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By itself, the Y253F mutation dysregulates the kinase, and transforms them to IL-3 independence, contributing to the resistance to Imatinib.The kinase dysregulation and Imatinib resistance induced in c-ABl by Y253F mutation seemed independent of one another.
Their results show that dysreguation of the Abl kinase activity by SH3 mutation (P112L) or by Bcr fusion (in Bcr-abl) increases the sensitivity to Imatinib. But that Y253F mutation conferred intrinsic
resistance to Im as an unphosphorylated protein (inactive form) with
IC50= 8.8 uM. (They had purified active from inactive forms).
The phosphorylated (or active) forms were significantly resistant to Imatinib with a 5-fold increase over that of unphosphorylated (inactive form).
So they also tested the Y253F in the active form, to ascertain that resistance to Im was not due to residual active form, by making a dual mutation Y253F and Y393F. (Y393 is the tyrosine that is phosphorylated in the active form. Note Y393F does not affect c-Abl sensitivity to IM nor its kinase activity)
The double mutant was still resistant to Im - this shows that Y253F reduces intrinsic sensitivity to IM indeoendent of phosphorylation of Y393.
The in vivo response of Ph+ cells to Im reflect a balance, wherein the IM acts to trap the Abl in the unphosphorylated form, and prevent reactivation by autophosphorylation.
They believe that the 5uM Im concentrations they had determined to significantly inhibit Abl with the y253F mutations, is theroetically achievable by dose escalation, based on Ph 1 pharmacokinetics data.
Although their detailed molecular studies were focused on the Y253F mutation, they expect the Y253H mutations to have similar responses. (I believe that the Y253H mutation is a bit better though, one must look at the possible H-interactions closely, to see if Histidine is capable of H interactions with the Asp 322 that would help increase intractions with Im which is Tyr 253's role. Phenylalanine without a group capable of the H-bond interaction would thus be expected to be more severe.)
Although this paper was discussing Y253 mutations, its study on purified active and inactive forms has further given me a better understanding of Im's phenomenal selectivity and efficacy.
Eva
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