Abstract B019: ASO-mediated NRAS knockdown overcomes gilteritinib late resistance in FLT3-AML
Authors: Joshi Sunil K. , Pittsenbarger Janét , Luo Xiaolin , Maazi Hadi , Revenko Alexey , Druker Brian J. , Traer Elie 
Published: May 2023

Abstract
The internal tandem duplication of the FLT3 receptor (FLT3-ITD) leads to constitutive receptor activation and is one of the most common mutations (~30%) in acute myeloid leukemia (AML), resulting in poor prognosis. Multiple FLT3 inhibitors (FLT3i) have been developed, including the potent FDA-approved FLT3i, gilteritinib. However, AML patients only respond to gilteritinib for approximately 6 months due to the emergence of drug resistance. Through an approach that integrated genomic, metabolomic, proteomic, and phosphoproteomic analyses with complementary genetic and pharmacologic screens, we demonstrated that gilteritinib resistance occurs in a stepwise manner with two distinct phases of early and late resistance (Joshi et al., Cancer Cell, 2021). While gilteritinib efficiently eliminates leukemia cells in the blood, residual cells survive within the bone marrow, where they are protected by extrinsic microenvironmental factors fostering early resistance. These lingering cells eventually develop intrinsic mutations in NRAS that allow them to resist gilteritinib treatment, leading to late resistance. Acquisition of mutations in the RAS pathway, particularly NRAS, are a common mode of late resistance (~11%) for many patients that relapse on FLT3i. Developing therapeutics to block NRAS activity remains a challenge with the lack of direct small-molecule inhibitors. Indirect approaches to inhibit mutant NRAS/MAPK pathway signaling have also not met much clinical success. Hence, therapeutic nucleic acid-based approaches, including antisense oligonucleotides (ASOs), may offer an alternative approach to target NRAS-driven cancers. We evaluated the effectiveness of a novel ASO targeting NRAS mRNA to restore gilteritinib sensitivity in resistant cultures in vitro and in primary AML patient samples ex vivo. We first assessed the potency of the NRAS ASO in both gilteritinib-sensitive and late resistant MOLM-14 AML cell lines (N=9) that expressed wild-type or mutant NRAS (G12S or G12D) via free uptake of ASO. NRAS expression was evaluated by qPCR after 48 hours. ASO treatment down-regulated mutant NRAS mRNA expression in a dose-dependent manner with an IC50 of &lt; 1 µM and decreased NRAS protein levels. No changes in NRAS mRNA or protein levels were observed with non-targeting ASO control. Decreased NRAS mRNA expression restored sensitivity to gilteritinib in late-resistant cultures, leading to decreased cell viability and increased apoptosis. A similar trend was observed in primary AML cells from patients (N=2) that relapsed on gilteritinib, where reduction of approximately 50% of mutant NRAS mRNA robustly sensitized patient cells to gilteritinib. In contrast, the combination of gilteritinib and the MEK inhibitor trametinib was not as effective in either cell cultures or patient samples. Our results suggest that ASO-mediated knockdown of NRAS may circumvent the development of resistance and improve the durability of FLT3i.
Citation Format: Sunil K. Joshi, Janét Pittsenbarger, Xiaolin Luo, Hadi Maazi, Alexey Revenko, Brian J. Druker, Elie Traer. ASO-mediated NRAS knockdown overcomes gilteritinib late resistance in FLT3-AML [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B019.