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G-protein Coupled Receptor Expression Patterns in Medulloblastoma Subgroups: Identifying and Exploiting Molecular Targets

2015-05-01 00:03:07

The University of Iowa; 2015 May; Doctoral Thesis


Kelsey Lynnea Whittier



Abstract



Medulloblastoma is the most common malignant brain tumor in children. Genetic profiling has identified four principle tumor subgroups; each subgroup is characterized by different initiating mutations, genetic and clinical profiles, and prognoses. The two most well-defined subgroups are caused by overactive signaling in the WNT and SHH mitogenic pathways; less is known about Groups 3 and 4 medulloblastomas. Identification of tumor subgroup using molecular classification is poised to become an important component of the medulloblastoma diagnosis and staging and will likely guide therapeutic options.



G-protein coupled receptors (GPCR) possess characteristics that make them ideal targets for molecular imaging and therapeutics. While expression patterns of many proteins in human medulloblastoma subgroups have been discerned, the expression pattern of GPCRs in medulloblastoma has not been investigated. We have found that clusters of medulloblastoma tumors arise based solely on differential GPCR expression patterns. Further, two of these clusters correspond with high fidelity to the WNT and SHH subgroups. Distinct over-expressed GPCRs emerge; for example, LGR5 and GPR64 are significantly and uniquely over-expressed in the WNT subgroup of tumors, while PTGER4 is over-expressed in the SHH subgroup. Uniquely under-expressed GPCRs were also observed. Our results identify GPCRs with potential to act as imaging and therapeutic targets; elucidating tumorigenic mechanisms is a secondary benefit to identifying differential GPCR expression patterns in medulloblastoma tumors.



Current imaging for diagnosis, staging, and measuring response to therapy for medulloblastoma patients relies heavily on MRI; single photon emission tomography (SPECT) using 111 In-DTPA-Octreotide targeting the somatostatin type 2 receptor (SSTR2) is also available. Positron emission tomography (PET) affords a more sensitive and specific imaging modality than SPECT; however, the most common tracer 18 FDG, is of limited usefulness for the delineation of brain tumors. Smoothened (SMO) is a GPCR that is overexpressed in a subset of medulloblastoma; we hypothesized that SMO overexpression could be exploited as a specific PET target in these tumors. Genentech generously provided the synthetically-derived small-molecule SMO ligand, GDC-0449, for use as the lead compound for development of a PET tracer. GDC-0449 has already been demonstrated to localize in brain tumors and has Cl-atoms incorporated in positions that are predicted to readily exchange with fluorine-18 to generate a fluorinated analog of the compound.



We have successfully fluorinated GDC-0449, with very high radiochemical purity. Binding assays reveal affinities of the fluorinated analog of GDC-0449 for SMO to be comparable to precursor GDC-0449, and biodistribution experiments demonstrate accumulation of the fluorinated compound in tumors. The fluorinated analog of GDC-0449 holds promise as a novel PET imaging agent in medulloblastoma, providing highly specific and sensitive imaging for use in diagnosis, staging and measurement of response-to-treatment.



CHAPTER 1 INTRODUCTION



Medulloblastoma is the most common malignant brain tumor in children and accounts for more deaths than any other central nervous system (CNS) disease. These tumors arise exclusively in the cerebellum,and represent dysregulation of cerebellar developmental pathways. Recent investigation has identified four molecularly and prognostically distinct medulloblastoma subgroups.



G-protein coupled receptors (GPCR) are key regulators and points of control in a large number of signaling pathways. GPCRs also possess characteristics that make them ideal targets for molecular imaging and therapeutics; including that they are located at the cell surface, their ligands bind with high affinity and specificity, and the receptor-ligand complex is endocytosed carrying the ligand into the tumor cell. Despite this functional promise, and while expression patterns of many proteins in human medulloblastoma subtypes have been discerned, the expression pattern of GPCRs in medulloblastoma has not been broadly investigated.



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