Investigation of the Biological Differences between Bcr-Abl Kinase Mutations Resistant to Imatinib.

Background: Bcr-Abl kinase domain (KD) mutations are a major cause of acquired resistance to imatinib. In some patients KD mutations have been detected prior to therapy, and P-loop mutations confer a particularly poor prognosis, suggesting an association with disease progression independent of imatinib therapy. We therefore compared the transforming potential of several frequent KD mutants with wild type (WT) Bcr-Abl in a variety of in vivo and in vitro assays.

Experimental: We compared the growth rate of BaF3 cells expressing WT or KD mutants of Bcr-Abl (Q252H, Y253F, E255K, T315I, M351T, F359V, H396P, H396R) under optimal (10% FBS) and low serum conditions (1% FBS) over an 18-day period. In 10% FBS, all mutants except Y253F, showed a decreased growth rate relative to WT. However in low serum all mutants except Q252H, M351T and F359V and H396P had an increased growth rate relative to WT. To further validate the differences in growth rates direct competition assays were done. Equal cell numbers of WT and mutant were plated in the same flask in media containing either 10% or 1% FBS. The proportion of mutant clone was monitored by PCR amplification of Bcr-Abl and direct sequencing. In 10% FBS only Y253F dominated WT. In contrast all mutants except M351T were able to outgrow WT in 1% FBS. To compare the transformation potency of mutant vs. WT Bcr-Abl in primary cells, WT and several mutants were introduced in the MIG1 retroviral vector. Bone marrow cells from Balb/c mice were infected with retroviral supernatant containing comparable numbers of infectious particles. For assessment of B cell transformation, transduced cells from non 5-FU treated mice were plated in Whitlock-Witte cultures under limiting dilution conditions. The trend for B-cell transformation potential was Y253F>E255K>WT>T315I>H396P>M351T. To compare the potency of the mutants for induction of myeloproliferative disease, lethally irradiated recipients were transplanted with transduced cells from 5-FU treated animals. The mice were sacrificed when the white blood cell count exceeded 200/nl or the animals appeared moribund. In WT animals disease latency was approximately 19 days with median survival 23 days. No significant differences between the mutants and WT were observed, although there was a trend toward longer survival in mice with the M351T and H396P mutations. To screen for biochemical differences between the mutants, we compared the activation of signaling pathways in BaF3 cells expressing mutant and WT Bcr-Abl, including Akt, Erk, Jnk, CrkL, Cbl, Stat3 and Stat5, but failed to detect differences. However, significant differences in the banding patterns were observed on phosphotyrosine blots from cellular lysates.

Conclusion: Our data suggest that the P-loop mutants Y253F and E255K have greater transforming potential than WT, whereas M351T and H396P are less potent. Our inability to detect significant differences in the murine leukemia disease model may be due to the rapid course of disease that precludes the detection of more subtle differences. Our data are consistent with the clinical observation of the poor prognosis of patients with P-loop mutations and the reversal to WT Bcr-Abl on imatinib withdrawal that has been observed in some patients with non P-loop mutations. In addition, the ability of certain mutants to outcompete WT under suboptimal conditions may allow cells harboring these mutations to populate less hospitable niches in the bone marrow, until selected by imatinib.