ARTS and small-molecule ARTS mimetics upregulate p53 levels by promoting the degradation of XIAP.

Mutations resulting in decreased activity of p53 tumor suppressor protein promote tumorigenesis. P53 protein levels are tightly regulated through the Ubiquitin Proteasome System (UPS). Several E3 ligases were shown to regulate p53 stability, including MDM2. Here we report that the ubiquitin E3 ligase XIAP (X-linked Inhibitors of Apoptosis) is a direct ligase for p53 and describe a novel approach for modulating the levels of p53 by targeting the XIAP pathway. Using in vivo (live-cell) and in vitro (cell-free reconstituted system) ubiquitylation assays, we show that the XIAP-antagonist ARTS regulates the levels of p53 by promoting the degradation of XIAP. XIAP directly binds and ubiquitylates p53. In apoptotic cells, ARTS inhibits the ubiquitylation of p53 by antagonizing XIAP. XIAP knockout MEFs express higher p53 protein levels compared to wild-type MEFs. Computational screen for small molecules with high affinity to the ARTS-binding site within XIAP identified a small-molecule ARTS-mimetic, B3. This compound stimulates apoptosis in a wide range of cancer cells but not normal PBMC (Peripheral Blood Mononuclear Cells). Like ARTS, the B3 compound binds to XIAP and promotes its degradation via the UPS. B3 binding to XIAP stabilizes p53 by disrupting its interaction with XIAP. These results reveal a novel mechanism by which ARTS and p53 regulate each other through an amplification loop to promote apoptosis. Finally, these data suggest that targeting the ARTS binding pocket in XIAP can be used to increase p53 levels as a new strategy for developing anti-cancer therapeutics.

Targeted Degradation of XIAP is Sufficient and Specific to Induce Apoptosis in MYCN-overexpressing High-risk Neuroblastoma.

XIAP, the most potent mammalian inhibitor of apoptosis protein (IAP), critically restricts developmental culling of sympathetic neuronal progenitors, and is correspondingly overexpressed in most MYCN-amplified neuroblastoma tumors. Because apoptosis-related protein in the TGFß signaling pathway (ARTS) is the only XIAP antagonist that directly binds and degrades XIAP, we evaluated the preclinical effectiveness and tolerability of XIAP antagonism as a novel targeting strategy for neuroblastoma. We found that antagonism of XIAP, but not other IAPs, triggered apoptotic death in neuroblastoma cells. XIAP silencing induced apoptosis while overexpression conferred protection from drug-induced apoptosis. From a screen of IAP inhibitors, first-in-class ARTS mimetic A4 was most effective against high-risk and high XIAP-expressing neuroblastoma cells, and least toxic toward normal liver- and bone marrow-derived cells, compared with pan-IAP antagonists. On target engagement assays and nuclear magnetic resonance spectroscopy, A4 was observed to degrade rather than inhibit XIAP, catalyzing rapid degradation of XIAP through the ubiquitin-proteasome pathway. In MYCN-amplified neuroblastoma patient-derived xenografts, A4 significantly prolonged survival as a single agent, and demonstrated synergism with standard-of-care agents to reduce their effective required doses 3- to 6-fold. Engagement and degradation of XIAP by ARTS mimetics is a novel targeting strategy for neuroblastoma that may be especially effective against MYCN-amplified disease with intrinsically high XIAP expression. First-in-class ARTS mimetic A4 demonstrates preclinical efficacy and warrants further development and study. SIGNIFICANCE: XIAP degradation is sufficient to kill MYCN-amplified neuroblastoma which overexpresses and relies on XIAP as a brake against cell death, without affecting normal cells.

Killing by Degradation: Regulation of Apoptosis by the Ubiquitin-Proteasome-System.

Apoptosis is a cell suicide process that is essential for development, tissue homeostasis and human health. Impaired apoptosis is associated with a variety of human diseases, including neurodegenerative disorders, autoimmunity and cancer. As the levels of pro- and anti-apoptotic proteins can determine the life or death of cells, tight regulation of these proteins is critical. The ubiquitin proteasome system (UPS) is essential for maintaining protein turnover, which can either trigger or inhibit apoptosis. In this review, we will describe the E3 ligases that regulate the levels of pro- and anti-apoptotic proteins and assisting proteins that regulate the levels of these E3 ligases. We will provide examples of apoptotic cell death modulations using the UPS, determined by positive and negative feedback loop reactions. Specifically, we will review how the stability of p53, Bcl-2 family members and IAPs (Inhibitor of Apoptosis proteins) are regulated upon initiation of apoptosis. As increased levels of oncogenes and decreased levels of tumor suppressor proteins can promote tumorigenesis, targeting these pathways offers opportunities to develop novel anti-cancer therapies, which act by recruiting the UPS for the effective and selective killing of cancer cells.

p53 induces ARTS to promote mitochondrial apoptosis.

Apoptosis related protein in TGF-ß signaling pathway (ARTS) was originally discovered in cells undergoing apoptosis in response to TGF-ß, but ARTS also acts downstream of many other apoptotic stimuli. ARTS induces apoptosis by antagonizing the anti-apoptotic proteins XIAP and Bcl-2. Here we identified the pro-apoptotic Sept4/ARTS gene as a p53-responsive target gene. Ectopic p53 and a variety of p53-inducing agents increased both mRNA and protein levels of ARTS, whereas ablation of p53 reduced ARTS expression in response to multiple stress conditions. Also, γ-irradiation induced p53-dependent ARTS expression in mice. Consistently, p53 binds to the responsive DNA element on the ARTS promoter and transcriptionally activated the promoter-driven expression of a luciferase reporter gene. Interestingly, ARTS binds to and sequesters p53 at mitochondria, enhancing the interaction of the latter with Bcl-XL. Ectopic ARTS markedly augments DNA damage stress- or Nutlin-3-triggered apoptosis, while ablation of ARTS preferentially impairs p53-induced apoptosis. Altogether, these findings demonstrate that ARTS collaborates with p53 in mitochondria-engaged apoptosis.

Inhibiting the inhibitors: Targeting anti-apoptotic proteins in cancer and therapy resistance.

The cytotoxic effect of anti-cancer drugs relies on their ability to induce programmed cell death known as apoptosis. Evading apoptosis is a common characteristic of cancer cells and it is linked to both carcinogenesis and anticancer drug resistance. To escape apoptosis, cancer cells often express high levels of anti-apoptotic proteins and become "addicted "to them for their survival. Consequently, anti-apoptotic proteins have emerged as attractive druggable targets for the development of cancer therapeutics. In this review we focus on two major anti-apoptotic protein families: IAPs (Inhibitor of Apoptosis) proteins and Bcl-2 (B-cell lymphoma-2) family members. We also discuss insights into the regulation of these proteins by natural antagonists, which has provided the conceptual basis for developing novel anti-cancer drugs. Significantly, the pro-apoptotic protein ARTS (apoptosis-related protein in the TGF-ß signaling pathway; Sept4_i2) acts as a dual antagonist of both X-linked inhibitor of apoptosis protein (XIAP) and Bcl-2. Because upregulation of anti-apoptotic proteins in response to cancer therapy contributes to drug resistance, targeted inhibition of these proteins is expected to enhance the efficacy of chemotherapy. Finally, we discuss the role of proteasome-mediated degradation in the regulation of apoptosis, and how this mechanism can be harnessed to develop small molecules that stimulate degradation of anti-apoptotic proteins for cancer therapy. This strategy has the potential to overcome drug resistance more effectively than mere inhibition. Therefore, this approach may allow use of lower drug concentrations and thereby reduce cytotoxicity and untoward side effects.

The pro-apoptotic ARTS protein induces neutrophil apoptosis, efferocytosis, and macrophage reprogramming to promote resolution of inflammation.

ARTS (Sept4_i2) is a pro-apoptotic protein and a product of the Sept4 gene. ARTS acts upstream of mitochondria to initiate caspase activation. ARTS induces apoptosis by specifically binding XIAP and allowing de-repression of active caspases required for Mitochondrial Outer Membrane Permeabilzation (MOMP). Moreover, ARTS promotes apoptosis by inducing ubiquitin-mediated degradation of both major anti-apoptotic proteins XIAP and Bcl-2. In the resolution phase of inflammation, the infiltrating leukocytes, which execute the acute innate response, undergo apoptosis and are subsequently cleared by phagocytic macrophages (i.e. efferocytosis). In this course, macrophages undergo reprogramming from inflammatory, to anti-inflammatory, and eventually to resolving macrophages that leave the injury sites. Since engulfment of apoptotic leukocytes is a key signaling step in macrophage reprogramming and resolution of inflammation, we hypothesized that a failed apoptosis in leukocytes in vivo would result in an impaired resolution process. To test this hypothesis, we utilized the Sept4/ARTS-/- mice, which exhibit resistance to apoptosis in many cell types. During zymosan A-induced peritonitis, Sept4/ARTS-/- mice exhibited impaired resolution of inflammation, characterized by reduced neutrophil apoptosis, macrophage efferocytosis and expression of pro-resolving mediators. This was associated with increased pro-inflammatory cytokines and reduced anti-inflammatory cytokines, secreted by resolution-phase macrophages. Moreover, ARTS overexpression in leukocytes in vitro promoted an anti-inflammatory behavior. Overall, our results suggest that ARTS is a key master-regulator necessary for neutrophil apoptosis, macrophage efferocytosis and reprogramming to the pro-resolving phenotype during the resolution of inflammation.

A small-molecule ARTS mimetic promotes apoptosis through degradation of both XIAP and Bcl-2.

Many human cancers over-express B cell lymphoma 2 (Bcl-2) or X-linked inhibitor of apoptosis (IAP) proteins to evade cell death. The pro-apoptotic ARTS (Sept4_i2) protein binds directly to both Bcl-2 and XIAP and promotes apoptosis by stimulating their degradation via the ubiquitin-proteasome system (UPS). Here we describe a small molecule, A4, that mimics the function of ARTS. Microscale thermophoresis assays showed that A4 binds XIAP, but not cellular inhibitor of apoptosis protein 1 (cIAP1). A4 binds to a distinct ARTS binding pocket in the XIAP-BIR3 (baculoviral IAP repeat 3) domain. Like ARTS, A4 stimulated poly-ubiquitylation and UPS-mediated degradation of XIAP and Bcl-2, but not cIAP1, resulting in caspase-9 and -3 activation and apoptosis. In addition, over-expression of XIAP rescued HeLa cells from A4-induced apoptosis, consistent with the idea that A4 kills by antagonizing XIAP. On the other hand, treatment with the SMAC-mimetic Birinapant induced secretion of tumour necrosis factor-α (TNFα) and killed ~50% of SKOV-3 cells, and addition of A4 to Birinapant-treated cells significantly reduced secretion of TNFα and blocked Birinapant-induced apoptosis. This suggests that A4 acts by specifically targeting XIAP. The effect of A4 was selective as peripheral blood mononuclear cells and normal human breast epithelial cells were unaffected. Furthermore, proteome analysis revealed that cancer cell lines with high levels of XIAP were particularly sensitive to the killing effect of A4. These results provide proof of concept that the ARTS binding site in XIAP is "druggable". A4 represents a novel class of dual-targeting compounds stimulating apoptosis by UPS-mediated degradation of important anti-apoptotic oncogenes.

Targeting XIAP for Promoting Cancer Cell Death-The Story of ARTS and SMAC.

Inhibitors of apoptosis (IAPs) are a family of proteins that regulate cell death and inflammation. XIAP (X-linked IAP) is the only family member that suppresses apoptosis by directly binding to and inhibiting caspases. On the other hand, cIAPs suppress the activation of the extrinsic apoptotic pathway by preventing the formation of pro-apoptotic signaling complexes. IAPs are negatively regulated by IAP-antagonist proteins such as Smac/Diablo and ARTS. ARTS can promote apoptosis by binding and degrading XIAP via the ubiquitin proteasome-system (UPS). Smac can induce the degradation of cIAPs but not XIAP. Many types of cancer overexpress IAPs, thus enabling tumor cells to evade apoptosis. Therefore, IAPs, and in particular XIAP, have become attractive targets for cancer therapy. In this review, we describe the differences in the mechanisms of action between Smac and ARTS, and we summarize efforts to develop cancer therapies based on mimicking Smac and ARTS. Several Smac-mimetic small molecules are currently under evaluation in clinical trials. Initial efforts to develop ARTS-mimetics resulted in a novel class of compounds, which bind and degrade XIAP but not cIAPs. Smac-mimetics can target tumors with high levels of cIAPs, whereas ARTS-mimetics are expected to be effective for cancers with high levels of XIAP.

Another one bites the dust; ARTS enables degradation of Bcl-2 by XIAP.

ARTS (Sept4_i2) is a pro-apoptotic mitochondrial tumor suppressor protein which binds to and causes degradation of XIAP (X-linked inhibitor of apoptosis). We recently showed that ARTS brings XIAP into close proximity to Bcl-2, creating a complex which enables degradation of both these major anti-apoptotic proteins and promotes apoptosis. The possible therapeutic implications are discussed here.

International conference on Cell Death in Cancer and Toxicology 2018 (CDCT-2018).

The International Conference on Cell Death in Cancer and Toxicology 2018 (February 20-22, 2018) provided an international forum for scientific collaborations across multiple disciplines in cancer, cell death, and toxicology. During the three-day symposium, researchers and clinicians shared recent advances in basic, clinical, and translational research in cancer. Several student poster abstracts were selected for platform talks and many young investigators participated in the meeting. Together, this highly interactive meeting showcased the rapid expansion in biomedical research in India and paved the way for future meetings on cell death and cancer throughout India.

PAX8 activates a p53-p21-dependent pro-proliferative effect in high grade serous ovarian carcinoma.

High grade serous carcinoma (HGSC) is the most common subtype of ovarian cancer and it is now widely accepted that this disease often originates from the fallopian tube epithelium. PAX8 is a fallopian tube lineage marker with an essential role in embryonal female genital tract development. In the adult fallopian tube, PAX8 is expressed in the fallopian tube secretory epithelial cell (FTSEC) and its expression is maintained through the process of FTSEC transformation to HGSC. We now report that PAX8 has a pro-proliferative and anti-apoptotic role in HGSC. The tumor suppressor gene TP53 is mutated in close to 100% of HGSC; in the majority of cases, these are missense mutations that endow the mutant p53 protein with potential gain of function (GOF) oncogenic activities. We show that PAX8 positively regulates the expression of TP53 in HGSC and the pro-proliferative role of PAX8 is mediated by the GOF activity of mutant p53. Surprisingly, mutant p53 transcriptionally activates the expression of p21, which localizes to the cytoplasm of HGSC cells where it plays a non-canonical, pro-proliferative role. Together, our findings illustrate how TP53 mutations in HGSC subvert a normal regulatory pathway into a driver of tumor progression.

Degradation of Bcl-2 by XIAP and ARTS Promotes Apoptosis.

We describe a mechanism by which the anti-apoptotic B cell lymphoma 2 (Bcl-2) protein is downregulated to induce apoptosis. ARTS (Sept4_i2) is a tumor suppressor protein that promotes cell death through specifically antagonizing XIAP (X-linked inhibitor of apoptosis). ARTS and Bcl-2 reside at the outer mitochondrial membrane in living cells. Upon apoptotic induction, ARTS brings XIAP and Bcl-2 into a ternary complex, allowing XIAP to promote ubiquitylation and degradation of Bcl-2. ARTS binding to Bcl-2 involves the BH3 domain of Bcl-2. Lysine 17 in Bcl-2 serves as the main acceptor for ubiquitylation, and a Bcl-2 K17A mutant has increased stability and is more potent in protection against apoptosis. Bcl-2 ubiquitylation is reduced in both XIAP- and Sept4/ARTS-deficient MEFs, demonstrating that XIAP serves as an E3 ligase for Bcl-2 and that ARTS is essential for this process. Collectively, these results suggest a distinct model for the regulation of Bcl-2 by ARTS-mediated degradation.

Parkin-mediated ubiquitination of mutant glucocerebrosidase leads to competition with its substrates PARIS and ARTS.

BACKGROUND: Parkinson's disease (PD) is a movement neurodegenerative disorder characterized by death of dopaminergic neurons in the substantia nigra pars compacta of the brain that leads to movement impairments including bradykinesia, resting tremor, postural instability and rigidity. Mutations in several genes have been associated with familial PD, such as parkin, pink, DJ-1, LRKK2 and α-synuclein. Lately, mutations in the GBA gene were recognized as a major cause for the development of PD.Mutations in the GBA gene, which encodes for lysosomal ß-glucocerebrosidase (GCase), lead to Gaucher disease (GD), an autosomal recessive sphingolipidosis characterized by accumulation of glucosylceramide, mainly in monocyte-derived cells. It is a heterogeneous disease, with Type 1 patients that do not present any primary neurological signs, and Type 2 or Type 3 patients who suffer from a neurological disease. The propensity of type 1 GD patients and carriers of GD mutations to develop PD is significantly higher than that of the non-GD population.We have shown in the past that parkin and mutant GCase, expressed in heterologous systems, interact with each other, and that normal but not mutant parkin mediates K48-dependent proteasomal degradation of mutant GCase variants. METHODS: We tested possible competition between mutant GCase and PARIS or ARTS on the E3 ubiquitin ligase parkin, using coimmunoprecipitation assays and quantitative real-time PCR. RESULTS: We show that endogenous mutant GCase variants associate with parkin and undergo parkin-dependent degradation. Mutant GCase competes with the known parkin substrates PARIS and ARTS, whose accumulation leads to apoptosis. Dopaminergic cells expressing mutant GCase are more susceptible to apoptotic stimuli than dopaminergic cells expressing normal GCase, present increased cleavage of caspase 3 and caspase 9 levels and undergo cell death. CONCLUSIONS: Our results imply that presence of mutant GCase leads to accumulation of parkin substrates like PARIS and ARTS, which may cause apoptotic death of cells.

Parkin promotes degradation of the mitochondrial pro-apoptotic ARTS protein.

Parkinson's disease (PD) is associated with excessive cell death causing selective loss of dopaminergic neurons. Dysfunction of the Ubiquitin Proteasome System (UPS) is associated with the pathophysiology of PD. Mutations in Parkin which impair its E3-ligase activity play a major role in the pathogenesis of inherited PD. ARTS (Sept4_i2) is a mitochondrial protein, which initiates caspase activation upstream of cytochrome c release in the mitochondrial apoptotic pathway. Here we show that Parkin serves as an E3-ubiquitin ligase to restrict the levels of ARTS through UPS-mediated degradation. Though Parkin binds equally to ARTS and Sept4_i1 (H5/PNUTL2), the non-apoptotic splice variant of Sept4, Parkin ubiquitinates and degrades only ARTS. Thus, the effect of Parkin on ARTS is specific and probably related to its pro-apoptotic function. High levels of ARTS are sufficient to promote apoptosis in cultured neuronal cells, and rat brains treated with 6-OHDA reveal high levels of ARTS. However, over-expression of Parkin can protect cells from ARTS-induced apoptosis. Furthermore, Parkin loss-of-function experiments reveal that reduction of Parkin causes increased levels of ARTS and apoptosis. We propose that in brain cells in which the E3-ligase activity of Parkin is compromised, ARTS levels increase and facilitate apoptosis. Thus, ARTS is a novel substrate of Parkin. These observations link Parkin directly to a pro-apoptotic protein and reveal a novel connection between Parkin, apoptosis, and PD.

Role of melanoma inhibitor of apoptosis (ML-IAP) protein, a member of the baculoviral IAP repeat (BIR) domain family, in the regulation of C-RAF kinase and cell migration.

Inhibitor of apoptosis (IAPs) proteins are characterized by the presence of evolutionarily conserved baculoviral inhibitor of apoptosis repeat (BIR) domains, predominantly known for their role in inhibiting caspases and, thereby, apoptosis. We have shown previously that multi-BIR domain-containing IAPs, cellular IAPs, and X-linked IAP can control tumor cell migration by directly regulating the protein stability of C-RAF kinase. Here, we extend our observations to a single BIR domain containing IAP family member melanoma-IAP (ML-IAP). We show that ML-IAP can directly bind to C-RAF and that ML-IAP depletion leads to an increase in C-RAF protein levels, MAPK activation, and cell migration in melanoma cells. Thus, our results unveil a thus far unknown role for ML-IAP in controlling C-RAF stability and cell migration.

Peptides mimicking the unique ARTS-XIAP binding site promote apoptotic cell death in cultured cancer cells.

PURPOSE: XIAP [X-linked inhibitor of apoptosis (IAP) protein] is the best characterized mammalian caspase inhibitor. XIAP is frequently overexpressed in a variety of human tumors, and genetic inactivation of XIAP in mice protects against lymphoma. Therefore, XIAP is an attractive target for anticancer therapy. IAP antagonists based on a conserved IAP-binding motif (IBM), often referred to as "Smac-mimetics," are currently being evaluated for cancer therapy in the clinic. ARTS (Sept4_i2) is a mitochondrial proapoptotic protein which promotes apoptosis by directly binding and inhibiting XIAP via a mechanism that is distinct from all other known IAP antagonists. Here, we investigated the ability of peptides derived from ARTS to antagonize XIAP and promote apoptosis in cancer cell lines. EXPERIMENTAL DESIGN: The ability of synthetic peptides, derived from the C-terminus of ARTS, to bind to XIAP, stimulate XIAP degradation, and induce apoptosis was examined. We compared the response of several cancer cell lines to different ARTS-derived peptides. Pull-down assays were used to examine binding to XIAP, and apoptosis was evaluated using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, caspase activation, and Western blot analyses of caspase substrates. RESULTS: The C-terminus of ARTS contains a unique sequence, termed ARTS-IBM (AIBM), which is important for binding to XIAP and cell killing. AIBM peptides can bind to XIAP-BIR3, penetrate cancer cells, reduce XIAP levels, and promote apoptosis. CONCLUSIONS: Short synthetic peptides derived from the C-terminus of ARTS are sufficient for binding to XIAP and can induce apoptosis in cancer cells. These results provide proof-of-concept for the feasibility of developing ARTS-based anticancer therapeutics.

X-linked Inhibitor of Apoptosis Protein promotes the degradation of its antagonist, the pro-apoptotic ARTS protein.

ARTS (Sept4_i2) is a mitochondrial pro-apoptotic tumor suppressor protein. In response to apoptotic signals, ARTS translocates to the cytosol where it promotes caspase activation through caspase de-repression and proteasome mediated degradation of X-linked Inhibitor of Apoptosis Protein (XIAP). Here we show that XIAP regulates the levels of ARTS by serving as its ubiquitin ligase, thereby providing a potential feedback mechanism to protect against unwanted apoptosis. Using both in vitro and in vivo ubiquitination assays we found that ARTS is directly ubiquitinated by XIAP. Moreover, we found that XIAP-induced ubiquitination and degradation is prevented by removal of the first four amino acids in the N-terminus of ARTS, which contains a single lysine residue at position 3. Thus, this lysine at position 3 is a likely target for ubiquitination by XIAP. Importantly, although the stabilized ARTS lacking its first 4 residues binds XIAP as well as the full length ARTS, it is more potent in promoting apoptosis than the full length ARTS. This suggests that increased stability of ARTS has a significant effect on its ability to induce apoptosis. Collectively, our data reveal a mutual regulatory mechanism by which ARTS and XIAP control each other's levels through the ubiquitin proteasome system.

Ripoptosome: a novel IAP-regulated cell death-signalling platform.

Recent studies have revealed that cell death stimuli can trigger programmed necrosis, necroptosis. Receptor-interacting serine-threonine kinase family RIP plays a crucial role in regulating the switch between apoptosis and necroptosis. Two studies now describe a novel RIP1 containing ~2 MDa 'Ripoptosome' complex assembled in the cytosol to mediate both apoptosis and necroptosis in response to genotoxic stress and TLR3 stimulation. Intriguingly, cIAPs and XIAP function as endogenous inhibitors of Ripoptosome by direct ubiquitination of its components.

ARTS-based anticancer therapy: taking aim at cancer stem cells.

Apoptosis related protein in TGF-ß signaling pathway (ARTS/septin 4 isoform 2) hereforth referred to as ARTS, was originally found to promote apoptosis induced by TGF-ß, but later was shown to promote apoptosis induced by a wide variety of apoptotic stimuli. In vivo and in vitro studies revealed that ARTS-induced apoptosis is mainly executed through direct binding and antagonizing XIAP. High levels of XIAP are found in many types of cancers and often correlate with poor prognosis. ARTS was shown to function as a tumor-suppressor protein in human patients and mouse-tumor models. In particular, Septin 4/ARTS-deficient mice have increased tumor susceptibility and contain increased numbers of stem cells (SCs) and progenitor cells, apparently owing to their resistance towards apoptosis. Based on these results we propose that loss of proapoptotic ARTS may act as the 'first hit' initiating tumorigenesis in two distinct ways. First, loss of ARTS-mediated apoptosis leads to increased numbers of normal SCs. Elevated numbers of normal SCs may lead to increased cancer risk due to higher numbers of cellular targets available for transforming mutations. Second, after these SCs acquire additional transforming mutations and become cancer SC (CSCs), they are more likely to survive in the absence of ARTS owing to increased resistance toward apoptosis. A combination of these two mechanisms, over time, is expected to significantly increase tumor risk. Because CSCs appear to share phenotypic markers with normal SCs, targeting the signaling pathways that affect normal SC development and maintenance can serve as a useful approach towards true eradication of cancer. In this article we describe the role of ARTS in apoptosis and cancer, with focus on its potential role as a CSC marker and as a potential target for anticancer and anti-CSC therapy.

Mechanism of the interaction between the intrinsically disordered C-terminus of the pro-apoptotic ARTS protein and the Bir3 domain of XIAP.

ARTS (Sept4_i2) is a mitochondrial pro-apoptotic protein that functions as a tumor suppressor. Its expression is significantly reduced in leukemia and lymphoma patients. ARTS binds and inhibits XIAP (X-linked Inhibitor of Apoptosis protein) by interacting with its Bir3 domain. ARTS promotes degradation of XIAP through the proteasome pathway. By doing so, ARTS removes XIAP inhibition of caspases and enables apoptosis to proceed. ARTS contains 27 unique residues in its C-terminal domain (CTD, residues 248-274) which are important for XIAP binding. Here we characterized the molecular details of this interaction. Biophysical and computational methods were used to show that the ARTS CTD is intrinsically disordered under physiological conditions. Direct binding of ARTS CTD to Bir3 was demonstrated using NMR and fluorescence spectroscopy. The Bir3 interacting region in ARTS CTD was mapped to ARTS residues 266-274, which are the nine C-terminal residues in the protein. Alanine scan of ARTS 266-274 showed the importance of several residues for Bir3 binding, with His268 and Cys273 contributing the most. Adding a reducing agent prevented binding to Bir3. A dimer of ARTS 266-274 formed by oxidation of the Cys residues into a disulfide bond bound with similar affinity and was probably required for the interaction with Bir3. The detailed analysis of the ARTS - Bir3 interaction provides the basis for setting it as a target for anti cancer drug design: It will enable the development of compounds that mimic ARTS CTD, remove IAPs inhibition of caspases, and thereby induce apoptosis.