A polymorphism in IRF4 affects human pigmentation through a tyrosinase-dependent MITF/TFAP2A pathway.

Sequence polymorphisms linked to human diseases and phenotypes in genome-wide association studies often affect noncoding regions. A SNP within an intron of the gene encoding Interferon Regulatory Factor 4 (IRF4), a transcription factor with no known role in melanocyte biology, is strongly associated with sensitivity of skin to sun exposure, freckles, blue eyes, and brown hair color. Here, we demonstrate that this SNP lies within an enhancer of IRF4 transcription in melanocytes. The allele associated with this pigmentation phenotype impairs binding of the TFAP2A transcription factor that, together with the melanocyte master regulator MITF, regulates activity of the enhancer. Assays in zebrafish and mice reveal that IRF4 cooperates with MITF to activate expression of Tyrosinase (TYR), an essential enzyme in melanin synthesis. Our findings provide a clear example of a noncoding polymorphism that affects a phenotype by modulating a developmental gene regulatory network.

Repeat expansions confer WRN dependence in microsatellite-unstable cancers.

The RecQ DNA helicase WRN is a synthetic lethal target for cancer cells with microsatellite instability (MSI), a form of genetic hypermutability that arises from impaired mismatch repair1-4. Depletion of WRN induces widespread DNA double-strand breaks in MSI cells, leading to cell cycle arrest and/or apoptosis. However, the mechanism by which WRN protects MSI-associated cancers from double-strand breaks remains unclear. Here we show that TA-dinucleotide repeats are highly unstable in MSI cells and undergo large-scale expansions, distinct from previously described insertion or deletion mutations of a few nucleotides5. Expanded TA repeats form non-B DNA secondary structures that stall replication forks, activate the ATR checkpoint kinase, and require unwinding by the WRN helicase. In the absence of WRN, the expanded TA-dinucleotide repeats are susceptible to cleavage by the MUS81 nuclease, leading to massive chromosome shattering. These findings identify a distinct biomarker that underlies the synthetic lethal dependence on WRN, and support the development of therapeutic agents that target WRN for MSI-associated cancers.

Pan-cancer genome and transcriptome analyses of 1,699 paediatric leukaemias and solid tumours.

Analysis of molecular aberrations across multiple cancer types, known as pan-cancer analysis, identifies commonalities and differences in key biological processes that are dysregulated in cancer cells from diverse lineages. Pan-cancer analyses have been performed for adult but not paediatric cancers, which commonly occur in developing mesodermic rather than adult epithelial tissues. Here we present a pan-cancer study of somatic alterations, including single nucleotide variants, small insertions or deletions, structural variations, copy number alterations, gene fusions and internal tandem duplications in 1,699 paediatric leukaemias and solid tumours across six histotypes, with whole-genome, whole-exome and transcriptome sequencing data processed under a uniform analytical framework. We report 142 driver genes in paediatric cancers, of which only 45% match those found in adult pan-cancer studies; copy number alterations and structural variants constituted the majority (62%) of events. Eleven genome-wide mutational signatures were identified, including one attributed to ultraviolet-light exposure in eight aneuploid leukaemias. Transcription of the mutant allele was detectable for 34% of protein-coding mutations, and 20% exhibited allele-specific expression. These data provide a comprehensive genomic architecture for paediatric cancers and emphasize the need for paediatric cancer-specific development of precision therapies.

Mcm2 hypomorph leads to acute leukemia or hematopoietic stem cell failure, dependent on genetic context.

Minichromosome maintenance proteins (Mcm2-7) form a hexameric complex that unwinds DNA ahead of a replicative fork. The deficiency of Mcm proteins leads to replicative stress and consequent genomic instability. Mice with a germline insertion of a Cre cassette into the 3'UTR of the Mcm2 gene (designated Mcm2Cre ) have decreased Mcm2 expression and invariably develop precursor T-cell lymphoblastic leukemia/lymphoma (pre-T LBL), due to 100-1000 kb deletions involving important tumor suppressor genes. To determine whether mice that were protected from pre-T LBL would develop non-T-cell malignancies, we used two approaches. Mice engrafted with Mcm2Cre/Cre Lin- Sca-1+ Kit+ hematopoietic stem/progenitor cells did not develop hematologic malignancy; however, these mice died of hematopoietic stem cell failure by 6 months of age. Placing the Mcm2Cre allele onto an athymic nu/nu background completely prevented pre-T LBL and extended survival of these mice three-fold (median 296.5 vs. 80.5 days). Ultimately, most Mcm2Cre/Cre ;nu/nu mice developed B-cell precursor acute lymphoblastic leukemia (BCP-ALL). We identified recurrent deletions of 100-1000 kb that involved genes known or suspected to be involved in BCP-ALL, including Pax5, Nf1, Ikzf3, and Bcor. Moreover, whole-exome sequencing identified recurrent mutations of genes known to be involved in BCP-ALL progression, such as Jak1/Jak3, Ptpn11, and Kras. These findings demonstrate that an Mcm2Cre/Cre hypomorph can induce hematopoietic dysfunction via hematopoietic stem cell failure as well as a "deletor" phenotype affecting known or suspected tumor suppressor genes.

Skp, Cullin, F-box (SCF)-Met30 and SCF-Cdc4-Mediated Proteolysis of CENP-A Prevents Mislocalization of CENP-A for Chromosomal Stability in Budding Yeast.

Restricting the localization of the histone H3 variant CENP-A (Cse4 in yeast, CID in flies) to centromeres is essential for faithful chromosome segregation. Mislocalization of CENP-A leads to chromosomal instability (CIN) in yeast, fly and human cells. Overexpression and mislocalization of CENP-A has been observed in many cancers and this correlates with increased invasiveness and poor prognosis. Yet genes that regulate CENP-A levels and localization under physiological conditions have not been defined. In this study we used a genome-wide genetic screen to identify essential genes required for Cse4 homeostasis to prevent its mislocalization for chromosomal stability. We show that two Skp, Cullin, F-box (SCF) ubiquitin ligases with the evolutionarily conserved F-box proteins Met30 and Cdc4 interact and cooperatively regulate proteolysis of endogenous Cse4 and prevent its mislocalization for faithful chromosome segregation under physiological conditions. The interaction of Met30 with Cdc4 is independent of the D domain, which is essential for their homodimerization and ubiquitination of other substrates. The requirement for both Cdc4 and Met30 for ubiquitination is specifc for Cse4; and a common substrate for Cdc4 and Met30 has not previously been described. Met30 is necessary for the interaction between Cdc4 and Cse4, and defects in this interaction lead to stabilization and mislocalization of Cse4, which in turn contributes to CIN. We provide the first direct link between Cse4 mislocalization to defects in kinetochore structure and show that SCF-mediated proteolysis of Cse4 is a major mechanism that prevents stable maintenance of Cse4 at non-centromeric regions, thus ensuring faithful chromosome segregation. In summary, we have identified essential pathways that regulate cellular levels of endogenous Cse4 and shown that proteolysis of Cse4 by SCF-Met30/Cdc4 prevents mislocalization and CIN in unperturbed cells.

Lineage of origin in rhabdomyosarcoma informs pharmacological response.

Lineage or cell of origin of cancers is often unknown and thus is not a consideration in therapeutic approaches. Alveolar rhabdomyosarcoma (aRMS) is an aggressive childhood cancer for which the cell of origin remains debated. We used conditional genetic mouse models of aRMS to activate the pathognomonic Pax3:Foxo1 fusion oncogene and inactivate p53 in several stages of prenatal and postnatal muscle development. We reveal that lineage of origin significantly influences tumor histomorphology and sensitivity to targeted therapeutics. Furthermore, we uncovered differential transcriptional regulation of the Pax3:Foxo1 locus by tumor lineage of origin, which led us to identify the histone deacetylase inhibitor entinostat as a pharmacological agent for the potential conversion of Pax3:Foxo1-positive aRMS to a state akin to fusion-negative RMS through direct transcriptional suppression of Pax3:Foxo1.

Characterization of genetically defined sporadic and hereditary type 1 papillary renal cell carcinoma cell lines.

Renal cell carcinoma (RCC) is not a single disease but is made up of several different histologically defined subtypes that are associated with distinct genetic alterations which require subtype specific management and treatment. Papillary renal cell carcinoma (pRCC) is the second most common subtype after conventional/clear cell RCC (ccRCC), representing ~20% of cases, and is subcategorized into type 1 and type 2 pRCC. It is important for preclinical studies to have cell lines that accurately represent each specific RCC subtype. This study characterizes seven cell lines derived from both primary and metastatic sites of type 1 pRCC, including the first cell line derived from a hereditary papillary renal carcinoma (HPRC)-associated tumor. Complete or partial gain of chromosome 7 was observed in all cell lines and other common gains of chromosomes 16, 17, or 20 were seen in several cell lines. Activating mutations of MET were present in three cell lines that all demonstrated increased MET phosphorylation in response to HGF and abrogation of MET phosphorylation in response to MET inhibitors. CDKN2A loss due to mutation or gene deletion, associated with poor outcomes in type 1 pRCC patients, was observed in all cell line models. Six cell lines formed tumor xenografts in athymic nude mice and thus provide in vivo models of type 1 pRCC. These type 1 pRCC cell lines provide a comprehensive representation of the genetic alterations associated with pRCC that will give insight into the biology of this disease and be ideal preclinical models for therapeutic studies.

Immunohistochemical detection of PAX-FOXO1 fusion proteins in alveolar rhabdomyosarcoma using breakpoint specific monoclonal antibodies.

Alveolar Rhabdomyosarcoma (ARMS) is an aggressive pediatric cancer with about 80% of cases characterized by either a t(1;13)(p36;q14) or t(2;13)(q35;q14), which results in the formation of the fusion oncogenes PAX7-FOXO1 and PAX3-FOXO1, respectively. Since patients with fusion-positive ARMS (FP-RMS) have a poor prognosis and are treated with an aggressive therapeutic regimen, correct classification is of clinical importance. Detection of the translocation by different molecular methods is used for diagnostics, including fluorescence in situ hybridization and RT-PCR or NGS based approaches. Since these methods are complex and time consuming, we developed specific monoclonal antibodies (mAbs) directed to the junction region on the PAX3-FOXO1 fusion protein. Two mAbs, PFM.1 and PFM.2, were developed and able to immunoprecipitate in vitro-translated PAX3-FOXO1 and cellular PAX3-FOXO1 from FP-RMS cells. Furthermore, the mAbs recognized a 105 kDa band in PAX3-FOXO1-transfected cells and in FP-RMS cell lines. The mAbs did not recognize proteins in fusion-negative embryonal rhabdomyosarcoma cell lines, nor did they recognize PAX3 or FOXO1 alone when compared to anti-PAX3 and anti-FOXO1 antibodies. We next evaluated the ability of mAb PFM.2 to detect the fusion protein by immunohistochemistry. Both PAX3-FOXO1 and PAX7-FOXO1 were detected in HEK293 cells transfected with the corresponding cDNAs. Subsequently, we stained 26 primary tumor sections and a rhabdomyosarcoma tissue array and detected both fusion proteins with a positive predictive value of 100%, negative predictive value of 98%, specificity of 100% and a sensitivity of 91%. While tumors are stained homogenously in PAX3-FOXO1 cases, the staining pattern is heterogenous with scattered positive cells only in tumors expressing PAX7-FOXO1. No staining was observed in stromal cells, embryonal rhabdomyosarcoma, and fusion-negative rhabdomyosarcoma. These results demonstrate that mAbs specific for the chimeric oncoproteins PAX3-FOXO1 and PAX7-FOXO1 can be used efficiently for simple and fast subclassification of rhabdomyosarcoma in routine diagnostics via immunohistochemical detection.

Engineered Bcor mutations lead to acute leukemia of progenitor B-1 lymphocyte origin in a sensitized background.

Approximately 10% of NUP98-PHF23 (NP23) mice develop an aggressive acute lymphoblastic leukemia of B-1 lymphocyte progenitor origin (pro-B1 ALL), accompanied by somatic frameshift mutations of the BCL6 interacting corepressor (Bcor) gene, most commonly within a 9-bp "hotspot" in Bcor exon 8. To determine whether experimentally engineered Bcor mutations would lead to pro-B1 ALL, we used clustered, regularly interspaced, short palindromic repeats-associated protein 9 to introduce a Bcor frameshift mutation into NP23 hematopoietic stem and progenitor cells through the use of Bcor small guide RNAs (Bcor sgRNAs). Recipient mice transplanted with NP23 bone marrow or fetal liver cells that had been transduced with a Bcor sgRNA developed pro-B1 ALL, characterized by a B-1 progenitor immunophenotype, clonal Igh gene rearrangement, and Bcor indel mutation, whereas control recipients did not. Similar to a subset of human B-cell precursor ALL, the murine pro-B1 ALL had acquired somatic mutations in Jak kinase genes. JAK inhibitors (ruxolitinib and tofacitinib) inhibited the growth of pro-B1 ALL cell lines established from Bcor sgRNA/NP23 recipients at clinically achievable concentrations (100 nM). Our results demonstrate that Bcor mutations collaborate with NP23 to induce pro-B1 ALL, and that JAK inhibitors are potential therapies for pro-B1 ALL.

Novel renal medullary carcinoma cell lines, UOK353 and UOK360, provide preclinical tools to identify new therapeutic treatments.

Renal medullary carcinoma (RMC) is a rare, aggressive disease that predominantly afflicts individuals of African or Mediterranean descent with sickle cell trait. RMC comprises 1% of all renal cell carcinoma diagnoses with a median overall survival of 13 months. Patients are typically young (median age-22) and male (male:female ratio of 2:1) and tumors are characterized by complete loss of expression of the SMARCB1 tumor suppressor protein. Due to the low incidence of RMC and the disease's aggressiveness, treatment decisions are often based on case reports. Thus, it is critical to develop preclinical models of RMC to better understand the pathogenesis of this disease and to identify effective forms of therapy. Two novel cell line models, UOK353 and UOK360, were derived from primary RMCs that both demonstrated the characteristic SMARCB1 loss. Both cell lines overexpressed EZH2 and other members of the polycomb repressive complex and EZH2 inhibition in RMC tumor spheroids resulted in decreased viability. High throughput drug screening of both cell lines revealed several additional candidate compounds, including bortezomib that had both in vitro and in vivo antitumor activity. The activity of bortezomib was shown to be partially dependent on increased oxidative stress as addition of the N-acetyl cysteine antioxidant reduced the effect on cell proliferation. Combining bortezomib and cisplatin further decreased cell viability both in vitro and in vivo that single agent bortezomib treatment. The UOK353 and UOK360 cell lines represent novel preclinical models for the development of effective forms of therapy for RMC patients.

Relationship of DNA methylation to mutational changes and transcriptional organization in fusion-positive and fusion-negative rhabdomyosarcoma.

Our previous study of DNA methylation in the pediatric soft tissue tumor rhabdomyosarcoma (RMS) demonstrated that fusion-positive (FP) and fusion-negative (FN) RMS tumors exhibit distinct DNA methylation patterns. To further examine the significance of DNA methylation differences in RMS, we investigated genome-wide DNA methylation profiles in discovery and validation cohorts. Unsupervised analysis of DNA methylation data identified novel distinct subsets associated with the specific fusion subtype in FP RMS and with RAS mutation status in FN RMS. Furthermore, the methylation pattern in normal muscle is most similar to the FN subset with wild-type RAS mutation status. Several biologically relevant genes were identified with methylation and expression differences between the two fusion subtypes of FP RMS or between the RAS wild-type and mutant subsets of FN RMS. Genomic localization studies showed that promoter and intergenic regions were hypomethylated and the 3' untranslated regions were hypermethylated in FP compared to FN tumors. There was also a significant difference in the distribution of PAX3-FOXO1 binding sites between genes with and without differential methylation. Moreover, genes with PAX3-FOXO1 binding sites and promoter hypomethylation exhibited the highest frequency of overexpression in FP tumors. Finally, a comparison of RMS model systems revealed that patient-derived xenografts most closely recapitulate the DNA methylation patterns found in human RMS tumors compared to cell lines and cell line-derived xenografts. In conclusion, these findings highlight the interaction of epigenetic changes with mutational alterations and transcriptional organization in RMS tumors, and contribute to improved molecular categorization of these tumors.

Provocative questions in osteosarcoma basic and translational biology: A report from the Children's Oncology Group.

Patients who are diagnosed with osteosarcoma (OS) today receive the same therapy that patients have received over the last 4 decades. Extensive efforts to identify more effective or less toxic regimens have proved disappointing. As we enter a postgenomic era in which we now recognize OS not as a cancer of mutations but as one defined by p53 loss, chromosomal complexity, copy number alteration, and profound heterogeneity, emerging threads of discovery leave many hopeful that an improving understanding of biology will drive discoveries that improve clinical care. Under the organization of the Bone Tumor Biology Committee of the Children's Oncology Group, a team of clinicians and scientists sought to define the state of the science and to identify questions that, if answered, have the greatest potential to drive fundamental clinical advances. Having discussed these questions in a series of meetings, each led by invited experts, we distilled these conversations into a series of seven Provocative Questions. These include questions about the molecular events that trigger oncogenesis, the genomic and epigenomic drivers of disease, the biology of lung metastasis, research models that best predict clinical outcomes, and processes for translating findings into clinical trials. Here, we briefly present each Provocative Question, review the current scientific evidence, note the immediate opportunities, and speculate on the impact that answered questions might have on the field. We do so with an intent to provide a framework around which investigators can build programs and collaborations to tackle the hardest problems and to establish research priorities for those developing policies and providing funding.

Imprints and DPPA3 are bypassed during pluripotency- and differentiation-coupled methylation reprogramming in testicular germ cell tumors.

Testicular germ cell tumors (TGCTs) share germline ancestry but diverge phenotypically and clinically as seminoma (SE) and nonseminoma (NSE), the latter including the pluripotent embryonal carcinoma (EC) and its differentiated derivatives, teratoma (TE), yolk sac tumor (YST), and choriocarcinoma. Epigenomes from TGCTs may illuminate reprogramming in both normal development and testicular tumorigenesis. Herein we investigate pure-histological forms of 130 TGCTs for conserved and subtype-specific DNA methylation, including analysis of relatedness to pluripotent stem cell (ESC, iPSC), primordial germ cell (PGC), and differentiated somatic references. Most generally, TGCTs conserve PGC-lineage erasure of maternal and paternal genomic imprints and DPPA3 (also known as STELLA); however, like ESCs, TGCTs show focal recurrent imprinted domain hypermethylation. In this setting of shared physiologic erasure, NSEs harbor a malignancy-associated hypermethylation core, akin to that of a diverse cancer compendium. Beyond these concordances, we found subtype epigenetic homology with pluripotent versus differentiated states. ECs demonstrate a striking convergence of both CpG and CpH (non-CpG) methylation with pluripotent states; the pluripotential methyl-CpH signature crosses species boundaries and is distinct from neuronal methyl-CpH. EC differentiation to TE and YST entails reprogramming toward the somatic state, with loss of methyl-CpH but de novo methylation of pluripotency loci such as NANOG Extreme methyl-depletion among SE reflects the PGC methylation nadir. Adjacent to TGCTs, benign testis methylation profiles are determined by spermatogenetic proficiency measured by Johnsen score. In sum, TGCTs share collective entrapment in a PGC-like state of genomic-imprint and DPPA3 erasure, recurrent hypermethylation of cancer-associated targets, and subtype-dependent pluripotent, germline, or somatic methylation.

Aneuploidy, TP53 mutation, and amplification of MYC correlate with increased intratumor heterogeneity and poor prognosis of breast cancer patients.

The clinical course of breast cancer varies from one patient to another. Currently, the choice of therapy relies on clinical parameters and histological and molecular tumor features. Alas, these markers are informative in only a subset of patients. Therefore, additional predictors of disease outcome would be valuable for treatment stratification. Extensive studies showed that the degree of variation of the nuclear DNA content, i.e., aneuploidy, determines prognosis. Our aim was to further elucidate the molecular basis of aneuploidy. We analyzed five diploid and six aneuploid tumors with more than 20 years of follow-up. By performing FISH with a multiplexed panel of 10 probes to enumerate copy numbers in individual cells, and by sequencing 563 cancer-related genes, we analyzed how aneuploidy is linked to intratumor heterogeneity. In our cohort, none of the patients with diploid tumors died of breast cancer during follow-up in contrast to four of six patients with aneuploid tumors (mean survival 86.4 months). The FISH analysis showed markedly increased genomic instability and intratumor heterogeneity in aneuploid tumors. MYC gain was observed in only 20% of the diploid cancers, while all aneuploid cases showed a gain. The mutation burden was similar in diploid and aneuploid tumors, however, TP53 mutations were not observed in diploid tumors, but in all aneuploid tumors in our collective. We conclude that quantitative measurements of intratumor heterogeneity by multiplex FISH, detection of MYC amplification and TP53 mutation could augment prognostication in breast cancer patients.

Genomic and metabolic characterization of a chromophobe renal cell carcinoma cell line model (UOK276).

Chromophobe renal cell carcinoma (ChRCC) represents 5% of all RCC cases and frequently demonstrates multiple chromosomal losses and an indolent pattern of local growth, but can demonstrate aggressive features and resistance to treatment in a metastatic setting. Cell line models are an important tool for the investigation of tumor biology and therapeutic drug efficacy. Currently, there are few ChRCC-derived cell lines and none is well characterized. This study characterizes a novel ChRCC-derived cell line model, UOK276. A large ChRCC tumor with regions of sarcomatoid differentiation was used to establish a spontaneously immortal cell line, UOK276. UOK276 was evaluated for chromosomal, mutational, and metabolic aberrations. The UOK276 cell line is hyperdiploid with a modal number of 49 chromosomes per cell, and evidence of copy-neutral loss of heterozygosity, as opposed to the classic pattern of ChRCC chromosomal losses. UOK276 demonstrated a TP53 missense mutation, expressed mutant TP53 protein, and responded to treatment with a small-molecule therapeutic agent, NSC319726, designed to reactivate mutated TP53. Xenograft tumors grew in nude mice and provide an in vivo animal model for the investigation of potential therapeutic regimes. The xenograft pathology and genetic analysis suggested that UOK276 was derived from the sarcomatoid region of the original tumor. In summary, UOK276 represents a novel in vitro and in vivo cell line model for aggressive, sarcomatoid-differentiated, TP53 mutant ChRCC. This preclinical model system could be used to investigate the novel biology of aggressive, sarcomatoid ChRCC and evaluate the new therapeutic regimes.

Somatic mutations in murine models of leukemia and lymphoma: Disease specificity and clinical relevance.

Malignant transformation is a multistep process that is dictated by the acquisition of multiple genomic aberrations that provide growth and survival advantage. During the post genomic era, high throughput genomic sequencing has advanced exponentially, leading to identification of countless cancer associated mutations with potential for targeted therapy. Mouse models of cancer serve as excellent tools to examine the functionality of gene mutations and their contribution to the malignant process. However, it remains unclear whether the genetic events that occur during transformation are similar in mice and humans. To address that, we chose several transgenic mouse models of hematopoietic malignancies and identified acquired mutations in these mice by means of targeted re-sequencing of known cancer-associated genes as well as whole exome sequencing. We found that mutations that are typically found in acute myeloid leukemia or T cell acute lymphoblastic leukemia patients are also common in mouse models of the respective disease. Moreover, we found that the most frequent mutations found in a mouse model of lymphoma occur in a set of epigenetic modifier genes, implicating this pathway in the generation of lymphoma. These results demonstrate that genetically engineered mouse models (GEMM) mimic the genetic evolution of human cancer and serve as excellent platforms for target discovery and validation.

Interferon-γ links ultraviolet radiation to melanomagenesis in mice.

Cutaneous malignant melanoma is a highly aggressive and frequently chemoresistant cancer, the incidence of which continues to rise. Epidemiological studies show that the major aetiological melanoma risk factor is ultraviolet (UV) solar radiation, with the highest risk associated with intermittent burning doses, especially during childhood. We have experimentally validated these epidemiological findings using the hepatocyte growth factor/scatter factor transgenic mouse model, which develops lesions in stages highly reminiscent of human melanoma with respect to biological, genetic and aetiological criteria, but only when irradiated as neonatal pups with UVB, not UVA. However, the mechanisms underlying UVB-initiated, neonatal-specific melanomagenesis remain largely unknown. Here we introduce a mouse model permitting fluorescence-aided melanocyte imaging and isolation following in vivo UV irradiation. We use expression profiling to show that activated neonatal skin melanocytes isolated following a melanomagenic UVB dose bear a distinct, persistent interferon response signature, including genes associated with immunoevasion. UVB-induced melanocyte activation, characterized by aberrant growth and migration, was abolished by antibody-mediated systemic blockade of interferon-γ (IFN-γ), but not type-I interferons. IFN-γ was produced by macrophages recruited to neonatal skin by UVB-induced ligands to the chemokine receptor Ccr2. Admixed recruited skin macrophages enhanced transplanted melanoma growth by inhibiting apoptosis; notably, IFN-γ blockade abolished macrophage-enhanced melanoma growth and survival. IFN-γ-producing macrophages were also identified in 70% of human melanomas examined. Our data reveal an unanticipated role for IFN-γ in promoting melanocytic cell survival/immunoevasion, identifying a novel candidate therapeutic target for a subset of melanoma patients.

Integrated genome-wide analysis of genomic changes and gene regulation in human adrenocortical tissue samples.

To gain insight into the pathogenesis of adrenocortical carcinoma (ACC) and whether there is progression from normal-to-adenoma-to-carcinoma, we performed genome-wide gene expression, gene methylation, microRNA expression and comparative genomic hybridization (CGH) analysis in human adrenocortical tissue (normal, adrenocortical adenomas and ACC) samples. A pairwise comparison of normal, adrenocortical adenomas and ACC gene expression profiles with more than four-fold expression differences and an adjusted P-value < 0.05 revealed no major differences in normal versus adrenocortical adenoma whereas there are 808 and 1085, respectively, dysregulated genes between ACC versus adrenocortical adenoma and ACC versus normal. The majority of the dysregulated genes in ACC were downregulated. By integrating the CGH, gene methylation and expression profiles of potential miRNAs with the gene expression of dysregulated genes, we found that there are higher alterations in ACC versus normal compared to ACC versus adrenocortical adenoma. Importantly, we identified several novel molecular pathways that are associated with dysregulated genes and further experimentally validated that oncostatin m signaling induces caspase 3 dependent apoptosis and suppresses cell proliferation. Finally, we propose that there is higher number of genomic changes from normal-to-adenoma-to-carcinoma and identified oncostatin m signaling as a plausible druggable pathway for therapeutics.

EWS-FLI1 employs an E2F switch to drive target gene expression.

Cell cycle progression is orchestrated by E2F factors. We previously reported that in ETS-driven cancers of the bone and prostate, activating E2F3 cooperates with ETS on target promoters. The mechanism of target co-regulation remained unknown. Using RNAi and time-resolved chromatin-immunoprecipitation in Ewing sarcoma we report replacement of E2F3/pRB by constitutively expressed repressive E2F4/p130 complexes on target genes upon EWS-FLI1 modulation. Using mathematical modeling we interrogated four alternative explanatory models for the observed EWS-FLI1/E2F3 cooperation based on longitudinal E2F target and regulating transcription factor expression analysis. Bayesian model selection revealed the formation of a synergistic complex between EWS-FLI1 and E2F3 as the by far most likely mechanism explaining the observed kinetics of E2F target induction. Consequently we propose that aberrant cell cycle activation in Ewing sarcoma is due to the de-repression of E2F targets as a consequence of transcriptional induction and physical recruitment of E2F3 by EWS-FLI1 replacing E2F4 on their target promoters.