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.

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.

Inhibition of ß-catenin-TCF1 interaction delays differentiation of mouse embryonic stem cells.

The ability of mouse embryonic stem cells (mESCs) to self-renew or differentiate into various cell lineages is regulated by signaling pathways and a core pluripotency transcriptional network (PTN) comprising Nanog, Oct4, and Sox2. The Wnt/ß-catenin pathway promotes pluripotency by alleviating T cell factor TCF3-mediated repression of the PTN. However, it has remained unclear how ß-catenin's function as a transcriptional activator with TCF1 influences mESC fate. Here, we show that TCF1-mediated transcription is up-regulated in differentiating mESCs and that chemical inhibition of ß-catenin/TCF1 interaction improves long-term self-renewal and enhances functional pluripotency. Genetic loss of TCF1 inhibited differentiation by delaying exit from pluripotency and conferred a transcriptional profile strikingly reminiscent of self-renewing mESCs with high Nanog expression. Together, our data suggest that ß-catenin's function in regulating mESCs is highly context specific and that its interaction with TCF1 promotes differentiation, further highlighting the need for understanding how its individual protein-protein interactions drive stem cell fate.

An RNAi-based chemical genetic screen identifies three small-molecule inhibitors of the Wnt/wingless signaling pathway.

Misregulated ß-catenin responsive transcription (CRT) has been implicated in the genesis of various malignancies, including colorectal carcinomas, and it is a key therapeutic target in combating various cancers. Despite significant effort, successful clinical implementation of CRT inhibitory therapeutics remains a challenging goal. This is, in part, because of the challenge of identifying inhibitory compounds that specifically modulate the nuclear transcriptional activity of ß-catenin while not affecting its cytoskeletal function in stabilizing adherens junctions at the cell membrane. Here, we report an RNAi-based modifier screening strategy for the identification of CRT inhibitors. Our data provide support for the specificity of these inhibitory compounds in antagonizing the transcriptional function of nuclear ß-catenin. We show that these inhibitors efficiently block Wnt/ß-catenin-induced target genes and phenotypes in various mammalian and cancer cell lines. Importantly, these Wnt inhibitors are specifically cytotoxic to human colon tumor biopsy cultures as well as colon cancer cell lines that exhibit deregulated Wnt signaling.

Characterization of a dominant-active STAT that promotes tumorigenesis in Drosophila.

Little is known about the molecular mechanisms by which STAT proteins promote tumorigenesis. Drosophila is an ideal system for investigating this issue, as there is a single STAT (Stat92E), and its hyperactivation causes overgrowths resembling human tumors. Here we report the first identification of a dominant-active Stat92E protein, Stat92E(DeltaNDeltaC), which lacks both N- and C-termini. Mis-expression of Stat92E(DeltaNDeltaC)in vivo causes melanotic tumors, while in vitro it transactivates a Stat92E-luciferase reporter in the absence of stimulation. These gain-of-function phenotypes require phosphorylation of Y(711) and dimer formation with full-length Stat92E. Furthermore, a single point mutation, an R(442P) substitution in the DNA-binding domain, abolishes Stat92E function. Recombinant Stat92E(R442P) translocates to the nucleus following activation but fails to function in all assays tested. Interestingly, R(442) is conserved in most STATs in higher organisms, suggesting conservation of function. Modeling of Stat92E indicates that R(442) may contact the minor groove of DNA via invariant TC bases in the consensus binding element bound by all STAT proteins. We conclude that the N- and C- termini function unexpectedly in negatively regulating Stat92E activity, possibly by decreasing dimer dephosphorylation or increasing stability of DNA interaction, and that Stat92E(R442) has a nuclear function by altering dimer:DNA binding.

Function of the wingless signaling pathway in Drosophila.

Signaling by the wingless pathway has been shown to govern numerous developmental processes. Much of our current understanding of wingless signaling mechanisms comes from studies conducted in Drosophila melanogaster, which offers superior experimental tractability for genetic and developmental studies. Wingless signaling is highly consequential during normal development and patterning of Drosophila. Its earliest identifiable role during development of Drosophila is in the embryonic segmentation cascade, wherein wingless functions as a segment polarity gene and serves to pattern each individual segment along the antero-posterior axis of the developing embryo. Subsequent developmental roles fulfilled by wingless include patterning the developing wings, legs, eyes, CNS, heart, and muscles. Each of these developmental contexts offers excellent systems to query mechanisms regulating different aspects of wingless signal transduction such as synthesis, secretion, reception, and transcription. This chapter presents a brief overview on the functions of wingless signaling during development of Drosophila melanogaster.

High-throughput RNAi screen in Drosophila.

Genetic and biochemical analyses in model systems such as the fruitfly, Drosophila melanogaster, have successfully identified several genes that play key regulatory roles in fundamental cellular and developmental processes. However, the analyses of the complete genome sequences of Drosophila, as well as of humans, now reveal that traditional methods have ascribed functions to only a fraction of the total predicted genes. Thus, the roles for many, as yet unidentified genes, in normal development and cancer remain to be discovered. The challenge presented by the various large-scale genome projects is how to derive biologically relevant information from the raw sequences. The past few years have witnessed a rapid growth in the development and implementation high-throughput screening (HTS) technologies that researchers are now using to discover "gene-function" in an unbiased, systematic, and time-efficient manner. In fact one of the most promising functional genomic approach that has emerged in the past few years is based on RNA-interference (RNAi), in which the introduction of double-stranded RNA (dsRNA) into cells or whole organisms has been shown to be an effective tool to suppress endogenous gene expression. The RNAi technology has made it feasible to query the function of every gene in the genome for their potential function in a given cell-biological process using cell-based assays. This chapter discusses the application, advantages, and limitations of this powerful technology in the identification of novel modulators of cell-signaling pathways as well as its future scope and utility in designing more efficient genome-scale screens.

MAPK regulation of maternal and zygotic Notch transcript stability in early development.

Spatiotemporal modulation of the evolutionarily conserved, intercellular Notch signaling pathway is important in the development of many animals. Examples include the regulation of neural-epidermal fate decisions in neurogenic ectoderm of Drosophila and somitogenesis in vertebrate presomitic mesoderm. In both these and most other cases, it appears that Notch-class transmembrane receptors are ubiquitously expressed. Modulation of the pathway is achieved primarily by the localized expression of the activating ligand or by alteration of receptor specificity through a glycosyl transferase. In contrast, we present this report of an instance where the abundance of the Notch-class mRNA itself is dynamically regulated. Taking advantage of the long cell cycle of the two-cell-stage embryo of the leech Helobdella robusta, we show that this regulation is achieved at the levels of both transcript stability and transcription. Moreover, MAPK signaling plays a significant role in regulating accumulation of the transcript by virtue of its effect on Hro-notch mRNA stability. Intracellular injection of heterologous reporter mRNAs shows that the Hro-notch 3' UTR, containing seven AU-rich elements, is key to regulating transcript stability. Thus, we show that regulation of the Notch pathway can occur at a previously underappreciated level, namely that of transcript stability. Given that AU-rich elements occur in the 3' UTR of Notch-class genes in Drosophila, human, and Caenorhabditis elegans, regulation of Notch signaling by modulation of mRNA levels may be operating in other animals as well.

Characterization of Notch-class gene expression in segmentation stem cells and segment founder cells in Helobdella robusta (Lophotrochozoa; Annelida; Clitellata; Hirudinida; Glossiphoniidae).

To understand the evolution of segmentation, we must compare segmentation in all three major groups of eusegmented animals: vertebrates, arthropods, and annelids. The leech Helobdella robusta is an experimentally tractable annelid representative, which makes segments in anteroposterior progression from a posterior growth zone consisting of 10 identified stem cells. In vertebrates and some arthropods, Notch signaling is required for normal segmentation and functions via regulation of hes-class genes. We have previously characterized the expression of an hes-class gene (Hro-hes) during segmentation in Helobdella, and here, we characterize the expression of an H. robusta notch homolog (Hro-notch) during this process. We find that Hro-notch is transcribed in the segmental founder cells (blast cells) and their stem-cell precursors (teloblasts), as well as in other nonsegmental tissues. The mesodermal and ectodermal lineages show clear differences in the levels of Hro-notch expression. Finally, Hro-notch is shown to be inherited by newly born segmental founder cells as well as transcribed by them before their first cell division.

Cell cycle-dependent expression of a hairy and Enhancer of split (hes) homolog during cleavage and segmentation in leech embryos.

We have cloned genes related to hairy and Enhancer of split (hes) from glossiphoniid leeches, Helobdella robusta and Theromyzon rude. In leech, segments arise sequentially in anteroposterior progression from a posterior growth zone that consists of five bilaterally paired embryonic stem cells called teloblasts. Each teloblast gives rise to segmental founder cells (primary blast cells) that contribute iterated sets of definitive progeny in each segment. Thus, in leech, the "segmentation clock," is closely identified with the cell cycle clock of the teloblasts. We have characterized normal expression patterns of mRNA and protein for the H. robusta hes-class gene (Hro-hes). Semiquantitative RT-PCR revealed that Hro-hes mRNA levels peak while the teloblasts are actively producing primary blast cells. RT-PCR, in situ hybridization and immunostaining revealed that Hro-hes is expressed as early as the first zygotic mitosis and throughout early development. Hro-hes is expressed in macromeres, pro-teloblasts, teloblasts and primary blast cells. HRO-HES protein is localized in the nuclei of cells expressing HRO-HES during interphase; nuclear HRO-HES is reduced during mitosis. In contrast, Hro-hes is transcribed during mitosis and its transcripts are associated with mitotic apparatus (MA). Thus, Hro-hes transcription cycles in antiphase to the nuclear localization of HRO-HES protein. These results indicate that Hro-hes expression, and thus possibly its biological activity, is linked to the cell cycle.