An instructive role for Interleukin-7 receptor α in the development of human B-cell precursor leukemia

Abstract

Kinase signaling fuels growth of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Yet its role in leukemia initiation is unclear and has not been shown in primary human hematopoietic cells. We previously described activating mutations in interleukin-7 receptor alpha (IL7RA) in poor-prognosis "ph-like" BCP-ALL. Here we show that expression of activated mutant IL7RA in human CD34⁺ hematopoietic stem and progenitor cells induces a preleukemic state in transplanted immunodeficient NOD/LtSz-scid IL2Rγ^null mice, characterized by persistence of self-renewing Pro-B cells with non-productive V(D)J gene rearrangements. Preleukemic CD34⁺CD10^highCD19⁺ cells evolve into BCP-ALL with spontaneously acquired Cyclin Dependent Kinase Inhibitor 2 A (CDKN2A) deletions, as commonly observed in primary human BCP-ALL. CRISPR mediated gene silencing of CDKN2A in primary human CD34⁺ cells transduced with activated IL7RA results in robust development of BCP-ALLs in-vivo. Thus, we demonstrate that constitutive activation of IL7RA can initiate preleukemia in primary human hematopoietic progenitors and cooperates with CDKN2A silencing in progression into BCP-ALL.

Fig. 1. CRLF2/IL7RA transduction alters B-lineage differentiation of human CB CD34⁺ progenitors transplanted in immune-deficient mice.

a B-lineage differentiation to immature/naive B cells (sIgM + ) of human CB CD34⁺ cells from the spleen (a) and BM (b) of engrafted mice expressing GFP (BB) [n = 20 (a), n = 21 (b)], CRLF2-GFP [n = 9 (a), n = 9 (b)], CRLF2-IL7RAwt [n = 10 (a), n = 10 (b), CRLF2-IL7RAins [n = 14 (a), n = 16 (b)], IL7RAwt-GFP [n = 9 (a), n = 11 (b)] and IL7RAins-GFP [n = 4 (a) n = 14 9 (b)]. Dot plots show sample scatter with mean +/− SEM. Each dot represents analysis of single mouse. Statistical analyses were performed using Kruskal–Wallis nonparametric test (Gaussian approximation) (a) P < 0.0001 Kruskal–Wallis statistic = 31.8 (b) P < 0.0001 Kruskal–Wallis statistic = 38.04. Gray linkers indicate statistically significant difference (*P < 0.05, **P < 0.01, ***P < 0.001) between groups in Dunn’s post hoc analysis significance level α = 0.05. Gating strategy Supplementary Fig. 3. c Mass cytometery analysis of human cells from BM of engrafted mice. Points represent mean values (BB n = 3, CRLF2-IL7RAins n = 3, IL7RAins n = 4). Statistical analysis was done by two-way ANOVA followed by Tukey test for multiple comparison corrections. Individual variances were computed for each B-cell subgroup with CI of 95% (α = 0.05). The asterisks indicate statistically significant difference compared with the backbone group (*P < 0.05, **P < 0.01, ****P < 0.0001). Definition of each B-cell subgroup is detailed in Supplementary Fig. 4. d, e V(D)J rearrangement analysis of CD10⁺ and CD19⁺ BB/CRLF2-IL7RAins-transduced cells sorted from BM of transplanted mice. Each dot represent a value of a single sample. lines represent mean +/− SEM of BB (n = 3) and CRLF2-IL7RAins (n = 3). Gray linkers indicate statistically significant difference (*P < 0.05, **P < 0.01, ***P < 0.001) between groups. Statistical analyses were performed using two-tailed t test. d Dot plot representing fraction of DJ rearranged of the total rearranged IgH loci in transduced cells P = 0.0035. e Dot plot representing ratio of nonproductive to total rearrangement in transduced cells P = 0.0006. Source data are provided as a Source Data file.

Fig. 2. Enhanced CD34⁺CD10⁺ expression and self-renewal of IL7RA-activated cells.

a Relative CD10⁺CD34⁺ population of engrafted transduced human CD19⁺ cells in BM (BB n = 11, CRLF2-IL7RAwt n = 4, CRLF2-IL7RAins n = 5, IL7RAwt-GFP n = 10, IL7RAins-GFP n = 14). b Flow cytometry immunophenotyping of engrafted backbone and CRLF2-IL7RAins or IL7RAins-transduced cells. Samples in the same row are from the same CB batch. Arrows indicate that the gated population was analyzed in the following scatters. Gating strategy for (a), (b) in Supplementary Fig. 3. c Percentage of human cells in BM of secondary recipient mice that were transplanted with BM cells of primary engrafted mice (BB n = 13, CRLF2-IL7RAwt n = 3, CRLF2-IL7RAins n = 8, IL7RAwt-GFP n = 9, IL7RAins-GFP n = 10—six points with a value of zero were out of the logarithmic graph range, but were included in the mean and SEM calculations). a, c Dot plots depict sample scatter with mean + /− SEM. Each dot represents the analysis of a single mouse. Gray linkers indicate a statistically significant difference (*P < 0.05) between groups. Statistical analyses were performed using Kruskal–Wallis nonparametric one-way ANOVA test with Dunn’s post hoc analysis. Source data are provided as a Source Data file.

Fig. 3. Secondary transplantation of IL7RA-activated human hematopoietic progenitors result in the development of clonal B-cell precursor leukemia.

a Flow cytometer scatter plot of human-engrafted cells in BM of the leukemic mouse. b Bar-graph of V–J rearrangements in leukemic population. The bars represent counts in the sequenced library of B-cell receptor rearrangements. Source data are provided as a Source Data file. c Genomic SNP array analysis of leukemic cells (Leukemia) and of BB-transduced engrafted cells from the corresponding cord blood (representing germline).

Fig. 4. Philadelphia-like ALL gene signature in bulk and scRNAseq analyses of activated IL7RA-engrafted cells.

a Scheme of sample acquisition for scRNAseq. b Transcriptome correlation t-SNE map after 10X scRNAseq. c Venn diagram of differentially expressed genes in bulk analysis of CD10⁺CD19⁺ and CD19⁺CD10^high cells of the preleukemic mouse and leukemia sample vs BB control sample Source data are provided as a Source Data file. d Relative expression of selected genes displayed on t-SNE map. e GSEA enrichment plot of preleukemic cells, leukemic cells and CRLF2-IL7RAins over BB differentially expressed genes aligned to Ph-like vs non Ph-like ranked gene list (f) AUCell analysis for single-cell expression of Ph-like (Ph) gene set projected on t-SNE map.

Fig. 5. CDKN2A disruption cooperates with IL7Rins for full leukemic transformation CD34 + human cord blood (CB) progenitors.

a Flow cytometer scatter plot of human-engrafted cells in BM of untransduced CB and leukemic mice. b Sanger sequencing electropherogram of gDNA from CD45⁺ cells of leukemic mice surrounding guide 2 targeting CDKN2A, demonstrating disruption of the locus in leukemic cells. c Scheme of leukemia development after aberrant activation of IL7RA.