ITPK1 Sensitizes Tumor Cells to IgA-dependent Neutrophil Killing In Vivo

Abstract

Neutrophils can efficiently trigger cytotoxicity toward 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.

FIGURE 1.

Characterization of in vivo model. (A) Schematic overview of experimental approach. CD47^+/+ hHer2⁺ Ba/F3 tumor cells (CFP⁺ or CFP⁺Kat⁺) were mixed with CD47^−/− hHer2⁺ Ba/F3 tumor cells (GFP⁺) in a 1:500 or 1:20,000 ratio, and 10⁷ cells were injected into hCD89^+/− transgenic mice or hCD89^−/− littermates. The hCD89^+/− and hCD89^−/− mice were then treated with anti-hHer2 IgA and PBS, respectively (first selection round). After 16 h, mice were sacrificed, and tumor cells and immune cells were quantified using flow cytometry. Purified tumor cells isolated from anti-hHer2 IgA-treated mice were expanded for 4 d in vitro, and 2 × 10⁷ cells were injected into hCD89^−/+ transgenic mice and hCD89^−/− littermates, which were treated with anti-hHer2 IgA or PBS, respectively (second selection round). After 16 h, mice were sacrificed, and tumor cells were quantified using flow cytometry. (B) Ratio of neutrophils (Ly-6G⁺) to macrophages (F4/80⁺) recovered in the i.p. lavage of hCD89^+/− transgenic mice and hCD89^−/− littermates treated with anti-hHer2 IgA or PBS, respectively. Dots represent individual mice. ***p = 0.0001, unpaired, two-tailed t test. (C) Number of target cells recovered from mice treated with anti-hHer2 IgA or PBS after the first in vivo selection round. ****p < 0.0001, unpaired, two-tailed t test. (D) Fraction of CD47^+/+ CFP⁺ Kat^−/− target cells (initially present at a 1:500 ratio) among all target cells recovered after the first in vivo selection round. Dots represent individual mice. *p = 0.0257, unpaired, two-tailed t test. (E) Number of target cells recovered from mice treated with anti-hHer2 IgA or PBS after the second in vivo selection round. Dots represent individual mice. ****p < 0.0001, unpaired, two-tailed t test. (F) Fraction of CD47^+/+ CFP⁺ Kat^−/− target cells (initially present at a 1:500 ratio) among all target cells recovered after the second in vivo selection round. Dots represent individual mice. **p = 0.0015, unpaired, two-tailed t test. (G) Fraction of CD47^+/+ CFP⁺ Kat⁺ target cells (initially present at a 1:20,000 ratio) among all target cells recovered after the second in vivo selection round. Dots represent individual mice. **p = 0.002, unpaired, two-tailed t test.

FIGURE 2.

Genome-wide in vivo CRISPR Cas9 screen reveals ITPK1 as a regulator of cancer cell sensitivity to IgA-mediated killing. (A) Schematic overview of pooled in vivo genome-wide CRISPR Cas9 screen (1, primary screen) and pooled in vivo focused CRISPR Cas9 screen (2, secondary screen). SensBa/F3 cells were transduced with a genome-wide (1, primary screen) or focused (2, secondary screen) sgRNA library to generate KO libraries. The resulting cell libraries were subsequently injected into hCD89^+/− transgenic mice or hCD89^−/− littermates at 2 × 10⁷ cells per mouse and treated with anti-hHer2 IgA or PBS, respectively. After 16 h, mice were sacrificed, genomic DNA (gDNA) was isolated from purified tumor cells, and sgRNA distributions were analyzed by deep sequencing. On the basis of observed sgRNA distributions, enrichment and depletion scores were generated using MAGeCK RRA (version 0.5.9.2). (B) Number of target cells isolated from mice treated with anti-hHer2 IgA or PBS in the genome-wide screen. Dots represent individual mice. ****p < 0.0001, unpaired, two-tailed t test. (C) Positive and negative MAGeCK RRA scores for each enriched and depleted gene from mice selected in primary screen. Cutoff for enrichment or depletion set at median positive RRA score. Genes contained in the focused screening library are marked in red and labeled. (D) Number of target cells isolated from mice treated with anti-hHer2 IgA or PBS in the focused screen. Dots represent individual mice. ****p < 0.0001, unpaired, two-tailed t test. (E) Ratio of average normalized sgRNA abundance (read counts) in tumor cells derived from anti-hHer2 IgA-treated mice versus PBS-treated mice in the focused screen. sgRNAs targeting ITPK1 are highlighted in red. sgRNAs targeting FERMT3 are highlighted in blue. Nontargeting control sgRNAs are depicted in gray. (F) Representation of the normalized sgRNA abundance of the two sgRNAs targeting ITPK1 in tumor cells recovered from mice treated with anti-hHer2 IgA or PBS in the focused screen. Dots represent individual mice or pooled samples as indicated in the Materials and Methods section.

FIGURE 3.

ITPK1 deletion reduces cancer cell sensitivity to IgA-mediated killing in vivo but not in vitro. (A) Ratio of sensBa/F3 tumor cells expressing ITPK1-targeting sgRNA (clone 2) over control cells expressing nontargeting sgRNA (NT, clone 2) recovered from hCD89^+/− transgenic mice or hCD89^−/− littermates after a 16-h treatment with anti-hHer2 IgA or PBS, respectively. Prior to treatment, mice were injected with an equal mixture of sensBa/F3 tumor cells with the previously indicated genotypes. Dots represent individual mice. ***p = 0.0001, unpaired, two-tailed t test. (B) Relative abundance of neutrophils (CD11b⁺Ly6G⁺) and macrophages (CD11b⁺F4/80⁺) in the i.p. lavage of hCD89^+/− transgenic mice treated for 16 h with anti-hHer2 IgA or hCD89^−/− littermates treated for 16 h with either anti-hHer2 IgA or PBS. Dots represent individual mice. Prior to treatment, mice were injected with an equal mixture of sensBa/F3 tumor cells expressing either ITPK1-targeting or nontargeting sgRNA. ****p < 0.0001, **p = 0.007, no indication = nonsignificant, one-way ANOVA. (C) Ratio of sensBa/F3 tumor cells expressing ITPK1-targeting sgRNA (clone 2) over control cells expressing nontargeting sgRNA (NT, clone 2) recovered from hCD89^+/− transgenic mice or hCD89^−/− littermates after a 6-h treatment with anti-hHer2 IgA or PBS, respectively. Prior to treatment, mice were injected with an equal mixture of sensBa/F3 tumor cells with the previously indicated genotypes. Dots represent individual mice. ****p < 0.0001, unpaired, two-tailed t test. (D) Relative abundance of neutrophils (CD11b⁺Ly6G⁺) and macrophages (CD11b⁺F4/80⁺) recovered from the i.p. lavage of hCD89^+/− transgenic mice or hCD89^−/− littermates after a 6-h treatment with anti-hHer2 IgA or PBS, respectively. Prior to treatment, mice were injected with an equal mixture of sensBa/F3 tumor cells expressing either ITPK1-targeting or nontargeting sgRNA. Dots represent individual mice. ****p < 0.0001, **p = 0.0005, no indication = nonsignificant, two-tailed t test. (E) Ratio of sensBa/F3 tumor cells expressing ITPK1-targeting sgRNA (clones 2 and 3), or sensBa/F3 tumor cells expressing both ITPK1-targeting sgRNA (clone 2) and ITPK1 OE, over sensBa/F3 tumor cells expressing nontargeting sgRNA (NT, clone 2) recovered from hCD89^+/− transgenic mice or hCD89^−/− littermates after a 16-h treatment with anti-hHer2 IgA or PBS, respectively. Prior to treatment, mice were injected with an equal mixture of sensBa/F3 tumor cells with the previously indicated genotypes. ****p < 0.0001, ***p = 0.0002, no indication = nonsignificant, one-way ANOVA. (F) Ratio of sensBa/F3 tumor cells expressing ITPK1-targeting sgRNA (clone 2) over sensBa/F3 tumor cells expressing nontargeting sgRNA (NT, clone 2) recovered from hCD89^+/− transgenic mice after 16-h treatment with anti-hHer2 IgA, anti-hHer2 IgG1, mIgG2a isotype, or PBS, respectively. Prior to treatment, mice were injected with an equal mixture of sensBa/F3 tumor cells with the previously indicated genotypes. Dots represent individual mice. ****p < 0.0001, no indication = nonsignificant, one-way ANOVA. (G) Ratio of CD47-reconstituted sensBa/F3 tumor cells expressing ITPK1-targeting sgRNA (clone 2) over CD47-reconstituted sensBa/F3 tumor cells expressing nontargeting sgRNA (NT, clone 2) recovered from hCD89^+/− transgenic mice after a 16-h treatment with anti-hHer2 IgA, anti-hHer2 IgG1, mIgG2a isotype, or PBS, respectively. Prior to treatment, mice were injected with an equal mixture of sensBa/F3 tumor cells with the previously indicated genotypes. Dots represent individual mice. ****p < 0.0001, no indication = nonsignificant, one-way ANOVA. (H) Sensitivity of WT (Ag-negative) Ba/F3 cells and sensBa/F3 cells expressing either nontargeting sgRNA (NT clone 2) or ITPK1-targeting sgRNA (clone 2), treated with the indicated concentrations of anti-hHer2 IgA, to bone marrow–derived mouse neutrophils in vitro at a 40:1 E:T ratio, as assessed by ⁵¹Cr-release assay. Dots represent mean of three technical replicates. *p = 0.0029, two-way ANOVA. (I) Sensitivity of hHer2⁺ Cas9⁺ K562 cells expressing either nontargeting sgRNA or ITPK1-targeting sgRNA, treated with the indicated concentrations of anti-hHer2 IgA, to peripheral blood–derived human neutrophils (two separate donors) in vitro at a 40:1 E:T ratio, as assessed by ⁵¹Cr-release assay. Dots represent mean of three technical replicates. *p = 0.0136, 0.0333, two-way ANOVA.

FIGURE 4.

ITPK1-linked cancer cell sensitivity requires an intact ATP-binding grasp. (A) Schematic overview of experimental setup. A total of 2.5 × 10⁷ WT (Ag-negative) Ba/F3 cells, sensBa/F3 cells expressing either nontargeting sgRNA (NT clone 2) or ITPK1-targeting sgRNA (clone 2), and sensBa/F3 cells expressing ITPK1-targeting sgRNA (clone 2) reconstituted with WT ITPK1 or with kinase-dead (D295A) ITPK1 were labeled and injected as mixtures into hCD89^+/− transgenic mice or hCD89^−/− littermates at an equal ratio. The hCD89^+/− transgenic mice and hCD89^−/− littermates were subsequently treated with anti-hHer2 IgA and PBS (control treatment), respectively. After 16 h, mice were sacrificed, and relative abundance of each target cell line was analyzed using flow cytometry. (B) Ratio of the aforementioned target cell lines over sensBa/F3 cells expressing nontargeting sgRNA in anti-hHer2 IgA and PBS-treated mice, respectively. Dots represent individual mice. ****p < 0.0001, ***p = 0.0005, no indication = nonsignificant, one-way ANOVA.

FIGURE 5.

ITPK1-linked cancer cell sensitivity depends on neutrophils. (A) Schematic overview of experimental setup. A total of 2.5 × 10⁷ WT (Ag-negative) Ba/F3 cells, sensBa/F3 cells expressing either nontargeting sgRNA (NT clone 2) or ITPK1-targeting sgRNA (clone 2), and sensBa/F3 cells expressing ITPK1-targeting sgRNA (clone 2) reconstituted with WT ITPK1 or with kinase-dead (D295A) ITPK1 were labeled and injected as equal mixtures into hCD89^+/− transgenic mice, hCD89^+/− transgenic mice pretreated with anti-Ly-6G (neutrophil depletion), hCD89^+/− transgenic mice pretreated with chlodronate (macrophage depletion), or hCD89^−/− littermates. Directly after tumor cell injection, hCD89^+/− transgenic mice and hCD89^−/− littermates were treated with anti-hHer2 IgA or PBS (control treatment), respectively. After 16 h, mice were sacrificed, and the relative abundance of each target cell line was analyzed using flow cytometry. (B) Ratio of sensBa/F3 cells expressing nontargeting sgRNA over WT Ba/F3 cells in each treatment group. Dots represent individual values. ****p < 0.0001, *p = 0.0177, no indication = nonsignificant, one-way ANOVA. (C) Ratio of cell populations described in (A) over sensBa/F3 cells expressing nontargeting sgRNA in PBS-treated mice without immune cell depletion (equivalent to Fig. 4B) or in anti-hHer2 IgA-treated mice depleted of neutrophils. Dots represent individual mice. ****p < 0.0001, ***p = 0.0005, one-way ANOVA. (D) Ratio of cell populations described in (A) over sensBa/F3 cells expressing nontargeting sgRNA in macrophage-depleted mice treated with PBS or with anti-hHer2 IgA. Dots represent individual mice. ****p < 0.0001, ***p = 0.0005, *p = 0.0129 (sg ITPK1 versus sg ITPK1 and ITPK1 OE), 0.0481 (sg ITPK1 and ITPK1 OE versus sg ITPK1 and ITPK1^D295A OE), one-way ANOVA.