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SAHA overcomes FLIP-mediated inhibition of SMAC mimetic-induced apoptosis in mesothelioma

Malignant pleural mesothelioma (MPM) is a highly pro-inflammatory malignancy that is rapidly fatal and increasing in incidence. Cytokine signaling within the pro-inflammatory tumor microenvironment makes a critical contribution to the development of MPM and its resistance to conventional chemotherapy approaches. SMAC mimetic compounds (SMCs) are a promising class of anticancer drug that are dependent on tumor necrosis factor alpha (TNFα) signaling for their activity. As circulating TNFα expression is significantly elevated in MPM patients, we examined the sensitivity of MPM cell line models to SMCs. Surprisingly, all MPM cell lines assessed were highly resistant to SMCs either alone or when incubated in the presence of clinically relevant levels of TNFα. Further analyses revealed that MPM cells were sensitized to SMC-induced apoptosis by siRNA-mediated downregulation of the caspase 8 inhibitor FLIP, an antiapoptotic protein overexpressed in several cancer types including MPM. We have previously reported that FLIP expression is potently downregulated in MPM cells in response to the histone deacetylase inhibitor (HDACi) Vorinostat (SAHA). In this study, we demonstrate that SAHA sensitizes MPM cells to SMCs in a manner dependent on its ability to downregulate FLIP. Although treatment with SMC in the presence of TNFα promoted interaction between caspase 8 and the necrosis-promoting RIPK1, the cell death induced by combined treatment with SAHA and SMC was apoptotic and mediated by caspase 8. These results indicate that FLIP is a major inhibitor of SMC-mediated apoptosis in MPM, but that this inhibition can be overcome by the HDACi SAHA.

Malignant pleural mesothelioma (MPM) is a highly pro-inflammatory malignancy that is rapidly fatal and increasing in incidence. Cytokine signaling within the pro-inflammatory tumor microenvironment makes a critical contribution to the development of MPM and its resistance to conventional chemotherapy approaches. SMAC mimetic compounds (SMCs) are a promising class of anticancer drug that are dependent on tumor necrosis factor alpha (TNFa) signaling for their activity. As circulating TNFa expression is significantly elevated in MPM patients, we examined the sensitivity of MPM cell line models to SMCs. Surprisingly, all MPM cell lines assessed were highly resistant to SMCs either alone or when incubated in the presence of clinically relevant levels of TNFa. Further analyses revealed that MPM cells were sensitized to SMC-induced apoptosis by siRNA-mediated downregulation of the caspase 8 inhibitor FLIP, an antiapoptotic protein overexpressed in several cancer types including MPM. We have previously reported that FLIP expression is potently downregulated in MPM cells in response to the histone deacetylase inhibitor (HDACi) Vorinostat (SAHA). In this study, we demonstrate that SAHA sensitizes MPM cells to SMCs in a manner dependent on its ability to downregulate FLIP. Although treatment with SMC in the presence of TNFa promoted interaction between caspase 8 and the necrosis-promoting RIPK1, the cell death induced by combined treatment with SAHA and SMC was apoptotic and mediated by caspase 8. These results indicate that FLIP is a major inhibitor of SMC-mediated apoptosis in MPM, but that this inhibition can be overcome by the HDACi SAHA. Malignant pleural mesothelioma (MPM) is a pro-inflammatory cancer associated with exposure to asbestos, which is rapidly fatal and is increasing in incidence. 1 High levels of pro-inflammatory cytokines associated with asbestos exposure have been linked to the initiation and progression of MPM. 2,3 Tumor necrosis factor alpha (TNFa) has been described as an early biomarker for MPM following exposure to asbestos. Circulating levels of TNFa are elevated in patients with MPM, and these levels are reduced following surgical debulking. 4,5 The majority of MPM patients present in the advanced stages where cisplatin and pemetrexed are the standardof-care chemotherapeutic agents, however, this combination therapy only modestly increases survival rates. Patients quickly relapse, and currently there is no standard-of-care treatment in the second line setting. Drug resistance is a major hurdle in the treatment of MPM. 6,7 Evasion of apoptosis is evident in many cancers, including mesothelioma, through the high expression of inhibitor of apoptosis proteins (IAPs). [8][9][10] High expression of cIAPs in MPM models mediates resistance to chemotherapy and correlates with poor prognosis in patients. 4 The defining features of cIAP1, cIAP2 and XIAP are their three baculovirus IAP repeat (BIR) domains and their RING zinc-finger domains. 11,12 The BIR domains are important for mediating protein-protein interactions, and the RING domain possesses E3 ubiquitin ligase activity. XIAP is the only IAP that directly inhibits caspases: XIAP binds to caspases 3 and 7 via the region between its BIR1 and BIR2 domains and via the BIR2 domain itself, thereby directly inhibiting caspase activity. In addition, the BIR3 domain of XIAP can bind to and prevent homodimerization of caspase 9, thereby preventing its activation. There is also evidence that XIAP may target caspases for ubiquitination and degradation by the proteasome via its RING domain. [13][14][15] The roles of cIAP1 and cIAP2 are more complex and involve regulation of signaling via TNF receptor 1 (TNFR1). 16,17 Following binding of the pro-inflammatory cytokine TNFa, TNFR1 recruits an adapter protein TRADD (TNFR-associated death domain) and receptor-interacting protein kinase 1 (RIPK1). TRADD recruits TRAF2 (TNFR-associated factor 2), and cIAP1 and cIAP2 to form a large membrane complex. At this complex (termed complex I), cIAP1 and cIAP2 act as E3 ubiquitin ligases adding lysine 63 (K63)-linked polyubiquitin chains to RIPK1. Polyubiquitinated RIPK1 then serves as a docking site for the downstream kinases that activate the canonical nuclear factor-kB (NF-kB, RelA/p50) signaling pathway. In the absence of cIAP1/2, RIPK1 is not polyubiquitinated and forms a second complex (complex IIb), which recruits Fas-associated via death domain (FADD), caspase 8 and FLIP. 18 Thus, by ubiquitinating RIPK1, cIAP1 and cIAP2 act as positive regulators of the generally antiapoptotic, pro-inflammatory canonical NF-kB signaling pathway and at the same time inhibit caspase 8 activation in response to ligation of TNFR1. Complex I can dissociate to form Complex IIa, a death inducing complex similar to Complex IIb, which is composed of TRADD, FADD, caspase 8 and FLIP. 19 FLIP is an antiapoptotic caspase 8 paralog, which prevents Complexes IIa and IIb from initiating apoptosis. Moreover, FLIP is the product of an NFkB target gene (CFLAR) and therefore is upregulated following TNFR1 ligation. 20 FLIP is mainly expressed as two splice forms: a long form FLIP(L) and a short form FLIP(S). FLIP inhibits apoptosis through binding to FADD and blocking caspase 8 activation in Complex IIa and IIb. Under certain conditions Complex II can induce cell death through an non-apoptotic, programmed necrosis pathway, which is dependent on RIPK1 and 3. 21 IAPs are recognized as attractive targets for anticancer therapeutics. Cellular IAP 1 (cIAP1), cIAP2 and X-linked IAP (XIAP) are the cellular targets for SMAC mimetic compounds (SMCs), which mimic the functions of the endogenous IAP antagonist SMAC. Second mitochondrial derived activator of caspases (SMAC) is released from the mitochondria by various stimuli and promotes apoptosis through its ability to antagonize IAP-mediated caspase inhibition. 22 SMCs bind to BIR2 and three domains of XIAP and neutralize its inhibition of caspases 3 and 7. SMCs also bind cIAP1 and cIAP2, resulting in a conformational change that leads to RING dimerization and activation of their E3 ligase activity, resulting in their rapid autoubiquitination and degradation. 23,24 As ligation of TNFR1 by TNFa in the absence of cIAP1 and cIAP2 rapidly leads to formation of the cell death inducing complex IIb, 25 we hypothesized that the high levels of TNFa associated with mesothelioma would make this disease particularly vulnerable to SMCs. We therefore investigated whether SMC-mediated inhibition of IAPs could use extracellular TNFa to drive apoptosis in mesothelioma.

Results
Effects of AT-IAP on cIAP and FLIP expression in MPM. To model the impact of SMCs in mesothelioma, we selected four MPM cell line models with different levels of cIAP1, cIAP2 and XIAP expression (Figure 1a). These cell line models also differed in their expression levels of FLIP(L), FLIP(S) and FADD, although expression of RIPK1 and procaspase eight were similar across the panel. Of note,  TNFa was undetectable in the culture medium of these cell lines under basal conditions and following treatment with SMCs (data not shown). The MPM cell lines also expressed TNFR1 on the cell surface to varying levels ( Figure 1b). In this study, we used a novel SMC (AT-IAP) from Astex Pharmaceuticals that is a monovalent inhibitor with nanomolar affinity for the BIR3 domain of both XIAP and cIAP1 (Supplementary Figure 1A). 26  MPM cell lines are insensitive to AT-IAP. To assess the sensitivity of MPM cells to AT-IAP, we conducted cell viability assays in the presence and absence of cotreatment with a physiologically relevant concentration of TNFa (10 ng/ml) in order to mimic the pro-inflammatory micro-environment of mesothelioma. 4 Surprisingly, none of the MPM cell lines were sensitive to AT-IAP at concentrations up to 10 mM, either alone, or in cells cocultured with TNFa ( Figure 2a). Similar results were obtained with several other SMCs (data not shown). To determine whether high expression of IAPs was responsible for resistance to AT-IAP, we used siRNA to individually downregulate cIAP1, cIAP2 and XIAP  Figure 3A). In Ren cells cocultured with TNFa, downregulation of cIAP1 and to a lesser extent XIAP and cIAP2 moderately increased sensitivity to AT-IAP ( Figure 2b); however, none of the other cell lines was sensitized to AT-IAP following siRNA-mediated IAP depletion, suggesting that high IAP expression is not responsible for resistance in these models.
FLIP regulates sensitivity to AT-IAP in MPM. SMCs are known to promote formation of TNFR complex IIa and b and the ripoptosome, [28][29][30] all of which are potentially activating platforms for the initiator caspase, procaspase 8. However, dimerization of procaspase 8 (a prerequisite for its activation) at these complexes can be inhibited by FLIP. Indeed, we found that caspase 8 and FLIP interacted in MPM cells following treatment with AT-IAP and TNFa and moreover, FLIP(L) was recovered in its p43-form indicative of its dimerization with and processing by procaspase 8 at TNFR complex IIb 31 (Figure 3a). Of note, RIPK1 also interacted with caspase 8 in AT-IAP/TNFa-treated cells (Figure 3a), but not in cells treated with AT-IAP alone or TNFa alone (Supplementary Figure 4A). Further evidence that this caspase 8/RIPK1 complex is complex IIb was obtained in cells in which the critical adapter protein for this complex FADD was silenced, as FADD downregulation abrogated the interaction between caspase 8 and RIPK1 (Supplementary Figure 4B).
To assess whether FLIP expression in MPM cells is responsible for their resistance to SMCs, we downregulated FLIP expression with siRNA and assessed sensitivity to AT-IAP in the presence and absence of TNFa A FLIP(L) and FLIP(S) dual-targeted siRNA and splice form-specific siRNAs were used (Figure 3b and Supplementary Figure 3B although the most potent effects were observed when both splice forms were simultaneously downregulated (Figure 3c(i)). Similar results were obtained in MM98 cells (Figure 3c(ii)). In H28 and JU77 MPM cells, downregulation of FLIP(S) failed to enhance AT-IAP/TNFa-induced cell death, whereas FLIP(L) downregulation significantly enhanced cell death (Figure 3ciii and iv). These differences between the cell lines may be related to their relative levels of FLIP(L) and FLIP(S) expression, as well as different levels of expression of other components of the TNFR1 signaling pathway (Figure 1a). Consistent with these findings, siRNA-mediated downregulation of either FLIP(L) or FLIP(S) enhanced caspase 3 processing and PARP cleavage in AT-IAP/TNFa-treated MPM Ren cells, although maximal levels of PARP cleavage were observed when both splice forms were simultaneously downregulated (Figure 4a). Also of note, treatment with TNFa alone induced PARP cleavage when either FLIP(S) or FLIP(L) were silenced and particularly when both splice forms were silenced, consistent with FLIP acting as an inhibitor of TNFR1 complex II-induced apoptosis. Somewhat surprisingly, downregulating FLIP did not significantly enhance apoptosis induced by AT-IAP alone (Figures 3c and 4a), suggesting that this combination treatment is not sufficient to drive complex IIb formation. Indeed, we found that FLIP silencing did not promote formation of complex IIb formation either in response to SMC or TNFa treatment (Supplementary Figure 4C). FLIP(L) but not FLIP(S) silencing significantly enhanced apoptosis in Ju77 MPM cells as assessed by flow cytometry, with maximal levels of apoptosis induced when both splice forms were downregulated (Figure 4b). AT-IAP in combination with TNFa in REN, MM98, JU77 and H28 cells efficiently activated caspase 8 (Supplementary Figure 5A) and caspase 3/7 (Supplementary Figure 5B) following FLIP silencing (Supplementary Figure 5C).
To further prove that it is FLIP's recruitment to complex IIb that modulates sensitivity to AT-IAP/TNFa, we utilized a FLIP mutant (F114A) that cannot interact with the critical adapter protein in the complex, FADD 32 (Figure 4c). When endogenous FLIP(L) and FLIP(S) proteins were depleted with a 5 0 -UTR-targeting siRNA (Supplementary Figure 4D), an exogenously expressed wild-type FLIP(S) protein was able to completely rescue the cell death induced by AT-IAP/TNFa treatment, however, the exogenously expressed F114A  Figure 5b). In H28 and Ju77 cells, the interaction of cisplatin with AT-IAP was less effective at reducing cell viability, with a greater-than-additive effect only observed when cells were treated with 10 mM cisplatin. Thus, increased sensitivity to AT-IAP and TNFa in these mesothelioma models correlated with extent of FLIP downregulation in response to cisplatin (Figures 5a and b). However, these results demonstrate that although cisplatin can induce downregulation of FLIP(S) in some MPM cell lines, it has little effect on FLIP(L) expression.
The HDAC inhibitor SAHA downregulates FLIP and sensitizes MPM cells to AT-IAP. We recently reported that HDAC inhibitors such as SAHA downregulate FLIP(S) and FLIP(L) expression in colorectal cancer 35 Figure 6). It was also apparent that SAHA treatment enhanced sensitivity to TNFa alone much more than to AT-IAP alone (Figure 6b), which is similar to the results obtained following FLIP silencing described  (Figure 7a). We have previously shown that SAHA is unable to downregulate exogenous FLIP expression in either of these two cell line models. 36 Consistent with FLIP downregulation being necessary for SAHA treatment to promote AT-IAP/ TNFa-induced cell death, overexpression of either FLIP(S) or FLIP(L) blocked the effects of SAHA and AT-IAP/TNFa cotreatment on cell death (Figure 7b). To directly prove that combined treatment with SAHA and AT-IAP/TNFa results in apoptosis induction rather than necroptosis, we cotreated cells with the pan-caspase inhibitor z-VAD-fmk and the necroptosis inhibitor Nec-1. z-VAD-fmk, but not Nec-1, blocked the cytotoxic effects of SAHA and AT-IAP/TNFa cotreatment indicating that the cell death induced is indeed apoptotic (Figure 7c). We further defined the mode of apoptosis induced by SAHA and AT-IAP/TNFa cotreatment to be caspase eight-dependent using siRNA to downregulate procaspase eight levels before treatment. In cells in which procaspase 8 was depleted, the apoptosis induced by SAHA and AT-IAP/TNFa cotreatment was completely abrogated (Figure 7d), indicating that the mechanism of cell death is caspase eight-dependent, consistent with a FLIP-dependent mechanism (Figure 7b). In further support of this mechanism, caspase 8 processing to its p43-and p18-forms was detected in control siRNA transfected, SAHA and AT-IAP/TNFa cotreated cells (Figure 7d, Western blot, lane 3).

Discussion
An inflammatory microenvironment makes a major contribution to cancer progression and drug resistance by promoting tumor cell survival. 37 Chronic inflammation associated with asbestos exposure is strongly linked with initiation and progression of MPM, and a number of studies have established important roles for pro-inflammatory cytokines in MPM tumorigenesis. 2,4 Circulating TNFa expression is significantly elevated in MPM patients and surgical debulking significantly reduces the levels of circulating TNFa in patients. 4 This is consistent with a substantial TNFa-rich inflammatory cell component associated with MPM. In this study, we examined the activity of a novel SMC in preclinical models of MPM to provide evidence for their clinical evaluation in this disease. In particular, we focused on the impact of TNFa on sensitivity to SMCs as we hypothesized that SMCs could convert this pro-inflammatory cytokine that is present in the mesothelioma microenvironment into a potent apoptosis inducing cytokine by downregulating cIAP1 and 2 and, thereby promoting breakdown of the pro-survival TNFR1 Complex I and formation of the pro-apoptotic Complex II. Thus, the central tenet of our approach was that inhibition of IAP function would divert microenvironment-derived, pro-inflammatory signaling from activating pro-tumorigenic NF-kB-driven responses towards apoptosis-promoting and anti-inflammatory signaling phenotypes.
Surprisingly, despite expressing all the pro-apoptotic components of TNFR1 Complex IIb, none of the mesothelioma cell lines examined was sensitive to SMCs, either alone or in combination with TNFa. Moreover, neither TNFa alone nor SMC alone was able to promote formation of TNFR1 Complex IIb; only cotreatment with both agents resulted in formation of this potentially cell death inducing complex. However, FLIP was also recruited to this complex in TNFa and SMC cotreated cells, and using RNAi approaches, we demonstrated that this recruitment blocked caspase 8 activation and initiation of apoptosis by this complex. Thus, FLIP is a major mediator of resistance to SMCs in mesothelioma cells as determined in cocultures with physiologically relevant levels of TNFa. Furthermore, we found that both splice forms were capable of blocking apoptosis induction in response to TNFa and SMC cotreatment. Although, TNFR1 complex IIb can induce programmed necrosis (necroptosis) under certain conditions (for example, if FLIP(S) is recruited rather than FLIP(L)), 28 we found that the mechanism of cell death induced when either FLIP splice form was downregulated in TNFa and SMC cotreated mesothelioma cells was apoptotic. These results indicate that in order for SMCs to unleash the pro-apoptotic potential of TNFa, mesothelioma cells must be cotreated with a FLIP inhibitor.
FLIP is a major antiapoptotic protein that as well as inhibiting TNFR1 complexes IIa, IIb and the ripoptosome, also blocks apoptosis induction in response to death ligands such as FasL and TRAIL and in response to various chemotherapeutic drugs in vitro and in vivo. As such, FLIP has been recognized as a potential therapeutic target for a range of cancers. However, because it lacks enzymatic activity and because there is a current lack of structural data, FLIP is a difficult therapeutic target. In addition, FLIP's antiapoptotic activity relies on its ability to compete with procaspase 8 for binding to FADD via its death effector domains (DEDs); as the DEDs of FLIP and procaspase 8 are highly similar, any small molecule that blocks the FLIP-FADD interaction may have the unwanted effect of blocking the procaspase 8-FADD interaction. Because of these difficulties, we and others have examined a number of approaches to targeting FLIP expression. 35,36,[38][39][40] Of these, HDAC inhibitors have the most consistent effects on FLIP expression, with pan-HDAC inhibitors such as vorinostat (SAHA) suppressing FLIP expression by both transcriptional and posttranscriptional mechanisms. 35,36 Moreover, we recently reported that SAHA potently downregulates FLIP expression in mesothelioma cell lines and three-dimensional spheroid models. 36 In this study, we found that SAHA treatment downregulated FLIP expression in 3/4 of the mesothelioma cell lines studied, and this downregulation resulted in induction of caspase 8-dependent apoptosis in cells cotreated with TNFa and SMC. In our previous study, we found that SAHA treatment downregulated FLIP expression in 7/7 cell lines. 36 Thus, SAHA is a potent repressor of FLIP expression in mesothelioma and is significantly more effective in this regard than the current standard-of-care, cisplatin. We therefore conclude that cotreatment with SAHA (or a similar HDAC inhibitor) and a SMC could be a promising therapeutic approach for the treatment of mesothelioma that will exploit the TNFa-rich, pro-inflammatory micro-environment to induce apoptosis via TNFR1 complex IIb (Figure 8). Moreover, circulating levels of TNFa would be a candidate biomarker for sensitivity to this novel combination treatment that could be assessed routinely through non-invasive means.  (Complex IIb) where it recruits FADD, FLIP and procaspase 8. SMCs can also block XIAP-mediated inhibition of caspases 3, 7 and 9. If present in sufficient amounts, FLIP will inhibit activation of caspase 8 at Complex IIb and thereby limit the activation of the downstream executioner caspases 3 and 7 with the result that the cells survive. (c) HDAC inhibitors such as SAHA efficiently target FLIP for degradation via the proteasome. Thus, when cells are cotreated with SAHA and SMC, procaspase 8 homodimers are recruited to Complex IIb, resulting in caspase 8 activation and subsequent activation of the executioner caspases and induction of apoptosis. The activation of the executioner caspases is further facilitated by SMC-mediated inhibition of XIAP