Mutant IL7R collaborates with MYC to induce T-cell acute lymphoblastic leukemia

Abstract

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive pediatric cancer. Amongst the wide array of driver mutations, 10% of T-ALL patients display gain-of-function mutations in the IL-7 receptor α chain (IL-7Rα, encoded by IL7R), which occur in different molecular subtypes of this disease. However, it is still unclear whether IL-7R mutational activation is sufficient to transform T-cell precursors. Also, which genes cooperate with IL7R to drive leukemogenesis remain poorly defined. Here, we demonstrate that mutant IL7R alone is capable of inducing T-ALL with long-latency in stable transgenic zebrafish and transformation is associated with MYC transcriptional activation. Additionally, we find that mutant IL7R collaborates with Myc to induce early onset T-ALL in transgenic zebrafish, supporting a model where these pathways collaborate to drive leukemogenesis. T-ALLs co-expressing mutant IL7R and Myc activate STAT5 and AKT pathways, harbor reduced numbers of apoptotic cells and remake tumors in transplanted zebrafish faster than T-ALLs expressing Myc alone. Moreover, limiting-dilution cell transplantation experiments reveal that activated IL-7R signaling increases the overall frequency of leukemia propagating cells. Our work highlights a synergy between mutant IL7R and Myc in inducing T-ALL and demonstrates that mutant IL7R enriches for leukemia propagating potential.

Fig. 1. IL7R mutational activation alone drives T-ALL in zebrafish.

a Stable transgenic rag2:RFP and rag2:IL7R^mut2-tdTomato zebrafish were followed for disease onset and progression. Representative images of stable transgenic zebrafish at 17 weeks of life. Panels are merged fluorescent and brightfield images. Scale bar, 2 mm. b Kaplan–Meier analysis of disease progression in stable transgenic zebrafish (Gehan-Breslow-Wilcoxon statistic). Number of animals analyzed per genotype is shown in parenthesis. c May-Grünwald and Wright-Giemsa stained cytospins of kidney marrow from wild-type fish and bulk leukemias of rag2:IL7R^mut2-tdTomato fish (left panels); Scale bar, 50 µm. Histological analysis of thymic cells from wild-type fish (n = 4) and primary T-ALLs (n ≥ 6); Hematoxylin and eosin-stained sections juxtaposed to immunohistochemistry for TUNEL (right panels). Arrowheads denote examples of positively stained cells. Scale bar equals 10 µm. Percent positive cells ± SEM are shown within each image panel. Asterisks denote significant differences as assessed by Student’s t test. d Immunoblot analysis of phosphorylated protein levels in normal rag2:RFP thymocytes and bulk leukemias or FACS-sorted T-ALL cells from stable transgenic animals (n = 8).

Fig. 2. Transcriptomic characterization of mutant IL7R-derived leukemias.

a Principal component analysis (PCA) plot of gene expression profiles from RNA sequencing of different zebrafish leukemias and control samples. b Heatmap representation showing expression of well-known T-, B- and Myeloid/NKL-cell associated genes, as well as common STAT5 target genes (adj. P < 0.05). WKM, whole kidney marrow. c TCR-β gene rearrangements in T-ALLs from stable mutant IL7R zebrafish compared with normal thymocytes from control zebrafish. Shown as dotplots and boxplots are the number of clonotypes of the TRB locus and the equitability value per sample, both based on productive rearrangements. Higher number of clonotypes indicates higher polyclonality. Higher equitability means the relative frequency of the different clonotypes in a given sample is more balanced, whereas a lower equitability value indicates unbalanced frequencies (i.e. one or a few clones predominate over the others).

Fig. 3. Mutant IL7R collaborates with Myc to accelerate T-ALL onset.

a Tu/AB-strain fish injected at the one-cell stage with either rag2:Myc alone or with rag2:IL7R^mut1 or rag2:IL7R^mut2. Animals were also co-injected with rag2:mCherry to visualize leukemia onset and progression. Representative images of transgenic mosaic zebrafish at 28 dpf; Panels are merged fluorescent and brightfield images; Scale bar, 1 mm. b Kaplan−Meier analysis (Gehan-Breslow-Wilcoxon test). Number of animals analyzed per genotype is shown in parenthesis. Red dots denote fish that developed leukemia from rag2:Myc + rag2:IL7R^mut1 injected fish, whereas black dots show leukemias developing in rag2:Myc + rag2:IL7R^mut2 fish. c May-Grünwald and Wright-Giemsa stained cytospins showing lymphoblast morphology (n ≥ 2 leukemias/genotype analyzed); Scale bar, 50 µm. Histological analysis of primary T-ALLs (n > 3 leukemias/genotype analyzed); Hematoxylin and eosin-stained sections juxtaposed to immunohistochemistry for TUNEL; Arrowheads denote examples of positively stained cells; Scale bar, 10 µm. Percent positive cells ± SEM are shown within each image panel. Asterisks denote significant differences between Myc and Myc + IL7Rmut leukemias as assessed by Student’s t test. d Immunoblot analysis of phosphorylated protein levels in bulk leukemias or FACS-sorted T-ALL cells (n ≥ 3/genotype).

Fig. 4. Myc + IL7R^mut induced leukemias display IL-7R-mediated signaling upregulation and are polyclonal.

a Heatmap representation showing expression of well-known T- and B-cell associated genes, as well as common STAT5 target genes (adj. P < 0.05). b Transcriptome data integration and gene set enrichment analysis show a significant enrichment of the IL-2/STAT5 signaling hallmark gene set in Myc + IL7R^mut derived leukemias when compared with Myc derived leukemias. c TCR-β gene rearrangements in Myc + IL7R^mut vs Myc derived T-ALLs. Shown as dotplots and boxplots are the number of clonotypes of the TRB locus and the equitability value per sample, both based on productive rearrangements.

Fig. 5. Mutant IL7R increases leukemia propagating potential in Myc-induced leukemias.

a CG1-strain fish injected at the one-cell stage with either rag2:Myc alone or with rag2:IL7R^mut2. Animals were also co-injected with rag2:mCherry or rag2:GFP to visualize leukemia onset and progression, respectively. Representative images of transgenic mosaic zebrafish at 44 and 39 dpf, respectively; Scale bar, 1 mm. b Kaplan–Meier analysis of disease progression (Gehan-Breslow-Wilcoxon test). Number of animals analyzed per genotype is shown in parenthesis. c Leukemia propagating cell (LPC) frequency was assessed using limiting dilution cell transplantation analysis and calculated using the ELDA software. Graph showing LPC frequency within Myc and Myc + IL7R^mut2 induced primary T-ALL. Each point represents a distinct primary leukemia generated in this manuscript (filled) and compared with LPC frequency from [38] and [39], denoted by X (Mann–Whitney test). In total, 16 of Myc-induced and 5 Myc + IL7R^mut2 T-ALLs were included in this analysis.