Background:RAS overexpression and activation is a common leukemogenic mechanism in acute myeloid leukemia (AML), which is characterized by HRAS, NRAS and KRAS mutations. However, after several decades of continuous research efforts, RAS direct inhibition with small molecules still remains a current challenge. Instead, interference with the enzymes involved in the post‐translational modification of RAS could be an alternative. Among these enzymes, the inhibition of isoprenylcysteine carboxylmethyltransferase (ICMT) deserves special attention considering that its inactivation blocks RAS proper localization and activity, and ameliorates phenotypes of RAS‐induced malignancies in vivo.Aims:Our working hypothesis will test the efficacy of a novel ICMT inhibitor (UCM‐1336) against RAS‐driven tumors, such as NRAS mutant AML.Methods:In order to identify new ICMT inhibitors, we carried out a screening of our in‐house library and performed docking studies to optimize the hit initially discovered. We have used confocal and immunofluorescence microscopy to confirm that the optimal compound (UCM‐1336) delocalizes all RAS‐isoforms (HRAS, NRAS and KRAS) from the membrane. Moreover, we analyzed UCM‐1336 effect in apoptosis and RAS downstream pathway (RAS, MEK and AKT) by western blot. Furthermore, in vitro MTT viability validation of six RAS‐dependent cell lines and controls (2 fibroblasts cell lines) was performed. Finally, we developed an in vivo assay in xenograft NSG mice transplanted with 1 million IV injected AML HL‐60 NRAS dependent cells, and after 1 week of engraftment the mice were treated with UCM‐1336 (25 mg/kg, intraperitoneally) for 15 days (3 cycles of 5 days of treatment followed by 2 days of rest). Results were analyzed by Kaplan‐Meier survival curve and bone marrow IHC of HL60 positive cells (human CD45 positive).Results:Initial high‐throughput screening followed by compound optimization strategies led to the identification of the new ICMT inhibitor UCM‐1336. Treatment of PC‐3 cells with 5 μM of UCM‐1336 significantly induced RAS mislocalization. Moreover, UCM‐1336 treatment decreased active RAS (GTP‐bound complex), the activity of its downstream MEK/ERK and PI3K/AKT signaling pathways, and activated autophagy and apoptosis (LC‐3 and PARP1 expression and CASP3 activity). In vitro, UCM‐1336 inhibited viability of RAS‐driven cancer cell lines (PANC1, MIA‐PaCa‐2, MDA‐MB‐231, SW620, SK‐Mel‐173 and HL60) with IC50 values between 2–12 uM. In addition, it showed selectivity versus control fibroblasts (>50 uM, NIH3T3 and 142BR). Finally, the treatment of NSG HL60 transplanted xenograft mice with UCM‐1336 led to a significant delay in tumor development and death and decreased HL60 infiltration in bone marrow compared to vehicle administration.Summary/Conclusion:UCM‐1336 is a novel ICMT inhibitor with capacity to kill RAS‐driven tumor cells in vitro and in vivo such as acute myeloid leukemia KRAS, HRAS and NRAS‐driven tumors.imageThis work was supported by grants from the Spanish MINECO (SAF2016–78792‐R, SAF2017–89672‐R).