Childhood acute lymphoblastic leukemia (ALL) can often be traced to a pre-leukemic clone carrying a prenatal genetic lesion. Postnatally acquired mutations then drive clonal evolution toward overt leukemia. The enzymes RAG1-RAG2 and AID, which diversify immunoglobulin-encoding genes, are strictly segregated in developing cells during B lymphopoiesis and peripheral mature B cells, respectively. Here we identified small pre-BII cells as a natural subset with increased genetic vulnerability owing to concurrent activation of these enzymes. Consistent with epidemiological findings on childhood ALL etiology, susceptibility to genetic lesions during B lymphopoiesis at the transition from the large pre-BII cell stage to the small pre-BII cell stage was exacerbated by abnormal cytokine signaling and repetitive inflammatory stimuli. We demonstrated that AID and RAG1-RAG2 drove leukemic clonal evolution with repeated exposure to inflammatory stimuli, paralleling chronic infections in childhood.
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We thank J.L. Wiemels and C. Gawad for encouragement and critical discussions; D.B. Kohn (University of California, Los Angeles) for the envelope and packaging vectors for lentivirus and retrovirus productions; and H. Hanenberg (Indiana University) for the plasmid pCL6-IRES-eGFP-wo. Supported by the National Cancer Institute of the US National Institutes of Health (R01CA137060, R01CA139032, R01CA157644, R01CA169458 and R01CA172558 to M.M.), the Leukemia and Lymphoma Society (1479-11, 6132-09, 6097-10 and 6221-12 to M.M.), the William Lawrence and Blanche Hughes Foundation, the California Institute for Regenerative Medicine (TR2-01816 to M.M.), Leukaemia, Lymphoma Research UK (M.F.G. and A.F.), Cancer Research UK (CRUK 18131 to M.M.) and The Wellcome Trust (WT105104/Z/14/Z to M.F.G. and WT101880AIA to M.M.).
The authors declare no competing financial interests.
Integrated supplementary information
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.
Supplementary Figure 2 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.).
Supplementary Figure 3 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.
Supplementary Figure 4 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.
Supplementary Figure 5 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.
Supplementary Figure 6 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 either transduced 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 sorted into 96well plates using a 488nm(525/50), 640nm(670/30), 561nm(582/15) and 355nm(450/50) configuration on an BD AriaII Sorter. 3D graphics were generated with WinList (Verity Software House).
Supplementary Figure 7 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).
Supplementary Figure 8 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 spleens were 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 mice that were injected with Aicda+/+ Rag1+/+ pre-B cells after repetitive stimulation with LPS and IL-7 withdrawal.
Supplementary Figure 9 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 by immunohistochemistry and leukemic infiltrates in spleen (top) and liver were visualized. H&E staining and immunohistochemistry 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.
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Swaminathan, S., Klemm, L., Park, E. et al. Mechanisms of clonal evolution in childhood acute lymphoblastic leukemia. Nat Immunol 16, 766–774 (2015). https://doi.org/10.1038/ni.3160
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