Comparison of the frequency and characterization of mutant BCR-ABL transcripts in CD34⁺ cells from IM responders and nonresponders. (A) BCR-ABL kinase domain transcript fragments in extracts of patients CD34⁺ cells were cloned and sequenced (10-30 clones per sample), and the frequency of mutant clones detected in each patients' sample was then calculated. Cross bars indicate the mean plus or minus SEM of data for each patient group. The difference between the frequencies of mutant transcripts detected in the IM nonresponders' cells (10 patients) and the 10 responders' cells (10 patients) is significant (P < .003). No mutations were found in CD34⁺ BM cells from 6 normal persons. (B) Locations of the single amino acid substitutions identified in relation to subdomains of the BCR-ABL kinase domain. The prevalence of each mutation among the 20 patients studied is shown as the proportion of patients in which each mutation was seen. Red letters indicate amino acid changes previously associated with IM resistance in patients. P-Loop indicates phosphate-binding loop; C-helix, catalytic domain; SH3 contact, Src homology 3 domain contact; IM binding site, imatinib binding site; SH2 domain, Src homology domain contact; and A-loop, activation loop. (C) Localization of the residue V304 based on the crystal structure of the c-ABL kinase domain in complex with IM (PDB code 1IEP).³²  Substitution of V304 with a polar residue aspartic acid (V304D) would perturb the conformation and/or structure of the helix that forms part of the IM-binding groove. (D) Frequency of various mutant BCR-ABL transcripts in CD34⁺ CML cells before and after IM treatment from 2 IM nonresponders calculated as described for panel A.