A Subset of Chronic Myeloid Leukemia (CML) Patients on ABL Kinase Inhibitor Therapy Develop Point Mutations outside the BCR-ABL Kinase Domain That Decrease Drug Sensitivity and May Have a Role in Disease Progression.
Authors: Sherbenou Daniel W. , Hantschel Oliver , Willis Stephanie , Press Richard D. , Druker Brian J. , Superti-Furga Giulio , Deininger Michael W. 
Published: October 2019

Abstract
Background : Point mutations in the kinase domain (KD) of BCR-ABL are the most common mechanism of drug resistance in CML patients treated with kinase inhibitors, including imatinib (IM), dasatinib and nilotinib. It has also been shown through in vitro mutagenesis screening that mutations outside the KD in the neighboring linker, SH2, SH3 and Cap domains can confer IM resistance (Azam et al. Cell, 2003). These domains in c-ABL have an autoinhibitory function on the KD and mutations result in an activated enzyme (Hantschel et al. Cell, 2003). Despite these observations, mutations in these regions have not yet been reported in CML patients being treated with ABL kinase inhibitors.
Methods: Bone marrow and peripheral blood samples were obtained following informed consent from CML patients in chronic-phase (CP, n=43), accelerated-phase (AP, n=18) and blast crisis (BC, n=8). Likewise, patients with all degrees of IM response were studied, including 29 with a complete cytogenetic response (CCR), 22 never achieving CCR (nonresponders) and 18 after sustained loss of CCR (relapse). Nested RT-PCR for BCR-ABL was performed using 2 sets of primers from the breakpoint to the end of the KD. Sequencing covered the Cap, SH3, SH2, linker and KD. Mutations found outside the KD were introduced into Ba/F3 cells for testing of drug sensitivity.
Results: Seven novel mutations outside the KD were discovered in 5 of 69 (7%) patients tested, 4 of which also had KD mutations. By comparison, KD mutations were found in 22 (32%). Novel mutations were in SH2 (T212R, S154N and A196V), 2 in the linker (N231D and N231I), 1 in SH3 (K84N) and 1 in the Cap (R47C). Of 2 tested thus far, T212R and N231D both confer resistance to IM, nilotinib and dasatinib in cell proliferation assays. Based on modeling of the amino acid changes in the crystal structure of c-ABL, T212R is hypothesized to act by stabilizing the active conformation of the kinase, whereas N231D is hypothesized to destabilize the autoinhibited form.
Conclusions: (1) We demonstrate that mutations in the Cap, SH3, SH2 and linker domains of BCR-ABL occur in a subset of patients on IM. (2) In each case these were associated with a second mutation (inside or outside the KD), suggesting potential cooperativity. (3) Preliminary data suggest that non-KD mutations may generate a ’super-activated’ BCR-ABL kinase, conferring decreased sensitivity to inhibitors and increased intrinsic activity that may contribute to disease progression.
Characteristics of patients with mutations outside the KD. Sex/Age Disease Stage (diagnosis-study) IM Response (at study) Time on IM (months) Current Response KD Mutation Result Non-KD Mutation Result Fold Increase in IM Cellular IC50 NA: Not available. Percentages indicate the mutated/total sequence. 52/F CP CCR 18 CCR C305S(15%) N231D(100%) 1.6(N231D) 84/F CP CCR 40 Relapse WT T212R(100%),  S154N(50%) 4.1(T212R) 38/F CP CCR 42 CCR H396R(100%) A196V(40%) NA 72/M BC Relapse 67 NA H396P(100%),  Q346R(50%) K84N(50%),  R47C(50%) NA 54/F AP CCR 20 CCR E292V(100%) N231I(60%) NA