SummaryExtracellular cues regulate cell fate, and this is mainly achieved through the engagement of specific transcriptional programs. The TORC1 and TORC2 complexes mediate the integration of nutritional cues to cellular behavior, but their interplay is poorly understood. Here, we use fission yeast to investigate how phosphatase activity participates in this interplay during the switch from proliferation to sexual differentiation. We find that loss of PP2A-B55Pab1 enhances the expression of differentiation-specific genes and leads to premature conjugation. pab1 deletion brings about a transcriptional profile similar to TORC1 inactivation, and deletion of pab1 overcomes the repression of differentiation genes in cells overexpressing TORC1. Importantly, we show that this effect is mediated by an increased TORC2-AKT (Gad8) signaling. Under nutrient-rich conditions, PP2A-B55Pab1 dephosphorylates Gad8 Ser546, repressing its activity. Conversely, TORC1 inactivation upon starvation leads to the inactivation of PP2A-B55Pab1 through the Greatwall-Endosulfin pathway. This results in the activation of Gad8 and the commitment to differentiation. Thus, PP2A-B55Pab1 enables a crosstalk between the two TOR complexes that controls cell-fate decisions in response to nutrient availability.

Vascular endothelial growth factor (VEGF) is critical for physiological and pathological angiogenesis. Within the tumor microenvironment, VEGF functions as an endothelial cell survival factor, permeability factor, mitogen, and chemotactic agent. The majority of these functions are mediated by VEGF-induced activation of VEGF receptor 2 (VEGFR2), a high affinity receptor tyrosine kinase expressed by endothelial cells and other cell types in the tumor microenvironment. VEGF can also ligate other cell surface receptors including VEGFR1 and neuropilin-1 and -2. However, the importance of VEGF-induced activation of these receptors in tumorigenesis is still unclear. We report the development and characterization of r84, a fully human monoclonal antibody that binds human and mouse VEGF and selectively blocks VEGF from interacting with VEGFR2 but does not interfere with VEGF∶VEGFR1 interaction. Selective blockade of VEGF binding to VEGFR2 by r84 is shown through ELISA, receptor binding assays, receptor activation assays, and cell-based functional assays. Furthermore, we show that r84 has potent anti-tumor activity and does not alter tissue histology or blood and urine chemistry after chronic high dose therapy in mice. In addition, chronic r84 therapy does not induce elevated blood pressure levels in some models. The ability of r84 to specifically block VEGF∶VEGFR2 binding provides a valuable tool for the characterization of VEGF receptor pathway activation during tumor progression and highlights the utility and safety of selective blockade of VEGF-induced VEGFR2 signaling in tumors.

Depletion of the Anaphase-Promoting Complex/Cyclosome (APC/C) activator Cdc20 arrests cells in metaphase with high levels of the mitotic cyclin (Cyclin B) and the Separase inhibitor Securin. In mammalian cells this arrest has been exploited for the treatment of cancer with drugs that engage the spindle assembly checkpoint and, recently, with chemical inhibitors of the APC/C. While most cells arrested in mitosis for prolonged periods undergo apoptosis, others skip cytokinesis and enter G1 with unsegregated chromosomes. This process, known as mitotic slippage, generates aneuploidy and increases genomic instability in the cancer cell. Here, we analyze the behavior of fission yeast cells arrested in mitosis through the transcriptional silencing of the Cdc20 homolog slp1. While depletion of slp1 readily halts cells in metaphase, this arrest is only transient and a majority of cells eventually undergo cytokinesis and show steady mitotic dephosphorylation. Notably, this occurs in the absence of Cyclin B (Cdc13) degradation. We investigate the involvement of phosphatase activity in these events and demonstrate that PP2A-B55Pab1 is required to prevent septation and, during the arrest, its CDK-mediated inhibition facilitates the induction of cytokinesis. In contrast, deletion of PP2A-B56Par1 completely abrogates septation. We show that this effect is partly due to this mutant entering mitosis with reduced CDK activity. Interestingly, both PP2A-B55Pab1 and PP2A-B56Par1, as well as Clp1 (the homolog of the budding yeast mitotic phosphatase Cdc14) are required for the dephosphorylation of mitotic substrates during the escape. Finally, we show that the mitotic transcriptional wave controlled by the RFX transcription factor Sak1 facilitates the induction of cytokinesis and also requires the activity of PP2A-B56Par1 in a mechanism independent of CDK.

While the established route for vaccines against the pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is intramuscular, it may be preferable to deliver vaccines intranasally to secure mucosal protection at the site of infection. This will limit the spread of the virus, ease administration and likely improve vaccine acceptance. Here, we report on a subunit vaccine platform, where the antigen is genetically fused to engineered human albumin. Upon intranasal delivery the subunit vaccines target the neonatal Fc receptor (FcRn) and induce both local and systemic antigen-specific antibody responses at magnitudes higher than after intramuscular delivery. We provide evidence that such needle-free vaccination induces production of antibodies with neutralizing capacity against SARS-CoV-2 or influenza A. Thus, the vaccine platform is particularly well suited for design of subunit vaccines against these and other infectious respiratory diseases.