Allosteric inhibition of RAN decreases mir-126 biogenesis in endothelial cells and controls Acute Myeloid Leukemia growth

Abstract RAS-related nuclear protein (RAN), a small GTPase of the RAS superfamily, cycles between active (GTP-bound) and inactive (GDP-bound) states and plays an oncogenic role by promoting cell proliferation, apoptosis resistance, and metastasis. We previously identified RAN as a key regulator of miR-126 biogenesis, a microRNA highly expressed in arterioles of bone marrow (BM) niche (PMID: 29505034). In acute myeloid leukemia (AML), arteriolar endothelial cells (ECs) produce and transfer miR-126 to LSCs, supporting their function and promoting disease progression (PMID: 34372909). These findings suggest that targeting the active GTP-bound form of RAN (RAN-GTP) may provide a novel therapeutic strategy to interfere with EC miR-126 biogenesis and in turn eliminate LSCs and disrupt AML progression. Distinct from other RAS family members, RAN lacks a C-terminal membrane-anchoring motif and instead contains a flexible, intrinsically disordered C-terminal region that autoinhibits its GTPase activity and regulates conformational transitions. In the GTP-bound state, RAN undergoes substantial structural rearrangements in its switch I/II regions and C terminus. To explore potential druggable sites within this dynamic region, we performed molecular dynamics (MD) simulations on the GDP-bound (inactive) conformation of RAN, which revealed a transient cryptic pocket in the C-terminal domain. Subsequent virtual ligand screening (VLS) identified candidate small molecules targeting this site. Despite the historical challenges of drugging small GTPases, these efforts led to the discovery of MAR-3.6.2, a novel allosteric inhibitor that binds the cryptic C-terminal pocket of RAN and blocks its activation. MD simulations and docking studies revealed that MAR-3.6.2 disrupts the interaction between RAN and its guanine nucleotide exchange factor RCC1, thereby preventing the conversion of RAN-GDP to its active GTP-bound form in the nucleus. In vitro, MAR-3.6.2 treatment led to nuclear retention of inactive RAN-GDP and reduced RAN/XPO5-mediated export of precursor miR-126 (pre–miR-126), limiting the production of mature miR-126 in ECs, AML cell lines, and primary AML blasts. Downregulation of miR-126 impaired EC function by disrupting actin polymerization, reducing gelatin degradation, and inhibiting 3D angiogenic sprouting—phenotypes recapitulated by miRisten, an oligonucleotide inhibitor of miR-126 (PMID: 34372909, 34686664), in HUVEC ECs. We previously reported that disrupting miR-126—either by directly in AML cells or blocking its transfer from ECs—significantly impairs AML and LSC proliferation and induces apoptosis (PMID: 34372909). Treatment with MAR-3.6.2 similarly suppressed AML growth: a 25 µM dose induced >60% apoptosis in MV4-11 and HL-60 AML cell lines within 24 hours and inhibited proliferation across all tested AML lines. In primary AML samples, 25 µM MAR-3.6.2 significantly suppressed proliferation of CD34⁺CD38⁻ blasts (enriched for LSCs; fold change = 0.5, n = 5) and increased apoptosis (fold change = 1.4, n = 5), while sparing normal CD34⁺CD38⁻ cells (enriched for HSCs). In colony-forming assays, MAR-3.6.2 significantly reduced colony formation in CD34⁺ AML blasts (28.4%, p = 0.05) without affecting colonies from normal CD34⁺ PBMCs. In vivo treatment of the MllPTD/WT/FLT3ITD/ITD AML mouse model with MAR-3.6.2 (25 mg/kg, IV, daily × 3 weeks) reduced leukemic burden as shown by decreased mCD45.2⁺ cells in peripheral blood (control: 92.6% vs. MAR-3.6.2: 86.2%, p = 0.005), smaller spleens, and extended survival (median: 34 vs. 29 days, p = 0.0002). In secondary transplants, mice receiving BM MNCs from MAR-3.6.2–treated donors showed reduced mCD45.2⁺ leukemia burden (73.0% vs. 64.2%, p = 0.04), smaller spleens, and prolonged survival (median: 35 vs. 26 days, p < 0.0001), confirming reduced LSC activity. In summary, MAR-3.6.2 is a novel small-molecule allosteric inhibitor of RAN GTPase that disrupts miR-126 expression and function in both ECs and AML cells, leading to potent anti-leukemic effects. Although the development of MAR-3.6.2 analogs or chemical modifications is ongoing to improve its properties, these findings support RAN as a druggable target and provide compelling preclinical evidence for MAR-3.6.2 as a promising therapeutic strategy for AML.