Sensitivity to Imatinib In BCR-ABL1-Positive Chronic Myeloid Leukemia Cells Depends on the Presence of Normal ABL1.
Authors: Dasgupta Yashodhara , Virgili Anna , Koptyra Mateusz , Nacheva Elisabeth P. , Skorski Tomasz 
Published: October 2019

Abstract
Abstract 3405

Background:
BCR-ABL1 encoding for oncogenic tyrosine kinase 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). The second (wild-type) ABL1 and BCR alleles in CML-CP cells remain intact on the non-rearranged homologues of chromosome 9 and 22, respectively. Accordingly, CML-CP cells at early stages express both forms of the ABL1 kinase, oncogenic BCR-ABL1 and normal ABL1. ABL1 tyrosine kinase inhibitors (TKIs) such as imatinib, dasatinib and nilotinib revolutionized the treatment of CML-CP, but they do not eradicate leukemia. Patients who do achieve complete cytogenetic remission (CCyR) may eventually stop responding and acquire resistance to TKIs, which may lead to disease relapse and malignant progression. Mutations in the sequence encoding BCR-ABL1 kinase have been detected in approximately 50% of CML-CP patients resistant to TKIs, but other factors contributing to this phenomenon are poorly characterized.

Results:
Here, we identified a novel mechanism of TKI resistance: loss of the remaining normal ABL1 allele resulting from cryptic deletion in the 9q34 region in the normal chromosome 9 [del(9)(q34)]. Using bacterial artificial chromosome probes (BACs) for dual color/dual probe fluorescent in situ hybridization (D-FISH), and oligonucleotide array comparative genomic hybridization (aCGH) we show that genomic deletion of ABL1 allele in non-translocated chromosome 9 acquired during TKI therapy in CML-CP patients was associated with resistance to imatinib and dasatinib. del(9)(q34) was detected in approximately 10% of the patients who initially failed to achieve CCyR within 12 months of TKI treatment. Moreover, BCR-ABL1-positive Abl1-/- murine leukemia cells were refractory to imatinib in comparison to BCR-ABL1–positive Abl1+/+ counterparts as indicated by persistent BCR-ABL1 –mediated tyrosine phosphorylation, lack of BCR-ABL1 protein degradation, increased cell survival and clonogenic activity. Expression of exogenous ABL1 kinase in BCR-ABL1–positive Abl1-/- cells restored their sensitivity to imatinib. These results provide direct evidence that Abl1 plays a crucial role in regulation of the sensitivity of BCR-ABL1-positive leukemia cells to imatinib. ABL1 is regarded as a cell cycle regulatory and pro-apoptotic protein, thus antagonistic to BCR-ABL1. The cell cycle inhibitory activity is independent of ABL1 kinase, whereas the pro-apoptotic function is dependent on its kinase activity. However, acquired resistance to TKIs caused by a loss of the wild-type ABL1 kinase does not appear to depend directly on the lack of ABL1-induced cell cycle arrest and/or apoptosis. In contrast to BCR-ABL1 –positive Abl1+/+ leukemia cells, imatinib exerted only modest effect on BCR-ABL1 kinase-dependent tyrosine phosphorylation and did not downregulate BCR-ABL1 protein in Abl1-/- leukemia cells, suggesting the role of Abl1 in cellular uptake of the drug and/or BCR-ABL1 degradation. Expression of imatinib cellular importer Oct-1 and cellular exporters Abcb1 and Abcg2 does not appear to favor the resistance to TKI in BCR-ABL1-positive Abl1-/- cells, but the impact of ABL1 on intracellular metabolism of imatinib cannot be excluded. In addition, expression of chaperone protein Hsp90, which protects BCR-ABL1 from proteasomal degradation, is not affected by Abl1. However, >3-fold downregulation of cathepsin B may be responsible for lack of degradation of BCR-ABL1 protein in imatinib-treated Abl1-/- cells.

Conclusions:
Altogether, it can be postulated that loss of expression of ABL1 kinase plays an important role in TKI resistance in CML. It can be achieved by interstitial deletion in chromosome 9 [del(9)(q34)] causing loss of normal ABL1 allele, which could be eventually combined with epigenetic silencing of the alternative ABL1 promoter retained in the CpG island of the BCR-ABL1 gene in t(9;22)(q34:q11). Detection of the del(9)(q34) is beyond the resolution of conventional karyotyping currently used to monitor TKI treatment response. In contrast, D-FISH using commercially available probes can identify such loss in both quiescent and dividing cells. In summary, downregulation of ABL1 caused by del(9)(q34) may serve as an important prognostic factor and have a significant impact on CML treatment.

Disclosures:
No relevant conflicts of interest to declare.