No abstract

Introduction: genetic interrogation of the KRAS signaling pathway in genetically engineered mouse models harboring a KRASG12V mutation has shown that RAF1 ablation promotes tumor regression, and is therefore highlighted as a promising therapeutic target. Recently, our group has been able to purify the full-length form of RAF1 in its native state and resolve part of its structure by Cryo-Electronic Microscopy. RAF1 is found as a component of a protein complex (RHC complex), including the chaperone HSP90 and its cochaperone CDC37, being this interaction crucial for RAF1 stability. In this work, we interrogate the role of CDC37-RAF1 interaction in the stability of RAF1 in order to identify effective approaches to degrade this kinase. Materials and Methods: by site-directed mutagenesis we generated a plethora of RAF1 mutants, altering key residues located in its CDC37-binding region. Then, the relative levels of the RAF1 mutant proteins in the isolated RHC complexes were assessed by mass spectrometry (MS) and compared to those formed by the wild type protein. To further evaluate the relevance of CDC37-RAF1 disruption, we designed small peptides covering the region of interaction. The binding affinity of these peptides was assessed by incubating the cochaperone with a nitrocellulosebound dodecapeptide array (PepScan) displaying the corresponding RAF1 sequences. Then, to assess the phenotypic effect of the selected peptides, we functionalized them for in vivo delivery, using a TAT cell penetrating motif. After treating cells with RAF1 specific and non-specific peptides, culture proliferation was measured. Results: according to MS results, some of the mutants resulted in RAF1 natural degradation, and same results were observed when the key residues involved in the interaction were mutated in CDC37 instead of RAF1. On the other hand, the treatment with CDC37-RAF1 interfering peptides selectively inhibited growth in lung cancer cell lines. These observations suggest that these RAF1-derived peptides may prevent RHC complex assembly, hindering RAF1 activity and thereby affecting cell proliferation. Conclusions: altogether, our results highlight the importance of chaperon association for RAF1 stability, raising the possibility that the interface between RAF1 and CDC37 may represent a vulnerable region, which could be targeted to induce the degradation of RAF1. Our preliminary data point out to the use of peptidomimetics as a potential pharmacological approach capable of reproducing the therapeutic results obtained in experimental mouse models of lung cancer upon ablation of RAF1 expression, representing a potential strategy to treat KRAS driven tumors.