Functional dissection of transcriptional regulation during normal and malignant T-cell development : an integrative (epi)genomic approach

T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive malignant disorder. While originally associated with poor prognosis, more recent intensified T-ALL therapy has led to remarkable improvements in survival of these patients. Unfortunately, these therapeutic schemes are associated with severe acute and long-term toxicities, thus demanding for further research in order to design more precision medicine oriented treatment. Importantly, for T-ALL patients with relapsed and refractory T-ALL, outcome remains extremely poor, thus urging further investigations to design therapies with further reduced relapse risk and/or novel treatment to cure relapsed cases. To shift towards this personalized medicine approach, a more profound understanding of the molecular basis of T-ALL progression is required. Several decades of genetic studies in T-ALL have uncovered a remarkable heterogeneous and complex landscape of combined oncogenic and loss-of-function mutations that contribute to malignant thymocyte transformation. One of the major challenges in T-ALL research is to unravel in detail how the diverse complement of oncogenes and tumor suppressors functionally contribute to T-ALL pathogenesis and response to therapy. To this end, I have studied the functional properties and cooperation of several key players that participate in normal and malignant T-cell development at the level of transcriptional regulatory networks. TLX1 is a major driver oncogene causing transformation of immature thymocytes towards T-ALL. Previous pioneering work partly uncovered its mode of action in relation to T-ALL formation, showing that ectopic overexpression of TLX1 in immature thymocytes causes repression of multiple T-ALL tumor suppressor genes. The study performed during this doctoral mandate, led to the observation of an unexpected antagonism between the TLX1 and NOTCH1 oncogenes, with activated TLX1 suppressing NOTCH1 and key NOTCH1 target genes. Based on this finding, we hypothesized that this unique interaction between both oncogenes could explain the presence of NOTCH1 mutations in most TLX1 driven T-ALL. Furthermore, the required cooperativity of NOTCH1 (pathway) activating mutations can also explain the very long latency of T-ALL development in a TLX1 driven leukemia mouse model (paper 1). In addition to NOTCH1 mutations, PHF6 loss-of-function mutations are also frequently observed, pointing at a further putative required cooperative genetic lesion for full-blown TLX1 driven T-ALL formation. Given the lack of insight into the normal cellular function of the epigenetic reader protein PHF6, I investigated its role during normal hematopoiesis and observed a profound effect of PHF6 loss on hematopoietic lineage development (paper 2). Moreover, in the context of TLX1 driven T-ALL formation, I identified the tyrosine kinase ‘interleukine-7 receptor’ (IL7R) as a robustly upregulated gene upon PHF6 knockdown. Given the role of IL7R signaling in survival of maturing thymocytes, this observation opens an exciting perspective that PHF6 loss is required as an essential cooperative event in TLX1 driven T-ALL pathogenesis by re-installment of TLX1 repressed IL7R expression. Importantly, in addition to paving the way for further animal modeling and mechanistic studies, this finding is also highly relevant in the context of design of novel therapies targeting IL7R downstream JAK-STAT signaling (paper 3). Until recently, transcriptional regulatory networks were mainly studied from a ‘gene-protein coding’ genomic viewpoint. Several studies have challenged this central dogma based on the proven role of non-coding RNAs in control of normal cellular behavior. Given this exciting new perspective on further expanding complexity of gene regulation during normal development and malignant transformation, I decided to study the role of such micro-RNAs (miRNAs) and long non-coding RNAs (lncRNAs) in T-ALL perturbed transcriptional networks. More specifically, I studied the role of miRNAs under control of TAL1 (paper 4), unraveled the landscape of lncRNAs implicated in the NOTCH1 signaling pathway (paper 5) and performed the first landscaping of the TLX1 lncRNAome (paper 6). In conclusion, my work has contributed to novel insights into transcriptional networks in normal and malignant T-cell development, revealing several novel nodes for therapeutic intervention in the pursuit of personalized medicine development in the field of T-ALL research

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

T-ALL and thymocytes : a message of noncoding RNAs

In the last decade, the role for noncoding RNAs in disease was clearly established, starting with microRNAs and later expanded towards long noncoding RNAs. This was also the case for T cell acute lymphoblastic leukemia, which is a malignant blood disorder arising from oncogenic events during normal T cell development in the thymus. By studying the transcriptomic profile of protein-coding genes, several oncogenic events leading to T cell acute lymphoblastic leukemia (T-ALL) could be identified. In recent years, it became apparent that several of these oncogenes function via microRNAs and long noncoding RNAs. In this review, we give a detailed overview of the studies that describe the noncoding RNAome in T-ALL oncogenesis and normal T cell development

Early and late effects of pharmacological ALK inhibition on the neuroblastoma transcriptome

Background: Neuroblastoma is an aggressive childhood malignancy of the sympathetic nervous system. Despite multi-modal therapy, survival of high-risk patients remains disappointingly low, underscoring the need for novel treatment strategies. The discovery of ALK activating mutations opened the way to precision treatment in a subset of these patients. Previously, we investigated the transcriptional effects of pharmacological ALK inhibition on neuroblastoma cell lines, six hours after TAE684 administration, resulting in the 77-gene ALK signature, which was shown to gradually decrease from 120 minutes after TAE684 treatment, to gain deeper insight into the molecular effects of oncogenic ALK signaling. Aim: Here, we further dissected the transcriptional dynamic profiles of neuroblastoma cells upon TAE684 treatment in a detailed timeframe of ten minutes up to six hours after inhibition, in order to identify additional early targets for combination treatment. Results: We observed an unexpected initial upregulation of positively regulated MYCN target genes following subsequent downregulation of overall MYCN activity. In addition, we identified adrenomedullin (ADM), previously shown to be implicated in sunitinib resistance, as the earliest response gene upon ALK inhibition. Conclusions: We describe the early and late effects of ALK inhibitor TAE684 treatment on the neuroblastoma transcriptome. The observed unexpected upregulation of ADM warrants further investigation in relation to putative ALK resistance in neuroblastoma patients currently undergoing ALK inhibitor treatment

A nanobody modulates the p53 transcriptional program without perturbing its functional architecture

The p53 transcription factor plays an important role in genome integrity. To perform this task, p53 regulates the transcription of genes promoting various cellular outcomes including cell cycle arrest, apoptosis or senescence. The precise regulation of this activity remains elusive as numerous mechanisms, e.g. posttranslational modifications of p53 and (non-)covalent p53 binding partners, influence the p53 transcriptional program. We developed a novel, non-invasive tool to manipulate endogenous p53. Nanobodies (Nb), raised against the DNA-binding domain of p53, allow us to distinctively target both wild type and mutant p53 with great specificity. Nb3 preferentially binds ‘structural’ mutant p53, i.e. R175H and R282W, while a second but distinct nanobody, Nb139, binds both mutant and wild type p53. The co-crystal structure of the p53 DNA-binding domain in complex with Nb139 (1.9 Å resolution) reveals that Nb139 binds opposite the DNA-binding surface. Furthermore, we demonstrate that Nb139 does not disturb the functional architecture of the p53 DNA-binding domain using conformation-specific p53 antibody immunoprecipitations, glutaraldehyde crosslinking assays and chromatin immunoprecipitation. Functionally, the binding of Nb139 to p53 allows us to perturb the transactivation of p53 target genes. We propose that reduced recruitment of transcriptional co-activators or modulation of selected post-transcriptional modifications account for these observations

Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes

Background: Transgenic zebrafish lines with the expression of a fluorescent reporter under the control of a cell-type specific promoter, enable transcriptome analysis of FACS sorted cell populations. RNA quality and yield are key determinant factors for accurate expression profiling. Limited cell number and FACS induced cellular stress make RNA isolation of sorted zebrafish cells a delicate process. We aimed to optimize a workflow to extract sufficient amounts of high-quality RNA from a limited number of FACS sorted cells from Tg(fli1a:GFP) zebrafish embryos, which can be used for accurate gene expression analysis. Results: We evaluated two suitable RNA isolation kits (theRNAqueous micro and the RNeasy plus micro kit) and determined that sorting cells directly into lysis buffer is a critical step for success. For low cell numbers, this ensures direct cell lysis, protects RNA from degradation and results in a higher RNA quality and yield. We showed that this works well up to 0.5x dilution of the lysis buffer with sorted cells. In our sort settings, this corresponded to 30,000 and 75,000 cells for the RNAqueous micro kit and RNeasy plus micro kit respectively. Sorting more cells dilutes the lysis buffer too much and requires the use of a collection buffer. We also demonstrated that an additional genomic DNA removal step after RNA isolation is required to completely clear the RNA from any contaminating genomic DNA. For cDNA synthesis and library preparation, we combined SmartSeq v4 full length cDNA library amplification, Nextera XT tagmentation and sample barcoding. Using this workflow, we were able to generate highly reproducible RNA sequencing results. Conclusions: The presented optimized workflow enables to generate high quality RNA and allows accurate transcriptome profiling of small populations of sorted zebrafish cells

In silico discovery of a FOXM1 driven embryonal signaling pathway in therapy resistant neuroblastoma tumors

Chemotherapy resistance is responsible for high mortality rates in neuroblastoma. MYCN, an oncogenic driver in neuroblastoma, controls pluripotency genes including LIN28B. We hypothesized that enhanced embryonic stem cell (ESC) gene regulatory programs could mark tumors with high pluripotency capacity and subsequently increased risk for therapy failure. An ESC miRNA signature was established based on publicly available data. In addition, an ESC mRNA signature was generated including the 500 protein coding genes with the highest positive expression correlation with the ESC miRNA signature score in 200 neuroblastomas. High ESC m(i)RNA expression signature scores were significantly correlated with poor neuroblastoma patient outcome specifically in the subgroup of MYCN amplified tumors and stage 4 nonamplified tumors. Further data-mining identified FOXM1, as the major predicted driver of this ESC signature, controlling a large set of genes implicated in cell cycle control and DNA damage response. Of further interest, re-analysis of published data showed that MYCN transcriptionally activates FOXM1 in neuroblastoma cells. In conclusion, a novel ESC m(i)RNA signature stratifies neuroblastomas with poor prognosis, enabling the identification of therapy-resistant tumors. The finding that this signature is strongly FOXM1 driven, warrants for drug design targeted at FOXM1 or key components controlling this pathway

PHF6 expression levels impact human hematopoietic stem cell differentiation

Transcriptional control of hematopoiesis involves complex regulatory networks and functional perturbations in one of these components often results in malignancies. Loss-of-function mutations in PHF6, encoding a presumed epigenetic regulator, have been primarily described in T cell acute lymphoblastic leukemia (T-ALL) and the first insights into its function in normal hematopoiesis only recently emerged from mouse modeling experiments. Here, we investigated the role of PHF6 in human blood cell development by performing knockdown studies in cord blood and thymus-derived hematopoietic precursors to evaluate the impact on lineage differentiation in well-established in vitro models. Our findings reveal that PHF6 levels differentially impact the differentiation of human hematopoietic progenitor cells into various blood cell lineages, with prominent effects on lymphoid and erythroid differentiation. We show that loss of PHF6 results in accelerated human T cell development through reduced expression of NOTCH1 and its downstream target genes. This functional interaction in developing thymocytes was confirmed in vivo using a phf6-deficient zebrafish model that also displayed accelerated developmental kinetics upon reduced phf6 or notch1 activation. In summary, our work reveals that appropriate control of PHF6 expression is important for normal human hematopoiesis and provides clues towards the role of PHF6 in T-ALL development