RAS -mutant clones drive extramedullary acute myeloid leukemia

Abstract Extramedullary acute myeloid leukemia (eAML) represents a clinically challenging manifestation of acute myeloid leukemia (AML), but its molecular drivers remain poorly defined. We performed targeted sequencing in 85 eAML biopsies, representing one of the largest molecular analyses of eAML to date. We detected mutations in RAS or RAS -modifying genes ( RAS MUT ; NRAS, KRAS, PTPN11, CBL , and NF1 ) in 41% of cases, representing a significant enrichment compared to bone marrow (BM) samples of more than 1300 AML patients not selected for eAML. Analysis of paired eAML and BM specimens revealed expansion and/or de-novo appearance of RAS MUT clones at the extramedullary site. Functional studies using primary murine leukemia cells and CRISPR/Cas9-engineered isogenic human leukemia cell lines demonstrated that RAS MUT increase the migration and invasion of leukemic cells compared to RAS -wildtype controls. Consistently, RAS MUT cells showed increased infiltration into the chorioallantoic membrane of chicken embryos and demonstrated enhanced extramedullary growth after injection into immunocompromised mice. RNA sequencing revealed increased expression of junctional adhesion molecule-like ( JAML ) and activation of PI3K/AKT signaling in RAS MUT cells. JAML silencing and pharmacologic AKT inhibition reversed the RAS MUT -driven effects on leukemic cell migration, demonstrating a causal role of the JAML-PI3K/AKT axis in RAS MUT -driven eAML formation. In conclusion, these findings delineate the molecular landscape of extramedullary AML and show that RAS MUT are enriched within this AML subform. They further demonstrate that RAS MUT actively contribute to leukemic tissue infiltration through activation of a RAS MUT -JAML-PI3K/AKT axis, highlighting AKT signaling as a potential therapeutic vulnerability in RAS MUT -associated eAML.