Causal variant-targeted ribonucleic acid treatment for congenital melanocytic nevi

In a recent study published in The Journal of Investigative Dermatology, researchers developed a genetic treatment targeting the causal NRAS c.181C>A mutation in congenital melanocytic naevus (CMN) using small interfering ribonucleic acid (siRNA) to suppress its expression and induce lesion clearance.

Study: RNA therapy for oncogenic NRAS-driven naevi induces apoptosis. Image Credit: Stock-Asso/Shutterstock.com

Background

RAS proteins are critical for cell divisions, differentiation, and apoptotic processes. Oncogenic RAS variations cause cancer, but they can also induce benign lesions such as thyroid nodules, colonic polyps, and melanocytic naevi.

Reversing these damages may lower cancer incidence, but their effects are difficult to combat using downstream system inhibitors. Severe CMN is a leading cause of illness and death in children.

Targeting the NRAS c.181C>A, p.(Q61K) pathogenic variation might be an effective precision medicine strategy for identifying disease persistence pathways.

About the study

In the present study, researchers created siRNAs that target the variant allele of CMN to cure lesions and offer a therapeutic method for cancer prevention.

The researchers cultured CMN naevus cells from eight children with CMN, obtaining their skin samples by punch biopsy or surgical excision. They used developed and optimized siRNA in human colorectal carcinoma (HCT116) cells.

They imaged the isolated primary CMN naevus cells using ptychographic imaging to analyze naevus cell form and behavior in four individuals, categorizing them using machine learning as multipolar, bipolar, non-polar, and big multinucleated probable senescent cells. Further, they performed flow cytometry and immunohistochemistry analyses.

The researchers conducted live apoptosis experiments and used two equations to evaluate siRNA candidates for in silico allelic discrimination. They performed a quantitative polymerase chain reaction (PCR), followed by Western blotting for analysis.

They performed transcriptome-wide RNA sequencing on paired C6072C and C8052C-WT cells with and without siRNAs 1, 8, and 15. They also tested the leading candidate siRNA, siNRAS^Q61K, on primary patient cells. They treated naevus cells with a single dosage of siNRAS^Q61K and examined the results after 48 hours.

To assess in vivo delivery, the researchers administered Cy5-tagged control siRNA encapsulated into receptor-targeted nanoparticles (RTNPs) intradermally into the Tyr:NRAS^Q61K murine dermis. They performed RNA sequencing on primary naevus cells before and after siNRAS^Q61K therapy to evaluate on- and off-target impacts.

The researchers investigated the expression of endoplasmic reticulum (ER)-stress-induced apoptosis regulators, including endoplasmic reticulum to nucleus signaling 1 (ERN1), eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3), activating transcription factor 4 (ATF4), (ATF6), and glucose-regulated protein 78 (GRP78).

The researchers examined genes with comparable target sequences and conducted pathway analyses. They used caspase-3/7 activation to evaluate apoptosis in four naevus patients during live imaging over seven days.

They compared siNRA^SQ61K therapy efficacy to the mitogen-activated protein kinase inhibitor (MEKi) trametinib, the sole medical therapy tested in CMN patients in vivo.

Results

The work demonstrated that siRNA targeting the recurrent causative variation considerably suppressed oncogenic and presently untreatable NRASQ61K in melanocytic naevi cells.

Treatment with siRNA significantly decreases the levels of ADP-ribosylation factor (Arl)-like protein 6-interacting protein 1 (ARL6IP1), a recognized inhibitor of ER stress-inflicted apoptosis previously unrelated to NRAS.

Packing targeted siRNA in self-assembling siNRAS^Q61K-RTNPs prevents siRNA deterioration, enabling the successful delivery of targeted siRNA into a humanized mouse model and allowing human skin explant administration.

The RTNPs caused a selective knockdown of variant humanized NRAS in vivo, supporting the potential for clinical trials in RAS-driven benign tumors.

In primary cells, a single dosage of siRNA induces an apoptotic cascade, unlike treatment with an MEK inhibitor. SiRNA8 showed a minimal off-target impact on RNAseq in HCT116 cells.

In primary CMN cell cultures, siNRAS^Q61K inhibited the production of the variant transcript and reduced NRAS variant allele mRNA levels without affecting conventional melanocytic markers.

Transcriptomic analysis revealed a significant increase in ERN1, a critical participant in similar pathways. However, the expression of other genes linked with ER-related stress (such as  ATF-4, 6, GRP78, and EIF2AK3) was unaltered, indicating that endoplasmic reticulum stress is not the primary reason for siNRASQ61K therapy-induced apoptosis.

The researchers found that apoptosis protection is critical for the survival of NRAS-driven melanocytic naevi and that treatment with allele-specific siRNA can eliminate this protection. The findings may constitute the foundation of cancer prevention, especially for high-risk individuals.

Targeted siRNA might serve as the cornerstone for therapeutic investigations for RAS-driven non-malignant tumors, demonstrating the potential for CMN resolution in individuals and a paradigm for preventative cancer treatment in benign tumors produced by oncogenic RAS.

The study suggests employing variational allele-specific therapies and receptor targeting to minimize dose. Trametinib reduced CMN cell growth within a day of treatment but did not induce apoptosis over time, whether administered alone or combined with siRNA controls.