{"doi":"10.1038/s42003-026-10026-0","title":"Allosteric inhibition of RAN decreases miR-126 biogenesis in endothelial cells and controls acute myeloid leukemia growth","abstract":"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 func","journal":"Communications Biology","year":2025,"id":584210,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.962,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1225939,"name":"Wenyuan Wei","orcid":"0009-0008-5146-8048","position":1,"is_corresponding":false},{"id":820356,"name":"HyunJun Kang","orcid":null,"position":2,"is_corresponding":false},{"id":272474,"name":"Ning Ma","orcid":"0000-0003-4653-7797","position":3,"is_corresponding":false},{"id":434047,"name":"S. Bhattacharya","orcid":"0000-0003-0483-2149","position":4,"is_corresponding":false},{"id":1497509,"name":"Genevieve Baker","orcid":null,"position":5,"is_corresponding":false},{"id":434046,"name":"Weidong Hu","orcid":"0000-0003-3911-5262","position":6,"is_corresponding":false},{"id":379475,"name":"Leying Zhang","orcid":"0000-0002-5285-0088","position":7,"is_corresponding":false},{"id":1229230,"name":"Jia Feng","orcid":"0000-0001-9968-2360","position":9,"is_corresponding":false},{"id":937046,"name":"Lianjun Zhang","orcid":"0000-0003-3151-9764","position":10,"is_corresponding":false},{"id":1463561,"name":"Bin Zhang","orcid":"0000-0002-4371-4746","position":11,"is_corresponding":false},{"id":453089,"name":"J. Jefferson P. Perry","orcid":"0000-0001-9889-3502","position":12,"is_corresponding":false},{"id":272488,"name":"Nagarajan Vaidehi","orcid":"0000-0001-8100-8132","position":13,"is_corresponding":false},{"id":651757,"name":"Le Xuan Truong Nguyen","orcid":"0000-0001-5464-0861","position":14,"is_corresponding":false},{"id":108574,"name":"Guido Marcucci","orcid":"0000-0002-3983-5908","position":15,"is_corresponding":false},{"id":890853,"name":"Melissa Valerio","orcid":"0000-0001-7897-2932","position":0,"is_corresponding":true}],"reference_count":27,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:59:11.978098Z","pmid":"41981249","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}