Hh Target Genes Research Articles

Increased Netrin downstream of overactive Hedgehog signaling disrupts optic fissure formation.

Uveal coloboma, a developmental eye defect, is caused by failed development of the optic fissure, a ventral structure in the optic stalk and cup where axons exit the eye and vasculature enters. The Hedgehog (Hh) signaling pathway regulates optic fissure development: loss-of-function mutations in the Hh receptor ptch2 produce overactive Hh signaling and can result in coloboma. We previously proposed a model where overactive Hh signaling disrupts optic fissure formation by upregulating transcriptional targets acting both cell- and non-cell-autonomously. Here, we examine the Netrin family of secreted ligands as candidate Hh target genes. We find multiple Netrin ligands upregulated in the zebrafish ptch2 mutant during optic fissure development. Using a gain-of-function approach to overexpress Netrin in a spatiotemporally specific manner, we find that netrin1a or netrin1b overexpression is sufficient to cause coloboma and disrupt wild-type optic fissure formation. We used loss-of-function alleles, CRISPR/Cas9 mutagenesis, and morpholino knockdown to test if loss of Netrin can rescue coloboma in the ptch2 mutant: loss of netrin genes does not rescue the ptch2 mutant phenotype. These results suggest that Netrin is sufficient but not required to disrupt optic fissure formation downstream of overactive Hh signaling in the ptch2 mutant.

PP2A phosphatase regulatory subunit PPP2R3C is a new positive regulator of the hedgehog signaling pathway

Cellular signaling pathways rely on posttranslational modifications (PTMs) to finely regulate protein functions, particularly transcription factors. The Hedgehog (Hh) signaling cascade, crucial for embryonic development and tissue homeostasis, is susceptible to aberrations that lead to developmental anomalies and various cancers. At the core of Hh signaling are Gli proteins, whose dynamic balance between activator (GliA) and repressor (GliR) states shapes cellular outcomes. Phosphorylation, orchestrated by multiple kinases, is pivotal in regulating Gli activity. While kinases in this context have been extensively studied, the role of protein phosphatases, particularly Protein Phosphatase 2A (PP2A), remains less explored.This study unveils a novel role for the B″gamma subunit of PP2A, PPP2R3C, in Hh signaling regulation. PPP2R3C interacts with Gli proteins, and its disruption reduces Hedgehog pathway activity as measured by reduced expression of Gli1/2 and Hh target genes upon Hh signaling activation, and reduced growth of a Hh signaling-dependent medulloblastoma cell line. Moreover, we establish an antagonistic connection between PPP2R3C and MEKK1 kinase in Gli protein phosphorylation, underscoring the intricate interplay between kinases and phosphatases in Hh signaling pathway. This study sheds light on the previously understudied role of protein phosphatases in Hh signaling and provides insights into their significance in cellular regulation.

New Smoothened ligands based on the purine scaffold as potential agents for treating pancreatic cancer

Aberrant activation of the Hedgehog (Hh) signalling pathway has been associated with the development and progression of pancreatic cancer. For this reason, blockade of Hh pathway by inhibitors targeting the G protein-coupled receptor Smoothened (SMO) has been considered as a therapeutic target for the treatment of this cancer. In our previous work, we obtained a new SMO ligand based on a purine scaffold (compound I), which showed interesting antitumor activity in several cancer cell lines. In this work, we report the design and synthesis of 17 new purine derivatives, some of which showed high cytotoxic effect on Mia-PaCa-2 (Hh-dependent pancreatic cancer cell lines) and low toxicity on non-neoplastic HEK-293 cells compared with gemcitabine, such as 8f, 8g and 8h (IC50 = 4.56, 4.11 and 3.08 μM, respectively). Two of these purines also showed their ability to bind to SMO through NanoBRET assays (pKi = 5.17 for 8f and 5.01 for 8h), with higher affinities to compound I (pKi = 1.51). In addition, docking studies provided insight the purine substitution pattern is related to the affinity on SMO. Finally, studies of Hh inhibition for selected purines, using a transcriptional functional assay based on luciferase activity in NIH3T3 Shh-Light II cells, demonstrated that 8g reduced GLI activity with a IC50 = 6.4 μM as well as diminished the expression of Hh target genes in two specific Hh-dependent cell models, Med1 cells and Ptch1−/− mouse embryonic fibroblasts. Therefore, our results provide a platform for the design of SMO ligands that could be potential selective cytotoxic agents for the treatment of pancreatic cancer.

CPLANE protein INTU regulates growth and patterning of the mouse lungs through cilia-dependent Hh signaling

Congenital lung malformations are fatal at birth in their severe forms. Prevention and early intervention of these birth defects require a comprehensive understanding of the molecular mechanisms of lung development. We find that the loss of inturned (Intu), a cilia and planar polarity effector gene, severely disrupts growth and branching morphogenesis of the mouse embryonic lungs. Consistent with our previous results indicating an important role for Intu in ciliogenesis and hedgehog (Hh) signaling, we find greatly reduced number of primary cilia in both the epithelial and mesenchymal tissues of the lungs. We also find significantly reduced expression of Gli1 and Ptch1, direct targets of Hh signaling, suggesting disruption of cilia-dependent Hh signaling in Intu mutant lungs. An agonist of the Hh pathway activator, smoothened, increases Hh target gene expression and tubulogenesis in explanted wild type, but not Intu mutant, lungs, suggesting impaired Hh signaling response underlying lung morphogenetic defects in Intu mutants. Furthermore, removing both Gli2 and Intu completely abolishes branching morphogenesis of the lung, strongly supporting a mechanism by which Intu regulates lung growth and patterning through cilia-dependent Hh signaling. Moreover, a transcriptomics analysis identifies around 200 differentially expressed genes (DEGs) in Intu mutant lungs, including known Hh target genes Gli1, Ptch1/2 and Hhip. Genes involved in muscle differentiation and function are highly enriched among the DEGs, consistent with an important role of Hh signaling in airway smooth muscle differentiation. In addition, we find that the difference in gene expression between the left and right lungs diminishes in Intu mutants, suggesting an important role of Intu in asymmetrical growth and patterning of the mouse lungs.

Increased Netrin downstream of overactive Hedgehog signaling disrupts optic fissure formation.

Uveal coloboma, a developmental eye defect, is caused by failed development of the optic fissure, a ventral structure in the optic stalk and cup where axons exit the eye and vasculature enters. The Hedgehog (Hh) signaling pathway regulates optic fissure development: loss-of-function mutations in the Hh receptor ptch2 produce overactive Hh signaling and can result in coloboma. We previously proposed a model where overactive Hh signaling disrupts optic fissure formation by upregulating transcriptional targets acting both cell- and non-cell-autonomously. Here, we examine the Netrin family of secreted ligands as candidate Hh target genes. We find multiple Netrin ligands upregulated in the zebrafish ptch2 mutant during optic fissure development. Using a gain-of-function approach to overexpress Netrin in a spatiotemporally specific manner, we find that netrin1a or netrin1b overexpression is sufficient to cause coloboma and disrupt wild-type optic fissure formation. We used loss-of-function alleles, CRISPR/Cas9 mutagenesis, and morpholino knockdown to test if loss of Netrin can rescue coloboma in the ptch2 mutant: loss of netrin genes does not rescue the ptch2 mutant phenotype. These results suggest that Netrin is sufficient but not required to disrupt optic fissure formation downstream of overactive Hh signaling in the ptch2 mutant.

Variants in EFCAB7 underlie nonsyndromic postaxial polydactyly.

Polydactyly is the most common limb malformation that occurs in 1.6-10.6 per one thousand live births, with incidence varying with ancestry. The underlying gene has been identified for many of the ~100 syndromes that include polydactyly. While for the more common form, nonsydromic polydactyly, eleven candidate genes have been reported. We investigated the underlying genetic cause of autosomal recessive nonsyndromic postaxial polydactyly in four consanguineous Pakistani families. Some family members with postaxial polydactyly also present with syndactyly, camptodactyly, or clinodactyly. Analysis of the exome sequence data revealed two novel homozygous frameshift deletions in EFCAB7: [c.830delG;p.(Gly277Valfs*5)]; in three families and [c.1350_1351delGA;p.(Asn451Phefs*2)] in one family. Sanger sequencing confirmed that these variants segregated with postaxial polydactyly, i.e., family members with postaxial polydactyly were found to be homozygous while unaffected members were heterozygous or wild type. EFCAB7 displays expressions in the skeletal muscle and on the cellular level in cilia. IQCE-EFCAB7 and EVC-EVC2 are part of the heterotetramer EvC complex, which is a positive regulator of the Hedgehog (Hh) pathway, that plays a key role in limb formation. Depletion of either EFCAB7 or IQCE inhibits induction of Gli1, a direct Hh target gene. Variants in IQCE and GLI1 have been shown to cause nonsyndromic postaxial polydactyly, while variants in EVC and EVC2 underlie Ellis van Creveld and Weyers syndromes, which include postaxial polydactyly as a phenotype. This is the first report of the involvement of EFCAB7 in human disease etiology.

Targeting the Hedgehog pathway with novel Gli1 hydrophobic tagging degraders

The Hedgehog/Glioma-associated oncogene (Hh/Gli) signaling pathway plays an essential role in embryonic development and tissue homeostasis. Aberrant regulation of this pathway has been linked to various human malignancies. Gli1, the downstream transcription factor of the Hh pathway, is the ultimate effector of the canonical Hh pathway and has been identified as a common regulator of several tumorigenic pathways prevalent in Hh-independent cancers. Thus Gli1 represents a unique and promising drug target for a wide range of cancers. However, the identification and development of small molecules that directly target Gli1 protein have progressed slowly, due to an insufficient efficacy and selectivity. Herein, we developed novel small-molecule Gli1 degraders based on the hydrophobic tagging (HyT) strategy. The Gli1 HyT degrader 8e potently inhibited the proliferation of Gli1-overexpressed HT29 colorectal cancer cells, induced Gli1 degradation with a DC50 value of 5.4 μM in HT29 and achieved 70% degradation at 7.5 μM in MEFPTCH1−/− and MEFSUFU−/−cell lines, via proteasome pathway. Compared to the canonical Hh antagonist Vismodegib, 8e exhibited much stronger potency in suppressing the mRNA expression of Hh target genes in Hh-overactivated MEFPTCH1−/− and Vismodegib resistant MEFSUFU−/− cells. Our study provides small molecule Gli1 degraders effectively interfering with both canonical and noncanonical Hh signaling and overcoming current Smoothened (SMO) antagonists resistance, which might pave a new avenue for developing therapeutic modalities targeting Hh/Gli1 signaling pathway.

CEP164-GLI2 association ensures the hedgehog signaling in pancreatic cancer cells

Hedgehog (Hh) signaling is involved in multiple biological events including development and cancers. It is processed through primary cilia, which are assembled from the mother centriole in most mammalian cells. Pancreatic ductal adenocarcinoma (PDAC) cells generally lose their primary cilia; thus, the Hh signaling pathway is postulated to be independent of the organelle in PDAC. We previously reported that the mother centriole-specific protein, centrosomal protein 164 (CEP164), is required for centriolar localization of the GLI2 transcription factor in Hh signaling and for suppressing the expression of Hh-target genes. In this study, we demonstrated the physical interaction between CEP164 and GLI2, and delineated their binding modes at the mother centriole. The ectopically expressed GLI2-binding region of CEP164 reduced the centriolar GLI2 localization and enhanced the expression of Hh-target genes in PDAC cells. Furthermore, similar phenotypes were observed in PDAC cells lacking primary cilia. These results suggest that the CEP164-GLI2 association at the mother centriole is responsible for controlling Hh signaling, independent of primary cilia in PDAC cells.

131 Hedgehog Signaling Mediates Astrocytic Response and Blood-Brain Barrier Repair Following Traumatic Central Nervous System Injury

INTRODUCTION: Injury to the central nervous system (CNS) provokes a multicellular response to facilitate restoration of tissue homeostasis. Astrocytes consolidate at the lesion site to form a “glial scar” which restricts leukocyte infiltration and repairs the blood-brain barrier (BBB). However, this “scar” has been considered an impediment to regeneration and functional recovery. Therefore, uncovering molecular regulators that may be therapeutically targeted to promote pro-regenerative astrocyte phenotypes is of great interest. METHODS: Genetic reporter and conditional mutant mice were used to monitor, constitutively activate, or ablate Hh signaling following stereotactically-induced penetrating cortical or spinal cord injury. Outcomes including reactive astrocyte proliferation and polarization, leukocyte restriction, and blood-brain barrier repair were assessed via immunohistochemistry. RESULTS: Astrocytic expression of the Hh target gene Gli1 was downregulated acutely following injury but restored in the subacute phase. Constitutive activation of Hh signaling reduced astrocytic proliferation (%BrdU/EdU+ = 9.6% vs. 51.0%, p<0.01) and polarization (L/W Ratio = 2.8 vs. 1.5, p < 0.01). Additionally, in the spinal cord, constitutive activation impaired migration and integration of reactive astrocytes into the white matter lesion site. In animals with intact Hh signaling, the BBB recovered by 28 days after injury as evidenced by low levels of plasma protein and leukocyte infiltration. In contrast, conditional ablation of Hh signaling from 14-16 days after injury resulted in persistent BBB defects at 28 days. CONCLUSIONS: Our results suggest that acute downregulation of Hh signaling is required to specify reactive astrocyte properties including polarization and cell cycle re-entry. However, restoration of Hh signaling, which occurs subacutely from 10-28 days after injury and coincides with glial scar maturation, is required for leukocyte restriction and BBB repair. Understanding the temporospatial dynamics of postinjury Hh signaling is essential to modeling future therapeutic targets.

Systematic analysis of cilia characteristics and Hedgehog signaling in five immortal cell lines.

Dysfunction of the primary cilium, a microtubule-based signaling organelle, leads to genetic conditions called ciliopathies. Hedgehog (Hh) signaling is mediated by the primary cilium in vertebrates and is therefore implicated in ciliopathies; however, it is not clear which immortal cell lines are the most appropriate for modeling pathway response in human disease; therefore, we systematically evaluated Hh in five commercially available, immortal mammalian cell lines: ARPE-19, HEK293T, hTERT RPE-1, NIH/3T3, and SH-SY5Y. Under proper conditions, all of the cell lines ciliated adequately for our subsequent experiments, except for SH-SY5Y which were excluded from further analysis. hTERT RPE-1 and NIH/3T3 cells relocalized Hh pathway components Smoothened (SMO) and GPR161 and upregulated Hh target genes in response to pathway stimulation. In contrast, pathway stimulation did not induce target gene expression in ARPE-19 and HEK293T cells, despite SMO and GPR161 relocalization. These data indicate that human hTERT RPE-1 cells and murine NIH/3T3 cells, but not ARPE-19 and HEK293T cells, are suitable for modeling the role of Hh signaling in ciliopathies.

Suppressor of Fused Regulation of Hedgehog Signaling is Required for Proper Astrocyte Differentiation.

Hedgehog signaling is essential for vertebrate development; however, less is known about the negative regulators that influence this pathway. Using the mouse P19 embryonal carcinoma cell model, suppressor of fused (SUFU), a negative regulator of the Hedgehog (Hh) pathway, was investigated during retinoic acid (RA)-induced neural differentiation. We found Hh signaling increased activity in the early phase of differentiation, but was reduced during terminal differentiation of neurons and astrocytes. This early increase in pathway activity was required for neural differentiation; however, it alone was not sufficient to induce neural lineages. SUFU, which regulates signaling at the level of Gli, remained relatively unchanged during differentiation, but its loss through CRISPR-Cas9 gene editing resulted in ectopic expression of Hh target genes. Interestingly, these SUFU-deficient cells were unable to differentiate toward neural lineages without RA, and when directed toward these lineages, they showed delayed and decreased astrocyte differentiation; neuron differentiation was unaffected. Ectopic activation of Hh target genes in SUFU-deficient cells remained throughout RA-induced differentiation and this was accompanied by the loss of Gli3, despite the presence of the Gli3 message. Thus, the study indicates the proper timing and proportion of astrocyte differentiation requires SUFU, likely acting through Gli3, to reduce Hh signaling during late-stage differentiation.

Ehrlichia SLiM ligand mimetic activates Hedgehog signaling to engage a BCL-2 anti-apoptotic cellular program.

Ehrlichia chaffeensis (E. chaffeensis) has evolved eukaryotic ligand mimicry to repurpose multiple cellular signaling pathways for immune evasion. In this investigation, we demonstrate that TRP120 has a novel repetitive short linear motif (SLiM) that activates the evolutionarily conserved Hedgehog (Hh) signaling pathway to inhibit apoptosis. In silico analysis revealed that TRP120 has sequence and functional similarity with Hh ligands and a candidate Hh ligand SLiM was identified. siRNA knockdown of Hh signaling and transcriptional components significantly reduced infection. Co-immunoprecipitation and surface plasmon resonance demonstrated that rTRP120-TR interacted directly with Hh receptor Patched-2 (PTCH2). E. chaffeensis infection resulted in early upregulation of Hh transcription factor GLI-1 and regulation of Hh target genes. Moreover, soluble recombinant TRP120 (rTRP120) activated Hh and induced gene expression consistent with the eukaryotic Hh ligand. The TRP120-Hh-SLiM (NPEVLIKD) induced nuclear translocation of GLI-1 in THP-1 cells and primary human monocytes and induced a rapid and expansive activation of Hh pathway target genes. Furthermore, Hh activation was blocked by an α-TRP120-Hh-SLiM antibody. TRP120-Hh-SLiM significantly increased levels of Hh target, anti-apoptotic protein B-cell lymphoma 2 (BCL-2), and siRNA knockdown of BCL-2 dramatically inhibited infection. Blocking Hh signaling with the inhibitor Vismodegib, induced a pro-apoptotic cellular program defined by decreased mitochondria membrane potential, significant reductions in BCL-2, activation of caspase 3 and 9, and increased apoptotic cells. This study reveals a novel E. chaffeensis SLiM ligand mimetic that activates Hh signaling to maintain E. chaffeensis infection by engaging a BCL-2 anti-apoptotic cellular program.

Modulating Intramuscular Fat Infiltration by Small Molecules

Skeletal muscle has a remarkable ability to regenerate upon injury. However, during aging and in diseases such as muscular dystrophies and diabetes, muscle tissue is replaced by fibrotic scar and fat tissue in a process called fatty fibrosis. Currently, there are no known treatments to prevent fatty fibrosis or the decline in muscle mass and function. Our lab and others have shown that ectopic activation of the Hedgehog (Hh) pathway accelerated muscle regeneration after an acute injury and preserved muscle function with age and disease. In addition, our lab demonstrated that Hh is a potent anti‐adipogenic signal and that activating Hh prevents intramuscular fat formation. However, the cellular and molecular mechanisms by which Hh balances fatty fibrosis and muscle regeneration are unclear. In this project, we use small molecules as a novel approach for modulating intramuscular fat infiltration via Hh signaling: Smoothened agonist (SAG) to activate and Vismodegib to inactivate Hh. We found that SAG (HHON) successfully activates the Hh pathway while Vismodegib (HHOFF) inactivates the pathway, as detected by RT‐qPCR analysis for the expression of key Hh target gene Gli1. Excitingly, by modulating the Hh pathway, SAG and Vismodegib decreased and increased fat infiltration, respectively. We are currently evaluating the effects of SAG and Vismodegib administration on muscle regeneration and fibrosis. Future experiments will focus on using small molecules to modulate the Hh pathway at different time points after injury, as well as looking to therapeutic applications in treating fatty fibrosis in conditions such as Duchenne muscular dystrophy. Together, the results of this project will help uncover the regulatory role and dynamics of Hh signaling in intramuscular fat infiltration, ultimately providing a starting point for developing powerful new therapeutics to treat fatty fibrosis.

Blocking chondrocyte hypertrophy in conditional Evc knockout mice does not modify cartilage damage in osteoarthritis.

Chondrocytes in osteoarthritic (OA) cartilage acquire a hypertrophic-like phenotype, where Hedgehog (Hh) signaling is pivotal. Hh overexpression causes OA-like cartilage lesions, whereas its downregulation prevents articular destruction in mouse models. Mutations in EVC and EVC2genes disrupt Hh signaling, and are responsible for the Ellis-van Creveld syndrome skeletal dysplasia. Since Ellis-van Creveld syndrome protein (Evc) deletion is expected to hamper Hh target gene expression we hypothesized that it would also prevent OA progression avoiding chondrocyte hypertrophy. Our aim was to study Evc as a new therapeutic target in OA, and whether Evc deletion restrains chondrocyte hypertrophy and prevents joint damage in an Evc tamoxifen induced knockout (EvccKO ) model of OA. For this purpose, OA was induced by surgical knee destabilization in wild-type (WT) and EvccKO adult mice, and healthy WT mice were used as controls (n=10knees/group). Hypertrophic markers and Hh genes were measured by qRT-PCR, and metalloproteinases (MMP) levels assessed by western blot. Human OA chondrocytes and cartilage samples were obtained from patients undergoing knee joint replacement surgery. Cyclopamine (CPA) was used for Hh pharmacological inhibition and IL-1 beta as an inflammatory insult. Our results showed that tamoxifen induced inactivation of Evc inhibited Hh overexpression and partially prevented chondrocyte hypertrophy during OA, although it did not ameliorate cartilage damage in DMM-EvccKO mice. Hh pathway inhibition did not modify the expression of proinflammatory mediators induced by IL-1 beta in human OA chondrocytes in culture. We found that hypertrophic-IHH-and inflammatory-COX-2-markers co-localized in OA cartilage samples. We concluded that tamoxifen induced inactivation of Evc partially prevented chondrocyte hypertrophy in DMM-EvccKO mice, but it did not ameliorate cartilage damage. Overall, our results suggest that chondrocyte hypertrophy per se is not a pathogenic event in the progression of OA.

Evaluation of Hedgehog Pathway Inhibition on Nevoid Basal Cell Carcinoma Syndrome Fibroblasts and Basal Cell Carcinoma-Associated Fibroblasts: Are Vismodegib and Sonidegib Useful to Target Cancer-Prone Fibroblasts?

Simple SummaryNevoid basal cell carcinoma syndrome (NBCCS) is a genetic disorder of autosomal dominant inheritance that dramatically predisposes a patient to the formation of basal cell carcinoma (BCC) due to causative mutations in several genes associated with the Hedgehog (Hh) pathway. Somatic mutations in this pathway are also associated with sporadic BCC, the most common form of skin cancer. Hh signalling extends its effect on tumorigenesis by modulating the tumour microenvironment in a paracrine fashion. Consistently, NBCCS fibroblasts could facilitate BCC occurrence. Here, we investigated vismodegib and sonidegib, two molecules currently used to target this pathway in cancer cells, as a therapeutic option against syndromic and BCC-associated fibroblasts.Activating mutations in the Hh pathway underlies the development of sporadic and familial skin BCC. For these oncogenic proliferations displaying ligand-independent activation of the intracellular pathway, two molecules have been approved for therapeutic purposes: vismodegib and sonidegib. Improper Hh signalling occurs in many human tumours also via a paracrine mechanism (ligand-dependent) in which the secretion of Hh ligands by stromal cells support tumour growth. On the other hand, the mobilization of neoplastic stroma by cancer cells is sustained by the activation of Hh signalling in surrounding fibroblasts suggesting a central role of this bidirectional crosstalk in carcinogenesis. Additionally, loss-of-function mutations in the PTCH1 gene in the context of NBCCS, an autosomal dominant disorder predisposing to multiple BCCs, determine tumour permissive phenotypes in dermal fibroblasts. Here, profiling syndromic and BCC-associated fibroblasts unveiled an extraordinary similarity characterized by overexpression of several Hh target genes and a marked pro-inflammatory outline. Both cell types exposed to Hh inhibitors displayed reversion of the tumour-prone phenotype. Under vismodegib and sonidegib treatment, the Wnt/β-catenin pathway, frequently over-active in tumour stroma, resulted down-regulated by pAKT-GSK3β axis and consequent increase of β-catenin turnover. Overall, this study demonstrated that vismodegib and sonidegib impacting on fibroblast tumour supportive functions might be considered in therapy for BCC independently to the mutation status of Hh components in neoplastic cells.

Abstract PR-014: Hedgehog represses angiogenesis in PDAC through a paracrine cascade mediated by Wif1

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is characterized by an extremely heterogeneous tumor microenvironment (TME) paired with a sparse and compromised vascular network. These features of the TME shape drug responses and contributes to PDAC’s distinctive chemotherapeutic resistance. Prior work demonstrated that inhibition of the Hedgehog (Hh) signaling using Smoothened (Smo) inhibitors relieved some of the local angiogenic suppression. However, the precise mechanisms through which the Hh pathway regulates angiogenesis has remained elusive. Subsequent studies evaluating different timepoints yielded conflicting data on the role of Hedgehog signaling in angiogenesis. In order to replicate the heterogeneity and spatial patterning of cells in pancreatic tumors, we developed a novel tumor explant system for short-term cultures of both murine and human PDAC. Briefly, we cultured intact 300 μm slices of PDAC tissues in a modified gelatin platform system with a novel culture medium informed by the metabolic composition of PDAC tumor interstitial fluid. This system replicates some of the nutrient/waste gradient of PDAC and enables tumors to be culture up to one week with good representation of multiple cell types. Studies in explants were complemented by work in the KPC genetically engineered model of PDAC. Comparison of short versus long-term Smo inhibition in KPC mouse pancreatic tumors revealed a robust increase in vessel density in short-term treatment, which is lost at later timepoints. Studies in murine and human PDAC explants employing different Smo inhibitors demonstrated increased endothelial tip cell formation upon two days of treatment, resulting in increased vessel density after four days. Computational analyses of human PDAC single cell RNAseq data identified candidate cellular subsets potentially involved in the signaling cascade. RNAseq of pre- and on-treatment tumor samples from KPC mice identified Wif1 as a candidate Hh target gene that effected these programs. Mechanistic studies in tumor explants reveal a complex, multi-step mechanism involving multiple cell types that led to suppression of angiogenesis in PDAC. These studies demonstrate how the tumor explant platform can elucidate complex intercellular signaling cascades and provide a candidate mechanism for the Hh-mediated suppression of angiogenesis in PDAC. Citation Format: Marie C. Hasselluhn, Amanda R. Decker, Alvaro Curiel Garcia, Carlo Maurer, Dafydd Thomas, Kenneth P. Olive. Hedgehog represses angiogenesis in PDAC through a paracrine cascade mediated by Wif1 [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-014.

Gli1+ progenitors mediate bone anabolic function of teriparatide via Hh and Igf signaling.

SUMMARYTeriparatide is the most widely prescribed bone anabolic drug in the world, but its cellular targets remain incompletely defined. The Gli1+ metaphyseal mesenchymal progenitors (MMPs) are a main source for osteoblasts in postnatal growing mice, but their potential response to teriparatide is unknown. Here, by lineage tracing, we show that teriparatide stimulates both proliferation and osteoblast differentiation of MMPs. Single-cell RNA sequencing reveals heterogeneity among MMPs, including an unexpected chondrocyte-like osteoprogenitor (COP). COP expresses the highest level of Hedgehog (Hh) target genes and the insulin-like growth factor 1 receptor (Igf1r) among all cell clusters. COP also expresses Pth1r and further upregulates Igf1r upon teriparatide treatment. Inhibition of Hh signaling or deletion of Igf1r from MMPs diminishes the proliferative and osteogenic effects of teriparatide. The study therefore identifies COP as a teriparatide target wherein Hh and insulin-like growth factor (Igf) signaling are critical for the osteoanabolic response in growing mice.

A PDZ Protein GIPC3 Positively Modulates Hedgehog Signaling and Melanoma Growth

The hedgehog (Hh) pathway is essential for animal development, but aberrant activation promotes cancer growth. In this study, we show that GIPC3, a PDZ domain-containing protein with putative adaptor protein function, positively modulates Hh target gene expression in normal fibroblasts and melanoma cells and supports melanoma tumor growth. Using overexpression and epistasis studies, we show that Gipc3 potentiates Hh transcriptional output and that it modulates GLI-dependent transcription independently of Sufu. Whereas we find that GIPC3 protein does not interact with Hh pathway components, Ingenuity Pathway Analyses of GIPC3-interacting proteins identified by coimmunoprecipitation and mass spectrometry show an association with cancer pathogenesis. Subsequent interrogation of The Cancer Genome Atlas and the Human Protein Atlas databases reveals GIPC3 upregulation in many cancers. Using expression screens in selected groups of GIPC3-upregulated cancers with reported Hh pathway activation, we find a significant positive correlation of GIPC3 expression with Hh pathway components GLI1, GLI2, and GPR161 in melanoma lines. Consistently, GIPC3 knockdown in melanoma lines significantly reduces GLI1 and GLI2 expression, cell viability, colony formation, and allograft tumor growth. Our findings highlight previously unidentified roles of GIPC3 in potentiating Hh response and melanoma tumorigenesis and suggest that GIPC3 modulation on Hh signaling may be targeted to reduce melanoma growth.

Phase I/II Trial of Glasdegib in Heavily Pre-Treated Patients with Primary or Secondary Myelofibrosis

Background: Characterized by the clonal proliferation of bone marrow stem cells and myeloid progenitors, patients with myelofibrosis (MF) have hepatosplenomegaly, abnormal blood counts, bleeding or thrombosis, and suffer debilitating disease-related symptoms. In preclinical models of MF, inhibitors of the Hedgehog (Hh) pathway were shown to inhibit growth and self-renewal of bone marrow stem/progenitor cells that differentiate into cells of the myeloid lineage and to reduce splenic fibrosis. Glasdegib is a potent and selective oral inhibitor of the Hh pathway, acting through the inhibition of Smoothened. Expression of Hh target genes has been shown to be upregulated in granulocytes isolated from MF patients. Here we present recent data from the lead-in cohort of a single arm, open-label phase 1b/2 trial of glasdegib in MF.Methods: Patients ≥18 years with primary/secondary MF previously-treated with ≥1 Janus Kinase inhibitor (JAKi) were enrolled and received glasdegib 100 mg orally once daily until there was no further clinical benefit. Primary endpoints include adverse events and laboratory abnormalities, as graded by National Cancer Institute Common Terminology Criteria for Adverse Events v 4.03. Secondary endpoints include the proportion of patients with spleen volume reduction ≥35% and the proportion of patients with hematologic improvement. All 21 subjects in the lead-in cohort provided plasma samples for pharmacokinetic (PK) analysis, with 19 subjects providing evaluable glasdegib samples (at steady state) for the purposes of glasdegib PK analysis.Results: Between October 2014 and October 2015, 21 patients were enrolled in this ongoing study. Mean age was 69 years (range 58-83) and the median duration of treatment with glasdegib was 85 days (range 22-512). Of the 21 subjects, 52% (n=11) were refractory patients who had shown an inadequate response to prior JAKi treatment. A total of five patients achieved a reduction in spleen volume, with a maximum reduction from baseline of 2.3% to 32.9% (Fig A). One patient on study had a positive anemia response, showing improvement from Grade (G) 3 to G2 and became transfusion independent while on treatment. One patient also had an improvement in absolute neutrophil counts (from G2 to G0). Dysgeusia (n=13, 62%), muscle spasms (n=12, 57%), alopecia (n=8, 38%), decreased appetite, fatigue (n=7 each, 33%), increased lipase, decreased weight (n=5 each, 24%), nausea, pyrexia, hyperuricemia, anemia (n=4 each, 19%), asthenia, constipation, cough, dehydration, prolonged electrocardiogram QT, decreased lymphocyte count, myalgia, pain in extremity, thrombocytopenia, and upper respiratory tract infection (n=3 each, 14%) occurred in ≥10% of patients. Despite the high frequency of muscle spasms, there were no significant changes in creatine kinase; only one patient showing a G1 increase compared with baseline. Following administration of 100 mg daily oral dose of glasdegib for 15 days, at steady state, the peak plasma concentrations (Cmax) generally occurred at 1 hour post dose (Fig B). The geometric mean (%CV of geometric means) values for AUCtau and Cmax were 13150 ng.hr/mL (50%) and 996.8 ng/mL (45%), respectively (Fig C). The pre-dose concentrations (Ctrough) following daily dosing were consistent over multiple cycles, with geometric mean Ctrough values of 204.1 ng/mL, 176.1 ng/mL and 189.5 ng/mL respectively, on Cycle 1 day 15, Cycle 2 day 1 and Cycle 3 day 1.Conclusions: Although some toxicities were observed, these data highlight the potential for glasdegib in patients with primary/secondary MF previously treated with JAKi. Following oral administration of 100 mg oral glasdegib once daily to steady state, peak plasma concentrations generally occurred at 1 hour post dose and glasdegib exposures were consistent over multiple cycles of dosing. Modest clinical activity was seen in some patients. [Display omitted] DisclosuresGerds:CTI BioPharma: Consultancy; Incyte: Consultancy. Tauchi:Pfizer Inc: Research Funding. Ritchie:Novartis: Consultancy, Research Funding; Incyte: Honoraria; Celgene: Speakers Bureau; Pfizer Inc: Honoraria. Deininger:Gilead: Research Funding; ARIAD: Consultancy; Ariad Pharmaceuticals, Bristol Myers Squibb, CTI BioPharma Corp, Gilead, Incyte, Novartis, Pfizer, Celgene, Blue Print, Galena: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy; Pfizer: Consultancy; BMS: Consultancy, Research Funding; Celgene: Research Funding; Novartis: Consultancy, Research Funding. Jamieson:Johnson and Johnson: Research Funding; GlaxoSmithKline: Research Funding; Celgene: Research Funding; Wintherix: Equity Ownership; Impact Biomedicines: Equity Ownership. Mesa:Novartis: Consultancy; Ariad: Consultancy; Galena: Consultancy; Incyte: Research Funding; Gilead: Research Funding; CTI: Research Funding; Promedior: Research Funding; Celgene: Research Funding. Heaney:Onconova Therapeutics: Research Funding; Novartis: Consultancy. Shaik:Pfizer Inc: Employment. Zhang:Pfizer Inc: Employment. DiRienzo:Pfizer Inc: Employment. Zeremski:Pfizer Inc: Employment. Chan:Pfizer Inc: Employment. Talpaz:Pfizer Inc: Consultancy, Other: Travel, Research Funding; ARIAD: Other: Travel, Research Funding.

Constitutive Hedgehog/GLI2 signaling drives extracutaneous basaloid squamous cell carcinoma development and bone remodeling.

Uncontrolled activation of the Hedgehog (Hh) signaling pathway, operating through GLI transcription factors, plays a central role in the pathogenesis of cutaneous basal cell carcinoma and contributes to the development of several malignancies arising in extracutaneous sites. We now report that K5-tTA;tetO-Gli2 bitransgenic mice develop distinctive epithelial tumors within their jaws. These tumors consist of large masses of highly proliferative, monomorphous, basaloid cells with scattered foci of keratinization and central necrosis, mimicking human basaloid squamous cell carcinoma (BSCC), an aggressive upper aerodigestive tract tumor. Like human BSCC, these tumors express epidermal basal keratins and differentiation-specific keratins within squamous foci. Mouse BSCCs express high levels of Gli2 and Hh target genes, including Gli1 and Ptch1, which we show are also upregulated in a subset of human BSCCs. Mouse BSCCs appear to arise from distinct epithelial sites, including the gingival junctional epithelium and epithelial rests of Malassez, a proposed stem cell compartment. Although Gli2 transgene expression is restricted to epithelial cells, we also detect striking alterations in bone adjacent to BSCCs, with activated osteoblasts, osteoclasts and osteal macrophages, indicative of active bone remodeling. Gli2 transgene inactivation resulted in rapid BSCC regression and reversal of the bone remodeling phenotype. This first-reported mouse model of BSCC supports the concept that uncontrolled Hh signaling plays a central role in the pathogenesis of a subset of human BSCCs, points to Hh/GLI2 signaling as a potential therapeutic target and provides a powerful new tool for probing the mechanistic underpinnings of tumor-associated bone remodeling.