M.I. Domínguez

Mutational loss of PTEN induces resistance to NOTCH1 inhibition in T-cell leukemia

Gain-of-function mutations in NOTCH1 are common in T-cell lymphoblastic leukemias and lymphomas (T-ALL), making this receptor a promising target for drugs such as γ-secretase inhibitors, which block a proteolytic cleavage required for NOTCH1 activation. However, the enthusiasm for these therapies has been tempered by tumor resistance and the paucity of information on the oncogenic programs regulated by oncogenic NOTCH1. Here we show that NOTCH1 regulates the expression of PTEN (encoding phosphatase and tensin homolog) and the activity of the phosphoinositol-3 kinase (PI3K)-AKT signaling pathway in normal and leukemic T cells. Notch signaling and the PI3K-AKT pathway synergize in vivo in a Drosophila melanogaster model of Notch-induced tumorigenesis, and mutational loss of PTEN is associated with human T-ALL resistance to pharmacological inhibition of NOTCH1. Overall, these findings identify transcriptional control of PTEN and regulation of the PI3K-AKT pathway as key elements of the leukemogenic program activated by NOTCH1 and provide the basis for the design of new therapeutic strategies for T-ALL.

Genetic inactivation of the polycomb repressive complex 2 in T cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an immature hematopoietic malignancy driven mainly by oncogenic activation of NOTCH1 signaling1. In this study we report the presence of loss-of-function mutations and deletions of EZH2 and SUZ12 genes, encoding critical components of the Polycomb Repressive Complex 2 (PRC2) complex2,3, in 25% of T-ALLs. To further study the role of the PRC2 complex in T-ALL, we used NOTCH1-induced animal models of the disease, as well as human T-ALL samples, and combined locus-specific and global analysis of NOTCH1-driven epigenetic changes. These studies demonstrated that activation of NOTCH1 specifically induces loss of the repressive mark lysine-27 tri-methylation of histone 3 (H3K27me3)4 by antagonizing the activity of the Polycomb Repressive Complex 2 (PRC2) complex. These studies demonstrate a tumor suppressor role for the PRC2 complex in human leukemia and suggest a hitherto unrecognized dynamic interplay between oncogenic NOTCH1 and PRC2 function for the regulation of gene expression and cell transformation.

The role of the PTEN/AKT pathway in NOTCH1-induced leukemia

Activating mutations in NOTCH1 are the most prominent genetic abnormality in T-cell acute Lymphoblastic Leukemia (T-ALL) and inhibition of NOTCH1 signaling with γ-secretase inhibitors (GSIs) has been proposed as targeted therapy in this disease. However, most T-ALL cell lines with mutations in NOTCH1 fail to respond to GSI therapy. Using gene expression profiling and mutation analysis we showed that mutational loss of PTEN is a common event in T-ALL and is associated with resistance to NOTCH inhibition. Furthermore, our studies revealed that NOTCH1 induces upregulation of the PI3K-AKT pathway via HES1, which negatively controls the expression of PTEN. This regulatory circuitry is evolutionary conserved from Drosophila to humans as demonstrated by the interaction of overexpression of Delta and Akt in a model of Notch-induced transformation in the fly eye. Loss of PTEN and constitutive activation of AKT in T-ALL induce increased glucose metabolism and bypass the requirement of NOTCH1 signaling to sustain cell growth. Importantly, PTEN-null/GSI resistant T-ALL cells switch their oncogene addiction from NOTCH1 to AKT and are highly sensitive to AKT inhibitors. These results should facilitate the development of molecular therapies targeting NOTCH1 and AKT for the treatment of T-ALL.

Epigenetic silencers and Notch collaborate to promote malignant tumours by Rb silencing

Cancer is both a genetic and an epigenetic disease. Inactivation of tumour-suppressor genes by epigenetic changes is frequently observed in human cancers, particularly as a result of the modifications of histones and DNA methylation. It is therefore important to understand how these damaging changes might come about. By studying tumorigenesis in the Drosophila eye, here we identify two Polycomb group epigenetic silencers, Pipsqueak and Lola, that participate in this process. When coupled with overexpression of Delta, deregulation of the expression of Pipsqueak and Lola induces the formation of metastatic tumours. This phenotype depends on the histone-modifying enzymes Rpd3 (a histone deacetylase), Su(var)3-9 and E(z), as well as on the chromodomain protein Polycomb. Expression of the gene Retinoblastoma-family protein (Rbf ) is downregulated in these tumours and, indeed, this downregulation is associated with DNA hypermethylation. Together, these results establish a mechanism that links the Notch–Delta pathway, epigenetic silencing pathways and cell-cycle control in the process of tumorigenesis.

Targeting Notch signalling by the conserved miR-8/200 microRNA family in development and cancer cells.

Notch signalling is crucial for the correct development and growth of numerous organs and tissues, and when subverted it can cause cancer. Loss of miR‐8/200 microRNAs (miRNAs) is commonly observed in advanced tumours and correlates with their invasion and acquisition of stem‐like properties. Here, we show that this miRNA family controls Notch signalling activation in Drosophila and human cells. In an overexpression screen, we identified the Drosophila miR‐8 as a potent inhibitor of Notch‐induced overgrowth and tumour metastasis. Gain and loss of mir‐8 provoked developmental defects reminiscent of impaired Notch signalling and we demonstrated that miR‐8 directly inhibits Notch ligand Serrate. Likewise, miR‐200c and miR‐141 directly inhibited JAGGED1, impeding proliferation of human metastatic prostate cancer cells. It has been suggested that JAGGED1 may also be important for metastases. Although in metastatic cancer cells, JAGGED1 modestly regulated ZEB1, the miR‐200cs target in invasion, studies in Drosophila revealed that only concurrent overexpression of Notch and Zfh1/ZEB1 induced tumour metastases. Together, these data define a new way to attenuate or boost Notch signalling that may have clinical interest.

Histone demethylase KDM5A is an integral part of the core Notch–RBP-J repressor complex

Timely acquisition of cell fates and the elaborate control of growth in numerous organs depend on Notch signaling. Upon ligand binding, the core transcription factor RBP-J activates transcription of Notch target genes. In the absence of signaling, RBP-J switches off target gene expression, assuring the tight spatiotemporal control of the response by a mechanism incompletely understood. Here we show that the histone demethylase KDM5A is an integral, conserved component of Notch/RBP-J gene silencing. Methylation of histone H3 Lys 4 is dynamically erased and re-established at RBP-J sites upon inhibition and reactivation of Notch signaling. KDM5A interacts physically with RBP-J; this interaction is conserved in Drosophila and is crucial for Notch-induced growth and tumorigenesis responses.

Two-step process for photoreceptor formation in Drosophila.

The formation of photoreceptor cells (PRCs) in Drosophila serves as a paradigm for understanding neuronal determination and differentiation. During larval stages, a precise series of sequential inductive processes leads to the recruitment of eight distinct PRCs (R1–R8). But, final photoreceptor differentiation, including rhabdomere morphogenesis and opsin expression, is completed four days later, during pupal development. It is thought that photoreceptor cell fate is irreversibly established during larval development, when each photoreceptor expresses a particular set of transcriptional regulators and sends its projection to different layers of the optic lobes. Here, we show that the spalt (sal) gene complex encodes two transcription factors that are required late in pupation for photoreceptor differentiation. In the absence of the sal complex, rhabdomere morphology and expression of opsin genes in the inner PRCs R7 and R8 are changed to become identical to those of outer R1–R6 PRCs. However, these cells maintain their normal projections to the medulla part of the optic lobe, and not to the lamina where outer PRCs project. These data indicate that photoreceptor differentiation occurs as a two-step process. First, during larval development, the photoreceptor neurons become committed and send their axonal projections to their targets in the brain. Second, terminal differentiation is executed during pupal development and the photoreceptors adopt their final cellular properties.

Organ Specification-Growth Control Connection: New In-sights From the Drosophila Eye-Antennal Disc

The eye–antennal disc of Drosophila is serving a guiding role in the studies of how eye identity is specified, as well as how the retina is patterned. However, this system also holds a great potential for studying the coordination between organ growth and specification when various distinct organs form from a common primordium. The eye–antennal disc gives origin not only to the compound eye but also to the head capsule, ocelli, maxillary palp, and antenna, and these organs develop bearing constant size proportions with each other. Here, we review recent results that have shed light on the mechanisms that control the specification and growth of organs of the eye–antennal disc and discuss how these controls are intertwined during the development of neighboring organs to ensure their constant shape and relative sizes. Developmental Dynamics 232:673–684, 2005.

Endocrine remodelling of the adult intestine sustains reproduction in Drosophila

The production of offspring is energetically costly and relies on incompletely understood mechanisms that generate a positive energy balance. In mothers of many species, changes in key energy-associated internal organs are common yet poorly characterised functionally and mechanistically. In this study, we show that, in adult Drosophila females, the midgut is dramatically remodelled to enhance reproductive output. In contrast to extant models, organ remodelling does not occur in response to increased nutrient intake and/or offspring demands, but rather precedes them. With spatially and temporally directed manipulations, we identify juvenile hormone (JH) as an anticipatory endocrine signal released after mating. Acting through intestinal bHLH-PAS domain proteins Methoprene-tolerant (Met) and Germ cell-expressed (Gce), JH signals directly to intestinal progenitors to yield a larger organ, and adjusts gene expression and sterol regulatory element-binding protein (SREBP) activity in enterocytes to support increased lipid metabolism. Our findings identify a metabolically significant paradigm of adult somatic organ remodelling linking hormonal signals, epithelial plasticity, and reproductive output. DOI: http://dx.doi.org/10.7554/eLife.06930.001

A brain circuit that synchronizes growth and maturation revealed through Dilp8 binding to Lgr3

Brain keeps body size and shape in check Animal systems show amazing left-right symmetry—think of how our legs or arms, or the legs or wings of an insect, are matched in size and shape. Environmental insults and growth defects can challenge these developmental programs. In order to limit the resultant variation, juvenile organisms buffer variability through homeostatic mechanisms, so that the correct final size is attained. Vallejo et al. report that the Drosophila brain mediates such homeostatic control via an insulin-like peptide Dilp8 binding to the relaxin hormone receptor Lgr3. Lgr3 neurons distribute this information to other neuronal populations to adjust the hormones ecdysone, insulin, and juvenile hormone in a manner that stabilizes body and organ size. Science, this issue p. 10.1126/science.aac6767 Drosophila Lgr3 defines a neural circuit for homeostatic regulation of body size. INTRODUCTION Animals have a remarkable capacity to maintain a constant size, even in the face of genetic and environmental perturbations. Size imperfections and asymmetries have an effect on fitness, potentially decreasing competitiveness, survival, and reproductive success. Therefore, immature animals must employ homeostatic mechanisms to counteract substantial size variations and withstand developmental growth perturbations caused by genetic errors, disease, environmental factors, or injury. Such mechanisms ensure that, despite inevitable variations, the appropriate final body size is attained. A better understanding of homeostatic size maintenance will afford insights into normal organ and organismal size control, as well as the developmental origin of anomalous random left-right asymmetries. RATIONALE The Drosophila insulin-like peptide Dilp8 has been shown to mediate homeostatic regulation. When growth is disturbed, Dilp8 is strongly activated and sexual maturation is postponed until the affected elements are recomposed; simultaneously, the growth of other organs is retarded during this process. This compensatory mechanism allows the growth of the affected tissues to catch up. It maintains the synchrony between organs so that the animals achieve the correct size, preserving proportionality and bilateral symmetry. However, the Dilp8 receptor and its site of action remain uncharacterized. RESULTS We found that Dilp8 binds to andactivates the relaxin leucine-rich repeat–containing G protein–coupled receptor Lgr3 to mediate homeostatic control through a pathway dependent on adenosine 3′,5′-monophosphate. Larvae that lack lgr3 in neurons alone do not respond to Dilp8, indicating that the homeostatic system is centered in the brain. Dilp8 delays reproductive maturation by suppressing the neurons releasing the prothoracicotropic hormone (PTTH), which projects to the prothoracic gland and regulates ecdysone production for growth termination. However, this modulation alone is insufficient to adjust growth and stabilize body size. We show that Dilp8-Lgr3 balances growth against the extended growth period by dampening the production of dilp3 and dilp5 by insulin-producing cells (IPCs) in the brain and inhibiting synthesis of the juvenile hormone (JH). We also identify two pairs of dorsomedial neurons in the pars intercerebralis that are necessary and sufficient to mediate the effects of Dilp8. Simultaneous detection of pre- and postsynaptic markers revealed that the Lgr3 neurons mediating this homeostatic control have extensive axonal arborizations. Genetic and GRASP (GFP reconstitution across synaptic partners) analyses demonstrate that these neurons are connected to both the IPCs and PTTH neurons critical for adjusting growth and maturation rate, respectively. Thus, through their extensive axonal arborizations, Lgr3 neurons function like a “neuronal hub”: They route peripheral information about growth status to other neuronal populations, thereby synchronizing damaged tissues and other (undamaged) ones and allocating additional development time so that each organ attains the correct size and maintains proportionality and symmetry. CONCLUSION We identified the relaxin receptor Lgr3 as a Dilp8 receptor and defined a brain circuit for homeostatic control of organismal and organ size in the face of perturbations. Lgr3 neurons that respond to Dilp8 signals directly input on the insulin-producing cells and the PTTH-producing neurons. As Lgr3 outputs, the modulation of these neuronal populations according to Dilp8 levels is critical to delay maturation and promote growth compensation in a manner that stabilizes body size. Without adequate Dilp8-Lgr3 signaling, the brain is incapable of stabilizing size between the distinct body parts, and we see left-right asymmetries and size variations that are greater than usual, reflecting developmental instability. Dilp8-Lgr3 neural circuit and outputs for body-size homeostasis. The brain detects growth status and anomalies via Dilp8 activation of the Lgr3 receptor in a pair of symmetric neurons. These neurons distribute this information to IPCs and PTTH neurons, which then trigger the hormonal responses that regulate size. Without Dilp8-Lgr3 homeostasis, the brain cannot correct variation, and identical body parts can display imperfect symmetry and size. Body-size constancy and symmetry are signs of developmental stability. Yet, it is unclear exactly how developing animals buffer size variation. Drosophila insulin-like peptide Dilp8 is responsive to growth perturbations and controls homeostatic mechanisms that coordinately adjust growth and maturation to maintain size within the normal range. Here we show that Lgr3 is a Dilp8 receptor. Through the use of functional and adenosine 3′,5′-monophosphate assays, we defined a pair of Lgr3 neurons that mediate homeostatic regulation. These neurons have extensive axonal arborizations, and genetic and green fluorescent protein reconstitution across synaptic partners show that these neurons connect with the insulin-producing cells and prothoracicotropic hormone–producing neurons to attenuate growth and maturation. This previously unrecognized circuit suggests how growth and maturation rate are matched and co-regulated according to Dilp8 signals to stabilize organismal size.