Deregulated Myc requires MondoA/Mlx for metabolic reprogramming and tumorigenesis

Abstract

Deregulated Myc transcriptionally reprograms cell metabolism to promote neoplasia. Here we show that oncogenic Myc requires the Myc superfamily member MondoA, a nutrient-sensing transcription factor, for tumorigenesis. Knockdown of MondoA, or its dimerization partner Mlx, blocks Myc-induced reprogramming of multiple metabolic pathways, resulting in apoptosis. Identification and knockdown of genes coregulated by Myc and MondoA have allowed us to define metabolic functions required by deregulated Myc and demonstrate a critical role for lipid biosynthesis in survival of Myc-driven cancer. Furthermore, overexpression of a subset of Myc and MondoA coregulated genes correlates with poor outcome of patients with diverse cancers. Coregulation of cancer metabolism by Myc and MondoA provides the potential for therapeutics aimed at inhibiting MondoA and its target genes.

Figure 1. Synthetic Lethal dependency of deregulated Myc on MondoA

(A) Simplified Myc network schematic showing competition for heterodimerization partners (black lines with double arrowhead) and genomic E-Box loci between network members (green and red lines indicate transcriptional activation and repression, respectively). (B–E) Relative viability of P3C1 (B), CB660 and its immortalized (I-CB660) or Ras-transformed (T-CB660) derivatives (D), or neuroblastoma (E) cell lines or cell number of P493-6 cells (C) transfected with indicated siRNAs normalized to the corresponding non-silencing control (siCtrl). A pro-apoptotic siRNA mix (siDeath) is included as a positive control for transfection efficiency. Shown are mean and SD (n=3). (F) Western blot analysis for MondoA, c-Myc and N-Myc across the indicated cell lines under indicated conditions. See also Figure S1 and Table S1.

Figure 2. Loss of MondoA blocks N-Myc-induced proliferation and tumorigenesis while potentiating apoptosis both in vitro and in vivo

(A) Growth curves of Tet21N cells, with or without N-Myc overexpression, treated with either siCtrl or siMonA. Shown are mean and SD, n=3. (B) Cell cycle analysis of cells from panel A at day 3. (C) Time course quantification of sub-G1 cell population. Shown are mean and SD, (n=3). (D) Western Blot analysis for the indicated proteins in Tet21N cells with or without N-Myc overexpression at day 3 post-transfection of either siCtrl or siMonA. (E, F) Representative colony morphology (E) and quantification of colony formation (F) of neuroblastoma cells in soft agar at day 14 after plating. Scale bar = 20 μm. CFU: Colony-forming units. Shown are mean and SEM, (n=9). (G) Subcutaneous growth of Tet21N cells stably expressing either shCtrl or shMonA in Nu/Nu mice and a representative animal at end point. (H) Final tumor weight and representative image of tumors at end point. For (G–H) Shown are mean and SEM (n=10). Scale bar = 1 cm. (I) Quantification of immunohistochemistry positivity for Ki67 and cleaved-caspase 3 (CC3) in tumors from (G) Shown are mean and SEM (n=12). See also Figure S2.

Figure 3. Transcriptional coordination between N-Myc and MondoA regulates metabolism and proliferation

(A) Heatmap generated by TreeView representing the median Z-score of all genes whose expression is altered for each condition. (B) Log fold change (Log-FC) of all, N-Myc upregulated, and N-Myc downregulated probes in the presence or absence of MondoA. Shown is a box plot indicating maximum (upper line), 3^rd quartile (top of box), median (line in box), 1^st quartile (bottom of box) and minimum (lower line). (C) Venn diagram of N-Myc (in the presence of MondoA) and MondoA (in the presence of N-Myc) regulated genes. (D) Gene Ontology (GO) Analysis: genes upregulated by N-Myc (white), MondoA (grey) or both (striped) for each category are shown. (E) Global expression profile of N-Myc and MondoA cooperatively upregulated genes among the four conditions. Genes encoding metabolic regulator as indicated with colored lines. (F) GO analysis of MondoA regulated genes (N-Myc-ON condition) (G) Global expression profile of MondoA upregulated genes involved in lipid and glucose metabolism as indicated with colored lines. See also Figure S3.

Figure 4. N-Myc and MondoA coordinately regulate metabolic and mitochondrial targets in Tet21N cells

(A) Western blot analysis of a subset of N-Myc and MondoA coordinately regulated or MondoA-dependent targets. (B) Western blot analysis of Tet21N cells under the indicated conditions in response to glucose stimulation. (C) Glutamine (Gln) uptake assay of Tet21N cells with the indicated treatment. Shown are mean and SD, n=3. (D) Oxygen consumption rate (OCR) assay of Tet21N cells with the indicated treatment. Shown are mean and SD (n=3). (E) Western blot analysis of the indicated proteins in siCtrl cells or cells with KD of Slc1A5 or Slc3A2. (F) Cells expressing indicated proteins or vector control were transfected with siCtrl or siMonA and viable cells were enumerated by trypan blue exclusion after 4 days. Shown are mean and SD, n=3. (G) Viability of cells in the presence or absence of Myc expression (normalized to the relative control siRNA) 4 days after transfection with the indicated siRNA. Shown are mean and SD (n=3). The red line indicates the control is set to 1. See also Figure S4 and Table S1.

Figure 5. MondoA suppresses N-Myc-induced metabolic stress

(A) Western blot analysis of Tet21N under the indicated conditions transfected with the indicated siRNA. (B) Western blot analysis of Tet21N cells for the indicated proteins (−/+ N-Myc and −/+ nutlin-3a). (C) Cell viability (Cell Titer Glo) and apoptosis (Caspase-Glo Reagent) quantification after nutlin-3a treatment under the indicated conditions. Shown are mean and SEM (n=5). (D) Viability assay of Tet21N cells expressing either a control or a p53 shRNA. Shown are mean and SD (n=3). (E) Viability assay of HCT116 p53^+/+ and p53^−/− cells after knockdown of MondoA, Mlx and Myc/MondoA target genes. Shown are mean and SEM (n=5). (F) Western blot analysis of the indicated apoptosis-related proteins in the Tet21N cells (−/+ N-Myc) under siCtrl or siMonA treatment. (G) Tet21N cells (−/+ N-Myc) expressing vector control (same as in Figure 4F) or BCL2 were transfected with siCtrl or siMonA and viable cells were enumerated by trypan blue exclusion after 4 days (left) and Western blot for Bcl2. Shown are mean and SD (n=3). See also Figure S6.

Figure 6. Metabolomic profiling of N-Myc and MondoA regulated pathways

(A) Heatmap depicting normalized and log-transformed metabolomic survey of 139 soluble metabolites under the four conditions. Color code for bar graphs in (A–E), white: siCtrl, blue: siCtrl+N-Myc, green: siMonA, and orange: siMonA+N-Myc. (B) Metabolite Set Enrichment Analysis (MSEA) of metabolite data from each condition compared to siCtrl. Shown is the −Log p Value for enrichment with a 0.05 p value indicated by the red line at 1.34. (C) GC-MS tracing of ¹³C₅-Gln for 24 hr in the Tet21N cells (−/+N-Myc) with siCtrl or siMonA treatment with the quantification of the mass isotopomers downstream of Gln indicated. Shown is mean and SD (n=3). (D) Quantification of total citrate (Cit) as in (C), normalized to relative cell number. Shown is mean and SD (n=3). (E) GC-MS tracing of ¹³C₅-Gln for 48 hr in the Tet21N cells as in (C), with contribution of ¹³C₅-Gln-derived acetyl-coA to palmitate indicated by m+(2n) isotopomers. The de novo fatty acid synthesis pathway is indicated, with enzymes shown in blue. Shown is mean and SD (n=3). (F) Relative caspase activity of Tet21N cells (−/+N-Myc) transfected with siCtrl or siMonA for 24 hr then treated with vehicle control (NT), ZOL, or C75 for 48 hr was measured by Caspase-Glo reagent. Shown is mean and SEM (n=5). (G) Tet21N cells were transfected with the indicated siRNA and treated with either vehicle (BSA), BSA+GGOH or BSA-conjugated Oleate 24 hr post-transfection and viability was assessed by Cell-Titer Glo reagent at 96 hr. Shown is mean and SEM (n=5). (H) Western blot analysis of cells treated as in (G) for the indicated proteins. All p values were calculated by ANOVA. See also Figure S6 and Table S2.

Figure 7. High expression of N-Myc and MondoA co-regulated metabolic genes correlates with poor prognosis

(A) Model of Myc-MondoA cooperation in regulation of cellular metabolism (left). Synthetic lethal condition upon activation of Myc and MondoA loss of function (right). Abbreviations: electron transport chain (ETC), tricarboxylic acid cycle (TCA) and reactive oxygen species (ROS). (B) Kaplan-Meier survival analysis based on the expression of selected Myc and MondoA-regulated genes. Red, blue and grey lines indicate the survival probability of patient with high (top 20%), low (bottom 20%) and intermediate (remaining 60%) cumulative expression of MLXIP, CAD, TFAM, CBS, SCD, PFAS, SLC3A2, and SLC1A5, respectively. Significance between the top and bottom 20% was determined by Wilcoxon model. Liver hepatocellular carcinoma (LIHC), lung squamous cell carcinoma and lung adenocarcinoma datasets combined (LUNG), colon adenocarcinoma (COAD), acute myeloid leukemia (LAML), and breast invasive carcinoma (BRCA). Gene datasets: neuroblastoma (Oberthuer 2006) and for rest, TCGA (http://cancergenome.nih.gov).