Figures and data in RAG1 and RAG2 non-core regions are implicated in leukemogenesis and off-target V(D)J recombination in BCR-ABL1-driven B-cell lineage lymphoblastic leukemia | eLife

Figures
Additional files

7 figures and 4 additional files

Figures

Figure 1 with 3 supplements

Download asset Open asset

RAG^c/cs give more aggressive leukemia in mice model of BCR-ABL1⁺ B-ALL.

(A) Kaplan–Meier survival curve for Rag^f/f (n = 8), Rag1^c/c (n = 6), and Rag2^c/c (n = 10) recipient mice. The survival was calculated by Mantel–Cox test (Rag^f/f vs Rag1^c/c, p < 0.0381, Rag^f/f vs Rag2^c/c, p < 0.0412). (B) The spleen weights of Rag^f/f, Rag1^c/c, and Rag2^c/c leukemic mice (Rag^f/f, n = 8, Rag1^c/c, n = 7, Rag2^c/c, n = 9; Rag^f/f vs Rag1^c/c p < 0.0001, Rag^f/f vs Rag2^c/c, p = 0.1352). (C) The numbers of spleen cell in Rag^f/f, Rag1^c/c, and Rag2^c/c leukemic mice (Rag^f/f, n = 7, Rag1^c/c, n = 8, Rag2^c/c, n = 13; Rag^f/f vs Rag1^c/c, p = 0.0047, Rag^f/f vs Rag2^c/c, p = 0.0180). (D) The percentage of GFP⁺ cells in peripheral blood (PB) (Rag^f/f, n = 6, Rag1^c/c, n = 6, Rag2^c/c, n = 6; Rag^f/f vs Rag1^c/c, p = 0.0003, Rag^f/f vs Rag2^c/c, p = 0.0035), bone marrow (BM, Rag^f/f, n = 5, Rag1^c/c, n = 5, Rag2^c/c, n = 6; Rag^f/f vs Rag1^c/c, p = 0.0341, Rag^f/f vs Rag2^c/c, p = 0.0008), and spleen (SP, Rag^f/f, n = 9, Rag1^c/c, n = 4, cRAG2, n = 9; Rag^f/f vs Rag1^c/c, p = 0.0016, Rag^f/f vs Rag2^c/c, p < 0.0001) of Rag^f/f, Rag1^c/c, and Rag2^c/c leukemic mice. (E) Representative flow cytometry plots of cell cycle arrest of leukemic cells in Rag^f/f, Rag1^c/c, and Rag2^c/c mice. In the graph, the percentages of each phase of the cell cycle are summarized below (Rag^f/f, n = 3, Rag1^c/c, n = 5, Rag2^c/c, n = 5; G0/G1, Rag^f/f vs Rag1^c/c, p = 0.0082, Rag^f/fvs Rag2 ^c/c, p = 0.0279; S, Rag^f/f vs Rag1 ^c/c, p = 0.0146, Rag^f/f vs Rag2 ^c/c, p = 0.0370; G2/M, Rag^f/f vs Rag1^c/c, p = 0.0134, Rag^f/f vs Rag2 ^c/c, p = 0.1507). In figures B, C, D, and J, error bars represent the mean ± standard deviation (s.d.), p values were calculated by Student’s t test and *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 1—figure supplement 1

Download asset Open asset

Construction of Rag^f/f, Rag1^c/c, and Rag2^c/c, BCR-ABL1⁺ B-ALL mice models using bone marrow transplantation.

In the establishment of BCR-ABL1⁺ B-ALL mice models, Rag^f/f, Rag1^c/c, and Rag2^c/c recipient mice after syngeneic lethal irradiation were transplanted with corresponding donor bone marrow cells transduced by MSCV-BCR-BAL1-IRES-GFP or MSCV-GFP retroviral supernatants. (A) Gross appearance of the spleen in Rag^f/f, Rag1^c/c, and Rag2^c/c leukemic mice and corresponding control mice. (B) Peripheral blood (PB) and bone marrow (BM) lymphoblastic cells were stained by Wright–Giemsa. The scale bars represent 10 µm. (C) Bone marrow cells from Rag^f/f, Rag1^c/c, and Rag2^c/c leukemic mice were examined by flow cytometry for the expression of green fluorescence protein (GFP) and CD19.

Figure 1—figure supplement 2

Download asset Open asset

Biological behavior of leukemia in Rag^f/f, Rag1^c/c, and Rag2^c/c BCR-ABL1⁺ B-ALL mouse model.

(A) BCR-ABL1 expressions in GFP⁺CD19⁺ leukemic cells were determined by western blotting. GAPDH protein was used as a loading control. The K562 and 293T cell lines served as the positive control and negative controls, respectively. (B) Survival of secondary transplant setting. Leukemia cells from primary recipients were recovered from the spleens and purified by GFP⁺ cell sorting. A total of 10⁵,10⁴, and 10³ GFP⁺ leukemia cells originating from Rag^f/f, Rag1^c/c, or Rag2^c/c BCR-ABL1⁺ B-ALL were transplanted into corresponding non-irradiated immunocompetent syngenetic recipient mice (RAG ^f/f, n = 3, Rag1^c/c, n = 3, Rag2^c/c, n = 3; survival days Rag ^f/f, 11–26 days, Rag1 ^c/c, 10–16 days, Rag2 ^c/c, 11–21 days in three different concentrations of GFP⁺ leukemic cells; Rag ^f/f vs Rag1 ^c/c, p = 0.0299, Rag^f/f vs Rag2 ^c/c, p = 0.2286 in concentrations of 10⁵, Rag^f/f vs Rag1 ^c/c, p = 0.0246, Rag^f/f vs Rag2^c/c, p = 0.0295 in concentrations of 10⁴, Rag^f/f vs Rag1^c/c, p = 0.0246, Rag^f/f vs Rag2^c/c, p = 0.0224 in concentrations of 10³, by Mantel–Cox test). (C) Apoptosis was measured by flow cytometry (Annexin V and 7-AAD). The Annexin V⁺ and 7-AAD⁻ cells were defined as early apoptotic cells, while Annexin V⁺ and 7-AAD⁺ cells were late apoptotic cells (Rag^f/f, n = 11, Rag1^c/c, n = 6, Rag2^c/c, n = 9; early apoptotic cells: Rag^f/f vs Rag1^c/c, p = 0.0002, Rag^f/f vs Rag2^c/c, p = 0.0026; late apoptotic cells, Rag ^f/f vs Rag1 ^c/c, p = 0.0026, Rag ^f/f vs Rag2 ^c/c, p < 0.0001). Error bars represent the mean ± standard deviation (s.d.), p values were calculated by Student’s t test and **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 1—figure supplement 2—source data 1 Original file for the western blot analysis in Figure 1—figure supplement 2A.: https://cdn.elifesciences.org/articles/91030/elife-91030-fig1-figsupp2-data1-v1.pdf
Download elife-91030-fig1-figsupp2-data1-v1.pdf

Figure 1—figure supplement 3

Download asset Open asset

The genetic pathways in Rag^f/f, Rag1^C/C, and Rag2^C/C BCR-ABL1⁺ lymphocytes.

mRNA sequence was performed in green fluorescence protein (GFP) and CD19 double positive cells. (A) Principal component analysis (PCA) showing the distribution of differentially expressed samples of Rag^f/f, Rag1^c/c, and Rag2^c/c, BCR-ABL1⁺ B-ALL. (B) Heatmap of representative different expressed genes related to non-homologous end repair. The scale ranges from minimum (blue) to medium (yellow) to maximum (red) relative expression. (C) Volcano plot depicting log2 (fold change) (x-axis) and −log10 (p value) (y-axis) for differentially expressed genes (FC >2, p < 0.05) in GFP⁺CD19⁺ leukemic cells sorted from Rag^f/f and Rag1^c/c, BCR-ABL1⁺ B-ALL mice; upregulated (red) and downregulated (blue). n = 3 per group. (D) The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was conducted to identify the differentially expressed genes in Rag1^c/c BCR-ABL1⁺ B-ALL. The top 15 pathways that exhibited significant differences were listed in this paragraph. The cell proliferation, apoptosis, and differentiation related pathway were highlighted in red squares. (E) Volcano plot depicting log2 (fold change) (x-axis) and −log10 (p value) (y-axis) for differentially expressed genes (FC >2, p < 0.05) in GFP⁺CD19⁺ leukemic cells sorted from Rag^f/f and Rag2^c/c, BCR-ABL1⁺ B-ALL mice; upregulated (red) and downregulated (blue). n = 3 per group. (F) KEGG analysis was conducted to identify the differentially expressed genes in Rag2^c/c BCR-ABL1⁺ ALL. The top 15 pathways that exhibited significant differences were listed in this paragraph. The cell proliferation, apoptosis, and differentiation related pathway were highlighted in red squares.

Figure 2 with 1 supplement

Download asset Open asset

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 Rag^f/f, Rag1^c/c, and Rag2^c/c leukemic bone marrow cells. The percentages of each phase of the B-cell stage are summarized in the bottom graph (Rag ^f/f, n = 9, Rag1 ^c/c, n = 4, Rag2 ^c/c, n = 9; Large-preB, Rag ^f/f, vs Rag1 ^c/c, p = 0.0349, Rag^f/f, vs Rag2^c/c, p = 0.0017; Small-pre-B, Rag ^f/f, vs Rag1^c/c, p = 0.0141, Rag ^f/f, vs Rag2 ^c/c, 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 (Rag^f/f, n = 11, Rag1^c/c, n = 9, Rag2^c/c, n = 8; Rag^f/f vs Rag1^c/c, p = 0.0312, Rag^f/f, vs Rag2^c/c, p = 0.0441). Error bars represent the mean ± standard deviation (s.d.), p values were calculated by Student’s t test and **p < 0.01, ***p < 0.001.

Figure 2—figure supplement 1

Download asset Open asset

VDJ recombination in leukemic cells with different genetic backgrounds.

(A, B) VDJ recombination was analyzed by genomic PCR in GFP⁺CD19⁺ cell’s DNA from Rag^f/f, Rag1^c/c, or Rag2^c/c leukemic cells. Genomic DNA from RAG1^−/− bone marrow cells and WT spleen was used as negative and positive control, respectively (DNA Marker: 1000 bp, 700 bp, 500 bp, 400 bp, 300 bp, 200 bp, 100 bp).

Figure 2—figure supplement 1—source data 1 Original file for the PCR analysis in Figure 2—figure supplement 1A.: https://cdn.elifesciences.org/articles/91030/elife-91030-fig2-figsupp1-data1-v1.pdf
Download elife-91030-fig2-figsupp1-data1-v1.pdf
Figure 2—figure supplement 1—source data 2 Original file for the PCR analysis in Figure 2—figure supplement 1B.: https://cdn.elifesciences.org/articles/91030/elife-91030-fig2-figsupp1-data2-v1.pdf
Download elife-91030-fig2-figsupp1-data2-v1.pdf

Figure 3 with 2 supplements

Download asset Open asset

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. The experiment was repeated under the same conditions three times. (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 (Rag^f/f, n = 3, Rag1^c/c, n = 3, Rag2^c/c, n = 3; Rag^f/f, vs Rag1 ^c/c, p = 0.0002, Rag ^f/f, vs Rag2 ^c/c, p = 0.5865). (C) Flow cytometry analysis of ɣ-H2AX levels in Rag^f/f, Rag1^c/c, and Rag2^c/c leukemic cells and the percentage of γ-H2AX-positive cell populations shown in the bottom graph (Rag ^f/f, n = 11, Rag1^c/c, n = 8, Rag2^c/c, n = 8; Rag^f/f, vs Rag1^c/c, p = 0.0505, Rag^f/f, vs Rag2^c/c, p = 0.0094). Error bars represent the mean ± standard deviation (s.d.), p values were calculated by Student’s t test and *p < 0.05, **p < 0.01.

Figure 3—source data 1 Original file for the western blot analysis in Figure 3A.: https://cdn.elifesciences.org/articles/91030/elife-91030-fig3-data1-v1.pdf
Download elife-91030-fig3-data1-v1.pdf

Figure 3—figure supplement 1

Download asset Open asset

RAG protein expression levels and schematic diagram of the recombinant substrate vector.

(A) RAG1/cRAG1 and RAG2/cRAG2 protein levels were compared by western blot and ImageJ software in Rag^f/f, Rag1^c/c, and Rag2^c/c, B-ALL cells. Error bars represent the mean ± standard deviation (s.d.). The p value was calculated by t test, **p < 0.01, *p < 0.05, ns p > 0.05. (B) The B-ALL cells were subjected to transformation with the recombinant substrate vector. In the event of expression of RAG recombinase in the leukemic cells, the recombination signal sequences (RSSs) flanking CD90.1 would be cleaved by RAG, thereby facilitating the positioning and expression of both CD90.1 and hCD4. In the absence of RAG expression, only hCD4 would be expressed.

Figure 3—figure supplement 2

Download asset Open asset

The relationship of RAG1 mRNA levels and survival of pediatric acute lymphoid leukemia.

The relationship of RAG1 mRNA levels and survival of pediatric acute lymphoid leukemia was research by cBioPortal (https://www.cbioportal.org/). RAG1 mRNA levels were studied in pediatric patients with ALL at the time of diagnosis. The patients were separated into two groups based on mRNA levels of RAG1 (mRNA expression z score relative to diploid sample, RNA sequence RPKM, RAG1 upregulated group, n = 8; RAG1 unaltered group, n = 146). The p values were calculated from the log-rank test, p = 0.0732.

Figure 4 with 1 supplement

Download asset Open asset

Structural alterations in BCR-ABL1⁺ B lymphocytes.

(A–C) Circos plot representation of all off-target recombination detected in the genome-wide analyses of Rag^f/f, Rag1^c/c, and Rag2^c/c leukemic cells.

Figure 4—figure supplement 1

Download asset Open asset

The criteria for identifying off-target recombination.

We adopted the criteria used in previous studies. First, a CAC must exist to the right (or GTG to the left) of both breakpoints, which includes the four RAG-mediated DNA fragmentation cases mentioned above, and second, it must occur within a specified distance from the breakpoint and the CAC distance-to-breakpoint value was set at 21 bp.

Figure 5

Download asset Open asset

Overview and characteristics of off-target recombination in BCR-ABL1⁺ B-ALL leukemic cells from Rag^f/f and Rag ^c/c mice.

(A) Exon–intron distribution profiles of 41 breakpoints generated by 24 structural variations (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 and N nucleotides of recombination signal sequence (RSS) to RSS and cryptic RSS (cRSS) to cRSS were calculated in BCR-ABL1⁺ B-ALL. (C) Hybrid joint percentage generated by either Rag^f/f, Rag1^c/c, or Rag2^c/c in BCR-ABL1⁺ B-ALL. It was 0, 100%, and 93% in Rag^f/f, Rag1^c/c, or Rag2^c/c 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 Rag^f/f, leukemic cells; 8 genes and 4.5 cancer genes average sample in Rag1^c/c leukemic cells; 3.3 genes and 0.3 cancer genes average sample in Rag2^c/c leukemic cells.

Figure 6

Download asset Open asset

The non-core regions have effects on RAG-binding accuracy and recombinat size in BCR-ABL1⁺ B lymphocyte.

(A) Sequence logos were used to compare the recombination signal sequence (RSS) and cryptic RSS (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 Rag^f/f, Rag1^c/c, and Rag2^c/c leukemic cells, respectively. The scale of recombinant size was categorized into three ranges: <1000, 1000–10,000, and >10,000 bp. The distribution of different recombinant sizes in Rag ^f/f, Rag1^c/c, and Rag2^c/c leukemic cells was presented in (B), while the number of different recombinant sizes in Rag ^f/f, Rag1^c/c, and Rag2^c/c leukemic cells is 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 Rag1^c/c and Rag2^c/c, 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 Rag1^c/c BCR-ABL1⁺ B-ALL.

Author response image 1

Download asset Open asset

Additional files

Supplementary file 1 Sequencing statistics.: https://cdn.elifesciences.org/articles/91030/elife-91030-supp1-v1.xlsx
Download elife-91030-supp1-v1.xlsx
Supplementary file 2 Abnormal rearrangements at Ig.: https://cdn.elifesciences.org/articles/91030/elife-91030-supp2-v1.xlsx
Download elife-91030-supp2-v1.xlsx
Supplementary file 3 RAG-mediated off-target genes.: https://cdn.elifesciences.org/articles/91030/elife-91030-supp3-v1.xlsx
Download elife-91030-supp3-v1.xlsx
MDAR checklist: https://cdn.elifesciences.org/articles/91030/elife-91030-mdarchecklist1-v1.pdf
Download elife-91030-mdarchecklist1-v1.pdf

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Mendeley

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

Xiaozhuo Yu
Wen Zhou
Xiaodong Chen
Shunyu He
Mengting Qin
Meng Yuan
Yang Wang
Woodvine Otieno Odhiambo
Yinsha Miao
Yanhong Ji

(2024)

RAG1 and RAG2 non-core regions are implicated in leukemogenesis and off-target V(D)J recombination in BCR-ABL1-driven B-cell lineage lymphoblastic leukemia

eLife 12:RP91030.

https://doi.org/10.7554/eLife.91030.3

Sign up for email alerts

Privacy notice