Meredith Leong

Serum and glucocorticoid-inducible kinase (SGK) is a target of the PI 3-kinase-stimulated signaling pathway

Serum and glucocorticoid‐inducible kinase (SGK) is a novel member of the serine/threonine protein kinase family that is transcriptionally regulated. In this study, we have investigated the regulatory mechanisms that control SGK activity. We have established a peptide kinase assay for SGK and present evidence demonstrating that SGK is a component of the phosphoinositide 3 (PI 3)‐kinase signaling pathway. Treatment of human embryo kidney 293 cells with insulin, IGF‐1 or pervanadate induced a 3‐ to 12‐fold activation of ectopically expressed SGK. Activation was completely abolished by pretreatment of cells with the PI 3‐kinase inhibitor, LY294002. Treatment of activated SGK with protein phosphatase 2A in vitro led to kinase inactivation. Consistent with the similarity of SGK to other second‐messenger regulated kinases, mutation of putative phosphorylation sites at Thr256 and Ser422 inhibited SGK activation. Cotransfection of PDK1 with SGK caused a 6‐fold activation of SGK activity, whereas kinase‐dead PDK1 caused no activation. GST‐pulldown assays revealed a direct interaction between PDK1 and the catalytic domain of SGK. Treatment of rat mammary tumor cells with serum caused hyperphosphorylation of endogenous SGK, and promoted translocation to the nucleus. Both hyperphosphorylation and nuclear translocation could be inhibited by wortmannin, but not by rapamycin.

Hyperosmotic Stress Stimulates Promoter Activity and Regulates Cellular Utilization of the Serum- and Glucocorticoid-inducible Protein Kinase (Sgk) by a p38 MAPK-dependent Pathway

We have established that the serum- and glucocorticoid-inducible protein kinase (Sgk) is a new component of the hyperosmotic stress response. Treatment of NMuMg mammary epithelial cells with the organic osmolyte, sorbitol, caused the stable accumulation of Sgk transcripts and protein after an approximately 4-h lag. Transient transfection of a series of sgk-CAT reporter plasmids containing either 5′ deletions or continuous 6-base pair substitutions identified a hyperosmotic stress-regulated element that is GC-rich and is necessary for the sorbitol stimulation ofsgk gene promoter activity. Gel shift analysis identified four major DNA-protein complexes in the hyperosmotic stress-regulated element that, by competition with excess consensus wild type and mutant oligonucleotides and by antibody supershifts, contains the Sp1 transcription factor. Several lines of evidence suggest that the p38 MAPK signaling pathway mediates the hyperosmotic stress stimulation of sgk gene expression. Treatment with pharmacological inhibitors of p38 MAPK or with a dominant negative form of MKK3, an upstream regulator of p38 MAPK, significantly reduced or ablated the sorbitol induction ofsgk promoter activity or protein production. Using anin vitro peptide transphosphorylation assay, sorbitol treatment activates either endogenous or exogenous Sgk that is localized to the cytoplasmic compartment. Thus, we propose that the stimulated expression of enzymatically active Sgk after sorbitol treatment is a newly defined component of the p38 MAPK-mediated response to hyperosmotic stress.

STING agonist formulated cancer vaccines can cure established tumors resistant to PD-1 blockade

Cyclic dinucleotide formulated cancer vaccine combined with PD-1 blockade can induce regression of tumors that do not express PD-L1 constitutively. A therapy that STINGs tumors Stimulator of interferon genes, or STING, is a receptor that is found on a variety of cell types and activates an immune response in response to cyclic dinucleotides. Fu et al. found that combining cyclic dinucleotides with a cellular cancer vaccine called STINGVAX was effective against multiple types of tumors in mouse models. The authors then modified the cyclic dinucleotides to strengthen their binding to human STING, increasing their antitumor activity. The authors also showed that treatment with STINGVAX caused cancer cells to up-regulate PD-L1, a protein that suppresses the immune response. Inhibiting the PD-L1 pathway in mice treated with STINGVAX was very effective at killing even poorly immunogenic tumors. Stimulator of interferon genes (STING) is a cytosolic receptor that senses both exogenous and endogenous cytosolic cyclic dinucleotides (CDNs), activating TBK1/IRF3 (interferon regulatory factor 3), NF-κB (nuclear factor κB), and STAT6 (signal transducer and activator of transcription 6) signaling pathways to induce robust type I interferon and proinflammatory cytokine responses. CDN ligands were formulated with granulocyte-macrophage colony-stimulating factor (GM-CSF)–producing cellular cancer vaccines—termed STINGVAX—that demonstrated potent in vivo antitumor efficacy in multiple therapeutic models of established cancer. We found that rationally designed synthetic CDN derivative molecules, including one with an Rp,Rp dithio diastereomer and noncanonical c[A(2′,5′)pA(3′,5′)p] phosphate bridge structure, enhanced antitumor efficacy of STINGVAX in multiple aggressive therapeutic models of established cancer in mice. Antitumor activity was STING-dependent and correlated with increased activation of dendritic cells and tumor antigen–specific CD8+ T cells. Tumors from STINGVAX-treated mice demonstrated marked PD-L1 (programmed death ligand 1) up-regulation, which was associated with tumor-infiltrating CD8+IFNγ+ T cells. When combined with PD-1 (programmed death 1) blockade, STINGVAX induced regression of palpable, poorly immunogenic tumors that did not respond to PD-1 blockade alone.

Rationale, progress and development of vaccines utilizing STING-activating cyclic dinucleotide adjuvants

A principal barrier to the development of effective vaccines is the availability of adjuvants and formulations that can elicit both effector and long-lived memory CD4 and CD8 T cells. Cellular immunity is the presumptive immune correlate of protection against intracellular pathogens: a group composed of bacteria, viruses and protozoans that is responsible for a staggering level of morbidity and mortality on a global scale. T-cell immunity is also correlated with clinical benefit in cancer, and the development of therapeutic strategies to harness the immune system to treat diverse malignancies is currently undergoing a renaissance. Cyclic dinucleotides (CDNs) are ubiquitous small molecule second messengers synthesized by bacteria that regulate diverse processes and are a relatively new class of adjuvants that have been shown to increase vaccine potency. CDNs activate innate immunity by directly binding the endoplasmic reticulum-resident receptor STING (stimulator of interferon genes), activating a signaling pathway that induces the expression of interferon-β (IFN-β) and also nuclear factor-κB (NF-κB) dependent inflammatory cytokines. The STING signaling pathway has emerged as a central Toll-like receptor (TLR) independent mediator of host innate defense in response to sensing cytosolic nucleic acids, either through direct binding of CDNs secreted by bacteria, or, as shown recently, through binding of a structurally distinct CDN produced by a host cell receptor in response to binding cytosolic double-stranded (ds)DNA. Although this relatively new class of adjuvants has to date only been evaluated in mice, newly available CDN-STING cocrystal structures will likely intensify efforts in this field towards further development and evaluation in human trials both in preventive vaccine and immunotherapy settings.

Constitutive Activation of the PrfA regulon enhances the potency of vaccines based on live-attenuated and killed but metabolically active Listeria monocytogenes strains.

ABSTRACT Recombinant vaccines derived from the facultative intracellular bacterium Listeria monocytogenes are presently undergoing early-stage clinical evaluation in oncology treatment settings. This effort has been stimulated in part due to preclinical results that illustrate potent activation of innate and adaptive immune effectors by L. monocytogenes vaccines, combined with efficacy in rigorous animal models of malignant and infectious disease. Here, we evaluated the immunologic potency of a panel of isogenic vaccine strains that varied only in prfA. PrfA is an intracellularly activated transcription factor that induces expression of virulence genes and encoded heterologous antigens (Ags) in appropriately engineered vaccine strains. Mutant strains with PrfA locked into a constitutively active state are known as PrfA* mutants. We assessed the impacts of three PrfA* mutants, G145S, G155S, and Y63C, on the immunologic potencies of live-attenuated and photochemically inactivated nucleotide excision repair mutant (killed but metabolically active [KBMA]) vaccines. While PrfA* substantially increased Ag expression in strains grown in broth culture, Ag expression levels were equivalent in infected macrophage and dendritic cell lines, conditions that more closely parallel those in the immunized host. However, only the prfA(G155S) allele conferred significantly enhanced vaccine potency to KBMA vaccines. In the KBMA vaccine background, we show that PrfA*(G155S) enhanced functional cellular immunity following an intravenous or intramuscular prime-boost immunization regimen. These results form the basis of a rationale for including the prfA(G155S) allele in future live-attenuated or KBMA L. monocytogenes vaccines advanced to the clinical setting.

Selective Targeting of Antitumor Immune Responses with Engineered Live-Attenuated Listeria monocytogenes

Improved immunization and ex vivo T-cell culture strategies can generate larger numbers and more potent tumor-specific effector cells than previously possible. Nonetheless, the capacity of these cells to eliminate established tumors is limited by their ability to efficiently enter tumor-bearing organs and mediate their effector function. In the current study, we show that the administration of an engineered organ-homing microbe selectively targets tumor-specific immune responses to metastases within that organ. Specifically, an attenuated Listeria monocytogenes strain, which preferentially infects the liver following systemic administration, dramatically enhances the activity of a cancer vaccine against liver metastases but not metastases in the lung. This enhanced activity results from both local recruitment of innate immune effectors as well as concentration and increased activation of vaccine-induced antitumor T cells within the liver. These findings show a general approach to focus systemic cancer immunotherapies to specific organs bearing tumor metastases by taking advantage of differential tropisms and the proinflammatory nature of microbes.

Impact of Preexisting Vector-Specific Immunity on Vaccine Potency: Characterization of Listeria monocytogenes-Specific Humoral and Cellular Immunity in Humans and Modeling Studies Using Recombinant Vaccines in Mice

ABSTRACT Recombinant live-attenuated Listeria monocytogenes is currently being developed as a vaccine platform for treatment or prevention of malignant and infectious diseases. The effectiveness of complex biologic vaccines, such as recombinant viral and bacterial vectors, can be limited by either preexisting or vaccine-induced vector-specific immunity. We characterized the level of L. monocytogenes-specific cellular and humoral immunity present in more than 70 healthy adult subjects as a first step to understanding its possible impact on the efficacy of L. monocytogenes-based vaccines being evaluated in early-phase clinical trials. Significant L. monocytogenes-specific humoral immunity was not measured in humans, consistent with a lack of antibodies in mice immunized with wild-type L. monocytogenes. Cellular immune responses specific for listeriolysin O, a secreted bacterial protein required for potency of L. monocytogenes-derived vaccines, were detected in approximately 60% of human donors tested. In mice, while wild-type L. monocytogenes did not induce significant humoral immunity, attenuated L. monocytogenes vaccine strains induced high-titer L. monocytogenes-specific antibodies when given at high doses used for immunization. Passive transfer of L. monocytogenes-specific antiserum to naïve mice had no impact on priming antigen-specific immunity in mice immunized with a recombinant L. monocytogenes vaccine. In mice with preexisting L. monocytogenes-specific immunity, priming of naïve T cells was not prevented, and antigen-specific responses could be boosted by additional vaccinations. For the first time, our findings establish the level of L. monocytogenes-specific cellular immunity in healthy adults, and, together with modeling studies performed with mice, they support the scientific rationale for repeated L. monocytogenes vaccine immunization regimens to elicit a desired therapeutic effect.

KBMA Listeria monocytogenes is an effective vector for DC-mediated induction of antitumor immunity

Vaccine strategies that utilize human DCs to enhance antitumor immunity have yet to realize their full potential. Approaches that optimally target a spectrum of antigens to DCs are urgently needed. Here we report the development of a platform for loading DCs with antigen. It is based on killed but metabolically active (KBMA) recombinant Listeria monocytogenes and facilitates both antigen delivery and maturation of human DCs. Highly attenuated KBMA L. monocytogenes were engineered to express an epitope of the melanoma-associated antigen MelanA/Mart-1 that is recognized by human CD8+ T cells when presented by the MHC class I molecule HLA-A*0201. The engineered KBMA L. monocytogenes induced human DC upregulation of costimulatory molecules and secretion of pro-Th1 cytokines and type I interferons, leading to effective priming of Mart-1-specific human CD8+ T cells and lysis of patient-derived melanoma cells. KBMA L. monocytogenes expressing full-length NY-ESO-1 protein, another melanoma-associated antigen, delivered the antigen for presentation by MHC class I and class II molecules independent of the MHC haplotype of the DC donor. A mouse therapeutic tumor model was used to show that KBMA L. monocytogenes efficiently targeted APCs in vivo to induce protective antitumor responses. Together, our data demonstrate that KBMA L. monocytogenes may be a powerful platform that can both deliver recombinant antigen to DCs for presentation and provide a potent DC-maturation stimulus, making it a potential cancer vaccine candidate.

Activation of immature hepatic NK cells as immunotherapy for liver metastatic disease.

NK cells can identify and eliminate emerging tumors due to altered expression of activating and inhibitory ligands on aberrant cells, a process that is greatly enhanced following NK cell activation. As a principal site of both tumor metastases and immature NK cells, the liver represents a unique anatomic location in which activation of the innate immune system could provide substantial therapeutic benefit. We describe here the NK cell-dependent destruction of a primary hepatic tumor following infection with an attenuated intracellular bacterium derived from Listeria monocytogenes. NK cell-mediated immunity correlated with the ordered migration and maturation of NK cells within the liver. Cytolytic activity was partially dependent on NKG2D-mediated tumor cell recognition, but surprisingly was still effective in the absence of type I IFN. Significantly, NK cell-mediated destruction of a primary hepatic tumor in infected mice led to long-lived CD4- and CD8 T cell-dependent tumor-specific adaptive immunity. These findings establish that activation and differentiation of immature NK cells using complex microbial stimuli can elicit potent anti-tumor activity within the liver, promote cross-presentation of tumor-derived Ags leading to long-lived systemic anti-tumor immunity, and suggests a paradigm for clinical intervention of liver metastatic carcinoma.

Modified STING-activating cyclic dinucleotide derivatives significantly enhance the anti-tumor activity of therapeutic vaccines

The STING signaling pathway has emerged as a central TLR-independent mediator of host innate defense in response to sensing cytosolic nucleic acids, either through direct binding of CDNs secreted by intracellular bacteria, or through binding of a structurally distinct CDN produced by a host cell receptor, cyclic GMP-AMP (cGAMP) synthase (cGAS), in response to binding cytosolic double-stranded DNA. Binding of CDNs to STING initiates a signaling cascade through the TBK-1/IRF-3 axis to induce type I interferon (IFN) and other co-regulated genes. Discovered recently, CDNs synthesized by cGAS and bacteria are structurally distinct. The cGAS product, termed non-canonical CDN, has a phosphate bridge with both 2’-5’ and 3’-5’ linkages that reportedly increases its affinity for STING by 200-fold. Here we show in a panel of human donors that the non-canonical linkage confers significantly increased activity of CDNs to activate PBMCs, as measured by expression of IFN and NF-κB dependent cytokines, as compared to CDNs with canonical 3’-5’ phosphate linkages. By conducting Immunogenicity-Structure Activity Relationship studies in vitro and in various animal models of infection and cancer, we selected synthetic derivatives of native CDNs produced by bacteria or eukaryotic cells with increased potency. CDN derivatives with sulfur atoms at non-bridging oxygens of the internucleotide phosphate bridge were resistant to digestion with phosphodiesterase. Surprisingly, R,R di-thio CDN diastereomers induced higher levels of IFN in vitro, and induced a higher magnitude of peak and memory antigen-specific CD4 and CD8 T cell responses correlated with protective immunity, as compared to either the R,S di-thio CDN diastereomer, or di-oxo CDN. CDNs based on adenosine nucleotides were comparatively independent of host cell permeabilization to activate STING signaling. We applied this finding to enhance the activity of immunotherapy regimens utilizing irradiated GM-CSF expressing tumor cell vaccines. Co-formulation with R,R dithio-c-di-AMP significantly inhibited tumor growth in several models, correlated with increased mobilization and activation of dendritic cells, increased tumor lymphocyte infiltration, and T cell immunity. Immunologic potency of CDNs was essentially lost in mice encoding a non-functional STING allele. Collectively, CDNs have high translation potential for diverse applications in clinical oncology.