Baozhi Chen

Small molecule–mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer

SUMMARY The pervasive influence of secreted Wnt signaling proteins in tissue homeostasis and tumorigenesis has galvanized efforts to identify small molecules that target Wnt-mediated cellular responses. By screening a diverse synthetic chemical library, we have discovered two novel classes of small molecules that disrupt Wnt pathway responses - whereas one class inhibits the activity of Porcupine (Porcn), a membrane-bound acyltransferase that is essential to the production of Wnt proteins, the other abrogates destruction of Axin proteins, suppressors of Wnt/β-catenin pathway activity. With these small molecules we establish a chemical genetic approach for studying Wnt pathway responses and stem cell function in adult tissue. We achieve transient, reversible suppression of Wnt/β-catenin pathway response in vivo, and establish a mechanism-based approach to target cancerous cell growth. The signal transduction mechanisms shown here to be chemically tractable additionally contribute to Wnt-independent signal transduction pathways and thus could be broadly exploited for chemical genetics and therapeutic goals.

Itraconazole and Arsenic Trioxide Inhibit Hedgehog Pathway Activation and Tumor Growth Associated with Acquired Resistance to Smoothened Antagonists

Recognition of the multiple roles of Hedgehog signaling in cancer has prompted intensive efforts to develop targeted pathway inhibitors. Leading inhibitors in clinical development act by binding to a common site within Smoothened, a critical pathway component. Acquired Smoothened mutations, including SMO(D477G), confer resistance to these inhibitors. Here, we report that itraconazole and arsenic trioxide, two agents in clinical use that inhibit Hedgehog signaling by mechanisms distinct from that of current Smoothened antagonists, retain inhibitory activity in vitro in the context of all reported resistance-conferring Smoothened mutants and GLI2 overexpression. Itraconazole and arsenic trioxide, alone or in combination, inhibit the growth of medulloblastoma and basal cell carcinoma in vivo, and prolong survival of mice with intracranial drug-resistant SMO(D477G) medulloblastoma.

Structure/Activity Relationship Studies of Small-Molecule Inhibitors of Wnt Response

Suppression of oncogenic Wnt-mediated signaling holds promise as an anti-cancer therapeutic strategy. We previously reported a novel class of small molecules (IWR-1/2, inhibitors of Wnt response) that antagonize Wnt signaling by stabilizing the Axin destruction complex. Herein, we present the results of structure-activity relationship studies of these compounds.

Genome-Wide RNAi Screen Reveals Disease-Associated Genes That Are Common to Hedgehog and Wnt Signaling

Loss of the kinase and tumor suppressor Stk11 (Lkb1) has opposite effects on Hh and Wnt signaling. Yin and Yang of Stk11 A screen to link positive regulators of the Hedgehog (Hh) pathway with human disease revealed a protein that sets the threshold of Hh and Wnt signaling, two pathways involved in embryonic development and adult tissue regeneration, as well as pathological conditions, such as cancer. The tumor suppressor Stk11 (also known as Lkb1) is a kinase that has been implicated in establishing cell polarity. Jacob et al. found that Stk11 was required for the formation of full-length primary cilia, to limit production of the Gli repressor, and thereby enhanced Hh signaling. In contrast, Stk11 restricted the availability of Disheveled, limiting Wnt signaling. Thus, the absence of Stk11 resulted in a higher threshold for Hh signaling but a lower threshold for Wnt signaling. The Hedgehog (Hh) and Wnt signal transduction pathways are master regulators of embryogenesis and tissue renewal and represent anticancer therapeutic targets. Using genome-wide RNA interference screening in murine cultured cells, we established previously unknown associations between these signaling pathways and genes linked to developmental malformations, diseases of premature tissue degeneration, and cancer. We identified functions in both pathways for the multitasking kinase Stk11 (also known as Lkb1), a tumor suppressor implicated in lung and cervical cancers. We found that Stk11 loss resulted in disassembly of the primary cilium, a cellular organizing center for Hh pathway components, thus dampening Hh signaling. Loss of Stk11 also induced aberrant signaling through the Wnt pathway. Chemicals that targeted the Wnt acyltransferase Porcupine or that restored primary cilia length by inhibiting the tubulin deacetylase HDAC6 (histone deacetylase 6) countered deviant pathway activities driven by Stk11 loss. Our study demonstrates that Stk11 is a critical mediator in both the Hh and the Wnt pathways, and our approach provides a platform to support the development of targeted therapeutic strategies.

Posaconazole, a Second-Generation Triazole Antifungal Drug, Inhibits the Hedgehog Signaling Pathway and Progression of Basal Cell Carcinoma

Deregulation of Hedgehog (Hh) pathway signaling has been associated with the pathogenesis of various malignancies, including basal cell carcinomas (BCC). Inhibitors of the Hh pathway currently available or under clinical investigation all bind and antagonize Smoothened (SMO), inducing a marked but transient clinical response. Tumor regrowth and therapy failure were attributed to mutations in the binding site of these small-molecule SMO antagonists. The antifungal itraconazole was demonstrated to be a potent SMO antagonist with a distinct mechanism of action from that of current SMO inhibitors. However, itraconazole represents a suboptimal therapeutic option due to its numerous drug–drug interactions. Here, we show that posaconazole, a second-generation triazole antifungal with minimal drug–drug interactions and a favorable side-effect profile, is also a potent inhibitor of the Hh pathway that functions at the level of SMO. We demonstrate that posaconazole inhibits the Hh pathway by a mechanism distinct from that of cyclopamine and other cyclopamine-competitive SMO antagonists but, similar to itraconazole, has robust activity against drug-resistant SMO mutants and inhibits the growth of Hh-dependent BCC in vivo. Our results suggest that posaconazole, alone or in combination with other Hh pathway antagonists, may be readily tested in clinical studies for the treatment of Hh-dependent cancers. Mol Cancer Ther; 15(5); 866–76. ©2016 AACR.

GLI1 Blockade Potentiates the Antitumor Activity of PI3K Antagonists in Lung Squamous Cell Carcinoma

Lung squamous cell carcinoma (SCC), strongly associated with smoking, is treated primarily with traditional cytotoxic chemotherapy due to a lack of FDA-approved targeted agents available. Here, we identify the Hedgehog pathway transcription factor GLI1 as a critical driver of lung SCC. Analysis of human lung cancer datasets showed that GLI1 mRNA was highly expressed in human lung SCC and portended a poor prognosis. Inhibition of GLI1 in human lung SCC cell lines suppressed tumor cell clonogenicity and proliferation in culture and in vivo Addition of SHH ligand, SMO antagonists, or other Hedgehog pathway agonists did not affect GLI1 expression in lung SCC cells. However, GLI1 expression was modulated by either inhibition or activation of the PI3K and MAPK pathways. Furthermore, in vivo growth of SCC harboring amplifications of the PI3K gene PIK3CA was attenuated by antagonizing GLI1 and PI3K. Thus, a combinatorial therapeutic strategy that targets the PI3K-mTOR pathway and GLI1 may lead to effective outcomes for PI3K pathway-dependent cancers, in contrast to recent results of human trials with single-agent PI3K antagonists. Cancer Res; 77(16); 4448-59. ©2017 AACR.

Abstract B03: Suppression of lung adenocarcinoma growth and metastasis by stromal Hedgehog pathway activation

Aberrant activation of the Hedgehog (Hh) signaling pathway drives the tumor growth of Gorlin-type cancers, but its role in other epithelial cancers, including non-small cell lung cancer (NSCLC), has been controversial. We used human lung adenocarcinoma (LAD) cell lines and autochthonous lung adenocarcinoma mouse models to elucidate the pathway’s role in lung cancer pathology. SHH and IHH is expressed heterogeneously among >35 tested LAD cell lines, although mostly in mutant Kras cells. SHH secreted from lung cancer cells activated the pathway in co-cultured fibroblasts in a paracrine, rather than autocrine, manner. Treatment with KAAD-cyclopamine, an Hh pathway antagonist, inhibited the growth of high SHH-expressing tumor cells when co-cultured with NIH-3T3 embryonic fibroblasts but not tumor cells alone, further reinforcing the paracrine nature of Hh pathway activation. However, the growth effect due to fibroblast pathway inhibition was diminished when co-cultured with normal lung fibroblasts. Analysis of potential Hh pathway target genes suggest that normal lung fibroblasts secrete ligands that induce a differentiation program in epithelial cells rather than ligands that induce mitogenic programs from NIH-3T3 fibroblasts. In Kras LSL-G12D/+ ;Trp53 fl/fl ; Shh fl/fl \(KPS) mice, genetic loss of SHH, surprisingly, had no effect on survival compared to Kras LSL-G12D/+ ;Trp53 fl/fl ;Shh +/+ (KP) mice. However, treatment with 5E1, an anti-SHH/IHH blocking antibody, 2 weeks after tumor induction in KP mice resulted in significantly worse survival with increased metastases compared to KP mice treated with control IgG 1 antibody. Furthermore, stromal Hh pathway abrogation by 5E1 led to increased tumor burden and decreased tumor vessel density compared to control mice. To account for the survival differences seen between KP mice with genetic SHH loss and those treated with 5E1, we analyzed KP tumors and found unexpectedly high levels of Ihh mRNA, compared to Shh mRNA, that have not been reported previously in normal or neoplastic murine lungs. Preliminary results using an in vivo CRISPR/Cas9 (pSECC) system to genetically ablate Ihh in Kras LSL-G12D/+ ;Trp53 fl/fl ;Rosa26 LSL-fluc/+ mice showed significant enhancement of tumor growth compared to control mice treated with sgGFP. These results suggest that IHH, rather than SHH, activates a tumor-suppressive program in lung stroma. These results correlate to human survival data of LAD with low lhh mRNA expression (KMPLotter). We are in the process of isolating and examining murine stromal cells from KP mice to identify the secreted stromal factors that limit tumor growth and metastases. Thus, stromal Hh pathway activation, in response to IHH secreted from lung cancer epithelia, suppresses LAD growth and metastases, increases tumor vessel growth, and prolongs survival. Citation Format: Sahba Kasiri, Baozhi Chen, Alexandra Wilson, Annika Reczek, Ummay Marriam, Zhiqun Zeng, Luc Girard, James Kim. Suppression of lung adenocarcinoma growth and metastasis by stromal Hedgehog pathway activation [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr B03.

Abstract 3017: Non-classical activation of GLI1 as a therapeutic target for squamous cell lung cancer

The Hedgehog (Hh) signaling pathway is critical for embryonic developmental processes and its deregulation is implicated in a wide variety of tumor types. However, the role of the Hh signaling pathway in the initiation and growth of non-small cell lung cancer is largely unknown. The purpose of this study is to investigate the role of GLI1, a major Hh pathway transcription factor, in lung squamous cell carcinoma (SCC) and to test the therapeutic potential of targeting GLI1. GLI1 expression in human SCC cell lines was evaluated by quantitative PCR and Western Blot. siRNA and shRNA of GLI1 in these cell lines were utilized in vitro and in vivo to test the requirement of GLI1 in tumor growth. Small molecule modulators of GLI1 were tested for their therapeutic potential. We have demonstrated that GLI1 has a critical role in SCC progression. GLI1 expression was significantly elevated in lung SCC compared to normal lung and lung adenocarcinoma patient specimens in several human genomic databases. Importantly, overexpression of GLI1 was correlated with poor overall survival in lung cancer patients. siRNA-mediated knock down of GLI1 in SCC cell lines decreased the expression of GLI1 target genes and caused a significant reduction in colony formation. Stable knock down of GLI1 in SCC cell lines caused a significant reduction in growth of xenograft tumors indicating the critical role of GLI1 in lung SCC progression. Inhibition or activation of SMO, an upstream component of Hh pathway, did not change GLI1 expression level in SCC cell lines. However, inhibition of PI3K/mTOR and MAPK signaling pathways down regulated GLI1 expression. These results suggested that GLI1 expression is dependent on PI3K/mTOR and MAPK pathway activity rather than Hh ligand. PI3K/mTOR inhibitor, or arsenic trioxide (ATO), a direct inhibitor of GLI proteins, significantly reduced GLI1 expression, proliferation, and clonogenicity in SCC cell lines. We have also observed a significant growth reduction in SCC xenografted tumors treated with combination of PI3K inhibitor and ATO. Our findings suggest that GLI1 is essential for lung SCC tumor progression. Furthermore, GLI1 expression in SCC is independent of Hh pathway ligand action and dependent on MAPK and PI3K pathway activity. Direct inhibition of GLI1 by repurposing ATO in combination with a PI3K inhibitor may represent a novel therapeutic strategy for lung SCC. Citation Format: Sahba Kasiri, Chunli Shao, Baozhi Chen, Alexandra Wilson, Paul Yenerall, Brenda Timmons, patrick dospoy, Suzie Hight, Luc Girard, Hui Tian, Carmen Behrens, Ignacio Wistuba, Adi Gazdar, James Kim. Non-classical activation of GLI1 as a therapeutic target for squamous cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3017.