N-acetylcysteine overcomes NF1 loss-driven resistance to PI3Kα inhibition in breast cancer.

A genome-wide PiggyBac transposon-mediated screen and a resistance screen in a PIK3CAH1047R-mutated murine tumor model reveal NF1 loss in mammary tumors resistant to the phosphatidylinositol 3-kinase α (PI3Kα)-selective inhibitor alpelisib. Depletion of NF1 in PIK3CAH1047R breast cancer cell lines and a patient-derived organoid model shows that NF1 loss reduces sensitivity to PI3Kα inhibition and correlates with enhanced glycolysis and lower levels of reactive oxygen species (ROS). Unexpectedly, the antioxidant N-acetylcysteine (NAC) sensitizes NF1 knockout cells to PI3Kα inhibition and reverts their glycolytic phenotype. Global phospho-proteomics indicates that combination with NAC enhances the inhibitory effect of alpelisib on mTOR signaling. In public datasets of human breast cancer, we find that NF1 is frequently mutated and that such mutations are enriched in metastases, an indication for which use of PI3Kα inhibitors has been approved. Our results raise the attractive possibility of combining PI3Kα inhibition with NAC supplementation, especially in patients with drug-resistant metastases associated with NF1 loss.

A high-throughput drug screen reveals means to differentiate triple-negative breast cancer.

Plasticity delineates cancer subtypes with more or less favourable outcomes. In breast cancer, the subtype triple-negative lacks expression of major differentiation markers, e.g., estrogen receptor α (ERα), and its high cellular plasticity results in greater aggressiveness and poorer prognosis than other subtypes. Whether plasticity itself represents a potential vulnerability of cancer cells is not clear. However, we show here that cancer cell plasticity can be exploited to differentiate triple-negative breast cancer (TNBC). Using a high-throughput imaging-based reporter drug screen with 9 501 compounds, we have identified three polo-like kinase 1 (PLK1) inhibitors as major inducers of ERα protein expression and downstream activity in TNBC cells. PLK1 inhibition upregulates a cell differentiation program characterized by increased DNA damage, mitotic arrest, and ultimately cell death. Furthermore, cells surviving PLK1 inhibition have decreased tumorigenic potential, and targeting PLK1 in already established tumours reduces tumour growth both in cell line- and patient-derived xenograft models. In addition, the upregulation of genes upon PLK1 inhibition correlates with their expression in normal breast tissue and with better overall survival in breast cancer patients. Our results indicate that differentiation therapy based on PLK1 inhibition is a potential alternative strategy to treat TNBC.

Hepatic stellate cells suppress NK cell-sustained breast cancer dormancy.

The persistence of undetectable disseminated tumour cells (DTCs) after primary tumour resection poses a major challenge to effective cancer treatment1-3. These enduring dormant DTCs are seeds of future metastases, and the mechanisms that switch them from dormancy to outgrowth require definition. Because cancer dormancy provides a unique therapeutic window for preventing metastatic disease, a comprehensive understanding of the distribution, composition and dynamics of reservoirs of dormant DTCs is imperative. Here we show that different tissue-specific microenvironments restrain or allow the progression of breast cancer in the liver-a frequent site of metastasis4 that is often associated with a poor prognosis5. Using mouse models, we show that there is a selective increase in natural killer (NK) cells in the dormant milieu. Adjuvant interleukin-15-based immunotherapy ensures an abundant pool of NK cells that sustains dormancy through interferon-γ signalling, thereby preventing hepatic metastases and prolonging survival. Exit from dormancy follows a marked contraction of the NK cell compartment and the concurrent accumulation of activated hepatic stellate cells (aHSCs). Our proteomics studies on liver co-cultures implicate the aHSC-secreted chemokine CXCL12 in the induction of NK cell quiescence through its cognate receptor CXCR4. CXCL12 expression and aHSC abundance are closely correlated in patients with liver metastases. Our data identify the interplay between NK cells and aHSCs as a master switch of cancer dormancy, and suggest that therapies aimed at normalizing the NK cell pool might succeed in preventing metastatic outgrowth.

Phenotypic landscape of intestinal organoid regeneration.

The development of intestinal organoids from single adult intestinal stem cells in vitro recapitulates the regenerative capacity of the intestinal epithelium1,2. Here we unravel the mechanisms that orchestrate both organoid formation and the regeneration of intestinal tissue, using an image-based screen to assay an annotated library of compounds. We generate multivariate feature profiles for hundreds of thousands of organoids to quantitatively describe their phenotypic landscape. We then use these phenotypic fingerprints to infer regulatory genetic interactions, establishing a new approach to the mapping of genetic interactions in an emergent system. This allows us to identify genes that regulate cell-fate transitions and maintain the balance between regeneration and homeostasis, unravelling previously unknown roles for several pathways, among them retinoic acid signalling. We then characterize a crucial role for retinoic acid nuclear receptors in controlling exit from the regenerative state and driving enterocyte differentiation. By combining quantitative imaging with RNA sequencing, we show the role of endogenous retinoic acid metabolism in initiating transcriptional programs that guide the cell-fate transitions of intestinal epithelium, and we identify an inhibitor of the retinoid X receptor that improves intestinal regeneration in vivo.

Phosphoinositide metabolism links cGMP-dependent protein kinase G to essential Ca²âº signals at key decision points in the life cycle of malaria parasites.

Many critical events in the Plasmodium life cycle rely on the controlled release of Ca²âº from intracellular stores to activate stage-specific Ca²âº-dependent protein kinases. Using the motility of Plasmodium berghei ookinetes as a signalling paradigm, we show that the cyclic guanosine monophosphate (cGMP)-dependent protein kinase, PKG, maintains the elevated level of cytosolic Ca²âº required for gliding motility. We find that the same PKG-dependent pathway operates upstream of the Ca²âº signals that mediate activation of P. berghei gametocytes in the mosquito and egress of Plasmodium falciparum merozoites from infected human erythrocytes. Perturbations of PKG signalling in gliding ookinetes have a marked impact on the phosphoproteome, with a significant enrichment of in vivo regulated sites in multiple pathways including vesicular trafficking and phosphoinositide metabolism. A global analysis of cellular phospholipids demonstrates that in gliding ookinetes PKG controls phosphoinositide biosynthesis, possibly through the subcellular localisation or activity of lipid kinases. Similarly, phosphoinositide metabolism links PKG to egress of P. falciparum merozoites, where inhibition of PKG blocks hydrolysis of phosphatidylinostitol (4,5)-bisphosphate. In the face of an increasing complexity of signalling through multiple Ca²âº effectors, PKG emerges as a unifying factor to control multiple cellular Ca²âº signals essential for malaria parasite development and transmission.

The malarial serine protease SUB1 plays an essential role in parasite liver stage development.

Transmission of the malaria parasite to its vertebrate host involves an obligatory exoerythrocytic stage in which extensive asexual replication of the parasite takes place in infected hepatocytes. The resulting liver schizont undergoes segmentation to produce thousands of daughter merozoites. These are released to initiate the blood stage life cycle, which causes all the pathology associated with the disease. Whilst elements of liver stage merozoite biology are similar to those in the much better-studied blood stage merozoites, little is known of the molecular players involved in liver stage merozoite production. To facilitate the study of liver stage biology we developed a strategy for the rapid production of complex conditional alleles by recombinase mediated engineering in Escherichia coli, which we used in combination with existing Plasmodium berghei deleter lines expressing Flp recombinase to study subtilisin-like protease 1 (SUB1), a conserved Plasmodium serine protease previously implicated in blood stage merozoite maturation and egress. We demonstrate that SUB1 is not required for the early stages of intrahepatic growth, but is essential for complete development of the liver stage schizont and for production of hepatic merozoites. Our results indicate that inhibitors of SUB1 could be used in prophylactic approaches to control or block the clinically silent pre-erythrocytic stage of the malaria parasite life cycle.

A tetracycline-repressible transactivator system to study essential genes in malaria parasites.

A major obstacle in analyzing gene function in apicomplexan parasites is the absence of a practical regulatable expression system. Here, we identified functional transcriptional activation domains within Apicomplexan AP2 (ApiAP2) family transcription factors. These ApiAP2 transactivation domains were validated in blood-, liver-, and mosquito-stage parasites and used to create a robust conditional expression system for stage-specific, tetracycline-dependent gene regulation in Toxoplasma gondii, Plasmodium berghei, and Plasmodium falciparum. To demonstrate the utility of this system, we created conditional knockdowns of two essential P. berghei genes: profilin (PRF), a protein implicated in parasite invasion, and N-myristoyltransferase (NMT), which catalyzes protein acylation. Tetracycline-induced repression of PRF and NMT expression resulted in a dramatic reduction in parasite viability. This efficient regulatable system will allow for the functional characterization of essential proteins that are found in these important parasites.

The alveolin IMC1h is required for normal ookinete and sporozoite motility behaviour and host colonisation in Plasmodium berghei.

Alveolins, or inner membrane complex (IMC) proteins, are components of the subpellicular network that forms a structural part of the pellicle of malaria parasites. In Plasmodium berghei, deletions of three alveolins, IMC1a, b, and h, each resulted in reduced mechanical strength and gliding velocity of ookinetes or sporozoites. Using time lapse imaging, we show here that deletion of IMC1h (PBANKA_143660) also has an impact on the directionality and motility behaviour of both ookinetes and sporozoites. Despite their marked motility defects, sporozoites lacking IMC1h were able to invade mosquito salivary glands, allowing us to investigate the role of IMC1h in colonisation of the mammalian host. We show that IMC1h is essential for sporozoites to progress through the dermis in vivo but does not play a significant role in hepatoma cell transmigration and invasion in vitro. Colocalisation of IMC1h with the residual IMC in liver stages was detected up to 30 hours after infection and parasites lacking IMC1h showed developmental defects in vitro and a delayed onset of blood stage infection in vivo. Together, these results suggest that IMC1h is involved in maintaining the cellular architecture which supports normal motility behaviour, access of the sporozoites to the blood stream, and further colonisation of the mammalian host.

A scalable pipeline for highly effective genetic modification of a malaria parasite.

In malaria parasites, the systematic experimental validation of drug and vaccine targets by reverse genetics is constrained by the inefficiency of homologous recombination and by the difficulty of manipulating adenine and thymine (A+T)-rich DNA of most Plasmodium species in Escherichia coli. We overcame these roadblocks by creating a high-integrity library of Plasmodium berghei genomic DNA (>77% A+T content) in a bacteriophage N15-based vector that can be modified efficiently using the lambda Red method of recombineering. We built a pipeline for generating P. berghei genetic modification vectors at genome scale in serial liquid cultures on 96-well plates. Vectors have long homology arms, which increase recombination frequency up to tenfold over conventional designs. The feasibility of efficient genetic modification at scale will stimulate collaborative, genome-wide knockout and tagging programs for P. berghei.

The centrosome neither persistently leads migration nor determines the site of axonogenesis in migrating neurons in vivo.

The position of the centrosome ahead of the nucleus has been considered crucial for coordinating neuronal migration in most developmental situations. The proximity of the centrosome has also been correlated with the site of axonogenesis in certain differentiating neurons. Despite these positive correlations, accumulating experimental findings appear to negate a universal role of the centrosome in determining where an axon forms, or in leading the migration of neurons. To further examine this controversy in an in vivo setting, we have generated cell type-specific multi-cistronic gene expression to monitor subcellular dynamics in the developing zebrafish cerebellum. We show that migration of rhombic lip-derived neurons is characterized by a centrosome that does not persistently lead the nucleus, but which is instead regularly overtaken by the nucleus. In addition, axonogenesis is initiated during the onset of neuronal migration and occurs independently of centrosome proximity. These in vivo data reveal a new temporal orchestration of organelle dynamics and provide important insights into the variation in intracellular processes during vertebrate brain differentiation.

The zebrafish cerebellar upper rhombic lip generates tegmental hindbrain nuclei by long-distance migration in an evolutionary conserved manner.

The upper rhombic lip (URL) of the developing mammalian cerebellum produces different neuronal cell types in a temporal sequence. The first neuronal populations arising from this proliferation zone include the progenitors of the parabrachial, parabigeminal, and laterodorsal-pedunculopontine tegmental hindbrain nuclei. By means of expression analysis, histology, and retrograde neuronal tracing, we have identified the zebrafish homologues of these nuclei, namely, the secondary gustatory/viscerosensory nucleus, the nucleus isthmi, and the superior reticular nucleus, respectively, in the embryonic and larval brain of a stable transgenic wnt1:Gal4-VP16-14 x UAS:GFP zebrafish strain. Combining time-lapse confocal imaging with individual cell tracing, we characterize the migratory behavior of these neuronal precursor populations in detail by revealing their migration path, velocity, and directionality. In addition, we identify neuronal progenitors of the secondary gustatory/viscerosensory nucleus and nucleus isthmi/superior reticular nucleus as belonging to the polysialic acid (PSA)-expressing cell population in the cerebellar plate that migrates in a PSA-dependent manner. Finally, we reveal that circuitries involved in the processing of sensory information (visual, gustatory, general viscerosensory) are already established in the zebrafish larva at day 4 of development. Also the wnt1-expressing pretectal neuronal precursors (not originating from the URL) sending mossy fiber-like projections into the cerebellar corpus are established at that time. In sum, our results show that the origin of neurons of some tegmental hindbrain nuclei, namely, nucleus isthmi/superior reticular nucleus and secondary gustatory/viscerosensory nucleus is in the URL, and that the temporal order of cell types produced by the URL and their developmental program are conserved among vertebrate species.

Complex I deficiency and dopaminergic neuronal cell loss in parkin-deficient zebrafish (Danio rerio).

Currently, only symptomatic therapy is available for Parkinson's disease. The zebrafish is a vertebrate animal model ideally suited for high throughput compound screening to identify disease-modifying compounds for Parkinson's disease. We have developed a zebrafish model for Parkin deficiency, the most commonly mutated gene in early onset Parkinson's disease. The zebrafish Parkin protein is 62% identical to its human counterpart with 78% identity in functionally relevant regions. The parkin gene is expressed throughout zebrafish development and ubiquitously in adult zebrafish tissue. Abrogation of Parkin activity leads to a significant decrease in the number of ascending dopaminergic neurons in the posterior tuberculum (homologous to the substantia nigra in humans), an effect enhanced by exposure to MPP+. Both light microscopic analysis and staining with the pan-neuronal marker HuC confirmed that this loss of dopaminergic neurons is not due to general impairment of brain development. Neither serotonergic nor motor neurons were affected, further emphasizing that the effect of parkin knockdown appears to be specific for dopaminergic neurons. Notably, parkin knockdown zebrafish embryos also develop specific reduction in the activity of the mitochondrial respiratory chain complex I, making this the first vertebrate model to share both important pathogenic mechanisms (i.e. complex I deficiency) and the pathological hallmark (i.e. dopaminergic cell loss) with human parkin-mutant patients. The zebrafish model is thus ideally suited for future drug screens and other studies investigating the functional mechanisms underlying neuronal cell death in early onset Parkinson's Disease. Additional electron microscopy studies revealed electron dense material in the t-tubules within the muscle tissue of parkin knockdown zebrafish. T-tubules are rich in L-type calcium channels, therefore our work might also provide a tentative link between genetically determined early onset Parkinson's disease and recent studies attributing an important role to these L-type calcium channels in late onset sporadic Parkinson's disease.

Polysialyltransferase expression is linked to neuronal migration in the developing and adult zebrafish.

Modulation of cell-cell adhesion is crucial for regulating neuronal migration and maintenance of structural plasticity in the embryonic and mature brain. Such modulation can be obtained by the enzymatic attachment of polysialic acid (PSA) to the neural cell adhesion molecule (NCAM) by means of the polysialyltransferases STX and PST. Thus, differential expression of STX and PST is likely to be responsible for varying functions of PSA-NCAM during neuronal differentiation, maintenance, plasticity, and regeneration. We have isolated the zebrafish homologues of STX (St8sia2) and PST (St8sia4) and demonstrate that their expression in the embryonic and adult nervous system is often confined to regions of neuronal migration. Moreover, in the adult cerebellum, the complementary expression pattern of both polysialyltransferases suggests a function in regulating cerebellar neuronal plasticity. Enzymatic removal of PSA in the embryonic cerebellum results in impaired neuronal migration, suggesting that PSA-NCAM is a key regulator of motility for cerebellar neuronal progenitors.

The zebrafish cerebellar rhombic lip is spatially patterned in producing granule cell populations of different functional compartments.

The upper rhombic lip, a prominent germinal zone of the cerebellum, was recently demonstrated to generate different neuronal cell types over time from spatial subdomains. We have characterized the differentiation of the upper rhombic lip derived granule cell population in stable GFP-transgenic zebrafish in the context of zebrafish cerebellar morphogenesis. Time-lapse analysis followed by individual granule cell tracing demonstrates that the zebrafish upper rhombic lip is spatially patterned along its mediolateral axis producing different granule cell populations simultaneously. Time-lapse recordings of parallel fiber projections and retrograde labeling reveal that spatial patterning within the rhombic lip corresponds to granule cells of two different functional compartments of the mature cerebellum: the eminentia granularis and the corpus cerebelli. These cerebellar compartments in teleosts correspond to the mammalian vestibulocerebellar and non-vestibulocerebellar system serving balance and locomotion control, respectively. Given the high conservation of cerebellar development in vertebrates, spatial partitioning of the mammalian granule cell population and their corresponding earlier-produced deep nuclei by patterning within the rhombic lip may also delineate distinct functional compartments of the cerebellum. Thus, our findings offer an explanation for how specific functional cerebellar circuitries are laid down by spatio-temporal patterning of cerebellar germinal zones during early brain development.

In vivo retrograde labeling of neurons in the zebrafish embryo or larva with rhodamine dextran.

INTRODUCTIONApplication of a fluorescent dye to a target area of axons or dendrites allows the identification of individual neurons projecting to the area and reconstruction of the tract of the neurite by retrograde labeling. For the red fluorescent dye tetramethylrhodamine dextran, the labeling occurs by active retrograde transport inside the cell. This protocol describes a method for applying tetramethylrhodamine dextran to living zebrafish embryos and larvae (2-7 dpf [days post-fertilization]) via microinjection. It may be adapted for use with green or yellow fluorescent dextran conjugates.

Translational repression of C. elegans p53 by GLD-1 regulates DNA damage-induced apoptosis.

p53 is a tumor suppressor gene whose regulation is crucial to maintaining genome stability and for the apoptotic elimination of abnormal, potentially cancer-predisposing cells. C. elegans contains a primordial p53 gene, cep-1, that acts as a transcription factor necessary for DNA damage-induced apoptosis. In a genetic screen for negative regulators of CEP-1, we identified a mutation in GLD-1, a translational repressor implicated in multiple C. elegans germ cell fate decisions and related to mammalian Quaking proteins. CEP-1-dependent transcription of proapoptotic genes is upregulated in the gld-1(op236) mutant and an elevation of p53-mediated germ cell apoptosis in response to DNA damage is observed. Further, we demonstrate that GLD-1 mediates its repressive effect by directly binding to the 3'UTR of cep-1/p53 mRNA and repressing its translation. This study reveals that the regulation of cep-1/p53 translation influences DNA damage-induced apoptosis and demonstrates the physiological importance of this mechanism.