Antineoplastic effects of liposomal short interfering RNA treatment targeting BLIMP1/PRDM1 in primary effusion lymphoma.

RNA interference (RNAi) has been suggested to represent a promising therapeutic approach in different disease settings. Primary effusion lymphoma (PEL) is a plasmablastic lymphoma consistently expressing B lymphocyte-induced maturation protein 1 (Blimp-1), a pivotal transcriptional regulator during terminal differentiation of B cells into plasma cells. Here we report, for the first time, that transient knockdown of the BLIMP1 gene (also known as PR Domain Containing 1 with ZNF Domain, or PRDM1) using small interfering RNA (siRNA) delivered by liposomes, induced remarkable killing in PEL cell lines. Furthermore, in a murine model of PEL, significantly prolonged survival was achieved by intraperitoneal treatment with such anti-BLIMP1 lipoplexes, while no vector-induced toxicity was observed. This effective and safe RNAi strategy, based on liposomal siRNA targeting a master transcription factor of post-germinal center B cells, may indeed be a potential treatment against plasmablastic lymphoma. Over the last decade, after the discovery of RNAi as a useful experimental tool for selective gene silencing, the possibility of harnessing siRNA also in disease treatment has been increasingly investigated. With regard to cancer treatment, several preclinical studies have shown promising therapeutic potential associated with siRNAmediated down-regulation of different tumor-relevant genes, both in solid neoplasms and hematologic malignancies. However, clinical applications of RNAi seem to be dependent on the use of optimal delivery systems. To date, while there are still safety concerns regarding siRNA-expressing viral vectors, the use of liposomal carriers has been suggested to be a safe and effective option, protecting siRNA from rapid degradation after administration, as well as efficiently delivering them into target cells. Recently, two clinical trials have provided major proof of concept for cationic liposome-mediated RNAi therapy, targeting abnormal genes implicated in transthyretin amyloidosis and hypercholesterolemia. PEL is an aggressive B-cell lymphoma, characterized by a plasma cell-like gene expression profile, driven by human herpesvirus-8/Kaposi sarcoma associated herpesvirus (HHV8/KSHV). PEL typically arises in serous body cavities of immunocompromised patients (mainly those infected with human immunodeficiency virus) or elderly subjects; it is manifested by pleural or peritoneal malignant effusions and has a poor prognosis. Indeed, it is often not feasible to administer standard chemotherapy or systemic antivirals (e.g. Cidofovir) to such fragile patients, who frequently have comorbidities and impaired organ function, and novel therapeutic strategies are, therefore, required for PEL. Interestingly, Godfrey et al. first proposed an RNAi-based approach to treat PEL, showing effective PEL inhibition in vitro and in vivo by lentiviral vectors encoding short hairpin RNA able to knockdown HHV8/KSHV-associated oncogenes. Aiming to expand RNAi strategies for PEL treatment, we previously tested different lipid-based nanocarriers for their ability to target PEL cell lines efficiently. In the present work, we investigated the in vitro and in vivo antineoplastic activity associated with liposomal siRNA-mediated knockdown of the BLIMP1/PRDM1 gene, which encodes Blimp-1, a transcription factor considered a crucial regulator of the transcriptional network in post-germinal center B-cell stages. For in vitro experiments, PEL cell lines (BCBL-1, HBL-6 and CRO-AP/3) were cultured in fetal calf serum-supplemented RPMI 1640 medium, as described elsewhere, and were treated with siRNA against BLIMP1/PRDM1 (siRNA sequences in the Online Supplementary Information) complexed with liposomes to form anti-BLIMP1 siRNA lipoplexes (formulated and characterized as previously described). The same liposomes, either loaded with scrambled oligonucleotides (i.e. mock siRNA, commercially provided together with validated siRNA) or empty (vehicle), as well as free anti-BLIMP1 siRNA without a vehicle, were used as negative controls. PEL viability and cell concentration were assessed daily by staining with annexin V/propidium iodide (Miltenyi Biotech, Bergisch Gladbach, Germany), in accordance with the manufacturer’s instructions, and by using an AcT8 automated cell counter (Beckman Coulter Inc., Brea, CA, USA), respectively. Caspase-3 activity was assayed (Calbiochem, EMD Biosciences, San Diego, CA, USA) as previously reported, to detect the activation of the apoptotic pathway. The cell cycle was analyzed using a commercial BrdU/7-AAD assay (BD Biosciences, San Jose, CA, USA), according to the manufacturer’s instructions. Quantitative real-time polymerase chain reaction and western blot assays were performed, as previously described, to evaluate mRNA levels and protein expression, respectively, for Blimp-1 and other relevant B-cell transcription factors. To investigate putative antineoplastic effects associated with BLIMP1 silencing in PEL and, at the same time, to define the most suitable carriers for such RNAi therapy, we preliminarily performed a screening set of in vitro experiments (data not shown), using different lipid-based formulations to deliver anti-BLIMP1 siRNA (50-200 nM) into PEL cell lines, and then we tested PEL viability at 24, 48 and 72 h after treatment, by using the annexin V/propidium iodide assay. Compared with controls, most formulations of anti-BLIMP1 siRNA-lipid lipoplexes were able to cause increased cell death in all PEL cell lines tested. In particular, we found that a single treatment with anti-BLIMP1 siRNA (100 nM), delivered by dioleoyl trimethylammonium propane (DOTAP) liposomes (i.e. anti-BLIMP1 siRNA/DOTAP lipoplexes, 1:100 molar ratio; mean encapsulation efficiency 85%, mean diameter 402±31 nm, polydispersivity index 0.13±0.02, ζ-potential 19±1 mV; transfection efficiency 60-80%), induced the most remarkable and consistent reduction of PEL viability and cellularity, while only mild toxicity was associated with the use of empty DOTAP liposomes (10 mM) (Figure 1, and Online Supplementary Figures S1 and S2). Of note, at 48 and 72 h after such treatment, we observed a mean decrease in PEL viability of 83% and 95%, as well as a mean cell loss equal to 79% and 91% of the number of cells treated, respectively (Figure 1A,B). All these results were statistically significant when compared with related controls (ANOVA test, P<0.001). In line with these findings, activation of caspase-3 (Online Supplementary Figure S3) and accumulation in the sub-G1 phase (data not shown) were detected in PEL cells treated with antiBLIMP1 siRNA/DOTAP lipoplexes, without evidence of cell-cycle arrest in G1 or G2 phase (no increase of either G1/S or G2/S ratios between treated cells and controls), thus suggesting that Blimp-1 inhibition induced apoptosis directly rather than having a cytostatic effect. In parallel, the effective knockdown of BLIMP1 after liposomal siRNA treatment was confirmed by quantitative realtime polymerase chain reaction analysis and western blot assays, showing a specific decrease in BLIMP1 transcrip-