JHU/CCR Fellowship in Molecular Targets and Drug Discovery Technologies Project Proposal Form (2013)
7/30/2012 8:42:48 PM
Doanld P Bottaro
BG 10-CRC RM 2-3952
10 CENTER DR
BETHESDA MD 20814
Modeling Acquired Resistance to Met-targeted Cancer Therapies
Target(s) of Interest:
Hepatocyte Growth Factor (HGF) HGF Receptor, Met
Application to the Molecular Targets Initiative:
Hepatocyte growth factor (HGF) is a secreted protein that stimulates mitogenesis, motogenesis, and morphogenesis in a wide spectrum of cellular targets. HGF and its receptor, Met, are essential for normal embryonic development, adult homeostasis and wound repair. Aberrant, uncontrolled activation of this pathway occurs in many human cancers; importantly, oncogenic HGF signaling in this setting promotes aggressive cellular invasiveness that is strongly linked to tumor metastasis, the predominant cause of deaths due to cancer. The prevalence of HGF pathway activation in cancer has driven rapid growth in drug development programs: at least 20 new agents are now in human clinical trials. As shown by experiences with other molecularly targeted therapeutics, intrinsic and acquired drug resistance is inevitable. Anticipating drug resistance is essential to successful treatment: understanding its earliest manifestations and identifying emerging escape mechanisms are needed to make timely and effective adjustments in treatment strategy. In collaboration with Amgen, we developed a cellular model of acquired resistance to the HGF-neutralizing antibody rilotumumab in glioblastoma. This model will be used to define the molecular basis of resistance, identify a resistance signature for recognizing its earliest manifestations in patients, and develop strategies to recover effective disease control.
Glioblastoma Multiforme (GBM) is an aggressive brain malignancy and the most common brain tumor in adults. The National Cancer Institute estimates that 23,000 adults will be diagnosed with brain and other nervous system tumors in 2012 and 13,700 of these diagnoses will result in death. At present, the average survival rate for GBM is less than 15 months. Despite a better understanding of the mechanisms that underlie the development of GBM gained in the last several decades, improving therapeutic efficacy remains a significant challenge in GBM treatment. Currently there are no effective long-term treatments for this disease. A wealth of evidence implicates the HGF/Met pathway in GBM progression and the number of HGF/Met pathway targeted therapeutics entering in clinical trials for GBM treatment is growing rapidly.
Acquired drug resistance is a long-standing problem of cancer therapeutics; for example, chemoresistance to temozolomide occurs in >90% of recurrent GBM cases. The issue has become even more vexing with the development of highly selective targeted agents; e.g. agents targeting the epidermal and hepatocyte growth factor (EGF and HGF, respectively) pathways; resistance to gefitinib and erlotinib has already been frequently demonstrated in lung adenocarcinomas. Thus, anticipating acquired resistance and understanding its basis may help us recognizing its earliest manifestations in patients and develop clinical strategies to prevent or circumvent its occurrence. HGF, through its receptor tyrosine kinase Met, regulates mitogenesis, motogenesis, and morphogenesis in a range of cellular targets during development and homeostasis. HGF/Met signaling also contributes to oncogenesis and tumor progression in many prevalent human malignancies, including glioblastoma. Rilotumumab (AMG102) is a fully human neutralizing monoclonal antibody against HGF tested in multiple Phase 2 clinical trials, including mono therapy in renal cell carcinoma and glioblastoma, as well as combination trials in gastric, colorectal and small cell lung cancers and castrate resistant prostate cancer.
Collaborative work with Amgen has focused on the generation of drug-resistant tumor cell lines as a way to anticipate, understand and potentially circumvent acquired drug resistance. The human GBM cell line U87-MG was selected for this task because tumorigenesis in this line is completely HGF-driven. Cells grown in continuous exposure to a high concentration of rilotumumab (600 nM) acquired resistance in 75 days. Growth rate, HGF secretion, Met content and Met activation state in the resistant cell line, named U87 MG/HNR for HGF neutralization resistant, are 10-fold, 10,000-fold, 10-fold and 80-fold higher than the parental cell values, respectively. Like the parental cell line, the HGF and MET coding sequences in U87 MG/HNR are wild type. The HGF promoter DATE region, where truncation reportedly increases HGF expression level, is also of normal length in both parental and resistant cell lines. Quantitative PCR studies to determine mRNA levels of all HGF isoforms revealed a dramatic increase in full-length HGF transcript. CGH array studies indicated amplification of both HGF and MET genes. Xenograft studies of U87 MG/HNR in mice performed at NCI and Amgen confirmed that tumor growth was resistant to rilotumumab, but interestingly, the resistant cell line and tumors remained sensitive to selective c-Met tyrosine kinase inhibitors, suggesting that resistance was acquired exclusively through increased HGF/Met pathway activity.
This project focuses on the discovery of signaling pathways that are highly mutated or deregulated in GBM, using the data sets generated by The Cancer Genome Atlas (TCGA) and a drug-resistant GBM cell line generated through a collaboration between our laboratory and Amgen. To date, TCGA has achieved comprehensive sequencing, characterization, and analysis of the genomic changes in the GBM. Results already obtained from comparative genomic hybridization (CGH) and gene expression microarray analysis of U87 MG/HNR will be used to identify cytogenetic abnormalities, common gene promoter elements, and in turn, regulatory networks that are altered in this experimental model of acquired drug resistance. This information will be compared with data from the TCGA pilot project to identify genetic events and regulatory pathway aberrations that are common to our experimental model and acquired drug resistance in GBM patients. A subset of these genetic and regulatory characteristics will be sought that provides a resistance signature for recognizing its earliest manifestations in patients. Finally, identifying functionally critical molecular targets and/or events that are susceptible to therapeutic intervention will provide a basis for developing strategies to minimize or prevent acquired resistance and to recover effective disease control.
Current Research Expertise and Projects:
Upon binding to the cell-surface receptor tyrosine kinase (TK) Met, HGF stimulates mitogenesis, motogenesis, and morphogenesis in a wide range of cellular targets. These pleiotropic actions are fundamentally important during development, homeostasis and tissue regeneration. HGF/Met signaling also contributes to oncogenesis and tumor progression in RCC, prostate cancer and bladder cancer, among others. The goals of the laboratory are to:  develop and evaluate pathway antagonists and potential mechanisms of acquired drug resistance through comparative assessment of commercially developed agents targeting HGF and/or Met;  develop HGF/Met pathway related biomarkers and methods for diagnosis, pharmacodynamics (PD), patient selection and molecular imaging; and  further define Met-driven oncogenic signaling pathways in urologic malignancies to identify critical and targetable effectors and events.
We have developed two-site electrochemiluminescent immunoassays to quantitate intact Met protein and phospho-Met (pMet) in cells and tissues, and soluble Met ectodomain (sMet) in fluids, for diagnostic and PD interrogation of the signaling pathway. Accumulating indirect evidence links malignancy, increased proteolytic activity, and proteolytic activation of HGF at the target cell surface with increased sMet production. There is also evidence that Met TK domain is activated as a consequence of ectodomain cleavage, and the degradation of this active TK fragment, that rapidly ensues in normal cells, may be disrupted in certain cancers, contributing to oncogenesis.
Of all GU malignancies, bladder cancer (CaB) is one of the most deadly and prevalent, it is also one of the most expensive cancers to treat due to urinary cytology, and cystoscopy. Reliable, safe and cost-effective alternatives to cystoscopy and cytology currently are now under investigation. We began a study of urinary sMet levels in CaB patients and found a significant difference between the median urinary sMet levels in CaB samples and those cystoscopically negative for CaB. Patients who underwent radical cystectomy had greater median sMet levels than patients with pTa and pT1 tumors, predictive of their more advanced local disease. Our findings suggest that sMet measurement, alone or possibly in combination with other markers, may provide the sensitivity and specificity needed to replace cystoscopy for the surveillance of advanced CaB.
To systematically investigate the potential utility of plasma sMet as a PD biomarker for drugs that directly target Met kinase activity, we measured the plasma levels of sMet and those of HGF, VEGF and soluble VEGFR2 (sVEGFR2) of patients in phase 1 and 2 clinical trials of GSK1363089, a TK inhibitor of VEGFR2 and Met (GSK?089). Although median tumor burden did not change significantly over the course of the study, significant positive correlations between plasma levels of sMet, VEGF-A and tumor burden were observed at baseline and at termination. Thus, the observed changes in plasma sMet and VEGF-A levels in this study are likely to be a biological response of target inhibition.
We have obtained the one-armed monoclonal anti-Met antibody MetMab and Met TK inhibitors from Genentech to develop MetMab for PET imaging. In parallel, we have obtained Met ectodomain-selective peptides from GE Healthcare for development as SPECT and optical imaging agents. Preliminary tumor xenograft studies were conducted using the human gastric carcinoma derived cell line MKN45; this cell line expresses Met at high levels resulting in constitutive pathway activation and tumorigenesis which can be inhibited by Met selective kinase inhibitors. The GE Met-directed peptide readily detected MKN45 cell tumors in most tissues except kidney, the principle site of probe clearance. Animals bearing MKN45 tumors received daily IP injections of the Met TK inhibitor PHA665752, resulting in reduction of tumor volume and lower peptide signal. In collaboration with Amgen, we also have identified highly Met-selective small molecule TK inhibitors that preferentially recognize the activated form of the kinase for development as radioisotopic fluorine-labeled PET imaging agents. These agents will be initially developed in xenograft studies of human tumor cells lines with constitutive Met activation before testing in animal models of paracrine HGF-driven oncogenicity.
Collaborating Labs/Sponsors (if applicable):
Drs. Donald Bottaro and Fabiola Cecchi, UOB/CCR/NCI and Drs. Karen Rex, Angela Coxon and Michael Damore, Research Oncology, Amgen
Interactions with collaborators at Amgen will be primarily for consultation although benchwork under their supervision is an option.
Fellow Research Plan:
The trainee will participate in the discovery of signaling pathways that are highly mutated or deregulated in GBM, using the data sets generated by The Cancer Genome Atlas (TCGA) and a drug-resistant GBM cell line generated through a collaboration between our laboratory and Amgen. To date, TCGA has achieved comprehensive sequencing, characterization, and analysis of the genomic changes in the GBM. The Fellow will learn data mining methods and apply these to results already obtained from comparative genomic hybridization (CGH) and gene expression microarray analysis of U87 MG/HNR to identify cytogenetic abnormalities, common gene promoter elements, and in turn, regulatory networks that are altered in our experimental model of acquired drug resistance. The Fellow will use bioinformatics methods to compare this information with data from the TCGA pilot project to identify genetic events and regulatory pathway aberrations that are common to our experimental model and acquired drug resistance in GBM patients. Additional genomic-bioinformatic analysis will be used to select a subset of these genetic and regulatory characteristics to generate a valid resistance signature for recognizing its earliest manifestations in patients. Finally, using regulatory modeling and bench experimentation, the Fellow will help identify functionally critical molecular targets and/or events that are susceptible to therapeutic intervention that will provide a basis for developing strategies to minimize or prevent acquired resistance and to recover effective disease control.