Comparative Proteomic Profiling of Refractory/Relapsed Multiple Myeloma Patient Plasma Cells Reveals Biomarkers and Pathways Involved in Bortezomib-Based-Therapy Resistance
Authors: Dytfeld Dominik , Luczak Magdalena , Przybylowicz-Chalecka Anna , Ratajczak Blazej , Nowicki Adam , Czerwinska-Rybak Joanna , Joks Monika , Szczepaniak Tomasz , Maciej Bochenek , Jakubowiak Andrzej , Komarnicki Mieczyslaw 
Published: October 2019

Abstract
Introduction: Despite the new drugs introduced to the treatment of multiple myeloma (MM) the disease remains incurable due to increasing resistance to therapy. Understanding biology of disease and identification of biomarkers of resistance is a key research challenge and may enable the individualization of the treatment.
Objective: The goal of this study was comparative proteomic profiling of pre-treatment plasma cells (PCs) from patients with refractory/relapsed MM (RRMM) subsequently treated with PAD (Bortezomib, Adriamycin, Dexamethasone) to identify biomarkers and signaling pathways differing PCs from patients who achieved CR and those with lower response. We wanted to expand recently published (Dytfeld et al, 2015) catalog of proteins and pathways involved in resistance to bortezomib-based therapy in newly-diagnosed multiple myeloma (NDMM).
Material and Methods: Comparative proteome analysis was performed on purified by negative selection (EasySep) pre-treatment PCs from 24 patients with RRMM qualified for PAD. PCs acquired from bone marrow specimens afterobtaining informed consent from patients were lysed, and protein was isolated. Trypsin digested peptides were labeled with iTRAQ (4-plex) reagents and subsequently analyzed using Q-Exactive hybrid quadrupole-Orbitrap mass spectrometer coupled to the chromatograph Dionex 3000 Ultimate nanoLC (Thermo Scientific). Proteins which accumulation in the analyzed subgroups differed by at least 50% were considered to differentiate. Potential biomarkers were validated using ELISA in the patients' sera. The dysregulated proteins were subjected to two bioinformatic tools: PANTHER and DAVID, for identifying enriched signaling pathways or networks. Only these suggested by both tools were considered significant.
Results and discussion: Out of 3821 proteins identified (FDR <1%), 325 showed significant differences in accumulation between patients who after treatment with PAD achieved CR and patients with worse response. TXNDC5 (peroxiredoxin activity) and PSME1 (proteasome component) found elevated in PCs from refractory patients were confirmed by ELISA in patients' sera (TXNDC5: 110.1pg/mL vs. 40.47, p =0.03; PSME1: 222.02 pg/ml vs. 141.99 pg/mL, p =0.03). Based on the log of p-value from Fisher's test and number of identified differential proteins, the most affected pathways constitute: proteasome pathway (55 proteins; p =3.5x10-19), oxidative phosphorylation (43 proteins; p =4.5x10-11), ribosome proteins (68 proteins; p =1.1x10-47), spliceosome proteins (46 proteins; p =1,5x10-13), translation initiation factor activity (24 proteins; p =1.16x10-11) and peroxiredoxin activity (7 proteins; p =3.7x10-6). All of them were up-regulated in resistant patients. The most affected pathways were previously identified in NDMM treated subsequently with VDD and RVD. Elevated proteasome activity in resistant patients may be a result of affected expression of proteins related to oxidative phosphorylation and enhanced production of reactive oxygen species connected with up-regulation of peroxisome activity proteins. As a consequence bortezomib may not inhibit proteasome sufficiently, thus prevents apoptosis. An alteration in the stoichiometry of components of the proteasome, ribosome, and spliceosome is the effect of genomic instability and thereby most cancers exhibit loss of genes responsible for tumor suppressor (Nijhawan et al, 2012).
Conclusion: We indicated biomarkers of resistance which may be a potential therapeutic targets and basis for individualization of MM therapy. We also showed pathways involved in resistance to bortezomib-based therapy in RRMM that confirms our previous finding in NDMM. Further studies are necessary to explore these pathways and define their role in multiple myeloma treatment.
Study was supported by research grant funded by Janssen Cilag.

Disclosures
Dytfeld: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Jakubowiak:Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SkylineDx: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: institutional funding for support of clinical trial conduct, Speakers Bureau; Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Karyopharm: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Onyx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SkylineDx: Membership on an entity's Board of Directors or advisory committees; Onyx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Karyopharm: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.