Exocytosis of polyubiquitinated proteins in bortezomib-resistant leukemia cells: a role for MARCKS in acquired resistance to proteasome inhibitors

// Niels E. Franke 1 , Gertjan L. Kaspers 1 , Yehuda G. Assaraf 2 , Johan van Meerloo 1, 3 , Denise Niewerth 1 , Floortje L. Kessler 3 , Pino J. Poddighe 4 , Jeroen Kole 9 , Serge J. Smeets 5 , Bauke Ylstra 5 , Chonglei Bi 6, 11 , Wee Joo Chng 6 , Terzah M. Horton 7 , Rene X. Menezes 8 , Renee J.P. Musters 9 , Sonja Zweegman 3 , Gerrit Jansen 10, * , Jacqueline Cloos 1, 3, * 1 Department of Pediatric Oncology/Hematology, VU University Medical Center, Amsterdam, The Netherlands 2 The Fred Wyszkowski Cancer Research Laboratory, Technion-Israel Institute of Technology, Haifa, Israel 3 Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands 4 Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands 5 Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands 6 Department of Experimental Therapeutics, Cancer Science Institute of Singapore, National University of Singapore, Singapore 7 Texas Children’s Cancer Center, Baylor College of Medicine, Houston, TX, USA 8 Department of Epidemiology and Biostatistics, VU University Medical Center, Amsterdam, The Netherlands 9 Department of Physiology, VU University, Amsterdam, The Netherlands 10 Department of Rheumatology, Amsterdam Rheumatology and immunology Center, VU University Medical Center, Amsterdam, The Netherlands 11 Current address: BGI-Shenzhen, Shenzhen, China * Joint senior authors Correspondence to: Jacqueline Cloos, email: j.cloos@vumc.nl Keywords: bortezomib, resistance, proteasome, MARCKS, leukemia Received: October 19, 2015      Accepted: July 26, 2016      Published: August 17, 2016 ABSTRACT PSMB5 mutations and upregulation of the β5 subunit of the proteasome represent key determinants of acquired resistance to the proteasome inhibitor bortezomib (BTZ) in leukemic cells in vitro . We here undertook a multi-modality (DNA, mRNA, miRNA) array-based analysis of human CCRF-CEM leukemia cells and BTZ-resistant subclones to determine whether or not complementary mechanisms contribute to BTZ resistance. These studies revealed signatures of markedly reduced expression of proteolytic stress related genes in drug resistant cells over a broad range of BTZ concentrations along with a high upregulation of myristoylated alanine-rich C-kinase substrate (MARCKS) gene expression. MARCKS upregulation was confirmed on protein level and also observed in other BTZ-resistant tumor cell lines as well as in leukemia cells with acquired resistance to other proteasome inhibitors. Moreover, when MARCKS protein expression was demonstrated in specimens derived from therapy-refractory pediatric leukemia patients ( n = 44), higher MARCKS protein expression trended ( p = 0.073) towards a dismal response to BTZ-containing chemotherapy. Mechanistically, we show a BTZ concentration-dependent association of MARCKS protein levels with the emergence of ubiquitin-containing vesicles in BTZ-resistant CEM cells. These vesicles were found to be extruded and taken up in co-cultures with proteasome-proficient acceptor cells. Consistent with these observations, MARCKS protein associated with ubiquitin-containing vesicles was also more prominent in clinical leukemic specimen with ex vivo BTZ resistance compared to BTZ-sensitive leukemia cells. Collectively, we propose a role for MARCKS in a novel mechanism of BTZ resistance via exocytosis of ubiquitinated proteins in BTZ-resistant cells leading to quenching of proteolytic stress.