Clonal Evolution and Blast Crisis Correlate with Enhanced Proteolytic Activity of Separase in BCR-ABL b3a2 Fusion Type CML under Imatinib Therapy

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.

c-MYB is a transcriptional regulator of ESPL1/Separase in BCR-ABL-positive chronic myeloid leukemia

“…In search for transcriptional mechanisms that could explain the IM-associated downregulation of Separase protein levels we analyzed the conditional context between c-MYB expression, Separase and IM treatment. Confirming and expanding previously published work we performed cell culture experiments on four human cell lines [ 13 , 35 ]. Of these, U937 cells served as model for leukemic but BCR-ABL-negative cells.…”

“…Recently, we have reported that enhanced rates of acquired chromosomal aberrations, clonal evolution and fast disease progression (time to BC) in CML patients undergoing long-term IM treatment correlate with enhanced proteolytic activity of Separase in the respective in vitro models [ 35 ]. Mechanistically, this was linked to a BCR-ABL-dependent regulatory feedback mechanism that posttranslationally stimulates Separase proteolytic activity after IM-induced decreases of Separase expression in b3a2 BCR-ABL fusion type CML cell lines [ 35 ]. To date, it was unclear what underlying transcriptional or translational mechanism may be involved in the IM-dependent regulation of Separase expression in BCR-ABL-positive cells.…”

Separase activity distribution can be a marker of major molecular response and proliferation of CD34+ cells in TKI-treated chronic myeloid leukemia patients

“…In previous work, we found that the posttranslationally stimulated separase proteolytic activity under imatinib treatment rendered b3a2 fusion type-related CML cells more prone to aneuploidy and clonal evolution, thereby confirming cytogenetic findings within a cohort of 1151 Philadelphia chromosome-positive chronic phase patients of the randomized CML-study IV that were examined on the incidence of newly arising additional chromosomal alterations (ACAs) under prolonged imatinib treatment. This points to an influence of high separase activity for the induction of gross genomic mutations in CML cells [30].…”

“…Moreover, unscheduled (cell cycle uncoupled) activation of separase can lead to aberrant high numbers of centrosomes (i.e., centrosome amplification) and subsequently to a defective mitotic spindle apparatus [25]. Both defects cause the emergence of aberrant karyotypes (aneuploidy), a hallmark of most advanced human malignancies [24,[26][27][28][29][30]. Overexpression of separase in the mammary gland of a MMTV-ESPL1 mouse model led to the development of highly aneuploid mammary carcinomas with high levels of chromosomal instability and aggressive disease phenotypes [31].…”

Separase: Function Beyond Cohesion Cleavage and an Emerging Oncogene

“…Overexpression of separase in transgenic MMTV-Espl1 mouse model leads to aggressive mammary adenocarcinomas with an 80% penetrance [Mukherjee et al, 2014b]. Around 5.4-fold increase in separase activity under imatinib treatment was observed in b3a2 cell lines [Haaß et al, 2015a]. Mutation in separase leads to increased genetic instability and predisposition to epithelial cancer in zebrafish [Shepard et al, 2007].…”