Investigating In Situ Expression of c-MYC and Candidate Ubiquitin-Specific Proteases in DLBCL and Assessment for Peptidyl Disruptor Molecule against c-MYC-USP37 Complex.

Diffuse Large B-Cell Lymphoma (DLBCL) is the most common form of non-Hodgkin's lymphoma (NHL). Elevated expression of c-MYC in DLBCL is associated with poor prognosis of the disease. In different cancers, c-MYC has been found regulated by different ubiquitin-specific proteases (USPs), but to date, the role of USPs in c-MYC regulation has not been investigated in DLBCL. In this study, in situ co expression of c-MYC and three candidates USPs, USP28, USP36 and USP37, have been investigated in both the ABC and GCB subtypes of DLBCL. This shows that USP37 expression is positively correlated with the c-MYC expression in the ABC subtype of DLBCL. Structurally, both c-MYC and USP37 has shown large proportion of intrinsically disordered regions, minimizing their chances for full structure crystallization. Peptide array and docking simulations has shown that N-terminal region of c-MYC interacts directly with residues within and in proximity of catalytically active C19 domain of the USP37. Given the structural properties of the interaction sites in the c-MYC-USP37 complex, a peptidyl inhibitor has been designed. Molecular docking has shown that the peptide fits well in the targeted site of c-MYC, masking most of its residues involved in the binding with USP37. The findings could further be exploited to develop therapeutic interventions against the ABC subtype of DLBCL.

Assessment of MYC/PTEN Status by Gene-Protein Assay in Grade Group 2 Prostate Biopsies.

This study leveraged a gene-protein assay to assess MYC and PTEN status at prostate cancer biopsy and examined the association with adverse outcomes after surgery. MYC gain and PTEN loss were simultaneously assessed by chromogenic in situ hybridization and immunohistochemistry, respectively, using 277 Grade Group 2 needle biopsies that were followed by prostatectomy. The maximal size of cribriform Gleason pattern 4 carcinoma (CRIB), the presence of intraductal carcinoma (IDC), and percentage of Gleason pattern 4 carcinoma at biopsy were also annotated. MYC gain or PTEN loss was present in 19% and 18% of biopsies, respectively, whereas both alterations were present in 9% of biopsies. Tumors with one or both alterations were significantly more likely to have non-organ-confined disease (NOCD) at radical prostatectomy. In logistic regression models, including clinical stage, tumor volume on biopsy, and presence of CRIB/IDC, cases with MYC gain and PTEN loss remained at higher risk for NOCD (odds ratio, 6.23; 95% CI, 1.74-24.55; P = 0.005). The area under the curve for a baseline model using CAPRA variables (age, prostate-specific antigen, percentage of core involvement, clinical stage) was increased from 0.68 to 0.69 with inclusion of CRIB/IDC status and to 0.75 with MYC/PTEN status. Dual MYC/PTEN status can be assessed in a single slide and is independently associated with increased risk of NOCD for Grade Group 2 biopsies.

Assessment of the WAP-Myc mouse mammary tumor model for spontaneous metastasis.

Breast cancer is the most common form of cancer in women. Despite significant therapeutic advances in recent years, breast cancer also still causes the greatest number of cancer-related deaths in women, the vast majority of which (> 90%) are caused by metastases. However, very few mouse mammary cancer models exist that faithfully recapitulate the multistep metastatic process in human patients. Here we assessed the suitability of a syngrafting protocol for a Myc-driven mammary tumor model (WAP-Myc) to study autochthonous metastasis. A moderate but robust spontaneous lung metastasis rate of around 25% was attained. In addition, increased T cell infiltration was observed in metastatic tumors compared to donor and syngrafted primary tumors. Thus, the WAP-Myc syngrafting protocol is a suitable tool to study the mechanisms of metastasis in MYC-driven breast cancer.

Custom G4 Microarrays Reveal Selective G-Quadruplex Recognition of Small Molecule BMVC: A Large-Scale Assessment of Ligand Binding Selectivity.

G-quadruplexes (G4) are considered new drug targets for human diseases such as cancer. More than 10,000 G4s have been discovered in human chromatin, posing challenges for assessing the selectivity of a G4-interactive ligand. 3,6-bis(1-Methyl-4-vinylpyridinium) carbazole diiodide (BMVC) is the first fluorescent small molecule for G4 detection in vivo. Our previous structural study shows that BMVC binds to the MYC promoter G4 (MycG4) with high specificity. Here, we utilize high-throughput, large-scale custom DNA G4 microarrays to analyze the G4-binding selectivity of BMVC. BMVC preferentially binds to the parallel MycG4 and selectively recognizes flanking sequences of parallel G4s, especially the 3'-flanking thymine. Importantly, the microarray results are confirmed by orthogonal NMR and fluorescence binding analyses. Our study demonstrates the potential of custom G4 microarrays as a platform to broadly and unbiasedly assess the binding selectivity of G4-interactive ligands, and to help understand the properties that govern molecular recognition.

Assessment of HER-2/neu, с-MYC and CCNE1 gene copy number variations and protein expression in endometrial carcinomas.

AIM: To analyze copy number variations of HER-2/neu, c-MYC and CCNE1 oncogenes and their protein expression in endometrioid endometrial carcinomas in relation to the degree of tumor progression and presence of a family history of cancer in cancer patients. MATERIALS AND METHODS: The study was conducted on endometrial cancer (EC) samples from 68 patients with I-II FIGO stages of disease. Copy number analysis of HER-2/neu, c-MYC and CCNE1 genes was performed by quantitative PCR. Protein expression was analyzed using immunohistochemistry. RESULTS: Assessment of copy number variations of HER-2/neu, c-MYC and CCNE1 genes revealed their amplification in the tumors of 18.8, 25.0 and 14.3% of EC patients, respectively. High expression of corresponding proteins was detected in 14.6, 23.5 and 65.6% of patients, respectively. It was established that HER-2/neu gene amplification is more common in the group of tumors of low differentiation grade than in moderate grade EC (35.7 and 5.5% of cases, respectively, p < 0.05). Also, high expression of c-Myc protein was more frequently observed in low differentiated tumors compared to the moderately differentiated EC (36.6 and 13.2% of cases, respectively, p < 0.05). Expression of HER-2/neu and cyclin E proteins was found to be dependent on the depth of tumor invasion into the myometrium. High expression of HER-2/neu protein was observed in 25.0 and 4.1% of EC patients with tumor invasion > ½ and < ½ of the myometrium, respectively, and cyclin E - in 86.7 and 46.6% of cases, respectively, p < 0.05. It was shown that among patients with a family history of cancer, a larger proportion of cases with high expression of c-Myc protein was observed compared to the group of patients with sporadic tumors (43.8 and 17.3%, respectively; p < 0.05). CONCLUSIONS: Amplification of HER-2/neu gene, along with high expression of c-Myc, HER-2/neu and cyclin E proteins, are associated with such indices of tumor progression as a low differentiation grade and deep myometrial invasion, suggesting the potential possibility of including these markers in the panel for determining the molecular EC subtype associated with an aggressive course of the disease. In a certain category of EC patients, there is a relationship between a family history of cancer and high expression of c-Myc protein.

Mild hypothermia affects the morphology and impairs glutamine-induced anabolic response in human primary myotubes.

The specific impact of reduced temperature on skeletal muscle adaptation has been poorly investigated. Cold water immersion, one situation leading to decreased skeletal muscle temperature, is commonly proposed to reduce the perception of fatigue and muscle soreness after strenuous exercise. In contrast, it may impair long-term benefits of resistance exercise training on muscle strength and hypertrophy. To date, the physiological factors responsible for this blunted muscle adaptation remain unclear. Here, we used a cell culture model of human primary myotubes to specifically investigate the intrinsic behavior of muscle cells during mild hypothermia (MH). Newly formed myotubes were exposed to either 37°C or 32°C to evaluate the effect of MH on myotube size and morphology, protein synthesis, and anabolic signaling. We also compared the glutamine (GLUT)-induced hypertrophic response between myotubes incubated at 32°C or 37°C. We showed that 48 h exposure to MH altered the cellular morphology (greater myotube area, shorter myosegments, myotubes with irregular shape) and impaired GLUT-induced myotube hypertrophy. Moreover, MH specifically reduced protein synthesis at 8 h. This result may be explained by an altered regulation of ribosome biogenesis, as evidenced by a lower expression of 45S pre-ribosomal RNA and MYC protein, and a lower total RNA concentration. Furthermore, MH blunted GLUT-induced increase in protein synthesis at 8 h, a finding consistent with an impaired activation of the mechanistic target of rapamycin pathway. In conclusion, this study demonstrates that MH impairs the morphology of human myotubes and alters the hypertrophic response to GLUT.

Activation of SRY accounts for male-specific hepatocarcinogenesis: Implication in gender disparity of hepatocellular carcinoma.

Sex affects the risk, treatment responses and outcome of many types of cancers. The mechanism of gender disparity in development of hepatocellular carcinoma (HCC) remains obscure. Sex-determining region on Y chromosome (SRY) was overexpressed in approximate 84% male patient HCC. Moreover, we are the first to generate a liver-specific transgenic (TG) murine model with overexpression of the male specific gene SRY. Subject to a single intraperitoneal injection N-nitrosodiethylamine (DEN) at day 14, TG and wildtype (WT) mice of both genders were sacrificed at different time points (6-13.5 months). Overexpression of SRY in male TG and ectopic expression of SRY in female TG livers promoted DEN-induced hepatocarcinogenesis compared to age- and sex-matched WT. This accelerated tumorigenesis in TG of both genders was a consequence of increased injury and inflammation, fibrosis, and compensatory enhancement in hepatocytes proliferation secondary to activation of downstream targets Sox9 and platelet-derived growth factor receptor α (PDGFRα)/phosphoinositide 3-kinase (PI3K)/Akt and c-myc/CyclinD1. In conclusion, activation of SRY and its downstream Sox9 and PDGFRα pathways are commonly involved in male hepatocarcinogenesis, which provides novel insights into gender disparity and sex-specific therapeutic strategies of HCC.

Increased ß­catenin and c-myc expression predict aggressive growth of non-functioning pituitary adenomas: An assessment using a tissue microarray-based approach.

Non-functional pituitary adenomas (NFPAs) account for 80% of pituitary adenomas with the majority of these exhibiting recurrences post-surgery. Overexpression of ß-catenin and c­myc is common in numerous invasive tumors. The present study sought to investigate the correlation of ß­catenin and c­myc expression levels with aggressive growth and recurrence of NFPAs, using immunohistochemical examination of tissue microarrays. Tissue microarrays comprised 212 NFPAs specimens and 10 healthy specimens as controls. NFPAs were categorized as non­aggressive or aggressive. Immunohistochemical examination was performed to determine the expression of ß­catenin and c­myc. Correlation of the expression levels of ß­catenin and c­myc with clinicopathological parameters, including aggressiveness and recurrence, were assessed by univariate, multivariate and logistic regression analysis. Increased expression of ß­catenin and c­myc was detected in the majority of aggressive NFPAs specimens (71.1 and 88.7%, respectively). There was a significant positive correlation between ß­catenin and c­myc expression and aggressiveness [P=0.001, Odds Ratio (OR)=4.011; P<0.001, OR=30.833]. Only ß­catenin expression demonstrated a significant correlation with recurrence in NFPAs (P=0.021, OR=2.571). ß­catenin and c­myc were demonstrated to be potential biomarkers for aggressive NFPAs and in the future, ß-catenin may serve as a marker for aggressive behavior and recurrence in NFPAs.

Protection against High-Fat-Diet-Induced Obesity in MDM2C305F Mice Due to Reduced p53 Activity and Enhanced Energy Expenditure.

The RPL11-MDM2 interaction constitutes a p53 signaling pathway activated by deregulated ribosomal biosynthesis in response to stress. Mice bearing an MDM2C305F mutation that disrupts RPL11-MDM2 binding were analyzed on a high-fat diet (HFD). The Mdm2C305F/C305F mice, although phenotypically indistinguishable from wild-type (WT) mice when fed normal chow, demonstrated decreased fat accumulation along with improved insulin sensitivity and glucose tolerance after prolonged HFD feeding. We found that HFD increases expression of c-MYC and RPL11 in both WT and Mdm2C305F/C305F mice; however, p53 was induced in WT but not in Mdm2C305F/C305F mice. Reduced p53 activity in HFD-fed Mdm2C305F/C305F mice resulted in higher levels of p53 downregulated targets GLUT4 and SIRT1, leading to increased biosynthesis of NAD+, and increased energy expenditure. Our study reveals a role for the RPL11-MDM2-p53 pathway in fat storage during nutrient excess and suggests that targeting this pathway may be a potential treatment for obesity.

Premalignant alteration assessment in liver-like tissue derived from embryonic stem cells by aristolochic acid I exposure.

The in vitro predictive evaluation of chemical carcinogenicity based on hepatic premalignance has so far not been established. Here, we report a novel approach to investigate the premalignant events triggered by human carcinogen aristolochic acid I (AAI) in the liver-like tissue derived from mouse embryonic stem cells. By AAI exposure, the liver-like tissue exhibited the paracrine interleukin-6 phenotypic characteristics. Hepatocytes expressed STAT3/p-STAT3, c-Myc and Lin28B in parallel. Some of them displayed the dedifferentiation characteristics, such as full of α-fetoprotein granules, increase in size, and nucleocytoplasmic shuttle of Oct4. When these cells were injected into mice, the xenografts mostly displayed the uniform area of hepatic-like tissue with malignant nuclei. The hepatic malignant markers, α-fetoprotein, cytokeratin 7 and cytokeratin 19, were co-expressed in albumin-positive areas, respectively. In conclusion, we established an approach to predict the hepatic premalignance triggered by carcinogen AAI. This premalignant assay system might aid to evaluate the effects of potential carcinogens in liver, and probably to screen the protecting against hepatocarcinogenic efficacy of pharmaceuticals in vitro.

Assessment of CD37 B-cell antigen and cell of origin significantly improves risk prediction in diffuse large B-cell lymphoma.

CD37 (tetraspanin TSPAN26) is a B-cell surface antigen widely expressed on mature B cells. CD37 is involved in immune regulation and tumor suppression but its function has not been fully elucidated. We assessed CD37 expression in de novo diffuse large B-cell lymphoma (DLBCL), and investigated its clinical and biologic significance in 773 patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) and 231 patients treated with CHOP. We found that CD37 loss (CD37-) in ∼60% of DLBCL patients showed significantly decreased survival after R-CHOP treatment, independent of the International Prognostic Index (IPI), germinal center B-cell-like (GCB)/activated B-cell-like (ABC) cell of origin, nodal/extranodal primary origin, and the prognostic factors associated with CD37-, including TP53 mutation, NF-κBhigh, Mychigh, phosphorylated STAT3high, survivinhigh, p63-, and BCL6 translocation. CD37 positivity predicted superior survival, abolishing the prognostic impact of high IPI and above biomarkers in GCB-DLBCL but not in ABC-DLBCL. Combining risk scores for CD37- status and ABC cell of origin with the IPI, defined as molecularly adjusted IPI for R-CHOP (M-IPI-R), or IPI plus immunohistochemistry (IHC; IPI+IHC) for CD37, Myc, and Bcl-2, significantly improved risk prediction over IPI alone. Gene expression profiling suggested that decreased CD20 and increased PD-1 levels in CD37- DLBCL, ICOSLG upregulation in CD37+ GCB-DLBCL, and CD37 functions during R-CHOP treatment underlie the pivotal role of CD37 status in clinical outcomes. In conclusion, CD37 is a critical determinant of R-CHOP outcome in DLBCL especially in GCB-DLBCL, representing its importance for optimal rituximab action and sustained immune responses. The combined molecular and clinical prognostic indices, M-IPI-R and IPI+IHC, have remarkable predictive values in R-CHOP-treated DLBCL.

Bimolecular Fluorescence Complementation (BiFC) Analysis of Protein-Protein Interactions and Assessment of Subcellular Localization in Live Cells.

Bimolecular fluorescence complementation (BiFC) is a fluorescence imaging technique used to visualize protein-protein interactions (PPIs) in live cells and animals. One unique application of BiFC is to reveal subcellular localization of PPIs. The superior signal-to-noise ratio of BiFC in comparison with fluorescence resonance energy transfer or bioluminescence resonance energy transfer enables its wide applications. Here, we describe how confocal microscopy can be used to detect and quantify PPIs and their subcellular localization. We use basic leucine zipper transcription factor proteins as an example to provide a step-by-step BiFC protocol using a Nikon A1 confocal microscope and NIS-Elements imaging software. The protocol given below can be readily adapted for use with other confocal microscopes or imaging software.

Enhanced Generation of Integration-free iPSCs from Human Adult Peripheral Blood Mononuclear Cells with an Optimal Combination of Episomal Vectors.

We previously reported the generation of integration-free induced pluripotent stem cells from adult peripheral blood (PB) with an improved episomal vector (EV) system, which uses the spleen focus-forming virus U3 promoter and an extra factor BCL-XL (B). Here we show an ∼100-fold increase in efficiency by optimizing the vector combination. The two most critical factors are: (1) equimolar expression of OCT4 (O) and SOX2 (S), by using a 2A linker; (2) a higher and gradual increase in the MYC (M) to KLF4 (K) ratio during the course of reprogramming, by using two individual vectors to express M and K instead of one. The combination of EV plasmids (OS + M + K + B) is comparable with Sendai virus in reprogramming efficiency but at a fraction of the cost. The generated iPSCs are indistinguishable from those from our previous approach in pluripotency and phenotype. This improvement lays the foundation for broad applications of episomal vectors in PB reprogramming.

Integrating understanding of epidemiology and genomics in B-cell non-Hodgkin lymphoma as a pathway to novel management strategies.

Non-Hodgkin lymphomas include a biologically and clinically heterogeneous group of cancers distinguished by genetics, histology, and treatment outcomes. New discoveries regarding the genomic alterations and epidemiological exposures associated with these lymphomas have enhanced our understanding of factors that contribute to lymphomagenesis for specific subtypes. We explore the impact of normal B-cell biology engineered for recognizing a wide variety of antigens on the development of specific lymphoma subtypes, review lymphoma genetics, and examine the epidemiology of B-cell NHLs including recent investigations of risk factors for particular lymphoma subtypes based on large pooled analyses. Burkitt lymphoma, an aggressive form of B-cell NHL involving translocation of the MYC gene and an immunoglobulin gene has been associated with a history of eczema, hepatitis C, and occupation as a cleaner. Increased risk of diffuse large B-cell lymphoma has been associated with increased young adult body mass index, history of B-cell-activating autoimmune diseases, hepatitis C, and several single nucleotide variants involving the human leukocyte antigen (HLA) region of chromosome 6 and non-HLA loci near EXOC2, PVT1, MYC, and NCOA1. Tumor sequencing studies suggest that multiple pathways are involved in the development of DLBCL. Additional studies of epidemiological exposures, genome wide associations, and tumor sequencing in follicular, lymphoplasmacytic, marginal zone, and mantle cell lymphoma demonstrate overlapping areas of increased risk factors and unique factors for specific subtypes. Integrating these findings is important for constructing comprehensive models of NHL pathogenesis, which could yield novel targets for therapy and strategies for lymphoma prevention in certain populations.

Comprehensive Assessment and Classification of High-Grade B-cell Lymphomas.

High-grade B-cell lymphomas (HGBCLs) are a heterogeneous group of neoplasms that include subsets of diffuse large B-cell lymphoma, Burkitt lymphoma, and lymphomas with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma. Morphologically indistinguishable HGBCLs may demonstrate variable clinical courses and responses to therapy. The morphologic evaluation and classification of these neoplasms must be followed by further genetic and immunophenotypic work-up. These additional diagnostic modalities lead to a comprehensive stratification of HGBCL that determines the prognosis and optimal therapy. This article reviews the well-established and emerging biomarkers that are most relevant to the clinical management of HGBCL.

Targeting megakaryocytic-induced fibrosis in myeloproliferative neoplasms by AURKA inhibition.

Primary myelofibrosis (PMF) is characterized by bone marrow fibrosis, myeloproliferation, extramedullary hematopoiesis, splenomegaly and leukemic progression. Moreover, the bone marrow and spleens of individuals with PMF contain large numbers of atypical megakaryocytes that are postulated to contribute to fibrosis through the release of cytokines, including transforming growth factor (TGF)-ß. Although the Janus kinase inhibitor ruxolitinib provides symptomatic relief, it does not reduce the mutant allele burden or substantially reverse fibrosis. Here we show through pharmacologic and genetic studies that aurora kinase A (AURKA) represents a new therapeutic target in PMF. Treatment with MLN8237, a selective AURKA inhibitor, promoted polyploidization and differentiation of megakaryocytes with PMF-associated mutations and had potent antifibrotic and antitumor activity in vivo in mouse models of PMF. Moreover, heterozygous deletion of Aurka was sufficient to ameliorate fibrosis and other PMF features in vivo. Our data suggest that megakaryocytes drive fibrosis in PMF and that targeting them with AURKA inhibitors has the potential to provide therapeutic benefit.

Quantitative determination of c-myc facilitates the assessment of prognosis of OSCC patients.

Myc genes are a family of proto-oncogenes whose proteins are implicated in the regulation of cell proliferation, differentiation and apoptosis, and in regulating the activity of genes involved in cell division. The aim of the present study was to establish a quantitative description of the expression of c-myc and evaluate its relationship with other clinical and prognostic factors, as well as to establish a multivariate survival prediction model. This is a retrospective study of 68 patients diagnosed with oral squamous cell carcinoma (OSCC). We constructed a tissue microarray for investigating the expression of c-myc by immunohistochemistry. Statistical analyses were carried out, and a multivariate model that predicts survival was established. The average expression of c-myc was 50.32 (SD, 26.05) with a range from 6.60 to 99.48; similar for initial and advanced tumor stages. Non-smoking patients had higher levels of c-myc, showing statistically significant differences (Kruskal-Wallis χ2=5.975; p=0.05). We found no statistically significant relationship between the quantitative expression of c-myc and any other clinical or pathological parameters. For each unit of increase of c-myc, the risk increased by 1.15 (p<0.001; HR, 1.150; 95% CI, 1062-1245). Further study of this protein, which may have a significant diagnostic, prognostic and therapeutic value is warranted. Its determination can be valuable when used together with other markers to assess the prognosis of OSCC patients.

Cellular MYCro economics: Balancing MYC function with MYC expression.

The expression levels of the MYC oncoprotein have long been recognized to be associated with the outputs of major cellular processes including proliferation, cell growth, apoptosis, differentiation, and metabolism. Therefore, to understand how MYC operates, it is important to define quantitatively the relationship between MYC input and expression output for its targets as well as the higher-order relationships between the expression levels of subnetwork components and the flow of information and materials through those networks. Two different views of MYC are considered, first as a molecular microeconomic manager orchestrating specific positive and negative responses at individual promoters in collaboration with other transcription and chromatin components, and second, as a macroeconomic czar imposing an overarching rule onto all active genes. In either case, c-myc promoter output requires multiple inputs and exploits diverse mechanisms to tune expression to the appropriate levels relative to the thresholds of expression that separate health and disease.