Poster S20 - Targeting the Hypoxic Tumor Response in Multiple Myeloma Using Tirapazamine-Loaded, BCMA-Targeted Nanoparticles Overcomes Drug Resistance and Prevents Metastasis, In Vitro and In Vivo.

Introduction: Multiple myeloma (MM), though rare, accounts for 1.8% of new cancer diagnoses in the United States yet remains a highly fatal disease. It is estimated that 35,730 new cases will be diagnosed in 2023, leading to 12,590 deaths. Despite encouraging responses in the upfront treatment setting, low responses are observed in relapsed patients due to development of drug resistance. We have previously shown that tumor hypoxia plays a significant role in drug resistance and metastasis in cancer in general, and MM in particular (Azab AK et al., 2012; Imai et al., 2017; Muz et al., 2015; Muz et al., 2020). Tirapazamine (TPZ) is a potent hypoxia-activated prodrug that causes single- and double-strand DNA breaks through the generation of free radicals under hypoxic conditions; however, it was found to have significant side effects in normal tissues in clinical trials. The goal of this study is to develop an MM-targeting nanoparticle-based drug delivery system to specifically deliver TPZ to MM cells and reverse their drug-resistant and metastatic potential. B cell maturation antigen (BCMA) is becoming a gold standard antigen for targeted T cell immunotherapies such as CAR T cells and bispecific T cell engagers in MM. We hypothesize that targeting TPZ-loaded nanoparticles to BCMA on MM cells will provide specific binding, uptake, and delivery of TPZ to MM cells, increasing its efficacy and reducing its side effects in other organs.

Materials and

Methods: Liposomes preparation and characterization: phosphor-lipids and fluorescent tracer DiD, with or without TPZ, were solubilized in chloroform, evaporated to a thin film, hydrated with PBS, and extruded using polycarbonate membranes. Streptavidin was conjugated to the amines on the surface of the liposomes, and biotinylated anti-BCMA or isotype (non-targeted) antibodies were conjugated to streptavidin. Liposome size, polydispersity index, and zeta potential were determined using Zetasizer. Encapsulation efficiency was determined by detecting free TPZ in the supernatant using HPLC. Binding and uptake: MM cell lines and primary cells were treated with increasing concentrations of BCMA-targeted or non-targeted liposomes for various times, washed, and their DiD fluorescence was measured by flow cytometry. Cell Toxicity: MM cell lines were treated with TPZ (free or liposomal) under normoxic or hypoxic (1% O2), and survival was determined using MTT assay. In addition, Immunoblotting, cell cycle, apoptosis, adhesion to stromal cells, and migration assays were performed with MM cell lines, which were pre-treated with or without TPZ under normoxic or hypoxic conditions, as we previously described. In vivo bio-distribution: non-targeted and BCMA-targeted liposomes were injected i.v. into MM-bearing mice, and 24 hours later, their blood and organs were extracted and analyzed by flow cytometry to detect the mean fluorescence intensity (MFI) of DiD. In vivo tumor progression: Four groups of MM-bearing mice were treated with (1) vehicle (PBS), (2) free TPZ, (3) TPZ-loaded non-targeted liposomes, and (4) TPZ-loaded BCMA-targeted liposomes. These treatments were injected i.v. once a week, followed by BLI imaging to assess tumor progression.

Results, Conclusions, and Discussions: The liposomes had a size of 178 nm, PDI of 0.125, and zeta potential of +2.1 mV, with a TPZ encapsulation efficiency of 59%.
TPZ was more efficacious in hypoxic MM cells compared to normoxic cells. Liposomal TPZ demonstrated similar efficacy to free TPZ in vitro, with similar IC50 values. Treatment with TPZ reduced HIF-1 protein levels under both normoxic and hypoxic conditions and decreased cell proliferation-related proteins p-AKT, p-mTOR, and p-S6R. TPZ decreased the cell cycle and induced G1 arrest in flow cytometry, reflected also by decreased pRb, CDK2, and CDK6 cell cycle-related protein expression levels. Additionally, TPZ increased apoptosis and pro-apoptotic signaling proteins cPARP and cCasp3. Moreover, TPZ increased the adhesion of hypoxic MM cells to the stromal cells. Notably, no substantial impact of TPZ on cell adhesion was observed under normoxic conditions. Furthermore, TPZ decreased in the hypoxia-induced migration in MM. In contrast, TPZ treatment did not exhibit a noticeable effect on cell migration in normoxia.

BCMA-targeted liposomes showed an 8-fold higher binding to MM cells compared to non-targeted liposomes, and the binding was in linear correlation with the expression of BCMA on MM cells. In vivo, bio-distribution of BCMA-targeted liposomes exhibited higher localization in both the bone marrow and liver compared to non-targeted liposomes, where MM cells were predominantly found. Within the same organ, distinguishing between the tumor microenvironment and the MM cell revealed high specificity of the BCMA-targeted liposomes to MM cells over other types of cells. Finally, TPZ-loaded BCMA-targeted liposomes decrease tumor progression in advanced tumor model, compared to TPZ-loaded non-targeted liposomes and to free TPZ. In addition, mice treated with BCMA-targeted liposomes showed lower weight loss than the other formulations, indicating lower side effects.

In conclusion, TPZ effectively targets hypoxic MM cells, reduces drug resistance, and prevents cell trafficking and metastasis in MM. TPZ encapsulated in liposomes targeting BCMA-expressed on MM cells enhanced TPZ’s anti-cancer effect in vitro and in vivo, and reduced side effects. This is a proof-of-concept study that TPZ-loaded BCMA-targeted liposomes are a promising strategy for future clinical applications such as the treatment of relapsed refractory MM patients.

Acknowledgements (Optional): This work is supported by a grant from the Cancer Prevention & Research of Texas (CPRIT # RR220088).