Deciphering and Targeting the Cancer Ubiquitylome

Protein ubiquitylation is a post-translational protein modification involved in a plethora of cellular processes, whereby ubiquitin moieties are transferred by ubiquitin ligases to distinct protein substrates. Genetic defects in the ubiquitin system are at the root of many acquired and hereditary diseases, including neurodegenerative, immune, and neoplastic disorders. Because of the complex biology and transient nature of ubiquitin modifications, the field has largely lacked comprehensive and unbiased approaches to detect ubiquitin changes in disease-relevant settings and related therapeutic approaches at the interface of ubiquitin ligases and their substrates. The overall goal of the Sinergia project is to develop and optimize approaches and tool compounds to detect and target dysregulated ubiquitylation in cancer by focusing on three recurrently mutated proteins within the ubiquitin posttranslational machinery: the ubiquitin ligase adaptor von Hippel-Lindau (VHL) and the deubiquitylating enzyme BRCA1 associated protein-1 (BAP1) in clear cell renal cell (RCC) cancer as well as the ubiquitin ligase adaptor speckle-type BTB/POZ protein (SPOP) in prostate and endometrial cancer. More specifically, we will take advantage of isobaric labeling to multiplex and quantify ubiquitin remnants in an unbiased manner in human tumor tissues and derived cell culture models. Subsequently, we aim to identify ubiquitin changes of relevant downstream effectors of tumorigenesis related to cancer-specific, recurrent alterations in VHL, BAP1 & SPOP by restoring the wild-type function of the protein. By applying multiplexed quantification of ubiquitin remnants on tumor tissue samples, we will further test whether specific ubiquitin sites or ubiquitin signatures correlate with histological subtypes and clinicopathological parameters of tumor aggressiveness. To develop new therapeutic avenues at the interface between ubiquitin ligases and their substrates and to chemically interrogate ubiquitylation with small molecule perturbation, we will model structural features of VHL & SPOP and their known or emerging substrates to design and synthetize small molecules that either modulate the function of the mutant ubiquitin ligase adaptors or target critical downstream effectors. The research proposal may establish an innovative and effective framework to decipher and target molecular mechanisms linked to dysregulated ubiquitylation, applicable to any disease type. To overcome the pertinent challenges in the field, we will use a multi-disciplinary approach that integrates existing expertise in proteomics-based ubiquitylation profiling and ubiquitin-related cancer biology with tissue and patient-derived cell line engineering, and structural computational and chemical-biophysical sciences.