The BCR-ABL^35INS insertion/truncation mutant is kinase-inactive and does not contribute to tyrosine kinase inhibitor resistance in chronic myeloid leukemia

Imatinib is an inhibitor of BCR-ABL, the tyrosine kinase that causes chronic myeloid leukemia (CML). Most newly diagnosed patients achieve durable remissions on imatinib therapy,^1,2  but 10%-15% fail to respond or relapse. The leading cause of imatinib resistance is reactivation of BCR-ABL because of kinase domain point mutations. Most BCR-ABL mutants are susceptible to alternative ABL tyrosine kinase inhibitor (TKI) therapies.^3-8  Sequencing of the BCR-ABL kinase domain in patients exhibiting signs of TKI treatment failure has also revealed the presence of alternatively spliced variants, including BCR-ABL^35INS, in which retention of 35 intronic nucleotides at the exon 8/9 splice junction introduces a stop codon after 10 intron-encoded residues.^9-13  The result is loss of the last 653 residues of BCR-ABL, including 22 native kinase domain residues.^10,12  Notably, the reported frequency of detection of the BCR-ABL^35INS mutant in cases of imatinib resistance (including instances in which a point mutation is concurrently detected in the BCR-ABL kinase domain) as detected by direct sequencing is ∼1%-2%,^10,14  although more sensitive quantitative assays have reported detection of very low levels of the mutant transcript at a considerably increased prevalence.¹⁴ 

Although BCR-ABL truncated immediately after the ABL kinase domain is fully transforming in a murine model of CML,¹⁵  we predicted BCR-ABL^35INS would lack kinase activity, because the mutation eliminates the last 2 helices of the ABL kinase domain and disrupts a complex set of interactions among noncontiguous residues.¹⁰  By contrast, recent reports have suggested that BCR-ABL^35INS confers TKI resistance in CML^9,12,14,16  and have proposed a BCR-ABL^35INS tailored clinical trial,¹⁶  but they have not addressed the mechanism for this or assessed BCR-ABL^35INS catalytic activity. We provide cell-based and biochemical studies of BCR-ABL^35INS and a retrospective analysis of its detection in the context of treatment and response in CML patients.

Ba/F3 cells cultured in standard media (RPMI 1640 media, 10% FBS, l-glutamine, penicillin-streptomycin; Invitrogen) containing IL-3 from WEHI-conditioned media were infected with retrovirus expressing BCR-ABL, BCR-ABL^35INS, or BCR-ABL^K271P/35INS (MSCV-IRES-GFP), and stable cell lines were sorted for GFP (FACSAria II; BD Biosciences). After IL-3 withdrawal, cells were counted daily.¹⁷ 

Ba/F3 parental cells and Ba/F3 cells expressing or coexpressing BCR-ABL, BCR-ABL^35INS, or BCR-ABL^K271P/35INS were boiled for 10 minutes in SDS-PAGE loading buffer. Lysates were separated on 4%-15% Tris-HCl gels, transferred, and immunoblotted with antibodies for the BCR N-terminus (3902; Cell Signaling Technology), ABL C-terminus (24-11; Santa Cruz Biotechnology), phospho-ABL (Y412 [1b numbering] and Y393 [1a numbering]; Cell Signaling Technology), or α-tubulin (T6074; Sigma-Aldrich).

Ba/F3 BCR-ABL cells were infected with retrovirus carrying BCR-ABL^35INS, BCR-ABL^K271P/35INS, or empty vector (MSCV-IRES-GFP), and cells were sorted by FACS for GFP. Resultant cell lines were plated in escalating concentrations of imatinib in quadruplicate, and proliferation was assessed after 72 hours. Analogous experiments were conducted with transfected, GFP-sorted K562 cells.

Autophosphorylation assays that used GST-ABL (residues 220-498), GST-ABL^35INS (220-474, then YFDNREERTR-STOP),^10,12  and GST-ABL^K271R/35INS were initiated with [γ-³²P]-ATP and quenched with SDS-PAGE loading buffer after 0-60 minutes, and proteins were separated on a 4%-15% Tris-HCl SDS-PAGE gel.⁵  Gels were imaged with a storage phosphor screen (Typhoon 9400; GE Healthcare). Transferred gels were immunoblotted with ABL antibody Ab-2 (Oncogene Science) to assess protein loading.

Peptide-substrate phosphorylation assays that used GST-ABL, GST-ABL^35INS, and GST-ABL^K271R/35INS and a peptide substrate (biotin-GGEAIYAAPFKK-amide; New England Peptides) were initiated with [γ-³²P]-ATP, quenched with guanidine hydrochloride (7M),⁵  spotted onto duplicate SAM2 Biotin Capture membranes (Promega), washed according to the manufacturer's instructions, and counted. Enzyme concentrations were matched on the basis of Bradford analysis.

Inclusion in the analysis required informed consent in accordance with the Declaration of Helsinki, a CML diagnosis, treatment with ABL TKIs, detection of BCR-ABL^35INS, and availability of clinical histories. All experiments with patient materials were approved by the Institutional Review Board of the Oregon Health and Science University (OHSU). Bone marrow or peripheral blood samples were collected at OHSU as clinically indicated during treatment. Direct BCR-ABL kinase domain sequencing was performed¹⁰  and reported by the OHSU Knight Diagnostic Laboratories or MolecularMD Corporation.

Modeling studies^9,14  and clinical reports^12,14,16  have implicated BCR-ABL^35INS as a potential mediator of resistance to ABL TKIs. However, critical mechanistic examination of BCR-ABL^35INS in vitro and clinical analysis in the context of response status and time of resistance are lacking.

We tested the ability of Ba/F3 cells that expressed BCR-ABL or BCR-ABL^35INS to grow without IL-3. As negative controls, we also tested parental Ba/F3 cells and Ba/F3 cells that expressed the BCR-ABL^35INS mutant coupled with an additional point mutation of residue K271 that disrupts a critical salt bridge with E286 and precludes adoption of the active conformation of the kinase (BCR-ABL^K271P/35INS).^18,19  Only Ba/F3 BCR-ABL cells were rendered growth factor independent (Figure 1A). Immunoblotting confirmed the absence of the C-terminus of ABL in Ba/F3 BCR-ABL^35INS cell lysate and further showed no phosphorylation of Y393, a critical activation loop residue that controls switching between active and inactive conformations^7,20-23  (Figure 1B). Lee et al¹⁴  reported that imatinib resistance depends on BCR-ABL^35INS expression level; however, we found that high-level coexpression of BCR-ABL^35INS in Ba/F3 BCR-ABL cells did not influence imatinib sensitivity (Figure 1C-D); analogous results were obtained with K562 cells transfected with BCR-ABL^35INS (supplemental Figure 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article). In addition,Y393 of BCR-ABL^35INS was not phosphorylated when coexpressed with native BCR-ABL, consistent with disruption of kinase domain architecture.

To evaluate catalytic competency, we compared GST-ABL, -ABL^35INS, and -ABL^K271R/35INS in autophosphorylation (Figure 1E) and peptide-substrate phosphorylation assays (Figure 1F). No autophosphorylation was detectable for ABL^35INS after 60 minutes, whereas full phosphorylation of ABL was reached by 15 minutes (Figure 1E). In the peptide-substrate assay, the extent of phosphorylation by ABL^35INS was < 2% of the total phosphorylation by native ABL (Figure 1F).

As reported previously, we detect the BCR-ABL^35INS mutant transcript by direct sequencing at our institution in ∼1.7% of all cases of suspected imatinib resistance.¹⁰  Analysis of 20 CML patients whose sequencing traces included evidence of BCR-ABL^35INS at least once revealed that BCR-ABL^35INS was demonstrably not the cause of disease progression or TKI resistance in 16 (80%) of 20 cases (Figure 2A; supplemental Table 1): 9 were responding to therapy at the time of BCR-ABL^35INS detection (Figure 2B), 3 experienced treatment failure because of TKI intolerance, 1 lost response on dasatinib interruption (patient 16) but recaptured major molecular response on dasatinib resumption, and 3 (patients 15, 18, and 20) harbored a predominant, concurrent point mutation that adequately explained TKI resistance (Figure 2C). Thus, only 4 (20%) of 20 patients (patients 13, 14, 17, and 19) harboring BCR-ABL^35INS at any point during therapy had this mutation exclusively detected at the time of resistance while undergoing ABL TKI therapy (Figure 2D).

It has been suggested that increased BCR-ABL^35INS expression may lead to imatinib resistance.¹⁴  However, we observed that BCR-ABL^35INS was detected as a minor component (≤ 20% of total signal) by direct sequencing in 18 of 20 patients; both occurrences as a major component (> 20% of total signal) were in imatinib responders (Figure 2A). Furthermore, in patients for whom serial sequence samples were available, BCR-ABL^35INS never emerged as a dominant clone (supplemental Table 1). Lastly, for all patients for whom sufficient material was available from sequencing time points at which BCR-ABL^35INS was detected, the same 35INS mutation was concurrently detected in native c-ABL (6/6 [100%]; supplemental Figure 2). Because of the retrospective nature of the present study and sample availability, this analysis could not be performed in the 2 patients who were imatinib responders and yet showed the BCR-ABL^35INS mutation as a major component by direct sequencing (patients 5 and 6). Others have detected variant ABL transcripts at similar frequencies in CML patients and healthy individuals, which implies the existence of alternative splicing mechanisms of ABL unrelated to TKI resistance.^11,24-26 

In total, the results of the present study demonstrate that BCR-ABL^35INS lacks the qualities of a functional tyrosine kinase at the cellular and biochemical level. An alternative possibility, that BCR-ABL^35INS is catalytically inactive but sequesters TKIs, is untenable on stoichiometric grounds and excluded by our experiments that demonstrated that coexpression of BCR-ABL and BCR-ABL^35INS in Ba/F3 cells does not mitigate imatinib sensitivity compared with Ba/F3 cells that express only BCR-ABL. Another possibility is that BCR-ABL^35INS heterodimerizes with BCR-ABL and maintains it in an imatinib-inaccessible active conformation; however, the present results demonstrate that BCR-ABL^35INS lacks the necessary kinase activity to maintain BCR-ABL in an activated state and that BCR-ABL^35INS is not susceptible to Y393 phosphorylation when expressed alone or with BCR-ABL. On the basis of our biochemical and cellular data and retrospective clinical analysis, we conclude that BCR-ABL^35INS is kinase-inactive, does not contribute to TKI resistance in vitro, does not explain or consistently track with time of resistance in CML patients, and should not be considered in treatment decisions.

The authors thank Jamshid Khorashad for critical evaluation of the manuscript and Oliver Hantochel for helpful discussions on BCR-ABL structural issues.

This work was supported by grants from the National Institutes of Health/National Cancer Institute (5 R01 CA65823), the Leukemia & Lymphoma Society (Specialized Center of Research 7393-06), and Howard Hughes Medical Institute.

National Institutes of Health

Contribution: T.O., M.S.Z., C.A.E., A.A., L.T.A., H.Y., A.S.C., F.Y., and J.T. performed research; T.O., M.S.Z., C.A.E., R.D.P., M.W.D., and B.J.D. designed research and analyzed data; R.D.P, V.M.R., and S.W. contributed vital reagents and analytical tools; T.O., M.S.Z., C.A.E., and B.J.D. wrote the paper; and all authors reviewed the manuscript.

Conflict-of-interest statement: V.M.R. is an employee of ARIAD Pharmaceuticals Inc; J.T. and S.W. are employees of MolecularMD Corporation; and OHSU and B.J.D. have a financial interest in MolecularMD. Technology used in this research has been licensed to MolecularMD; this potential conflict of interest has been reviewed and managed by the OHSU Conflict of Interest in Research Committee and the Integrity Program Oversight Council. The remaining authors declare no competing financial interests.

Correspondence: Thomas O'Hare, Huntsman Cancer Institute, University of Utah, 2000 Circle of Hope, Salt Lake City, UT 84112; e-mail: Thomas.OHare@hci.utah.edu.