Establishing and characterising murine CALM/AF10-MF leukaemias

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Results

Validation of stable expression cell lines

One of the primary objectives of this study was to constitutively express the CALM/AF10 (C/A) or the CALM/AF10 minimal fusion (C/A-MF) in murine bone marrow cells using the retroviral transduction/transplantation technique. For this purpose, we sought to use the previously established stable producer cell lines, E86-MIG-C/A and E86-MIG-C/A-MF that produce retroviral particles that contain C/A and the C/A-MF, respectively. Dr. Aniruddha Deshpande, at Ludwig Maximilian University, Munich, Germany, established these cell lines (189). CALM/AF10 and the minimal fusion gene were cloned by Dr. Aniruddha Deshpande into the Murine Stem Cell Virus (MSCV)-IRES-GFP (pMIG) vector (Figure 3.1-1) (189). The vector contains long terminal repeats (LTR) for integration of the retroviral DNA into the host genome, ampicillin as a selection marker and GFP as a fluorescent marker. Further, it is a bicistronic vector and has an internal ribosome entry site (IRES) allowing the expression of two proteins, the gene of interest and the GFP marker, from one mRNA. The vector has been shown to effectively transduce haematopoietic cells. Moreover, the expression of GFP as a marker is useful in tracking and purification of cells expressing the vector and hence the gene of interest.
For generating the producer cell lines, pMIG vectors expressing CALM/AF10 and CALM/AF10-MF were transiently transfected into phoenix-ECO, which is a retrovirus producer cell line and used for the generation of ecotropic retroviruses. The phoenix-ECO cells transiently shed the retrovirus into the surrounding medium. Viral conditioned medium (VCM) from the phoenix-ECO cells was collected and used to stably transduce the E86 cell line, which is another ecotropic retroviral producer/packaging cell line that can be used for delivery of genes of interest into eukaryotic cells (191). We also had an E86 cell line that produced viral particles with the empty pMIG vector (E86-MIG) as a control. Before using the E86-MIG-C/A and E86-MIG-C/A-MF expression cell lines to transduce murine bone marrow cells in our study, we wanted to determine the efficiency of transduction of the retrovirally conditioned supernatant from each of the cell lines.

Efficiency of transduction of E86-CALM/AF10 and E86-CALM/AF10-MF

In order to test the transduction efficiency of retroviruses produced by the E86-MIG-C/A and E86-MIG-C/A-MF cell lines, the culture medium from each of these cell lines was collected and assayed on a mouse fibroblast cell line, NIH3T3. Different dilutions of the viral conditioned culture medium from the E86 cells were used to transduce NIH3T3 cells, in the presence of protamine sulphate as a cofactor (refer section 2.2.1.5). 48 hours post transduction, the percentage of GFP positive NIH3T3 cells was determined using flow cytometry. Each assay was repeated three times and the E86-MIG cell line was used as a positive control. On average, the E86-MIG-C/A cell line had a transduction efficiency of 5.6%; the E86-MIG-C/A-MF had an efficiency of 30%, whereas the E86-MIG cell line had a transduction efficiency of 70.8% as shown in the figure below (Figure 3.1-2).
From the results it is evident that the E86-MIG-C/A-MF cell line has higher transduction efficiency compared to the E86-MIG-C/A. In a previous study, Deshpande and colleagues have shown that in a murine bone marrow transplantation model, the expression of the CALM/AF10 minimal fusion leads to the development of an aggressive leukaemia with a similar latency and phenotype as that of full-length CALM/AF10 (137). Hence, we decided to confirm the presence and expression of C/A-MF in the E86-C/A-MF cell line and use this cell line and construct for all further experiments.

PCR amplification and sequencing of the CALM/AF10-minimal fusion gene

In order to confirm the presence and the sequence of the CALM/AF10 minimal fusion gene in the E86-MIG-C/A-MF cell line, we isolated DNA from the cell line and amplified the fusion gene by PCR using the pMIG forward and pMIG reverse primers (section 2.1.9). The amplification product was 1.2Kb in size (Figure 3.1-3) and was confirmed to be CALM/AF10-MF by Sanger sequencing. The C/A-MF is 993bp in length and contains nucleotides 1515 to 2261, corresponding to the C-terminal of CALM (NM_007166.3) and the region spanning from nucleotide 2107 to 2352 of the OM-LZ domain (octapeptide motif-leucine zipper) of AF10 (NM_001195626.1) (Section 8.1).

Detection of the CALM/AF10-MF protein in the E86-C/A-MF cell line

The integrity and expression of the C/A-MF fusion protein was confirmed by Western blotting. The C/A-MF fusion gene is tagged with a FLAG epitope (section 8.1) and thus protein expression can be detected using an antibody against the FLAG-tag. Whole cell lysates were prepared from the E86-MIG-C/A-MF cell line and E86-MIG-BCR/ABL (E86-MIG-B/A) cell line, which does not express the FLAG epitope, to be used as a negative control. The proteins in the cell lysates were resolved by SDS-PAGE and analysed by Western blotting. The blot was probed with an anti-FLAG antibody, and a 37kDa (expected size of C/A-MF) protein was clearly observed in the E86-MIG-C/A-MF cells and not in the E86-MIG-B/A cells. The blot was then stripped and incubated with an antibody against β-actin (expected size ~42kDa) as a loading control (Figure 3.1-4). The results from the Western blot clearly demonstrated that the E86-MIG-C/A-MF cell line expressed the FLAG-tagged C/A-MF protein.

Summary

The results from this chapter confirmed that virally conditioned supernatant from the E86-MIG-C/A-MF cell line has higher retroviral transduction efficiency than supernatant from the E86-MIG-C/A (full length) cell line. Further, the presence and expression of the C/A-MF transgene and the corresponding protein in the E86-MIG-C/A-MF cell line was validated by PCR amplification and Western blot analysis. Hence, we decided to use the E86-MIG-CALM/AF10-minimal fusion cells to establish a murine bone marrow transplantation model, in order to study and characterise CALM/AF10 induced leukaemias.

Establishing and characterising murine CALM/AF10-MF leukaemias

As described earlier, the mouse is an irreplaceable model organism to study the underlying mechanisms of cancer and is well suited for the in vivo modelling of human AML (158). The murine bone marrow transplantation (MBMT) model that makes use of retroviral vectors for gene transfer into primary haematopoietic cells provides a rapid and robust method for analysing the role of genetic lesions and their contribution to the pathogenesis of AML (160). We thus sought to establish a MBMT model using CALM/AF10-minimal fusion (C/A-MF) as a driver oncogene with the aim of recapitulating the human disease, characterising the phenotype and identifying secondary mutations that might contribute to the pathogenesis of AML.

Establishing the primary murine bone marrow transplantation model

In order to express C/A-MF in the murine model, bone marrow cells were isolated from 5-FU treated donor mice (C57/Bl6 strain) and retrovirally transduced using the E86-MIG-C/A-MF cell line as described in 2.2.2. The percentage of transduced bone marrow cells was determined by analysing the cells for expression of GFP using flow cytometry. A total of 1×106 bone marrow cells (combination of transduced and untransduced cells) were injected into lethally irradiated syngeneic recipients (primary transplants) via tail vein injections. Bone marrow cells transduced using the E86-MIG cell line were used as empty vector controls.
In total, 25 C/A-MF and 30 control (MIG) mice were transplanted. 22 of the 25 C/A-MF mice and 27 of the 30 controls survived the transplantation procedure. Six mice had to be euthanized within the first two weeks after transplantation due to bone marrow (BM) failure. A summary of the primary transplant experiments is given in Table 3.2-1.
We further wanted to check if the transduced bone marrow cells transplanted into the lethally irradiated recipients had engrafted successfully. We thus analysed the peripheral blood (from the tail) of each of the C/A-MF and MIG transplanted mice 4-6 weeks after transplantation, for the expression of GFP by flow cytometry. The percentage of transduced cells detected in each of the primary C/A-MF and MIG transplanted mice were plotted (Figure 3.2-1) and interestingly, the percentage of GFP positive cells were found to be significantly higher in the peripheral blood of the C/A-MF transplanted mice compared to MIG transplanted controls.
This might indicate that the expression of C/A-MF provides a proliferative advantage to the cells in vivo.
The percentage of transduced cells in the peripheral blood of MIG and C/A-MF mice was detected by flow cytometry. 4 -6 weeks after transplantation, the percentage of GFP positive cells in the C/A-MF primary mice was significantly higher that of the MIG controls (p-value <0.0001). The graph was constructed and unpaired t test was performed using GraphPad prism® (192).
The figure above shows that we successfully performed bone marrow transplantation with retrovirally transduced primary bone marrow cells that express either CALM/AF10 minimal fusion and GFP or just GFP (empty MIG vector). The engrafted MIG and C/A-MF mice were observed closely for signs and symptoms of disease, including ruffled fur, a hunched back, paleness in the feet and ears and inactiveness or lethargy.

Expression of CALM/AF10-MF leads to aggressive disease

Of the 22 C/A-MF mice that had engrafted, 21 developed symptoms of leukaemia and were euthanized when found to be moribund and one mouse was found dead in the cage. Prior to euthanizing the mice (N=21), peripheral blood was collected from the tail veins and used for performing erythrocyte and leukocyte counts. The mice were euthanized by CO2 asphyxiation and blood was collected via cardiac puncture for isolation of peripheral blood mononuclear cells (PBMCs). Liver, lung, kidney, spleen as well as the femur, tibia and pelvic bone were collected. A complete post mortem analysis was performed and on analysis the mice were found to have symptoms of leukaemia including splenomegaly, leukocytosis and anaemia as described in the following sections. For the one mouse that was found dead in the cage, a complete post mortem analysis could not be performed due to autolysis (10 Tx.3_C/A-MF#1). However, the spleen was collected and the weight, size and morphology were noted.
The time taken for the development of the disease varied considerably among the C/A-MF transplanted mice (Table 3.2-2) and the median latency was 91 days after transplantation. In comparison, none of the control (MIG) mice were found to develop leukaemia. However, 8 of the 27 MIG mice had to euthanized for reasons other than leukaemia, while 19 of the MIG mice remained healthy a year after transplantation, as shown in the survival curve (Figure 3.2-2).
The mice transplanted with C/A-MF transduced bone marrow cells (C/A-MF primary Tx.) developed leukaemia with a median latency of 91 days, whereas none of the MIG controls (MIG Tx.) developed leukaemia. The survival curve was plot using the GraphPad prism® software (192). Each step in the plot corresponds to the death of a mouse. Only mice that died of leukaemia have been considered. The dots represent censored events, i.e. animals that could not be analysed or were euthanized for reasons other than leukaemia, including those that did not engraft and were culled due to BM failure.

CALM/AF10-MF positive mice show splenomegaly

Splenomegaly, or an enlarged spleen, is often associated with haematological malignancies. In leukaemias, splenomegaly is caused by the infiltration of malignant cells into the spleen. Thus, an abnormal weight, size and/or morphology of the spleen are indicators of an underlying disorder. The spleen of a healthy C57/Bl6 mouse, on average, weighs between 80 to 120mg and measures about 1 to 1.5cm. In comparison to the weight and size of healthy spleens, 19 of the C/A-MF transplanted mice (including one that was found dead in the cage) were found to have enlarged spleens with weights ranging from 243 to 980mg and sizes ranging from 2 to 3 cm (Figure 3.2-3). Two of the remaining three mice had a slightly enlarged spleen, weighing approximately 147mg each, whereas the spleen of one mouse was within the normal range, weighing 102mg and measuring 1.4 cm. The morphology, weight and size of the spleens from the MIG transplanted mice were found to be similar to that of normal mice, as shown in the figure below.
Spleens from a healthy (wild type) mouse, a MIG transplanted mouse and three primary C/A-MF positive mice. b and c) Analysis of the spleen weights and sizes from wild type (N=6), MIG (N=20) and primary C/A-MF mice (N=21). The C/A-MF mice had significantly enlarged spleens as compared to the wild type mice (p-value=0.0006). The spleens from the MIG mice were similar in weight and size to those from the wild type, healthy controls (p-value >0.99). One-way ANOVA followed by Dunn’s multiple comparison tests were performed using the GraphPad prism® software (192). Note: A log 10 scale was used for the analysis of weight of the spleens (b).

CALM/AF10-MF mice have an abnormal blood count

Primary bone marrow disorders, including leukaemia, lead to abnormal blood cell counts. Patients with AML often present with leukocytosis and anaemia. Leukocytosis is defined as an elevated white blood cell (WBC)/leukocyte count, while anaemia refers to an unusually low level of red blood cells (RBC)/erythrocytes. We collected the peripheral blood from the tails of moribund mice and performed WBC and RBC counts. The normal leukocyte counts in the peripheral blood of healthy mice range from 5 x 103/μl to 12 x 103/μl, while the normal erythrocyte counts range from 7 x106/μl to 13 x106/μl. We found that most of the CALM/AF10-MF positive mice showed a significant increase in the number of white blood cells (mean WBC count of 99.7×103/μl) (Figure 3.2-4), and a decrease in the number of red blood cells (mean RBC count of 2.6 x106/μl) (Figure 3.2-5) compared to the wild type and MIG transplanted mice.
WBC counts were performed using peripheral blood collected from the tail vein, at the time of euthanizing the mice. The WBC counts of the leukaemic C/A-MF primary mice were significantly higher than that of the wild type controls (p-value = 0.0002) and the MIG transplanted mice (p-value = 0.0011). One way ANOVA followed by Dunn’s multiple comparison test was performed using GraphPad prism® (192). Note– A log 10 scale was used for the Y-axis.
RBC counts were performed using peripheral blood collected from the tail vein, at the time of euthanizing the mice. The RBC counts from the primary C/A-MF mice were significantly reduced compared to the wild type controls (p-value = 0.0003) or the MIG transplanted mice (0.0040). One-way ANOVA and Dunn’s multiple comparison tests were performed using GraphPad prism®

CALM/AF10-MF induces two distinct leukaemia phenotypes

Flow cytometry is a powerful tool to identify and study heterogeneous populations of cells on the basis of the expression of various cell surface antigens (193). In order to characterise the C/A-MF positive leukaemias, cell suspensions of bone marrow (BM), splenocytes and peripheral blood mononuclear cells (PBMCs) from each of the leukaemic mice were stained with lineage specific antibodies and analysed by flow cytometry as described in section 2.2.3.4. Cell populations from four healthy, wild type mice were used as controls. For immunophenotyping the different cell populations (myeloid cells, B-cells, T-cells and stem cells), we used a panel of antibodies in three different cocktails. The antibody panel and the dilutions used for each antibody are listed in Table 3.2-3.
Different gating strategies were used for the analysis of the FACS data. Gates refer to an area of selection placed around a population of cells with common characteristics or similar cell surface expression. Before looking at the expression of different cell surface markers, we first wanted to exclude dead cells from our analysis. All our samples were stained with a live/dead fixable blue dye that allowed us to distinguish between the live and dead cell populations. Thus, we first gated on the live cells (Figure 3.2-6) and used this population for further examination.
Since we were primarily interested in the GFP (and hence C/A-MF expressing) positive cell populations, the next step was to gate on GFP positive cells within the live cell compartment. We analysed a total of 21 C/A-MF positive mice and found 19 of the 21 (Tu2-Tu12 and Tu14-Tu21) samples to be highly positive for GFP, with a mean GFP-positivity of 96.0%, 91.7% and 92.8% in the BM, spleen and peripheral blood, respectively (Figure 3.2-7a). One leukaemic sample (Tu13) had only 6%, 6.5% and 4% GFP positive cells in the BM, peripheral blood and spleen, respectively. However, this sample was also classified with the other GFP positive leukaemias (due to the phenotype as explained later in this section). One primary C/A-MF transplanted mouse (Tu1) seemed to have lost its GFP expression with time (97.2% GFP negative cells at the time of euthanasia- Figure 3.2-7b) but had developed leukaemic symptoms 156 days post transplantation.

Table of Contents
Abstract 
Acknowledgements 
Table of Contents
List of Figures 
List of Tables
Abbreviations
1 Introduction
1.1 The genetic basis of cancer
1.2 Oncogenes and tumour suppressor genes
1.3 Haematopoiesis
1.4 Acute myeloid leukaemia
1.5 The t(10;11)(p13;q14)
1.6 CALM, AF10 and CALM/AF10
1.7 Modelling AML
1.8 Next generation sequencing
1.9 Aim
2 Materials and Methods
2.1 Materials
2.2 Methods
3 Results 
3.1 Validation of stable expression cell lines
3.2 Establishing and characterising murine CALM/AF10-MF leukaemias
3.3 Identifying potential co-operating mutations by whole exome sequencing .
3.4 Studying clonal evolution in the murine CALM/AF10-MF leukaemias
3.5 Determining the frequency of leukaemia stem cells
4 Discussion
4.1 Establishing a clinically relevant, CALM/AF10 driven model of AML
4.2 Expression of CALM/AF10-MF leads to development of leukaemia with a short median  latency and 100% penetrance
4.3 Expression of C/A-MF leads to development of myeloid and biphenotypic leukaemias
4.4 The C/A-MF bone marrow transplantation model recapitulates the genetic heterogeneity observed in human leukaemias .
4.5 V(D)J rearrangements and clonality of the disease
4.6 Integration sites as markers of clonality
4.7 Mutational patterns of clonal evolution
4.8 Characterising the leukaemia stem cell
5 Future Directions 
5.1 Transcriptome analysis of the murine leukaemias
5.2 Gene-editing experiments to functionally validate the identified driver mutations
5.3 Using the leukaemia models for pre-clinical studies
5.4 Monitoring leukaemias in vivo
5.5 Further characterisation of leukaemia stem cells
6 Significance 
7 References

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Recapitulating Genetic Diversity, Clonal Evolution and Stem Cell Dynamics of Human Acute Leukaemia in a Mouse Model using the CALM/AF10 Fusion

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