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

Birgit Spiess,Susanne Saussele,Alice Fabarius,Helga Kleiner,Wolfgang Seifarth,Wolf-Karsten Hofmann,Johanna Flach

doi:10.1007/s00277-020-04007-4

Abstract

Separase, a cysteine endopeptidase, is a key player in mitotic sister chromatid separation, replication fork dynamics, and DNA repair. Aberrant expression and/or altered separase proteolytic activity are associated with aneuploidy, tumorigenesis, and disease progression. Since genomic instability and clonal evolution are hallmarks of progressing chronic myeloid leukemia (CML), we have comparatively examined separase proteolytic activity in TKI-treated chronic phase CML. Separase proteolytic activity was analyzed on single cell level in 88 clinical samples and in 14 healthy controls by a flow cytometric assay. In parallel, BCR-ABL1 gene expression and replication fork velocity were measured by qRT-PCR and DNA fiber assays, respectively. The separase activity distribution (SAD) value indicating the occurrence of MNCs with elevated separase proteolytic activity within samples was found to positively correlate with BCR-ABL1 gene expression levels and loss of MMR (relapse) throughout routine BCR-ABL1 monitoring. Analyses of CD34+ cells and MNCs fractionized by flow cytometric cell sorting according to their separase activity levels (H- and L-fractions) revealed that CD34+ cells with elevated separase activity levels (H-fractions) displayed enhanced proliferation/viability when compared with cells with regular (L-fraction) separase activity (mean 3.3-fold, p = 0.0011). BCR-ABL1 gene expression positivity prevailed in MNC H-fractions over L-fractions (42% vs. 8%, respectively). Moreover, expanding CD34+ cells of H-fractions showed decreased replication fork velocity compared with cells of L-fractions (p < 0.0001). Our data suggests an association between high separase activity, residual BCR-ABL1 gene expression, and enhanced proliferative capacity in hematopoietic cells within the leukemic niche of TKI-treated chronic phase CML.

Highlights

Improved therapy regimen employing first, second, and third-generation tyrosine kinase inhibitors (TKI) directed at Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.Ann Hematol (2020) 99:991–1006 oncoprotein in chronic myeloid leukemia (CML) stem cells is inhibited by TKI treatment without affecting CML stem cell survival [7, 8]
Elevated separase activity distribution (SAD) values are found in PBMNC fractions of CML patients without Major molecular remission (MMR) when compared with corresponding cells of patients in MMR or deep molecular remissions (MR) and to healthy controls
In order to investigate the pathophysiological context between the occurrence of hematopoietic leukemic stem cell (LSC)-enriched progenitor cells (CD34+) with elevated levels of separase activity and disease progression, we have fractionized and comparatively analyzed separase activity–positive cells derived from diagnostic samples of a total of 88 CML patients and from 14 healthy control donors

Summary

Introduction

There, separase acts locally to cleave distinct cohesin molecules and to facilitate homology-directed repair (HDR) thereby preventing oncogenic transformation [19,20,21,22]. Due to these interphase activities, separase has been recently proposed to be active throughout the cell cycle on a low level and there may be a mere increase in separase activity during metaphase contrary to the common believes that separase is inactive throughout cell cycle except for metaphase/anaphase [23]

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