Functional interactions among members of the MAX and MLX transcriptional network during oncogenesis.

The transcription factor MYC and its related family members MYCN and MYCL have been implicated in the etiology of a wide spectrum of human cancers. Compared to other oncoproteins, such as RAS or SRC, MYC is unique because its protein coding region is rarely mutated. Instead, MYC's oncogenic properties are unleashed by regulatory mutations leading to unconstrained high levels of expression. Under both normal and pathological conditions MYC regulates multiple aspects of cellular physiology including proliferation, differentiation, apoptosis, growth and metabolism by controlling the expression of thousands of genes. How a single transcription factor exerts such broad effects remains a fascinating puzzle. Notably, MYC is part of a network of bHLHLZ proteins centered on the MYC heterodimeric partner MAX and its counterpart, the MAX-like protein MLX. This network includes MXD1-4, MNT, MGA, MONDOA and MONDOB proteins. With some exceptions, MXD proteins have been functionally linked to cell cycle arrest and differentiation, while MONDO proteins control cellular metabolism. Although the temporal expression patterns of many of these proteins can differ markedly they are frequently expressed simultaneously in the same cellular context, and potentially bind to the same, or similar DNA consensus sequence. Here we review the activities and interactions among these proteins and propose that the broad spectrum of phenotypes elicited by MYC deregulation is intimately connected to the functions and regulation of the other network members. Furthermore, we provide a meta-analysis of TCGA data suggesting that the coordinate regulation of the network is important in MYC driven tumorigenesis. This article is part of a Special Issue entitled: Myc proteins in cell biology and pathology.

A Pilot Study of Clinical Targeted Next Generation Sequencing for Prostate Cancer: Consequences for Treatment and Genetic Counseling.

BACKGROUND: Targeted next generation sequencing (tNGS) is increasingly used in oncology for therapeutic decision-making, but is not yet widely used for prostate cancer. The objective of this study was to determine current clinical utility of tNGS for prostate cancer management. METHODS: Seven academic genitourinary medical oncologists recruited and consented patients with prostate cancer, largely with unusual clinical and/or pathologic features, from 2013 to 2015. UW-OncoPlex was performed on formalin-fixed, paraffin-embedded (FFPE) primary tumors and/or metastatic biopsies. Results were discussed at a multidisciplinary precision tumor board prior to communicating to patients. FFPE tumor DNA was extracted for tNGS analysis of 194 cancer-associated genes. Results, multidisciplinary discussion, and treatment changes were recorded. RESULTS: Forty-five patients consented and 42 had reportable results. Findings included mutations in genes frequently observed in prostate cancer. We also found alterations in genes where targeted treatments were available and/or in clinical trials. 4/42 (10%) cases, change in treatment directly resulted from tNGS and multidisciplinary discussion. In 30/42 (71%) cases additional options were available but not pursued and/or were pending. Notably, 10/42 (24%) of patients harbored suspected germline mutations in moderate or high-penetrance cancer risk genes, including BRCA2, TP53, ATM, and CHEK2. One patient's tumor had bi-allelic MSH6 mutation and microsatellite instability. In total, 34/42 (81%) cases resulted in some measure of treatment actionability. Limitations include small size and limited clinical outcomes. CONCLUSIONS: Targeted NGS tumor sequencing may help guide immediate and future treatment options for men with prostate cancer. A substantial subset had germline mutations in cancer predisposition genes with potential clinical management implications for men and their relatives. Prostate 76:1303-1311, 2016. © 2016 Wiley Periodicals, Inc.

The non-random clustering of non-synonymous substitutions and its relationship to evolutionary rate.

BACKGROUND: Protein sequences are subject to a mosaic of constraint. Changes to functional domains and buried residues, for example, are more apt to disrupt protein structure and function than are changes to residues participating in loops or exposed to solvent. Regions of constraint on the tertiary structure of a protein often result in loose segmentation of its primary structure into stretches of slowly- and rapidly-evolving amino acids. This clustering can be exploited, and existing methods have done so by relying on local sequence conservation as a signature of selection to help identify functionally important regions within proteins. We invert this paradigm by leveraging the regional nature of protein structure and function to both illuminate and make use of genome-wide patterns of local sequence conservation. RESULTS: Our hypothesis is that the regional nature of structural and functional constraints will assert a positive autocorrelation on the evolutionary rates of neighboring sites, which, in a pairwise comparison of orthologous proteins, will manifest itself as the clustering of non-synonymous changes across the amino acid sequence. We introduce a dispersion ratio statistic to test this and related hypotheses. Using genome-wide interspecific comparisons of orthologous protein pairs, we reveal a strong log-linear relationship between the degree of clustering and the intensity of constraint. We further demonstrate how this relationship varies with the evolutionary distance between the species being compared. We provide some evidence that proteins with a history of positive selection deviate from genome-wide trends. CONCLUSIONS: We find a significant association between the evolutionary rate of a protein and the degree to which non-synonymous changes cluster along its primary sequence. We show that clustering is a non-redundant predictor of evolutionary rate, and we speculate that conflicting signals of clustering and constraint may be indicative of a historical period of relaxed selection.

Arming oHSV with ULBP3 drives abscopal immunity in lymphocyte-depleted glioblastoma.

Oncolytic viruses induce local tumor destruction and inflammation. Whether virotherapy can also overcome immunosuppression in noninfected tumor areas is under debate. To address this question, we have explored immunologic effects of oncolytic herpes simplex viruses (oHSVs) in a genetically engineered mouse model of isocitrate dehydrogenase (IDH) wild-type glioblastoma, the most common and most malignant primary brain tumor in adults. Our model recapitulates the genomics, the diffuse infiltrative growth pattern, and the extensive macrophage-dominant immunosuppression of human glioblastoma. Infection with an oHSV that was armed with a UL16-binding protein 3 (ULBP3) expression cassette inhibited distant tumor growth in the absence of viral spreading (abscopal effect) and yielded accumulation of activated macrophages and T cells. There was also abscopal synergism of oHSVULBP3 with anti-programmed cell death 1 (anti-PD-1) against distant, uninfected tumor areas; albeit consistent with clinical trials in patients with glioblastoma, monotherapy with anti-PD-1 was ineffective in our model. Arming oHSV with ULBP3 led to upregulation of antigen processing and presentation gene sets in myeloid cells. The cognate ULBP3 receptor NKG2D, however, is not present on myeloid cells, suggesting a noncanonical mechanism of action of ULBP3. Overall, the myeloid-dominant, anti-PD-1-sensitive abscopal effect of oHSVULBP3 warrants further investigation in patients with IDH wild-type glioblastoma.

Genomic distinctions between metastatic lower and upper tract urothelial carcinoma revealed through rapid autopsy.

BACKGROUND: Little is known about the genomic differences between metastatic urothelial carcinoma (LTUC) and upper tract urothelial carcinoma (UTUC). We compare genomic features of primary and metastatic UTUC and LTUC tumors in a cohort of patients with end stage disease. METHODS: We performed whole exome sequencing on matched primary and metastatic tumor samples (N=37) from 7 patients with metastatic UC collected via rapid autopsy. Inter- and intra-patient mutational burden, mutational signatures, predicted deleterious mutations, and somatic copy alterations (sCNV) were analyzed. RESULTS: We investigated 3 patients with UTUC (3 primary samples, 13 metastases) and 4 patients with LTUC (4 primary samples, 17 metastases). We found that sSNV burden was higher in metastatic LTUC compared to UTUC. Moreover, the APOBEC mutational signature was pervasive in metastatic LTUC and less so in UTUC. Despite a lower overall sSNV burden, UTUC displayed greater inter- and intra-individual genomic distances at the copy number level between primary and metastatic tumors than LTUC. Our data also indicate that metastatic UTUC lesions can arise from small clonal populations present in the primary cancer. Importantly, putative druggable mutations were found across patients with the majority shared across all metastases within a patient. CONCLUSIONS: Metastatic UTUC demonstrated a lower overall mutational burden but greater structural variability compared to LTUC. Our findings suggest that metastatic UTUC displays a greater spectrum of copy number divergence from LTUC. Importantly, we identified druggable lesions shared across metastatic samples, which demonstrate a level of targetable homogeneity within individual patients.

Copy number profiling across glioblastoma populations has implications for clinical trial design.

Background: Copy number alterations form prognostic molecular subtypes of glioblastoma with clear differences in median overall survival. In this study, we leverage molecular data from several glioblastoma cohorts to define the distribution of copy number subtypes across random cohorts as well as cohorts with selection biases for patients with inherently better outcome. Methods: Copy number subtype frequency was established for 4 glioblastoma patient cohorts. Two randomly selected cohorts include The Cancer Genome Atlas (TCGA) and the German Glioma Network (GGN). Two more selective cohorts include the phase II trial ARTE in elderly patients with newly diagnosed glioblastoma and a multi-institutional cohort focused on paired resected initial/recurrent glioblastoma. The paired initial/recurrent cohort also had exome data available, which allowed for evaluation of multidimensional scaling analysis. Results: Smaller selective glioblastoma cohorts are enriched for copy number subtypes that are associated with better survival, reflecting the selection of patients who do well enough to enter a clinical trial or who are deemed well enough to undergo resection at recurrence. Adding exome data to copy number data provides additional data reflective of outcome. Conclusions: The overall outcome for diffuse glioma patients is predicted by DNA structure at initial tumor resection. Molecular signature shifts across glioblastoma populations reflect the inherent bias of patient selection toward longer survival in clinical trials. Therefore it may be important to include molecular profiling, including copy number, when enrolling patients for clinical trials in order to balance arms and extrapolate relevance to the general glioblastoma population.

Multidimensional scaling of diffuse gliomas: application to the 2016 World Health Organization classification system with prognostically relevant molecular subtype discovery.

Recent updating of the World Health Organization (WHO) classification of central nervous system (CNS) tumors in 2016 demonstrates the first organized effort to restructure brain tumor classification by incorporating histomorphologic features with recurrent molecular alterations. Revised CNS tumor diagnostic criteria also attempt to reduce interobserver variability of histological interpretation and provide more accurate stratification related to clinical outcome. As an example, diffuse gliomas (WHO grades II-IV) are now molecularly stratified based upon isocitrate dehydrogenase 1 or 2 (IDH) mutational status, with gliomas of WHO grades II and III being substratified according to 1p/19q codeletion status. For now, grading of diffuse gliomas is still dependent upon histological parameters. Independent of WHO classification criteria, multidimensional scaling analysis of molecular signatures for diffuse gliomas from The Cancer Genome Atlas (TCGA) has identified distinct molecular subgroups, and allows for their visualization in 2-dimensional (2D) space. Using the web-based platform Oncoscape as a tool, we applied multidimensional scaling-derived molecular groups to the 2D visualization of the 2016 WHO classification of diffuse gliomas. Here we show that molecular multidimensional scaling of TCGA data provides 2D clustering that represents the 2016 WHO classification of diffuse gliomas. Additionally, we used this platform to successfully identify and define novel copy-number alteration-based molecular subtypes, which are independent of WHO grading, as well as predictive of clinical outcome. The prognostic utility of these molecular subtypes was further validated using an independent data set of the German Glioma Network prospective glioblastoma patient cohort.

Deregulated Myc requires MondoA/Mlx for metabolic reprogramming and tumorigenesis.

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.

Molecular portraits of epithelial, mesenchymal, and hybrid States in lung adenocarcinoma and their relevance to survival.

Epithelial-to-mesenchymal transition (EMT) is a key process associated with tumor progression and metastasis. To define molecular features associated with EMT states, we undertook an integrative approach combining mRNA, miRNA, DNA methylation, and proteomic profiles of 38 cell populations representative of the genomic heterogeneity in lung adenocarcinoma. The resulting data were integrated with functional profiles consisting of cell invasiveness, adhesion, and motility. A subset of cell lines that were readily defined as epithelial or mesenchymal based on their morphology and E-cadherin and vimentin expression elicited distinctive molecular signatures. Other cell populations displayed intermediate/hybrid states of EMT, with mixed epithelial and mesenchymal characteristics. A dominant proteomic feature of aggressive hybrid cell lines was upregulation of cytoskeletal and actin-binding proteins, a signature shared with mesenchymal cell lines. Cytoskeletal reorganization preceded loss of E-cadherin in epithelial cells in which EMT was induced by TGFß. A set of transcripts corresponding to the mesenchymal protein signature enriched in cytoskeletal proteins was found to be predictive of survival in independent datasets of lung adenocarcinomas. Our findings point to an association between cytoskeletal and actin-binding proteins, a mesenchymal or hybrid EMT phenotype and invasive properties of lung adenocarcinomas.

An overview of MYC and its interactome.

This review is intended to provide a broad outline of the biological and molecular functions of MYC as well as of the larger protein network within which MYC operates. We present a view of MYC as a sensor that integrates multiple cellular signals to mediate a broad transcriptional response controlling many aspects of cell behavior. We also describe the larger transcriptional network linked to MYC with emphasis on the MXD family of MYC antagonists. Last, we discuss evidence that the network has evolved for millions of years, dating back to the emergence of animals.

A novel N-terminal domain may dictate the glucose response of Mondo proteins.

Glucose is a fundamental energy source for both prokaryotes and eukaryotes. The balance between glucose utilization and storage is integral for proper energy homeostasis, and defects are associated with several diseases, e.g. type II diabetes. In vertebrates, the transcription factor ChREBP is a major component in glucose metabolism, while its ortholog MondoA is involved in glucose uptake. Both MondoA and ChREBP contain five Mondo conserved regions (MCRI-V) that affect their cellular localization and transactivation ability. While phosphorylation has been shown to affect ChREBP function, the mechanisms controlling glucose response of both ChREBP and MondoA remain elusive. By incorporating sequence analysis techniques, structure predictions, and functional annotations, we synthesized data surrounding Mondo family proteins into a cohesive, accurate, and general model involving the MCRs and two additional domains that determine ChREBP and MondoA glucose response. Paramount, we identified a conserved motif within the transactivation region of Mondo family proteins and propose that this motif interacts with the phosphorylated form of glucose. In addition, we discovered a putative nuclear receptor box in non-vertebrate Mondo and vertebrate ChREBP sequences that reveals a potentially novel interaction with nuclear receptors. These interactions are likely involved in altering ChREBP and MondoA conformation to form an active complex and induce transcription of genes involved in glucose metabolism and lipogenesis.

Evolution of the Max and Mlx networks in animals.

Transcription factors (TFs) are essential for the regulation of gene expression and often form emergent complexes to perform vital roles in cellular processes. In this paper, we focus on the parallel Max and Mlx networks of TFs because of their critical involvement in cell cycle regulation, proliferation, growth, metabolism, and apoptosis. A basic-helix-loop-helix-zipper (bHLHZ) domain mediates the competitive protein dimerization and DNA binding among Max and Mlx network members to form a complex system of cell regulation. To understand the importance of these network interactions, we identified the bHLHZ domain of Max and Mlx network proteins across the animal kingdom and carried out several multivariate statistical analyses. The presence and conservation of Max and Mlx network proteins in animal lineages stemming from the divergence of Metazoa indicate that these networks have ancient and essential functions. Phylogenetic analysis of the bHLHZ domain identified clear relationships among protein families with distinct points of radiation and divergence. Multivariate discriminant analysis further isolated specific amino acid changes within the bHLHZ domain that classify proteins, families, and network configurations. These analyses on Max and Mlx network members provide a model for characterizing the evolution of TFs involved in essential networks.