Targeting apoptosis in cancer therapy

Abstract

For over three decades, a mainstay and goal of clinical oncology has been the development of therapies promoting the effective elimination of cancer cells by apoptosis. This programmed cell death process is mediated by several signalling pathways (referred to as intrinsic and extrinsic) triggered by multiple factors, including cellular stress, DNA damage and immune surveillance. The interaction of apoptosis pathways with other signalling mechanisms can also affect cell death. The clinical translation of effective pro-apoptotic agents involves drug discovery studies (addressing the bioavailability, stability, tumour penetration, toxicity profile in non-malignant tissues, drug interactions and off-target effects) as well as an understanding of tumour biology (including heterogeneity and evolution of resistant clones). While tumour cell death can result in response to therapy, the selection, growth and dissemination of resistant cells can ultimately be fatal. In this Review, we present the main apoptosis pathways and other signalling pathways that interact with them, and discuss actionable molecular targets, therapeutic agents in clinical translation and known mechanisms of resistance to these agents.

Figure 1.. Overview of apoptosis signaling pathways and pro-survival, immune, or tumor microenvironment influences that impact on them.

Intrinsic mitochondrial apoptosis involves proapoptotic BCL-2 family members (e.g. BAX or BAK) located in the mitochondrial outer membrane promoting mitochondrial outer membrane permeability (MOMP), cytochrome c release, caspase activation and apoptosis. Cytochrome c binds APAF1, and in an ATP-dependent process, the multimeric apoptosome is formed. Procaspase 9 binds to the apoptosome, gets cleaved and activated, and then activates caspase 3 . Members of the IAP family including XIAP negatively regulate caspase activation and they can be inactivated by SMAC. Anti-apoptotic members of the BCL-2 family (e.g. BCL-2, BCL-XL, MCL1, Bcl-w, A1) inhibit the proapoptotic BCL-2 family members (e.g. BAX and BAK). BH3-only proteins such as BIM, BAD, BID, PUMA and NOXA can inhibit MCL1. It is important to note that within the intrinsic apoptosis pathway, mitochondrial permeabilization represents the point of commitment to cell death (irrespective of caspase activity). The extrinsic apoptosis pathway is depicted at the cell membrane as a trimeric TNF family receptor (Fas-R, TRAIL-R1, TRAIL-R2, TNF-R1, TNF-R2) that is bound by the respective ligand with proteins FADD and caspase 8 recruited to form the death-inducing signaling complex (DISC). Caspase 8 or 10 activation at the DISC results in Bid cleavage which provides cross-talk to the mitochondria in Type II cells which amplifies the death signal at the level of cytochrome c leading to apoptosis. c-FLIP binds to caspase 8 and prevents its activation. Prototypical growth factor activated receptor tyrosine kinases (RTKs) are shown in the cell membrane as dimeric proteins (e.g. members of the ERBB family such as EGFR, Her2, ERBB3) with downstream events involving PI3K and Akt activation. The RAS-MAPK pathway is also depicted. These pro-survival pathways also promote cell proliferation and negatively impact on the intrinsic cell death pathway, in part, through phosphorylation of BCL-2 family members. TNF and TRAIL receptors can activate NFkB which transcriptionally activates anti-apoptotic genes in the nucleus thereby attenuating cell death and promoting cell survival. Immune mechanisms can activate cell death through the extrinsic pathway, e.g. through TRAIL produced by Natural Killer (NK) cells in response to Interferon action, or through the blockade of cell death ligands, e.g. PD-L1. Immune cells such as cytotoxic T-cells or NK cells also produce granzyme which is a serine protease that induces apoptosis in target cells such as cancer cells. A protein called perforin forms pores in target cells to mediate granzyme-induced cell death. Various other components of the tumor microenvironment including stroma, fibroblasts and physical factors can impact cell death and cell survival pathways in ways that impact on drug responses. Drugs targeting endothelial cells can deprive tumor cells of nutrients leading to cell death. DNA damage response (e.g. those involving checkpoint kinases such as ATM/ATR, Chk1/Chk2) and cell stress response pathways (e.g. the integrated tress response leading to ATF4 and CHOP activation) ultimately activate proapoptotic genes that induce cell death, in addition to p53. Among the transcribed genes following p53 activation, Bax, Puma, Noxa, DR5, and FasL are noteworthy, but it should also be noted that many kinase inhibitors ultimately signal transcriptional activation of Bim. Additional cellular processes (e.g. autophagy, anoikis, ferroptosis, regulated necrosis) can cross-talk with apoptosis signaling pathways and are impacted by cancer therapeutics including those used in combinations with proapoptotic agents.

Figure 2.. Cancer therapeutic approaches targeting apoptosis pathways.

Death receptors 4 and 5 (DR4, DR5) can be activated by agonist antibodies such as mapatumumab, conatumumab, Apo2L/TRAIL, ABBV-621 and GEN1029, fusion protein of recombinant TRAIL with IgG1 (MM-201) or pegylated recombinant TRAIL (TLY012). BH3 mimetics bind to anti-apoptosis BCL-2 family members (BCL-2, BCL-XL, MCL1) leading to activation of BAX and BAK that form pores in the outer mitochondrial membrane resulting in release of cytochrome C. Selective inhibitors of BCL-2 (venetoclax, S55746, APG-2575), BCL-XL (ABBV-155) and MCL1 (AMG-176, S64315, AZD5991) have reached clinical trials. Modulation of growth signaling pathways also induces apoptosis and combinations of MEK inhibitors with navitoclax (BCL-2/BCL-XL inhibitor) are being investigated in clinical trials. Pro-survival pathways such as AKT and MEK inhibit apoptosis by phosphorylation of proapoptotic proteins such as BAD (BCL-2-associated agonist of cell death) and BIM (BCL-2-interacting mediator of cell death). Strategies targeting the p53 pathway to induce apoptosis include MDM2 inhibitors (i.e., idasanutlin, AMG-232, APG-115, DS-302b, BI-907828, ALRN-6924). The ONC201 which has a three-ring imipridone structure has been found to bind to dopamine receptors DRD2 and DRD3 as well as the mitochondrial caseinolytic protease P (CIpP) leading to activation of integrated stress response protein ATF4 ^,. ATF4 activation by ONC201 leads to CHOP-dependent DR5 upregulation and cell death ^,. The relationship between ClpP and the other pathways engaged by ONC201 remains under study although the binding appears to be upstream of ATF4 activation (Lanlan Zhou and W.S.E-D., unpublished observations). SMAC (second mitochondria-derived activator of caspase) is released from mitochondria together with cytochrome c. Cytochrome s forms the apoptosome complex with caspase 9 and APAF-1 (apoptotic protease-activating factor 1). Inhibitors of apoptosis proteins (IAP) prevent caspase activation and this is blocked by SMAC or Omi/HtrA2 which is also released from mitochondria. SMAC mimetics and inhibitors of IAP (i.e., LCL161, birinapant, Debio 1143) have transitioned to clinical trials. tBid: truncated BH-3 interacting domain death agonist; BCL-XL: B cell lymphoma extra-large; MCL1: myeloid cell leukemia 1; BAX: BCL-2 associated X protein; BAK: BCL-2 antagonist/killer; MDM2: mouse double minute 2 protein; DRL: death receptor ligand; ER: endoplasmic reticulum. PERK: PKR-like ER kinase; ATF4: Activating transcription factor 4; CHOP: CCAAT-enhancer-binding protein homologous protein.