Kinase and phosphatase overexpression drives tumorigenesis and drug resistance. We previously developed a mass-cytometry-based single-cell proteomics approach that enables quantitative assessment of overexpression effects on cell signaling. Here, we applied this approach in a human kinome- and phosphatome-wide study to assess how 649 individually overexpressed proteins modulated cancer-related signaling in HEK293T cells in an abundance-dependent manner. Based on these data, we expanded the functional classification of human kinases and phosphatases and showed that the overexpression effects include non-catalytic roles. We detected 208 previously unreported signaling relationships. The signaling dynamics analysis indicated that the overexpression of ERK-specific phosphatases sustains proliferative signaling. This suggests a phosphatase-driven mechanism of cancer progression. Moreover, our analysis revealed a drug-resistant mechanism through which overexpression of tyrosine kinases, including SRC, FES, YES1, and BLK, induced MEK-independent ERK activation in melanoma A375 cells. These proteins could predict drug sensitivity to BRAF-MEK concurrent inhibition in cells carrying BRAF mutations.
Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.

Vasoactive intestinal peptide (VIP) is a neuroendocrine peptide hormone that has potent anti-inflammatory activities. VIP signaling through its receptor VPAC1 on T cells leads to reduced proliferation and a reduction in pro-inflammatory cytokine secretion. We report here that inhibition of the VIP pathway with a peptide antagonist significantly enhances a T-cell-dependent, autologous anti-leukemia response in murine models of acute myeloid leukemia and T lymphoblastic leukemia. Subcutaneous administration of the VIP antagonist, VIPhyb, resulted in reduced tumor burden and significantly enhanced survival (30-50% survival) over vehicle-treated controls (0-20% survival). The T cells in mice treated with VIPhyb expressed lower levels of the co-inhibitory PD-1 and secreted higher levels of IFNγ. Furthermore, T cells from VIPhyb-treated survivors were protective against C1498 following adoptive transfer. These data highlight the potential for the VIP pathway as a novel target for immunomodulation in settings of hematological malignancies.

A biochemical system and method have been developed to enable the quantitative measurement of cytoplasmic versus nuclear localization within cells in whole blood. Compared with the analyses of nuclear localization by western blot or fluorescence microscopy, this system saves a lot of time and resources by eliminating the necessity of purification and culturing steps, and generates data that are free from the errors and artifacts associated with using tumor cell lines or calculating nuclear signals from 2D images. This user-friendly system enables the analysis of cell signaling within peripheral blood cells in their endogenous environment, including measuring the kinetics of nuclear translocation for transcription factors without requiring protein modifications. We first demonstrated the efficiency and specificity of this system for targeting nuclear epitopes, and verified the results by fluorescence microscopy. Next, the power of the technique to analyze LPS-induced signaling in peripheral blood monocytes was demonstrated. Finally, both FoxP3 localization and IL-2-induced STAT5 signaling in regulatory T cells were analyzed. We conclude that this system can be a useful tool for enabling multidimensional molecular-biological analyses of cell signaling within endogenous peripheral blood cells by conventional flow cytometry. © 2017 International Society for Advancement of Cytometry.
© 2017 International Society for Advancement of Cytometry.

The spleen is involved in fluid volume regulation, immune responses, and hematopoiesis. Yet, the composition of the fluid phase within the spleen microenviroment, the migratory routes of lymphocytes as well as the splenic response to bacterial endotoxin is incomplete. To address these issues, we isolated postnodal lymph in rats by cannulating an efferent lymphatic draining the spleen, and assessed the secretion of signaling substances during a septic response induced by LPS. Spleen lymph flow increased 8-fold after LPS exposure. The spleen exhibited a permeable microvasculature with low sieving of macromolecules that was absent after exposure to LPS. Furthermore, after LPS exposure the spleen contributed significantly to the production of pro- and anti-inflammatory cytokines, and experiments in splenectomized rats suggested it may induce a protracted inflammation because of a dominant role in IL-6 production. A significant amount of lymphocytes exited via lymphatics draining the spleen in control rats. LPS-induced inflammation resulted in increased T cell and reduced B cell subset fractions, and gave a significant increase in CD4(+) and CD8(+) subset T cell efflux and a reduced B cell efflux in spleen lymph. Exposure of leukocytes to the spleen microenvironment affected their signaling status, and by phosphorylation specific flow cytometry we could identify STAT3 and CREB as important mediators in the cellular signaling occurring during endotoxemia. We conclude that analysis of spleen lymph may unravel immune cell migration patterns and local signaling, and immune cells exit via lymph having acquired specific activation signatures after exposure to the spleen microenvironment.