Smoothened Inhibitor Research Articles

Melanoma of the Breast with Smoothened (SMO) Mutation: Case Report and review of the Literature

We report the case of a 75 year-old female with past history of ampullary adenocarcinoma presenting with a rapidly enlarging breast mass, initially misclassified on fine needle aspiration as a probable sarcoma, which was ultimately diagnosed as melanoma on resection in the absence of a known cutaneous primary lesion. Next-generation sequencing (NGS) of the tumor revealed a mutation in the Smoothened oncogene (SMO) of unknown significance and wild-type BRAF. To our knowledge, SMO mutation in melanoma of any site has not been previously reported, though the effectiveness of SMO inhibitors has been studied in both in vivo and in vitro models of melanoma. Currently, these inhibitors have not been studied in SMO mutant melanoma. The patient declined further therapy after resection due to multiple comorbidities. She expired two years after presenting with the breast mass from complications of high grade urothelial carcinoma.

Abstract P5-07-03: Small molecule inhibition of smoothened in triple negative breast cancer-associated fibroblasts depletes cancer stem cells and sensitizes to cytotoxic chemotherapy in mice and humans

Abstract The cellular and molecular basis of stromal cell recruitment, activation and crosstalk in carcinomas is poorly understood, limiting the development of targeted anti-stromal therapies. In mouse models of triple negative breast cancer (TNBC), we use single cell genomics to show that Hh ligand produced by neoplastic cells reprograms cancer-associated fibroblast (CAF) gene expression, driving tumor growth and metastasis. Hh-activated CAFs upregulated expression of FGF5 and deposition of fibrillar collagen, leading to FGFR and FAK activation in adjacent neoplastic cells and the acquisition of a stem-like, drug-resistant phenotype. Treatment with smoothened inhibitors (SMOi) reversed these phenotypes. Stromal treatment of TNBC patient-derived xenograft (PDX) models with SMOi downregulated the expression of cancer stem cell markers and sensitized tumors to docetaxel, leading to markedly improved survival and reduced metastatic burden. In the phase I clinical trial EDALINE, 3 patients with metastatic TNBC derived clinical benefit from combination therapy with the SMOi Sonedigib and docetaxel chemotherapy, with one patient experiencing a complete radiological response. Responders also exhibited high baseline FGFR activation and ECM deposition, suggesting a mechanism of action consistent with findings from the animal models. These studies identify Hh signaling to CAFs as a novel mediator of cancer stem cell plasticity and drug resistance and an exciting new therapeutic target in TNBC. Citation Format: Swarbrick A, Cazet A, Hui M, O'Toole S, Lim E, Martin M. Small molecule inhibition of smoothened in triple negative breast cancer-associated fibroblasts depletes cancer stem cells and sensitizes to cytotoxic chemotherapy in mice and humans [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-07-03.

A CK1α Activator Penetrates the Brain and Shows Efficacy Against Drug-resistant Metastatic Medulloblastoma.

Although most children with medulloblastoma are cured of their disease, Sonic Hedgehog (SHH) subgroup medulloblastoma driven by TRP53 mutations is essentially lethal. Casein kinase 1α (CK1α) phosphorylates and destabilizes GLI transcription factors, thereby inhibiting the key effectors of SHH signaling. We therefore tested a second-generation CK1α activator against TRP53-mutant, MYCN-amplified medulloblastoma. The ability of this CK1α activator to block SHH signaling was determined in vitro using GLI reporter cells, granular precursor primary cultures, and PATCHED1 (PTCH1)-mutant sphere cultures. While in vivo efficacy was tested using 2 different medulloblastoma mouse models: PTCH1 and ND2:SMOA1. Finally, the clinical relevance of CK1α activators was demonstrated using a TRP53-mutant, MYCN-amplified patient-derived xenograft. SSTC3 inhibited SHH activity in vitro, acting downstream of the vismodegib target SMOOTHENED (SMO), and reduced the viability of sphere cultures derived from SHH medulloblastoma. SSTC3 accumulated in the brain, inhibited growth of SHH medulloblastoma tumors, and blocked metastases in a genetically engineered vismodegib-resistant mouse model of SHH medulloblastoma. Importantly, SSTC3 attenuated growth and metastasis of orthotopic patient-derived TRP53-mutant, MYCN-amplified, SHH subgroup medulloblastoma xenografts, increasing overall survival. Using a newly described small-molecule, SSTC3, we show that CK1a activators could address a significant unmet clinical need for patients with SMO inhibitor-resistant medulloblastoma, including those harboring mutations in TRP53.

Loss of Primary Cilia Drives Switching from Hedgehog to Ras/MAPK Pathway in Resistant Basal Cell Carcinoma

Basal cell carcinomas (BCCs) rely on Hedgehog (HH) pathway growth signal amplification by the microtubule-based organelle, the primary cilium. Despite naive tumor responsiveness to Smoothened inhibitors (Smoi), resistance in advanced tumors remains common. Although the resistant BCCs usually maintain HH pathway activation, squamous cell carcinomas with Ras/MAPK pathway activation also arise, and the molecular basis of tumor type and pathway selection are still obscure. Here, we identify the primary cilium as a critical determinant controlling tumor pathway switching. Strikingly, Smoothened inhibitor-resistant BCCs have an increased mutational load in ciliome genes, resulting in reduced primary cilia and HH pathway activation compared with naive or Gorlin syndrome patient BCCs. Gene set enrichment analysis of resistant BCCs with a low HH pathway signature showed increased Ras/MAPK pathway activation. Tissue analysis confirmed an inverse relationship between primary cilia presence and Ras/MAPK activation, and primary cilia removal in BCCs potentiated Ras/MAPK pathway activation. Moreover, activating Ras in HH-responsive cell lines conferred resistance to both canonical (vismodegib) and noncanonical (atypical protein kinase C and MRTF inhibitors) HH pathway inhibitors and conferred sensitivity to MAPK inhibitors. Our results provide insights into BCC treatment and identify the primary cilium as an important lineage gatekeeper, preventing HH-to-Ras/MAPK pathway switching.

Targeting the cross-talk between the hedgehog and NF-κB signaling pathways in multiple myeloma

Multiple myeloma (MM) is an incurable plasma cell malignancy. Aberrant activation of the Hedgehog (Hh) and NF-κB signaling pathways is observed in MM and plays a pivotal role in the development of MM by promoting myeloma cell growth, survival, and drug resistance. In this study, we found that the Sonic Hh (SHh) ligand in the bone marrow microenvironment is responsible for the enhancement of NF-κB activity in MM cell lines NCI-H929 and U266. Notably, we discovered that Hh signaling regulates NF-κB through its classical pathway (SHh/PTCH1/SMO/GLI1) in MM cells. Meanwhile, non-classical pathway by SMO recruitment of TRAF6 to ubiquitination is also involved in it. Moreover, the SMO inhibitor cyclopamine enhances the cytotoxic effects of bortezomib in MM cell lines. Our study reveals the cross-talk between Hh members and the NF-κB pathway in the myeloma cells and provides a theoretical basis for combined utilization of Hh members and proteasome inhibition in MM.

Promotion of osteogenesis by bioactive glass–ceramic coating: Possible involvement of the Hedgehog signaling pathway

PurposeBioactive glass–ceramic (BGC) coatings have been extensively studied and clinically used as bone substitute materials because of their osteogenesis, osteoinduction, and osteoconduction characteristics. Although the Hedgehog (Hh) signaling pathway plays an important role in skeletal development, the relationship between BGC coatings and the Hh signaling pathway is unknown. MethodsIn this study, a BGC coating is fabricated by furnace sintering, and its surface is investigated by a scanning electron microscope (SEM) and a transmission electron microscope (TEM). Furthermore, the expression of Ki67 is evaluated using immunofluorescence, and osteogenesis-related factors and Hh signaling pathway molecules on the BGC coating are examined by real-time reverse transcription polymerase chain reaction (real-time RT-PCR) and Western blotting in bone marrow mesenchymal stem cells (BMSCs). ResultsThe SEM and the TEM show that the BGC coating surface is smooth, without cracks, and composed of particles with mesoporous structure. The expression of Ki67 positive BMSCs of the BGC group is higher than that of the control group. Real-time RT-PCR and Western blotting assay reveal that the expression levels of osteoblast-related genes (BMP2, Osteocalcin, ALP, Runx2) and Hh signaling pathway molecules (Gli1, Smo) are much higher for the BGC coating group than those for the control group. Furthermore, after treating with Smo inhibitor cyclopamine, the Smo and Gli1 expressions in BMSCs are dramatically down-regulation for the BGC coating compared to those for the control group. Both mRNA and protein expression levels of osteogenesis-related factors was downregulated after treating Smo inhibitor cyclopamine in BMSCs with the BGC coating. ConclusionsThe BGC coatings promote osteogenesis probably via the Hh signaling pathway, which provides a theory reference for future clinical application of bone formation.

Efficacy of Hedgehog inhibitors in acute myeloid leukemia and establishment of valid biomarkers

Hedgehog (Hh) signaling pathway plays an important role in embryonic patterning and organ development. Its aberrant activation is involved in many types of cancers and is required for the maintenance of patients with leukemic stem cell (LSC). Clinical phase trials of SMO (a positive regulator in the Hh signaling pathway) inhibitors are currently underway in hematologic malignancies, such as acute myeloid leukemia (AML).

Hedgehog pathway permissive conditions allow generation of immortal cell lines from granule cells derived from cancerous and non-cancerous cerebellum.

Cerebellar granule cell progenitors (GCPs) undergo proliferation in the post-natal cerebellum that is dependent on sonic hedgehog (SHH) signalling. Deregulated SHH signalling leads to type 2 medulloblastoma (MB). In this work, a novel cell culture protocol is described, which is suitable for the establishment and long-term maintenance of GCP-derived cells. This method is first applied to SHH pathway active MB cells from Atoh1-cre;Ptch1FL/FL tumours, which leads to the generation of neurosphere-like cell lines expressing GCP markers and an active SHH signalling pathway. These cells also show high sensitivity to the Smoothened inhibitor vismodegib, therefore recapitulating the SHH pathway requirement for survival shown by type 2 MB. Analysis of culture supplements reveals that bFGF and fetal bovine serum act as inhibitors of the SHH pathway and therefore preclude generation of cell lines that are relevant to the study of the SHH pathway. Consequently, these insights are transferred from the context of MB to non-transformed, post-natal day 7 cerebellum-derived cellular explants. In contrast to other, previously used methods, these GCP cultures proliferate indefinitely and depend on SHH pathway activation, either by means of the small molecule SAG or through genetic ablation of Ptch1. This culture method therefore leads to the generation of immortal neurosphere-like cell lines, that are named murine SAG-dependent spheres (mSS). Despite long-term culture, mSS cells remain dependent on continuous stimulation of the SHH pathway. Further, mSS cells maintain their lineage after extensive periods in vitro, as demonstrated by their differentiation towards the neural lineage. Herein a simple method for the generation of immortal cell lines from murine cerebella is defined. These lines can be maintained indefinitely through hedgehog pathway activation and maintain the GCP lineage.

The long non-coding RNA LncHDAC2 drives the self-renewal of liver cancer stem cells via activation of Hedgehog signaling

Liver cancer is the second leading cause of cancer death worldwide. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer in adults. The aim of this study was to define the role of the long non-coding RNA lncHDAC2 in the tumorigenesis of HCC. CD13+CD133+ cells (hereafter called liver cancer stem cells [CSCs]) and CD13-CD133- cells (referred to as non-CSCs) were sorted from 3 primary HCC tumor tissues and followed by transcriptome microarray. The expression and function of lncHDAC2 were further assessed by northern blot, sphere formation and xenograft tumor models. LncHDAC2 is highly expressed in HCC tumors and liver CSCs. LncHDAC2 promotes the self-renewal of liver CSCs and tumor propagation. In liver CSCs, lncHDAC2 recruits the NuRD complex onto the promoter of PTCH1 to inhibit its expression, leading to activation of Hedgehog signaling. Moreover, HDAC2 expression levels are positively related to HCC severity and PTCH1 levels are negatively related to HCC severity. Additionally, the Smo inhibitor cyclopamine was shown to impair the self-renewal of liver CSCs and suppress tumor propagation. Our findings reveal that lncHDAC2 promotes the self-renewal of liver CSCs and tumor propagation by activating the Hedgehog signaling pathway. Downregulating lncHDAC2 is a promising antitumor strategy in HCC. Liver cancer stem cells harbor high tumor-initiating potential and confer resistance to typical therapies, but the mechanism underlying their self-renewal remains elusive. LncHDAC2 augments the self-renewal of these cells, promoting tumor propagation. In liver cancer stem cells, lncHDAC2 activates Hedgehog signaling to initiate liver tumorigenesis. Therefore, lncHDAC2 and the Hedgehog signaling pathway may serve as biomarkers and potential drug targets for hepatocellular carcinoma.

Targeting the Oncoprotein Smoothened by Small Molecules: Focus on Novel Acylguanidine Derivatives as Potent Smoothened Inhibitors.

Hedgehog-GLI (HH) signaling was originally identified as a critical morphogenetic pathway in embryonic development. Since its discovery, a multitude of studies have reported that HH signaling also plays key roles in a variety of cancer types and in maintaining tumor-initiating cells. Smoothened (SMO) is the main transducer of HH signaling, and in the last few years, it has emerged as a promising therapeutic target for anticancer therapy. Although vismodegib and sonidegib have demonstrated effectiveness for the treatment of basal cell carcinoma (BCC), their clinical use has been hampered by severe side effects, low selectivity against cancer stem cells, and the onset of mutation-driven drug resistance. Moreover, SMO antagonists are not effective in cancers where HH activation is due to mutations of pathway components downstream of SMO, or in the case of noncanonical, SMO-independent activation of the GLI transcription factors, the final mediators of HH signaling. Here, we review the current and rapidly expanding field of SMO small-molecule inhibitors in experimental and clinical settings, focusing on a class of acylguanidine derivatives. We also discuss various aspects of SMO, including mechanisms of resistance to SMO antagonists.

Inhibition of the Hedgehog Pathway Decreases the Quiescent CD34+CD38- Population in Acute Myeloid Leukemia

IntroductionAcute myeloid leukemia (AML) is a clonal disease with a reduced life expectancy due to a high relapse rate. One explanation is that leukemic stem cells (LSC) evade the action of conventional chemotherapy due to their quiescent state. Several mechanisms have been proposed that regulate their quiescence, however, by analogy with normal hematopoietic stem cells, a key role may be carried out by the signaling pathways Notch and Hedgehog (Hh). The objectives of this study are to analyze the role of Notch and Hh pathways in the quiescence of LSC and to verify if the pharmacological inhibition of the Notch and Hh pathways decreases the percentage of quiescent LSC. In this way, LSCs would be sensitized to chemotherapy treatments and the high relapse rate of AML could be reduced.MethodsExpression of GLI1, a transcription factor of the Hh signaling pathway and NOTCH Internal Cleaved Domain (NICD) were analyzed in the hematopoietic stem and progenitor cells of four patients diagnosed with AML. The selection of quiescent fraction was performed by flow cytometry using anti-CD34-FITC, anti-CD117-PerCP, anti-CD45PE-Cy7, anti-CD38-APC-Cy7 and anti-KI67-BV510 antibodies. KI67 negative cells were considered quiescent. The activation of NOTCH and Hh pathways was studied using rabbit anti-GLI, anti-NICD primary antibodies and anti-rabbit BV421 secondary antibodies. Results were expressed in median (range) or mean ± standard deviation.Dose-response curves of inhibitors of the Notch pathway (BMS-906024, inhibitor of γ-secretase), Hh pathway (BMS-833923, SMO inhibitor and GANT61, GLI inhibitor), and cytarabine (AraC) were made to study drug potency in the OCI-AML3 cell line. We also analyzed its synergistic behavior in combination with Arac by calculating the combination index (CI) of each of them. These experiments were conducted in triplicate and values were expressed as the mean ± standard deviation.Finally, the effect of BMS-833923 and BMS-906024 on the quiescence of the CD34+CD38- cells of two patients diagnosed with AML was studied by flow cytometry.The paired samples t-test was used in the statistical analysis of GLI and NICD expression between G0 and proliferating cells and in the statistical analysis of the decrease of quiescent cells due to Notch and Hh inhibitors.ResultsFirst of all, hematopoietic and progenitor cells were quantified in four AML patients: the median of the percentage of CD34+CD38- cells with respect to total cells in the bone marrow of the AML patient studied was 1.1% (range: 0.12%-9.05%), within which 71.50% (range: 64.30%-88.43%) are quiescent. Interestingly, we found a trend for a higher expression of NOTCH signaling pathway in the proliferating CD34+CD38- cells (relative median fluorescent intensity (MFI) = 1.91 (range: 1.51-3.34)) compared to the quiescent fraction (relative MFI=1.55 (range: 1.18-1.94); p=0.105). But no differences were found in expression of GLI1.Before studying the effect on cellular quiescence of Notch and Hh inhibitors in monotherapy and in combination with AraC, we evaluated their effect on cell viability. The most potent drug studied was AraC (IC50 = 4.055 μM), followed by inhibitors of the Hh pathway (IC50 BMS-833923 = 5.041 μM; IC50 GANT61 = 7.042 μM) on the OCI-AML3 cell line. In contrast, the γ-secretase inhibitor (BMS-906024) showed no effect. Moreover, it was found that the combination 0.8 μM AraC plus 8 μM BMS-833923 was the most synergistic (CI = 0.53, 15% viability with respect to DMSO control).Subsequently, the effect of the SMO inhibitor on the quiescent CD34+CD38- cells of two patients diagnosed with CD34+ AML was analyzed: BMS-833923 decreased the percentage of quiescent CD34+CD38- cells by 88.5±16.3% in monotherapy (p=0.083) and in presence of AraC by 85.8 ± 21.2% (p=0.113) (figure 1).ConclusionThe use of SMO inhibitors for the treatment of AML is promising because it increases the sensitivity of leukemic cells to chemotherapy and facilitates their action by reducing the percentage of quiescent LSC. This could mean a decrease in the probability of relapse in patients with AML treated with Hh inhibitors. These results were derived from an ongoing project and more patients are being studied in order to confirm the explained results.This work is partially funded by the Madrid Association of Hematology and Hemotherapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Addition of the SMO Inhibitor Sonidegib to Azacitidine in Patients with Higher Risk Myelodysplastic Syndrome (MDS) Who Failed to Respond or Lost Response to AZA Alone: Results of a Phase 1-2 Add-on Study By the GFM

Background: although azacitidine (AZA) is an established frontline therapy for HR-MDS, approximately 40% of the patients fail to respond and virtually all of the responders will ultimately relapse. We previously reported the poor outcome of patients who failed AZA treatment for whom a median survival of 6 months is expected (Prebet, J Clin Oncol. 2011;29(24):3322). Overexpression of SMO a member of the hedgehog pathways has been observed in HR-MDS. Preclinical data have shown the activity of hedgehog pathway inhibitors and suggested synergism with Hypomethylating agents (HMA) (Zou, PLoSONE 2015; 10(8):e0136843). To harness potential synergism, we designed an add-on trial in which sonidegib (LDE-225), an oral inhibitor, was added to AZA in patients who failed AZA monotherapy.Patients and methods:Eligible patients should have IPSS Int-2 or HR-MDS or AML, (to have 10% to 30% marrow blasts), have received a minimum of 6 cycles of AZA monotherapy and to have failed to achieve a response to AZA (defined as CR, CRi, PR or HI as per IWG criteria) in the absence of marrow progression or have relapsed after achieving a response. Patients were scheduled to receive sonidegib daily from day 1 to 28 of 28-day cycles while continuing on AZA at the maximum previously tolerated dose (ie 50 to 75 mg/m²/d for 5-7 days). Two sonidegib dose levels were evaluated in the phase 1 part of the trial (400 and 800 mg/d) according to a classical 3+3 scheme.Results:23 patients, median age 76, were included in the study. One withdrew consent before starting the study treatment and is not included in the analysis. Characteristics of the remaining 22 patients are summarized in the Table. At the 800 mg/d dose level, 3 pts experienced DLT (grade 4 ASAT elevation (1 pt); grade 4 CPK elevation (2 pts)). The recommended dose for the expansion phase was 400 mg/d and no DLT was observed. 19 patients received 400 mg/d. Pts received a median of 3.5 cycles [IQR 2 ; 5], and 5 pts (23 %) received ≥ 6 cycles. All of them were withdrawn from study: 5 (26%) for disease progression (including 1 pt who progressed before starting cycle 1 and did not received treatment), 5 (26%) for death (all due to infection in the context of active disease) and 9 for miscellaneous reasons. 25 SAEs were reported including 23 infections, 1 intra cranial hemorrhage and 1 urinary retention. The most frequent non-hematologic toxicity was infection that occurred in 16/22 (73%) pts. In addition, 243 non hematologic toxicities of any grade were recorded in 20 pts for a total of 83 cycles, all but 3 of grade 1-2, including GI, cutaneous and musculo-skeletal toxicities and fatigue. Hospitalization was required in 45% of the pts. Only 3 responses were observed (1 CR, 1 marrow CR and 1 PR). Response durations were 1, 9 and 15 months .With a median follow-up of 23 months, 13 pts have died and the median overall survival is 6.8 months.ConclusionSonidegib at the dose of 400 mg/d can be given in combination with azacitidine with an acceptable toxicity. With a response rate of only 13%, our results indicate that the addition of sonidegib failed to reverse resistance to azacitidine in pts with higher risk MDS. This contrasts with the positive effect of the combination of glasdegib, another hedgehog pathway inhibitor with cytarabine in pts with AML and MDS (Cortes, Blood 2016 128:99). Besides potential differences in drug potency, our results may highlight the difficulty to identify active agents in the difficult-to-treat patient population selected after HMA therapy failure. The recommended dose-schedule of AZA-sonidegib established here could be used for further investigation in previously untreated patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Tumidulin, a Lichen Secondary Metabolite, Decreases the Stemness Potential of Colorectal Cancer Cells.

Lichens produce various unique chemicals that are used in the pharmaceutical industry. To screen for novel lichen secondary metabolites that inhibit the stemness potential of colorectal cancer cells, we tested acetone extracts of 11 lichen samples collected in Chile. Tumidulin, isolated from Niebla sp., reduced spheroid formation in CSC221, DLD1, and HT29 cells. In addition, mRNA expressions and protein levels of cancer stem markers aldehyde dehydrogenase-1 (ALDH1), cluster of differentiation 133 (CD133), CD44, Lgr5, and Musashi-1 were reduced after tumidulin treatment. Tumidulin decreased the transcriptional activity of the glioma-associated oncogene homolog zinc finger protein (Gli) promoter in reporter assays, and western blotting confirmed decreased Gli1, Gli2, and Smoothened (SMO) protein levels. Moreover, the tumidulin activity was not observed in the presence of Gli and SMO inhibitors. Together, these results demonstrate for the first time that tumidulin is a potent inhibitor of colorectal cancer cell stemness.

A slow-cycling LGR5 tumour population mediates basal cell carcinoma relapse after therapy.

Basal cell carcinoma (BCC) is the most frequent cancer in humans and results from constitutive activation of the Hedgehog pathway1. Several Smoothened inhibitors (Smoi) are used to treat Hedgehog-mediated malignancies, including BCC and medulloblastoma2. Vismodegib, a Smoi, leads to BCC shrinkage in the majority of the BCC patients3, but the mechanism by which it mediates BCC regression is currently unknown. Here, we used two different genetically engineered mouse models4 to investigate the mechanisms by which Smoi mediates tumor regression. We found that vismodegib mediates BCCs regression by inhibiting hair follicle-like fate and promoting the differentiation of tumour cells (TCs). However, a small population of TCs persists and is responsible for tumour relapse following treatment discontinuation, mimicking the situation found in humans5. In both mouse and human BCC, this persisting slow-cycling tumour population expresses Lgr5 and is characterised by active Wnt signalling. Lgr5 lineage ablation or Wnt signalling inhibition together with vismodegib leads to BCC eradication. Our study reveals that vismodegib induces tumour regression by promoting tumour differentiation, and demonstrates that the synergy between Wnt and Smoothened inhibitors constitutes a clinically relevant strategy to overcome tumour relapse in BCC.

A cell identity switch allows residual BCC to survive Hedgehog pathway inhibition.

Despite the efficacy of Hedgehog pathway inhibitors in the treatment of basal cell carcinoma (BCC)1, residual disease persists in some patients and may contribute to relapse when treatment is discontinued2. Here, to study the effect of the Smoothened inhibitor vismodegib on tumour clearance, we have used a Ptch1-Trp53 mouse model of BCC3 and found that mice treated with vismodegib harbour quiescent residual tumours that regrow upon cessation of treatment. Profiling experiments revealed that residual BCCs initiate a transcriptional program that closely resembles that of stem cells of the interfollicular epidermis and isthmus, whereas untreated BCCs are more similar to the hair follicle bulge. This cell identity switch was enabled by a mostly permissive chromatin state accompanied by rapid Wnt pathway activation and reprogramming of super enhancers to drive activation of key transcription factors involved in cellular identity. Accordingly, treatment of BCC with both vismodegib and a Wnt pathway inhibitor reduced the residual tumour burden and enhanced differentiation. Our study identifies a resistance mechanism in which tumour cells evade treatment by adopting an alternative identity that does not rely on the original oncogenic driver for survival.

Different Response of Ptch Mutant and Ptch Wildtype Rhabdomyosarcoma Toward SMO and PI3K Inhibitors

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma with poor prognosis. RMS frequently show Hedgehog (HH) pathway activity, which is predominantly seen in the embryonal subtype (ERMS). They also show activation of Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling. Here we compared the therapeutic effectiveness and the impact on HH target gene expression of Smoothened (SMO) antagonists with those of the PI3K inhibitor pictilisib in ERMS with and without mutations in the HH receptor Patched1 (PTCH). Our data demonstrate that growth of ERMS showing canonical Hh signaling activity due to Ptch germline mutations is efficiently reduced by SMO antagonists. This goes along with strong downregulation of the Hh target Gli1. Likewise Ptch mutant tumors are highly responsive toward the PI3K inhibitor pictilisib, which involves modulation of AKT and caspase activity. Pictilisib also modulates Hh target gene expression, which, however, is rather not correlated with its antitumoral effects. In contrast, sporadic ERMS, which usually express HH target genes without having PTCH mutation, apparently lack canonical HH signaling activity. Thus, stimulation by Sonic HE (SHH) or SAG (Smoothened agonist) or inhibition by SMO antagonists do not modulate HH target gene expression. In addition, SMO antagonists do not provoke efficient anticancer effects and rather exert off-target effects. In contrast, pictilisib and other PI3K/AKT/mTOR inhibitors potently inhibit cellular growth. They also efficiently inhibit HH target gene expression. However, of whether this is correlated with their antitumoral effects it is not clear. Together, these data suggest that PI3K inhibitors are a good and reliable therapeutic option for all ERMS, whereas SMO inhibitors might only be beneficial for ERMS driven by PTCH mutations.

Targeting stromal remodeling and cancer stem cell plasticity overcomes chemoresistance in triple negative breast cancer

The cellular and molecular basis of stromal cell recruitment, activation and crosstalk in carcinomas is poorly understood, limiting the development of targeted anti-stromal therapies. In mouse models of triple negative breast cancer (TNBC), Hedgehog ligand produced by neoplastic cells reprograms cancer-associated fibroblasts (CAFs) to provide a supportive niche for the acquisition of a chemo-resistant, cancer stem cell (CSC) phenotype via FGF5 expression and production of fibrillar collagen. Stromal treatment of patient-derived xenografts with smoothened inhibitors (SMOi) downregulates CSC markers expression and sensitizes tumors to docetaxel, leading to markedly improved survival and reduced metastatic burden. In the phase I clinical trial EDALINE, 3 of 12 patients with metastatic TNBC derived clinical benefit from combination therapy with the SMOi Sonidegib and docetaxel chemotherapy, with one patient experiencing a complete response. These studies identify Hedgehog signaling to CAFs as a novel mediator of CSC plasticity and an exciting new therapeutic target in TNBC.

YAP-TEAD signaling promotes basal cell carcinoma development via a c-JUN/AP1 axis.

The mammalian Hippo signaling pathway, through its effectors YAP and TAZ, coerces epithelial progenitor cell expansion for appropriate tissue development or regeneration upon damage. Its ability to drive rapid tissue growth explains why many oncogenic events frequently exploit this pathway to promote cancer phenotypes. Indeed, several tumor types including basal cell carcinoma (BCC) show genetic aberrations in the Hippo (or YAP/TAZ) regulators. Here, we uncover that while YAP is dispensable for homeostatic epidermal regeneration, it is required for BCC development. Our clonal analyses further demonstrate that the few emerging Yap-null dysplasia have lower fitness and thus are diminished as they progress to invasive BCC Mechanistically, YAP depletion in BCC tumors leads to effective impairment of the JNK-JUN signaling, a well-established tumor-driving cascade. Importantly, inthis context, YAP does not influence canonical Wnt or Hedgehog signaling. Overall, we reveal Hippo signaling as an independent promoter of BCCpathogenesis and thereby a viable target for drug-resistant BCC.

Abstract 3704: Efficacy of DR5-targeted immunoliposomes in pancreatic cancer models primed with SMO inhibitors

Abstract Background: Pancreatic cancer (PaCA) is a deadly disease with 5-year survival of 6%. Poor prognosis is attributed to low tumor microvessel density, poor perfusion, and amplified stroma, constituting a drug delivery barrier. Tumor priming using NVP-LDE225, an inhibitor of sonic hedgehog (sHH) signaling, can compromise the stromal drug delivery barrier by mediating stromal thinning, and discharge of tumor interstitial pressure (IFP) and decompression of tumor vasculature, resulting in increased perfusion and deposition of drug-containing nanoparticles into tumors. However, convective fluid efflux may increase as interstitial tumor pressure declines during priming, increasing drug washout. We hypothesize that a targeting nanoparticle delivery system will increase drug retention during priming, thereby increasing efficacy. Approach: Patient-derived xenograft (PDX) PaCA tumors possessing varied stromal architecture and degrees of desmoplasia were implanted s.c. in SCID mice. They ranged from low- to high desmoplasia and varied in microvessel density. The sHH inhibitor (sHHI) NVP-LDE225 was administered p.o. for 10 days, and fluorescent 80nm sterically-stabilized liposomes (SSL) were administered at intervals to probe tumor vascular perfusion/permeability. Efficacy was investigated with SSL liposomes containing doxorubicin (SSL-DXR), optionally coupled covalently with Death Receptor 5 monoclonal antibody AMG655 (SSL-DXR-DR5), which binds the pro-apoptotic DR5 receptor and initiates the extrinsic apoptotic pathway. Results: Quantitative fluorescence microscopy showed that sHH inhibitor treatment mediated a temporal window of enhanced permeability/perfusion and deposition of SSL that varied with the amount of desmoplasia in PDX models, which are otherwise highly impermeant to SSL. Quantitative IFP measurements using the “wick and needle” technique showed reduction of IFP paralleled establishment of the priming window. In the absence of sHH inhibitor priming, SSL-DXR (2 mg/kg), SSL-DXR-DR5 (2 mg/kg of DXR), and free DR5 (dose-matched to SSL-DXR-DR5 protein) were administered to evaluate the effect of tumor priming on therapeutic efficacy. Any DR5-containing treatment was curative for MiaPaCA-2 tumors (high DR5 expression). The response of Panc-1 tumors (low DR5 expression) was greatest with SSL-DXR-DR5. Tumor responses with the PDX model #12424, (high DR5 expression) were greater in sHHI-treated animals compared to controls. SSL-DXR + free DR5 mediated a transient inhibition of tumor progression, whereas a durable inhibition of tumor progression was observed with SSL-DXR-DR5 immunoliposomes. Conclusions: Efficacy of SSL-DXR-DR5 immunoliposomes was superior to other treatments in two cell line-based tumors, and tumor response varied with DR5 receptor density. Efficacy in low permeability/perfusion PDX tumors was greatest following tumor priming by sHH inhibitors. Citation Format: Darren Chan, Charlene Minx, Jun Wang, Arindam Sen, Michael Johnston, Wen Wee Ma, Christopher J. Scott, Robert M. Straubinger. Efficacy of DR5-targeted immunoliposomes in pancreatic cancer models primed with SMO inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3704.

21 Defining the cell populations responsible for skin cancer initiation and relapse following therapy

IntroductionThe identification of specific cell type from which cancer arises and the cancer cell population that resists upon therapy leading to tumour relapse constitute the main topics of our research. We use the basal cell carcinoma (BCC), the most frequent cancer in humans, as a cancer model for our studies.Material and methodsWe used lineage tracing approach to elucidate the cell at the origin of BCC and study the cancer cell population responsible for relapse following therapy discontinuation.Results and discussionsTo uncover the cancer cell of origin in BCC and the changes in the cellular dynamics that lead to tumour initiation, we assessed the impact of oncogenic hedgehog signalling activation in distinct cell populations and their capacity to induce BCC. We found that only stem cells, and not progenitors, were competent to initiate tumour formation upon oncogenic hedgehog signalling. Interestingly, this difference was due to the hierarchical organisation of tumour growth in oncogene-targeted stem cells, characterised by an increase of symmetric self-renewing divisions and a higher p53-dependent resistance to apoptosis, leading to rapid clonal expansion and progression into invasive tumours.11 Sánchez-Danés A, Hannezo E, Larsimont JC, Liagre M, Youssef KK, Simons BD, Blanpain C. Defining the clonal dynamics leading to mouse skin tumour initation. Nature. 2016 Jul 8;536(7616):298–303. doi: 10.1038/nature19069To study the cancer cell population that mediates BCC relapse upon therapy, we treated two different genetic BCC mouse models with a Smoothened inhibitor (Smoi), the most commonly drug used to treat locally advanced and metastatic BCC. The mechanism by which Smoi leads BCC regression and emergence of resistant tumour cells are currently unknown. We found that Smoi mediates BCC regression by promoting epidermal differentiation and that during the course of Smoi administration, some BCC become resistant to therapy mimicking the situation found in humans. We identify Lgr5 as a marker expressed in the resistant tumour cell population upon Smoi administration.Finally, we demonstrate that combination of Smoi administration with Lgr5 lineage ablation leads to BCC eradication.ConclusionWe demonstrated that stem cells cells are competent to initiate basal cell carcinoma, whereas, committed progenitors resistant.We demonstrated that Smoi administration in combination with Lgr5 lineage ablation leads to BCC eradication, constituting a clinically promising approach to overcome resistance to therapy and cure BCC.