Elucidation of molecular basis of osteolytic bone lesions in advanced multiple myeloma

Abstract

Osteolytic bone lesion is a major cause of lower quality of life and poor prognosis in patients with multiple myeloma (MM), but molecular pathogenesis of the osteolytic process in MM remains elusive. Fms-like tyrosine kinase 3 ligand (FLT3L) was reported to be elevated in bone marrow (BM) and blood of patients with advanced MM who often show osteolysis. Here, we investigated a functional link of FLT3L to osteolytic process in MM. We recruited 86, 306, and 52 patients with MM, acute myeloid leukemia (AML), and acute lymphoblastic leukemia (ALL), respectively. FLT3L levels of patients with hematologic malignancies were measured in BM-derived plasma and found to be significantly higher in MM than in AML or ALL, which rarely show osteolysis. FLT3L levels were further elevated in MM patients with bone lesion compared with patients without bone lesion. In vitro cell-based assays showed that the administration of FLT3L to HEK293T, HeLa, and U2OS cells led to an increase in the DKK1 transcript level through STAT3 phosphorylation at tyrosine 705. WNT reporter assay showed that FLT3L treatment reduced WNT signaling and nuclear translocation of β-catenin. These results collectively show that the FLT3L-STAT3-DKK1 pathway inhibits WNT signaling-mediated bone formation in MM, which can cause osteolytic bone lesion. Finally, transcriptomic profiles revealed that FLT3L and DKK1 were predominantly elevated in the hyperdiploidy subtype of MM. Taken together, FLT3L can serve as a promising biomarker for predicting osteolytic bone lesion and also a potential therapeutic target to prohibit the progression of the osteolytic process in MM with hyperdiploidy.

Figure 1.

Plasma expression of Fms-like tyrosine kinase 3 ligand is elevated in multiple myeloma patients. (A) The plasma level of Fms-like tyrosine kinase 3 ligand (FLT3L) in bone marrow (BM) is significantly higher in multiple myeloma (MM) (N=86; median 161.8 pg/mL, interquartile range [IQR] 73.96-233.5) than in acute myeloid leukemia (AML) (N=306; median 28.16, IQR 8.48-107.3), acute lymphoblastic leukemia (ALL) (N=52; median 46.11, IQR 12.52-172.8), and monoclonal gammopathy of undetermined significance (MGUS) / smoldering multiple myeloma (SMM) (N=42). (B) The plasma level of FLT3L in BM of MM shows a linear correlation with the percentage of plasma cell / total nucleated cells in BM aspirates at the time of initial diagnosis (N=43). (C) The plasma level of FLT3L in BM is higher in MM with osteolytic bone lesion (N=27, median 194.70, IQR, 152.52-242.27) than in MM without bone lesion (N=16, median 141.62 pg/mL, IQR, 109.57-215.98) at the time of initial diagnosis (P=0.0047). (D) Kaplan-Meier survival curves for MM patients with low and high FLT3L expression levels. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.

Figure 2.

Fms-like tyrosine kinase 3 ligand enhances Dick Kopf-related protein 1 expression via STAT3 signaling. (A) The mRNA expression of Dick Kopf-related protein 1 (DKK1) and Fms-like tyrosine kinase 3 ligand (FLT3L) obtained from HEK293T, HeLa, and U2OS cells treated with 40 ng/mL rhFLT3L for from two to approximately four days, as analyzed by quantitative real-time polymerase chain reaction (RT-qPCR) (N=4). (B) Western blot analysis of phosphorylation of STAT3, total STAT3 and DKK1 under treatment with rhFLT3L (40 ng/mL) in HEK293T and MOLP8 cells for three days. (C) In HEK293T on treatment with rhFLT3L (40 ng/ mL), Stattic (10 μM), or both for three days, protein level of phosphorylated STAT3 and total STAT3 and mRNA level of DKK1 and FLT3L were analyzed by western blotting and RT-qPCR, respectively (N=8). Statistical analysis was performed by one-way ANOVA with Dunnett’s multiple comparison. Error bars indicate the standard deviation of 3 independent replicates. *P<0.05, **P<0.01, ***P<0.001, ns: not significant (P>0.05).

Figure 3.

Fms-like tyrosine kinase 3 ligand-induced Dick Kopf-related protein 1 attenuates WNT/b-catenin signaling. (A) Conditioned media (CM) obtained from HEK293T transiently transfected with DNA construct, such as Dick Kopf-related protein 1 (DKK1), Fms-like tyrosine kinase 3 ligand (FLT3L), or both were treated into newly HEK293T cells for three days. Expression of b-catenin protein from cell lysates was analyzed by western blot assay. (B) EV (empty vector) and DKK1 transiently expressing HEK293T cells were treated with rhFLT3L (40 ng/mL) for three days. Nuclear and cytoplasmic fraction from the cell pellets was separated by Extraction kit. Protein levels of b-catenin and GAPDH (cytosol) and Lamin A/C (Nucleus) as loading controls were determined by western blotting. (C) Reporter gene assay for b-catenin-mediated transcriptional activation was conducted in HEK293T transiently transfected with plasmids carrying EV or DKK1 under treatment of rhFLT3L (40 ng/mL) for three days (N=4). (D) Alkaline phosphatase staining and (E) activity were evaluated with MC3T3-E1 cells treated with mock, rhFLT3L, rhBMP2 or rhDKK1 for seven days (N=6). (F) Relative mRNA expression level of DKK1 was determined by qRT PCR. mRNA was prepared from the MC3T3-E1 cells treated with mock, rhFLT3L, rhBMB2 or rhDKK1 for seven days (n=3). Error bars indicate the standard deviation of at least 3 independent replicates. ALP: alkaline phosphatase **P<0.01, ***P<0.001, ****P<0.0001, ns: not significant (P>0.05).

Figure 4.

Fms-like tyrosine kinase 3 ligand and Dick Kopf-related protein 1 are highly expressed in plasma cells of multiple myeloma patients with hyperdiploidy subtype. (A) Boxplots showing distributions of log₂-fold-changes of mRNA expression levels of Fms-like tyrosine kinase 3 ligand (FLT3L) and Dick Kopf-related protein 1 (DKK1) across 7 subtypes (CD1, CD2, HY, LB, MF, MS and PR) reported by Zhan et al. Log₂-fold changes were computed with respect to the median mRNA expression value in all samples. (B) Scatterplot showing log₂-fold changes of mRNA expression levels between FLT3L (x-axis) and DKK1 (y-axis) in patients (N=116) of the hyperdiploidy (HY) subtype. Spearman’s correlation between FLT3L and DKK1 (top) and the percentage of patients in each quadrant are displayed in the HY subtype. Median expression value for each gene was used to define high expression of the gene. (C) Gene ontology biological processes (GOBP) and pathways represented by the signature genes for the HY subtype. (D) GOBP and pathways represented by the up-regulated (left) or down-regulated (right) genes in the HY subgroup with high expression of both FLT3L and DKK1 (FLT3L^HDKK1^H) compared to the other subgroups. Significance (P-value) of the GOBP and pathways being enriched by the genes is displayed as -log₁₀(P-value). Cut-off of enrichment P-value (0.05) is indicated by the line. Pathways used to build a network model are highlighted. GPCR: G protein coupled receptors.