Ribo Guo

Interfering with Resistance to Smoothened Antagonists by Inhibition of the PI3K Pathway in Medulloblastoma

Resistance of medulloblastoma to Smo antagonists can be delayed or prevented by specific drug combinations. An End Run Against Tumor Resistance Cancer cells are as clever as microbes. Mustering their considerable abilities to rapidly replicate and evolve, both cancer cells and bacteria quickly develop resistance to the drugs we use to fight them. Modern medicine confronts a growing population of pathogens that cannot be treated by our usual antibiotics, and oncologists must be prepared with second- and third-line therapies, because tumors that retreat from initial drug treatments often return with renewed vigor. Buonamici et al. confront this problem in their study of a new class of cancer therapeutic agents now in clinical trials—antagonists of a membrane protein called Smoothened (Smo). The Smo receptor normally regulates a developmental pathway but is abnormally activated in medulloblastoma (a malignant brain tumor) and basal cell carcinoma of the skin. Medulloblastomas in mice respond well to these Smo antagonists but soon become resistant, these authors find. If, however, an inhibitor of the phosphatidylinositol 3-kinase (PI3K) signaling pathway is added to the initial drug cocktail, resistance is delayed or even prevented. In some cancers, the Smo receptor is active even when its ligand is absent, conferring dependence of the tumor on the downstream Hedgehog signaling pathway, which ultimately regulates gene expression through the Gli transcription factors. Treatment of Smo-addicted tumors in mice with Smo antagonists ultimately leads to development of resistance, although tumor growth is inhibited for a while. The authors found that the tumors eluded the drug in several ways: The genes for the Gli transcription factors were sometimes amplified, compensating for loss of pathway stimulation. In other resistant tumors, there were point mutations in the Smo receptor itself that allowed reactivation of the pathway. In yet another group of tumors, by examining which genes were up-regulated, the authors found activation of a completely different signaling pathway—the PI3K pathway. Further experiments in medulloblastoma-bearing mice revealed that resistance could be delayed or even prevented by including a PI3K inhibitor along with the Smo antagonist in the initial treatment that tumor-bearing animals received. The PI3K inhibitor alone had no effect. By looking at resistance mechanisms to Smo antagonists before the drug is used in the clinic, the results of this study will better arm oncologists against the molecular defenses that cancers may commandeer to evade this drug. And by identifying a drug combination that delays or even combats development of resistance when used as a first-line treatment in clinical trials, these results could ultimately improve the lives of patients with medulloblastoma or other cancers that depend on Smo for their survival. The malignant brain cancer medulloblastoma is characterized by mutations in Hedgehog (Hh) signaling pathway genes, which lead to constitutive activation of the G protein (heterotrimeric guanosine triphosphate–binding protein)–coupled receptor Smoothened (Smo). The Smo antagonist NVP-LDE225 inhibits Hh signaling and induces tumor regression in animal models of medulloblastoma. However, evidence of resistance was observed during the course of treatment. Molecular analysis of resistant tumors revealed several resistance mechanisms. We noted chromosomal amplification of Gli2, a downstream effector of Hh signaling, and, more rarely, point mutations in Smo that led to reactivated Hh signaling and restored tumor growth. Analysis of pathway gene expression signatures also, unexpectedly, identified up-regulation of phosphatidylinositol 3-kinase (PI3K) signaling in resistant tumors as another potential mechanism of resistance. Probing the relevance of increased PI3K signaling, we demonstrated that addition of the PI3K inhibitor NVP-BKM120 or the dual PI3K-mTOR (mammalian target of rapamycin) inhibitor NVP-BEZ235 to the initial treatment with the Smo antagonist markedly delayed the development of resistance. Our findings may be useful in informing treatment strategies for medulloblastoma.

A crucial requirement for Hedgehog signaling in small cell lung cancer

Small-cell lung cancer (SCLC) is an aggressive neuroendocrine subtype of lung cancer for which there is no effective treatment. Using a mouse model in which deletion of Rb1 and Trp53 in the lung epithelium of adult mice induces SCLC, we found that the Hedgehog signaling pathway is activated in SCLC cells independently of the lung microenvironment. Constitutive activation of the Hedgehog signaling molecule Smoothened (Smo) promoted the clonogenicity of human SCLC in vitro and the initiation and progression of mouse SCLC in vivo. Reciprocally, deletion of Smo in Rb1 and Trp53-mutant lung epithelial cells strongly suppressed SCLC initiation and progression in mice. Furthermore, pharmacological blockade of Hedgehog signaling inhibited the growth of mouse and human SCLC, most notably following chemotherapy. These findings show a crucial cell-intrinsic role for Hedgehog signaling in the development and maintenance of SCLC and identify Hedgehog pathway inhibition as a therapeutic strategy to slow the progression of disease and delay cancer recurrence in individuals with SCLC.

1-Amino-4-benzylphthalazines as Orally Bioavailable Smoothened Antagonists with Antitumor Activity

Abnormal activation of the Hedgehog (Hh) signaling pathway has been linked to several types of human cancers, and the development of small-molecule inhibitors of this pathway represents a promising route toward novel anticancer therapeutics. A cell-based screen performed in our laboratories identified a new class of Hh pathway inhibitors, 1-amino-4-benzylphthalazines, that act via antagonism of the Smoothened receptor. A variety of analogues were synthesized and their structure-activity relationships determined. This optimization resulted in the discovery of high affinity Smoothened antagonists, one of which was further profiled in vivo. This compound displayed a good pharmacokinetic profile and also afforded tumor regression in a genetic mouse model of medulloblastoma.

Maintenance of adenomatous polyposis coli (APC)-mutant colorectal cancer is dependent on Wnt/β-catenin signaling

Persistent expression of certain oncogenes is required for tumor maintenance. This phenotype is referred to as oncogene addiction and has been clinically validated by anticancer therapies that specifically inhibit oncoproteins such as BCR-ABL, c-Kit, HER2, PDGFR, and EGFR. Identifying additional genes that are required for tumor maintenance may lead to new targets for anticancer drugs. Although the role of aberrant Wnt pathway activation in the initiation of colorectal cancer has been clearly established, it remains unclear whether sustained Wnt pathway activation is required for colorectal tumor maintenance. To address this question, we used inducible β-catenin shRNAs to temporally control Wnt pathway activation in vivo. Here, we show that active Wnt/β-catenin signaling is required for maintenance of colorectal tumor xenografts harboring APC mutations. Reduced tumor growth upon β-catenin inhibition was due to cell cycle arrest and differentiation. Upon reactivation of the Wnt/β-catenin pathway colorectal cancer cells resumed proliferation and reacquired a crypt progenitor phenotype. In human colonic adenocarcinomas, high levels of nuclear β-catenin correlated with crypt progenitor but not differentiation markers, suggesting that the Wnt/β-catenin pathway may also control colorectal tumor cell fate during the maintenance phase of tumors in patients. These results support efforts to treat human colorectal cancer by pharmacological inhibition of the Wnt/β-catenin pathway.

Discovery of NVP-LEQ506, a Second-Generation Inhibitor of Smoothened

Inhibition of the Hedgehog (Hh) pathway targeting the Smoothened receptor has proven therapeutic benefit for the treatment of Hh-dependent cancers. Lead optimization provided a novel type of Smoothened inhibitor based on a pyridazine core resulting in the clinical compound NVP-LEQ506. This new agent combines high intrinsic potency and good pharmacokinetic properties resulting in excellent efficacy in preclinical rodent tumor models of medulloblastoma. Activity against a Smo mutant conferring resistance observed in a clinical trial with a competitor compound suggests additional therapeutic potential.

Design and Optimization of Potent and Orally Bioavailable Tetrahydronaphthalene Raf Inhibitors

Inhibition of mutant B-Raf signaling, through either direct inhibition of the enzyme or inhibition of MEK, the direct substrate of Raf, has been demonstrated preclinically to inhibit tumor growth. Very recently, treatment of B-Raf mutant melanoma patients with a selective B-Raf inhibitor has resulted in promising preliminary evidence of antitumor activity. This article describes the design and optimization of tetrahydronaphthalene-derived compounds as potent inhibitors of the Raf pathway in vitro and in vivo. These compounds possess good pharmacokinetic properties in rodents and inhibit B-Raf mutant tumor growth in mouse xenograft models.

Abstract C149: The PKC inhibitor Sotrastaurin selectively inhibits the growth of GNAQ mutant uveal melanoma.

GNAQ mutations are early and frequent events in the pathology of Uveal Melanoma. These mutations hyper-activate PLCb-PKC-ERK1/2 signaling resulting in PKC dependence. Targeting GNAQ mutant Uveal Melanoma Cells with the PKC inhibitor AEB071 (Sotrastaurin) leads to pathway modulation and growth inhibition. The Myristoylated Alanine-rich C-kinase Substrate (MARCKS) is directly phosphorylated by PKC and can be used as a readout for PKC activation. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C149. Citation Format: Rosemary Barrett, Ribo Guo, Jing Yuan, Vesselina Cooke, Anthony Vattay, Joshua Korn, Guiqing Liang, Xin Li, Ramu Thiruvamoor, Andrew Wylie, Anka Bric, Nicholas Keen, William Sellers. The PKC inhibitor Sotrastaurin selectively inhibits the growth of GNAQ mutant uveal melanoma. [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 C149.

Abstract 1976: The discovery of mechanisms of resistance to SMO antagonists and the therapeutic implications

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Smoothened (Smo) is a G-protein coupled receptor (GPCR)-like molecule that activates the Hedgehog (Hh) signal transduction pathway. In the resting state, the 12-pass transmembrane protein Patched (Ptch) inhibits Smo activity. When Ptch inhibition is attenuated, Smo signals via a cytosolic complex of proteins leading to activation of the Gli family of transcription factors. Genetic activation of the Hh pathway at or upstream of Smo is linked to tumorigenesis in several cancers. In particular, somatic mutations in Ptch and Smo leading to constitutive pathway activation are found in sporadic medulloblastoma (MB) and basal cell carcinoma (BCC). Evidence suggests that antagonists of Smo may abrogate the tumorigenic phenotype engendered by Ptch inactivation. NVP-LDE225 is a potent and selective orally available Smo antagonist that robustly inhibits Smo-dependent signaling in vitro and in vivo. NVP-LDE225 exerted dose-related anti-tumor activity in vivo in several genetically defined MB models that are driven by mutations in Ptch leading to near complete tumor regression and Hh pathway inhibition. However, following long-term continuous dosing of NVP-LDE225 in medulloblastoma allograft models, evidence of resistance to NVP-LDE225 was observed. Here, we describe our efforts to proactively identify mechanisms of resistance to targeted therapy of Smo. Genome-wide DNA- and RNA-profiling of resistant tumors revealed distinct resistance mechanisms allowing tumors to evade the inhibitory effects of Smo antagonists. Chromosomal amplification of Gli2, a downstream effector of Hh signaling, was identified as one mechanism leading to restoration of pathway signaling despite adequate drug exposure. Additional mining of the gene expression data for pathway signatures that are selectively deregulated in resistant tumors identified increased phosphatidylinositol-3-kinase (PI3K) signaling as another potential resistance mechanism. Probing the functional relevance of increased PI3K signaling, we showed that the combination of NVP-LDE225 with the dual PI3K/mTor inhibitor NVP-BEZ235 markedly delayed the development of resistance. Our findings have important clinical implications for future treatment strategies in medulloblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1976.