FBXO3-mediated DUSP9 ubiquitination reprograms MAPK signaling to eradicate tyrosine kinase inhibitor-resistant CML stem cells

Discovering new therapeutic targets to overcome tyrosine kinase inhibitors (TKIs) resistance and clear leukemic stem cells (LSCs) is an urgent clinical need for chronic myeloid leukemia (CML) treatment. In the present study, we report that the F-box protein 3 (FBXO3) is upregulated in CD34+ CML stem cells from patients with TKI treatment failure and is regulated by BCR-ABL. Single-cell RNA sequencing (scRNA-seq) analysis indicated that FBXO3+ HSCs from CML patients have significant LSC signatures. Genetic depletion of FBXO3 significantly increased apoptosis and decreased proliferation of both imatinib-sensitive/resistant CML cell lines and CML stem cells in vitro and in vivo, with minimal effects on normal CD34+ HSCs. Mechanistically, FBXO3 interacts with DUSP9 and mediates its ubiquitination. DUSP9 knockdown partially counteracts the elimination of CML stem cells mediated by FBXO3 depleting in vitro and in vivo. RNA-seq results indicates that FBXO3-mediated ubiquitination of DUSP9 activates MAPK signaling pathway in CML cells. Moreover, FBXO3 inhibitor alone or in combination with imatinib significantly clear CML stem cells and overcome TKI resistance in murine and human CML, with minimal effects on normal hematopoiesis. Overall, our findings uncover novel roles of FBXO3 in CML progression and provide a rationale for combining FBXO3 inhibitors with TKIs to induce durable elimination of CML-LSCs.