GLI Inhibitor Research Articles

Abstract B022: Tumoral GLI1 controls the resident innate and adaptive tumor immune microenvironment in triple negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) accounts for 15-20% of breast cancer (BC) cases but due to its aggressive nature and the lack of targeted treatment options, represents a disproportionately higher number of BC-related deaths. Compared to other BC subtypes, TNBCs are more inflamed with M2-like tumor-associated macrophages (TAMs) and cytotoxic T-lymphocytes (CTLs) which are associated with poor and good prognosis, respectively. Clinical trials have proven that immunogenic cancers can be effectively treated through immune checkpoint blockade (ICB), a treatment strategy that prevents cancer cells from silencing CTL-mediated attack. However, CTLs can be suppressed by M2-like TAMs which can limit ICB efficacy and support tumor growth. The hedgehog (HH) signaling pathway is highly activated in TNBC and higher expression of GLI1 is associated with worse overall survival. Recent pan-cancer analyses highlight a significant correlation between activated HH signaling and characteristics of immune evasion. We aim to clarify how activated HH signaling regulates resident innate and adaptive immune cells in the tumor microenvironment (TME) in TNBC. Methods: Primary K14-cre; Brca1 fl/fl ; P53 fl/fl (KBP) murine TNBC cells were transfected with CRISPR/Cas9 + sgRNA targeting GLI1. After isolating single cell clones and confirming GLI1 knockout (KO), KBP and KBP-GLI1-KO cells were orthotopically injected into the mammary fat pad of syngeneic immune-competent female mice and allowed to grow for 6-8 weeks. Tumor volume was measured once tumors were palpable, and tumors were harvested once the largest tumor reached ethical endpoint. Tumors were weighed at harvest and fluorescence activated cell sorting (FACS) was performed to determine if there was an effect on the composition of immune populations. Results: At harvest, KBP-GLI1-KO tumors were delayed in growth and significantly smaller than KBP tumors. FACS analysis showed that GLI1-KO tumors had increased CD4+ and CD8+ T cells and a reduced amount of Foxp3+ CD4+ Tregs. Furthermore, GLI1-KO tumors had significantly fewer F4/80+ CD11b+ TAMs within the TME. Amongst TAM populations, there was reduced CD206+ M2-like TAMs and an increase in CD80+ M1-like TAMs upon loss of GLI1. Similar trends were observed when KBP allografts were treated with the GLI1 inhibitor, GANT61. Immunohistochemical and RNAseq analysis are ongoing. Similar experiments with other KBP-GLI1-KO clones are currently being done. Conclusion: Here, we show that tumoral GLI1 not only promotes tumor growth, but also supports an immunosuppressive TME in TNBC. Abolishing GLI1 converted tumors to a pro-inflammatory phenotype marked by an increase in CD4+ and CD8+ T cells and a reduction in CD206+ M2-like TAMs. Future experiments are aimed at revealing the mechanisms by which GLI1 regulates immune cell recruitment to the TME and testing the effect of combination immunotherapy. Overall, these preliminary findings identify GLI1 as a potential therapeutic target to limit tumor growth and promote anti-tumor immunity. Citation Format: Aidan J Gray, Wanda Marini, Kiichi Murakami, Michael Reedijk. Tumoral GLI1 controls the resident innate and adaptive tumor immune microenvironment in triple negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr B022.

MiR-875-5p suppresses Gli1 to alter the hedgehog signaling pathway, which in turn has hepatocellular cancer-related tumor suppressing properties

BackgroundOne of the most prevalent cancers worldwide is HCC, which has put patient health at risk. Increasing evidence indicated that messenger RNAs (mRNAs) played significant roles in modulating tumorigenesis. It has been established that Gli1 acts as an oncogene in a number of malignancies. However, more research was necessary to understand the Gli1 regulation mechanism in HCC. MethodsMicroarray technology was used to evaluate the expression of mRNAs. RT-qPCR was utilized to evaluate Gli1 and miR-875-5p expression. To investigate the role of Gli1, tests using CCK-8, EdU, transwell, immunofluorescence, and Western blot analysis was performed. RIP, RNA pull down, and luciferase reporter assays were employed to verify the interaction between Gli1 and miR-875-5p. ResultsIn tissues and cells of HCC, Gli1 expression appeared to be upregulated, especially in metastatic samples and advanced stages of the disease. A worse outcome was predicted by elevated Gli1 expression. Additionally, in HCC, Gli1 inhibition impeded the growth, migration, and development of the EMT. Since miR-875-5p was shown to have a molecular target in Gli1, miR-875-5p mediated the negative regulation of Gli1. In HCC tissues, its expression pattern was less prominent. In HCC tissues, there was an inverse relationship between Gli1 expression and miR-875-5p expression. Overexpressing Gli1 helped to partially counteract the suppression of HCC migration, proliferation, and EMT formation by miR-875-5p overexpression. ConclusionsMiR-875-5p in HCC suppresses tumors by downregulating Gli1, which supplies a novel treatment for HCC patients.

Combined targeting of Hedgehog/GLI1 and Wnt/β-catenin pathways in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a rare and aggressive form of non-Hodgkin lymphoma. Challenges in its treatment include relapse, drug resistance, and a short survival period. The Hedgehog/GLI1 (Hh/GLI1) and Wnt/β-catenin pathways are crucial in cancer cell proliferation, survival, and drug resistance, making them significant targets for anticancer research. This study aimed to assess the effectiveness of combining inhibitors for both pathways against MCL and investigate the underlying molecular mechanisms. The co-expression of key proteins from the Hh/GLI1 and Wnt/β-catenin pathways was observed in MCL. Targeting the Hh/GLI1 pathway with the GLI1 inhibitor GANT61 and the Wnt/β-catenin pathway with the CBP/β-catenin transcription inhibitor ICG-001, dual-target therapy was demonstrated to synergistically suppressed the activity of MCL cells. This approach promoted MCL cell apoptosis, induced G0/G1 phase blockade, decreased the percentage of S-phase cells, and enhanced the sensitivity of MCL cells to the drugs adriamycin and ibrutinib. Both GANT61 and ICG-001 downregulated GLI1 and β-catenin while upregulating GSK-3β expression. The interaction between Hh/GLI1 and Wnt/β-catenin pathways was mediated by GANT61-dependent Hh/GLI1 inhibition. Moreover, GLI1 knockdown combined with ICG-001 synergistically induced apoptosis and increased drug sensitivity of MCL cells to doxorubicin and ibrutinib. GANT61 attenuated the overexpression of β-catenin and decreased the inhibition of GSK-3β in MCL cells. Overall, the combined targeting of both the Hh/GLI1 and Wnt/β-catenin pathways was more effective in suppressing proliferation, inducing G0/G1 cycle retardation, promoting apoptosis, and increasing drug sensitivity of MCL cells than mono treatments. These findings emphasize the potential of combinatorial therapy for treating MCL patients.

MTORC1 hampers Hedgehog signaling in Tsc2 deficient cells.

The mTORC1-complex is negatively regulated by TSC1 and TSC2. Activation of Hedgehog signaling is strictly dependent on communication between Smoothened and the Hedgehog-signaling effector and transcription factor, GLI2, in the primary cilium. Details about this communication are not known, and we wanted to explore this further. Here we report that in Tsc2 -/- MEFs constitutively activated mTORC1 led to mis-localization of Smoothened to the plasma membrane, combined with increased concentration of GLI2 in the cilia and reduced Hedgehog signaling, measured by reduced expression of the Hedgehog target gene, Gli1 Inhibition of mTORC1 rescued the cellular localization of Smoothened to the cilia, reduced the cilia concentration of GLI2, and restored Hedgehog signaling. Our results reveal evidence for a two-step activation process of GLI2. The first step includes GLI2 stabilization and cilium localization, whereas the second step includes communication with cilia-localized Smoothened. We found that mTORC1 inhibits the second step. This is the first demonstration that mTORC1 is involved in the regulation of Hedgehog signaling.

GLI1 confers resistance to PARP inhibitors by activating the DNA damage repair pathway.

Identifying the mechanisms of action of anticancer drugs is an important step in the development of new drugs. In this study, we established a comprehensive screening platform consisting of 68 oncogenes (MANO panel), encompassing 243 genetic variants, to identify predictive markers for drug efficacy. Validation was performed using drugs that targeted EGFR, BRAF, and MAP2K1, which confirmed the utility of this functional screening panel. Screening of a BRCA2-knockout DLD1 cell line (DLD1-KO) revealed that cells expressing SMO and GLI1 were resistant to olaparib. Gene set enrichment analysis identified genes associated with DNA damage repair that were enriched in cells overexpressing SMO and GLI1. The expression of genes associated with homologous recombination repair (HR), such as the FANC family and BRCA1/2, was significantly upregulated by GLI1 expression, which is indicative of PARP inhibitor resistance. Although not all representative genes of the nucleotide excision repair (NER) pathway were upregulated, NER activity was enhanced by GLI1. The GLI1 inhibitor was effective against DLD1-KO cells overexpressing GLI1 both in vitro and in vivo. Furthermore, the combination therapy of olaparib and GLI1 inhibitor exhibited a synergistic effect on DLD1-KO, suggesting the possible clinical application of GLI1 inhibitor targeting cancer with defective DNA damage repair. This platform enables the identification of biomarkers associated with drug sensitivity, and is a useful tool for drug development.

Identification of Novel GANT61 Analogs with Activity in Hedgehog Functional Assays and GLI1-Dependent Cancer Cells.

Aberrant activation of hedgehog (Hh) signaling has been implicated in various cancers. Current FDA-approved inhibitors target the seven-transmembrane receptor Smoothened, but resistance to these drugs has been observed. It has been proposed that a more promising strategy to target this pathway is at the GLI1 transcription factor level. GANT61 was the first small molecule identified to directly suppress GLI-mediated activity; however, its development as a potential anti-cancer agent has been hindered by its modest activity and aqueous chemical instability. Our study aimed to identify novel GLI1 inhibitors. JChem searches identified fifty-two compounds similar to GANT61 and its active metabolite, GANT61-D. We combined high-throughput cell-based assays and molecular docking to evaluate these analogs. Five of the fifty-two GANT61 analogs inhibited activity in Hh-responsive C3H10T1/2 and Gli-reporter NIH3T3 cellular assays without cytotoxicity. Two of the GANT61 analogs, BAS 07019774 and Z27610715, reduced Gli1 mRNA expression in C3H10T1/2 cells. Treatment with BAS 07019774 significantly reduced cell viability in Hh-dependent glioblastoma and lung cancer cell lines. Molecular docking indicated that BAS 07019774 is predicted to bind to the ZF4 region of GLI1, potentially interfering with its ability to bind DNA. Our findings show promise in developing more effective and potent GLI inhibitors.

MODL-14. TARGETING GL1/GL2-DNMT1-UHRF1 EPIGENETIC COMPLEX DISRUPTION VIA BET INHIBITION IMPAIRS SHH MB DEVELOPMENT

Abstract BACKGROUND Sonic Hedgehog Medulloblastoma (SHH MB) shares 30% among all molecular subtypes of medulloblastomas (MBs). Constitutive activation of SHH signaling in the cerebellum’s granular cell precursors (GCPs) is critical for this MB development. When SHH receptor PATCHED-1 (PTCH1) is mutated, stimulation of this receptor by SHH ligand binding leads to derepression of heterotrimeric GTP-binding protein–coupled receptor Smoothened (SMO) eventually altering the activity, location, and stability of the three downstream glioma associated oncogene (GLI) family members: GLI1, GLI2, and GLI3. GLI1 and GLI2 mostly function as transcriptional activators, while GLI3 mainly plays a repressor role. The bromodomain and extra-terminal domain (BET) family of proteins comprises four conserved members (Brd2, Brd3, Brd4, and Brdt), which are critical regulators of GLI1 and GLI2-mediated transcription. METHODS In the present study, we pharmacologically inhibited BETs with a previously described BET inhibitor, BMS986158, and a novel BET inhibitor, UM002, discovered by our lab. We treated human SHH MB cells (ONS76 cells) in vitro and ex vivo in mouse SHH MB cells (from Ptch1 conditional knockout mice) with minimum effective concentrations of these inhibitors. We screened for GLI1/GLI2-DNMT1-UHRF1 epigenetic complex components expressions by immunoblotting and assessed the effectiveness of inhibiting their interactions by performing PLA assays. RESULTS In this study, we show that pharmacological inhibition of BETs leads to attenuated GLI1 and GLI2 levels. This GLI1 and GLI2 inhibition eventually disrupts the previously reported GL1/GL2, DNMT1-UHRF1 epigenetic complex, which is crucial for SHH MB development. A previously described BET inhibitor, BMS986158, and a novel BET inhibitor, UM002, synthesized and characterized by our lab can inhibit BETs. This could be a potential therapeutic approach targeting GLI-associated epigenetic complexes to treat SHH medulloblastoma.

PDLIM3 knockdown promotes ferroptosis in endometriosis progression via inducing Gli1 degradation and blocking Hedgehog signaling pathway.

Current evidence suggests that there is no completely effective method for endometriosis (EMS) without trauma due to diverse adverse effects. Reliable evidence illustrates that inhibiting ferroptosis is a potential strategy for EMS. We sufficiently verified that the expression of endogenous protein PDZ and LIM domain 3 (PDLIM3) was significantly increased in EMS. PDLIM3 knockdown reduced primary ectopic endometrial stromal cells' (EESCs) viability and migration, and elevated ferroptosis signaling indicators including Fe2+, malondialdehyde (MDA), and reactive oxygen species (ROS) in EESCs. Mechanistic studies revealed that inhibition of PDLIM3 accelerated glioma-associated oncogene-1 (Gli1) degradation and further deactivated Hedgehog signaling. Gli1 inhibitor, GANT61, abrogated the impact of PDLIM3 deletion on EESC growth, migration, and ferroptosis. In vivo experiments suggested that PDLIM3 reduction repressed the growth of endometrial lesions. Likewise, repression of PDLIM3 promoted ferroptosis and attenuated Hedgehog signaling in endometrial lesions. Collectively, silencing of PDLIM3 facilitates ferroptosis in EMS by inducing Gli1 degradation and blocking Hedgehog signaling. It may provide an alternative strategy for developing therapeutic agents of EMS in the future.

Sonic Hedgehog potentiates BMP9-induced osteogenic differentiation of mesenchymal stem cells

Bone morphogenetic protein 9 (BMP9) has remarkable potential to induce the differentiation of mesenchymal stem cells (MSCs) towards the osteoblastic lineage. Additionally, research suggests that certain growth factors have the ability to potentiate BMP9-induced osteogenic differentiation of MSCs. Sonic Hedgehog (Shh) plays an indispensable role in the regulation of skeletal development. The objective of this research was to assess the potential influence of Shh on BMP9-induced osteogenic differentiation of MSCs. Our findings indicated that Shh effectively enhanced BMP9-induced early and late osteogenic differentiation of MSCs, and increased BMP9-induced expression/transcriptional activity of osteogenesis-related transcription factors. Besides, it was observed that Shh promoted BMP9-induced ectopic bone formation of MSCs in vivo. Moreover, BMP9 was able to facilitate the repair of bone defects in rats, while Shh further accelerated this reparative process. Mechanistically, Shh enhanced the activation of the Smad1/5/8 signaling pathway which was induced by BMP9. Furthermore, GANT-61, an inhibitor of Gli1 and Gli2, attenuated the enhancing effect of Shh on BMP9-induced osteogenic differentiation of MSCs. Collectively, the co-administration of BMP9 and Shh may present a promising therapeutic approach for the treatment of fracture nonunion, delayed fracture healing, and bone defects.

Abstract 6773: Tissue engineered colorectal cancer microspheres for drug screening

Abstract Three-dimensional (3D) disease models have garnered widespread interest for use in later stages of the drug discovery process, such as preclinical efficacy and toxicology studies, due to their pathophysiologically relevant properties. However, there is a need and opportunity for 3D cancer models to be used earlier in the drug screening process. To meet this need, the 3D models must strike a balance between throughput, which includes scalability and uniformity, and physiological relevance, such as the ability to modulate key attribute of the tumor microenvironment. Here we report the creation of 3D colorectal cancer (CRC) tissue models, referred to as VivoSpheres, and demonstrated their relevance to cancer drug screening. The VivoSphere production platform couples tissue engineering toolkits with microfluidics, enabling the scalable production of engineered cancer microspheres. The model supports the long-term maintenance of the cancer cell phenotype. In a preliminary study, we were able to generate more physiologically relevant drug responses. We formed CRC VivoSpheres by encapsulating HT-29 CRC cells within poly(ethylene glycol)-fibrinogen hydrogel microspheres using our previously developed microfluidic platform. CRC VivoSpheres were rapidly produced with high cell densities (20 × 106 cells/ml) and high uniformity on day 0 with a coefficient of variation (COV) < 7%. This high uniformity was maintained for 15 days (COV ≤ 10%), which is critical for long-term dose studies. The cells maintained high viability and showed high proliferative capability with a significant increase in colony size and expression of Ki67 up to day 29. The encapsulated cells maintained the CRC phenotype over time with the expression of CD44 (cancer stem cell marker) and CK20 (CRC marker). After establishing shipping conditions that maintained cell viability for remote use, the HT-29 VivoSpheres were shipped to the oncology team at Southern Research for drug testing. The CRC VivoSpheres were treated with DMSO, GANT61, and SRI-38832, the latter two of which are GLI1 inhibitors. Phase contrast images and western blot were used to assess the response of CRC VivoSpheres to the treatments. Oncogenic GLI1 transcription activity and NBS1 overexpression have been found to contribute to chemotherapeutic resistance, negating the anti-tumor effects of 5-fluorouracil. While 2D cultured HT-29s responded to treatment with GANT61, HT-29 VivoSpheres continued to express NBS1 following GANT1 treatment, but downregulated NBS1 in response to the GLI1 inhibitor SRI-38832, which is the same response Southern Research has seen in in vivo tumor models. In conclusion, we have developed tissue-engineered 3D CRC models that hold promise for use in drug screening. These models have demonstrated an initial capability to reproduce the CRC phenotype and mimic in vivo drug response. Citation Format: Yuan Tian, Mohammadjafar Hashemi, Benjamin Anbiah, Ruowen Zhang, Iman Hassani, Rebecca Boohaker, Elizabeth Lipke. Tissue engineered colorectal cancer microspheres for drug screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6773.

Hedgehog signalling is involved in acquired resistance to KRASG12C inhibitors in lung cancer cells

Although KRASG12C inhibitors have shown promising activity in lung adenocarcinomas harbouring KRASG12C, acquired resistance to these therapies eventually occurs in most patients. Re-expression of KRAS is thought to be one of the main causes of acquired resistance. However, the mechanism through which cancer cells re-express KRAS is not fully understood. Here, we report that the Hedgehog signal is induced by KRASG12C inhibitors and mediates KRAS re-expression in cancer cells treated with a KRASG12C inhibitor. Further, KRASG12C inhibitors induced the formation of primary cilia and activated the Hedgehog-GLI-1 pathway. GLI-1 binds to the KRAS promoter region, enhancing KRAS promoter activity and KRAS expression. Inhibition of GLI using siRNA or the smoothened (Smo) inhibitor suppressed re-expression of KRAS in cells treated with a KRASG12C inhibitor. In addition, we demonstrate that KRASG12C inhibitors decreased Aurora kinase A (AURKA) levels in cancer cells, and inhibition of AURKA using siRNA or inhibitors led to increased expression levels of GLI-1 and KRAS even in the absence of KRAS inhibitor. Ectopic expression of AURKA attenuated the effect of KRASG12C inhibitors on the expression of GLI-1 and re-expression of KRAS. Together, these findings demonstrate the important role of AURKA, primary cilia, and Hedgehog signals in the re-expression of KRAS and therefore the induction of acquired resistance to KRASG12C inhibitors, and provide a rationale for targeting Hedgehog signalling to overcome acquired resistance to KRASG12C inhibitors.

The deubiquitinating enzyme USP44 suppresses hepatocellular carcinoma progression by inhibiting Hedgehog signaling and PDL1 expression

Hepatocellular carcinoma (HCC) is one of the deadliest malignancies in the world. Research into the key genes that maintain the malignant behavior of cancer cells is crucial for the treatment of HCC. Here, we identified ubiquitin‐specific peptidase 44 (USP44), a member of the deubiquitinase family, as a novel regulator of HCC progression. The tumor suppressive function of USP44 was evaluated in a series of in vitro and in vivo experiments. Through quantitative proteomics examination, we demonstrated that USP44 inhibits HCC PDL1 expression by downregulating the Hedgehog (Hh) signaling pathway. Mechanistically, we found that USP44 directly interacts with Itch, an E3 ligase involved in Hh signaling, and promotes the deubiquitination and stabilization of Itch. These events result in the proteasomal degradation of Gli1 and subsequent inactivation of Hh signaling, which ultimately suppresses PDL1 expression and the progression of HCC. Furthermore, the HCC tissue microarray was analyzed by immunohistochemistry to evaluate the pathological relevance of the USP44/Itch/Gli1/PDL1 axis. Finally, the Gli1 inhibitor GANT61 was found to act in synergy with anti-PDL1 therapy. Overall, USP44 can act as a suppressive gene in HCC by modulating Hh signaling, and co-inhibition of Gli1 and PDL1 might be an effective novel combination strategy for treating HCC patients.

RCC2 promotes prostate cancer cell proliferation and migration through Hh/GLI1 signaling pathway and cancer stem-like cells

BackgroundRegulator of chromosome condensation 2 (RCC2) was a telophase disk-binding protein on mitosis, and functions as an oncogene in many human cancers. However, its role on prostate cancer (PCa) was unknown. The goal of this study is to explore the function of RCC 2 on PCa development.MethodsThe expression of RCC2 and its methylation level, its correlation with lymph node metastasis or disease-free survival (DFS) was analyzed using TCGA database. The effect of RCC2 on PCa cell proliferation, migration and invasion were detected using CCK-8, cell colony formation, Transwell and wood healing assays. RNA-seq and GSEA analysis were used to search the downstream genes and pathways of RCC2 in mediated PCa progression. Western blot was used to detect the proteins in PCa cells transfected with indicated siRNAs or plasmids.ResultsRCC2 had high expression and low promoter methylation level in PCa, and its expression was correlated with regional node metastasis and disease-free survival. Cell proliferation, migration, invasion and EMT of PCa cells in vitro were greatly enhanced after RCC2 overexpression, while the RCC2 knockdown suppressed these processes. RNA-seq and GSEA results showed the Hedgehog signaling regulator Gli1 and Gli3 were involved in RCC2 knockdown DU145 cells. Gli1 was also a marker of cancer stem-like cells (CSCs). Mechanistically, RCC2 induced cell growth, EMT, CSCs markers through Gli1; inhibiting Gli1 expression using siGli1 or GLI inhibitor suppressed cell progression in vitro and tumor growth in vivo.ConclusionIn summary, RCC2 promoted PCa development through Hh/Gli1 signaling pathway via regulating EMT and CSCs.

Hedgehog signaling and the glioma-associated oncogene in cancer radioresistance.

Tumor radioresistance remains a key clinical challenge. The Hedgehog (HH) signaling pathway and glioma-associated oncogene (GLI) are aberrantly activated in several cancers and are thought to contribute to cancer radioresistance by influencing DNA repair, reactive oxygen species production, apoptosis, autophagy, cancer stem cells, the cell cycle, and the tumor microenvironment. GLI is reported to activate the main DNA repair pathways, to interact with cell cycle regulators like Cyclin D and Cyclin E, to inhibit apoptosis via the activation of B-cell lymphoma-2, Forkhead Box M1, and the MYC proto-oncogene, to upregulate cell stemness related genes (Nanog, POU class 5 homeobox 1, SRY-box transcription factor 2, and the BMI1 proto-oncogene), and to promote cancer stem cell transformation. The inactivation of Patched, the receptor of HH, prevents caspase-mediated apoptosis. This causes some cancer cells to survive while others become cancer stem cells, resulting in cancer recurrence. Combination treatment using HH inhibitors (including GLI inhibitors) and conventional therapies may enhance treatment efficacy. However, the clinical use of HH signaling inhibitors is associated with toxic side effects and drug resistance. Nevertheless, selective HH agonists, which may relieve the adverse effects of inhibitors, have been developed in mouse models. Combination therapy with other pathway inhibitors or immunotherapy may effectively overcome resistance to HH inhibitors. A comprehensive cancer radiotherapy with HH or GLI inhibitor is more likely to enhance cancer treatment efficacy while further studies are still needed to overcome its adverse effects and drug resistance.

RBIO-04. INHIBITING THE SHH PATHWAY AND PARP1 IN COMBINATION ENHANCES THE RADIATION SENSITIVITY OF PEDIATRIC SONIC HEDGEHOG MEDULLOBLASTOMA

Abstract Medulloblastoma accounts for 20-25% of all pediatric brain tumors. These patients undergo craniospinal and focal radiation therapy during their treatment, resulting in negative long-term consequences such as bone and tissue hypoplasia and decreased neurocognitive functioning. One of the most concerning effects is the significantly increased risk for secondary radiation induced cancers later in life. Thus, there is a critical need for novel approaches to radiation therapy that can effectively target and kill tumor cells while minimizing long lasting toxic effects. Here, we investigate a novel approach to radiation therapy that relies on the combination of two classes of therapeutic agents, Sonic Hedgehog (SHH) inhibitors and Poly (ADP-ribose) Polymerase (PARP) inhibitors. The SHH pathway plays a key role in medulloblastoma tumorigenesis and affects the radiation sensitivity of various cancer types such as basal cell carcinoma. The PARP family, specifically PARP1, plays a key role in DNA damage repair and affects the radiation sensitivity of pediatric brain cancers such as high-grade glioma and medulloblastoma. We hypothesize that combining these approaches by inhibiting Smo and Gli1 in the SHH pathway, and PARP1 simultaneously, will enhance medulloblastoma tumor cell killing following radiation therapy. We found that in vitro treatment with Sonidegib, GANT61, and Veliparib alone and in combination with was sufficient to significantly decrease tumor cell viability and self-renewal as measured by clonogenic survival assays (p < 0.01). 24 hours following these targeted treatments, 2 Gy of radiation was administered which resulted in a greater degree of decreased cell viability and self-renewal (p < 0.01), as well as in increased cell death. Together, this data suggests that inhibition of Smo, Gli1, and PARP1 either alone as single agents, or in combination is a promising therapeutic strategy for increasing the radiation sensitivity of pediatric SHH medulloblastoma.

Co-targeting FAK and Gli1 inhibits the tumor-associated macrophages-released CCL22-mediated esophageal squamous cell carcinoma malignancy.

Esophageal squamous cell carcinoma (ESCC) is a frequently seen esophageal tumor type in China. Activation of signaling proteins and relevant molecular mechanisms in ESCC are partially explored, impairing the antitumor efficiency of targeted therapy in ESCC treatment. Tumor-associated macrophages (TAMs)-released C-C motif chemokine 22 (CCL22) can activate intratumoral focal adhesion kinase (FAK), thus promoting the progression of ESCC. Here, we demonstrated that highly secreted CCL22 by TAMs (CCL22-positive TAMs) induced ESCC cell stemness and invasion through facilitating transcriptional activity of intratumoral glioma-associated oncogene 1 (Gli1), a downstream effector for Hedgehog (HH) pathway. Mechanistically, FAK-activated protein kinase B (AKT) mediated Gli1 phosphorylation at its Ser112/Thr115/Ser116 sites and released Gli1 from suppressor of fused homolog, the endogenous inhibitor of Gli1 to activate downstream stemness-associated factors, such as SRY-box transcription factor 2 (SOX2), Nanog homeobox (Nanog), or POU class 5 homeobox (OCT4). Furthermore, inhibition of FAK activity by VS-4718, the FAK inhibitor, enhanced antitumor effect of GDC-0449, the HH inhibitor, both in xenografted models and in vitro assays. Clinically, CCL22/Gli1 axis is used to evaluate ESCC prognosis. Overall, our study establishes the communication of FAK with HH pathway and offers the novel mechanism related to Gli1 activation independent of Smoothened as well as the rationale for the anti-ESCC combination treatment.

GANT61, an inhibitor of Gli1, inhibits the proliferation and migration of hepatocellular carcinoma cells.

Constitutive activation of Hedgehog (Hh) signaling has been implicated in many cancers including hepatocellular carcinoma (HCC). Among them, the terminal glioma-associated oncogene homolog 1 (Gli1) regulates the expression of critical genes in the Hh pathway. The current study aims to evaluate the anti-HCC effect of the Gli1 inhibitor, GANT61. In vitro analysis including cell counting kit-8 (CCK-8) assay, flow cytometry, and migration and invasion assay were adopted to evaluate the effect of GANT61 on HCC cell lines. In vivo, xenograft studies were also performed to verify the effect of GANT61 on HCC. By CCK-8 assay, we found that GANT61 could significantly reduce the growth of HCC cell lines Huh7 and hemophagocytic lymphohistiocytosis (HLE), and their IC50 concentrations were 4.481 and 6.734 μM, respectively. Flow cytometry shows that GANT61 induced cell cycle arrest in the G2/M phase and accelerated apoptosis of both HLE and Huh7 cells. While migration and invasion assay shows that GANT61 weakens cells' migration and invasion ability. Besides that, GANT61 inhibits the expression of Gli1, FoxM1, CyclinD1, and Bcl-2, upregulates the level of Bax protein, and also reverses the epithelial-mesenchymal transition program by downregulating the expression of Vimentin and N-Cadherin and upregulating the expression of epithelial E-Cadherin expression. Furthermore, GANT61 inhibits the growth of subcutaneous xenografts of Huh7 cells in nude mice. Overall, this study suggests that Gli1 is a potential target for therapy and GANT61 shows promising therapeutic potential for future treatment in HCC.

CSF3R T618I Collaborates With RUNX1-RUNX1T1 to Expand Hematopoietic Progenitors and Sensitizes to GLI Inhibition.

Activating colony-stimulating factor-3 receptor gene (CSF3R) mutations are recurrent in acute myeloid leukemia (AML) with t(8;21) translocation. However, the nature of oncogenic collaboration between alterations of CSF3R and the t(8;21) associated RUNX1-RUNX1T1 fusion remains unclear. In CD34+ hematopoietic stem and progenitor cells from healthy donors, double oncogene expression led to a clonal advantage, increased self-renewal potential, and blast-like morphology and distinct immunophenotype. Gene expression profiling revealed hedgehog signaling as a potential mechanism, with upregulation of GLI2 constituting a putative pharmacological target. Both primary hematopoietic cells and the t(8;21) positive AML cell line SKNO-1 showed increased sensitivity to the GLI inhibitor GANT61 when expressing CSF3R T618I. Our findings suggest that during leukemogenesis, the RUNX1-RUNXT1 fusion and CSF3R mutation act in a synergistic manner to alter hedgehog signaling, which can be exploited therapeutically.

Vemurafenib activates the sonic hedgehog pathway and promotes thyroid cancer stem cell self-renewal.

B-Raf kinase inhibitors such as vemurafenib (PLX4032) and dabrafenib have limited therapeutic efficacy on BRAF-mutated thyroid cancer. Cancer stem cells (CSCs) play important roles in tumor recurrence, drug resistance, and metastasis. Whether CSCs play a role in dampening the antitumor activity of B-Raf kinase inhibitors remains unknown. Here, we report that vemurafenib (PLX4032) induced the expression of several stemness-related genes including Gli1, Snail, BMI1, and SOX2 in two anaplastic thyroid cancer cell lines, SW1736 and 8505C, but decreased the expression of these genes in A375 cells, a human melanoma cell line. PLX4032 promoted thyroid cancer stem cell self-renewal, as evidenced by increased numbers of aldehyde dehydrogenase-positive cells and thyrospheres. Mechanistically, PLX4032 activates the PI-3 and mitogen-activated protein kinase pathways through HER3 to cross-activate Gli1, a transcription factor of the sonic hedgehog (Shh) pathway. GANT61, a specific inhibitor of Gli1, blocked the expression of the stemness-related genes in PLX4032-treated thyroid cancer cells in vitro and in vivo in two thyroid cancer xenograft models. GANT61 treatment alone weakly inhibited SW1736 tumor growth but enhanced the antitumor activity of PLX4032 when used in combination. Our study provides mechanistic insights into how thyroid cancer poorly responds to B-Raf kinase inhibitors and suggests that targeting B-Raf and the Shh pathway in combination may overcome thyroid cancer drug resistance.

Smoothened mediates medaka spermatogonia proliferation via Gli1-Rgcc-Cdk1 axis†.

The proliferation of spermatogonia directly affects spermatogenesis and male fertility, but its underlying molecular mechanisms are poorly understood. In this study, Smoothened (Smo), the central transducer of Hedgehog signaling pathway, was characterized in medaka (Oryzias latipes), and its role and underlying mechanisms in the proliferation of spermatogonia were investigated. Smo was highly expressed in spermatogonia. In ex vivo testicular organ culture and a spermatogonial cell line (SG3) derived from medaka mature testis, Smo activation promoted spermatogonia proliferation, while its inhibition induced apoptosis. The expression of glioma-associated oncogene homolog 1 (gli1) and regulator of cell cycle (rgcc) was significantly upregulated in SG3 after Smo activation. Furthermore, Gli1 transcriptionally upregulated the expression of rgcc, and Rgcc overexpression rescued cell apoptosis caused by Smo or Gli1 inhibition. Co-immunoprecipitation assay indicated that Rgcc could interact with cyclin-dependent kinase 1 (Cdk1) to regulate the cell cycle of spermatogonia. Collectively, our study firstly reveals that Smo mediates the proliferation of spermatogonia through Gli1-Rgcc-Cdk1 axis. In addition, Smo and Gli1 are necessary of the survival of spermatogonia. This study deepens our understanding of spermatogonia proliferation and survival at the molecular level, and provides insights into male fertility control and reproductive disease treatment.