A pan-tumor-siRNA aptamer chimera to block nonsense-mediated mRNA decay inflames and suppresses tumor progression

Abstract

Immune-checkpoint blockade (ICB) therapy has changed the clinical outcome of many types of aggressive tumors, but there still remain many cancer patients that do not respond to these treatments. There is an unmet need to develop a feasible clinical therapeutic platform to increase the rate of response to ICB. Here we use a previously described clinically tested aptamer (AS1411) conjugated with SMG1 RNAi (AS1411-SMG1 aptamer-linked siRNA chimeras [AsiCs]) to inhibit the nonsense-mediated RNA decay pathway inducing tumor inflammation and improving response to ICB. The aptamer AS1411 shows binding to numerous mouse and human tumor cell lines tested. AS1411 induces tumor cytotoxicity in long incubation times, which allows for the use of the aptamer as a carrier to target the RNAi inhibition to the tumor. The AS1411-SMG1 AsiCs induce a strong antitumor response in local and systemic treatment in different types of tumors. Finally, AS1411-SMG1 AsiCs are well tolerated with no detected side effects.

Keywords: NMD; RNAi delivery; aptamer; cancer immunotherapy; oligonucleotides: therapies and applications.

Graphical abstract

Figure 1

AS1411 binds and triggers tumor cytotoxicity in human and murine tumor cells (A) AS1411 aptamer binding to murine cancer cell lines of colon carcinoma (CT26), melanoma (B16/F10), breast cancer (4T1), and pancreatic cancer (Panc02). (B) AS1411 binds to human tumor cells: melanoma (AXBI, ARST), hepatocellular carcinoma (JHH6), colorectal adenocarcinoma (SW480), and breast cancer (MDA-MB-231). (C) Cytotoxic effect of AS1411 measured by MTS in human and mouse tumor cell lines. IC₅₀ is indicated for each cell line. n = 3. Data shown are mean ± SEM.

Figure 2

AS1411-siRNA SMG1 AsiC downregulates NMD via SMG1 silencing in tumor cells and improves the stability of potential NMD-regulated neoantigens (A) Schematic of AS1411-siRNA SMG1 AsiC. AS1411 is shown as G4 monomer conformation. Thymidines within the G4 are not represented for graphical simplification. 2′-Fluoro-modified nucleotides are indicated in green, and siRNA guide and passenger strands are specified with arrows. (B) AS1411-SMG1 AsiCs inhibit SMG1 mRNA by free uptake in CT26 cells. AS1411-AsiC was added twice at 24 and 48 h, and SMG1 mRNA was quantified by qRT-PCR. n = 3. (C) psiCHECK reporter assay to validate target inhibition of SMG1 in left, Panc02 cells and right, B16/F10. Cells transfected with psiCHECK luciferase reported plasmid containing the SMG1 target were treated as in (B) with AS1411-AsiC. SMG1 downregulation was proportional to the Renilla signal and normalized with Firefly luciferase. n = 3. (D) Western blot to validate AS1411 AsiC SMG1 silencing by free uptake. Treatment schedule followed was the same as in (B). (E) AS1411-SMG1 free uptake inhibits NMD in SIIN-BG-ter-expressing B16/F10 cells. This stabilizes SIINFEKL mRNA and leads to peptide presentation triggering OT-I lymphocytes activation. (F) B16/F10 expressing a β-Globin-SIINFEKL-PTC39 plasmid were treated with AS1411-SMG1 AsiC as in Figure 2C and then co-cultured with OT-I splenocytes. Supernatants were analyzed by IFN-γ ELISA. n = 3. Data shown are mean ± SEM. p < 0.05(^∗), p < 0.01(^∗∗), p < 0.001(^∗∗∗), and p < 0.0001 (^∗∗∗∗).

Figure 3

Intratumoral administration of AS1411-SMG1 AsiC significantly reduces tumor growth (A) Treatment schedule. CT26 cells were implanted into the right flank of Balb/c mice and AS1411-AsiC was injected intratumorally on days 4, 6, 8, 11, 14, and 16. Tumors were resected at day 20. (B) CT26 tumors were resected and weighed on day 20. n = 5–6. (C) Treatment schedule. B16/F10 cells were implanted into the right flank of C57/BL6 mice. AS1411-AsiC was injected intratumorally on days 1, 2, 3, 10, 14, and 15. On day 16, tumors were resected. (D) B16/F10 tumor weight on day 16. n = 5–6. Data shown are mean ± SEM. p < 0.05(^∗), p < 0.01(^∗∗), p < 0.001(^∗∗∗), and p < 0.0001 (^∗∗∗∗).

Figure 4

T cell infiltration increases in the B16/F10 tumor model after AS1411-SMG1 AsiC treatment. B16/F10 cells were injected into the right flank of C57/BL6 mice Mice were treated with six doses (300 pmol per dose) of AS1411-SMG1, AS1411-control, or vehicle (see Figure 3D for detailed treatment schedule). Tumors were resected on day 16 to analyze the lymphocyte infiltrate by flow cytometry. T cell populations were gated in the CD45⁺ Live cells (Zombie Green negative population) and quantified in percentage. (A) CD3⁺CD8⁺. (B) quantification of CD3⁺CD4⁺ T cells. (C) T regulatory lymphocytes (CD4⁺FOXP3⁺). (D) CD8⁺: T regulatory cells coefficient. n = 4–6. Data shown are mean ± SEM. p < 0.05(^∗), p < 0.01(^∗∗), p < 0.001(^∗∗∗), and p < 0.0001 (^∗∗∗∗).

Figure 5

Antitumor effect of AS1411-SMG1 AsiC administered systemically (A) Treatment schedule. B16/F10 cells were implanted in C57/BL6 mice. 300 pmol of AS1411 AsiCs or scramble aptamer were administered intravenously on the indicated days. (B) AS1411-SMG1 treatment showed a highly significant antitumor effect compared with controls. n = 7–10. (C) Treatment schedule. 4T1 cells were injected in Balb/c mice. Mice were injected with CTLA-4 and PD-1 (100 μg of each antibody or 200 μg of isotype control antibody) on the days shown. (D) Additive effect of CTLA-4 + PD-1 checkpoint blockade in 4T1 murine breast cancer model. NMD inhibition with chimera showed a similar effect with the immune-checkpoint blockade therapy. AS1411-control per se had almost no significant effect. A significant improvement in antitumor effect was observed in the combination of AS1411-SMG1 CTLA-4 + PD-1 compared to both monotherapies. n = 6–10. Data shown are mean ± SEM. p < 0.05(^∗), p < 0.01(^∗∗), p < 0.001(^∗∗∗), and p < 0.0001 (^∗∗∗∗).

Figure 6

Evaluation of immune-mediated side effects of AS1411-SMG1 AsiC in mice Tumor-free C57/BL6 mice were treated with therapeutic doses used in Figure 5C of AS1411-SMG1 AsiC, anti-CTLA-4 (clone 9H10), and anti-PD-1 (clone rmp1-14) antibodies or three doses of 200 μg of anti 4-1BB antibody (clone 3H3) as control immune-mediated toxicity. n = 3. (A–C) Liver samples were analyzed by flow cytometry to study toxicity-induced inflammation. Total CD3⁺ (A) were quantified in the whole immune population (CD45⁺). CD4 (B) T cells were analyzed in the CD3⁺ cluster as well as CD8 lymphocytes (C). (D) Evidence of splenomegaly as side effect of systemic inflammation. (E) Hematoxylin and eosin staining of liver samples of treated mice. Abnormal infiltration of lymphocytes was detected in the samples of 4-1BB-treated mice as expected (indicated with dashed lines in the bottom right panel). No other major histological changes were observed. Data shown are mean ± SEM.