Combinatorial effects of microRNAs to suppress the Myc oncogenic pathway by María J. Bueno, Marta Gómez de Cedrón, Gonzalo Gómez-López, Ignacio Pérez de Castro, Lorena Di Lisio, Santiago Montes-Moreno, Nerea Martínez, Manuel Guerrero, Ruth Sánchez-Martínez, Javier Santos, David G. Pisano, Miguel Angel Piris, José Fernández-Piqueras, and Marcos Malumbres Blood Volume 117(23):6255-6266 June 9, 2011 ©2011 by American Society of Hematology

General overview of the transcriptional and genetic alterations in γ-irradiation-induced lymphomas. General overview of the transcriptional and genetic alterations in γ-irradiation-induced lymphomas. (A) Myc is a central node in the molecular interactions between up-regulated (red) or down-regulated (blue) proteins, including cell cycle regulators (Cdk or Ran pathways), protein synthesis molecules (Rpl and Rps proteins), and other signaling cascades. (See also supplemental Data for further details.) (B) Transcriptional profiling of miRNAs in normal thymuses (H1, H2, M1, and M3) or T-cell lymphomas. Unsupervised clustering of these data clearly discriminates normal thymuses vs tumor samples. Only significantly deregulated miRNA genes are shown. Blue shadows in miRNA names indicate the presence of a CpG island upstream of the corresponding human or mouse miRNA genes. Green shadows indicate that the CpG island is only present in the mouse sequence. (C) Summary from the comparative genome hybridization analysis of γ-irradiation-induced T-cell lymphomas showing the chromosomal position of down-regulated miRNA genes. Major DNA losses (green bars) and gains (red bars) are indicated to the right of the corresponding chromosomes. The location of Myc in chromosome 15 is also indicated. María J. Bueno et al. Blood 2011;117:6255-6266 ©2011 by American Society of Hematology

GIs between overexpressed genes and down-regulated miRNA genes. GIs between overexpressed genes and down-regulated miRNA genes. GIs are defined as the number of down-regulated miRNA genes that can potentially target specific sequences in all possible 3′-UTRs of the overexpressed gene indicated. (See supplemental Data for further details.) Myc is the overexpressed gene with the highest number of GIs in γ-irradiation-induced T-cell lymphomas. Thirteen of the 41 miRNA genes down-regulated in these tumors can potentially target the Myc 3′-UTR. Other genes potentially targeted by multiple miRNAs, such as Ncor2 (11 GI) and Kkbp3 (10 GI), are indicated in the figure. Only genes up-regulated in irradiation-induced lymphomas with more than or equal to 7 GI are represented. María J. Bueno et al. Blood 2011;117:6255-6266 ©2011 by American Society of Hematology

Control of Myc expression by miRNAs. Control of Myc expression by miRNAs. (A) Potential target sites for mature miRNAs in the mouse Myc 3′-UTR. Only mature miRNAs produced by miRNA genes down-regulated in γ-irradiation-induced lymphomas are shown in this scheme. Gene nomenclature is also indicated when different from the mature form (eg, mir-125b-1 is the gene that generates miR-125–3p). (B) Luciferase activity of a reporter construct carrying the Myc 3′-UTR downstream of the luciferase gene. The construct was cotransfected with a vector expressing each of the indicated miRNA precursors. All data are normalized versus the luciferase levels generated by scramble sequences. (C) Effect of miRNA genes on Myc protein levels. Transfection with miRNA genes was performed as described earlier in the Figure 3 legend, but cells were processed for immunoblot analysis for Myc or 2 different Myc targets, Mcm4 and Cyclin B1. A vertical line has been inserted to indicate repositioned gel lanes. The relative levels of Myc proteins were normalized using α-tubulin (α-tub.) as a loading control. (D) Luciferase activity of wild-type (wt) or mutant (mut) Myc 3′-UTRs in the presence of scrambled sequences (S) or the corresponding miRNAs. wt, indicates wild-type Myc 3′-UTR sequence; mut, single mutants for the indicated miRNA; and mut2, double mutant for the miR-125b-3p target sites. (E) Luciferase assays of wild-type Myc 3′-UTRs in the presence of pools of group 1 (G1; mir-132, mir-125b-1, let-7e, let-7-a, and mir-154), group 2 (G2; mir-301a, mir-148a, and mir-134), or group 3 (G3; mir-26b, mir-150, mir-207, and mir-223) miRNAs. In these pools, the sum of all miRNA vectors also equals 10 μg as in the scramble vectors or the previous assays. (F) Effect of G1–3 pools on Myc protein levels. Transfection with miRNA genes was performed as described earlier in the Figure 3 legend, but cells were processed for immunoblot analysis for Myc. The relative levels of Myc proteins were normalized using α-tubulin (α-tub.) as a loading control. María J. Bueno et al. Blood 2011;117:6255-6266 ©2011 by American Society of Hematology

Genetic interactions between protein-coding genes and miRNAs in BL Genetic interactions between protein-coding genes and miRNAs in BL. (A) Heat map of significantly down-regulated miRNAs in BL compared with normal lymph nodes. Genetic interactions between protein-coding genes and miRNAs in BL. (A) Heat map of significantly down-regulated miRNAs in BL compared with normal lymph nodes. (B) GSEA for down-regulated miRNAs targeting the MYC 3′-UTR. (C) Genetic interactions (GI) between up-regulated protein-coding genes and down-regulated miRNAs in BL. Only protein-coding genes with GI ≥ 6 are shown. María J. Bueno et al. Blood 2011;117:6255-6266 ©2011 by American Society of Hematology

Control of human MYC by multiple miRNAs. Control of human MYC by multiple miRNAs. (A) Schematic representation of the human MYC 3′-UTR and the localization of target sites for the miRNAs down-regulated in BLs. (B) Luciferase reporter assays to test the effect of the indicated miRNAs in the MYC 3′-UTR. Vectors expressing scrambled sequences (S) or mir-22 (not predicted to target MYC) were used as controls. (C) Mutagenesis of miRNA target sites in the human MYC 3′-UTR. Luciferase activity in the presence of the wild-type (wt) or a mutated (mut) MYC 3′-UTR in which 3 positions of the seed sequence have been mutated for each specific miRNA target site indicated. These constructs were assayed in the presence of vectors expressing the indicated miRNAs or scrambled sequences. (D) Effect of the indicated miRNAs in the protein levels of MYC in Raji BL cells. The pool contains an equimolar mixture of all indicated miRNAs in which the sum of all these miRNAs equals 10 μg as in the scramble vectors or the previous assays. María J. Bueno et al. Blood 2011;117:6255-6266 ©2011 by American Society of Hematology

Significant effect of mouse or human down-regulated miRNAs on Myc targets. Significant effect of mouse or human down-regulated miRNAs on Myc targets. (A) GIs between Myc target genes and down-regulated miRNA genes in mouse irradiation-induced lymphomas. Peripheral yellow nodes represent down-regulated miRNA genes (as in Figure 2), whereas Myc targets are distributed as a circle around Myc (the interaction between Myc and Myc targets is not shown for clarity). A total of 72 Myc targets are potentially targeted by down-regulated miRNA genes, and only genes with more than or equal to 3 GI are shown. A complete list of these interactions is provided in the Supplemental data. (B) Similar analysis of the GI between MYC target genes and down-regulated miRNAs in BLs. Peripheral yellow nodes represent down-regulated miRNA genes (as in Figure 7), whereas MYC targets are distributed as a circle around MYC. María J. Bueno et al. Blood 2011;117:6255-6266 ©2011 by American Society of Hematology

A model of the GIs between Myc, its targets, and miRNAs in normal and tumor cells. A model of the GIs between Myc, its targets, and miRNAs in normal and tumor cells. In normal cells, multiple miRNAs are expressed that can repress Myc or Myc targets. miRNA genes are not repressed by Myc because of the low levels of Myc signaling in normal cells. Tumor cells acquire diverse genetic or epigenetic alterations that result in the overexpresssion of Myc and Myc targets to facilitate tumor development through diverse cellular processes. On one hand, Myc can be amplified and overexpressed. On the other hand, miRNAs are silenced by genetic (loss of heterozygosity), epigenetic (hypermethylation), or regulatory (repression by Myc) mechanisms. Because of the low levels of these miRNAs, signaling by Myc and Myc targets is enhanced resulting in dramatic deregulation of the cell cycle, protein translation, and metabolism among other cellular processes. This model is mostly based on the results obtained by Chang et al19 on the repression of miRNAs by Myc and the control of Myc by miRNAs reported here. María J. Bueno et al. Blood 2011;117:6255-6266 ©2011 by American Society of Hematology