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. 2013 Aug 8;122(6):1007-16.
doi: 10.1182/blood-2013-03-489823. Epub 2013 Jun 18.

Dysregulated signaling pathways in the development of CNTRL-FGFR1-induced myeloid and lymphoid malignancies associated with FGFR1 in human and mouse models

Mingqiang Ren  1 , Haiyan Qin, Eiko Kitamura, John K Cowell
Affiliations

Dysregulated signaling pathways in the development of CNTRL-FGFR1-induced myeloid and lymphoid malignancies associated with FGFR1 in human and mouse models

Mingqiang Ren et al. Blood. .

Abstract

Myeloid and lymphoid neoplasm associated with FGFR1 is an aggressive disease, and resistant to all the current chemotherapies. To define the molecular etiology of this disease, we have developed murine models of this disease, in syngeneic hosts as well as in nonobese diabetic/severe combined immunodeficiency/interleukin 2Rγ(null) mice engrafted with transformed human CD34+ hematopoietic stem/progenitor cells. Both murine models mimic the human disease with splenohepatomegaly, hypercellular bone marrow, and myeloproliferative neoplasms that progresses to acute myeloid leukemia. Molecular genetic analyses of these model mice, as well as primary human disease, demonstrated that CNTRL-FGFR1, through abnormal activation of several signaling pathways related to development and differentiation of both myeloid and T-lymphoid cells, contribute to overt leukemogenesis. Clonal evolution analysis indicates that myeloid related neoplasms arise from common myeloid precursor cells that retain potential for T-lymphoid differentiation. These data indicate that simultaneously targeting these pathways is essential to successfully treating this almost invariably lethal disease.

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Figures

Figure 1
Figure 1
The CNTRL-FGFR1 fusion kinase induces BaF3 cell transformation. (A) Scheme for construction of the MIEG3-CNTRL-FGFR1. (B) Flow cytometry analysis shows that almost all BaF3 cells transformed by CNTRL-FGFR1 are viable and express GFP after withdrawal of IL-3. (C) Cell proliferation assays demonstrate increased IL-3–independent growth of CNTRL-FGFR1-BaF3 cells. (D) Western blot analysis shows the presence of the CNTRL-FGFR1 fusion protein in stably transformed BaF3 cells.
Figure 2
Figure 2
Phenotypic analysis of CNTRL-FGFR1 transduced and transplanted mice. (A) Comparison of splenic weight between leukemic and normal mice. (B) Comparison of survival from 3 chimeric BM transduction and transplanted mouse models. (C) Flow cytometric analysis (see “Methods”) of cells in BM, PB, SP, and thymus (Thy) from 2 mice with T-LBL shows a CD4+CD8+ phenotype. (D) Flow cytometry analysis shows that cells from a representative mouse that developed AML have a B220+Gr1+Mac1+ immunophenotype in both GFP+ and GFP− cell populations.
Figure 3
Figure 3
CNTRL-FGFR1 neoplasms are transplantable and originate from oligo- or monoclonal hematopoietic stem/progenitor cells. (A) Kaplan-Meier analysis of primary and secondary recipients shows no significant difference between the primary and secondary transplants. (B) Schematic representation showing the relative location of the PCR primers used to analyze the Tcrb locus (top). Gel electrophoresis of PCR products shows DJ arrangement of Tcrb in 2 representative, serially transplanted CNTRL-FGFR1 mice (bottom). DNA from BM displays 1 large band reflecting no rearrangement. DNA from normal Thy and lymph node (LN) shows several smaller bands resulting from rearrangements. DNA from Thy and LNs from 2 leukemic mice (#2, #5) shows oligo- or monoclonality. Specifically, DNA from 2 cell lines (CEP2A and CEP5A) shows only 1 predominant band (arrow), indicating that these lymphoma cells were monoclonal. (C) RT-PCR analysis shows the specific transcriptional levels of B-lineage genes in sorted myeloid cells (Gr1+Mac1+) or B cells (B220+CD19+) from normal (Nor) BALB/c mice as well as in sorted AML cells (Gr1+Mac1+ B220+) from 2 leukemic mice. The B-lymphoid cell line BBC2 is shown as a positive control. (D) Genomic PCR analysis of IgH rearrangement showing the germline configuration in DNA from the tail and polyclonal rearrangements in the B cells sorted from 3 normal BALB/c mouse splenocytes. The B220+Gr1+Mac1+ AML cells have the same pattern as normal Gr1+Mac1+ myeloid cells.
Figure 4
Figure 4
Mutational activation of Notch1 and deletion of Tcra are etiologically associated with the development of T-cell leukemia/lymphoma. (A) Array CGH analysis of CEP2A (top) and CEP5A (bottom) cells shows almost identical chromosome changes in the 2 cell lines, including 14qC2 segmental loss as well as gain of chr7, chr10, and chr15. (B) Detailed view of murine Tcra deletion in both CEP2A and CEP5A cell lines. (C) Genomic PCR confirms loss of Tcra in the CEP2A and CEP5A cells. (D) Flow cytometric analysis reveals that both CEP2A and CEP5A cells are negative for cell-surface Tcra. (E) Western blot analysis, using antibodies against the activated Notch1 (Val 1774 antibody, Cell Signaling Technologies), showing that activated Notch1 (different bands resulting from different truncating mutations) is present in LN isolated from the primary (1°) and secondary (2°) T-LBL mice compared with 3 murine FGFR1-related neoplasm cell lines. A spleen sample from #3 AML mouse is used as a negative control. (F) Schematic of the Notch1 gene showing the relative locations of the primers used to analyze the deletion mutants (top). A 5′ deletion (brackets) creates a 500-base pair fragment using the P1/P2 primers, as shown in LN from different T-LBL mice. In normal cells, the wild-type 11.5-kb fragment cannot be amplified. (G) γ-Secretase inhibitors DAPT and Comp E significantly inhibit CEP2A and CEP5A cell growth in vitro at micromolar concentrations.
Figure 5
Figure 5
Gene expression analysis by RNA-seq demonstrates dysregulation of multiple genes associated with myeloid cell development. (A) Hierarchical cluster analysis generated by Euclidean distance and pairwise complete-linkage analysis of unsupervised RNA-seq data. (B) Heat map (left) and Gene Set Enrichment Analysis (right) show genes related to myeloid and T-lymphoid cell development are up-regulated (red) in sorted AML cells (B220+Gr1+Mac1+) compared with sorted normal myeloid cells (Gr1+Mac1+) from normal BALB/c mice (P < .05). (C) Leading-edge analysis of RNA-seq data sets demonstrates gene expression patterns in leukemic cells characteristic of both myeloid and T-lymphoid lineage cells. (D) Quantitative RT-PCR analysis confirms the transcription level changes for selected genes in AML cells measured by RNA-seq shown in (B). (E) Schematic summary of differentially expressed genes related to stages of myeloid development.
Figure 6
Figure 6
Human CD34+ progenitor cells transduced and xenotransplanted in NSG mice develop into AML. (A) Schematic representation of the experimental approach to generate the human CD34+ progenitor mouse model carrying the CNTRL-FGFR1 fusion gene. (B) Kaplan-Meier survival analysis of primary recipients following engraftment of either the MIEG3 control vector or CNTRL-FGFR1-transduced human CD34+ progenitor cells. (C) Representative flow cytometry analysis of BM, PB, SP, and liver (LV) cells from a primary recipient mouse. (D) Quantitative RT-PCR analysis shows the comparison of gene expression levels in CNTRL-FGFR1 mice (CNTRL) and 1 CNTRL-FGFR1 patient compared with an MIEG3-NSG (MIEG3) mouse and normal healthy human PB mononuclear cells (Nor. PBMN), respectively. (E) Western blot analysis shows the gene expression levels of activated FGFR1, MYC, and GFI1 in leukemic mouse spleens compared with normal human PBMN. (F) Flow cytometry analysis of FLT3 and KIT expression on the cell surface from the CNTRL-FGFR1-NSG (CNTRL) and control MIEG3-NSG (MIEG3) mice. (G) Cell viability assays show the synergistic effect of ponatinib (Pon) and JQ1 on cell growth inhibition in different primary leukemic mouse splenocytes and normal PBMN cells. (H) Western blot analysis shows the MYC, FGFR1, or FGFROP2-FGFR1 fusion protein levels in 4 human AML cell lines (left). Cell viability assays show the synergistic effect of Pon and JQ1 on cell growth inhibition is only seen in KG-1 cells that carry an FGFR1 rearrangement (right).
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