Long non-coding RNAs defining major subtypes of B cell precursor acute lymphoblastic leukemia

BACKGROUND: Long non-coding RNAs (lncRNAs) have emerged as a novel class of RNA due to its diverse mechanism in cancer development and progression. However, the role and expression pattern of lncRNAs in molecular subtypes of B cell acute lymphoblastic leukemia (BCP-ALL) have not yet been investigated. Here, we assess to what extent lncRNA expression and DNA methylation is driving the progression of relapsed BCP-ALL subtypes and we determine if the expression and DNA methylation profile of lncRNAs correlates with established BCP-ALL subtypes. METHODS: We performed RNA sequencing and DNA methylation (Illumina Infinium microarray) of 40 diagnosis and 42 relapse samples from 45 BCP-ALL patients in a German cohort and quantified lncRNA expression. Unsupervised clustering was applied to ascertain and confirm that the lncRNA-based classification of the BCP-ALL molecular subtypes is present in both our cohort and an independent validation cohort of 47 patients. A differential expression and differential methylation analysis was applied to determine the subtype-specific, relapse-specific, and differentially methylated lncRNAs. Potential functions of subtype-specific lncRNAs were determined by using co-expression-based analysis on nearby (cis) and distally (trans) located protein-coding genes. RESULTS: Using an integrative Bioinformatics analysis, we developed a comprehensive catalog of 1235 aberrantly dysregulated BCP-ALL subtype-specific and 942 relapse-specific lncRNAs and the methylation profile of three subtypes of BCP-ALL. The 1235 subtype-specific lncRNA signature represented a similar classification of the molecular subtypes of BCP-ALL in the independent validation cohort. We identified a strong correlation between the DUX4-specific lncRNAs and genes involved in the activation of TGF-β and Hippo signaling pathways. Similarly, Ph-like-specific lncRNAs were correlated with genes involved in the activation of PI3K-AKT, mTOR, and JAK-STAT signaling pathways. Interestingly, the relapse-specific lncRNAs correlated with the activation of metabolic and signaling pathways. Finally, we found 23 promoter methylated lncRNAs epigenetically facilitating their expression levels. CONCLUSION: Here, we describe a set of subtype-specific and relapse-specific lncRNAs from three major BCP-ALL subtypes and define their potential functions and epigenetic regulation. The subtype-specific lncRNAs are reproducible and can effectively stratify BCP-ALL subtypes. Our data uncover the diverse mechanism of action of lncRNAs in BCP-ALL subtypes defining which lncRNAs are involved in the pathogenesis of disease and are relevant for the stratification of BCP-ALL subtypes

Epigenetic regulation of neuroblastoma development

In recent years, technological advances have enabled a detailed landscaping of the epigenome and the mechanisms of epigenetic regulation that drive normal cell function, development and cancer. Rather than merely a structural entity to support genome compaction, we now look at chromatin as a very dynamic and essential constellation that is actively participating in the tight orchestration of transcriptional regulation as well as DNA replication and repair. The unique feature of chromatin flexibility enabling fast switches towards more or less restricted epigenetic cellular states is, not surprisingly, intimately connected to cancer development and treatment resistance, and the central role of epigenetic alterations in cancer is illustrated by the finding that up to 50% of all mutations across cancer entities affect proteins controlling the chromatin status. We summarize recent insights into epigenetic rewiring underlying neuroblastoma (NB) tumor formation ranging from changes in DNA methylation patterns and mutations in epigenetic regulators to global effects on transcriptional regulatory circuits that involve key players in NB oncogenesis. Insights into the disruption of the homeostatic epigenetic balance contributing to developmental arrest of sympathetic progenitor cells and subsequent NB oncogenesis are rapidly growing and will be exploited towards the development of novel therapeutic strategies to increase current survival rates of patients with high-risk NB

SINGLE CELL LINEAGE TRACING REVEALS MECHANISMS OF TUMOR INITIATION AND CHEMORESISTANCE IN SMALL CELL LUNG CANCER

Small Cell Lung Cancer (SCLC) is a devastating disease characterized by a very low two-year survival rate and almost universal acquisition of chemoresistance. Nearly all patients have tumors driven by functional inactivation of the tumor suppressors Rb and p53, but despite the uniform origins of this tumor, not all patients are genetically or phenotypically identical. SCLC can be subtyped into four unique molecular subtypes, determined by the expression of ASCL1, NEUROD1, POU2F3, or YAP1. These subtypes are plastic, and subtype switching after chemotherapy has been documented. Without the understanding of how tumor heterogeneity arises, we cannot solve the challenge of chemoresistance in SCLC. In recent years, a powerful new tool in studying tumor heterogeneity has emerged. Genetic barcoding allows for the identification and tracking of individual tumor populations by inserting a small genetic sequence (“barcode”) into the genome of tumor cells. As the cells divide, the barcode is passed on and a high-resolution lineage map is constructed. Here, genetic barcoding is used for the first time in SCLC, combined with single-cell RNA sequencing in a genetically engineered mouse model and a xenograft model of SCLC. In the mouse model of SCLC, tumors were sequenced at early, middle, and late stages of tumor development, as well as chemoresistant tumors. While no barcodes were detected by scRNA-seq, valuable information about the process of tumor development in SCLC is observed. I identify two cellular populations (“early” and “late”) that arise during tumor development. A notable difference in the two populations is the expression of genes corresponding to members of the AP-1 network. The AP-1 network was validated to be critical for tumorigenesis in SCLC. Barcoded SCLC xenografts and chemoresistant xenografts belonging to two SCLC subtypes were generated. scRNA-seq revealed increased transcriptomic plasticity following chemotherapy treatment in SCLC-A xenografts but not SCLC-N xenografts. The Cancer Testis Antigens PAGE5 and GAGE2A were identified and validated as mediators of chemoresistance in SCLC. This work represents the first application of genetic barcoding in SCLC and identifies actionable drug targets for future development

A PROTEOMIC ANALYSIS OF NEOPLASTIC PROGRESSION IN BREAST CANCER

The utilization of high-throughput -omics strategies, such as proteomics, in the analysis of breastcancer will function to define central molecular characteristics across a disease that is associatedwith a high degree of molecular heterogeneity. Data reported herein details the investigation ofkey subjects in breast cancer biology focused on the characterization of endogenous andexperimentally-induced disease biology characteristics utilizing the application of LC-MS basedproteomic analyses of both in vitro models of breast cancer as well as primary clinical samples.Results include a combined global and functional proteomic strategy to identify governingfunctional roles for mutually, differentially abundant proteins observed across three divergentcell line models of breast cancer. Further, evidence is presented which provides insights into theregulatory activity of the breast cancer-associated microRNA (miR-145) in several cell linemodels of breast cancer in which expression of this microRNA has been restored. Lastly, robustanalyses are detailed focused on the identification of differential protein characteristics indicativeof disease stage as well as of recurrent disease in breast cancer derived from proteomic analysisof formalin-fixed, paraffin embedded (FFPE) clinical samples. These studies contribute to thefield of proteomics in the form of 1) providing robust experimental workflows directed towardsinvestigation of functional themes and associated functional targets in large protein data sets 2)detailing strategies for navigating the application of proteomic analysis to microRNA targetdiscovery and 3) further development and utilization of methodologies towards the proteomicanalysis of clinical, FFPE tissue samples. Furthermore, these studies benefit the breast cancercommunity on several fronts including 1) the elucidation of provocative protein candidateswhich warrant further investigation for their role in regulating disease mechanisms underlyingvbreast cancer biology and 2) through the discovery of diagnostic markers indicative of discretesubtypes and stages of disease progression in breast cancer. The results reported herein detaildisease-specific protein abundance characteristics associated with neoplastic progression inbreast cancer that will benefit further expansion of the basic biological understanding of thisdisease and describes novel proteins for further evaluation as biomarker candidates for thediagnosis of breast cancer

The inflammatory infiltrate of high-grade serous carcinoma omental metastasis

PhDThe aim of this thesis is to investigate the role of inflammatory infiltrates and chemokines in metastasis of high-grade serous ovarian cancer, HGSC, to the omentum using human tissue biopsies and a 3- dimensional (3D) cell culture model. In ten patients with metastatic HGSC, omental tumour deposits contained a prominent leukocyte infiltrate of CD3+ T cells (9% of total cells) and CD68+ macrophages (11% of total cells). The presence of CD68+ macrophages showed a significant positive correlation with tumour cell proliferation analysed by Ki67 expression. Four ovarian cancer cell lines were co-cultured on a 3D model mimicking the microenvironment of the omentum for two weeks. The model was composed of collagen embedded human fibroblasts covered in a confluent layer of human primary mesothelial cells. The mesothelial cells in the 3D model significantly increased the growth (p = 0.002) and invasion (p = 0.0004) of the ovarian cancer cells. CXCL12 is the macrophage chemoattractant and ligand for the major chemokine receptor expressed on ovarian cancer cells. An association between CXCL12 and extracellular matrix remodelling was identified in two independent gene expression microarrays of ovarian cancer biopsies. The expression of CXCL12 in the HGSC omental metastases measured by quantitative Real Time-PCR positively correlated with decorin expression. Antibody mediated neutralisation of CXCL12 reduced growth (p = 0.012) and invasion (p = 0.029) in the 3D model. Mimicking an infiltrate of CD68+ macrophages in this multicellular 3D in vitro system also produced measurable changes in inflammatory cytokine and chemokine expression. There is currently a demand for more accurate models of HGSC and a necessity to study its metastasis that presents itself as the major clinical problem in patients. Therefore the development of this 3D model to mimic tumour-promoting inflammation in HGSC metastasis will provide researchers with an essential tool for testing novel therapeutic strategies.This work was supported by the BBSRC (Biotechnology and Biological Sciences Research Council) and AstraZeneca in an Industrial Case Studentship, grant number BB/G017867/1

Inflammatory Breast Cancer: The Immune Perspective

Inflammatory breast cancer (IBC) is the most insidious form of locally advanced disease. Although rare and less than 2% of all breast cancer, IBC is responsible for up to 10% of all breast cancer deaths. Despite the name, very little is known about the role of inflammation or immune mediators in IBC. Therefore, we analyzed blood samples from IBC patients and non-IBC patients, as well as healthy donor controls to establish an IBC-specific profile of peripheral blood leukocyte phenotype and function of T cells and dendritic cells and serum inflammatory cytokines. Emerging evidence suggests that host factors in the microenviromement may interact with underlying IBC genetics to promote the aggressive nature of the tumor. An integral part of the metastatic process involves epithelial to mesenchymal transition (EMT) where primary breast cancer cells gain motility and stem cell-like features that allow distant seeding. Interestingly, the IBC consortium microarray data found no clear evidence for EMT in IBC tumor tissues. It is becoming increasingly evident that inflammatory factors can induce EMT. However, it is unknown if EMT-inducing soluble factors secreted by activated immune cells in the IBC microenvironment canπ account for the absence of EMT in studies of the tumor cells themselves. We hypothesized that soluble factors from immune cells are capable of inducing EMT in IBC. We tested the ability of immune conditioned media to induce EMT in IBC cells. We found that soluble factors from activated immune cells are able to induce the expression of EMT-related factors in IBC cells along with increased migration and invasion. Specifically, the pro-inflammatory cytokines TNF-α, IL-6 and TGF-β were able to induce EMT and blocking these factors in conditioned media abated the induction of EMT. Surprisingly, unique to IBC cells, this process was related to increased levels of E-cadherin expression and adhesion, reminiscent of the characteristic tightly packed tumor emboli seen in IBC samples. This data offers insight into the unique pathology of IBC by suggesting that tumor immune interactions in the tumor microenvironment contribute to the aggressive nature of IBC implying that immune induced inflammation can be a novel therapeutic target. Specifically, we showed that soluble factors secreted by activated immune cells are capable of inducing EMT in IBC cells and may mediate the persistent E-cadherin expression observed in IBC. This data suggests that immune mediated inflammation may contribute to the highly aggressive nature of IBC and represents a potential therapeutic target that warrants further investigation