Drug tolerance development of mouse Bcr/Abl pre-B ALL cells on irradiated MEFs

Although cure rates for acute lymphoblastic leukemia (ALL) have increased, development of resistance to drugs and patient relapse are common. The environment in which the leukemia cells are present during the drug treatment is known to provide significant survival benefit. Here, we have modeled this process by culturing murine Bcr/Abl-positive acute lymphoblastic leukemia cells in the presence of stroma while treating them with a moderate dose of two unrelated drugs, the farnesyltransferase inhibitor lonafarnib and the tyrosine kinase inhibitor nilotinib. This results in an initial large reduction in cell viability of the culture and inhibition of cell proliferation. However, after a number of days, cell death ceases and the culture becomes drug-tolerant, enabling cell division to resume. We used gene expression profiling to analyze changes in the transcriptome of these leukemia cells over a 3-4 week period, taking samples at the start, the point at which most of the leukemia cells had been eradicated while a small percentage survived, and at the end when the cells were proliferating again. We used two different pre-B ALL cell lines 8093 and Bin-2, derived from two BCR/ABL transgenic mice. 8093 is a leukemia on an inbred f11 C57Bl/6J background whereas Bin2 was derived from ALL on a mixed genetic background. Each was treated with the tyrosine kinase inhibitor nilotinib = AMN107 (20 nM for 8093, 50 nM for Bin-2, abbreviated with nil or n) or with the farnesyltransferase inhibitor (FTI) Lonafarnib/SCH66336 (1 mM for 8093, 0.25 mM for Bin-2; abbreviated with lon or l) in the presence of an irradiated mouse embryonic fibroblast feeder layer. Cells loosely attached to the top of the feeder layer or present in the medium were harvested for RNA isolation. Except where noted, all samples were taken in biological triplicates (separate plates). Samples were taken at t=0 (begin/start); on day 3 for Bin-2 (nil and lon) when the viability was 5-10% based on Trypan blue exclusion, on d4 for 8093 (lon, viability 20%) or d3 (nil, 5-10% viability) at the midpoint when cells start to develop resistance, and on d30 (Bin-2 x lon), d21 (Bin-2 x nil), d26 (8093 x lon) and d 20 (8093 x nil) when the viability of the culture was completely restored (around 90% viability), and the cells started proliferating agin in the presence of the drug. Cells were kept in the presence of drug throughout the entire treatment, which was added fresh with medium changes. The same feeder layer was kept during the entire period.