Page 194 - Haematologica Vol. 109 - July 2024

ARTICLE - FLT3L promotes osteolysis in multiple myeloma
D. Shin et al.
and mortality in MM patients, are induced by an imbalance in bone remodeling characterized by the suppressed osteo- blast formation and function, increased osteoclast forma- tion and activity, as well as direct cell interactions between MM cells and osteoclasts, and the release of cytokines and growth factors.9 One of the key mechanisms contributing to the development of osteolytic bone lesions is the activation of osteoclasts resulting in enhanced bone resorption and subsequent bone loss.10 However, the molecular mechanisms underlying the pathogenesis of MM-induced osteolytic bone lesions are still not fully understood.
The putative mechanism of bone osteolysis in intercellular interactions between myeloma cells and osteoclast has been extensively studied. Cytokines of IL-6, IL-1b, TNF-α, MIP1α, and receptor activator of nuclear factor κ-B ligand (RANKL)11 generated in the bone marrow (BM) microenviron- ment accentuate the interactions between myeloma cells and BM stromal cells, which enhance osteoclastic activity. In contrast, Dick Kopf-related protein 1 (DKK1), an extracel- lular antagonist of WNT signaling, is known to contribute to the formation of osteolytic bone lesion by depressing osteoblastic activity.12 Specifically, the high expression levels of DKK1 in the BM microenvironment have been associated with osteolytic bone lesions in MM patients13 and regulation of the WNT signaling pathway. Recent studies have investi- gated the molecular mechanisms underlying osteolytic bone lesions in MM, providing critical insights into the disease’s pathogenesis. The cross-talk between MM cells and the BM microenvironment closely modulates the WNT/b-catenin signaling pathway, which promotes osteoblast activity and suppresses osteoclast differentiation.14 Many studies have demonstrated the importance of the WNT/b-catenin pathway in healthy bone development through canonical WNT signaling participation in the regulation of osteoblast differentiation.15 MM cells secrete WNT antagonists that disrupt BM regu- lation of osteoblastic differentiation, leading to osteolytic lesions.16 Dysregulation of WNT pathway by DKK1, secreted from MM cells, affects the BM microenvironment, leading to altered bone homeostasis and osteolytic bone disease.14 Several studies have investigated the expression pattern and functions of DKK1 in MM, revealing significantly higher DKK1 expression in MM patients compared to healthy con- trols, which is associated with poor prognosis.17 Additionally, preclinical studies have demonstrated that targeting DKK1 using monoclonal antibodies or small molecule inhibitors can inhibit MM cell proliferation and induce apoptosis both in vitro and in vivo.18
Recently, Fms-like tyrosine kinase 3 ligand (FLT3L), a cyto- kine that plays a crucial role in hematopoiesis and immune regulation, has been reported to be elevated in BM and blood of patients with MM,19 and correlated with vascular endothelial growth factor (VEGF) which may reflect the in- creased proliferative tumor activity based on the association between FLT3L level and Ki-67 positivity in MM.20 FLT3L binds to the FLT3 receptor, which is expressed on hematopoiet-
ic stem cells and myeloid progenitor cells.21 In MM, FLT3L has been shown to promote the proliferation and survival of malignant plasma cells,20 as well as the recruitment of immunosuppressive cells to the tumor microenvironment.22 Inhibiting FLT3L using a small molecule inhibitor has re- duced proliferation and increased apoptosis in MM cells.23 Similarly, targeting FLT3L with antibody-based therapy has demonstrated significant antitumor activity in MM.24 While emerging evidence highlights the crucial role of FLT3L in MM pathogenesis and its potential as a therapeutic target, further studies are needed to fully understand the underly- ing mechanisms of FLT3L function in MM and develop more effective FLT3L-targeted therapies for the disease. There- fore, we investigated the link of FLT3L to bone osteolysis in MM and explored the molecular mechanism in view of the osteolytic phenomenon in MM.
Methods
Primary sample
The patients who had been diagnosed with hematologic malignancies at Seoul National University Hospital from March 2004 to December 2012 were enrolled in this study. BM-derived blood samples from patients with leukemia or MM were used to measure FLT3L. Primary samples were obtained with informed consent (Seoul National University Hospital [SNUH] Institutional Review Board [IRB] N. 1902- 047-1008). The study was approved by the SNUH IRB (N. 1902-047-1008).
Cell lines and reagents
HEK293T, HeLa, U2OS, and MOLP8 cells were used to inves- tigate the effect of FLT3L on intracellular signaling. HEK293T, HeLa, and U2OS cells were grown in high-glucose DMEM, and MOLP8 cells were cultured in RPMI. Culture media were supplemented with 10% FBS and 100 U/mL penicillin-strep- tomycin (all Gibco; Grand Island, NY, USA). Cells were main- tained in a humidified 5% CO2 at 37°C. Recombinant human FLT3L ligand (rhFLT3L) was purchased from R&D SYSTEMS (Minneapolis, MN, USA). Stattic (Sigma-Aldrich; St. Louis, MO, USA), a STAT3 inhibitor, was pre-treated for one hour before treatment with rhFLT3L in HEK293T cells.
Measurement of FLT3L
Plasma was extracted from patients’ BM-derived blood by gradient centrifugation, and used to measure FLT3L. FLT3L level was measured using human FLT3L pre-coated enzyme-linked immunosorbent assay (ELISA) kit (Biogems, Westlake Village, CA, USA) according to the manufacturer’s protocol. The standard curve was created by generating a 4-parameter logistic (4-PL) regression for the concentra- tions versus measured intensities. The concentration in the sample was taken as an estimate of the measured intensity using the standard curve.
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