Mutations in Hedgehog (Hh) pathway genes, leading to constitutive activation of Smoothened (Smo), occur in medulloblastoma. Antagonists of Smo induce tumor regression in mouse models of medulloblastoma and hold great promise for treating this disease. However, acquired resistance has emerged as a challenge to targeted therapeutics and may limit their anti-cancer efficacy. Here, we describe novel mechanisms of acquired resistance to Smo antagonists in medulloblastoma. NVP-LDE225, a potent and selective Smo antagonist, inhibits Hh signaling and induces tumor regressions in allograft models of medulloblastoma that are driven by mutations of Patched (Ptch), a tumor suppressor in the Hh pathway. However, evidence of resistance was observed during the course of treatment. Molecular analysis of resistant tumors revealed distinct resistance mechanisms. Chromosomal amplification of Gli2, a downstream effector of Hh signaling, or more rarely point mutations in Smo led to reactivated Hh signaling and restored tumor growth. Unexpectedly, analysis of pathway gene-expression signatures selectively deregulated in resistant tumors identified increased phosphoinositide 3-kinase (PI3K) signaling as another potential resistance mechanism. Probing the functional relevance of increased PI3K signaling, we demonstrated that the combination of NVP-LDE225 with the PI3K class I inhibitor NVP-BKM120 or 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.

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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 Wntdriven cancer.antibody therapeutics | cancer T he Wnt/β-catenin pathway regulates diverse biological processes during development and tissue homeostasis by modulating the protein stability of β-catenin (1-3). In the absence of extracellular Wnt proteins, cytoplasmic β-catenin is associated with the β-catenin destruction complex and degraded by ubiquitinmediated proteolysis. Wnt signals are transduced by two distinct receptors, the serpentine receptor Frizzled (Frz) and the singlespan transmembrane proteins LRP5 or LRP6. Wnt proteins promote the assembly of the Frz-LRP5/6 signaling complex and induce phosphorylation of LRP5 or LRP6. Phosphorylated LRP5 or LRP6 inactivates the β-catenin degradation complex, allowing stabilized β-catenin to enter the nucleus, bind to the TCF transcription factors, and act as a transcriptional coactivator.The extracellular domain of LRP5 or LRP6 contains four YWTD-type β-propeller domains each followed by an EGF-like domain and an LDLR domain. Each propeller contains six YWTD motifs that form a six-bladed β-propeller structure (4). Biochemical studies suggest that Wnt proteins physically interact with both Frz and LRP6 and induce the formation of an Frz-

Tumor cells display fundamental changes in metabolism and nutrient uptake in order to utilize additional nutrient sources to meet their enhanced bioenergetic requirements. Glutamine (Gln) is one such nutrient that is rapidly taken up by tumor cells to fulfill this increased metabolic demand. A vital step in the catabolism of glutamine is its conversion to glutamate by the mitochondrial enzyme glutaminase (GLS). This study has identified GLS a potential therapeutic target in breast cancer, specifically in the basal subtype that exhibits a deregulated glutaminolysis pathway. Using inducible shRNA mediated gene knockdown, we discovered that loss of GLS function in triple-negative breast cancer (TNBC) cell lines with a deregulated glutaminolysis pathway led to profound tumor growth inhibition in vitro and in vivo. GLS knockdown had no effect on growth and metabolite levels in non-TNBC cell lines. We rescued the anti-tumor effect of GLS knockdown using shRNA resistant cDNAs encoding both GLS isoforms and by addition of an α-ketoglutarate (αKG) analog thus confirming the critical role of GLS in TNBC. Pharmacological inhibition of GLS with the small molecule inhibitor CB-839 reduced cell growth and led to a decrease in mammalian target of rapamycin (mTOR) activity and an increase in the stress response pathway driven by activating transcription factor 4 (ATF4). Finally, we found that GLS inhibition synergizes with mTOR inhibition, which introduces the possibility of a novel therapeutic strategy for TNBC. Our study revealed that GLS is essential for the survival of TNBC with a deregulated glutaminolysis pathway. The synergistic activity of GLS and mTOR inhibitors in TNBC cell lines suggests therapeutic potential of this combination for the treatment of vulnerable subpopulations of TNBC.

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Members of the hypoxia-inducible factor (HIF) family of transcription factors regulate the cellular response to hypoxia. In non-small cell lung cancer (NSCLC), high HIF2α levels correlate with decreased overall survival, and inhibition of either the protein encoded by the canonical HIF target gene VEGF or VEGFR2 improves clinical outcomes. However, whether HIF2α is causal in imparting this poor prognosis is unknown. Here, we generated mice that conditionally express both a nondegradable variant of HIF2α and a mutant form of Kras (Kras G12D ) that induces lung tumors. Mice expressing both Hif2a and Kras G12D in the lungs developed larger tumors and had an increased tumor burden and decreased survival compared with mice expressing only Kras G12D . Additionally, tumors expressing both Kras G12D and Hif2a were more invasive, demonstrated features of epithelial-mesenchymal transition (EMT), and exhibited increased angiogenesis associated with mobilization of circulating endothelial progenitor cells. These results implicate HIF2α causally in the pathogenesis of lung cancer in mice, demonstrate in vivo that HIF2α can promote expression of markers of EMT, and define HIF2α as a promoter of tumor growth and progression in a solid tumor other than renal cell carcinoma. They further suggest a possible causal relationship between HIF2α and prognosis in patients with NSCLC.

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