Activating epidermal growth factor receptor (EGFR) mutations are common in many cancers including glioblastoma. However, clinical responses to EGFR inhibitors are infrequent and short-lived. We show that the Src family kinases (SFK) Fyn and Src are effectors of oncogenic EGFR signaling, enhancing invasion and tumor cell survival in vivo. Expression of a constitutively active EGFR mutant, EGFRvIII, resulted in activating phosphorylation and physical association with Src and Fyn, promoting tumor growth and motility. Gene silencing of Fyn and Src limited EGFR-and EGFRvIIIdependent tumor cell motility. The SFK inhibitor dasatinib inhibited invasion, promoted tumor regression, and induced apoptosis in vivo, significantly prolonging survival of an orthotopic glioblastoma model expressing endogenous EGFRvIII. Dasatinib enhanced the efficacy of an anti-EGFR monoclonal antibody (mAb 806) in vivo, further limiting tumor growth and extending survival. Examination of a large cohort of clinical samples showed frequent coactivation of EGFR and SFKs in glioblastoma patients. These results establish a mechanism linking EGFR signaling with Fyn and Src activation to promote tumor progression and invasion in vivo and provide rationale for combined anti-EGFR and anti-SFK targeted therapies. [Cancer Res 2009;69(17):6889-98]

Key Points• Decitabine treatment of in vitro expanded primary AML samples leads to global hypomethylation.• Highly methylated CpGs are most affected by decitabineinduced hypomethylation, with little influence on transcriptional activity.Acute myeloid leukemia (AML) is characterized by dysregulated gene expression and abnormal patterns of DNA methylation; the relationship between these events is unclear. Many AML patients are now being treated with hypomethylating agents, such as decitabine (DAC), although the mechanisms by which it induces remissions remain unknown. The goal of this study was to use a novel stromal coculture assay that can expand primary AML cells to identify the immediate changes induced by DAC with a dose (100nM) that decreases total 5-methylcytosine content and reactivates imprinted genes (without causing myeloid differentiation, which would confound downstream genomic analyses). Using array-based technologies, we found that DAC treatment caused global hypomethylation in all samples (with a preference for regions with higher levels of baseline methylation), yet there was limited correlation between changes in methylation and gene expression. Moreover, the patterns of methylation and gene expression across the samples were primarily determined by the intrinsic properties of the primary cells, rather than DAC treatment. Although DAC induces hypomethylation, we could not identify canonical target genes that are altered by DAC in primary AML cells, suggesting that the mechanism of action of DAC is more complex than previously recognized. (Blood. 2013;121(9):1633-1643) IntroductionAcute myeloid leukemia (AML) is a clonal hematopoietic neoplasm characterized by maturation arrest in the myeloid lineage. Treatment typically consists of induction chemotherapy with an anthracycline and cytarabine with the goal of achieving a complete remission, 1,2 followed by consolidation therapy. Despite these measures, the mortality rate of AML is still very high. Although recent genomic advances have improved our understanding of AML pathogenesis and risk stratification 3,4 the overall outcome is still dismal for most patients, and alternative treatment strategies are needed.One alternative approach for the treatment of AML and myelodysplastic syndromes (MDS) is the use of hypomethylating agents, including the cytosine analogs 5-azacytidine (AZA) and 5-aza-2Ј-deoxycytidine (decitabine; DAC). 5,6 These nucleosides are distinct from other cytosine analogs because they contain a pyrimidine ring modification that results in the covalent trapping of the maintenance DNA methyltransferase (DNMT1). This effect is cell-cycle dependent, because AZA and DAC must first be modified and incorporated into newly synthesized DNA; this leads to passive hypomethylation of DNA as cells divide, because of the depletion and degradation of DNMT1. 7,8 Exposure to these drugs is also associated with cellular differentiation 9,10 and cytotoxicity at higher doses. 11 Although the impact of DAC and AZA on acute myeloid leukemia cell lines ha...

Molecularly targeted therapies are transforming the care of patients with malignant gliomas, including glioblastoma, the most common malignant primary brain tumor of adults. With an arsenal of small molecule inhibitors and antibodies that target key components of the signal transduction machinery that are commonly activated in gliomas, neuro-oncologists and neurosurgeons are poised to transform the care of these patients. Yet, the successful application of targeted therapies remains a challenge. Strategies are lacking for directing kinase inhibitor or other pathway-specific therapies to individual patients most likely to benefit. In addition, response to targeted agents is determined not only by the presence of the key mutant kinases, but also by other critical changes in the molecular circuitry of cancer cells; e.g. such as loss of key tumor suppressor proteins, the selection for kinase resistant mutants and the deregulation of feedback loops. Understanding these networks, and studying them in patients, will be critical for developing rational combination therapies to suppress resistance for malignant glioma patients. Here we review the current status of molecular targeted therapies for malignant gliomas. We focus initially on identifying some of the insights learned so far from targeting the EGFR/PI3K/Akt/mTOR signaling pathway in patients and on how this has lead towards a re-conceptualization of some of the new challenges and new directions for targeted treatment. We describe how new advances from the world of genomics have the potential to transform our approach towards targeted therapies, and describe how a deeper understanding of the complex nature of cancer, its adeptness at re-wiring molecular circuitry to evade targeted agents, has raised new challenges and identified new leads.

Acute promyelocytic leukemia (APL) is initiated by the PML-RARA fusion oncogene and has a characteristic expression profile that includes high levels of the Notch ligand JAG1. In this study, we used a series of bioinformatic, in vitro, and in vivo assays to assess the role of Notch signaling in human APL samples, and in a PML-RARA knockin mouse model of APL (Ctsg-PML-RARA). We identified a Notch expression signature in both human primary APL cells and in Kit+Lin−Sca1+ (KLS) cells from pre-leukemic Ctsg-PML-RARA mice. Both genetic and pharmacologic inhibition of Notch signaling abrogated the enhanced self-renewal seen in hematopoietic stem/progenitor cells (HSPCs) from pre-leukemic Ctsg-PML-RARA mice, but had no influence on cells from age-matched wildtype mice. In addition, 6 of 9 murine APL tumors tested displayed diminished growth in vitro when Notch signaling was inhibited pharmacologically. Finally, we found that genetic inhibition of Notch signaling with a dominant negative MAML protein reduced APL growth in vivo in a subset of tumors. These findings expand the role of Notch signaling in hematopoietic diseases, and further define the mechanistic events important for PML-RARA-mediated leukemogenesis.

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