Oncogenic PKA signaling increases c-MYC protein expression through multiple targetable mechanisms

  1. Gary KL Chan
  2. Samantha Maisel
  3. Yeonjoo C Hwang
  4. Bryan C Pascual
  5. Rebecca RB Wolber
  6. Phuong Vu
  7. Krushna C Patra
  8. Mehdi Bouhaddou
  9. Heidi L Kenerson
  10. Huat C Lim
  11. Donald Long
  12. Raymond S Yeung
  13. Praveen Sethupathy
  14. Danielle L Swaney
  15. Nevan J Krogan
  16. Rigney E Turnham
  17. Kimberly J Riehle
  18. John D Scott
  19. Nabeel Bardeesy
  20. John D Gordan  Is a corresponding author
  1. Division of Hematology/Oncology, Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, United States
  2. Quantitative Biosciences Institute (QBI), University of California San Francisco, United States
  3. Department of Medicine, Harvard Medical School, United States
  4. Massachusetts General Hospital Cancer Center, United States
  5. Department of Cellular and Molecular Pharmacology, University of California San Francisco, United States
  6. J. David Gladstone Institute, United States
  7. Department of Surgery and Northwest Liver Research Program, University of Washington, United States
  8. Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, United States
  9. Department of Pharmacology, University of Washington Medical Center, United States
10 figures, 1 table and 8 additional files

Figures

Recurrent PKA activating somatic alterations in human cancer.

(A) Pathway illustrations of different PKA activating genomic alterations in order from top: PRKACA amplification, DNAJB1-PRKACA fusion, PRKACA activating mutation, and PRKAR1A inactivation or …

Kinome profiling to identify signaling nodes downstream of PRKACA.

(A) Global phosphorylation changes in 639V with induction of 3xFLAG-PRKACA or 3xFLAG-PRKAR1AG325D; VASP is shown as a positive control for PKA activation, technical replicates shown. (B) Change in …

Figure 3—figure supplement 1
Effects of PKA inhibition on FLX1 cell proliferation.

(A) Impact of individual PRKACA-targeting siRNAs on relative cell confluence in FLX1. Confirmation of knockdown shown on right. Experiment was done in duplicate, the representative results shown …

Figure 4 with 1 supplement
c-MYC alters transcription and proliferation in PKA-dependent cell models.

(A) RNASEQ data from FLX1 cells after 48 hr treatment with four pooled siRNA against PRKACA. PRKACA, ornithine decarboxylase (ODC) and Cyclin D1 (CCND1) are highlighted. (B) Gene set enrichment …

Figure 4—figure supplement 1
Data supporting c-MYC effects on transcription and proliferation.

(A) Confirmation of PRKACA and MYC knockdown for expression analysis in Figure 4E and proliferation data in Figure 4F; shown with mean of technical replicates ± SD. (B) Relative confluence of …

Figure 5 with 2 supplements
AURKA and GSK3B regulate c-MYC in PKA-dependent cell models.

(A) Summary data from the FLX1 cell line treated with 352 kinase inhibitors from an advanced clinical compound library at 2 μM for 120 hr. The targets of selected compounds with a z-score ≥2 are …

Figure 5—figure supplement 1
Signaling effects on c-MYC in PKA-driven cells.

(A) Colo741 cells treated with dose curves of multiple AURKA inhibitors for 72 hr. Relative cell viability was measured by CTG assay vs. untreated control samples. Results are the mean ± SEM of …

Figure 5—figure supplement 2
Proteasome-independent PKA effects on c-MYC, (A) immunoblots showing the change of c-MYC protein in FLX1 cells after treatment with 50 μM forskolin (FSK)/3-isobutyl-1-methylxanthine (IBMX) and/or 20 μM MG132 for 2 hr.

(B) Immunoblots showing the change of c-MYC level in engineered FLX1 cells with doxycycline (dox)-inducible 3xFLAG- PRKAR1AG325D after dox for 24 hr and/or 20 μM MG132 for 2 hr. (C) Li-Cor western …

PKA signaling supports translation initiation.

(A) Gene set enrichment analysis (GSEA) of significantly altered phosphoproteins following doxycycline (dox) induction of PKA in proteomics data from this study or chemical PKA stimulation in our …

Figure 7 with 1 supplement
PKA effects on c-MYC are blocked by eIF4A inhibition.

(A) Immunoblots showing the c-MYC protein levels in FLX1 cells after treatment with 100 nM rocaglamide or zotatifin for 24 hr and/or 50 μM forskolin (FSK)/3-isobutyl-1-methylxanthine (IBMX) for 4 …

Figure 7—figure supplement 1
Data supporting reversal of PKA effects on c-MYC by eIF4A inhibition.

(A) Immunoblot showing the effect of 48 hr of non-targeting control (NTC) or four pooled PRKACA siRNA on endogenous c-MYC or overexpressed c-MYC lacking a 5’UTR. Doxycycline (dox) treatment was 1 …

Author response image 1
PKA effects on cell cycle distribution in FLX1.

(A) FLX1 parental cells were treated with DMSO or 50 μM FSK/IBMX for 4 hours, with BrdU added for the last 20 minutes. Cells were then stained for nuclear content with 7-AAD and active DNA synthesis …

Author response image 2
FLX1 and Colo741 with dox-induced 3xFLAG-c-MYC lacking a 5’UTR and including the stabilizing T58A mutation.

Dox treatment was 1 μG/mL for 40 hours prior to harvest with zotatifin or DMSO for 24 hours. Of note, the MYCT58A allele was toxic in both cells but particularly in FLX1, resulting in reduced …

Author response image 3
FSK/IBMX and zotatifin effects on other translationally regulated transcripts.

(A) Time course of FSK/IBMX in Colo741 and FLX1 showing increased expression of ERBB2; kinetics are somewhat different between the two cell lines. (B) Effect of 24 hour treatment with 100 nM …

Tables

Author response table 1
CRISPR
PRKACAMYC
Average-0.24-1.46
639V-0.14-0.67
Colo741-0.35-1.38
siRNA
PRKACAMYC
Average-0.15-0.48
Colo741-1.38-0.35

Additional files

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