Page 199 - Haematologica Vol. 109 - July 2024

ARTICLE - FLT3L promotes osteolysis in multiple myeloma
D. Shin et al.
respectively). Taken together, these data suggest that the FLT3L level can serve as a promising factor that reflects both osteolytic bone lesion and prognosis in MM.
FLT3L enhances DKK1 expression via STAT3 signaling
It has been reported that expression of the DKK1 gene, a soluble inhibitor for WNT signaling, is significantly elevated in plasma cells from patients presenting with MM, and the enhanced DKK1 expression in turn leads to defects in osteo- blast differentiation.12 However, the molecular basis of how DKK1 expression is enhanced in MM has not been determined. As both FLT3L and DKK1 are increased in MM patients, we hypothesized that expression of DKK1 might be positively reg- ulated by FLT3 signaling. To test this hypothesis, we treated FLT3L to HEK293T, HeLa, and U2OS cells and measured the DKK1 transcription levels using RT-qPCR. Intriguingly, cells treated with FLT3L exhibited the increased DKK1 transcript levels (Figure 2A). The DKK1 transcript level increased over time during the culture of up to four days. Consistent with previous reports,26,27 we confirmed that FLT3L transcript is also enhanced upon FLT3L treatment (Figure 2A, right).28 Next, we attempted to elucidate the molecular basis of the increased DKK1 expression through FLT3L signaling. FLT3L signaling was reported to be mediated by STAT3 phosphor- ylation.29,30 We consistently found that STAT3 was activated by phosphorylation at tyrosine 705 in HEK293T and MOLP8, an MM cell line, upon treatment of FLT3L (Figure 2B). Again we were able to observe enhanced DKK1 expression in re- sponse to FLT3L treatment by western blot analysis (Figure 2B). In order to confirm that the enhanced DKK1 expression is mediated by STAT3 phosphorylation, we treated the cell with STAT3 signaling inhibitor, Stattic, and found that treat- ment of Stattic significantly reduced FLT3L-mediated STAT3 phosphorylation (Figure 2C, top). RT-qPCR showed that the inhibition of STAT3 results in a significant reduction in DKK1 expression (Figure 2C, bottom). These results suggest that FLT3L signaling activates the DKK1 transcription through STAT3 phosphorylation.
FLT3L-induced DKK1 attenuates WNT signaling to reduce osteogenesis
We next tested whether the enhanced DKK1 expression im- pairs WNT signaling, which can result in reduced osteogenic activity. To this end, we examined the b-catenin activity in HEK293T cells in response to DKK1 and/or FLT3L. It was seen that DKK1 actually reduced the b-catenin protein level, which became further exacerbated when FLT3L was added (Figure 3A). Correspondingly, FLT3L treatment led to reduced nuclear translocation of b-catenin (Figure 3B). To further confirm this FLT3L-mediated reduction of WNT signaling at the cellular level, we performed a WNT reporter assay using the construct in which the expression of luciferase was under the promoter containing b-catenin responsible elements. As expected, the reporter activity was reduced on FLT3L treatment, which was much further reduced when
DKK1 was added (Figure 3C). In order to validate the impact of FLT3L on osteoblast differentiation at the cellular level, we performed alkaline phosphatase (ALP) staining analysis, which has been widely used for evaluating osteogenesis. We found a significant reduction in ALP staining and activity in the mouse pre-osteoblast cell line, MC3T3-E1, follow- ing treatment with FLT3L or DKK1, while the addition of BMP2 enhanced the osteoblast differentiation (Figure 3D, E). In addition, RT-qPCR with the cells in the same setting showed that treatment of FLT3L significantly enhanced DKK1 expression at the transcriptional level (Figure 3F), demon- strating the detrimental effect of FLT3L on osteogenesis. To further explore the relationship of the FLT3L-DKK1 pathway with osteolytic bone lesions, we next measured DKK1 level by ELISA in a subgroup of MM patients whose BM plas- ma samples were available (N=35). FLT3L and DKK1 levels tended to show a positive correlation in MM patients with osteolytic bone lesion (Adjusted [Adj] R2 0.0922, P=0.1809), but a negative correlation in MM patients without osteolytic bone lesion (Adj R2 = -0.0183) (Online Supplementary Figure S2A, B). Next, we explored whether FLT3L would be also implicated in osteoclastogenesis, which could lead to bone loss. To this end, we established a TRAP assay using mouse BM and found that FLT3L treatment does not affect osteo- clastogenesis (Online Supplementary Figure S3A, B). Taken together, these findings demonstrate that osteolytic bone lesions mediated by the FLT3L-DKK1 pathway is due to the reduced osteogenesis through the inhibited WNT signaling, and not to the enhanced osteoclastogenesis.
FLT3L and DKK1 are highly expressed in malignant plasma cells with hyperdiploidy
It has been reported that patients with hyperdiploidy (HY) showed the elevated level of DKK1, but only about 59% of these patients had osteolytic bone lesions.12 This suggests that only a subset of patients show osteolytic bone lesion mediated by the FLT3L-DKK1 pathway. To explore this sub- set of patients, we obtained the previously reported mRNA expression profiles of plasma cells from 414 patients with MM (GSE265822 in GEO database). When the gene expression profiles from all patients were used, we found virtually no correlation (Spearman’s correlation = -0.02) in mRNA ex- pression levels of FLT3L and DKK1 (Online Supplementary Figure S4A). We then examined mRNA expression patterns of FLT3L and DKK1 across the previously defined 7 subtypes of MM patients and found that the HY subtype showed the highest median mRNA expression level of both FLT3L and DKK1 (Figure 4A). Moreover, the HY subtype showed the larg- est percentage (43.10%) of patients showing high (≥ median expression level in all samples) expression levels of both FLT3L and DKK1 compared with the other subtypes (Figure 4B, Online Supplementary Figure S4B). Correspondingly, the HY subtype had the smallest percentage (6.03%) of patients with low expression levels of FLT3L and DKK1. These results suggest that HY subtype may represent a subset of patients
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