Abstract
Background: Drug resistance and progressive bone destruction are the primary causes of morbidity and mortality in multiple myeloma (MM). Critically, the pathogenesis of MM is intrinsically linked to the bone marrow microenvironment (BME), which provides essential survival, growth, and drug-resistance signals to the tumor cells. Proteasome inhibitors (PIs) like bortezomib are a mainstay of MM therapy, inhibiting cancer growth and exerting bone anabolic effects. However, drug-resistant relapses ultimately negate their benefit. Sphingolipid dysregulation has been implicated in conferring PI resistance. The oral sphingosine 1-phosphate receptor modulator, fingolimod (FTY720), improves bone health in multiple sclerosis and is well-tolerated long-term. The potential of combining fingolimod with bortezomib for MM treatment remains unexplored. Repurposing safe oral drug may facilitate clinical trial in myeloma and safe cost
We hypothesized that fingolimod, at a clinically achievable dose, would synergize with bortezomib to indirectly inhibit MM growth primarily through modulation of the bone marrow microenvironment, enhancing its anabolic effects and disrupting tumor-supportive signals.
Method: We investigated the effects of fingolimod alone and combined with bortezomib on tumor growth and osteoblastic differentiation using: 1) In vitro assays with human MM cell lines and primary MM cells, 2) Ex vivo co-cultures of human MM cells with mouse bone explants (modeling the critical tumor-BME interaction), and 3) In vivo intratibial xenograft models in NOD-SCID mice (luciferase secreted RPMI-8226 cells).
Results: Fingolimod exhibited direct growth inhibition of MM cells at an IC50 of 3-6 µM, but these concentrations were toxic to bone cells (osteoblasts, osteocytes, osteoclasts) and explants. At clinically relevant doses (10-100 nM, comparable to blood levels in MS patients), fingolimod alone showed no significant cytotoxicity against MM or bone cells but potently stimulated osteoblast differentiation, increasing alkaline phosphatase and osteocalcin mRNA while promoting robust nodular calcification in pre-osteoblasts. When combined with bortezomib (3 nM), fingolimod further enhanced calcification beyond either drug alone. In mouse bone explants, fingolimod (10-100 nM) demonstrated dual effects: increasing osteoblast markers (alkaline phosphatase, osteocalcin mRNA) and osteocyte differentiation (DMP1 mRNA) while simultaneously suppressing osteoclast markers (ACP5/TRAcP, cathepsin K mRNA), contrasting with zoledronic acid's exclusive osteoclast inhibition. In MM-bone explant co-cultures modeling tumor engraftment, MM cells induced osteolytic lesions with elevated osteoclast gene expression (ACP5, cathepsin K). While 100 nM fingolimod alone minimally affected tumor growth, its combination with bortezomib (3 nM) significantly inhibited tumor growth versus bortezomib alone (p<0.05), with profound suppression of osteoclast genes and enhanced osteoblast/osteocyte markers (osteocalcin, DMP1).
In both the intratibial (local BME engraftment), and subcutaneous (plasmacytoma) xenografts, fingolimod monotherapy (5 mg/kg, 3x/week) showed marginal effect on tumor burden, whereas bortezomib monotherapy (0.25 mg/kg, 2x/week) reduced burden by 25-30%. The combination achieved >80% tumor reduction (p<0.01), reduced osteolytic lesions in the intratibial model, and uniquely increased vertebral bone mineral density distant from tumor site, indicating systemic reversal of myeloma-induced osteolysis. RNA sequencing revealed mechanistic synergy: fingolimod suppressed tumor-intrinsic sphingolipid (SPHK2, SGPL1) and autophagy (ATG5, LC3B) pathways while counteracting bortezomib-induced resistance signatures (BCL2, MCL1, IL6, TNFα).
Conclusions: Fingolimod, at clinically achievable doses, synergizes with bortezomib through dual mechanisms: 1) direct disruption of sphingolipid-mediated survival pathways in tumor cells 2) remodeling the BME by coupled osteoclast inhibition/osteoblast activation, including systemic osteo-protection, suggesting neutralization of circulating osteolytic factors. Future work includes validation in PDX models, sphingolipid-risk biomarker development, and Phase Ib trials in relapsed/refractory MM with bone disease. Fingolimod's established safety profile as an oral multiple sclerosis therapy enables rapid clinical translation.
