Integrative methylome-transcriptome analysis unravels cancer cell vulnerabilities in infant MLL-rearranged B cell acute lymphoblastic leukemia
Juan Ramón Tejedor, Clara Bueno, Meritxell Vinyoles, Paolo Petazzi, Antonio Agraz-Doblas, Isabel Cobo, Raúl Torres-Ruiz, Gustavo F. Bayón, Raúl F. Pérez, Sara López-Tamargo, Francisco Gutierrez-Agüera, Pablo Santamarina-Ojeda, Manuel Ramírez-Orellana, Michela Bardini, Giovanni Cazzaniga, Paola Ballerini, Pauline Schneider, Ronald W. Stam, Ignacio Varela, Mario F. Fraga, Agustín F. Fernández, Pablo Menéndez
Juan Ramón Tejedor, Clara Bueno, Meritxell Vinyoles, Paolo Petazzi, Antonio Agraz-Doblas, Isabel Cobo, Raúl Torres-Ruiz, Gustavo F. Bayón, Raúl F. Pérez, Sara López-Tamargo, Francisco Gutierrez-Agüera, Pablo Santamarina-Ojeda, Manuel Ramírez-Orellana, Michela Bardini, Giovanni Cazzaniga, Paola Ballerini, Pauline Schneider, Ronald W. Stam, Ignacio Varela, Mario F. Fraga, Agustín F. Fernández, Pablo Menéndez
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Research Article Genetics Oncology

Integrative methylome-transcriptome analysis unravels cancer cell vulnerabilities in infant MLL-rearranged B cell acute lymphoblastic leukemia

Abstract

B cell acute lymphoblastic leukemia (B-ALL) is the most common childhood cancer. As predicted by its prenatal origin, infant B-ALL (iB-ALL) shows an exceptionally silent DNA mutational landscape, suggesting that alternative epigenetic mechanisms may substantially contribute to its leukemogenesis. Here, we have integrated genome-wide DNA methylome and transcriptome data from 69 patients with de novo MLL-rearranged leukemia (MLLr) and non-MLLr iB-ALL leukemia uniformly treated according to the Interfant-99/06 protocol. iB-ALL methylome signatures display a plethora of common and specific alterations associated with chromatin states related to enhancer and transcriptional control in normal hematopoietic cells. DNA methylation, gene expression, and gene coexpression network analyses segregated MLLr away from non-MLLr iB-ALL and identified a coordinated and enriched expression of the AP-1 complex members FOS and JUN and RUNX factors in MLLr iB-ALL, consistent with the significant enrichment of hypomethylated CpGs in these genes. Integrative methylome-transcriptome analysis identified consistent cancer cell vulnerabilities, revealed a robust iB-ALL–specific gene expression–correlating dmCpG signature, and confirmed an epigenetic control of AP-1 and RUNX members in reshaping the molecular network of MLLr iB-ALL. Finally, pharmacological inhibition or functional ablation of AP-1 dramatically impaired MLLr-leukemic growth in vitro and in vivo using MLLr-iB-ALL patient–derived xenografts, providing rationale for new therapeutic avenues in MLLr-iB-ALL.

Authors

Juan Ramón Tejedor, Clara Bueno, Meritxell Vinyoles, Paolo Petazzi, Antonio Agraz-Doblas, Isabel Cobo, Raúl Torres-Ruiz, Gustavo F. Bayón, Raúl F. Pérez, Sara López-Tamargo, Francisco Gutierrez-Agüera, Pablo Santamarina-Ojeda, Manuel Ramírez-Orellana, Michela Bardini, Giovanni Cazzaniga, Paola Ballerini, Pauline Schneider, Ronald W. Stam, Ignacio Varela, Mario F. Fraga, Agustín F. Fernández, Pablo Menéndez

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Figure 8

FOSL2 contributes to the modulation of the MLL-AF4+ methylome.

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FOSL2 contributes to the modulation of the MLL-AF4+ methylome. (A) Weste...
(A) Western blot confirming FOSL2 knockout in 2 clones of CRISPR/Cas9-edited SEM cells. Total protein refers to the loading control measured by Ponceau staining. (B) Violin plot displaying the average methylation landscape of SEM-WT and SEM-FOSL2KO cells ***P < 0.001, 2-sided Wilcoxon’s rank sum test. (C) Histogram indicating the number of hyper- and hypomethylated CpG sites between SEM-FOSL2KO and SEM-WT cells (mean β difference > 0.40). (D) HOMER motif enrichment analysis highlighting the top enriched TFBS identified in the context of hypermethylated CpGs in SEM-FOSLKO cells. (E) Plot illustrating the enrichment analysis obtained from gene expression data from SEM-FOSL2KO and SEM-WT and the gene set corresponding to the AP-1 pathway. The NES and the significance scores were calculated using the preranked mode of GSEA. (F) Violin plots displaying the distribution of gene expression changes (log2 fold change of SEM-FOSL2KO versus SEM-WT cells) of FOSL2 target genes (number of annotated dmCpGs > 3, n = 107) or a random subset on genes (n = 500), including all genes with detectable expression in the RNA-Seq data set. ***P < 0.001, 2-sided Wilcoxon’s rank sum test. (G) Barplots representing RNA-Seq expression results (in FPKM) of NUDT21 and FSCN1 genes in a pool of SEM-WT and SEM-FOSL2KO cells. (H) Plots depicting the genomic localization and β value score of the interrogated MethylationEPIC probes in the vicinity of NUDT21 and FSCN1 genes. Colors denote the methylation landscape of the indicated groups, and MLL-AF4+ iB-ALL patient samples were included for proper comparisons. Significant dmCpGs (mean β difference > 0.40) are highlighted in red. Bottom panel displays the genomic context of the aforementioned probes and genes, including the location of FOSL2 motifs (GTRD database) and CpG islands. TSSs are highlighted in orange.