Fig 2: FAM167A and surface DSG1 levels are associated with BCR-ABL-independent TKI resistance in CML patients. A qRT-PCR analysis of FAM167A mRNA expression in CD34+ stem/progenitor cells from imatinib-responsive CML patients (S) at the time of diagnosis (n = 26) and at follow-up (n = 26), in imatinib-resistant CML patients (R) without BCR-ABL mutation at the time of diagnosis (n = 12) and at follow-up (n = 11), and in imatinib-resistant CML patients harboring a BCR-ABL mutation at the time of diagnosis (n = 10) and at follow-up (n = 12). Imatinib resistance and BCR-ABL mutation status were determined at follow-up. The level of FAM167A was normalized to that of GAPDH. B Relative surface expression of DSG1 (MFI) in CD34+ stem/progenitor cells from imatinib-responsive CML patients at the time of diagnosis (n = 8) and at follow-up (n = 8), and from imatinib-resistant CML patients without BCR-ABL mutation at the time of diagnosis (n = 10) and at follow-up (n = 7), as measured via flow cytometry. C Proposed model showing involvement of the FAM167A-induced noncanonical NF-?B pathway in BCR-ABL-independent TKI resistance. Unpaired two-tailed t-test; *P < 0.05, ***P < 0.001. NS, not significant

Fig 3: FAM167A regulates NIK binding and ubiquitination through DSG1. A NF-?B luciferase reporter activity in K562R cells after DSG1 knockdown using recombinant lentiviruses encoding two different DSG1-specific shRNAs. B Immunoblot analysis of p100/p52 in K562R cells after DSG1 knockdown. C NF-?B luciferase reporter activity in K562R cells 24 h after transfection of a plasmid encoding DSG1, or both DSG1 and Erbin, with or without treatment with 2 µg/ml anti-FAM167A neutralizing antibody. D Co-immunoprecipitation analysis of DSG1, Erbin, and NIK in K562S and K562R cells. E Co-immunoprecipitation analysis of DSG1 and NIK in K562R cells after treatment with 2 µg/ml of an anti-FAM167A neutralizing antibody or an isotype control for 3 h. F Immunoblot analysis of NIK ubiquitination with an anti-ubiquitin antibody after immunoprecipitation of NIK from K562R cells treated with 2 µg/ml of an anti-FAM167A neutralizing antibody or an isotype control plus 10 µM MG132 for 3 h. G Schematic model of the FAM167A-induced noncanonical NF-?B pathway. GAPDH was used as an internal standard (B, D-F). Ub, ubiquitination. Data are representative of two (B) or three (A, C-F) independent experiments (error bars, s.d. of duplicate (B) or triplicate (A, C-E) samples). Unpaired two-tailed t-test; *P < 0.05, **P < 0.01, ***P < 0.001