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

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. FBXO3, an important member of the FBXO family, is one of the most important E3 ubiquitin ligases and is widely involved in tumor development. However, the role and underlying mechanism of FBXO3 in CML LSCs remain unknown. In this study, we aim to explore the effect of FBXO3 on the growth, proliferation, apoptosis in IM-sensitive/resistant CML cell lines and stemness of CML LSCs, and to screen the candidate substrate proteins of FBXO3 in CML.

We used single-cell RNA sequencing to compare FBXO3 expression between healthy individuals and CML patients, then confirmed the results with western blot and qRT-PCR. We inhibited its expression using shRNA and sgRNA in CML cell lines, assessing effects on cell growth (counting and soft agar assays), apoptosis, and protein levels (western blot). For in vivo validation, we established nude mouse subcutaneous tumor model, CML-CDX (Cell line-derivative xenograft) model and BCR-ABL-induced CML mouse model to evaluate FBXO3's impact on CML cells. Candidate substrate proteins of FBXO3 in CML were screened out through tandem affinity purification mass spectrometry experiments, and Co-IP experiments, half-life experiments and in vitro ubiquitination experiments were carried out to verify whether FBXO3 interacted with the substrate protein and mediated its ubiquitination degradation. We also generated FBXO3 domain mutants to determine key regions regulating substrate stability. Rescue experiments confirmed FBXO3's role in CML via substrate ubiquitination and degradation. RNA-seq were used to identify downstream pathways and validated them experimentally. Functional experiments related to growth and apoptosis and BCR-ABL-induced CML mouse model were carried out to verify whether the FBXO3 inhibitor BC1215 had an inhibitory effect on CML in vitro and vivo.

FBXO3 expression is elevated in CML patients, correlating with GEO data and specimen verification. Knockdown of BCR-ABL or IM treatment reduces FBXO3. Silencing FBXO3 in CML cell lines and CD34⁺ cells impairs growth, colony formation, self-renewal, and increases apoptosis. In vivo, FBXO3 inhibition reduces tumor volume, leukemia burden, leukemia stem/progenitor cell ratios, and prolongs survival without affecting normal hematopoietic stem cells. FBXO3 targets DUSP9 for ubiquitination-mediated degradation, as confirmed by co-IP and half-life studies. Mutation of FBXO3's ΔApaG domain abolishes this regulation. DUSP9 deletion partially reverses FBXO3 knockdown's inhibitory effects on CML growth and apoptosis. RNA-seq indicates FBXO3-DUSP9 functions via the MAPK pathway, with DUSP9 knockdown promoting p38 and JNK activation. BC1215 also inhibits CML cell growth and apoptosis, and in combination with IM, it significantly improves survival and reduces LSCs in a CML mouse model.

FBXO3 is upregulated in CML, regulated by BCR-ABL, and crucial for CML stem cell survival. Inhibition of FBXO3 increases apoptosis and reduces growth in both imatinib-sensitive and -resistant CML cells and stem cells, without affecting normal HSCs. Mechanistically, FBXO3 deficiency leads to decreased DUSP9 ubiquitination and activation of apoptosis pathways. DUSP9 knockdown partially reverses FBXO3 depletion's effects on CML stem cells and cell growth. Importantly, the FBXO3 inhibitor BC1215 combined with imatinib shows enhanced therapeutic efficacy for CML stem cells.

Chronic myeloid leukemia, TKIs resistance, leukemia stem cells, FBXO3, DUSP9, MAPK signaling pathway