title: Exocytosis of polyubiquitinated proteins in bortezomib-resistant leukemia cells: a role for MARCKS in acquired resistance to proteasome inhibitors.
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.
authors.affiliation: Department of Pediatric Oncology/Hematology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Niels E
authors.initials: NE
authors.lastname: Franke
authors.affiliation: Department of Pediatric Oncology/Hematology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Gertjan L
authors.initials: GL
authors.lastname: Kaspers
authors.affiliation: The Fred Wyszkowski Cancer Research Laboratory, Technion-Israel Institute of Technology, Haifa, Israel.
authors.forename: Yehuda G
authors.initials: YG
authors.lastname: Assaraf
authors.affiliation: Department of Pediatric Oncology/Hematology, VU University Medical Center, Amsterdam, The Netherlands.|Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Johan
authors.initials: J
authors.lastname: van Meerloo
authors.affiliation: Department of Pediatric Oncology/Hematology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Denise
authors.initials: D
authors.lastname: Niewerth
authors.affiliation: Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Floortje L
authors.initials: FL
authors.lastname: Kessler
authors.affiliation: Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Pino J
authors.initials: PJ
authors.lastname: Poddighe
authors.affiliation: Department of Physiology, VU University, Amsterdam, The Netherlands.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Kole
authors.affiliation: Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Serge J
authors.initials: SJ
authors.lastname: Smeets
authors.affiliation: Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Bauke
authors.initials: B
authors.lastname: Ylstra
authors.affiliation: Department of Experimental Therapeutics, Cancer Science Institute of Singapore, National University of Singapore, Singapore.|Current address: BGI-Shenzhen, Shenzhen, China.
authors.forename: Chonglei
authors.initials: C
authors.lastname: Bi
authors.affiliation: Department of Experimental Therapeutics, Cancer Science Institute of Singapore, National University of Singapore, Singapore.
authors.forename: Wee Joo
authors.initials: WJ
authors.lastname: Chng
authors.affiliation: Texas Children's Cancer Center, Baylor College of Medicine, Houston, TX, USA.
authors.forename: Terzah M
authors.initials: TM
authors.lastname: Horton
authors.affiliation: Department of Epidemiology and Biostatistics, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Rene X
authors.initials: RX
authors.lastname: Menezes
authors.affiliation: Department of Physiology, VU University, Amsterdam, The Netherlands.
authors.forename: Renée J P
authors.initials: RJ
authors.lastname: Musters
authors.affiliation: Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Sonja
authors.initials: S
authors.lastname: Zweegman
authors.affiliation: Department of Rheumatology, Amsterdam Rheumatology and immunology Center, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Gerrit
authors.initials: G
authors.lastname: Jansen
authors.affiliation: Department of Pediatric Oncology/Hematology, VU University Medical Center, Amsterdam, The Netherlands.|Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Cloos
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D000069286:Bortezomib / Q000494:pharmacology*; D045744:Cell Line, Tumor; D055028:Comparative Genomic Hybridization; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D005089:Exocytosis; D020869:Gene Expression Profiling; D015973:Gene Expression Regulation, Leukemic; D053263:Gene Regulatory Networks; D006801:Humans; D007938:Leukemia / Q000235:genetics / Q000378:metabolism* / Q000401:mortality / Q000628:therapy; D035683:MicroRNAs / Q000235:genetics; D000076250:Myristoylated Alanine-Rich C Kinase Substrate / Q000235:genetics / Q000378:metabolism*; D011379:Prognosis; D061988:Proteasome Inhibitors / Q000494:pharmacology*; D025801:Ubiquitin / Q000378:metabolism; D054875:Ubiquitination; D015854:Up-Regulation
chemical_list: D000970:Antineoplastic Agents; D035683:MicroRNAs; D061988:Proteasome Inhibitors; D025801:Ubiquitin; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D000069286:Bortezomib
keywords: MARCKS; bortezomib; leukemia; proteasome; resistance