Abstract B027: An Innovative cell-penetrating peptide-based RNAi Therapy to Overcome B-Raf/MEK Inhibitor Resistance in Melanoma
Authors: Tuttolomondo Martina , Terp Mikkel Green , Brienza Chiara , Gjerstorff Morten Frier , Ditzel Henrik Jørn 
Published: November 2024

Abstract
Small-molecule-targeted therapies have revolutionized cancer treatment, including for B-RafV600E-mutated melanoma, where B-Raf and combined B-Raf/MEK inhibitors (BRAFi/MEKi) prolong progression-free survival and overall survival. However, resistance to these therapies quickly develops, driven by mechanisms such as the expression of alternative spliced isoforms of B-RafV600E and mutated MEK1 variants. The need for novel strategies to overcome such resistance is critical. In this study, we developed and evaluated a novel RNAi-based therapeutic using our designed cell-penetrating peptide HE25 to specifically target and silence resistance-associated spliced isoforms of B-RafV600E, restoring the efficacy of existing treatments. In a recently published work (Tuttolomondo et al., 2024), we demonstrated that HE25 effectively reduced SARS-CoV-2 replication when complexed with siRNA targeting the virus's RNA-dependent RNA polymerase. HE25 facilitated siRNA internalization via multiple receptors involved in viral entry, including ACE2, integrins, and NRP1, through endocytosis, highlighting its versatility as a delivery platform with potential applications beyond cancer research. Initially, we established a vemurafenib-resistant A375 melanoma cell line (A375-VR), which revealed an alternatively spliced B-RafV600E isoform driving resistance to vemurafenib. Through cDNA Sanger sequencing, we confirmed that this alternatively spliced isoform lacks the regulatory domain, leading to constitutive B-Raf activation and rendering vemurafenib ineffective. Given these findings, we hypothesized that specifically silencing this isoform with our siRNA-HE25 complex could synergize with B-Raf inhibition, thereby overcoming vemurafenib resistance. The B-RafV600E-targeting siRNA encapsulated within HE25 significantly inhibited the proliferation of both A375 and A375-VR cells in vitro, with no toxic effects on non-cancerous melanocytes. In vivo, B-RafV600E siRNA-HE25 treatment significantly reduced tumor growth in A375 melanoma xenografts compared to a non-targeting siRNA control. We also developed A375-VTR cells resistant to combined vemurafenib and MEK inhibitor trametinib. This cell line also expressed the shortened B-RafV600E isoform and its silencing by the B-RafV600E siRNA/HE25 system was confirmed by Western blotting. Indeed, in A375-VTR cells, the combination of vemurafenib and B-RafV600E siRNA/HE25 was more effective than the combination of vemurafenib and trametinib. Our strategy holds promise for personalized cancer treatment by targeting resistance-related mutations or alternatively spliced isoforms. Future work will optimize this approach for broader clinical use and explore its efficacy in combination with existing therapies. These findings suggest that siRNA-based therapeutics via HE25 offer a promising solution for overcoming resistance in B-RafV600E-mutated melanoma and resistance mechanisms in other cancer types. This strategy should enable tailored treatments with broad applicability across cancer types.
Citation Format: Martina Tuttolomondo, Mikkel Green Terp, Chiara Brienza, Morten Frier Gjerstorff, Henrik Jørn Ditzel. An Innovative cell-penetrating peptide-based RNAi Therapy to Overcome B-Raf/MEK Inhibitor Resistance in Melanoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B027.