Targeting Apoptosis to Treat Multiple Sclerosis – Therapeutics Based on Downregulation of the Inhibitor of Apoptosis Family

The clinical course of MS has been divided into four major categories – relapsing-remitting (RR), secondaryprogressive (SP), primary progressive (PP), and benign. Patients with RRMS have clinical relapses every few months or years, with intervening periods of clinical stability. RRMS is twice as prevalent in females than males.⁶ Although rare, late-onset MS with initial symptom presentation in patients over 50 years of age ⁷ and early-onset MS in children under the age of 10 years, as young as 11 months of age have also been documented.^8–10 In contrast to RRMS, patients with SPMS display progressive deterioration between relapses. RRMS patients may convert to SPMS over time, characterized by a gradual decline in neurological function.¹¹ Approximately 15% of MS patients have PPMS characterized by late onset and the relentless deterioration of neurological function following disease onset.¹² There are currently no effective treatments for PPMS.¹³ In addition to RR, SP and PP MS, there is a benign form of the disease that affects approximately 10% to 20% of RRMS patients. Benign MS is arbitrarily defined in RRMS patients who, after more than 15 years following initial diagnosis, are still mobile and show only mild deficits (expanded disability status scale (EDSS) = 2).Typically, these patients show little or no progression after their initial attack. Moreover, patients with an EDSS score of = 2 and disease duration of more than 10 years tend to maintain a low EDSS disability score for an additional 10 years. Benign MS requires no therapeutic intervention and is generally not diagnosed until 10 to 15 years from disease onset.¹⁴

Cellular Immunology of MS
Although the precise cause of MS remains unknown, this auto-immune disease is considered to be T-cellmediated, ^15,16 and can be modeled in both rodents and non-human primates by experimental auto-immune encephalomyelitis (EAE).^17,18 The clinical manifestations and neuropathology of EAE parallel many of the cardinal features of MS.¹⁹ Like MS, EAE is predominantly a Th1-cell-mediated auto-immune disease. In the EAE model, animals are immunized with a specific myelin antigen and adjuvant that triggers an immune response against the recognized antigen. The first clinical signs of EAE generally become apparent 10 days post-induction, beginning with walking deficits and eventually progressing to ascending paralysis. Multiple myelin antigens, such as myelin oligodendrocyte glycoprotein (MOG), proteolipid protein (PLP), and myelin basic protein (MBP), all induce EAE. The combination of myelin antigen and animal species/strain often predicts the clinical course of EAE that can either be chronic, acute, or relapsing-remitting.²⁰

While CD4+ T-cells specific for myelin antigens are thought to initiate and exacerbate MS through the secretion of pro-inflammatory cytokines, activated macrophages also migrate into the CNS and release a variety of factors that directly cause nerve and tissue damage.^21–23 Activated macrophages and microglia are consistently found in demyelinated plaques in MS patients,²² and probably serve as the major antigenpresenting cells (APCs) in the CNS.²¹ Moreover, the extent of axonal damage in demyelinated lesions of the CNS and the persistence of neurological deficits in MS are directly correlated with the number of activated macrophages/microglia and CD8+ T-cells within the margins of sclerotic plaques.²³ Mice immunized with MOG35-55 to induce EAE possess APCs that display heightened immune activity compared with controls.²⁴ In addition, minor changes in MHC class II molecules have had varying effects on the onset and severity of EAE.²⁵ In humans, a single nucleotide polymorphism in the type III promoter of the MHC class II transactivator (MHC2TA) has been identified that is associated with increased susceptibility for MS.²⁶ Taken together, these results suggest that blocking both APC activation and antigen presentation may have therapeutic value in MS.

A role for auto-reactive B-cells in the pathogenesis of MS is also suggested by two lines of evidence – elevated myelin-specific antibodies and the increased presence of auto-reactive B-lymphocytes to myelin and oligodendrocyte antigens in MS patients relative to controls.^27,28 Antibodies specific to several myelin antigens, including MBP and PLP, have also been identified in EAE29,30 further indicating the validity of this animal model.

Failed Apoptosis as a Disease Mechanism in MS
Apoptosis is an important mechanism in immune system regulation, responsible for the elimination of auto-reactive T- and B-cells (lymphocytes) and macrophages from the circulation and preventing their entry into the CNS.³¹ It has been hypothesized that a failure of auto-reactive T- and B-lymphocytes, as well as activated macrophages, to undergo apoptosis contributes to the pathogenesis of MS.^32,33 Consistent with this hypothesis, expression of members of the inhibitors of apoptosis (IAP) family of anti-apoptotic proteins such as X chromosome-linked inhibitor of apoptosis (XIAP), Human IAP-1 (HIAP-1), and Human IAP-2 (HIAP-2) are elevated in mitogen stimulated T-cells from MS patients relative to neurologically healthy control subjects.34–39 It should be emphasized that while recent evidence from Sharief et al. suggests that the IAPs are involved in MS, these findings are based primarily on IAP expression in mitogen (phytohemagglutinin (PHA))-stimulated T-cells from RRMS patients. By comparison, the authors have recently expanded these findings to untreated peripheral blood mononuclear (PBMN) and T-cells in other forms of MS. In more aggressive forms of MS, higher levels of XIAP expression were found in PBMN cells while both HIAP-1 and HIAP-2 were elevated in resting T-cells relative to healthy controls.⁴⁰

The IAP family of anti-apoptotic genes encodes proteins that directly bind and inactivate initiator and effector caspases, a group of cysteinyl proteases that mediate initiation and execution of apoptosis.⁴¹ First discovered in baculovirus, the IAPs are well conserved in eukaryotes, ranging from yeast to humans.To date, eight human IAPs have been identified, including XIAP, which is expressed ubiquitously in most fetal and adult tissue.^41,42 XIAP, like many of the IAPs, possesses three highly conserved domains of approximately 70 amino acids in length, known as baculoviral IAP repeats, or BIR domains, which are essential for anti-apoptotic activity.XIAP, HIAP-1, and HIAP-2 all possess three BIR domains that mediate suppression of caspase-3 and -7 – the two most potent effector caspases.^42,45 In addition to BIR domains, many IAPs also possess a carboxy-terminal ring zinc finger motif that has ubiquitin ligase activity that targets caspases for degradation by the proteosome.⁴¹ Members of the B-cell lymphoma-2 (BCL-2) family are also potent inhibitors of apoptosis; however, BCL-2 proteins can only block apoptosis by preventing the release of cytochrome c from mitochondria. Cytochrome c binds to apoptotic protease activating factor-1 (APAF-1) and caspase-9 forming the apoptosome.The apoptosome in turn activates caspase-3, through to caspase-9, resulting in programmed cell death. Transgenic mice that overexpress BCL-2 in T-cells display a more severe form of EAE during the chronic phase, suggesting that increased survival of auto-reactive T-cells enhances EAE pathogenesis.⁴³ In contrast to BCL-2, the IAPs directly block upstream triggers of apoptosis known as death receptors that initiate Fas-mediated activation of caspase-8, the extrinsic cell death pathway.⁴⁴ Like BCL-2, the IAPs also block the intrinsic cell death pathway,mediated by the release of pro-apoptotic factors from the mitochondria.^41,45 Consequently, the anti-apoptotic activities of the IAPs can be distinguished from members of the BCL-2 family by their ability to block both of these cell death pathways. Elevated expression of XIAP, HIAP-1 and HIAP-2 in patients with active MS correlates with clinical features of disease activity, deficits in Fas-mediated cell death, and T-cell resistance to apoptosis.³⁸ By contrast, protein expression of BCL- 2 in activated T-cells is similar in MS patients and normal controls.38 Basal levels of XIAP, HIAP-1 and HIAP-2 in B lymphocytes and macrophages obtained from MS patients are currently unknown, although it has been shown that expression of BCL-2 proteins in B-cells of RRMS patients, as well as patients with systemic inflammatory diseases, is elevated relative to normal control subjects.46 In macrophages, upregulation of XIAP also increases cell survival, while XIAP knockdown, using antisense oligonucleotides, decreases cell viability.⁴⁷ Taken together, these findings suggest that impaired apoptosis resulting from elevated IAP expression contributes to MS.

IAP Downregulation as a Novel Treatment for MS
The goal of current MS therapies is to lengthen the time between relapses and thereby slow or perhaps even halt disease progression. Much of the work in the EAE model has led to the development of novel therapeutic strategies for treating MS.⁴⁸ There are currently three categories of approved therapies for long-term treatment of RRMS, including three different preparations of interferon-beta (IFN-?; Avonex, Betaseron and Rebif), glatiramer-acetate (Copaxone), and mitoxantrone (Novantrone).^49,50 IFN-? has been shown to lengthen the time between relapses in individuals with MS. One mechanism underlying the propensity of IFN-? to lengthen the time between relapses in MS patients is its ability to promote the elimination of auto-reactive T- and B-cells through the reduction of anti-apoptotic proteins. IFN-? has been shown to reduce expression of the anti-apoptotic proteins XIAP, HIAP-1 and HIAP-2 in mitogen stimulated T-cells from MS patients, suggesting that IFN-? drugs may improve the symptoms of MS by promoting the elimination of auto-reactive T-cells through IAP downregulation.³⁷

The anti-apoptotic activity of XIAP is opposed by a physical interaction with XIAP-associated factor-1 (XAF-1)^41,51 (see Figure 1). IFN-? has been shown to increase the expression of XAF-1 mRNA in a number of melanoma and myeloma cell lines, as well as dendritic and natural killer cells.^52,53 A specific role for XAF-1 in programmed cell death was confirmed by showing that XAF-1 over-expression sensitized cells normally resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, whereas inhibition of XAF-1 expression prevented the sensitizing effects of IFN-?.⁵³ Many cancer cell lines undergo apoptosis when exposed to TRAIL; however, several cancer cell lines that are resistant to TRAIL-induced apoptosis can be sensitized to this apoptotic trigger by exposure to IFN- ?. In this case, there is a corresponding increase in levels of XAF-1 protein.⁵³ The ability of IFN-? to sensitize auto-reactive T-cells to TRAIL-induced apoptosis therefore appears to be mediated by the induction of XAF-1. In EAE, using TRAIL blockade, the disease is inhibited and activation of auto-reactive T-cells is prevented.⁵⁴ In addition,TRAIL has been correlated with IFN-? responsiveness and is shown to influence disease severity in a murine model of EAE.⁵⁵ If similar mechanisms are at play in MS, it is possible that induction of XAF-1 in auto-reactive T-cells may be indicative of clinical responsiveness to IFN-?. In MS patients who do not respond to IFN-?, there may be a failure of IFN-? to induce XAF-1 in auto-reactive T-cells.

IAP Associated Poteins as Diagnostic Markers in MS
IFN-? treatment has been associated with the development of neutralizing antibodies in MS patients six to 15 months following the initiation of treatment.⁵⁶ The predictive value of such antibodies for IFN-? responsiveness remains controversial ^56,57 and there is an urgent need for more robust and reliable diagnostic markers.⁵⁸ To this end, complementary deoxyribonucleic acid (cDNA) microarray studies have been performed to identify genes responsive to IFN-? in PBMN cells from MS patients.⁵⁹ This work has led to retrospective studies reporting a positive correlation between expression of TRAIL mRNA in PBMN cells or levels of this pro-apoptotic protein in the serum of MS patients responsive to IFN-? therapy.⁶⁰ These findings encouraged the authors to explore the possibility that clinical disability, disease progression and responsiveness to IFN-? may be predicted by the expression profile of apoptosis-related genes such as XIAP, HIAP-1, HIAP-2 and XAF-1 in immune cells from MS patients. Moreover, since XAF-1 has been implicated in the signaling mechanisms that mediate the apoptotic actions of IFN-?, the authors hypothesize that levels of XAF-1 in auto-reactive immune cells may represent a reliable biochemical index of IFN-? unresponsiveness. In comparison with T-cell apoptosis, little is known about the effects of IFN-? on XIAP, HIAP-1, HIAP-2, and XAF-1 levels in auto-reactive B-cells or macrophages. By stark contrast, it has been reported that IFN-? treatment does not alter increased BCL-2 protein levels in auto reactive B-cells in patients with RRMS,⁴⁸ hence, the IAPs may be more relevant to disease course than members of the BCL-2 family. Therapeutic Prospects for IAP Downregulation in MS
In summary, the authors hypothesize that expression levels of the IAP family of anti-apoptotic genes may represent novel diagnostic markers predictive of disease severity, subtype, and IFN-? responsiveness in MS. In support of this proposal, knock-down of XIAP expression in PBMN cells by systemic administration of an antisense oligonucleotide (AEG35156) against XIAP has recently been reported to reduce the severity of EAE in a murine model. Furthermore, AEG35156 dramatically decreased the severity of EAE even when administration of this compound was delayed until the animals showed early signs of paralysis.⁶¹ It is now well established that knock-down of XIAP expression with AEG35156 also lowers the resistance of cancer cells to apoptotic triggers.^62–65 This compound is currently in Phase II clinical trials in the UK for the treatment of leukemia. Success of this study would pave the way for clinical trials designed to assess the efficacy of XIAP knock-down in the treatment of MS.