A SUMOylation-dependent transcriptional subprogram is required for Myc-driven tumorigenesis

Abstract

Myc is an oncogenic transcription factor frequently dysregulated in human cancer. To identify pathways supporting the Myc oncogenic program, we used a genome-wide RNA interference screen to search for Myc-synthetic lethal genes and uncovered a role for the SUMO-activating enzyme (SAE1/2). Loss of SAE1/2 enzymatic activity drives synthetic lethality with Myc. Inactivation of SAE2 leads to mitotic catastrophe and cell death upon Myc hyperactivation. Mechanistically, SAE2 inhibition switches a transcriptional subprogram of Myc from activated to repressed. A subset of these SUMOylation-dependent Myc switchers (SMS genes) is required for mitotic spindle function and to support the Myc oncogenic program. SAE2 is required for growth of Myc-dependent tumors in mice, and gene expression analyses of Myc-high human breast cancers suggest that low SAE1 and SAE2 abundance in the tumors correlates with longer metastasis-free survival of the patients. Thus, inhibition of SUMOylation may merit investigation as a possible therapy for Myc-driven human cancers.

Figure 1. Genome-wide screen for Myc-Synthetic Lethal (MySL) candidates

a) Identification of Myc-Synthetic Lethal (MySL) genes. Myc-ER HMECs were transduced with a genome-wide library of retroviral shRNAs in triplicate. At population doubling 0(PD0), cells were cultured +/− Myc -ER induction for 12 population doublings(PD12). To identify MySL candidates, relative barcode abundance from both conditions was compared to initial PD0 samples via barcode microarrays. b) Identification of MySL shRNAs. The Myc-selective effect of all shRNAs from the genome-wide library are graphed (y-axis represents median difference between Myc-ER-on and Myc-ER-off groups (log2)), with a ratio < −1.0 indicating a decrease of at least twofold. shRNAs are shown on the x -axis (rank ordered by MySL effect). c) Multiple cellular processes are required to tolerate the Myc oncogenic state. MySL candidates were analyzed for gene ontology (GO; Z-scores for enriched cellular components and processes). d) MySL proteins engage in a highly connected interaction network that regulates the mitotic spindle. Protein-protein interactions between the top 100 MySL proteins were analyzed via HPRD. Green indicates a MySL protein, blue indicates a protein with a known role in mitotic spindle function, red indicates a MySL protein with a known role in spindle function, and gray indicates a protein that interacts with a MySL protein.

Figure 2. The E1 SUMO-activating enzyme is required to tolerate oncogenic MYC in HMECs

a) Inactivation of SAE2, SAE1, or UBE2I is synthetically lethal with Myc hyper-activation. Myc-ER HMECs were infected with control, SAE2, SAE1, or UBE2I-targeting shRNAs and cultured −/+ Myc-ER induction. Cell number was quantified after 6 d. Data are represented as relative change in doubling time in Myc-On vs Myc-Off states and normalized to control shRNA (Average of at least 8 replicates for each shRNA). Representative images demonstrating altered morphology and decreased cell number in Myc/shSAE2 cells. b) Total protein SUMOylation is decreased upon SAE2 knockdown. Myc-ER HMECs infected with control or SAE2-shRNA encoding virus were analyzed for SUMO1, SUMO2/3, and Vinculin (loading control) protein levels. c) and d) SAE2 catalytic activity is required to tolerate Myc hyper-activation. Myc-ER HMECs transduced with a doxycycline (dox)-inducible shRNA targeting the SAE2 UTR (pInducer11-mir-SAE2 UTR-eGFP) were subsequently infected with virus expressing GFP, SAE2 WT or SAE2 C173S cDNAs. Western blots were performed to confirm depletion of SAE2 (c). Cells were cultured in −/+ Dox and −/+ Myc-ER-induction, and cell number was quantified after 8 d. The Y-axis indicates the relative change in growth of shSAE2 expressing cells due to Myc induction in the presence of the indicated transgenes (average of 8 replicates) (d).

Figure 3. Inactivation of SAE2 switches the Myc transcriptional program and dysregulates mitotic fidelity and cell viability

a) Ectopic Myc activation and SAE2 inactivation leads to increase in G2/M cells and aberrant chromosomal content. Myc-ER HMECs transduced with inducible shSAE2 were cultured −/+ Myc-ER-induction (24 h) and −/+ shSAE2 induction. Cells were analyzed for DNA content by flow cytometry (quantification of cells with >2N DNA, right panel). b) Depletion of SAE2 induces apoptosis in cooperation with Myc hyper-activation. pINDUCER-mir-SAE2-eGFP Myc-ER HMECs were cultured −/+ Myc-ER-induction and −/+ shSAE2 induction (48 h). The cells were analyzed for apoptosis (Annexin-V) by flow cytometry. c) and d) Myc-SAE2 genetic interaction leads to defects in the mitotic spindle. Myc-ER HMECs transduced with inducible shRNA-SAE2 were cultured −/+ Myc-ER-induction (16 h) and −/+ shSAE2 induction. Cells were stained for Tubulin (green) and phospho-H3 (red) to visualize mitotic defects. Images from c) were quantified for both total and abnormal mitotic events d). Data are represented as percent abnormal mitoses (at least 100 mitotic events counted per condition; p -values from Fisher’s exact test). Scale bar=5uM e) Loss of SAE2 alters the transcriptional response to Myc. HMECs expressing Myc-ER and dox-inducible SAE2-shRNA were analyzed by gene expression profiling −/+ Myc-ER-induction and −/+ SAE2-shRNA induction. All mRNAs altered by Myc-ER-induction (p < 0.05, 2-fold) are shown. The effect of Myc-ER-induction on mRNA levels in the absence or presence of shRNA-SAE2-induction are shown (left and right panels, respectively). mRNAs that change their response to Myc in the presence or absence of shSAE2 are termed “Sumoylation-dependent Myc switchers,” or SMS genes. f) Loss of SAE2 alters Myc control of spindle-regulatory genes. The effect of Myc in the absence or presence of shSAE2 (blue and red bars, respectively) is shown for top 4 of 17 SMS genes with known roles in spindle integrity and function (see Fig. S9B for list of 17). g) SMS genes are required to tolerate Myc hyper-activation. Myc-ER HMECs transduced with shRNAs targeting the indicated SMS genes were cultured −/+ Myc-ER-induction for 6 d. Cell numbers were counted and analyzed as in Fig 2A.

Figure 4. The E1 SUMO-activating enzyme is required to support MYC-dependent human breast cancers

a) Myc-dependency in breast cancer cells. Breast cancer-derived cell lines infected with control-or Myc-shRNA lentivirus were analyzed for clonogenic growth. Macroscopic colonies were quantified and normalized to control-shRNA infected cells for each cell line. b) Inactivation of SAE2 inhibits clonogenicity in Myc-dependent breast cancer cells. Breast cancer-derived cell lines infected with dox-inducible control-or SAE2 -shRNA lentivirus were analyzed for clonogenic growth +/− dox. c) Inactivation of SAE2 inhibits tumorigenicity of Myc-dependent tumors. Myc-dependent (SUM159 and MDA-MB-231, left and middle panels, respectively) or Myc-independent (MCF7, right panel) breast cancer cells infected with dox-inducible SAE2-targeting shRNA lentivirus were transplanted into nude mice. Recipient animals were treated +/− dox and xenograft volume was measured over time. d) Low SUMO-Activating-Enzyme expression correlates with patient metastasis-free survival selectively in Myc-high breast cancers. The expression of SAE1/SAE2 is inversely correlated with increased metastasis-free survival in patients with Myc-high tumors (p=0.001, log-rank test). Tumors with the highest and lowest tertile of Myc mRNA expression were considered “Myc high” and “Myc low,” respectively. Patients with the highest-and lowest -tertile of SAE1/SAE2 mRNA expression are shown as blue and red lines, respectively.