Supplementary MaterialsSupplementary Information 41467_2020_20259_MOESM1_ESM. cells sensitive or resistant to imatinib in vitro and in vivo. The knockout of significantly inhibits BCR-ABL and BCR-ABLT315I-induced CML in mice with the reduction of Lin?Sca1+c-Kit+ CML stem/progenitor cells. Mechanistic studies show that stabilizing Y-box binding protein 1 contributes to USP47-mediated DNA damage repair in CML cells. Inhibiting USP47 by “type”:”entrez-protein”,”attrs”:”text”:”P22077″,”term_id”:”134707″,”term_text”:”P22077″P22077 exerts cytotoxicity to CML cells with or without TKI resistance in vitro and in vivo. Moreover, “type”:”entrez-protein”,”attrs”:”text”:”P22077″,”term_id”:”134707″,”term_text”:”P22077″P22077 eliminates leukemia stem/progenitor cells in CML mice. Together, targeting USP47 is a promising strategy to overcome TKI resistance and eradicate bio-THZ1 leukemia stem/progenitor cells in CML. oncogenic fusion gene that encodes the chimeric BCR-ABL protein with constitutive bio-THZ1 kinase activity1,2. The introduction of imatinib (IM) in 2001, a tyrosine kinase inhibitor (TKI) that targets BCR-ABL, revolutionized the prognosis of CML3. However, TKI resistance, including BCR-ABL-dependent and -independent resistance, is a major problem in IM-based CML treatment. The BCR-ABL-dependent mechanism is mainly mediated through the mutation of the bio-THZ1 ABL kinase domain, BCR-ABL overexpression, or MDR1 upregulation4C7. To overcome IM resistance, the second generation of bio-THZ1 TKIs, such as dasatinib and nilotinib, Rabbit Polyclonal to OR9Q1 has been developed8. For the gatekeeper mutation T315I, which confers resistance to all first- and second-generation TKIs, the third-generation TKI ponatinib was developed9. Nevertheless, the toxicity of ponatinib limits its use in some patients10. On the other hand, the bio-THZ1 underlying mechanisms of BCR-ABL-independent TKI resistance are still not well understood. It has been reported that the leukemia stem cells (LSCs) in CML are insensitive to TKI in a BCR-ABL-independent manner, thereby leading to relapse and minimal residual disease (MRD)11. Additionally, the aberrant activation of the PI3K and RAS/MAPK signaling pathways also contributes to BCR-ABL-independent TKI resistance12C15. Hence, identifying promising drug targets that overcome TKI resistance via both mechanisms is urgently required to provide new possibilities for CML treatment. To date, ~100 kinds of deubiquitinating enzymes (DUBs) have been identified16. DUBs remove ubiquitin conjugates from their substrates, thereby altering their stabilities, localizations, or activities17. Accumulating evidence shows that DUBs are promising targets for cancer treatment, including hematopoietic malignancies. For instance, USP10 is involved in the pathogenesis of FLT3-ITD-positive leukemia18; targeting USP1 and USP7 is effective in multiple myeloma cells19,20; and USP37 can stabilize PLZF/RARA in acute promyelocytic leukemia21. Although USP9X has been demonstrated to be involved in the survival of CML, how DUBs are related to CML pathogenesis is largely unexplored22,23. Ubiquitin-specific peptidase 47 (USP47) is a member of the USP subfamily of DUBs24. Similar to other USPs, USP47 regulates cellular activities by removing ubiquitin conjugates from diverse substrates and, thereby, altering their stabilities, localizations, or activities. Specifically, USP47 deubiquitinates and stabilizes MAPK25, DNA polymerase (Pol)26, E-cadherin27, -catenin28, SNAIL29, YAP30, -Trcp31, and katanin-p6032. Hence, USP47 is involved in cell proliferation33, cell survival31, DNA damage repair26, NLRP3 inflammasome activation34, and epithelial-mesenchymal transition29. USP47 plays an important role in cancers such as gastric cancer, medulloblastoma, and colorectal cancer35C37; however, its role in CML remains unexplored. In this study, we reveal the critical role of USP47 in the pathogenesis of CML. Specifically, we demonstrate that USP47 is highly expressed in primary CML cells and promotes cell proliferation, while knockout significantly prolongs the survival of BCR-ABL and BCR-ABLT315I-induced CML mice by reducing leukemia stem/progenitor cells. We further demonstrate that USP47 facilitates DNA damage repair by regulating a novel substrate, Y-box binding protein 1 (YB-1). Moreover, we find that “type”:”entrez-protein”,”attrs”:”text”:”P22077″,”term_id”:”134707″,”term_text”:”P22077″P22077, a USP47 inhibitor38, substantially eliminates TKI-sensitive cells, TKI-resistant cells, leukemia stem/progenitor cells, and MRD in CML. We propose that USP47 is a promising target to overcome TKI resistance in CML treatment. Results BCR-ABL regulates USP47 through RAS/ERK and STAT5 pathway in CML To screen the potential DUBs involved in the pathogenesis of CML, we compared the expression of DUBs in primary CML cells (chronic phase, being the most upregulated one (Fig.?1a). Consistent with this result, the USP47 protein level is significantly.