Roger Abounader - US grants

DESCRIPTION (provided by applicant): In malignant human gliomas, the tyrosine kinase receptor c-Met and its ligand hepatocyte growth factor (HGF) are frequently overexpressed and the tumor suppressor PTEN is frequently mutated. In the previous grant, we uncovered multiple mechanistic and functional interactions between PTEN and c-Met pathway and found that c-Met activation and PTEN loss have additive effects on glioma malignancy. In this competitive renewal, we propose a continuation of the analysis of PTEN/c-Met in gliomas with a focus on novel mechanistic interactions between PTEN and c-Met pathway and on testing new preclinical therapeutic agents and strategies. In aim #1, we will study the regulation of PTEN phosphorylation and half-life by HGF that we recently discovered. The findings will uncover a new mechanism of HGF-induced malignancy and of PTEN post-translational regulation. In aim #2, we will test the hypothesis that PTEN downregulates c-Met expression via a new pathway involving p53 and microRNA-34a. The findings could explain the concurrent PTEN loss and c-Met overexpression in glioblastoma and uncover a new mechanism of c-Met expression regulation. In aim #3, we will determine the effects of PTEN expression on the in vivo therapeutic efficiency of anti-HGF monoclonal antibodies and a new orally bioavailable small molecule inhibitor of c-Met. We will also assess the therapeutic value of combining anti-HGF or anti-c-Met therapies with mTOR inhibitors that counteract the effects of PTEN loss in PTEN- mutated xenografts. The translational experiments will be performed using primary glioma cell-derived and glioma stem cell-derived xenografts. Successful completion of the proposed experiments will provide new mechanistic insights into the regulation of PTEN and c-Met and uncover previously unknown molecular interactions between them. The findings will also provide necessary and timely information for the use in a clinical setting of clinically applicable anti-HGF and anti-c-Met therapies also in combination with mTOR inhibitors. The use of clinically applicable agents in primary and glioma stem cell animal models will confer high validity and preclinical significance to the obtained results. PUBLIC HEALTH RELEVANCE: We previously found that increase of the oncogene c-Met and loss of the tumor suppressor PTEN have additive effects on the growth of glioma brain tumors. We propose to study new mechanisms with which c-Met and PTEN regulate each other in gliomas. We also propose to test the effects of PTEN on new clinically useful drugs that target c-Met and also test new drugs and drug combinations for glioma therapies. The results will lead to the development of new glioma therapeutic strategies and provide important information for the use of the new drugs in the clinic.

DESCRIPTION (provided by applicant): PTEN and p53 are the most frequently mutated tumor suppressors in human cancer, including gliomas. Recent evidence shows that wild-type PTEN and wild-type p53 (wt-p53) enhance each other's tumor suppressive functions. Wt-p53 induces PTEN gene transcription and wt-PTEN protects wt-p53 protein from degradation. We recently found, for the first time, that PTEN has unexpected tumor promoting properties in some glioma cells and tumor xenografts. We have preliminary evidence that PTEN acquires these unexpected tumor promoting properties by enhancing the half-life and oncogenic effects of gain-of-function p53 mutants (mut-p53). Based on these findings, we formulate the following novel hypothesis: PTEN tumor suppressor can exhibit tumor promoting properties in the setting of gain-of-function mut-p53. Therefore, therapeutic strategies that aim at restoring PTEN expression or function could lead to varying effects that depend on the mutational status of p53. To test this hypothesis and its prognostic, mechanistic, functional and therapeutic implications, we propose the following studies: In aim #1, we will use a large number of banked human glioblastoma specimens to determine the association between the combined PTEN/p53 mutational status and clinical outcome. In aim #2, we will investigate the mechanism through which PTEN regulates mut-p53 protein levels and function. In aim #3, we will assess the in vivo effects of restoring PTEN to glioma tumors with varying p53 mutational status. In aim #4, we will determine if small molecule modulators of p53 can reverse the tumor promoting effects of PTEN in mut-p53 cells and tumors. The results from all aims will be assessed for their consistency with the hypothesis. Successful completion of the studies proposed in this application would: 1) establish the combined PTEN/p53 status as a prognostic parameter (aim 1), 2) uncover previously unknown mechanistic and functional interactions between PTEN and mut-p53 (aim 2), 3) determine conditions and strategies for a successful therapeutic restoration of PTEN (aim 3), and 4) have important clinical implications for the use of small molecule modulators of p53 by identifying a subset of tumors that are more sensitive to these drugs and providing a rationale for their combination with PTEN restoration (aim 4).

DESCRIPTION (provided by applicant): Receptor tyrosine kinase (RTK) pathways are deregulated in a majority of glioblastoma (GBM), the most common and most deadly primary malignant brain tumor. Consequently, a number of clinically applicable RTK inhibitors have been developed. However, these inhibitors failed to significantly improve the clinical outcomes of GBM patients. The main reasons for this failure are signal redundancy due to co-activation of several RTKs and compensatory mechanisms that lead to resistance to RTK inhibition. In this competitive renewal application, we build on our previous findings and propose to explore new mechanisms and strategies for improving the efficacy of MET and other RTK inhibitions in GBM therapy. More specifically, we propose to comprehensively uncover the factors that determine sensitivity and resistance to MET inhibition with clinically applicable drugs and use the acquired knowledge to test new and more efficient combination therapies. We also propose to explore two conceptually novel approaches for RTK targeting in GBM therapy that are based on two recent exciting discoveries from our lab. We propose four specific aims. In Aim 1, we will identify the not well known factors that determine sensitivity to MET inhibition. We will use RNA-seq, reverse phase antibody arrays, and PCR to comprehensively identify the genetic and molecular factors that determine responsiveness to clinically applicable small molecule kinase inhibitor (crizotinib) and MET neutralizing antibody (MetMAb) in GBM cells, stem cells, animal models and MetMAb clinical trial-derived human tumors. In Aim 2, we will comprehensively elucidate the mechanisms of resistance to MET inhibition by the above drugs and develop new combination therapies that overcome resistance. In Aim 3, we will study and develop ligand pre-treatment as a new strategy for improving the efficacy of MET, EGFR and PDGFR inhibitors. This is based on a recent intriguing discovery form our lab that shows that short-term pre-treatment of RTKs with their respective ligands enhances the anti-tumor effects of their inhibitors. In Aim 4, we will explore the role of microRNA-134 (miR- 134) in RTK signaling and experimental therapy. This is based on the recent discovery in our lab of miR-134 as a new tumor suppressive hub that mediates the effects of RTKs in GBM and that is required for the anti-tumor effects of their inhibitors. For Aims 3 and 4, we will develop and test new pre-clinical approaches for the systemic and local deliveries of RTK ligands and microRNAs to GBM xenografts using focused ultrasound and microbubbles/nanoparticles as well as convection enhanced delivery. Altogether, successful completion of the proposed studies would lead to a better understanding of the mechanisms of MET and RTK-induced malignancy and to the development of novel and more efficient RTK targeting strategies for GBM therapy.

ABSTRACT Glioblastoma (GBM) is the most common and most deadly primary malignant brain tumor. Despite the most advanced treatment with combinations of surgery, radiotherapy and chemotherapy, GBM is associated with a median life expectancy of only ~15 months. Targeted molecular therapies are arguably one of the most promising approaches to achieving more effective future GBM therapies. A major challenge facing such an approach is the simultaneous deregulation of multiple molecules in any given single tumor, as demonstrated by The Cancer Genome Atlas (TCGA) and other published research. Because of this co-deregulation, it is not surprising that molecular monotherapies have failed to achieve significant improvements in GBM clinical outcomes. Several lines of evidence suggest that the simultaneous targeting of multiple deregulated molecules and pathways is required to achieve better therapies. Based on preliminary evidence, we hypothesize that there exist ?master regulatory microRNAs? (miRNAs) that simultaneously regulate multiple deregulated molecules in GBM. The goal of this application is to discover, investigate, and therapeutically exploit such miRNAs. We believe that studying them will provide new information on the mechanisms of gene expression (de)regulation in GBM and that restoring or inhibiting them can be exploited for therapy. We propose three specific aims. In aim 1, we will use a novel screening approach, PAR-CLIP, in combination with smRNA-seq and TCGA gene expression data analysis to uncover global miRNA targets and identify single miRNAs (designated master regulatory miRNAs) that simultaneously target and regulate the largest number of deregulated molecules in GBM. In aim 2, we will investigate the functions and modes of action of these master regulatory miRNAs and validate their expressions and targets in human GBM specimens. In aim 3, we will test miRNAs as novel experimental therapeutic agents or targets in GBM. Thereby, we will develop and use novel potentially clinically applicable local and systemic delivery agents and approaches including brain penetrating nanoparticles (BPN), convection-enhanced delivery (CED) and focused ultrasound with microbubbles (FUS-MB). Successful completion of the proposed studies would establish the first compendium of miRNA targets in GBM, generate new knowledge on the (de)regulation of gene expression by miRNAs and their effects on GBM malignancy, and develop novel technologies for the exploitation of novel master regulatory miRNAs in GBM therapy.