In:
Blood, American Society of Hematology, Vol. 128, No. 22 ( 2016-12-02), p. 3247-3247
Abstract:
Multiple myeloma (MM) is the most frequent cancer to involve the skeleton with patients developing osteolytic bone lesions due to hyperactivation of osteoclasts (OCL) resulting in severe bone pain, pathological fractures and enhanced mortality. These bone lesions rarely heal even after therapeutic remission due to MM-induced suppression of bone marrow stromal cells (BMSC) differentiation into functional osteoblasts (OB), enhancing their support of MM growth and survival. Although new therapies for MM have greatly improved progression-free survival and overall survival, MM remains incurable for most patients. EZH2is the methyltransferase subunit of the Polycomb Repressive Complex 2 (PRC2) catalyzing the tri-methylation of histone-3 lysine-27 (H3K27me3), which induces gene repression. We previously reported that MM cells induce increased recruitment of EZH2 to the Runx2 gene in the murine pre-OB cell line MC4 resulting in H3K27me3-mediated repression of Runx2, thereby causing repression of OB differentiation. In the present study, we show that the repressive H3K27me3 levels are elevated with reduced H3K9 acetylation on the Runx2 promoter in MM patient derived BMSC. Using the selective small molecule inhibitor GSK126 to block EZH2 activity, we investigated if the MM-induced epigenetic repression of Runx2 is reversible. We found that GSK126 enhances the active architecture at the Runx2 promoter in both BMSC from MM patients and MM-treated MC4 cells, and rescues the osteogenic potential and mineralization capability. 5TGM1 MM cells exhibited reduced adhesion to MC4 pre-OB pre-treated with GSK126 as compared to vehicle in an in vitro adhesion assay, suggesting that EZH2 inhibition will decrease pre-OB support for MM cells. In addition to the direct osteo-anabolic effects of EZH2 inhibition on OB differentiation, we investigated if GSK126 treatment affected the pro-inflammatory, myeloma-amplified OCL differentiation that results in enhanced osteolysis in MM. We reported that expansion of bone marrow monocytes (BMM), which are OCL precursors, in the presence of MM1.S-conditioned media, significantly enhanced formation of TRAP-positive multinucleated OCL. Here we show that MM-conditioned media significantly increased EZH2 and OCL marker genes NFATc1, RANK, OSCAR, Cathepsin K, and DC-STAMP mRNA and enhanced RANKL-induced formation of OCL, which was blocked by GSK126. We found that EZH2 and the corresponding H3K27me3 levels increase during the initial 24h of RANKL-induced osteoclastogenesis and that GSK126 inhibition during the first 24h of RANKL treatment was necessary and sufficient to decrease formation of mature OCL. Thus, GSK126 is likely to both protect the bone from new lytic lesions and help heal the existing osteolytic lesions in MM. We found that GSK126 treatment (48h) inhibited proliferation of human (MM1.S, RPMI, U266, H929, and JJN3) and murine (5TGM1) MM cell lines, and primary 138+MM patient cells. We analyzed the interaction of GSK126 and bortezomib using IC50 constant ratio drug combinations based on the Chou & Talalay method using the CompuSyn program. We found that GSK126 synergizes with bortezomib to inhibit cell viability of these MM cell lines and activated apoptosis via induction of activated caspase-3. We used 5TGM1-GLuc-GFP cells, which secrete Guassia luciferase (GLuc), to measure MM cell viability in a longitudinal drug treatment study. GLuc levels in media correlated with cell proliferation, which was inhibited by single and combination doses of GSK126 and bortezomib, further demonstrating the synergism of these drugs. GSK126 decreased EZH2 mRNA and protein levels in MM cells, which is a cell-type specific effect, since we did not observe EZH2 protein alteration in GSK126 treated OCL progenitors. Our in vitro study suggests that selective inhibition of the epigenetic modifier EZH2 using GSK126 would positively regulate the bone microenvironment by improving the osteogenic potential of MM-exposed BMSC and suppressing OCL formation, and would directly decrease MM cell survival, particularly in combination with bortezomib. These data suggest that targeting EZH2 activity alone or in combination with bortezomib may prove a valuable therapeutic strategy to improve bone health and limit disease progression in MM patients. Disclosures Roodman: Amgen: Consultancy.
Type of Medium:
Online Resource
ISSN:
0006-4971
,
1528-0020
DOI:
10.1182/blood.V128.22.3247.3247
Language:
English
Publisher:
American Society of Hematology
Publication Date:
2016
detail.hit.zdb_id:
1468538-3
detail.hit.zdb_id:
80069-7
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