CRISPR-based functional genomics to dissect the regulation of multiple myeloma cell responses to perturbations of mutant RAS function: biological and therapeutic implications.

• Applicant: Dr. Torsten Steinbrunn
• Subject Area: Hematology, Oncology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 442740310

The RAS oncogene is mutated in up to 40% of multiple myeloma (MM) patients and mediates tumor cell growth and survival. In my previous work, I documented the dependence of MM cells on respective mutated KRAS or NRAS isoforms by suppressing their expression via RNA interference and causing MM cell death selectively in RAS-mutated cells. My studies indicated that potentially RAS-associated pathways (e.g. RAF/MAPK, the GTPase RAL, PI3K/Akt) mediate MM cell survival, but these effects were independent from mutant RAS. Therefore, direct inhibition of oncogenic RAS constitutes a key translational goal, which so far could not be reached due to lack of direct pharmacological RAS inhibitors. However, novel small molecule inhibitors were recently developed to specifically target mutated KRAS(G12C) and preliminary studies from Prof. Mitsiades’ lab indicate that these compounds exhibit potent activity against MM cells harboring this mutation. Therefore, these inhibitors enable us to decipher RAS-dependent functional contexts, mechanisms of resistance and protein interactions. Their results will also be relevant for further RAS-mutations in MM cells and translationally actionable as soon as novel inhibitors for currently undruggable RAS mutations become available.We will apply a series of clustered regularly interspaced short palindromic repeats (CRISPR)-based techniques to perform genome-scale loss-of-function (LOF) and gain-of-function (GOF) studies to identify genes that confer sensitization vs. resistance of MM cells in the context of pharmacological or genetic RAS inhibition. Candidate genes identified in these studies will be integrated in sub-genome sgRNA libraries and validated in focused CRISPR experiments. To account for signaling from the bone marrow (BM) micromilieu, these studies will be conducted in vitro in MM cell cultures vs. their co-culture with BM stromal cells from MM patients as well as in vivo using a “humanized“ BM-like mouse model. Next, we will perform dual sgRNA library-based CRISPR-studies, in which instead of one individual gene, pairwise combinations of two genes per cell will be abrogated (LOF) or activated (GOF). This will allow for specific predictions about RAS-dependent signaling interactions (cross-talk, feedback) and rational therapeutic combinations (synergism, antagonism) in MM.Using a biotin-labeled protein identification assay (BioID) in MM cells with undruggable RAS mutations (e.g. G12D, Q61H), we will identify interaction partners within close proximity (to oncogenic RAS, even if they are not bound or only transiently present. We expect that we can subsequently validate novel therapeutic targets using focused CRISPR-analysis to characterize them as potential combination partners or mediators of resistance.I will continue to develop these platforms after my return to Germany to apply them to a plethora of existing genomic data at a functional level with direct translational relevance even beyond mutant RAS and MM.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professor Constantine Mitsiades, Ph.D.