CLL Xenograft Models to Address the Impact of Single Gene Loss (ATM) on Proliferation and Cytotoxic Response

Abstract 2439

Chronic lymphocytic leukaemia (CLL) is a malignancy characterised by gradual accumulation of mature B cells in peripheral lymphoid organs. This accumulation is a consequence of both prolonged survival but also active proliferation restricted to a small population of tumour cells. Access to the proliferating tumour population in CLL patients is limited and there is a need for establishment of human CLL xenograft models that can recapitulate human disease. Several xenograft models of CLL have been already established, both with primary tumours and CLL cell lines, and they exhibit either a limited length of engraftment or tissue distribution that only partially recapitulates CLL disease. Furthermore, there is an absence of CLL xenograft models that address the impact of a single gene loss that confers a poor clinical outcome. The purpose of this study was to a) establish a range of CLL xenografts that exhibit different kinetics and distribution of CLL engraftment b) develop individual xenograft models with isogenic CLL cell lines, with and without the principal DNA damage response gene ATM whose loss confers chemoresistance, c) obtain simultaneous engraftment of differentially labelled ATM null and ATM wild type isogenic CLL cells and d) demonstrate that such CLL xenograft models can be used to measure the cytotoxic impact of new treatments. Initially, we addressed the kinetics of engraftment and tissue distribution of the following CLL cell lines: MEC-1 (Stacchini A at al. Leuk Res. 1999; 23:127–36), HG3 (Klein and Rosen, in prep), CII (Najfeld V et al.Int J Cancer. 1980;26:543–549) and PGA (Lewin N et al. Int J Cancer. 1988;41:892–895) administered intravenously into NOD/SCID mice. Engraftment and tissue distribution varied considerably between the different cell lines, with CII recapitulating CLL most closely. Detectable levels of peripheral blood engraftment of all cell lines occurred within 2–4 weeks. Mice engrafted with HG3 cells rapidly succumbed to CLL engraftment, surviving for a maximum of 6 weeks, whilst engraftment of other CLL cell lines was more prolonged (PGA up to 13 weeks; CII up to 16 weeks; MEC-1 up to 21 weeks). MEC-1 and CII cells were found to reside mainly in haemopoietic organs such as spleen, bone marrow and blood of injected animals, whereas HG3 and PGA showed a wider distribution that included kidneys, liver, uterus, gastrointestinal tract, bladder and neck. We subsequently established a subcutaneous model of the CLL cell line CII that both recapitulated CLL disease by dissemination into lymphoid organs but also allowed direct measurement of tumour size. Solid subcutaneous tumours were evident within 2 weeks and engraftment in the peripheral blood, spleen and bone marrow was demonstratable within 5 days of subcutaneous cell injection. In order to recapitulate the effect of ATM loss on engraftment and proliferation we simultaneously injected isogenic cell line pairs (MEC-1, HG3, CII), with and without stable ATM knock down. Isogenic cell line pairs were differentially labelled with either SNARF (ATM wild-type) or CFSE (ATM null), mixed in equal numbers (3×10⁶) and intravenously injected into multiple NOD/SCID mice. The relative proportion of each cell type in the peripheral blood was assessed weekly by FACS analysis and using an IVIS whole body imager. We observed a predominance of ATM null CLL cells (MEC-1, HG3, CII) in the peripheral blood and spleen within three weeks of engraftment, whereas equivalent levels of ATM null and control isogenic cells were found in the bone marrow. Finally, to determine whether the CLL xenografts could serve as a valid models for testing new CLL treatments, the effect of a PARP inhibitor (Olaparib) on tumour load and animal survival was examined in a MEC-1 xenograft with stable ATM knockdown. Daily oral administration of olaparib (100mg/kg/mouse) to mice engrafted with ATM null MEC-1 cells compared to the vehicle-treated animals resulted in a reduction in the tumour load both in the bone marrow and to a lesser extent the spleen with concurrent lengthening of overall survival. We conclude that xenograft models with isogenic CLL cell lines provide a robust system where the impact of a single gene loss on engraftment, proliferation and treatment response can be addressed.