MoCoLo: a testing framework for motif co-localization.

Sequence-level data offers insights into biological processes through the interaction of two or more genomic features from the same or different molecular data types. Within motifs, this interaction is often explored via the co-occurrence of feature genomic tracks using fixed-segments or analytical tests that respectively require window size determination and risk of false positives from over-simplified models. Moreover, methods for robustly examining the co-localization of genomic features, and thereby understanding their spatial interaction, have been elusive. We present a new analytical method for examining feature interaction by introducing the notion of reciprocal co-occurrence, define statistics to estimate it and hypotheses to test for it. Our approach leverages conditional motif co-occurrence events between features to infer their co-localization. Using reverse conditional probabilities and introducing a novel simulation approach that retains motif properties (e.g. length, guanine-content), our method further accounts for potential confounders in testing. As a proof-of-concept, motif co-localization (MoCoLo) confirmed the co-occurrence of histone markers in a breast cancer cell line. As a novel analysis, MoCoLo identified significant co-localization of oxidative DNA damage within non-B DNA-forming regions that significantly differed between non-B DNA structures. Altogether, these findings demonstrate the potential utility of MoCoLo for testing spatial interactions between genomic features via their co-localization.

TTSBBC: triplex target site biomarkers and barcodes in cancer.

The technology of triplex-forming oligonucleotides (TFOs) provides an approach to manipulate genes at the DNA level. TFOs bind to specific sites on genomic DNA, creating a unique intermolecular triple-helix DNA structure through Hoogsteen hydrogen bonding. This targeting by TFOs is site-specific and the locations TFOs bind are referred to as TFO target sites (TTS). Triplexes have been observed to selectively influence gene expression, homologous recombination, mutations, protein binding, and DNA damage. These sites typically feature a poly-purine sequence in duplex DNA, and the characteristics of these TTS sequences greatly influence the formation of the triplex. We introduce TTSBBC, a novel analysis and visualization platform designed to explore features of TTS sequences to enable users to design and validate TTSs. The web server can be freely accessed at https://kowalski-labapps.dellmed.utexas.edu/TTSBBC/.

NBBC: a non-B DNA burden explorer in cancer.

Alternate (non-B) DNA-forming structures, such as Z-DNA, G-quadruplex, triplex have demonstrated a potential role in cancer etiology. It has been found that non-B DNA-forming sequences can stimulate genetic instability in human cancer genomes, implicating them in the development of cancer and other genetic diseases. While there exist several non-B prediction tools and databases, they lack the ability to both analyze and visualize non-B data within a cancer context. Herein, we introduce NBBC, a non-B DNA burden explorer in cancer, that offers analyses and visualizations for non-B DNA forming motifs. To do so, we introduce 'non-B burden' as a metric to summarize the prevalence of non-B DNA motifs at the gene-, signature- and genomic site-levels. Using our non-B burden metric, we developed two analyses modules within a cancer context to assist in exploring both gene- and motif-level non-B type heterogeneity among gene signatures. NBBC is designed to serve as a new analysis and visualization platform for the exploration of non-B DNA, guided by non-B burden as a novel marker.

A Case Series Exploration of Multi-Regional Expression Heterogeneity in Triple-Negative Breast Cancer Patients.

Extensive intratumoral heterogeneity (ITH) is believed to contribute to therapeutic failure and tumor recurrence, as treatment-resistant cell clones can survive and expand. However, little is known about ITH in triple-negative breast cancer (TNBC) because of the limited number of single-cell sequencing studies on TNBC. In this study, we explored ITH in TNBC by evaluating gene expression-derived and imaging-derived multi-region differences within the same tumor. We obtained tissue specimens from 10 TNBC patients and conducted RNA sequencing analysis of 2-4 regions per tumor. We developed a novel analysis framework to dissect and characterize different types of variability: between-patients (inter-tumoral heterogeneity), between-patients across regions (inter-tumoral and region heterogeneity), and within-patient, between-regions (regional intratumoral heterogeneity). We performed a Bayesian changepoint analysis to assess and classify regional variability as low (convergent) versus high (divergent) within each patient feature (TNBC and PAM50 subtypes, immune, stroma, tumor counts and tumor infiltrating lymphocytes). Gene expression signatures were categorized into three types of variability: between-patients (108 genes), between-patients across regions (183 genes), and within-patients, between-regions (778 genes). Based on the between-patient gene signature, we identified two distinct patient clusters that differed in menopausal status. Significant intratumoral divergence was observed for PAM50 classification, tumor cell counts, and tumor-infiltrating T cell abundance. Other features examined showed a representation of both divergent and convergent results. Lymph node stage was significantly associated with divergent tumors. Our results show extensive intertumoral heterogeneity and regional ITH in gene expression and image-derived features in TNBC. Our findings also raise concerns regarding gene expression based TNBC subtyping. Future studies are warranted to elucidate the role of regional heterogeneity in TNBC as a driver of treatment resistance.

Genetic heterogeneity within collective invasion packs drives leader and follower cell phenotypes.

Collective invasion, the coordinated movement of cohesive packs of cells, has become recognized as a major mode of metastasis for solid tumors. These packs are phenotypically heterogeneous and include specialized cells that lead the invasive pack and others that follow behind. To better understand how these unique cell types cooperate to facilitate collective invasion, we analyzed transcriptomic sequence variation between leader and follower populations isolated from the H1299 non-small cell lung cancer cell line using an image-guided selection technique. We now identify 14 expressed mutations that are selectively enriched in leader or follower cells, suggesting a novel link between genomic and phenotypic heterogeneity within a collectively invading tumor cell population. Functional characterization of two phenotype-specific candidate mutations showed that ARP3 enhances collective invasion by promoting the leader cell phenotype and that wild-type KDM5B suppresses chain-like cooperative behavior. These results demonstrate an important role for distinct genetic variants in establishing leader and follower phenotypes and highlight the necessity of maintaining a capacity for phenotypic plasticity during collective cancer invasion.

Prognostic significance of an invasive leader cell-derived mutation cluster on chromosome 16q.

BACKGROUND: Intratumoral heterogeneity is defined by subpopulations with varying genotypes and phenotypes. Specialized, highly invasive leader cells and less invasive follower cells are phenotypically distinct subpopulations that cooperate during collective cancer invasion. Because leader cells are a rare subpopulation that would be missed by bulk sequencing, a novel image-guided genomics platform was used to precisely select this subpopulation. This study identified a novel leader cell mutation signature and tested its ability to predict prognosis in non-small cell lung cancer (NSCLC) patient cohorts. METHODS: Spatiotemporal genomic and cellular analysis was used to isolate and perform RNA sequencing on leader and follower populations from the H1299 NSCLC cell line, and it revealed a leader-specific mutation cluster on chromosome 16q. Genomic data from patients with lung squamous cell carcinoma (LUSC; n = 475) and lung adenocarcinoma (LUAD; n = 501) from The Cancer Genome Atlas were stratified by 16q mutation cluster (16qMC) status (16qMC+ vs 16qMC-) and compared for overall survival (OS), progression-free survival (PFS), and gene set enrichment analysis (GSEA). RESULTS: Poorer OS, poorer PFS, or both were found across all stages and among early-stage patients with 16qMC+ tumors within the LUSC and LUAD cohorts. GSEA revealed 16qMC+ tumors to be enriched for the expression of metastasis- and survival-associated gene sets. CONCLUSIONS: This represents the first leader cell mutation signature identified in patients and has the potential to better stratify high-risk NSCLC and ultimately improve patient outcomes.

Flt3L Treatment of Bone Marrow Donors Increases Graft Plasmacytoid Dendritic Cell Content and Improves Allogeneic Transplantation Outcomes.

A higher number of donor plasmacytoid dendritic cells (pDCs) is associated with increased survival and reduced graft-versus-host disease (GVHD) in human recipients of unrelated donor bone marrow (BM) grafts, but not granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood grafts. We show that in murine models, donor BM pDCs are associated with increased survival and decreased GVHD compared with G-CSF-mobilized pDCs. To increase the content of pDCs in BM grafts, we studied the effect of FMS-like tyrosine kinase 3 ligand (Flt3L) treatment of murine BM donors on transplantation outcomes. Flt3L treatment (300 µg/kg/day) resulted in a schedule-dependent increase in the content of pDCs in the BM. Mice treated on days -4 and -1 had a >5-fold increase in pDC content without significant changes in numbers of HSCs, T cells, B cells, and natural killer cells in the BM graft. In an MHC-mismatched murine transplant model, recipients of Flt3L-treated T cell-depleted (TCD) BM (TCD F-BM) and cytokine-untreated T cells had increased survival and decreased GVHD scores with fewer Th1 and Th17 polarized T cells post-transplantation compared with recipients of equivalent numbers of untreated donor TCD BM and T cells. Gene array analyses of pDCs from Flt3L-treated human and murine donors showed up-regulation of adaptive immune pathways and immunoregulatory checkpoints compared with pDCs from untreated BM donors. Transplantation of TCD F-BM plus T cells resulted in no loss of the graft-versus-leukemia (GVL) effect compared with grafts from untreated donors in 2 murine GVL models. Thus, Flt3L treatment of BM donors is a novel method for increasing the pDC content in allografts, improving survival, and decreasing GVHD without diminishing the GVL effect.

Distinct Gene Expression Profiles Define Anaplastic Grade in Retinoblastoma.

Morbidity and mortality associated with retinoblastoma have decreased drastically in recent decades, in large part owing to better prediction of high-risk disease and appropriate treatment stratification. High-risk histopathologic features and severe anaplasia both predict the need for more aggressive treatment; however, not all centers are able to assess tumor samples easily for the degree of anaplasia. Instead, identification of genetic signatures that are able to distinguish among anaplastic grades and thus predict high- versus low-risk retinoblastoma would facilitate appropriate risk stratification in a wider patient population. A better understanding of genes dysregulated in anaplasia also would yield valuable insights into pathways underlying the development of more severe retinoblastoma. Here, we present the histopathologic and gene expression analysis of 28 retinoblastoma cases using microarray analysis. Tumors of differing anaplastic grade show clear differential gene expression, with significant dysregulation of unique genes and pathways in severe anaplasia. Photoreceptor and nucleoporin expression in particular are identified as highly dysregulated in severe anaplasia and suggest particular cellular processes contributing to the development of increased retinoblastoma severity. A limited set of highly differentially expressed genes also are able to predict severe anaplasia accurately in our data set. Together, these data contribute to the understanding of the development of anaplasia and facilitate the identification of genetic markers of high-risk retinoblastoma.

Gliosarcoma: distinct molecular pathways and genomic alterations identified by DNA copy number/SNP microarray analysis.

PURPOSE: Gliosarcoma is a histologic variant of glioblastoma (GBM), and like GBM carries a poor prognosis. Median survival is less than one (1) year with less than 5% of patients alive after 5 years. Although there is no cure, standard treatment includes surgery, radiation and chemotherapy. While very similar to GBM, gliosarcoma exhibits several distinct differences, morphologically and molecularly. Therefore, we report a comprehensive analysis of DNA copy number changes in gliosarcoma using a cytogenomic DNA copy number (CN) microarray (OncoScan®). METHODS: Cytogenomic DNA copy number microarray (OncoScan®) was performed on 18 cases of gliosarcoma. MetaCore™ enrichment was applied to the array results to detect associated molecular pathways. RESULTS: The most frequent alteration was copy number loss, comprising 57% of total copy number changes. The number of losses far exceeded the number of amplifications (***, < 0.001) and loss of heterozygosity events (***, < 0.001). Amplifications were infrequent (4.6%), particularly for EGFR. Chromosomes 9 and 10 had the highest number of losses; a large portion of which correlated to CDKN2A/B loss. Copy number gains were the second most common alteration (26.2%), with the majority occurring on chromosome 7. MetaCore™ enrichment detected notable pathways for copy number gains including: HOXA, Rho family of GTPases, and EGFR; copy number loss including: WNT, NF-kß, and CDKN2A; and copy number loss of heterozygosity including: WNT and p53. CONCLUSIONS: The pathways and copy number alterations detected in this study may represent key drivers in gliosarcoma oncogenesis and may provide a starting point toward targeted oncologic analysis with therapeutic potential.

Modulating native GABAA receptors in medulloblastoma with positive allosteric benzodiazepine-derivatives induces cell death.

PURPOSE: Pediatric brain cancer medulloblastoma (MB) standard-of-care results in numerous comorbidities. MB is comprised of distinct molecular subgroups. Group 3 molecular subgroup patients have the highest relapse rates and after standard-of-care have a 20% survival. Group 3 tumors have high expression of GABRA5, which codes for the α5 subunit of the γ-aminobutyric acid type A receptor (GABAAR). We are advancing a therapeutic approach for group 3 based on GABAAR modulation using benzodiazepine-derivatives. METHODS: We performed analysis of GABR and MYC expression in MB tumors and used molecular, cell biological, and whole-cell electrophysiology approaches to establish presence of a functional 'druggable' GABAAR in group 3 cells. RESULTS: Analysis of expression of 763 MB tumors reveals that group 3 tumors share high subgroup-specific and correlative expression of GABR genes, which code for GABAAR subunits α5, ß3 and γ2 and 3. There are ~ 1000 functional α5-GABAARs per group 3 patient-derived cell that mediate a basal chloride-anion efflux of 2 × 109 ions/s. Benzodiazepines, designed to prefer α5-GABAAR, impair group 3 cell viability by enhancing chloride-anion efflux with subtle changes in their structure having significant impact on potency. A potent, non-toxic benzodiazepine ('KRM-II-08') binds to the α5-GABAAR (0.8 µM EC50) enhancing a chloride-anion efflux that induces mitochondrial membrane depolarization and in response, TP53 upregulation and p53, constitutively phosphorylated at S392, cytoplasmic localization. This correlates with pro-apoptotic Bcl-2-associated death promoter protein localization. CONCLUSION: GABRA5 expression can serve as a diagnostic biomarker for group 3 tumors, while α5-GABAAR is a therapeutic target for benzodiazepine binding, enhancing an ion imbalance that induces apoptosis.