Pieter Rondou

A cooperative microRNA-tumor suppressor gene network in acute T-cell lymphoblastic leukemia (T-ALL)

The importance of individual microRNAs (miRNAs) has been established in specific cancers. However, a comprehensive analysis of the contribution of miRNAs to the pathogenesis of any specific cancer is lacking. Here we show that in T-cell acute lymphoblastic leukemia (T-ALL), a small set of miRNAs is responsible for the cooperative suppression of several tumor suppressor genes. Cross-comparison of miRNA expression profiles in human T-ALL with the results of an unbiased miRNA library screen allowed us to identify five miRNAs (miR-19b, miR-20a, miR-26a, miR-92 and miR-223) that are capable of promoting T-ALL development in a mouse model and which account for the majority of miRNA expression in human T-ALL. Moreover, these miRNAs produce overlapping and cooperative effects on tumor suppressor genes implicated in the pathogenesis of T-ALL, including IKAROS (also known as IKZF1), PTEN, BIM, PHF6, NF1 and FBXW7. Thus, a comprehensive and unbiased analysis of miRNA action in T-ALL reveals a striking pattern of miRNA-tumor suppressor gene interactions in this cancer.

The need for transparency and good practices in the qPCR literature

Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.

The H3K27me3 demethylase UTX is a gender-specific tumor suppressor in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of leukemia that is mainly diagnosed in children and shows a skewed gender distribution toward males. In this study, we report somatic loss-of-function mutations in the X-linked histone H3K27me3 demethylase ubiquitously transcribed X (UTX) chromosome, in human T-ALL. Interestingly, UTX mutations were exclusively present in male T-ALL patients and allelic expression analysis revealed that UTX escapes X-inactivation in female T-ALL lymphoblasts and normal T cells. Notably, we demonstrate in vitro and in vivo that the H3K27me3 demethylase UTX functions as a bona fide tumor suppressor in T-ALL. Moreover, T-ALL driven by UTX inactivation exhibits collateral sensitivity to pharmacologic H3K27me3 inhibition. All together, our results show how a gender-specific and therapeutically relevant defect in balancing H3K27 methylation contributes to T-cell leukemogenesis.

The dopamine D4 receptor: biochemical and signalling properties.

Dopamine is an important neurotransmitter that regulates several key functions in the brain, such as motor output, motivation and reward, learning and memory, and endocrine regulation. Dopamine does not mediate fast synaptic transmission, but rather modulates it by triggering slow-acting effects through the activation of dopamine receptors, which belong to the G-protein-coupled receptor superfamily. Besides activating different effectors through G-protein coupling, dopamine receptors also signal through interaction with a variety of proteins, collectively termed dopamine receptor-interacting proteins. We focus on the dopamine D4 receptor, which contains an important polymorphism in its third intracellular loop. This polymorphism has been the subject of numerous studies investigating links with several brain disorders, such as attention-deficit hyperactivity disorder and schizophrenia. We provide an overview of the structure, signalling properties and regulation of dopamine D4 receptors, and briefly discuss their physiological and pathophysiological role in the brain.

BTB Protein KLHL12 Targets the Dopamine D4 Receptor for Ubiquitination by a Cul3-based E3 Ligase

Dopamine receptors belong to the superfamily of G-protein-coupled receptors and are subdivided into D1-type (D1 and D5) and D2-type (D2, D3, and D4) receptors. The D4 receptor has a remarkable polymorphism in its third intracellular loop, which is under intensive investigation and which has been associated with, among other conditions, attention deficit hyperactivity disorder. Here, we demonstrate that KLHL12, a BTB-Kelch protein, specifically binds to this polymorphic region of the D4 receptor through its Kelch domain. Moreover, we show that KLHL12 also interacts with Cullin3 and thereby functions as an adaptor to target the D4 receptor to an E3 ubiquitin ligase complex. By ubiquitination assays in eukaryotic cells, we further demonstrate that overexpression of KLHL12 strongly promotes ubiquitination of the D4 receptor. In addition, we show that also other dopamine receptor subtypes undergo basal ubiquitination, but this is not affected by KLHL12. These data are the first to show ubiquitination of dopamine receptors and the first to identify a protein specifically interacting with the D4 polymorphism, thereby building up an E3 ligase complex with substrate specificity toward the D4 receptor.

MicroRNA-193b-3p acts as a tumor suppressor by targeting the MYB oncogene in T-cell acute lymphoblastic leukemia

The MYB oncogene is a leucine zipper transcription factor essential for normal and malignant hematopoiesis. In T-cell acute lymphoblastic leukemia (T-ALL), elevated MYB levels can arise directly through T-cell receptor-mediated MYB translocations, genomic MYB duplications or enhanced TAL1 complex binding at the MYB locus or indirectly through the TAL1/miR-223/FBXW7 regulatory axis. In this study, we used an unbiased MYB 3′untranslated region–microRNA (miRNA) library screen and identified 33 putative MYB-targeting miRNAs. Subsequently, transcriptome data from two independent T-ALL cohorts and different subsets of normal T-cells were used to select miRNAs with relevance in the context of normal and malignant T-cell transformation. Hereby, miR-193b-3p was identified as a novel bona fide tumor-suppressor miRNA that targets MYB during malignant T-cell transformation thereby offering an entry point for efficient MYB targeting-oriented therapies for human T-ALL.

Novel biological insights in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive type of blood cancer that accounts for about 15% of pediatric and 25% of adult acute lymphoblastic leukemia (ALL) cases. It is considered as a paradigm for the multistep nature of cancer initiation and progression. Genetic and epigenetic reprogramming events, which transform T-cell precursors into malignant T-ALL lymphoblasts, have been extensively characterized over the past decade. Despite our comprehensive understanding of the genomic landscape of human T-ALL, leukemia patients are still treated by high-dose multiagent chemotherapy, potentially followed by hematopoietic stem cell transplantation. Even with such aggressive treatment regimens, which are often associated with considerable acute and long-term side effects, about 15% of pediatric and 40% of adult T-ALL patients still relapse, owing to acquired therapy resistance, and present with very dismal survival perspectives. Unfortunately, the molecular mechanisms by which residual T-ALL tumor cells survive chemotherapy and act as a reservoir for leukemic progression and hematologic relapse remain poorly understood. Nevertheless, it is expected that enhanced molecular understanding of T-ALL disease biology will ultimately facilitate a targeted therapy driven approach that can reduce chemotherapy-associated toxicities and improve survival of refractory T-ALL patients through personalized salvage therapy. In this review, we summarize recent biological insights into the molecular pathogenesis of T-ALL and speculate how the genetic landscape of T-ALL could trigger the development of novel therapeutic strategies for the treatment of human T-ALL.

Dopamine D4 receptor oligomerization – contribution to receptor biogenesis

Dopamine D4 receptors (D4Rs) are G protein‐coupled receptors that play a role in attention and cognition. In the present study, we investigated the dimerization properties of this receptor. Western blot analysis of the human D4.2R, D4.4R and D4.7R revealed the presence of higher molecular weight immunoreactive bands, which might indicate the formation of receptor dimers and multimers. Homo‐ and heterodimerization of the receptors was confirmed by co‐immunoprecipitation and bioluminescence resonance energy transfer studies. Although dimerization of a large number of G protein‐coupled receptors has been described, the functional importance often remains to be elucidated. Folding efficiency is rate‐limiting for D4R biogenesis and quality control in the endoplasmic reticulum plays an important role for D4R maturation. Co‐immunoprecipitation and immunofluorescence microscopy studies using wild‐type and a nonfunctional D4.4R folding mutant show that oligomerization occurs in the endoplasmic reticulum and that this plays a role in the biogenesis and cell surface targeting of the D4R. The different polymorphic repeat variants of the D4R display differential sensitivity to the chaperone effect. In the present study, we show that this is also reflected by bioluminescence resonance energy transfer saturation assays, suggesting that the polymorphic repeat variants have different relative affinities to form homo‐ and heterodimers. In summary, we conclude that D4Rs form oligomers with different affinities and that dimerization plays a role in receptor biogenesis.

ZEB2 drives immature T-cell lymphoblastic leukaemia development via enhanced tumour-initiating potential and IL-7 receptor signalling

Early T-cell precursor leukaemia (ETP-ALL) is a high-risk subtype of human leukaemia that is poorly understood at the molecular level. Here we report translocations targeting the zinc finger E-box-binding transcription factor ZEB2 as a recurrent genetic lesion in immature/ETP-ALL. Using a conditional gain-of-function mouse model, we demonstrate that sustained Zeb2 expression initiates T-cell leukaemia. Moreover, Zeb2-driven mouse leukaemia exhibit some features of the human immature/ETP-ALL gene expression signature, as well as an enhanced leukaemia-initiation potential and activated Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signalling through transcriptional activation of IL7R. This study reveals ZEB2 as an oncogene in the biology of immature/ETP-ALL and paves the way towards pre-clinical studies of novel compounds for the treatment of this aggressive subtype of human T-ALL using our Zeb2-driven mouse model.

The Notch driven long non-coding RNA repertoire in T-cell acute lymphoblastic leukemia

Genetic studies in T-cell acute lymphoblastic leukemia have uncovered a remarkable complexity of oncogenic and loss-of-function mutations. Amongst this plethora of genetic changes, NOTCH1 activating mutations stand out as the most frequently occurring genetic defect, identified in more than 50% of T-cell acute lymphoblastic leukemias, supporting a role as an essential driver for this gene in T-cell acute lymphoblastic leukemia oncogenesis. In this study, we aimed to establish a comprehensive compendium of the long non-coding RNA transcriptome under control of Notch signaling. For this purpose, we measured the transcriptional response of all protein coding genes and long non-coding RNAs upon pharmacological Notch inhibition in the human T-cell acute lymphoblastic leukemia cell line CUTLL1 using RNA-sequencing. Similar Notch dependent profiles were established for normal human CD34+ thymic T-cell progenitors exposed to Notch signaling activity in vivo. In addition, we generated long non-coding RNA expression profiles (array data) from ex vivo isolated Notch active CD34+ and Notch inactive CD4+CD8+ thymocytes and from a primary cohort of 15 T-cell acute lymphoblastic leukemia patients with known NOTCH1 mutation status. Integration of these expression datasets with publicly available Notch1 ChIP-sequencing data resulted in the identification of long non-coding RNAs directly regulated by Notch activity in normal and malignant T cells. Given the central role of Notch in T-cell acute lymphoblastic leukemia oncogenesis, these data pave the way for the development of novel therapeutic strategies that target hyperactive Notch signaling in human T-cell acute lymphoblastic leukemia.