Establishing Murine AML Models Suitable for the Study of Clonal Evolution, Leukaemia Stem Cells and the Testing of New Treatment Strategies

Abstract

The stepwise acquisition of genetic changes and selection of the fittest clones result in clonal heterogeneity in leukaemia. In this study, we characterised a previously established murine bone marrow transplantation leukaemia model (MBMTLM) driven by the CALM-AF10 minimal fusion gene (CA-MF; the leukaemogenic part of the CALM-AF10 fusion gene). We had previously determined the leukaemia stem cell (LSC) frequency (LSC-F) of the primary leukaemia using limiting dilution assay (LDA) and established serially bulk transplanted leukaemias. Additional LDAs, showed that the LSC-F of our leukaemia did not change during serial transplantation. However, we observed varying latencies and varying B220 expression in the tertiary LDA leukaemias. We showed that the LSC-F of the B220-positive compartment of a leukaemia with high B220 expression was similar to the LSC-F of the B220-negative compartment of a leukaemia with low B220 expression. Therefore, unlike previous studies, which suggested that LSCs in CALMAF10- driven leukaemias are enriched in the B220-positive compartment, our results indicate that the LSCs in CALM-AF10 leukaemias can also be found enriched in the B220-negative compartment of the same leukaemia. Splinkerette and whole-exome sequencing analyses demonstrated that, although the primary leukaemia had originated from a single transduced cell, this leukaemia had undergone clonal evolution and had three distinct subclones. We showed that the subclones differed in their proliferative potential and B220 expression levels. Our results suggest a complex and dynamic clonal architecture with high heterogeneity in our CA-MF-driven MBMTLM. Moreover, using RNA-seq analysis, we found aberrant expression of genes reported to be dysregulated in CALM-AF10 patients, suggesting that our model recapitulates human CALM-AF10 leukaemias. To study the interaction of the immune system with leukaemia, we established a transplantable immunocompetent CA-MF MBMTLM expressing SIINFEKL, an immunogenic model antigen. Using this model, we observed that an intact immune system could eliminate a large number of AML cells if the cells express an immunogenic antigen. We also established a transplantable immunocompetent CA-MF MBMTLM expressing luciferase in the leukaemia cells, which can be used to track leukaemia development in vivo using non-invasive bioluminescent imaging in the living mouse.