A novel binding pocket in the D2 domain of protein tyrosine phosphatase mu (PTPmu) guides AI screen to identify small molecules that modulate tumour cell adhesion, growth and migration.

Approximately 40% of people will get cancer in their lifetime in the US, and 20% are predicted to die from the condition when it is invasive and metastatic. Targeted screening for drugs that interact with proteins that drive cancer cell growth and migration can lead to new therapies. We screened molecular libraries with the AtomNet® AI-based drug design tool to identify compounds predicted to interact with the cytoplasmic domain of protein tyrosine phosphatase mu. Protein tyrosine phosphatase mu (PTPmu) is proteolytically downregulated in cancers such as glioblastoma generating fragments that stimulate cell survival and migration. Aberrant nuclear localization of PTPmu intracellular fragments drives cancer progression, so we targeted a predicted drug-binding site between the two cytoplasmic phosphatase domains we termed a D2 binding pocket. The function of the D2 domain is controversial with various proposed regulatory functions, making the D2 domain an attractive target for the development of allosteric drugs. Seventy-five of the best-scoring and chemically diverse computational hits predicted to interact with the D2 binding pocket were screened for effects on tumour cell motility and growth in 3D culture as well as in a direct assay for PTPmu-dependent adhesion. We identified two high-priority hits that inhibited the migration and glioma cell sphere formation of multiple glioma tumour cell lines as well as aggregation. We also identified one activator of PTPmu-dependent aggregation, which was able to stimulate cell migration. We propose that the PTPmu D2 binding pocket represents a novel regulatory site and that inhibitors targeting this region may have therapeutic potential for treating cancer.

Small molecule antagonists of PTPmu identified by artificial intelligence-based computational screening block glioma cell migration and growth.

PTPmu (PTPµ) is a member of the receptor protein tyrosine phosphatase IIb family that participates in both homophilic cell-cell adhesion and signaling. PTPmu is proteolytically downregulated in glioblastoma generating extracellular and intracellular fragments that have oncogenic activity. The intracellular fragments, in particular, are known to accumulate in the cytoplasm and nucleus where they interact with inappropriate binding partners/substrates generating signals required for glioma cell migration and growth. Thus, interfering with these fragments is an attractive therapeutic strategy. To develop agents that target these fragments, we used the AI-based AtomNetⓇ model, a drug design and discovery tool, to virtually screen molecular libraries for compounds able to target a binding pocket bordered by the wedge domain, a known regulatory motif located within the juxtamembrane portion of the protein. Seventy-four high-scoring and chemically diverse virtual hits were then screened in multiple cell-based assays for effects on glioma cell motility (scratch assays) and growth in 3D culture (sphere assays), and PTPmu-dependent adhesion (Sf9 aggregation). We identified three inhibitors (247678835, 247682206, 247678791) that affected the motility of multiple glioma cell lines (LN229, U87MG, and Gli36delta5), the growth of LN229 and Gli36 spheres, and PTPmu-dependent Sf9 aggregation. Compound 247678791 was further shown to suppress PTPmu enzymatic activity in an in vitro phosphatase assay, and 247678835 was able to inhibit the growth of human glioma tumors in mice. We propose that these three compounds are PTPmu-targeting agents with therapeutic potential for treating glioblastoma.

Artificial Intelligence-Based Computational Screening and Functional Assays Identify Candidate Small Molecule Antagonists of PTPmu-Dependent Adhesion.

PTPmu (PTPµ) is a member of the receptor protein tyrosine phosphatase IIb family that participates in cell-cell adhesion and signaling. PTPmu is proteolytically downregulated in glioblastoma (glioma), and the resulting extracellular and intracellular fragments are believed to stimulate cancer cell growth and/or migration. Therefore, drugs targeting these fragments may have therapeutic potential. Here, we used the AtomNet® platform, the first deep learning neural network for drug design and discovery, to screen a molecular library of several million compounds and identified 76 candidates predicted to interact with a groove between the MAM and Ig extracellular domains required for PTPmu-mediated cell adhesion. These candidates were screened in two cell-based assays: PTPmu-dependent aggregation of Sf9 cells and a tumor growth assay where glioma cells grow in three-dimensional spheres. Four compounds inhibited PTPmu-mediated aggregation of Sf9 cells, six compounds inhibited glioma sphere formation/growth, while two priority compounds were effective in both assays. The stronger of these two compounds inhibited PTPmu aggregation in Sf9 cells and inhibited glioma sphere formation down to 25 micromolar. Additionally, this compound was able to inhibit the aggregation of beads coated with an extracellular fragment of PTPmu, directly demonstrating an interaction. This compound presents an interesting starting point for the development of PTPmu-targeting agents for treating cancer including glioblastoma.

Physically-cross-linked poly(vinyl alcohol) cell culture plate coatings facilitate preservation of cell-cell interactions, spheroid formation, and stemness.

We employed aqueous solutions of highly-hydrolyzed (>99+%) poly(vinyl alcohol), PVA, to coat plastic dishes as a method to efficiently induce three-dimensional (3D) culturing of cells. The coatings were prepared by simple evaporation of 3 wt/vol% solutions of PVA in water and require no additional processing steps after air drying under sterile conditions. The coating allows spheroids to form in solution. Spheroid formation is usually preferable to two-dimensional (2D) culturing as it creates a more realistic ex vivo model of some human tissues and tumors. Using PVA-coated cell culture plates, we demonstrated that we can grow reproducibly sized spheroids using several human glioma cell lines, including LN229, U87 MG, and Gli36, and the embryonic kidney cell line, 293T. Spheroids formed on PVA-coated plates grow as well as on other commercially-available, low-attachment plates, and have excellent optical imaging properties. As spheroids, LN229 cells express markers of cancer stem cells. Finally, we confirmed that spheroids generated on PVA-coated plates are sensitive to molecular perturbations, as increased expression of the cell adhesion molecule PTPµ significantly increased the size of spheroids. The PVA hydrogel layer is an effective tool for creating a more realistic ex vivo culture system than traditional 2D culture and can be used to generate cell spheroids for potential application in drug screening and personalized medicine for diseases such as cancer.

In vivo germ line stem cell migration: a mouse model.

A stem cell niche is a specialized tissue environment that controls the proliferation and differentiation of its resident stem cells. The functions of these structures have been well characterized in adult organisms. In particular, the bone marrow stem cell niche in mammals has been amenable to analysis because of the ability of transplanted hematopoietic cells to home and to recolonize the bone marrow of an irradiated host. Despite clues from adult models, it remains unclear how stem cells become partitioned into appropriate niches during embryonic development. To examine the earliest steps in niche formation, we created an organ culture system to observe the development of primordial germ cells (PGCs), a migratory stem cell population that will eventually give rise to the gametes. Using this assay, we can watch PGCs as they migrate to colonize the developing gonads and can introduce growth factor agonists or antagonists to test the function of proteins that regulate this process. This provides an unprecedented opportunity to identify the cellular and molecular interactions required for the formation of the germ cell niche.

KHDC1B is a novel CPEB binding partner specifically expressed in mouse oocytes and early embryos.

mRNAs required for meiotic maturation and early embryonic development are stored in growing oocytes. These transcripts are translationally repressed until hormonal cues trigger ovulation. Errors in translation underlie some cases of human infertility and are associated with ovarian germ cell tumors. However, it remains unclear how maternal transcripts are kept quiescent in mammals. This study describes a potential translational regulator, KHDC1B. KHDC1B is a member of a small family of KH-domain containing proteins specific to eutherian mammals. Two family members, KHDC1A and 1B, are highly expressed in oocytes. KHDC1A and 1B bind polyU agarose and form oligomers like other KH-domain proteins. The functions of these proteins were tested by expression in Xenopus embryos. KHDC1A caused cell death, whereas KHDC1B caused cleavage arrest. This arrest phenotype was rescued by coexpression of the mouse translational regulator cytoplasmic polyadenylation binding protein 1 (mCPEB1). Coimmunoprecipitation and coimmunostaining experiments confirmed the functional interaction between KHDC1B and mCPEB1. Finally, KHDC1B levels and binding partners were shown to fluctuate with the cell cycle. KHDC1B, via its interaction with mCEPB1, may regulate translation of mRNA targets required for oocyte maturation.

BMP signaling controls formation of a primordial germ cell niche within the early genital ridges.

Stem cells are necessary to maintain tissue homeostasis and the microenvironment (a.k.a. the niche) surrounding these cells controls their ability to self-renew or differentiate. For many stem cell populations it remains unclear precisely what cells and signals comprise a niche. Here we identify a possible PGC niche in the mouse genital ridges. Conditional ablation of Bmpr1a was used to demonstrate that BMP signaling is required for PGC survival and migration as these cells colonize the genital ridges. Reduced BMP signaling within the genital ridges led to increased somatic cell death within the mesonephric mesenchyme. Loss of these supporting cells correlated with decreased levels of the mesonephric marker, Pax2, as well as a reduction in genes expressed in the coelomic epithelium including the putative PGC chemo-attractants Kitl and Sdf1a. We propose that BMP signaling promotes mesonephric cell survival within the genital ridges and that these cells support correct development of the coelomic epithelium, the target of PGC migration. Loss of BMP signaling leads to the loss of the PGC target resulting in reduced PGC numbers and disrupted PGC migration.

Nanotome cluster bombardment to recover spatial chemistry after preparation of biological samples for SIMS imaging.

A C(60)(+) cluster ion projectile is employed for sputter cleaning biological surfaces to reveal spatio-chemical information obscured by contamination overlayers. This protocol is used as a supplemental sample preparation method for time of flight secondary ion mass spectrometry (ToF-SIMS) imaging of frozen and freeze-dried biological materials. Following the removal of nanometers of material from the surface using sputter cleaning, a frozen-patterned cholesterol film and a freeze-dried tissue sample were analyzed using ToF-SIMS imaging. In both experiments, the chemical information was maintained after the sputter dose, due to the minimal chemical damage caused by C(60)(+) bombardment. The damage to the surface produced by freeze-drying the tissue sample was found to have a greater effect on the loss of cholesterol signal than the sputter-induced damage. In addition to maintaining the chemical information, sputtering is not found to alter the spatial distribution of molecules on the surface. This approach removes artifacts that might obscure the surface chemistry of the sample and are common to many biological sample preparation schemes for ToF-SIMS imaging.

Inhibition of HMG CoA reductase reveals an unexpected role for cholesterol during PGC migration in the mouse.

BACKGROUND: Primordial germ cells (PGCs) are the embryonic precursors of the sperm and eggs. Environmental or genetic defects that alter PGC development can impair fertility or cause formation of germ cell tumors. RESULTS: We demonstrate a novel role for cholesterol during germ cell migration in mice. Cholesterol was measured in living tissue dissected from mouse embryos and was found to accumulate within the developing gonads as germ cells migrate to colonize these structures. Cholesterol synthesis was blocked in culture by inhibiting the activity of HMG CoA reductase (HMGCR) resulting in germ cell survival and migration defects. These defects were rescued by co-addition of isoprenoids and cholesterol, but neither compound alone was sufficient. In contrast, loss of the last or penultimate enzyme in cholesterol biosynthesis did not alter PGC numbers or position in vivo. However embryos that lack these enzymes do not exhibit cholesterol defects at the stage at which PGCs are migrating. This demonstrates that during gestation, the cholesterol required for PGC migration can be supplied maternally. CONCLUSION: In the mouse, cholesterol is required for PGC survival and motility. It may act cell-autonomously by regulating clustering of growth factor receptors within PGCs or non cell-autonomously by controlling release of growth factors required for PGC guidance and survival.

BMP signaling regulates PGC numbers and motility in organ culture.

Members of the bone morphogenetic protein (BMP) family play diverse roles in multiple developmental processes. However, in the mouse, mutations in many BMPs, BMP receptors and signaling components result in early embryonic lethality making it difficult to analyze the role of these factors during organogenesis or tissue homeostasis in the adult. To bypass this early lethality, we used an organ culture system to study the role of BMPs during primordial germ cell (PGC) migration. PGCs are the embryonic precursors of the sperm and eggs. BMPs induce formation of primordial germ cells within the proximal epiblast of embryonic day 7.5 (E7.5) mouse embryos. PGCs then migrate via the gut to arrive at the developing gonads by E10.5. Addition of BMP4 or the BMP-antagonist Noggin to transverse slices dissected from E9.5 embryos elevated PGC numbers or reduced PGC numbers, respectively. Noggin treatment also slowed and randomized PGC movements, resulting in a failure of PGCs to colonize the urogenital ridges (UGRs). Based on p-Smad1/5/8 staining, migratory PGCs do not respond to endogenous BMPs. Instead, the somatic cells of the urogenital ridges exhibit elevated p-Smad1/5/8 staining revealing active BMP signaling within the UGRs. Noggin treatment abrogated p-Smad staining within the UGRs and blocked localized expression of Kitl, a cytokine known to regulate the survival and motility of PGCs and Id1, a transcription factor expressed within the UGRs. We propose that BMP signaling regulates PGC migration by controlling gene expression within the somatic cells along the migration route and within the genital ridges.

Steel factor controls midline cell death of primordial germ cells and is essential for their normal proliferation and migration.

During germ-cell migration in the mouse, the dynamics of embryo growth cause many germ cells to be left outside the range of chemoattractive signals from the gonad. At E10.5, movie analysis has shown that germ cells remaining in the midline no longer migrate directionally towards the genital ridges, but instead rapidly fragment and disappear. Extragonadal germ cell tumors of infancy, one of the most common neonatal tumors, are thought to arise from midline germ cells that failed to die. This paper addresses the mechanism of midline germ cell death in the mouse. We show that at E10.5, the rate of apoptosis is nearly four-times higher in midline germ cells than those more laterally. Gene expression profiling of purified germ cells suggests this is caused by activation of the intrinsic apoptotic pathway. We then show that germ cell apoptosis in the midline is activated by down-regulation of Steel factor (kit ligand) expression in the midline between E9.5 and E10.5. This is confirmed by the fact that removal of the intrinsic pro-apoptotic protein Bax rescues the germ-cell apoptosis seen in Steel null embryos. Two interesting things are revealed by this: first, germ-cell proliferation does not take place in these embryos after E9.0; second, migration of germ cells is highly abnormal. These data show first that changing expression of Steel factor is required for normal midline germ cell death, and second, that Steel factor is required for normal proliferation and migration of germ cells.

The roles of FGF signaling in germ cell migration in the mouse.

Fibroblast growth factor (FGF) signaling is thought to play a role in germ cell behavior. FGF2 has been reported to be a mitogen for primordial germ cells in vitro, whilst combinations of FGF2, steel factor and LIF cause cultured germ cells to transform into permanent lines of pluripotent cells resembling ES cells. However, the actual function of FGF signaling on the migrating germ cells in vivo is unknown. We show, by RT-PCR analysis of cDNA from purified E10.5 germ cells, that germ cells express two FGF receptors: Fgfr1-IIIc and Fgfr2-IIIb. Second, we show that FGF-mediated activation of the MAP kinase pathway occurs in germ cells during their migration, and thus they are potentially direct targets of FGF signaling. Third, we use cultured embryo slices in simple gain-of-function experiments, using FGF ligands, to show that FGF2, a ligand for FGFR1-IIIc, affects motility, whereas FGF7, a ligand for FGFR2-IIIb, affects germ cell numbers. Loss of function, using a specific inhibitor of FGF signaling, causes increased apoptosis and inhibition of cell shape change in the migrating germ cells. Lastly, we confirm in vivo the effects seen in slice cultures in vitro, by examining germ cell positions and numbers in embryos carrying a loss-of-function allele of FGFR2-IIIb. In FGFR2-IIIb(-/-) embryos, germ cell migration is unaffected, but the numbers of germ cells are significantly reduced. These data show that a major role of FGF signaling through FGFR2-IIIb is to control germ cell numbers. The data do not discriminate between direct and indirect effects of FGF signaling on germ cells, and both may be involved.

Primordial germ cell migration.

Mutational and antisense screens in Drosophila and zebrafish, and transcriptional profiling and time-lapse analysis in the mouse, have contributed greatly to our understanding of PGC development. In all three systems, the behavior of PGCs is controlled by growth factors which signal through G-protein coupled receptors and/or tyrosine kinase receptors. Additionally, regulated cell-cell and cell-substrate adhesion is important for PGC motility. Finally, localized growth factors may control PGC survival and consequently PGC position. Chemotaxis, regulated adhesion and cell survival are important for multiple migration processes which occur during development and disease. PGC migration shares these features.

Primordial germ cell migration in the chick and mouse embryo: the role of the chemokine SDF-1/CXCL12.

As in many other animals, the primordial germ cells (PGCs) in avian and reptile embryos are specified in positions distinct from the positions where they differentiate into sperm and egg. Unlike in other organism however, in these embryos, the PGCs use the vascular system as a vehicle to transport them to the region of the gonad where they exit the blood vessels and reach their target. To determine the molecular mechanisms governing PGC migration in these species, we have investigated the role of the chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) in guiding the cells towards their target in the chick embryo. We show that sdf-1 mRNA is expressed in locations where PGCs are found and towards which they migrate at the time they leave the blood vessels. Ectopically expressed chicken SDF-1alpha led to accumulation of PGCs at those positions. This analysis, as well as analysis of gene expression and PGC behavior in the mouse embryo, suggest that in both organisms, SDF-1 functions during the second phase of PGC migration, and not at earlier phases. These findings suggest that SDF-1 is required for the PGCs to execute the final migration steps as they transmigrate through the blood vessel endothelium of the chick or the gut epithelium of the mouse.

Transcriptional profiling identifies genes differentially expressed during and after migration in murine primordial germ cells.

Mouse primordial germ cells (PGCs) are migratory until they colonize the genital ridges, assemble with the somatic tissue, and start to differentiate into oocytes or spermatogonia. Using cell transplantation experiments, we show here that germ cells isolated during migration (at E10.5) will migrate actively to the genital ridges, whereas post-migratory PGCs isolated from E12.5 embryos are non-motile even when transferred into a permissive environment (e.g. E10.5 host tissue). Major transcriptional changes must take place between E10.5 and E12.5 that convert germ cells from a migratory to a non-migratory state. To identify the genes involved, we have performed transcriptional profiling of motile and non-motile populations of PGCs. We have identified 55 transcripts that are expressed in E10.5 PGCs at levels at least 3 x their expression at E12.5, and 48 transcripts with the reciprocal expression levels. Additionally, 309 transcripts were found to be expressed in both populations. Many of the E10.5 transcripts encode proteins involved in controlling cytoskeletal and adhesive interactions implicated in cell motility. Many of the E12.5 transcripts encode proteins implicated in germ cell differentiation.

The chemokine SDF1/CXCL12 and its receptor CXCR4 regulate mouse germ cell migration and survival.

In mouse embryos, germ cells arise during gastrulation and migrate to the early gonad. First, they emerge from the primitive streak into the region of the endoderm that forms the hindgut. Later in development, a second phase of migration takes place in which they migrate out of the gut to the genital ridges. There, they co-assemble with somatic cells to form the gonad. In vitro studies in the mouse, and genetic studies in other organisms, suggest that at least part of this process is in response to secreted signals from other tissues. Recent genetic evidence in zebrafish has shown that the interaction between stromal cell-derived factor 1 (SDF1) and its G-protein-coupled receptor CXCR4, already known to control many types of normal and pathological cell migrations, is also required for the normal migration of primordial germ cells. We show that in the mouse, germ cell migration and survival requires the SDF1/CXCR4 interaction. First, migrating germ cells express CXCR4, whilst the body wall mesenchyme and genital ridges express the ligand SDF1. Second, the addition of exogenous SDF1 to living embryo cultures causes aberrant germ cell migration from the gut. Third, germ cells in embryos carrying targeted mutations in CXCR4 do not colonize the gonad normally. However, at earlier stages in the hindgut, germ cells are unaffected in CXCR4(-/-) embryos. Germ cell counts at different stages suggest that SDF1/CXCR4 interaction also mediates germ cell survival. These results show that the SDF1/CXCR4 interaction is specifically required for the colonization of the gonads by primordial germ cells, but not for earlier stages in germ cell migration. This demonstrates a high degree of evolutionary conservation of part of the mechanism, but also an area of evolutionary divergence.

GP130, the shared receptor for the LIF/IL6 cytokine family in the mouse, is not required for early germ cell differentiation, but is required cell-autonomously in oocytes for ovulation.

GP130 is the shared receptor for members of the IL6 family of cytokines. Members of this family have been shown to enhance the survival of migratory (E10.5) or postmigratory (E12.5) murine primordial germ cells (PGCs) in culture; however, it is uncertain what role these cytokines play during PGC development in vivo. We have examined PGC numbers in E13.5 GP130-deficient mouse embryos and found that males exhibited a slight decrease in PGC numbers; females were normal. Also, we used the Cre-loxP system to inactive GP130 specifically in germ cells and found that this resulted in a fertility defect in females. These animals were found to have a slight reduction in the number of primary follicles and a major defect in ovulation. This data suggests that GP130 is required in female germ cells for their normal function, but is dispensable in male germ cells.