Combination therapy in a xenograft model of glioblastoma: enhancement of the antitumor activity of temozolomide by an MDM2 antagonist

OBJECTIVE Improvement in treatment outcome for patients with glioblastoma multiforme (GBM) requires a multifaceted approach due to dysregulation of numerous signaling pathways. The murine double minute 2 (MDM2) protein may fulfill this requirement because it is involved in the regulation of growth, survival, and invasion. The objective of this study was to investigate the impact of modulating MDM2 function in combination with front-line temozolomide (TMZ) therapy in GBM. METHODS The combination of TMZ with the MDM2 protein-protein interaction inhibitor nutlin3a was evaluated for effects on cell growth, p53 pathway activation, expression of DNA repair proteins, and invasive properties. In vivo efficacy was assessed in xenograft models of human GBM. RESULTS In combination, TMZ/nutlin3a was additive to synergistic in decreasing growth of wild-type p53 GBM cells. Pharmacodynamic studies demonstrated that inhibition of cell growth following exposure to TMZ/nutlin3a correlated with: 1) activation of the p53 pathway, 2) downregulation of DNA repair proteins, 3) persistence of DNA damage, and 4) decreased invasion. Pharmacokinetic studies indicated that nutlin3a was detected in human intracranial tumor xenografts. To assess therapeutic potential, efficacy studies were conducted in a xenograft model of intracranial GBM by using GBM cells derived from a recurrent wild-type p53 GBM that is highly TMZ resistant (GBM10). Three 5-day cycles of TMZ/nutlin3a resulted in a significant increase in the survival of mice with GBM10 intracranial tumors compared with single-agent therapy. CONCLUSIONS Modulation of MDM2/p53-associated signaling pathways is a novel approach for decreasing TMZ resistance in GBM. To the authors' knowledge, this is the first study in a humanized intracranial patient-derived xenograft model to demonstrate the efficacy of combining front-line TMZ therapy and an inhibitor of MDM2 protein-protein interactions

Preclinical characterization of the CDK4/6 inhibitor LY2835219: in-vivo cell cycle-dependent/independent anti-tumor activities alone/in combination with gemcitabine

The G1 restriction point is critical for regulating the cell cycle and is controlled by the Rb pathway (CDK4/6-cyclin D1-Rb-p16/ink4a). This pathway is important because of its inactivation in a majority of human tumors. Transition through the restriction point requires phosphorylation of retinoblastoma protein (Rb) by CDK4/6, which are highly validated cancer drug targets. We present the identification and characterization of a potent CDK4/6 inhibitor, LY2835219. LY2835219 inhibits CDK4 and CDK6 with low nanomolar potency, inhibits Rb phosphorylation resulting in a G1 arrest and inhibition of proliferation, and its activity is specific for Rb-proficient cells. In vivo target inhibition studies show LY2835219 is a potent inhibitor of Rb phosphorylation, induces a complete cell cycle arrest and suppresses expression of several Rb-E2F-regulated proteins 24 hours after a single dose. Oral administration of LY2835219 inhibits tumor growth in human tumor xenografts representing different histologies in tumor-bearing mice. LY2835219 is effective and well tolerated when administered up to 56 days in immunodeficient mice without significant loss of body weight or tumor outgrowth. In calu-6 xenografts, LY2835219 in combination with gemcitabine enhanced in vivo antitumor activity without a G1 cell cycle arrest, but was associated with a reduction of ribonucleotide reductase expression. These results suggest LY2835219 may be used alone or in combination with standard-of-care cytotoxic therapy. In summary, we have identified a potent, orally active small-molecule inhibitor of CDK4/6 that is active in xenograft tumors. LY2835219 is currently in clinical development. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10637-014-0120-7) contains supplementary material, which is available to authorized users

Multiple molecular interactions redundantly contribute to RB-mediated cell cycle control

BACKGROUND: The G1-S phase transition is critical to maintaining proliferative control and preventing carcinogenesis. The retinoblastoma tumor suppressor is a key regulator of this step in the cell cycle. RESULTS: Here we use a structure–function approach to evaluate the contributions of multiple protein interaction surfaces on pRB towards cell cycle regulation. SAOS2 cell cycle arrest assays showed that disruption of three separate binding surfaces were necessary to inhibit pRB-mediated cell cycle control. Surprisingly, mutation of some interaction surfaces had no effect on their own. Rather, they only contributed to cell cycle arrest in the absence of other pRB dependent arrest functions. Specifically, our data shows that pRB–E2F interactions are competitive with pRB–CDH1 interactions, implying that interchangeable growth arrest functions underlie pRB’s ability to block proliferation. Additionally, disruption of similar cell cycle control mechanisms in genetically modified mutant mice results in ectopic DNA synthesis in the liver. CONCLUSIONS: Our work demonstrates that pRB utilizes a network of mechanisms to prevent cell cycle entry. This has important implications for the use of new CDK4/6 inhibitors that aim to activate this proliferative control network

Vascular Abnormalities in the Neck Associated with Intracranial Aneurysms

ABSTRACT In 102 cases of ruptured intracranial aneurysms, the cervical as well as the cranial areas were explored by angiography. Loops in the course of the cervical vessels were present in 28 patients; features of fibromuscular dysplasia were present in 31 patients; and in 50 patients, no abnormalities were observed. In 7 patients, both cervical anomalies were present. Loops were associated with single aneurysms (95%), located primarily on the anterior communicating artery (76%), predominantly in men (M/F = 1.6). Aneurysms with fibromuscular dysplasia affected women more than men (F/M = 7), were frequently multiple (58%), and were located mainly on the intracranial part of the internal carotid and vertebral arteries (51%). These data suggest new concepts of aneurysm formation from inherited diseases and should permit the detection of some aneurysms before rupture.</jats:p

Vascular Abnormalities in the Neck Associated with Intracranial Aneurysms

ABSTRACT In 102 cases of ruptured intracranial aneurysms, the cervical as well as the cranial areas were explored by angiography. Loops in the course of the cervical vessels were present in 28 patients; features of fibromuscular dysplasia were present in 31 patients; and in 50 patients, no abnormalities were observed. In 7 patients, both cervical anomalies were present. Loops were associated with single aneurysms (95%), located primarily on the anterior communicating artery (76%), predominantly in men (M/F = 1.6). Aneurysms with fibromuscular dysplasia affected women more than men (F/M = 7), were frequently multiple (58%), and were located mainly on the intracranial part of the internal carotid and vertebral arteries (51%). These data suggest new concepts of aneurysm formation from inherited diseases and should permit the detection of some aneurysms before rupture