Ectoenzymes and Their Products As Communicators in the Human Myeloma Niche
Authors: Horenstein Alberto L. , Ferri Valentina , Bolzoni Marina , Quarona Valeria , Chillemi Antonella , Zaccarello Gianluca , Giuliani Nicola , Malavasi Fabio 
Published: October 2019

Abstract
Development of human multiple myeloma (MM) in the bone niche derives from multiple factors. Besides cells and soluble factors, hypoxia triggers anaerobic glycolysis, reduces ATP and simultaneously increases NAD+. This condition likely influences the ectoenzymatic pathways working in the niche. ATP is the substrate for the CD39/CD73 pathway leading to adenosine (Ado) production. NAD+ activates an alternative pathway, where Ado is obtained by the articulated actions of the CD38/CD203a/CD73 axis.
The working hypothesis is that Ado plays a significant role in tumor immune escape and growth in closed systems, such as the myeloma niche. The two distinct pathways were confirmed by immunohistochemistry and immunofluorescence on cells derived from the bone marrow (BM) myeloma niche, where they operate in a discontinuous way.
The first step of the study was to design a simple and reliable assay for detecting Ado at nanomolar levels in the fluids of the niche, taking into account i) the very short half-life of Ado, ii) the degradation of Ado to inosine, and iii) the fast cellular uptake of Ado. The technique adopted relied on HPLC with UV detection, which simultaneously monitored nucleotide and nucleoside concentrations. Ado was identified by retention time and by enzymatic peak shift assay using adenosine deaminase. The HPLC assay showed a good linearity for Ado (R2=0.9972) as well as a low value of detection (5 ng/ml) and quantitation (10 ng/ml).
BM plasma derived from osteomedullary biopsies from monoclonal gammopathy of unknown significance (MGUS) (3), from smoldering MM (3) and from symptomatic MM (27) were immediately treated with acetonitrile to extract Ado and avoid its degradation or use by the cells present in the biopsy.
The results demonstrate that Ado is detectable in BM plasma, taken as representative of the niche, where all the products converge. Results are summarized in the Table.
Table Adenosine ( µ M) MGUS(n=3) SMM(n=3) MM(n=27) Mean value 19 34 59 Limit values 9.58 – 28.33 17.05 – 46.50 24.88 - 193.07 Range value 18.75 29.45 168.19
The variability in the levels of Ado detected suggest that they are the result of multiple heterotypic interactions taking place between myeloma cells on the one side, and osteoblasts (OB), osteoclasts (OC) and stromal cells (SC) on the other. The individual contributions of the most important components of the niche were assessed by using cell and cell line models, which consented the design of selected co-cultures. Human myeloma cells (JJN3 and KSM12BM lines) were challenged by: i) osteoblasts (HOBIT and HOB01 lines); ii) fully differentiated human osteoclasts (PBMCs isolated from BM samples of MM patients exposed to MCSF and RANKL) and iii) stromal cells (HS5 line). Ado levels were assayed as described.
Co-cultures of MM with OCs displayed a significant increase in Ado levels (88%±10). On the contrary, co-cultures of MM with OBs and SCs lines were characterized by decreased Ado levels (41%±12 and 29%±14, respectively).
Schematically, this study indicates that significant levels of Ado are present in BM, which robustly increase in MM conditions. This conclusion was made possible by the use of an ad hoc designed HPLC assay, which defines adenine nucleotides, nucleosides and their metabolites. Extracellular Ado concentrations ranged from 10-200 µM in the biopsies from different monoclonal gammopathies disorders. This observation supports the view that bone and MM are equipped with the ability to maintain Ado at micromolar levels, with a profile that may appear characteristic of different pathological conditions.
An appealing hypothesis is that myeloma cells usurp the normal organization of bone, replacing it with a niche that maximizes local growth and protection from immune defenses. Relevant for translational medicine is that Ado levels in the myeloma niche might be an early indicator of an aggressive form of the disease. Consequently, Ado levels could become a valuable marker of disease progression in plasma cell dyscrasias.

Disclosures
Giuliani: Celgene Italy: Research Funding. Malavasi:Jensen Research & Development: Consultancy, Research Funding.