A light-gated transcriptional recorder for detecting cell-cell contacts.

Technologies for detecting cell-cell contacts are powerful tools for studying a wide range of biological processes, from neuronal signaling to cancer-immune interactions within the tumor microenvironment. Here, we report TRACC (Transcriptional Readout Activated by Cell-cell Contacts), a GPCR-based transcriptional recorder of cellular contacts, which converts contact events into stable transgene expression. TRACC is derived from our previous protein-protein interaction recorders, SPARK (Kim et al., 2017) and SPARK2 (Kim et al., 2019), reported in this journal. TRACC incorporates light gating via the light-oxygen-voltage-sensing (LOV) domain, which provides user-defined temporal control of tool activation and reduces background. We show that TRACC detects cell-cell contacts with high specificity and sensitivity in mammalian cell culture and that it can be used to interrogate interactions between neurons and glioma, a form of brain cancer.

Receptor tyrosine kinases activate heterotrimeric G proteins via phosphorylation within the interdomain cleft of Gαi.

The molecular mechanisms by which receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major signaling hubs in eukaryotes, independently relay signals across the plasma membrane have been extensively characterized. How these hubs cross-talk has been a long-standing question, but answers remain elusive. Using linear ion-trap mass spectrometry in combination with biochemical, cellular, and computational approaches, we unravel a mechanism of activation of heterotrimeric G proteins by RTKs and chart the key steps that mediate such activation. Upon growth factor stimulation, the guanine-nucleotide exchange modulator dissociates Gαi•ßγ trimers, scaffolds monomeric Gαi with RTKs, and facilitates the phosphorylation on two tyrosines located within the interdomain cleft of Gαi. Phosphorylation triggers the activation of Gαi and inhibits second messengers (cAMP). Tumor-associated mutants reveal how constitutive activation of this pathway impacts cell's decision to "go" vs. "grow." These insights define a tyrosine-based G protein signaling paradigm and reveal its importance in eukaryotes.

Convergence of Wnt, growth factor, and heterotrimeric G protein signals on the guanine nucleotide exchange factor Daple.

Cellular proliferation, differentiation, and morphogenesis are shaped by multiple signaling cascades, and their dysregulation plays an integral role in cancer progression. Three cascades that contribute to oncogenic potential are those mediated by Wnt proteins and the receptor Frizzled (FZD), growth factor receptor tyrosine kinases (RTKs), and heterotrimeric G proteins and associated GPCRs. Daple is a guanine nucleotide exchange factor (GEF) for the G protein Gαi Daple also binds to FZD and the Wnt/FZD mediator Dishevelled (Dvl), and it enhances ß-catenin-independent Wnt signaling in response to Wnt5a-FZD7 signaling. We identified Daple as a substrate of multiple RTKs and non-RTKs and, hence, as a point of convergence for the three cascades. We found that phosphorylation near the Dvl-binding motif in Daple by both RTKs and non-RTKs caused Daple/Dvl complex dissociation and augmented the ability of Daple to bind to and activate Gαi, which potentiated ß-catenin-independent Wnt signals and stimulated epithelial-mesenchymal transition (EMT) similarly to Wnt5a/FZD7 signaling. Although Daple acts as a tumor suppressor in the healthy colon, the concurrent increased abundance of Daple and epidermal growth factor receptor (EGFR) in colorectal tumors was associated with poor patient prognosis. Thus, the Daple-dependent activation of Gαi and the Daple-dependent enhancement of ß-catenin-independent Wnt signals are not only stimulated by Wnt5a/FZD7 to suppress tumorigenesis but also hijacked by growth factor-activated RTKs to enhance tumor progression. These findings identify a cross-talk paradigm among growth factor RTKs, heterotrimeric G proteins, and the Wnt/FZD pathway in cancer.

Intratumoral treatment of smaller mouse neuroblastoma tumors with a recombinant protein consisting of IL-2 linked to the hu14.18 antibody increases intratumoral CD8+ T and NK cells and improves survival.

Hu14.18-IL2 is an immunocytokine (IC) consisting of human IL-2 linked to hu14.18 mAb, which recognizes GD2 disialoganglioside. Phase II clinical trials of intravenous-hu14.18-IL2 (IV-IC) in neuroblastoma and melanoma are underway, and have already demonstrated activity in neuroblastoma. In our Phase II trial, lower neuroblastoma burden at the time of treatment was associated with a greater likelihood of clinical response to IV-IC. We have previously shown that intratumoral-hu14.18-IL2 (IT-IC) compared to IV-IC results in enhanced local and systemic antitumor activity in tumor-bearing mice. We utilized a mouse model to investigate the impact of tumor burden on hu14.18-IL2 treatment efficacy in IV- versus IT-treated animals. Studies presented here describe the analyses of tumor burden at the initiation of treatment and its effects on treatment efficacy, survival, and tumor-infiltrating leukocytes in A/J mice bearing subcutaneous NXS2 neuroblastoma. We show that smaller tumor burden at treatment initiation is associated with increased infiltration of NK and CD8+ T cells and increased overall survival. NXS2 tumor shrinkage shortly after completion of the 3 days of hu14.18-IL2 treatment is necessary for long-term survival. This model demonstrates that tumor size is a strong predictor of hu14.18-IL2-induced lymphocyte infiltration and treatment outcome.

Intratumoral hu14.18-IL-2 (IC) induces local and systemic antitumor effects that involve both activated T and NK cells as well as enhanced IC retention.

hu14.18-IL-2 (IC) is an immunocytokine consisting of human IL-2 linked to hu14.18 mAb, which recognizes the GD2 disialoganglioside. Phase 2 clinical trials of i.v. hu14.18-IL-2 (i.v.-IC) in neuroblastoma and melanoma are underway and have already demonstrated activity in neuroblastoma. We showed previously that intratumoral hu14.18-IL-2 (IT-IC) results in enhanced antitumor activity in mouse models compared with i.v.-IC. The studies presented in this article were designed to determine the mechanisms involved in this enhanced activity and to support the future clinical testing of intratumoral administration of immunocytokines. Improved survival and inhibition of growth of both local and distant tumors were observed in A/J mice bearing s.c. NXS2 neuroblastomas treated with IT-IC compared with those treated with i.v.-IC or control mice. The local and systemic antitumor effects of IT-IC were inhibited by depletion of NK cells or T cells. IT-IC resulted in increased NKG2D receptors on intratumoral NKG2A/C/E⁺ NKp46⁺ NK cells and NKG2A/C/E⁺ CD8⁺ T cells compared with control mice or mice treated with i.v.-IC. NKG2D levels were augmented more in tumor-infiltrating lymphocytes compared with splenocytes, supporting the localized nature of the intratumoral changes induced by IT-IC treatment. Prolonged retention of IC at the tumor site was seen with IT-IC compared with i.v.-IC. Overall, IT-IC resulted in increased numbers of activated T and NK cells within tumors, better IC retention in the tumor, enhanced inhibition of tumor growth, and improved survival compared with i.v.-IC.

Tumor-associated myeloid cells can be activated in vitro and in vivo to mediate antitumor effects.

Tumor growth is often accompanied by the accumulation of myeloid cells in the tumors and lymphoid organs. These cells can suppress T cell immunity, thereby posing an obstacle to T cell-targeted cancer immunotherapy. In this study, we tested the possibility of activating tumor-associated myeloid cells to mediate antitumor effects. Using the peritoneal model of B16 melanoma, we show that peritoneal cells (PEC) in tumor-bearing mice (TBM) had reduced ability to secrete nitric oxide (NO) following in vitro stimulation with interferon gamma and lipopolysaccharide, as compared to PEC from control mice. This reduced function of PEC was accompanied by the influx of CD11b(+) Gr-1(+) myeloid cells to the peritoneal cavity. Nonadherent PEC were responsible for most of the NO production in TBM, whereas in naïve mice NO was mainly secreted by adherent CD11b(+) F4/80(+) macrophages. Sorted CD11b(+) Gr-1(-) monocytic and CD11b(+) Gr-1(+) granulocytic PEC from TBM had a reduced ability to secrete NO following in vitro stimulation (compared to naïve PEC), but effectively suppressed proliferation of tumor cells in vitro. In vivo, treatment of mice bearing established peritoneal B16 tumors with anti-CD40 and CpG resulted in activation of tumor-associated PEC, reduction in local tumor burden and prolongation of mouse survival. Inhibition of NO did not abrogate the antitumor effects of stimulated myeloid cells. Taken together, the results indicate that in tumor-bearing hosts, tumor-associated myeloid cells can be activated to mediate antitumor effects.