cRAGs give more aggressive leukemia in mice model of BCR-ABL1+ B-ALL (A) Kaplan-Meier survival curve for fRAG (n=8), cRAG1 (n=6), and cRAG2 (n=10) recipient mice. The survival was calculated by Mantel–Cox test (P<0.0425). (B) The spleen weights of fRAG, cRAG1 and cRAG2 leukemic mice (fRAG, n=8, cRAG1, n=7, cRAG2, n=9; fRAG vs cRAG1, P<0.0001, fRAG vs cRAG2, P=0.1352). (C) The spleen cell numbers of fRAG, cRAG1 and cRAG2 leukemic mice (fRAG, n=7, cRAG1, n=8, cRAG2, n=13; fRAG vs cRAG1, P=0.0047, fRAG vs cRAG2, P=0.0180). (D) The percentage of GFP+ cells in peripheral blood (PB, fRAG, n=6, cRAG1, n=6, cRAG2, n =6; fRAG vs cRAG1, P=0.0003, fRAG vs cRAG2, P=0.0035), bone marrow (BM, fRAG, n=5, cRAG1, n=5, cRAG2, n=6; fRAG vs cRAG1, P=0.0341, fRAG vs cRAG2, P=0.0008), and spleen (SP, fRAG, n=9, cRAG1, n=4, cRAG2, n=9; fRAG vs cRAG1, P=0.0016, fRAG vs cRAG2, P<0.0001) of fRAG, cRAG1 and cRAG2 leukemic mice. (E) Representative flow cytometry plots of cell cycle arrest of leukemic cells in fRAG, cRAG1 and cRAG2 mice. In the graph, the percentages of each phase of the cell cycle are summarized below (fRAG, n=3, cRAG1, n=5, cRAG2, n=5; G0/G1, fRAG vs cRAG1, P=0.0082, fRAG vs cRAG2, P=0.0279; S, fRAG vs cRAG1, P=0.0146, fRAG vs cRAG2, P=0.0370; G2/M, fRAG vs cRAG1, P=0.0134, fRAG vs cRAG2, P=0.1507). In figures B, C, D and J, error bars represent the mean ± s.d., P values were calculated by Student’s t test and *P < 0.05, **P < 0.01, ***P < 0.001.

The non-core RAG region loss corresponds to a less mature cell surface phenotype (A) Flow cytometry analysis of the B cell markers CD19, BP-1, B220, and CD43 on BCR-ABL1-transformed fRAG, cRAG1 and cRAG2 leukemic bone marrow cells. The percentages of each phase of the B cell stage are summarized in the bottom graph (fRAG, n=9, cRAG1, n=4, cRAG2, n=9; Large-preB, fRAG vs cRAG1, P=0.0349, fRAG vs cRAG2, P=0.0017; Small-pre-B, fRAG vs cRAG1, P=0.0141, fRAG vs cRAG2, P=0.0005). The expression of the cytoplasmic μ chain was analyzed by flow cytometry. Representative samples are shown in (B), and the results from multiple samples analyzed in independent experiments are summarized in the bottom graph as the fraction of cells expressing cytoplasmic factors (fRAG, n=11, cRAG1, n=8, cRAG2, n=8; fRAG vs cRAG1, P=0.3020, fRAG vs cRAG2, P=0.2267). Error bars represent the mean ± s.d., P values were calculated by Student’s t test and *P < 0.05, **P < 0.01, ***P < 0.001.

The non-core RAG region loss highlights genomic DNA damage (A) Western blotting analysis showed RAG1 and RAG2 expression in GFP+CD19+ leukemic cells originating from BCR-ABL1+ B-ALL in different genetic backgrounds. (B) Rearrangement substrate retrovirus was transduced into leukemic cells. Flow cytometry was used to analyze the percentage of CD90.1 and hCD4 positive cells, and the percentage populations are shown in the bottom graph (fRAG, n=3, cRAG1, n=3, cRAG2, n=3; fRAG vs cRAG1, P=0.0002, fRAG vs cRAG2, P=0.5865). (C) Flow cytometry analysis of LH2AX levels in fRAG, cRAG1 and cRAG2 leukemic cells and the percentage of L-H2AX-positive cell populations shown in the bottom graph (fRAG, n=11, cRAG1, n=8, cRAG2, n=8; fRAG vs cRAG1, P=0.0505, fRAG vs cRAG2, P=0.0094). Error bars represent the mean ± s.d., P values were calculated by Student’s t test and *P < 0.05, **P < 0.01, ***P < 0.001.

Structural alterations in BCR-ABL1+ B lymphocytes

(A-C) Circos plot representation of all off-target recombination detected in the genome-wide analyses of fRAG, cRAG1 and cRAG2 leukemic cells. See also Table S3.

Overview and characteristics of off-target recombination in BCR-ABL1+ B-ALL leukemic cells from fRAG and cRAG mice (A)Exon-intron distribution profiles of 42 breakpoints generated by 24 SVs. Gene body includes exon (n = 9; 17.3%) and intron (n = 20; 38.5%). Flanking sequence includes 3’UTR (n = 6; 11.5%), 5’UTR (n = 2; 3.8%), promoter (n = 6; 11.5%), and downstream (n = 9; 17.3%). (B) the off-target recombination was filtered and verified by whole genomic sequence and PCR respectively. P nucleotides and N nucleotides of RSS to RSS and cRSS to cRSS were calculated in BCR-ABL1+ B-ALL. (C) Hybrid joint percentage generated by either fRAG, cRAG1 or cRAG2 in BCR-ABL1+ B-ALL. It was 0, 100%, and 93% in fRAG, cRAG1 or cRAG2 leukemic cells respectively. (D)The 24 off-target recombination genes were retrieved by COSMIC Cancer Gene Census (http://cancer.sanger.ac.uk/census/). 0.5 genes and 0.5 cancers gene average sample in fRAG leukemic cells; 8 genes and 4.5 cancer genes average sample in cRAG1 leukemic cells; 3.3 genes and 0.3 cancer genes average sample in cRAG2 leukemic cells.

The non-core regions have effects on RAG binding accuracy and recombinat size in BCR-ABL1+B lymphocytes (A) Sequence logos were used to compare the RSS and cRSS in Ig loci and non-Ig loci. Top panel: V(D)J recombination at Ig locus; the next three panels: RAG-mediated off-target recombination at non-Ig locus from fRAG, cRAG1 and cRAG2 leukemic cells respectively. The scale of recombinant size was categorized into three ranges: <1000bp, 1000-10000bp, and >10000bp. The distribution of different recombinant sizes in fRAG, cRAG1, and cRAG2 leukemic cells was presented in (B), while the number of different recombinant sizes in fRAG, cRAG1, and cRAG2 leukemic cells was displayed in (C). (D) A schematic depiction of the mechanism of cRAG-accelerated off-target V(D)J recombination was provided. Both RAG1 and RAG2’s non-core region deletion decreases RAG binding accuracy in cRAG1 and cRAG2, BCR-ABL1+ B ALL. Additionally, RAG1’s non-core region deletion significantly reduces the size and scale of off-target V(D)J recombination in cRAG1, BCR-ABL1+ B ALL.