Iryna Lebedyeva

Ceramide activates lysosomal cathepsin B and cathepsin D to attenuate autophagy and induces ER stress to suppress myeloid-derived suppressor cells

Myeloid-derived suppressor cells (MDSCs) are immune suppressive cells that are hallmarks of human cancer. MDSCs inhibit cytotoxic T lymphocytes (CTLs) and NK cell functions to promote tumor immune escape and progression, and therefore are considered key targets in cancer immunotherapy. Recent studies determined a key role of the apoptosis pathways in tumor-induced MDSC homeostasis and it is known that ceramide plays a key role in regulation of mammalian cell apoptosis. In this study, we aimed to determine the efficacy and underlying molecular mechanism of ceramide in suppression of MDSCs. Treatment of tumor-bearing mice with LCL521, a lysosomotropic inhibitor of acid ceramidase, significantly decreased MDSC accumulation in vivo. Using a MDSC-like myeloid cell model, we determined that LCL521 targets lysosomes and increases total cellular C16 ceramide level. Although MDSC-like cells have functional apoptosis pathways, LCL521-induced MDSC death occurs in an apoptosis- and necroptosis-independent mechanism. LCL521 treatment resulted in an increase in the number of autophagic vesicles, heterolysosomes and swollen ERs. Finally, concomitant inhibition of cathepsin B and cathepsin D was required to significantly decrease LCL521-induced cell death. Our observations indicate that LCL521 targets lysosomes to activate cathepsin B and cathepsin D, resulting in interrupted autophagy and ER stress that culminates in MDSC death. Therefore, a ceramidase inhibitor is potentially an effective adjunct therapeutic agent for suppression of MDSCs to enhance the efficacy of CTL-based cancer immunotherapy.

Multi-kinase inhibitors can associate with heat shock proteins through their NH2-termini by which they suppress chaperone function

We performed proteomic studies using the GRP78 chaperone-inhibitor drug AR-12 (OSU-03012) as bait. Multiple additional chaperone and chaperone-associated proteins were shown to interact with AR-12, including: GRP75, HSP75, BAG2; HSP27; ULK-1; and thioredoxin. AR-12 down-regulated in situ immuno-fluorescence detection of ATP binding chaperones using antibodies directed against the NH2-termini of the proteins but only weakly reduced detection using antibodies directed against the central and COOH portions of the proteins. Traditional SDS-PAGE and western blotting assessment methods did not exhibit any alterations in chaperone detection. AR-12 altered the sub-cellular distribution of chaperone proteins, abolishing their punctate speckled patterning concomitant with changes in protein co-localization. AR-12 inhibited chaperone ATPase activity, which was enhanced by sildenafil; inhibited chaperone – chaperone and chaperone – client interactions; and docked in silico with the ATPase domains of HSP90 and of HSP70. AR-12 combined with sildenafil in a GRP78 plus HSP27 –dependent fashion to profoundly activate an eIF2α/ATF4/CHOP/Beclin1 pathway in parallel with inactivating mTOR and increasing ATG13 phosphorylation, collectively resulting in formation of punctate toxic autophagosomes. Over-expression of [GRP78 and HSP27] prevented: AR-12 –induced activation of ER stress signaling and maintained mTOR activity; AR-12 –mediated down-regulation of thioredoxin, MCL-1 and c-FLIP-s; and preserved tumor cell viability. Thus the inhibition of chaperone protein functions by AR-12 and by multi-kinase inhibitors very likely explains why these agents have anti-tumor effects in multiple genetically diverse tumor cell types.

The Lectin-like Domain of TNF Increases ENaC Open Probability through a Novel Site at the Interface between the Second Transmembrane and C-terminal Domains of the α-Subunit.

Regulation of the epithelial sodium channel (ENaC), which regulates fluid homeostasis and blood pressure, is complex and remains incompletely understood. The TIP peptide, a mimic of the lectin-like domain of TNF, activates ENaC by binding to glycosylated residues in the extracellular loop of ENaC-α, as well as to a hitherto uncharacterized internal site. Molecular docking studies suggested three residues, Val567, Glu568, and Glu571, located at the interface between the second transmembrane and C-terminal domains of ENaC-α, as a critical site for binding of the TIP peptide. We generated Ala replacement mutants in this region of ENaC-α and examined its interaction with TIP peptide (3M, V567A/E568A/E571A; 2M, V567A/E568A; and 1M, E571A). 3M and 2M ENaC-α, but not 1M ENaC-α, displayed significantly reduced binding capacity to TIP peptide and to TNF. When overexpressed in H441 cells, 3M mutant ENaC-α formed functional channels with similar gating and density characteristics as the WT subunit and efficiently associated with the β and γ subunits in the plasma membrane. We subsequently assayed for increased open probability time and membrane expression, both of which define ENaC activity, following addition of TIP peptide. TIP peptide increased open probability time in H441 cells overexpressing wild type and 1M ENaC-α channels, but not 3M or 2M ENaC-α channels. On the other hand, TIP peptide-mediated reduction in ENaC ubiquitination was similar in cells overexpressing either WT or 3M ENaC-α subunits. In summary, this study has identified a novel site in ENaC-α that is crucial for activation of the open probability of the channel, but not membrane expression, by the lectin-like domain of TNF.

Intravenous Formulation of HET0016 Decreased Human Glioblastoma Growth and Implicated Survival Benefit in Rat Xenograft Models

Glioblastoma (GBM) is a hypervascular primary brain tumor with poor prognosis. HET0016 is a selective CYP450 inhibitor, which has been shown to inhibit angiogenesis and tumor growth. Therefore, to explore novel treatments, we have generated an improved intravenous (IV) formulation of HET0016 with HPßCD and tested in animal models of human and syngeneic GBM. Administration of a single IV dose resulted in 7-fold higher levels of HET0016 in plasma and 3.6-fold higher levels in tumor at 60 min than that in IP route. IV treatment with HPßCD-HET0016 decreased tumor growth, and altered vascular kinetics in early and late treatment groups (p < 0.05). Similar growth inhibition was observed in syngeneic GL261 GBM (p < 0.05). Survival studies using patient derived xenografts of GBM811, showed prolonged survival to 26 weeks in animals treated with focal radiation, in combination with HET0016 and TMZ (p < 0.05). We observed reduced expression of markers of cell proliferation (Ki-67), decreased neovascularization (laminin and αSMA), in addition to inflammation and angiogenesis markers in the treatment group (p < 0.05). Our results indicate that HPßCD-HET0016 is effective in inhibiting tumor growth through decreasing proliferation, and neovascularization. Furthermore, HPßCD-HET0016 significantly prolonged survival in PDX GBM811 model.

HET0016 decreases lung metastasis from breast cancer in immune-competent mouse model

Distant metastasis is the primary cause of death in the majority of the cancer types. Recently, much importance has been given to tumor microenvironment (TME) in the development of invasive malignant tumors, as well as the metastasis potential. The ability of tumor cells to modulate TME and to escape immune-mediated attack by releasing immunosuppressive cytokines has become a hallmark of breast cancer. Our study shows the effect of IV formulation of HET0016 (HPßCD-HET0016) a selective inhibitor of 20-HETE synthesis, administered intravenously in immune-competent in vivo mouse model of murine breast cancer. 4T1 luciferase positive cells were implanted to the mammary fat pad in Balb/c mice. Treatment started on day 15, and was administered for 5 days a week for 3 weeks. The development of metastasis was detected via optical imaging. Blood, spleen, lungs, bone marrow and tumor were collected for flow cytometry, to investigate changes in myeloid-derived suppressive cells (MDSCs) populations and endothelial phenotype. Tumor and lungs were collected for protein analysis. Our results show that HPßCD-HET0016: (1) decreased tumor volume and lung metastasis compared to the vehicle group; (2) reduced migration and invasion of tumor cells and levels of metalloproteinases in the lungs of animals treated with HPßCD-HET0016 via PI3K/AKT pathway; and (3) decreased expression of pro-inflammatory cytokines, growth factors and granulocytic MDSCs population in the lung microenvironment in treated animals. Thus, HPßCD-HET0016 showed potential in treating lung metastasis in a preclinical mouse model and needs further investigations on TME.

CXCR2-Expressing Tumor Cells Drive Vascular Mimicry in Antiangiogenic Therapy–Resistant Glioblastoma

BACKGROUND: Glioblastoma (GBM) was shown to relapse faster and displayed therapeutic resistance to antiangiogenic therapies (AATs) through an alternative tumor cell-driven mechanism of neovascularization called vascular mimicry (VM). We identified highly upregulated interleukin 8 (IL-8)-CXCR2 axis in tumor cells in high-grade human glioma and AAT-treated orthotopic GBM tumors. METHODS: Human GBM tissue sections and tissue array were used to ascertain the clinical relevance of CXCR2-positive tumor cells in the formation of VM. We utilized U251 and U87 human tumor cells to understand VM in an orthotopic GBM model and AAT-mediated enhancement in VM was modeled using vatalanib (anti-VEGFR2) and avastin (anti-VEGF). Later, VM was inhibited by SB225002 (CXCR2 inhibitor) in a preclinical study. RESULTS: Overexpression of IL8 and CXCR2 in human datasets and histological analysis was identified as a bonafide candidate to validate VM through in vitro and animal model studies. AAT-treated tumors displayed a higher number of CXCR2-positive GBM-stem cells with endothelial-like phenotypes. Stable knockdown of CXCR2 expression in tumor cells led to decreased tumor growth as well as incomplete VM structures in the animal models. Similar data were obtained following SB225002 treatment. CONCLUSIONS: The present study suggests that tumor cell autonomous IL-8-CXCR2 pathway is instrumental in AAT-mediated resistance and VM formation in GBM. Therefore, CXCR2 can be targeted through SB225002 and can be combined with standard therapies to improve the therapeutic outcomes in clinical trials.

Crystal structure of ethyl 2-methyl-5,10-dioxo-4-phenyl-5,10-di­hydro-4H-11-thia-1,4a-di­aza­benzo[b]fluorene-3-carb­oxy­late

The dihydropyrimidine ring adopts a twist-boat conformation while the quinone ring is slightly non-planar. In the crystal, molecules are linked by weak C—H⋯O and C—H⋯S hydrogen bonds and C—H⋯π interactions. In addition, a short intermolecular S⋯N contact occurs.

Abstract 2327: IFNgamma regulates PD-L1 expression through activating IRF1 transcription in tumor cells

The emerging clinical success of checkpoint blockade immunotherapy in human cancer patients has highlighted the critical importance of PD-L1, a ligand for the T cell inhibitory receptor PD-1, as a molecular target in cancer immunotherapy. PD-L1 is constitutively expressed in tumor cells and is also inducible by inflammatory cytokines such as IFNgamma. It has been proposed that tumor cells may sense the elevated IFNgamma from activated host T cells as a “threat” in the tumor microenvironment and adapt it by up-regulating PD-L1. The aim of this study is to elucidate the molecular mechanism underlying IFNgamma regulation of PD-L1 expression in tumor cells. We observed that PD-L1 is constitutively expressed, albeit at low level, in multiple types of cancer cells, including pancreatic, colon, breast, and sarcoma cells. IFNgamma treatment dramatically increased PD-L1 expression level and IFNgamma up-regulates PD-L1 expression through the Jak-STAT1 signaling pathway in vitro and in vivo. Chromatin immunoprecipitation assay did not identify IFNgamma-activated pSTAT1 binding to the pd-l1 promoter. Instead, we observed that IFNgamma activates IRF1 transcription and IRF1 is required for IFNgamma-induced PD-L1 expression. Chromatin immunoprecipitation analysis shows that pSTAT1 is associated with the irf1 but not the pd-l1 promoter. Analysis of the irf1 promoter DNA sequence revealed a pSTAT1-binding consensus sequence, and electrophoretic mobility shift assay indicates that pSTAT1 directly binds to this DNA element of the irf1 promoter. Furthermore, we demonstrated that IRF1 is associated with the pd-l1 promoter chromatin near the irf1 transcription initiation site. The pd-l1 promoter region contains a putative IRF1-binding consensus sequence and electrophoretic mobility shift assay shows that IRF1 binds to this DNA element of the pd-l1 promoter region. Taken together, our data indicate that IFNgamma activates pSTAT1 that binds to the irf1 promoter to activate irf1 transcription. IFNgamma-induced IRF1 then binds to the pd-l1 promoter to activate pd-l1 transcription in tumor cells. Citation Format: Chunwan Lu, Amy V. Paschall, Priscilla S. Redd, Iryna Lebedyeva, Kebin Liu. IFNgamma regulates PD-L1 expression through activating IRF1 transcription in tumor cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2327.

Benzotriazole-Based Strategies Toward Peptidomimetics, Conjugates, and Other Peptide Derivatives

Benzotriazole-mediated routes to peptidomimetics and peptide conjugates have been discussed in detail. The Katritzky group developed a benzotriazole methodology toward the activation of carbonyl groups of amino acids and modified analogs, which allowed the synthesis of cyclic peptides, azapeptides, azidopeptides, aminoxypeptides, oxyazapeptides, depsipeptides, and isopeptides. High-yielding reactions of N-, O-, S-, and C-acylated nucleophiles with activated aminoacyl or peptidoyl benzotriazole derivatives have also been reported. Benzotriazole methodology enabled the efficient incorporation of bioactive moieties into peptides, peptidomimetics, amino acids, and other carbonyl-containing compounds. Predominant number of reported products retained chiral purity. Some of the products displayed promising biological activities such as anticancer and antibacterial activity along with the improved stability under physiological conditions.