Ramon J. Whitson

Epigenetic targeting of Hedgehog pathway transcriptional output through BET bromodomain inhibition

Hedgehog signaling drives oncogenesis in several cancers, and strategies targeting this pathway have been developed, most notably through inhibition of Smoothened (SMO). However, resistance to Smoothened inhibitors occurs by genetic changes of Smoothened or other downstream Hedgehog components. Here we overcome these resistance mechanisms by modulating GLI transcription through inhibition of bromo and extra C-terminal (BET) bromodomain proteins. We show that BRD4 and other BET bromodomain proteins regulate GLI transcription downstream of SMO and suppressor of fused (SUFU), and chromatin immunoprecipitation studies reveal that BRD4 directly occupies GLI1 and GLI2 promoters, with a substantial decrease in engagement of these sites after treatment with JQ1, a small-molecule inhibitor targeting BRD4. Globally, genes associated with medulloblastoma-specific GLI1 binding sites are downregulated in response to JQ1 treatment, supporting direct regulation of GLI activity by BRD4. Notably, patient- and GEMM (genetically engineered mouse model)-derived Hedgehog-driven tumors (basal cell carcinoma, medulloblastoma and atypical teratoid rhabdoid tumor) respond to JQ1 even when harboring genetic lesions rendering them resistant to Smoothened antagonists. Altogether, our results reveal BET proteins as critical regulators of Hedgehog pathway transcriptional output and nominate BET bromodomain inhibitors as a strategy for treating Hedgehog-driven tumors with emerged or a priori resistance to Smoothened antagonists.

RAS/MAPK activation drives resistance to Smo inhibition, metastasis and tumor evolution in Shh pathway-dependent tumors

Aberrant Shh signaling promotes tumor growth in diverse cancers. The importance of Shh signaling is particularly evident in medulloblastoma and basal cell carcinoma (BCC), where inhibitors targeting the Shh pathway component Smoothened (Smo) show great therapeutic promise. However, the emergence of drug resistance limits long-term efficacy, and the mechanisms of resistance remain poorly understood. Using new medulloblastoma models, we identify two distinct paradigms of resistance to Smo inhibition. Sufu mutations lead to maintenance of the Shh pathway in the presence of Smo inhibitors. Alternatively activation of the RAS-MAPK pathway circumvents Shh pathway dependency, drives tumor growth, and enhances metastatic behavior. Strikingly, in BCC patients treated with Smo inhibitor, squamous cell cancers with RAS/MAPK activation emerged from the antecedent BCC tumors. Together, these findings reveal a critical role of the RAS-MAPK pathway in drug resistance and tumor evolution of Shh pathway-dependent tumors.

Advanced Treatment for Basal Cell Carcinomas

Basal cell carcinomas (BCCs) are very common epithelial cancers that depend on the Hedgehog pathway for tumor growth. Traditional therapies such as surgical excision are effective for most patients with sporadic BCC; however, better treatment options are needed for cosmetically sensitive or advanced and metastatic BCC. The first approved Hedgehog antagonist targeting the membrane receptor Smoothened, vismodegib, shows remarkable effectiveness on both syndromic and nonsyndromic BCCs. However, drug-resistant tumors frequently develop, illustrating the need for the development of next-generation Hedgehog antagonists targeting pathway components downstream from Smoothened. In this article, we will summarize available BCC treatment options and discuss the development of next-generation antagonists.

Growth differentiation factor-15 (GDF-15) suppresses in vitro angiogenesis through a novel interaction with connective tissue growth factor (CCN2)

Growth differentiation factor‐15 (GDF‐15) and the CCN family member, connective tissue growth factor (CCN2), are associated with cardiac disease, inflammation, and cancer. The precise role and signaling mechanism for these factors in normal and diseased tissues remains elusive. Here we demonstrate an interaction between GDF‐15 and CCN2 using yeast two‐hybrid assays and have mapped the domain of interaction to the von Willebrand factor type C domain of CCN2. Biochemical pull down assays using secreted GDF‐15 and His‐tagged CCN2 produced in PC‐3 prostate cancer cells confirmed a direct interaction between these proteins. To investigate the functional consequences of this interaction, in vitro angiogenesis assays were performed. We demonstrate that GDF‐15 blocks CCN2‐mediated tube formation in human umbilical vein endothelial (HUVEC) cells. To examine the molecular mechanism whereby GDF‐15 inhibits CCN2‐mediated angiogenesis, activation of αVβ3 integrins and focal adhesion kinase (FAK) was examined. CCN2‐mediated FAK activation was inhibited by GDF‐15 and was accompanied by a decrease in αVβ3 integrin clustering in HUVEC cells. These results demonstrate, for the first time, a novel signaling pathway for GDF‐15 through interaction with the matricellular signaling molecule CCN2. Furthermore, antagonism of CCN2 mediated angiogenesis by GDF‐15 may provide insight into the functional role of GDF‐15 in disease states. J. Cell. Biochem. 114: 1424–1433, 2013.

Noncanonical hedgehog pathway activation through SRF–MKL1 promotes drug resistance in basal cell carcinomas

Hedgehog pathway–dependent cancers can escape Smoothened (SMO) inhibition through mutations in genes encoding canonical hedgehog pathway components; however, around 50% of drug-resistant basal cell carcinomas (BCCs) lack additional variants of these genes. Here we use multidimensional genomics analysis of human and mouse drug-resistant BCCs to identify a noncanonical hedgehog activation pathway driven by the transcription factor serum response factor (SRF). Active SRF along with its coactivator megakaryoblastic leukemia 1 (MKL1) binds DNA near hedgehog target genes and forms a previously unknown protein complex with the hedgehog transcription factor glioma-associated oncogene family zinc finger-1 (GLI1), causing amplification of GLI1 transcriptional activity. We show that cytoskeletal activation through Rho and the formin family member Diaphanous (mDia) is required for SRF–MKL-driven GLI1 activation and for tumor cell viability. Remarkably, nuclear MKL1 staining served as a biomarker in tumors from mice and human subjects to predict tumor responsiveness to MKL inhibitors, highlighting the therapeutic potential of targeting this pathway. Thus, our study illuminates, for the first time, cytoskeletal-activation-driven transcription as a personalized therapeutic target for combatting drug-resistant malignancies.

Reduced expression of GDF-15 is associated with atrophic inflammatory lesions of the prostate.

Accumulating evidence suggests that chronic prostatic inflammation may lead to prostate cancer development. Growth differentiation factor‐15 (GDF‐15) is highly expressed in the prostate and has been associated with inflammation and tumorigenesis.

Tumor-Derived Suppressor of Fused Mutations Reveal Hedgehog Pathway Interactions

The Hedgehog pathway is a potent regulator of cellular growth and plays a central role in the development of many cancers including basal cell carcinoma (BCC). The majority of BCCs arise from mutations in the Patched receptor resulting in constitutive activation of the Hedgehog pathway. Secondary driver mutations promote BCC oncogenesis and occur frequently due to the high mutational burden resulting from sun exposure of the skin. Here, we uncover novel secondary mutations in Suppressor of Fused (SUFU), the major negative regulator of the Hedgehog pathway. SUFU normally binds to a Hedgehog transcriptional activator, GLI1, in order to prevent it from initiating transcription of Hedgehog target genes. We sequenced tumor-normal pairs from patients with early sporadic BCCs. This resulted in the discovery of nine mutations in SUFU, which were functionally investigated to determine whether they help drive BCC formation. Our results show that four of the SUFU mutations inappropriately activate the Hedgehog pathway, suggesting they may act as driver mutations for BCC development. Indeed, all four of the loss of function SUFU variants were found to disrupt its binding to GLI, leading to constitutive pathway activation. Our results from functional characterization of these mutations shed light on SUFU’s role in Hedgehog signaling, tumor progression, and highlight a way in which BCCs can arise.

Patching up our tumor signaling knowledge.

The tumor suppressor Patched1 (Ptch1) possesses well-described roles in regulating sonic hedgehog (SHH) signaling in the skin and preventing the formation of basal cell carcinomas (BCCs). In this issue, Kang et al. extend their previous work to show that a naturally occurring allele of Ptch1 found in FVB mice promotes early squamous cell carcinoma (SCC) growth without aberrant activation of the SHH pathway. The study reveals new roles for Ptch1 that lie at the nexus between BCC and SCC formation.

Initial in vitro functional characterization of serum exosomal microRNAs from patients with metastatic basal cell carcinoma

Basal cell carcinoma (BCC) is the most common of human malignancies, but the mechanism by which metastasis occurs is poorly understood1. Serum exosomes, 30-100 nanometer membrane-bound vesicles containing regulatory microRNAs (miRs), are emerging as a mechanism for cancer metastasis in melanoma2, breast3, prostate4, and lung cancers5, but the role of exosomes in BCC metastasis is not known. In this pilot study, we assessed for differences in exosomal composition in BCC patients with and without metastasis, and whether these differences confer functional changes in vitro. This article is protected by copyright. All rights reserved.

Abstract B6: Epigenetic regulation of Hedgehog pathway transcriptional output by BET bromodomain proteins

Aberrant activation of Hedgehog signaling drives oncogenesis in several types of cancer. As a result, there has been significant interest in developing therapeutic strategies targeting this pathway, most notably through inhibition of Smoothened. Though Smoothened inhibitors have shown efficacy in several cancer clinical trials, the initial enthusiasm for these inhibitors has been tempered by emergence of resistance and a priori resistance, often via mutation of Smoothened itself or through dysregulation of downstream components of the Hedgehog signaling axis. Here we reveal a strategy that overcomes these resistance mechanisms by targeting the far downstream transcriptional mediators of Hedgehog signaling through inhibition of the BET bromodomain protein, BRD4. We show that knockdown of BRD4 or treatment with the BET bromodomain inhibitor, JQ1, dramatically inhibits transcription of GLI1 and other Hedgehog target genes upon ligand-mediated or genetic activation of the Hedgehog pathway. We confirm the inhibitory effect of JQ1 occurs downstream of SMO and SUFU and verify by chromatin immunoprecipitation that Brd4 directly occupies the GLI1 and GLI2 promoters, with a substantial decrease in the engagement of these genomic sites upon treatment with JQ1. We observe a corresponding downregulation of genes associated with medulloblastoma-specific GLI1 binding sites upon exposure to JQ1, confirming the direct regulation of GLI1 by BET bromodomain proteins. Finally, using patient- and GEMM-derived cell lines of Hedgehog-driven cancer (basal cell carcinoma, medulloblastoma and ATRT), we show that JQ1 decreases Hh pathway output and proliferation, even in cell lines resistant to Smoothened inhibitors. These results expand the role of BET bromodomain inhibitors to targeting Hedgehog-driven cancers and highlight a strategy that overcomes the limitation of Hedgehog pathway inhibitors currently in clinical use. Citation Format: Yujie Tang, Simone Schubert, Jun Qi, Brian Nguyen, Sabran Masoud, Nujsaunusi Vue, Brianna Balansay, Furong Yu, Scott X. Atwood, Ramon J. Whitson, Anitha Ponnuswami, Spencer Chen, Sharareh Gholamin, Woo J. Pamelyn, Michelle Monje-Diesseroth, Sekyung Oh, Alex Lee, Jean Y. Tang, Rob Wechsler-Reya, Anthony E. Oro, James E. Bradner, Yoon-Jae Cho. Epigenetic regulation of Hedgehog pathway transcriptional output by BET bromodomain proteins. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B6.