Furnari, Frank B.

Abstract The clinical efficacies of molecularly targeted glioblastomas therapies have been vastly disappointing. The multitudes of resistance mechanisms suggest that glioblastomas possess highly dynamic molecular circuits grounded in functional redundancy. Emerging data suggests that the expression of functionally redundant oncogenes induced similar forms of cellular stress, requiring hyper-activation of common compensatory pathways to ensure cell viability. Targeting these pathways, therefore, afford potential opportunities for tumor ablation while by-passing the redundancy of oncogenic circuitry. To explore this paradigm, we carried out a siRNA screen to identify synthetic lethal partners of the oncogenic Epidermal Growth Factor Receptor variant III (EGFRvIII) in glioblastoma cells and identified Polo-Like Kinase 1 (PLK1). Treatment using a PLK1 inhibitor, BI2536, or siRNAs induced preferential toxicity to the EGFRvIII expressing glioblastoma cells in multiple in vitro (serum and neurosphere lines) and in vivo models (heterotopic and orthotopic xenograft models). Consistent with the heightened PLK1 requirement, EGFRvIII expressing glioblastomas harbored increased levels of the p-Thr210 PLK1 (an activated form of PLK1). Inhibition of PLK1 by BI2536 treatment induced an increase in the proportion of cells that co-stained for p-Histone H3 and γH2AX foci, suggesting accumulation of mitotic DNA damage. This effect was exacerbated by EGFRvIII expression, implicating induction of mitotic DNA damage as a major contributor to the observed synthetic lethality. Consistent with this observation, EGFRvIII expression induced the formation of aberrant mitosis as well as prolonged mitotic progression. Further supporting an essential role for PLK1 in suppressing DNA damage accumulation, BI2536 treatment significantly enhanced the tumoricidal effect of the DNA damaging chemotherapy, temozolomide. Mechanistically, inhibition of PLK1 suppressed the expression of Rad51, the accumulation of pS14 Rad51 (an active form of Rad51), as well as overall homologous recombination efficiency in vitro. We validated the clinical pertinence of these results using three clinically annotated glioblastoma databases (TCGA, REMEBMRANDT, CGGA). In all three datasets, increased expression of a PLK1 signature consistently associated with increased expression of HR genes and lowered gene expression signature associated with DNA damage accumulation. Supporting our proposed paradigm, the tumoricidal effect of BI2536 was universally observed in a panel of eight murine ink4a/arf (-/-) EGFRvIII expressing glioblastoma clones that developed resistance to EGFR inhibitors by distinct and independent mechanisms. In aggregate, our results support the essential role of PLK1 in suppressing mitotic DNA damage and provide a novel framework for glioblastoma therapy. Citation Format: Ying Shen, Masayuki Nitta, Jie Li, Diahnn Futalan, Tyler Steed, Zack Taich, Jeffrey M. Treiber, Deanna Stevens, Mark A. Schroeder, Jann N. Sarkaria, Hong-Zhuan Chen, Tao Jiang, Bob S. Carter, Fumiko Esashi, Jill Wakosky, Frank Furnari, Webster K. Cavenee, Arshad Desai, Clark C. Chen. Modulation of mitotic DNA damage as a paradigm for glioblastoma therapy. [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 2737. doi:10.1158/1538-7445.AM2014-2737

Glioblastoma, the most common primary malignant cancer of the brain, is characterized by rapid tumor growth and infiltration of tumor cells throughout the brain. These traits cause glioblastomas to be highly resistant to current therapies with a resultant poor prognosis. Although aberrant oncogenic signaling driven by signature genetic alterations, such as EGF receptor (EGFR) gene amplification and mutation, plays a major role in glioblastoma pathogenesis, the responsible downstream mechanisms remain less clear. Here, we report that EGFRvIII (also known as ΔEGFR and de2-7EGFR), a constitutively active EGFR mutant that is frequently co-overexpressed with EGFR in human glioblastoma, promotes tumorigenesis through Src family kinase (SFK)-dependent phosphorylation of Dock180, a guanine nucleotide exchange factor for Rac1. EGFRvIII induces phosphorylation of Dock180 at tyrosine residue 722 (Dock180 Y722 ) and stimulates Rac1-signaling, glioblastoma cell survival and migration. Consistent with this being causal, siRNA knockdown of Dock180 or expression of a Dock180 Y722F mutant inhibits each of these EGFRvIII-stimulated activities. The SFKs, Src, Fyn, and Lyn, induce phosphorylation of Dock180 Y722 and inhibition of these SFKs by pharmacological inhibitors or shRNA depletion markedly attenuates EGFRvIII-induced phosphorylation of Dock180 Y722 , Rac1 activity, and glioblastoma cell migration. Finally, phosphorylated Dock180 Y722 is coexpressed with EGFRvIII and phosphorylated Src Y418 in clinical specimens, and such coexpression correlates with an extremely poor survival in glioblastoma patients. These results suggest that targeting the SFK-p-Dock180 Y722 -Rac1 signaling pathway may offer a novel therapeutic strategy for glioblastomas with EGFRvIII overexpression.

Abstract 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 EGFRvIII-dependent 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]