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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE37286 | GEO | 2012/09/30

SECONDARY ACCESSION(S): PRJNA159405

REPOSITORIES: GEO

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Project description: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.

Project description:Aplidin (plitidepsin) is a novel antitumor agent isolated from the Mediterranean tunicate Aplidium albicans. Aplidin has shown impressive in vitro and in vivo activity against various cancer cell lines. Based on the results from several laboratories Aplidin has recently been granted orphan drug designation for the treatment of acute lymphoblastic leukemia (ALL) and Multiple Myeloma (MM) by the European Commision and Food and Drug Administration. Aplidin has selective cytotoxicity in vitro towards leukemia cells as compared to hematopoeitic progenitors and lacks cross-resistance with other known cytotoxic drugs with the exception of gemcitabine (Leukemia, 2003, Bresters, Broekhuizen). Here, a systematic study of drug combinations with Aplidin, for possible use in hematological malignancies was undertaken. The combination of cytarabine (AraC) and Aplidin was found to be synergistic in three cell lines tested: CCRF-CEM, a human T-cell lymphoblastic leukemia cell line, K562, a acute mylelogenous leukemia cell line and SKI-DLCL, a human Diffuse Large Cell Lymphoma cell line. The combination was further tested in the CCRF-CEM and the SKI-DLCL xenograft model in SCID mice. In an attempt to understand the basis of Aplidin action (alone and in combination) on leukemia and lymphoma cells in vitro and in vivo, gene expression profiling was carried out. Results indicate that multiple cellular pathways are effected by treatment with Aplidine including MAPK signaling, WNT signaling, cell cycle and ATP synthesis. Additionally we have found that that cells lacking functional mitochondria are resistant to Aplidin. Mitochondrial membrane potential and reactive oxygen spicies production was increased following Aplidin treatment but the overall synthesis of ATP was decreased. Addition of AraC to Aplidin resulted in depolarization of mitochondrial membrane and even further increase of ROS production. AraC alone did not affect the cellular ATP pool thus significantly potentiating Aplidin induced apoptosis. Our studies suggest that cancer cell mitochondria may be a target of Aplidin. Based on these studies, a phase I study of the combination of Aplidin and AraC is planned for the treatment of patients with relapsed leukemia. Overall design. Overall goal of the experiment was to obtain molecular insight into the mechanism of action of Aplidin alone as well as its combination with AraC. SKI-DLCL cells were treated in vitro with IC50 doses for Aplidin, AraC or Aplidin+AraC combination. Each samples was done in biological triplicates. Total number of samples was 12. Alternatively, SKI-DLCL cells were injected into scid mice and when the tumor was pulpable animals were given a single dose of Aplidin 1mg/kg and AraC 50mg/kg in combination. On day eight after drugs administration tumors were harvested and total RNA was isolated. No replicates are available. Data was validated using semi-quantitative RT-PCR

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

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