Tet2-deficient abnormal B-cells are BCR-inhibition sensitive. (A) Abnormal B-cell phenotype and frequencies in the peripheral blood of mice transplanted with TCL1A transduced Tet2^+/+ or Tet2^−/− hematopoietic progenitors. Dot plots show a representative example of B220 and CD19 expression analysis in the GFP⁺ total leukocytes 4 months after transplantation. Diagrams depict the average percentages of the B220^low population in the blood of n = 3 mice per group 4 months after transplantation. Error bars represent means ± standard deviation (SD). (B) Tet2 deficiency enhances the tumor phenotype of TCL1A (GFP⁺) overexpressing B cells, as is shown by abnormal cell numbers and CD5 membrane expression level. (Upper left) B220 CD19 expression in gated GFP⁺ cells expressing TCL1A in primary and secondary recipient mice engrafted with BM cells overexpressing TCL1A in a Tet2^+/+ or Tet2^−/− background. (Lower left) Average ± SD of the median fluorescence intensity of the B220 antigen at the cell surface of CD19⁺ B cells in each group. (Upper right) Expression of CD5 and IgM on the gated B cells as in the upper-left contour plots. A representative contour plot is shown for each group. (Lower right) Average ± SD of the median fluorescence intensity of the CD5 antigen at the cell surface of CD19⁺ B cells in each group. (C) Ibrutinib-mediated BTK inhibition results in growth reduction and increase of surface IgM expression on Tet2-deficient B cells. Dose effects of ibrutinib on the B-cell population of Tet2-deficient or wild-type cell lines are derived from independent primary mice: 2 tumoral B-cell lines, one obtained from a spontaneous B-cell malignancy development in a Tet2^−/− mouse, and the other obtained from a mouse from a BMT experiment of wild-type Tet2^+/+ BM cells transduced by the TCL1A oncogene; and a T-cell line obtained from a BMT experiment of wild-type Tet2^+/+ BM cells transduced by the BRAFG469R oncogene. Diagrams depict variable cell proportion in comparison with the nontreated cells. Cells were counted 3 days after treatment with the indicated inhibitor concentration. (D) Surface expression level (mean fluorescence intensity) of IgM on B-cell population (IgM⁺B220⁺) observed after staining of the 2 B-cell lines cultivated as in panel C. (E) Splenocytes from Tet2^−/− mice were treated with ibrutinib and fludarabine for 18 hours ex vivo. Cells were stained with CD19-BV510, B220-allophycocyanin (APC)-Cy7, annexin V–APC, and 7-aminoactinomycin D (7-AAD). Gated B220^lowCD19⁺ splenic cells were analyzed for annexin V and 7-AAD staining. Histograms show the mean ± SD of the percentages of viable cells (annexin V and 7-AAD double negative) in gated B220^lowCD19⁺ populations from spleens of Tet2^−/− mice (n = 2). *P < .05 (unpaired Student t test). MFI, mean fluorescence intensity.