The Notch signaling pathway mediates cell fate decisions1,2 and is tumor suppressive or oncogenic depending on the context2,3. During lung development, Notch pathway activation inhibits the differentiation of precursor cells to a neuroendocrine (NE) fate4–6. In small cell lung cancer (SCLC), an aggressive NE lung cancer7, loss-of-function NOTCH mutations and the inhibitory effects of ectopic Notch activation indicate that Notch signaling is tumor suppressive8,9. Here, we show that Notch signaling can be both tumor suppressive and pro-tumorigenic in SCLC. Endogenous activation of the Notch pathway results in a NE to non-NE fate switch in 10-50% of tumor cells in a mouse model of SCLC and in human tumors. This switch is mediated in part by Rest/Nrsf, a transcriptional repressor that inhibits NE gene expression. Non-NE Notch-active SCLC cells are slow growing, consistent with a tumor suppressive role for Notch, but these cells are also relatively chemoresistant and provide trophic support to NE tumor cells, consistent with a pro-tumorigenic role. Importantly, Notch blockade in combination with chemotherapy suppresses tumor growth and delays relapse. Thus, SCLC tumors generate their own microenvironment via activation of Notch signaling in a subset of tumor cells, and the presence of these cells may serve as a biomarker for the use of Notch pathway inhibitors in combination with chemotherapy in select SCLC patients.

The Wnt/β-catenin pathway, which signals through the Frizzled (Fzd) receptor family and several coreceptors, has long been implicated in cancer. Here we demonstrate a therapeutic approach to targeting the Wnt pathway with a monoclonal antibody, OMP-18R5. This antibody, initially identified by binding to Frizzled 7, interacts with five Fzd receptors through a conserved epitope within the extracellular domain and blocks canonical Wnt signaling induced by multiple Wnt family members. In xenograft studies with minimally passaged human tumors, this antibody inhibits the growth of a range of tumor types, reduces tumor-initiating cell frequency, and exhibits synergistic activity with standard-of-care chemotherapeutic agents.differentiation | cancer stem cell | pancreatic | breast | lung

Purpose: The Notch pathway plays an important role in both stem cell biology and cancer. Dysregulation of Notch signaling has been reported in several human tumor types. In this report, we describe the development of an antibody, OMP-59R5 (tarextumab), which blocks both Notch2 and Notch3 signaling. Experimental Design: We utilized patient-derived xenograft tumors to evaluate antitumor effect of OMP-59R5. Immunohistochemistry, RNA microarray, real-time PCR, and in vivo serial transplantation assays were employed to investigate the mechanisms of action and pharmacodynamic readouts. Results: We found that anti-Notch2/3, either as a single agent or in combination with chemotherapeutic agents was efficacious in a broad spectrum of epithelial tumors, including breast, lung, ovarian, and pancreatic cancers. Notably, the sensitivity of anti-Notch2/3 in combination with gemcitabine in pancreatic tumors was associated with higher levels of Notch3 gene expression. The antitumor effect of anti-Notch2/3 in combination with gemcitabine plus nab-paclitaxel was greater than the combination effect with gemcitabine alone. OMP-59R5 inhibits both human and mouse Notch2 and Notch3 function and its antitumor activity was characterized by a dual mechanism of action in both tumor and stromal/vascular cells in xenograft experiments. In tumor cells, anti-Notch2/3 inhibited expression of Notch target genes and reduced tumor-initiating cell frequency. In the tumor stroma, OMP-59R5 consistently inhibited the expression of Notch3, HeyL, and Rgs5, characteristic of affecting pericyte function in tumor vasculature. Conclusions: These findings indicate that blockade of Notch2/3 signaling with this cross-reactive antagonist antibody may be an effective strategy for treatment of a variety of tumor types. Clin Cancer Res; 21(9); 2084–95. ©2015 AACR.

Activating mutations in c-KIT are associated with gastrointestinal stromal tumors, mastocytosis, and acute myeloid leukemia. In attempting to establish a murine model of human KIT D816V (hKIT D816V )-mediated leukemia, we uncovered an unexpected relationship between cellular transformation and intracellular trafficking. We found that transport of hKIT D816V protein was blocked at the endoplasmic reticulum in a species-specific fashion. We exploited these species-specific trafficking differences and a set of localization domain-tagged KIT mutants to explore the relationship between subcellular localization of mutant KIT and cellular transformation. The protein products of fully transforming KIT mutants localized to the Golgi apparatus and to a lesser extent the plasma membrane. Domain-tagged KIT D816V targeted to the Golgi apparatus remained constitutively active and transforming. Chemical inhibition of intracellular transport demonstrated that Golgi localization is sufficient, but plasma membrane localization is dispensable, for downstream signaling mediated by KIT mutation. When expressed in murine bone marrow, endoplasmic reticulum-localized hKIT D816V failed to induce disease in mice, while expression of either Golgi-localized HyKIT D816V or cytosol-localized, ectodomain-deleted KIT D816V uniformly caused fatal myeloproliferative diseases. Taken together, these data demonstrate that intracellular, non-plasma membrane receptor signaling is sufficient to drive neoplasia caused by mutant c-KIT and provide the first animal model of myelomonocytic neoplasia initiated by human KIT D816V .The c-Kit gene is the mammalian homolog of the HardyZuckerman 4 feline sarcoma virus-transforming sequence (5), maps to the murine white spotted (W) locus (12), and encodes a type III receptor tyrosine kinase (RTK) (26, 31) sharing strong sequence similarity to other type III RTK members FLT3, FMS, and platelet-derived growth factor receptor (32). The KIT receptor is expressed in hematopoietic stem cells, mast cells, neural crest-derived melanocytes, and germ cells. Immunoprecipitation studies of Kit expression demonstrated two protein products, 160 kDa and 140 kDa, detectable in Kit-expressing cells due to different posttranslational glycosylation (32). Binding to KIT ligand (stem cell factor [SCF]) leads to receptor dimerization, activation of the intrinsic receptor kinase, and initiation of a spectrum of downstream signaling cascades responsible for various cellular responses, such as proliferation, migration, and survival (7).Gain-of-function mutations in c-KIT, causing constitutive, ligand-independent activation of the receptor, were first identified in neoplastic mast cell lines of human, mouse, and rat origins (11,36,37). Activating mutations in the human c-KIT gene (KIT) occur in association with systemic mastocytosis (24), gastrointestinal stromal tumors (33), germ cell tumors (29), and acute myeloid leukemia (AML) (1, 15). Among these mutations, D816V is the most frequent mutation. The wildtype KIT receptor is widely exp...

The t(8;21)(q22;q22) translocation, occurring in 40% of patients with acute myeloid leukemia (AML) of the FAB-M2 subtype (AML with maturation), results in expression of the RUNX1-CBF2T1 [AML1-ETO (AE)] fusion oncogene. AML͞ETO may contribute to leukemogenesis by interacting with nuclear corepressor complexes that include histone deacetylases, which mediate the repression of target genes. However, expression of AE is not sufficient to transform primary hematopoietic cells or cause disease in animals, suggesting that additional mutations are required. Activating mutations in receptor tyrosine kinases (RTK) are present in at least 30% of patients with AML. To test the hypothesis that activating RTK mutations cooperate with AE to cause leukemia, we transplanted retrovirally transduced murine bone marrow coexpressing TEL-PDGFRB and AE into lethally irradiated syngeneic mice. These mice (19͞19, 100%) developed AML resembling M2-AML that was transplantable in secondary recipients. In contrast, control mice coexpressing with TEL-PDGFRB and a DNA-binding-mutant of AE developed a nontransplantable myeloproliferative disease identical to that induced by TEL-PDGFRB alone. We used this unique model of AML to test the efficacy of pharmacological inhibition of histone deacetylase activity by using trichostatin A and suberoylanilide hydroxamic acid alone or in combination with the tyrosine kinase inhibitor, imatinib mesylate. We found that although imatinib prolonged the survival of treated mice, histone deacetylase inhibitors provided no additional survival benefit. These data demonstrate that an activated RTK can cooperate with AE to cause AML in mice, and that this system can be used to evaluate novel therapeutic strategies.T he t(8;21)(q22;q22) translocation, which fuses the RUNX1 (AML1͞PEBP␣͞CBFA2) gene on chromosome 21 with the ETO (MTG8) gene on chromosome 8, is a common mutation associated with cases of acute myeloid leukemia (AML) of the FAB-M2 subtype (AML with maturation) (1). Expression of the resulting AML1-ETO (AE) fusion gene is detected in 40% of M2-AML patients and 12% of all newly diagnosed cases of AML (2, 3). The correlation between AE expression and the leukemic phenotype strongly suggests a causative role for AE in transformation. AE transcripts have been detected in nonneoplastic progenitors from AML patients in remission, suggesting that the translocation is an early event in the leukemogenic process (4). Furthermore, t(8;21) translocation and AE expression can be detected in neonatal Guthrie blood spots, implying an in utero origin of the translocation preceding development of AML in children by as much as 10 years (5,6).Several murine models have demonstrated that AE alone is not sufficient to induce leukemia. Mice expressing an inducible AE transgene in bone marrow cells remained disease-free for a normal life span of 24 mo (7). When expression of AE was targeted to the myeloid lineage by using the human MRP8 promoter, again the mice had no discernable phenotype (8). However, when additional random mu...