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Letters to the Editor
CD
Figure 3. Loss of Ikaros in BCR-ABL1 transformed B1 progenitors of adult and fetal origin results in aggressive leukemia. (A) Bone marrow (BM) B1 progenitor cells were retrovirally transduced with BCR-ABL1-IRES-TdTomato (BCR-ABL) or the empty vector (pMITo). NSG mice were intraveously injected with an equivalent of 104 TdTo+ cells as indicated. Recipient BM and spleen cells were analyzed by flow cytometry for CD19+TdTo+ cells when the mice showed signs of sickness ("sick" conditional knockout [cKO] BCR-ABL=29 days, sick wild-type [WT] BCR-ABL=56 days after transplantation) or at the corresponding "healthy" timepoints. The graph on the right shows the survival of NSG mice transplanted with cKO B1 progenitors pMITo+ (control), WT B1 progenitors BCR-ABL1+ (WT) or cKO B1 pro- genitors BCR-ABL1+ (cKO). The graph represents the results of 4 independent experiments with 16 WT, 14 cKO and 3 control mice. MS: median survival. (B) FL B1 progenitors were transduced with a BCR-ABL1-IRES-TdTomato construct. NSG mice were injected with an equivalent of 104 TdTo+ cells. BM and spleen cells from sick mice were analyzed for CD19+TdTo+ cells. The survival curve of 2 independent experiments with 8 WT and 7 cKO mice is shown on the right. MS: medi- an survival. Ikf/fMb-1-Cre and NSG mice were housed in specific pathogen-free (SPF) conditions or kept in single-ventilated cages. All experiments were approved (APAFIS reference #9742-2017042718317841 v6T). (C) Vh usage of pediatric (upper graphs) and adult (lower graphs) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) samples were analyzed according to Griffin et al11 and classified into B1-like (B1) and non-B1-like (nonB1), and further divided into samples with normal or deleted IKZF1. Numbers in the bars represent the % patients with the indicated IKZF1 status. Numbers at the bottom of the bars indicate the numbers of patients analyzed. (D) Pediatric BCP-ALL samples with KMT2A rearrangements were grouped according to IKZF1 status and displayed as a function of patient age. Pediatric data were obtained from the Genetics Department of the Robert Debré Hospital (Paris, France), and adult data from the Genome and Cancer Department of the Saint Louis Hospital (Paris, France). Informed consent was obtained according to the Helsinki Declaration.
analyzed them for IKZF1 deletions (gross chromosomal deletions of chromosome 7, -7p, intragenic deletions) (Figure 3C). In both children and adult samples, the ratio of mutant to functional IKZF1 samples was similar between B1- and non-B1-like BCP-ALL.11 Since IKZF1 alterations are frequent in BCR-ABL1+ BCP-ALL, we checked if BCR-ABL1+ leukemias were enriched in B1- like cases. Interestingly, there was a significant enrich- ment of BCR-ABL1+ cases within the B1-like group when compared with the non-B1 group in both pediatric (21% vs. 13.4%) and adult (67% vs. 45%) patients (P<0.05 in both cases; hypergeometric test). Among the pediatric samples, 35 came from infants <1 year of age, and most (34/35) contained KMT2A rearrangements, considered to occur in utero;12 four of these samples exhibited an IKZF1 deletion (Figure 3D). Thus, our results in mice and humans showed that Ikaros loss is associated with BCP-ALL development in B cells of both fetal and adult origin.
In conclusion, our study indicates that murine B1-cell development can be divided into phenotypically discrete stages that resemble the Hardy fractions6A-C' currently used to evaluate B2-cell differentiation. These newly identified populations will allow further investigation into the requirements of B1-cell development and the factors involved. We show that Ikaros is required at the fraction C' stage of B1-cell differentiation as observed for B2 progenitors, suggesting similar regulation of gene expression, particularly in antagonizing genes regulated by IL-7/STAT5.8 Our results also suggest that Ikaros functions as a tumor suppressor by repressing STAT5 activity in B-cell progenitors of fetal and adult origin. Whether the B1-cell equivalent exists in humans is still unclear. However, human fetal B cells and murine B1
progenitors are alike in some ways. Infant and pediatric BCP-ALL cells often co-express lymphoid and myeloid markers;13 similarly, murine B1 cells have been reported to be bipotent and can develop into both B cells and macrophages.14 While our results do not address the validity of human B1 cells, we found IKZF1 alterations in both B1- and non-B1-like leukemias of children and adults, including those with fetus-associated ETV6- RUNX1 translocations and KMT2A rearrangements. Thus, Ikaros loss-of-function is associated with BCP-ALL development in patients of all ages. It will be interesting in future studies to determine if B1- and non-B1-like cases are associated with different outcomes, and if this parameter could improve the risk classification of BCP- ALL patients.
Célestine Simand,1,2,3,4,5 Céline Keime,1,2,3,4 Aurélie Caye,6,7 Chloé Arfeuille,6,7Marie Passet,8 Rathana Kim,8 Hélène Cavé,6,7 Emmanuelle Clappier,8 Philippe Kastner,1,2,3,4,9 Susan Chan1,2,3,4# and Beate Heizmann1,2,3,4#
1Institut de Génétique et de Biologie Moléculaire et Cellulaire
(IGBMC), Illkirch; 2Institut National de la Santé et de la Recherche
Médicale, INSERM U1258, Illkirch; 3Centre National de la
Recherche Scientifique, CNRS UMR7104, Illkirch; 4Université de
Strasbourg, Illkirch; 5Service d’Hématologie, Institut de Cancérologie
Strasbourg Europe, ICANS, Strasbourg; 6Département de Génétique,
Assistance Publique des Hôpitaux de Paris (AP-HP), Hôpital Robert
7
Debré, Paris; Institut de Recherche Saint-Louis, INSERM
UMR_S1131, Université de Paris, Paris; 8Université de Paris, Laboratory of Hematology, AP-HP, Hôpital Saint-Louis, Paris and 9Faculté de Médecine, Université de Strasbourg, Strasbourg, France.
#SC and BH contributed equally as co-senior authors
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