Smoothened Inhibitor Research Articles

The Hedgehog/GLI signaling pathway activates transcription of Slug (Snail2) in melanoma cells.

In melanoma and other cancers, invasion, epithelial-to-mesenchymal transition, metastasis and cancer stem cell maintenance are regulated by transcription factors including the Snail family. Slug (Snail2) protein generally supports migration and apoptosis resistance. However, its role in melanoma is not completely understood. The present study investigated the transcriptional regulation of the SLUG gene in melanoma. It demonstrated that SLUG is under the control of the Hedgehog/GLI signaling pathway and is activated predominantly by the transcription factor GLI2. The SLUG gene promoter contains a high number of GLI-binding sites. Slug expression is activated by GLI factors in reporter assays and inhibited by GANT61 (GLI inhibitor) and cyclopamine (SMO inhibitor). SLUG mRNA levels are lowered by GANT61 as assessed by reverse transcription-quantitative PCR. Chromatin immunoprecipitation revealed abundant binding of factors GLI1-3 in the four subregions of the proximal SLUG promoter. Notably, melanoma-associated transcription factor (MITF) is an imperfect activator of the SLUG promoter in reporter assays, and downregulation of MITF had no effect on endogenous Slug protein levels. Immunohistochemical analysis confirmed the above findings and showed MITF-negative regions in metastatic melanoma that were positive for GLI2 and Slug. Taken together, the results demonstrated a previously unrecognized transcriptional activation mechanism of the SLUG gene, which may represent its main regulation of expression in melanoma cells.

Novel Approach to the Hedgehog Signaling Pathway: Combined Treatment of SMO and PTCH Inhibitors

Purpose: Abnormal Sonic Hedgehog signaling Pathway (Shh) activation is crucial for development of cancer stem cells, neoplastic growth and epithelial-mesenchymal transition processes in adulthood. Activation of Hedgehog signaling pathway may induces the changes in cilia found in the cell membrane, iniciates the Gli1 transcription factor that is translocated to the cell nucleus and finally, the target genes are transcribed. In this study, invastigation of the antiproliferative, anti-invasive and antimigrative effect of the combined use of robotnikinin (Ptch1 antagonist) and vismodegib (Smo inhibitor) on the hedgehog signaling pathway was aimed. 
 
 Material and Methods: After demonstarting the presence of the hedgehog signaling pathway in the glioblastoma cell line U87-MG, the effect of the combined use of the robotnikinin and the vismodegib on the hedgehog signaling pathway was investigated. In-vitro cell proliferation, migration, and invasion analysis of the combination of antagonist and inhibitor and in silico drug-likeness analysis were performed.
 
 Results: Two different combinations of robotnikinin and vismodegib were tested. In vitro studies show that the combined use of agents in combined treatments of Smo and Ptch1is more effective than their individual usage.
 
 Conclusion: Inhibition of the hedgehog signaling pathway with specific inhibitors and antagonists is considered an innovative strategy for cancer therapy.

Targeting the Hedgehog Pathway in Rhabdomyosarcoma.

Aberrant activation of the Hedgehog (Hh) signalling pathway is known to play an oncogenic role in a wide range of cancers; in the particular case of rhabdomyosarcoma, this pathway has been demonstrated to be an important player for both oncogenesis and cancer progression. In this review, after a brief description of the pathway and the characteristics of its molecular components, we describe, in detail, the main activation mechanisms that have been found in cancer, including ligand-dependent, ligand-independent and non-canonical activation. In this context, the most studied inhibitors, i.e., SMO inhibitors, have shown encouraging results for the treatment of basal cell carcinoma and medulloblastoma, both tumour types often associated with mutations that lead to the activation of the pathway. Conversely, SMO inhibitors have not fulfilled expectations in tumours-among them sarcomas-mostly associated with ligand-dependent Hh pathway activation. Despite the controversy existing regarding the results obtained with SMO inhibitors in these types of tumours, several compounds have been (or are currently being) evaluated in sarcoma patients. Finally, we discuss some of the reasons that could explain why, in some cases, encouraging preclinical data turned into disappointing results in the clinical setting.

Combined Inhibition of Smoothened and the DNA Damage Checkpoint WEE1 Exerts Antitumor Activity in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is characterized by resistance to chemotherapy and a poor prognosis. Therefore, treatments that can effectively suppress tumor growth are urgently needed. Aberrant activation of hedgehog (HH) signaling has been implicated in several cancers, including those of the hepatobiliary tract. However, the role of HH signaling in intrahepatic CCA (iCCA) has not been completely elucidated. In this study, we addressed the function of the main transducer Smoothened (SMO) and the transcription factors (TFs) GLI1 and GLI2 in iCCA. In addition, we evaluated the potential benefits of the combined inhibition of SMO and the DNA damage kinase WEE1. Transcriptomic analysis of 152 human iCCA samples showed increased expression of GLI1, GLI2, and Patched 1 (PTCH1) in tumor tissues compared with nontumor tissues. Genetic silencing of SMO, GLI1, and GLI2 inhibited the growth, survival, invasiveness, and self-renewal of iCCA cells. Pharmacologic inhibition of SMO reduced iCCA growth and viability in vitro, by inducing double-strand break DNA damage, leading to mitotic arrest and apoptotic cell death. Importantly, SMO inhibition resulted in the activation of the G2-M checkpoint and DNA damage kinase WEE1, increasing the vulnerability to WEE1 inhibition. Hence, the combination of MRT-92 with the WEE1 inhibitor AZD-1775 showed increased antitumor activity in vitro and in iCCA xenografts compared with single treatments. These data indicate that combined inhibition of SMO and WEE1 reduces tumor burden and may represent a strategy for the clinical development of novel therapeutic approaches in iCCA.

A Potent Antagonist of Smoothened in Hedgehog Signaling for Epilepsy.

Epilepsy is one of the common encephalopathies caused by sudden abnormal discharges of neurons in the brain. About 30% of patients with epilepsy are insensitive and refractory to existing antiseizure medications. The sonic hedgehog signaling pathway is essential to the development and homeostasis of brain. Aberrant sonic hedgehog signaling is increased in refractory epileptic lesions and may involve the etiology of epilepsy. Thus, new inhibitors of Smoothened, a key signal transducer of this signaling pathway are urgently need for refractory epilepsy. We have established a high-throughput screening platform and discovered several active small molecules targeting Smoothened including TT22. Here we show that the novel Smoothened inhibitor TT22 could block the translocation of βarrestin2-GFP to Smoothened, reduce the accumulation of Smoothened on primary cilia, displace Bodipy-cyclopamine binding to Smoothened, and inhibit the expression of downstream Gli transcription factor. Moreover, TT22 inhibits the abnormal seizure-like activity in neurons. Furthermore, we demonstrated that FDA-approved Smoothened inhibitor GDC-0449 and LDE-225 are able to inhibit abnormal seizure-like activity in neurons. Thus, our study suggests that targeting the sonic hedgehog signaling with new small-molecule Smoothened inhibitors might provide a potential new therapeutic avenue for refractory epilepsy.

De novo design of dual-target JAK2, SMO inhibitors based on deep reinforcement learning, molecular docking and molecular dynamics simulations

Triple-negative breast cancer (TNBC) and HER2-positive breast cancer are particularly aggressive and the effectiveness of current therapies for them is limited. TNBC lacks effective therapies and HER2-positive cancer is often resistant to HER2-targeted drugs after an initial response. The recent studies have demonstrated that the combination of JAK2 inhibitors and SMO inhibitors can effectively inhibit the growth and metastasis of TNBC and HER2-positive drug resistant breast cancer cells. In this study, deep reinforcement learning was used to learn the characteristics of existing small molecule inhibitors of JAK2 and SMO, and to generate a novel library of small molecule compounds that may be able to inhibit both JAK2 and SMO. Subsequently, the molecule library was screened by molecular docking and a total of 7 compounds were selected out as dual inhibitors of JAK2 and SMO. Molecular dynamics simulations and binding free energies showed that the top three compounds stably bound to both JAK2 and SMO proteins. The binding free energies and hydrogen bond occupancy of key amino acids indicate that A8976 and A10625 has good properties and could be a potential dual-target inhibitor of JAK2 and SMO.

Abstract C032: Combination CAF/myeloid targeting in PDAC

Abstract Immunotherapy has revolutionized clinical care for many cancers, yet these treatments fail to control disease in many patients and new strategies are needed to improve anti-tumor immunity and enhance response rates. There is great need for an increased understanding of the cellular crosstalk within tumors and the identification of stromal and immune populations involved in shaping the tumor microenvironment (TME). Local immunosuppression (LIS) is one of the striking hallmarks of Pancreatic Ductal Adenocarcinoma (PDAC), a disease that is highly resistant to existing immunotherapies. Oncogenic Kras activation in tumor cells promotes the invasion and proliferation of tumor-supporting stromal cells, while excluding cancer-targeted cytotoxic T cells. LIS is mediated by multiple subtypes of cancer-associated fibroblasts (CAFs) and myeloid cells resident within the tumor parenchyma. Multiple prior attempts to reverse LIS in PDAC by targeting individual stromal cell populations have been unsuccessful, alluding to the complexity of stromal crosstalk within the TME. The stromal diversity of PDAC complicates investigating paracrine cascades involving multiple cell types. To decipher diverse drug effects on altering the TME, we employ in vivo studies in mouse models recapitulating the human disease, as well as a novel tumor explant model that enables the short-term culture of intact human or murine PDAC. Importantly, PDAC explants maintain their histopathological architecture and cellular diversity over time. This medium-throughput platform allows for testing of multiple drugs and mechanistic hypotheses in the native PDAC TME. We show in preliminary data that Smoothened inhibition (SMOi) decreases proliferation and activity of myCAFs, but provokes the expansion of CD11b-positive myeloid cells in vivo. Thus, we hypothesize that LIS in PDAC is maintained by a delicate balance between myCAFs and myeloid cells, preventing effective T cell invasion. Single cell RNA-seq data comparing ctrl vs. SMOi-treated murine PDAC elucidates stromal subpopulations involved in the LIS phenotype and guides the identification of myeloid subtypes emerging after SMOi. Strikingly, we demonstrated that simultaneous SMOi and targeting myeloid cells via anti-Gr1 or CCR1 inhibition (CCR1i) significantly elevates cytotoxic T cell numbers within the TME. We are currently investigating whether the activity of these T cells may be further potentiated through combination with immunomodulatory agents. By testing various treatment combination in the same TME, we will identify the best synergistic effects for future immunotherapy approaches in human PDAC. In summary, we are elucidating the complex mechanism behind LIS in PDAC by employing our novel explant culture system alongside in vivo studies. We aim to develop a translatable regimen to neutralize LIS, reactivating the cytotoxic T cells in the tumor periphery to invade, proliferate, and attack cancer cells. Citation Format: Marie C. Hasselluhn, Lukas J. Vlahos, Dafydd Thomas, Alvaro Curiel Garcia, Amanda R. Decker, Tanner C. Dalton, Stephen A. Sastra, Carmine F. Palermo, Andrea Califano, Kenneth P. Olive. Combination CAF/myeloid targeting in PDAC [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C032.

EXTH-78. CRISPR/CAS9 KNOCKOUT SCREENS UNRAVEL EPIGENETIC REGULATORS AS DRUGGABLE DEPENDENCIES FOR SONIC HEDGEHOG MEDULLOBLASTOMA

Abstract Sonic hedgehog medulloblastoma (SHH-MB) is a malignant, highly heterogeneous brain tumor entity, accounting for 30% of all MBs in the pediatric population and is characterized by aberrant activation of the canonical SHH signaling pathway. Although current therapies targeting Smoothened (Smo) have proven a promising treatment approach for SHH-MB patients, pre-existing or acquired resistance impedes its clinical efficacy. Therefore, novel targeted approaches that overcome mechanisms of resistance are urgently needed. Here, we performed a genome-wide CRISPR/Cas9 knockout screen in a murine and a human SHH-MB cell line, SMB21 and DAOY, respectively, in order to decipher tumor-specific genetic essentialities. Our data demonstrate that SMB21 cells highly depend on key mediators of the SHH pathway, such as Gli2) for their proliferation, as opposed to DAOY cells, suggesting that the latter does not represent a faithful model of SHH-MB. Among other dependencies for SHH-MB are members of the epigenetic machinery such as Dnmt1) and Smarca5). Pharmacologically, we show that DNMT1 inhibition is efficacious at clinically relevant concentrations against Smo inhibitor- sensitive, as well as resistant SHH-MB cell lines. By performing RNA sequencing of SMB21 cells, we identified early and late alterations in global gene expression induced by DNMT1 inhibition, including decreased expression of positive regulators of SHH signaling. An additional knockout drug screen in SMB21 cells unraveled synthetic lethal interactors for DNMT1 inhibitors, as validated in vitro) by drug combination treatments. Further global DNA methylation profiling in SMB cells will help to define the molecular basis of sensitivity to DNMT1 inhibitors in SHH-MB. Last but not least, genetic ablation of epigenetic regulators and combinatorial treatments using DNMT1 inhibition will be investigated in in vivo) established mouse models of SHH-MB. Summarizing, our data indicate the potential of inhibiting epigenetic regulators as novel therapeutic avenues in SHH-MB sensitive, as well as resistant to Smo inhibition.

DNAR-13. SONIDEGIB AND GANT61 INDUCE DNA DAMAGE IN PEDIATRIC SONIC HEDGEHOG MEDULLOBLASTOMA

Abstract Medulloblastoma is the most common malignant brain tumor in children, accounting for 20-25% of all pediatric brain tumors. The current standard of care includes both craniospinal and focal radiation therapy which leads to long term consequences in the pediatric population such as bone and tissue hypoplasia, decreased neurocognitive functioning, as well as an increased risk for secondary radiation induced cancers. Thus, there is a need for novel approaches to radiation therapy that can effectively target and kill tumor cells while minimizing radiation toxicity in healthy tissue in the pediatric patient population. The dysregulation and constitutive activation of the Sonic Hedgehog (SHH) signaling pathway accounts for ~30% of all medulloblastomas. In addition to playing a role in tumorigenesis, the SHH pathway is known to play a role in the radiation sensitivity and risk of tumor recurrence. We hypothesize that inhibiting the SHH pathway at Smoothened (Smo) or Gli1 will increase DNA damage, resulting in increased cell killing following radiation therapy. We found that treatment of medulloblastoma cells with the drug Sonidegib or GANT61 decreased the expression of the respective targets Smo and Gli1. Both drugs also significantly decreased expression of FANCD2, a DNA damage repair gene essential for homologous recombination. Pretreatment with Sonidegib or GANT61 resulted functionally with decreased tumor cell viability and self-renewal following radiation. These data suggest that Smo and Gli1 inhibition are promising targets for increasing the radiation sensitivity of pediatric SHH medulloblastoma.

MODL-33. AN ANIMAL MODEL OF GLI2-AMPLIFIED MEDULLOBLASTOMA

Abstract Medulloblastoma (MB) is the most common malignant brain tumor affecting children. Among the known subtypes of MB, GLI2-amplified SHH-MB associated with P53 mutations has the worst prognosis, and a 5-year survival rate of less than 30%. Moreover, GLI2-amplified MBs are non-responsive to SMO inhibitors, the only targeted treatment option available for SHH-MB. To address this treatment gap, there is an urgent need to identify novel potential therapeutic targets, an effort that requires better understanding of key cellular and molecular mechanisms underlying GLI2-amplified MB. To that end, we generated a mouse model of GLI2-amplified MB and identified GL12 as the critical driver of tumorigenesis, and granule cell progenitors (GCPs) as the cells of tumor origin. We further found that GLI2 specifically drives embryonic, but not neonatal, Math1+ GCPs to form SHH-MB tumors. An scRNA-seq analysis revealed MAPK pathway activation specifically in embryonic Math1+ GCPs. The MAPK pathway was activated in mouse and human GLI2-driven MB tumors, and a MEK/ERK inhibitor significantly inhibited GLI2-driven MB in vivo. Based on these findings, we hypothesize that GLI2-driven MB originates from a specific population of Math1+ GCPs during a discrete temporal interval in cerebellar development, and that targeting MAPK/MEK/ERK pathway components represents a novel approach to treatment of GLI2-amplified MB.

RF01 | PMON168 Hedgehog-Induced Somatostatin Receptor Expression is Inhibited by the Activation of the Glucocorticoid Receptor Signaling Pathway in Human Pituitary Adenoma Organoids

Abstract Background Cushing's disease (CD) is an endocrine disorder caused by an ACTH-secreting pituitary adenoma that is associated with increased morbidity and mortality. Because somatostatin inhibits pituitary ACTH secretion, the somatostatin receptor subtypes (SSTR) 2 and 5 have been targets of medical therapies for CD. The SSTR5 is most consistently and strongly expressed in corticotroph pituitary tumors but the SSTR dynamics in CD during treatment and responses to cortisol level changes are far from being understood. Studies in adult stomach demonstrated that the Hedgehog (Hh) signaling pathway is critical for the regulation of somatostatin and SSTR signaling. While Hh signaling is known to be essential during the embryonic development of the pituitary and in the adult gland, the mechanism by which Hh signaling regulates SSTR expression in CD is unknown. Hypothesis Activation of glucocorticoid receptor (NR3C1, GR) results in the inhibition of Hh transcription factor GLI1 leading to reduced SSTR expression. Methods We developed a human pituitary adenoma organoid model (hPITOiPSC) from induced pluripotent stem cells treated with the glucocorticoid receptor (GR) antagonist mifepristone, and co-treated them with or without SSTR agonists pasireotide or octreotide. In a separate series of experiments, the role of Hh transcription factor GLI1 was identified using hPITOiPSC treated with mifepristone with or without GANT61 (GLI inhibitor) or ketoconazole (Smoothened, SMO inhibitor). CRISPR-Cas9 gene editing of hPITOiPSC pituitary organoids were used to model the development of pituitary corticotroph adenomas in the presence of BRAF, USP48 and USP8 mutations. Human pituitary adenoma tissue harvested fresh during pituitary surgery was used to generate pituitary corticotroph subtype adenoma organoids (hPITOs). Dose responses using standard of care drugs were performed using the hPITOs. Results 1) At baseline SSTR2 was abundantly expressed within hPITOiPSC. Mifepristone induced significant increases in ACTH secretion and POMC expression that correlated with induced SSTR2 and 5. Mifepristone-induced SSTR2 and 5 expression was significantly inhibited in the presence of GANT61, whereas SMO inhibitor ketoconazole had a minimal effect on mifepristone-induced SSTR2/5, POMC expression and ACTH secretion. Dexamethasone alone significantly inhibited both GLI1 and SSTR2 and 5. 2) While pituitary organoids that were differentiated from control iPSCs (iPSCCtrl) expressed all major hormone-producing cell lineages, there was a significant increase in ACTH expression with loss of PIT1, GH, FSH, LH and PRL in iPCSs expressing mutated BRAF, USP48 and USP8. Organoids expressing a mutation in BRAF had significantly higher SSTR2 expression levels compared to controls. 3) HPITOs generated from pituitary adenomas of CD patients showed differential organoid responses to pasireotide, mifepristone, and ketoconazole. Conclusion Within pituitary adenomas, the SSTR is a transcriptional target of Gli1, and this response is blocked by the activation of the GR. These data form the basis of combination therapy for CD with mifepristone and pasireotide. Presentation: Saturday, June 11, 2022 1:37 p.m. - 1:42 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Vismodegib, a sonic hedgehog signalling blockade, ameliorates ovalbumin and ovalbumin/lipopolysaccharide-induced airway inflammation and asthma phenotypical models

AimsAsthmatics exhibit clinical fluctuations between manageable and treatment-resistant phenotypes as a worldwide socioeconomic health burden. Sonic Hedgehog (Shh) genes mediate regulatory pulmonary cell renewal in adults and contribute to the pathogenesis of high phenotypic asthma which depends mainly on T helper-2 (Th-2) cells and related cytokines. However, the exact pathophysiological roles of Shh molecular signalling in the Th-17-dependent low phenotypic allergic airway inflammation and asthma are not evidenced previously. Main methodsOvalbumin (OVA) and OVA/lipopolysaccharide (LPS)-sensitized and challenged BALB/c mice were enrolled currently to assess the Shh signalling proteins. Furthermore, the effects of vismodegib, a Smo inhibitor, on the modulation of Shh signalling were compared to dexamethasone. The asthma phenotypes were confirmed by serum total immunoglobulin-E (IgE), bronchoalveolar lavage (BAL) fluid white blood cell counts, lung interleukins, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β1, and histopathological changes, and scoring. Key findingsMice challenged with OVA or OVA/LPS showed upregulated lung Shh, patched (Ptch1), smoothened (Smo), and Gli1 proteins. Vismodegib in the two experimental phenotypes of asthma showed reduced airway inflammation and remodelling. Additionally, vismodegib reduced the eosinophilia and neutrophilia reported in high and low asthma types, respectively. Moreover, vismodegib and dexamethasone exhibited negative feedback control throughout the enhanced Shh signalling cascades, including Shh, Ptch1, and Gli1 in several asthma models. SignificanceIn conclusion, Shh signalling partially elucidates the OVA/LPS-challenged mice with severe asthma, which proposes a new promising molecular therapeutic target. Furthermore, Smo inhibition by vismodegib has therapeutic potential in both experimental eosinophilic and neutrophilic allergic airway diseases.

What's New in Musculoskeletal Basic Science.

What's New in Musculoskeletal Basic Science.

Design, synthesis and biological evaluation of novel 4-aminopiperidine derivatives as SMO/ERK dual inhibitors

The hedgehog (Hh) pathway is tightly related with the formation, metastasis and recurrence of various cancers, which makes it a perfect anticancer target. Smoothened (SMO) is one of its key members. Three drugs targeting the Hh pathway have been successfully used in clinic, and they are all known as SMO inhibitors. However, serious drug resistant problem has limited their clinical application. The interaction of oncogenic ERK pathway with the Hh pathway in multiple ways has been proved as one of the main factors that result in drug resistance. Dual inhibition of the Hh and ERK pathways has displayed synergistic suppression to cancer cells overexpressing both pathways. Herein, we designed and synthesized a series of novel 4-aminopiperidine derivatives as SMO/ERK dual inhibitors, and evaluated their biological activities. The results showed that compounds I-13 displayed strong inhibitory activities towards both SMO and ERK, and it also exhibited significant cytotoxicity against human cholangiocarcinoma RBE cells which overexpress both the Hh and ERK pathways. All the results indicate that compound I-13 is a promising anticancer candidate as a SMO/ERK dual inhibitor.

Towards Precision Oncology: The Role of Smoothened and Its Variants in Cancer.

The G protein-coupled receptor Smoothened (Smo) is a central signal transducer of the Hedgehog (Hh) pathway which has been linked to diverse forms of tumours. Stimulated by advancements in structural and functional characterisation, the Smo receptor has been recognised as an important therapeutic target in Hh-driven cancers, and several Smo inhibitors have now been approved for cancer therapy. This receptor is also known to be an oncoprotein itself and its gain-of-function variants have been associated with skin, brain, and liver cancers. According to the COSMIC database, oncogenic mutations of Smo have been identified in various other tumours, although their oncogenic effect remains unknown in these tissues. Drug resistance is a common challenge in cancer therapies targeting Smo, and data analysis shows that healthy individuals also harbour resistance mutations. Based on the importance of Smo in cancer progression and the high incidence of resistance towards Smo inhibitors, this review suggests that detection of Smo variants through tumour profiling could lead to increased precision and improved outcomes of anti-cancer treatments.

28P SHH pathway-targeted therapy in a relapsed medulloblastoma

Recurrent medulloblastoma is a deleterious disease with a 5-year survival of less than 5% and no recognized standard treatment. In recent years, we have learned about the molecular characterization of the different types of medulloblastoma with four subgroups also appearing different subgroups, each with its oncogenic pathway activation. SHH activation is the hallmark of medulloblastoma group 2 and several molecules have been developed to block this signaling mechanism, including SMO inhibitors, that are approved for the treatment of basal cell carcinoma.

Sonic Hedgehog signaling pathway is expressed in normal human adrenal cortices and up-regulated in adrenocortical carcinomas

The Sonic Hedgehog (Shh) signaling pathway is involved in mouse adrenal growth and differentiation. However, it remains unclear whether this pathway plays a similar role in human especially because we previously showed that this organ displays a significant capacity for tissue regeneration from differentiated cells. The expression of the components of the pathway was examined in 38 normal adult adrenal glands and 23 adrenocortical carcinomas (ACCs). We found that Shh is widely expressed in steroidogenic cells. Shh-positive cells also expressed the Shh receptor Patched-1 as well as the receptor-like signal transducer Smoothened. Moreover, expression of the transcription factors Gli1, Gli2 and Gli3 was detected in the nucleus of the steroidogenic cells. Our data identified Shh pathway as a new autocrine/paracrine loop in human steroidogenic cells. However, we cannot exclude that a subpopulation of cells may correspond to stem/progenitor cells since non-steroidogenic cells located in the capsule express components of the Shh pathway. ACCs exhibited an increased expression of all components of Shh pathway with the exception of Gli2 indicating a possible involvement in the tumorigenic process. In addition, the Smoothened inhibitor NVP-LDE225 potently inhibited tumor growth and reduced cortisol and aldosterone secretion by H295R cells. We also showed that LDE225 acts by targeting mitochondria leading to a decrease in transmembrane potential and an increase in ROS production. Collectively, our findings show that Shh pathway is expressed in normal human steroidogenic cells and is activated in carcinomas. Shh may be a promising target for the treatment of this aggressive cancer.

ABT-737 suppresses aberrant Hedgehog pathway and overcomes resistance to smoothened antagonists by blocking Gli.

Abnormally activated Hedgehog (Hh) pathway has been linked to multiple types of cancers including medulloblastoma (MB). Current Hh-targeted drug development projects mainly focus on antagonizing the upstream oncoprotein Smoothened (Smo). However, the effectiveness of Smo inhibitors is compromised by primary and acquired resistance, which is caused by mutations of Smo or other downstream components. Here, we conducted a cellular screening of small-molecule compounds and identified ABT-737 as a selective Hh inhibitor resulting in active suppression of human Hh-dependent MB cells. Mechanistically, ABT-737 suppressed Hh signals far-downstream of Smo and Sufu at Gli transcriptional effector level. In line with this, ABT-737 potentially inhibited wild-type and drug-resistant mutant Smo. More importantly, ABT-737 also delayed the growth of drug-refractory Hh-dependent MB xenografts derived from genetically engineered mouse model in vivo. These findings identify ABT-737 as a therapeutical substance for cancers with excessive Hh signaling activity, especially for those with primary or acquired resistance to Smo inhibitors in clinic.

32283 A phase I study of CX-4945 administered orally twice daily to patients with advanced basal cell carcinoma

Background: Smoothened inhibitors (SMOi) targeting the hedgehog (Hh) pathway have been approved for the treatment of patients with locally advanced basal cell carcinoma (laBCC) or metastatic basal cell carcinoma (mBCC). Unfortunately, resistance against SMOi can develop. Targeting the signaling cascade downstream of SMO could obviate this issue. Casein kinase 2 (CK2) affects the terminal component of the Hh signaling pathway by promoting Gli2 stability and Gli2’s interaction with target genes. Given the interplay between CK2 and Gli-2, CX-4945 may provide benefit for BCC patients with resistance or intolerance to SMOi. Methods: A phase I trial to explore various treatment durations of CX-4945 was designed for patients with laBCC or mBCC; with endpoints including safety and objective response rate. Major eligibility criteria include progression after SMOi. The first phase of the trial uses a 3+3 design to test the tolerance of a CX-4945 dose of 1000 mg BID for a duration of 28 days continuously. Upon determining the RP2D, a dose-expansion phase will further evaluate CX-4945 in 10 patients with laBCC. Results: As of September 1, 2021, 15 patients with BCC were enrolled. The most common grade 3 treatment-related adverse event was GI related, diarrhea (13%). No grade 4 events were reported. Among the 13 patients who were evaluable for response at end of cycle 2, 2 patients (15%) demonstrated a partial response. The expansion phase is currently enrolling patients. Complete safety and efficacy data will be presented after completion of phase 1.

495TiP A phase II study evaluating the safety and efficacy of ENV-101 (taladegib) in patients with advanced solid tumors harboring PTCH1 loss of function mutations

ENV-101 is a Hh pathway inhibitor (Smo inhibitor) originally developed as a treatment for patients with advanced solid tumors. Clinical responses have been reported in patients with gene mutations affecting PTCH1 treated with other Hh pathway inhibitors. Endeavor is studying ENV-101 in advanced cancer patients with solid tumors harboring PTCH1 loss of function mutations. This study aims to evaluate dose as well as the efficacy and safety of ENV-101, a potent Hh pathway inhibitor, in patients with refractory advanced solid tumors characterized by loss of function mutations in the PTCH1 gene.