(F-207) Bone Marrow Vascular Microenvironment Combination RNAi Nanotherapy for Multiple Myeloma

Introduction:: Multiple myeloma (MM) is an incurable hematologic cancer characterized by the accumulation of abnormal plasma cells within the bone marrow. It is well documented that the bone marrow niche provides nutrients to myeloma cells, promoting their homing, proliferation, transcellular migration, and acquired drug resistance [1]. Specifically, extracellular cyclophilin A (CyPA), secreted by bone marrow endothelium, acts as a chemoattractant enhancing the expression of CD147, its receptor, on the surface of MM cells which enhances their ability to extravasate [2]. In addition, the E-selectin (ES) receptor is constitutively expressed by bone marrow endothelial cells, promoting the rolling and adhesion of myeloma cells to the vasculature, contributing to subsequent MM dissemination to distant bone marrow sites (Figure 1a) [3]. Thus, simultaneous inhibition of CyPA and ES could be a therapeutic strategy to avoid MM spreading. However, specific- and direct-inhibition of CyPA and constitutive ES on bone marrow endothelial cells by small molecules has been elusive, and blocking antibodies induces endothelial cell activation, which can promote further MM dissemination, respectively. Here, we engineered a lipid-polymer nanoparticle (NP) to use RNA interference (RNAi) therapy, co-delivering CyPA and ES siRNAs, to inhibit their expressions within the vessels of the bone marrow, as a strategy to evade drug resistance in MM.

Materials and Methods:: NPs were synthesized by combining an RNA phase with an ethanol phase containing polymer-lipids and a polyethylene glycol (PEG)-lipid conjugate via controlled mixing in a microfluidic device [4]. ES knockdown was investigated using the mouse endothelial cell line bEnd.3 and was confirmed using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). Adhesion and transmigration assays were performed, where myeloma (MM.1S) cells were co-cultured with either untreated or NP-treated endothelial monolayers. The number of cells was determined using confocal microscopy or a fluorescent reader, respectively. To probe the therapeutic efficacy of the presented combination RNAi strategy in vivo, a survival study was assessed where siRNAs encoded for either CyPA (siCyPA) and/or ES (siES) were encapsulated within the NP. These were administered intravenously to tumor-bearing Nod/SCID mice, either alone or in combination with bortezomib, an FDA-approved MM therapy.


Results, Conclusions, and Discussions:: Using RT-qPCR, effective knockdown of ES on bEnd.3 monolayers was confirmed in a dose-response matter (Figure 1b). Further, the number of MM.1S cells adhered to (GFP+) or transmigrated through bEnd.3 monolayers (Figure 1c, 1d), reveals that siES-NPs induce potent gene silencing in endothelial cells in vitro, preventing MM.1S cells from extravasate to distant bone marrow sites. Lastly, to assess the efficacy of this strategy in vivo, extended survival of mice up to 100 days was observed when mice were treated with NPs co-encapsulating siCyPA and siES in combination with bortezomib, compared to 65 days when treated with one or the other (Figure 2e). These findings imply that simultaneous silencing of CyPA and ES is vital to induce bortezomib therapy sensitivity to myeloma cells in vivo. These data present the first vascular microenvironment combination RNAi therapy for MM as means to evade its spreading, and overcome drug resistance, which can potentially be applied to target any vascular bed within the body to treat other malignancies.


Acknowledgements (Optional): :

References (Optional): : [1] Kumar, S. K. et al., Nat Rev Dis Primers (2017).
[2] Zhu D., et al., Nat Med (2015).
[3] Natoni, A. et al., Leukemia (2017).
[4] Chen, D. et al., J Am Chem Soc (2012).