Page 127 - Haematologica August 2018

OTSSP167 has activity in myeloma bone disease
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Figure 3. Established osteoclast cultures are sensitive to OTSSP167. A) Representative images of TRAP-stained RAW264.7-derived osteoclast cultures treated with a range of OTSSP167 concentrations. Treatment of these cultures was initiated after the final day of osteoclast differentiation (day 5). B) Representative images of Von Kossa-stained bone matrix resorption assays of huOC and RAW264.7-derived osteoclast cultures treated with a range of OTSSP167 concentrations. Treatment of these cultures was initiated when mature osteoclasts appeared (early: day 5, late: day 7). C) Quantification of osteoclast numbers per field of view (N.OC/FOV) in RAW264.7-derived cultures treated with OTSSP167 starting at day 5. D) Quantification of the matrix resorption area in RAW264.7-derived osteoclast cultures treated with OTSSP167 starting at day 5 or day 7. E) Real-time PCR analysis of osteoclast differentiation markers following OTSSP167 treatment starting at day 5 of RAW264.7-derived osteoclast cultures. F) ROS generation by RAW264.7-derived osteoclasts treated with OTSSP167 starting on day 5. All data are represented as mean +/- standard error. *: P<0.05, **: P<0.01, ***: P<0.001.
other hand, we detected an increase in EZH2 mRNA and protein levels, while the phosphorylation level of FOXM1 decreased (Online Supplementary Figure S1B). Finally, we observed no difference in actin ring formation in mature osteoclasts following OTSSP167 treatment (Figure 2F).
Established osteoclast cultures are sensitive to OTSSP167
In order to confirm that the effects of OTSSP167 on osteoclast function are not solely due to a decrease in monocyte viability, we initiated treatment of mature RAW264.7 osteoclasts after the final day of differentiation (day 5). In this setting, treatment with up to 10 nM OTSSP167 had no effect on the number of osteoclasts (Figure 3A and 3C). Initiation of OTSSP167 treatment on day 5 or day 7 of in vitro matrix resorption assays markedly reduced bone matrix resorption (Figure 3B and 3D). Mature osteoclasts appeared at day 5 in these cultures and the presence of mature osteoclasts at the end of both DMSO- and OTSSP167-treated cultures was confirmed (not shown). Twenty-four hour treatment of mature osteo- clasts had no impact on NFATc1 or TRAP mRNA levels (Figure 3E). However, we found OTSSP167 greatly reduced CTSK expression and greatly increased calcitonin receptor (CALCR) expression by mature osteoclasts (Figure 3E). Finally, as multiple reports indicate that both osteoclast differentiation and bone resorption by osteo- clasts depend on the generation of reactive oxygen species (ROS),23,24 we assessed whether OTSSP167 could hamper the generation of ROS by mature osteoclasts and this was indeed the case (Figure 3F).
OTSSP167 stimulates osteoblast function
Contrary to osteoclasts, MELK, EZH2 and FOXM1 expression decreased during OB formation indicating an
inhibitory role in OB development (Figure 4A). These data suggest an opposing role for these molecules in OB forma- tion which is in line with the results obtained with OTSSP167. Treatment of BMSC-TERT with OTSSP167 for 24 hours resulted in decreased MELK, EZH2 and FOXM1 protein levels (Figure 4B). Although monocyte viability was considerably reduced following OTSSP167 treatment in the low nanomolar range, bone marrow stro- mal cell viability was unaffected at these concentrations (IC50: 800.2 nM) (Figure 4C). Accordingly, no G2/M cell cycle arrest was detected (Online Supplementary Figure S1A). Functionally, OTSSP167 increased collagen deposi- tion by osteoblasts (Figure 4D and 4E) and induced a marked increase in matrix mineralization (Figure 4D and 4F). Finally, we determined the effect of OTSSP167 on osteoblast marker gene expression and found that osterix (OSX) expression was increased. Conversely RUNX2, osteopontin (OPN) and interleukin-6 (IL-6) expression were decreased (Figure 4G).
Preclinical activity of OTSSP167 in multiple myeloma bone disease
Our in vitro data indicate that OTSSP167 blocks osteo- clast activity while stimulating osteoblast activity. Therefore, we assessed the potential of OTSSP167 as a novel therapy for MMBD in vivo in the 5TGM.1 MM model. The effects of OTSSP167 on myeloma develop- ment are given and discussed in detail in a separate publi- cation.7 OTSSP167 treatment prevented the development of MMBD and this effect was similar at all dosing sched- ules tested (Figure 5A). OTSSP167 reduced the number of cortical perforations in MM-bearing mice (Figure 5B), without affecting cortical thickness (Ct.Th) (Online Supplementary Figure S1E). The loss of trabecular bone vol- ume (Tb.BV/TV) that occurs in MM-bearing mice com-
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