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.

Fig 1. Protein and activity levels of p210BCR-ABL and Separase expression in cell lines under investigation.

Protein levels of p210BCR-ABL (A), pCrkL (B) and Separase (C) based on densitometric evaluation of immunostained Western blots were normalized to Actin as loading control. Abl-related TK activity (BCR-ABL + c-ABL) was measured as pCrkL/Actin (B). Analyses were performed on protein lysates derived from p210BCR-ABL-positive (KCL-22, BV-173, LAMA-84, K562) and-negative non-malignant cells (NHDF, UROtsa). KCL-22 and BV-173 carry the bcr-abl breakpoint variant b2a2, whereas LAMA-84 and K562 display the b3a2 fusion gene variant. All values refer to that of K562 (= 100%).

Fig 2. Separase proteolytic activity and levels of master Separase proteolytic activity regulators in bcr-abl-negative (A) and–positive cell lines (B, C) treated with IM.

Cells were treated with IM for times and doses [μM] given on top. Separase proteolytic activity was quantified using an in vitro fluorometric assay and was given as relative fluorescence units/Actin (RFU/Actin). Analyses were performed on protein lysates derived from bcr-abl-negative (A) control cells (NHDF, UROtsa) and from p210BCR-ABL-positive cells with the bcr-abl breakpoint variant b2a2 (B) (KCL-22, BV-173) and variant b3a2 (C) (LAMA-84, K562). Each data point corresponds to one single experiment. The protein levels of Separase, Securin, and CyclinB1 based on densitometric evaluation of immunostained Western blots were normalized to Actin as loading control. The corresponding Western blot images are cropped sections derived from stripped and reprobed Western blot immunostainings used for acquisition of densitometric data shown in Table 4. At least triplicate Western blot experiments were performed, one representative composite is depicted. Only significant p-values as calculated between treated and untreated cells were shown (see Table 4 for summarized Δ-values). Abbreviations: RFU, relative fluorescence units; IM, imatinib.

Fig 3. Separase expression and proteolytic activity after espl1 silencing by RNAi.

BV-173 (b2a2) and LAMA-84 (b3a2) cells were treated with negative control siRNA (control) and espl1-specific siRNA (siRNA) for 48h. Consecutively, Separase expression was analysed on transcript level (qRT-PCR), protein level (Western blot immunostaining) and proteolytic activity level (fluorometric assay). All protein analyses were performed as described in legend to Fig 1. In qRT-PCR analysis the house-keeping gene gus (beta-glucuronidase) served as internal standard. Abbreviations: RFU, relative fluorescence units. As for interpretation, 11.7% of Separase protein (middle panel, p<0.0001) performs 118.5% proteolytic activity in BV-173 cells (right panel, p = 0.0427) corresponding to an about 10fold posttranslational activation of present Separase molecules (118.5%/11.7% = 10.1).