Truncating PREX2 mutations activate its GEF activity and alter gene expression regulation in NRAS-mutant melanoma.

PREX2 (phosphatidylinositol-3,4,5-triphosphate-dependent Rac-exchange factor 2) is a PTEN (phosphatase and tensin homolog deleted on chromosome 10) binding protein that is significantly mutated in cutaneous melanoma and pancreatic ductal adenocarcinoma. Here, genetic and biochemical analyses were conducted to elucidate the nature and mechanistic basis of PREX2 mutation in melanoma development. By generating an inducible transgenic mouse model we showed an oncogenic role for a truncating PREX2 mutation (PREX2(E824)*) in vivo in the context of mutant NRAS. Using integrative cross-species gene expression analysis, we identified deregulated cell cycle and cytoskeleton organization as significantly perturbed biological pathways in PREX2 mutant tumors. Mechanistically, truncation of PREX2 activated its Rac1 guanine nucleotide exchange factor activity, abolished binding to PTEN and activated the PI3K (phosphatidyl inositol 3 kinase)/Akt signaling pathway. We further showed that PREX2 truncating mutations or PTEN deletion induces down-regulation of the tumor suppressor and cell cycle regulator CDKN1C (also known as p57(KIP2)). This down-regulation occurs, at least partially, through DNA hypomethylation of a differentially methylated region in chromosome 11 that is a known regulatory region for expression of the CDKN1C gene. Together, these findings identify PREX2 as a mediator of NRAS-mutant melanoma development that acts through the PI3K/PTEN/Akt pathway to regulate gene expression of a cell cycle regulator.

Investigational agent MLN9708/2238 targets tumor-suppressor miR33b in MM cells.

miRs play a critical role in tumor pathogenesis as either oncogenes or tumor-suppressor genes. However, the role of miRs and their regulation in response to proteasome inhibitors in multiple myeloma (MM) is unclear. In the current study, miR profiling in proteasome inhibitor MLN2238-treated MM.1S MM cells shows up-regulation of miR33b. Mechanistic studies indicate that the induction of miR33b is predominantly via transcriptional regulation. Examination of miR33b in patient MM cells showed a constitutively low expression. Overexpression of miR33b decreased MM cell viability, migration, colony formation, and increased apoptosis and sensitivity of MM cells to MLN2238 treatment. In addition, overexpression of miR33b or MLN2238 exposure negatively regulated oncogene PIM-1 and blocked PIM-1 wild-type, but not PIM-1 mutant, luciferase activity. Moreover, PIM-1 overexpression led to significant abrogation of miR33b- or MLN2238-induced cell death. SGI-1776, a biochemical inhibitor of PIM-1, triggered apoptosis in MM. Finally, overexpression of miR33b inhibited tumor growth and prolonged survival in both subcutaneous and disseminated human MM xenograft models. Our results show that miR33b is a tumor suppressor that plays a role during MLN2238-induced apoptotic signaling in MM cells, and these data provide the basis for novel therapeutic strategies targeting miR33b in MM.

Integrative analysis of gene and miRNA expression profiles with transcription factor-miRNA feed-forward loops identifies regulators in human cancers.

We describe here a novel method for integrating gene and miRNA expression profiles in cancer using feed-forward loops (FFLs) consisting of transcription factors (TFs), miRNAs and their common target genes. The dChip-GemiNI (Gene and miRNA Network-based Integration) method statistically ranks computationally predicted FFLs by their explanatory power to account for differential gene and miRNA expression between two biological conditions such as normal and cancer. GemiNI integrates not only gene and miRNA expression data but also computationally derived information about TF-target gene and miRNA-mRNA interactions. Literature validation shows that the integrated modeling of expression data and FFLs better identifies cancer-related TFs and miRNAs compared to existing approaches. We have utilized GemiNI for analyzing six data sets of solid cancers (liver, kidney, prostate, lung and germ cell) and found that top-ranked FFLs account for ∼20% of transcriptome changes between normal and cancer. We have identified common FFL regulators across multiple cancer types, such as known FFLs consisting of MYC and miR-15/miR-17 families, and novel FFLs consisting of ARNT, CREB1 and their miRNA partners. The results and analysis web server are available at http://www.canevolve.org/dChip-GemiNi.

The dChip survival analysis module for microarray data.

BACKGROUND: Genome-wide expression signatures are emerging as potential marker for overall survival and disease recurrence risk as evidenced by recent commercialization of gene expression based biomarkers in breast cancer. Similar predictions have recently been carried out using genome-wide copy number alterations and microRNAs. Existing software packages for microarray data analysis provide functions to define expression-based survival gene signatures. However, there is no software that can perform survival analysis using SNP array data or draw survival curves interactively for expression-based sample clusters. RESULTS: We have developed the survival analysis module in the dChip software that performs survival analysis across the genome for gene expression and copy number microarray data. Built on the current dChip software's microarray analysis functions such as chromosome display and clustering, the new survival functions include interactive exploring of Kaplan-Meier (K-M) plots using expression or copy number data, computing survival p-values from the log-rank test and Cox models, and using permutation to identify significant chromosome regions associated with survival. CONCLUSIONS: The dChip survival module provides user-friendly way to perform survival analysis and visualize the results in the context of genes and cytobands. It requires no coding expertise and only minimal learning curve for thousands of existing dChip users. The implementation in Visual C++ also enables fast computation. The software and demonstration data are freely available at http://dchip-surv.chenglilab.org.

Reprogramming of bivalent chromatin states in NRAS mutant melanoma suggests PRC2 inhibition as a therapeutic strategy.

The dynamic evolution of chromatin state patterns during metastasis, their relationship with bona fide genetic drivers, and their therapeutic vulnerabilities are not completely understood. Combinatorial chromatin state profiling of 46 melanoma samples reveals an association of NRAS mutants with bivalent histone H3 lysine 27 trimethylation (H3K27me3) and Polycomb repressive complex 2. Reprogramming of bivalent domains during metastasis occurs on master transcription factors of a mesenchymal phenotype, including ZEB1, TWIST1, and CDH1. Resolution of bivalency using pharmacological inhibition of EZH2 decreases invasive capacity of melanoma cells and markedly reduces tumor burden in vivo, specifically in NRAS mutants. Coincident with bivalent reprogramming, the increased expression of pro-metastatic and melanocyte-specific cell-identity genes is associated with exceptionally wide H3K4me3 domains, suggesting a role for this epigenetic element. Overall, we demonstrate that reprogramming of bivalent and broad domains represents key epigenetic alterations in metastatic melanoma and that EZH2 plus MEK inhibition may provide a promising therapeutic strategy for NRAS mutant melanoma patients.

The effects of MicroRNA deregulation on pre-RNA processing network in multiple myeloma.

Over the last few years, a detailed map of genetic and epigenetic lesions that underlie multiple myeloma (MM) has been created. Regulation of microRNA (miR)-dependent gene expression and mRNA splicing play significant roles in MM pathogenesis; however, to date an interplay between these processes is not yet delineated. Here we investigated miR-mediated regulation of splicing networks at the transcriptome level. Our studies show that a significant number (78%) of miRs which are either up- or down-regulated in patient CD138+ MM cells, but not in healthy donors (HD) CD138+ plasma cells (PC), target genes involved in early stages of pre-mRNA splicing. We also identified deregulated miRs that target core splicing factors (SF) and modifiers (SM, enhancers/silencers) which cause altered splicing in MM. Our studies suggest that Let-7f, in combination other miRs which are frequently and significantly deregulated in patients with overt MM, targets genes that regulate intron excision. Importantly, deregulated expression of certain miRs in MM promote increased intron retention, a novel characteristic of the MM genome, by inducing deregulated expression of the genes that regulate the splicing network. Our studies, therefore, provide the rationale for therapeutically targeting deregulated miRs to reverse aberrant splicing and improve patient outcome in MM.

KMT2D Deficiency Impairs Super-Enhancers to Confer a Glycolytic Vulnerability in Lung Cancer.

Epigenetic modifiers frequently harbor loss-of-function mutations in lung cancer, but their tumor-suppressive roles are poorly characterized. Histone methyltransferase KMT2D (a COMPASS-like enzyme, also called MLL4) is among the most highly inactivated epigenetic modifiers in lung cancer. Here, we show that lung-specific loss of Kmt2d promotes lung tumorigenesis in mice and upregulates pro-tumorigenic programs, including glycolysis. Pharmacological inhibition of glycolysis preferentially impedes tumorigenicity of human lung cancer cells bearing KMT2D-inactivating mutations. Mechanistically, Kmt2d loss widely impairs epigenomic signals for super-enhancers/enhancers, including the super-enhancer for the circadian rhythm repressor Per2. Loss of Kmt2d decreases expression of PER2, which regulates multiple glycolytic genes. These findings indicate that KMT2D is a lung tumor suppressor and that KMT2D deficiency confers a therapeutic vulnerability to glycolytic inhibitors.

Enhancer Reprogramming Confers Dependence on Glycolysis and IGF Signaling in KMT2D Mutant Melanoma.

Histone methyltransferase KMT2D harbors frequent loss-of-function somatic point mutations in several tumor types, including melanoma. Here, we identify KMT2D as a potent tumor suppressor in melanoma through an in vivo epigenome-focused pooled RNAi screen and confirm the finding by using a genetically engineered mouse model (GEMM) based on conditional and melanocyte-specific deletion of KMT2D. KMT2D-deficient tumors show substantial reprogramming of key metabolic pathways, including glycolysis. KMT2D deficiency aberrantly upregulates glycolysis enzymes, intermediate metabolites, and glucose consumption rates. Mechanistically, KMT2D loss causes genome-wide reduction of H3K4me1-marked active enhancer chromatin states. Enhancer loss and subsequent repression of IGFBP5 activates IGF1R-AKT to increase glycolysis in KMT2D-deficient cells. Pharmacological inhibition of glycolysis and insulin growth factor (IGF) signaling reduce proliferation and tumorigenesis preferentially in KMT2D-deficient cells. We conclude that KMT2D loss promotes tumorigenesis by facilitating an increased use of the glycolysis pathway for enhanced biomass needs via enhancer reprogramming, thus presenting an opportunity for therapeutic intervention through glycolysis or IGF pathway inhibitors.

Histone demethylase JARID1C inactivation triggers genomic instability in sporadic renal cancer.

Mutations in genes encoding chromatin-remodeling proteins are often identified in a variety of cancers. For example, the histone demethylase JARID1C is frequently inactivated in patients with clear cell renal cell carcinoma (ccRCC); however, it is largely unknown how JARID1C dysfunction promotes cancer. Here, we determined that JARID1C binds broadly to chromatin domains characterized by the trimethylation of lysine 9 (H3K9me3), which is a histone mark enriched in heterochromatin. Moreover, we found that JARID1C localizes on heterochromatin, is required for heterochromatin replication, and forms a complex with established players of heterochromatin assembly, including SUV39H1 and HP1α, as well as with proteins not previously associated with heterochromatin assembly, such as the cullin 4 (CUL4) complex adaptor protein DDB1. Transcription on heterochromatin is tightly suppressed to safeguard the genome, and in ccRCC cells, JARID1C inactivation led to the unrestrained expression of heterochromatic noncoding RNAs (ncRNAs) that in turn triggered genomic instability. Moreover, ccRCC patients harboring JARID1C mutations exhibited aberrant ncRNA expression and increased genomic rearrangements compared with ccRCC patients with tumors endowed with other genetic lesions. Together, these data suggest that inactivation of JARID1C in renal cancer leads to heterochromatin disruption, genomic rearrangement, and aggressive ccRCCs. Moreover, our results shed light on a mechanism that underlies genomic instability in sporadic cancers.