Purpose: Despite high tumor mutationburden, immune checkpoint blockade has limited efficacy in SCLC. We hypothesized that poly (ADP-ribose) polymerase inhibition could render SCLC more susceptible to immune checkpoint blockade.Methods: A single-arm, phase II trial (NCT02484404) enrolled patients with relapsed SCLC who received durvalumab, 1500 mg every 4 weeks, and olaparib, 300 mg twice a day. The primary outcome was objective response rate. Correlative studies included mandatory collection of pretreatment and during-treatment biopsy specimens, which were assessed to define SCLC immunephenotypes: desert (CD8-positive T-cell prevalence low), excluded (CD8-positive T cells in stroma immediately adjacent/within tumor), and inflamed (CD8-positive T cells in direct contact with tumor).Results: A total of 20 patients were enrolled. Their median age was 64 years, and most patients (60%) had platinumresistant/refractory disease. Of 19 evaluable patients, two were observed to have partial or complete responses

Vpr and selected mutants were used in a Saccharomyces cerevisiae two-hybrid screen to identify cellular interactors. We found Vpr interacted with 14-3-3 proteins, a family regulating a multitude of proteins in the cell. Vpr mutant R80A, which is inactive in cell cycle arrest, did not interact with 14-3-3. 14-3-3 proteins regulate the G 2 /M transition by inactivating Cdc25C phosphatase via binding to the phosphorylated serine residue at position 216 of Cdc25C. 14-3-3 overexpression in human cells synergized with Vpr in the arrest of cell cycle. Vpr did not arrest efficiently cells not expressing 14-3-3. This indicated that a full complement of 14-3-3 proteins is necessary for optimal Vpr function on the cell cycle. Mutational analysis showed that the C-terminal portion of Vpr, known to harbor its cell cycle-arresting activity, bound directly to the C-terminal part of 14-3-3, outside of its phosphopeptide-binding pocket. Vpr expression shifted localization of the mutant Cdc25C S216A to the cytoplasm, indicating that Vpr promotes the association of 14-3-3 and Cdc25C, independently of the presence of serine 216. Immunoprecipitations of cell extracts indicated the presence of triple complexes (Vpr/14-3-3/Cdc25C). These results indicate that Vpr promotes cell cycle arrest at the G 2 /M phase by facilitating association of 14-3-3 and Cdc25C independently of the latter's phosphorylation status.

Nuclear export of intron-containing human immunodeficiency virus type 1 RNA is mediated by the viral Rev protein. Rev is a nucleocytoplasmic transport protein that directly binds to its cis-acting Rev-responsive element RNA. Rev function depends on its ability to multimerize. The in vivo dynamics and the subcellular dependence of this process are still largely unexplored. To visualize and quantitatively analyze the mechanism of Rev multimeric assembly in live cells, we used high resolution in vivo fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching. By using two different dynamic FRET approaches (acceptor photobleaching and donor bleaching time measurements), we observed a strong Rev-Rev interaction in the nucleoli of living cells. Most interestingly, we could also detect Rev multimerization in the cytoplasm; however, FRET efficiency in the cytoplasm was significantly lower than in the nucleolus. By using fluorescence recovery after photobleaching, we investigated the mobility of Rev within the nucleolus. Mathematical modeling of the fluorescence recovery after photobleaching recoveries enabled us to extract relative association and dissociation constants and the diffusion coefficient of Rev in the nucleolus. Our results show that Rev multimerizes in the nucleolus of living cells, suggesting an important role of the nucleolus in nucleocytoplasmic transport.The replication of the human immunodeficiency virus, type 1 (HIV-1), 1 is regulated in a temporal manner by its viral mRNA expression. Control of HIV RNA expression is complex and involves the interplay of cis-acting viral transactivators and several cellular proteins. Rev is a viral trans-activator protein that controls the differential expression of viral proteins at the post-transcriptional level by allowing the accumulation in the cytoplasm of unspliced and singly spliced viral RNA containing the Rev-responsive element (RRE) (1-7). Rev interacts directly with a purine-rich stem-loop within RRE (6,8), and through multimerization additional Rev molecules bind throughout the RRE. In the absence of Rev, the only viral RNA species that accumulate in the cytoplasm are multiply spliced and encode for the viral regulatory proteins. In the presence of Rev, the incompletely spliced mRNAs accumulate in the cytoplasm to serve as templates for viral structural protein synthesis or as viral genomes. Rev activity is therefore essential for virus replication.Rev is a small protein of 116 amino acids or 18 kDa. Its cellular localization is nucleolar, but it has been shown to shuttle continuously between the nucleus and the cytoplasm (1, 9, 10). Nuclear localization is mediated by a short stretch of basic amino acids characterized by eight arginine residues located near the amino terminus of Rev that serves both as nuclear localization signal (NLS) and as an RNA binding domain (5,11,12). It is flanked on both sides by sequences that contribute to Rev oligomerization on the RRE but have no detectable role in RRE binding. The NLS of Re...