Figure 5.
TP53 loss impairs killing induced by an MCL-1 inhibitor. (A) Genome-wide recessive CRISPR screenings to identify genes critical for the action of MCL-1 inhibition. Three independent Cas9-expressing Eµ-Myc mouse lymphoma cell lines were transduced with a library of sgRNAs targeting the mouse genome. These infected cells were expanded and treated with the MCL-1 inhibitor S63845 at doses that killed >99% of cells. The remaining cells that survived were left to expand for 3 to 4 days before DNA was extracted, and targeted amplicon deep sequencing was performed on the MiSeq to identify sgRNAs enriched in cells resistant to MCL-1 inhibitor treatment. (B) Loss of Trp53 and Bax were the top hits for positive selection with the MCL-1i. Enrichment of sgRNAs targeting the indicated genes in 3 independently derived Eµ-Myc lymphoma cell lines treated with the MCL-1i S63845. Eµ-Myc lymphoma cell lines 1-3: AH15A, AF47A, and 560, respectively. (C) The growth of paired Trp53 WT or Trp53 KO Eµ-Myc 1, Eµ-Myc 2, or Eµ-Myc 3 lymphoma cell lines treated continuously with a suboptimal (IC30) dose (supplemental Table 4) of MCL-1i (or control treated) was monitored over 20 days by flow cytometric analysis. See Figure 2C for the experimental outline. (D) Growth competition assay experiments similar to those with mouse Eµ-Myc lymphoma cell lines (panel C) were undertaken with the indicated human cancer-derived cell lines. (E) NSG mice received transplants of an equal number of TP53 WT and TP53 KO MOLM-13 cells. Three days after transplantation, the mice were treated with vehicle or MCL-1i (25 mg/kg once weekly) for 2 weeks. The proportions of TP53 WT (GFP+) and TP53 KO (BFP+) human (CD45+) cells in the peripheral blood were enumerated by flow cytometry. ***P = 2.3 × 10−3 vehicle vs MCL-1i treatment of TP53 KO cells. Data are means ± SD of 5 animals per group from a representative experiment (n = 3 independent experiments). (F) Activation of BAX (detected by antibody 6A7) or BAK (detected by antibody G317-2) in Trp53 WT, Trp53 KO, or Noxa/Puma/Bim KO Eµ-Myc 2 cells were determined by flow cytometry 6 hours after treatment with 150 nM of MCL-1i. Data are representative of ≥3 independent experiments.

TP53 loss impairs killing induced by an MCL-1 inhibitor. (A) Genome-wide recessive CRISPR screenings to identify genes critical for the action of MCL-1 inhibition. Three independent Cas9-expressing Eµ-Myc mouse lymphoma cell lines were transduced with a library of sgRNAs targeting the mouse genome. These infected cells were expanded and treated with the MCL-1 inhibitor S63845 at doses that killed >99% of cells. The remaining cells that survived were left to expand for 3 to 4 days before DNA was extracted, and targeted amplicon deep sequencing was performed on the MiSeq to identify sgRNAs enriched in cells resistant to MCL-1 inhibitor treatment. (B) Loss of Trp53 and Bax were the top hits for positive selection with the MCL-1i. Enrichment of sgRNAs targeting the indicated genes in 3 independently derived Eµ-Myc lymphoma cell lines treated with the MCL-1i S63845. Eµ-Myc lymphoma cell lines 1-3: AH15A, AF47A, and 560, respectively. (C) The growth of paired Trp53 WT or Trp53 KO Eµ-Myc 1, Eµ-Myc 2, or Eµ-Myc 3 lymphoma cell lines treated continuously with a suboptimal (IC30) dose (supplemental Table 4) of MCL-1i (or control treated) was monitored over 20 days by flow cytometric analysis. See Figure 2C for the experimental outline. (D) Growth competition assay experiments similar to those with mouse Eµ-Myc lymphoma cell lines (panel C) were undertaken with the indicated human cancer-derived cell lines. (E) NSG mice received transplants of an equal number of TP53 WT and TP53 KO MOLM-13 cells. Three days after transplantation, the mice were treated with vehicle or MCL-1i (25 mg/kg once weekly) for 2 weeks. The proportions of TP53 WT (GFP+) and TP53 KO (BFP+) human (CD45+) cells in the peripheral blood were enumerated by flow cytometry. ***P = 2.3 × 10−3 vehicle vs MCL-1i treatment of TP53 KO cells. Data are means ± SD of 5 animals per group from a representative experiment (n = 3 independent experiments). (F) Activation of BAX (detected by antibody 6A7) or BAK (detected by antibody G317-2) in Trp53 WT, Trp53 KO, or Noxa/Puma/Bim KO Eµ-Myc 2 cells were determined by flow cytometry 6 hours after treatment with 150 nM of MCL-1i. Data are representative of ≥3 independent experiments.

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