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. 2019 Jun;26(6):1138-1155.
doi: 10.1038/s41418-018-0192-6. Epub 2018 Sep 20.

Systematic genetic mapping of necroptosis identifies SLC39A7 as modulator of death receptor trafficking

Astrid Fauster #  1 , Manuele Rebsamen #  2 , Katharina L Willmann  1   3 , Adrian César-Razquin  1 , Enrico Girardi  1 , Johannes W Bigenzahn  1 , Fiorella Schischlik  1 , Stefania Scorzoni  1 , Manuela Bruckner  1 , Justyna Konecka  1 , Katrin Hörmann  1 , Leonhard X Heinz  1 , Kaan Boztug  1   3   4   5 , Giulio Superti-Furga  6   7
Affiliations

Systematic genetic mapping of necroptosis identifies SLC39A7 as modulator of death receptor trafficking

Astrid Fauster et al. Cell Death Differ. 2019 Jun.

Abstract

Regulation of cell and tissue homeostasis by programmed cell death is a fundamental process with wide physiological and pathological implications. The advent of scalable somatic cell genetic technologies creates the opportunity to functionally map such essential pathways, thereby identifying potential disease-relevant components. We investigated the genetic basis underlying necroptotic cell death by performing a complementary set of loss-of-function and gain-of-function genetic screens. To this end, we established FADD-deficient haploid human KBM7 cells, which specifically and efficiently undergo necroptosis after a single treatment with either TNFα or the SMAC mimetic compound birinapant. A series of unbiased gene-trap screens identified key signaling mediators, such as TNFR1, RIPK1, RIPK3, and MLKL. Among the novel components, we focused on the zinc transporter SLC39A7, whose knock-out led to necroptosis resistance by affecting TNF receptor surface levels. Orthogonal, solute carrier (SLC)-focused CRISPR/Cas9-based genetic screens revealed the exquisite specificity of SLC39A7, among ~400 SLC genes, for TNFR1-mediated and FAS-mediated but not TRAIL-R1-mediated responses. Mechanistically, we demonstrate that loss of SLC39A7 resulted in augmented ER stress and impaired receptor trafficking, thereby globally affecting downstream signaling. The newly established cellular model also allowed genome-wide gain-of-function screening for genes conferring resistance to necroptosis via the CRISPR/Cas9-based synergistic activation mediator approach. Among these, we found cIAP1 and cIAP2, and characterized the role of TNIP1, which prevented pathway activation in a ubiquitin-binding dependent manner. Altogether, the gain-of-function and loss-of-function screens described here provide a global genetic chart of the molecular factors involved in necroptosis and death receptor signaling, prompting further investigation of their individual contribution and potential role in pathological conditions.

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Conflict of interest statement

The Superti-Furga laboratory at CeMM has an active research collaboration with Boehringer Ingelheim (Research Collaboration Agreement BICeMM 238114). The remaining authors declare that they have no conflict of interests.

Figures

Fig. 1
Fig. 1
KBM7 FADDˉ cells undergo necroptosis upon treatment with TNFα or the SMAC mimetic birinapant. a Cell viability of KMB7 wildtype and KBM7 FADD- cells treated for 24 h with 100 ng/ml TNFα, 1 µM SMAC mimetic birinapant, 50 µM Nec-1s and 10 µM z-VAD-FMK as indicated. b Cell viability of KBM7 FADD- cells treated for 24 h with 100 ng/ml TNFα, 10 µM SMAC mimetic or 100 ng/ml TNFα and 1 µM SMAC mimetic, and the indicated inhibitors. Cell viability was assessed using a luminescence-based readout for ATP (CellTiter Glo) and normalized to untreated control. Data represent mean value ± s.d. of two independent experiments performed in triplicates
Fig. 2
Fig. 2
Haploid genetic screens in KBM7 FADD- cells identify genes required for necroptosis. a–c Circos plots of haploid genetic screens in KBM7 FADD- cells with necroptosis induction by 10 µM SMAC mimetic birinapant (a) 100 ng/ml TNFα (b) and 1 µM SMAC mimetic and 100 ng/ml TNFα combined (c). Each dot represents a mutagenized gene identified in the resistant cell population, dot size corresponds to the number of independent insertions identified for each gene and distance from center indicates the significance of enrichment compared to an unselected control data set. Hits with an adjusted p-value < 10‒10 are labeled. d Bubble plot depicting the top hits over all three screens ranked according to adjusted p-value in the TNFα screen. Bubble size corresponds to the number of independent insertions identified and color gradient reflects the significance of enrichment. e, f Multi-color competition assay (MCA) of KBM7 FADD- SpCas9 cells transduced with a GFP marker (GFP+) and sgRNAs targeting either SLC39A7 or RIPK1 (e), SP1 or TNFR2 (f), or Renilla luciferase (sgRen) as control, against cells transduced with sgRen and an mCherry marker (mCherry+). The cell populations were mixed at 1:1 ratio, treated with SMAC mimetic (1 µM) or TNFα (10 ng/ml), and analyzed after 14 days by flow cytometry. Data represent mean value ± s.d. of two independent experiments performed in duplicates, n.d. (not determined) indicates wells with no outgrowth
Fig. 3
Fig. 3
Loss of SLC39A7 mediates resistance to TNFα-induced cell death by diminishing TNFR1 surface expression. a Growth curve for KBM7 FADD- sgRen or SLC39A7- cells. Equal cell numbers were seeded and cell growth monitored over 7 days. Data represent mean value ± s.d. of five independent experiments; statistical analysis by t-test, *P < 0.01. b Cell viability in KBM7 FADD- sgRen or SLC39A7- cells treated overnight (16 h) with TNFα (10 ng/ml), SMAC mimetic (0.5 µM), and Nec-1s (50 µM) as indicated. Cell viability was assessed using a luminescence-based readout for ATP (CellTiter Glo). Data represent mean value ± s.d. of two independent experiments performed in triplicates. c KBM7 FADD- SpCas9 (empty, sgRen or SLC39A7-) cells were stimulated for the time indicated with TNFα (10 ng/ml). Cells were then lysed and subjected to immunoblotting with the indicated antibodies. d Flow cytometry analysis for TNFR1 surface expression. KBM7 TNFRSF1A- cells serve as negative control for background staining. Data shown are representative of two independent experiments. e KBM7 FADD- SpCas9 (empty, sgRen or SLC39A7-) cells were treated for 7 h with Brefeldin A (0.5 µM), Tunicamycin (2 µM), Thapsigargin (0.5 µM), MG-132 (10 µM) or DMSO as control. Cells were then lysed and subjected to immunoblotting with the indicated antibodies. f KBM7 TNFRSF1A-, KBM7 FADD- and KBM7 FADD- SLC39A7- cells were lysed and subjected to immunoblotting with the indicated antibodies. g KBM7 FADD- SpCas9 sgRen or SLC39A7- cell lysates were incubated for 1 h at 37 °C in presence or absence of PNGaseF or EndoH, respectively, and analysed by immunoblot with the indicated antibodies. Immunoblots shown are representative of two independent experiments. Asterisk (*) indicates non-specific band
Fig. 4
Fig. 4
Orthogonal genetic screens and surface marker analysis specify SLC39A7 loss-of-function phenotype on receptor trafficking. a Spider plot summarizing the results of 8 independent CRISPR/Cas9 screens in KBM7 wildtype, KBM7 FADD-, or HAP1 cells using an SLC-specific gRNA library. Each plot section represents one screen with the indicated stimuli. Screen analysis was performed by identifying differentially enriched sgRNAs using DESeq2 and then aggregating sgRNAs to genes using Gene Set Enrichment Analysis. Identified hits are ranked according to the adjusted p-value of enrichment (–log10(padj)), bubble size indicates the number of significantly enriched sgRNAs and color the average log2 fold-change (aLFC) of the enriched sgRNAs. Screens were performed in duplicate. SLC39A7 is highlighted in orange. No gene was identified in KBM7 wildtype cells upon TRAIL treatment. b MCA of Jurkat E6.1 SpCas9 cells transduced with a GFP marker (GFP+) and sgRNAs targeting either SLC39A7 or Renilla luciferase (sgRen) as control, against cells transduced with sgRen and an mCherry marker (mCherry+). The cell populations were mixed at 1:1 ratio, treated with TRAIL (20 ng/ml) or FASL (1 ng/ml), and analyzed after 14 days by flow cytometry. Data represent mean value ± s.d. of two independent experiments performed in duplicates, n.d. (not determined) indicates wells with no outgrowth. c Flow cytometry analysis for TRAILR1 (left) and TRAILR2 (right) surface expression in KBM7 FADD-, KBM7 FADD- sgRen or KBM7 FADD- SLC39A7- cells. Data shown are representative of two independent experiments
Fig. 5
Fig. 5
SLC39A7 transporter function is required to relieve ER stress, restore TNFR1 surface expression and induce cell death. a KBM7 FADD- SLC39A7- cells lentivirally transduced with either V5-tagged SLC39A7 or GFP were lysed and immunoblotted with the indicated antibodies, KBM7 FADD- sgRen cells serve as control. Immunoblots shown are representative of two independent experiments, asterisk (*) indicates non-specific band. b Flow cytometry analysis for TNFR1 surface expression in KBM7 FADD- SLC39A7- cells reconstituted with either V5-tagged SLC39A7 or GFP. KBM7 FADD- sgRen and empty KBM7 FADD- SLC39A7- cells serve as positive and negative control, respectively. Data shown are representative of two independent experiments. c Cell viability in KBM7 FADD- SLC39A7- cells reconstituted with either V5-tagged SLC39A7 or GFP, KBM7 FADD- sgRen and empty KBM7 FADD- SLC39A7- serve as controls. Cells were treated overnight (16 h) with TNFα (10 ng/ml), SMAC mimetic (0.5 µM), or a combination thereof. Cell viability was assessed using a luminescence-based readout for ATP (CellTiter Glo). Data represent mean value ± s.d. of two independent experiments performed in triplicates. d Cell viability in KBM7 FADD- SLC39A7- cells stably reconstituted with GFP or the indicated V5-tagged SLC39A7 constructs. Cells were treated as indicated for 24 h and cell viability was assessed as in c. Data represent mean value ± s.d. of two independent experiments performed in triplicates. e KBM7 FADD- SLC39A7- or KBM7 FADD- cells stably reconstituted with the specified constructs were lysed and subjected to immunoblotting with the indicated antibodies. Asterisk (*) indicates non-specific band
Fig. 6
Fig. 6
Genome-scale gene activation screens identify regulators of necroptosis. a, b Circos plots of genome-scale SAM screens in KBM7 FADD- SAM cells with necroptosis induction by TNFα (a) or SMAC mimetic birinapant (b) for 72 h. For each stimulus, screens were performed at low and high concentrations (TNFα: 10 ng/ml (green) or 100 ng/ml (purple); birinapant: 0.1 µM (blue) or 1 µM (orange)). Screen analysis was performed by identifying differentially enriched sgRNAs using DESeq2 and then aggregating sgRNAs to genes using Gene Set Enrichment Analysis. Identified hits are ranked according to the adjusted p-value of enrichment (–log10(padj). Bubble size corresponds to the average log2 fold-change (aLFC) of enrichment, color indicates the number of significantly enriched sgRNAs. Screens were performed in duplicate except the high concentration of birinapant in simplicate. c, d Cell viability in KBM7 FADD- SAM cells transduced with sgRNA targeting TNIP1, BIRC3 or Renilla luciferase (Ren). Cells were treated as indicated for 72 h and viability was assessed using a luminescence-based readout for ATP (CellTiter Glo). Data represent mean value ± s.d. of two independent experiments performed in triplicates. e KBM7 FADD- SAM cells transduced with the specified sgRNAs were lysed and subjected to immunoblotting with the indicated antibodies. Asterisk (*) indicates non-specific band
Fig. 7
Fig. 7
TNIP1 interferes with TNFα-induced necroptosis and RIPK1 activation in a ubiquitin binding–dependent manner. a, b Cell viability (a) and immunoblot (b) of KBM7 FADD- cells stably expressing the specified V5-tagged TNIP1 constructs or GFP. In a cells were treated as indicated for 24 h and viability was assessed using a luminescence-based readout for ATP (CellTiter Glo). Data represent mean value ± s.d. of two independent experiments performed in triplicates. (c-d) KBM7 FADD- cells stably expressing the specified TNIP1 constructs were stimulated for the time indicated with TNFα (100 ng/ml in c; 10 ng/ml in d). Cells were then lysed and subjected to immunoblotting with the indicated antibodies. Data shown are representative of at least two independent experiments
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