precursor ALL. · Nature Immunology: doi:10.1038/ni.3160 Supplementary Figure 1 High levels of AICDA and RAG1 mRNA at the time of diagnosis predict poor clinical outcomes for patients

Nature Immunology: doi:10.1038/ni.3160

Supplementary Figure 1

High levels of AICDA and RAG1 mRNA at the time of diagnosis predict poor clinical outcomes for patients with B cell precursor ALL. (a) Left: Comparison of overall survival probabilities of ALL patients segregated into two categories based on their median AICDA expression levels (ECOG, n=215). P value was calculated by logrank test. Center: Comparison of overall survival probabilities of ALL patients segregated into two categories based on their median RAG1 expression levels (ECOG, n=215). P value calculated by logrank test. Right: Comparison of relapse-free survival probabilities of ALL patients segregated into two categories based on their median RAG1 expression levels (ECOG, n=215). P value calculated by logrank test. (b) Comparison of AICDA expression levels at diagnosis and relapse in matched sample pairs (P9906 COG, n=49) of childhood ALL patients. P-value was calculated using paired Wilcoxon two sided t test.



Aicda and Rag are upregulated during early B cell development upon loss of IL-7R signaling (small pre-BII cell stage).

(a) Quantitative RT-PCR showing Aicda mRNA levels upon reconstitution of Blnk into Blnk-/- pre-B cells (n=3, mean ± s.d.). (b) Aicda mRNA levels measured by qRT-PCR before and after 24 hours of IL-7 withdrawal (n=3, mean ± s.d.). (c)


Rag1 mRNA levels measured by qRT-PCR (n=3, mean ± s.d.) before and after IL-7 withdrawal in mouse pre-B cells. (d) Aicda mRNA levels measured by qRT-PCR after retroviral expression of a constitutively active form of Foxo3a (Foxo3aCA) or empty vector (EV) in pre-B cells, in the presence or absence of IL-7 (n=3, mean ± s.d.).



Small pre-BII cells from Aicda-GFP and Aicda-Cre × Rosa 26-LSL-eYFP reporter mice respond to inflammatory signals from LPS by upregulating Aicda.


(a) Change in percentage of Aicda-GFP+ cells with time, in the presence and absence of LPS, before and after differentiation to small pre-BII stage. One representative experiment out of three is shown. (b) Aicda-GFP pre-B cells upregulate expression of Aicda, Rag1 and Rag2 at the small pre-BII stage in the context of inflammatory signals like LPS (GFP+ κLC+ cells). One representative experiment out of three is shown. (c, d) Change in percentage of Aicda-Cre eYFP+ cells with time, in the presence and absence of LPS, before and after differentiation to the small pre-BII stage. Experiments from two independent bone marrows are shown.



Evidence for concurrent activity of RAG1-RAG2 and AID in single pre-B cell clones.

Diagrammatic representation of cooperation between AID (somatic hypermutation), and RAG1-RAG2 activities (VH replacement) in the clonal evolution of a pediatric pre-B ALL patient.



Cooperation among RAG1 and RAG2 and AID promotes clonal evolution towards pre-B ALL.

Schematic: Loss of IL-7R signaling at small pre-BII makes a pre-B cell vulnerable to acquisition of genetic changes by activation of AID, RAG1 and RAG2.



Flow cytometry to sorting human cord blood B cell clones transduced to express AID (iRFP670), RAG1 (eGFP) and RAG2(dsRedE2).

Lentiviral vectors encoding Aicda (pCL6-Aicda-IRES-iRFP670-wo), Rag1 (pCL6-Rag1-IRES-eGFP-wo), Rag2 (pCL6-Rag2-IRES-dsRedExpress2-wo) and the corresponding empty vector (EV) controls were introduced into EBV-transformed human CD19+ cord blood B cells by the transduction protocol described in Materials and Methods. Cells were eithertransduced with EVs, Aicda alone, Rag1 and Rag2 combination or Aicda, Rag1 and Rag2. After 4 days, living EBV cord blood B cells, stained with DAPI, that were triple positive for eGFP, iRFP670 and dsRedExpress2 were single cell sortedinto 96well plates using a 488nm(525/50), 640nm(670/30), 561nm(582/15) and 355nm(450/50) configuration on an BDAriaII Sorter. 3D graphics were generated with WinList (Verity Software House).



Verification of the overexpression of AID, RAG1 and RAG1 alone or in combination in human cord blood B cell clones by fluorescence microscopy

Lentiviral vectors encoding Aicda (pCL6-Aicda-IRES-iRFP670-wo), Rag1 (pCL6-Rag1-IRES-eGFP-wo), Rag2 (pCL6-Rag2-IRES-dsRedExpress2-wo) and the corresponding empty vector (EV) controls were introduced into EBV-transformed human CD19+ cord blood B cells by the transduction protocol described in Materials and Methods. Cells were either transduced with EVs, Aicda alone, Rag1 and Rag2 combination or Aicda, Rag1 and Rag2. The transduction of the cells for Rag1 (eGFP) and Rag2 (dsRedExpress2) was verified by immunofluorescence microscopy. Transduction of Aicda (iRFP670) was verified by flow cytometry (Figure S6).



AID and RAG are required for the leukemic transformation of ETV6-RUNX1 pre-B cell clones in the context of repeated inflammatory stimulation.

Mice that had become terminally ill (Aicda+/+ Rag1+/+ No IL-7+LPS group) were sacrificed and bone marrow and spleenswere analyzed by flow cytometry. Verification of leukemia as the cause of terminal illness was carried out by flow cytometry measurement of the percentage of CD19+/ ETV6-RUNX1 GFP+ cells in the bone marrow and spleen of all the sacrificed mice in the group of LPS and IL-7 withdrawal.

mice that were injected with Aicda+/+ Rag1+/+ pre-B cells after repetitive stimulation with



Immunohistochemical analysis of ETV6-RUNX1 pre-B ALL infiltration in congenic recipient mice.

Mice that had become terminally ill (Aicda+/+

Rag1+/+

No IL7+LPS group) were sacrificed and bone marrow and spleens were analyzed by flow cytometry. Pre-B ALL as the cause of terminal illness was verified byimmunohistochemistry and leukemic infiltrates in spleen (top) and liver were visualized. H&E staining andimmunohistochemistry for CD19

+ pre-B cell infiltration (and isotype control staining) were performed on

spleen and liver sections of all the sacrificed mice in the group of mice that were injected with Aicda+/+

Rag1+/+

pre-B cells after repetitive stimulation with LPS and IL7 withdrawal.

Table S1: Mutation frequency of VH region in pre-B cells from human bone marrow.

Average hypermutation frequency of VH region = 26.2 x 10-3 bp

Clone VH DH JH Mut Freq (x10-3

bp)

1 VH1-3 D1-7 JH4 34.3

2 VH1-24 D1-26 JH4 2.9

3 VH1-18 D2-15 JH4 37.1

4 VH1-2 D2-2 JH6 25.7

5 VH1-24 D2-2 JH6 57.1

6 VH1-69 D2-2 JH6 8.6

7 VH1-18 D2-2 JH6 68.6

8 VH1-8 D2-2 JH6 17.1

9 VH1-46 D3-10 JH6 37.1

10 VH1-8 D3-22 JH4 34.3

11 VH1-46 D3-22 JH5 0.0

12 VH1-58 D3-22 JH5 34.3

13 VH1-46 D3-3 JH5 11.4

14 VH1-2 D3-9 JH4 14.3

VH1-2 D3-9 JH4 8.6

VH1-2 D3-9 JH4 11.4

15 VH1-46 D4-17 JH4 22.9

16 VH1-2 D4-17 JH4 31.4

17 VH1-3 D5-12 JH6 14.3

18 VH1-24 D5-24 JH4 22.9


Do

no

r 1

D

on

or

2

Don

or

3

Table S2: SHM and CSR of VH region in pre-B cells from fetal liver.

Average hypermutation frequency of VH region = 14.1 x 10-3 bp

VH DH JH Constant Mut freq (x10-3bp) JH Constant Region

VH1-46 D4-17 JH4 Cµ→Cγ1 20.0 TGACTACTGG CAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCC

VH3-23 D1-14 JH4 Cµ→Cα 25.7 GACTACTGG CATCCCCGACCAGCCCCAAGGTCTTCCCGCTGAGCCT

VH3-23 D1-20 JH4 Cµ 0.0 ACTACTTTGACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA

VH3-23 D1-7 JH6 Cµ 14.3 CTACTACTACGGTATGGACGTCTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA

VH3-23 D3-10 JH4 Cµ 14.3 GACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA

VH3-23 D3-3 JH4 Cµ→Cγ1 17.1 TTGACTACTGG AGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCT

VH3-23 D4-11 JH6 Cµ→Cγ3 8.6 TTACTACTACTACTGCGGTATGGACGTCTGG GGCGCCCTGCTCCAGGAGCACCTCTGGGGGCACAGCG

VH3-23 D6-19 JH4 Cµ 17.1 TGACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA



VH3-30 D6-13 JH6 Cµ→Cγ1 45.7 CTACTACAACGGTATGGACGTCTGG CCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACA

VH3-30-3 D1-26 JH6 Cµ→Cγ1 14.3 TACTACTCCTACTACGGTATGGACGTCTGG TCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCT

VH3-33 D6-19 JH5 Cµ 8.6 AACTGGTTCGACCCCTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA


VH4-34 D6-13 JH5 Cµ 14.3 ACCCCTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA

VH4-39 D3-10 JH4 Cµ 11.4 TACTTTGACTACTGG GGGGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA

VH6-1 D2-8 JH2 n.d. 14.3 ACTGGTACTTCGATCTCTGG

VH3-11 D3-9 JH3 Cµ→Cγ1 8.6 GCTTTTGATATCTGG CCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACC

VH3-30 D6-6 JH4 Cµ 11.4 TTTGACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA

VH3-30-3 D6-13 JH4 Cµ 11.4 TTTGACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA

VH3-33 D7-27 JH4 Cµ 11.4 ACTTTGACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA


VH3-7 D2-15 JH3 Cµ 2.9 GATGCTTTTGATATCTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA


VH3-73 D4-23 JH4 Cµ→Cγ1 17.1 TTGACTACTGG CCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACC


VH4-34 D4-11 JH3 Cµ→Cγ1 25.7 GATGCTTTAGATATCTGG CTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCC

VH4-59 D2-15 JH3 Cµ→Cγ1 8.6 GATGCTTTTGATGTCTGG CCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACC

VH4-59 D6-19 JH4 Cµ 20.0 TTTGACTACTGG GAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA

VH6-1 D3-10 JH3 Cµ 5.7 GCTTTTGATATCTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA

VH6-1 D6-13 JH5 Cµ 14.3 ACTGGTTCGACCCCTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA

VH6-1 D7-27 JH4 Cµ 11.4 TACGTTGACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA



VH1-69 D3-10 JH6 Cµ 2.9 ACTACTACTACTACGGTATGGACGTCTGG GGGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA

VH2-5 D6-13 JH4 Cµ→Cγ1 77.1 CTTTGACTACTGG CCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACC


VH3-11 D3-10 JH1 Cµ 5.7 TGAATACTTCCAGCACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA

VH3-11 D3-10 JH4 Cµ 17.1 TTGACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA

VH3-11 D6-19 JH4 Cµ 8.6 TACTTTGACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA

VH3-23 D1-1 JH4 Cµ 8.6 CTTTGACTACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA


VH3-23 D1-26 JH6 Cµ 34.3 ACGGTATGGACGTCTGG GGGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA



VH3-33 D3-10 JH2 Cµ 8.6 ACTGGTACTTCGATCTCTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA

VH3-33 D3-9 JH6 Cµ 8.6 TACTACTACTACGGTATGGACGTCTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA

VH3-66*03 D6-19 JH1 Cµ 2.9 GCTGAATACTTCCAGCACTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA

VH3-7 D1-26 JH4 Cµ→Cγ1 20.0 ACTACTGG CCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACC

VH3-7 D2-2 JH5 Cµ 8.6 AACTGGTTCGACCCCTGG GGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA

VH3-7 D6-19 JH6 Cµ 14.3 ATTACTACTACTACTACGGTATGGACGTCTGG GGGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA

VH3-74 D2-8 JH4 Cµ→Cγ1 8.6 CTTTGACTACTGG CCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACC

VH6-1 D1-26 JH3 Cµ 8.6 GCTTTTGATATCTGG GAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTA


Table S3: SHM and VH replacement in ETV6-RUNX1 ALL patients (n = 13). 7/13 cases positive for VH

replacement. Cryptic RSSs are underlined.

Average mutation frequency of VH = 6.4 x 10-3 bp

Average mutation frequency of JH = 34.5 x 10-3 bp

Patient Allele VH DH JH

VH Mut

Freq

(x10-3

bp)

JH Mut

Freq

(x10-3

bp) VH-N DH-N-JH

REH 1 VH3-15 D3-10 JH6 8.0 66.9 AGCCGTGTATTACTGTACCACAGGG ATGGTTCGGGGAGTTATTATACTACTACTACGGTATGGACGTCTGG

LAX13 1 VH3-33 D3-22 JH6 8.2 96.8 GCTGTGTATTACTGTGCGAGAGA CTCTCAGAGTACTACTACGGTATGGACGTCTGG

PD4009a 1 VH1-69 D3-3 JH6 5.5 80.6 GCCGTGTATTACTGTGCGAGAGAGGCGAGTCCTCCCGAGAGATTGAGGGCGGGG ACGATTTTTGGAGTGGTTATTCTACTACTACGGTATGGACGTCTGG

VH2-70 D3-3 JH6 0.0 0.0 CAGCCACGTATTACTGTGCACGGCCCCGCGAGTCCTCCCGAGAGATTGAGGGCGGGG ACGATTTTTGGAGTGGTTATTCTACTACTACGGTATGGACGTCTGG

VH3-66 D3-3 JH6 0.0 0.0 ACGGCTGTGTATTACTGTGCGAGTCCTCCCGAGAGATTGAGGGCGGGG ACGATTTTTGGAGTGGTTATTCTACTACTACGGTATGGACGTCTGG

PD4020a 1 VH5-10 D3-10 JH4 12.9 122.4 CTCGGACACCGCCATGTATTACTCCTAGCGAGACACCTT TTACTATGGTTCGGGGAGTTATTATAACTACTGG

2 VH1-3 D6-6 JH4 15.3 272.5 ACACGGCTGTGTATTACTGTGCGTGGGCC TATAGCAGCTACCCGG

VH2-5 D6-6 JH4 0.0 0.0 CACAGCCACATATTACTGTGCACTCACGTGGGCC TATAGCAGCTACCCGG

VH3-21/VH3-48 D6-6 JH4 0.0 0.0 GCTGTGTATTACTGTGCGAGAGAGAGTGGGCC TATAGCAGCTACCCGG

VH3-23 D6-6 JH4 0.0 0.0 GCCTTGTATTACTGTGCAAGAGAGGGCGTGGGCC TATAGCAGCTACCCGG

VH3-30 D6-6 JH4 0.0 0.0 GACACGGCTGTGTATTACTGTGCCGGAAGCGACC TATAGCAGCTACCCGG

VH3-33 D6-6 JH4 0.0 0.0 GCTGTGTATTACTGTGCGAGAGATTCTCTTCGTGGGCC TATAGCAGCTACCCGG

VH3-7 D6-6 JH4 0.0 0.0 GGCCGTGTATTACTGTGCGAGAGCAGGGTGGGCC TATAGCAGCTACCCGG

PD4021a 1 VH2-70 D2-2 JH5 4.3 0.0 CCACGTATTACTGTGCACGGATAGT ATAATGTAGTAGTACCAGCTGCTATACAACTGGTTCGACCCCTGG

PD4038a 1 VH2-70 D3-16 JH4 3.8 145.8 GCCACGTATTACTGTGCACGGAT GGGGAGAGACGCGAGACCCATCGAGAGATCGGGGCGAAAGAGGCTACTGG

VH3-66 D3-16 JH4 0.0 0.0 ACACGGCTGTGTATTACTGTGCGTTAG GGGGAGAGACGCGAGACCCATCGAGAGATCGGGGCGAAAGAGGCTACTGG

VH4-61 D3-16 JH4 0.0 0.0 GGCCGTGTATTACTGTGCGAGAG GGGGAGAGACGCGAGACCCATCGAGAGATCGGGGCGAAAGAGGCTACTGG

2 VH2-70 D5-12 JH4 6.9 155.1 GACACAGCCACGTATTACTGTGCCTATTCGACCCCGCGAGACTAGAGACGCGAGACCCATCGA GAGATCGGGGCGAAAGAGGCTACTGG

VH3-64 D5-12 JH4 0.0 0.0 GGACATGGCTGTGTATTACTGTGGGGGGCGGCGCGAGACCCATCGA GAGATCGGGGCGAAAGAGGCTACTGG

VH3-66 D5-12 JH4 0.0 0.0 ACGGCTGTGTATTACTGTGCGAGGACCCTCCGAGAGACGCGAGACCCATCGA GAGATCGGGGCGAAAGAGGCTACTGG

VH3-72 D5-12 JH4 0.0 0.0 GCCGTGTATTACTGTGCTAGAGAAGAAAGACGCGAGACCCATCGA GAGATCGGGGCGAAAGAGGCTACTGG

VH4-59 D5-12 JH4 0.0 0.0 GCCGTGTATTACTGTGCGAGAGACGCGAGACCCATCGA GAGATCGGGGCGAAAGAGGCTACTGG

VH4-61 D5-12 JH4 0.0 0.0 GGCCGTGTATTACTGTGCGAGAGTCCCTCCTAGCCCATCGA GAGATCGGGGCGAAAGAGGCTACTGG

PD3952a 1 VH2-70D D3-22 JH6 18.0 32.3 CACGTATTACTGTGCACGGATACTCAGC GTATTACTATGATAGTAGTGGTTATTCCCAAGGCATTACTACTACTACTACGGTATGGACGTCTGG

VH3-30/VH3-33 D3-22 JH6 0.0 0.0 TGAGGACACGGCTGTGAGGACTA ---------TGATAGTAGTGGTTATTCCCAAGGCATTACTACTACTACTACGGTATGGACGTCTGG

VH3-66 D3-22 JH6 0.0 0.0 CGGCTGTGTATTACTGTGCGAGAGAGCGGAC GTATTACTATGATAGTAGTGGTTATTCCCAAGGCATTACTACTACTACTACGGTATGGACGTCTGG

VH3-73 D3-22 JH6 0.0 0.0 GCCGTGTATTACTGTACTAGACAGAGC GTATTACTATGATAGTAGTGGTTATTCCCAAGGCATTACTACTACTACTACGGTATGGACGTCTGG

VH4-55 D3-22 JH6 0.0 0.0 ACGGCCGTGTATTACTGTGCGAGC GTATTACTATGATAGTAGTGGTTATTCCCAAGGCATTACTACTACTACTACGGTATGGACGTCTGG

2 VH3-21 D3-22 JH6 0.0 0.0 CACGGCTGTGTATTACTGTGCGAAAAGCG ATTACTATGATAGTAGTGGTGAACCATACTACTACGGTATGGACGTCTGG

PD3961a 1 VH3-30 D2-2 JH6 19.8 83.0 GGCTGTGTATTACTGTGCGAAAGGGGGG GGATATTGTAGTAGTACCAGCTGCTATGGGGGGCTACTACTACTACGGTATGGACGTCTGG

VH3-7 D2-2 JH6 0.0 0.0 GGCTGTGTATTACTGTGCGAAAGGGGGG GGATATTGTAGTAGTACCAGCTGCTATGGGGGGCTACTACTACTACGGTATGGACGTCTGG

VH4-34 D2-2 JH6 0.0 0.0 GCTGTGTATTACTGTGCGAAAGGGGGG GGATATTGTAGTAGTACCAGCTGCTATGGGGGGCTACTACTACTACGGTATGGACGTCTGG

2 VH3-7 D7-27 JH4 22.9 71.4 GACACGGCTGTGTATTACTGTGC CTCAGAGCGGCTACTGG

PD4022a 1 VH1-2 JH6 16.1 100.7

2 VH3-7 D2-2 JH6 9.3 57.2 GGCTGTGTATTACTGTGCGAGAGTGGG CCACCTCTACTACTACTACGGTATGGACGTCTGG

PD4024a 1 VH2-70 D3-10 JH5 4.8 82.9 GCCACGTATTACTGTGCACGGATCCCTGACTC ATTACTATGGTTCAGGGAGTCTGGTTCGACCCCTGG

VH3-71 D3-10 JH5 0.0 0.0 CGGCTGTGTATTACTGTGCGAGAAGTC ATTACTATGGTTCAGGGAGTCTGGTTCGACCCCTGG

PD4036a 1 VH6-1 D3-10 JH6 4.2 48.4 GCTGTGTATTACTGTGCAAGAGAGTT GTATTACTATGGTTCGGGGAGTTAGGGGGGTAAGTACTACTACTACTACATGGACGTCTGG

2 VH3-30/VH3-33/ VH3-7 D2-21 JH6 116.0 50.4 AAGGACACGGCCATGTATTACTGTACCAGAGAGGTCCCCCG GTGGTGGTGACTGCTATTCCTCTACTACTACGGTATGGACGTCTGG

PD4037a 1 VH1-69 D2-2 JH1 3.6 19.2 GCCGTGTATTACTGTGCGAGAGACCC GATATTGTAGTAGTACCAGCTGCTATACCCTGAATACTTCCAGCACTGG

VH2-70 D2-2 JH1 0.0 0.0 CAGCCACGTATTACTGTGCACGGCCTTGACGAGAGATCCCC GATATTGTAGTAGTACCAGCTGCTATACCCTGAATACTTCCAGCACTGG

VH2-70D D2-2 JH1 0.0 0.0 ACAGCCACGTATTACTGTGCCCGCCGGACGAGAGATCCCC GATATTGTAGTAGTACCAGCTGCTATACCCTGAATACTTCCAGCACTGG

VH3-69-1 D2-2 JH1 0.0 0.0 GCTGTGTATTACTGTGCGAGAGAAGATAGATGAGACATCCCC GATATTGTAGTAGTACCAGCTGCTATACCCTGAATACTTCCAGCACTGG

920527-4278 1 VH6-1 JH6 0.0 34.5


Table S4: SHM and VH replacement in Ph-negative ALL patients (n = 58). 2/58 cases positive for VH

replacement in non-ETV6-RUNX1 leukemic subgroups.

Average mutation frequency of VH = 14 x 10-3 bp






Table S4a: SHM and VH replacement leukemia patients with common MLL and TCF3-PBX1

rearrangements (n = 5). Cryptic RSSs are underlined.

Table S4b: SHM and VH replacement in hyperdiploid leukemia patients (n = 17). Cryptic RSSs are

underlined.

Table S4c: SHM and VH replacement in leukemia patients with uncommon karyotypes (n = 10).

Cryptic RSSs are underlined.

Patient Cytogenetics VH DH JH

Mut Freq

VH (x10-3

bp)

Mut Freq

JH (x10-3

bp) VH-N DH-N-JH

P902486/96 MLL-AF4 VH5-a D2-15 JH6 7.8 150.9 CGCCATGTATTACTGTGCGAGAC TATTGTAGTGGTGGTAGCTGCCGCCGAGTACTACTACTACGGTATGGACGTCTGG

P901941/96 MLL-ENL VH2-5 D3-10 JH6 0.0 241.9 CACATATTACTGTGCACACAGATGGGATTT TATTACTATGGTTCGGGGAGTTATTATACCCTTACTACTACTACGGTATGGACGTCTGG

P902032/95 TCF3-PBX1 VH1-8 JH4 12.3 0.0

VH3-66 JH4 8.3 0.0

P900469/95 TCF3-PBX1 VH6-1 D1-26 JH6 0.0 132.1 CTGTGTATTACTGTGCAAGAGAT GTAGAGTGGGAGCTACTTACTACTACTACGGTATGGACGTCTGG

Patient 20 MYC-IGH VH3-74 D6-13 JH4 55.6 83.3 CTCTATATTACTGTGTAAGAGACGGAG ATACCACCAGTTGGTCTTTTGACTACTGG


Mut Freq

VH (x10-3

bp)

Mut Freq

JH (x10-3

bp) VH-N DH-N-JH

B273/98 Hyperdiploid VH2-5 D2-15 JH4 7.4 210.5 ACATATTACTGTGCACACAGACCAAATTCCTTCTCGA GTAGTGGTGGTAGCTGCTTGAGACTACTGG

B2033/97 Hyperdiploid VH1-58 D3-3 JH5 11.7 73.2 CCGTGTATTACTGTGCGGCAGAAG TACGATTTTTGGAGTGGTTATTATATCAACTGGTTCGACCCCTGG

P90100/96 Hyperdiploid VH3-9 D3-3 JH6 12.2 132.1 CTTGTATTACTGTGCAAAAGAT GTATTACGATTTTTGGAGTGGTTATTATACTACTACTACGGTATGGACGTCTGG

P901756/96 Hyperdiploid VH2-5 D5-12 JH5 15.0 285.7 ACATATTACTGTGCACACAGACAAGGGG ATAGTGGCTACCCGTATAAAGACCTGG

B960/97jfr1224 Hyperdiploid VH4-34 D3-16 JH4 0.0 184.2 GCTGTGTATTACTGTGCGAGAGTCTAGGTC TGATTACGTTTGGGGGAGTTATCGATTGAGATCTGACTACTGG

c901275/94 Hyperdiploid VH6-1 JH6 4.7 200.0

c27012-91 Hyperdiploid VH1-3 D6-6 JH4 0.0 261.9 CTGTGTATTACTGTGCGAGAGAGG ATAGCAGCTCGCCCTTAAACTGG

VH6-1 D2-8 JH6 0.0 277.8 CTGTGTATTACTGTGCAAGAGAGC TGTACTAATGGTGTATGCTATAAGAGGAAAACGGTATGGACGTCTGG

P901348/95 Hyperdiploid VH4-34 D7-27 JH4 4.2 184.2 CTGTGTATTACTGTGCGAGAGGAG CTAACTGGGGTGACCGTGGGCCGACTACTGG

P902781/96 Hyperdiploid VH3-23 D2-2 JH6 4.1 113.2 CCGTATATTACTGTGCGAAAGACCTCCGGG AGTAGTACCAGCTGCTATTACTACTACTACGGTATGGACGTCTGG

B675/97 Hyperdiploid VH4-34 D6-19 JH4 0.0 52.6 GGCTGTGTATTACTGTGCGAGATGCCGATCCTTCT GTATAGCAGTGGCTGGTACTTTGACTACTGG

P90131/96 Hyperdiploid VH3-53 D4-23 JH4 4.1 105.3 CCGTGTATTACTGTGCGAGAGAA ACTGTGGGGATTCCCCTTTGACTACTGG

P902300/95 Hyperdiploid VH6-1 JH6 33.0 172.4

P900978/95 Hyperdiploid VH3-9 D1-26 JH4 8.2 256.4 TTGTATTACTGTGCAAAAGATAATAGATAGA TGGGAGCTACTACTTTGACTACTGG

c900162/94 Hyperdiploid VH3-30 D3-10 JH6 4.1 264.2 GCTGTGTATTACTGTGCGAGAGGAGGG GGTTTCGGGATACTACGGTATGGACGTCTGG

Patient 5 Hyperdiploid VH3-13 JH5 24.6 196.1

Patient 10 Hyperdiploid VH3-7 D3-10 JH5 85.1 16.0 CGGCTGTGTATTACTGTGCGAG GGGGACAACTGGTTCGACCCCTGG

VH3-66 D2-15 JH4 73.7 166.7 GCTGTGTATTACTGTGCGAGAGTT TGGTATCAGCTAATGCCCCGGGACTCCTGG

VH4-34 49.3 0.0

Patient 18 Hyperdiploid VH4-39 D3-10 JH6 13.7 80.6 GCGGACACGGCTGTGTATTACCCCCCCGTAAGTGG TTATTAACGTTCGGGTCTTTGGCTACTACTACTACGGTATGGACGTCTGG

VH1-2 D2-2 JH6 31.0 112.9 ACGGCCGTGTATTACTGTGCGACACGGTGTTGATAGC TATTGTAGTAGTACCAGCTGCTATAGGTTTCGGCTACTACTACGGTATGGACGTGTGG


Mut Freq

VH (x10-3

bp)

Mut Freq

JH (x10-3

bp) VH-N DH-N-JH

hösten-95 Other VH1-3 D7-27 JH4 16.5 210.5 CTGTGTATTACTGTGCGAGAGATTGGGT TAACTGGGGATGACTACTGG

B1057/97 Other VH3-74 JH4 24.5 285.7

P900667/96 Other VH3-33 D2-2 JH6 8.2 150.9 GGCTGTGTATTACTGTGCGAGACCCC AGGAGAAACAGAATACTACTACTACGGTATGGACGTCTGG

VH4-4 D4-11 JH4/JH6 33.5 138.9 GCGGACACGGCCGTGTACAGTA CTACATGGACGTCTGG

c900891/94 Other VH6-1 JH6 4.2 0.0

c900024/94 Other VH2-70 3.7 0.0

P901693/95 Other VH3-21 8.2 0.0

c26170/87 Other VH3-23 D3-10 JH4 0.0 250.0 ACGGCCGTATATTACTGTGCGA GAGGTATTACGATTTTTGATGGG

Patient 3 Other VH4-61 D3-3 JH4 27.5 62.5 GCCCTGTATTACTGTGCGGGAG TTTGGGGCTGGGCTACTTCCTTTGACTACTGG

Patient 17 Other VH4-34 D3-22 JH4 42.1 104.2 GGCTGTTTATTACTGTGCGAGATGCGG CTATCATAGTAGTGGTTATTACTACGGACCGCCGTTCACTACTGG

Patient 23 Other VH3-64D D1-14 JH5 69.4 102.6 CACGCCTGTGTATTACTGTGTG CCACGGG

VH7-4-1 D6-19 JH2 45.5 18.9 TGTCCTCTATTACTGTGCGAGAGTCAGCC ATAGTAGTGGCTCCCCACTACTGGTACTTCGATCTCTGG

VH1-18 D3-3 JH6 45.5 112.9 CCGTGTATTACTGTGCGAGAGACAGACAGAC TACGATTTTTGGAGTGGTTATTAAAGTTGACTACTACTACGGTATGGACGTCTGG




Table S4: SHM and VH replacement in Ph-negative leukemia patients (n = 58). 2/58 cases positive for

VH replacement in non-ETV6-RUNX1 leukemic subgroups.

Table S4d: SHM and VH replacement leukemia patients with normal or undetermined karyotypes (n =26).

Cryptic RSSs are underlined.


Mut Freq

VH (x10-3

bp)

Mut Freq

JH (x10-3

bp) VH-N DH-N-JH

B192/98 Normal VH4-34 D2-2 JH4 12.6 333.3 CTGTGTATTACTGTGCGAGAGGCCCACCTGGATATTCGC TGTAGTAGTACCAGCTGCTATGGTTCGGGGAGTTATTATTTGACTACTGG

P902775/95 Normal VH3-33 JH6 0.0 277.8

Patient 1 n.d. VH7-81 D3-22 JH6 115.8 129.0 GAGGACACTGCCGGCAGGTCCAACCCA AGTGGTTATTACCCAGCCCTACTACTACTACATGGACGTCTGG

Patient 29 n.d. VH3-71 D1-26 JH5 27.4 19.6 AGGACACGGCCGTGTTCCGGTGGG GTAGGAACTACGTCGGGCCCCCATCCGGAGTGAAAACTGGTTCGACCCCTGG

Ber 10 n.d. VH1-8 JH4/JH5 38.2 228.6

VH1-8 34.7 0.0

VH1-8 JH4/JH5 34.7 58.8

VH1-8 JH4/ JH5 38.2 0.0

Ber 71 n.d. VH1-8 D2-15 JH6 0.0 32.3 CCGTGTATTACTGTGCGAGAGGGGGG ----ATTGTAGTGGTGGTAGCTGCTACTCGGGGGCCCCTCACTACTACTACTACTACATGGACGTCTGG

VH1-2 D2-15 JH6 0.0 64.5 CCGTGTATTACTGTGCGAGAGAGTTTCTAT GGATATTGTAGTGGTGGTAGCTGCTACTCGGGGGCCCCTCACTACTACTACTACTACATGGACGTCTGG

VH1-2 D2-15 JH6 0.0 32.3 CGGCCGTGTATTACTGTGCGAGCCTG GGGTATTGTAGTGGTGGTAGCTGCTACTCGGGGGCCCCTCACTACTACTACTACTACATGGACGTCTGG

VH1-2 D2-15 JH6 0.0 129.0 CCGTGTATTACTGTGCGAGAGA AGGATATTGTAGTGGTGGTAGCTGCTACTCGGGGGCCCCTCACTACTACTACTACATGGACGTCTGG

Ber 77 n.d. VH1-69 D6-19 JH4 0.0 41.7 CGGCCGTGTATTACTGTGCGAGCCGAC GTATAGCAGTGGCTAACCTCAACTTTGACTACTGG

VH3-30 3.5 0.0

VH1-18 D3-10 JH3 24.3 0.0 CCGTGTATTACTGTGCGAGAGACACGACCCC ATTACTATGGTTCGGGGAGAATTTTGATGCTTTTGATATCTGG

VH1-8 D3-3 JH4 0.0 125.0 CCGTGTATTACTGTGCGAGAGG TTTTCGGACAGCAGAGGACCTTGGCTACTGG

Ber 82 n.d. VH3-11 D3-22 JH6 0.0 32.3 CTGTGTATTACTGTGCGAGAGAGGGGGAGGGTTTTT GTATTACTATGATAGTAGTGGTTATTACTACCCCTTATTACTACTACTACTACGGTATGGACGTCTGG

VH3-13 D3-22 JH6 0.0 0.0 CTGTGTATTACTGTGCAAGAGAGGGTGATCGGGGGAGGGTTTTT GTATTACTATGATAGTAGTGGTTATTACTACCCCTTATTACTACTACTACTACGGTATGGACGTCTGG

VH3-13 D3-22 JH6 0.0 32.3 CTGTGTATTACTGTGCAAGAGAGGGTGATCGGGGGAGGGTTTTT GTATTACTATGATAGTAGTGGTTATTACTACCCCTTATTACTACTACTACTACGGTATGGACGTCTGG

Ber 100 n.d. VH1-18 D2-21 JH4 0.0 187.5 GCCGTGTATTACTGTGCGAGAGCCTCT GTGGTGACTGCTATGGGGTACTGG

Ber 101 n.d. VH1-45 D3-16 JH5 3.5 156.9 ACACAGCCATGTATTACTGTGCC TGGGGGGCGGTTCGACCCCTGG

VH3-30 D5-18 JH4 10.5 20.8 ACACGGCTGTGTATTACTGTGCCCCCTCCCCCCC CAGCTATGGTTACCTACTTTGACTACTGG

VH1-18 D3-22 JH3 48.6 120.0 CCATGTATAACTGTGCGAGAGAGCCGC GATATTAGTGCTTCTTACCGTTTGATATCTGG

Ber 107 n.d. VH3-23 D4-11 JH4 0.0 104.2 GCCGTATATTACTGTGCGAAAGGCTTGCCTTTGAAGAC TGACTTCAAAGATTGACTACTGG

VH1-2 D3-22 JH3 0.0 20.0 CCGTGTATTACTGTGCGAGAGAGCGATCCTTAGAAAG TTACTATGATAGTAGTGGTAATGCTTTTGATATCTGG

Ber 111 n.d. VH1-2 D5-18 JH4 0.0 145.8 CCGTGTATTACTGTGCGAGAGAA GGATACAGCTATGGTGCCTGG

VH3-9 D3-22 JH4 0.0 62.5 GGCCTTGTATTACTGTGCAAAACCTT TATTACTATGATAGTAGTGGTTATTAAAGGGTGCTTTGACTACTGG

Ber 117 n.d. VH1-69 D3-22 JH4 0.0 62.5 CACGGCCGTGTATTACTGTGCGGGGT ATAGTAGTGGTTATTACTCGGCCCCGGGGGACTTTGACTACTGG

VH1-8 D2-2 JH5 13.9 0.0 GCCGTGTATTACTGTGCGAGAGAACGACACTTT TACTGTAGTAGTCCCGGCTGCTATACGGGCGGCCCTCGTGCACAACTGGTTCGACCCCTGG

VH7-4-1 D3-22 JH4 24.3 187.5 GCCGTCTACTACTGTGCGAGAGTAAC TAGTAGTTACCTAGCCCTCTGG

VH1-69 D2-15 JH5 3.5 98.0 CCGTGTATTACTGTGCGAGAGAGCGCTGA GGTGGTAGCTGCTACTCACTGGTTCGACCCCTGG

Ber 127 n.d. VH1-69 D3-10 JH6 6.9 16.1 CCGTGTATTACTGTGCGAGAGAG ATGGTTCGGGGAGTTATTATCTACTGGGAACTACTACTACTACTACGGTATGGACGTCTGG

VH3-13 D4-17 JH6 3.5 32.3 GCTGTGTATTACTGTGCAAGAGCTTA TACGGTGACTACGGGGATTACTACTACTACTACGGTATGGACGTCTGG

VH1-18 D2-8 JH6 0.0 96.8 CGGCCGTGTATTACTGTGCGAGGG CTGGGGGTGGGCCATCACCCTACTACTACGGTATGGACGTCTGG

Ber 137 n.d. VH1-8 D6-19 JH6 0.0 161.3 CCGTGTATTACTGTGCGAGAGG GGTATAGCAGTGGCTGGTACCACATGGACGTCTGG

VH1-2 D3-22 JH6 0.0 209.7 CGGCCGTGTATTACTGTGCGAGTATCGGGG ATTACTATGATAGTAGTGGTTACGTATTAGACATGGACGTCTGG

VH1-2 D2-15 JH4 59.0 104.2 CCGTTTATTACTGTGCGAGAGATCCAC TAGTCTTGGCTTTGGCTACTGG

VH1-2 D3-10 JH4 3.5 145.8 CCGTGTATTACTGTGCGACAGAAAC TATTACTATGGTTCGCTACATGGACGTCTGG

VH1-2 D2-15 JH4 86.8 95.2 GCCATATTTTACTGTGCGACAGTGGAGGCGGTCGCAG TGTAGTGGTGGTAGCTGCTCAATCTCTACTACTACGGTATGGACGTCTGG

VH3-30 D3-10 JH5 6.9 39.2 CTGTGTATTACTGTGCGAAAGAAGGA GGTTCAGGGAGTTTTCTAACTGGTTCGACCCCTGG

VH1-69 D3-3 JH6 0.0 158.7 CCGTGTATTACTGTGCGAGAGAAGGTGTCGGAG ACGATTTTTGGAGTGGTTATTATGGGGCGGCGGGCTACTACTACGGTATGGACGTCTGG

VH1-2 D5-18 JH4 3.5 104.2 ACACGGCCGTGTATTACTGTGCCCTAGGGG GGATACAGCTATGGTACCCTTTTGACTACTGG

Ber 72 n.d. VH1-18 D3-3 JH4 0.0 83.3 GCCGTGTATTACTGTGCGAGAGTGGGACCGAGG GGAGGGGTTTACTTTGACTACTGG

VH6-1 D6-19 JH6 0.0 193.5 CGGCTGTGTATTACTGTGCAAGTGGGGAGGC GTATAGCAGTGGCTGGATATACGGTATGGACGTCTGG

VH3-15 D2-2 JH4 0.0 208.3 CCGTGTATTACTGTACCACAGA TTGTAGTAGTACCAGCTGCTATGCCCACTTGTAGACTGG

VH3-15 D2-2 JH4 0.0 166.7 CCGTGTATTACTGTACCACAGA TTGTAGTAGTACCAGCTGCTATGCCCACTTGTAGACTGG

Ber 75 n.d. VH1-NL1 D3-22 JH4 66.2 104.2 TCGTGTATTCCTGTGTGAGAGAG TACTATGATAGTAGTGGTTGTCTAGCCGGACTACTGG

VH3-30 D1-1 JH4 0.0 270.8 GCTGTGTATTACTGTGCGAAAGTA GGTAGAATAAGCGACCCCGGGGTCTGAGGCGGTACTGG

VH1-2 D3-22 JH2 49.8 113.2 CGGCCGTGCATTACTGTGCGAGGTGCAAAGCATG TGATAGAAGTGATTATCTTACGCGACCCCTATTACAACGACACGTACTTCGATCTCTGG

Ber 83 n.d. VH1-46 D5-24 JH4 0.0 83.3 CGGCCGTGTATTACTGTGCGAGGG GTAGAGATGGCTACAATTATAACTACTGG

VH3-53 D3-9 JH6 3.5 129.0 GCCGTGTATTACTGTGCGAGAGGGGGGG ATTACGATATTTTGACTGGTTATTATGCCCGTACCACTACTACGGTATGGACGTCTGG

VH1-2 D6-19 JH4 0.0 125.0 CCGTGTATTACTGTGCGAGAGATCCC ATAGCAGTGGCTGGTACTCCACCGACTACTGG

VH1-2 D2-2 JH4 0.0 0.0 GGCCGTGTATTACTGTGCGAGATCCATTTCT CTGCTAGACAAGTCGACCCATAAACTAACTACTTTGACTACTGG

Ber 94 n.d. VH1-2 D3-22 JH5 0.0 98.0 CCGTGTATTACTGTGCGAGAGAGGGGC TATTACTATGATAGTAGTGGTTATATCTAAGGGGGGTGGTTCGACCCCTGG

Ber 102 n.d. VH6-1 JH6 37.0 0.0 GGACAGGGGGGAACCTGGGGGT ATGGACGTCTGG

VH1-8 D2-15 JH5 3.5 98.0 CGGCCGTGTATTACTGTGCGAGTTGCCCCC GTGGTAGCTGCTACTTACAGGGGTTCGACCCCTGG

VH6-1 D3-16 JH4 0.0 130.4 GCTGTGTATTACTGTGCAAGAGCCGGAACCATTAAACGATATTTTGACTGGTTATTATAAA GTATTATGATTACATTTGGGGGAGTTATCGTTTCTCTGACTACTGG

Ber 103 n.d. VH1-45 D2-21 JH5 3.5 78.4 GCCATGTATTACTGTGCAAGATTTTT GTGACTGCCCCCAGGGGGTCTGGTTCGACCCCTGG

VH1-45 D2-21 JH5 6.9 78.4 GCCATGTATTACTGTGCAAGATTTTT GTGACTGCCCCCAGGGGGTCTGGTTCGACCCCTGG

Ber 113 n.d. VH1-2 D6-6 JH6 114.6 161.3 GGCCGTATATTACTGCGCGAGA AGCAGTTCGTCGACCATTTTCTACCACGGCATGGACGTCTGG

VH1-8 D1-26 JH6 0.0 145.2 GGCCGTGTATTACTGTGCGAGACCCGTCCTC AGTGGGAGCTACTTCTACGGTATGGACGTCTGG

VH3-15 D6-13 JH1 61.2 230.8 CAGCCGTCTATTACTGTACCACGTACAGGTTTGGGAAC ATAGCAGCAGCTCTCAGTGTAAAGAAGTGG

Ber 115 n.d. VH1-8 D3-9 JH5 3.5 98.0 GGACACGGCCGTGTATTACTGTTCCTTTCGGCGGAGAATTGG ATTACGATATTTTGACTGGTTATGTACTGGTTCGACCCCTGG

VH1-69 D1-1 JH6 52.1 193.5 GGCCGTGTATTACTGTGCGAGAT CACCTGGAGCGCAAGGGAACTACTACAATATGGACGTCTGG

VH1-18 D1-26 JH4 41.7 83.3 GGCCGTGTATTACTGTGCGAGATTAA GTTTAGTGGGAGTTTCCATTGACTACTGG

VH3-30 D1-26 JH5 0.0 235.3 ACGGCTGTGTATTACTGTGCGAACGACGC TACAGTGGGAGCTACTACGG

Ber 129 n.d. VH1-3 D6-13 JH1 0.0 134.6 CTGTGTATTACTGTGCGAGAGAGTG ATAGCAGCGGCTGGTACTACTTCCAGCACTGG

VH1-8 D2-2 JH5 41.7 39.2 CCGTGTATTATTGTGCGAGAGGGAGG TTGTACTAGTACCAGTTGCTAAGTCTACAGTCCGGGTATAGCAGTGGCTGGCCTGAACTGGTTCGACCCCTGG

Ber 131 n.d. VH6-1 D2-2 JH6 0.0 161.3 GCTGTGTATTACTGTGCAAGAGGGGCG TATTGTAGTAGTACCAGCTGCTATGATGGGTGGGGTACTACGGTATGGACGTCTGG

VH3-53 D6-19 JH6 0.0 177.4 ACACGGCCGTGTATTACTGTGCCCGCT TAGAAGGGGCCTACTACGGTATGGACGTCTGG

VH3-53 D6-19 JH6 0.0 145.2 ACACGGCCGTGTATTACTGTGCCCGCT TAGAAGGGGCCTACTACGGTATGGACGTCTGG

Ber 125 n.d. VH3-13 D3-9 JH5 0.0 39.2 CGGCTGTGTATTACTGTGCAAGTCGGGGGG GATATTTTGACTGGTTATTAAGGGGGAAACTGGTTCGACCCCTGG

VH1-69 D6-6 JH4 34.7 166.7 CCGTGTATTACTGTGCGAGAGAC GTAGAGCCCCTAGTAGGCGGGGGTCTGGACGTCTGG

VH1-69 D3-16 JH4 0.0 125.0 CCGTGTATTACTGTGCGAGAGAGGAGAGT TATTATGATTACGTTTGGGGGAGCCTATGGGCTTTTGACTACTGG


Table S5: Clonal Evolution in MLL-AF4 patientCell Allele 1 Allele 2

VH DH JH VH DH JH

Mut

Freq VH

(x10-3

bp)

Mut

Freq JH

(x10-3

bp) VH-N DH-N-JH

1 D3-15 JH6 VH4-31 D2-2 JH6-3 3.3 40.0 TGTGCGAGAGATGGGGTTTGAGC GGGTATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG




5 D3-15 JH6 VH1-2 D2-2 JH6-3 40.0 100.0 TGTGTTCTTCCCTAGGA ---TATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG





8 D3-15 JH6 VH3-13 D2-2 JH6-3 3.3 60.0 TGTGCAAGAGAGGGTTTGAGC GGGTATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG



11 D3-15 JH6 VH7-4 D2-2 JH6-3 20.0 60.0 TGTGCGAGAGGGTTTGAGC GGGTATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG





18 D3-15 JH6 VH2-5 D2-2 JH6-3 3.3 20.0 TGTGCACACCCCCCCGGGGGGGGTTTGAGC GGGTATTGTAGTAGTACCAGCTGCTACTACTACTACTACTTACATGGACGTCTGG









Cell Allele 1 Allele 2

VH DH JH VH DH JH

Mut

Freq VH

(x10-3

bp)

Mut

Freq JH

(x10-3

bp) VH-N DH-N-JH

27 Germline VH3-7 D3-22 JH6-2 3.3 20.0 TGTGCGAGAGACG TATTACTATGATAGTAGTGGTTATTACCAGGGTATTACTACTACTACTACTTACATGGACGTCTGG

28 Germline VH3-30 D3-22 JH6-2 3.3 0.0 TGTGCAAGAGATA TATTACTATGATAGTAGTGGTTATTACCAGGGTATTACTACTACTACTACTTACATGGACGTCTGG

29 Germline VH3-30 D3-22 JH6-2 10.0 0.0 TGTGCGAG TATTACTATGATAGTAGTGGTTATTACCAGGGTATTACTACTACTACTACTTACATGGACGTCTGG



32 D3-15 JH6 VH6-1 D3-22 JH6-2 0.0 0.0 TGTGCAAGAGATCCGTATAGCAGTGGCTGG TATTACTATGATAGTAGTGGTTATTACCAGGGTATTACTACTACTACTACTTACATGGACGTCTGG







25

Supplementary Table 6: Characteristics of ETV6-RUNX1 patient samples used for IGH sequencing studies in

Supplementary Table 3.

Ref No.

ETV6-

RUNX1

sample

Diagnosis

age

(months)

BM

blasts

(%)

ETV6-RUNX1 breakpoint

Sample used

for IGH

sequencing

51 REH 180 100

46(44-47)<2n>X, -X, +16, del(3)(p22),

t(4;12;21;16)(q32;p13;q22;q24.3)-inv(12)(p13q22),

t(5;12)(q31-q32;p12), der(16)t(16;21)(q24.3;q22)

sideline with inv(5)der(5)(p15q31),+18

carries t(12;21) and del(12) producing respective

ETV6-RUNX1 fusion and deletion of residual ETV6

Cell line

LAX13 96 80 47,XX,t(12;15)(p13;q15),+21[6]/46,XX[14] Xenograft

(Passage 3)

23 PD4009a 44 96 chr12:g.o12024743_chr21:36311593bkpt

chr12:g.12024661_chr21:o36311621bkpt Diagnostic

23 PD4020a 49 85 Diagnostic

23 PD4021a 58 80 chr12:g.o12031874_chr21:36356100bkpt Diagnostic



23 PD3952a 30 63 chr12:g.12035173_chr21:o36347548bkpt Diagnostic





23 PD4036a 30 96 chr12:g.o12034799_chr21:36420621bkpt Diagnostic



43 920527-4278 n.d. n.d. 46,XY,t(2;14)(q23;q32); t(12;21) Diagnostic


26

Supplementary Table 7: Characteristics of patient samples used for IGH sequencing studies in

Supplementary Table 4.

Ref No. Leukemia

sample

Diagnosis

age

(months)

Karyotype Cytogenetics Sample used for

IGH sequencing

43 aP902486/96 n.d. 46,XY,t(4;11)(q21;q23) MLL-AF4 Diagnostic

43 aP901941/96 n.d. 46,XX,t(11;19)(q23;p13) MLL-ENL Diagnostic

43 aP902032/95 n.d. 46,XX,t(1;19)(q23;p13) TCF3-PBX1 Diagnostic

43 aP900469/95 n.d. 46,XY,t(1;15)(q11;p11),t(1;19)(q2

3;p11),del(6)(q21) TCF3-PBX1 Diagnostic

43 bPatient 20 144 46,XY,del(2)(p2),t(8;14)(q24;q32) MYC-IGH Diagnostic

43 aB273/98 n.d. 55,XY,+X,+4,+5,+6,-

10,+14,+18,+21,inc[4] Hyperdiploid Diagnostic

43 aB2033/97

n.d. 54-

55,XX,+X,+4,+6,+?9,+14,+15,+17

,+21

Hyperdiploid Diagnostic

43 aP90100/96 n.d. 51-55,XX,+6,+14,+21,+22,inc Hyperdiploid Diagnostic

43 aP901756/96 n.d. 55,XX,+6,+14,+18,+19,+21,+21,in

c Hyperdiploid Diagnostic

43 aB960/97jfr12

24

n.d. 52-

55,XY,+X,+4,+6,+?14,+17,+21,+2

1


43 ac901275/94 n.d. 55,XX,+X,+3,+6,+10,+14,+18,inc Hyperdiploid Diagnostic

43 ac27012-91 n.d. 56,XX,+X,+X,+3,+4,+9,+10,+13+

18,+19,+21 Hyperdiploid Diagnostic

43 aP901348/95

n.d. 53-

55,XX,+X,+4,+5,+6,+14,+17,+21,

+21,+mar


43 aP902781/96 n.d. 51-

54,XX,+X,+6,+8,+14,+17,+18,inc Hyperdiploid Diagnostic

43 aB675/97

n.d. 50-

56,+X,+4,+6,+10,+17,+18,+18,+2

1,inc


43 aP90131/96 n.d. 57-61,XX,+2,+3,+6,+8,+10,+14,-

16,-17+19,+20,+21,+22,inc Hyperdiploid Diagnostic


27

43 aP902300/95

n.d. 54,XY,+X,+4,del(10)(p11),+21,+2

1,+4mar/52-

55,XY,+X,+4,+13,+19,+21,+4mar


43 aP900978/95 n.d. 54,XX,+X,+7,+18,+21,+4mar Hyperdiploid Diagnostic

43 ac900162/94

n.d. 59,XXY,+Y,-1,-2,-3,-7,-8,-9,-10,-

11,-12,-13,-16,-19,-20,+21,-

22,+2mar


52 bPatient 5 36 High hyperdiploid clone Hyperdiploid Diagnostic

52 bPatient 10 48 High hyperdiploid clone with

del(6)(q2) Hyperdiploid Diagnostic

52 bPatient 18 84 High hyperdiploid clone with

trisomy 14 Hyperdiploid Diagnostic

43 ahösten-95 n.d. 46,XX,del(1)(q?) Other Diagnostic

43 aB1057/97

n.d. 47,XY,t(1;12)(p32;p12),add(6)(q2

1),t(7;7)(q22;q36),add(11)(q21),m

ar,inc

Other Diagnostic

43 aP900667/96 n.d. 46,XY,t(3;12)(p11;p13)[15]/46,XY

[23] Other Diagnostic

43 ac900891/94 n.d. 47-48,XX,+X,+?21 Other Diagnostic

43 ac900024/94 n.d. 46,XY,der(12)t(12;14)(p12;q11) Other Diagnostic

43 aP901693/95 n.d. 46,XY,del(9)(p11),del(17)(p11)[20

] Other Diagnostic

43 ac26170/87 n.d. 46,XX,t(1;12)(p31;?q)),del(11)(q1

4) Other Diagnostic

52 bPatient 3 24 46,XY,t(9;15)(p2;q1) Other Diagnostic

52 bPatient 17 72 46,XX,del(6)(q2?q5) Other Diagnostic

52 bPatient 23 168 46,XY,t(17;22)(q25;q11) Other Diagnostic

43 aP902775/95 n.d. 46,XX Normal Diagnostic

43 aB192/98 n.d. 46,XX Normal Diagnostic

52 bPatient 1 24 n.d. N/A Diagnostic

52 bPatient 29 252 n.d. N/A Diagnostic

Ber 10 n.d. n.d. N/A Diagnostic




28





















29

Supplementary Table 8: Antibodies used in this study.

Immunoblot

Antigen Clone ID Specificity Dilution Source

AID L7E7 Mouse/Human 1:1000 Cell Signaling Technology

β-Actin C4 Mouse/Human 1:10,000 Santa Cruz Biotechnology

FOXO4 9472 Mouse/Human 1:1000 Cell Signaling Technology

RAG-1 Mouse/Human 1:1000 A kind gift from David

Schatz Laboratory

STAT5 3H7 Mouse/Human 1:1000 Cell Signaling Technology

Phospho-Y694 STAT5 14H2 Mouse/Human 1:1000 Cell Signaling Technology

RAG-1 EPRAGR1 Mouse 1:1000 Abcam

RAG-2 EPRAGR239 Mouse 1:1000 Abcam

Flow cytometry

Surface Antigen Clone ID Specificity Source

CD19 1D3 Mouse BD

IL-7Rα A7R34 Mouse eBioscience

Igκ Light Chain 187.1 Mouse BD

IL-2Rα (CD25) 7D4 Mouse BD

B220 RA3-6B2 Mouse/Human BD

c-Kit 2B8 Mouse BD

IgM R6-60.2 Mouse BD

CD19 HIB19 Human Biolegend

Igκ Light Chain G20-193 Human BD

Igλ Light Chain JDC-12 Human BD


30

Supplementary Table 9: Sequences of oligonucleotide primers used in this study.

Quantitative RT-PCR

Hprt_F 5’-GGGGGCTATAAGTTCTTTGC-3’

Hprt_R 5’-TCCAACACTTCGAGAGGTCC-3’

Rag2_F 5’-GCAGATGGTAACAGTGGGTC-3’

Rag2_R 5’-ATTGCAGGCTTCAGTTTGAG-3’

Rag1_F 5’-TAACAACCAAGCTGCAGACA-3’

Rag1_R 5’-CCTCTGAGGAATCCTTCTCC-3’

Aicda_F 5’- AAATGTCCGCTGGGCCAA-3’

Aicda_R 5’- CATCGACTTCGTACAAGGG-3’

AICDA F 5’–TCCTTTTCACTGGACTTTGG–3’

AICDA R 5’–GACTGAGGTTGGGGTTCC–3’

COX6B F 5’-AACTACAAGACCGCCCCTTT-3’

COX6B R 5’-GCAGCCAGTTCAGATCTTCC-3’

Somatic hypermutation of human VH region

Forward primer mix (VH mix)

VH1_F 5’-CAGTCTGGGGCTGAGGTGAAGA-3’

VH2_F 5’-GTCCTRCGCTGGTGAAACCCACACA-3’

VH3_F 5’-GGGGTCCCTGAGACTCTCCTGTGCAG-3’

VH4_F 5’-GACCCTGTCCCTCACCTGCRCTGTC-3’

VH5_F 5’-AAAAAGCCCGGGGAGTCTCTGARGA-3’

VH6_F 5’-ACCTGTGCCATCTCCGGGGACAGTG-3’

Reverse primer mix (JH mix)

3’JH1.2.4.5_R 5’-ACCTGAGGAGACGGTGACCAGGGT-3’

3’JH3_R 5’-ACCTGAAGAGACGGTGACCATTGT-3’

3’JH6_R 5’-ACCTGAGGAGACGGTGACCGTGGT-3’


31

Supplementary Table 10a: Retroviral vectors used in the study.

Constitutive expression Inducible activation

MSCV Cre-IRES-GFP

MSCV Blnk-IRES-GFP35 MSCV-ERT2-Puro

MSCV BlnkY96F-IRES-GFP35 MSCV-Cre-ERT2-Puro53 (Addgene)

MSCV ETV6-RUNX1-IRES-GFP

MSCV ETV6-RUNX1 ∆RHD-IRES-GFP

MSCV Foxo1CA-IRES-GFP35

MSCV IRES-GFP35

MSCV Foxo3aCA IRES-CD9054

MSCV IRES-CD9054

Supplementary Table 10b: Lentiviral vectors used in the study.

Constitutive expression

pCL6-Aicda-IRES-iRFP670-wo

pCL6-Rag1-IRES-eGFP-wo

pCL6-Rag2-IRES-dsRedExpress2-wo

Transfections of the above retroviral and lentiviral constructs were performed as discussed in the online methods

section.


Documents

precursor ALL. · Nature Immunology: doi:10.1038/ni.3160 Supplementary Figure 1 High levels of AICDA and RAG1 mRNA at the time of diagnosis predict poor clinical outcomes for patients