The Role of FAS Receptor Methylation in Osteosarcoma Metastasis.

Osteosarcoma is the most frequent primary malignant bone tumor with an annual incidence of about 400 cases in the United States. Osteosarcoma primarily metastasizes to the lungs, where FAS ligand (FASL) is constitutively expressed. The interaction of FASL and its cell surface receptor, FAS, triggers apoptosis in normal cells; however, this function is altered in cancer cells. DNA methylation has previously been explored as a mechanism for altering FAS expression, but no variability was identified in the CpG island (CGI) overlapping the promoter. Analysis of an expanded region, including CGI shores and shelves, revealed high variability in the methylation of certain CpG sites that correlated significantly with FAS mRNA expression in a negative manner. Bisulfite sequencing revealed additional CpG sites, which were highly methylated in the metastatic LM7 cell line but unmethylated in its parental non-metastatic SaOS-2 cell line. Treatment with the demethylating agent, 5-azacytidine, resulted in a loss of methylation in CpG sites located within the FAS promoter and restored FAS protein expression in LM7 cells, resulting in reduced migration. Orthotopic implantation of 5-azacytidine treated LM7 cells into severe combined immunodeficient mice led to decreased lung metastases. These results suggest that DNA methylation of CGI shore sites may regulate FAS expression and constitute a potential target for osteosarcoma therapy, utilizing demethylating agents currently approved for the treatment of other cancers.

Actionability classification of variants of unknown significance correlates with functional effect.

Genomically-informed therapy requires consideration of the functional impact of genomic alterations on protein expression and/or function. However, a substantial number of variants are of unknown significance (VUS). The MD Anderson Precision Oncology Decision Support (PODS) team developed an actionability classification scheme that categorizes VUS as either "Unknown" or "Potentially" actionable based on their location within functional domains and/or proximity to known oncogenic variants. We then compared PODS VUS actionability classification with results from a functional genomics platform consisting of mutant generation and cell viability assays. 106 (24%) of 438 VUS in 20 actionable genes were classified as oncogenic in functional assays. Variants categorized by PODS as Potentially actionable (N = 204) were more likely to be oncogenic than those categorized as Unknown (N = 230) (37% vs 13%, p = 4.08e-09). Our results demonstrate that rule-based actionability classification of VUS can identify patients more likely to have actionable variants for consideration with genomically-matched therapy.

A functional genomic approach to actionable gene fusions for precision oncology.

Fusion genes represent a class of attractive therapeutic targets. Thousands of fusion genes have been identified in patients with cancer, but the functional consequences and therapeutic implications of most of these remain largely unknown. Here, we develop a functional genomic approach that consists of efficient fusion reconstruction and sensitive cell viability and drug response assays. Applying this approach, we characterize ~100 fusion genes detected in patient samples of The Cancer Genome Atlas, revealing a notable fraction of low-frequency fusions with activating effects on tumor growth. Focusing on those in the RTK-RAS pathway, we identify a number of activating fusions that can markedly affect sensitivity to relevant drugs. Last, we propose an integrated, level-of-evidence classification system to prioritize gene fusions systematically. Our study reiterates the urgent clinical need to incorporate similar functional genomic approaches to characterize gene fusions, thereby maximizing the utility of gene fusions for precision oncology.

Combined inhibition of DDR1 and CDK4/6 induces synergistic effects in ER-positive, HER2-negative breast cancer with PIK3CA/AKT1 mutations.

Molecular alterations in the PI3K/AKT pathway occur frequently in hormone receptor-positive breast tumors. Patients with ER-positive, HER2-negative metastatic breast cancer are often treated with CDK4/6 inhibitors such as palbociclib in combination with endocrine therapy. Although this is an effective regimen, most patients ultimately progress. The purpose of this study was identifying synthetic lethality partners that can enhance palbociclib's antitumor efficacy in the presence of PIK3CA/AKT1 mutations. We utilized a barcoded shRNA library to determine critical targets for survival in isogenic MCF7 cells with PIK3CA/AKT1 mutations. We demonstrated that the efficacy of palbociclib is reduced in the presence of PIK3CA/AKT1 mutations. We also identified that the downregulation of discoidin domain receptor 1 (DDR1) is synthetically lethal with palbociclib. DDR1 knockdown and DDR1 pharmacological inhibitor decreased cell growth and inhibited cell cycle progression in all cell lines, while enhanced the sensitivity of PIK3CA/AKT1 mutant cells to palbociclib. Combined treatment of palbociclib and 7rh further induced cell cycle arrest in PIK3CA/AKT1 mutant cell lines. In vivo, 7rh significantly enhanced palbociclib's antitumor efficacy. Our data indicates that DDR1 inhibition can augment cell cycle suppressive effect of palbociclib and could be effective strategy for targeted therapy of ER-positive, HER2-negative breast cancers with PI3K pathway activation.

Clinical and Functional Characterization of Atypical KRAS/NRAS Mutations in Metastatic Colorectal Cancer.

PURPOSE: Mutations in KRAS/NRAS (RAS) predict lack of anti-EGFR efficacy in metastatic colorectal cancer (mCRC). However, it is unclear if all RAS mutations have similar impact, and atypical mutations beyond those in standard guidelines exist. EXPERIMENTAL DESIGN: We reviewed 7 tissue and 1 cell-free DNA cohorts of 9,485 patients to characterize atypical RAS variants. Using an in vitro cell-based assay (functional annotation for cancer treatment), Ba/F3 transformation, and in vivo xenograft models of transduced isogenic clones, we assessed signaling changes across mutations. RESULTS: KRAS exon 2, extended RAS, and atypical RAS mutations were noted in 37.8%, 9.5%, and 1.2% of patients, respectively. Among atypical variants, KRAS L19F, Q22K, and D33E occurred at prevalence ≥0.1%, whereas no NRAS codon 117/146 and only one NRAS codon 59 mutation was noted. Atypical RAS mutations had worse overall survival than RAS/BRAF wild-type mCRC (HR, 2.90; 95% confidence interval, 1.24-6.80; P = 0.014). We functionally characterized 114 variants with the FACT assay. All KRAS exon 2 and extended RAS mutations appeared activating. Of 57 atypical RAS variants characterized, 18 (31.6%) had signaling below wild-type, 23 (40.4%) had signaling between wild-type and activating control, and 16 (28.1%) were hyperactive beyond the activating control. Ba/F3 transformation (17/18 variants) and xenograft model (7/8 variants) validation was highly concordant with FACT results, and activating atypical variants were those that occurred at highest prevalence in clinical cohorts. CONCLUSIONS: We provide best available evidence to guide treatment when atypical RAS variants are identified. KRAS L19F, Q22K, D33E, and T50I are more prevalent than many guideline-included RAS variants and functionally relevant.

Comprehensive assessment of computational algorithms in predicting cancer driver mutations.

BACKGROUND: The initiation and subsequent evolution of cancer are largely driven by a relatively small number of somatic mutations with critical functional impacts, so-called driver mutations. Identifying driver mutations in a patient's tumor cells is a central task in the era of precision cancer medicine. Over the decade, many computational algorithms have been developed to predict the effects of missense single-nucleotide variants, and they are frequently employed to prioritize mutation candidates. These algorithms employ diverse molecular features to build predictive models, and while some algorithms are cancer-specific, others are not. However, the relative performance of these algorithms has not been rigorously assessed. RESULTS: We construct five complementary benchmark datasets: mutation clustering patterns in the protein 3D structures, literature annotation based on OncoKB, TP53 mutations based on their effects on target-gene transactivation, effects of cancer mutations on tumor formation in xenograft experiments, and functional annotation based on in vitro cell viability assays we developed including a new dataset of ~ 200 mutations. We evaluate the performance of 33 algorithms and found that CHASM, CTAT-cancer, DEOGEN2, and PrimateAI show consistently better performance than the other algorithms. Moreover, cancer-specific algorithms show much better performance than those designed for a general purpose. CONCLUSIONS: Our study is a comprehensive assessment of the performance of different algorithms in predicting cancer driver mutations and provides deep insights into the best practice of computationally prioritizing cancer mutation candidates for end-users and for the future development of new algorithms.

Comprehensive Molecular Characterization of the Hippo Signaling Pathway in Cancer.

Hippo signaling has been recognized as a key tumor suppressor pathway. Here, we perform a comprehensive molecular characterization of 19 Hippo core genes in 9,125 tumor samples across 33 cancer types using multidimensional "omic" data from The Cancer Genome Atlas. We identify somatic drivers among Hippo genes and the related microRNA (miRNA) regulators, and using functional genomic approaches, we experimentally characterize YAP and TAZ mutation effects and miR-590 and miR-200a regulation for TAZ. Hippo pathway activity is best characterized by a YAP/TAZ transcriptional target signature of 22 genes, which shows robust prognostic power across cancer types. Our elastic-net integrated modeling further reveals cancer-type-specific pathway regulators and associated cancer drivers. Our results highlight the importance of Hippo signaling in squamous cell cancers, characterized by frequent amplification of YAP/TAZ, high expression heterogeneity, and significant prognostic patterns. This study represents a systems-biology approach to characterizing key cancer signaling pathways in the post-genomic era.

AKT isoform-specific expression and activation across cancer lineages.

BACKGROUND: Aberrant AKT activation is prevalent across human cancer lineages, providing an important therapeutic target. AKT comprises three isoforms that mediate critical non-redundant, even opposing functions in cancer pathophysiology. Therefore, targeting specific AKT isoforms in particular cancers may be more effective than pan-AKT inhibition while avoiding disadvantages of pan-AKT inhibition. Currently, AKT isoform-specific expression and activation in cancer are not clearly characterized. METHODS: We systematically characterized AKT isoform-specific expression and activation in 211 cancer cell lines derived from different lineages and genetic backgrounds using a reverse-phase protein array platform. RESULTS: We found that phosphorylation, but not expression, of AKT1 and AKT2 was coordinated in most but not all cells. Different cancer lineages displayed differential AKT1 and AKT2 expression and phosphorylation. A PIK3CA hotspot mutation H1047R but not E545K was associated with selective activation of AKT2 but not AKT1. CONCLUSIONS: Our study identified and validated AKT isoform-specific expression and phosphorylation in certain cell lines and demonstrated that genetic changes can affect AKT isoform-specific activation. These results provide a more precise understanding of AKT isoform-specific signaling and, in addition, facilitate AKT isoform targeting for personalized cancer therapies.

Comprehensive Characterization of Cancer Driver Genes and Mutations.

Identifying molecular cancer drivers is critical for precision oncology. Multiple advanced algorithms to identify drivers now exist, but systematic attempts to combine and optimize them on large datasets are few. We report a PanCancer and PanSoftware analysis spanning 9,423 tumor exomes (comprising all 33 of The Cancer Genome Atlas projects) and using 26 computational tools to catalog driver genes and mutations. We identify 299 driver genes with implications regarding their anatomical sites and cancer/cell types. Sequence- and structure-based analyses identified >3,400 putative missense driver mutations supported by multiple lines of evidence. Experimental validation confirmed 60%-85% of predicted mutations as likely drivers. We found that >300 MSI tumors are associated with high PD-1/PD-L1, and 57% of tumors analyzed harbor putative clinically actionable events. Our study represents the most comprehensive discovery of cancer genes and mutations to date and will serve as a blueprint for future biological and clinical endeavors.

Systematic Functional Annotation of Somatic Mutations in Cancer.

The functional impact of the vast majority of cancer somatic mutations remains unknown, representing a critical knowledge gap for implementing precision oncology. Here, we report the development of a moderate-throughput functional genomic platform consisting of efficient mutant generation, sensitive viability assays using two growth factor-dependent cell models, and functional proteomic profiling of signaling effects for select aberrations. We apply the platform to annotate >1,000 genomic aberrations, including gene amplifications, point mutations, indels, and gene fusions, potentially doubling the number of driver mutations characterized in clinically actionable genes. Further, the platform is sufficiently sensitive to identify weak drivers. Our data are accessible through a user-friendly, public data portal. Our study will facilitate biomarker discovery, prediction algorithm improvement, and drug development.

Hypermethylation of NF-κB-Activating Protein-Like (NKAPL) Promoter in Hepatocellular Carcinoma Suppresses Its Expression and Predicts a Poor Prognosis.

BACKGROUND AND AIM: Hepatocellular carcinoma (HCC) is a complicated disease with low survival rate partially due to frequent recurrence and no efficient therapy. Promoter hypermethylation of tumor suppressor genes has been demonstrated as one of the molecular mechanisms contributing to tumorigenesis and progression in HCC. This study aims to investigate regulation of NKAPL expression by promoter methylation and its clinical relevance as a biomarker for HCC. METHODS: We measured mRNA expression of NKAPL in 5 HCC cell lines and a cohort of 62 pairs of primary HCC tumor and their adjacent non-cancer liver tissues. NKAPL protein expression on HCC cell lines and clinical samples was assessed by Western blot and immunohistochemistry, respectively. Association analyses between NKAPL expression and clinicopathologic characteristics in the cohort were conducted. Methylation statuses of NKAPL promoter in 18 pairs of tumor and adjacent non-tumor HCC samples were studied using methylation-specific PCR. Biological functions of NKAPL in HCC were investigated by ectopic expression of NKAPL in HCC cells, and cell viability and cell cycle analyses were performed. RESULTS: Our present study showed suppressed expression and promoter hypermethylation are common events in HCC. Demethylation experiment in HCC cells demonstrated that the NKAPL expression was regulated by promoter methylation. In addition, high methylation level of NKAPL and its low expression predict poor outcome. Furthermore, ectopic expression of NKAPL in the HCC cells inhibited cell growth. CONCLUSIONS: Our findings suggest that methylation of NKAPL is a frequent event and is a potential prognosis biomarker in HCC.

Rational combination therapy with PARP and MEK inhibitors capitalizes on therapeutic liabilities in RAS mutant cancers.

Mutant RAS has remained recalcitrant to targeted therapy efforts. We demonstrate that combined treatment with poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors and mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitors evokes unanticipated, synergistic cytotoxic effects in vitro and in vivo in multiple RAS mutant tumor models across tumor lineages where RAS mutations are prevalent. The effects of PARP and MEK inhibitor combinations are independent of BRCA1/2 and p53 mutation status, suggesting that the synergistic activity is likely to be generalizable. Synergistic activity of PARP and MEK inhibitor combinations in RAS mutant tumors is associated with (i) induction of BIM-mediated apoptosis, (ii) decrease in expression of components of the homologous recombination DNA repair pathway, (iii) decrease in homologous recombination DNA damage repair capacity, (iv) decrease in DNA damage checkpoint activity, (v) increase in PARP inhibitor-induced DNA damage, (vi) decrease in vascularity that could increase PARP inhibitor efficacy by inducing hypoxia, and (vii) elevated PARP1 protein, which increases trapping activity of PARP inhibitors. Mechanistically, enforced expression of FOXO3a, which is a target of the RAS/MAPK pathway, was sufficient to recapitulate the functional consequences of MEK inhibitors including synergy with PARP inhibitors. Thus, the ability of mutant RAS to suppress FOXO3a and its reversal by MEK inhibitors accounts, at least in part, for the synergy of PARP and MEK inhibitors in RAS mutant tumors. The rational combination of PARP and MEK inhibitors warrants clinical investigation in patients with RAS mutant tumors where there are few effective therapeutic options.

YAP/TAZ-Mediated Upregulation of GAB2 Leads to Increased Sensitivity to Growth Factor-Induced Activation of the PI3K Pathway.

The transcription regulators YAP and TAZ function as effectors of the HIPPO signaling cascade, critical for organismal development, cell growth, and cellular reprogramming, and YAP/TAZ is commonly misregulated in human cancers. The precise mechanism by which aberrant YAP/TAZ promotes tumor growth remains unclear. The HIPPO tumor suppressor pathway phosphorylates YAP and TAZ, resulting in cytosolic sequestration with subsequent degradation. Here, we report that the PI3K/AKT pathway, which is critically involved in the pathophysiology of endometrial cancer, interacts with the HIPPO pathway at multiple levels. Strikingly, coordinate knockdown of YAP and TAZ, mimicking activation of the HIPPO pathway, markedly decreased both constitutive and growth factor-induced PI3K pathway activation by decreasing levels of the GAB2 linker molecule in endometrial cancer lines. Furthermore, targeting YAP/TAZ decreased endometrial cancer tumor growth in vivo In addition, YAP and TAZ total and phosphoprotein levels correlated with clinical characteristics and outcomes in endometrial cancer. Thus, YAP and TAZ, which are inhibited by the HIPPO tumor suppressor pathway, modify PI3K/AKT pathway signaling in endometrial cancer. The cross-talk between these key pathways identifies potential new biomarkers and therapeutic targets in endometrial cancer. Cancer Res; 77(7); 1637-48. ©2017 AACR.

Transcriptome analysis of different developmental stages of amphioxus reveals dynamic changes of distinct classes of genes during development.

Vertebrates diverged from other chordates approximately 500 million years ago and have adopted several modifications of developmental processes. Amphioxus is widely used in evolutionary developmental biology research, such as on the basic patterning mechanisms involved in the chordate body plan and the origin of vertebrates. The fast development of next-generation sequencing has advanced knowledge of the genomic organization of amphioxus; however, many aspects of gene regulation during amphioxus development have not been fully characterized. In this study, we applied high-throughput sequencing on the transcriptomes of 13 developmental stages of Chinese amphioxus to gain a comprehensive understanding of transcriptional processes occurring from the fertilized egg to the adult stage. The expression levels of 3,423 genes were significantly changed (FDR ≤ 0.01). All of these genes were included in a clustering analysis, and enrichment of biological functions associated with these clusters was determined. Significant changes were observed in several important processes, including the down-regulation of the cell cycle and the up-regulation of translation. These results should build a foundation for identifying developmentally important genes, especially those regulatory factors involved in amphioxus development, and advance understanding of the developmental dynamics in vertebrates.

The draft genome, transcriptome, and microbiome of Dermatophagoides farinae reveal a broad spectrum of dust mite allergens.

BACKGROUND: A sequenced house dust mite (HDM) genome would advance our understanding of HDM allergens, a common cause of human allergies. OBJECTIVE: We sought to produce an annotated Dermatophagoides farinae draft genome and develop a combined genomic-transcriptomic-proteomic approach for elucidation of HDM allergens. METHODS: A D farinae draft genome and transcriptome were assembled with high-throughput sequencing, accommodating microbiome sequences. The allergen gene structures were validated by means of Sanger sequencing. The mite's microbiome composition was determined, and the predominant genus was validated immunohistochemically. The allergenicity of a ubiquinol-cytochrome c reductase binding protein homologue was evaluated with immunoblotting, immunosorbent assays, and skin prick tests. RESULTS: The full gene structures of 20 canonical allergens and 7 noncanonical allergen homologues were produced. A novel major allergen, ubiquinol-cytochrome c reductase binding protein-like protein, was found and designated Der f 24. All 40 sera samples from patients with mite allergy had IgE antibodies against rDer f 24. Of 10 patients tested, 5 had positive skin reactions. The predominant bacterial genus among 100 identified species was Enterobacter (63.4%). An intron was found in the 13.8-kDa D farinae bacteriolytic enzyme gene, indicating that it is of HDM origin. The Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed a phototransduction pathway in D farinae, as well as thiamine and amino acid synthesis pathways, which is suggestive of an endosymbiotic relationship between D farinae and its microbiome. CONCLUSION: An HDM genome draft produced from genomic, transcriptomic, and proteomic experiments revealed allergen genes and a diverse endosymbiotic microbiome, providing a tool for further identification and characterization of HDM allergens and development of diagnostics and immunotherapeutic vaccines.

RET fusion as a novel driver of medullary thyroid carcinoma.

INTRODUCTION: Oncogenic RET tyrosine kinase gene fusions and activating mutations have recently been identified in lung cancers, prompting initiation of targeted therapy trials in this disease. Although RET point mutation has been identified as a driver of tumorigenesis in medullary thyroid carcinoma (MTC), no fusions have been described to date. OBJECTIVE: We evaluated the role of RET fusion as an oncogenic driver in MTC. METHODS: We describe a patient who died from aggressive sporadic MTC < 10 months after diagnosis. Her tumor was evaluated by means of next-generation sequencing, including an intronic capture strategy. RESULTS: A reciprocal translocation involving RET intron 12 was identified. The fusion was validated using a targeted break apart fluorescence in situ hybridization probe, and RNA sequencing confirmed the existence of an in-frame fusion transcript joining MYH13 exon 35 with RET exon 12. Ectopic expression of fusion product in a murine Ba/F3 cell reporter model established strong oncogenicity. Three tyrosine kinase inhibitors currently used to treat MTC in clinical practice blocked tumorigenic cell growth. CONCLUSION: This finding represents the report of a novel RET fusion, the first of its kind described in MTC. The finding of this potential novel oncogenic mechanism has clear implications for sporadic MTC, which in the majority of cases has no driver mutation identified. The presence of a RET fusion also provides a plausible target for RET tyrosine kinase inhibitor therapies.

A comprehensive evaluation of biomarkers predictive of response to PI3K inhibitors and of resistance mechanisms in head and neck squamous cell carcinoma.

The PI3K/AKT/mTOR pathway is frequently activated in head and neck squamous cell carcinoma (HNSCC), but pathway inhibition has variable efficacy. Identification of predictive biomarkers and mechanisms of resistance would allow selection of patients most likely to respond and novel therapeutic combinations. The purpose of this study was to extend recent discoveries regarding the PI3K/AKT/mTOR pathway in HNSCC by more broadly examining potential biomarkers of response, by examining pathway inhibitors with a diverse range of targets, and by defining mechanisms of resistance and potential combination therapies. We used reverse-phase protein arrays (RPPA) to simultaneously evaluate expression of 195 proteins; SNP array to estimate gene copy number; and mass array to identify mutations. We examined altered signaling at baseline and after pathway inhibition. Likewise, we examined the activation of the PI3K/AKT/mTOR pathway in HNSCC tumors by RPPA. Cell lines with PIK3CA mutations were sensitive to pathway inhibitors, whereas amplification status did not predict sensitivity. While we identified a set of individual candidate biomarkers of response to pathway inhibitors, proteomic pathway scores did not correlate with amplification or mutation and did not predict response. Several receptor tyrosine kinases, including EGFR and ERK, were activated following PI3K inhibition in resistant cells; dual pathway inhibition of PI3K and EGFR or MEK demonstrated synergy. Combined MEK and PI3K inhibition was markedly synergistic in HRAS-mutant cell lines. Our findings indicate that clinical trials of single-agent PI3K/AKT/mTOR pathway inhibitors in selected populations and of PI3K/EGFR or PI3K/MEK inhibitor combinations are warranted; we plan to conduct such trials.

A pan-cancer proteomic perspective on The Cancer Genome Atlas.

Protein levels and function are poorly predicted by genomic and transcriptomic analysis of patient tumours. Therefore, direct study of the functional proteome has the potential to provide a wealth of information that complements and extends genomic, epigenomic and transcriptomic analysis in The Cancer Genome Atlas (TCGA) projects. Here we use reverse-phase protein arrays to analyse 3,467 patient samples from 11 TCGA 'Pan-Cancer' diseases, using 181 high-quality antibodies that target 128 total proteins and 53 post-translationally modified proteins. The resultant proteomic data are integrated with genomic and transcriptomic analyses of the same samples to identify commonalities, differences, emergent pathways and network biology within and across tumour lineages. In addition, tissue-specific signals are reduced computationally to enhance biomarker and target discovery spanning multiple tumour lineages. This integrative analysis, with an emphasis on pathways and potentially actionable proteins, provides a framework for determining the prognostic, predictive and therapeutic relevance of the functional proteome.