Appearance of tuft cells during prostate cancer progression.

Tuft cells are chemosensory epithelial cells that increase in number following infection or injury to robustly activate the innate immune response to alleviate or promote disease. Recent studies of castration resistant prostate cancer and its subtype, neuroendocrine prostate cancer, revealed Pou2f3+ populations in mouse models. The transcription factor Pou2f3 is a master regulator of the tuft cell lineage. We show that tuft cells are upregulated early during prostate cancer development, and their numbers increase with progression. Cancer-associated tuft cells in the mouse prostate express DCLK1, COX1, COX2, while human tuft cells express COX1. Mouse and human tuft cells exhibit strong activation of signaling pathways including EGFR and SRC-family kinases. While DCLK1 is a mouse tuft cell marker, it is not present in human prostate tuft cells. Tuft cells that appear in mouse models of prostate cancer display genotype-specific tuft cell gene expression signatures. Using bioinformatic analysis tools and publicly available datasets, we characterized prostate tuft cells in aggressive disease and highlighted differences between tuft cell populations. Our findings indicate that tuft cells contribute to the prostate cancer microenvironment and may promote development of more advanced disease. Further research is needed to understand contributions of tuft cells to prostate cancer progression.

Transcriptional Repression by FoxM1 Suppresses Tumor Differentiation and Promotes Metastasis of Breast Cancer.

The transcription factor Forkhead box M1 (FoxM1) is overexpressed in breast cancers and correlates with poor prognosis. Mechanistically, FoxM1 associates with CBP to activate transcription and with Rb to repress transcription. Although the activating function of FoxM1 in breast cancer has been well documented, the significance of its repressive activity is poorly understood. Using CRISPR-Cas9 engineering, we generated a mouse model that expresses FoxM1-harboring point mutations that block binding to Rb while retaining its ability to bind CBP. Unlike FoxM1-null mice, mice harboring Rb-binding mutant FoxM1 did not exhibit significant developmental defects. The mutant mouse line developed PyMT-driven mammary tumors that were deficient in lung metastasis, which was tumor cell-intrinsic. Single-cell RNA-seq of the tumors revealed a deficiency in prometastatic tumor cells and an expansion of differentiated alveolar type tumor cells, and further investigation identified that loss of the FoxM1/Rb interaction caused enhancement of the mammary alveolar differentiation program. The FoxM1 mutant tumors also showed increased Pten expression, and FoxM1/Rb was found to activate Akt signaling by repressing Pten. In human breast cancers, expression of FoxM1 negatively correlated with Pten mRNA. Furthermore, the lack of tumor-infiltrating cells in FoxM1 mutant tumors appeared related to decreases in pro-metastatic tumor cells that express factors required for infiltration. These observations demonstrate that the FoxM1/Rb-regulated transcriptome is critical for the plasticity of breast cancer cells that drive metastasis, identifying a prometastatic role of Rb when bound to FoxM1. SIGNIFICANCE: This work provides new insights into how the interaction between FoxM1 and Rb facilitates the evolution of metastatic breast cancer cells by altering the transcriptome.

Cysteine oxidation of copper transporter CTR1 drives VEGFR2 signalling and angiogenesis.

Vascular endothelial growth factor receptor type 2 (VEGFR2, also known as KDR and FLK1) signalling in endothelial cells (ECs) is essential for developmental and reparative angiogenesis. Reactive oxygen species and copper (Cu) are also involved in these processes. However, their inter-relationship is poorly understood. Evidence of the role of the endothelial Cu importer CTR1 (also known as SLC31A1) in VEGFR2 signalling and angiogenesis in vivo is lacking. Here, we show that CTR1 functions as a redox sensor to promote angiogenesis in ECs. CTR1-depleted ECs showed reduced VEGF-induced VEGFR2 signalling and angiogenic responses. Mechanistically, CTR1 was rapidly sulfenylated at Cys189 at its cytosolic C terminus after stimulation with VEGF, which induced CTR1-VEGFR2 disulfide bond formation and their co-internalization to early endosomes, driving sustained VEGFR2 signalling. In vivo, EC-specific Ctr1-deficient mice or CRISPR-Cas9-generated redox-dead Ctr1(C187A)-knockin mutant mice had impaired developmental and reparative angiogenesis. Thus, oxidation of CTR1 at Cys189 promotes VEGFR2 internalization and signalling to enhance angiogenesis. Our study uncovers an important mechanism for sensing reactive oxygen species through CTR1 to drive neovascularization.

Sequential Activation of Guide RNAs to Enable Successive CRISPR-Cas9 Activities.

Currently, either highly multiplexed genetic manipulations can be delivered to mammalian cells all at once or extensive engineering of gene regulatory sequences can be used to conditionally activate a few manipulations. Here, we provide proof of principle for a new system enabling multiple genetic manipulations to be executed as a preprogrammed cascade of events. The system leverages the programmability of the S. pyogenes Cas9 and is based on flexible arrangements of individual modules of activity. The basic module consists of an inactive single-guide RNA (sgRNA)-like component that is converted to an active state through the effects of another sgRNA. Modules can be arranged to bring about an algorithmic program of sequential genetic manipulations without the need for engineering cell-type-specific promoters or gene regulatory sequences. With the expanding diversity of available tools that use spCas9, this sgRNA-based system provides multiple levels of interfacing with mammalian cell biology.

Netrin-1 promotes naive pluripotency through Neo1 and Unc5b co-regulation of Wnt and MAPK signalling.

In mouse embryonic stem cells (mESCs), chemical blockade of Gsk3α/ß and Mek1/2 (2i) instructs a self-renewing ground state whose endogenous inducers are unknown. Here we show that the axon guidance cue Netrin-1 promotes naive pluripotency by triggering profound signalling, transcriptomic and epigenetic changes in mESCs. Furthermore, we demonstrate that Netrin-1 can substitute for blockade of Gsk3α/ß and Mek1/2 to sustain self-renewal of mESCs in combination with leukaemia inhibitory factor and regulates the formation of the mouse pluripotent blastocyst. Mechanistically, we reveal how Netrin-1 and the balance of its receptors Neo1 and Unc5B co-regulate Wnt and MAPK pathways in both mouse and human ESCs. Netrin-1 induces Fak kinase to inactivate Gsk3α/ß and stabilize ß-catenin while increasing the phosphatase activity of a Ppp2r2c-containing Pp2a complex to reduce Erk1/2 activity. Collectively, this work identifies Netrin-1 as a regulator of pluripotency and reveals that it mediates different effects in mESCs depending on its receptor dosage, opening perspectives for balancing self-renewal and lineage commitment.

Cryo-EM structures reveal coordinated domain motions that govern DNA cleavage by Cas9.

The RNA-guided Cas9 endonuclease from Streptococcus pyogenes is a single-turnover enzyme that displays a stable product state after double-stranded-DNA cleavage. Here, we present cryo-EM structures of precatalytic, postcatalytic and product states of the active Cas9-sgRNA-DNA complex in the presence of Mg2+. In the precatalytic state, Cas9 adopts the 'checkpoint' conformation with the HNH nuclease domain positioned far away from the DNA. Transition to the postcatalytic state involves a dramatic ~34-Å swing of the HNH domain and disorder of the REC2 recognition domain. The postcatalytic state captures the cleaved substrate bound to the catalytically competent HNH active site. In the product state, the HNH domain is disordered, REC2 returns to the precatalytic conformation, and additional interactions of REC3 and RuvC with nucleic acids are formed. The coupled domain motions and interactions between the enzyme and the RNA-DNA hybrid provide new insights into the mechanism of genome editing by Cas9.

Intracellular Ca2+ Homeostasis and Nuclear Export Mediate Exit from Naive Pluripotency.

Progression through states of pluripotency is required for cells in early mammalian embryos to transition away from heightened self-renewal and toward competency for lineage specification. Here, we use a CRISPR mutagenesis screen in mouse embryonic stem cells (ESCs) to identify unexpected roles for nuclear export and intracellular Ca2+ homeostasis during the exit out of the naive state of pluripotency. Mutation of a plasma membrane Ca2+ pump encoded by Atp2b1 increased intracellular Ca2+ such that it overcame effects of intracellular Ca2+ reduction, which is required for naive exit. Persistent self-renewal of ESCs was supported both in Atp2b1-/-Tcf7l1-/- double-knockout ESCs passaged in defined media alone (no LIF or inhibitors) and in wild-type cells passaged in media containing only calcitonin and a GSK3 inhibitor. These new findings suggest a central role for intracellular Ca2+ in safeguarding naive pluripotency.

Complementary Wnt Sources Regulate Lymphatic Vascular Development via PROX1-Dependent Wnt/ß-Catenin Signaling.

Wnt/ß-catenin signaling is necessary for lymphatic vascular development. Oscillatory shear stress (OSS) enhances Wnt/ß-catenin signaling in cultured lymphatic endothelial cells (LECs) to induce expression of the lymphedema-associated transcription factors GATA2 and FOXC2. However, the mechanisms by which OSS regulates Wnt/ß-catenin signaling and GATA2 and FOXC2 expression are unknown. We show that OSS activates autocrine Wnt/ß-catenin signaling in LECs in vitro. Tissue-specific deletion of Wntless, which is required for the secretion of Wnt ligands, reveals that LECs and vascular smooth muscle cells are complementary sources of Wnt ligands that regulate lymphatic vascular development in vivo. Further, the LEC master transcription factor PROX1 forms a complex with ß-catenin and the TCF/LEF transcription factor TCF7L1 to enhance Wnt/ß-catenin signaling and promote FOXC2 and GATA2 expression in LECs. Thus, our work defines Wnt sources, reveals that PROX1 directs cell fate by acting as a Wnt signaling component, and dissects the mechanisms of PROX1 and Wnt synergy.

Enhanced Bacterial Immunity and Mammalian Genome Editing via RNA-Polymerase-Mediated Dislodging of Cas9 from Double-Strand DNA Breaks.

The ability to target the Cas9 nuclease to DNA sequences via Watson-Crick base pairing with a single guide RNA (sgRNA) has provided a dynamic tool for genome editing and an essential component of adaptive immune systems in bacteria. After generating a double-stranded break (DSB), Cas9 remains stably bound to DNA. Here, we show persistent Cas9 binding blocks access to the DSB by repair enzymes, reducing genome editing efficiency. Cas9 can be dislodged by translocating RNA polymerases, but only if the polymerase approaches from one direction toward the Cas9-DSB complex. By exploiting these RNA-polymerase/Cas9 interactions, Cas9 can be conditionally converted into a multi-turnover nuclease, mediating increased mutagenesis frequencies in mammalian cells and enhancing bacterial immunity to bacteriophages. These consequences of a stable Cas9-DSB complex provide insights into the evolution of protospacer adjacent motif (PAM) sequences and a simple method of improving selection of highly active sgRNAs for genome editing.

TCF7L1 suppresses primitive streak gene expression to support human embryonic stem cell pluripotency.

Human embryonic stem cells (hESCs) are exquisitely sensitive to WNT ligands, which rapidly cause differentiation. Therefore, hESC self-renewal requires robust mechanisms to keep the cells in a WNT inactive but responsive state. How they achieve this is largely unknown. We explored the role of transcriptional regulators of WNT signaling, the TCF/LEFs. As in mouse ESCs, TCF7L1 is the predominant family member expressed in hESCs. Genome-wide, it binds a gene cohort involved in primitive streak formation at gastrulation, including NODAL, BMP4 and WNT3 Comparing TCF7L1-bound sites with those bound by the WNT signaling effector ß-catenin indicates that TCF7L1 acts largely on the WNT signaling pathway. TCF7L1 overlaps less with the pluripotency regulators OCT4 and NANOG than in mouse ESCs. Gain- and loss-of-function studies indicate that TCF7L1 suppresses gene cohorts expressed in the primitive streak. Interestingly, we find that BMP4, another driver of hESC differentiation, downregulates TCF7L1, providing a mechanism of BMP and WNT pathway intersection. Together, our studies indicate that TCF7L1 plays a major role in maintaining hESC pluripotency, which has implications for human development during gastrulation.

A Novel l-Asparaginase with low l-Glutaminase Coactivity Is Highly Efficacious against Both T- and B-cell Acute Lymphoblastic Leukemias In Vivo.

Acute lymphoblastic leukemia (ALL) is the most common type of pediatric cancer, although about 4 of every 10 cases occur in adults. The enzyme drug l-asparaginase serves as a cornerstone of ALL therapy and exploits the asparagine dependency of ALL cells. In addition to hydrolyzing the amino acid l-asparagine, all FDA-approved l-asparaginases also have significant l-glutaminase coactivity. Since several reports suggest that l-glutamine depletion correlates with many of the side effects of these drugs, enzyme variants with reduced l-glutaminase coactivity might be clinically beneficial if their antileukemic activity would be preserved. Here we show that novel low l-glutaminase variants developed on the backbone of the FDA-approved Erwinia chrysanthemi l-asparaginase were highly efficacious against both T- and B-cell ALL, while displaying reduced acute toxicity features. These results support the development of a new generation of safer l-asparaginases without l-glutaminase activity for the treatment of human ALL.Significance: A new l-asparaginase-based therapy is less toxic compared with FDA-approved high l-glutaminase enzymes Cancer Res; 78(6); 1549-60. ©2018 AACR.

DDB2 Is a Novel Regulator of Wnt Signaling in Colon Cancer.

Deregulation of the Wnt/ß-catenin signaling pathway drives the development of colorectal cancer, but understanding of this pathway remains incomplete. Here, we report that the damage-specific DNA-binding protein DDB2 is critical for ß-catenin-mediated activation of RNF43, which restricts Wnt signaling by removing Wnt receptors from the cell surface. Reduced expression of DDB2 and RNF43 was observed in human hyperplastic colonic foci. DDB2 recruited EZH2 and ß-catenin at an upstream site in the Rnf43 gene, enabling functional interaction with distant TCF4/ß-catenin-binding sites in the intron of Rnf43 This novel activity of DDB2 was required for RNF43 function as a negative feedback regulator of Wnt signaling. Mice genetically deficient in DDB2 exhibited increased susceptibility to colon tumor development in a manner associated with higher abundance of the Wnt receptor-expressing cells and greater activation of the downstream Wnt pathway. Our results identify DDB2 as both a partner and regulator of Wnt signaling, with an important role in suppressing colon cancer development. Cancer Res; 77(23); 6562-75. ©2017 AACR.

Method for Dual Viral Vector Mediated CRISPR-Cas9 Gene Disruption in Primary Human Endothelial Cells.

Human endothelial cells (ECs) are widely used to study mechanisms of angiogenesis, inflammation, and endothelial permeability. Targeted gene disruption induced by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-Associated Protein 9 (Cas9) nuclease gene editing is potentially an important tool for definitively establishing the functional roles of individual genes in ECs. We showed that co-delivery of adenovirus encoding EGFP-tagged Cas9 and lentivirus encoding a single guide RNA (sgRNA) in primary human lung microvascular ECs (HLMVECs) disrupted the expression of the Tie2 gene and protein. Tie2 disruption increased basal endothelial permeability and prevented permeability recovery following injury induced by the inflammatory stimulus thrombin. Thus, gene deletion via viral co-delivery of CRISPR-Cas9 in primary human ECs provides a novel platform to investigate signaling mechanisms of normal and perturbed EC function without the need for clonal expansion.

Co-incident insertion enables high efficiency genome engineering in mouse embryonic stem cells.

CRISPR/Cas9 nucleases have enabled powerful, new genome editing capabilities; however, the preponderance of non-homologous end joining (NHEJ) mediated repair events over homology directed repair (HDR) in most cell types limits the ability to engineer precise changes in mammalian genomes. Here, we increase the efficiency of isolating precise HDR-mediated events in mouse embryonic stem (ES) cells by more than 20-fold through the use of co-incidental insertion (COIN) of independent donor DNA sequences. Analysis of on:off-target frequencies at the Lef1 gene revealed that bi-allelic insertion of a PGK-Neo cassette occurred more frequently than expected. Using various selection cassettes targeting multiple loci, we show that the insertion of a selectable marker at one control site frequently coincided with an insertion at an unlinked, independently targeted site, suggesting enrichment of a sub-population of HDR-proficient cells. When individual cell events were tracked using flow cytometry and fluorescent protein markers, individual cells frequently performed either a homology-dependent insertion event or a homology-independent event, but rarely both types of insertions in a single cell. Thus, when HDR-dependent selection donors are used, COIN enriches for HDR-proficient cells among heterogeneous cell populations. When combined with a self-excising selection cassette, COIN provides highly efficient and scarless genome editing.

Transcription factor 7-like 1 is involved in hypothalamo-pituitary axis development in mice and humans.

Aberrant embryonic development of the hypothalamus and/or pituitary gland in humans results in congenital hypopituitarism (CH). Transcription factor 7-like 1 (TCF7L1), an important regulator of the WNT/ß-catenin signaling pathway, is expressed in the developing forebrain and pituitary gland, but its role during hypothalamo-pituitary (HP) axis formation or involvement in human CH remains elusive. Using a conditional genetic approach in the mouse, we first demonstrate that TCF7L1 is required in the prospective hypothalamus to maintain normal expression of the hypothalamic signals involved in the induction and subsequent expansion of Rathke's pouch progenitors. Next, we reveal that the function of TCF7L1 during HP axis development depends exclusively on the repressing activity of TCF7L1 and does not require its interaction with ß-catenin. Finally, we report the identification of two independent missense variants in human TCF7L1, p.R92P and p.R400Q, in a cohort of patients with forebrain and/or pituitary defects. We demonstrate that these variants exhibit reduced repressing activity in vitro and in vivo relative to wild-type TCF7L1. Together, our data provide support for a conserved molecular function of TCF7L1 as a transcriptional repressor during HP axis development in mammals and identify variants in this transcription factor that are likely to contribute to the etiology of CH.

Regulation of Tcf7l1 DNA binding and protein stability as principal mechanisms of Wnt/ß-catenin signaling.

Wnt/ß-catenin signal transduction requires direct binding of ß-catenin to Tcf/Lef proteins, an event that is classically associated with stimulating transcription by recruiting coactivators. This molecular cascade plays critical roles throughout embryonic development and normal postnatal life by affecting stem cell characteristics and tumor formation. Here, we show that this pathway utilizes a fundamentally different mechanism to regulate Tcf7l1 (formerly named Tcf3) activity. ß-catenin inactivates Tcf7l1 without a switch to a coactivator complex by removing it from DNA, which leads to Tcf7l1 protein degradation. Mouse genetic experiments demonstrate that Tcf7l1 inactivation is the only required effect of the Tcf7l1-ß-catenin interaction. Given the expression of Tcf7l1 in pluripotent embryonic and adult stem cells, as well as in poorly differentiated breast cancer, these findings provide mechanistic insights into the regulation of pluripotency and the role of Wnt/ß-catenin in breast cancer.

Stage-specific regulation of reprogramming to induced pluripotent stem cells by Wnt signaling and T cell factor proteins.

Wnt signaling is intrinsic to mouse embryonic stem cell self-renewal. Therefore, it is surprising that reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is not strongly enhanced by Wnt signaling. Here, we demonstrate that active Wnt signaling inhibits the early stage of reprogramming to iPSCs, whereas it is required and even stimulating during the late stage. Mechanistically, this biphasic effect of Wnt signaling is accompanied by a change in the requirement of all four of its transcriptional effectors: T cell factor 1 (Tcf1), Lef1, Tcf3, and Tcf4. For example, Tcf3 and Tcf4 are stimulatory early but inhibitory late in the reprogramming process. Accordingly, ectopic expression of Tcf3 early in reprogramming combined with its loss of function late enables efficient reprogramming in the absence of ectopic Sox2. Together, our data indicate that the stepwise process of reprogramming to iPSCs is critically dependent on the stage-specific control and action of all four Tcfs and Wnt signaling.

Wnt signaling regulates the lineage differentiation potential of mouse embryonic stem cells through Tcf3 down-regulation.

Canonical Wnt signaling plays a rate-limiting role in regulating self-renewal and differentiation in mouse embryonic stem cells (ESCs). We have previously shown that mutation in the Apc (adenomatous polyposis coli) tumor suppressor gene constitutively activates Wnt signaling in ESCs and inhibits their capacity to differentiate towards ecto-, meso-, and endodermal lineages. However, the underlying molecular and cellular mechanisms through which Wnt regulates lineage differentiation in mouse ESCs remain to date largely unknown. To this aim, we have derived and studied the gene expression profiles of several Apc-mutant ESC lines encoding for different levels of Wnt signaling activation. We found that down-regulation of Tcf3, a member of the Tcf/Lef family and a key player in the control of self-renewal and pluripotency, represents a specific and primary response to Wnt activation in ESCs. Accordingly, rescuing Tcf3 expression partially restored the neural defects observed in Apc-mutant ESCs, suggesting that Tcf3 down-regulation is a necessary step towards Wnt-mediated suppression of neural differentiation. We found that Tcf3 down-regulation in the context of constitutively active Wnt signaling does not result from promoter DNA methylation but is likely to be caused by a plethora of mechanisms at both the RNA and protein level as shown by the observed decrease in activating histone marks (H3K4me3 and H3-acetylation) and the upregulation of miR-211, a novel Wnt-regulated microRNA that targets Tcf3 and attenuates early neural differentiation in mouse ESCs. Our data show for the first time that Wnt signaling down-regulates Tcf3 expression, possibly at both the transcriptional and post-transcriptional levels, and thus highlight a novel mechanism through which Wnt signaling inhibits neuro-ectodermal lineage differentiation in mouse embryonic stem cells.

Tcf7l1 prepares epiblast cells in the gastrulating mouse embryo for lineage specification.

The core gene regulatory network (GRN) in embryonic stem cells (ESCs) integrates activities of the pro-self-renewal factors Oct4 (Pou5f1), Sox2 and Nanog with that of an inhibitor of self-renewal, Tcf7l1 (Tcf3). The inhibitor function of Tcf7l1 causes dependence on extracellular Wnt/ß-catenin signaling activity, making its embryonic role within the ESC GRN unclear. By analyzing intact mouse embryos, we demonstrate that the function of Tcf7l1 is necessary for specification of cell lineages to occur concomitantly with the elaboration of a three-dimensional body plan during gastrulation. In Tcf7l1(-/-) embryos, specification of mesoderm is delayed, effectively uncoupling it from the induction of the primitive streak. Tcf7l1 repressor activity is necessary for a rapid switch in the response of pluripotent cells to Wnt/ß-catenin stimulation, from one of self-renewal to a mesoderm specification response. These results identify Tcf7l1 as a unique factor that is necessary in pluripotent cells to prepare them for lineage specification. We suggest that the role of Tcf7l1 in mammals is to inhibit the GRN to ensure the coordination of lineage specification with the dynamic cellular events occurring during gastrulation.

Wnt pathway regulation of embryonic stem cell self-renewal.

Embryonic stem cells (ESCs) can generate all of the cell types found in the adult organism. Remarkably, they retain this ability even after many cell divisions in vitro, as long as the culture conditions prevent differentiation of the cells. Wnt signaling and ß-catenin have been shown to cause strong effects on ESCs both in terms of stimulating the expansion of stem cells and stimulating differentiation toward lineage committed cell types. The varied effects of Wnt signaling in ESCs, alongside the sometimes unconventional mechanisms underlying the effects, have generated a fair amount of controversy and intrigue regarding the role of Wnt signaling in pluripotent stem cells. Insights into the mechanisms of Wnt function in stem cells can be gained by examination of the causes for seemingly opposing effects of Wnt signaling on self-renewal versus differentiation.