Dysregulation of apoptosis can result in inappropriate suppression of cell death, as occurs in the development of some cancers, or in failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain neurodegenerative disorders, such as spinal muscular atrophy (SMA). Recently, we isolated a candidate gene, encoding neuronal apoptosis inhibitor protein (NAIP), for SMA. This gene is homologous to two baculovirus inhibitor of apoptosis proteins (Cp-IAP and Op-IAP) and is partly deleted in individuals with type I SMA. A second SMA candidate gene encoding survival motor neuron (SMN), which is contiguous with the NAIP locus on 5q13.1, was also reported. Here we demonstrate a NAIP-mediated inhibition of apoptosis induced by a variety of signals, and have identified three additional human complementary DNAs and a Drosophila melanogaster sequence that are also homologous to the baculovirus IAPs. The four open reading frames (ORFs) possess three baculoviral inhibition of apoptosis protein repeat (BIR) domains and a carboxy-terminal RING zinc-finger. The human iap genes have a distinct but overlapping pattern of expression in fetal and adult tissues. These proteins significantly increase the number of known apoptotic suppressors.

The spinal muscular atrophies (SMAs), characterized by spinal cord motor neuron depletion, are among the most common autosomal recessive disorders. One model of SMA pathogenesis invokes an inappropriate persistence of normally occurring motor neuron apoptosis. Consistent with this hypothesis, the novel gene for neuronal apoptosis inhibitory protein (NAIP) has been mapped to the SMA region of chromosome 5q13.1 and is homologous with baculoviral apoptosis inhibitor proteins. The two first coding exons of this gene are deleted in approximately 67% of type I SMA chromosomes compared with 2% of non-SMA chromosomes. Furthermore, RT-PCR analysis reveals internally deleted and mutated forms of the NAIP transcript in type I SMA individuals and not in unaffected individuals. These findings suggest that mutations in the NAIP locus may lead to a failure of a normally occurring inhibition of motor neuron apoptosis resulting in or contributing to the SMA phenotype.

rogrammed cell death (apoptosis) plays a critical part in regulating cell turnover during embryogenesis, metamorphosis, tissue homeostasis and viral infection 1 . Dysregulation of apoptosis occurs in such pathologies as cancer, autoimmunity, immunodeficiency and neurodegeneration. Proteins of the inhibitor-ofapoptosis (IAP) family are intrinsic cellular suppressors of apoptosis and are represented by highly conserved members found from insect viruses to mammals 2-4 . The most potent mammalian IAP is the X-linked IAP, or XIAP 5 , whose mechanism of action involves direct inhibition of caspases 3 and 7, key proteases of the apoptotic cascade 6 . Cellular control of XIAP expression should be fundamental to a cell's ability to modulate its responses to apoptotic stimuli. However, XIAP messenger RNA is expressed in most tissues and cells at fairly constant levels 5 , indicating that translational control of XIAP levels may be an important regulatory mechanism. Here we characterize the primary genomic structure and function of XIAP, and show that XIAP expression is controlled at the translational level, specifically through an internal ribosome-entry site (IRES).Several features of XIAP mRNA indicate that it may be translationally regulated, including an unusually long 5′ untranslated region (UTR) (>5.5 kilobases (kb) for murine and >1.6 kb for human XIAP transcripts) with predicted complex secondary structure and numerous potential translation start sites upstream of the authentic initiation codon. This UTR would be expected to present a significant obstacle to efficient translation by conventional ribosome scanning 7 . An alternative mechanism of translation initiation, mediated through the IRES, has been identified in picornaviruses and in a few cellular mRNAs 8 . Thus we tested whether the 5′ UTR of XIAP mRNA could be involved in translation initiation from reporter-based bicistronic mRNA transcripts encoding β-galactosidase and chloramphenicol aceytyltransferase (CAT) (for example, see ref. 9). (Translation of β-galactosidase is driven by the 5′ mRNA methylguanosine cap.) Both human and mouse XIAP 5′ UTRs directed translation of the second cistron (encoding CAT) at 150-fold higher levels than those produced without the 5′ UTR or with the 5′ UTR in reverse orientation, suggesting the presence of an IRES (Fig. 1a). No activity was detected when using the identical DNA segments cloned into a promoterless construct, confirming P Figure 1 The 5′ UTR of mouse and human XIAP mRNA contains functional IRES elements. a, DNA segments corresponding to the indicated regions of the 5′ UTR of human (h) or mouse (m) XIAP transcripts were inserted (in the indicated directions) into the XhoI site of the linker region (LR) of the bicistronic plasmid pβgal/CAT (where βgal is β-galactosidase); HeLa cells were transfected with these plasmids (2 µg each). The promoterless CAT reporter plasmid pCATbasic/UTR was constructed by inserting the indicated 5′ UTR region into the pCATbasic vector, and in this case HeLa cells were co-transfected ...

To expand our current array of safe and potent oncolytic viruses, we screened a variety of wild-type (WT) rhabdoviruses against a panel of tumor cell lines. Our screen identified a number of viruses with varying degrees of killing activity. Maraba virus was the most potent of these strains. We built a recombinant system for the Maraba virus platform, engineered a series of attenuating mutations to expand its therapeutic index, and tested their potency in vitro and in vivo. A double mutant (MG1) strain containing both G protein (Q242R) and M protein (L123W) mutations attenuated Maraba virus in normal diploid cell lines, yet appeared to be hypervirulent in cancer cells. This selective attenuation was mediated through interferon (IFN)-dependent and -independent mechanisms. Finally, the Maraba MG1 strain had a 100-fold greater maximum tolerable dose (MTD) than WT Maraba in vivo and resulted in durable cures when systemically administered in syngeneic and xenograft models. In summary, we report a potent new oncolytic rhabdovirus platform with unique tumor-selective attenuating mutations.

The cellular inhibitor of apoptosis 2 (cIAP2/HIAP1) is a potent inhibitor of apoptotic death. In contrast to the other members of the IAP family, cIAP2 is transcriptionally inducible by nuclear factor-B in response to multiple triggers. We demonstrate here that cIAP2 ؊/؊ mice exhibit profound resistance to lipopolysaccharide (LPS)-induced sepsis, specifically because of an attenuated inflammatory response. We show that LPS potently upregulates cIAP2 in macrophages and that cIAP2 ؊/؊ macrophages are highly susceptible to apoptosis in a LPS-induced proinflammatory environment. Hence, cIAP2 is critical in the maintenance of a normal innate immune inflammatory response.

Stable expression of short-hairpin RNAs (shRNAs) directed against the X-linked inhibitor of apoptosis (XIAP) resulted in the generation of three MDA-MB-231 cell lines (XIAP shRNA cells) with reductions in XIAP mRNA and protein levels 485% relative to MDA-MB-231 cells stably transfected with the U6 RNA polymerase III promoter alone (U6 cells). This RNA interference (RNAi) approach dramatically sensitized these cells to killing by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Importantly, loss of XIAP also sensitized the cells to killing by taxanes but had no additional effects on killing by carboplatin and doxorubicin. The increased sensitivity of the XIAP shRNA cells to killing by TRAIL and taxanes correlated with enhanced caspase cleavage and activation, including caspase-8, and robust processing of poly(ADP-ribose) polymerase and BID compared to U6 cells. Additionally, increasing XIAP levels by adenovirus-mediated expression protected both XIAP shRNA and U6 cells from TRAIL killing in a dose-dependent manner. The effects observed by stable RNAi with respect to TRAIL sensitization were also achieved following downregulation of XIAP in Panc-1 cells treated with a second-generation, mixed-backbone antisense oligonucleotide, AEG 35156/GEM640. These data indicate that reducing XIAP protein expression by either RNAi or antisense approaches increases cancer cell susceptibility to functionally diverse chemotherapeutic agents and supports the notion that downregulation of XIAP in vivo may synergize with disease-relevant chemotherapeutic regimes, including TRAIL and taxanes, to increase the effectiveness of antineoplastic agents.

To identify therapeutic opportunities for oncolytic viral therapy, we conducted genome-wide RNAi screens to search for host factors that modulate rhabdoviral oncolysis. Our screens uncovered the endoplasmic reticulum (ER) stress response pathways as important modulators of rhabdovirus-mediated cytotoxicity. Further investigation revealed an unconventional mechanism whereby ER stress response inhibition preconditioned cancer cells, which sensitized them to caspase-2-dependent apoptosis induced by a subsequent rhabdovirus infection. Importantly, this mechanism was tumor cell specific, selectively increasing potency of the oncolytic virus by up to 10,000-fold. In vivo studies using a small molecule inhibitor of IRE1α showed dramatically improved oncolytic efficacy in resistant tumor models. Our study demonstrates proof of concept for using functional genomics to improve biotherapeutic agents for cancer.

Despite improvements in chemotherapy and radical surgical debulking, peritoneal carcinomatosis (PC) remains among the most common causes of death from abdominal cancers. Immunotherapies have been effective for selected solid malignancies, but their potential in PC has been little explored. Here, we report that intraperitoneal injection of an infected cell vaccine (ICV), consisting of autologous tumor cells infected ex vivo with an oncolytic Maraba MG1 virus expressing IL12, promotes the migration of activated natural killer (NK) cells to the peritoneal cavity in response to the secretion of IFNγ-induced protein-10 (IP-10) from dendritic cells. The recruitment of cytotoxic, IFNγ-secreting NK cells was associated with reduced tumor burden and improved survival in a colon cancer model of PC. Even in mice with bulky PC (tumors > 8 mm), a complete radiologic response was demonstrated within 8 to14 weeks, associated with 100% long-term survival. The impact of MG1-IL12-ICV upon NK-cell recruitment and function observed in the murine system was recapitulated in human lymphocytes exposed to human tumor cell lines infected with MG1-IL12. These findings suggest that an MG1-IL12-ICV is a promising therapy that could provide benefit to the thousands of patients diagnosed with PC each year. Cancer Immunol Res; 5(3); 211–21. ©2017 AACR.