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Chapter 57 Pharmacology and Molecular Mechanisms of Antineoplastic Agents for Hematologic Malignancies 871

to mutually cleave each other. Processing of caspase 8 removes the inhibiting its activation. Overexpression of XIAP inhibits anticancer
DED-containing prodomain, thus releasing the activated protease drug (including ara-C)–induced caspase activity and apoptosis. In
into the cytosol, where it can cleave and activate other downstream contrast, downregulation of XIAP sensitizes cancer cells to apoptosis
procaspases. In certain types of cells (type I), enough caspase 8 is induced by chemotherapeutic drugs. The antiapoptotic activity of
activated by the activation of the death receptor to cause apoptosis. NFκB has also been shown to be mediated by the induction of IAPs.
In other cell types, such as hepatocytes (type II), a sufficient level of The first four amino acids of the mature SMAC, Ala-Val-Pro-Ile
caspase 8 activation is not achieved and the apoptotic signal is ampli- (AVPI), bind to the BIR domain of IAP proteins. The four amino
fied by the mitochondrial pathway. The active caspase 8 cleaves the acids (AVPI) of SMAC that bind to the BIR3 domain of XIAP are
cytosolic p22 Bid into a BH3-only domain-containing, proapoptotic, similar to the XIAP-binding sequence of active caspase 9. In addition,
truncated p15 tBid fragment, which translocates to the mitochondria SMAC has been shown to form a stable complex with the BIR2
and triggers the release of cyt c into the cytosol. Cleavage by caspase domain of XIAP. SMAC binds to the linker-BIR2 domain and pre-
8 has been shown to cause exposure of a glycine residue on p15 Bid sumably disrupts its inhibition of the active caspase 3 and caspase 7
that is myristoylated, thereby targeting p15 Bid to the mitochondria. by steric hindrance. On induction of apoptosis, another mitochon-
Thus, N-myristoylation acts as an activating switch that enhances drial protein, Omi/HtrA2 (a serine protease), is released from mito-
tBid-induced release of cyt c and apoptosis. The ability to cleave Bid chondria and can induce caspase-independent cell death. Additionally,
may not be limited to caspase 8. Other caspases, such as caspase 3, like SMAC, the mature Omi protein contains a conserved IAP-
as well other proteases, such as granzyme B and lysosomal proteases, binding motif (AVPS) at its N-terminus. Omi complexes with XIAP
have been shown to activate Bid. This indicates that Bid probably at high stoichiometry and promotes apoptosis by neutralizing the
serves to amplify the caspase cascade rather than to initiate it (see Fig. caspase-inhibitory effect of XIAP. Recently, Omi has also been shown
57.9). to process XIAP and promote its proteasomal degradation.
Fas ligation has also been shown to initiate an alternative pathway The Bcl-2 family of proteins is divided into three subfamilies.
leading to apoptosis, involving activation of JNK/SAPK. After liga- Members of the subfamily that include Bcl-2, Bcl-x L , and Mcl-1
tion, Fas recruits an adaptor protein called Daxx that interacts with inhibit apoptosis (see Fig. 57.9). The Bax subfamily members that
the apoptosis signal-regulating kinase 1 (ASK1), activating the tran- promote apoptosis, including Bax and Bak, share with Bcl-2 three of
scription factors AP-1 and ATF-2. After activation, ASK-1 launches the four Bcl-2 homology domains, BH1–BH3. A C-terminus hydro-
a phosphorylation cascade that culminates in the activation of JNK. phobic tail is responsible for the localization of these proteins to the
Activated JNK phosphorylates substrates such as c-Jun, p53, and a outer membranes of the mitochondria and ER. The third BH3-only
pro-death Bcl-2 member Bim. This, in turn, has been shown to subfamily of proteins that includes Bid, Bim, and Bad also promotes
trigger mitochondrial or death receptor-initiated apoptotic apoptosis by binding and inactivating prosurvival Bcl-2 family
signaling. members. The prosurvival Bcl-2 family members, to a variable degree,
are bound to the membranes of the mitochondria, the ER, and the
nuclear envelope. In the event of an apoptotic stimulus, BH3-only
Mitochondria-Initiated Apoptotic Signaling proteins require Bax and Bak to trigger mitochondrial apoptotic
signaling. For example, the exposed BH3 domain of tBid oligomer-
Mitochondria sequester a potent cocktail of proapoptotic proteins. izes with Bak and Bax, causing their mitochondrial membrane
These proteins promote apoptosis by activating caspases (e.g., by cyt insertion and cyt c release. In response to apoptotic stimuli, Bax and
c), by inducing DNA fragmentation (e.g., by endonuclease G or Bak undergo conformational change and form membrane-associated
apoptosis-inducing factor [AIF]), or by neutralizing cytosolic inhibi- homo-oligomers, disrupting the outer membrane of the mitochondria
tors of apoptosis (IAPs; e.g., by SMAC or Omi/HtrA2). On induction and ER and releasing the pro-death molecules into the cytosol.
of apoptosis, unlike other proapoptotic proteins, mitochondrial AIF Besides tBid, Humanin peptide has been shown to activate Bax. Bcl-2
translocates to the nucleus to induce DNA fragmentation and apop- can block these events involving Bax activation by titrating tBid or
tosis. Cyt c is a well-known component of the mitochondrial electron Bim and/or heterodimerization with Bax and preventing mitochon-
transfer chain. Cyt c is released from the mitochondria during drial permeabilization.
wt
apoptosis. After release into the cytosol, cyt c binds to apoptosis- Induction of P53 in cells triggers apoptosis by transcriptional
activating factor (Apaf-1), a cytosolic adapter protein that contains a activation of prodeath effectors, including Bax, Noxa, Puma, Apaf-1,
caspase recruitment domain (CARD), a nucleotide-binding domain, and DR5, and by transcriptional repression of Bcl-2 and IAPs. p53-
and multiple WD-40 repeats. Binding of cyt c to Apaf-1 increases its dependent apoptosis has also been shown to occur in the absence of
affinity for dATP or ATP by approximately 10-fold. It also triggers any gene transcription or translation. In response to apoptotic stimu-
the oligomerization of Apaf-1 into a multimeric Apaf-1–cyt c lus such as irradiation, p53 translocates to the mitochondria, where
complex, also called the apoptosome. This exposes the CARD domain it directly induces permeabilization of the outer membrane by
of Apaf-1, which recruits several molecules of procaspase 9, inducing forming complexes with the protective Bcl-2, resulting in the release
their autoactivation. Only the caspase 9 bound to the apoptosome is of cyt c into the cytosol. The E2F transcription factor, normally
able to efficiently cleave and activate a downstream executioner restrained by the Rb tumor suppressor to inhibit cell proliferation,
caspase, caspase 3. has been shown to induce apoptosis though p53-dependent and
SMAC/DIABLO is a 25-kDa mitochondrial protein that is p53-independent mechanisms. These mechanisms include transcrip-
released by mitochondria into the cytosol during apoptosis. SMAC tional activation of ARF (the alternate reading frame product of the
contains a mitochondria-targeting sequence at its N-terminus. This INK4a/ARF tumor suppressor locus) or p73 (a member of the p53
sequence is removed on import into the mitochondria, generating family), repression of Mcl-1, and inducing the levels of caspase
the mature SMAC protein. In the mitochondria-initiated and proenzymes.
common effector pathways of apoptosis, the processing and proteo-
lytic activity of caspase 9 followed by caspases 3 and 7 are inhibited Selective Antiapoptotic Agents or Strategies
by the IAP family of proteins. IAP family members include XIAP,
cIAP1, cIAP2, and survivin. All IAPs contain at least one baculovirus
repeat (BIR) domain, although some contain three. Another region, BCL2 Family of Proteins as Targets for Anticancer
the RING domain, has ubiquitin ligase activity and promotes the Drug Design
self-degradation of IAPs through proteasomes in response to some
apoptotic stimuli. Furthermore, during Fas death receptor-mediated The antiapoptotic protein BCL2 was originally identified during
apoptosis, XIAP is cleaved by activated caspase 3 into the N-terminal the process of the discovery of the t(14;18) in human follicular
BIR1 and 2 and BIR3-RING finger fragments. The BIR3 domain lymphoma. Overexpression of BCL2 has been reported in both
binds and sequesters the monomeric and inactive caspase 9, thereby hematologic and solid malignancies and to mediate resistance to