Abstract 1697: Signaling pathways involved in melanoma cell functions and dedifferentiation induced by inflammatory cytokines
Authors: Cerniglia Michael , Campbell Katie M. , Schwerdtfeger Julianne , Martignier Christophe , Gilfillan Connie B. , Schaeuble Karin , Cheung-Lau Gardenia , Teper Yaroslav , Maneiro David Berger , Guemes-Aragon Miriam , Garcia Alejandro J. , Speiser Daniel E. , Ribas Antoni , Comin-Anduix Begoña 
Published: September 2020

Abstract
INTRODUCTION: Melanoma-acquired resistance against T cell-based immunotherapy may be mediated by alterations of their responses to inflammatory cytokines. However, the cytokine signaling pathways in cancer cells remain poorly understood.
METHODS: 8 melanoma cell lines were exposed to interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) over a time course. Signaling molecules and their phosphorylated state were subsequently analyzed using mass cytometry. Briefly, cells were incubated with IFNγ (220 IU/mL), TNFα (1000 IU/mL), and both cytokines (220 IU/mL IFNγ + 200 IU/mL TNFα) for up to 48 hours. Staining was done with antibodies specific for (1) phosphorylated - STAT1(Y701), STAT3(Y707), AKT(S437), ERK1/2(T202/Y204), IKK(S176/180), NK-kbetap65 (S536), JNK (T183/T22), P38 (T180/Y182), Rb(S807/Y182), cMYC (S62), S6 (S235/S236), Histone3 (S28) and (2) non-phosphorylated - cPARP, β-catenin, CyclinB1, Ki67. Cells were read on a Helios Mass cytometer and de-barcoded using PREMESSA (R package). FlowJo was used to normalize time and eliminate calibration beads. Viable cell files were introduced to Cytobank where fold change (FC) with respect to time 0 of each protein was calculated with median intensity. The FC was exported to R for final analysis.
RESULTS: All cell lines were sensitive to IFNγ/TNFα, with detectable (phosphorylated) signaling molecules. No synergistic effect was observed at any dose for IFNγ+TNFα. For the following data, numbers contained within parentheses represent the mean range of FC control vs treatment or mean standard error. pSTAT1 was significantly activated at all time points for IFNγ ([0.8, 1.0] vs [1.8, 3.6] p&lt;0.02) and for IFNγ+TNFα ([1.9, 3.5] p&lt;0.03). For TNFα it was activated at 48h ([3.1±0.9 vs 1.9±0.2] p=0.003). pJNK was significantly activated for IFNγ at time 0.25h and 0.5h ([0.9, 1.0] vs [1, 5] p&lt;0.01); for TNFα at 0.25h and 1h ([0.9, 1.0] vs [1.5, 1.6] p&lt;0.02); and for IFNγ+TNFα at 0.25h and 1h ([0.9, 1.0] vs 1.5 p&lt;0.007). pNF-KBp65 was only significantly different for IFNγ+TNFα at 24h ([0.9±0.0 vs 11.6±0.2] p=0.025). The other time points followed a similar trend to activation. pSTAT3 was significantly activated for IFNγ at time 0.25h and 24h ([0.6, 0.9] vs [1.7, 2.1] p&lt;0.04); for IFNγ+TNFα at time 0.25h and 24h ([0.6, 0.9] vs [1.9, 2.0] p&lt;0.02); and for TNFα at 0.25h and 0.5h ([0.5, 0.6] vs [1.3, 1.5] p&lt;0.02). pERK was significantly activated for IFNγ at 0.25h ([0.9±0.2 vs 1.6±0.3] p=0.04). β-catenin presented no difference in FC across cell lines.
CONCLUSIONS: Our data show evidence for functional IFNγ/TNFα signaling in most melanoma cells. However, unexpected signaling was observed for some cell lines, possibly reflecting interpatient heterogeneity. Additional studies are necessary to understand the roles of cytokine signaling in cancer immunotherapy.
Citation Format: Michael Cerniglia, Katie M. Campbell, Julianne Schwerdtfeger, Christophe Martignier, Connie B. Gilfillan, Karin Schaeuble, Gardenia Cheung-Lau, Yaroslav Teper, David Berger Maneiro, Miriam Guemes-Aragon, Alejandro J. Garcia, Daniel E. Speiser, Antoni Ribas, Begoña Comin-Anduix. Signaling pathways involved in melanoma cell functions and dedifferentiation induced by inflammatory cytokines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1697.