P01.05 Deciphering the function of the ubiquitin-proteasome-system in regulating the immune checkpoint protein B7-H3 (CD276) in non-small cell lung cancer

Background Cancer cells use the expression of immune checkpoint proteins on their surface to evade responses. Targeting these checkpoints with antibodies has substantially advanced cancer therapy in past years, especially treatment lung cancer. However, prognosis metastatic patients still remains poor and be leading cause death worldwide. Further therapeutic concepts are therefore urgently needed. It been shown that protein levels PD-L1, a member B7 family, is regulated by ubiquitin-proteasome system (UPS). Ubiquitin-ligases (E3-ligases) deubiquitinating enzymes regulate cell considered promising potential drug targets. Inhibiting increase might anti-cancer response. Here, we investigate whether another family member, B7-H3, UPS non-small (NSCLC). Materials Methods B7-H3 NSCLC lines patient samples was evaluated using mRNASeq data from open databases. Immunoblotting FACS were used analyse total endogenous different under normal growth conditions response various inhibitors (MG-132, Chloroquine (CQ) Cycloheximide (CHX)). Immunoprecipitation FLAG-tagged followed TUBE IP ubiquitin-binding beads in-vivo ubiquitylation assays based co-overexpression HA-tagged ubiquitin and/or K48/K63-linkages specific together or K526R mutant HEK-93T performed ubiquitination B7-H3. Mass spectrometry analysis FLAG-purified identify possible interaction partners. Results Database revealed higher than healthy tissue. We found highly expressed RNA levels. Treatments either proteasomal (MG-132) lysosomal degradation alone showed only minor effects abundance. CHX H1437 decreased over time this decrease recovered adding MG-132 CQ suggesting both lysosome as well proteasome involved In vivo assay K48 K63 ubiquitination. purified E3-ligase Trim21, which recently identified ligase PD-L1 lines, partner. experiments planned validate result other UPS-related post-translational level regulation. Conclusions Our indicate lines. With further experiments, aim stabilize surface. Pharmacological inhibition such reduce checkpoint’s anti-tumour Disclosure Information M. Kurz: None. L. Rieger: P. Giansanti: B. Kuster: F. Bassermann: