Smac Mimetic Research Articles

Self-synthesized second mitochondria-derived activator of caspase (SMAC) mimetic TP-WY-1345 enhances the radiosensitivity of NSCLC cells H1299 by targeting anti-apoptotic protein cIAP1

ObjectiveTo explore the toxicity and cellular uptake of the self-synthesized second mitochondria-derived activator of caspase (SMAC) mimetic TP-WY-1345 polypeptide and its radiosensitizing effect on non-small cell lung cancer (NSCLC). MethodsA fluorescence microscope was used to observe the uptake efficiency of TP-WY-1345 by human NSCLC cells H1299. Toxicity of the TP-WY-1345 peptide in normal cells was examined in human embryonic lung fibroblasts MRC5. CCK-8 and clone formation experiments were performed to detect the radiosensitizing effect of TP-WY-1345 in the H1299 ​cells. The AutoDock Vina simulation software was employed to predict the binding efficiency of TP-WY-1345 to the inhibitor of apoptosis proteins (IAPs). Moreover, Western blot and qPCR experiments were carried out to determine the protein and gene expressions, and the flow cytometry (FCM) technique was used to detect the apoptosis level under different conditions. ResultsTP-WY-1345 can be self-synthesized, and significant green fluorescence was observed in H1299 2 ​h and 6 ​h after incubation with TP-WY-1345. The cell counting and CCK-8 results showed that 10 ​μmol/L and 20 ​μmol/L TP-WY-1345 did not produce a significant toxic effect on the MRC5 cells (P ​> ​0.05). Compared with the single ionizing radiation group, the cell viability and clone formation of H1299 ​cells were significantly inhibited after treatment with TP-WY-1345 at a concentration of 50 ​μmol/L (P ​< 0.05). The sensitivity enhancement ratio (SER) was calculated to be 1.14. Moreover, the binding efficiency of TP-WY-1345 to cIAP1 protein was predicted to be −7.3, and this strong binding force was demonstrated by Western blot. TP-WY-1345 inhibited the protein and mRNA expressions of cIAP1 and further increased the apoptosis level of the H1299 ​cells at 24 ​h ​(P ​< ​0.01) and 48 ​h after radiation (P ​< ​0.05). ConclusionSMAC mimetic TP-WY-1345 can enter the H1299 ​cells and produce a radiosensitizing effect by increasing the level of radiation-induced apoptosis of the cells. There, TP-WY-1345 is expected to become a new generation of radiosensitizing drugs.

Construction and validation of a novel tumor necrosis factor-related apoptosis-inducing ligand mutant MuR5S4-TR.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively kill tumor cells but has no significant effect on normal cells. However, the use of TRAIL is limited for resistance by more than 50% of the tumor cell lines. It's very important to develop a more efficient form of TRAIL for cancer treatment. The N-terminal in soluble fragments (114-281aa) of TRAIL was redesigned to construct a novel TRAIL mutant-MuR5S4-TR. The Cell Counting Kit-8 method to explore the antitumor effects. The potential mechanisms were also explored. Novel TRAIL mutant with cell-penetrating peptides (CPP) like and Second mitochondria-derived activator of caspases (Smac) like structure-MuR5S4-TR was successfully constructed. The prokaryotic expression system was successfully built, and the MuR5S4-TR was purified and reconfirmed by western blot. MuR5S4-TR could enhance the antitumor effects of TRAIL in most of the cancer cell lines significantly, NCI-H460 lung cancer cell line, for instance. After MuR5S4-TR treatment, the expressions of death receptor 4 (DR4), DR5, Caspase-8, and cleaved Caspase-3 were remarkably increased, however, there was no significant difference in X-linked inhibitor of apoptosis expression. We constructed a novel TRAIL mutant with CPP-like and Smac-like structure-MuR5S4-TR. The MuR5S4-TR showed significantly stronger antitumor effects than TRAIL in many tumor cell lines. The MuR5S4-TR showed strong antitumor effects both in vitro and in vivo. This preliminary study implies that MuR5S4-TR may be a more efficient form of TRAIL for cancer therapy.

LCL161 enhances expansion and survival of engineered anti-tumor T cells but is restricted by death signaling.

The genesis of SMAC mimetic drugs is founded on the observation that many cancers amplify IAP proteins to facilitate their survival, and therefore removal of these pathways would re-sensitize the cells towards apoptosis. It has become increasingly clear that SMAC mimetics also interface with the immune system in a modulatory manner. Suppression of IAP function by SMAC mimetics activates the non-canonical NF-κB pathway which can augment T cell function, opening the possibility of using SMAC mimetics to enhance immunotherapeutics. We have investigated the SMAC mimetic LCL161, which promotes degradation of cIAP-1 and cIAP-2, as an agent for delivering transient costimulation to engineered BMCA-specific human TAC T cells. In doing so we also sought to understand the cellular and molecular effects of LCL161 on T cell biology. LCL161 activated the non-canonical NF-κB pathway and enhanced antigen-driven TAC T cell proliferation and survival. Transcriptional profiling from TAC T cells treated with LCL161 revealed differential expression of costimulatory and apoptosis-related proteins, namely CD30 and FAIM3. We hypothesized that regulation of these genes by LCL161 may influence the drug's effects on T cells. We reversed the differential expression through genetic engineering and observed impaired costimulation by LCL161, particularly when CD30 was deleted. While LCL161 can provide a costimulatory signal to TAC T cells following exposure to isolated antigen, we did not observe a similar pattern when TAC T cells were stimulated with myeloma cells expressing the target antigen. We questioned whether FasL expression by myeloma cells may antagonize the costimulatory effects of LCL161. Fas-KO TAC T cells displayed superior expansion following antigen stimulation in the presence of LCL161, suggesting a role for Fas-related T cell death in limiting the magnitude of the T cell response to antigen in the presence of LCL161. Our results demonstrate that LCL161 provides costimulation to TAC T cells exposed to antigen alone, however LCL161 did not enhance TAC T cell anti-tumor function when challenged with myeloma cells and may be limited due to sensitization of T cells towards Fas-mediated apoptosis.

Effect of Butylphthalide Capsules on Smac and XIAP Expression in Rats after Ischemia Reperfusion

IntroductionLittle is known about the protective effects of butylphthalide on cerebral ischemia-reperfusion injury. This study aims to investigate the impact on the second mitochondrial-derived activator of Caspases (Smac) and X-linked inhibitor of apoptosis protein (XIAP) expression in the ischemic semidark area using a rat model of carotid artery stenosis. MethodsThirty Sprague–Dawley rats were randomly divided into the sham-operated group, carotid stenosis model controls, low-dose (20 mg/kg), medium-dose (40 mg/kg), and high-dose (80 mg/kg) butylphthalide groups. The neurological function was scored by the balance beam test (BBT). The morphological changes of brain tissue were detected by Hematoxylin-eosin (HE) staining, with apoptosis detected by Terminal Deoxynucleotidyl Transferase mediated dUTP Nick-End Labeling (TUNEL) staining. Smac and XIAP protein expression were detected by immunohistochemistry (IHC). The expressions of Smac and XIAP mRNA were detected by real-time quantitative polymerase chain reaction (RT-qPCR). ResultsHE showed that neuronal loss, nuclear consolidation, and vacuolar degeneration were significantly reduced in the medium and high-dose butylphthalide groups compared with the model controls. The BBT scores and apoptotic index were significantly lower in the medium and high doses of butylphthalide compared with the model controls. RT-qPCR and IHC showed that Smac, XIAP mRNA and protein expressions in the ischemic hemispheric region were significantly reduced in low, medium, and high doses of butylphthalide compared with the model controls (P < 0.05), showing some concentration effect. ConclusionsButylphthalide can significantly reduce Smac and XIAP mRNA and protein expression, inhibit neuronal apoptosis induced by ischemia-reperfusion injury in rats with carotid stenosis, and exert neuroprotective effects.

Drug response profiles in patient-derived cancer cells across histological subtypes of ovarian cancer: real-time therapy tailoring for a patient with low-grade serous carcinoma

Many efforts are underway to develop novel therapies against the aggressive high-grade serous ovarian cancers (HGSOCs), while our understanding of treatment options for low-grade (LGSOC) or mucinous (MUCOC) of ovarian malignancies is not developing as well. We describe here a functional precision oncology (fPO) strategy in epithelial ovarian cancers (EOC), which involves high-throughput drug testing of patient-derived ovarian cancer cells (PDCs) with a library of 526 oncology drugs, combined with genomic and transcriptomic profiling. HGSOC, LGSOC and MUCOC PDCs had statistically different overall drug response profiles, with LGSOCs responding better to targeted inhibitors than HGSOCs. We identified several subtype-specific drug responses, such as LGSOC PDCs showing high sensitivity to MDM2, ERBB2/EGFR inhibitors, MUCOC PDCs to MEK inhibitors, whereas HGSOCs showed strongest effects with CHK1 inhibitors and SMAC mimetics. We also explored several drug combinations and found that the dual inhibition of MEK and SHP2 was synergistic in MAPK-driven EOCs. We describe a clinical case study, where real-time fPO analysis of samples from a patient with metastatic, chemorefractory LGSOC with a CLU-NRG1 fusion guided clinical therapy selection. fPO-tailored therapy with afatinib, followed by trastuzumab and pertuzumab, successfully reduced tumour burden and blocked disease progression over a five-year period. In summary, fPO is a powerful approach for the identification of systematic drug response differences across EOC subtypes, as well as to highlight patient-specific drug regimens that could help to optimise therapies to individual patients in the future.

Killing in self-defense: proapoptotic drugs to eliminate intracellular pathogens.

Intracellular infections rely on host cell survival for replication and have evolved several mechanisms to prevent infected cells from dying. Drugs that promote apoptosis, a noninflammatory form of cell death, can dysregulate these survival mechanisms to kill infected cells via a mechanism that resists the evolution of drug resistance. Two such drug classes, known as SMAC mimetics and BH3 mimetics, have shown preclinical efficacy at mediating clearance of liver-stage malaria and chronic infections such as hepatitis-B virus and Mycobacterium tuberculosis. Emerging toxicity and efficacy data have reinforced the broad applicability of these drugs and form the foundations for preclinical and clinical studies into their various usage cases.

PP 5.4 – 00184 Optimization of Smac Mimetics as HIV-1 Latency Reversing Agents

PP 5.4 – 00184 Optimization of Smac Mimetics as HIV-1 Latency Reversing Agents

Combining the SMAC mimetic LCL161 with Gemcitabine plus Cisplatin therapy inhibits and prevents the emergence of multidrug resistance in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a highly lethal gastrointestinal malignancy that has one of the worst prognoses among solid tumors. The combination of Gemcitabine + Cisplatin (GEM/CIS) remains the standard first-line treatment for advanced stage CCA. However, this drug combination yields only a modest objective response rate, and in cases that initially respond to this treatment, drug resistance commonly rapidly develops. To improve the efficiency of GEM/CIS therapy for CCA, a thorough understanding of the mechanism of GEM/CIS resistance in CCA is required. To that end - in this study, we developed several acquired GEM/CIS-resistant CCA cell lines and we screened those cell lines for acquired vulnerability. The screening process revealed that subset of CCA with GEM/CIS resistance acquired vulnerability to the small-molecule second mitochondrial-derived activator of caspases (SMAC) mimetics LCL161 and Birinapant. The observed acquired vulnerability was found to be associated with upregulation of an inhibitor of apoptosis protein 2 (cIAP2), a known target of SMAC mimetics. LCL161 or cIAP2-shRNA downregulated cIAP2 and restored the sensitivity to GEM/CIS in GEM/CIS-resistant CCA cell lines and in in vivo GEM/CIS-resistant xenograft models. A strong synergic effect was observed when LCL161 was added to GEM/CIS. Interestingly, this synergism was also observed in drug-naïve CCA cell lines, xenografts, and patient-derived organoids. This triplet therapy also prevented the emergence of multidrug-resistant CCA in in vitro and in vivo models. Our findings suggest that activation of cIAP2 allows CCA to escape GEM/CIS, and that suppression of cIAP2 reestablishes the apoptotic profile of CCA, thus restoring its vulnerability to GEM/CIS. The results of this study indicate that combining the SMAC mimetic LCL161 with GEM/CIS inhibits and prevents the emergence of multidrug resistance in CCA.

X-linked inhibitor of apoptosis protein represents a promising therapeutic target for relapsed/refractory ALL.

Acute lymphoblastic leukemia (ALL) represents the most frequent malignancy in children, and relapse/refractory (r/r) disease is difficult to treat, both in children and adults. In search for novel treatment options against r/r ALL, we studied inhibitor of apoptosis proteins (IAP) and Smac mimetics (SM). SM-sensitized r/r ALL cells towards conventional chemotherapy, even upon resistance against SM alone. The combination of SM and chemotherapy-induced cell death via caspases and PARP, but independent from cIAP-1/2, RIPK1, TNFα or NF-κB. Instead, XIAP was identified to mediate SM effects. Molecular manipulation of XIAP invivo using microRNA-30 flanked shRNA expression in cell lines and patient-derived xenograft (PDX) models of r/r ALL mimicked SM effects and intermediate XIAP knockdown-sensitized r/r ALL cells towards chemotherapy-induced apoptosis. Interestingly, upon strong XIAP knockdown, PDX r/r ALL cells were outcompeted invivo, even in the absence of chemotherapy. Our results indicate a yet unknown essential function of XIAP in r/r ALL and reveal XIAP as a promising therapeutic target for r/r ALL.

Induction of Breast Cancer Cell Apoptosis by TRAIL and Smac Mimetics: Involvement of RIP1 and cFLIP.

Smac mimetics are a group of compounds able to facilitate cell death in cancer cells. TNF-related apoptosis-inducing ligand (TRAIL) is a death receptor ligand currently explored in combination with Smac mimetics. The molecular mechanisms determining if the combination treatment results in apoptosis are however not fully understood. In this study, we aimed to shed light on these mechanisms in breast cancer cells. Three breast cancer cell lines, MDA-MB-468, CAMA-1 and MCF-7, were used to evaluate the effects of Smac mimetic LCL-161 and TRAIL using cell death assays and Western blot. The combination treatment induces apoptosis and caspase-8 cleavage in MDA-MB-468 and CAMA-1 but not in MCF-7 cells and downregulation of caspase-8 blocked apoptosis. Downregulation, but not kinase inhibition, of receptor-interacting protein 1 (RIP1) suppressed apoptosis in CAMA-1. Apoptosis is preceded by association of RIP1 with caspase-8. Downregulating cellular FLICE-like inhibitory protein (c-FLIP) resulted in increased caspase cleavage and some induction of apoptosis by TRAIL and LCL-161 in MCF-7. In CAMA-1, c-FLIP depletion potentiated TRAIL-induced caspase cleavage and LCL-161 did not increase it further. Our results lend further support to a model where LCL-161 enables the formation of a complex including RIP1 and caspase-8 and circumvents c-FLIP-mediated inhibition of caspase activation.

Interaction of LATS1 with SMAC links the MST2/Hippo pathway with apoptosis in an IAP-dependent manner

Metastatic malignant melanoma is the deadliest skin cancer, and it is characterised by its high resistance to apoptosis. The main melanoma driving mutations are part of ERK pathway, with BRAF mutations being the most frequent ones, followed by NRAS, NF1 and MEK mutations. Increasing evidence shows that the MST2/Hippo pathway is also deregulated in melanoma. While mutations are rare, MST2/Hippo pathway core proteins expression levels are often dysregulated in melanoma. The expression of the tumour suppressor RASSF1A, a bona fide activator of the MST2 pathway, is silenced by promoter methylation in over half of melanomas and correlates with poor prognosis. Here, using mass spectrometry-based interaction proteomics we identified the Second Mitochondria-derived Activator of Caspases (SMAC) as a novel LATS1 interactor. We show that RASSF1A-dependent activation of the MST2 pathway promotes LATS1-SMAC interaction and negatively regulates the antiapoptotic signal mediated by the members of the IAP family. Moreover, proteomic experiments identified a common cluster of apoptotic regulators that bind to SMAC and LATS1. Mechanistic analysis shows that the LATS1-SMAC complex promotes XIAP ubiquitination and its subsequent degradation which ultimately results in apoptosis. Importantly, we show that the oncogenic BRAFV600E mutant prevents the proapoptotic signal mediated by the LATS1-SMAC complex while treatment of melanoma cell lines with BRAF inhibitors promotes the formation of this complex, indicating that inhibition of the LATS1-SMAC might be necessary for BRAFV600E-driven melanoma. Finally, we show that LATS1-SMAC interaction is regulated by the SMAC mimetic Birinapant, which requires C-IAP1 inhibition and the degradation of XIAP, suggesting that the MST2 pathway is part of the mechanism of action of Birinapant. Overall, the current work shows that SMAC-dependent apoptosis is regulated by the LATS1 tumour suppressor and supports the idea that LATS1 is a signalling hub that regulates the crosstalk between the MST2 pathway, the apoptotic network and the ERK pathway.

Systematic Exploration of Passive Permeability in Tetrapeptides with Hydrogen-Bond-Accepting Amino Acid Side Chains.

We synthesized and experimentally tested the passive permeability of more than thirty tetrapeptides mimicking the N-terminus of the pro-apoptotic protein Smac (Second mitochondria-derived activator of caspases). Each peptide bore one or two unnatural Hydrogen Bond Acceptor-bearing Amino Acid (HBA-AA) residues, such that intramolecular hydrogen bonding with proximal backbone amide N-H donors is feasible. Passive permeability of the synthetic peptides was determined using the parallel artificial membrane permeability assay (PAMPA). Experimental permeability values were found to span three orders of magnitude, providing useful empirical guidance for the design of more permeable Smac mimetics specifically, and peptidic ligands generally.

Modulating mitofusins to control mitochondrial function and signaling

Mitofusins reside on the outer mitochondrial membrane and regulate mitochondrial fusion, a physiological process that impacts diverse cellular processes. Mitofusins are activated by conformational changes and subsequently oligomerize to enable mitochondrial fusion. Here, we identify small molecules that directly increase or inhibit mitofusins activity by modulating mitofusin conformations and oligomerization. We use these small molecules to better understand the role of mitofusins activity in mitochondrial fusion, function, and signaling. We find that mitofusin activation increases, whereas mitofusin inhibition decreases mitochondrial fusion and functionality. Remarkably, mitofusin inhibition also induces minority mitochondrial outer membrane permeabilization followed by sub-lethal caspase-3/7 activation, which in turn induces DNA damage and upregulates DNA damage response genes. In this context, apoptotic death induced by a second mitochondria-derived activator of caspases (SMAC) mimetic is potentiated by mitofusin inhibition. These data provide mechanistic insights into the function and regulation of mitofusins as well as small molecules to pharmacologically target mitofusins.

Host HSPD1 Translocation from Mitochondria to the Cytoplasm Induced by Streptococcus suis Serovar 2 Enolase Mediates Apoptosis and Loss of Blood-Brain Barrier Integrity.

Streptococcus suis serovar 2 (S. suis serovar 2) is a zoonotic pathogen that causes meningitis in pigs and humans, and is a serious threat to the swine industry and public health. Understanding the mechanism(s) by which S. suis serovar 2 penetrates the blood–brain barrier (BBB) is crucial to elucidating the pathogenesis of meningitis. In a previous study, we found that expression of the virulence factor enolase (Eno) by S. suis serovar 2 promotes the expression of host heat shock protein family D member 1 (HSPD1) in brain tissue, which leads to the apoptosis of porcine brain microvascular endothelial cells (PBMECs) and increased BBB permeability, which in turn promotes bacterial translocation across the BBB. However, the mechanism by which HSPD1 mediates Eno-induced apoptosis remains unclear. In this study, we demonstrate that Eno promotes the translocation of HSPD1 from mitochondria to the cytoplasm, where HSPD1 binds to β-actin (ACTB), the translocated HSPD1, and its interaction with ACTB led to adverse changes in cell morphology and promoted the expression of apoptosis-related proteins, second mitochondria-derived activator of caspases (Smac), and cleaved caspase-3; inhibited the expression of X-linked inhibitor of apoptosis protein (XIAP); and finally promoted cell apoptosis. These results further elucidate the role of HSPD1 in the process of Eno-induced apoptosis and increased BBB permeability, increasing our understanding of the pathogenic mechanisms of meningitis, and providing a framework for novel therapeutic strategies.

Abstract 1068: Anti-DR5 agonist IgM antibody IGM-8444 combined with SMAC mimetic birinapant induces strong synergistic tumor cytotoxicity

Abstract Apoptosis is induced through extrinsic and intrinsic signaling pathways. Extrinsic apoptosis can be activated through multimerization of death receptor 5 (DR5), a tumor necrosis factor (TNF) receptor family member highly expressed in many cancers. However, cellular resistance mechanisms within the intrinsic pathway may limit DR5 activity, including inhibitor of apoptosis proteins (IAPs) that block caspase activity or promote pro-survival NFκB signaling. Second mitochondria-derived activator of caspases (SMAC) is an endogenous bivalent IAP antagonist. Birinapant, a bivalent SMAC mimetic that binds and degrades IAPs, has been evaluated through Phase 2, demonstrating good safety and on target activity but minimal efficacy as a monotherapy. We hypothesized that simultaneously targeting the extrinsic apoptotic pathway with IGM-8444, an anti-DR5 multivalent IgM agonist, and the intrinsic apoptotic pathway with birinapant could enhance tumor cell apoptosis. Human cancer cell lines including triple negative breast cancer (TNBC), head and neck, ovarian, colorectal, lung, and sarcomas, were screened for sensitivity to IGM-8444 and birinapant combination. Strong synergistic cytotoxicity was observed in vitro in 36/45 (80%) cancer cell lines, as measured by both Bliss synergy score and maximal killing. IGM-8444 and birinapant combination also induced synergistic cytotoxicity in cells with acquired resistance to DR5 agonist antibodies. By contrast, IGM-8444 and birinapant did not kill primary human hepatocytes in vitro, demonstrating the potential clinical safety for this combination. In vivo, the IGM-8444 and birinapant combination dose-dependently reduced tumor growth in a MDA-MB-231 TNBC model, with 8/10 tumor-free mice at the highest dose of birinapant tested. By comparison, birinapant combination with an anti-DR5 IgG agonist showed a modest response. IGM-8444 and birinapant also showed significant anti-tumor responses in additional cell line and patient-derived xenograft models, including 7/9 tumor-free animals in a HT-1080 fibrosarcoma model and 9/10 complete responses in a EBC-1 lung squamous cell lung carcinoma model. Lastly, pharmacodynamic biomarkers including cIAP1 degradation, caspase activation, and caspase-cleaved cytokeratin 18 in tumor and serum correlated with anti-tumor response. In summary, combined targeting of the extrinsic and intrinsic apoptotic pathways with IGM-8444 and birinapant respectively enhances tumor cytotoxicity in multiple preclinical models. The combination of IGM-8444 with birinapant is currently under evaluation in a Phase 1 study in patients with relapsed and/or refractory solid cancers (NCT04553692). Citation Format: Beatrice T. Wang, Melanie Desbois, Susan E. Calhoun, Thomas J. Matthew, Poonam Yakkundi, Ling Wang, Xingjie Chen, Tasnim Kothambawala, Miho Oyasu, Maya F. Kotturi, Genevive Hernandez, Xiaohan Liu, Marvin S. Peterson, Eric W. Humke, Bruce A. Keyt, Angus M. Sinclair. Anti-DR5 agonist IgM antibody IGM-8444 combined with SMAC mimetic birinapant induces strong synergistic tumor cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1068.

Abstract LB098: Integrative approaches to modulate antigen presentation and boost cancer immune response

Abstract Immune checkpoint blockade (ICB) therapy revolutionized cancer treatment, but patients with impaired MHC-I and/or MHC-II expression show inferior response. We observed differential expression patterns for MHC-I and MHC-II in cancer cells and applied multiple approaches to examine their regulatory mechanisms. To identify the modulators of MHC-I, we combined FACS-based genome-wide CRISPR screens with data-mining from public data. We identified TRAF3, a suppressor of the NFkB pathway, as a negative regulator of MHC-I. The Traf3-knockout gene expression signature is associated with better survival in ICB-naïve patients with cancer and better ICB response. We then screened for drugs with similar transcriptional effects as this signature and identified Second Mitochondria-derived Activator of Caspase (SMAC) mimetics. We experimentally validated that the SMAC mimetic birinapant upregulates MHC-I, sensitizes cancer cells to T cell-dependent killing, and adds to ICB efficacy. In contrast to MHC-I, the expression of MHC-II shows dramatic intra- and inter-sample heterogeneity. To identify the regulators of MHC-II, we conducted data-mining of the transcriptomic and proteomic data from Cancer Cell Line Encyclopedia (CCLE) and multiple melanoma clinical cohorts. We found that a higher cancer-cell-intrinsic MHC-II level is significantly associated with better anti-PD-1 response, and that the Hippo signaling pathway can regulate MHC-II expression in melanoma cells. Our findings provide preclinical rationales for increasing cancer cell MHC-I/MHC-II expression to enhance sensitivity to immunotherapy. Citation Format: Shengqing Gu, Wubing Zhang, Xiaoqing Wang, Peng Jiang, Gordon J. Freeman, Scott J. Rodig, Henry Long, Benjamin E. Gewurz, F. Stephen Hodi, X. Shirley Liu, Myles Brown. Integrative approaches to modulate antigen presentation and boost cancer immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB098.

Abstract 4193: Lymphotoxin-alpha-armed oncolytic VSV-GP synergizes with SMAC mimetics to induce enhanced tumor cell death and regression

Abstract Introduction: Oncolytic viruses (OVs), which selectively replicate in and destroy tumor cells, form a highly promising class of cancer therapeutics with a strong immune stimulatory potential. VSV-GP, a chimeric Vesicular Stomatitis Virus (VSV) pseudotyped with the glycoprotein of the lymphocytic choriomeningitis virus (LCMV) can selectively replicate and express therapeutic cargos in the tumor microenvironment. Lymphotoxin alpha (LTα) - a member of the TNF superfamily - may present as a potent therapeutic cargo based on its immune stimulatory and cell death modulatory characteristics.In this study, we explore the immune promoting and tumor cell death inducing properties of VSV-GP-LTα, a next generation variant with an NF-κB facilitated mode of action. We further investigate clinically relevant combination therapies with “second mitochondria-derived activator of caspases” mimetic compounds (SMCs). Experimental Procedures: Viral fitness, cargo expression and test substance quality were assessed using growth kinetic, ELISA, RT-qPCR and titration assays. Therapeutic efficacy and safety of VSV-GP-LTα, with or without SMC co-treatment, were assessed in vivo in syngeneic C57BL/6 mice bearing subcutaneous LLC1-IFNAR-/- or B16F10-OVA tumors. To shed further light into the VSV-GP-LTα treatment effects, we applied immunohistochemistry, flow cytometry, multiplex ELISA and Nanostring® assays. Data Summary: VSV-GP-LTα induced stronger anti-tumor effects and increased overall survival compared to VSV-GP. It was also better tolerated than VSV-GP armed with TNF-α, a therapeutic cargo from the same class. Histological and gene expression analysis confirmed widespread cell killing within the tumor and activation of innate and adaptive immune response markers. However, further characterization of the adaptive immune compartment did not show significant changes induced by the expressed LTα. Building on the observed therapeutic strengths of VSV-GP-LTα, we further explored a treatment combination with SMC. This resulted in a therapeutic synergy with improved treatment-induced tumor cell death, especially in a less permissive tumor setting. Conclusion: Together, here we show that arming VSV-GP with LTα resulted in a strong inflammation of the tumor microenvironment and enhanced levels of tumor cell death induction. This effect could be further enhanced by the co-treatment with SMC. Citation Format: Bart Spiesschaert, Philipp Müller, Katharina Angerer, Krishna Das, Tobias Nolden, Carles Urbiola, Judy Ng, Fabian Heinemann, Birgit Stierstorfer, Patrik Erlmann, Guido Wollmann. Lymphotoxin-alpha-armed oncolytic VSV-GP synergizes with SMAC mimetics to induce enhanced tumor cell death and regression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4193.

Suppressed XIAP and cIAP expressions in human brain cancer stem cells using BV6- and GDC0152-encapsulated nanoparticles

BackgroundSmac mimetics functioned against inhibitors of apoptosis proteins (IAP) often fail to achieve sufficient ability in glioblastoma multiforme (GBM) treatment due to the obstruction of the blood-brain barrier (BBB). MethodsBV6- and GDC0152-encapsulated solid lipid nanoparticles (SLNs) with surface transferrin (Tf) and folic acid (FA) (BV6-GDC0152-Tf-FA-SLNs) were developed to downregulate IAP in U87MG cells and human brain cancer stem cells (HBCSCs) for GBM treatment. Significant findingsAn increase in stearic acid (SA) level enlarged SLNs with improved entrapment of BV6 and GDC0152. 1H NMR study revealed hydrogen bonding between SA and the two IAP antagonists, supporting controlled release of BV6 and GDC0152 from SLNs. Increasing sodium dodecyl sulfate concentration reduced the size of SLNs, and raised the particle stability. Conjugated Tf and FA on SLNs favored permeating the BBB as corroborated from decreased transendothelial electrical resistance, and raised BBB permeability of propidium iodide, BV6 and GDC0152. The ability of BV6-GDC0152-Tf-FA-SLNs to target IAP in U87MG cells and HBCSCs was validated from the downregulated XIAP and cIAP-1 expressions and upregulated caspase-3 expression in immunofluorescence staining, flow cytometry and western blot analysis. Hence, Tf- and FA-grafted SLNs can be an effective colloidal delivery system to deliver BV6 and GDC0152 across the BBB, facilitate IAP targeting, and enhanced drug bioavailability of the two Smac mimetics in GBM cells for potential clinical trials.

Crotonate sensitizes IAPi in the disruption of latent HIV by modulating the ncNF‐κB signaling pathway at the step of p100 cleavage into p52

BackgroundAntiretroviral therapy (ART) effectively suppresses HIV‐1 (HIV) replication to an undetectable level in the periphery; however, the treatment is life‐long and HIV reservoirs cannot be eliminated by ART alone in people living with HIV (PLWH). The major obstacle that impedes HIV eradication in ART‐treated PLWH is the latent viral reservoir harbors in the resting CD4+ T cells. Recently, the activation of non‐canonical (nc) NF‐κB pathway via IAP inhibitor (IAPi) or the SMAC mimetic (SMACm) compounds has raised as an attractive mechanism to disrupt HIV from latency. IAPi/SMACm targets IAPs to activate NF‐κB inducing kinase (NIK) followed by phosphorylation of IkappaB kinase (IKK), leading to the cleavage of p100 into p52. The latter forms a complex with RelB to stimulate transcription of its target genes after translocation into the nucleus. Previously, we discovered that crotonylation stimulates HIV latency reversal that elicited by PKC/NF‐κB agonist. We hypothesize that there may exist a functional interaction between protein crotonylation and ncNF‐κB signaling, which may enhance IAPi‐induced latency reversal to optiomally disrupt HIV latency for a cure of HIV.MethodsMultiple HIV latency models were used, including Jurkat cell derived HIV latency models, the U1 promonocytic model of latency, the primary CD4+ T cell model of HIV latency and the resting CD4+ T cells isolated from ART‐suppressed PLWH. HIV expression was determined by flow cytometry and/or real‐time PCR. Protein expression and cleavage was determined by Western blot.ResultsWe found that IAPi/SMACm can disrupt latent HIV. However, its ability to reactivate latent HIV varied among different HIV latency models, with a lowest efficacy in the primary CD4+ T cell model of latency. Interestingly, IAPi/SMACm‐induced disruption of latent HIV was greatly enhanced in the presence of crotonate in all these in vitro and ex vivo models. This was possibly mediated by the enhancement of p100 cleavage into p52 in the ncNF‐κB signaling pathway.ConclusionsWe propose that a blockage of p100 cleavage prevents an efficient activation of ncNF‐κB signaling pathway in the latently infected CD4+ T cells in PLWH, which could be relieved by targeting IAP proteins to induce p100 cleavage for the activation of ncNF‐κB signaling. Crotonylation, which was recently defined by us as a new epigenetic mark for the active HIV transcription, potentiates the reactivation of HIV from latency by further releasing the blockage at the p100 cleavage step, thereby enhancing ncNF‐κB activation to disrupt HIV latency. These data indicate that crotonylation could be exploited to develop novel strategies for HIV cure or remission in the future.

Morphological Selectivity of a Protein Self-Assembly System with a Repertoire of Diverse Interaction Modes.

Multiple metal chelating sites were incorporated onto the second mitochondria-derived activator of caspase (SMAC) building blocks. The combination of different binding sites generated a repertoire of diverse binding modes, among which two different microfilament types (small and large) with distinct patterns were selected under thermodynamic control. Furthermore, the two microfilaments exhibited a pronounced secondary assembly trend due to the potential noncovalent interactions on the protein surfaces. Coupled with stereoselectivity, they presented a strong self-recognition effect and underwent two distinct reassembly patterns. That is, the large filaments self-associated in pairs to form "interlocked chain" structures, while the small ones twisted to form protein helical bundles. This work represents one of the few studies of selective self-assembly of self-assembled protein assemblies. Such an idea may provide inspiration for constructing more sophisticated protein architectures in the future.