Research ArticleCancer

Off-target toxicity is a common mechanism of action of cancer drugs undergoing clinical trials

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Science Translational Medicine  11 Sep 2019:
Vol. 11, Issue 509, eaaw8412
DOI: 10.1126/scitranslmed.aaw8412
  • Fig. 1 Cell competition assays to test the essentiality of putative cancer dependencies.

    (A) Schematic of the CRISPR-based cell competition assays used in this paper (18). (B) Cell competition assays comparing guides targeting AAVS1 and ROSA26 (nonessential, negative control genes), RPA3 and PCNA (pan-essential positive control proteins), and Aurora A, Aurora B, and ERCC3 (inhibitor-validated cancer dependencies). Full results from these competition experiments are included in data file S2. (C) Cell competition assays for the cell type–specific cancer dependencies BRAF and ESR1. (D) Western blot analysis of A375 populations transduced with the indicated gRNAs. (E) Cell competition assays with gRNAs targeting HDAC6, MAPK14, PAK4, PBK, or PIM1 in four different cancer cell lines.

  • Fig. 2 Generating and analyzing single cell–derived KO clones of putative cancer dependencies.

    (A) Schematic of the two-guide strategy used to generate clonal KO cell lines. (B) Western blot analysis of single cell–derived A375 KO clones. ab, antibody. (C) Proliferation assays for HDAC6, MAPK14, PAK4, PBK, and PIM1 KO clones. (D) Representative images of A375 and DLD1 Rosa26 or MAPK14-KO clones grown in soft agar. Scale bar, 2 mm. (E) Quantification of colony formation in control or KO A375, DLD1, and HCT116 clones. Boxes represent the 25th, 50th, and 75th percentiles of colonies per field, and the whiskers represent the 10th and 90th percentiles. For each assay, colonies were counted in at least 15 fields under a 10× objective.

  • Fig. 3 Target-independent cell killing by multiple anticancer drugs.

    (A) Western blot analysis for caspase-3 in A375 and HCT116 cells. (B) Seven-point dose-response curves of Rosa26 and CASP3-KO A375 and HCT116 cells in the presence of two putative caspase-3 activators: 1541B and PAC-1. (C) Seven-point dose-response curves of Rosa26 and HDAC6-KO A375 and DLD1 cells in the presence of two putative HDAC6 inhibitors: ricolinostat and citarinostat. (D) Seven-point dose-response curves of Rosa26 and MAPK14-KO A375 and DLD1 cells in the presence of two putative MAPK14 inhibitors: ralimetinib and SCIO-469. (E) Seven-point dose-response curves of Rosa26 and PBK-KO A375 and DLD1 cells in the presence of two putative PBK inhibitors: OTS514 and OTS964. (F) Seven-point dose-response curves of Rosa26 and PIM1-KO A375 and DLD1 cells in the presence of a putative PIM1 inhibitor: SGI-1776. (G) Seven-point dose-response curves of Rosa26 and PAK4-KO A375 and HCT116 cells in the presence of a putative PAK4 inhibitor: PF-3758309.

  • Fig. 4 Discovery of CDK11 as the in cellulo target of the mischaracterized anticancer drug OTS964.

    (A) A schematic of the strategy to use the highly mutagenic HCT116 cell line to isolate mutations that confer OTS964 resistance. (B) Sanger sequencing validation of two heterozygous mutations in the CDK11B kinase domain. (C) Constructs used to introduce the G579S mutation into CDK11B via CRISPR-mediated HDR. Yellow arrowhead indicates the site of Cas9 cleavage. Red bar indicates the G579S substitution, and blue bars indicate silent mutations introduced to prevent recutting after HDR. (D) Crystal violet staining of cancer cells transfected with the indicated constructs and then cultured in a lethal concentration of OTS964. (E) Seven-point dose-response curves of Rosa26, PBK-KO, and CDK11BG579S clones grown in varying concentration of OTS964. (F) Titration experiments reveal that OTS964 binds to CDK11B with a KD of 40 nM. (G) Pancreatic cancer cell line MiaPaca-2 was transduced with guides specific for CDK11A, guides specific for CDK11B, or guides that harbored cut sites in both genes. (H) A375 H2B-mCherry cells (left) or A375 H2B-mCherry cells that express CDK11BG579S (right) were arrested at G1/S with a double-thymidine block and then were released into normal medium or medium containing OTS964. The percentage of mitotic cells in each population was scored every hour. (I) Representative images of the experiments in (H), 9 hours after release from thymidine. Scale bar, 25 μm.

  • Table 1 Anticancer drugs and drug targets.

    TargetDrugNo. of cancer clinical trials
    CASP31541BPreclinical
    PAC-13
    HDAC6Citarinostat5
    Ricolinostat10
    MAPK14 (p38α)Ralimetinib5
    SCIO-4693
    PAK4PF-037583091
    PBK (TOPK)OTS514Preclinical
    OTS964Preclinical
    PIM1SGI-17762

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/509/eaaw8412/DC1

    Materials and Methods

    Fig. S1. Drug target ablation with CRISPR-Cas9.

    Fig. S2. Cell competition assays in multiple cancer types.

    Fig. S3. Verification of drug target-KOs.

    Fig. S4. Knocking out the verified genetic dependency MEK1 in A375 melanoma cells.

    Fig. S5. MDA-MB-231 clonal analysis.

    Fig. S6. Analysis of homolog gene expression in CRISPR-KO clones.

    Fig. S7. Analysis of homolog gene expression in published RNA-seq experiments.

    Fig. S8. Assessing putative cancer dependencies in whole-genome CRISPR and RNAi screens.

    Fig. S9. Targeting several putative cancer dependencies with CRISPRi.

    Fig. S10. Lack of sensitivity to several clinical chemotherapy agents in putative cancer dependency KOs.

    Fig. S11. Target-independent toxicity of RNAi reagents previously used to investigate several putative cancer dependencies.

    Fig. S12. Using CRISPR to validate the MOA of several anticancer drugs.

    Fig. S13. Off-target toxicity of two caspase-3–activating compounds in CASP3-KO clones.

    Fig. S14. Target-independent cancer cell killing in single-agent and combination therapy experiments.

    Fig. S15. Off-target toxicity of two putative HDAC6-inhibiting compounds in HDAC6-KO ovarian cancer clones.

    Fig. S16. A mutation in the xDFG residue of CDK11B in OTS964-resistant clones.

    Fig. S17. Requirement for CDK11 activity for progression through mitosis.

    Data file S1. Literature supporting the designation of HDAC6, MAPK14, PAK4, PBK, and PIM1 as cancer genetic dependencies and CASP3 as a drug target.

    Data file S2. Cell competition assay results.

    Data file S3. Sources of the cell lines used in this manuscript.

    Data file S4. CRISPR gRNA sequences.

    Data file S5. CRISPRi gRNA sequences.

    Data file S6. Quantitative polymerase chain reaction primers.

    Data file S7. Antibody sources and concentrations.

    Data file S8. Drugs and drug sources.

    Movie S1. A375 cells expressing H2B-mCherry released from a double-thymidine block into normal medium.

    Movie S2. A375 cells expressing H2B-mCherry released from a double-thymidine block into medium with 25 nM OTS964.

    Movie S3. A375 cells expressing H2B-mCherry released from a double-thymidine block into medium with 100 nM OTS964.

    Movie S4. A375CDK11B-G579S cells expressing H2B-mCherry released from a double-thymidine block into medium with 100 nM OTS964.

    References (70257)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Drug target ablation with CRISPR-Cas9.
    • Fig. S2. Cell competition assays in multiple cancer types.
    • Fig. S3. Verification of drug target-KOs.
    • Fig. S4. Knocking out the verified genetic dependency MEK1 in A375 melanoma cells.
    • Fig. S5. MDA-MB-231 clonal analysis.
    • Fig. S6. Analysis of homolog gene expression in CRISPR-KO clones.
    • Fig. S7. Analysis of homolog gene expression in published RNA-seq experiments.
    • Fig. S8. Assessing putative cancer dependencies in whole-genome CRISPR and RNAi screens.
    • Fig. S9. Targeting several putative cancer dependencies with CRISPRi.
    • Fig. S10. Lack of sensitivity to several clinical chemotherapy agents in putative cancer dependency KOs.
    • Fig. S11. Target-independent toxicity of RNAi reagents previously used to investigate several putative cancer dependencies.
    • Fig. S12. Using CRISPR to validate the MOA of several anticancer drugs.
    • Fig. S13. Off-target toxicity of two caspase-3–activating compounds in CASP3-KO clones.
    • Fig. S14. Target-independent cancer cell killing in single-agent and combination therapy experiments.
    • Fig. S15. Off-target toxicity of two putative HDAC6-inhibiting compounds in HDAC6-KO ovarian cancer clones.
    • Fig. S16. A mutation in the xDFG residue of CDK11B in OTS964-resistant clones.
    • Fig. S17. Requirement for CDK11 activity for progression through mitosis.
    • References (70257).

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Literature supporting the designation of HDAC6, MAPK14, PAK4, PBK, and PIM1 as cancer genetic dependencies and CASP3 as a drug target.
    • Data file S2 (Microsoft Excel format). Cell competition assay results.
    • Data file S3 (Microsoft Excel format). Sources of the cell lines used in this manuscript.
    • Data file S4 (Microsoft Excel format). CRISPR gRNA sequences.
    • Data file S5 (Microsoft Excel format). CRISPRi gRNA sequences.
    • Data file S6 (Microsoft Excel format). Quantitative polymerase chain reaction primers.
    • Data file S7 (Microsoft Excel format). Antibody sources and concentrations.
    • Data file S8 (Microsoft Excel format). Drugs and drug sources.
    • Movie S1 (.mov format). A375 cells expressing H2B-mCherry released from a double-thymidine block into normal medium.
    • Movie S2 (.mov format). A375 cells expressing H2B-mCherry released from a double-thymidine block into medium with 25 nM OTS964.
    • Movie S3 (.mov format). A375 cells expressing H2B-mCherry released from a double-thymidine block into medium with 100 nM OTS964.
    • Movie S4 (.mov format). A375CDK11B-G579S cells expressing H2B-mCherry released from a double-thymidine block into medium with 100 nM OTS964.

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