Autotaxin impedes anti-tumor immunity by suppressing chemotaxis and tumor infiltration of CD8+ T cells

Artículo publicado en revista Cell Reports, 2021 Nov 16;37(7):110013. doi: 10.1016/j.celrep.2021.110013.Autotaxin (ATX; ENPP2) produces lysophosphatidic acid (LPA) that regulates multiple biological functions via cognate G protein-coupled receptors LPAR1-6. ATX/LPA promotes tumor cell migration and metastasis via LPAR1 and T cell motility via LPAR2, yet its actions in the tumor immune microenvironment remain unclear. Here, we show that ATX secreted by melanoma cells is chemorepulsive for tumor-infiltrating lymphocytes (TILs) and circulating CD8+ T cells ex vivo, with ATX functioning as an LPA-producing chaperone. Mechanistically, T cell repulsion predominantly involves Gα12/13-coupled LPAR6. Upon anti-cancer vaccination of tumor-bearing mice, ATX does not affect the induction of systemic T cell responses but, importantly, suppresses tumor infiltration of cytotoxic CD8+ T cells and thereby impairs tumor regression. Moreover, single-cell data from melanoma tumors are consistent with intratumoral ATX acting as a T cell repellent. These findings highlight an unexpected role for the pro-metastatic ATX-LPAR axis in suppressing CD8+ T cell infiltration to impede anti-tumor immunity, suggesting new therapeutic opportunities.This work was supported by private funding to W.H.M. and grants from the Dutch Cancer Society (NKI 2013-5951 and 10764 to I.V. and NKI 2017-10894 to J.B. and I.V.), the German Research Foundation (DFG) (ME 4924/1-1 to A. Mazzocca), and the NIH (P30 GM127211 to A.J.M.). E.M.-R. is supported by a ‘‘Ramo ´n y Cajal’’ Award (RYC2019-027950-I) from Ministerio de Ciencia e Innovacio´n (MICINN), Spain

Cell-line dependent effects of hypoxia prior to irradiation in squamous cell carcinoma lines

Purpose: To assess the impact of hypoxia exposure on cellular radiation sensitivity and survival of tumor cells with diverse intrinsic radiation sensitivity under normoxic conditions. Materials and methods: Three squamous cell carcinoma (SCC) cell lines, with pronounced differences in radiation sensitivity, were exposed to hypoxia prior, during or post irradiation. Cells were seeded in parallel for colony formation assay (CFA) and stained for γH2AX foci or processed for western blot analysis. Results: Hypoxia during irradiation led to increased cellular survival and reduced amount of residual γH2AX foci in all the cell lines with similar oxygen enhancement ratios (OER SKX: 2.31, FaDu: 2.44, UT-SCC5: 2.32), while post-irradiation hypoxia did not alter CFA nor residual γH2AX foci. Interestingly, prolonged exposure to hypoxia prior to irradiation resulted in differential outcome, assessed as Hypoxia modifying factor (HMF) namely radiosensitization (SKX HMF: 0.76), radioresistance (FaDu HMF: 1.54) and no effect (UT SCC-5 HMF: 1.1). Notably, radiosensitization was observed in the ATM-deficient SKX cell line while UT SCC-5 and to a lesser extent also FaDu cells showed radiation- and hypoxia-induced upregulation of ATM phosphorylation. Across all the cell lines Rad51 was downregulated whereas phosphor-DNA-PKcs was enhanced under hypoxia for FaDu and UTSCC-5 and was delayed in the SKX cell line. Conclusion: We herein report a key role of ATM in the cellular fitness of cells exposed to prolonged moderate hypoxia prior to irradiation. While DNA damage response post-irradiation seem to be mainly driven by non-homologous end joining repair pathway in these conditions, our data suggest an important role for ATM kinase in hypoxia-driven modification of radiation response

Combined Inactivation of Pocket Proteins and APC/C^Cdh1 by Cdk4/6 Controls Recovery from DNA Damage in G1 Phase

Most Cyclin-dependent kinases (Cdks) are redundant for normal cell division. Here we tested whether these redundancies are maintained during cell cycle recovery after a DNA damage-induced arrest in G1. Using non-transformed RPE-1 cells, we find that while Cdk4 and Cdk6 act redundantly during normal S-phase entry, they both become essential for S-phase entry after DNA damage in G1. We show that this is due to a greater overall dependency for Cdk4/6 activity, rather than to independent functions of either kinase. In addition, we show that inactivation of pocket proteins is sufficient to overcome the inhibitory effects of complete Cdk4/6 inhibition in otherwise unperturbed cells, but that this cannot revert the effects of Cdk4/6 inhibition in DNA damaged cultures. Indeed, we could confirm that, in addition to inactivation of pocket proteins, Cdh1-dependent anaphase-promoting complex/cyclosome (APC/CCdh1) activity needs to be inhibited to promote S-phase entry in damaged cultures. Collectively, our data indicate that DNA damage in G1 creates a unique situation where high levels of Cdk4/6 activity are required to inactivate pocket proteins and APC/CCdh1 to promote the transition from G1 to S phase

Combined Inactivation of Pocket Proteins and APC/CCdh1 by Cdk4/6 Controls Recovery from DNA Damage in G1 Phase

Most Cyclin-dependent kinases (Cdks) are redundant for normal cell division. Here we tested whether these redundancies are maintained during cell cycle recovery after a DNA damage-induced arrest in G1. Using non-transformed RPE-1 cells, we find that while Cdk4 and Cdk6 act redundantly during normal S-phase entry, they both become essential for S-phase entry after DNA damage in G1. We show that this is due to a greater overall dependency for Cdk4/6 activity, rather than to independent functions of either kinase. In addition, we show that inactivation of pocket proteins is sufficient to overcome the inhibitory effects of complete Cdk4/6 inhibition in otherwise unperturbed cells, but that this cannot revert the effects of Cdk4/6 inhibition in DNA damaged cultures. Indeed, we could confirm that, in addition to inactivation of pocket proteins, Cdh1-dependent anaphase-promoting complex/cyclosome (APC/CCdh1) activity needs to be inhibited to promote S-phase entry in damaged cultures. Collectively, our data indicate that DNA damage in G1 creates a unique situation where high levels of Cdk4/6 activity are required to inactivate pocket proteins and APC/CCdh1 to promote the transition from G1 to S phase