Partially or Fully BCR-ABL Independent Mechanisms of in Vitro Resistance to Ponatinib

Four BCR-ABL+ CML cell lines (K562, AR230, BV173, and 32D(BCR-ABL)) were maintained in liquid culture containing ponatinib (0.1 nM) for 10 days. The ponatinib concentration was increased in small increments for a minimum of 90 days, yielding corresponding ponatinib-resistant cell lines. BCR-ABL kinase domain sequencing of sensitive and resistant cells confirmed BCR-ABL to be unmutated. Real-time qPCR was used to compare the expression of BCR-ABL in ponatinib-sensitive and -resistant cell lines. Immunoblot analysis (total and tyrosine-phosphorylated BCR-ABL) was used to the compare levels of BCR-ABL protein and to determine whether resistance to ponatinib corresponded with reduced (partially BCR-ABL-independent) or complete inhibition of BCR-ABL tyrosine phosphorylation (fully BCR-ABL-independent). Cell proliferation assays were performed on resistant and sensitive cell lines in the presence of ponatinib, nilotinib, and dasatinib. A small-molecule inhibitor screen composed of >90 cell-permeable inhibitors that collectively target the majority of the tyrosine kinome as well as other kinases (Blood 116, 2010, abstract 2754) is currently being applied to the 32D(BCR-ABL)^R cell line in the presence of 24 nM ponatinib to assess synthetic lethality, with results analyzed using a companion drug sensitivity algorithm. As a second strategy to generate resistant lines, N-ethyl-N-nitrosourea (ENU) mutagenesis was done to investigate BCR-ABL kinase domain-mediated resistance in myeloid K562, AR230, BV173, and 32D(BCR-ABL) cells. After ENU exposure, cells were washed and cultured in 96-well plates with escalating ponatinib.

The four BCR-ABL+ cell lines initially grew in the presence of 0.1 nM but not 0.5 nM ponatinib. Upon gradual exposure to escalating ponatinib, each of the cell lines exhibited a degree of adaptation to growth in the presence of the inhibitor (range: 10 to 240-fold). Real-time qPCR showed a modest two-fold increase in BCR-ABL expression level in K562^R, AR230^R and BV173^R cell lines relative to the respective parental lines. Based on immunoblot analysis, cell lines segregated into two categories of ponatinib resistance: partially (K562^R and AR230^R) or fully BCR-ABL-independent (BV173^R and 32D(BCR-ABL)^R). Cell proliferation assays showed that ponatinib resistant cell lines also exhibited resistance to nilotinib and dasatinib. The 32D(BCR-ABL)^R cell line exhibited a level of ponatinib resistance comparable to that of the Ba/F3 BCR-ABL^E255V cell line, which carries the most ponatinib-resistant BCR-ABL mutation. BCR-ABL tyrosine phosphorylation was efficiently blocked by low concentrations of ponatinib (<5 nM) in the 32D(BCR-ABL)^R cell line, yet these cells remained viable in the presence of up to 24 nM ponatinib. The effects of providing a second kinase inhibitor along with ponatinib (24 nM) in order to probe for synthetic lethality are under study. Possible involvement of a second, moderately ponatinib-sensitive target is suggested by the sharp ponatinib maximum at 24 nM; even 26 nM ponatinib is toxic to 32D(BCR-ABL)^R cells. Thus far, ENU mutagenesis screens in human CML cell lines failed to yield resistant clones and only a few were recovered from the murine 32D(BCR-ABL)^R cell line (3/1440 wells; the only BCR-ABL mutant recovered was BCR-ABL^L387F).

The ponatinib resistant, BCR-ABL+ cell lines described here exhibit either a partially or fully BCR-ABL independent mechanism of resistance. The molecular details of both processes will be reported, with an emphasis on the striking level of resistance (240-fold over starting conditions) exhibited by the 32D(BCR-ABL)^R cell line. Our in vitro results indicate that BCR-ABL independent mechanisms may contribute to ponatinib resistance in myeloid CML cells.

No relevant conflicts of interest to declare.