Hh Inhibitors Research Articles

Phase I trial of the hedgehog (Hh) inhibitor, LDE225, in combination with etoposide and cisplatin (EP) for initial treatment of extensive stage small cell lung cancer (ES-SCLC).

7602 Background: SCLC relies on the Hh developmental pathway for tumor initiation and progression. Pharmacologic blockade with Hh inhibitors blocks these processes. We performed a phase I study to ...

Hedgehog signaling and radiation induced liver injury: a delicate balance.

Radiation-induced liver disease (RILD) is a major limitation of radiation therapy (RT) for the treatment of liver cancer. Emerging data indicate that hedgehog (Hh) signaling plays a central role in liver fibrosis and regeneration after liver injury. Here, we review the potential role of Hh signaling in RILD and propose the temporary use of Hh inhibition during liver RT to radiosensitize HCC tumor cells and inhibit their progression, while blocking the initiation of the radiation-induced fibrotic response in the surrounding normal liver.

Tu1222 Non-Steroidal Anti-Inflammatory Drugs-Induced Small Intestinal Damage Is Associated With Autophagy and Apoptosis

were decreased 1.5-fold (± 0.2, p<0.02) and 7.1-fold (± 1.4, p<0.01) in the setting of acute Hh inhibition. Finally, mice exposed to LDE treatment for 6 days were predisposed to significant, damaging colitis secondary to 3% DSS challenge as evidenced by increased weight loss (35% vs 15%), increased histological damage and inflammatory infiltrate when compared to solvent treated controls exposed to DSS. Our initial evaluation of the effect of Hh targeted therapies on the gastrointestinal tract indicates that inhibiting epithelial derived Hh signaling leads to a pro-inflammatory immune environment thereby predisposing the GI tract to inflammatory mediated damage when challenged. While targeting the Hh pathway for treatment of malignancies is attractive and powerful, understanding the resultant effect on mucosal immune homeostasis is a critical component to developing safe and tolerable therapies. 1 Sekulic et al NEJM 2012; 366:2171-79. 2 Chang et al. J. Am Acad Dermatol. 2013 Nov 1, S0190-9622 (13). 3 Keating GM Drugs 2012 72(11)1535-41.

Tu1220 Rebamipide Inhibits Indomethacin-Induced Experimental Small Intestinal Injury and the Exacerbation of the Injury Induced by Proton Pump Inhibitor: Possible Involvement of Intestinal Microbiota Modulation by Rebamipide

were decreased 1.5-fold (± 0.2, p<0.02) and 7.1-fold (± 1.4, p<0.01) in the setting of acute Hh inhibition. Finally, mice exposed to LDE treatment for 6 days were predisposed to significant, damaging colitis secondary to 3% DSS challenge as evidenced by increased weight loss (35% vs 15%), increased histological damage and inflammatory infiltrate when compared to solvent treated controls exposed to DSS. Our initial evaluation of the effect of Hh targeted therapies on the gastrointestinal tract indicates that inhibiting epithelial derived Hh signaling leads to a pro-inflammatory immune environment thereby predisposing the GI tract to inflammatory mediated damage when challenged. While targeting the Hh pathway for treatment of malignancies is attractive and powerful, understanding the resultant effect on mucosal immune homeostasis is a critical component to developing safe and tolerable therapies. 1 Sekulic et al NEJM 2012; 366:2171-79. 2 Chang et al. J. Am Acad Dermatol. 2013 Nov 1, S0190-9622 (13). 3 Keating GM Drugs 2012 72(11)1535-41.

Suppression of Hedgehog signalling promotes pro‐tumourigenic integrin expression and function

Aberrant Hedgehog (Hh) signalling has been reported in a number of malignancies, particularly basal cell carcinoma (BCC) of the skin. Clinical trials of Hh inhibitors are underway in many cancers, and these have produced significant clinical benefit in BCC patients, although regrowth of new, or clinically aggressive, variants, as well as development of secondary malignancies, has been reported. αvβ6 integrin is expressed in many cancers, where it has been shown to correlate with an aggressive tumour phenotype and poor prognosis. We have previously reported αvβ6 up-regulation in aggressive, morphoeic BCC variants, where it modulates the stromal response and induces invasion. To examine a possible link between Hh and αvβ6 function, we generated BCC models, overexpressing Gli1 in immortalized keratinocytes (NTert1, HaCaT). Unexpectedly, we found that suppressing Gli1 significantly increased αvβ6 expression. This promoted tumour cell motility and also stromal myofibroblast differentiation through integrin-dependent TGF-β1 activation. Gli1 inhibited αvβ6 expression by suppressing TGF-β1-induced Smad2/3 activation, blocking a positive feedback loop maintaining high αvβ6 levels. A similar mechanism was observed in AsPC1 pancreatic cancer cells expressing endogenous Gli1, suggesting a common mechanism across tumour types. In vitro findings were supported using human clinical samples, where we showed an inverse correlation between αvβ6 and Gli1 expression in different BCC subtypes and pancreatic cancers. In summary, we show that expression of Gli1 and αvβ6 inversely correlates in tumours in vivo, and Hh targeting up-regulates TGF-β1/Smad2/3-dependent αvβ6 expression, promoting pro-tumourigenic cell functions in vitro. These results have potential clinical significance, given the reported recurrence of BCC variants and secondary malignancies in patients treated by Hh targeting.

Structure-activity relationships for vitamin D3-based aromatic a-ring analogues as hedgehog pathway inhibitors.

A structure-activity relationship study for a series of vitamin D3-based (VD3) analogues that incorporate aromatic A-ring mimics with varying functionality has provided key insight into scaffold features that result in potent, selective Hedgehog (Hh) pathway inhibition. Three analogue subclasses containing (1) a single substitution at the ortho or para position of the aromatic A-ring, (2) a heteroaryl or biaryl moiety, or (3) multiple substituents on the aromatic A-ring were prepared and evaluated. Aromatic A-ring mimics incorporating either single or multiple hydrophilic moieties on a six-membered ring inhibited the Hh pathway in both Hh-dependent mouse embryonic fibroblasts and cultured cancer cells (IC50 values 0.74-10 μM). Preliminary studies were conducted to probe the cellular mechanisms through which VD3 and 5, the most active analogue, inhibit Hh signaling. These studies suggested that the anti-Hh activity of VD3 is primarily attributed to the vitamin D receptor, whereas 5 affects Hh inhibition through a separate mechanism.

Hedgehog pathway as a potential treatment target in human cholangiocarcinoma.

Innovative treatment concepts targeting essential signaling pathways may offer new chances for patients suffering from cholangiocarcinoma (CCC). For that, we performed a systematic molecular genetic analysis concerning the Hedgehog activity in human CCC samples and analyzed the effect of Hh inhibition on CCC cells in vitro and in vivo. Activation of the Hh pathway was analyzed in 50 human CCC samples using quantitative polymerase chain reaction (qPCR). The efficacy of Hh inhibition using cyclopamine and BMS-833923 was evaluated in vitro. In addition, the effect of BMS-833923, alone or in combination with gemcitabine, was analyzed in vivo in a murine subcutaneous xenograft model. Expression analysis revealed a significant activation of the Hh-signaling pathway in nearly 50% of CCCs. Hh inhibition resulted in a significant decrease in cell proliferation of CCC cells. Moreover, a distinct inhibition of tumor growth could be seen as a result of a combined therapy with BMS-833923 and gemcitabine in CCC xenografts. The results of our study suggest that the Hh pathway plays a relevant role at least in a subset of human CCC. Inhibition of this pathway may represent a possible treatment option for CCC patients in which the Hh pathway is activated.

Effects of inhibition of hedgehog signaling on cell growth and migration of uveal melanoma cells

The Hedgehog (Hh) signaling pathway has been demonstrated to play a critical role in controlling embryonic development, tissue patterning, wound healing and a variety of cell functions. Aberrant activation of Hh signaling is implicated in the pathogenesis of many human cancers, and in angiogenesis. However, the role of this pathway in uveal melanoma (UM) carcinogenesis remains unknown. In this study, we investigated the effects of Hh inhibition using the specific Smoothened (Smo) antagonist cyclopamine to block Hh signaling in cultured human UM cell lines expressing Hh signaling components. Cyclopamine treatment effectively increased apoptosis and inhibited cell proliferation, migration, and epithelial-to-mesenchymal transition (EMT) by downregulating the Hh final arbiter glioblastoma 1 (Gli1), which regulates the transcription of target genes in the nucleus. Changes in gene and protein expression levels were detected by real-time PCR and by western blotting and immunocytochemistry, respectively. Cell cycle and apoptosis regulation induced by cyclopamine were demonstrated by flow cytometry. In addition, the migration capability of UM cells was reduced, as demonstrated by transwell migration and scratch assays. The effects of Hh inhibition on the levels of angiogenesis factors secreted by UM cells were examined by tube-formation assay. Conclusion: Blocking the Hh pathway by cyclopamine decreased cell viability, migration, EMT, and angiogenesis, increased apoptosis, and induced G1 phase cell cycle arrest in UM cells. Collectively, these results provide the first evidence of the significance of Gli1 activation downstream of Smo as a therapeutic target and the potential value of cyclopamine for the treatment of human UM.

Reoxygenation from chronic hypoxia promotes metastatic processes in pancreatic cancer through the Hedgehog signaling

Pancreatic ductal adenocarcinoma (PDAC) is among the most deadly types of malignancies because of its high ability to metastasize. PDAC is thought to be under hypoxic condition. Therefore, to investigate the mechanism of metastatic processes, chronic-hypoxia-resistant PDAC cells were newly generated under hypoxic condition for 3–6 months and reoxygenation experiments were performed using these chronic-hypoxia-resistant PDAC cells in in vivo-mimicking conditions. Proliferation, invasiveness and tumorigenicity in PDAC cells were significantly increased by reoxygenation. A Hedgehog (Hh) signaling component, Gli1, was significantly increased by reoxygenation. Gli1 knockdown inhibited reoxygenation-induced increases in proliferation and tumorigenicity and decreased invasiveness through suppression of matrix metalloproteinase (MMP) 2 and MMP9. Moreover, inhibition of Sonic Hh and Smoothened abrogated reoxygenation induced increases in proliferation and invasiveness. These results suggest that metastatic processes in PDAC are induced through activation of the Hh signaling pathway. Therefore, the Hh signaling pathway may be a therapeutic target for refractory PDAC in metastatic processes induced by reoxygenation.

A phase II study of vismodegib, a hedgehog (Hh) pathway inhibitor, combined with gemcitabine and nab-paclitaxel (nab-P) in patients (pts) with untreated metastatic pancreatic ductal adenocarcinoma (PDA).

257 Background: The Hh pathway is overexpressed in PDA tumors. Pre-clinically, Hh inhibitors have demonstrated a reduction in pancreatic cancer stem cells (pCSC) and stroma. Vismodegib, an oral small-molecule antagonist of the Hh pathway, has previously been safely combined with Gemcitabine chemotherapy. Methods: Pts with untreated, metastatic PDA were treated with Gemcitabine (1000 mg/m2) + nab-P (125 mg/m2) on days 1, 8 and 15 of 28 days cycle. Vismodegib (150mg PO daily) was started on the second cycle. All drugs were continued until disease progression or unacceptable toxicities. Primary endpoint was progression-free survival (PFS). Secondary endpoints were overall survival (OS), response rate (RR), and toxicity. Pre and post treatment tumor biopsies were obtained from primary or metastatic lesions. Results: 59 patients have been enrolled at 3 sites. Median age 60 (range 42-86); ECOG PS 0/1: 23 (40%)/ 34 (60%); male/female 32 (54%)/ 27 (46%). Estimated median PFS and OS in ITT population was 5.5 and 10 mo respectively (95% CI: 5.2-5.9 / 7.3-11). Of the 49 pts evaluable for response to date, 1 (2%) had CR, 20 (41%) had PR, 21 (43%) had SD and 7 (14%) had PD. Common Gr ≥3 toxicities: neutropenia 37.5% (n=21), anemia 21.4% (n=12), neuropathy 16.1% (n=9) and fatigue 9.4% (n=5). All patients with partial response had response within the primary pancreatic tumor. CA19-9 declines of &gt;70% occurred in 57% of patients with measurable levels. Conclusions: Addition of Vismodegib to Gemcitabine/nab-P is well tolerated in patients with untreated PDA. This trial is ongoing to complete 80 patients. Blood and tumor tissue biomarker analyses for stem cells, Hh signaling and stromal activity are ongoing and will be reported in ASCO GI 2014. Clinical trial information: NCT01088815.

Physalin H from Solanum nigrum as an Hh signaling inhibitor blocks GLI1–DNA-complex formation

Hedgehog (Hh) signaling plays an important role in embryonic development, cell maintenance and cell proliferation. Moreover, Hh signaling contributes to the growth of cancer cells. Physalins are highly oxidized natural products with a complex structure. Physalins (1–7) were isolated from Solanum nigrum (Solanaceae) collected in Bangladesh by using our cell-based assay. The isolated physalins included the previously reported Hh inhibitors 5 and 6. Compounds 1 and 4 showed strong inhibition of GLI1 transcriptional activity, and exhibited cytotoxicity against cancer cell lines with an aberrant activation of Hh signaling. Compound 1 inhibited the production of the Hh-related proteins patched (PTCH) and BCL2. Analysis of the structures of different physalins showed that the left part of the physalins was important for Hh inhibitory activity. Interestingly, physalin H (1) disrupted GLI1 binding to its DNA binding domain, while the weak inhibitor physalin G (2) did not show inhibition of GLI1-DNA complex formation.

Hedgehog signaling pathway as a new therapeutic target in pancreatic cancer

Pancreatic cancer is one of the most aggressive and difficult cancers to treat. Despite numerous research efforts, limited success has been achieved in the therapeutic management of patients with this disease. In the current review, we focus on one component of morphogenesis signaling, Hedgehog (Hh), with the aim of developing novel, effective therapies for the treatment of pancreatic cancer. Hh signaling contributes to the induction of a malignant phenotype in pancreatic cancer and is responsible for maintaining pancreatic cancer stem cells. In addition, we propose a novel concept linking Hh signaling and tumor hypoxic conditions, and discuss the effects of Hh inhibitors in clinical trials. The Hh signaling pathway may represent a potential therapeutic target for patients with refractory pancreatic cancer.

Abstract OT2-6-12: A randomized pacebo-controlled phase II trial evaluating the effect of hedgehog inhibitor LDE225 on bone marrow disseminated tumor cells in women with early stage estrogen receptor negative and HER2 negative breast cancer

Abstract Background: 20% of invasive breast cancers are triple negative (TN). Chemotherapy improves the outcome for a subset of these patients. Recurrence rates of 40-50% have been reported in those patients who do not achieve a complete pathological response to neoadjuvant therapy. Currently, there are no targeted therapies to TN cancers. Data suggest that micrometastases or disseminated tumor cells (DTCs) that persist despite chemotherapy are enriched with cells that have stem cell-like features. The Hedgehog (Hh) signaling pathway, which is involved in modulating epithelial-mesenchymal transition and maintenance of breast cancer stem cells, is an attractive therapeutic target for patients with TN tumors. In preclinical models, we have shown that Hh inhibition reduced breast cancer growth and metastasis. Trial Design: This is a placebo-controlled, double blinded, randomized phase II trial in early stage TN breast cancer patients with detectable DTCs in their bone marrow (BM) at the completion of all therapy. LDE225 is administered at 400 mg orally daily for a 28 day cycle for 20 cycles. After 6 cycles, BM will be collected for analysis. Specific Aims: The primary endpoint is the elimination of DTCs after 6 cycles of LDE225. Additional endpoints include: 2-year disease-free survival (DFS) and overall survival (OS), effect on BM Ptch1 gene expression, toxicity profile of LDE225, DFS and OS in patients with no DTCs at screening versus DTC-positive patients in the placebo arm. Eligibility: Patients with ER-/Her2- stage I-III invasive breast cancers who have completed all therapy and have detectable DTCs, as defined by a molecular based assay for DTCs performed in a CLIA-licensed facility are eligible. Statistical Methods: Based on preliminary data, 60% of patients in the LDE225 arm are expected to be Ptch1 positive, and 50% of these patients are expected eliminate DTCs after 6 cycles of treatment. The expected clearance rate in the remaining 40% of the LDE225 arm is 5% with an overall clearance rate of 32%. In the placebo arm, the expected clearance rate is 5% regardless of Ptch1 status. A sample of 68 patients, 34 in each treatment arm, will have power ∼ 0.8 at a 0.05 significance level, after adjusting for one interim analysis, to detect a difference in DTC clearance rate of 32% in the LDE225 arm versus 5% in the placebo arm. Assuming that 40% of patients screened have detectable DTCs, approximately 170 patients will be screened to identify 68 with detectable DTCs. Enrollment will be suspended for 3 months after the first 6 patients have been randomized to LDE225 therapy to allow time to document toxicity and adverse events. One interim analysis will be conducted after 50% of the patients have completed 6 months on study. The proportion of patients who are DTC-positive at 6 months will be estimated. The power of the study power to achieve its primary goal will be calculated conditional on results obtained to that point. The significance level of the final analysis will be adjusted to 0.048 in order to maintain an overall 0.05 significance level. Accrual: Accrual is anticipated to begin August 2013. Target = 34 per arm. NCT01757327. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr OT2-6-12.

Repair-associated inflammation in nonalcoholic fatty liver disease

The mechanisms that drive non-alcoholic fatty liver disease (NAFLD) progression from simple steatosis to non-alcoholic steatohepatitis (NASH) and NASH-fibrosis and/or cirrhosis are complex. Recent studies suggest that the liver progenitor cell (ie liver stem cell) population expands during chronic liver injury, and is an essential component of the repair process. Hedgehog (Hh) is a developmental morphogen that has an important role in the adult tissue repair (and progenitor) response. Accumulating data in mice and human show that resurrection of the Hh pathway occurs during progressive NAFLD, and that activity of this pathway correlates with NASH-fibrosis stage. Importantly, Hh ligands secreted by dying (or stressed) hepatocytes, hepatic stellate cells (i.e. myofibroblasts), cholangiocytes and recruited immune cells can act on neighbouring cells to perpetuate the fibrogenic response. Intriguingly, Hh ligands can also stimulate cholangiocytes to secrete chemokines that recruit immune cell subsets (such as natural killer T cells), which could explain why fibrosis generally occurs in the context of chronic inflammation (i.e. fibrosis-associated inflammatory response). Finally, the administration of Hh inhibitors led to reduced fibrosis in a model of NASH. Future studies are needed to evaluate the utility of these inhibitors in other models of chronic liver disease. If successful, this could pave the way for the development of new therapy for patients with NASH, because Hh pathway inhibitors have now been licensed for use in patients with advanced basal cell carcinoma.

Nanomedicines of Hedgehog Inhibitor and PPAR-γ Agonist for Treating Liver Fibrosis

Hedgehog (Hh) and peroxisome proliferator-activated receptor gamma (PPAR-γ) are major signaling pathways involved in the pathogenesis of liver fibrosis. Since Hh inhibitor, vismodegib (GDC) and PPAR-γ agonist, rosiglitazone (RSG) have poor water solubility, our objective was to formulate biodegradable polymeric nanoparticles encapsulating GDC and RSG for treating liver fibrosis. Methoxy-polyethylene-glycol-b-poly(carbonate-co-lactide) [mPEG-b-p(CB-co-LA)] was synthesized and characterized using (1)H NMR. Nanoparticles were prepared using this polymer by emulsification/solvent evaporation method to encapsulate GDC and RSG either alone or in combination. Nanoparticles were characterized for particle size, drug loading, drug release, and anti-fibrotic efficacy after tail vein injection into common bile duct ligated (CBDL) fibrotic rats. mPEG-b-p(CB-co-LA) copolymer has molecular weight of 30,000Da as determined by (1)H NMR. Nanoparticles were monodisperse with a mean particle size of 120-130nm. Drug loading was 5% and 2% w/w for GDC and RSG, respectively. Nanoparticles carrying both GDC and RSG were formulated at half of their individual drug loading. Systemic administration of drug loaded nanoparticles protected liver injury in CBDL rats by suppressing the activation of hepatic stellate cells, and decreasing inflammatory cytokines. Polymeric nanoparticles for co-delivery of Hh inhibitor and PPAR-γ agonist have the potential to treat liver fibrosis by intervening complex fibrotic cascade.

Improved Efficacy Of Combination Of JAK2 and Hedgehog Inhibitors In Myelofibrosis

The JAK1/2 dual kinase inhibitor INC424 (ruxolitinibª, Jakafi¨) has been recently approved for the treatment of intermediate or high-risk myelofibrosis (MF) and is in late-stage clinical trials for polycythemia vera (PV). Although JAK inhibitors have been shown to improve patient constitutional symptoms and reduce splenomegaly, JAK inhibitor monotherapy does not significantly reduce mutant allele burden in the majority of MPN patients. Furthermore, the therapeutic window of JAK inhibitors is limited due to the essential role of the JAK-STAT signaling pathway in normal hematopoiesis, which has been observed in the clinic where these inhibitor have been associated with dose limiting toxicities including anemia and thrombocytopenia. Therefore, there is a need to identify additional pathways that might be involved in the development and maintenance of MPN mutant clones, which could then be targeted in combination with JAK2 for improved therapeutic benefit. Recent studies have shown that the hedgehog (Hh) signaling pathway has important roles in normal hematopoiesis as well as in the pathogenesis of myeloid malignancies. We therefore investigated whether the Hh pathway was activated in MPN patients and whether it represented a therapeutic target in MPN preclinical models.We observed 20-100 fold increase in expression of Hh target genes including Gli1 and Ptch1 by quantitative PCR in granulocytes isolated from MPN patients as compared to normal controls. We also observed activation of the pathway in a murine bone marrow transplant (BMT) model of PMF using a Gli-luciferase reporter system. These data demonstrate the Hh pathway is active in primary MPN patient samples and in preclinical, models of PMF.Small molecule inhibitors of Hh signaling have shown promising results in Phase I clinical trials in patients with myeloid malignancies. In this study, we tested the effects of LDE225 (Sonidegib), a selective Smo inhibitor in combination with INC424 in a murine BMT model of ET/MF. Mice were treated with vehicle, 90mg/kg INC424 or a combination of INC424 and 80mg/kg LDE225 for 28 days. We observed a significant reduction in WBC (32.74K/ul vs. 9.85K/ul, p<0.02) and platelets (1243.6 K/ul vs. 913.25K/ul, p<0.02) in mice treated with INC424 + LDE225 as compared to INCB18424 monotherapy (Figure 1). Combination treatment also resulted in a reduction of mutant allele burden in the bone marrow, which is not observed with INC424 or LDE225 treatment alone. Notably, we observed a significant reduction in bone marrow fibrosis with the addition of LDE225 to INC424 in this model compared to INC424 monotherapy. In summary, combination treatment of Hh and JAK inhibitors demonstrates increased efficacy in MPN. Thus, combination studies with JAK and Hh inhibitors are warranted in the clinical setting to improve outcomes for MPN patients. [Display omitted] Reduction in blood counts and spleen weights observed in combination of INC424 and LDE225 as compared to INC424 monotherapy. Disclosures:Rose:Novartis Pharmaceutical: Employment. Amakye:Novartis Pharmaceutical: Employment.

Treatment With Hedgehog Inhibitor, PF-04449913, Attenuates Leukemia-Initiation Potential In Acute Myeloid Leukemia Cells

BackgroundAberrant activation of the Hedgehog (Hh) signaling pathway is involved in a variety of cancers, and required for maintenance of the leukemic stem cell (LSC) populations in several experimental systems. Cumulative evidence suggests that dormant self-renewing LSC contribute to acute myeloid leukemia (AML) propagation and relapse by evading conventional chemotherapies that target cycling cells. PF-04449913 (PF) is a novel oral small molecule inhibitor that selectively binds and targets Smoothened (SMO), a membrane protein regulating the Hh pathway. Treatment with PF has shown promising Results regarding safety, tolerability, and early signs of efficacy in a phase 1 dose-escalation study of hematologic malignancies including AML (Jamieson C, et al. ASH, 2011). On the basis of these encouraging Results, phase 1b and phase 2 studies of PF in combination with chemotherapies have been planned in patients with AML. However, the detailed mechanisms and biomarkers remain to be elucidated in AML therapy with Hh pathway inhibitors. Research Aims and MethodsWe used AML cell lines and patient-derived primary AML cells in order to evaluate the efficacy and elucidate detailed mechanisms and biomarkers in the Hh antagonist, PF treatment. Using the co-culturing system with HS-5 stromal cells, the colony assay system, and the immunodeficient NOD/SCID/IL2rgnull (NOG) mouse model serially xenotransplanted with primary AML cells, we examined the effects of PF on LSC population and AML propagation. ResultsUsing FACS sorting and RQ-PCR assays of AML patient-derived primary cells, the Hh signaling pathway was activated more in CD34-positive cells than CD34-negative cells. Ex vivo-treatment with PF inhibited proliferation and induced minimal cell death in leukemia cell lines and primary AML cells. However, in vivo-treatment with PF attenuated leukemia-initiation potential in AML cells through the serial transplantation system, while limiting reduction of tumor burden in the primary leukemia system. Also in the colony-assay system using primary AML cells, treatment with PF reduced serially colony formation. In MOLM-14 cells, treatment with PF down-regulated mRNA encoding downstream effector GLIs and GLI-targeting molecules in the canonical Hh pathway using RQ-PCR assays, and decreased nuclear expression of GLI-2 using immunofluorescence assays. In addition, treatment with PF remarkably decreased the quiescent (Hoechst-33342low/Pyronin-Ylow) cell population and increased cycling cell population. In the in vivo-NOG mouse system, comprehensive Gene Set Enrichment Analysis (GSEA) revealed that PF treatment modulated cell cycle regulation and self-renewal signaling in primary AML cells. Moreover, combined treatment with PF abrogated resistance to Ara-C in AML cell lines co-cultured with HS-5 stromal cells and sensitized primary AML cells to Ara-C in the colony-assay system. We are also investigating toxicity for normal cord blood cells with PF treatment. ConclusionsOur findings imply that selective Hh inhibitor, PF treatment can attenuate the leukemia-initiation potential in AML cells by modulation of cell cycle regulation and self-renewal signaling, and can also improve AML therapy through sensitizing dormant LSC to chemotherapy and overcoming the resistance in the bone marrow microenvironment. Disclosures:Kiyoi:Kyowa Hakko Kirin Co. Ltd.: Research Funding; Novartis Pharma: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Bristol-Myers Squibb: Research Funding.

Inhibition of Hedgehog and Androgen Receptor Signaling Pathways Produced Synergistic Suppression of Castration-Resistant Prostate Cancer Progression

Metastatic prostate cancer is initially treated with androgen ablation therapy, which causes regression of androgen-dependent tumors. However, these tumors eventually relapse resulting in recurrent castration-resistant prostate cancer (CRPC). Currently, there is no effective therapy for CRPC and the molecular mechanisms that lead to the development of CRPC are not well understood. Here, we evaluated the hypothesis that combined inhibition of Hedgehog (Hh) and androgen receptor (AR) signaling will synergistically attenuate the growth of CRPC in vitro and in vivo. Androgen deprivation induced full-length androgen receptor protein levels in CRPC cells, but decreased its nuclear localization and transcriptional activity. However, androgen deprivation also increased a truncated form of androgen receptor (lacking ligand-binding domain) that possessed transcriptional activity in CRPC cells. Androgen deprivation also promoted the expression of Hh signaling components in CRPC cells, xenograft tumors, and the prostate glands of castrated mice. Importantly, although inhibition of either Hh or androgen receptor signaling alone was only moderately effective in blocking CRPC cell growth, combination of an Hh pathway inhibitor and a noncompetitive androgen receptor inhibitor synergistically suppressed the growth of CRPC cells in vitro and in vivo. Finally, noncompetitive inhibition of androgen receptor, but not competitive inhibition, was effective at limiting the activity of truncated androgen receptor leading to the inhibition of CRPC. Combined therapy using Hh inhibitors and a non-competitive AR inhibitor may limit CRPC growth.

Abstract PR05: Resistance to Hedgehog inhibitor through Smoothened receptor mutation in basal cell carcinoma.

Abstract The inappropriate activation of Hedgehog (Hh) pathway is involved in the pathogenesis of skin basal cell carcinoma (BCC). Loss of function of the transmembrane receptor Patched (PTCH1), or gain of function of the Smoothened (SMO), Sonic Hh (SHH), and GLI family transcription factors can be ligand-independent oncogenic drivers of this disease. The activity of Vismodegib, a small molecule inhibitor of the Hh pathway that binds and inhibits SMO, is currently being evaluated in clinical trials against advanced BCC. Here, we report two cases of BCC, the first showing primary resistance to Vismodeginb treatment and the second characterized by a dramatic response to Vismodegib treatment (150 mg/day) followed by insurgence of secondary resistance. Formalin-fixed specimens of pre-treatment primary tumors and, for the second case, recurrence arisen during Vismodegib regimen were investigated for the mutational status of the serpentine receptor SMO by sequencing (3500DX Genetic Analyzer). In the case of primary resistance, SMO sequencing revealed the presence of the missense point mutation G497W while in the case of secondary resistance the D473Y mutant isoform of SMO receptor was found in the recurrence specimen. This substitution was not detected in the corresponding primary tumor specimen. In silico simulations of the wild type and mutated SMO receptor proteins in complex with Vismodegib showed that two different mechanisms are at work leading to primary and secondary inhibitor resistance, respectively. In the case of G497W (primary resistance), this residue is in a distal position with respect to the receptor drug binding site; however, the mutant residue reflects in a rearrangement of the corresponding loop ultimately leading in a partial obstruction to inhibitor entry. In the case of D473Y (secondary resistance), this residue is involved with other two residues (R400, H470) in a peculiar hydrogen bonds network, and hence appears to play a crucial role in stabilizing Vismodegib binding to the receptor. The mutation D473Y alters this equilibrium leading to a substantial decrease in receptor affinity for the inhibitor. Accordingly, the G497W and the D473Y mutations in SMO could represent two paradigm of primary and acquired resistance to Vismodegib in BCC, driven by different mechanism of SMO/Vismodegib interactions. Further ongoing elucidation of such mechanisms will help in developing therapeutic strategies able to overcome both type of Hh inhibitors resistance. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PR05. Citation Format: Paolo Bossi, Federica Perrone, Barbara Cortellazzi, Lisa Licitra, Valentina Dal Col, Erik Laurini, Maurizio Fermeglia, Sabrina Pricl. Resistance to Hedgehog inhibitor through Smoothened receptor mutation in basal cell carcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PR05.

Histone Acetyltransferase PCAF Is Required for Hedgehog–Gli-Dependent Transcription and Cancer Cell Proliferation

The Hedgehog (Hh) signaling pathway plays an important role in embryonic patterning and development of many tissues and organs as well as in maintaining and repairing mature tissues in adults. Uncontrolled activation of the Hh-Gli pathway has been implicated in developmental abnormalities as well as in several cancers, including brain tumors like medulloblastoma and glioblastoma. Inhibition of aberrant Hh-Gli signaling has, thus, emerged as an attractive approach for anticancer therapy; however, the mechanisms that mediate Hh-Gli signaling in vertebrates remain poorly understood. Here, we show that the histone acetyltransferase PCAF/KAT2B is an important factor of the Hh pathway. Specifically, we show that PCAF depletion impairs Hh activity and reduces expression of Hh target genes. Consequently, PCAF downregulation in medulloblastoma and glioblastoma cells leads to decreased proliferation and increased apoptosis. In addition, we found that PCAF interacts with GLI1, the downstream effector in the Hh-Gli pathway, and that PCAF or GLI1 loss reduces the levels of H3K9 acetylation on Hh target gene promoters. Finally, we observed that PCAF silencing reduces the tumor-forming potential of neural stem cells in vivo. In summary, our study identified the acetyltransferase PCAF as a positive cofactor of the Hh-Gli signaling pathway, leading us to propose PCAF as a candidate therapeutic target for the treatment of patients with medulloblastoma and glioblastoma.