Kurihara, Noriyoshi

Abstract The bone marrow microenvironment in MM is characterized by the presence of upregulated osteoclast activity (OCL) and increased angiogenesis. We have recently demonstrated that the HSP90 inhibitor 17-AAG (provided by the NCI) and the mTOR inhibitor rapamycin (LC Laboratories, MA) have synergistic inhibitory activity on MM cells. The objective of this study was to determine the effect of rapamycin, 17-AAG and the combination on OCL formation and angiogenesis. Rapamycin (0.01–100nM), 17-AAG (10–1000nM) and the combination was tested using an in vitro human OCL formation assay and a human angiogenesis assay (AngioKit, TCS Cellworks, UK). Nonadherent human marrow mononuclear cells (1 x 105/100 μL) were plated in 96-well plates in the presence or absence of DMSO, rapamycin, 17-AAG or the combination. RANKL (100ng/ml) and MCSF (20ng/ml) were added to all wells except control media and MCSF. After 3 weeks, cells were fixed, and the number of OCL-like multinucleated cells were scored. To test the effect of the agents on early OCL precursors, we added the inhibitory agents on days 1, 7 or 14 of the culture. The AngioKit is comprised of human endothelial cells in a 24 well plate. The endothelial cells proliferate and then migrate through the matrix to form tubular structures and anastomosing tubules by 2 weeks. Two control wells were treated with VEGF (+ve control) and two with suramin (−ve control). The optimized medium and test samples were replaced on days 4, 7, and 9 after initial treatment. On day 11, cultures were fixed and stained with antibodies to CD31 to detect vessel formation. The degree of tubule formation was quantitated using computerized image analysis (Angiosys, TCS Cellworks, UK). Single agent rapamycin (20–100nM) inhibited OCL formation by 35% as compared to control in all tested doses indicating that PI3K/mTOR is an important regulator of OCL formation. The effect was similar on day 7 and day 14 indicating that this pathway is important for early and late OCL formation. 17-AAG 100–600nM significantly inhibited OCL formation with 100nM 17-AAG inducing 12% OCL formation as compared to control, while 300 and 600nM completely abrogated OCLs (0% OCLs). This effect was similar at day 7. However, when 17-AAG was added on day 14, it only induced 50–60% reduction in OCL formation indicating that 17-AAG affects early OCL formation. The combination of the two agents completely inhibited OCL formation on day 1 and 7 and led to a 65% reduction in OCLs when added on day 14 of the culture. Rapamycin showed a marked decrease in angiogenesis (similar to the negative control suramin), even at the lowest level tested of 0.01nM. 17-AAG demonstrated some inhibition of angiogenesis at 10 nM, and completely abrogated angiogenesis at 500–1000nM. In summary, rapamycin and 17-AAG inhibit OCL formation and angiogenesis. The effect of 17-AAG was on early OCL formation while rapamycin was on both early and late OCL. These results are contradictory to previous data indicating that 17-AAG increases OCL activity in MM. Rapamycin had a significant inhibitory effect on angiogenesis even at low doses. These results demonstrate that the use of rapamycin analogues and 17-AAG in clinical trials may have a therapeutic effect, not only on MM cells, but also on the bone marrow microenvironment. Supported in part by an ASH Scholar Award and an MMRF grant.

Abstract Marrow stromal cells in the bone microenvironment of multiple myeloma (MM) patients play a critical role in promoting both tumor growth and bone destruction. Binding of MM cells to marrow stromal cells, through VCAM-1 on stromal cells and α4β1 integrin on MM cells, results in increased production of TNF-α, receptor activator of NF-KB ligand (RANKL) and IL-6 by marrow stromal cells. These factors in turn increase osteoclast (OCL) formation and the growth of MM cells. Adhesive interaction between MM cells and marrow stromal cells also decrease the sensitivity of MM cells to chemotherapeutic agents. Many of the downstream effects of these adhesive interactions are mediated through the NF-KB, p38 MAPK and JNK signaling pathways. The adapter protein sequestosome-1 (p62) sits at the crossroads of these signaling pathways and mediates the effects of cytokines and other factors that activate NF-KB, p38 MAPK and JNK. However, the role that increased signaling through p62 plays in MM tumor growth and bone destruction is unknown. It is our hypothesis that increased signaling through p62 in marrow stromal cells is necessary for their capacity to increase the growth of MM cells and OCL formation in MM, and thus p62 may be an attractive therapeutic target for MM. To test these hypotheses, we established long-term Dexter-type marrow cultures to isolate marrow stromal cells from MM patients and normals and measured signaling through p62 and PKCζ activation in MM marrow. We found significantly elevated levels of phosho-PKCζ, total PKCζ and VCAM-1 in MM stromal cells. The enhanced IL-6 production resulted from increased p38 MAPK activity and the increased VCAM-1 expression was NF-KB dependent. We then examined the effects of blocking p62 activity in primary patients and normal stromal cells with p62 siRNA (10μg). We confirmed that p62 expression was decreased by at least 90% in both these stromal cells by Western blot analysis. Stromal cells expressing p62siRNA or control siRNA were cultured with or without MM1.S cells for 3 days in separate experiments. Knocking-down p62 in MM derived marrow stromal cells significantly decreased the levels of PKCζ, VCAM-1 and IL-6 in marrow stromal cells and markedly decreased stromal cell support of MM cell growth and OCL formation. Similarly, marrow stromal cells from p62−/− mice produced much lower levels of IL-6, TNF-α, VCAM-1 and RANKL and minimally supported MM cell growth and OCL formation compared to normal cells. Thus, increased signaling through p62 in marrow stromal cells in patients with MM plays an important role in the increased tumor growth and OCL formation in MM, and support p62 as an attractive therapeutic target for MM.

Adhesive interactions between multiple myeloma (MM) cells and marrow stromal cells activate multiple signaling pathways including nuclear factor κB (NF-κB), p38 mitogen-activated protein kinase (MAPK), and Jun N-terminal kinase (JNK) in stromal cells, which promote tumor growth and bone destruction. Sequestosome-1 (p62), an adapter protein that has no intrinsic enzymatic activity, serves as a platform to facilitate formation of signaling complexes for these pathways. Therefore, we determined if targeting only p62 would inhibit multiple signaling pathways activated in the MM microenvironment and thereby decrease MM cell growth and osteoclast formation. Signaling through NF-κB and p38 MAPK was increased in primary stromal cells from MM patients. Increased interleukin-6 (IL-6) production by MM stromal cells was p38 MAPK-dependent while increased vascular cell adhesion molecule-1 (VCAM-1) expression was NF-κB–dependent. Knocking-down p62 in patient-derived stromal cells significantly decreased protein kinase Cζ (PKCζ), VCAM-1, and IL-6 levels as well as decreased stromal cell support of MM cell growth. Similarly, marrow stromal cells from p62−/− mice produced much lower levels of IL-6, tumor necrosis factor-α (TNF-α), and receptor activator of NF-κB ligand (RANKL) and supported MM cell growth and osteoclast formation to a much lower extent than normal cells. Thus, p62 is an attractive therapeutic target for MM.