GLI Binding Research Articles

Abstract LB200: Defining the mechanisms of cancer specific transcription factor, tGLI1

Abstract Glioblastoma (GBM) is the most common malignant central nervous system tumor in adults with a five-year survival rate of less than 7%. First identified in GBM, truncated GLI1 (tGLI1), an alternative splicing isoform of GLI1, has emerged as a cancer specific GLI1 variant as tGLI1 is highly expressed in both GBM cell lines and primary specimens but not in normal tissue. Despite an in-frame loss of 41 amino acids, the tGLI1 protein has retained all known GLI1 functional domains and appears to respond to sonic hedgehog in a similar manner as GLI1. However, tGLI1 operates as a gain of function transcription factor with the ability to bind to and activate genes unique from GLI1 to promote invasion, migration, angiogenesis, and stemness in both GBM and breast cancer. Despite the advancements in our understanding of tGLI1, a genome wide DNA binding pattern of tGLI1 has not been established and the mechanism by which tGLI1 gains access to these genes is unknown. To establish a more robust understanding of the differential DNA binding patterns of GLI1 and tGLI1, we carried out ChIP-sequencing (ChIP-seq) and found only 14% of GLI1 and tGLI1 binding sites are shared. These results suggest GLI1 and tGLI1 have even more unique binding pattens across the entire genome that previously thought. However, the underlying mechanism for why GLI1 and tGLI1 bind different regions of the genome remains elusive. To determine whether variations in protein interactions of GLI1 and tGLI1 contribute to the unique binding pattern of tGLI1, we carried out immunoprecipitation (IP) followed by mass spectrometry (mass spec). Results identified 45 GLI1 associated proteins and 52 tGLI1 associated proteins with only a 29% overlap between the two. Furthermore, gene ontology analysis revealed 43% of the tGLI1-specific associated proteins are involved in processes related to RNA binding, processing, and metabolism. From these RNA-related proteins, we validated an interaction between tGLI1 and the protein NONO. NONO, which can bind both RNA and DNA, is involved in a range of molecular processes including paraspeckle formation and transcriptional regulation. In GBM, high NONO expression correlated with poor patient outcomes and the mesenchymal subtype. Interestingly, we observed that NONO, along with another essential paraspeckle component, SFPQ, had a greater interaction with tGLI1 compared to GLI1. Furthermore, co-expressing NONO with tGLI1 significantly increased clonogenic growth as well as CD44 gene transactivation using a luciferase reporter, suggesting it may play a role in tGLI1-driven stem-like properties. Furthermore, co-expression of NONO and tGLI1 increased the nuclear localization of both proteins. Together, these results show NONO may enhance tGLI1 nuclear localization, transcriptional activity, and exacerbate tGLI1-driven phenotypes. These effects of NONO could also contribute to the differential binding pattern between GLI1 and tGLI1. Citation Format: Haddie DeHart, Nolan Gregg, Richard Carpenter. Defining the mechanisms of cancer specific transcription factor, tGLI1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB200.

MRCKα/β positively regulates Gli protein activity

Posttranslational modifications (PTMs) are key regulatory events for the majority of signaling pathways. Transcription factors are often phosphorylated on multiple residues, which regulates their trafficking, stability, or transcriptional activity. Gli proteins, transcription factors that respond to the Hedgehog pathway, are regulated by phosphorylation, but the sites and the kinases involved have been only partially described. We identified three novel kinases: MRCKα, MRCKβ, and MAP4K5 which physically interact with Gli proteins and directly phosphorylate Gli2 on multiple sites. We established that MRCKα/β kinases regulate Gli proteins, which impacts the transcriptional output of the Hedgehog pathway. We showed that double knockout of MRCKα/β affects Gli2 ciliary and nuclear localization and reduces Gli2 binding to the Gli1 promoter. Our research fills a critical gap in our understanding of the regulation of Gli proteins by describing their activation mechanisms through phosphorylation.

GLI1 interaction with p300 modulates SDF1 expression in cancer-associated fibroblasts to promote pancreatic cancer cells migration.

Carcinoma-associated fibroblasts (CAFs) play an important role in the progression of multiple malignancies. Secretion of cytokines and growth factors underlies the pro-tumoral effect of CAFs. Although this paracrine function has been extensively documented, the molecular mechanisms controlling the expression of these factors remain elusive. In this study, we provide evidence of a novel CAF transcriptional axis regulating the expression of SDF1, a major driver of cancer cell migration, involving the transcription factor GLI1 and histone acetyltransferase p300. We demonstrate that conditioned media from CAFs overexpressing GLI1 induce the migration of pancreatic cancer cells, and this effect is impaired by an SDF1-neutralizing antibody. Using a combination of co-immunoprecipitation, proximity ligation assay and chromatin immunoprecipitation assay, we further demonstrate that GLI1 and p300 physically interact in CAFs to co-occupy and drive SDF1 promoter activity. Mapping experiments highlight the requirement of GLI1 N-terminal for the interaction with p300. Importantly, knockdowns of both GLI1 and p300 reduce SDF1 expression. Further analysis shows that knockdown of GLI1 decreases SDF1 promoter activity, p300 recruitment, and levels of its associated histone marks (H4ac, H3K27ac, and H3K14ac). Finally, we show that the integrity of two GLI binding sites in the SDF1 promoter is required for p300 recruitment. Our findings define a new role for the p300-GLI1 complex in the regulation of SDF1, providing new mechanistic insight into the molecular events controlling pancreatic cancer cells migration.

RNA-seq and ChIP-seq Identification of Unique and Overlapping Targets of GLI Transcription Factors in Melanoma Cell Lines.

Simple SummaryThe majority of melanomas show hyperactivation of the MAPK signaling pathway, most often through mutations in BRAF and NRAS. Despite significant progress in therapy, targeting this signaling pathway solely has not been the solution for stopping the progression of this disease. Recently, researchers recognized the involvement of the Hedgehog-GLI (HH-GLI) signaling pathway in melanoma and its crosstalk with the MAPK pathway. In order to identify new HH-GLI-regulated targets that could be involved in the crosstalk with the MAPK pathway, we performed RNA sequencing and ChIP sequencing on three melanoma cell lines. By combining RNA-seq and ChIP-seq results, we successfully validated 15 novel targets of GLI proteins in melanoma cell lines. These findings will contribute to a better understanding of the GLI code and its role in melanoma.Background: Despite significant progress in therapy, melanoma still has a rising incidence worldwide, and novel treatment strategies are needed. Recently, researchers have recognized the involvement of the Hedgehog-GLI (HH-GLI) signaling pathway in melanoma and its consistent crosstalk with the MAPK pathway. In order to further investigate the link between the two pathways and to find new target genes that could be considered for combination therapy, we set out to find transcriptional targets of all three GLI proteins in melanoma. Methods: We performed RNA sequencing on three melanoma cell lines (CHL-1, A375, and MEL224) with overexpressed GLI1, GLI2, and GLI3 and combined them with the results of ChIP-sequencing on endogenous GLI1, GLI2, and GLI3 proteins. After combining these results, 21 targets were selected for validation by qPCR. Results: RNA-seq revealed a total of 808 differentially expressed genes (DEGs) for GLI1, 941 DEGs for GLI2, and 58 DEGs for GLI3. ChIP-seq identified 527 genes that contained GLI1 binding sites in their promoters, 1103 for GLI2 and 553 for GLI3. A total of 15 of these targets were validated in the tested cell lines, 6 of which were detected by both RNA-seq and ChIP-seq. Conclusions: Our study provides insight into the unique and overlapping transcriptional output of the GLI proteins in melanoma. We suggest that our findings could provide new potential targets to consider while designing melanoma-targeted therapy.

Derivatives of (R)-3-(5-Furanyl)carboxamido-2-aminopropanoic Acid as Potent NMDA Receptor Glycine Site Agonists with GluN2 Subunit-Specific Activity.

NMDA receptors mediate glutamatergic neurotransmission and are therapeutic targets due to their involvement in a variety of psychiatric and neurological disorders. Here, we describe the design and synthesis of a series of (R)-3-(5-furanyl)carboxamido-2-aminopropanoic acid analogues 8a-s as agonists at the glycine (Gly) binding site in the GluN1 subunit, but not GluN3 subunits, of NMDA receptors. These novel analogues display highly variable potencies and agonist efficacies among the NMDA receptor subtypes (GluN1/2A-D) in a manner dependent on the GluN2 subunit. Notably, compound 8p is identified as a potent partial agonist at GluN1/2C (EC50 = 0.074 μM) with an agonist efficacy of 28% relative to activation by Gly and virtually no agonist activity at GluN1/2A, GluN1/2B, and GluN1/2D. Thus, these novel agonists can modulate the activity of specific NMDA receptor subtypes by replacing the full endogenous agonists Gly or d-serine (d-Ser), thereby providing new opportunities in the development of novel therapeutic agents.

Therapeutic Targeting of GLI1 in Acute Myeloid Leukemia

Hematopoietic stem and progenitor cell (HSPCs) proliferation are tightly regulated during normal homeostasis. Recent data suggests that aberrantly activated signaling pathways such as Hedgehog (Hh), Notch, and Wnt occur during early hematopoietic oncogenesis. Gli genes (Gli1-3), known as Gli-code, encode Cys2-His2 zinc finger transcription factors governing the expression of pro-survival genes at the distal end of the Hh pathway (Hh→PTCH1→SMO→GLI). These genes are tightly regulated in embryonic development. They are a common node of dysregulated activation through which many oncogenic signals, such as Hh, FLT3, PI3K-AKT, TGFβ, and RAS, converge. Hh-signaling critical for embryogenesis is mostly inactive in adult tissues; however, aberrant activation of the Hh/Gli has been implicated in cancer progression and chemotherapy resistance in AML. Activation of SMO triggers an intracellular signaling cascade leading to the formation of active Gli transcription factors which functionally converge on Gli1. In addition to canonical PTCH/SMO-dependent regulation of Gli1, we have shown that Gli-signaling axis can be activated independent of Hh-signaling (Fig.1).The specificity of the Gli pathway in leukemic cells suggests that Gli inhibition may have clinical utility. Most efforts have been devoted towards therapeutic targeting upstream of the Gli Hh-signaling through PTCH/SMO. These efforts remained mostly ineffective due to convergence of parallel oncogenic signals at Gli1 irrespective of Hh-signaling status.We have focused our structure-guided small molecule design efforts to develop potent Gli1-inhibitors, a nodal point of the convergence of various oncogenic signals (Fig.1). Our Gli1 inhibitor prevents both Gli1 binding to DNA and transcription of Gli-target genes, inducing cell death specifically in leukemic cells while sparing normal CD34+ hematopoietic cells. Our inhibitor binds on the Gli1-DNA interface between Zn-finger 2 and 3 (KD= 14nM) and is >200-fold more effective than the previously reported Gant61 Gli1 (KD=3.2µM) at inducing cell death of leukemic cells in semi-solid methylcellulose-based media. We performed a colony forming assay on CD34+ bone marrow cells derived from RAS mutant patients in the presence and abscence of our highly potent iGli1 inhibitor. iGLi1 demonstarted high potency (IC50, 50nM) in preventing colony formation in a dose dependent manner while no cytotoxic effect was observed on normal bone marrow cells. The LD50 for normal bone marrow cells was 10µM providing a therapeutic index of more than 200. Cellular mRNA sequencing analysis was also performed in MOLM13 and MV4-11 cells to confirm the downregulation of Gli1 target genes. Consistent with the Gli1 inhibition, iGli1 treatment of these AML cells led to the downregulation of Gli1 target genes Gli1, Gli2, PTCH1, Cyclin1/2, P21, P27, and IKK-β. iGli1 inhibited the binding of Gli to DNA and rapidly inhibits transcription of critical Gli-dependent target genes. Furthermore, inhibition of Gli1 resulted in the stalling of RNA Pol II, leading to redistribution of pause and pause-release factors DSIF/NELF and PTEFb, respectively. We also observed that iGli1 treatment of MOLM13 or THP1 cells arrests cell cycle progression in S-phase. Inhibition of Gli-dependent transcription by iGli1 leads to accumulation of R-loops (RNA:DNA hybrids) that cause a robust DNA damage, inhibition of DNA replication, S-phase progression, and subsequent apoptosis.The efficacy of iGli1 was also compared to standard care used in the treatment of AML (Cytarabine and Idarubicin under in vitro conditions against MOLM-13, THP-1, K562, and MV4-11). We observed that iGli1 was superior to cytarabine/idarubicin treatment in cell culture model system as a single agent. In addition, ex vivo exposure of MV4-11 cells with iGli1 completely shut down the tumor formation in NSG mice in a xenograft model.In summary, we have shown that Gli1 is not only a strong target to treat AML patients but also an excellent approach for developing a novel therapeutic for patients where Gli1 expression and activity is high, such as patients with RAS mutations. The Gli1 inhibitior is highly effctive as a single agent against highly aggressive form of AML with an excellent therapeutic index. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Constitutive GLI1 expression in chondrosarcoma is regulated by major vault protein via mTOR/S6K1 signaling cascade

Hedgehog signaling plays a pivotal role in embryonic pattern formation and diverse aspects of the postnatal biological process. Perturbation of the hedgehog pathway and overexpression of GLI1, a downstream transcription factor in the hedgehog pathway, are highly relevant to several malignancies including chondrosarcoma (CS). We previously found that knocking down expression of GLI1 attenuates the disrupted Indian hedgehog (IHH) signal pathway and suppresses cell survival in human CS cells. However, the underlying mechanisms regulating the expression of GLI1 are still unknown. Here, we demonstrated the implication of GLI1 in SMO-independent pathways in CS cells. A GLI1 binding protein, major vault protein (MVP), was identified using the affinity purification method. MVP promoted the nuclear transport and stabilization of GLI1 by compromising the binding affinity of GLI1 with suppressor of fused homolog (SUFU) and increased GLI1 expression via mTOR/S6K1 signaling cascade. Functionally, knockdown of MVP suppressed cell growth and induced apoptosis. Simultaneous inhibition of MVP and GLI1 strongly inhibits the growth of CS in vitro and in vivo. Moreover, IHC results showed that MVP, GLI1, and P-p70S6K1 were highly expressed and positively correlated with each other in 71 human CS tissues. Overall, our findings revealed a novel regulating mechanism for HH-independent GLI1 expression and provide a rationale for combination therapy in patients with advanced CS.

CRISPR editing of the GLI1 first intron abrogates GLI1 expression and differentially alters lineage commitment.

GLI1 is one of three GLI family transcription factors that mediate Sonic Hedgehog signaling, which plays a role in development and cell differentiation. GLI1 forms a positive feedback loop with GLI2 and likely with itself. To determine the impact of GLI1 and its intronic regulatory locus on this transcriptional loop and human stem cell differentiation, we deleted the region containing six GLI binding sites in the human GLI1 intron using CRISPR/Cas9 editing to produce H1 human embryonic stem cell (hESC) GLI1‐edited clones. Editing out this intronic region, without removing the entire GLI1 gene, allowed us to study the effects of this highly complex region, which binds transcription factors in a variety of cells. The roles of GLI1 in human ESC differentiation were investigated by comparing RNA sequencing, quantitative‐real time PCR (q‐rtPCR), and functional assays. Editing this region resulted in GLI1 transcriptional knockdown, delayed neural commitment, and inhibition of endodermal and mesodermal differentiation during spontaneous and directed differentiation experiments. We found a delay in the onset of early osteogenic markers, a reduction in the hematopoietic potential to form granulocyte units, and a decrease in cancer‐related gene expression. Furthermore, inhibition of GLI1 via antagonist GANT‐61 had similar in vitro effects. These results indicate that the GLI1 intronic region is critical for the feedback loop and that GLI1 has lineage‐specific effects on hESC differentiation. Our work is the first study to document the extent of GLI1 abrogation on early stages of human development and to show that GLI1 transcription can be altered in a therapeutically useful way.

MiR-214-3p-Sufu-GLI1 is a novel regulatory axis controlling inflammatory smooth muscle cell differentiation from stem cells and neointimal hyperplasia

BackgroundInflammatory smooth muscle cells (iSMCs) generated from adventitial stem/progenitor cells (AdSPCs) have been recognised as a new player in cardiovascular disease, and microRNA-214-3p (miR-214-3p) has been implicated in mature vascular SMC functions and neointimal hyperplasia. Here, we attempted to elucidate the functional involvements of miR-214-3p in iSMC differentiation from AdSPCs and unravel the therapeutic potential of miR-214-3p signalling in AdSPCs for injury-induced neointimal hyperplasia.MethodsThe role of miR-214-3p in iSMC differentiation from AdSPCs was evaluated by multiple biochemistry assays. The target of miR-214-3p was identified through binding site mutation and reporter activity analysis. A murine model of injury-induced arterial remodelling and stem cell transplantation was conducted to study the therapeutic potential of miR-214-3p. RT-qPCR analysis was performed to examine the gene expression in healthy and diseased human arteries.ResultsmiR-214-3p prevented iSMC differentiation/generation from AdSPCs by restoring sonic hedgehog-glioma-associated oncogene 1 (Shh-GLI1) signalling. Suppressor of fused (Sufu) was identified as a functional target of miR-214-3p during iSMC generation from AdSPCs. Mechanistic studies revealed that miR-214-3p over-expression or Sufu inhibition can promote nuclear accumulation of GLI1 protein in AdSPCs, and the consensus sequence (GACCACCCA) for GLI1 binding within smooth muscle alpha-actin (SMαA) and serum response factor (SRF) gene promoters is required for their respective regulation by miR-214-3p and Sufu. Additionally, Sufu upregulates multiple inflammatory gene expression (IFNγ, IL-6, MCP-1 and S100A4) in iSMCs. In vivo, transfection of miR-214-3p into the injured vessels resulted in the decreased expression level of Sufu, reduced iSMC generation and inhibited neointimal hyperplasia. Importantly, perivascular transplantation of AdSPCs increased neointimal hyperplasia, whereas transplantation of AdSPCs over-expressing miR-214-3p prevented this. Finally, decreased expression of miR-214-3p but increased expression of Sufu was observed in diseased human arteries.ConclusionsWe present a previously unexplored role for miR-214-3p in iSMC differentiation and neointima iSMC hyperplasia and provide new insights into the therapeutic effects of miR-214-3p in vascular disease.

Hedgehog-GLI signalling promotes chemoresistance through the regulation of ABC transporters in colorectal cancer cells

Colorectal cancer (CRC) is a leading cause of cancer death. Chemoresistance is a pivotal feature of cancer cells leading to treatment failure and ATP-binding cassette (ABC) transporters are responsible for the efflux of several molecules, including anticancer drugs. The Hedgehog-GLI (HH-GLI) pathway is a major signalling in CRC, however its role in chemoresistance has not been fully elucidated. Here we show that the HH-GLI pathway favours resistance to 5-fluorouracil and Oxaliplatin in CRC cells. We identified potential GLI1 binding sites in the promoter region of six ABC transporters, namely ABCA2, ABCB1, ABCB4, ABCB7, ABCC2 and ABCG1. Next, we investigated the binding of GLI1 using chromatin immunoprecipitation experiments and we demonstrate that GLI1 transcriptionally regulates the identified ABC transporters. We show that chemoresistant cells express high levels of GLI1 and of the ABC transporters and that GLI1 inhibition disrupts the transporters up-regulation. Moreover, we report that human CRC tumours express high levels of the ABCG1 transporter and that its expression correlates with worse patients’ prognosis. This study identifies a new mechanism where HH-GLI signalling regulates CRC chemoresistance features. Our results indicate that the inhibition of Gli1 regulates the ABC transporters expression and therefore should be considered as a therapeutic option in chemoresistant patients.

Sonic Hedgehog-Gli1 Signaling and Cellular Retinoic Acid Binding Protein 1 Gene Regulation in Motor Neuron Differentiation and Diseases.

Cellular retinoic acid-binding protein 1 (CRABP1) is highly expressed in motor neurons. Degenerated motor neuron-like MN1 cells are engineered by introducing SODG93A or AR-65Q to model degenerated amyotrophic lateral sclerosis (ALS) or spinal bulbar muscular atrophy neurons. Retinoic acid (RA)/sonic hedgehog (Shh)-induced embryonic stem cells differentiation into motor neurons are employed to study up-regulation of Crabp1 by Shh. In SODG93A or AR-65Q MN1 neurons, CRABP1 level is reduced, revealing a correlation of motor neuron degeneration with Crabp1 down-regulation. Up-regulation of Crabp1 by Shh is mediated by glioma-associated oncogene homolog 1 (Gli1) that binds the Gli target sequence in Crabp1′s neuron-specific regulatory region upstream of minimal promoter. Gli1 binding triggers chromatin juxtaposition with minimal promoter, activating transcription. Motor neuron differentiation and Crabp1 up-regulation are both inhibited by blunting Shh with Gli inhibitor GANT61. Expression data mining of ALS and spinal muscular atrophy (SMA) motor neurons shows reduced CRABP1, coincided with reduction in Shh-Gli1 signaling components. This study reports motor neuron degeneration correlated with down-regulation in Crabp1 and Shh-Gli signaling. Shh-Gli up-regulation of Crabp1 involves specific chromatin remodeling. The physiological and pathological implication of this regulatory pathway in motor neuron degeneration is supported by gene expression data of ALS and SMA patients.

GLI transcriptional repression regulates tissue-specific enhancer activity in response to Hedgehog signaling.

Transcriptional repression needs to be rapidly reversible during embryonic development. This extends to the Hedgehog pathway, which primarily serves to counter GLI repression by processing GLI proteins into transcriptional activators. In investigating the mechanisms underlying GLI repression, we find that a subset of GLI binding regions, termed HH-responsive enhancers, specifically loses acetylation in the absence of HH signaling. These regions are highly enriched around HH target genes and primarily drive HH-specific transcriptional activity in the mouse limb bud. They also retain H3K27ac enrichment in limb buds devoid of GLI activator and repressor, indicating that their activity is primarily regulated by GLI repression. Furthermore, the Polycomb repression complex is not active at most of these regions, suggesting it is not a major mechanism of GLI repression. We propose a model for tissue-specific enhancer activity in which an HDAC-associated GLI repression complex regulates target genes by altering the acetylation status at enhancers.

Abstract A103: Targeting GLI1-mediated NBS1 transcription overcomes 5-FU resistance

Abstract Resistance to radiation and chemotherapy in colorectal patients is considered one of the major contributors of refractory disease and disease progression. Dysregulation or aberrant activation of the SHH/GLI1 signaling pathway, common to both colorectal cancer and other cancer subtypes, influences the integrity of the DNA damage response (DDR) pathway, which may impact tumor response to chemo- and radiation therapies. The DDR pathway is initiated in part by the recognition of a DNA lesion by a sensor protein and the recruitment NBS1 to that lesion. Subsequent repair is initiated by the Mre11, Rad50, NBS1 (MRN) complex formation. In this study, we investigated the expression of GLI1 and NBS1 in tissues samples from 188 patients diagnosed with colorectal cancer by immunohistochemistry and analyzed the clinical significance and prognostic relevance of elevated GLI1-NBS1 co-expression. GLI1 expression was positively associated with the NBS1 expression, and high expression of both in the biopsied specimens significantly correlated with poor patient survival. The result of a series of biochemical experiments directly links GLI1 transcriptional activity to the DNA damage repair pathway. First, NBS1 mRNA levels and protein were found to be reduced upon treatment with GANT61, a GLI inhibitor, indicating that GLI activity was required for NBS1 transcription. Second, overexpression of NBS1 in HT29 cells rescued GANT61-induced cell death by measure of cleaved caspase-3, indicating a significant role of induced DNA damage in the mechanism of GANT61-induced cell death in oncogenic GLI1 cancers. Third, this was verified by ChIP analysis of GLI1 binding to the NBS1 promotor region containing a putative GLI binding sequence. The efficacy of GLI1 inhibition as therapeutic strategy was then tested in parallel with 5-Fluorouracil (5-FU), a component of the standard-of-care therapy FOLFOX. In vitro, colorectal cell lines with GLI1-driven NBS1 expression demonstrated strong 5-FU resistance. These cells were re-sensitized to 5-FU by silencing the expression of NBS1 with siRNA, or with combination therapy involving 5-FU and GANT61. This suggests that a first-in class chemotherapeutic strategy may provide additional treatment options for patients demonstrating 5-FU resistance during the course of their chemotherapy. While GANT61 is commonly used to inhibit GLI in vitro, it exhibits poor pharmacokinetic properties and is not a viable drug for the clinic. To that end, we present a novel GLI1 antagonist SR38832, which exhibits a more stable pharmacokinetic profile than GANT61 in xenograft mouse models and lower IC50 in colon cancer cell lines. In a human colorectal cancer xenograft mouse model, SR38832 significantly inhibited both tumor growth and proliferation. Not only has the identification of SR38832 provided an in vivo tool to study GLI oncogenic activity, but SR38832 is also structural lead to be optimized through an iterative medicinal chemistry approach. Citation Format: Ruowen Zhang, Justin Avery, Jinlu Ma, Theresa Nguyen, Mark J Suto, Bo Xu, Rebecca J Boohaker. Targeting GLI1-mediated NBS1 transcription overcomes 5-FU resistance [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A103. doi:10.1158/1535-7163.TARG-19-A103

GLI2 Modulated by SUFU and SPOP Induces Intestinal Stem Cell Niche Signals in Development and Tumorigenesis

Gut mesenchyme provides key stem cell niche signals such as Wnt ligands, but how these signals areregulated is unclear. Because Hedgehog (Hh) signaling is critical for gut mesenchymal development and tumorigenesis, we investigated Hh-mediated mechanisms by analyzing mice deleted for key negative regulators of Hh signaling, Sufu and/or Spop, in the gut mesenchyme, and demonstrated their dosage-dependent roles. Although these mutants exhibit abnormal mesenchymal cell growth and functionally defective muscle layers, villification is completed with proper mesenchymal clustering, implying a permissive role for Hh signaling. These mesenchymal defects are partially rescued by Gli2 reduction. Consistent with increased epithelial proliferation caused by abnormal Hh activation in development, Sufu reduction promotes intestinal tumorigenesis, whereas Gli2 heterozygosity suppresses it.Our analyses of chromatin and GLI2 binding genomic regions reveal its transcriptional regulation of stem cell niche signals through enhancers, providing mechanistic insight into the intestinal stem cell niche in development and tumorigenesis.

Regulation of GLI1 by cis DNA elements and epigenetic marks

GLI1 is one of three transcription factors (GLI1, GLI2 and GLI3) that mediate the Hedgehog signal transduction pathway and play important roles in normal development. GLI1 and GLI2 form a positive-feedback loop and function as human oncogenes. The mouse and human GLI1 genes have untranslated 5′ exons and large introns 5′ of the translational start. Here we show that Sonic Hedgehog (SHH) stimulates occupancy in the introns by H3K27ac, H3K4me3 and the histone reader protein BRD4. H3K27ac and H3K4me3 occupancy is not significantly changed by removing BRD4 from the human intron and transcription start site (TSS) region. We identified six GLI binding sites (GBS) in the first intron of the human GLI1 gene that are in regions of high sequence conservation among mammals. GLI1 and GLI2 bind all of the GBS in vitro. Elimination of GBS1 and 4 attenuates transcriptional activation by GLI1. Elimination of GBS1, 2, and 4 attenuates transcriptional activation by GLI2. Eliminating all sites essentially eliminates reporter gene activation. Further, GLI1 binds the histone variant H2A.Z. These results suggest that GLI1 and GLI2 can regulate GLI1 expression through protein-protein interactions involving complexes of transcription factors, histone variants, and reader proteins in the regulatory intron of the GLI1 gene. GLI1 acting in trans on the GLI1 intron provides a mechanism for GLI1 positive feedback and auto-regulation. Understanding the combinatorial protein landscape in this locus will be important to interrupting the GLI positive feedback loop and providing new therapeutic approaches to cancers associated with GLI1 overexpression.

PRMT7 methylates and suppresses GLI2 binding to SUFU thereby promoting its activation.

Cellular senescence is implicated in aging or age-related diseases. Sonic hedgehog (Shh) signaling, an inducer of embryonic development, has recently been demonstrated to inhibit cellular senescence. However, the detailed mechanisms to activate Shh signaling to prevent senescence is not well understood. Here, we demonstrate that Protein arginine methyltransferase 7 (PRMT7) promotes Shh signaling via GLI2 methylation which is critical for suppression of cellular senescence. PRMT7-deficient mouse embryonic fibroblasts (MEFs) exhibited a premature cellular senescence with accompanied increase in the cell cycle inhibitors p16 and p21. PRMT7 depletion results in reduced Shh signaling activity in MEFs while PRMT7 overexpression enhances GLI2-reporter activities that are sensitive to methylation inhibition. PRMT7 interacts with and methylates GLI2 on arginine residues 225 and 227 nearby a binding region of SUFU, a negative regulator of GLI2. This methylation interferes with GLI2-SUFU binding, leading to facilitation of GLI2 nuclear accumulation and Shh signaling. Taken together, these data suggest that PRMT7 induces GLI2 methylation, reducing its binding to SUFU and increasing Shh signaling, ultimately leading to prevention of cellular senescence.

Transcriptional repression of the tyrosinase-related protein 2 gene by transforming growth factor-β and the Kruppel-like transcription factor GLI2

BackgroundTyrosinase-Related Protein 2 (TRP2) is an enzyme involved in melanogenesis, that also exerts proliferative, anti-apoptotic and immunogenic functions in melanoma cells. TRP2 transcription is regulated by the melanocytic master transcription factor MITF. GLI2, a transcription factor that acts downstream of Hedgehog signaling, is also a direct transcriptional target of the TGF-β/SMAD pathway that contributes to melanoma progression and exerts transcriptional antagonistic activities against MITF. ObjectivesTo characterize the molecular events responsible for TGF-β and GLI2 repression of TRP2 expression. MethodsIn silico promoter analysis, transient cell transfection experiments with 5′-end TRP2 promoter deletion constructs, chromatin immuno-precipitation, and site-directed promoter mutagenesis were used to dissect the molecular mechanisms of TRP2 gene regulation by TGF-β and GLI2. ResultsWe demonstrate that TGF-β and GLI2-specific TRP2 repression involves direct mechanisms that occur in addition to MITF downregulation by TGF-β and GLI2. We identify two functional GLI2 binding sites within the TRP2 promoter that are critical for TGF-β and GLI2 responsiveness, one of them overlapping a CREB binding site. GLI2 and CREB competing for the same cis-element is associated with opposite transcriptional outcome. ConclusionOur results further refine the understanding of how TGF-β and GLI2 control the phenotypic plasticity of melanoma cells. In particular, we identify critical GLI2-binding cis-elements within the TRP2 promoter region that allow for its transcriptional repression independently from MITF concomitant downregulation.

Hand2 Functions to Synergistically Activate Gli Target Genes in Mandibular Neural Crest Cells

The Hedgehog (Hh) pathway has been well‐studied and implicated in neural crest cell (NCC) survival and facial patterning; however, the mechanism(s) by which the transcription factors (TFs) of the pathway, the Gli TFs, orchestrate Hh signal transduction remains elusive. For example, Gli ChIP‐seq data from NCCs in the developing mandibular prominence (MNP) reveal that the presence of a peak is not sufficient to predict a Gli “target.” In light of these data and previous studies suggesting Gli proteins interact with co‐factors, we performed genome‐wide analyses to identify motifs that frequently appear near Gli binding regions (GBRs). These analyses revealed that E‐box motifs (bHLH TF binding sites) were significantly enriched (p=0.001) within Gli ChIP‐seq peaks compared to random sequences. Additionally, ChIP‐seq peaks for Hand2, a bHLH TF known to be necessary for MNP development, were 111‐fold enriched within Gli peaks. A comparison of Hand2f/f;Wnt1‐Cre and Gli2f/f;Gli3f/f;Wnt1‐Cre MNP phenotypes shows striking similarities, which in conjunction with the significant overlap of Gli and Hand2 co‐occupied genomic regions suggests these two proteins may work in tandem to influence Gli target gene expression. Interrogation of DNA motifs within genomic locations occupied by Gli and Hand2 together versus those occupied by Gli alone revealed the existence of two distinct types of GBRs: a ‘canonical’ GBR defined by the presence of a medially located A (GACCACCC), and a ‘deviant’ GBR defined by a T in the medial position (GACCTCCC). Interestingly, the ‘canonical’ GBR is 19‐fold enriched (p<10−136) in Gli3 peaks without a Hand2 peak, whereas it is only 3‐fold enriched (p<10−47) in Gli3 peaks that contain a Hand2 peak. To test if these distinct GBRs convey biological relevance, we identified canonical and deviant GBRs within Gli and Hand2 ChIP‐seq peaks of Gli targets important for mandibular development and linked them to reporter constructs transfected into 09–1 cells. We found that Hand2 and Gli utilize a deviant GBR to synergistically activate Gli targets including Foxd1 and Foxd2. In sum, our work establishes insight into the existence of a sequence‐dependent mechanism for Gli target gene regulation in which molecular accomplices may be necessary for optimal transcription of Gli targets in NCCs of the developing MNP.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Direct repression of anoctamin 1 (ANO1) gene transcription by Gli proteins.

The Ca2+-activated Cl- channel, anoctamin 1 (Ano1, also known as transmembrane protein 16A) contributes to intestinal pacemaking, fluid secretion, cellular excitability, and tissue development. The human ANO1 promoter contains binding sites for the glioma-associated oncogene (Gli) proteins. We investigated regulation of ANO1 transcription by Gli. ANO1 promoter activity was determined using a luciferase reporter system. Binding and functional effects of Glis on ANO1 transcription and expression were demonstrated by chromatin immunoprecipitation, small interfering RNA knockdown, PCR, immunolabeling, and recordings of Ca2+-activated Cl- currents in human embryonic kidney 293 (HEK293) cells. Results from previous genome-wide association studies were used to test ANO1 promoter polymorphisms for association with disease. Gli1 and Gli2 bound to the promoter and repressed ANO1 transcription. Repression depended on Gli binding to a site close to the ANO1 transcriptional start site. Mutation of this site prevented Gli binding and transcriptional repression. Knockdown of Gli expression and inhibition of Gli activity increased expression of ANO1 RNA and Ca2+-activated Cl- currents in HEK293 cells. A single-nucleotide polymorphism prevented Gli binding and showed association with irritable bowel syndrome. We conclude that Gli1 and Gli2 repress ANO1 by a novel mechanism that is independent of Gli cleavage and that has a role in gastrointestinal function.-Mazzone, A., Gibbons, S. J., Eisenman, S. T., Strege, P. R., Zheng, T., D'Amato, M., Ordog, T., Fernandez-Zapico, M. E., Farrugia, G. Direct repression of anoctamin 1 (ANO1) gene transcription by Gli proteins.

Unique and overlapping GLI1 and GLI2 transcriptional targets in neoplastic chondrocytes.

Excessive Hedgehog (Hh) signaling in chondrocytes is sufficient to cause formation of enchondroma-like lesions which can progress to chondrosarcoma. To elucidate potential underlying mechanisms, we identified GLI1 and GLI2 target genes in human chondrosarcoma. Using chromatin immunoprecipitation (ChIP) sequencing and microarray data, in silico analyses were conducted to identify and characterize unique and overlapping GLI1 and GLI2 binding regions in neoplastic chondrocytes. After overlaying microarray data from human chondrosarcoma, 204 upregulated and 106 downregulated genes were identified as Hh-responsive Gli binding targets. After overlaying published Gli ChIP-on-chip data from mouse, 48 genes were identified as potential direct downstream targets of Hedgehog signaling with shared GLI binding regions in evolutionarily conserved DNA elements. Among these was BMP2, pointing to potential cross-talk between TGF beta signaling and Hh signaling. Our identification of potential target genes that are unique and common to GLI1 and GLI2 in neoplastic chondrocytes contributes to elucidating potential pathways through which Hh signaling impacts cartilage tumor biology.