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OTSSP167 has activity in myeloma bone disease
clast differentiation (illustrated in Online Supplementary Figure S1C). OTSSP167 decreased MELK protein levels in RAW264.7 cells, which agrees with previous studies on various cell types.25,26 Of note, OTSSP167 can have off-tar- get effects, which could partially account for the effects of OTSSP167 on osteoclast and osteoblast function.27,28 In accordance with previous reports implicating MELK in G2/M transition and proliferation, OTSSP167 decreased the viability of monocytes due to G2/M cell cycle arrest.26,27,29,30 The decreased progenitor cell viability result- ed in a decreased osteoclast differentiation following OTSSP167 treatment. It is difficult to differentiate between an anti-proliferative effect on progenitor cells and an anti-osteoclast differentiation effect. We observed that 2 pathways, known to interact with MELK, were affected by OTSSP167: i) the transcriptional factor FOXM1 which is implicated in proliferation and ii) the EZH2-IRF8-NFATC1 axis, involved in osteoclast differen- tiation. Unexpectedly, EZH2 levels increased, which can be explained by the strong link between cell cycle arrest and CDK1/2-dependent EZH2 phosphorylation on differ- ent residues (T487), which can either disrupt the binding of EZH2 to other partners of the polycomb repressive complex31 or target it for ubiquitin-mediated degrada- tion.32 EZH2 thus becomes less functional, which results is a decline in H3K27 trimethylation and a de-repression of EZH2 target genes (in our case IRF8).33 When mature osteoclasts were treated with OTSSP167, their numbers remained unaltered but matrix resorption was drastically decreased, further corroborating that OTSSP167 has a direct effect on osteoclast activity. Together, our data indi- cate that OTSSP167 inhibits osteoclast function by ham- pering monocytic progenitor viability as well as by direct- ly inhibiting mature osteoclast function.
MELK, EZH2 and FOXM1 mRNA levels decreased dur- ing osteoblast differentiation, consistent with an inhibito- ry role of MELK and downstream factors such as EZH2 on osteoblast function.16,17 Contrary to osteoclasts and vari- ous malignant cells, BMSC-TERT viability was not affect- ed following OTSSP167 treatment at similar concentra- tions. OTSSP167 treatment increased collagen deposition and strongly stimulated mineralization activity of osteoblasts in vitro and this coincided with an increase in OSX levels but a decrease in RUNX2, OPN and IL-6 mRNA levels. The pro-mineralization activity of OTSSP167 is likely mediated by EZH2 as treatment with an EZH2 inhibitor showed a similar effect.16 However, the decrease in RUNX2 expression following OTSSP167 treat- ment does not correspond with the described role of EZH2 as a suppressor of RUNX2 transcription.16 Alternative mechanisms of OTSSP167-induced osteoblast maturation include a marked decrease in the expression of OPN, a non-collagenous bone matrix protein that inhibits matrix mineralization34 and IL-6,34 a potent growth factor for MM cells, but also a negative regulator of osteoblast differentiation35 and inducer of bone resorption.36 Deregulation of these genes in conjunction with the above-mentioned upregulation of OSX, a master regulator of mineralization, likely mediates the pro-osteogenic activity of OTSSP167.
The regulation of MELK expression and activity by upstream signaling pathways remains poorly understood including in bone cells. E2F137 and FOXM110 have been shown to regulate MELK gene transcription, the former in osteoblastic MC3T3-E1 cells. Of note, E2F1 has been
implicated in increased osteoclastogenesis and osteoblast activity.38,39 MELK both regulates and is regulated by one family of MAP kinases, the c-Jun NH(2)-terminal kinases (JNK2), that acts downstream of the RANK-receptor.40 Interestingly, JNK2 does not seem to be required for osteo- clast differentiation, but rather appears to be involved in osteoclast survival.41
Given the promising in vitro data, we assessed whether OTSSP167 would affect the development of MMBD in the murine 5TGM.1 MM model. Although this model reflects human myeloma and associated bone disease in an immunocompetent setting, it should be noted that MM growth in this model progresses rapidly. This cell line allows in vivo studies, however, it also shows a BM-inde- pendent growth in vitro and its murine origin may not reflect all the human aspects of myeloma disease (from cytogenetic and molecular point of view). We have previ- ously shown that the dosing schemes we used dose- dependently decreased MM tumor load.7 OTSSP167 com- pletely prevented the development of MMBD at all doses, with no difference between the treatment groups. Both the development of lytic cortical lesions and the large loss of trabecular bone were completely prevented in MM- bearing mice treated with OTSSP167. Importantly, this effect also occurred at an OTSSP167 concentration which had no effect on MM tumor load (7.5 mg/kg/2d), indicat- ing that OTSSP167 has a direct effect on bone cells and does not solely reduce MMBD by reducing MM tumor load. In fact, given the lower concentration of OTSSP167 needed to achieve an anti-MMBD effect, our data suggest that OTSSP167 could exert its anti-MM effect in part by normalizing bone homeostasis. Indeed, the reduced osteo- clast numbers following OTSSP167 treatment likely result in reduced myeloma pro-survival factor levels and increased myeloma anti-proliferative factor levels, respec- tively. Upon treatment with OTSSP167, we found decreased mRNA expression levels of insulin-like growth factor 1 (IGF-1), osteopontin, a proliferation-inducing lig- and (APRIL) and interleukin-10 (IL-10) (Online Supplementary Figure S1D).
In conclusion, this study provides a novel approach for the treatment of MMBD. The maintenance of bone ana- bolic activity by OTSSP167 is promising and warrants fur- ther investigation. Reducing MM patient morbidity and mortality via the combined anti-MM and anti-MMBD effect of OTSSP167 holds great clinical promise and our results warrant similar studies in other cancers with bone involvement.
Acknowledgments
The authors would like to thank the GIGA-imaging platform for their excellent technical assistance.
Funding
JM and ML are Télévie PhD candidates. The Wilhelminen Cancer Research Institute is supported by the Austrian Forum against Cancer. The laboratory of Hematology was supported by Foundation Against Cancer, the Fonds National de la Recherche Scientifique (F.N.R.S., Belgium) and the Fonds spéciaux de la Recherche (University of Liege). Elodie Duray (research fellow), Erwan Plougonven (post-doctoral researcher) and Frédéric Baron (senior research associate) have a mandate supported by the FNRS. Roy Heusschen is a Télévie postdoctoral research associ- ate. Jo Caers is a post-doctorate clinical specialist funded by the Belgian Foundation against Cancer.
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