Cavenee, Webster K.

PURPOSE GBM AGILE (ClinicalTrials.gov identifier: NCT03970447 ) is a phase II/III Bayesian adaptive platform registration trial testing multiple arms against a common control; the primary end point is overall survival (OS). Regorafenib, a multikinase inhibitor, showed OS benefit in recurrent (RD) glioblastoma in the phase II REGOMA trial and entered GBM AGILE as the first investigational arm. METHODS Patient subtypes included in the regorafenib arm of GBM AGILE were newly diagnosed unmethylated (NDU) and RD glioblastoma. Prospective defined sets of subtypes, or arm signatures, were NDU, RD, and all (NDU + RD). As the first investigational arm in GBM AGILE, regorafenib was equally randomized to the control arm. Treatment in the control arm is temozolomide + radiotherapy (in newly diagnosed) or lomustine (in RD). Efficacy was assessed by OS hazard ratio (HR), arm/control, and demonstrated when the Bayesian probability of benefit (HR <1.00) was ≥98%. Analysis was performed monthly for limited efficacy, which occurs when the Bayesian predictive power is <25% for all signatures, and determines stopping enrollment. Follow-up continued for 12 months after accrual stopped. RESULTS When the predictive power was <25% in all predefined signatures for regorafenib, accrual stopped for limited efficacy. The final analysis did not demonstrate OS improvement in the regorafenib arm in RD nor NDU glioblastoma. Median HRs were 1.05 (NDU), 1.07 (RD), and 1.07 (all) with final probabilities of benefit (HR <1.00) of 0.421 (NDU), 0.312 (RD), and 0.296 (all). Regorafenib was associated with increased toxicity relative to control. CONCLUSION GBM AGILE did not show superiority of regorafenib over control in RD (lomustine) or NDU (temozolomide + radiotherapy) glioblastoma, yet caused increased toxicities. Regorafenib has been removed from National Comprehensive Cancer Network guidelines as a treatment option for RD.

Abstract GBM AGILE(NCT03970447) is a phase 2/3 Bayesian adaptive registration platform trial testing multiple therapies against a common control. Paxalisib, a PI3K/mTOR inhibitor, is the 3rd arm in the trial to conclude evaluation. METHODS Paxalisib was open to patients with newly diagnosed unmethylated (NDU) and recurrent (RD) glioblastoma, with three possible signatures: NDU, RD, and All(NDU+RD). Arm enrollment occurred Dec2020 through May2022. Control patients were enrolled from study initiation (July2019) and were treated with temozolomide(NDU) or lomustine(RD). GBM AGILE investigational arms have 1 or 2 stages, with adaptive randomization in stage 1 and fixed randomization if arms continue to stage 2. Efficacy is based on OS hazard ratio(HR) of Arm/Control. Efficacy goal is final Bayesian probability ≥ 98% for HR&lt;1.00 in combined Stages. An Arm continues to Stage 2 if Bayesian predictive power (PP) ≥ 0.8. An Arm stops accruing in Stage 1 if it reaches maximal sample size(N) or does not meet a minimum efficacy threshold (PP&lt;0.25 for all signatures when N&gt;50). Clinical cut-off is 12 months after accrual stops. The maximum N for paxalisib was approximately 150(Stage 1) and 50(Stage 2). RESULTS After paxalisib reached &gt;150 patients in Stage 1, accrual stopped [Paxalisib/control N 54/75(NDU), 100/188(RD)]. Neither PP thresholds for moving to Stage 2 nor stopping for minimum efficacy were met. Following an unplanned public disclosure that the arm did not continue to stage 2, clinical cut-off for final analysis (planned May2023) was updated to public disclosure date (August2022). At final analysis mean HRs were 0.89(NDU), 1.25(RD), 1.05(All), probabilities of HR&lt;1.00 of 0.72(NDU), 0.076(RD), 0.398(All). Model estimated median OS paxalisib/control (months) were 14.77/13.84(NDU) and 8.58/10.06(RD). CONCLUSION Paxalisib did not show survival benefit over cumulative control in final primary analysis; additional secondary analyses are being considered. GBM AGILE continues to rapidly and efficiently assess therapies in ND and RD glioblastoma.

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

Significance We have identified a lethal subtype of gastric cancer (GC) that is characterized by high levels of clonal heterogeneity and TP53 (tumor protein P53) mutation. We have also uncovered key novel mutations in the targetable NRG1 (neuregulin-1) and ERBB4 (V-Erb-B2 avian erythroblastic leukemia viral oncogene homolog 4) ligand-receptor pair and identified BRCA2 (breast cancer 2, early onset) mutations as new genetic markers to predict better survival for GC. Our study represents a novel approach for GC personalized medicine and identified novel clinical actionable therapies for GC therapy.

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]