Associating somatic mutation with clinical outcomes through kernel regression and optimal transport

Somatic mutations in cancer patients are inherently sparse and potentially high dimensional. Cancer patients may share the same set of deregulated biological processes perturbed by different sets of somatically mutated genes. Therefore, when assessing the associations between somatic mutations and clinical outcomes, gene-by-gene analysis is often under-powered because it does not capture the complex disease mechanisms shared across cancer patients. Rather than testing genes one by one, an intuitive approach is to aggregate somatic mutation data of multiple genes to assess their joint association with clinical outcomes. The challenge is how to aggregate such information. Building on the optimal transport method, we propose a principled approach to estimate the similarity of somatic mutation profiles of multiple genes between tumor samples, while accounting for gene–gene similarities defined by gene annotations or empirical mutational patterns. Using such similarities, we can assess the associations between somatic mutations and clinical outcomes by kernel regression. We have applied our method to analyze somatic mutation data of 17 cancer types and identified at least five cancer types, where somatic mutations are associated with overall survival, progression-free interval, or cytolytic activity.     

Combined analysis of murine and human microarrays and ChIP analysis reveals genes associated with the ability of MYC to maintain tumorigenesis

The MYC oncogene has been implicated in the regulation of up to thousands of genes involved in many cellular programs including proliferation, growth, differentiation, self-renewal, and apoptosis. MYC is thought to induce cancer through an exaggerated effect on these physiologic programs. Which of these genes are responsible for the ability of MYC to initiate and/or maintain tumorigenesis is not clear. Previously, we have shown that upon brief MYC inactivation, some tumors undergo sustained regression. Here we demonstrate that upon MYC inactivation there are global permanent changes in gene expression detected by microarray analysis. By applying StepMiner analysis, we identified genes whose expression most strongly correlated with the ability of MYC to induce a neoplastic state. Notably, genes were identified that exhibited permanent changes in mRNA expression upon MYC inactivation. Importantly, permanent changes in gene expression could be shown by chromatin immunoprecipitation (ChIP) to be associated with permanent changes in the ability of MYC to bind to the promoter regions. Our list of candidate genes associated with tumor maintenance was further refined by comparing our analysis with other published results to generate a gene signature associated with MYC-induced tumorigenesis in mice. To validate the role of gene signatures associated with MYC in human tumorigenesis, we examined the expression of human homologs in 273 published human lymphoma microarray datasets in Affymetrix U133A format. One large functional group of these genes included the ribosomal structural proteins. In addition, we identified a group of genes involved in a diverse array of cellular functions including: BZW2, H2AFY, SFRS3, NAP1L1, NOLA2, UBE2D2, CCNG1, LIFR, FABP3, and EDG1. Hence, through our analysis of gene expression in murine tumor models and human lymphomas, we have identified a novel gene signature correlated with the ability of MYC to maintain tumorigenesis.     

根据在癌突变谱中的共突变基因对识别癌基因

癌是一种涉及多基因变异的遗传异质性疾病,涉及多种生物学功能通路中不同基因的遗传变异。因此,识别癌基因是一项富有挑战性的工作。提出通过寻找在癌样本中突变显著共发生的基因筛选候选癌基因的方法。应用该方法,通过分析蛋白激酶基因在癌组织中的突变谱数据,发现了167个显著共发生突变的基因对,包含85个基因。分析这167个基因对发现:(1)发生共突变的基因富集已知的癌基因;(2)共突变基因对倾向于共扰动与癌症相关的通路对。以上结果提示,在癌样本中显著共发生突变的基因倾向于候选癌基因;在癌发生过程中起重要作用的基因倾向于协同扰动不同的癌相关细胞生物学过程。     

Stochastic dynamics of metastasis formation

Tumor metastasis accounts for the majority of deaths in cancer patients. The metastatic behavior of cancer cells is promoted by mutations in many genes, including activation of oncogenes such as RAS and MYC. Here, we develop a mathematical framework to analyse the dynamics of mutations enabling cells to metastasize. We consider situations in which one mutation is necessary to confer metastatic ability to the cell. We study different population sizes of the main tumor and different somatic fitness values of metastatic cells. We compare mutations that are positively selected in the main tumor with those that are neutral or negatively selected, but faster at forming metastases. We study whether metastatic potential is the property of all (or the majority of) cells in the main tumor or only the property of a small subset. Our theory shows how to calculate the expected number of metastases that are formed by a tumor.     

MYC-induced apoptosis in mammary epithelial cells is associated with repression of lineage-specific gene signatures

Apoptosis caused by deregulated MYC expression is a prototype example of intrinsic tumor suppression. However, it is still unclear how supraphysiological MYC expression levels engage specific sets of target genes to promote apoptosis. Recently, we demonstrated that repression of SRF target genes by MYC/MIZ1 complexes limits AKT-dependent survival signaling and contributes to apoptosis induction. Here we report that supraphysiological levels of MYC repress gene sets that include markers of basal-like breast cancer cells, but not luminal cancer cells, in a MIZ1-dependent manner. Furthermore, repressed genes are part of a conserved gene signature characterizing the basal subpopulation of both murine and human mammary gland. These repressed genes play a role in epithelium and mammary gland development and overlap with genes mediating cell adhesion and extracellular matrix organization. Strikingly, acute activation of oncogenic MYC in basal mammary epithelial cells is sufficient to induce luminal cell identity markers. We propose that supraphysiological MYC expression impacts on mammary epithelial cell identity by repressing lineage-specific target genes. Such abrupt cell identity switch could interfere with adhesion-dependent survival signaling and thus promote apoptosis in pre-malignant epithelial tissue.     

Negative regulation of L-arabinose metabolism in Bacillus subtilis: characterization of the araR (araC) gene.

The Bacillus subtilis araC locus, mapped at about 294 degrees on the genetic map, was defined by mutations conferring an Ara- phenotype to strains bearing the metabolic araA, araB, and araD wild-type alleles (located at about 256 degrees on the genetic map) and by mutants showing constitutive expression of the three genes. In previous work, it has been postulated that the gene in which these mutations lie exerts its effect on the ara metabolic operon in trans, and this locus was named araC by analogy to the Escherichia coli regulatory gene. Here, we report the cloning and sequencing of the araC locus. This region comprises two open reading frames with divergently arranged promoters, the regulatory gene, araC, encoding a 41-kDa polypeptide, and a partially cloned gene, termed araE, which most probably codes for a permease involved in the transport of L-arabinose. The DNA sequence of araC revealed that its putative product is very similar to a number of bacterial negative regulators (the GalR-LacI family). However, a helix-turn-helix motif was identified in the N-terminal region by its identity to the consensus signature sequence of another group of repressors, the GntR family. The lack of similarity between the predicted primary structure of the product encoded by the B. subtilis regulatory gene and the AraC regulator from E. coli and the apparently different modes of action of these two proteins lead us to propose a new name, araR, for this gene. The araR gene is monocistronic, and the promoter region contains -10 and -35 regions (as determined by primer extension analysis) similar to those recognized by RNA polymerase containing the major vegetative cell sigma factor sigmaA. An insertion-deletion mutation in the araR gene leads to constitutive expression of the L-arabinose metabolic operon. We demonstrate that the araR gene codes for a negative regulator of the ara operon and that the expression of araR is repressed by its own product.     

Study of dosage compensation in Drosophila

Using a sensitive RT-QPCR assay, we analyzed the regulatory effects of sex and different dosage compensation mutations in Drosophila. To validate the assay, we showed that regulation for several genes indeed varied with the number of functional copies of that gene. We then confirmed that dosage compensation occurred for most genes we examined in male and female flies. Finally, we examined the effects on regulation of several genes in the MSL pathway, presumed to be involved in sex-dependent determination of regulation. Rather than seeing global alterations of either X chromosomal or autosomal genes, regulation of genes on either the X chromosome or the autosomes could be elevated, depressed, or unaltered between sexes in unpredictable ways for the various MSL mutations. Relative dosage for a given gene between the sexes could vary at different developmental times. Autosomal genes often showed deranged regulatory levels, indicating they were in pathways perturbed by X chromosomal changes. As exemplified by the BR-C locus and its dependent Sgs genes, multiple genes in a given pathway could exhibit coordinate regulatory modulation. The variegated pattern shown for expression of both X chromosomal and autosomal loci underscores the complexity of gene expression so that the phenotype of MSL mutations does not reflect only simple perturbations of genes on the X chromosome.     

Finding Driver Pathways in Cancer: Models and Algorithms

ABSTRACT: BACKGROUND: Cancer sequencing projects are now measuring somatic mutations in large numbers of cancer genomes. A key challenge in interpreting these data is to distinguish driver mutations, mutations important for cancer development, from passenger mutations that have accumulated in somatic cells but without functional consequences. A common approach to identify genes harboring driver mutations is a single gene test that identifies individual genes that are recurrently mutated in a significant number of cancer genomes. However, the power of this test is reduced by: (1) the necessity of estimating the background mutation rate (BMR) for each gene; (2) the mutational heterogeneity in most cancers meaning that groups of genes (e.g. pathways), rather than single genes, are the primary target of mutations. RESULTS: We investigate the problem of discovering driver pathways, groups of genes containing driver mutations, directly from cancer mutation data and without prior knowledge of pathways or other interactions between genes. We introduce two generative models of somatic mutations in cancer and study the algorithmic complexity of discovering driver pathways in both models. We show that a single gene test for driver genes is highly sensitive to the estimate of the BMR. In contrast, we show that an algorithmic approach that maximizes a straightforward measure of the mutational properties of a driver pathway successfully discovers these groups of genes without an estimate of the BMR. Moreover, this approach is also successful in the case when the observed frequencies of passenger and driver mutations are indistinguishable, a situation where single gene tests fail. CONCLUSIONS: Accurate estimation of the BMR is a challenging task. Thus, methods that do not require an estimate of the BMR, such as the ones we provide here, can give increased power for the discovery of driver genes.     

Finding co-mutated genes and candidate cancer genes in cancer genomes by stratified false discovery rate control

Finding candidate cancer genes playing causal roles in carcinogenesis is an important task in cancer research. The non-randomness of the co-mutation of genes in cancer samples can provide statistical evidence for these genes' involvement in carcinogenesis. It can also provide important information on the functional cooperation of gene mutations in cancer. However, due to the relatively small sample sizes used in current high-throughput somatic mutation screening studies and the extraordinary large-scale hypothesis tests, the statistical power of finding co-mutated gene pairs based on high-throughput somatic mutation data of cancer genomes is very low. Thus, we proposed a stratified FDR (False Discovery Rate) control approach, for identifying significantly co-mutated gene pairs according to the mutation frequency of genes. We then compared the identified co-mutated gene pairs separately by pre-selecting genes with higher mutation frequencies and by the stratified FDR control approach. Finally, we searched for pairs of pathways annotated with significantly more between-pathway co-mutated gene pairs to evaluate the functional roles of the identified co-mutated gene pairs. Based on two datasets of somatic mutations in cancer genomes, we demonstrated that, at a given FDR level, the power of finding co-mutated gene pairs could be increased by pre-selecting genes with higher mutation frequencies. However, many true co-mutation between genes with lower mutation rates will still be missed. By the stratified FDR control approach, many more co-mutated gene pairs could be found. Finally, the identified pathway pairs significantly overrepresented with between-pathway co-mutated gene pairs suggested that their co-dysregulations may play causal roles in carcinogenesis. The stratified FDR control strategy is efficient in identifying co-mutated gene pairs and the genes in the identified co-mutated gene pairs can be considered as candidate cancer genes because their non-random co-mutations in cancer genomes are highly unlikely to be attributable to chance.     

AID-targeting and hypermutation of non-immunoglobulin genes does not correlate with proximity to immunoglobulin genes in germinal center B cells

Upon activation, B cells divide, form a germinal center, and express the activation induced deaminase (AID), an enzyme that triggers somatic hypermutation of the variable regions of immunoglobulin (Ig) loci. Recent evidence indicates that at least 25% of expressed genes in germinal center B cells are mutated or deaminated by AID. One of the most deaminated genes, c-Myc, frequently appears as a translocation partner with the Ig heavy chain gene (Igh) in mouse plasmacytomas and human Burkitt's lymphomas. This indicates that the two genes or their double-strand break ends come into close proximity at a biologically relevant frequency. However, the proximity of c-Myc and Igh has never been measured in germinal center B cells, where many such translocations are thought to occur. We hypothesized that in germinal center B cells, not only is c-Myc near Igh, but other mutating non-Ig genes are deaminated by AID because they are near Ig genes, the primary targets of AID. We tested this "collateral damage" model using 3D-fluorescence in situ hybridization (3D-FISH) to measure the distance from non-Ig genes to Ig genes in germinal center B cells. We also made mice transgenic for human MYC and measured expression and mutation of the transgenes. We found that there is no correlation between proximity to Ig genes and levels of AID targeting or gene mutation, and that c-Myc was not closer to Igh than were other non-Ig genes. In addition, the human MYC transgenes did not accumulate mutations and were not deaminated by AID. We conclude that proximity to Ig loci is unlikely to be a major determinant of AID targeting or mutation of non-Ig genes, and that the MYC transgenes are either missing important regulatory elements that allow mutation or are unable to mutate because their new nuclear position is not conducive to AID deamination.     

Elements of the maize A1 promoter required for transactivation by the anthocyanin B/C1 or phlobaphene P regulatory genes.

The extensive genetic and molecular characterization of the flavonoid pathway's structural and regulatory genes has provided some of the most detailed knowledge of gene interactions in plants. In maize flavonoid biosynthesis, the A1 gene is independently regulated in the anthocyanin and phlobaphene pathways. Anthocyanin production requires the expression of the C1 or PI and R or B regulatory genes, whereas phlobaphene production requires only the P regulatory gene. By deletion analysis of the A1 promoter, we show that the sequences between -123 and -88 are critical for activation by anthocyanin and phlobaphene regulatory genes. Linker-scanner mutations indicated that the -123 to -100 region is more important for transactivation by the P protein. The -98 to -88 region is more important for B/C1 transactivation and shows a strong homology with the region of the Bz1 anthocyanin structural gene promoter shown to be activated by B/C1 and not by P. We identified a 14-bp consensus sequence that is also present in the promoters of three other genes in the anthocyanin pathway, and we propose a model for how the flavonoid regulatory proteins interact with the promoters of the structural genes.     

Linkage in cultured Chinese hamster cells of two genes, emtB and leuS, involved in protein synthesis and isolation of cell lines with mutations in three linked genes

We have determined via segregation analyses from appropriate hybrids that two genes involved in protein synthesis, one encoding for a ribosomal protein (emtB) and one encoding for leucyl-tRNA synthetase (leuS), cosegregate at a very high frequency and are linked in both Chinese hamster ovary and lung cells. In contrast, the emtA locus, defined by a second complementation group of emetine-resistant mutants which also have alterations affecting protein synthesis and probably the ribosome, is not linked to leuS. In addition, we have determined that a third gene, one that can be altered to give rise to chromate resistance, is syntenic with emtB and leuS. We have selected cell lines with mutations in each of these three linked genes and have shown that the three loci cosegregate at a high frequency. Because the mutations in these three linked genes provide easily distinguishable phenotypes, these cell lines should provide a powerful tool for examining several important questions concerning mitotic recombination in somatic cells.