Understanding the proliferative and self-renewal potential of different leukaemic stem cell populations

Abstract

Although contemporary treatment cures most children with ALL, with survival rates in the region of 90%, treatment outcomes in certain cytogenetic subgroups remain poor. Patients with such abnormalities have a greatly increased risk of relapse and re-treatment responses are poor. Understanding the mechanisms of resistance in these patients is therefore a priority in the search for improvements in chemotherapy. The translocation t(17;19)(q22;p13) is a rare cytogenetic abnormality, which occurs in less than 1% of childhood ALL and is associated with a very poor prognosis and chemotherapy resistance. The translocation results in the fusion of E2A on chromosome 17 with HLF on chromosome 19. It has been commonly assumed that, following relapse, ALL cells will divide more rapidly than at presentation. To test this hypothesis, competitive transplantation studies were performed using paired presentation and relapse samples from a case of t(17;19)-positive ALL in a NSG xenograft mouse model. Surprisingly mice engrafted with the presentation cells survived significantly shorter (p<0.05, logrank test) than those with relapse cells, indicating the aggressiveness and rapid proliferation of presentation cells, while the survival curves of mice engrafted with various proportions of presentation and relapse cells had intermediate levels of survival. Administration of dexamethasone prolonged the survival of mice engrafted with the presentation cells and those with various proportions of cells, with no effect on survival of mice with relapse cells. Flow cytometry analysis showed that a high percentage (70%-95%) of human leukaemic cells engrafted in bone marrow and spleen across all groups tested. RT-PCR amplification identified this t(17;19) case to be a Type 1 E2A/HLF fusion. In order to determine the genes responsible for chemo-insensitivity, whole genome SNP array analysis was performed on the matched presentation and relapse samples which demonstrated deletion of glucocorticoid receptor (NR3C1) gene in relapse cells which is not seen in presentation cells, confirmed by fluorescence in situ hybridisation. This deletion was used to identify the relapse cells (R) in mice engrafted with mixtures of presentation and relapse cells. FISH analysis showed the percentages of presentation cells (P) were higher than the ratios of the cell initially transplanted. In concordance, real-time PCR analysis showed high levels of NR3C1 in all mix cells ratios. These results indicated that presentation cells proliferate more rapidly and outgrow relapse cells in competitive clonal repopulation experiments. Dexamethasone treatment reduced the percentages of presentation cells in all mix populations with high significance (p<0.01, t-test) in the 30%-P+70%-R ratio. Levels of NR3C1 in all mix cell populations were significantly depleted (p<0.05, t-test) by dexamethasone. To be able to physically track the proliferation of leukemic cells populations in vivo and to follow progression of the disease, two lentiviruses pSLIEW (expressing firefly luciferase and green fluorescent protein) and pSRLICW (expressing Renilla luciferase and mCherry fluorescent protein) were generated. pSRLICW was just successfully generated but has not yet been tested. Lentivirus pSLIEW functionality was validated by transducing various leukaemic cell lines. By assessing the percentage of green fluorescence (gfp) cells, transduction efficiencies in SEM and RS4;11 (ALL cell lines) were 45% and 5% respectively, whereas transduction efficiencies in Kasumi-1 and SKNO-1 (AML cell lines) were 72% and 98% respectively, higher than the ALL cell lines. Transduced SEM and Kasumi-1 lines were sorted for higher gfp populations for xenograft purposes. Real time bioluminescence imaging on mice xenografted with sorted transduced SEM cells showed rapid progression of the disease in the systemic while Kasumi-1 cells xenografted in mice produced localised tumours and progressed much slower. These lentiviral-transduced cell lines xenografts have proven the in vivo monitoring capability by real time luminescence imaging. The information gained from this project study provides novel insights into the mechanisms of relapse in childhood ALL. The growth characteristics of the leukaemic blasts should be considered in assigning patients to different therapeutic options.