Research ArticleCELL BIOLOGY

PBRM1 and the glycosylphosphatidylinositol biosynthetic pathway promote tumor killing mediated by MHC-unrestricted cytotoxic lymphocytes

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Science Advances  27 Nov 2020:
Vol. 6, no. 48, eabc3243
DOI: 10.1126/sciadv.abc3243
  • Fig. 1 Identification of positive regulators of TALL-104–mediated tumor killing in a genome-wide haploid genetic screen.

    (A) TALL-104 cells (CLs) kill HAP1 cells (tumor cells) but not normal cells. HAP1 cells and human PBMCs grown on a 24-well plate were treated with the indicated ratios of TALL-104 cells for 8 hours. The cells were stained with propidium iodide (PI) and measured by flow cytometry. Error bars indicate SD (n = 3). P values were calculated using Student’s t test. ***P < 0.001. n.s., P > 0.05. (B) Illustration of the genome-wide haploid genetic screen aiming to identify tumor-intrinsic genes required for TALL-104 cytotoxicity. (C) Bubble graph showing significant hits from the haploid genetic screen. The y axis depicts the −log10 of P values for the gene hits in the TALL-104selected population as compared to a published unselected control (62) using Fisher’s exact test. Dashed line indicates the cutoff of significance. We set a P value cutoff of 1 × 10−5 to account for multiple hypothesis testing. In addition, for genes with P values less than 1 × 10−10, we considered a gene as a hit only if it also had strong enrichment for sense-strand intron insertions based on the binomial test (P value cutoff: 1 × 10−5). The x axis depicts the chromosomal positions of the genes. The size of a circle is scaled according to the number of unique inactivating gene-trap insertions within the gene. Circles are colored according to the annotated or predicted functions of the gene products. Genes that did not reach the cutoff are shown in gray. Full datasets of the screen are included in table S1.

  • Fig. 2 Discovery of additional positive regulators of TALL-104–mediated killing in a modifier screen.

    (A) Flow cytometry measurements of the surface levels of CD59, a GPI-AP used as a marker for the GPI pathway (CD59 is not involved in CL-mediated killing). PIGP KO HAP1 cells were generated using CRISPR-Cas9 genome editing. A lack of surface CD59 in pooled PIGP KO cells indicates that the GPI pathway was abolished. WT, wild-type. (B) Screen hits from the modifier haploid genetic screen using the PIGP KO HAP1 cells. The screen was performed as described in Fig. 1. The y axis depicts the −log10 of P values for the gene hits in the TALL-104 selected population compared to the unselected control using Fisher’s exact test. We set a P value cutoff of 1 × 10−5 to account for multiple hypothesis testing. In addition, we considered a gene as a hit only if it also had strong enrichment for sense-strand intron insertions based on the binomial test (P value cutoff: 1 × 10−5). The x axis depicts the chromosomal positions of the genes. The size of the circle is scaled according to the number of unique inactivating gene-trap insertions with the gene. Circles are colored according to the annotated or predicted functions of the gene products. Dashed line indicates the cutoff of significance. Genes that did not reach the cutoff are shown in gray. Full datasets are included in table S2. (C) Lists of genes identified in the primary and modifier screens. Genes linked to cancer or CL killing are highlighted in bold. Cancer association is based on the COSMIC database.

  • Fig. 3 Identification of negative regulators of TALL-104–mediated tumor killing.

    (A) Visual representations of gene-trap insertions in CFLAR and MGA in the passage control (no TALL-104 selection), the primary screen, and the modifier screen. The gray boxes indicate exons, while the gray lines indicate introns. The gray arrows depict the directions of transcription. Chromosomal locations of inactivating, sense gene-trap insertions are shown in red, while chromosomal locations of neutral, antisense insertions are shown in blue. GSP is a new scoring metric based on the numbers of unique sense (inactivating) or antisense (neutral) gene-trap insertion within the introns of a candidate gene. GSP = [log2(S/A) × log10(S × A)], where “S” and “A” represent the numbers of sense and antisense gene-trap insertions, respectively. (B) Top: Color key of the heatmap. Bottom: Heatmap showing the genes with significantly negative GSP scores in both the primary and modifier screens but not in the passage control. The complete lists of genes are included in fig. S8 and tables S5 and S6. The GSP scores of the genes were quantile-normalized. Genes with significantly negative GSP scores in both screens were clustered using the Euclidean distance metric. Dashed gray lines represent the sample mean, while the solid gray lines represent each hit’s GSP score relative to the sample mean. The color of each bar represents the GSP score. Genes associated with cancer or CL killing are highlighted in bold. Cancer association is based on the COSMIC database. (C) Summary of the negative regulators identified in the primary and modifier screens based on annotated or predicted gene function. Full datasets are shown in tables S5 and S6.

  • Fig. 4 The GPI biosynthetic pathway is essential to CL activation and cytolytic granule secretion.

    (A) A mixed population of WT and PIGP KO HAP1 cells was either untreated or treated with TALL-104 cells. Negative surface staining of CD55, a GPI-AP not involved in CL-mediated killing, was used as a marker for PIGP KO cells. CD55+ WT cells and CD55 PIGP KO cells were analyzed by flow cytometry. (B) Percentage of WT and PIGP KO HAP1 cells in the passage control or TALL-104–treated population. (C) Percentage of WT and PIGP KO 786-O cells in the passage control or TALL-104–treated population. CD55+ WT cells and CD55 PIGP KO 786-O cells were quantified by flow cytometry after each round of treatment. Data in (B) and (C) are presented as mean values (n = 3). (D) Normalized surface levels of immune regulators on WT and PIGP KO HAP1 cells. (E) TALL-104 degranulation presented as normalized surface levels of LAMP1 on TALL-104 cells. (F) Normalized surface levels of TRAIL on TALL-104 cells. (G) Normalized surface levels of CD69 on TALL-104 cells. Data in (D) to (G) are presented as means ± SD (n = 3). P values were calculated using Student’s t test. n.s., P > 0.05. ***P < 0.001.

  • Fig. 5 PBRM1 regulates ULBP expression in target cells and promotes cytolytic granule secretion in CLs.

    (A) Visual representations of gene-trap insertions in the PBRM1 gene in the screens. The gray boxes indicate exons, and the gray lines indicate introns. The gray arrows depict the direction of transcription. (B) Immunoblot showing PBRM1 expression in WT HAP1 cells and a clonal PBRM1 KO HAP1 cell line. PBRM1 mRNA expression in these cells is shown in fig. S13. (C) WT (GFP+) and PBRM1 KO (GFP) cells in mixed populations were quantified by flow cytometry after three rounds of passage or TALL-104 treatment. (D) Percentage of WT and PBRM1 KO HAP1 cells in the passage control or TALL-104–treated population. Data are presented as mean values (n = 3). (E) Percentage of propidium iodide–positive WT and PBRM1 KO HAP1 cells after treatment with primary human NK cells. (F) Mean surface levels of immune regulators on WT and PBRM1 KO HAP1 cells. (G) TALL-104 degranulation presented as normalized surface levels of LAMP1 on TALL-104 cells after incubation with target cells. (H) NK-92 degranulation presented as normalized surface levels of LAMP1 in NK-92 cells. Data in (E) to (H) are presented as means ± SD (n = 3). P values were calculated using Student’s t test. n.s., P > 0.05. *P < 0.05, **P < 0.01, ***P < 0.001.

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