Tumour-intrinsic endomembrane trafficking by ARF6 shapes an immunosuppressive microenvironment that drives melanomagenesis and response to checkpoint blockade therapy.

Tumour-host immune interactions lead to complex changes in the tumour microenvironment (TME), impacting progression, metastasis and response to therapy. While it is clear that cancer cells can have the capacity to alter immune landscapes, our understanding of this process is incomplete. Herein we show that endocytic trafficking at the plasma membrane, mediated by the small GTPase ARF6, enables melanoma cells to impose an immunosuppressive TME that accelerates tumour development. This ARF6-dependent TME is vulnerable to immune checkpoint blockade therapy (ICB) but in murine melanoma, loss of Arf6 causes resistance to ICB. Likewise, downregulation of ARF6 in patient tumours correlates with inferior overall survival after ICB. Mechanistically, these phenotypes are at least partially explained by ARF6-dependent recycling, which controls plasma membrane density of the interferon-gamma receptor. Collectively, our findings reveal the importance of endomembrane trafficking in outfitting tumour cells with the ability to shape their immune microenvironment and respond to immunotherapy.

ARF6-dependent endocytic trafficking of the Interferon-γ receptor drives adaptive immune resistance in cancer.

Adaptive immune resistance (AIR) is a protective process used by cancer to escape elimination by CD8+ T cells. Inhibition of immune checkpoints PD-1 and CTLA-4 specifically target Interferon-gamma (IFNγ)-driven AIR. AIR begins at the plasma membrane where tumor cell-intrinsic cytokine signaling is initiated. Thus, plasma membrane remodeling by endomembrane trafficking could regulate AIR. Herein we report that the trafficking protein ADP-Ribosylation Factor 6 (ARF6) is critical for IFNγ-driven AIR. ARF6 prevents transport of the receptor to the lysosome, augmenting IFNγR expression, tumor intrinsic IFNγ signaling and downstream expression of immunosuppressive genes. In murine melanoma, loss of ARF6 causes resistance to immune checkpoint blockade (ICB). Likewise, low expression of ARF6 in patient tumors correlates with inferior outcomes with ICB. Our data provide new mechanistic insights into tumor immune escape, defined by ARF6-dependent AIR, and support that ARF6-dependent endomembrane trafficking of the IFNγ receptor influences outcomes of ICB.

Small GTPase ARF6 controls VEGFR2 trafficking and signaling in diabetic retinopathy.

The devastating sequelae of diabetes mellitus include microvascular permeability, which results in retinopathy. Despite clinical and scientific advances, there remains a need for new approaches to treat retinopathy. Here, we have presented a possible treatment strategy, whereby targeting the small GTPase ARF6 alters VEGFR2 trafficking and reverses signs of pathology in 4 animal models that represent features of diabetic retinopathy and in a fifth model of ocular pathological angiogenesis. Specifically, we determined that the same signaling pathway utilizes distinct GEFs to sequentially activate ARF6, and these GEFs exert distinct but complementary effects on VEGFR2 trafficking and signal transduction. ARF6 activation was independently regulated by 2 different ARF GEFs - ARNO and GEP100. Interaction between VEGFR2 and ARNO activated ARF6 and stimulated VEGFR2 internalization, whereas a VEGFR2 interaction with GEP100 activated ARF6 to promote VEGFR2 recycling via coreceptor binding. Intervening in either pathway inhibited VEGFR2 signal output. Finally, using a combination of in vitro, cellular, genetic, and pharmacologic techniques, we demonstrated that ARF6 is pivotal in VEGFR2 trafficking and that targeting ARF6-mediated VEGFR2 trafficking has potential as a therapeutic approach for retinal vascular diseases such as diabetic retinopathy.

ARF6 Is an Actionable Node that Orchestrates Oncogenic GNAQ Signaling in Uveal Melanoma.

Activating mutations in Gαq proteins, which form the α subunit of certain heterotrimeric G proteins, drive uveal melanoma oncogenesis by triggering multiple downstream signaling pathways, including PLC/PKC, Rho/Rac, and YAP. Here we show that the small GTPase ARF6 acts as a proximal node of oncogenic Gαq signaling to induce all of these downstream pathways as well as ß-catenin signaling. ARF6 activates these diverse pathways through a common mechanism: the trafficking of GNAQ and ß-catenin from the plasma membrane to cytoplasmic vesicles and the nucleus, respectively. Blocking ARF6 with a small-molecule inhibitor reduces uveal melanoma cell proliferation and tumorigenesis in a mouse model, confirming the functional relevance of this pathway and suggesting a therapeutic strategy for Gα-mediated diseases.

The small GTPase ARF6 stimulates ß-catenin transcriptional activity during WNT5A-mediated melanoma invasion and metastasis.

ß-Catenin has a dual function in cells: fortifying cadherin-based adhesion at the plasma membrane and activating transcription in the nucleus. We found that in melanoma cells, WNT5A stimulated the disruption of N-cadherin and ß-catenin complexes by activating the guanosine triphosphatase adenosine diphosphate ribosylation factor 6 (ARF6). Binding of WNT5A to the Frizzled 4-LRP6 (low-density lipoprotein receptor-related protein 6) receptor complex activated ARF6, which liberated ß-catenin from N-cadherin, thus increasing the pool of free ß-catenin, enhancing ß-catenin-mediated transcription, and stimulating invasion. In contrast to WNT5A, the guidance cue SLIT2 and its receptor ROBO1 inhibited ARF6 activation and, accordingly, stabilized the interaction of N-cadherin with ß-catenin and reduced transcription and invasion. Thus, ARF6 integrated competing signals in melanoma cells, thereby enabling plasticity in the response to external cues. Moreover, small-molecule inhibition of ARF6 stabilized adherens junctions, blocked ß-catenin signaling and invasiveness of melanoma cells in culture, and reduced spontaneous pulmonary metastasis in mice, suggesting that targeting ARF6 may provide a means of inhibiting WNT/ß-catenin signaling in cancer.

Increased expression of multifunctional serine protease, HTRA1, in retinal pigment epithelium induces polypoidal choroidal vasculopathy in mice.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly. Wet AMD includes typical choroidal neovascularization (CNV) and polypoidal choroidal vasculopathy (PCV). The etiology and pathogenesis of CNV and PCV are not well understood. Genome-wide association studies have linked a multifunctional serine protease, HTRA1, to AMD. However, the precise role of HTRA1 in AMD remains elusive. By transgenically expressing human HTRA1 in mouse retinal pigment epithelium, we showed that increased HTRA1 induced cardinal features of PCV, including branching networks of choroidal vessels, polypoidal lesions, severe degeneration of the elastic laminae, and tunica media of choroidal vessels. In addition, HTRA1 mice displayed retinal pigment epithelium atrophy and photoreceptor degeneration. Senescent HTRA1 mice developed occult CNV, which likely resulted from the degradation of the elastic lamina of Bruch's membrane and up-regulation of VEGF. Our results indicate that increased HTRA1 is sufficient to cause PCV and is a significant risk factor for CNV.

KIF21A mutations in two Chinese families with congenital fibrosis of the extraocular muscles (CFEOM).

PURPOSE: Two Chinese families (XT and YT) with congenital fibrosis of the extraocular muscles (CFEOM) were identified. The purpose of this study was to determine if previously described Homo sapiens kinesin family member 21A (KIF21A) mutations were responsible for CFEOM in these two Chinese pedigrees. METHODS: Clinical characterization and genetic studies were performed. Microsatellite genotyping for linkage to the CFEOM1 and CFEOM3 loci was performed. The probands were screened for KIF21A mutations by bidirectional direct sequencing. Once a mutation was detected in the proband, all other participating family members and 100 unrelated control normal individuals were screened for the mutation. RESULTS: All affected individuals in family XT shared the common manifestations of CFEOM1. Family YT had two affected individuals, a mother and a daughter. The daughter had CFEOM1, while her mother never had congential ptosis but did have limited extraocular movements status post strabismus surgery. Haplotype analysis revealed that pedigree XT was linked to the 12q CFEOM1 locus and the affected memberes harbored the second most common missense mutation in KIF21A (2,861G>A, R954Q). Family YT harbored the most common missense de novo mutation in KIF21A (2,860C>T, R954W). Both of these mutations have been previously described. CONCLUSIONS: The observation of these two KIF21A mutations in a Chinese pedigree underscores the homogeneity of these mutations as a cause of CFEOM1 and CFEOM3 across ethnic divisions.

Clinical characterization and genetic mapping of North Carolina macular dystrophy.

North Carolina macular dystrophy (NCMD) is an autosomal dominant macular disease, was mapped to 6q14-q16.2, the disease-causing gene has yet not been identified. It shares phenotypic similarity with age-related macular degeneration including drusen and choroidal neovascularization. We collected six families with NCMD including 75 members, and conducted clinical characterization and genetic mapping for these families. Forty-five patients were diagnosed as NCMD; all six NCMD families were mapped to MCDR1 locus using genetic linkage analysis. MCDR1 interval was refined to 3 cM (1.8mb) between D6S1716 to D6S1671 via fine mapping using microsatellite markers in these six families, all eleven annotated genes within the interval were analyzed by mutation screening in coding regions, no mutation was found, suggesting a potential novel gene or a new pathological mechanism causing NCMD. The refinement of MCDR1 locus will aid the disease-causing gene identification. Functional studies of NCMD genes should provide important insights into pathogenetic mechanisms of NCMD and age-related macular degeneration.

DNA microarray analysis of the heat- and cold-shock stimulons in Yersinia pestis.

DNA microarray was used as a tool to define the heat- and cold-shock stimulons in Yersinia pestis. Heat shock dramatically enhanced the transcription of genes encoding major heat-shock proteins (MHSPs) that are important for cell survival against the heat. Many other genes were also greatly up-regulated, but their roles in heat-shock response need to be elucidated. Meanwhile, heat shock retarded most of the metabolic processes, i.e. RNA transcription, protein translation, aerobic respiration, energy metabolism, small molecule metabolism, peptidoglycan biosynthesis, sulfate uptake and cysteine biosynthesis. In response to cold shock, Y. pestis has evolved complex adaptive mechanisms by elevating the transcription of a specific set of genes whose protein products are designed to prevent or eliminate cold-induced DNA or RNA structuring, to remodel cell membrane components for maintenance of normal functions, to elevate the energy generation for ensuring ATP-dependent responses during cold adaptation and to synthesize or transport compatible solutes such as cryoprotectants, and at the same time, by repressing the mRNA level of certain genes whose protein products are not needed for bacterial growth at low temperatures, such as the MHSPs. These results provide a set of new candidate genes for hypothesis-based investigations of their roles in stress response, host adaptation and pathogenicity of this deadly pathogen.