ANKRD28 interacts with DOCK180 to activate Rac1-STAT3-Myc signaling and sustain stemness of CD34+ leukemia stem cells in acute myeloid leukemia.

Leukemia stem cell (LSC) stemness maintenance is a core cause of relapse and therapeutic resistance in acute myeloid leukemia (AML). ANKRD28, an ankyrin repeat domain‑containing gene, has been implicated in malignant phenotypes across various tumors, but its role in regulating AML LSC stemness remains unclear. GEO datasets (GSE9476, GSE30029) and public databases (GEPIA2, TIMER, CCLE) were analyzed to characterize ANKRD28 expression and prognostic relevance in AML and LSCs. ANKRD28 expression in CD34+ LSCs from newly diagnosed AML patients was assessed by RT‑qPCR and Western blot. Effects on proliferation, cell cycle, apoptosis, and stemness markers were evaluated using colony formation, CCK‑8, EdU incorporation, flow cytometry, and Western blot. A patient‑derived xenograft (PDX) model was used to assess in vivo tumorigenic capacity. Downstream signaling was explored via GEPIA2 correlation analysis, Western blot, and rescue experiments. Co‑immunoprecipitation (Co‑IP), Rac1 activity assays, and nuclear‑cytoplasmic fractionation were performed to elucidate the underlying mechanism. ANKRD28 was overexpressed in AML tissues, CD34+ cells, and CD34⁺‑enriched cell lines, and was associated with FLT3 mutations, relapse, and poor prognosis. Its expression was positively correlated with LSC markers. CD34+ANKRD28+ cells exhibited greater colony‑forming capacity than CD34+ANKRD28- cells. ANKRD28 knockdown inhibited LSC proliferation, induced apoptosis, caused cell cycle arrest, and downregulated stemness markers, whereas overexpression exerted opposite effects. In vivo, ANKRD28 knockdown prolonged survival, reduced LSC infiltration, and alleviated histopathological damage. Mechanistically, ANKRD28 directly bound DOCK180, activated Rac1, promoted STAT3 nuclear translocation and transcriptional activation, and upregulated Myc expression. ANKRD28 is overexpressed in AML CD34+ LSCs and sustains stem cell‑like properties via the DOCK180‑Rac1‑STAT3‑Myc axis, serving as a potential therapeutic target for AML.