In vitro characterization of BRAF(V600E) splicing variants: BRAF-220 and BRAF-204

BRAF is a kinase that can be found mutated (V600E) in several cancer types, such as melanoma, and, because of its cancer driver effect, it has been studied as therapeutic target. Irrespective of its mutational status, BRAF(V600E) is always expressed as a pool of at least two transcripts: the canonical one, called BRAF-ref (“reference”, ENST00000646891.2, BRAF-220), and BRAF-X1 (ENST00000496384.7, BRAF-204), being more phylogenetically conserved and 4-8 fold more expressed than the ref. From the mRNA point of view, -220 and -204 harbor 3’UTRs of different sequence and length (~4000nt vs ~7000nt), and, at the protein level, they differ for the last 5-6 amino acids at the C-terminal (-220: -GYGAFPVH vs -204: -GYGEFAAFK). Considering that the most studied BRAF(V600E) is the -220 isoform, while the most expressed is the conserved -204, with my PhD research I aimed to exploit state-of-the-art tools for understanding differences and similarities between the two variants, a crucial aspect to improve BRAF(V600E) targeting for melanoma patient treatment. For this purpose, an RNAi/CRISPR based system has been successfully set up to achieve, in an inducible fashion, the over-expression of only one BRAFV600E splicing variant at the time: through RNAi I’m able to induce the expression of a BRAF-targeting shRNA that results in the knock-down of cells’ endogenous BRAF (both -220 and -204 isoforms) and, at the same time, re-introduce BRAFV600E (-220, -204, or -DeltaCTerm (-∆CT) lacking the last 5-6 amino acids) expression, through CRISPR/Cas9-mediated insertion of the construct (consisting in BRAFV600E-220/-204/-∆CT CDS under the control of an inducible promoter) in the AAVS1 genomic locus. This complex system is currently being exploited to look for differences between BRAFV600E-220 and -204 (and -∆CT) proteins, starting with investigating whether they have different kinase activity, hence a different impact on MAPK signaling. I’m also interested in studying the role of the different C-term in determining proteins interaction with different partner proteins, for this purpose Co-Immunoprecipitation experiments will be performed followed by mass spectrometry. Finally, the system is being used to assess the different impact of BRAFV600E-220 and -204 on the cell’s profile, in terms of changes in the transcriptome, as well as in the miRNome, and the kinome. During my research, I also investigated changes in cells’ behavior when endogenous BRAF-204/BRAF-220 mRNA ratio is biased through alternative splicing modulators. Two oligonucleotides, one ASO (SplBRAF09) and a Morpholino (m204), having almost the same target sequence (just shifted of 1 nucleotide) on BRAF pre-mRNA, were tested in melanoma cell lines and showed to be able to promote BRAF(V600E)-220 expression against -204’s. From the phenotypic point of view, SplBRAF09 leads to a statistically significant decrease in cells’ clonogenicity; in addition, in 501Mel pigmentable melanoma cells appeared pigmented after 72h from SplBRAF09 transfection. However, no impact on clonogenicity has been observed with m204 in 501Mel, neither on cells’ pigmentation. Whether the different chemistries of the two tested oligos, or the 1 nucleotide shift between their target sequences are responsible for these unmatched results is currently under investigation: confirming SplBRAF09-related phenotypes could contribute to enrich the current knowledge regarding the balance between BRAF(V600E) isoforms, with all the therapeutic implications that could consequently arise. Finally, thanks to Co-IP studies, I was able to confirm how, irrespective of their C-term sequences, wild-type BRAF protein variants are able to homo- and hetero-dimerize in human cells (-∆CT as well); however, the recovery of different amount of each possible dimers combinations in Co-IPs suggests different affinity among the variants and/or different stability of the dimeric complexes. This aspect is currently under investigation: the highly flexible C-term could indirectly participate to BRAF variants affinity and/or dimers stabilization through interplay with other interactors in the cell. In conclusion, the results of this research not only will enrich the current knowledge on the expression regulation and interactors of one of the strongest oncogenes to be known in cancer biology, but it will also be crucial for achieving a more precise and effective targeting for oncologic patients’ treatment that considers isoform-specific features.