Ether lipids influence cancer cell fate by modulating iron uptake.

Cancer cell fate has been widely ascribed to mutational changes within protein-coding genes associated with tumor suppressors and oncogenes. In contrast, the mechanisms through which the biophysical properties of membrane lipids influence cancer cell survival, dedifferentiation and metastasis have received little scrutiny. Here, we report that cancer cells endowed with a high metastatic ability and cancer stem cell-like traits employ ether lipids to maintain low membrane tension and high membrane fluidity. Using genetic approaches and lipid reconstitution assays, we show that these ether lipid-regulated biophysical properties permit non-clathrin-mediated iron endocytosis via CD44, leading directly to significant increases in intracellular redox-active iron and enhanced ferroptosis susceptibility. Using a combination of in vitro three-dimensional microvascular network systems and in vivo animal models, we show that loss of ether lipids also strongly attenuates extravasation, metastatic burden and cancer stemness. These findings illuminate a mechanism whereby ether lipids in carcinoma cells serve as key regulators of malignant progression while conferring a unique vulnerability that can be exploited for therapeutic intervention.

Targeted therapies prime oncogene-driven lung cancers for macrophage-mediated destruction.

Macrophage immune checkpoint inhibitors, such as anti-CD47 antibodies, show promise in clinical trials for solid and hematologic malignancies. However, the best strategies to use these therapies remain unknown, and ongoing studies suggest they may be most effective when used in combination with other anticancer agents. Here, we developed a novel screening platform to identify drugs that render lung cancer cells more vulnerable to macrophage attack, and we identified therapeutic synergy exists between genotype-directed therapies and anti-CD47 antibodies. In validation studies, we found the combination of genotype-directed therapies and CD47 blockade elicited robust phagocytosis and eliminated persister cells in vitro and maximized anti-tumor responses in vivo. Importantly, these findings broadly applied to lung cancers with various RTK/MAPK pathway alterations-including EGFR mutations, ALK fusions, or KRASG12C mutations. We observed downregulation of ß2-microglobulin and CD73 as molecular mechanisms contributing to enhanced sensitivity to macrophage attack. Our findings demonstrate that dual inhibition of the RTK/MAPK pathway and the CD47/SIRPa axis is a promising immunotherapeutic strategy. Our study provides strong rationale for testing this therapeutic combination in patients with lung cancers bearing driver mutations.

A maternally programmed intergenerational mechanism enables male offspring to make piRNAs from Y-linked precursor RNAs in Drosophila.

In animals, PIWI-interacting RNAs (piRNAs) direct PIWI proteins to silence complementary targets such as transposons. In Drosophila and other species with a maternally specified germline, piRNAs deposited in the egg initiate piRNA biogenesis in the progeny. However, Y chromosome loci cannot participate in such a chain of intergenerational inheritance. How then can the biogenesis of Y-linked piRNAs be initiated? Here, using Suppressor of Stellate (Su(Ste)), a Y-linked Drosophila melanogaster piRNA locus as a model, we show that Su(Ste) piRNAs are made in the early male germline via 5'-to-3' phased piRNA biogenesis initiated by maternally deposited 1360/Hoppel transposon piRNAs. Notably, deposition of Su(Ste) piRNAs from XXY mothers obviates the need for phased piRNA biogenesis in sons. Together, our study uncovers a developmentally programmed, intergenerational mechanism that allows fly mothers to protect their sons using a Y-linked piRNA locus.

A complex of Wnt/planar cell polarity signaling components Vangl1 and Fzd7 drives glioblastoma multiforme malignant properties.

Targeting common oncogenic drivers of glioblastoma multiforme (GBM) in patients has remained largely ineffective, raising the possibility that alternative pathways may contribute to tumor aggressiveness. Here we demonstrate that Vangl1 and Fzd7, components of the non-canonical Wnt planar cell polarity (Wnt/PCP) signaling pathway, promote GBM malignancy by driving cellular proliferation, migration, and invasiveness, and engage Rho GTPases to promote cytoskeletal rearrangements and actin dynamics in migrating GBM cells. Mechanistically, we uncover the existence of a novel Vangl1/Fzd7 complex at the leading edge of migrating GBM cells and propose that this complex is critical for the recruitment of downstream effectors to promote tumor progression. Moreover, we observe that depletion of FZD7 results in a striking suppression of tumor growth and latency and extends overall survival in an intracranial mouse xenograft model. Our observations support a novel mechanism by which Wnt/PCP components Vangl1 and Fzd7 form a complex at the leading edge of migratory GBM cells to engage downstream effectors that promote actin cytoskeletal rearrangements dynamics. Our findings suggest that interference with Wnt/PCP pathway function may offer a novel therapeutic strategy for patients diagnosed with GBM.

Vangl-dependent Wnt/planar cell polarity signaling mediates collective breast carcinoma motility and distant metastasis.

BACKGROUND: In light of the growing appreciation for the role of collective cell motility in metastasis, a deeper understanding of the underlying signaling pathways will be critical to translating these observations to the treatment of advanced cancers. Here, we examine the contribution of Wnt/planar cell polarity (Wnt/PCP), one of the non-canonical Wnt signaling pathways and defined by the involvement of the tetraspanin-like proteins Vangl1 and Vangl2, to breast tumor cell motility, collective cell invasiveness and mammary tumor metastasis. METHODS: Vangl1 and Vangl2 knockdown and overexpression and Wnt5a stimulation were employed to manipulate Wnt/PCP signaling in a battery of breast cancer cell lines representing all breast cancer subtypes, and in tumor organoids from MMTV-PyMT mice. Cell migration was assessed by scratch and organoid invasion assays, Vangl protein subcellular localization was assessed by confocal fluorescence microscopy, and RhoA activation was assessed in real time by fluorescence imaging with an advanced FRET biosensor. The impact of Wnt/PCP suppression on mammary tumor growth and metastasis was assessed by determining the effect of conditional Vangl2 knockout on the MMTV-NDL mouse mammary tumor model. RESULTS: We observed that Vangl2 knockdown suppresses the motility of all breast cancer cell lines examined, and overexpression drives the invasiveness of collectively migrating MMTV-PyMT organoids. Vangl2-dependent RhoA activity is localized in real time to a subpopulation of motile leader cells displaying a hyper-protrusive leading edge, Vangl protein is localized to leader cell protrusions within leader cells, and actin cytoskeletal regulator RhoA is preferentially activated in the leader cells of a migrating collective. Mammary gland-specific knockout of Vangl2 results in a striking decrease in lung metastases in MMTV-NDL mice, but does not impact primary tumor growth characteristics. CONCLUSIONS: We conclude that Vangl-dependent Wnt/PCP signaling promotes breast cancer collective cell migration independent of breast tumor subtype and facilitates distant metastasis in a genetically engineered mouse model of breast cancer. Our observations are consistent with a model whereby Vangl proteins localized at the leading edge of leader cells in a migrating collective act through RhoA to mediate the cytoskeletal rearrangements required for pro-migratory protrusion formation.

Derepression of Y-linked multicopy protamine-like genes interferes with sperm nuclear compaction in D. melanogaster.

Across species, sperm maturation involves the dramatic reconfiguration of chromatin into highly compact nuclei that enhance hydrodynamic ability and ensure paternal genomic integrity. This process is mediated by the replacement of histones by sperm nuclear basic proteins, also referred to as protamines. In humans, a carefully balanced dosage between two known protamine genes is required for optimal fertility. However, it remains unknown how their proper balance is regulated and how defects in balance may lead to compromised fertility. Here, we show that a nucleolar protein, modulo, a homolog of nucleolin, mediates the histone-to-protamine transition during Drosophila spermatogenesis. We find that modulo mutants display nuclear compaction defects during late spermatogenesis due to decreased expression of autosomal protamine genes (including Mst77F) and derepression of Y-linked multicopy Mst77F homologs (Mst77Y), leading to the mutant's known sterility. Overexpression of Mst77Y in a wild-type background is sufficient to cause nuclear compaction defects, similar to modulo mutant, indicating that Mst77Y is a dominant-negative variant interfering with the process of histone-to-protamine transition. Interestingly, ectopic overexpression of Mst77Y caused decompaction of X-bearing spermatids nuclei more frequently than Y-bearing spermatid nuclei, although this did not greatly affect the sex ratio of offspring. We further show that modulo regulates these protamine genes at the step of transcript polyadenylation. We conclude that the regulation of protamines mediated by modulo, ensuring the expression of functional ones while repressing dominant-negative ones, is critical for male fertility.

Functional Annotation Routines Used by ABRF Bioinformatics Core Facilities - Observations, Comparisons, and Considerations.

The functional annotation of gene lists is a common analysis routine required for most genomics experiments, and bioinformatics core facilities must support these analyses. In contrast to methods such as the quantitation of RNA-Seq reads or differential expression analysis, our research group noted a lack of consensus in our preferred approaches to functional annotation. To investigate this observation, we selected 4 experiments that represent a range of experimental designs encountered by our cores and analyzed those data with 6 tools used by members of the Association of Biomolecular Resource Facilities (ABRF) Genomic Bioinformatics Research Group (GBIRG). To facilitate comparisons between tools, we focused on a single biological result for each experiment. These results were represented by a gene set, and we analyzed these gene sets with each tool considered in our study to map the result to the annotation categories presented by each tool. In most cases, each tool produces data that would facilitate identification of the selected biological result for each experiment. For the exceptions, Fisher's exact test parameters could be adjusted to detect the result. Because Fisher's exact test is used by many functional annotation tools, we investigated input parameters and demonstrate that, while background set size is unlikely to have a significant impact on the results, the numbers of differentially expressed genes in an annotation category and the total number of differentially expressed genes under consideration are both critical parameters that may need to be modified during analyses. In addition, we note that differences in the annotation categories tested by each tool, as well as the composition of those categories, can have a significant impact on results.

Targeted therapies prime oncogene-driven lung cancers for macrophage-mediated destruction.

Macrophage immune checkpoint inhibitors, such as anti-CD47 antibodies, show promise in clinical trials for solid and hematologic malignancies. However, the best strategies to use these therapies remain unknown and ongoing studies suggest they may be most effective when used in combination with other anticancer agents. Here, we developed a novel screening platform to identify drugs that render lung cancer cells more vulnerable to macrophage attack, and we identified therapeutic synergy exists between genotype-directed therapies and anti-CD47 antibodies. In validation studies, we found the combination of genotype-directed therapies and CD47 blockade elicited robust phagocytosis and eliminated persister cells in vitro and maximized anti-tumor responses in vivo. Importantly, these findings broadly applied to lung cancers with various RTK/MAPK pathway alterations-including EGFR mutations, ALK fusions, or KRASG12C mutations. We observed downregulation of ß2-microglobulin and CD73 as molecular mechanisms contributing to enhanced sensitivity to macrophage attack. Our findings demonstrate that dual inhibition of the RTK/MAPK pathway and the CD47/SIRPa axis is a promising immunotherapeutic strategy. Our study provides strong rationale for testing this therapeutic combination in patients with lung cancers bearing driver mutations.

Leading edge competition promotes context-dependent responses to receptor inputs to resolve directional dilemmas in neutrophil migration.

Maintaining persistent migration in complex environments is critical for neutrophils to reach infection sites. Neutrophils avoid getting trapped, even when obstacles split their front into multiple leading edges. How they re-establish polarity to move productively while incorporating receptor inputs under such conditions remains unclear. Here, we challenge chemotaxing HL60 neutrophil-like cells with symmetric bifurcating microfluidic channels to probe cell-intrinsic processes during the resolution of competing fronts. Using supervised statistical learning, we demonstrate that cells commit to one leading edge late in the process, rather than amplifying structural asymmetries or early fluctuations. Using optogenetic tools, we show that receptor inputs only bias the decision similarly late, once mechanical stretching begins to weaken each front. Finally, a retracting edge commits to retraction, with ROCK limiting sensitivity to receptor inputs until the retraction completes. Collectively, our results suggest that cell edges locally adopt highly stable protrusion/retraction programs that are modulated by mechanical feedback.

Zonated leucine sensing by Sestrin-mTORC1 in the liver controls the response to dietary leucine.

The mechanistic target of rapamycin complex 1 (mTORC1) kinase controls growth in response to nutrients, including the amino acid leucine. In cultured cells, mTORC1 senses leucine through the leucine-binding Sestrin proteins, but the physiological functions and distribution of Sestrin-mediated leucine sensing in mammals are unknown. We find that mice lacking Sestrin1 and Sestrin2 cannot inhibit mTORC1 upon dietary leucine deprivation and suffer a rapid loss of white adipose tissue (WAT) and muscle. The WAT loss is driven by aberrant mTORC1 activity and fibroblast growth factor 21 (FGF21) production in the liver. Sestrin expression in the liver lobule is zonated, accounting for zone-specific regulation of mTORC1 activity and FGF21 induction by leucine. These results establish the mammalian Sestrins as physiological leucine sensors and reveal a spatial organization to nutrient sensing by the mTORC1 pathway.

Synthesis of 2-BMIDA Indoles via Heteroannulation: Applications in Drug Scaffold and Natural Product Synthesis.

A Pd-catalyzed heteroannulation approach for the synthesis of C2 borylated indoles is reported. The process allows access to highly functionalized 2-borylated indole scaffolds with complete control of regioselectivity. The utility of the process is demonstrated in the synthesis of borylated sulfa drugs and in the concise synthesis of the Aspidosperma alkaloid Goniomitine.

WASP integrates substrate topology and cell polarity to guide neutrophil migration.

To control their movement, cells need to coordinate actin assembly with the geometric features of their substrate. Here, we uncover a role for the actin regulator WASP in the 3D migration of neutrophils. We show that WASP responds to substrate topology by enriching to sites of inward, substrate-induced membrane deformation. Superresolution imaging reveals that WASP preferentially enriches to the necks of these substrate-induced invaginations, a distribution that could support substrate pinching. WASP facilitates recruitment of the Arp2/3 complex to these sites, stimulating local actin assembly that couples substrate features with the cytoskeleton. Surprisingly, WASP only enriches to membrane deformations in the front half of the cell, within a permissive zone set by WASP's front-biased regulator Cdc42. While WASP KO cells exhibit relatively normal migration on flat substrates, they are defective at topology-directed migration. Our data suggest that WASP integrates substrate topology with cell polarity by selectively polymerizing actin around substrate-induced membrane deformations in the front half of the cell.

Directional reorientation of migrating neutrophils is limited by suppression of receptor input signaling at the cell rear through myosin II activity.

To migrate efficiently to target locations, cells must integrate receptor inputs while maintaining polarity: a distinct front that leads and a rear that follows. Here we investigate what is necessary to overwrite pre-existing front-rear polarity in neutrophil-like HL60 cells migrating inside straight microfluidic channels. Using subcellular optogenetic receptor activation, we show that receptor inputs can reorient weakly polarized cells, but the rear of strongly polarized cells is refractory to new inputs. Transient stimulation reveals a multi-step repolarization process, confirming that cell rear sensitivity to receptor input is the primary determinant of large-scale directional reversal. We demonstrate that the RhoA/ROCK/myosin II pathway limits the ability of receptor inputs to signal to Cdc42 and reorient migrating neutrophils. We discover that by tuning the phosphorylation of myosin regulatory light chain we can modulate the activity and localization of myosin II and thus the amenability of the cell rear to 'listen' to receptor inputs and respond to directional reprogramming.

Optogenetic control of receptors reveals distinct roles for actin- and Cdc42-dependent negative signals in chemotactic signal processing.

During chemotaxis, neutrophils use cell surface G Protein Coupled Receptors to detect chemoattractant gradients. The downstream signaling system is wired with multiple feedback loops that amplify weak inputs and promote spatial separation of cell front and rear activities. Positive feedback could promote rapid signal spreading, yet information from the receptors is transmitted with high spatial fidelity, enabling detection of small differences in chemoattractant concentration across the cell. How the signal transduction network achieves signal amplification while preserving spatial information remains unclear. The GTPase Cdc42 is a cell-front polarity coordinator that is predictive of cell turning, suggesting an important role in spatial processing. Here we directly measure information flow from receptors to Cdc42 by pairing zebrafish parapinopsina, an optogenetic G Protein Coupled Receptor with reversible ON/OFF control, with a spectrally compatible red/far red Cdc42 Fluorescence Resonance Energy Transfer biosensor. Using this toolkit, we show that positive and negative signals downstream of G proteins shape a rapid, dose-dependent Cdc42 response. Furthermore, F-actin and Cdc42 itself provide two distinct negative signals that limit the duration and spatial spread of Cdc42 activation, maintaining output signals local to the originating receptors.

Genetically Defined Syngeneic Mouse Models of Ovarian Cancer as Tools for the Discovery of Combination Immunotherapy.

Despite advances in immuno-oncology, the relationship between tumor genotypes and response to immunotherapy remains poorly understood, particularly in high-grade serous tubo-ovarian carcinomas (HGSC). We developed a series of mouse models that carry genotypes of human HGSCs and grow in syngeneic immunocompetent hosts to address this gap. We transformed murine-fallopian tube epithelial cells to phenocopy homologous recombination-deficient tumors through a combined loss of Trp53, Brca1, Pten, and Nf1 and overexpression of Myc and Trp53 R172H, which was contrasted with an identical model carrying wild-type Brca1. For homologous recombination-proficient tumors, we constructed genotypes combining loss of Trp53 and overexpression of Ccne1, Akt2, and Trp53 R172H, and driven by KRAS G12V or Brd4 or Smarca4 overexpression. These lines form tumors recapitulating human disease, including genotype-driven responses to treatment, and enabled us to identify follistatin as a driver of resistance to checkpoint inhibitors. These data provide proof of concept that our models can identify new immunotherapy targets in HGSC. SIGNIFICANCE: We engineered a panel of murine fallopian tube epithelial cells bearing mutations typical of HGSC and capable of forming tumors in syngeneic immunocompetent hosts. These models recapitulate tumor microenvironments and drug responses characteristic of human disease. In a Ccne1-overexpressing model, immune-checkpoint resistance was driven by follistatin.This article is highlighted in the In This Issue feature, p. 211.

Formation of Human Neuroblastoma in Mouse-Human Neural Crest Chimeras.

Neuroblastoma (NB), derived from the neural crest (NC), is the most common pediatric extracranial solid tumor. Here, we establish a platform that allows the study of human NBs in mouse-human NC chimeras. Chimeric mice were produced by injecting human NC cells carrying NB relevant oncogenes in utero into gastrulating mouse embryos. The mice developed tumors composed of a heterogenous cell population that resembled that seen in primary NBs of patients but were significantly different from homogeneous tumors formed in xenotransplantation models. The human tumors emerged in immunocompetent hosts and were extensively infiltrated by mouse cytotoxic T cells, reflecting a vigorous host anti-tumor immune response. However, the tumors blunted the immune response by inducing infiltration of regulatory T cells and expression of immune-suppressive molecules similar to escape mechanisms seen in human cancer patients. Thus, this experimental platform allows the study of human tumor initiation, progression, manifestation, and tumor-immune-system interactions in an animal model system.

Human iPSC-derived microglia assume a primary microglia-like state after transplantation into the neonatal mouse brain.

Microglia are essential for maintenance of normal brain function, with dysregulation contributing to numerous neurological diseases. Protocols have been developed to derive microglia-like cells from human induced pluripotent stem cells (hiPSCs). However, primary microglia display major differences in morphology and gene expression when grown in culture, including down-regulation of signature microglial genes. Thus, in vitro differentiated microglia may not accurately represent resting primary microglia. To address this issue, we transplanted microglial precursors derived in vitro from hiPSCs into neonatal mouse brains and found that the cells acquired characteristic microglial morphology and gene expression signatures that closely resembled primary human microglia. Single-cell RNA-sequencing analysis of transplanted microglia showed similar cellular heterogeneity as primary human cells. Thus, hiPSCs-derived microglia transplanted into the neonatal mouse brain assume a phenotype and gene expression signature resembling that of resting microglia residing in the human brain, making chimeras a superior tool to study microglia in human disease.

Optimizing Systems for Cas9 Expression in Toxoplasma gondii.

CRISPR-Cas9 technologies have enabled genome engineering in an unprecedented array of species, accelerating biological studies in both model and nonmodel systems. However, Cas9 can be inherently toxic, which has limited its use in some organisms. We previously described the serendipitous discovery of a single guide RNA (sgRNA) that helped overcome Cas9 toxicity in the apicomplexan parasite Toxoplasma gondii, enabling the first genome-wide loss-of-function screens in any apicomplexan. Even in the presence of the buffering sgRNA, low-level Cas9 toxicity persists and results in frequent loss of Cas9 expression, which can affect the outcome of these screens. Similar Cas9-mediated toxicity has also been described in other organisms. We therefore sought to define the requirements for stable Cas9 expression, comparing different expression constructs and characterizing the role of the buffering sgRNA to understand the basis of Cas9 toxicity. We find that viral 2A peptides can substantially improve the selection and stability of Cas9 expression. We also demonstrate that the sgRNA has two functions: primarily facilitating integration of the Cas9-expression construct following initial genome targeting and secondarily improving long-term parasite fitness by alleviating Cas9 toxicity. We define a set of guidelines for the expression of Cas9 with improved stability and selection stringency, which are directly applicable to a variety of genetic approaches in diverse organisms. Our work also emphasizes the need for further characterizing the effects of Cas9 expression.IMPORTANCEToxoplasma gondii is an intracellular parasite that causes life-threatening disease in immunocompromised patients and affects the developing fetus when contracted during pregnancy. Closely related species cause malaria and severe diarrhea, thereby constituting leading causes for childhood mortality. Despite their importance to global health, this family of parasites has remained enigmatic. Given its remarkable experimental tractability, T. gondii has emerged as a model also for the study of related parasites. Genetic approaches are important tools for studying the biology of organisms, including T. gondii As such, the recent developments of CRISPR-Cas9-based techniques for genome editing have vastly expanded our ability to study the biology of numerous species. In some organisms, however, CRISPR-Cas9 has been difficult to implement due to its inherent toxicity. Our research characterizes the basis of the observed toxicity, using T. gondii as a model, allowing us to develop approaches to aid the use of CRISPR-Cas9 in diverse species.

Dynamic changes in ORC localization and replication fork progression during tissue differentiation.

BACKGROUND: Genomic regions repressed for DNA replication, resulting in either delayed replication in S phase or underreplication in polyploid cells, are thought to be controlled by inhibition of replication origin activation. Studies in Drosophila polytene cells, however, raised the possibility that impeding replication fork progression also plays a major role. RESULTS: We exploited genomic regions underreplicated (URs) with tissue specificity in Drosophila polytene cells to analyze mechanisms of replication repression. By localizing the Origin Recognition Complex (ORC) in the genome of the larval fat body and comparing this to ORC binding in the salivary gland, we found that sites of ORC binding show extensive tissue specificity. In contrast, there are common domains nearly devoid of ORC in the salivary gland and fat body that also have reduced density of ORC binding sites in diploid cells. Strikingly, domains lacking ORC can still be replicated in some polytene tissues, showing absence of ORC and origins is insufficient to repress replication. Analysis of the width and location of the URs with respect to ORC position indicates that whether or not a genomic region lacking ORC is replicated is controlled by whether replication forks formed outside the region are inhibited. CONCLUSIONS: These studies demonstrate that inhibition of replication fork progression can block replication across genomic regions that constitutively lack ORC. Replication fork progression can be inhibited in both tissue-specific and genome region-specific ways. Consequently, when evaluating sources of genome instability it is important to consider altered control of replication forks in response to differentiation.

Parallel High-Resolution Imaging of Leukocyte Chemotaxis Under Agarose with Rho-Family GTPase Biosensors.

Neutrophils are key early responders in the innate immune response that use chemotaxis, the directed migration along chemical gradients, to reach sites of infection or inflammation. This process requires integrating inputs from cell surface receptors with the cell's polarity and motility signaling network, in which highly dynamic and interconnected signaling by Rho-family GTPases plays a central role. To understand this fundamentally important behavior, we describe a high-resolution, under-agarose chemotaxis assay for use with neutrophil-like cell lines (HL-60 or PLB-985) or with primary neutrophils. We also describe how to use optical uncaging of chemoattractants to stimulate cells in this assay. These techniques are compatible with epifluorescence, total internal reflection fluorescence (TIRF), and confocal microscopy. Additionally, we cover how to measure the activities of Rho-family GTPases in this context using Förster resonance energy transfer (FRET)-based biosensors. The specific experimental steps outlined in this chapter include how to (1) set up the under-agarose assay, (2) optically pattern chemoattractant gradients, (3) image cells, and (4) conduct basic image analysis for FRET biosensors.