Germline ETV6 mutation promotes inflammation and disrupts lymphoid development of early hematopoietic progenitors

Abstract

Germline mutations in ETV6 are associated with a syndrome of thrombocytopenia and leukemia predisposition, and ETV6 is among the most commonly mutated genes in leukemias, especially childhood B-cell acute lymphoblastic leukemia. However, the mechanisms underlying disease caused by ETV6 dysfunction are poorly understood. To address these gaps in knowledge, using CRISPR/Cas9, we developed a mouse model of the most common recurrent, disease-causing germline mutation in ETV6. We found defects in hematopoiesis related primarily to abnormalities of the multipotent progenitor population 4 (MPP4) subset of hematopoietic progenitor cells and evidence of sterile inflammation. Expression of ETV6 in Ba/F3 cells altered the expression of several cytokines, some of which were also detected at higher levels in the bone marrow of the mice with Etv6 mutation. Among these, interleukin-18 and interleukin-13 abrogated B-cell development of sorted MPP4 cells, but not common lymphoid progenitors, suggesting that inflammation contributes to abnormal hematopoiesis by impairing lymphoid development. These data, along with those from humans, support a model in which ETV6 dysfunction promotes inflammation, which adversely affects thrombopoiesis and promotes leukemogenesis.

Figure 1.. Mice with h.P214L-ETV6 have impaired hematopoiesis.

A. Etv6^wt/wt (WT), Etv6^wt/mt (HT), and Etv6^mt/mt (HO) mice were treated with 5FU (150 mg/kg IP). Peripheral blood was periodically analyzed with complete blood counts and flow cytometry. Numbers of B220⁺ and Gr1⁺ cells in the peripheral blood are demonstrated. (n=4 per genotype from a single experiment; *P<0.05, **P<0.01; Mixed-effects model of repeated measures with Dunnett correction for multiple comparisons.). B, C, D. Whole BM from WT, HT, and HO mice was serially transplanted into lethally irradiated Rag1^−/− recipients. Peripheral blood was analyzed by flow cytometry every 4 weeks. The percentage of live B220⁺ (B), CD3⁺ (C), and Gr1/Mac1⁺ (D) cells are depicted over time. E, F, G, H. Whole BM from WT, HT, and HO mice was mixed 1:1 with whole BM from B6.SJL (BoyJ) mice and serially transplanted into lethally irradiated Rag1^−/− recipients. Peripheral blood was analyzed by flow cytometry every 4 weeks. Graphs present the compiled data from 13 primary recipients and 7 secondary recipients per group from 3 independent experiments. (*P<0.05, **P<0.01, ***P<0.001 and P>0.05; Mixed-effects model of repeated measures with Dunnett correction for multiple comparisons.) The percentage of live CD45.2⁺ cells among all (E), B220⁺ (F), CD3⁺ (G), and Gr1/Mac1⁺ (H) cells are depicted.

Figure 2.. Mice with h.P214L-Etv6 have reduced proportion of MPP4 cells in the bone marrow.

Bone marrow from Etv6^wt/wt (WT), Etv6^wt/mt (HT), and Etv6^mt/mt (HO) was analyzed by multiparameter flow cytometry. A. Gating strategy for analyses of hematopoietic stem and progenitor cell populations, after gating on singlets and live cells. B. The percentage of hematopoietic stem and progenitor cell populations is depicted (*P<0.05; **P<0.01; ANOVA with Dunnett’s multi-comparison test; n ≥ 13/genotype compiled from 3 independent experiments).

Figure 3.. Impaired lymphoid potential of early hematopoietic progenitor cells from mice with h.P214L-Etv6.

Hematopoietic stem and progenitor cell populations were sorted from Etv6^wt/wt (WT), Etv6^wt/mt (HT), and Etv6^mt/mt (HO) mice. A. Sorted populations were used for colony forming assays in methylcellulose. The number of colonies, as a percentage of WT is depicted. (*P<0.05; **P<0.01; ***P<0.001; ANOVA with Dunnett’s multiple comparison test.) B. Sorted MPP4 cells were cultured in conditions to promote B cell development. Dot plots of flow cytometry data from days 6, 12 and 18. Data are representative of 3 independent experiments with very similar results. C. Sorted MPP4 cells were transplanted into lethally irradiated recipients. Controls (Ctl) were injected with PBS without cells. The relative proportions of B220⁺ (Left) and Mac1⁺ and/or Gr1⁺ (Right) cells, compared to WT, in the spleens of mice 10 days after transplantation are depicted (**P<0.01; Kruskall-Wallis ANOVA with Dunn’s multiple comparison test: n ≥ 7/genotype compiled from 3 independent experiments).

Figure 4.. Inflammation-related gene expression changes due to h.P214L-Etv6 in MPP4 cells.

LT-HSC, MPP4 and CLP cells from Etv6^wt/wt and Etv6^mt/mt mice were sorted prior to RNA-seq. A. Representative enrichment plots of gene sets from MPP4 with nominal P<0.05. B. Hierarchical clustering and heatmaps of genes in the Inflammatory Response gene set from LT-HSC, MPP4 and CLP populations.

Figure 5.. h.P214L-Etv6 alters the secretion of cytokines which impair MPP4 differentiation to B cells.

A. The supernatant of Ba/F3 cells transduced with empty vector, wild type ETV6 or P214L ETV6 expressing plasmids was analyzed by Luminex for cytokine levels. Relative cytokine levels are depicted as a heatmap (***P<0.001; ****P<0.0001, ANOVA with Dunnett’s multiple comparison test). B. MPP4 cells from WT mice were sorted and cultured in conditions to promote B cell development. The percentage of B220⁺ cells after 6 (Top) and 12 (Bottom) days is depicted (**P<0.01; ***P<0.001; ****P<0.0001; ANOVA with Dunnett’s multiple comparison test; data are compiled from 3 independent experiments performed in triplicate).