Chronic myeloid leukemia in chronic phase (CML-CP) cells that harbor oncogenic BCR-ABL1 and normal ABL1 allele often become resistant to the ABL1 kinase inhibitor imatinib. Here, we report that loss of the remaining normal ABL1 allele in these tumors, which results from cryptic interstitial deletion in 9q34 in patients who did not achieve a complete cytogenetic remission (CCyR) during treatment, engenders a novel unexpected mechanism of imatinib resistance. BCR-ABL1–positive Abl1−/− leukemia cells were refractory to imatinib as indicated by persistent BCR-ABL1–mediated tyrosine phosphorylation, lack of BCR-ABL1 protein degradation, increased cell survival, and clonogenic activity. Expression of ABL1 kinase, but not a kinase-dead mutant, restored the antileukemic effects of imatinib in ABL1-negative chronic myelogenous leukemia (CML) cells and in BCR-ABL1–positive Abl1−/− murine leukemia cells. The intracellular concentration of imatinib and expression of its transporters were not affected, although proteins involved in BCR-ABL1 degradation were downregulated in Abl1−/− cells. Furthermore, 12 genes associated with imatinib resistance were favorably deregulated in Abl1−/− leukemia. Taken together, our results indicate that loss of the normal ABL1 kinase may serve as a key prognostic factor that exerts major impact on CML treatment outcomes. Cancer Res; 71(16); 5381–6. ©2011 AACR.

BCR-ABL1 results from t(9;22)(q34;q11) reciprocal translocation or variants generating the Philadelphia chromosome (Ph), which initiates chronic myeloid leukemia in chronic phase (CML-CP) cells. The second (wild-type) ABL1 allele remains intact on the nonrearranged homologue of chromosome 9, and CML-CP cells at early stages express both forms of the ABL1 kinase, oncogenic BCR-ABL1 and normal ABL1 (1).

ABL1 and BCR-ABL1 can exert opposite effects on a variety of cellular functions (2). For example, BCR-ABL1 can act upstream and downstream of cytochrome c to inhibit apoptosis. In contrast, ABL1 kinase may facilitate apoptosis by stimulation of p73, p53, and caspase 9.

Tyrosine kinase inhibitor (TKI) imatinib revolutionized the treatment of BCR-ABL1–positive leukemias (3). The incidence of a continuous complete cytogenetic remission (CCyR) in CML-CP patients treated for 12 months with the drug was 66% (4). Mutations within the kinase domain of BCR-ABL1, overexpression of LYN kinase, and loss of p53 and BCR-ABL1 amplification were implicated in the lack of achieving CCyR (5).

Here we show that loss of expression of normal ABL1 kinase due to cryptic deletion in remaining normal chromosome 9 [del(9q34)] reduced the sensitivity of BCR-ABL1 leukemia cells to imatinib and may contribute to drug resistance in chronic myeloid leukemia (CML) patients.

Chromosome and whole genome analysis of CML-CP samples

Bone marrow cells (BMC) of CML-CP patients who failed to achieve CCyR within 12 months of TKI treatment were obtained after informed consent and analyzed at presentation and at 3 monthly intervals by G-banding and dual color/dual fusion probe FISH (D-FISH) with a range of bacterial artificial chromosome (BAC) probes to detect the loss of normal 9q34 (6). DNA from BMCs of patients 1 and 3 was also analyzed by array comparative genomic hybridization (aCGH) using DNA Analytics (105K Agilent) and Formatter software (7). All genome addresses are derived from NCBI36/hg18 (March 2006) of the Human Genome. Additional information about the patients and cytogenetic and molecular results are described in Supplementary Materials and Supplementary Table S1.

Cells

Abl1+/− mice were kindly obtained from Dr. A.J. Koleske (Department of Molecular Biophysics and Biochemistry, Yale University) and bred to obtain −/− and +/+ littermates. Animal studies were approved by the Institutional Animal Care and Use Committee at Temple University. p210BCR-ABL1–positive growth factor–independent leukemia cells were generated by retroviral infection of Abl1+/+ and Abl1−/− BMCs with pMIG-BCR-ABL1-IRES-GFP retroviral construct as previously described (8). CML-BP LAMA84R and KCL22 cell lines were described before (9, 10). LAMA84R and KCL22 cells, and green fluorescent protein (GFP+) BCR-ABL1–positive Abl1−/− leukemia cells were infected with pKI retroviral construct encoding YFP-ABL1 fusion protein or kinase-dead YFP-ABL1 (K290R) mutant (kindly obtained from Dr. Koleske). GFP, yellow fluorescent protein (YFP), and YFP/GFP-positive cells were sorted and expanded in growth factor–free conditions.

Sensitivity to imatinib

Cells were treated with imatinib (Novartis Pharma) and evaluated by clonogenic assay as described before (8). TKI-resistant BCR-ABL1 kinase mutations were not detected in cells used for these experiments.

Imatinib retention

Radiolabeled drug uptake was carried out using 14C-labeled imatinib (Novartis) as previously described with modifications (11). Briefly, 2 × 106 cells were incubated with 1.6 μmol/L 14C-labeled imatinib (3,052 MBq/mg) at 37°C for 2 hours. After incubation, the cells were washed twice with ice-cold PBS and incubated in culture medium at 37°C for another 15 minutes. Cell pellet was then solubilized in 50 μL of distilled water and radioactivity was counted using β-counter (Perkin Elmer).

Protein expression

Total cell lysates were analyzed by Western blotting using primary antibodies recognizing ABL1, Abcb1, CHIP, and tubulin (Calbiochem); phosphotyrosine (Upstate), Bag1, Cbl, and GFP (Santa Cruz Biotechnology); Oct-1 (Novus Biologicals); Abcg2, HSP90, and cathepsin B (Abcam Inc.); and Hsc70 (Enzo Life Sciences International, Inc.) as described previously (8).

Genome-wide expression array

Affymetrix Mouse gene 1.0ST array containing 28,815 probe sets (Affymetrix) was used to measure mRNA expression levels. Affymetrix arrays were processed by Partek Genomic Suite at the Penn Molecular Profiling Facility–Bioinformatics Group (University of Pennsylvania) to determine whether a given transcript was present and if there were consistently significant differences between BCR-ABL1–positive Abl−/− and BCR-ABL1–positive Abl+/+ BMC based on 3 separate experiments. Genes were considered to have a significant differential expression between the 2 groups when displayed a False Discovery Rate (FDR) not exceeding 5% and a cut-off value more than 1-fold (upregulated or downregulated).

Using G-banding, D-FISH, and aCGH, we detected that 3 CML patients who initially failed to achieve CCyR within 12 months of TKI treatment acquired a cryptic deletion in 9q34 region in the normal chromosome 9 [del(9q34)] resulting in the loss of normal ABL1 allele (Figs. 1 and 2, Supplementary Table S1 and Fig. S1). Two of these are among 21 CML-CP patients without CCyR on first line TKI, we analyzed in the lab. Importantly, in addition to the cryptic loss at 9q34.1, all 3 patients showed karyotype evolution—from the presence of a second Ph (Fig. 2A) to multiple numerical and structural aberrations (Supplementary Table S1).

In patient 1, FISH analysis revealed an aberrant signal pattern due to a missing ABL1 signal, which was mapped to the morphologically normal chromosome 9 indicating a cryptic deletion [Fig. 1A (iii) and 1B]. In patient 2, the deletion was detectable by G-banding and assessed as del(9q31;q34) (Supplementary Table S1). In patient 3, the loss of ABL1 was revealed by aCGH and confirmed by FISH mapping (Fig. 1B, Supplementary Fig. S1). aCGH results for the 9q34 region in patient 1 (blue) and patient 3 (red) show the extent of the cryptic deletions [Fig. 1B (i)]. The common loss is defined proximally by the ABL1 breakpoint in patient 3 and distally by the telomeric breakpoint in patient 1 [arrow in Figs. 1B (i), and 2C and D, and Supplementary Fig. S1]. The estimated size of the common genome loss is 567 kb, which includes ABL1 exons a2 to a11 together with downstream sequences encompassing the LAMC3 and NUP214 genes. These deletions differ significantly from the deletions at der(9), where the genome loss involves only ABL1 exons 1a and 1b and spans toward the centromere [Fig. 1A (ii) and B (ii)].

The observed loss of the wild allele of ABL1 is an evolutionary event as evidenced by the presence of Ph(+) cells with and without ABL1 deletion in patient 1 (Fig. 2B). Furthermore, it is the ABL1-deficient cell clone that sustains the disease progression by acquiring a second copy of Ph (Fig. 2A, B, and D). Our observation combined with other report that inhibition of Abl1 kinase compromises genomic stability suggests that loss of ABL1 not only decreases imatinib sensitivity but also promotes accumulation of chromosomal aberrations (12).

Cryptic deletions in 9q34 causing the loss of normal ABL1 allele may be underreported in CML-CP patients probably because they would be missed unless either D-FISH or aCGH have been used for monitoring therapy response. In concordance, similar del(9q34) was found in several CML-BP cell lines (Supplementary Table S2). To detect loss of the ABL1 signal from the normal 9 homologue, FISH using a BCR-ABL1-ASS tricolor dual fusion translocation probe could be recommended (Supplementary Fig. S2), which produces unique signals for the translocation products and the normal nonrearranged loci at 9q34 and 22q11. Importantly, identification of del(9q34) using any of the 2 double fusion BCR-ABL1 probe sets is as reliable on interphase cells as on chromosome preparations. In contrast, the popular ES-FISH probe creates in BCR-ABL1–positive cell with ABL1 loss a signal pattern (1R1G1F) that cannot reliably differentiate the “wild” ABL1 allele from the ABL1-BCR fusion (Supplementary Fig. S2) thus misreporting del(9q34) as deletions at der(9).

To prove that loss of ABL1 directly contributes to imatinib resistance, BCR-ABL1 was expressed in Abl1−/− and +/+ BMCs. The absence of Abl1 reduced the sensitivity of BCR-ABL1 leukemia cells to imatinib whereas expression of YFP-ABL1 fusion kinase, but not the kinase-dead YFP-ABL1(K290R) mutant, in BCR-ABL1–positive Abl1/ leukemia cells restored antileukemia effect of the drug (Fig. 3A). YFP-ABL1, but not YFP-ABL1 (K290R), also increased imatinib sensitivity in drug-resistant CML-BP cell line LAMA84R (ABL1-negative, Supplementary Table S2; Fig. 3B). Because BCR-ABL1 gene amplification and overexpression of the multidrug resistance P-glycoprotein was observed in LAMA84R cells, loss of ABL1 may collaborate with other genetic/epigenetic abnormalities to promote drug resistance in CML-BP (9). Moreover, expression of YFP-ABL1 kinase in KCL22 CML-BP cells [relatively low ABL1 expression (10)] increased their sensitivity to imatinib suggesting that increased BCR-ABL1:ABL1 ratio observed during the course of disease can also limit the effect of imatinib (Fig. 3C; ref. 13).

The fact that ABL1 kinase may regulate the sensitivity of CML cells to imatinib is rather unexpected because BCR-ABL1 and ABL1 kinases are equally sensitive to imatinib in vitro (3). However, inhibition of intracellular ABL1 kinase usually requires higher concentration of the drug in comparison to BCR-ABL1 kinase; in addition ABL1 may work in a kinase-independent manner (14, 15). Moreover, imatinib-induced inhibition of BCR-ABL1 kinase is associated with release of ABL1 from the complex with 14-3-3 sigma, which promotes ABL1 relocation to the nucleus (triggers p73-dependent apoptosis), to the mitochondrial membranes (causes the loss of mitochondrial membrane potential), and to the complex with caspase 9 (activates caspase cascade) (2, 16). In summary, the presence of ABL1 kinase may exert a significant impact on anti-CML effect of imatinib. This speculation is supported by the observation that expression of YFP-ABL1 fusion kinase, but not its kinase-dead K290M mutant restored sensitivity to imatinib in BCR-ABL1–positive Abl1−/− leukemia cells and LAMA84R (ABL1-negative) CML-CP cells (Fig. 3A and B).

In Abl1−/− leukemia cells, imatinib displayed reduced capability to inhibit BCR-ABL1 kinase-mediated tyrosine phosphorylation and to induce BCR-ABL1 protein degradation in comparison with Abl1+/+ counterparts and Abl1−/− leukemia reconstituted with YFP-ABL1 (Fig. 4A). Moreover, genome-wide array confirmed imatinib-resistant signature of BCR-ABL1–positive Abl1/ cells by detecting deregulated expression of 12 genes previously reported in imatinb-resistant CML patients (Fig. 4B; refs. 17, 18). Intracellular retention of imatinib, and expression of drug importer Oct-1 and exporters Abcb1 and Abcg2 seem unaffected by Abl1 (Fig. 4C and D), but the impact of Abl1 on metabolism of imatinib cannot be excluded. On the other hand, more than 10-fold downregulation of Cbl E3 ligase, which induce ubiquitin-dependent degradation of “mature” BCR-ABL1 protein, and/or more than 3-fold downregulation of cathepsin B, which cleaves BCR-ABL1 may be responsible for lack of degradation of BCR-ABL1 protein in imatinib-treated Abl1/ cells (Fig. 4D; refs. 19, 20). Hsc70, Bag1, and E3 ligase CHIP responsible for degradation of “immature” BCR-ABL1 protein, and chaperone protein Hsp90 protecting BCR-ABL1 from proteasomal degradation, are not affected by Abl1 (Fig. 4D; ref. 19). Downregulation of BCR-ABL1 in imatinib-treated CD34+ CML-CP cells was implicated in regulating their sensitivity to the drug (20).

Altogether, it can be postulated that loss of expression of ABL1 kinase may contribute to imatinib resistance in CML-CP patients which do not achieve CCyR during 12 months on imatinib and eventually progress to CML-BP. ABL1 loss in CML-CP can be achieved by interstitial deletion in chromosome 9 [del(9q34)] causing a loss of normal ABL1 allele (this report), which may be combined with epigenetic silencing of the alternative ABL1 promoter retained in t(9;22) (13). Therefore, detection of del(9q34) may serve as an important prognostic factor and have a significant impact on CML treatment.

No potential conflicts of interest were disclosed.

The authors thank H. Mazzullo, J. Howard-Reeves, D. Brazma, C. Grace, and K. Gancheva for their contribution to the cytogenetic and aCGH analysis, L. Foroni for the BCR-ABL1 mutation analysis, and Dr. J. Tobias for help with statistical analysis of the microarray data.

Supported by the grants from NIH/NCI CA123014 (T. Skorski), Leukemia and Lymphoma Research Fund 05098 (E.P. Nacheva), and 1M19/NK1W/2009 (T. Stoklosa) and 1M19/WB1/2010 (E. Glodkowska-Mrowka) from Medical University of Warsaw.

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Supplementary data