Smac

[Tabita Knezevic]

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Small-molecule inhibitor of apoptosis (IAP) antagonists, called Smac mimetic compounds (SMCs), sensitize tumours to TNF-α-induced killing while simultaneously blocking TNF-α growth-promoting activities. SMCs also regulate several immunomodulatory properties within immune cells. We report that SMCs synergize with innate immune stimulants and immune checkpoint inhibitor biologics to produce durable cures in mouse models of glioblastoma in which single agent therapy is ineffective. The complementation of activities between these classes of therapeutics is dependent on cytotoxic T-cell activity and is associated with a reduction in immunosuppressive T-cells. Notably, the synergistic effect is dependent on type I IFN and TNF-α signalling. Furthermore, our results implicate an important role for TNF-α-producing cytotoxic T-cells in mediating the anti-cancer effects of immune checkpoint inhibitors when combined with SMCs. Overall, this combinatorial approach could be highly effective in clinical application as it allows for cooperative and complimentary mechanisms in the immune cell-mediated death of cancer cells.

Evasion of apoptosis and avoidance of immune attack represent two key hallmarks of cancer1. Members of the inhibitor of apoptosis (IAP) gene family play important interconnecting roles in both of these characteristic pathways of tumorigenesis2, providing a critical nexus in the targeting of cancer. Small-molecule antagonists of the IAPs, known as Smac mimetic compounds (SMCs), are in clinical development for cancer therapy3. SMCs were found to exert immunological effects leading to the eradication of tumours4,5. Mechanistically, SMCs bind to cellular IAP 1 and 2 (cIAP1 and cIAP2), which induces the auto-ubiquitination and subsequent proteasomal-mediated degradation of these IAPs6. At higher doses, SMCs can antagonize X-linked IAP (XIAP), de-repressing the ability of XIAP to inhibit pro-apoptotic caspases. These three IAPs are E3 ubiquitin ligases that control diverse signalling pathways through post-translational ubiquitination reactions, including pathways central to immunity7. The SMC drug-sustained loss of IAPs has important consequences. First, SMC-mediated antagonism of the IAPs sensitizes cancer cells to death ligands originating from the immune system by switching tumour necrosis factor alpha (TNF-α) from a survival factor to a potent death factor, leading to death through the ripoptosome or the necrosome8,9. Second, the loss of the cIAPs activates the alternative nuclear factor kappa B (NF-κB) pathway through the stabilization of NF-κB-inducing kinsase (NIK) in cells10. NIK is a target of the cIAPs, wherein NIK is constitutively ubiquitinated and degraded. However, on binding of a TNF superfamily ligand to their cognate receptor, such as CD137 (aka, 4-1BB), the cIAPs are sequestered and degraded, thereby allowing for the accumulation of NIK and activation of the alternative NF-κB pathway10,11.

In general, tumours are resistant to the induction of apoptosis due to the p53-mediated adaptations of the intrinsic mitochondrial cell death pathway to damaging DNA lesions and prior chemotherapy treatments12. In contrast, the extrinsic cell death pathway, which responds to death ligands from the immune system, is typically intact in cancer cells12. Thus, the extrinsic pathway provides an avenue to exploit for the induction of cancer cell death. However, tumours have evolved other means to suppress immune attack such as by upregulating T-cell co-inhibitory molecules, typified by Programmed death-ligand (PD-L1, a.k.a., CD274), on the cancer cell surface. The recent clinical successes for antibody-based biologics, called immune checkpoint inhibitors (ICIs), which target molecules like programmed cell death protein 1 (PD-1, a.k.a. CD279), have demonstrated remarkable efficacy13,14,15. ICIs overcome the countervailing immune checkpoint blockade and promote the immune system to attack tumour cells. However, these drugs are not without limitations: a notable example is the appearance of limiting toxicities related to the induction of autoimmunity.

Here, we investigate the efficacy of targeting cIAP1 and cIAP2 with a SMC in combination with an immunotherapy agent for the treatment of glioblastoma. We demonstrate that SMCs and ICIs combine to form an effective immunotherapy for the treatment in mouse models of this deadly brain cancer, and for other cancers, such as mammary carcinoma and multiple myeloma/plasmacytoma. In addition to the synergy that we have found with innate immune stimulants, our results uncover a second important mechanism by which SMCs exert their anti-cancer effects, specifically through the potentiation of cytotoxic T-cell (CTL) activity against tumours, which is amplified with an ICI.

The virus-induced immune effects are mediated in part by type I IFNs. We show here that CT-2A cells are partially sensitive to combined SMC and recombinant IFN-α in vitro (Fig. 2e). We observed that the intracranial administration of SMC resulted in even more profound degradation of the IAP proteins in CT-2A brain tumours (Supplementary Fig. 6). For in vivo studies, we used a form of recombinant IFN-α that consists of a hybrid of human isoforms IFN-α B and IFN-α D, which displays potent antiviral activity among a broad range of species24. A single co-administration of SMC and IFN-α significantly extended mouse survival and resulted in a 50% durable cure rate (Fig. 2f). Long-term survivors displayed no overt physical or behavioural defects from the single or combined intracranial treatments of SMC, poly(I:C) or IFN-α (Fig. 2c,f; Supplementary Fig. 7). Furthermore, as we observed a transient increase of intracranial TNF-α within the brain on systemic VSVΔ51 infection or treatment with poly(I:C) (Fig. 2b), we sought to determine whether systemic administration of recombinant IFN-α alongside with SMC treatment would be efficacious in the CT-2A glioblastoma model. Similar to the combination of SMC and VSVΔ51, the combination of IFN-α administered i.p. with oral gavage of SMC resulted in durable cures in 55% of the mice (Fig. 2g). These results suggest that the presence of a transient inflammatory environment in the brain is tolerable and indicate that indirect and other direct (intracranial) routes of combination treatment administration may be feasible.

The innate immune system is a key player in the SMC-mediated death of tumour cells16. Nevertheless, fundamental questions remain as to the contributory role of the adaptive immune system in this SMC combination approach. Furthermore, a potential pitfall of the proposed use of oncolytic viruses or other immunostimulatory agents in combination with SMC treatment could be the increase in expression of checkpoint inhibitor ligands on cancer cells, thereby negating CTL-mediated attack of tumours25. Flow cytometry analysis revealed that treatment of glioma cells with recombinant type I IFN or infection with VSVΔ51, but not treatment with TNF-α, resulted in the increased surface expression of PD-L1 and major histocompatibility complex (MHC) I markers. Moreover, there was no significant impact on the expression of these tumour surface molecules by SMC treatment (Fig. 3a; Supplementary Fig. 8).

Interestingly, mice previously cured of orthotopic EMT6 mammary carcinomas by combined SMC treatments were completely resistant to tumour engraftment when rechallenged with EMT6 cells (Fig. 3b). However, another syngeneic cell line, 4T1, that shares the major histocompatibility proteins, was not rejected from these cured mice. We found that mice cured with intracranial CT-2A tumours were also resistant to tumour engraftment of CT-2A cells injected either subcutaneously or intracranially (Fig. 3c). We next evaluated the cytotoxic potential of CD8 T-cells from cured mice via an ELISpot assay. Stimulation of CD8+ T-cells from cured mice, but not cells isolated from naive mice, with CT-2A cells revealed the presence of specific reactive T-cells, as demonstrated by enhanced IFN-γ and Granzyme B (GrzB) production (Fig. 4a). The inclusion of anti-PD-1 blocking antibodies further increased the expression of IFN-γ and GrzB. Similar results were observed with mice cured of EMT6 tumours (Supplementary Fig. 9). Collectively, these results suggest the generation of a robust and specific long-term tumour immunity using SMC combination therapy.

To determine whether the increased levels of PD-1+ CD8+ T-cells may be a negative modulator for SMC efficacy, we also assessed blocking the checkpoint target, PD-1, as well as CTLA-4, in combination with SMC using two mouse models of glioblastoma. The systemic administration of anti-PD-1 or anti-CTLA4 antibodies demonstrated no activity on their own (Fig. 4d,e). In contrast, the combination of anti-PD-1 and SMC significantly extended survival and resulted in 71% and 33% durable cure rates in the CT-2A and GL261 models, respectively. Furthermore, when combined with a SMC, the anti-PD-1 biologic was superior to the anti-CTLA4 biologic in the CT-2A model (71% versus 43%) (Fig. 4d). There are two structural classes of SMCs: monomers and dimers. Monomeric SMCs consist of a single chemical molecule that binds to the BIR domains of the IAPs while dimeric SMCs consist of two SMC molecules connected by a linker allowing for cooperative binding and/or tethering of IAPs. A clinically advanced SMC, LCL161, is the focus of most of our studies, and is a potent monomer. We next sought to assess whether another clinically advanced dimeric SMC similarly synergizes with an ICI for the treatment of glioblastoma. Similar to our previous results, we observed a significant increase in survival of mice bearing intracranial CT-2A tumours when treated with anti-PD-1 and the dimer SMC, Birinapant (Fig. 4f). As the combined blockade of PD-1 or CTLA-4 is beneficial for patients with melanoma28, we sought to determine whether the combination of anti-PD-1 and anti-CTLA-4 would similarly significantly enhance SMC therapy. Consistent with a previous report, the combination of antibodies targeting PD-1 and CTLA-4 was effective at inducing durable cures in a mouse model of cancer27, as we observed an overall survival rate of 67% (Fig. 4g). Strikingly, the inclusion of SMC treatment with anti-PD-1 and anti-CTLA-4 together resulted in a 100% durable cure rate.

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