inv(16) and NPM1^mut AMLs engraft human cytokine knock-in mice

Biologically less aggressive, so-called favorable-risk acute myeloid leukemia (AML) constitutes up to 40% of newly diagnosed cases of AML and is relatively more common in patients younger than 60 years.¹  This risk group is currently defined by 4 distinct cytogenetic and molecular constellations according to the European LeukemiaNet 2010 guidelines,^2,3  that is, AML with t(8;21), inv(16)/t(16;16), isolated nucleophosmin (NPM1) mutation (cytogenetically normal without recurrent FLT3 mutations), or mutated CEBPA. Upon state-of-the-art therapy, these patients benefit from significantly longer survival when compared with intermediate- or adverse-risk cases. However, only 66% of younger patients and 33% of older patients with favorable-risk AML are alive 3 years after diagnosis.¹  Studying the biology of these entities in in vivo model systems has been inherently difficult. In fact, faithful xenoengraftment of human AML to immunodeficient mice has been limited to higher-risk cases,⁴  and there is no published mouse model supporting robust engraftment of lower-risk disease.⁵  We recently developed humanized mice expressing human cytokines central to myeloid development and hematopoietic stem cell maintenance (macrophage colony-stimulating factor [M-CSF], interleukin-3 [IL-3], granulocyte-macrophage colony-stimulating factor [GM-CSF], and thrombopoietin) under their endogenous promoter, as well as human SIRPα as a transgene. These mice have displayed superior human healthy CD34⁺ cell engraftment and myelopoiesis.^6-11  We hypothesized that this human myelopoiesis-supportive environment may also permit xenoengraftment of human favorable-risk AML.

Humanized cytokine knock-in (KI) mice were generated as reported previously.^6-11  Bone marrow aspirate was collected from patients with newly diagnosed AML after obtaining informed consent. The study was approved by the Cantonal Ethics Committee Zurich. Human blast cells were purified by density gradient centrifugation followed by immunomagnetic bead depletion of lymphocytes. Newborn mice were sublethally irradiated and intrahepatically injected with 1 × 10⁶ cells per mouse. Mice were analyzed 16 to 24 weeks after transplantation (Figure 1A). All animal experiments were approved by the Cantonal Veterinary Office. For further details, see supplemental Methods (available on the Blood Web site).

Our studies focused on 2 entities that together make up 60% of favorable-risk AML cases encountered in the clinical setting, namely AML with isolated mutations in the nucleophosmin 1 gene and AML with inv(16)(p13.1q22). Flow cytometric analysis (supplemental Figure 1) of bone marrow and blood of cytokine KI vs NSG mice, the current standard xenotransplantation model,¹²  16 to 24 weeks after transplantation revealed profound differences: average engraftment, defined as the proportion of cells expressing human CD45 among all cells expressing human and murine CD45, was 36% in the bone marrow of cytokine KI mice compared with only 10% in NSG mice, across all patient samples transplanted in this study (Figure 1B; supplemental Table 1; supplemental Figures 2A and 3). A similar pattern was observed in the peripheral blood, where human engraftment reached 10% in KI mice compared with >2% in NSG mice (Figure 1C; supplemental Figure 2B). In line with engraftment levels, engraftment efficacy, that is, overall engraftment vs no engraftment (defined as ≥1% hCD45⁺/all CD45⁺ cells¹ ), was higher in cytokine KI mice (supplemental Table 2). Importantly, average engraftment in cytokine KI mice approached bone marrow and peripheral blast count observed in human AML patients at diagnosis, whereas NSG engraftment levels were significantly lower (Figure 1B-C). AML engraftment in cytokine KI mice was found to be independent of expression of the SIRPα transgene in mice expressing all 4 cytokines (supplemental Figure 4).

Next, we set out to define the phenotype of favorable-risk AML engrafted in MI(S)TRG mice, the most advanced type of human cytokine KI mice,¹⁰  in comparison with NSG mice, utilizing animals matched by donor and time of analysis. Because virtually no engraftment of inv(16) AML could be observed in NSG mice, this comparative analysis was limited to NMP1^mut AML. This subtype showed a trend toward higher bone marrow engraftment in MI(S)TRG vs NSG mice, and the difference in the peripheral blood reached statistical significance (Figure 1D-E). Engraftment of NMP1^mut AML in MI(S)TRG mice faithfully reproduced the original leukemia-associated phenotype by flow cytometry, and the same leukemia in NSG mice displayed loss of CD117 (c-kit) expression (supplemental Figure 5A-B), suggesting an environmentally driven alteration of the initial AML blast population or selective support of nondominant cell populations in the xenograft. Because of low engraftment efficacy in NSG mice, future studies will need to determine whether this finding is a general feature of NPM1^mut AML cases or if individual AML cases within this risk group might behave differently. As an independent measure of engraftment of the diseased clone, we also assessed expression of the mutant NPM1 allele. NPM1^mut expression was more than threefold higher in MI(S)TRG mice compared with NSG mice, suggesting more faithful AML engraftment in MI(S)TRG mice (Figure 1F). Performing targeted next-generation sequencing in patient and engrafted cytokine KI mouse bone marrow (supplemental Figure 6A-B), we found a comparable allelic frequency ranging from 38% to 51% for NPM1 mutation (TCTG insertion at position chr5:170837542) in the patient’s leukemia (PID250) and 3 engrafted mice, and no reads mapped to a nontransplanted cytokine KI mouse serving as control (Figure 1G). Of note, even the composition of subclones of the NPM1 mutation and additional identified mutations was constant between the analyzed patient and respective cytokine KI recipient mice (Figure 1H; supplemental Figure 6C-D).

In contrast to NPM1^mut AML engraftment, a striking difference in quantitative inv(16) AML engraftment was observed between cytokine KI and NSG mice, both by aggregate and split-sample analysis (Figure 2A). This could be verified by molecular analysis (Figure 2B) and microscopy, which demonstrated marrow infiltration by large granulated blasts with cytoplasmatic vacuolization in both patient and transplanted MISTRG recipients, whereas NSG recipients displayed murine hematopoiesis only (Figure 2C); we did not observe bone marrow eosinophilia, which might be because of the lack of additional human cytokines, particularly human IL-5.^11,13  Serial transplantation further validated engraftment of an inv(16) leukemia-initiating cell population in cytokine KI mice (Figure 2D; supplemental Figure 7). To dissect the contribution of respective single human KI cytokines to inv(16) AML propagation, we analyzed engraftment in Rag2^−/−gc^−/− mice carrying, in addition to a human SIRPα transgene, subsequent additions of KI genes for human thrombopoietin (STRG), M-CSF and thrombopoietin (MSTRG), and M-CSF, IL-3, GM-CSF, and thrombopoietin (MI(S)TRG). STRG mice, as NSG mice, did not support engraftment. In contrast, addition of M-CSF in MSTRG mice led to robust engraftment of inv(16) AML, whereas addition of IL-3 and GM-CSF in MI(S)TRG mice resulted in only marginal, if any, improvement of engraftment over MSTRG mice (Figure 2A). Of note, KI of human M-CSF has previously been shown to be sufficiently cross-reactive to rescue the murine M-CSF knockout phenotype.⁹  We thus reasoned that the engraftment advantage observed in MSTRG mice was likely because of human M-CSF effects on blasts and not because of reduction in mouse macrophages. Together, this establishes a central role for M-CSF in maintenance of inv(16) AML.

To further decipher the molecular basis of M-CSF dependency observed in inv(16) AML, we profiled cytokine receptor expression for thrombopoietin, M-CSF, IL-3, and GM-CSF on inv(16) AML blasts in comparison with NPM1^mut AML and an aggregate of nonfavorable-risk AML cases, using microarray data from an international cohort study.¹⁴  inv(16) AML showed significantly higher expression of each of the 4 receptors compared with NPM1^mut AML and significantly higher expression of M-CSFR and GM-CSFR compared with nonfavorable-risk AML, with the overall highest difference in expression observed for M-CSFR (Figure 2E). Moreover, gene set enrichment analysis¹⁵  demonstrated enrichment of M-CSF-inducible genes¹⁶  in inv(16) AML compared with NPM1^mut AML (Figure 2F) and nonfavorable-risk AML cases (supplemental Figure 8), confirming increased signaling through the M-CSF pathway. Of note, core-binding factor is a known regulator of M-CSFR transcription,^17,18  yet the functional consequences for inv(16) (CBFβ-MYH11) driven leukemogenesis are incompletely understood.¹⁹ 

In sum, we here describe a faithful and highly efficacious xenotransplantation model of favorable-risk, specifically NPM1^mut and, importantly, inv(16) AML. We further demonstrate a strong dependency of inv(16) AML on M-CSF, a finding that might provide a basis for therapy optimization studies informed by an improved understanding of favorable-risk AML biology.

The authors are indebted to all patients and their physicians who participated in the study. The authors also thank the staff of the Hematology laboratory at University Hospital Zurich for expert technical assistance.

This study was supported by a Hanne-Liebermann fellowship (J.M.E.), the Swiss National Science Foundation (310030_146528/1), the University of Zurich Clinical Research Priority Program Human Hemato-Lymphatic Diseases (M.G.M.), the Bill and Melinda Gates Foundation, and the National Institutes of Health, National Cancer Institute (grant CA156689) (R.A.F. and M.G.M.).

Contribution: J.M.E. and P.J.R. designed research, performed experiments, analyzed data, and wrote the manuscript; L.V.K., R.M., U.W., Y.S., and J.S.G. performed experiments and analyzed data; N.W.-V.v.W., C.F., A.R., V.L., E.D., A.P.T., and D.S. performed experiments; and R.A.F. and M.G.M. directed the study and wrote the manuscript.

Conflict-of-interest disclosure: R.A.F. and M.G.M. filed patent applications on genetically modified mice and the use thereof. MISTRG mice were donated for public access to the Jackson Laboratory. The remaining authors declare no competing financial interests.

Correspondence: Markus G. Manz, Hematology, University Hospital and University of Zurich, Raemistr 100, CH-8091 Zurich, Switzerland; e-mail: markus.manz@usz.ch.