The Angiopoietin-2 and TIE Pathway as a Therapeutic Target for Enhancing Antiangiogenic Therapy and Immunotherapy in Patients with Advanced Cancer.

Despite significant advances made in cancer treatment, the development of therapeutic resistance to anticancer drugs represents a major clinical problem that limits treatment efficacy for cancer patients. Herein, we focus on the response and resistance to current antiangiogenic drugs and immunotherapies and describe potential strategies for improved treatment outcomes. Antiangiogenic treatments that mainly target vascular endothelial growth factor (VEGF) signaling have shown efficacy in many types of cancer. However, drug resistance, characterized by disease recurrence, has limited therapeutic success and thus increased our urgency to better understand the mechanism of resistance to inhibitors of VEGF signaling. Moreover, cancer immunotherapies including immune checkpoint inhibitors (ICIs), which stimulate antitumor immunity, have also demonstrated a remarkable clinical benefit in the treatment of many aggressive malignancies. Nevertheless, the emergence of resistance to immunotherapies associated with an immunosuppressive tumor microenvironment has restricted therapeutic response, necessitating the development of better therapeutic strategies to increase treatment efficacy in patients. Angiopoietin-2 (ANG2), which binds to the receptor tyrosine kinase TIE2 in endothelial cells, is a cooperative driver of angiogenesis and vascular destabilization along with VEGF. It has been suggested in multiple preclinical studies that ANG2-mediated vascular changes contribute to the development and persistence of resistance to anti-VEGF therapy. Further, emerging evidence suggests a fundamental link between vascular abnormalities and tumor immune evasion, supporting the rationale for combination strategies of immunotherapy with antiangiogenic drugs. In this review, we discuss the recent mechanistic and clinical advances in targeting angiopoietin signaling, focusing on ANG2 inhibition, to enhance therapeutic efficacy of antiangiogenic and ICI therapies. In short, we propose that a better mechanistic understanding of ANG2-mediated vascular changes will provide insight into the significance of ANG2 in treatment response and resistance to current antiangiogenic and ICI therapies. These advances will ultimately improve therapeutic modalities for cancer treatment.

Osmotic Shock-Triggered Assembly of Highly Charged, Nanoparticle-Supported Membranes.

Spherical nanoparticle-supported lipid bilayers (SSLBs) combine precision nanoparticle engineering with biocompatible interfaces for various applications, ranging from drug delivery platforms to structural probes for membrane proteins. Although the bulk, spontaneous assembly of vesicles and larger silica nanoparticles (>100 nm) robustly yields SSLBs, it will only occur with low charge density vesicles for smaller nanoparticles (<100 nm), a fundamental barrier in increasing SSLB utility and efficacy. Here, through whole mount and cryogenic transmission electron microscopy, we demonstrate that mixing osmotically loaded vesicles with smaller nanoparticles robustly drives the formation of SSLBs with high membrane charge density (up to 60% anionic lipid or 50% cationic lipid). We show that the osmolyte load necessary for SSLB formation is primarily a function of absolute membrane charge density and is not lipid headgroup-dependent, providing a generalizable, tunable approach toward bulk production of highly curved and charged SSLBs with various membrane compositions.

Angiopoietin-2-Dependent Spatial Vascular Destabilization Promotes T-cell Exclusion and Limits Immunotherapy in Melanoma.

T-cell position in the tumor microenvironment determines the probability of target encounter and tumor killing. CD8+ T-cell exclusion from the tumor parenchyma is associated with poor response to immunotherapy, and yet the biology that underpins this distinct pattern remains unclear. Here we show that the vascular destabilizing factor angiopoietin-2 (ANGPT2) causes compromised vascular integrity in the tumor periphery, leading to impaired T-cell infiltration to the tumor core. The spatial regulation of ANGPT2 in whole tumor cross-sections was analyzed in conjunction with T-cell distribution, vascular integrity, and response to immunotherapy in syngeneic murine melanoma models. T-cell exclusion was associated with ANGPT2 upregulation and elevated vascular leakage at the periphery of human and murine melanomas. Both pharmacologic and genetic blockade of ANGPT2 promoted CD8+ T-cell infiltration into the tumor core, exerting antitumor effects. Importantly, the reversal of T-cell exclusion following ANGPT2 blockade not only enhanced response to anti-PD-1 immune checkpoint blockade therapy in immunogenic, therapy-responsive mouse melanomas, but it also rendered nonresponsive tumors susceptible to immunotherapy. Therapeutic response after ANGPT2 blockade, driven by improved CD8+ T-cell infiltration to the tumor core, coincided with spatial TIE2 signaling activation and increased vascular integrity at the tumor periphery where endothelial expression of adhesion molecules was reduced. These data highlight ANGPT2/TIE2 signaling as a key mediator of T-cell exclusion and a promising target to potentiate immune checkpoint blockade efficacy in melanoma. SIGNIFICANCE: ANGPT2 limits the efficacy of immunotherapy by inducing vascular destabilization at the tumor periphery to promote T-cell exclusion.

Angiopoietin-2 blockade suppresses growth of liver metastases from pancreatic neuroendocrine tumors by promoting T cell recruitment.

Improving the management of metastasis in pancreatic neuroendocrine tumors (PanNETs) is critical, as nearly half of patients with PanNETs present with liver metastases, and this accounts for the majority of patient mortality. We identified angiopoietin-2 (ANGPT2) as one of the most upregulated angiogenic factors in RNA-Seq data from human PanNET liver metastases and found that higher ANGPT2 expression correlated with poor survival rates. Immunohistochemical staining revealed that ANGPT2 was localized to the endothelial cells of blood vessels in PanNET liver metastases. We observed an association between the upregulation of endothelial ANGPT2 and liver metastatic progression in both patients and transgenic mouse models of PanNETs. In human and mouse PanNET liver metastases, ANGPT2 upregulation coincided with poor T cell infiltration, indicative of an immunosuppressive tumor microenvironment. Notably, both pharmacologic inhibition and genetic deletion of ANGPT2 in PanNET mouse models slowed the growth of PanNET liver metastases. Furthermore, pharmacologic inhibition of ANGPT2 promoted T cell infiltration and activation in liver metastases, improving the survival of mice with metastatic PanNETs. These changes were accompanied by reduced plasma leakage and improved vascular integrity in metastases. Together, these findings suggest that ANGPT2 blockade may be an effective strategy for promoting T cell infiltration and immunostimulatory reprogramming to reduce the growth of liver metastases in PanNETs.

CDK4/6 initiates Rb inactivation and CDK2 activity coordinates cell-cycle commitment and G1/S transition.

External signaling controls cell-cycle entry until cells irreversibly commit to the cell cycle to ensure faithful DNA replication. This process is tightly regulated by cyclin-dependent kinases (CDKs) and the retinoblastoma protein (Rb). Here, using live-cell sensors for CDK4/6 and CDK2 activities, we propose that CDK4/6 initiates Rb inactivation and CDK2 activation, which coordinates the timing of cell-cycle commitment and sequential G1/S transition. Our data show that CDK4/6 activation induces Rb inactivation and thereby E2F activation, driving a gradual increase in CDK2 activity. We found that rapid CDK4/6 inhibition can reverse cell-cycle entry until CDK2 activity reaches to high levels. This suggests that high CDK2 activity is required to initiate CDK2-Rb positive feedback and CDK4/6-indpendent cell-cycle progression. Since CDK2 activation also facilitates initiation of DNA replication, the timing of CDK2-Rb positive feedback is coupled with the G1/S transition. Our experiments, which acutely increased CDK2 activity by cyclin E1 overexpression, indicate that cells commit to the cell cycle before triggering DNA replication. Together, our data suggest that CDK4/6 inactivates Rb to begin E2F and CDK2 activation, and high CDK2 activity is necessary and sufficient to generate a bistable switch for Rb phosphorylation before DNA replication. These findings highlight how cells initiate the cell cycle and subsequently commit to the cell cycle before the G1/S transition.

α-Synuclein Sterically Stabilizes Spherical Nanoparticle-Supported Lipid Bilayers.

While it is generally accepted that neuronal protein α-synuclein binds to highly curved and highly charged lipid membranes, its biological function beyond binding remains unknown despite its fundamental link to Parkinson's disease. Herein, we utilize spherical nanoparticle lipid bilayers (SSLBs) to recapitulate the charge and curvature limit of membrane organelles with which α-synuclein associates and probe how α-synuclein affects SSLB structure and dynamics as a proxy for interorganelle interactions. Small-angle X-ray scattering and X-ray photon correlation spectroscopy reveal our SSLBs form aggregates that are clearly broken up by the addition of α-synuclein, a clear indication that α-synuclein confers steric stabilization to membrane surfaces.