Research ArticleCancer

Application of a MYC degradation screen identifies sensitivity to CDK9 inhibitors in KRAS-mutant pancreatic cancer

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Science Signaling  16 Jul 2019:
Vol. 12, Issue 590, eaav7259
DOI: 10.1126/scisignal.aav7259
  • Fig. 1 Validation of a MYC degradation reporter.

    (A) Overview of the GPS-MYC vector. LTR, long terminal repeat. (B) Confocal images of GPS-MYC cells to determine EGFP-MYC subcellular localization to the nucleus, which was visualized by DAPI staining. Scale bars, 20 μm. (C) GPS-MYC cells were treated with CHX for the indicated times, and EGFP-MYC and MYC abundance was measured by immunoblotting (left). The half-lives of EGFP-MYC and endogenous MYC were calculated by fitting the data to a one-phase decay curve. *P < 0.05. (D) GPS-MYC cells were treated with MG132 and CHX for 6 hours, and EGFP and DsRed abundance was measured by flow cytometry. All data are representative of at least three independent experiments.

  • Fig. 2 Optimization of a MYC degradation screen.

    (A) Schematic of the GPS-MYC screen. (B) GPS-MYC cells were treated with vehicle alone (DMSO) or with MG132 or CHX for 6 hours and analyzed on an IntelliCyt iQue Screener. The data presented are from the first two and last two wells of each control, representing the beginning (0 min) and the end (45 min) of the assay. Data are from one plate and are representative of at least three independent experiments. (C) The GPS-MYC screen was run in duplicate. Data were normalized to control DMSO (blue circles, 0% stabilization) and MG132 (green circles, 100% stabilization), and hits were determined by a cutoff of a 30% average stabilization of the two replicates. The circles representing the hits are shown in purple. The circle size is proportional to the number of events per well. Stabilizing compounds that were evaluated in the paper are labeled in the graph. (D) As described in (C), except the data were normalized to control DMSO (blue circles, 0% destabilization) and CHX (red circles, 100% destabilization). Destabilizing compounds evaluated in the paper are indicated.

  • Fig. 3 MYC degradation screen identifies a compound that stabilizes MYC protein.

    (A) GPS-MYC cells were treated with 20 μM UNC10112731 for 6 hours, and EGFP and DsRed intensities were measured by flow cytometry. Data are from the GPS-MYC screen. (B and C) GPS-MYC cells were treated with increasing concentrations of UNC10112731 for 6 hours and EGFP and DsRed intensities were measured by flow cytometry (B) or immunoblotting (C). (D) KRAS-mutant PDAC cell lines were treated for 6 hours with UNC10112731, and MYC protein abundance was measured by immunoblotting (top), with quantitation by densitometry relative to vehicle control (bottom). *P < 0.05, **P < 0.01, ***P < 0.001 by t test. (E) MIA PaCa-2 cells were treated for 6 hours with UNC10112731, and MYC mRNA expression was measured by quantitative PCR (qPCR). MYC mRNA expression was normalized to that of GAPDH mRNA. a.u., arbitrary units. (F) Kinase selectivity of UNC10112731 as described previously (30). (G) MIA PaCa-2 cells were treated for 6 hours with the indicated compounds, and MYC protein abundance was measured by immunoblot. All data are representative of at least three independent experiments. Data in (D) are presented as means ± SD.

  • Fig. 4 UNC10112785 drives MYC protein loss.

    (A) Chemical structure of UNC10112785. The red circle indicates the position at which different analogs were synthesized. (B) GPS-MYC cells were treated with 20 μM UNC10112785 for 6 hours, and EGFP and DsRed intensity was measured by flow cytometry. Data from the GPS-MYC screen. (C) GPS-MYC cells were treated with UNC10112785 for 6 hours, and EGFP-MYC and MYC abundance was measured by immunoblotting. (D) PDAC cells were treated with UNC10112785, and MYC protein abundance was measured by immunoblot. (E) Kinase selectivity of UNC10112785 as described previously (30). (F) MIA PaCa-2 cells were treated for 6 hours with the indicated compounds, and MYC protein abundance was measured by immunoblot. All data are representative of at least three independent experiments.

  • Fig. 5 Inhibition of CDK9 by UNC10112785 drives MYC loss.

    (A) MIA PaCa-2 cells were treated for 1 hour with different concentrations UNC10112785, after which they were lysed and applied to the MIBs column. Kinases were eluted from the column and identified and quantified by LC/MS as described in Materials and Methods. Bars to the left of center line indicate kinases reduced after compound addition. Data are from one experiment, which served to identify hits to validate with reproducibility and explore further. (B) MIA PaCa-2 cells were treated with UNC10112785, and CDK8/19 inhibition was measured by the abundance of phosphorylated STAT1 (pSTAT1). (C and D) MIA PaCa-2 cells were treated with the indicated compounds for 6 hours, and CDK8/19 or CDK9 inhibition was measured by pSTAT1 (C) and pPol II (D). (E) MIA PaCa-2 cells were treated for 72 hours with the indicated compounds, and cell proliferation was measured by cell count, normalized to that cells treated with the vehicle control (DMSO). 2D, two-dimensional. (F) MIA PaCa-2 cells suspended in 3% agarose were treated for 72 hours with the indicated compounds, and cell proliferation was measured by alamarBlue, normalized to cells treated with vehicle (DMSO). All data are representative of at least three independent experiments unless otherwise indicated. Data in (E) and (F) are presented as means ± SD.

  • Fig. 6 CDK9 regulates MYC protein stability through phosphorylation of Ser62.

    (A) MIA PaCa-2 cells were treated with UNC5668. MYC mRNA expression was measured by qPCR and normalized to that of BACT mRNA. (B) MIA PaCa-2 cells were treated with UNC5668 in the presence or absence of MG132 for the indicated concentrations and duration, and MYC protein abundance was measured by immunoblot. (C) Recombinant CDK9 and MYC were incubated in the presence of ATP. After separation by SDS-PAGE, the band corresponding to MYC was excised and analyzed by LC/MS. MYC protein sequence shown in red are phosphorylated peptides, and red circles indicate phosphorylated residues. The first 182 amino acids of MYC are shown. Data are from one experiment as a basis for further validation performed subsequently here. (D) Tandem MS/MS spectrum for MYC peptide containing Ser62. (E) MIA PaCa-2 cells were treated for 30 min with the indicated compounds and pMYC Ser62 and total MYC abundance was measured by immunoblot. (F) MIA PaCa-2 cells stably expressing MYC proteins were treated with the indicated compounds for 2 hours and MYC abundance was measured by immunoblot. Blots are representative of five independent experiments. Other data are representative of at least three independent experiments unless otherwise indicated. Data in (A) are presented as means ± SD. WT, wild type; EV, empty vector.

  • Fig. 7 CDK9 and MEK5-ERK5 comprise distinct signaling mechanisms that regulate MYC Ser62 phosphorylation and stability.

    (A) MIA PaCa-2 cells were treated for 6 hours with XMD8-92 (ERK5i) and UNC5668 (CDK9i), and ERK5 and CDK9 signaling was measured by immunoblotting for the indicated proteins. (B) MIA PaCa-2 cells were treated with KRAS siRNA for 24 hours and CDK9 signaling was measured by immunoblotting for the indicated proteins. (C) HPNE cells were treated for 6 hours with the indicated concentrations of UNC10112785, and MYC abundance was measured by immunoblotting for the indicated proteins. Data are representative of at least three independent experiments.

  • Table 1 Biochemical profiling of UNC10112785.

    The activity of UNC10112785 was tested against a panel of kinases that had previously been identified in either the published PKIS data or the MIB-MS assay using SelectScreen Kinase Profiling (Thermo Fisher Scientific).

    KinaseAssay typeIC50 (nM)% Max
    CDK19/cyclin CLanthaScreen binding1.05100
    CDK8/cyclin CLanthaScreen
    binding
    2.67104
    CDK9 (inactive)LanthaScreen
    binding
    7.65105
    CDK9/cyclin KLanthaScreen
    binding
    19.9103
    CDK19 (inactive)LanthaScreen
    binding
    82.9100
    DYRK1BZ-Lyte17496
    DYRK1AZ-Lyte28492
    KIAZ-Lyte50088
    HIPK1 (MYAK)Z-Lyte53995
    MAP2K6 (MKK6)LanthaScreen
    binding
    225065
    CAMKK1 (CAMKKA)LanthaScreen
    binding
    419059
    MAP3K3 (MEKK3)LanthaScreen
    binding
    758052
    STK32C (YANK3)LanthaScreen
    binding
    962046

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/590/eaav7259/DC1

    Text S1. Synthesis of analogs.

    Fig. S1. Kinase specificity of compounds identified in the GPS-MYC screen.

    Fig. S2. Inhibition of CDK9 but not CDK8/19 is responsible for MYC loss induced by UNC10112785.

    Fig. S3. UNC5668 regulation of MYC abundance involves Ser62.

    Table S1. Structures and cellular activities of UNC10112785 analogs.

    Data file S1. Results from the GPS-MYC screen, with specificity data of PKIS compounds.

  • The PDF file includes:

    • Text S1. Synthesis of analogs.
    • Fig. S1. Kinase specificity of compounds identified in the GPS-MYC screen.
    • Fig. S2. Inhibition of CDK9 but not CDK8/19 is responsible for MYC loss induced by UNC10112785.
    • Fig. S3. UNC5668 regulation of MYC abundance involves Ser62.
    • Table S1. Structures and cellular activities of UNC10112785 analogs.
    • Legend for data file S1

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Results from the GPS-MYC screen, with specificity data of PKIS compounds.

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