Retinoblastoma protein and E2-promoter binding factor (E2F) family members are important regulators of G1-S phase progression. Deregulated E2F also sensitizes cells to apoptosis, but this aspect of E2F function is poorly understood. Studies of E2F-induced apoptosis have mostly been carried out in tissue culture cells, and the analysis of the factors that are important for this process has been restricted to the testing of a few candidate genes. Using Drosophila as a model system, we have generated tools that allow genetic modifiers of E2F-dependent apoptosis to be identified in vivo and developed assays that allow effects on E2F-induced apoptosis to be studied in cultured cells. Genetic interactions show that dE2F1-dependent apoptosis in vivo involves dArk/Apaf1 apoptosome-dependent activation of both initiator and effector caspases and is sensitive to levels of Drosophila inhibitor of apoptosis-1 (dIAP1). Using these approaches, we report the surprising finding that apoptosis inhibitor-5/antiapoptosis clone-11 (Api5/Aac11) is a critical determinant of dE2F1-induced apoptosis in vivo and in vitro. This functional interaction occurs in multiple tissues, is specific to E2F-induced apoptosis, and is conserved from flies to humans. Interestingly, Api5/Aac11 acts downstream of E2F and suppresses E2F-dependent apoptosis without generally blocking E2F-dependent transcription. Api5/Aac11 expression is often upregulated in tumor cells, particularly in metastatic cells. We find that depletion of Api5 is tumor cell lethal. The strong genetic interaction between E2F and Api5/Aac11 suggests that elevated levels of Api5 may be selected during tumorigenesis to allow cells with deregulated E2F activity to survive under suboptimal conditions. Therefore, inhibition of Api5 function might offer a possible mechanism for antitumor exploitation.
Abstract Molecular and immunologic studies analyzing tumor samples have failed to find a robust and reliable predictive marker of responsiveness to immune checkpoints. Exosomes are circulating microvesicles that contain a subtranscriptome of their cell of origin. They are produced by tumor and immune cells and have been shown to be mediators of immune responses in cancer. To examine the role of peripheral-blood (PB) derived exosomal transcriptomic signatures, we performed microarray analysis on N=99 samples from both PB-derived exosomes and tumor biopsies derived from N=39 (N=25 responders, N=14 non-responders) patients undergoing aPD1 immunotherapy for metastatic melanoma prior to and throughout the course of treatment. We observed increased expression of adaptive immunity, innate immunity, and antigen-presentation pathways in responders versus nonresponders in both exosomal and tumor samples prior to and during the course of treatment. We further observed that PB-derived exosomal expression profiles are highly concordant with tumor-specific expression profiles; however, they are significantly enriched in genes related to both innate and adaptive immune pathways relative to tumor expression profiles, suggesting that PB-derived exosomes share both immune-derived and tumor-derived signatures. A time-series analysis of the exosomal profiles during treatment showed significant differences in chemokine and cytokine signaling time dynamics between responders and non-responders, with IL-12 and type 1 interferon signaling pathways being the most impacted. Due to these findings, we hypothesized that exosomal profiles may serve as a predictive and prognostic tool for checkpoint blockade immunotherapy success. By utilizing markers derived from differential gene expression analysis, we were able to construct a performant machine learning classifier that can predict immunotherapy success using only pre-treatment PB-derived exosomal expression signatures. In a preliminary analysis, our model is able to achieve an auROC of 0.938 when evaluated on a N=24 (N=16 responders, N=8 non-responders) cohort using leave-one-out cross-validation, suggesting that PB-derived exosomal signatures may be predictive of immunotherapy success in metastatic melanoma. Citation Format: Alvin Shi, Jessica A. Cintolo-Gonzalez, Isabel Chien, Dennis T. Frederick, Roman Alpatov, William Michaud, Deborah Plana, David Panka, Ryan Corcoran, Keith Flaherty, Ryan Sullivan, Manolis Kellis, Genevieve Boland. Exosomal transcriptomic signatures tracks and predicts response to checkpoint blockade immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B25.
CDC23 is required in Saccharomyces cerevisiae for cell cycle progression through the G2/M transition. The CDC23 gene product contains tandem, imperfect repeats, termed tetratricopeptide repeats, (TPR) units common to a protein family that includes several other nuclear division CDC genes. In this report we have used mutagenesis to probe the functional significance of the TPR units within CDC23. Analysis of truncated derivatives indicates that the TPR block of CDC23 is necessary for the function or stability of the polypeptide. In-frame deletion of a single TPR unit within the repeat block proved sufficient to inactivate CDC23 in vivo, though this allele could rescue the temperature-sensitive defect of a cdc23 point mutant by intragenic complementation. By both in vitro and in vivo mutagenesis techniques, 17 thermolabile cdc23 alleles were produced and examined. Fourteen alleles contained single amino acid changes that were found to cluster within two distinct mutable domains, one of which encompasses the most canonical TPR unit found in CDC23. In addition, we have characterized CDC23 as a 62-kDa protein (p62cdc23) that is localized to the yeast nucleus. Our mutagenesis results suggest that TPR blocks form an essential domain within members of the TPR family.
Abstract Early in the current pandemic, the D614G mutation arose in the Spike protein of SARS-CoV-2 and quickly became the dominant variant globally. Mounting evidence suggests D614G enhances viral entry. Here we use a direct competition assay with single-cycle viruses to show that D614G outcompetes the wildtype. We developed a cell line with inducible ACE2 expression to confirm that D614G more efficiently enters cells with ACE2 levels spanning the different primary cells targeted by SARS-CoV-2. Using a new assay for crosslinking and directly extracting Spike trimers from the pseudovirus surface, we found an increase in trimerization efficiency and viral incorporation of D614G protomers. Our findings suggest that D614G increases infection of cells expressing a wide range of ACE2, and informs the mechanism underlying enhanced entry. The tools developed here can be broadly applied to study other Spike variants and SARS-CoV-2 entry, to inform functional studies of viral evolution and vaccine development.
Background. The number of patients waiting for heart transplant far exceeds the number of hearts available. Donation after circulatory death (DCD) combined with machine perfusion can increase the number of transplantable hearts by as much as 48%. Emerging studies also suggest machine perfusion could enable allograft “reconditioning” to optimize outcomes. However, a detailed understanding of the energetic substrates and metabolic changes during perfusion is lacking. Methods. Metabolites were analyzed using 1-dimensional 1 H and 2-dimensional 13 C- 1 H heteronuclear spectrum quantum correlation nuclear magnetic resonance spectroscopy on serial perfusate samples (N = 98) from 32 DCD hearts that were successfully transplanted. Wilcoxon signed-rank and Kruskal-Wallis tests were used to test for significant differences in metabolite resonances during perfusion and network analysis was used to uncover altered metabolic pathways. Results. Metabolite differences were observed comparing baseline perfusate to samples from hearts at time points 1–2, 3–4, and 5–6 h of perfusion and all pairwise combinations. Among the most significant changes observed were a steady decrease in fatty acids and succinate and an increase in amino acids, especially alanine, glutamine, and glycine. This core set of metabolites was also altered in a DCD porcine model perfused with a nonblood-based perfusate. Conclusions. Temporal metabolic changes were identified during ex vivo perfusion of DCD hearts. Fatty acids, which are normally the predominant myocardial energy source, are rapidly depleted, while amino acids such as alanine, glutamine, and glycine increase. We also noted depletion of ketone, β-hydroxybutyric acid, which is known to have cardioprotective properties. Collectively, these results suggest a shift in energy substrates and provide a basis to design optimal preservation techniques during perfusion.
