Investigating leukaemic propagation in childhood acute lymphoblastic leukaemia

Abstract

Childhood acute lymphoblastic leukaemia (ALL) does not possess a propagating cell hierarchy, at least as defined by B-cell precursor immunophenotype. Indeed, many, or even all, leukaemic blasts may have the potential to propagate the disease. This unusual characteristic mirrors the substantial capacity for clonal expansion demonstrated by fully differentiated normal lymphoid cells. This Fellowship aimed to investigate the genetic programmes underlying the propagation of acute lymphoblastic leukaemia. An initial candidate approach confirmed the expression of PIWIL2, a gene critical to the maintenance of germline stem cells, in both cell line and primary ALL. Knockdown of PIWIL2 resulted in reduced cellular proliferation and significant prolongation of doubling time in two ALL cell lines, SEM (MLL/AF4) and 697 (E2A/PBX1). Unexpectedly, PIWIL2 was also found to be expressed in peripheral lymphoid cells from healthy donors, but not terminally differentiated cells of myeloid origin, suggesting that PIWIL2 may have a previously unidentified function in both normal and malignant lymphoid cells. A second project has developed an in vitro genome-wide RNAi screen to identify candidate genes involved in the clonal propagation of ALL. This project has assessed a serial re-plating assay using feeder cell co-culture to provide a surrogate niche environment. Initial results have demonstrated the feasibility of such an approach. The benefit of using a co-culture re-plating assay, as compared to a standard suspension culture approach, remains under investigation. ii Finally, this Fellowship developed a protocol for the lentiviral transduction of patient-derived leukaemic blasts and cloned and validated a novel lentiviral vector capable of in vitro analysis, in vivo disease monitoring and RNAi. With these, it will now be possible to validate candidate leukaemic propagation genes in vivo, using primary leukaemic material. The results of these studies will provide candidates for the development of novel therapeutic agents for children with ALL.