CCAAT/enhancer-binding protein alpha (CEBPA) gene haploinsufficiency does not alter hematopoiesis or induce leukemia in Lck-CALM/AF10 transgenic mice

Although rare, CALM/AF10 is a chromosomal rearrangement found in immature T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia, and mixed phenotype acute leukemia of T/myeloid lineages with poor prognosis. Moreover, this translocation is detected in 50% of T-ALL patients with gamma/delta T cell receptor rearrangement, frequently associated with low expression of transcription factor CCAAT/enhancer-binding protein alpha (CEBPA). However, the relevance of CEBPA low expression for CALM/AF10 leukemogenesis has not yet been evaluated. We generated double mutant mice, which express the Lck-CALM/AF10 fusion gene and are haploinsufficient for the Cebpa gene. To characterize the hematopoiesis, we quantified hematopoietic stem cells, myeloid progenitor cells, megakaryocyte-erythrocyte progenitor cells, common myeloid progenitor cells, and granulocyte-macrophage progenitor cells. No significant difference was detected in any of the progenitor subsets. Finally, we tested if Cebpa haploinsufficiency would lead to the expansion of Mac-1+/B220+/c-Kit+ cells proposed as the CALM/AF10 leukemic progenitor. Less than 1% of bone marrow cells expressed Mac-1, B220, and c-Kit with no significant difference between groups. Our results showed that the reduction of Cebpa gene expression in Lck-CALM/AF10 mice did not affect their hematopoiesis or induce leukemia. Our data corroborated previous studies suggesting that the CALM/AF10 leukemia-initiating cells are early progenitors with lymphoid/myeloid differentiating potential.


Introduction
The CALM/AF10 fusion gene derived from the t(10;11) (p13;q14) chromosomal translocation can be found in Tcell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML) and mixed phenotype acute leukemia (MPAL) of T/myeloid lineages (1)(2)(3)(4)(5). The leukemogenic activity of CALM/AF10 was demonstrated in several murine models. Mice transplanted with murine bone marrow (BM) cells expressing CALM/AF10 fusion transcripts developed an aggressive form of biphenotypic leukemia (6). Accordingly, Caudell et al. (7) generated transgenic mice (TM) expressing CALM/AF10 under the control of the vav promoter (a pan-hematopoietic vector) and approximately 40-50% of TM developed leukemia at the median age of 12 months. The leukemic blasts isolated from the BM and spleen of vav-CALM/AF10 TM co-expressed Mac-1, B220, and CD24 with variable expression of CD117 in the absence of other B-cell markers, thus resembling human MPAL with involvement of B and myeloid lineages. However, Abdelali et al. (8) screened 665 patients with T-ALL and identified CALM-AF10 in 30/431 (7%) patients aged 16 years and over, and in 15/234 (6%) of those aged up to 15 years of age.
Aberrant expression of T-cell lineage markers on AML blasts has been associated with mutations or epigenetic changes of the CEBPA gene or its promoter (9). CEBPA encodes for a transcription factor of crucial relevance for granulocytic differentiation (10). In addition to mutations in the CEBPA gene, which were detected in approximately in 6 À 15% of de novo AML and in 15 À 18% of AML with normal karyotypes (11), silencing of CEBPA expression due to promoter hypermethylation was found in a small subset of AML patients with myeloid/T-lymphoid features (12). In the study by Terriou et al. (13), half of the CEBPA methylated T-ALL cases were CALM/AF10 positive (5/10), suggesting that the search of CALM/AF10 in CEBPA methylated AML could help distinguish between the immature T-ALL and myeloperoxidase negative AML. Based on the existing data showing the aberrantly low expression of the CEBPA gene in patients with acute leukemia harboring the CALM/AF10 fusion gene, we decided to evaluate in vivo if haploinsufficiency of CEBPA predisposes CALM/AF10 + hematopoietic cells to develop leukemia.

Generation of double mutant mice
Mice haploinsufficient for the Cebpa gene (Cebpa +/-) were kindly provided by Prof. Daniel G. Tenen (National University of Singapore) and were crossed to Lck-CALM/ AF10 TM, in which the expression of the fusion gene is under the control of the lck regulatory region. The Lck-CALM/AF10 TM were kindly provided by Prof. Stefan K Bohlander (University of Auckland). The phenotype of parental lines has been previously described (14). The offspring was genotyped by conventional polymerase chain reaction (PCR) ( Table 1).

Serial analysis of hematological counts
Animals aged 3-15 months were monitored once every three months for peripheral blood counts. Leishmanstained blood smears were used for differential leukocyte counts. All animal experiments were performed after the approval of the Animal Care and Use Committee of the Medical School of Ribeirão Preto of the University of São Paulo (Protocol number: 144/2013) in strict accordance with institutional guidelines regulated by the National Council of Animal Experimentation (CONCEA-Brazil). All experimental procedures were performed under isoflurane anesthesia, and all efforts were made to minimize suffering.

Histopathological analysis
Vertebrae and spleens from animals used for flow cytometry analysis were first fixed in PBS with 4% paraformaldehyde and processed for paraffin-embedded sectioning at 5 mm in thickness, and then stained with hematoxylin & eosin.

Results
After a median follow-up of 15 months, no significant difference in the median values of leukocyte counts, red blood cell counts, hemoglobin concentrations, and platelet counts were detected among the four genotypes. Figure 1A-D shows the main hematological parameters of peripheral blood observed in last sample collection during monitoring, but similar results were found at all timepoints. Moreover, there was no change in the percentage of erythroid precursors, granulocytes, and B and T lymphocytes in the BM (Figure 1E-H). Of note, no case of leukemia was detected among 23 mice of the CA + /WT group and the 33 animals of the CA + /Cebpa +/group. The number of animals was adequate for the objectives and followed the good practice guide for the use of animals in research. Our results were in agreement with Krause (14) who did not detect leukemia in Lck-CALM/ AF10 TM. In the present study, CALM/AF10 expression was driven by the Lck proximal promoter region. During normal T-cell ontogenesis, Lck transcription is regulated by two distinct promoter elements. The proximal promoter is active almost exclusively in thymocytes and becomes inactive later during T-cell maturation and has been used experimentally to drive the expression of the transgene to T-cell lineage (16)(17). However, Bell and Bhandoola (18) demonstrated that the earliest thymic progenitors have not only lymphoid but also myeloid potential during hematopoiesis, providing alternative phenotypes for a lineage specification of a potential leukemic transformation driven by an lck-CALM/AF10 translocation. In our model, CALM/AF10 expression occurred in hematopoietic progenitors with restricted capacity of differentiation and our results suggest that CALM/AF10 may not be leukemogenic in this cell compartment. Reinforcing this hypothesis, Caudell et al. (7) generated a murine model in which the expression of CALM/AF10 was targeted to multipotent progenitors using the vav promoter, and reported that approximately 50% of vav-CALM/AF10 TM developed leukemia. The leukemic blasts showed myeloid differentiation associated with lymphoid features, thus resembling human MPAL. However, the long latency of the disease in this model further argues that additional events are necessary for the development of leukemia.

Discussion
In order to fully characterize the hematopoiesis in WT/WT, CA + /WT, WT/Cebpa +/-, CA + /Cebpa +/mice, we examined whether there were genotype-specific differences in BM HSC and myeloid progenitors (MP, MEP, CMP, and GMP). Frequencies of HSC, MP, MEP, CMP, and GMP did not differ between groups (Figure 2A). In addition, BM and spleen did not show differences between the groups on histopathological analysis (Figure 3).
Decreased expression of CEBPA plays a role in acute promyelocytic leukemia (APL) leukemogenesis. Guibal et al. reported that Cebpa +/mice did not develop leukemia nor present abnormal cell counts, but double mutants human cathepsin G (hCG)-PML/RARA Cebpa +/developed APL at a higher frequency, decreased latency, and had a significantly shorter survival rate than hCG-PML/ RARA TM. Furthermore, the APL-initiating cells showed decreased mRNA and protein expression of CEBPA compared with normal promyelocytes (19).
Deshpande et al. (6) showed that, in a transplantation model of murine CALM/AF10 + leukemia, the myeloid leukemia cells were frequently B220 + , with clonal B cell receptor (BCR) rearrangement and lacked c-Kit expression, demonstrating that these AML cells had lymphoid characteristics. In contrast, in the study of Caudell et al. (7), only 50% of the AML from vav-CALM/AF10 TM were B220 + and exhibited clonal BCR rearrangement, with variable expression of c-Kit. Nevertheless, both studies showed that Mac-1 + /B220 + cells lacked specific B-cell markers. Dutta et al. (20) studied the role of CALM/AF10 specifically in B cell development and function in Mb1-Cre or CD19-Cre mice and demonstrated that expression of CALM/AF10 in the B cell compartment did not lead to leukemia. Together, these authors show evidence that the cell of origin of CALM/AF10 AML with lymphoid characteristics is an early progenitor not derived from B-cell lineage. In order to test if there is an expansion of the rare population of hematopoietic progenitor cells (Mac-1 + / B220 + ) followed or not by the expression of c-Kit in the BM of double mutant mice CA + /Cebpa +/-, we quantified the frequency of these cells in our model. Less than 1% of BM cells expressed Mac-1, B220, and c-Kit with no significant difference between groups ( Figure 2B).
In summary, our results showed that the reduction of Cebpa expression did not contribute to leukemogenesis in Lck-CALM/AF10 mice and did not affect hematopoiesis.
In future experiments, it will be important to determine if decreased Cebpa expression can modify leukemia frequency or promote the expansion of specific cell subsets in vav-CALM/AF10/Cebpa +/double mutant mice. Nevertheless, our data support the hypothesis that the normal counterpart of the CALM/AF10 + leukemia-initiating cells is a hematopoietic stem cell or an early progenitor with myeloid and lymphoid potential.