Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - BCM-UPS (Dissecting the role of the ubiquitin proteasome system in the pathogenesis and therapy of B-cell malignancies)

Teaser

Genome instability has been appreciated as one of the central cellular properties that enables the acquisition of the recognized hallmarks of cancer. At the same time, DNA damaging therapies such as ionizing radiation and chemotherapeutics are major treatment modalities used...

Summary

Genome instability has been appreciated as one of the central cellular properties that enables the acquisition of the recognized hallmarks of cancer. At the same time, DNA damaging therapies such as ionizing radiation and chemotherapeutics are major treatment modalities used in oncology. Therefore, cancer as a disease as well as its treatment are closely linked with DNA damage at multiple levels. To maintain genomic stability, cells have evolved a complex network, which coordinates cell cycle regulation with the DNA damage response (DDR) and apoptotic signalling. Previous progress in our understanding of the DDR has distinguished posttranslational modifications as central regulatory modes. The role of protein phosphorylation in cell cycle control and the DDR has been relatively well established. By contrast, the role of ubiquitylation, particularly as part of the ubiquitin proteasome system (UPS), is only recently emerging.

Cullin/RING ubiquitin ligases (CRLs) are modular enzymes that represent the largest subfamily of ubiquitin ligases comprising approximately 250 members. Prominent members include the prototypical SCF (Skp1/Cul1/F-box) complexes and CRL4-DDB1 ligases, which are characterized by variable substrate recruiting receptors, the F-box proteins and DCAFs (DDB1 Cul4 associated factor), respectively. While the vast majority of CRLs has not been linked to any substrates or biological activities, individual characterized members play key roles in cell cycle control, DNA damage response and tumourigenesis. Similar to other covalent modifications, ubiquitylation is reversible. The human genome harbours approximately 90 deubiquitylating enzymes (DUBs), which cleave ubiquitin off substrates and terminate ubiquitin-dependent signalling. Notably, several DUBs have been critically implicated in DNA damage response.

Faithfull cell cycle progression and DNA damage response is particularly relevant to B-cell malignancies, where in the B-cell of origin, DNA double strand breaks (DSBs) are a physiological process during the formation and revision of their antigen receptors. These DNA breaks must be resolved to generate functional B cells, and a failure of this process puts cells at risk for the formation of lymphoma and plasma cell malignancies. Certain forms of mature B-cell neoplasms respond well to available immuno-chemotherapeutic regimens, resulting in cure rates of around 40% in aggressive entities like DLBCL or long-term response rates in indolent entities like follicular lymphoma (survival of around 70% at 10 years). In explicit contrast, mantle cell lymphoma (MCL) and multiple myeloma (MM) cannot be cured and have the lowest median survival rates of all B-cell malignancies with an estimated 3-5 years for MCL and 4-6 years for MM. MCL localizes to the mantle region of secondary follicles, and accounts for around 8% of all NHL. MM, the overall second most frequent hematologic malignancy, results from the clonal expansion of plasma cells. Progressive levels of genomic instability reflect hallmarks of MCL and MM that result in multiple chromosomal abnormalities, including aneuploidy, important for progression of the disease and resistance to therapy.

The previous clinical success of proteasome inhibitors (e.g. Bortezomib, Carfilzomib) in the treatment of MM and MCL has highlighted the importance of the ubiquitin-proteasome system in these diseases and cancer in general, particularly in light of the importance of the UPS in the DNA damage response. Moreover, immunomodulatory drugs (IMiDs) like thalidomide and its derivatives Lenalidomide and Pomalidomide which demonstrate high efficacy in newly diagnosed and relapsed/refractory MM and MCL, exert their anti-tumour activity by targeting the UPS. Therefore, the identification of disease-specific aberrant pathways of the UPS would be predicted to significantly contribute to a better understanding of these diseases, and, even more importantly, define target structures whose inhi

Work performed

In Aim 1 we characterize candidate orphan CRLs and DUBs, for which we have obtained substantial evidence as to their role as oncogenes and tumour suppressors in MM (FBXO3, USP24), MCL (FBXO25), or MM and MCL (CRBN), respectively. Within this aim, these candidates are functionally dissected with regard to both their physiological and pathophysiological functions using human disease relevant tissue culture models.
In Aim 2 we elucidate the global role of CRLs and DUBs in the pathophysiology and evolution of MM and MCL using defined genetic screens that apply the CRISPR/Cas9 system. Here, custom designed gRNA libraries that cover 250 CRLs and 90 DUBs are utilized.
In Aim 3 we apply proteome wide unbiased functional proteomics based on specialized affinity- and non-affinity based protein purification strategies and quantitative mass-spectrometry to identify the relevant substrates of CRLs/DUBs identified in Aim 2.
Finally, in Aim 4, we analyze promising CRL/DUB candidates and their respective substrates generated in Aims 1-3 in murine transplant/xenograft models and defined patient cohorts with available clinical follow up data to obtain a true translational validation.

Final results

We expect that our work and the techniques applied will delineate the role of the UPS in MM and MCL, and will unravel novel oncogenic mechanisms and novel vulnerabilities, which may also be applicable to other haematological and solid tumour entities.