Functional validation of novel drug targets in multiple myeloma by use of a targeted shRNA library

• Applicant: Professorin Dr. Franziska Jundt
• Subject Area: Hematology, Oncology
• Term: from 2017 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 370022528

Despite impressive improvement in the treatment of multiple myeloma (MM) during the past decade through the use of target-specific agents, there is still a need for the identification of novel substances since in most cases MM remains an incurable disease. Most MM patients suffer from relapses even after many years due to intrinsic and acquired pharmacological resistance. It has been clearly established that aberrant Notch signaling is a master regulator of onset, progression and drug resistance in MM. Current strategies for Notch inhibition involve (1) blockade of receptor activation/cleavage through gamma secretase inhibitors (GSI) of which Notch is no exclusive target and (2) Notch1-directed antibodies. Thus far these strategies have been hampered by side effects such as skin disorders and gastrointestinal toxicities most probably due to wild-type Notch inhibition in nondiseased cells. We hypothesize that parts of these negative side effects might be avoided by selective blocking of critical downstream Notch effectors which mediate drug resistance in MM cells. The goal of this project is the identification and functional validation of yet unknown druggable Notch targets in MM by use of a high-throughput functional shRNA screening technology.In preliminary work we performed gene expression profiling employing RNAseq analysis to reveal Notch target genes both in engineered MM cells with low and inducible Notch signaling activity and in highly Notch-activated MM cells after Notch inhibition through GSI. In collaboration with Martin Eilers´s group we most recently generated a shRNA library that specifically targets Notch downstream effectors in MM cells. In this project we will now functionally screen candidate target genes for a role in MM survival and resistance to drugs such as melphalan, bortezomib and lenalidomide (WP1). Two different constitutively active forms of Notch1 (NdeltaE and NIC) will allow for identification of cytoplasmic as well as nuclear interaction partners (WP1). Functional validation of critical effectors of the shRNA screens will be achieved by use of chemical inhibitors (small molecule inhibitors), if available, and by nanogel-mediated transfer of target-specific shRNAs in MM cells (WP2). In-vivo response will be tested in two MM mouse models: (1) our newly established BALB/c-MOPC315.BM and (2) the VK*MYC model (WP3). For quantitative determination of bone disease (trabecular and cortical bone morphology, density and porosity) we use in collaboration with Bettina Willie´s group in Montreal (Canada) in-vivo microCT analysis and conventional histomorphometry (WP3). The knowledge gained from these experiments will aid in the development of novel strategies for treating MM patients with intrinsic and acquired drug resistance.

• DFG Programme: Research Grants