Myeloma Cell Expression of Candidate Genes for Osteolytic Bone Disease - Over-Expression of DKK1 Correlates with Clinical Bone Involvment.
Authors: Haaber Jacob , Knudsen Lene M. , Dahl Inger M. , Abildgaard Niels , Lodahl Marianne , Rasmussen Thomas 
Published: October 2019

Abstract
The etiology of the osteolytic bone disease (OBD) in multiple myeloma (MM) is not fully understood but seems to involve interactions between myeloma cells and the bone marrow microenvironment. Over-expression of the osteoclast activation cytokine, receptor activator of nuclear factor-κB ligand (RANKL), and decreased expression of the inhibitor of RANKL, called osteoprotegerin (OPG), is believed to be important. Macrophage inflammatory proteins are also reported to play a role in osteoclast activation. Recently, the role of osteoblast inactivation in OBD has come into focus, especially the importance of over expression of dickkopf 1 (DKK1), an inhibitor of the Wnt signalling pathway that induces osteoblastogenesis and osteoblast activation.
Micro array profiling studies in MM have also introduced other candidate genes. Besides DKK1, over expression of CKS2, PSME2 and DHFR have been reported.
We examined 9 candidate genes involved in OBD: RANKL, RANK, OPG, MIP1α, PTHrp, DKK1, CKS2, PSME2 and DHFR. Bone marrow (BM) aspirates from 170 newly diagnosed, un-treated MM patients and from 9 healthy volunteers were collected over a period of 12 years. Median age in the MM group was 62 years (33–83 years), male/ female ratio 1:1.25. 62 % of the patients had advanced disease at diagnosis (Durie Salmon stage III), 30 % of the patients had no, 28% limited, and 42 % advanced bone lesions.
The immune phenotype of the myeloma cells were characterised by flowcytometry and afterwards used in fluorescence activated cell sorting (FACS) of the myeloma cells using a FACS Aria (Becton Dickinson immune systems) to ≥98% purity.
For global amplification 100 PCs were FACS-sorted directly to PCR tubes, followed by global reverse transcription (RT)-PCR, generating a cDNA archive. By using a polyadenylating step we generated a 5′-oligo(dT)-transcript-poly(A)-3′ cDNA, that is finally amplified by PCR using a sequence independent X-(dT) 24 primer.
Gene expression of the analysed genes and a housekeeping gene (β-actin) were performed by real-time quantitative polymerase chain reaction (RQ-PCR), using specific designed oligo primers and Taqman probes.
Only over-expression of DKK1 in highly purified plasma cells was correlated to the degree of OBD in the studied population. We found expression of DKK1 in myeloma cells in about 42 % (71/170) of the samples. Expression of DKK1 correlated (p<0,002) to OBD, and the DKK1 expression levels were observed to increase with more advanced bone disease.
No, or only a small percentage of the PC expressed RANK, RANKL, OPG, MIP1α or PTHrp1. CKS2, PSME2 and DHFR were expressed in varied degrees, but we observedno correlation to either OBD or disease stage of disease.
In conclsion, our data showed a close connection between over-expression of DKK1 and OBD, whereas we were not able to confirm any correlation between OBD and myeloma cell expression of the genes CKS2, PSME2 and DHFR. We found no evidence for myeloma cell expression of RANKL, RANK, OPG, MIP1α, and PTHrp.