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. 2014 Oct;4(10):1182-1197.
doi: 10.1158/2159-8290.CD-13-0900. Epub 2014 Aug 6.

Development of siRNA Payloads to Target KRAS-mutant Cancer

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Free PMC article

Development of siRNA Payloads to Target KRAS-mutant Cancer

Tina L Yuan et al. Cancer Discov. .
Free PMC article

Abstract

RNAi is a powerful tool for target identification and can lead to novel therapies for pharmacologically intractable targets such as KRAS. RNAi therapy must combine potent siRNA payloads with reliable in vivo delivery for efficient target inhibition. We used a functional "Sensor" assay to establish a library of potent siRNAs against RAS pathway genes and to show that they efficiently suppress their targets at low dose. This reduces off-target effects and enables combination gene knockdown. We administered Sensor siRNAs in vitro and in vivo and validated the delivery of KRAS siRNA alone and siRNA targeting the complete RAF effector node (A/B/CRAF) as promising strategies to treat KRAS-mutant colorectal cancer. We further demonstrate that improved therapeutic efficacy is achieved by formulating siRNA payloads that combine both single-gene siRNA and node-targeted siRNAs (KRAS + PIK3CA/B). The customizable nature of Sensor siRNA payloads offers a universal platform for the combination target identification and development of RNAi therapeutics.

Significance: To advance RNAi therapy for KRAS-mutant cancer, we developed a validated siRNA library against RAS pathway genes that enables combination gene silencing. Using an in vivo model for real-time siRNA delivery tracking, we show that siRNA-mediated inhibition of KRAS as well as RAF or PI3K combinations can impair KRAS-mutant colorectal cancer in xenograft models.

Figures

Figure 1
Figure 1. Functionally validated Ras pathway RNAi libraries for potent and specific gene silencing
(A) The Sensor assay enables the generation of functionally validated shRNA libraries. The potency of candidate shRNAs was biologically probed in a pooled assay by quantifying knockdown of a Venus reporter cDNA fused to the shRNA’s cognate target site. (B-F) Human Ras set (hRas) Sensor assay results. Comparable results were obtained for the mouse Ras set (mRas, Table S1 and S2). (B) Correlations in read numbers of technical vector pool replicates (V1, V2), and biological duplicates (R1, R2) at different selection stages of the Sensor assay (S3, Sort 3; S5, Sort 5; r, Pearson correlation coefficient). (C) Correlations in read numbers between the initial vector library (mean of technical duplicates) and the endpoint populations after the indicated sorts (geometric mean of biological replicates), and the final Sensor scores. (D) Sensor scores of control shRNAs of strong (orange), intermediate (blue) or weak (yellow) potency. (E) Rank correlation between input (Algorithm rank) and output library (Sensor rank), showing that the Sensor rank is not predicted by the informatics tool (ρ, Spearman rank correlation coefficient). (F) Sensor shRNA scores for a selection of direct “Ras effector” genes (for full list see Table S2). The dotted line indicates a threshold for potent shRNAs, based on known controls. 65 shRNAs/gene; controls are represented multiple times for better visibility.
Figure 2
Figure 2. Sensor siRNA potency and KRAS siRNA sensitivity in CRC lines
(A) SW1116 cells expressing an shRNA-resistant HA-KRASG12V cDNA were infected with one of three KRAS shRNAs or a negative control (sh.Ren.713), and knockdown of endogenous KRAS protein was measured. (B-C) U2OS cells were transfected with varying concentrations of Sensor siRNAs against KRAS (B) or RAF kinases (C) for 72h and knockdown of endogenous protein was measured (See Figure S2A for quantification). (D) Top-ranked siRNAs against selected Ras pathway genes were transfected at 2nM into U2OS cells, and mRNA levels were measured by RT-qPCR 72h post-transfection. (E) Colorectal cancer cell lines with the indicated KRAS and BRAF mutational status were transfected with siKRAS_234 or siKRAS_355 at 5nM, and cell viability was correlated between the two siRNAs. Four of the 5 KRAS mutant lines were sensitive to KRAS depletion whereas KRAS WT lines were resistant. (F) SW1116 cells carrying a doxycycline-inducible siRNA-resistant HA-KRASG12V construct were treated ± doxycycline (100ng/ml) 60 hours prior to siRNA transfection. Cell lysates were collected 48h post-siRNA transfection for western blot analysis. (G) SW1116 cells carrying doxycycline-inducible HA-KRASG12V or HA-KRASWT constructs were treated ± doxycycline (100ng/ml) 60 hours prior to siRNA transfection. Cell viability (normalized to uninduced cells) was assessed 5 days post-siRNA transfection.
Figure 3
Figure 3. Low dose shRNA and siRNA minimizes off-target effects
(A) Trp53−/− MEFs were infected at single- or high-copy with one of 6 Trp53 shRNAs. RNA deep-sequencing showed that high-copy shRNA transduction resulted in increased levels of mature Trp53 shRNAs in all cases. Endogenous microRNA expression was unperturbed (see also S3A, S3B, S3E and Table S3 for details; r, Pearson correlation coefficient). (B) Microarray analysis was performed on Trp53−/− MEFs not expressing any shRNA (WT and empty vector) or transduced at single- or high-copy with one of 6 Trp53 shRNAs. Shown are the top 1500 genes that are sequence-independently up- and down-regulated across all 6 shRNAs. Significant perturbations were observed only at high-copy transduction (p<0.05). For more details see Figure S3C and S3D. (C) Gene expression changes were analyzed in Kras−/− mouse embryonic fibroblasts transfected with Sensor siKRAS_234 or siKRAS_355 at various concentrations after 72h. Unsupervised clustering of significantly downregulated genes (>2-fold) showed a similar gene expression pattern between control cells and cells transfected with low concentrations (0.2 and 2 nM) of siRNAs. More profound gene perturbation occurred in cells transfected with high concentrations (20 and 50 nM) of siRNAs (see also Figure S3F-H).
Figure 4
Figure 4. Sensor siRNAs can be used in high-order combinations
(A) U2OS cells were transfected with Sensor siRNAs targeting MAPK pathway genes. Each siRNA was transfected at 2nM ± a non-targeting siNEG for a total of 14nM siRNA. Target protein knockdown was assessed by western blot 72h post-transfection. (B) Viability of CRC cells was measured 5 days post-siRNA transfection or treatment with PLX4032 (1□M). (C) Apoptosis of CRC cells was assessed by measuring caspase-3/7 activity 72h post-siRNA transfection. Apoptosis was adjusted by viability and normalized to a siNEG-transfected control. (D) CRC cells were treated for 72h with 5nM of the indicated siRNA(s) ± siNEG for a total of 30nM siRNA. Where indicated, cells were treated with AZD6244 (1□M), BEZ235 (1□M) or PLX4032 (10□M for SW48, Caco2, SW620 and SW1116; 1□M for RKO and LS411N) for 24h. Lysates were analyzed for pMEK, pERK, pAKT and pS6 levels with the ViBE bioanalyzer (ND, not determined).
Figure 5
Figure 5. SW620 xenografts treated with varying doses of siKRAS
(A) Three doses of NP(siKRAS+siDsRed) were prepared by varying the ratios of siKRAS_234 and siANC_22. Nanoparticles were administered via tail vein injection on days 1, 3, 8, 10 and 15, beginning when tumors reached 100mm3. (B) NP treatment of siKRAS at 4mg/kg was able to significantly slow tumor growth compared to control siANC_22 treated tumors. (C) At day 19, whole tumor lysate was analyzed by western blot for KRAS expression and markers of MAPK signaling and cell cycle progression. (D) Tumor volume and relative tumor viability were measured daily with optical imaging of EGFP fluorescence. Raw EGFP images of 2 representative tumors from each treatment group are shown (left), where NP-siRNAs were injected on day 1 and day 3. Viability of transduced cells (right) is monitored by concomitant tracking of DsRed and EGFP. Concurrent drops in DsRed and EGFP signals (synchronous arrowheads) illustrate siRNA-induced lethality, while DsRed drops that do not induce EGFP drops (opposing arrowheads) indicated no siRNA effect on viability. (E) Tumors were analyzed by immunohistochemistry for pERK and pS6 levels. (F) Immunohistochemistry staining and quantification of Ki67 and cleaved caspase-3 (CC3) positive cells in tumors (***p<0.001; ****p<0.0001).
Figure 6
Figure 6. SW1116 xenografts treated with siKRAS or combination siA/B/C-RAF
(A) Four NP treatments delivering 2mg/kg of each siRNA species were prepared as depicted. Nanoparticles were administered via tail vein injection on days 1, 3, 5, 8, 10, 12 and 15, beginning when tumors reached 100mm3. (B) siKRAS and siA/B/C-RAF treatment slowed tumor growth to a similar extent, compared to siANC_22 and siB/C-RAF treated tumors. The inset (bottom) shows growth of all individual tumors analyzed. (C) At day 17, whole tumor lysate was analyzed by western blot for KRAS and RAF isoform expression. (D) Tumor volume and relative viability were measured daily with optical imaging in the EGFP channel, and DsRed fluorescence tracked nanoparticle delivery, as described in Figure 5C. NP-siRNAs were injected on days 8, 10 and 12. (E) Tumors were analyzed by immunohistochemistry for Ki67 and cleaved caspase-3 positive cells (**p<0.01; ***p<0.001; ****p<0.0001).
Figure 7
Figure 7. SW620 xenografts treated with combination siKRAS+siPIK3CA/B
(A) Four NP treatments delivering 2mg/kg of each siRNA species were prepared as depicted. Nanoparticles were administered via tail vein injection on days 1, 3, 5, 8, 10, and 12, beginning when tumors reached 70-100mm3. (B) siKRAS and siPIK3CA/B treatment induced mild tumor regression and slowed the rate of tumor growth during the first week of treatment compared to siANC_22-treated tumors. siKRAS+PIK3CA/B treatment potentiated the growth inhibition throughout the course of treatment. The inset (bottom) shows growth of all individual tumors analyzed. (C) Whole tumor lysate was collected on day 15 and analyzed by western blot for KRAS, p110□ and p110□ expression. (D) Tumor volume and relative viability were measured daily with optical imaging in the DsRed channel, and EGFP fluorescence tracked nanoparticle delivery, as described in Figure 5C. NP-siRNAs were injected on days 1, 3 and 5. (E) Tumors were analyzed by immunohistochemistry for Ki67 and cleaved caspase-3 positive cells (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

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