key: cord-0310910-s82t77bv
authors: Liu, Lin; Sandow, Jarrod J.; Leslie Pedrioli, Deena M.; Silke, Natasha; Hu, Zhaoqing; Morrish, Emma; Chau, Diep; Kratina, Tobias; Kueh, Andrew J.; Hottiger, Michael O.; Webb, Andrew I.; Lalaoui, Najoua; Silke, John
title: Tankyrase-mediated ADP-ribosylation is a novel regulator of TNF-induced death
date: 2021-02-09
journal: bioRxiv
DOI: 10.1101/2021.02.09.430424
sha: 504a127c9eeb9528396f5e97bce11bfd598d31cb
doc_id: 310910
cord_uid: s82t77bv

Tumor necrosis factor (TNF) is an inflammatory cytokine that, upon binding to its receptor TNFR1, can drive cytokine production, cell survival, or cell death and is a major component of an organism’s anti-pathogen repetoire1,2. TNF stimulation leads to the formation of two distinct signalling complexes, a well-defined membrane bound complex (complex 1), and a less well characterised cytosolic death inducing complex (complex 2). Using mass spectrometry, we identified the ADP-ribosyltransferase, tankyrase-1 (TNKS1/TNKS/ARTD5/PARP5a) as a novel native complex 2 component. Following a TNF-induced death stimulus TNKS1 is recruited to complex 2, resulting in complex 2 poly(ADP-ribosyl)ation (PARylation). Tankyrase inhibitors sensitise cells to TNF-induced death, which is correlated with increased complex 2 assembly. Tankyrase-mediated PARylation promotes recruitment of the E3 ligase RNF146 and RNF146 deficiency or proteasome inhibition results in increased levels of complex 2, suggesting that RNF146 causes proteasomal degradation of complex 2. Several viruses express ADP-ribose binding macrodomain proteins, and expression of the SARS-CoV-2 or VEEV macrodomain markedly sensitises cells to TNF-induced death. This suggests that ADP-ribosylation serves as yet another mechanism to detect pathogenic interference of TNF signalling and retaliate with an inflammatory cell death.

Tumor necrosis factor (TNF)/TNFR1 signalling helps coordinate an anti-pathogen response by 35 promoting transcriptional upregulation and secretion of other cytokines and inflammatory mediators 36 [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] . To counter this, pathogens have evolved mechanisms to disrupt signalling from the membrane 37 bound complex 1 that nucleates around TNFR1 2,18 . This in turn has prompted an evolutionary arms 38 race whereby disruption of the transcriptional response can provoke TNF-induced cell death via a 39 secondary cytosolic complex 2, containing RIPK1, FADD and caspase-8 3,5,16,19-32 . Dysregulation of 40 TNF signalling has been implicated in a diverse range of inflammatory and auto-immune diseases 41 [33] [34] [35] , stimulating research that has generated a detailed understanding of complex 1 and the 42 TNF/TNFR1 transcriptional response. Compelling evidence showing that TNF-induced cell death is 43 also pathogenic has stimulated the development of drugs to block the cell death response 16,35-37 , but 44 a correspondingly detailed insight into the composition and regulation of complex 2 is lacking. 45 46

To identify TNFR1 complex 2 components, we generated and validated both N-and C-terminally 48 3x FLAG tagged murine caspase-8 constructs (Extended Data Fig. 1a-c) . These tagged constructs 49 allowed us to immunoprecipitate caspase-8 with a number of controls that increase the chance of 50 identifying true hits. Complex 2 formation was induced by treating cells with TNF (T), Smac-51 mimetic (S) to impair the transcriptional response and the pan-caspase inhibitor emricasan/IDN-52 6556 (I) to stabilise complex 2 38-40 . As expected, mass spectrometry analysis of the caspase-8 53 C3FLAG immunoprecipitate from TSI treated Mouse Dermal Fibroblasts (MDFs) revealed 54 enrichment of known complex 2 components, including RIPK1, RIPK3, A20, TRADD and FADD 55 ( Fig. 1a; Supplementary Data 1, sheet 1) . We also identified a previously unreported complex 2 56 protein, tankyrase-1 (TNKS/TNKS1/ARTD5/PARP5a) ( Fig. 1a; Supplementary Data 1, sheet 1) . 57 TNKS1 is an ADP-ribosyltransferase of the ARTD family 41,42 (Extended Data Fig. 1d) , and has 58 not previously been implicated in regulating TNF-induced cell death. To explore the physiological 59 significance of this finding we generated both N-and C-terminally 3x FLAG tagged caspase-8 60 (Casp8 N3FLAG and Casp8 C3FLAG ) knock-in mice using CRISPR/Cas9 technology (Extended Data 61 Fig. 1e-f ). Bone marrow derived macrophages (BMDMs) and MDFs generated from heterozygote 62 knock-in mice were treated with TSI and caspase-8 was immunoprecipitated ± FLAG peptide 63 spiking. As expected, cleaved caspase-8, FADD and RIPK1 were immunoprecipitated together with 64 caspase-8 upon TSI from both Casp8 +/N3FLAG and Casp8 +/C3FLAG cells although we precipitated 65 slightly more of these proteins from Casp8 +/C3FLAG cells (Fig. 1b, Extended Data Fig. 1g) . 66

Consistently we also observed higher levels of TNKS1 co-precipitating with caspase-8 C3FLAG 67 (Fig. 1b, Extended Data Fig. 1g) . In contrast, we did not observe PARP1/ARTD1, the most widely 68 WWE domains are found in many E3 ubiquitin ligases 55 , including HUWE1 and TRIP12. The 137 critical residues for PAR binding are conserved in most WWE domains and HUWE1 and TRIP12 138 WWE domains specifically interact with PAR chains 56 . To determine whether there might be some 139 specificity to the complex 2 interaction, we performed a PAR pulldown assay using GST-HUWE1, 140 -TRIP12 and -RNF146 WWE fusion proteins (Extended Data Fig. 2e) . GST-RNF146 WWE was 141 more efficient than GST-HUWE1 WWE which in turn was far more efficient than GST-TRIP12, at 142 precipitating complex 2 components, suggesting that there may be some specificity and indicating 143 that the RNF146 WWE is optimal for PARylated complex 2 purification (Extended Data Fig. 2e) . 144 145

Thus far, our data suggested that TNKS1 is a functional component of complex 2 and complex 2 147 undergoes PARylation and also hinted that ADP-ribosylation might limit caspase-8 activation. To 148 explore this further we treated WT BMDMs with increasing doses IWR-1 and measured TNF-149 induced cell death by flow cytometry. Consistent with our earlier Western blot analyses (Fig. 2) , 150

BMDMs were rendered increasingly sensitive to TNF plus Smac-mimetic-induced apoptosis (TS) 151 1,15,30,31,38,57,58 and TSI-induced necroptosis 34,59-70 by increasing doses of IWR-1 ( Fig. 3a-b) . This 152 sensitisation was reversed by inhibition of RIPK1 kinase activity with necrostatin-1s, suggesting 153 that tankyrase inhibition sensitised cells to TNF-induced cell death in a RIPK1 kinase-dependent 154 manner ( Fig. 3a-b) . Inhibition or depletion of tankyrases also sensitized MDFs to TS-induced death 155 (Extended Data Fig. 3a-b) , but consistent with the lack of TNKS1 in TNF+CHX-induced complex 156 2 (Extended Data Fig. 1m) , inhibition of tankyrases did not affect TNF+CHX-induced cell death 157 (Extended Data Fig. 3a) . Another tankyrase inhibitor, Az6102 51 , also increased sensitivity to 158 TNF-induced death, while the PARP1/2 inhibitor, olaparib, did not (Extended Data Fig. 3c ). 159

Consistent with the increased cell death, increasing IWR-1 concentrations increased the levels of 160 cleaved caspase-8 and caspase-3 (Fig. 3c) observed in TS treated BMDMs and phospho-RIPK3 and 161 phospho-MLKL in TSI treated cells (Fig. 3d) . The clinical Smac-mimetic birinapant kills leukemic 162 cells in a TNF-dependent manner 30,31,38 , and consistent with this, and our previous data, MLL-163 AF9/NRas G12D cells were dramatically sensitised to both apoptotic and necroptotic cell death by 164 increasing doses of IWR-1 (Extended Data Fig. 3d) . 165

To determine why cells were more sensitive to TNF-induced cell death when tankyrase activity was 167 inhibited we immunoprecipitated complex 2 from Casp8 C3FLAG/C3FLAG BMDMs and MEFs treated 168 with TSI ± IWR-1. By selecting a TSI dose that induced only low levels of caspase-8 activation, we 169 were able to show that tankyrase inhibition dramatically increased the amount of complex 2 that 170 could be immunoprecipitated by anti-FLAG beads, suggesting that tankyrase-mediated ADP-171 ribosylation reduces the stability of complex 2 (Fig. 3e, Extended Data Fig. 3e) . Typically, 172 complex 2 is difficult to purify unless a caspase inhibitor, such as emricasan/IDN-6556, is used to 173 stabilise it 3,38 . However, this makes it difficult to test whether tankyrase inhibition increases 174 complex 2 formation in the absence of a caspase inhibitor. To circumvent this issue, we took 175 advantage of the fact that complex 2 can be isolated more readily from cells expressing an 176 uncleavable form of RIPK1 71 . We therefore treated Ripk1 D325A/+ heterozygous MDFs with TS ± 177 IWR-1 and immunoprecipitated RIPK1 and found that tankyrase inhibition also increased the 178 amount of complex 2 that could be purified from these cells and sensitised them to TNF-induced 179 cell death in a dose dependent manner ( Fig. 3f-g, Extended Data Fig. 3f) . 180

The tankyrase-RNF146 axis regulates the stability of complex 2 and TNF-induced death 182

Tankyrases regulate a number of other signalling pathways 48,72-75 , and the most well-studied is the 183 Wnt pathway where tankyrase-mediated ADP-ribosylation of Axin recruits the E3 ligase RNF146 184 via its WWE motif. RNF146 then ubiquitylates Axin causing its recruitment to and degradation by 185 the proteasome 50,56,76-78 . Given the increased stability of complex 2 in the presence of tankyrase 186

inhibitor IWR-1 that we observed, we hypothesized that tankyrase-mediated ADP-ribosylation of 187 complex 2 might function analogously to recruit RNF146 and promote its proteasomal degradation. 188

In accord with this hypothesis RNF146 was recruited to complex 2 immunoprecipitated from 189

Casp8 +/C3FLAG heterozygote MEFs treated with TSI (Fig. 4a) . Furthermore, there was a reduction in 190 the precipitation of ubiquitylated complex 2 components using a GST-UBA fusion protein, when 191 cells were treated with IWR-1 (Fig. 4b) . Consistent with the idea that proteasomal mediated 192 degradation limits complex 2 levels, we observed a striking increase in the amount of ubiquitylated 193 complex 2 when cells were treated with the proteasomal inhibitor MG132 (Fig. 4c) . To avoid the 194 possibility that constitutive loss of RNF146 affected cell viability, we generated stable Dox 195 inducible RNF146 shRNA expressing cells and immunoprecipitated RIPK1 in the presence or 196 absence of Dox. Similarly to the proteasome inhibitor experiment, we saw that there was a stark 197 increase in the levels of complex 2 in the cells with reduced levels of RNF146 when compared with 198 control shRNA expressing cells (Fig. 4d) , and as expected shRNF146 expressing cells were more 199 sensitive to TNF-induced cell death (Fig. 4e, Extended Data Fig. 4) . TNF is an important part of the mammalian anti-pathogen armamentarium and as a consequence is 203 frequently targeted by pathogens which produce proteins that interfere with the pathway 2 . The TNF 204 pathway has however several mechanisms to respond to interference and one of those is to trigger 205 cell death. This begs the question whether ADP-ribosylation of complex 2 also serves to control for 206 interference and whether the increased death that we observed when tankyrase activity is inhibited 207 might mimic some form of pathogen manipulation. A number of viruses, including Coronaviruses, 208 express evolutionarily conserved MacroD type macrodomains 79,80 , similar to that of Af1521 that we 209 used to precipitate complex 2, that are able to bind to mono-ADP-ribosylated proteins or to the end 210 of poly-ADP-Ribose chains and in some cases have been shown to remove ADP-ribose from mono-211 ADP-ribosylated proteins 81-85 . We therefore asked whether inducible expression of the 212 macrodomain from SARS-CoV-2 or a closely related VEEV macrodomain might affect TNF-213 induced cell death. Consistent with the idea that ADP-ribosylation of complex 2 could serve as a 214 checkpoint to detect perturbations in TNF signalling we found that expression of both these viral 215 macrodomains markedly increased the sensitivity of cells to TNF-induced cell death (Fig. 5) . 216

We show that the ability of TNF to induce cell death is regulated by tankyrase-mediated 218

PARylation. Interestingly, while TNKS1 was readily recruited to complex 2 upon Smac-mimetic 219 treatment, it was not detectable in complex 2 assembled in response to cycloheximide. This 220 suggests that ADP-ribosylation is a context sensitive regulator and since RIPK1 involvement is a 221 major difference in these two complexes, it suggests RIPK1 might be directly involved. 222 223 Tankyrase 1 & 2 regulate a number of signalling pathways and one possibility is that the 224 PARylation-mediated by tankyrases might allow different signalling pathways to interact and co-225 ordinate with one another. In particular there is evidence linking TNF signalling with the Wnt and 226 GSK3 signalling pathways as well as cell cycle and cell division, all of which are known to be 227 regulated by tankyrases 46,86,87 . Indeed, specific and potent tankyrase inhibitors, such as IWR-1, 228

were developed to block Wnt signalling in cancers yet clearly sensitise cells to TNF killing and this 229 unintended activity might increase the efficacy of these drugs in tumors with an inflammatory 230 component. Furthermore, it has been noted that some cancers are sensitive to these inhibitors 231 without apparently affecting Wnt signalling thus opening up the possibility that sensitivity to TNF 232 might be an additional predictive biomarker to consider when using these drugs. induced cell death, this suggests that ADP-ribosylation may serve as yet another mechanism to 240 allow TNF to retaliate against a dangerous infection by inducing cell death. This idea is supported 241 by the observation that PARP-10, a mono-ADP-ribosyltransferase, inhibits IL-1β/TNF-induced NF-242 κB signalling 90 .Given the broad involvement of ADP-ribosylation in other signalling pathways 91,92 , 243 one intriguing possibility is that pathogens select for the ability to interfere with ADP-ribosylation 244 to target these pathways and that ADP-ribosylation has been co-opted into the TNF response to 245 control for the integrity of these pathways rather than of the TNF pathway alone. 246 were treated with TSI (as in a) ± IWR-1 (10 μM). Western blot analysis of complex 2 and lysates 288 using the indicated antibodies is shown. 289 290 d, Enrichment of PARylated complex 2 using GST-WWE in a sequential pulldown analysis. 291

Casp8 C3FLAG/C3FLAG BMDMs were treated with TSI (as in a) and complex 2 was 292 immunoprecipitated using anti-FLAG M2 affinity beads. Immunoprecipitants were eluted with 3x 293 FLAG peptides followed by ± PARG treatment at 37°C for 3 hours before being subjected to GST-294 WWE pulldown. Western blot analysis of lysates and sequential pulldown using the indicated 295 antibodies is shown. 296 297

Filled arrowheads alone indicate potential tankyrase species. Empty arrowheads alone denote 298 unmodified RIPK1 that is purified non-specifically by either Sepharose anti-PAR (a) or Sepharose with TNF (10 ng/mL) + Smac-mimetic (500 nM) (TS) ± IWR-1 (250nM, 500nM, 1 μM, 2 μM, 5 306 μM) ± Nec-1s (10 μM) for 24 hours. Graphs show mean ± SEM, n=3 biologically independent 307 repeats. Comparisons were performed with a Student's t test whose values are denoted as *p ≤ 0.05, 308 ***p ≤ 0.001 and ****p ≤ 0.0001. 309 310

b, Level of cell death assessed by PI positive cells. WT BMDMs were treated with TNF (10 ng/mL) 311 + Smac-mimetic (10 nM) + caspase inhibitor (5 μM) (TSI) ± IWR-1 (250nM, 500nM, 1 μM, 2 μM, 312 5 μM) ± Nec-1s (10 μM) for 16 hours. Graphs show mean ± SEM, n=3 biologically independent 313 repeats. Comparisons were performed with a Student's t test whose values are denoted as *p ≤ 0.05, 314 **p ≤ 0.01 and ****p ≤ 0.0001. 315 316 c, Western blot analysis of cell lysates from WT BMDMs using indicated antibodies is shown. 317

Cells were treated with TNF (10 ng/mL) + Smac-mimetic (500 nM) (TS) ± IWR-1 (250nM, 318 500nM, 1 μM, 2 μM, 5 μM) for 8 hours. 319 320 d, Western blot analysis of cell lysates from WT BMDMs using indicated antibodies is shown. 321

Cells were treated with TNF (10 ng/mL) + Smac-mimetic (20 nM) + caspase inhibitor (5 μM) 322

(TSI) ± IWR-1 (250nM, 500nM, 1 μM, 2 μM, 5 μM) for 8 hours. 323 324 e, TNF-induced complex 2 immunoprecipitation using anti-FLAG M2 affinity beads. Western blot 325 analysis of complex 2 and lysates from Casp8 C3FLAG/C3FLAG BMDMs using the indicated antibodies 326 is shown. Cells were treated with TNF (10 ng/mL) + Smac-mimetic (50 nM) + caspase inhibitor (5 327 μM) (TSI) ± IWR-1 (5 μM) for 1.5 hours before being subjected to anti-FLAG 328

immunoprecipitation. 329 330

f, TNF-induced complex 2 immunoprecipitation using anti-RIPK1 antibody. Western blot analysis 331 of complex 2 and lysates from Ripk1 D325A/+ heterozygous MDFs using the indicated antibodies is 332

shown. Cells were treated with TNF (50 ng/mL) + Smac-mimetic (100 nM) ± IWR-1 (5 μM) for 2 333 hours before being subjected to anti-RIPK1 immunoprecipitation. Level of cell death assessed by PI positive cells. WT MEFs expressing Dox-inducible shLuciferase 543 or shRNF146 were pre-treated with ± Dox (1 μg/mL) for 48 hours. Cells were then subjected to 544

Western blot analysis or treated with TNF (100 ng/mL) + Smac-mimetic (25 nM) (TS) ± Dox 545 (1μg/mL) for another 12 hours. 546 547

Blots are representative of two independent experiments. 548 549

Graphs show mean ± SD throughout, n = 2 independent biological repeats. The number of independent experiments for each dataset is stipulated in the respective figure  789 legend. Comparisons were performed with a Student's t test whose values are represented in the 790

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