Abstract

Abstract 65

Background

Specific oncogenes when overexpressed in lymphoma are associated with poor prognosis. Small interfering ribonucleic acids (siRNAs) can be custom designed to inhibit target oncogenes, but delivery of siRNA into lymphoma cells is a difficult task. Aptamers are synthetic in vitro selected nucleic acids that bind with high specificty to their target molecules. B cell activating factor (BAFF) enhances the maturation and survival of B cells via binding to its receptor, BAFF-R, which is expressed on the surface of B cell lymphoid malignancies. We have designed an aptamer against BAFF-R (R1) as a delivery vehicle for siRNA by covalently linking it to an siRNA molecule that inhibits oncogene target STAT3 (si-STAT3). We aim to downregulate STAT3 mRNA both in vitro (primary lymphoma cells) and in vivo (mouse xenograft models) with our aptamer/siRNA construct (R1-STAT3).

Methods

In vitro experiments used 2 mantle cell lymphoma cell (MCL) lines (Jeko-1, Z138), one diffuse large B cell lymphoma (DLBCL) cell line (Daudi), one negative control cell line (CEM, no BAFF-R expression), and primary lymphoid tumors. Primary lymphoid tumors were obtained from patients with at least 15% peripheral blood lymphoma involvement (chronic lymphoid leukemia-CLL, marginal zone lymphoma-MZL, and MCL). The selection and synthesis of aptamers used SELEX, gel shift, and filter binding assays. The visualization of aptamers utilized Z-axis confocal microscopy and Cy3 labeled aptamers. siRNA against STAT3 was generated using standard algorithms. Conjugation of aptamer to siRNA was done by two separate methods (chimera and stick, Zhou 2009). Downregulation of STAT3 was accessed by qRT-PCR and western blot analysis.

We used NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice and MCL cell lines for in vivo mouse xenograft experiments. One million Z138 cells transduced with lentiviral vectors expressing luciferase were injected subcutaneously into the flanks of mice. Tumor engraftment was visualized by palpation and Xenogen imaging. Mice were then treated with R1 alone, R1-STAT3, and a negative control via intratumoral injection. Dosage and schedule of administration were consistent for all experimental groups. Mice were then sacrificed and subcutaneous tumors analyzed for STAT3 expression. We also injected one million Z138 cells transduced with a lentiviral vector expressing luciferase intravenously via tail and visualized engraftment by Xenogen imaging.

Results

Confocal microscopy showed easy entry of Cy3 labeled R1 aptamers and aptamer/STAT3 siRNA (R1-STAT3) conjugate into lymphoma cell lines (MCL and DLBCL), and primary lymphoma cells (CLL and MCL), but not into CEM cells. Intravenous injection of R1 showed biodistribution of R1 in lung/liver/spleen/BM and tumor site. qRT-PCR for R1 showed the highest concentration of R1 in tumor sites as compared to spleen/liver (4 log increase). These results show R1 aptamer can selectively target BAFF-R expressing lymphomas in vitro and in vivo. Table 1 shows R1-STAT3 conjugates downregulate STAT3 mRNA and STAT3 protein in MCL cells lines, but has no effect on CEM cells. Subcutaneous injection of MCL cells caused subcutaneous tumor formation which was easily palpable. We then performed Intratumoral injection of R1 and R1-STAT3 into subcutaneous tumors and analyzed STAT3 mRNA expression. Table 1 shows downregulation of STAT3 mRNA with R1-STAT3 as compared to R1 injection alone (83% downregulation). These results shows R1-STAT3 can efficiently downregulate STAT3 mRNA in vivo as well as in vitro.

Conclusions

BAFF-R functions as an efficient delivery vehicle for siRNA therapy in BAFF-R expressing B cell lymphomas. The aptamer/siRNA construct selectively and efficiently downregulates target oncogenes in primary lymphoma cells and in mouse xenograft models.

Table 1.

Functional assay of R1 and R1-STAT3

Assay typeDaudiMCL pt sample (1)CLL pt samples (2)Jeko-1Z138CEMMCL Mouse Xenograft
Confocal microscopy + entry + entry + entry + entry + entry No entry N/A 
R1 alone        
Confocal microscopy R1-STAT 3 + entry + entry + entry + entry + entry No entry N/A 
qRT-PCR (STAT3 mRNA) N/A N/A N/A 100% 100% 100% 100% 
R1 alone        
qRT-PCR (STAT3 mRNA) N/A N/A N/A 38%+3.6% 21.6%+1.1% 91%+8.05% 17%+10.5% 
R1-STAT3        
Western Blot (STAT3 protein) R1-STAT3 N/A N/A N/A 67% 35% N/A N/A 
Assay typeDaudiMCL pt sample (1)CLL pt samples (2)Jeko-1Z138CEMMCL Mouse Xenograft
Confocal microscopy + entry + entry + entry + entry + entry No entry N/A 
R1 alone        
Confocal microscopy R1-STAT 3 + entry + entry + entry + entry + entry No entry N/A 
qRT-PCR (STAT3 mRNA) N/A N/A N/A 100% 100% 100% 100% 
R1 alone        
qRT-PCR (STAT3 mRNA) N/A N/A N/A 38%+3.6% 21.6%+1.1% 91%+8.05% 17%+10.5% 
R1-STAT3        
Western Blot (STAT3 protein) R1-STAT3 N/A N/A N/A 67% 35% N/A N/A 
Disclosures:

No relevant conflicts of interest to declare.

Author notes

*

Asterisk with author names denotes non-ASH members.