inv(16) and NPM1mut AMLs engraft human cytokine knock-in mice

Abstract

Favorable-risk human acute myeloid leukemia (AML) engrafts poorly in currently used immunodeficient mice, possibly because of insufficient environmental support of these leukemic entities. To address this limitation, we here transplanted primary human AML with isolated nucleophosmin (NPM1) mutation and AML with inv(16) in mice in which human versions of genes encoding cytokines important for myelopoiesis (macrophage colony-stimulating factor [M-CSF], interleukin-3, granulocyte-macrophage colony-stimulating factor, and thrombopoietin) were knocked into their respective mouse loci. NPM1^mut AML engrafted with higher efficacy in cytokine knock-in (KI) mice and showed a trend toward higher bone marrow engraftment levels in comparison with NSG mice. inv(16) AML engrafted with high efficacy and was serially transplantable in cytokine KI mice but, in contrast, exhibited virtually no engraftment in NSG mice. Selected use of cytokine KI mice revealed that human M-CSF was required for inv(16) AML engraftment. Subsequent transcriptome profiling in an independent AML patient study cohort demonstrated high expression of M-CSF receptor and enrichment of M-CSF inducible genes in inv(16) AML cases. This study thus provides a first xenotransplantation mouse model for and informs on the disease biology of inv(16) AML.

Figure 1

Humanized cytokine KI mice support robust engraftment of favorable-risk AML and closely model disease biology. (A) Schematic representation of experimental setup. Scatter plots depict blast count in patients with inv(16) or NPM1^mut AML (left columns) and human engraftment in transplanted NSG or cytokine KI mice, in the bone marrow (B) and peripheral blood (C) (mean ± standard deviation [SD], N = 15-45 per group, 1-way ANOVA P < .0001 for both bone marrow and peripheral blood). Data stem from >20 independent experiments. Scatter plots compare human engraftment in NSG and MI(S)TRG mice transplanted with NPM1^mut AML in the bone marrow (D) and peripheral blood (E) (mean ± SD, N = 19-36 per group). (F) Mean expression of NPM1A^mut allele normalized to PBGD in engrafted NSG and MISTRG mice (mean ± SD, N = 4 per group). (G) Allelic frequency of NPM1 mutation (TCTG insertion at position chr5:170837542) in patient (PID250), transplanted cytokine KI mice (n = 3) and untransplanted cytokine KI mouse (ctrl) when mapping to HG19:chr5:170837542 (human reference genome). (H) Frequency of subclones of NPM1 insertion at HG19:chr5:170837542 in patient (PID250) and 3 cytokine KI mice.

Figure 2

M-CSF is required for engraftment of AML with inv(16). (A) Scatter plot compares human engraftment in the bone marrow of mice of the indicated strains transplanted with inv(16) AML. Split samples are indicated by blue (PID330) and red (PID420) symbols (mean ± SD; N = 8-16 per group; Mann-Whitney U test P < .0001 for aggregate NSG vs cytokine KI, P = .01 for PID330, P = .03 for PID420; 1-way ANOVA P = .0002). (B) Mean expression of inv(16), type A normalized to ABL1 in transplanted mice of the indicated strains (mean ± SD, N = 4-7 per group, 1-way ANOVA P < .0001). (C) Brightfield microscopy images depict bone marrow aspirate smear from PID420 at initial diagnosis (left) and cytospins from NSG (middle) and MISTRG (right) mice transplanted with PID420 AML. Bars represent 20 µm. Data are representative of N = 6. (D) Serial transplantation: cartoon shows experimental setup; dot plots show immunophenotype of engrafted human cells. Data are representative of 2 independent experiments. (E) Bar graphs depict expression of indicated cytokine receptors in indicated AML subgroups (non-FR, nonfavorable risk) by microarray analysis (mean ± SD; N = 32-298 per group; 1-way ANOVA P < .0001 for c-Mpl, M-CSF receptor [M-CSFR], and GM-CSF receptor [GM-CSFR]; P = .02 for IL-3Rα). (F) Gene set enrichment analysis of M-CSF inducible genes in inv(16) compared with NPM1A^mut AML (N = 33 for inv(16), N = 46 for NPM1A^mut; enrichment score 0.72, significant at nominal P value <5%).