Noncysteine mutations in IL7R and CRLF2 are transforming leukemogenic mutations. (A) Predicted transmembrane domain (TMD) of IL7R and CRLF2 with location of cysteine mutations (orange box) and noncysteine mutations (green box) and alignment of WT and mutated TMD sequences. Numbers show the positions of nucleotides and corresponding amino acids. The inserted nucleotides and amino acids are shown in red. (B) Expression of CRLF2^InsEIM mutation. The mutated allele is expressed in the RNA from diagnosis whereas only the normal allele is found in the remission DNA sample because CRLF2 is expressed only in leukemic B-cell as a result of a chromosomal rearrangement.⁸  (C,D) Cytokine withdrawal assay of BaF3 cells transduced with mutated or WT IL7R or CRLF2. (E) Overall survival of mice (7 mice in each group, 3 experiments) injected intravenously with 1-2*10⁶ BaF3 cells expressing IL7R, WT or mutant, or IL7R^WT and CRLF2^insEIM compared by Kaplan-Meier analysis P ≤ .01. (F) Representative flow cytometric analysis of blood from mice injected intravenously with 1-2*10⁶ BaF3 cells expressing IL7R^V253G. (G) Lymphoma tumor (top) observed in a mouse injected subcutaneously with 1-2*10⁶ BaF3 cells expressing IL7R^{Ins EKV}-GFP. Representative GFP expression from lymphoma cells (bottom). (H) Representative analysis of cells from spleen and bone marrow from mice injected intravenously with 1-2*10⁶ BaF3 cells expressing IL7R^{Ins EKV} and sacrificed at day 14. (I) Representative analysis of cells from spleen and bone marrow from mice injected intravenously with 1-2*10⁶ BaF3 cells coexpressing the IL7R^WT and CRLF2^WT or CRLF2^insEIM mutant, and sacrificed at day 16. Del, deletion; Ins, insertion; WT, wild type.