Tumour cells evade immune surveillance by upregulating the surface expression of programmed death-ligand 1 (PD-L1), which interacts with programmed death-1 (PD-1) receptor on T cells to elicit the immune checkpoint response. Anti-PD-1 antibodies have shown remarkable promise in treating tumours, including metastatic melanoma. However, the patient response rate is low. A better understanding of PD-L1-mediated immune evasion is needed to predict patient response and improve treatment efficacy. Here we report that metastatic melanomas release extracellular vesicles, mostly in the form of exosomes, that carry PD-L1 on their surface. Stimulation with interferon-γ (IFN-γ) increases the amount of PD-L1 on these vesicles, which suppresses the function of CD8 T cells and facilitates tumour growth. In patients with metastatic melanoma, the level of circulating exosomal PD-L1 positively correlates with that of IFN-γ, and varies during the course of anti-PD-1 therapy. The magnitudes of the increase in circulating exosomal PD-L1 during early stages of treatment, as an indicator of the adaptive response of the tumour cells to T cell reinvigoration, stratifies clinical responders from non-responders. Our study unveils a mechanism by which tumour cells systemically suppress the immune system, and provides a rationale for the application of exosomal PD-L1 as a predictor for anti-PD-1 therapy.

The exocyst is an octameric protein complex that is implicated in the tethering of secretory vesicles to the plasma membrane prior to SNARE-mediated fusion. Spatial and temporal control of exocytosis through the exocyst has a crucial role in a number of physiological processes, such as morphogenesis, cell cycle progression, primary ciliogenesis, cell migration and tumor invasion. In this Cell Science at a Glance poster article, we summarize recent works on the molecular organization, function and regulation of the exocyst complex, as they provide rationales to the involvement of this complex in such a diverse array of cellular processes.

Adamantanes (amantadine and rimantadine) have been used to prevent and treat influenza A virus infections for many years; however, resistance to these drugs has been widely reported in the world. To investigate the frequency and distribution of M2 gene mutations in adamantane-resistant influenza variants circulated in the world between 1902 and 2013, 31251 available M2 protein sequences from different HA-subtype influenza A viruses (H1–H17) were analyzed and adamantane resistance-associated mutations were compared (L26F, V27A, A30T, A30V, S31N, G34E, and L38F). We find that 45.2% (n = 14132) of influenza A (H1–H17) viruses circulating globally were resistant to adamantanes, and the vast majority of resistant viruses (95%) bear S31N mutations. Whereas, only about 1% have V27A mutations and other mutations (L26F, A30T, G34E, and L38F) were extremely rare (their prevalence appeared to be < 0.2%). Our results confirm that H1, H3, H5, H7, H9, and H17 subtype influenza A viruses exhibit high-level resistance to adamantanes. In contrast, the appearance of adamantane-resistant mutants in H2, H4, H6, H10, and H11 subtypes was rare. However, no adamantane resistance viruses were identified among other HA subtypes (H8, H12–H16). Our findings indicate that the frequency and distribution of adamantane-resistant influenza variants varied among different HA subtypes, host species, years of isolation, and geographical areas. This comprehensive study raises concerns about the increasing prevalence of adamantane-resistant influenza A viruses and highlights the importance of monitoring the emergence and worldwide spread of adamantane-resistant variants.

The exocyst complex regulates the last steps of exocytosis, which is essential to organisms across kingdoms. In humans, its dysfunction is correlated with several significant diseases, such as diabetes and cancer progression. Investigation of the dynamic regulation of the evolutionarily conserved exocyst-related processes using mutants in genetically tractable organisms such as Arabidopsis thaliana is limited by the lethality or the severity of phenotypes. We discovered that the small molecule Endosidin2 (ES2) binds to the EXO70 (exocyst component of 70 kDa) subunit of the exocyst complex, resulting in inhibition of exocytosis and endosomal recycling in both plant and human cells and enhancement of plant vacuolar trafficking. An EXO70 protein with a C-terminal truncation results in dominant ES2 resistance, uncovering possible distinct regulatory roles for the N terminus of the protein. This study not only provides a valuable tool in studying exocytosis regulation but also offers a potentially new target for drugs aimed at addressing human disease.T he EXO70 (exocyst component of 70 kDa) protein is a component of the evolutionarily conserved octameric exocyst complex that tethers post-Golgi vesicles to the plasma membrane before SNARE-mediated membrane fusion (1). As an important component of the exocyst complex that mediates exocytosis, EXO70 regulates, for example, neurite outgrowth, epithelial cell polarity establishment, cell motility, and cell morphogenesis in animal cells (2-6). In plants, EXO70 proteins participate in polarized pollen tube growth, root hair growth, deposition of cell wall material, cell plate initiation and maturation, defense, and autophagy (7-12). In humans, EXO70 mediates the trafficking of the glucose transporter Glut4 to the plasma membrane that is stimulated by insulin and involved in the development of diabetes (13). A specific isoform of human EXO70 is also involved in cancer cell invasion (13-15). Endosidin2 (ES2) was identified from a plantbased chemical screen as an inhibitor of trafficking. We demonstrate that the target of ES2 is the EXO70 subunit of the exocyst and that ES2 is active in plants and mammalian systems. Significantly, no inhibitor of the exocyst complex has been reported, yet such compounds could be important for understanding the basic mechanisms of exocyst-mediated processes, for modifying secretion in biotechnological applications, and for the development of potential new drugs with higher affinity and more potent activity to control exocyst-related diseases. Results ES2 InhibitsTrafficking to the Plasma Membrane. ES2 is a previously identified plant endomembrane trafficking disruptor (Fig. 1A) that inhibits polarized growth of pollen tubes in a dose-dependent manner (Fig. S1 A and B) (16). Arabidopsis seedlings grown on media containing ES2 have shorter roots and fewer and shorter root hairs and are less sensitive to gravity stimulation (Fig. S1 C-G). ES2 disrupted the trafficking of proteins that are actively recycled between the plasma membrane and endosome...

Targeted BRAF inhibition (BRAFi) and combined BRAF and MEK inhibition (BRAFi+MEKi) therapies have significantly improved clinical outcomes in patients with metastatic melanoma. Unfortunately, the efficacy is beset by the acquisition of drug resistance1–6. Here we investigated molecular mechanisms underlying acquired resistance to BRAFi (BRAFi resistance, “BR”) and BRAFi+MEKi (combination therapy resistance, “CR”). Consistent with previous studies, BR is mediated by ERK pathway re-activation. CR is, however, mediated by mechanisms independent of re-activation of ERK in many therapy-resistant cell lines and clinical samples. p21-activated kinases (PAKs) become activated in acquired drug resistant cells and play a pivotal role in mediating both BR and CR. Our screening using reverse phase protein array (RPPA) revealed distinct mechanisms by which PAKs mediate BR and CR. In BR, PAKs phosphorylate CRAF and MEK to reactivate ERK. In CR, PAKs regulate JNK and β-catenin phosphorylation, mTOR pathway activation, and inhibit apoptosis, thereby bypassing ERK. Together, our results provide new insights into molecular mechanisms underlying acquired drug resistance to current targeted therapies, and may help to direct novel drug development efforts to overcome acquired drug resistance.