ITPK1 sensitizes tumor cells to IgA-dependent neutrophil killing in vivo [CRISPR Epistasis]
Neutrophils can efficiently trigger cytotoxicity towards tumor cells and other target cells upon engagement of the IgA receptor CD89. However, the cell-intrinsic factors that influence the induction of cell death upon exposure to neutrophil effector mechanisms in vivo remain largely unknown. To uncover genetic regulators that influence target cell sensitivity to IgA-induced neutrophil-mediated killing, we used a human CD89 (hCD89) transgenic mouse model in which IgA-mediated killing of Her2-positive CD47-deficient murine target cells is mediated by neutrophils. Using a genome-wide in vivo screening approach, we demonstrate that deletion of the gene encoding inositol-tetrakisphosphate 1 kinase, ITPK1, increases survival of target cells in anti-Her2 IgA-treated mice. Moreover, we show that this effect depends on neutrophil activity and on the ITPK1 kinase domain. Notably, ITPK1 deficiency did not measurably impact survival of IgA-opsonized target cells in in vitro systems, underscoring the importance of in vivo screening systems to uncover physiologically relevant regulators of neutrophil killing. Overall design: Epistasis_Screen: A set of 76 genes involved in cell death processes was assembled based on literature research, including gene-ontology terms related to apoptosis, necroptosis, ferroptosis, pyroptosis and TNF-mediated cell death. Furthermore, genes reported to be involved in cytokine-induced cell-death were included. An sgRNA library targeting these 76 genes (3 sgRNAs per gene) was ordered as an oligonucleotide pool (IDT DNA) and cloned into the lenti-Guide-Puro vector (Addgene #52963). SgRNA sequences were picked using CRISPick (Broad Institute GPP Webportal). In order to generate sensBa/F3 cells deficient of ITPK1, sensBa/F3 cells were electroporated (Lonza #V4XP-3024) (Program E0-117) with sgRNA targeting ITPK1 (in lenti-Guide-Puro, Addgene #52963) and selected for 48h in 10?g/mL puromycin. Next single cell clones were screened for ITPK1 inactivation by DNA sequencing and western blot analysis, and for lentiviral integration. Six clones with ITPK1 deletions, but without lentiviral integration were pooled. Subsequently, both sensBa/F3 cells and sensBa/F3 ITPK1 KO cells were transduced with the sgRNA library targeting the 76 cell death-related genes. Next, the resulting sensBa/F3 and sensBa/F3 ITPK1 KO cell death libraries were cultured for 10 days to remove cells containing knockouts of essential genes, and sgRNA distributions were assessed using Illumina MiSeq sequencing. The expanded sensBa/F3 cell death libraries were subsequently stained with CellTrace (CT) violet or yellow (ThermoFisher #C34554), respectively and pooled at a 1:1 ratio. Thereafter, the stained cell pool was intraperitoneally injected into hCD89-transgenic mice (n=8, 20M cells each) that were subsequently treated with anti-hHer2 IgA or PBS (n=4 each). After 16h, mice were sacrificed and cells obtained by intraperitoneal lavage, and stained with Near-IR Dead Cell Staining Kit (ThermoFisher #L10119). Subsequently, viable tumor cells obtained from each mouse were sorted into CT violet- and yellow-positive populations. Next, genomic DNA of each sample was obtained using DirectPCR Lysis reagent (Viagen Biotech #301-C) according to the manufacturer's instructions. SgRNA sequences were subsequently amplified using NebNext High-Fidelity 2X PCR Master Mix (New England Biolabs #M0541S), following the manufacturer's instructions, using primers containing sample barcodes. After PCR, prepared libraries were pooled at equimolar amounts and sgRNA library distributions were analyzed by Illumina MiSeq sequencing. In brief, sequence reads were aligned to a reference library, reads containing mismatches removed, and sgRNA distributions quantified and scored using MAGeCK RRA (version 0.5.6) (27). Downstream analysis focused on comparison of the positive log2-fold changes of sgRNAs between treated and untreated mice in either ITPK1-proficient or -deficient backgrounds.