Abstract
Unbalanced (major route) additional cytogenetic aberrations (ACA) at diagnosis of chronic myeloid leukemia (CML) indicate an increased risk of progression and shorter survival. Moreover, newly arising ACA under imatinib treatment and clonal evolution are considered features of acceleration and define failure of therapy according to the European LeukemiaNet (ELN) recommendations. On the basis of 1151 Philadelphia chromosome positive chronic phase patients of the randomized CML-study IV, we examined the incidence of newly arising ACA under imatinib treatment with regard to the p210BCR-ABL breakpoint variants b2a2 and b3a2. We found a preferential acquisition of unbalanced ACA in patients with b3a2 vs. b2a2 fusion type (ratio: 6.3 vs. 1.6, p = 0.0246) concurring with a faster progress to blast crisis for b3a2 patients (p = 0.0124). ESPL1/Separase, a cysteine endopeptidase, is a key player in chromosomal segregation during mitosis. Separase overexpression and/or hyperactivity has been reported from a wide range of cancers and cause defective mitotic spindles, chromosome missegregation and aneuploidy. We investigated the influence of p210BCR-ABL breakpoint variants and imatinib treatment on expression and proteolytic activity of Separase as measured with a specific fluorogenic assay on CML cell lines (b2a2: KCL-22, BV-173; b3a2: K562, LAMA-84). Despite a drop in Separase protein levels an up to 5.4-fold increase of Separase activity under imatinib treatment was observed exclusively in b3a2 but not in b2a2 cell lines. Mimicking the influence of imatinib on BV-173 and LAMA-84 cells by ESPL1 silencing stimulated Separase proteolytic activity in both b3a2 and b2a2 cell lines. Our data suggest the existence of a fusion type-related feedback mechanism that posttranslationally stimulates Separase proteolytic activity after therapy-induced decreases in Separase protein levels. This could render b3a2 CML cells more prone to aneuploidy and clonal evolution than b2a2 progenitors and may therefore explain the cytogenetic results of CML patients.
Highlights
The BCR-ABL tyrosine kinase (TK) formed by the balanced translocation t(9;22)(q34;q11) is the “key player” in the pathogenesis of chronic myeloid leukemia (CML)
Genomic instability and aneuploidy are hallmarks of the progressing CML and concur with BCR-ABL mutations encoding resistance to TK inhibitor (TKI) and/or development of additional chromosomal aberrations (ACA) in addition to the Philadelphia chromosome (Ph) (= clonal evolution). [6,7] About 35% of patients in chronic phase (CP) develop resistance or intolerance to IM and frequently undergo clonal evolution. [8,9] While approximately 10–12% of patients in CML CP display ACA at diagnosis, this proportion of patients rises to approximately 30% and 80% in accelerated phase (AP) and blast crisis (BC), respectively. [10,11] Recently, we have shown that major route ACA at diagnosis are associated with a negative impact on survival and signify progression to AP and BC
Since chromosomal missegregations are the first line consequences of defective mitotic spindles concurring with supernumerous centrosomes, the observed ACA were further classified with respect to the types “balanced” or “unbalanced” (Table 2)
Summary
The BCR-ABL tyrosine kinase (TK) formed by the balanced translocation t(9;22)(q34;q11) is the “key player” in the pathogenesis of chronic myeloid leukemia (CML). [17] Recently, in a long-term in vitro study on a CML CP model we have established the functional link of p210BCR-ABL TK activity with centrosome amplification and clonal evolution. [31,35] Recently, striking functional evidence for the oncogenic activity of deregulated Separase was provided by Pati and coworkers who have generated a transgenic MMTV-Espl mouse model that overexpresses Separase protein in the mammary gland These mice developed aggressive and highly aneuploid mammary carcinomas with high levels of CIN and cell cycle defects including multiple centrosomes and multinucleated cells. [38,39,40,41] The finding that espl1/Separase acts as an oncogene/-protein in various cancers including CML renders this protease a key target to unravel the molecular mechanisms involved in the development of centrosome amplification and clonal evolution in IM-treated CML. As a known promoter of aneuploidy and clonal evolution hyperactive Separase is an excellent candidate for explaining the cytogenetic in vivo data
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