Genomic DNA isolated from K562-NIL[r] cells.
K562-NIL[r] cells are 15-fold more resistant to NILOTINIB than parental K562 cells: In vitro IC50s are 87 nM vs 5.9 nM, respectively.
Genomic DNA (at least 100 µg) isolated from proliferating K562-NIL[r] cells established after 8 months of continuous in vitro exposure of parental K562 cells to NILOTINIB.
K562-NIL[r] cells are 15-fold more resistant to NILOTINIB than the parent K562 cells. The NILOTINIB IC50s for K562-NIL[r] and parental cells are 87 nM and 5.9 nM, respectively (MTT assay).
Phenotype: CD1-, CD2-, CD3-, CD4-, CD5-, CD7-, CD8-, CD9+, CD11b(+), CD13+, CD14-, CD15+ CD28-, CD10-, CD19-, CD20-, CD21-, CD22-, CD25, CD33+, CD34-, CD36-, CD38-, CD41+, CD42b-, CD44+, CD45+, CD56-, CD57-, CD61(+), CD65+, CD71+, CD80+, CD86-, CD117(+), CD122+, CD132+, CD235a/GlyA+, PPO-, vWF-, HLA-DR-, TdT-, EPO-R+, TCRα/β-, TCRγ/δ- (summarized from Drexler and Wehler).
Genotype: (COSMIC database, October 2014):
Mutated genes: 97 point mutations, inserts and deletions reported. Key lesions are the BCR/ABL1 fusion and CDK2NA mutation (c.151_457del307).
K562-NIL[r] cells authenticated by STR analysis to verify both resistant and parent cells are K562.
Target: Inhibits the tyrosine kinase domains of ABL1, BCR-ABL1, KIT, LCK, EPHA3, EPHA8, DDR1, DDR2, PDGFRB, MAPK11 and ZAK.
Alternative ID: Tasigna, AMN107; CAS #: 641571-10-0 (free base); Mol. weight: 529.5245 g/mol; Supplier: LC Labs; Lot #: BNL-108.
Genomic DNA was purified from a culture of proliferating K562-NIL[r] cells derived from a K562-NIL[r] Working Cell Bank (WCB) using DNAzol reagent (Invitrogen) and ethanol precipitation.
Cell Line Development: Parental K562 cells from a cryopreserved WCB were seeded in RPMI-1640 tissue culture medium containing serum and inorganic supplements. Cells were incubated at 37°C in an atmosphere of 5% CO2 in humidified air. The resistant cell line was established after 8 months of culture during which cells were incubated with a progressively increasing concentration of NILOTINIB. A Master Cell Bank (MCB) of K562-NIL[r] cells was then cryopreserved. A WCB of K562-NIL[r] cells was prepared from Master Cell Bank (MCB) cells cultured in the presence of NILOTINIB.
Genomic DNA isolated from K562-NIL[r] cells
1. Identify genetic or epigenetic changes associated with resistance
Genomic DNA from K562-NIL[r] cells may be used to identify gene mutations or epigenetic changes associated with resistance to NILOTINIB. Cells bearing the native BCR/ABL1 locus may provide a more clinically-relevant model to explore mutation development than cells (e.g. Ba/F3) transduced by BCR/ABL1 (1,2).
2. Drug development to overcome NILOTINIB resistance
Identification of a gene mutation or epigenetic change associated with resistance to NILOTINIB may enable drug development aimed at inhibiting expression of the mutated gene, inhibiting function of the mutated gene product, or reversal of the epigenetic change.
RNA and cell lysate preparations from K562-NIL[r] cells are also available to further characterize molecular changes associated with NILOTINIB resistance.
ChemoScreen can test client compounds for inhibitory activity against proliferating K562-NIL[r] cells in vitro or in a rodent tumor model. Alternatively, clients can license K562-NIL[r] cells from ChemoScreen.
3. Diagnostic test for NILOTINIB resistance
Identification of the molecular basis for resistance to NILOTINIB facilitates development of a diagnostic test for resistance to NILOTINIB.
4. Development of intellectual property
Identification of a gene mutation or epigenetic change acquired during development of resistance to NILOTINIB in vitro might enable development of a proprietary position to support development of technologies for diagnosis or treatment of NILOTINIB-resistant tumors.
Relevance of acquired resistance in vitro versus in a patient
There is extensive literature reporting common genetic changes associated with resistance to a chemotherapeutic compound acquired in vitro with those occurring in patients treated with the same compound. This means that K562-NIL[r] cells may provide a relevant model of acquired resistance to NILOTINIB. This model bearing endogenous BCR/ABL1 might also confer a more clinically-relevant mutation profile compared with models using transduced BCR/ABL1.