Therapeutic targeting of SLC6A8 creatine transporter suppresses colon cancer progression and modulates human creatine levels.

Colorectal cancer (CRC) is a leading cause of cancer mortality. Creatine metabolism was previously shown to critically regulate colon cancer progression. We report that RGX-202, an oral small-molecule SLC6A8 transporter inhibitor, robustly inhibits creatine import in vitro and in vivo, reduces intracellular phosphocreatine and ATP levels, and induces tumor apoptosis. RGX-202 suppressed CRC growth across KRAS wild-type and KRAS mutant xenograft, syngeneic, and patient-derived xenograft (PDX) tumors. Antitumor efficacy correlated with tumoral expression of creatine kinase B. Combining RGX-202 with 5-fluorouracil or the DHODH inhibitor leflunomide caused regressions of multiple colorectal xenograft and PDX tumors of distinct mutational backgrounds. RGX-202 also perturbed creatine metabolism in patients with metastatic CRC in a phase 1 trial, mirroring pharmacodynamic effects on creatine metabolism observed in mice. This is, to our knowledge, the first demonstration of preclinical and human pharmacodynamic activity for creatine metabolism targeting in oncology, thus revealing a critical therapeutic target.

Regulation of embryonic and adult neurogenesis by Ars2.

Neural development is controlled at multiple levels to orchestrate appropriate choices of cell fate and differentiation. Although more attention has been paid to the roles of neural-restricted factors, broadly expressed factors can have compelling impacts on tissue-specific development. Here, we describe in vivo conditional knockout analyses of murine Ars2, which has mostly been studied as a general RNA-processing factor in yeast and cultured cells. Ars2 protein expression is regulated during neural lineage progression, and is required for embryonic neural stem cell (NSC) proliferation. In addition, Ars2 null NSCs can still transition into post-mitotic neurons, but fail to undergo terminal differentiation. Similarly, adult-specific deletion of Ars2 compromises hippocampal neurogenesis and results in specific behavioral defects. To broaden evidence for Ars2 as a chromatin regulator in neural development, we generated Ars2 ChIP-seq data. Notably, Ars2 preferentially occupies DNA enhancers in NSCs, where it colocalizes broadly with NSC regulator SOX2. Ars2 association with chromatin is markedly reduced following NSC differentiation. Altogether, Ars2 is an essential neural regulator that interacts dynamically with DNA and controls neural lineage development.

LXR/ApoE Activation Restricts Innate Immune Suppression in Cancer.

Therapeutic harnessing of adaptive immunity via checkpoint inhibition has transformed the treatment of many cancers. Despite unprecedented long-term responses, most patients do not respond to these therapies. Immunotherapy non-responders often harbor high levels of circulating myeloid-derived suppressor cells (MDSCs)-an immunosuppressive innate cell population. Through genetic and pharmacological approaches, we uncovered a pathway governing MDSC abundance in multiple cancer types. Therapeutic liver-X nuclear receptor (LXR) agonism reduced MDSC abundance in murine models and in patients treated in a first-in-human dose escalation phase 1 trial. MDSC depletion was associated with activation of cytotoxic T lymphocyte (CTL) responses in mice and patients. The LXR transcriptional target ApoE mediated these effects in mice, where LXR/ApoE activation therapy elicited robust anti-tumor responses and also enhanced T cell activation during various immune-based therapies. We implicate the LXR/ApoE axis in the regulation of innate immune suppression and as a target for enhancing the efficacy of cancer immunotherapy in patients.

Transcriptional repression of Bmp2 by p21(Waf1/Cip1) links quiescence to neural stem cell maintenance.

Relative quiescence and self renewal are defining features of adult stem cells, but their potential coordination remains unclear. Subependymal neural stem cells (NSCs) lacking cyclin-dependent kinase (CDK) inhibitor (CKI) 1a (p21) exhibit rapid expansion that is followed by their permanent loss later in life. Here we demonstrate that transcription of the gene encoding bone morphogenetic protein 2 (Bmp2) in NSCs is under the direct negative control of p21 through actions that are independent of CDK. Loss of p21 in NSCs results in increased levels of secreted BMP2, which induce premature terminal differentiation of multipotent NSCs into mature non-neurogenic astrocytes in an autocrine and/or paracrine manner. We also show that the cell-nonautonomous p21-null phenotype is modulated by the Noggin-rich environment of the subependymal niche. The dual function that we describe here provides a physiological example of combined cell-autonomous and cell-nonautonomous functions of p21 with implications in self renewal, linking the relative quiescence of adult stem cells to their longevity and potentiality.

BEND6 is a nuclear antagonist of Notch signaling during self-renewal of neural stem cells.

The activity of the Notch pathway revolves around a CSL-class transcription factor, which recruits distinct complexes that activate or repress target gene expression. The co-activator complex is deeply conserved and includes the cleaved Notch intracellular domain (NICD) and Mastermind. By contrast, numerous CSL co-repressor proteins have been identified, and these are mostly different between invertebrate and vertebrate systems. In this study, we demonstrate that mammalian BEND6 is a neural BEN-solo factor that shares many functional attributes with Drosophila Insensitive, a co-repressor for the Drosophila CSL factor. BEND6 binds the mammalian CSL protein CBF1 and antagonizes Notch-dependent target activation. In addition, its association with Notch- and CBF1-regulated enhancers is promoted by CBF1 and antagonized by activated Notch. In utero electroporation experiments showed that ectopic BEND6 inhibited Notch-mediated self-renewal of neocortical neural stem cells and promoted neurogenesis. Conversely, knockdown of BEND6 increased NSC self-renewal in wild-type neocortex, and exhibited genetic interactions with gain and loss of Notch pathway activity. We recapitulated all of these findings in cultured neurospheres, in which overexpression and depletion of BEND6 caused reciprocal effects on neural stem cell renewal and neurogenesis. These data reveal a novel mammalian CSL co-repressor in the nervous system, and show that the Notch-inhibitory activity of certain BEN-solo proteins is conserved between flies and mammals.

Widespread and extensive lengthening of 3' UTRs in the mammalian brain.

Remarkable advances in techniques for gene expression profiling have radically changed our knowledge of the transcriptome. Recently, the mammalian brain was reported to express many long intergenic noncoding (lincRNAs) from loci downstream from protein-coding genes. Our experimental tests failed to validate specific accumulation of lincRNA transcripts, and instead revealed strongly distal 3' UTRs generated by alternative cleavage and polyadenylation (APA). With this perspective in mind, we analyzed deep mammalian RNA-seq data using conservative criteria, and identified 2035 mouse and 1847 human genes that utilize substantially distal novel 3' UTRs. Each of these extends at least 500 bases past the most distal 3' termini available in Ensembl v65, and collectively they add 6.6 Mb and 5.1 Mb to the mRNA space of mouse and human, respectively. Extensive Northern analyses validated stable accumulation of distal APA isoforms, including transcripts bearing exceptionally long 3' UTRs (many >10 kb and some >18 kb in length). The Northern data further illustrate that the extensions we annotated were not due to unprocessed transcriptional run-off events. Global tissue comparisons revealed that APA events yielding these extensions were most prevalent in the mouse and human brain. Finally, these extensions collectively contain thousands of conserved miRNA binding sites, and these are strongly enriched for many well-studied neural miRNAs. Altogether, these new 3' UTR annotations greatly expand the scope of post-transcriptional regulatory networks in mammals, and have particular impact on the central nervous system.

Ars2 maintains neural stem-cell identity through direct transcriptional activation of Sox2.

Fundamental questions remain unanswered about the transcriptional networks that control the identity and self-renewal of neural stem cells (NSCs), a specialized subset of astroglial cells that are endowed with stem properties and neurogenic capacity. Here we report that the zinc finger protein Ars2 (arsenite-resistance protein 2; also known as Srrt) is expressed by adult NSCs from the subventricular zone (SVZ) of mice, and that selective knockdown of Ars2 in cells expressing glial fibrillary acidic protein within the adult SVZ depletes the number of NSCs and their neurogenic capacity. These phenotypes are recapitulated in the postnatal SVZ of hGFAP-cre::Ars2(fl/fl) conditional knockout mice, but are more severe. Ex vivo assays show that Ars2 is necessary and sufficient to promote NSC self-renewal, and that it does so by positively regulating the expression of Sox2. Although plant and animal orthologues of Ars2 are known for their conserved roles in microRNA biogenesis, we unexpectedly observed that Ars2 retains its capacity to promote self-renewal in Drosha and Dicer1 knockout NSCs. Instead, chromatin immunoprecipitation revealed that Ars2 binds a specific region within the 6-kilobase NSC enhancer of Sox2. This association is RNA-independent, and the region that is bound is required for Ars2-mediated activation of Sox2. We used gel-shift analysis to refine the Sox2 region bound by Ars2 to a specific conserved DNA sequence. The importance of Sox2 as a critical downstream effector is shown by its ability to restore the self-renewal and multipotency defects of Ars2 knockout NSCs. Our findings reveal Ars2 as a new transcription factor that controls the multipotent progenitor state of NSCs through direct activation of the pluripotency factor Sox2.

Vascular niche factor PEDF modulates Notch-dependent stemness in the adult subependymal zone.

We sought to address the fundamental question of how stem cell microenvironments can regulate self-renewal. We found that Notch was active in astroglia-like neural stem cells (NSCs), but not in transit-amplifying progenitors of the murine subependymal zone, and that the level of Notch transcriptional activity correlated with self-renewal and multipotency. Moreover, dividing NSCs appeared to balance renewal with commitment via controlled segregation of Notch activity, leading to biased expression of known (Hes1) and previously unknown (Egfr) Notch target genes in daughter cells. Pigment epithelium-derived factor (PEDF) enhanced Notch-dependent transcription in cells with low Notch signaling, thereby subverting the output of an asymmetrical division to the production of two highly self-renewing cells. Mechanistically, PEDF induced a non-canonical activation of the NF-kappaB pathway, leading to the dismissal of the transcriptional co-repressor N-CoR from specific Notch-responsive promoters. Our data provide a basis for stemness regulation in vascular niches and indicate that Notch and PEDF cooperate to regulate self-renewal.

A combined ex/in vivo assay to detect effects of exogenously added factors in neural stem cells.

We describe a protocol developed/modified by our group for the ex vivo and in vivo assessment of the response to a soluble factor of murine neural stem cells from the adult sub-ventricular zone (SVZ). The procedure includes several experimental options that can be used either independently or in combination. Potential factor effects on self-renewal, survival and proliferation are assayed by means of neurosphere cultures, with the factor administered directly in vitro to the culture plates (Step 1) or infused in vivo immediately before tissue dissociation (Step 3). We also use bromodeoxiuridine (BrdU) retention to label slowly dividing cells in vivo and subsequently perform two different types of experiments. In one set of experiments, the factor is added to primary cultures of stem cells obtained from the BrdU-pulsed animals and effects are tested on label-retaining cells after immunocytochemistry (Step 2). In another set, prolonged intraventricular infusion of the factor in BrdU-pulsed animals is followed by immunohistochemical analysis of BrdU labeling in the intact SVZ (Step 4). The minimum estimated time for the full combined procedure is 45 d.

Pigment epithelium-derived factor is a niche signal for neural stem cell renewal.

Adult stem cells are characterized by self-renewal and multilineage differentiation, and these properties seem to be regulated by signals from adjacent differentiated cell types and by extracellular matrix molecules, which collectively define the stem cell "niche." Self-renewal is essential for the lifelong persistence of stem cells, but its regulation is poorly understood. In the mammalian brain, neurogenesis persists in two germinal areas, the subventricular zone (SVZ) and the hippocampus, where continuous postnatal neuronal production seems to be supported by neural stem cells (NSCs). Here we show that pigment epithelium-derived factor (PEDF) is secreted by components of the murine SVZ and promotes self-renewal of adult NSCs in vitro. In addition, intraventricular PEDF infusion activated slowly dividing stem cells, whereas a blockade of endogenous PEDF decreased their cycling. These data demonstrate that PEDF is a niche-derived regulator of adult NSCs and provide evidence for a role for PEDF protein in NSC maintenance.