caspase pathway, negating apoptosis,⁶² in addition to accounting for the untoward inflammatory actions of this cytokine. Several antiapoptotic genes are among the direct transcriptional targets of NF-κB (REL)-family proteins, including the genes encoding c-FLIP, c-IAP2, Bcl-X_L, and Bfl-1.

In this regard, the c-IAP1 and c-IAP2 proteins were first identified because of their association with TNF receptor complexes. These IAPs bind the kinase Rip1 via their BIR3 domains, mediating noncanonical ubiquitination of Rip1 via interactions of atypical UBCs with their RING domains and possibly also indirectly via interactions of the E3 ligase TRAF2, which binds their BIR1 domains. The noncanonical ubiquitination of Rip1 is required for TNFR1-mediated NF-κB activation and suppression of cytokine-induced apoptosis. The c-IAP1 and c-IAP2 proteins also control the “alternative” pathway for NF-κB activation via yet another mechanism, which involves classical lysine 48 mediated polyubiquitination of the kinase Nik.

Additional members of the IAP family not described here (Survivin, Apollon/Bruce, ML-IAP, etc.) also have interesting mechanisms of interacting with components of cell-death pathways and they also can have other roles beyond cell-death regulation. For example, XIAP, c-IAP1, and c-IAP2 have other documented cellular activities, which include, for example, their interactions with kinases (e.g., Rip2) or kinase-binding adapter proteins (TAB/Tak) involved in processes such as innate immunity and morphogenesis. In these circumstances, the most relevant activity of IAPs appears to be their noncanonical E3 ligase activity, as well as a protein scaffold role where they serve as platforms for assembling multiprotein complexes. Additional roles for IAP family members include cell division, where, for example, the Survivin protein plays a fundamental role in chromosome segregation and cytokinesis.

Several of the IAPs are opposed by proteins released from mitochondria, SMAC and HtrA2. SMAC and Htra2 bind BIR domains on IAPs, thus displacing caspases and other associated proteins. In many cases, SMAC binding to IAPs induces their polyubiquitination and proteasomal degradation. Thus, factors that cause MOMP take the breaks off the caspases by eliminating various IAP family proteins.

Various receptor-mediated signal transduction pathways converge on the core components of the cell death machinery outlined above, including receptors for growth factors, lymphokines and cytokines. Some examples illustrating the intimate links between receptor-mediated signal transduction and apoptosis pathways are provided here (Fig. 15–4).

LYMPHOKINES

Many lymphokine receptors signal via Jak/STAT pathways. STAT family transcription factors are known to stimulate transcription of the BCL-X gene as at least one of their mechanisms of suppressing apoptosis. Examples with relevance to hematopoietic system include erythropoietin-mediated stimulation of survival of erythrocyte precursors and interleukin (IL)-3 and IL-7-mediasted stimulation of survival of B-lymphocyte progenitors (pro/pre–B-cells). Additionally, Jak family nonreceptor protein tyrosine kinases are capable of stimulating phosphatidylinositol 3’-kinase (PI3K) activity,⁶³ which, in turn, causes activation of Akt family kinases. The murine gene encoding Akt was first discovered by virtue of its similarity to the v-akt oncogene found in some murine leukemia viruses and through its activation in thymomas caused by retrovirus insertions near the c-akt gene.⁶⁴ Humans contain three AKT genes. Akt can phosphorylate multiple proteins within the core apoptosis machinery. For example, the proapoptotic Bcl-2 family member Bad is a target of Akt, where phosphorylation of Bad causes its sequestration by 14–3–3 family proteins, thus inhibiting Bad from heterodimerizing with Bcl-X_L.⁶⁵ Akt also can phosphorylate human caspase-9, blocking apoptosis downstream of mitochondria.⁶⁶ Another substrate of Akt that is relevant to apoptosis is forkhead transcription factors (FKHD). Some FKHD family members appear to control apoptosis, perhaps by affecting transcription of the gene encoding FasL.⁶⁷ Phosphorylation of FKHD by Akt prevents its entry into the nucleus.

CYTOKINES

Several cytokines stimulate the activation of NF-κB (REL) family transcription factors. NF-κB directly binds the promoters and induces expression of several antiapoptotic genes, including the BCL-2 family members BCL-X and BFL-1, the IAP-family member C-IAP2, and C-FLIP.⁶² Thus, elevations in NF-κB activity can increase cellular resistance to apoptosis, affecting (1) the Intrinsic (mitochondrial) pathway through elevations in antiapoptotic Bcl-2 family proteins, (2) the extrinsic (TNF family DR) pathway through upregulation of c-Flip, and (3) downstream common pathways involving effector caspases as a result of overexpression of c-IAP2.

The first example of NF-κB involvement in malignancy was provided by studies of the avian Rev-T retrovirus, a transforming retrovirus that causes rapidly fatal lymphomas in young chickens and which carries the v-Rel oncogene. The cellular homologue of this viral oncogene is C-REL, which encodes the p65 subunit of NF-κB. Amplification of the C-REL gene has been reported in non-Hodgkin lymphomas (NHLs), occurring particularly in diffuse large B-cell lymphoma and commonly associated with extranodal presentation.⁶⁸ Other genetic alterations associated with dysregulation of NF-κB include chromosomal translocations involving the I-κB family member BCL-3 in B-CLL.⁶⁹ I-κB family proteins bind and sequester NF-kB complexes, preventing the transcription factor from entering the nucleus.⁷⁰ Typically, I-κB is regulated by ubiquitin-mediated turnover by the 26S proteasome. Mutations in I-κB thus may enhance NF-κB activity, either by producing unstable proteins or reducing the affinity of I-κB for NF-κB. The anticancer activity of drugs that inhibit the proteasome, approved for multiple myeloma, may be attributable in part to suppression of I-κB degradation, thereby inhibiting NF-κB induction.⁷¹

GENOTOXIC STRESS

Gamma-radiation and many DNA-damaging anticancer drugs potently stimulate apoptosis of hematopoietic cells. The principal mediator of apoptosis induced by genotoxic stress is p53. The p53 protein is a tetrameric transcription factor, whose levels are controlled by the E3 ligase Mdm2. This transcription factor directly induces expression of BH3-only proteins Noxa (APR), Bid, and PUMA,^72,73,74 thus linking p53 to the death machinery. Additionally, p53 directly binds and transcriptionally activates the human BAX gene promoter. Loss of p53 activity occurs in many human malignancies by a variety of mechanism, including gene deletion, gene mutations that result in mutant p53 proteins lacking transcriptional activity, and MDM2 gene amplification. Small molecule drugs that block Mdm2 protein interaction with p53 have shown promising preclinical activity against hematopoietic malignancies.

Interestingly, in addition to its role as a nuclear transcription factor, evidence has emerged suggesting that p53 may promote apoptosis also via nontranscriptional mechanisms under some circumstances. Specifically, a cytoplasmic pool of p53 reportedly associates with mitochondria, directly inducing activation of the Bax and inhibiting Bcl-2 and Bcl-X_L.⁷⁵ Importantly, even mutant p53 is capable of activating this cell-death pathway, raising hopes of finding pharmacologic interventions that would entice mutant p53 to attack mitochondria and trigger apoptosis of cancer cells in which this important tumor-suppressor gene product has suffered somatic mutations that inactive its nuclear (transcriptional) functions.

Either insufficient or excessive apoptosis plays important roles in a wide diversity of hematologic diseases. Convincing evidence has gathered to support a major role for insufficient apoptosis in the context of most (if not all) hematologic malignancies, where defects in apoptosis prolong cell life span and thereby promote cell accumulation, as well as complementing the proapoptotic effects of certain oncogenes (e.g., C-MYC; CYCLIN-D1), permitting growth factor independent cell survival, and promoting resistance to chemotherapy, radiation, and immune-mediated cell killing. Defects in apoptosis also seem to underlie some aspects of autoimmunity, where a failure to eradicate autoreactive lymphocytes occurs. Conversely, excessive apoptosis has been implicated in the depletion of CD4+ T-lymphocytes seen in chronic HIV infection, bacteria-mediated killing of macrophages, myelodysplastic disorders where marrow failure occurs resulting in anemia and failed myelopoiesis, and many other conditions. Some examples of the human diseases that have been linked to genomic alterations of apoptosis genes are highlighted here, without an attempt to be comprehensive.

ALTERNATIONS IN APOPTOSIS GENES IN HEMATOLOGIC MALIGNANCIES

Defects in apoptosis (cell death) are recognized as one of the hallmarks of essentially all cancers. Not surprisingly, therefore, myriad examples of alternations in genes regulating the core apoptosis machinery have been delineated in human cancers. Hematologic malignancies have, in fact, revealed many of the first and most striking examples of the critical importance of cell death as a constraint to inappropriate cell expansion and accumulation. Here, some illustrative examples are provided without an attempt to be comprehensive, particularly focusing on the families of apoptosis-regulating genes outlined above with emphasis on genomic alterations. Additionally, myriad epigenetic mechanisms modulate the expression of apoptosis genes in hematologic malignancies, which are not covered here.

BCL-2 FAMILY

The Bcl-2 family derives its name from discovery of the founding member as a result of its involvement in B-cell lymphomas and leukemias. The human BCL-2 gene is involved in t(14;18) chromosomal translocations found commonly in NHLs. In this regard, the BCL-2 gene normally resides on chromosome 18, but it becomes merged with the immunoglobulin heavy-chain (IgH) locus on chromosome 14, probably as a result of aberrant actions of the V-D-J gene recombination machinery responsible for antibody generation in B-cells. In this context, the BCL-2 gene becomes deregulated in its expression via the powerful cis-acting enhancer elements of the IgH locus. Chromosomal translocations activating BCL-2 occur in most indolent NHLs (especially follicular B-cell lymphomas) as well as a substantial proportion of aggressive NHLs (perhaps commonly arising from progression of a previously undiagnosed low-grade B-lymphoma). Gene amplification provides another mechanism for BCL-2 gene dysregulation, and is reported in nearly 20 percent of aggressive B-NHLs, particularly diffuse large B-cell lymphomas (DLBCLs). Thus, by studying the cytogenetics of B-cell malignancies, the world’s first example of an antiapoptotic gene (BCL-2) was discovered.

Additionally, loss of genes encoding microRNAs (miRNAs) that posttranscriptionally suppress BCL-2 by inducing Bcl-2 mRNA degradation accounts for the widespread dysregulation of BCL-2 gene in B-cell chronic lymphocytic leukemia (B-CLL). In this context, approximately 90 percent of B-CLLs have homozygous loss of function mutations or deletions involving miRNA15 and miRNA16 on chromosome 13q14, thus derepressing BCL-2 gene expression. This somatic loss of miRNA15 and miRNA16 genes in CLL was the first example of miRNA genes operating as tumor suppressors.

Genetic lesions responsible for dysregulation of other members of the Bcl-2 family have also been identified in various types of hematologic and nonhematologic malignancies. Among these somatic genetic mechanisms is the amplification of the BCL-X (BCL2L2) or MCL-1 (BCL2L3) gene loci, which occurs probably in one in 10 human solid tumors. Conversely, homozygous gene mutations that inactivate the proapoptotic BAX gene have also been identified in occasional hematopoietic malignancies and some solid tumors. In this regard, deletions or inactivating mutations in the tumor-suppressor p53 also reduce the expression of several proapoptotic Bcl-2 family genes, including the BAX, PUMA, NOXA, and BID genes, which are direct transcriptional targets of p53 (see above).⁷⁶ The incidence of p53 gene deletions and mutations varies among hematologic malignancies, ranging from rare (<5 percent) in T-cell leukemias and low-grade B-cell lymphomas to frequent (>30 percent) in disorders such as high-grade B-cell lymphomas, Burkitt lymphomas, and relapsed/aggressive acute lymphocytic leukemia (ALL), chronic lymphocytic leukemias (CLLs) that have progressed to Richter syndrome, and chronic myelogenous leukemias (CML) in blast crisis.⁷⁷

INHIBITORS OF APOPTOSIS

Genomic lesions involving the IAP family genes are also associated with hematologic malignancies. For example, in marginal zone mucosa-associated lymphoid tissue (MALT) B-cell lymphoma, the most common of the extranodal NHLs, t(11;18)(q21;q21) chromosomal translocations occur frequently.⁷⁸ These translocations fuse the three BIR domains of c-IAP2 with portions of the gene encoding MALT1, a caspase-like protein. The predominant mechanism by which the resulting c-IAP2/MALT1 fusion protein suppresses apoptosis appears to be hyperactivation of NF-κB. In this regard, the BIR1 domain of c-IAP1 binds NF-κB–inducing E3 ligase TRAF2, and this interaction has been shown to be critical for NF-κB induction by c-IAP2/MALT1 fusion proteins. Additionally, the C-terminal region of MALT1 also binds a related NF-κB–inducing E3 ligase, TRAF6, making additional contributions to NF-κB stimulation. Interestingly, TRAF2 gene amplification has also been described in a significance number of human malignancies.

CASPASES

Inactivating mutations have been described in a variety of cancers. In approximately 15 percent of NHLs, mutations that alter the activity of caspase-10 have been reported. The resulting mutant caspase-10 proteins may operate as dominant-negative inhibitors of DR-mediated apoptosis.⁷⁹ Although exhaustive analysis has not been performed to date, overall mutations inactivating caspase-encoding genes appear to be relatively rare in hematopoietic malignancies, though epigenetic silencing may be more common.

TUMOR NECROSIS FACTOR FAMILY DEATH RECEPTORS

Somatic mutations in the FAS (CD95) gene have been found in multiple myelomas (MMs) and NHLs.⁸⁰ Missense mutations within the DD of Fas (CD95) were associated with retention of the wild-type allele, suggesting a dominant-negative mechanism, whereas missense mutations outside the DD were associated with allelic loss.⁸⁰ The observation that tumor-suppressor p53 can induce transcription of the DRs Fas (CD95) and DR5 (TRAILR2) in some types of tumor cells,^81,82 suggests an additional cancer-relevant mechanism by which reductions in the expression TNF family DRs could occur in human malignancies, namely, secondarily to genomic lesions that inactivate p53 or that cause overexpression of endogenous p53 antagonists.⁸³

OTHER GENOMICALLY BASED DISEASES INVOLVING APOPTOSIS GENES

Hereditary deficiency of XIAP is a very strong risk factor for early onset inflammatory bowel disease (IBD). This function of XIAP is probably not related to its role in apoptosis, but rather stems from the function of XIAP as a component of NACHT and Leucine rich repeat domain-containing receptor (NLR) family protein complexes involved in innate immunity. Causative mutations have been identified in the FAS (CD95) gene of humans in patients with autoimmune lymphoproliferative syndrome (ALPS), also known as Canale-Smith syndrome.⁸⁴ Thus, the Fas/Fas ligand (FasL) system plays a critical role in lymphocyte homeostasis in vivo. At least some of the mutant Fas proteins found in humans with ALPS have been shown to operate as trans-dominant inhibitors of wild-type Fas, probably explaining the dominant inheritance pattern of this disorder. Likewise, germline mutations in Fas and FasL have been discovered as the underlying basis for the lymphoproliferative autoimmune phenotype of lpr/lpr and gdl/gdl strain mice, respectively.^85,86 In contrast to humans, however, the mutations in the fas gene of lpr-strain mice produce disease with a recessive inheritance pattern.⁸⁵ FasL, like most TNF family members, is a trimer, and the receptor also forms trimers and probably higher-order oligomers, thus explaining why some Fas mutants display dominant-negative effects on wild-type Fas while others do not. Indeed, mutant versions of Fas from some patients with hereditary ALPS have been demonstrated to antagonize wild-type Fas,⁸⁷ probably forming mixed oligomers of wild-type and mutant molecules. Additionally, mutations in caspase-10 gene that produce altered proteins that interfere with Fas-induced apoptosis have been identified in patients with ALPS.⁸⁸

These examples of autoimmune disorders associated with hereditary alternations in apoptosis-regulatory genes highlight the intricate linkages between cell death regulation and host–pathogen interactions. Many components of the apoptosis machinery play important roles in aspects of innate and adaptive immunity, possibly reflecting the notion that altruistic cell suicide may be the best defense against pathogens for multicellular organisms. Moreover, a subfamily of the caspases (e.g., caspases 1, 4, and 5 in humans) has as its primary role the proteolytic processing and activation of inflammatory cytokines (pro–IL-1β, pro–IL-18, etc.), with apoptosis representing a secondary function that is observed mostly in the context of excessive stimulation.

Programmed cell-death mechanisms play critical roles in hematopoietic and immune cell homeostasis. Elaboration of the complex cell-death pathways and the networks of proteins that modulate these pathways have provided insights into the underlying mechanisms of cell life–death decisions, although many mechanistic details remain still to be revealed. In several cases, the available information has already sparked efforts to translate the base of information into therapeutic strategies. Among the experimental therapeutics targeting components of the cell death machinery that have entered clinical testing are small molecule inhibitors of Bcl-2 family and IAP family proteins, as well as large molecule modulators of TNF family DRs. Compounds that modulate upstream inputs into cell death pathways are also advancing through clinical evaluations, such as small molecule inhibitors of Mdm2 (which cause p53 protein accumulation) and small molecule inhibitors of Akt family kinases. Many other targets within the cell-death pathways are the subject of preclinical drug discovery efforts today. The stage is thus set for continued progress in manipulating cell-death pathways for therapeutic benefit of multiple diseases, including disorders of the hematopoietic system.