Previously, we used gene expression profiling (GEP) on whole-bone biopsies (BX) and plasma cells (PCs) of newly diagnosed multiple myeloma (MM) patients and age-matched controls to identify 62 non-PC-related genes that were significantly differentially expressed in bone biopsies of MM. CYR61/CCN1 was the most significantly overexpressed gene in BX in subsets of MGUS and MM patients. We hypothesize that alteration of the microenvironment may be central to the conversion of MGUS to MM and that abnormal expression of CYR61 may precede this conversion. CYR61 has been shown to promote proliferation, migration, adhesion and angiogenesis and is expressed in many cell types. Within the bone, CYR61 is induced during osteoblast differentiation by endothelin-1, (Clines et al., 2007) and by Wnt3a (Si et al., 2006). CYR61 also inhibits the formation of multinucleate osteoclasts in vitro (Crockett et al., 2007). To investigate the role of CYR61 in MM pathogenesis CYR61 protein production in MM was analyzed in sera of 192 newly diagnosed myeloma patients by ELISA. In agreement with that observed by GEP, CYR61 is significantly elevated in a subset of MM patients (144/192) at diagnosis compared with healthy donors (p < 0.001). In Total Therapy 3 (TT3) patients low CYR61 (< 500 pg/ml) is associated with poor overall survival (p < 0.05) compared with levels greater than 500 pg/ml. CYR61 levels were also significantly elevated in MGUS, Waldenstrom's macroglobulinemia and smoldering/indolent myeloma compared with healthy donors (p < 0.001). Patient samples were grouped into the 8 GEP-defined MM molecular subtypes (Zhan et al., 2006) and CYR61 levels were significantly different from healthy donors in all subtypes with the exception of MY that typically is associated with a better prognosis. Analysis of CYR61 levels in TT3 patients 48h post-Velcade (Vel) revealed an inverse correlation between change in CYR61 levels post-Vel and 80-gene risk in TT3a patients (p < 0.01). Likewise, the change in CYR61 levels from diagnosis to remission is also inversely correlated with risk (p < 0.01). To better understand the mechanism by which loss of CYR61 might negatively affect risk we examined the GEP from MM Bx and CD138-selected plasma cells to compare gene signatures in patients with low CYR61 (Q1) and high CYR61 (Q4). Those genes that exhibited ≥ 2-fold difference were investigated further. Patients with low CYR61 differentially expressed 179 probesets in MMBX including elevated SFRP2 (4.1-fold), a Wnt inhibitor, and COL2A1 (3.5-fold) which are associated with myeloma bone disease and osteoarthritis, respectively. SFRP2 is also elevated (4.3-fold) in MMPCs (194 probesets elevated) along with TNFSF10 (2.1-fold) indicative of the HY subtype and FGFR3 (2.1-fold) which is elevated in the MS subtype. In patients with high CYR61, many muscle-specific genes were elevated in MM BX and may indicate a predominance of osteocytes. Additionally, BGLAP, whose loss is associated with poor survival in myeloma, is elevated (2-fold) in MM BX (171 probesets) with high CYR61 and EDN1, an activator of CYR61/CCN1, is elevated (2.8-fold) in MM PCs (111 probesets) with high CYR61. The test the effect of CYR61 on cell growth, H929 cells were exposed to rCYR61 which caused a dose- and time-dependent suppression of cell growth and survival. To examine the role of CYR61 in the MM ME, H929 cells transduced with empty vector, wild type CYR61 or CYR61 cDNA with a mutated αvβ3 binding site were injected into implanted bones in SCID-hu mice. CYR61-induced effects on bone have been shown to be mediated by the αvβ3 integrin, presumably through osteoblasts. X-rays of the implanted bone and serum measurement of hIg levels were taken weekly for 5 weeks. Quantitation of change in bone mineral density (BMD) over the course of the experiment showed that CYR61 inhibits osteolysis (p<0.04), whereas CYR61 that is incapable of binding αvβ3 attenuates this effect. These data indicate that the αvβ3 integrin mediates CYR61-induced inhibition of osteolysis in MM. Additionally, quantitation of serum hIg demonstrated that CYR61 significantly reduces MM tumor growth (p<0.04) and this is not effected by mutation of the avb3 binding site. Taken together these data suggest that loss of CYR61 in myeloma may be indicative of a dysfunctional ME and thus serve as a biomarker to monitor the MM ME. Finally, therapeutics that upregulate CCN1 will be tested as a novel treatment for myeloma bone disease.
Shaughnessy:Myeloma Health, Celgene, Genzyme, Novartis: Consultancy, Employment, Equity Ownership, Honoraria, Patents & Royalties.
Asterisk with author names denotes non-ASH members.