ITPK1 sensitizes tumor cells to IgA-dependent neutrophil killing in vivo [CRISPR focused]

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: For the focused screen, sensBa/F3 cells were lentivirally transduced with the focused sgRNA library in the lenti-Guide-Puro vector (Addgene #52963) at a transduction rate of approximately 50% and a coverage of >2000 fold. The focused library targets 35 genes with 2 sgRNAs each, and with target genes selected based on observed enrichment scores in a primary screen (and on RNA-expression levels. In addition, high scoring genes encoding certain transcription factors (e.g. Rhox11 and Gtf2e2) were excluded, and a number of presumed interactors of top-scoring genes (i.e. Kctd10, Cul3, and Wipf2 with Itsn2) were included. Following transduction, the cells were cultured for >7 days in the presence of 5ug/ml puromycin. Before injection into mice, a library reference sample was taken. Next, hCD89-transgenic mice and littermate controls were injected intraperitoneally (i.p.) with 2x107 cells in 200uL PBS. Directly after target cell injection, mice received PBS (control treatment) or 100ug anti-hHer2 IgA1 antibody in PBS by intraperitoneal injection. 16h after injection, mice were euthanized and the peritoneal cavity was washed with PBS containing 5 mM EDTA to obtain target cells and murine immune cell infiltrates. To estimate target cell killing efficiency by flow cytometry, a small aliquot was taken from each sample and pooled per treatment group (PBS or anti-hHer2 IgA1). Next, cells were stained with APC-conjugated anti-hHer2 and anti-hCD19 antibodies and target cells were positively selected for by MACS using anti-APC magnetic beads (MiltenyiBiotec #130-090-855), according to the manufacturer's protocol. Thereafter, DNA was isolated from enriched target cells using the DNeasy Blood & Tissue Kit (QIAGEN #69504). In the focused screen, 2 of the DNA samples derived from the treated group were pooled. SgRNA sequences in the pooled or unpooled sample groups were amplified using NEBNext High-Fidelity 2X PCR Master Mix (New England Biolabs #M0541S), following the manufacturer's instructions and using primers containing sample barcodes. After PCR, prepared libraries were pooled at equimolar amounts and sgRNA distributions were analyzed by deep sequencing using the Illumina HiSeq2500 platform. In brief, sequence reads were aligned to the Brie mouse library, reads containing mismatches were removed, sgRNA distributions were quantified, and genes were scored using MAGeCK RRA (version 0.5.6) (27).