Qunyan Yu

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.

Inhibition of tumorigenesis driven by different Wnt proteins requires blockade of distinct ligand-binding regions by LRP6 antibodies

Disregulated Wnt/β-catenin signaling has been linked to various human diseases, including cancers. Inhibitors of oncogenic Wnt signaling are likely to have a therapeutic effect in cancers. LRP5 and LRP6 are closely related membrane coreceptors for Wnt proteins. Using a phage-display library, we identified anti-LRP6 antibodies that either inhibit or enhance Wnt signaling. Two classes of LRP6 antagonistic antibodies were discovered: one class specifically inhibits Wnt proteins represented by Wnt1, whereas the second class specifically inhibits Wnt proteins represented by Wnt3a. Epitope-mapping experiments indicated that Wnt1 class-specific antibodies bind to the first propeller and Wnt3a class-specific antibodies bind to the third propeller of LRP6, suggesting that Wnt1- and Wnt3a-class proteins interact with distinct LRP6 propeller domains. This conclusion is further supported by the structural functional analysis of LRP5/6 and the finding that the Wnt antagonist Sclerostin interacts with the first propeller of LRP5/6 and preferentially inhibits the Wnt1-class proteins. We also show that Wnt1 or Wnt3a class-specific anti-LRP6 antibodies specifically block growth of MMTV-Wnt1 or MMTV-Wnt3 xenografts in vivo. Therapeutic application of these antibodies could be limited without knowing the type of Wnt proteins expressed in cancers. This is further complicated by our finding that bivalent LRP6 antibodies sensitize cells to the nonblocked class of Wnt proteins. The generation of a biparatopic LRP6 antibody blocks both Wnt1- and Wnt3a-mediated signaling without showing agonistic activity. Our studies provide insights into Wnt-induced LRP5/6 activation and show the potential utility of LRP6 antibodies in Wnt-driven cancer.

Selective blockade of the hydrolysis of the endocannabinoid 2-arachidonoylglycerol impairs learning and memory performance while producing antinociceptive activity in rodents

Monoacylglycerol lipase (MAGL) represents a primary degradation enzyme of the endogenous cannabinoid (eCB), 2-arachidonoyglycerol (2-AG). This study reports a potent covalent MAGL inhibitor, SAR127303. The compound behaves as a selective and competitive inhibitor of mouse and human MAGL, which potently elevates hippocampal levels of 2-AG in mice. In vivo, SAR127303 produces antinociceptive effects in assays of inflammatory and visceral pain. In addition, the drug alters learning performance in several assays related to episodic, working and spatial memory. Moreover, long term potentiation (LTP) of CA1 synaptic transmission and acetylcholine release in the hippocampus, two hallmarks of memory function, are both decreased by SAR127303. Although inactive in acute seizure tests, repeated administration of SAR127303 delays the acquisition and decreases kindled seizures in mice, indicating that the drug slows down epileptogenesis, a finding deserving further investigation to evaluate the potential of MAGL inhibitors as antiepileptics. However, the observation that 2-AG hydrolysis blockade alters learning and memory performance, suggests that such drugs may have limited value as therapeutic agents.

Anti–miR-21 Suppresses Hepatocellular Carcinoma Growth via Broad Transcriptional Network Deregulation

Hepatocellular carcinoma (HCC) remains a significant clinical challenge with few therapeutic options available to cancer patients. MicroRNA 21-5p (miR-21) has been shown to be upregulated in HCC, but the contribution of this oncomiR to the maintenance of tumorigenic phenotype in liver cancer remains poorly understood. We have developed potent and specific single-stranded oligonucleotide inhibitors of miR-21 (anti-miRNAs) and used them to interrogate dependency on miR-21 in a panel of liver cancer cell lines. Treatment with anti–miR-21, but not with a mismatch control anti-miRNA, resulted in significant derepression of direct targets of miR-21 and led to loss of viability in the majority of HCC cell lines tested. Robust induction of caspase activity, apoptosis, and necrosis was noted in anti–miR-21-treated HCC cells. Furthermore, ablation of miR-21 activity resulted in inhibition of HCC cell migration and suppression of clonogenic growth. To better understand the consequences of miR-21 suppression, global gene expression profiling was performed on anti–miR-21-treated liver cancer cells, which revealed striking enrichment in miR-21 target genes and deregulation of multiple growth-promoting pathways. Finally, in vivo dependency on miR-21 was observed in two separate HCC tumor xenograft models. In summary, these data establish a clear role for miR-21 in the maintenance of tumorigenic phenotype in HCC in vitro and in vivo. Implications: miR-21 is important for the maintenance of the tumorigenic phenotype of HCC and represents a target for pharmacologic intervention. Mol Cancer Res; 13(6); 1009–21. ©2015 AACR.

Identification of the endosomal sorting complex required for transport-I (ESCRT-I) as an important modulator of anti-miR uptake by cancer cells

Mechanisms of unassisted delivery of RNA therapeutics, including inhibitors of microRNAs, remain poorly understood. We observed that the hepatocellular carcinoma cell line SKHEP1 retains productive free uptake of a miR-21 inhibitor (anti-miR-21). Uptake of anti-miR-21, but not a mismatch (MM) control, induces expression of known miR-21 targets (DDAH1, ANKRD46) and leads to dose-dependent inhibition of cell growth. To elucidate mechanisms of SKHEP1 sensitivity to anti-miR-21, we conducted an unbiased shRNA screen that revealed tumor susceptibility gene 101 (TSG101), a component of the endosomal sorting complex required for transport (ESCRT-I), as an important determinant of anti-proliferative effects of anti-miR-21. RNA interference-mediated knockdown of TSG101 and another ESCRT-I protein, VPS28, improved uptake of anti-miR-21 in parental SKHEP1 cells and restored productive uptake to SKHEP1 clones with acquired resistance to anti-miR-21. Depletion of ESCRT-I in several additional cancer cell lines with inherently poor uptake resulted in improved activity of anti-miR-21. Finally, knockdown of TSG101 increased uptake of anti-miR-21 by cancer cells in vivo following systemic delivery. Collectively, these data support an important role for the ESCRT-I complex in the regulation of productive free uptake of anti-miRs and reveal potential avenues for improving oligonucleotide free uptake by cancer cells.

Oncogenic dependency on β-catenin in liver cancer cell lines correlates with pathway activation

Hepatocellular carcinoma (HCC) represents a serious public health challenge with few therapeutic options available to cancer patients.Wnt/β-catenin pathway is thought to play a significant role in HCC pathogenesis. In this study, we confirmed high frequency of CTNNB1 (β-catenin) mutations in two independent cohorts of HCC patients and demonstrated significant upregulation of β-catenin protein in the overwhelming majority of HCC patient samples, patient-derived xenografts (PDX) and established cell lines. Using genetic tools validated for target specificity through phenotypic rescue experiments, we went on to investigate oncogenic dependency on β-catenin in an extensive collection of human HCC cells lines. Our results demonstrate that dependency on β-catenin generally tracks with its activation status. HCC cell lines that harbored activating mutations in CTNNB1 or displayed elevated levels of non-phosphorylated (active) β-catenin were significantly more sensitive to β-catenin siRNA treatment than cell lines with wild-type CTNNB1 and lower active β-catenin. Finally, significant therapeutic benefit of β-catenin knock-down was demonstrated in established HCC tumor xenografts using doxycycline-inducible shRNA system. β-catenin downregulation and tumor growth inhibition was associated with reduction in AXIN2, direct transcriptional target of β-catenin, and decreased cancer cell proliferation as measured by Ki67 staining. Taken together, our data highlight fundamental importance of aberrant β-catenin signaling in the maintenance of oncogenic phenotype in HCC.

Abstract 4787: Targeting miR-21 in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) remains a significant unmet medical need with few therapeutic options available. Micro RNA 21 (miR-21) has been shown to be upregulated in HCC, however, contribution of this onco-miR to the maintenance of tumorigenic phenotype in liver cancer remains poorly understood. We have developed potent and specific single-stranded oligonucleotide inhibitors of miR-21 (anti-miR-21) and used them to interrogate dependency on miR-21 in a panel of 20 commercially available HCC cell lines in vitro. Upon lipid-mediated transfection, anti-miR-21, but not its mismatched (MM) control, caused significant de-repression of known direct targets of miR-21 (ANKRD46, DDAH1, RECK1) and inhibited survival of a large subset of HCC cell lines. Treatment of these sensitive HCC cell lines with anti-miR-21 resulted in dose- and time-dependent induction of caspase 3/7 activity. In contrast, non-responder HCC cell lines failed to significantly upregulate caspase activity and maintained viability in the presence of anti-miR compound. Further analysis of responder cell lines revealed robust induction of cell death, inhibition of cell migration and suppression of clonogenic growth upon treatment with miR-21 inhibitor. To better understand the consequences of miR-21 suppression in HCC, we carried out global gene expression profiling of anti-miR-21 treated sensitive liver cancer cells. Striking enrichment in miR-21 targets was noted among upregulated transcripts. Gene ontology analysis identified key cellular processes affected by miR-21 inhibition, including deregulation of metabolic pathways. In addition to the induction of direct miR-21 targets, cyclin H was found to be significantly downregulated upon miR-21 inhibition in the majority of responder cell lines. We hypothesize that inhibition of cyclin H expression, while an indirect effect of miR-21 suppression, could contribute to the activity of anti-miR-21 compounds. In summary, our data suggest that inhibition of miR-21 merits further investigation in the treatment of hepatocellular carcinoma. Citation Format: Sonya Zabludoff, Timothy Wagenaar, Francisco Adrian, Charles Allerson, Heike Arlt, Raffaele Baffa, Bal Bhat, Hui Cao, Scott Davis, Carlos Garcia-Echeverria, Kathrin Heermeier, Shih-Min Huang, Lan Jiang, Eric Marcusson, Christiane Metz-Weidmann, Adam Pavlicek, Jack Pollard, Jennifer Rocnik, Sabine Scheidler, Chaomei Shi, Fangxian Sun, Tatiana Tolstykh, Qunyan Yu, Gang Zheng, Dmitri Wiederschain. Targeting miR-21 in hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4787. doi:10.1158/1538-7445.AM2014-4787

Abstract 1796: SAR302503: A Jak2 inhibitor with antitumor activity in solid tumor models

Numerous studies have recognized the critical role of STAT3 in malignant transformation and tumor progression. Constitutive STAT3 activation is frequently found in cancer cell lines and tumor samples and it is usually linked to the presence of IL-6. Autocrine or paracrine IL-6 loops have been described to provide tumor cells with the ability to proliferate, survive, migrate and metastasize. At the molecular level, IL-6 binding to its receptor results in activation of Jak/STAT3 signaling and other signaling cascades, namely PI3K/Akt, MEK/ERK1-2, with a well established role in cancer. We have evaluated the ability of SAR302503, a selective Jak2 inhibitor entering a PhIII clinical trial in myelofibrosis patients, to block these pathways in a panel of ∼20 tumor cell lines representing different cancer types (prostate, breast, lung, colorectal, pancreas, hepatocellular, etc). A 45 minutes treatment with different concentrations of SAR302503 (0.1-10 µM) was sufficient to block both basal or IL-6 induced STAT3 phosphorylation in a dose dependent manner. Compound concentrations equal or greater than 0.1 µM were able to reduce the phosphorylation levels of STAT3 by an extent greater than 50% in all the cell lines included in the study. The impact of SAR302503 in tumor cell proliferation and survival was evaluated using different assays including clonogenic assays. Complete inhibition of colony formation was achieved at concentrations of SAR302503 below 1 µM in most of the cell lines. The antitumor activity of SAR302503 was evaluated in mice xenotransplanted subcutaneously with DU145 human prostate cancer cells. Oral administration of SAR302503 for 10 days resulted in significant dose dependent tumor growth inhibition, near to stasis at the highest dose (T/C=19% at 100 mg/kg, bid). In summary, we demonstrate that SAR302503 negatively impacts the proliferation and survival of different solid tumor cells and our data supports a role for a selective Jak2 inhibition in treating solid tumors with activated Jak/STAT signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1796. doi:1538-7445.AM2012-1796