Viruses interact with numerous host factors to facilitate viral replication and to dampen antiviral defense mechanisms. We currently have a limited mechanistic understanding of how SARS-CoV-2 binds ...host factors and the functional role of these interactions. Here, we uncover a novel interaction between the viral NSP3 protein and the fragile X mental retardation proteins (FMRPs: FMR1, FXR1-2). SARS-CoV-2 NSP3 mutant viruses preventing FMRP binding have attenuated replication in vitro and reduced levels of viral antigen in lungs during the early stages of infection. We show that a unique peptide motif in NSP3 binds directly to the two central KH domains of FMRPs and that this interaction is disrupted by the I304N mutation found in a patient with fragile X syndrome. NSP3 binding to FMRPs disrupts their interaction with the stress granule component UBAP2L through direct competition with a peptide motif in UBAP2L to prevent FMRP incorporation into stress granules. Collectively, our results provide novel insight into how SARS-CoV-2 hijacks host cell proteins and provides molecular insight into the possible underlying molecular defects in fragile X syndrome.
Synopsis
The SARS-CoV-2 NSP3 protein binds directly to fragile X mental retardation proteins (FMRPs) to support viral replication. NSP3 binding disrupts FMRP interaction with the stress granule protein UBAP2L, thereby preventing FMRP localization to these structures.
The SARS-CoV-2 NSP3 protein binds to the KH domains of FMRPs through a short peptide motif.
Engineered SARS-CoV-2 viruses unable to bind FMRPs have reduced replication.
NSP3 binding to FMRPs disrupts their localization to stress granules through competition with UBAP2L.
The SARS-CoV-2 NSP3 protein binds directly to fragile X mental retardation proteins (FMRPs) to support viral replication. NSP3 binding disrupts FMRP interaction with the stress granule protein UBAP2L, thereby preventing FMRP localization to these structures.
Abstract
Purpose: The purpose of this study was to identify mechanisms of resistance to BRAF targeted therapy using proteomics together with genomics and transcriptomics in patients with BRAFV600E ...mutated solid tumors.
Experimental procedures: A total of nine patients with BRAFV600E mutated advanced solid tumors (5 with colorectal cancer, 2 with neuroendocrine carcinoma, 1 with cholangiocarcinoma and 1 with breast cancer) treated with BRAF targeted therapy (BRAF inhibitor in combination with either MEK inhibitor and/or EGRF antibody) as part of the Copenhagen Prospective Personalized Oncology study, were included in this study. Tumor biopsies at baseline and at disease progression were analyzed with whole exome/genome sequencing (WES/WGS), transcriptomics (RNA sequencing) and proteomics. Genomic variants were analyzed together with changes in protein expression. Three filtering steps were used to identify potential resistance mechanisms from the proteomics measurements. Proteins were filtered for 1) proteins with a high shift in abundance between baseline and progression 2) proteins with known associations to the patient’s primary cancer based on text-mining and 3) proteins in pathways where BRAF is also involved.
Results: Alterations conferring resistance were identified in 2 out of 9 patients when comparing data from WES/WGS and RNA sequencing at baseline and at disease progression (one patient with PTBP2-BRAF fusion and one with NRAS mutation). Genomic or transcriptomic alterations conferring resistance were not detected in the remaining 7 patients. Notably, there was not a single protein shared between all 9 patients after our filtering, but when comparing overlap on pathway level, six pathways related to RAF and MAPK signaling were affected in all 9 patients. Particularly, paradoxical activation of RAF signaling seems to be an interesting candidate to explain resistance, where formation of RAF dimer structures happens as a response to treatment with BRAF inhibitors, selectively inhibiting BRAF monomer structures (i.e. BRAFV600E).
Conclusion: With a multiomic approach using proteomics together with genomics and transcriptomics, potential mechanisms of resistance were detected in all patients at disease progression, where resistance to BRAF targeted therapies had occurred. Six mechanisms of resistance were shared independently of diagnosis and BRAF inhibitor combination regimes. The potential of integrating proteomics with genomics and transcriptomics is promising and may potentially guide therapy for future patients with treatment resistant BRAFV600E mutated solid tumors.
Citation Format: Martina Eriksen, Annelaura Bach Nielsen, Filip Mundt, Josephine Kerzel Duel, Matthias Mann, Ulrik Lassen, Christina Westmose Yde, Camilla Qvortrup, Martin Højgaard, Iben Spanggaard, Kristoffer Staal Rohrberg. Multiomics detect potential mechanisms of resistance to BRAF targeted therapy in patients with BRAFV600E mutated solid tumors abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3254.
