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Modeling the influence of stromal microenvironment in the selection of ENU-induced BCR-ABL1 mutants by tyrosine kinase inhibitors.

Aggoune D, Tosca L, Sorel N, Bonnet ML, Dkhissi F, Tachdjian G, Bennaceur-Griscelli A, Chomel JC, Turhan AG - Oncoscience (2014)

Bottom Line: These drugs are ineffective on the T315I gatekeeper substitution, which remains sensitive to 3(rd) generation TKI ponatinib.Using array-comparative genomic hybridization experiments, we found an increased number of variations (involving some recurrent chromosome regions) in clones cultured on MS-5 feeder.Overall, our study suggests that the hematopoietic niche could play a crucial role in conferring resistance to ponatinib, by providing survival signals and favoring genetic instability.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U935, F-86000 Poitiers, France.

ABSTRACT
Tyrosine kinase inhibitors (TKIs) have profoundly changed the natural history of chronic myeloid leukemia (CML). However, acquired resistance to imatinib, dasatinib or nilotinib (1(st) and 2(nd) generation TKIs), due in part to BCR-ABL1 kinase mutations, has been largely described. These drugs are ineffective on the T315I gatekeeper substitution, which remains sensitive to 3(rd) generation TKI ponatinib. It has recently been suggested that the hematopoietic niche could protect leukemic cells from targeted therapy. In order to investigate the role of a stromal niche in mutation-related resistance, we developed a niche-based cell mutagenesis assay. For this purpose, ENU (N-ethyl-N-nitrosourea)-exposed UT-7 cells expressing non-mutated or T315I-mutated BCR-ABL1 were cultured with or without murine MS-5 stromal cells and in the presence of imatinib, dasatinib, nilotinib, or ponatinib. In the assays relative to 1(st) and 2(nd) generation TKIs, which were performed on non-mutated BCR-ABL1 cells, our data highlighted the increasing efficacy of the latter, but did not reveal any substantial effect of the niche. In ponatinib assays performed on both non-mutated and T315I-mutated BCR-ABL1 cells, an increased number of resistant clones were observed in the presence of MS-5. Present data suggested that T315I mutants need either compound mutations (e.g. E255K/T315I) or a stromal niche to escape from ponatinib. Using array-comparative genomic hybridization experiments, we found an increased number of variations (involving some recurrent chromosome regions) in clones cultured on MS-5 feeder. Overall, our study suggests that the hematopoietic niche could play a crucial role in conferring resistance to ponatinib, by providing survival signals and favoring genetic instability.

No MeSH data available.


Related in: MedlinePlus

ENU mutagenized resistant clones from UT-7-11 cells treated with 1st and 2nd generation TKIs (imatinib, nilotinib and dasatinib) with or without the stromal cell line MS-5(A) Clones resistant to imatinib at a concentration of 2μM. (B) Clones resistant to nilotinib at a concentration of 75nM. (C) Clones resistant to dasatinib at a concentration of 10 nM. Each bar represents the number of clones of the indicated KD mutation. * Only one clone carried a compound mutation (G250E+E275K) and was counted in both bars in this condition. ** No outgrowth was observed in this condition; 24 clones were tested after 14 days of culture in methylcellulose.
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Figure 1: ENU mutagenized resistant clones from UT-7-11 cells treated with 1st and 2nd generation TKIs (imatinib, nilotinib and dasatinib) with or without the stromal cell line MS-5(A) Clones resistant to imatinib at a concentration of 2μM. (B) Clones resistant to nilotinib at a concentration of 75nM. (C) Clones resistant to dasatinib at a concentration of 10 nM. Each bar represents the number of clones of the indicated KD mutation. * Only one clone carried a compound mutation (G250E+E275K) and was counted in both bars in this condition. ** No outgrowth was observed in this condition; 24 clones were tested after 14 days of culture in methylcellulose.

Mentions: For 1st and 2nd generation TKIs, the mutation profile appeared almost similar whether or not MS-5 was present. A majority of clones harbored p-loop mutations (G250E, Q252H, Y253H, E255K) or the T315I substitution (Fig. 1). Some other mutations (E275K, E279K, E281K, H396R) were rarely detected. In the nilotinib assay, clones from methylcellulose culture (with MS-5) appeared somewhat enriched in T315I (Fig. 1B). Concerning dasatinib selection, a predominance of the gatekeeper mutation was observed with or without MS-5 feeder (Fig. 1C). Mutation profiles with UT-7-315 and ponatinib selection displayed interesting data. As expected, all resistant clones carried the T315I substitution. In many cases, in addition to the gatekeeper mutation, direct sequencing detected BCR-ABL1 KD mutations located within the p-loop (G250E, Y253F/H, E255K) or elsewhere (E279K, F359C, L384M). When UT-7-315 cells were cultured in the absence of MS-5, clones with compound mutation (T315I + another mutation) represented the majority of resistant clones (Fig. 2A). Conversely, in the presence of MS-5 cells, resistant clones mainly harbored the T315I mutation alone. Consequently, the mutation profiles appeared different according to MS-5 condition. Finally, resistant UT-7-11 cells selected by ponatinib in MS-5 cocultures carried the gatekeeper substitution alone in most cases, whereas no outgrowth was observed without MS-5 (Fig. 2B). Compound mutations were found in only two clones (T315I/G250E, T315I/E255K).


Modeling the influence of stromal microenvironment in the selection of ENU-induced BCR-ABL1 mutants by tyrosine kinase inhibitors.

Aggoune D, Tosca L, Sorel N, Bonnet ML, Dkhissi F, Tachdjian G, Bennaceur-Griscelli A, Chomel JC, Turhan AG - Oncoscience (2014)

ENU mutagenized resistant clones from UT-7-11 cells treated with 1st and 2nd generation TKIs (imatinib, nilotinib and dasatinib) with or without the stromal cell line MS-5(A) Clones resistant to imatinib at a concentration of 2μM. (B) Clones resistant to nilotinib at a concentration of 75nM. (C) Clones resistant to dasatinib at a concentration of 10 nM. Each bar represents the number of clones of the indicated KD mutation. * Only one clone carried a compound mutation (G250E+E275K) and was counted in both bars in this condition. ** No outgrowth was observed in this condition; 24 clones were tested after 14 days of culture in methylcellulose.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4295758&req=5

Figure 1: ENU mutagenized resistant clones from UT-7-11 cells treated with 1st and 2nd generation TKIs (imatinib, nilotinib and dasatinib) with or without the stromal cell line MS-5(A) Clones resistant to imatinib at a concentration of 2μM. (B) Clones resistant to nilotinib at a concentration of 75nM. (C) Clones resistant to dasatinib at a concentration of 10 nM. Each bar represents the number of clones of the indicated KD mutation. * Only one clone carried a compound mutation (G250E+E275K) and was counted in both bars in this condition. ** No outgrowth was observed in this condition; 24 clones were tested after 14 days of culture in methylcellulose.
Mentions: For 1st and 2nd generation TKIs, the mutation profile appeared almost similar whether or not MS-5 was present. A majority of clones harbored p-loop mutations (G250E, Q252H, Y253H, E255K) or the T315I substitution (Fig. 1). Some other mutations (E275K, E279K, E281K, H396R) were rarely detected. In the nilotinib assay, clones from methylcellulose culture (with MS-5) appeared somewhat enriched in T315I (Fig. 1B). Concerning dasatinib selection, a predominance of the gatekeeper mutation was observed with or without MS-5 feeder (Fig. 1C). Mutation profiles with UT-7-315 and ponatinib selection displayed interesting data. As expected, all resistant clones carried the T315I substitution. In many cases, in addition to the gatekeeper mutation, direct sequencing detected BCR-ABL1 KD mutations located within the p-loop (G250E, Y253F/H, E255K) or elsewhere (E279K, F359C, L384M). When UT-7-315 cells were cultured in the absence of MS-5, clones with compound mutation (T315I + another mutation) represented the majority of resistant clones (Fig. 2A). Conversely, in the presence of MS-5 cells, resistant clones mainly harbored the T315I mutation alone. Consequently, the mutation profiles appeared different according to MS-5 condition. Finally, resistant UT-7-11 cells selected by ponatinib in MS-5 cocultures carried the gatekeeper substitution alone in most cases, whereas no outgrowth was observed without MS-5 (Fig. 2B). Compound mutations were found in only two clones (T315I/G250E, T315I/E255K).

Bottom Line: These drugs are ineffective on the T315I gatekeeper substitution, which remains sensitive to 3(rd) generation TKI ponatinib.Using array-comparative genomic hybridization experiments, we found an increased number of variations (involving some recurrent chromosome regions) in clones cultured on MS-5 feeder.Overall, our study suggests that the hematopoietic niche could play a crucial role in conferring resistance to ponatinib, by providing survival signals and favoring genetic instability.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U935, F-86000 Poitiers, France.

ABSTRACT
Tyrosine kinase inhibitors (TKIs) have profoundly changed the natural history of chronic myeloid leukemia (CML). However, acquired resistance to imatinib, dasatinib or nilotinib (1(st) and 2(nd) generation TKIs), due in part to BCR-ABL1 kinase mutations, has been largely described. These drugs are ineffective on the T315I gatekeeper substitution, which remains sensitive to 3(rd) generation TKI ponatinib. It has recently been suggested that the hematopoietic niche could protect leukemic cells from targeted therapy. In order to investigate the role of a stromal niche in mutation-related resistance, we developed a niche-based cell mutagenesis assay. For this purpose, ENU (N-ethyl-N-nitrosourea)-exposed UT-7 cells expressing non-mutated or T315I-mutated BCR-ABL1 were cultured with or without murine MS-5 stromal cells and in the presence of imatinib, dasatinib, nilotinib, or ponatinib. In the assays relative to 1(st) and 2(nd) generation TKIs, which were performed on non-mutated BCR-ABL1 cells, our data highlighted the increasing efficacy of the latter, but did not reveal any substantial effect of the niche. In ponatinib assays performed on both non-mutated and T315I-mutated BCR-ABL1 cells, an increased number of resistant clones were observed in the presence of MS-5. Present data suggested that T315I mutants need either compound mutations (e.g. E255K/T315I) or a stromal niche to escape from ponatinib. Using array-comparative genomic hybridization experiments, we found an increased number of variations (involving some recurrent chromosome regions) in clones cultured on MS-5 feeder. Overall, our study suggests that the hematopoietic niche could play a crucial role in conferring resistance to ponatinib, by providing survival signals and favoring genetic instability.

No MeSH data available.


Related in: MedlinePlus