Smoothened Inhibitor Research Articles

PI3K Promotes Basal Cell Carcinoma Growth Through Kinase-Induced p21 Degradation.

Basal cell carcinoma (BCC) is a locally invasive epithelial cancer that is primarily driven by the Hedgehog (HH) pathway. Advanced BCCs are a critical subset of BCCs that frequently acquire resistance to Smoothened (SMO) inhibitors and identifying pathways that bypass SMO could provide alternative treatments for patients with advanced or metastatic BCC. Here, we use a combination of RNA-sequencing analysis of advanced human BCC tumor-normal pairs and immunostaining of human and mouse BCC samples to identify a PI3K pathway expression signature in BCC. Pharmacological inhibition of PI3K activity in BCC cells significantly reduces cell proliferation and HH signaling. However, treatment of Ptch1fl/fl; Gli1-CreERT2 mouse BCCs with the PI3K inhibitor BKM120 results in a reduction of tumor cell growth with no significant effect on HH signaling. Downstream PI3K components aPKC and Akt1 showed a reduction in active protein, whereas their substrate, cyclin-dependent kinase inhibitor p21, showed a concomitant increase in protein stability. Our results suggest that PI3K promotes BCC tumor growth by kinase-induced p21 degradation without altering HH signaling.

Modulating Hedgehog Signaling Reduces MAPK, TGF-β, and Marrow Fibrosis in a Transgenic Mouse Model of JAK2V617F Associated Myeloproliferative Disease

Treatment of JAK2V617F driven myeloproliferative neoplasms (MPN) with FDA approved drug Ruxolitinib (JAK inhibitor, JAKi) has shown limited benefits. The failure of JAKi to eradicate JAK2V617F disease has led to the search for other pathways as potential therapeutic targets for this disease. Hedgehog (Hh) pathway inhibitors targeting Smoothened (SMO) have shown promising activity in MPN by reducing bone marrow fibrosis but are inadequate as single agents.In the present study we tested the effects of SMO inhibitor, PF-04449913 in transgenic mouse model of myeloproliferative disease (vavJak2V617F, gifted by Joe Zhao, University of Oklahoma Health Sciences Center). JAK2V617F transgenic mice exhibit a number of phenotypes that recapitulate those observed in MPN including bone marrow fibrosis and splenomegaly.Treatment with SMO inhibitor, PF-04449913, reduces splenomegaly. JAK2V617F mice showed pronounced splenomegaly compared to WT mice and treatment with SMO inhibitor resulted in a significant decrease in spleen size (Figure 1A). This was also associated with a drop in IL-4 and IL-7 in the peripheral blood (data not shown).SMO inhibitor reduces JAK2V617F allelic burden. In the next set of experiment we made mixed chimeras by transplanting one million JAK2V617F or WT bone marrow into lethally irradiated normal CD45.1 congenic hosts. Transplanted recipient mice were treated with PF-04449913 for one month and assessed for changes in peripheral blood chimerism. Treatment with PF-04449913 resulted in no change in WT engraftment. However, we found that PF-04449913 reduces the JAK2V617F allele burden in 3/5 mice.JAKV617F activates Hh signaling. Proteins were isolated from the spleens of 8-10 week JAK2V617F or WT mice. Western blot analyses demonstrate that JAK2V617F splenocytes express high SHH levels. We next measured levels of the GLI activator (GLI1) and the GLI repressor (GLI3R). Activation of Hh signaling is associated with an increase in GLI1 and a decrease in GLI3R. Treatment of the mice with PF-04449913 by daily gavage at 100mg/kg reduced GLI1 and increased GLI3R.JAK2V617F is associated with increased pERK, AKT, NF-κB activation. Oh and colleagues recently remonstrated the MPN were associated with activation of MAPK and NF-κB signaling pathways (Fisher et al., Leukemia, 2017). To understand how Hh signaling interacted with these pathways, we assessed ERK, AKT, and NF-κB activity by western blotting. JAK2V617F mice showed increased pERK, AKT and NFKB signaling. Treatment with PF-04449913 reduced pERK and AKT levels, confirming that both ERK and AKT are downstream of SMO, while it had little effect on IκBα levels suggesting that NF-κB activation occurs upstream of SMO.SMO inhibitor blocks bone marrow fibrosis by reducing TGF-β signaling. Numerous studies have implicated TGF-β in the pathogenesis of fibrosis. We analyzed the bone marrow of patients with JAK2V617F MPN and found them to express high TGF-β levels (Figure 1B). These findings were recapitulated in the JAK2V617F transgenic mice with levels of TGF-β in JAK2V617F splenocytes 15-fold greater than WT mice. Treatment with PF-04449913 normalized the levels of TGF-β (Figure 1C). Using a OP-9 co-culture system we demonstrated that JAK2V617F splenocytes robustly induced pSMAD2, which was completely blocked by PF-04449913 (Figure 1D). To assess for changes in bone marrow fibrosis, mice at 27 weeks were started on daily treatment with PF-04449913 for one month. Treatment with PF-04449913 blocks bone marrow fibrosis as demonstrated by decreased reticulin staining and reduced hydroxyproline. (Figure 1E-F).In conclusion, these data are the first to report that the Smoothened inhibitor, PF-04449913 modulates TGF-β and MAPK signaling in MPN and this results in decreased splenomegaly and marrow fibrosis. These results provide critical insight into the mechanism of action of SMO inhibitors in JAK2V617F associated MPN, and support the rational development of SMO and JAK inhibitor combinations in MPN.Figure Legend(A) SMO antagonist, PF-04449913 reduces splenomegaly. (B) Bone marrow of JAK2V617F MPN patient express high TGF-β levels. (C) Splenocytes of JAK2V617F transgenic mice express high TGF-β levels and PF-04449913 treatment normalizes TGF-β levels. (D) JAK2V617F induces Hh dependent TGF-β/pSMAD2 signaling in stroma. (E-F) PF-04449913 blocks fibrosis as measured by reticulin staining and quantification of Hydroxyproline. [Display omitted] DisclosuresO'Connell:Astex Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees; Otsuka: Membership on an entity's Board of Directors or advisory committees. Merchant:Pfizer: Consultancy, Research Funding.

Aberrant Hedgehog Signaling in Myelodysplastic Syndrome Accelerates Leukemic Transformation Via Smoothened-Dependent and Smoothened-Independent Gli1 Activation

Introduction: Myelodysplastic syndromes (MDS) are malignant hematopoietic stem cell disorders with increased risk of transformation to secondary acute myeloid leukemia (sAML) that carries uniformly dismal prognosis. The exact mechanisms driving this process remain obscure. Aberrant Hedgehog (Hh) signaling has been demonstrated in a number of solid tumors and hematologic malignancies. An emerging body of literature suggests the importance of Hh signaling in leukemic transformation of MDS. Moreover, a recent Phase II clinical trial using Smoothened (SMO) inhibitor with low-dose chemotherapy resulted in significant prolongation of overall survival (Lancet et al. Blood 2016). Thus, we sought to examine the clinical impact of aberrant Hh activation in MDS and further explore the mechanism of Hh-driven leukemic transformation.Methods/Results:To determine the pathway activity, we quantified the level of GLI1 in CD34+ cells from patients with MDS and healthy controls in published databases (N=17 and N=159, respectively; GSE58831) and while absent in controls, GLI1 was significantly upregulated in MDS patients (p<0.05). Analysis of serial samples revealed Hh activation at disease progression in 67% of patients (4/6). In addition, GLI1 levels were significantly higher in poor-risk AML with complex cytogenetics comparing to favorable risk disease (p<0.001, TCGA).To further evaluate the impact of of Hh activation in MDS progression, we crossed Nup98-HoxD13 (NHD13) mice with mice conditionally expressing the constitutively active mutant of Smoothened (SmoM2). Double transgenic NHD13/SmoM2 mice succumbed to aggressive myeloid leukemia (median survival of NHD13/SmoM2 and NHD13 controls, 3 months vs. 13 months, respectively, p<0.0001) characterized by leukocytosis (p<0.05), splenomegaly (p<0.0001) and significant expansion of immature myeloid progenitors in peripheral blood (p<0.0001) and bone marrow (p<0.0001). Analysis of hematopoietic stem/progenitor (HSPC) compartment revealed expansion of phenotypic granulocyte macrophage progenitors (GMP) (6244 vs. 131487 mean GMPs/femur , in NHD13 v. NHD13/SmoM2 respectively, p<0.05). To determine whether Hh activation resulted in aberrant self-renewal in GMP compartment, we carried out an in vivo repopulation assay. While NHD13 GMPs showed only transient engraftment, characteristic of non-self-renewing progenitors, NHD13/SmoM2 GMPs robustly engrafted and significantly expanded over time, and all recipient animals developed aggressive leukemia within 2-3 months. The engraftment was sustained through secondary transplantation. Our results suggest that Hh activation resulted in abnormal self-renewal in normally non-self-renewing progenitors.To delineate the mechanism leading to aberrant activation of self-renewal we compared the transcriptome of GMPs isolated from NHD13/SmoM2 and NHD13 controls. In addition to expected Hh activation, Gene Set Enrichment Analysis (GSEA) showed enrichment in gene signatures associated with other developmental pathways such as Wnt or TGF-β signaling as well as EWS/FLI1-protooncogen signature. Interestingly, these pathways have been found to act via direct activation of GLI1 .Using human AML (TF-1) and MDS (MDS-L) cell lines we confirmed that GLI1 may be activated in the absence of SMO. GLI1 knock-down and/or inhibition with GANT61 resulted in growth arrest (p<0.05, both) and significantly lower clonogenic potential in vitro (p<0.05, both) more so than upstream SMO inhibition.Conclusions: Our data suggest that aberrant Hh/GLI1 signaling is frequent in MDS and marks disease progression and leukemic transformation. In some cases, GLI1 can be activated in a SMO-independent manner and therapeutic strategies focused on targeting downstream Gli transcription factors may be more effective than currently used Smo inhibitors. DisclosuresNo relevant conflicts of interest to declare.

Upregulation of Alternatively Glycosylated Smoothened (SMO) Is Involved in Doxorubicin (DXR) Resistance in Diffuse Large B-Cell Lymphoma

▪Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy in adults. Chemoresistance remains the most urgent clinical challenge in the management of DLBCL patients. The salvage therapy options for patients with relapsed disease are poor (responses rates less than 20%) and survival is usually short. Therefore, the development of less toxic yet effective therapeutic approaches for DLBCL patients will address a significant immediate medical need. Smoothened (SMO), a Frizzled-class G-protein-coupled-receptor (GPCR), is activated by cholesterol and regulated indirectly by Hedgehog (Hh) ligands through the release of PTCH1-mediated suppression. SMO contains an N-terminal extracellular segment with the cysteine-rich domain (CRD), a heptahelical transmembrane domain (TMD), and a C-terminal intracellular segment. Although SMO localizes in cilia to function as Hh signal transducer, our data support that SMO is also a constituent of lipid rafts, and plays a fundamental role in lipid raft organization in DLBCL. SMO in doxorubicin (DXR) resistant cell lines shows increased glycosylation of its extracellular segments and drug resistance is sensitive to modulation of cellular cholesterol levels, both features associated with protein localization to lipid raft and lipid raft composition, a poorly understood aspect in drug resistance. To improve our current therapeutic approaches for lymphoma patients we need to increase our understanding of the cellular mechanisms that contribute to tumor progression and drug resistance.We established a DXR-resistant cell line (SUDHL4) through long-term exposure to escalating doses of DXR (DXR-resistant cell line: virtually no cell death up to a DXR concentration of 120 nM; parental cell line DXR-IC50: 40nM). We observed that DXR-resistant SUDHL4 cells were also resistant to escalating doses of vincristine in comparison with the parental (sensitive) cells. Studies on the response of DXR resistant lymphoma cells to DXR confirmed high levels of DNA damage in resistant cells (measured by phosphorylation of histone H2AX) confirming that DXR resistance was not due to increase drug efflux. In addition, in this experimental model, both parental and DXR-resistant lymphoma cells have mutated TP53 (missense mutation p.V157A) showing that TP53 deficiency was not a factor in inducing DXR resistance.Several lines of evidence support a direct role of SMO in the DXR-resistance observed in DLBCL cells. First, DXR-resistant lymphoma cells in comparison with the parental cells showed a strong increase in SMO mRNA, but not its canonical downstream target of signaling, the transcription factor GLI1. Concordantly, SMO protein levels were substantially elevated in DXR-resistant cells (as detected by immunofluorescence and western blot). Moreover, upregulation of the expression of SMO was detected by immunohistochemistry in 4 of 5 relapsed DLBCL tumors in comparison with the matched tumors at the time of the initial diagnosis and before chemotherapy treatment. Second, we found that SMO is localized in the lipid rafts and mediates the assembly of a TRAF6 E3 ligase-dependent machinery that enhances AKT signaling. Additionally, we observed that SMO is highly glycosylated in DXR-resistant SUDHL4 cells but not in the parental cell line; previously, it has been found that SMO has 4 potential N-glycosilation sites that are dispensable for canonical Hh signaling but needed to induce a non-canonical SMO signal through Gα1. Finally, depletion of SMO using shRNA and a lentiviral-tetracycline inducible system and pharmacologically inhibition of SMO using the classic SMO inhibitor (cyclopamine-KAAD) or a cholesterol synthesis inhibitor (lovastatin) re-established sensitivity to DXR in DXR-resistant cells. Interestingly, lovastatin did not modify DXR sensitivity in the parental cell line. In summary, this study suggests a novel function of SMO in the development of DXR-resistance in DLBCL and opens new opportunities to regain or enhance DXR sensitivity through targeting this novel mechanism. DisclosuresNo relevant conflicts of interest to declare.

Combining Effects of the SMO Inhibitor and BCL-2 Inhibitor in MDS-Derived Induced Potent Stem Cells (iPSC)

Background:Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders characterized by no efficient hematopoiesis and frequent progression to acute myeloid leukemia (AML). MDS clones acquire an apoptotic resistance upon disease progression, which coincides with the appearance of an elevated number of myeloid blasts and increased likelihood of disease acceleration into AML. Induced pluripotent stem cells (iPSCs) are promising for studying stem cell signaling pathways. When reprogramming is performed with MDS cells, MDS-derived iPSCs offer a unique system for modeling MDS pathogenesis and developing high throughput drug screening. Glasdegib is a potent and selective hedgehog pathway inhibitor that act by binding Smoothened (SMO) and blocking signal transduction. We have previously shown the persistent effects of glasdegib on long term self-renewing MDS-initiating cells (J Cancer Sci Ther. 2017, 9:479), however, quick clinical efficacy of glasdegib is only limitted. In the present study, we investigated the combining effects of venetolax (ABT-199), the BCL2-selective inhibitor, and glasdegib on phenotypically immature cell population of MDS-derived iPSCs.Methods:We generated iPSCs from bone marrow mononuclear cells of three MDS patients (MDS-MLD, MDS-EB1 and MDS-EB2 by WHO classification). Karyotyping analysis revealed that MDS-derived iPSCs have identical abnormalities to primary MDS cells. In vitro re-differentiation of MDS-iPSCs was performed with differentiation media (30 ng/ml VEGF, 30 ng/ml BMP-4, 40 ng/ml SCF, 50 ng/ml Activin) for 4 days. At day 14, a single cell suspension expressing CD34+CD38- was achieved with hematopoietic cytokine (300 ng/ml Flt-3 ligand, 10 ng/ml IL-3, 10 ng/ml IL-6, 50 ng/ml G-CSF, 25 ng/ml BMP-4).Results:We generated the phenotypically immature cell population (CD34+CD38-CD90+CD45+) and mature cell population (CD34-CD45+) from MDS-derived iPSCs. The single treatment of venetoclax significantly reduced the numbers of mature cell population (CD34-CD45+) from MDS-derived iPSCs. Although the single treatment of venetoclax or glasdegib alone have minimum effect on immature cell population (CD34+CD38-CD90+CD45+), co-treatment with venetoclax and glasdegib significantly reduce the numbers of cell population (CD34+CD38- CD90+CD45+) (p<0.001). Further, NOD/SCID mice were injected with MDS-derived iPSCs, and then treated with venetoclax and glasdegib on day 21 for 7 days. Co-treatments with venetoclax and glasdegib significantly reduced the population of CD34+CD38- CD90+ cells compared with venetoclax or glasdegib as single agents. Also co-treatment with venetoclax and glasdegib significantly reduced the colony formations of mature erythroid, granulocyte-macrophade, and mixed of these hematopoietic cells derived from MDS-EB2-iPSCs for CFC assay To identify the mechanisms that limit the MDS-EB2-derived immature cell population by venetoclax and glasdegib, CD34+CD38-CD90+CD45+ cell fractions from MDS-iPSCs were cultured with venetoclax and glasdegib for 72 hrs. Co-treatment with venetoclax and glasdegib reduced the expression of BMI1, c-Myc and nanog. Finally, we investigate the engraftment potential of MDS-EB1 derived iPSCs treated with venetoclax. One million of CD34+ cells derived from MDS-EB1 iPSCs treated with venetoclax were intravenously transplanted into NOD/SCID mouse. Transient engraft was observed from control mice, however, co-treatment of venetoclax never shows the engraftment.Conclusion:Our preclinical results indicate that the combination with venetoclax and glasdegib have potential as an important option for controlling the immature MDS cell population. It is expected that the combination with venetoclax and glasdegib may become extremely useful therapeutic interventions to obtain the quick clinical efficacy for MDS patients. DisclosuresTauchi:Novartis pharma KK: Research Funding, Speakers Bureau; Bristol-Myers Squibb KK: Speakers Bureau; Celegen KK: Speakers Bureau; Phizer KK: Research Funding, Speakers Bureau; Otsuka pharmaceutical KK: Speakers Bureau. Ohyashiki:Kyowa Hakko Kirin KK: Honoraria; Kyowa Hakko Kirin KK: Honoraria; Chugai pharmaceutical KK: Research Funding; Bristol-Myers Squibb KK: Honoraria, Research Funding, Speakers Bureau; Taiho pharmaceutical KK: Honoraria; Alexion: Honoraria; MSD KK: Honoraria; Nippon Shinyaku: Honoraria, Research Funding, Speakers Bureau; MSD KK: Honoraria; Taiho pharmaceutical KK: Honoraria; Otsuka pharmaceutical KK: Honoraria; Jansen pharmaceutical KK: Honoraria, Research Funding; Fujirebio KK: Honoraria; Dainippon Sumitomo KK: Honoraria, Research Funding, Speakers Bureau; Nippon Shinyaku: Honoraria, Research Funding, Speakers Bureau; Celegen KK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Chugai pharmaceutical KK: Research Funding; Alexion: Honoraria; Phizer KK: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb KK: Honoraria, Research Funding, Speakers Bureau; Fujirebio KK: Honoraria; Dainippon Sumitomo KK: Honoraria, Research Funding, Speakers Bureau; Celegen KK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis pharma KK: Honoraria, Research Funding, Speakers Bureau; Otsuka pharmaceutical KK: Honoraria; Jansen pharmaceutical KK: Honoraria, Research Funding; Novartis pharma KK: Honoraria, Research Funding, Speakers Bureau; Phizer KK: Honoraria, Research Funding, Speakers Bureau.

Phase I/IB Study of Azacitidine and Hedgehog Pathway Inhibition with Sonidegib (LDE225) in Myeloid Malignancies

Background: Lower intensity therapies for patients (pts) with myeloid malignancies (AML, MDS, CMML, MPNs) are beneficial, but suboptimal despite the emergence of potentially active new agents. Even after an initial response to hypomethylating agents (HMAs) [5-Azacitidine (AZA) or decitabine], the majority of patients fail treatment. In large-scale RNAi screens we identified Hedgehog pathway (HP) genes as a potential rational combination to overcome a priori and acquired HMA resistance. Herein we report the first analysis of the final study results of the oral smoothened (SMO) inhibitor, Sonidegib (LDE225) in combination with AZA in 63 pts treated on a Phase 1/1B (Ph1/1B) trial.Methods: Ph1 (3+3) dose escalation and Ph1B (expansion cohort) study to determine the MTD (Ph1) and best overall response (Ph1B) of Sonidegib (SON) combined with AZA. 2nd endpoints included AEs, DOR, TTP, OS, time to AML for MDS pts and QOL. Pts with untreated and relapsed/refractory (rel/ref) AML, CMML, MDS, MDS/MPN overlap (MDS/MPN) or myelofibrosis (MF) were eligible. SON was started at dose level (DL) 0 at 400mg oral daily continuously (28 day cycle) and AZA at 75mg/m2 x 7 days or days 5-2-2 with dose adjustment per label. Dose-limiting toxicity (DLT) in Ph1 was assessed after 42 days at steady-state concentrations of SON. Pharmacokinetics were not performed.Results:Study population:From 05/2014 to 05/2017 (data cut-off) 63 pts were enrolled and followed for outcome: 10 pts were treated on the Ph1 and 52 in Ph1B part of the trial (1 pt withdrew). Median age was 72.5 years (range 39-86) and 66% pts were male. ECOG 0, 1 and 2 are 34%, 61% and 5% respectively. 45% (n=28) were newly diagnosed and 55% (n=34) rel/ref pts. 38 pts (61%) had AML and 13 of the 15 newly diagnosed/untreated AML pts had 2nd AML due to AHD; 23/38 pts had rel/ref AML. For all AML pts 21/38 were intermediate risk and 16/38 had unfavorable cytogenetics (1/38 unknown). 18 pts (29%) had MDS, all in the Intermediate, High or Very High risk status (IPSS-R), 4 (6.5%) had CMML and 2 pts MF.Dose levels, treatment administration and follow up: 6 pts were treated at DL0 SON 400mg/day continuously with AZA and 1 DLT of CPK elevation observed, while another pt had grade 4 neutropenia (at that time included in DLT definition, later removed). Due to overall tolerance of the combination at DL0 and investigator assessment, despite only 1/6 formal DLTs, DL -1 at SON 200mg x28 days was explored without further DLT (n=4 pts) and 52 pts accrued on Ph1B. For the 56 pts treated at the MTD dose of SON 200mg (Ph1B=52 and Ph1=4 pts), after a median of 14.5 months follow up, 218 cycles have been administered (median 3 cycles-range 1-25 ms). Median doses for SON and AZA were 200mg and 75mg/m2 respectively. 12.5% and 18% of pts had dose adjustments and 71.4% (i.e. missed only 1 dose) and 18% dose omissions at least once for SON and AZA respectively. 19/56 (34%) of pts are alive.Safety and Tolerability : Of 56 pts treated at MTD dose100% experienced at least one grade (gr) 3 AE and 52 (93%) had a gr 4 AE regardless of attribution. 96% and 91% had a gr 3 and gr 4 Heme AE and 73% and 20% had a gr 3 and gr 4 Non-Hem AE respectively. Gr 3/4 AEs were as expected for either drug and the diseases under study and were: thrombocytopenia (n=51), neutropenia (47), anemia (43), leukopenia (41). Most common gr 1/2 AEs were: fatigue (n=48), constipation (29), nausea/cough (each 24), insomnia (19), diarrhea (18), abdominal pain (16). 4/56 pts died on study deemed not drug related (cardiac arrest, MOF, neoplasm, sudden death).Efficacy: With a median follow up of 14.5 ms the best response outcome for untreated AML/MDS patients was 23.1% and for rel/ref 7.1%. However, the rate of SD (NR) was remarkably high particularly in the rel/ref AML population at 76% (16/21 pts, 2 not yet assessed). This high disease control rate was reflected in an OS of 7.6 ms (4.6-NE) in rel/ref AML, the majority of which failed previous HMAs .Conclusion: In this, to date largest SMO inhibitor/HMA combination trial, the oral SMO inhibitor SON was safe with full dose AZA in untreated and previously treated AML, MDS/CMML and MF pts. Although remission rates are not superior to single agent AZA in the heavily pre-treated and advanced myeloid malignancy population, the OS and lack of progression are encouraging, particular in the rel/ ref AML population indicating that SMO/Hh pathway inhibition may add to HMA efficacy. Predictive biomarker studies are ongoing. DisclosuresMesa:Ariad: Consultancy; Galena Biopharma, Inc.: Consultancy; CTI BioPharma Corp.: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Incyte Corporation: Research Funding; Promedico: Research Funding; Gilead Sciences, Inc.: Research Funding; Celgene Corporation: Research Funding.

Resveratrol-mediated neurorestoration after cerebral ischemic injury - Sonic Hedgehog signaling pathway

AimsResveratrol pretreatment can decrease ischemic cerebral injury and enhance proliferation of neural stem cells via mediation of Sonic Hedgehog signaling. However, it is relatively little known about whether neurorestorative effects of resveratrol are mediated by Shh signaling in ischemic cerebral injury. The present study tests whether the Shh signaling pathway mediates resveratrol to promote neurorestoration of ischemic cerebral injury. Materials and methodsRats or neurons before middle cerebral artery occlusion/reperfusion (MCAO/R) or oxygen-glucose deprivation/reoxygenation (OGD/R) injury were pretreated with resveratrol. Immunohistochemistry is used to be determined BrdU+/DCX+, BrdU+/Nestin+ and BrdU+/NG2+ cell (markers of new proliferated neural stem/progenitor and oligodendrocyte precursor cell, respectively), BrdU+/MAP2+ and BrdU+/CNPase+ cell (markers of new mature neuron and oligodendrocyte, respectively), BrdU+/TUNEL+ cell (marker of apoptosis for new proliferated cell), SY, NF200, Iba-1 and GFAP (markers of synaptogenesis, axon, microglia and astrocyte, respectively). Shh and Gli-1 mRNAs were detected by RT-PCR assay. Iba-1, GFAP, Shh and Gli-1 proteins were detected by Western blot. Key findingsResveratrol pretreatment significantly reduced neurological deficit scores, promoted proliferation, differentiation, migration and survival of neural stem/progenitor and oligodendrocyte precursor cells, inhibited astrocyte and microglia activation, strengthened synaptophysin and NF200 expression, at the same time, promoted neurite outgrowth of neurons. Meanwhile, expression levels of Shh and Gli-1 proteins were significantly increased and Gli-1 translocated into the nucleus. However, cyclopamine, a Smo inhibitor, canceled the above effects of resveratrol. ConclusionsIt may be mediated, at least partly, by the Shh signaling pathway that resveratrol pretreament promote neurorestoration of ischemic cerebral injury.

Drug Delivery Systems for Hedgehog Inhibitors in the Treatment of SHH-Medulloblastoma.

Medulloblastoma (MB) is a highly aggressive pediatric tumor of the cerebellum. Hyperactivation of the Hedgehog (HH) pathway is observed in about 30% of all MB diagnoses, thereby bringing out its pharmacological blockade as a promising therapeutic strategy for the clinical management of this malignancy. Two main classes of HH inhibitors have been developed: upstream antagonists of Smoothened (SMO) receptor and downstream inhibitors of GLI transcription factors. Unfortunately, the poor pharmacological properties of many of these molecules have limited their investigation in clinical trials for MB. In this minireview, we focus on the drug delivery systems engineered for SMO and GLI inhibitors as a valuable approach to improve their bioavailability and efficiency to cross the blood–brain barrier (BBB), one of the main challenges in the treatment of MB.

Basal epidermis collective migration and local Sonic hedgehog signaling promote skeletal branching morphogenesis in zebrafish fins

Teleost fish fins, like all vertebrate limbs, comprise a series of bones laid out in characteristic pattern. Each fin's distal bony rays typically branch to elaborate skeletal networks providing form and function. Zebrafish caudal fin regeneration studies suggest basal epidermal-expressed Sonic hedgehog (Shh) promotes ray branching by partitioning pools of adjacent pre-osteoblasts. This Shh role is distinct from its well-studied Zone of Polarizing Activity role establishing paired limb positional information. Therefore, we investigated if and how Shh signaling similarly functions during developmental ray branching of both paired and unpaired fins while resolving cellular dynamics of branching by live imaging. We found shha is expressed uniquely by basal epidermal cells overlying pre-osteoblast pools at the distal aspect of outgrowing juvenile fins. Lateral splitting of each shha-expressing epidermal domain followed by the pre-osteoblast pools precedes overt ray branching. We use ptch2:Kaede fish and Kaede photoconversion to identify short stretches of shha+basal epidermis and juxtaposed pre-osteoblasts as the Shh/Smoothened (Smo) active zone. Basal epidermal distal collective movements continuously replenish each shha+domain with individual cells transiently expressing and responding to Shh. In contrast, pre-osteoblasts maintain Shh/Smo activity until differentiating. The Smo inhibitor BMS-833923 prevents branching in all fins, paired and unpaired, with surprisingly minimal effects on caudal fin initial skeletal patterning, ray outgrowth or bone differentiation. Staggered BMS-833923 addition indicates Shh/Smo signaling acts throughout the branching process. We use live cell tracking to find Shh/Smo restrains the distal movement of basal epidermal cells by apparent ‘tethering’ to pre-osteoblasts. We propose short-range Shh/Smo signaling promotes these heterotypic associations to couple instructive basal epidermal collective movements to pre-osteoblast repositioning as a unique mode of branching morphogenesis.

Smoothened inhibition leads to decreased cell proliferation and suppressed tissue fibrosis in the development of benign prostatic hyperplasia

Benign prostatic hyperplasia (BPH) is a common disease in aging males. It has been proven that the Hedgehog (HH) is implied as an effective and fundamental regulatory growth factor signal for organogenesis, homeostasis, and regeneration. Smoothened (SMO), as the major control point of HH signals, activates aberrantly in most human solid tumors. However, the specific function of SMO and its downstream glioma-associated oncogene (GLI) family in BPH has not been well understood. Here, we first revealed that the SMO cascade was upregulated in BPH tissues and was localized in both the stromal and the epithelium compartments of human prostate tissues. Cyclopamine, as a specific SMO inhibitor, was incubated with BPH-1 and WPMY-1, and intraperitoneally injected into a BPH rat model established by castration with testosterone supplementation. SMO inhibition could induce cell apoptosis, cell cycle arrest at the G0/G1 phase, and a reduction of tissue fibrosis markers, both in vitro and in vivo. Finally, a tissue microarray, containing 104 BPH specimens, was constructed to analyze the correlations between the expression of SMO cascade and clinical parameters. The GLI2 was correlated positively with nocturia and negatively with fPSA. The GLI3 was in a positive relationship with International Prostate Symptom Score and nocturia. In conclusion, our study suggested that SMO cascade could play important roles in the development of BPH and it might be rediscovered as a promising therapeutic target for BPH.

Targeting non-canonical activation of GLI1 by the SOX2-BRD4 transcriptional complex improves the efficacy of HEDGEHOG pathway inhibition in melanoma

Despite the development of new targeted and immune therapies, the prognosis of metastatic melanoma remains bleak. Therefore, it is critical to better understand the mechanisms controlling advanced melanoma to develop more effective treatment regimens. Hedgehog/GLI (HH/GLI) signaling inhibitors targeting the central pathway transducer Smoothened (SMO) have shown to be clinical efficacious in skin cancer; however, several mechanisms of non-canonical HH/GLI pathway activation limit their efficacy. Here, we identify a novel SOX2-BRD4 transcriptional complex driving the expression of GLI1, the final effector of the HH/GLI pathway, providing a novel mechanism of non-canonical SMO-independent activation of HH/GLI signaling in melanoma. Consistently, we find a positive correlation between the expression of GLI1 and SOX2 in human melanoma samples and cell lines. Further, we show that combined targeting of canonical HH/GLI pathway with the SMO inhibitor MRT-92 and of the SOX2-BRD4 complex using a potent Proteolysis Targeted Chimeras (PROTACs)-derived BRD4 degrader (MZ1), yields a synergistic anti-proliferative effect in melanoma cells independently of their BRAF, NRAS, and NF1 mutational status, with complete abrogation of GLI1 expression. Combination of MRT-92 and MZ1 strongly potentiates the antitumor effect of either drug as single agents in an orthotopic melanoma model. Together, our data provide evidence of a novel mechanism of non-canonical activation of GLI1 by the SOX2-BRD4 transcriptional complex, and describe the efficacy of a new combinatorial treatment for a subset of melanomas with an active SOX2-BRD4-GLI1 axis.

Steroidal alkaloids isolated from Veratrum grandiflorum Loes. as novel Smoothened inhibitors with anti-proliferation effects on DAOY medulloblastoma cells

Constitutive activation of Hedgehog (Hh) pathway is intimately related with the occurrence and development of several malignancies, such as medulloblastoma (MB) and other tumors. Therefore, small molecular inhibitors of Hh pathway are urgently needed. In this study, three new steroidal alkaloids, ⊿5 (20R, 24R) 23-oxo-24-methylsolacongetidine, ⊿5 (20S, 24R) 23-oxo-24-methylsolacongetidine and veralinine 3-O-α-l-rhamnopyranosyl-(1 → 2)-β-D-glucopyranoside, together with six known alkaloids, 20-epi-verazine, verazine, protoverine 15-(l)-2′-methylbutyrate, jervine, veramarine and β1-chaconine, were isolated and determined from Veratrum grandiflorum Loes. The dual-luciferase bioassay indicated that all compounds exhibited significant inhibitions of Hh pathway with IC50 values of 0.72–14.31 μM against Shh-LIGHT 2 cells. To determine whether these Hh pathway inhibitors act with the Smoothened (Smo) protein, which is an important oncoprotein and target for this pathway, BODIPY-cyclopamine (BC) competitive binding assay was preferentially performed. Compared with BC alone, all compounds obviously reduced the fluorescence intensities of BC binding with Smo in Smo-overexpression HEK293T cells through fluorescence microscope and flow cytometer. By directly interacting with Smo, it revealed that they were actually novel natural Smo inhibitors. Then, their anti-tumor effects were investigated against the human MB cell line DAOY, which is a typical pediatric brain tumor cells line with highly expressed Hh pathway. Interestingly, most of compounds had slight proliferation inhibitions on DAOY cells after treatment for 24 h same as vismodegib, while β1-chaconine showed the strongest inhibitory effect on the growth of DAOY with IC50 value of 5.35 μM. In conclusion, our studies valuably provide several novel natural Smo inhibitors for potential targeting treatment of Hh-dependent tumors.

077 Isoform-specific aPKC maintains Hedgehog signaling in the absence of primary cilia

Primary cilia loss is a common feature of advanced cancers. While primary cilia are necessary to initiate Hedgehog (HH)-driven cancers, how HH pathway activity is maintained in advanced cancers devoid of primary cilia is unclear. Here, we find that HH-driven basal cell carcinoma (BCC) and medulloblastoma accumulate mutations in the Alström and Usher syndrome genes. Loss of Alström and Usher syndrome gene expression, which are common underlying causes of deafness and blindness, suppresses ciliogenesis and HH signaling. Loss of primary cilia also enhances atypical protein kinase C iota/lambda (aPKC) expression, a GLI1 kinase necessary for advanced BCC growth. We show that aPKC expression is inversely correlated with primary ciliogenesis and that superficial BCCs display less primary cilia and higher aPKC expression, with the opposite true in nodular BCC subtypes. Surprisingly, a constitutively active isoform of aPKC drives HH pathway activity but not full-length protein. Overexpression of the constitutively active aPKC variant can maintain HH pathway activity in the absence of primary cilia and can drive resistance to the SMO antagonist vismodegib regardless of cilia status. Our results suggest tumors enhance isoform-specific expression of aPKC to prevent mutation-induced cessation of tumor growth and that aPKC may serve as a biomarker for SMO inhibitor sensitivity.

Reflectance Confocal Microscopy Identification of Subclinical Basal Cell Carcinoma after Vismodegib Treatment: Report of a Case.

Although surgery represents the treatment of choice for the majority of basal cell carcinomas, different therapies are required to treat the advanced ones. Vismodegib and sonidegib are the two oral Smoothened (Smo) inhibitors approved for the treatment of advanced basal cell carcinoma. Clinical detection of subclinical basal cell carcinoma during and after vismodegib treatment could be difficult, requiring the use of dermoscopy and reflectance confocal microscopy as noninvasive diagnostic methods. We report the case of a 62-year-old woman with a locally advanced basal cell carcinoma successfully treated with vismodegib in which dermoscopy and reflectance confocal microscopy showed their superiority in detecting subclinical recurrent basal cell carcinoma developed on scar tissue after complete regression of the tumor.

Inhibition of the FACT Complex Targets Aberrant Hedgehog Signaling and Overcomes Resistance to Smoothened Antagonists.

Hedgehog signaling is aberrantly activated in hematologic malignancies and solid tumors, and targeting it is a promising therapeutic strategy against these cancers. Resistance to clinically available hedgehog-targeted Smoothened inhibitor (SMOi) drugs has become a critical issue in hedgehog-driven cancer treatment. Our previous studies identified inhibition of BET and CDK7 as two epigenetic/transcriptional-targeted therapeutic strategies for overcoming SMOi resistance, providing a promising direction for anti-hedgehog drug development. To uncover additional strategies for inhibiting aberrant hedgehog activity, here we performed CRISPR-Cas9 screening with an single-guide RNA library targeting epigenetic and transcriptional modulators in hedgehog-driven medulloblastoma cells, combined with tumor dataset analyses. Structure specific recognition protein 1 (SSRP1), a subunit of facilitates chromatin transcription (FACT) complex, was identified as a hedgehog-induced essential oncogene and therapeutic target in hedgehog-driven cancer. The FACT inhibitor CBL0137, which has entered clinical trials for cancer, effectively suppressed in vitro and in vivo growth of multiple SMOi-responsive and SMOi-resistant hedgehog-driven cancer models. Mechanistically, CBL0137 exerted anti-hedgehog activity by targeting transcription of GLI1 and GLI2, which are core transcription factors of the hedgehog pathway. SSRP1 bound the promoter regions of GLI1 and GLI2, while CBL0137 treatment substantially disrupted these interactions. Moreover, CBL0137 synergized with BET or CDK7 inhibitors to antagonize aberrant hedgehog pathway and growth of hedgehog-driven cancer models. Taken together, these results identify FACT inhibition as a promising epigenetic/transcriptional-targeted therapeutic strategy for treating hedgehog-driven cancers and overcoming SMOi resistance. SIGNIFICANCE: This study identifies FACT inhibition as an anti-hedgehog therapeutic strategy for overcoming resistance to Smoothened inhibitors and provides preclinical support for initiating clinical trials of FACT-targeted drug CBL0137 against hedgehog-driven cancers.

Understanding the Hedgehog Signaling Pathway in Acute Myeloid Leukemia Stem Cells: A Necessary Step toward a Cure

Simple SummaryThe Hedgehog signaling pathway is related to the cell cycle. In particular, it is considered to play a fundamental role in the quiescence of leukemic stem cell (i.e., a temporary resting state without cell replication). Leukemic stem cells are the cells supposed to give rise to the relapses of the leukemia. Therefore, the Hedgehog pathway must be understood to improve the current treatments against acute myeloid leukemia and avoid the relapse of the disease. In this review, we gather the present knowledge about the physiological Hedgehog pathway function, the aberrant activation of Hedgehog in leukemia, and highlight the lack of evidence regarding some aspects of this important pathway. Finally, we summarize the acute myeloid leukemia treatments targeting this signaling pathway.A better understanding of how signaling pathways govern cell fate is fundamental to advances in cancer development and treatment. The initialization of different tumors and their maintenance are caused by the deregulation of different signaling pathways and cancer stem cell maintenance. Quiescent stem cells are resistant to conventional chemotherapeutic treatments and, consequently, are responsible for disease relapse. In this review we focus on the conserved Hedgehog (Hh) signaling pathway which is involved in regulating the cell cycle of hematopoietic and leukemic stem cells. Thus, we examine the role of the Hh signaling pathway in normal and leukemic stem cells and dissect its role in acute myeloid leukemia. We explain not only the connection between illness and the signaling pathway but also evaluate innovative therapeutic approaches that could affect the outcome of patients with acute myeloid leukemia. We found that many aspects of the Hedgehog signaling pathway remain unknown. The role of Hh has only been proven in embryo and hematopoietic stem cell development. Further research is needed to elucidate the role of GLI transcription factors for therapeutic targeting. Glasdegib, an SMO inhibitor, has shown clinical activity in acute myeloid leukemia; however, its mechanism of action is not clear.

The mammalian Hedgehog pathway is modulated by ANP32 proteins

Medulloblastoma (MB) is the most common malignant brain tumor in children. Transcriptional profiling has so far delineated four major MB subgroups of which one is driven by uncontrolled Hedgehog (Hh) signaling (SHH-MB). This pathway is amenable to drug targeting, yet clinically approved compounds exclusively target the transmembrane component Smoothened (SMO). Unfortunately, drug resistance against SMO inhibitors is encountered frequently, making the identification of novel Hh pathway components mandatory, which could serve as novel drug targets in the future. Here, we have used MB as a tool to delineate novel modulators of Hh signaling and have identified the Acidic Nuclear Phosphoprotein 32 (ANP32) family of proteins as novel regulators. The expression of all three family members (ANP32A, ANP32B, ANP32E) is increased in Hh-induced MB and their expression level is negatively associated with overall survival in SHH-MB patients. Mechanistically, we could find that ANP32 proteins function as positive modulators of mammalian Hh signaling upstream of GLI transcription factors. These findings add hitherto unknown regulators to the mammalian Hh signaling cascade and might spur future translational efforts to combat Hh-driven malignancies.

Identification of a potent antagonist of smoothened in hedgehog signaling

BackgroundHedgehog signaling is essential to the regulation of embryonic development, tissue homeostasis, and stem cell self-renewal, making it a prime target for developing cancer therapeutics. Given the close link between aberrant Hedgehog signaling and cancers, many small molecular compounds have been developed to inhibit Smoothened, a key signal transducer of this pathway, for treating cancer and several such compounds have been approved by the United States Food and Drug Administration (GDC-0449 and LDE-225). However, acquired drug resistance has emerged as an important obstacle to the effective use of these first generation Hedgehog pathway blockers. Thus, new Smoothened inhibitors that can overcome such resistance is an urgent need going forward.ResultsWe established the Smoothened/βarrestin2-GFP high-throughput screening platform based on the mechanistic discovery of Hedgehog signaling pathway, and discovered several active small molecules targeting Smoothened including 0025A. Here we show that 0025A can block the translocation of βarrestin2-GFP to Smoothened, displace Bodipy-cyclopamine binding to wild-type Smoothened or mutant Smoothened-D473H, reduce the accumulation of Smo on primary cilia and the expression of Gli upon Hedgehog stimulation. In addition, we show that 0025A can effectively suppress hair follicle morphogenesis and hair growth in mice.ConclusionsOur results demonstrate that 0025A is a potent antagonist targeting Smoothened wild-type and mutant receptors in the Hedgehog signaling pathway and may provide a new therapy for refractory cancers.

Epigallocatechin-3-gallate inhibits proliferation and triggers apoptosis in colon cancer via the hedgehog/phosphoinositide 3-kinase pathways.

The present study evaluated whether epigallocatechin-3-gallate (EGCG) effectively attenuates tumor growth in colon cancer cells and in the xenografts of nude mice and investigated the underlying mechanisms by focusing on the sonic hedgehog (Shh) and phosphoinositide 3-kinase (PI3K) pathways. Three kinds of colon cancer cells and BALB/c nude mice were used to evaluate the antiproliferative effect of EGCG. The apoptosis, migration, and invasion of colon cancer cells were analyzed to explore the toxicity effect of EGCG on colon cancer cells. Western blotting was used to demonstrate the expression levels of related proteins. The results showed that EGCG exhibited an antiproliferative effect against colon cancer cells in a dose-dependent manner with low toxicity against normal colon epithelial cells. Administration of EGCG caused significant apoptosis and inhibited the migration and invasion of colon cancer cells. The toxic effect of EGCG on colon cancer cells was accompanied by downregulation of the Shh and PI3K/Akt pathways. In addition, EGCG reduced tumor volume and weight without affecting the body weight of nude mice and inhibited the activation of the Shh and PI3K/AKT pathways in tumor tissue. Further study showed that purmorphamine (smoothened (Smo) agonist) or insulin like growth factor-1 (IGF-1, PI3K agonist) partly abolished the effect of EGCG on cell proliferation, migration, and apoptosis. Cyclopamine (Smo inhibitor) and LY294002 (PI3K inhibitor) showed the similar toxic effects as EGCG on colon cancer cells. In conclusion, EGCG inhibited colon tumor growth via downregulation of the Shh and PI3K pathways and may be a potential chemotherapeutic agent against colon cancer.

NCTD Retards AML HL60 Cell Proliferation via Targeting Hedgehog/SOX2 axis

To investigate the effect of norcantharidin (NCTD) to proliferation of leukemia cells through disrupting key regulators of sonic Hedgehog (SHH) pathway and its downstream transcription factor SOX2. CCK8 was used to detected the HL60 and NB4 cells after inhibited by NCTD, SMO and GLI1 inhibitor for 24 hours. Expression level of SMO, GLI1 and SOX2 in HL60 cells with NCTD treatment was detected by immunoblot. HL60 cells were transfected with pcDNA3.1 plasmid expressing GLI1 or SOX2. Empty vector and pcDNA3. 1-EGFP were divided into negative and positive control group, respectively. The expression of exogenous GLI1 or SOX2 in HL60 cells was confirmed by immunoblot, and growth curve of HL60 cell was checked by CCK8. Proliferation of genetic modified HL60 cells treated by various dose of NCTD was detected. NCTD, SMO/GLI1 inhibitors could inhibit the proliferation of NB4 and HL60 cells in a dose-dependent manner. Compared with solvent (DMSO)-treated control group, NCTD remarkably decreased protein level of SMO, GLI1 and SOX2. GLI1 and SOX2 were overexpressed in HL60 cells as compared with pcDNA3.1 empty vector-transfected group. Growth curve demonstrated significant proliferative advantage of GLI1/SOX2-transfected cells. CCK8 assay indicated that GLI1/SOX2-overexpressed HL60 cells were more resistant to NCTD treatment. NCTD attenuates HL60 proliferation via targeting the Hedgehog/SOX2 axis.