KRAS siRNA Research Articles

Abstract 1080: Kras downregulation by nanoparticle delivery of siRNA reduces tumor growth in mice

Abstract Introduction Small-interfering RNA (siRNA) is an attractive option for delivering precision therapy to cancer patients because of the potential for highly-specific control of gene expression. Attempts to utilize siRNA in vivo have been hampered by its short circulating half-life, limited cellular uptake, and cellular confinement within endosomes. To overcome these barriers, we packaged siRNA within serum-stable, cell-penetrating, and endosomolytic peptide-based nanoparticles (NPs) to deliver siRNA to human and mouse pancreas and colorectal cancers. In a small pilot study, twice weekly administration of KRAS siRNA-NP to mice bearing human colorectal cancers significantly reduced tumor growth compared to controls. Methods NP uptake was assessed in cell lines utilizing fluorescent siRNA-NPs in combination with fluorescence microscopy and flow cytometry. Cell lines were incubated with KRAS-siRNA NP and KRAS knockdown was assessed by quantitative PCR. Mice bearing tumors derived from these cell lines were injected intravenously with fluorescent NP, and localization and uptake were assessed with an in vivo imaging system (IVIS) and flow cytometry. Immune deficient mice were inoculated subcutaneously with a human colorectal cancer and half were treated for 4 weeks with twice weekly IV injections of KRAS siRNA-NP (1 nmol KRAS siRNA per dose). Results On average, fluorescent siRNA was detected in more than 93% of human and mouse pancreas and colorectal cancer cells (n = 5 cell lines) after 24 hours of incubation. Exposure of colorectal cancer cells to KRAS siRNA-NP in vitro produced a conservatively-estimated 70% downregulation of KRAS. Tumors derived from all cell lines were strongly fluorescent 2 hours after IV injection of fluorescent NP and signal persisted beyond 30 hours. The liver and kidneys also expressed fluorescent signal by IVIS. 86% of tumor cells derived from tumors exposed to fluorescent-NP by IV injection expressed fluorescent signal 24 hours post-injection. Only 18% and 14% of liver and kidney cells, respectively, were fluorescent. Compared to control mice, tumor size was reduced in mice bearing colorectal cancer that received KRAS-siRNA NP IV injections. Eighteen days after initiation of treatment, tumor volumes in the control and treatment groups were 229.5 mm3 and 460.25 mm3 respectively (p < 0.05). Hematologic, liver and kidney function tests revealed no differences between the treatment and control groups. Conclusions Peptide NPs are rapidly and efficiently taken up by human and mouse pancreas and colorectal cancer cells both in vitro and in vivo. Incubation of cancer cells with peptide NP-packaged KRAS-siRNA reduces KRAS expression in vitro. Treatment of tumor-bearing mice with KRAS-siRNA NP leads to a statistically significant reduction in tumor growth compared to control mice. This delivery platform overcomes traditional limitations of siRNA and may be generalizable to target other putative drivers of tumor progression. Citation Format: Matthew Strand, Hua Pan, Xiuli Zhang, Julie Grossman, Peter Goedegebuure, Timothy Fleming, William E. Gillanders, Samuel A. Wickline, Ryan C. Fields. Kras downregulation by nanoparticle delivery of siRNA reduces tumor growth in mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1080.

A single nucleotide polymorphism in the 3'-untranslated region of the KRAS gene disrupts the interaction with let-7a and enhances the metastatic potential of osteosarcoma cells.

The objective of the present study was to explore the molecular mechanism with which a single nucleotide polymorphism (rs61764370) interferes with the interaction between the 3'-untranslated region(3'-UTR) of Kirsten rat sarcoma viral oncogene homolog(KRAS) and let-7a, and its association with the metastasis of osteosarcoma(OS). In this study, we confirmed that KRAS is a target of let-7a in OS cells, and the introduction of rs61764370 minor allele into KRAS 3'-UTR significantly compromised the microRNA(miRNA)/mRNA interaction using a luciferase reporter system. Additionally, a total of 36OS tissue samples of three different genotypes (TT,22; TG,10; GG,4) were obtained, and the expression of let-7a and KRAS was determined. We showed that let-7a mRNA expression was similar between each group whereas the mRNA and protein expression of KRAS in the TT genotype group was significantly lower than that in the GT or GG genotype groups. Moreover, we identified a negative regulatory relationship between let-7a and KRAS. Furthermore, we demonstrated that let-7a and KRAS interfered with the viability, invasiveness and migration of OS cells genotyped as TT. In the OScells genotyped as TG, let-7a exerted minimal effects, and the effect of KRAS siRNA remained. Taken together, the findings of the present study demonstrated that the KRAS3'-UTRrs61764370 polymorphism interfered with miRNA/mRNA interaction, and showed that the minor allele was associated with an elevated risk of developing metastatic disease in OS.

Impact of Cellular Genetic Make-up on Colorectal Cancer Cell Lines Response to Ellagic Acid: Implications of Small Interfering RNA.

K-Ras activation is an early event in colorectal carcinogenesis and associated mutations have been reported in about 40% of colorectal cancer patients. These mutations have always been responsible for enhancing malignancy and silencing them is associated with attenuation of tumorigenicity. Among downstream effectors are the RAF/MEK/ERK and the PI3K/Akt signaling pathways. PI3K/Akt signaling leads to reduction of apoptosis, stimulated cell growth and enhanced proliferation. Ellagic acid (EA), a naturally occurring antioxidant, has recently emerged as a promising anti-cancer agent. To evaluate the impact of cellular genetic make- up of two colon cancer cell lines with different genetic backgrounds, HCT-116 (K-Ras-/p53+) and Caco-2 (K-Ras+/ p53-), on response to potential anti-tumour effects of EA. In addition, the influence of K-Ras silencing in HCT- 116 cells was investigated. Cellular proliferation, morphology and cell cycle analysis were carried out in addition to Western blotting for detecting total Akt and p-Akt (at Thr308 and Ser473) in the presence and absence of different concentrations of EA. Cell proliferation was also assessed in cells transfected with different concentrations of K-Ras siRNA or incubated with ellagic acid following transfection. The results of the present study revealed that EA exerts anti-proliferative and dose-dependent pro-apoptotic effects. Cytostatic and cytotoxic effects were also observed. p-Akt (at Thr308 and Ser473) was downregulated. Moreover, EA treatment was found to (i) reduce K-Ras protein expression; (ii) in cells transfected with siRNA and co-treated with EA, pronounced anti-proliferative effects as well as depletion of p-Akt (at Thr308) were detected. Cellular genetic makeup (K-Ras-/p53-) was not likely to impose limitations on targeting EA in treatment of colon cancer. EA had a multi-disciplinary pro-apoptotic anti-proliferative approach, having inhibited Akt phosphorylation, induced cell cycle arrest and showed an anti-proliferative potential in HCT-116 cells (expressing mutant K-Ras).

KRAS gene amplification to define a distinct molecular subgroup of gastroesophageal adenocarcinoma.

74 Background: KRAS mutation is rare ( < 5%) in gastroesophageal cancer (GEC). However, the incidence of KRAS gene amplification (amp+), consequent protein levels, and prognostic and/or therapeutic implications are unknown. Methods: 410 GEC samples and 30 cell lines were assessed for KRAS gene copy number (GCN) by fluorescence in situ hybridization (FISH) (n = 90), Kras expression by selected reaction monitoring mass spectrometry (Kras-SRM-MS) (n = 393), and Kras-SRM level evaluated for correlation with KRAS amp+ status (n = 73). Survival analysis was performed comparing KRAS amp+ versus non-amp+ patients. When possible, concurrent 315 gene next-generation sequencing was also performed. Four KRAS-amplified xenograft lines (CAT-2,12,14,15) were established from malignant effusions. Tumorigenic activity of KRAS amp+ lines (CAT lines, MKN-1) were assessed using MTT and soft agar assays in vitro and subcutaneous xenograft models, compared to non-amp+ lines. Inhibitory assays were performed using KRAS siRNA and CRIPSR, and commercial inhibitors targeting downstream effectors MEK and/or PIK3CA. Results: KRAS FISH revealed clustered gene amp+ in 28.9% (26/90); these patients had worse prognosis than non-amp+ patients. GCN significantly correlated with Kras expression. All KRAS amp+ cell lines significantly overexpressed Kras protein and were tumorigenic in xenograft subcutaneous models. KRAS siRNA and KRAS CRISPR of KRAS amp+ cell lines demonstrated inhibition in MTT viability and soft agar assays, compared to appropriate controls, and demonstrated significant and durable xenograft growth reduction. Conversely, inhibition using MEK and/or PI3K inhibitors demonstrated only transient growth reduction in vivo. Conclusions: KRAS gene amp+ was observed in a large subset (26%) of GEC patients, which correlated with extreme expression by mass spectrometry. Established xenograft lines serve as models to investigate therapeutic strategies for KRAS amp+ patients. Inhibition using MEK/PIK3CA inhibitors provided transient benefit for KRAS amp+ tumors while durable inhibition was observed with Kras protein knockdown, suggesting potential benefit from novel siRNA therapeutics currently in development.

Reversal of Mutant KRAS-Mediated Apoptosis Resistance by Concurrent Noxa/Bik Induction and Bcl-2/Bcl-xL Antagonism in Colon Cancer Cells.

KRAS mutations are frequently detected in human colorectal cancer and contribute to de novo apoptosis resistance and ultimately therapeutic failure. To overcome KRAS-mediated apoptosis resistance, the irreversible proteasome inhibitor, carfilzomib, was evaluated and found to potently induce Noxa, which was dependent upon c-Myc, and Bik. Isogenic mutant versus wild-type KRAS carcinoma cells showed elevated Bcl-xL, confirmed by KRAS siRNA or ectopic expression. Upregulated Bcl-xL by mutant KRAS was mediated by ERK as indicated by ERK knockdown. Bcl-xL expression was regulated at the level of mRNA and protein as shown using actinomycin D and cyclohexamide, respectively. Suppression of Bcl-xL by shRNA sensitized mutant KRAS cells to carfilzomib. Concurrent Bcl-xL antagonism by the BH3 mimetic ABT-263 combined with carfilzomib synergistically enhanced apoptosis that was dependent on Bax or p53, and was attenuated by Noxa or Bik shRNA. In support of this strategy, ectopically expressed Noxa enhanced apoptosis by ABT-263. Carfilzomib-induced Noxa and Bik sequestered Mcl-1 and ABT-263 released Bik and Bak from Bcl-xL, suggesting a mechanism for drug synergy. These preclinical findings establish mutant KRAS-mediated Bcl-xL upregulation as a key mechanism of apoptosis resistance in KRAS-mutant colorectal cancer. Furthermore, antagonizing Bcl-xL enabled carfilzomib-induced Noxa and Bik to induce synergistic apoptosis that reversed KRAS-mediated resistance. This novel study reveals a promising treatment strategy to overcome apoptosis resistance in KRAS-mutant colorectal cancer by concurrent upregulation of Noxa/Bik and antagonism of Bcl-xL.

49 Reversal of Mutant KRAS-Mediated Apoptosis Resistance by Concurrent Bcl-xL Antagonism and Induction of Pro-Apoptotic BH3-Only Proteins in Colorectal Carcinoma Cells

Background. KRAS mutations, detected in at least 40% of human colorectal cancers (CRC), contribute to de novo apoptosis resistance and ultimately treatment failure. Aims. To study the mechanism of KRAS-mediated apoptosis resistance, we examined proand anti-apoptotic Bcl-2 family proteins in isogenic mutant vs wild type KRAS colorectal cancer (CRC) cells. Experimental Methods. Isogenic KRAS mutant CRC cells (HCT116, DLD1) and KRAS mutant SW620 cells were analyzed for Bcl-2 family proteins by immunoblotting. Cells were incubated with ABT-263 (1 μM), carfilzomib (0-50nM) or their combination; apoptosis was measured by annexin V+ labeling and caspase cleavage. Drug synergy was determined by calculation of a combination index. Protein-protein interaction was examined by immunoprecipitation. Knockdown of ERK or KRAS was achieved by siRNA. Ectopic expression of mutant KRAS or Noxa was achieved by a constitutive or doxycyline-inducible expression system, respectively. Results. We found that mutant KRAS cells showed upregulation of Bcl-xL that was attenuated by KRAS siRNA or reproduced by ectopic expression of mutant KRAS. Upregulation of Bcl-xL by mutant KRAS was mediated by ERK shown using EKR siRNA. Bcl-xL upregulation occurred at the level of mRNA and protein shown using actinomycin D and cyclohexamide, respectively. While BH3 mimetic agents can antagonize Bcl-2/BclxL, they are generally insufficient to overcome apoptosis resistance in solid tumors. Therefore, we evaluated a strategy of concurrent Bcl-xL antagonism and induction of pro-apoptotic BH3-only proteins. Carfilzomib, an irreversible proteasome inhibitor, was shown to induce Noxa and Bik proteins but failed to reverse mutant KRAS-mediated apoptosis resistance. However, concurrent antagonism of Bcl-xL by the BH3 mimetic, ABT-263, combined with carfilzomib was shown to synergistically enhance apoptosis in isogenic mutant KRAS CRC cell lines (Figure). In support of this strategy, we mimicked Noxa induction, seen with carfilzomib, using a doxycycline-inducible expression system. Ectopic Noxa was shown to markedly enhance apoptosis by ABT-263. A mechanism for drug synergy was suggested by the finding that Noxa and Bik proteins can sequester anti-apoptotic Mcl-1, while ABT-263 was able to release Bik and Bak from binding to Bcl-xL. Conclusion. Our novel findings establish mutant KRAS-mediated Bcl-xL upregulation as a key mechanism of apoptosis resistance. Antagonism of Bcl-xL enabled carfilzomib-induced Noxa and Bik to induce synergistic apoptosis that reversed KRAS-mediated resistance. Together, these preclinical data suggest a promising therapeutic strategy to overcome apoptosis resistance in KRAS mutant CRC cells.

Biodegradable nanoparticle-mediated K-ras down regulation for pancreatic cancer gene therapy.

RNA interference (RNAi) targeting the K-ras oncogene mutation in pancreatic cancer mediated by small interfering RNA (siRNA) transfection is a very promising treatment. However, the rapid degradation and negative charge of naked siRNAs restrict their direct delivery into cells. In this contribution, we propose a safe and effective transmembrane transport nanocarrier formulation based on a newly developed biodegradable charged polyester-based vector (BCPV) for K-ras siRNA delivery into pancreatic cancer cells. Our results have shown that these biodegradable and biocompatible vectors are able to transfect siRNAs targeting mutant K-ras into MiaPaCa-2 cells with high transfection and knockdown efficiency. More importantly, the RNAi process initiated a cascade gene regulation of the downstream proteins of K-ras associated with cell proliferation, migration, invasion and apoptosis. We observed that after the mutant K-ras siRNA transfection, the growth, migration and invasion of the MiaPaCa-2 cells were significantly reduced; also, the apoptosis of the pancreatic cancer cells was promoted. Although in vivo testing data are limited, we propose that the BCPV based nanoparticle formulation could be a promising candidate as non-viral vectors for gene therapy in clinical settings.

Assembling Mn:ZnSe quantum dots-siRNA nanoplexes for gene silencing in tumor cells.

In this work, we demonstrate the use of manganese doped zinc selenide QDs (Mn:ZnSe d-dots) for gene delivery in vitro. Specifically, the d-dots were prepared as nanoplexes for facilitating the intracellular delivery of small interfering RNA (siRNA) molecules to pancreatic cancer cells (Panc-1), thereby inducing sequence-specific silencing of oncogenic K-Ras mutations in pancreatic carcinoma. For nanoplex preparation, a layer-by-layer (LBL) assembling method was adopted to modify the d-dot surface with cationic polymer poly(allylamine hydrochloride) (PAH) or polyethylenimine (PEI) for generating positive surface potential for complexing with K-Ras siRNA molecules. Owing to the unique and stable PL properties of the d-dots, siRNA transfection and the subsequent intracellular release profile from the d-dot/polymer-siRNA nanoplexes were monitored by fluorescence imaging. Quantitative results from flow cytometry study suggested that a high gene transfection efficiency was achieved. The expression of the mutant K-Ras mRNA in Panc-1 cells was observed to be significantly suppressed upon transfecting them with the nanoplex formulation. More importantly, cell viability studies showed that the d-dot/PAH nanoplexes were biocompatible and non-toxic even at concentrations as high as 160 μg mL(-1). Furthermore, the amine-terminated surface could be further modified to obtain multiple bio-functions. Based on these results, we envision that the designed d-dot nanoplexes can be developed as a flexible nanoplatform for both fundamental and practical clinical research applications.

Abstract B47: Therapeutic KRAS silencing in lung and colon cancer models

Abstract Introduction: Despite being amongst the most common oncogenes in human cancer, to date there are no effective clinical options for inhibiting KRAS activity. We investigated whether systemically delivered KRAS siRNAs using biocompatible nanoparticles has therapeutic potential in KRAS mutated lung and colorectal cancer models. Methods: We identified two KRAS siRNA sequences with notable potency in knocking-down KRAS expression independent of nucleotide substitution. Using human lung (A549; KRASG12S) and colon (HCP1; KRASG12D) adenocarcinoma cell lines, we assessed anti-proliferative effects of KRAS silencing with MTT assays in vitro. For in vivo experiments, we used a nano-liposomal delivery platform, DOPC, for systemic delivery of siRNAs. We used luciferase-labeled A549 cells to create an orthotopic lung cancer model in athymic nude mice. One week after cell injection, we began weekly IVIS imaging and treatments to assess efficacy of systemic KRAS siRNA with and without cisplatin (CDDP) chemotherapy. Proliferation and apoptosis indices were assessed using Ki-67 and caspase-3 IHC, respectively. Using a patient-derived xenograft colon cancer cell line, HCP1, we performed several in vivo experiments to assess KRAS siRNA effects on down-stream signaling, primary tumor growth, and experimental metastasis. Results: For both KRAS siRNA sequences, greater than 90% knock-down of KRAS was observed by western blot; ∼40-60% reductions in cell viability was observed in both A549 and HCP1 (p<0.001). In the lung cancer model, following four weeks of systemic therapy, the KRAS siRNA treatment group demonstrated an 81% reduction in luminescence compared to mice in the NC siRNA group (p=0.008). In addition, only in the KRAS siRNA treatment groups did we observe significant reductions in primary tumor size (KRAS siR: 50%, p=0.003; NC siR+CDDP: 6%, p=0.24; KRAS siR+CDDP: 73%, p=0.008), number of distant metastases (KRAS siR: 71%, p=0.07; NC siR+CDDP: 0%, p=0.49; KRAS siR+CDDP: 81%, p=0.02) and aggregate mass of distant metastases (KRAS siR: 77%, p=0.0002; NC siR+CDDP: 11%, p=0.85; KRAS siR+CDDP: 74%, p=0.009). KRAS siRNA significantly reduced the frequency of mediastinal metastases (NC siR: 100% versus KRAS siR: 25%, p=0.002). Compared with control siRNA, significant reductions in Ki-67 indices were seen in all treatment groups (KRAS siR: 34%, NC siR+CDDP: 52%, KRAS siR+CDDP: 36%; all p<0.0001), while significant increases in caspase-3 activity was only seen in the CDDP treatment groups (NC siR+CDDP: 2-fold increase, p=0.03; KRAS siR+CDDP: 5.5-fold increase, p=0.0007). Using the HCP1 colon cancer model, KRAS siRNA treatment reduced tumor KRAS and pERK expression levels at 48 and 96 hours after systemic delivery. Consistent with these effects, as compared with NC siRNA, treatment with KRAS siRNAs reduced HCP1 subcutaneous tumor growth by 69% (p<0.05). To assess effects of KRAS siRNA on liver metastases, HCP1 cells were delivered by intra-splenic injection followed by splenectomy the following day. Following 3 weeks of treatment with KRAS siRNAs, we observed reduced aggregate liver metastatic tumor burden compared to controls (73%, p=0.014). Notably, no mice in any of the therapeutic experiments exhibited any overt signs of toxicity from KRAS siRNA treatment, such as decreased feeding habits, activity, or total body weight. Discussion: Our studies demonstrate a proof-of-concept approach to therapeutic KRAS targeting using nanoparticle delivery of siRNA. This study highlights the potential translational impact of therapeutic RNA interference, which may have broad applications in oncology, especially for traditional “undruggable” targets. Citation Format: Chad Pecot, Sherry Wu, Seth Bellister, Rajat Bhattacharya, Anshumaan Maharaj, Cristian Rodriguez-Aguayo, Vianey Gonzalez-Villasana, Rajesha Rupaimoole, Gabriel Lopez-Berestein, Lee M. Ellis, Anil Sood. Therapeutic KRAS silencing in lung and colon cancer models. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B47. doi: 10.1158/1557-3125.RASONC14-B47

Therapeutic silencing of KRAS using systemically delivered siRNAs.

Despite being among the most common oncogenes in human cancer, to date, there are no effective clinical options for inhibiting KRAS activity. We investigated whether systemically delivered KRAS siRNAs have therapeutic potential in KRAS-mutated cancer models. We identified KRAS siRNA sequences with notable potency in knocking down KRAS expression. Using lung and colon adenocarcinoma cell lines, we assessed antiproliferative effects of KRAS silencing in vitro. For in vivo experiments, we used a nanoliposomal delivery platform, DOPC, for systemic delivery of siRNAs. Various lung and colon cancer models were used to determine efficacy of systemic KRAS siRNA based on tumor growth, development of metastasis, and downstream signaling. KRAS siRNA sequences induced >90% knockdown of KRAS expression, significantly reducing viability in mutant cell lines. In the lung cancer model, KRAS siRNA treatment demonstrated significant reductions in primary tumor growth and distant metastatic disease, while the addition of CDDP was not additive. Significant reductions in Ki-67 indices were seen in all treatment groups, whereas significant increases in caspase-3 activity were only seen in the CDDP treatment groups. In the colon cancer model, KRAS siRNA reduced tumor KRAS and pERK expression. KRAS siRNAs significantly reduced HCP1 subcutaneous tumor growth, as well as outgrowth of liver metastases. Our studies demonstrate a proof-of-concept approach to therapeutic KRAS targeting using nanoparticle delivery of siRNA. This study highlights the potential translational impact of therapeutic RNA interference, which may have broad applications in oncology, especially for traditional "undruggable" targets.

Abstract A55: KRAS gene amplification is a distinct molecular subgroup of gastroesophageal adenocarcinoma that may benefit from combined RAS/RAF/MEK/ERK and PI3K/PTEN/AKT/mTOR pathway inhibition

Abstract Background: Given the prevelance of chromosomal instability and gene amplification (amp+) as driver mechanisms in gastroesophageal adenocarcinoma (GEC), we assessed the incidence of KRAS gene amp+ and gene copy number (GCN) along with protein expression, at baseline and after molecular targeted therapy in GEC cell lines/tissues. KRAS undergoes activating mutation in various cancers. KRAS mutation is rare (<5%) in GEC. KRAS is a downstream effector of HER2 and MET tyrosine kinases, and KRAS upregulation may be a resistance mechanism to targeted anti-HER2/anti-MET therapy. We assessed KRAS amp+ in a series of 81 GEC samples and 19 GEC cell lines. We established a KRAS amp+ model to evaluate potential therapeutic strategies. Methods: 81 GEC samples and 19 GEC cell lines were assessed for KRAS GCN by fluorescence in situ hybridization (FISH) using HER2 scoring criteria. KRAS expression was assessed by immunohistochemistry (IHC) and selected reaction monitoring mass spectrometry (SRM-MS) using a novel mulit-plex (GEC-plex) assay for formalin fixed paraffin embedded (FFPE) samples. Patient samples with HER2+ status treated with trastuzuamb as well as a cohort of patients treated with anti-MET therapy were available for pre/post treatment assessment. A KRAS-amplified cell line (Cat2) was established from ascites fluid and grown in vitro and screened for KRAS mutation using deep next-gerenation sequencing to determine if KRAS mutant alleles were present. Tumorigenic activity of Cat2, and another amplified line, MKN1, were assessed using standard proliferation assays in vitro, and an intraperitoneal (IP) murine model assessing time to ascites development (TTA), with/without anti-MEK, anti-PI3K/mTOR, or combinations thereof. Cat2 and MKN1 were also treated with KRAS siRNA and compared to siRNA control in vitro. Results: Assessment of KRAS GCN revealed clustered wild type gene amp+ in 16% (13/81) of FFPE samples, while KRAS mutation was observed in 7% (6/81) (mutually exclusive). KRAS amp+ was found in newly diagnosed metastatic patients (12/55, 22%) more than locally advanced (1/26, 4%) (p=0.06). GCN correlated with expression as determined by IHC and the MS GEC-plex. Patients treated with anti-HER2 or anti-MET therapy were observed to have developed KRAS amp+ in post-treatment biopsies at progressive disease as compared to pre-treatment biopsy. In the cell line, Cat2, KRAS was amp+ (by FISH, mean copies/nucleus: 43.2) and extremely overexpressed by IHC and MS (60 fold over KRAS non-amp+ samples); KRAS was not mutated. In vitro, KRAS siRNA substantially inhibited KRAS amp+ lines versus siRNA control (>80%). We observed diminished KRAS amp+ growth/proliferation both in vitro and in vivo using combined MEK and AKT pathway inhibitors over either alone, and compared to various inhibitor controls (anti-EGFR, anti-MET, anti-HER2). Conclusions: In this series, we observed KRAS wild type gene amp+ to be present in a subset (16%) of GEC patients at diagnosis, correlating with very high protein expression. KRAS amp+ was present after treatment with trastuzumab in HER2+ patients, and also after anti-MET therapy. These data suggest that KRAS amp+ represents a molecular subset with advanced disease at diagnosis. The observation of acquired KRAS amp+ after targeted therapies may be a resistance mechanism to anti-HER and anti-MET inhibitors. Inhibition using combined MEK/AKT pathway inhibitors, and proof-of-principle siRNA, warrants further investigation for KRAS amp+ tumors. Citation Format: Les Henderson, Peng Xu, Brittany Rambo, Wei-Li Liao, Todd Hembrough, Daniel Catenacci. KRAS gene amplification is a distinct molecular subgroup of gastroesophageal adenocarcinoma that may benefit from combined RAS/RAF/MEK/ERK and PI3K/PTEN/AKT/mTOR pathway inhibition. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A55. doi: 10.1158/1557-3125.RASONC14-A55

Stable and Efficient Transfection of siRNA for Mutated KRAS Silencing Using Novel Hybrid Nanoparticles

siRNA is currently the most widely studied form of RNAi, and it has promising therapeutic potential not just in cancer but also in other diseases such as autoimmune and infectious diseases. However, efficient delivery of siRNA to target cells is being limited by lack of an effective delivery system that ensures efficient transfection into cells while protecting the encapsulated siRNA from nuclease. We hypothesized that a hybrid nanoparticle system composed of human IgG and poloxamer-188, a stealth polymer, will efficiently deliver mutated KRAS siRNA to A549 cells, leading to an efficient knockdown of mutated siRNA while protecting the siRNA from serum nuclease. We also hypothesized that the nanoparticles will not elicit an immunostimulatory effect in murine macrophages and also avoid clearance by macrophages. These nanoparticles were found to efficiently deliver siRNA to the cytoplasm and nuclease of A549 cells in a controlled and sustained manner while avoiding recycling by endosomes. An effective knockdown of mutated KRAS was achieved, which subsequently led to an increased sensitivity to erlotinib. These nanoparticles successfully avoided uptake by murine macrophages and reduced immune responses normally associated with siRNA/nanoparticle therapy. These results demonstrate that the novel hybrid nanoparticles could potentially serve as a platform for efficient delivery of siRNA to cells for stable gene knockdown.

Development of siRNA payloads to target KRAS-mutant cancer.

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. 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.

Inhibiting K-ras signaling reserves the epithelial-mesenchymal transition of pancreatic cancer cells and its mechanisms.

To investigate the effects of K-ras siRNA on pancreatic cancer cells and the expression levels of GLI1, E-cadherin and vimentin in pancreatic cancer cells transfected with K-ras siRNA. Ppancreatic cancer cells PANC-1 were transfected with K-ras siRNA. Growth inhibition ratio of the cells were measured by MTT assay, apoptosis was detected by flow cytometery, expression level of GLI1, E-cadherin and vimentin were detected by Western blot. The expression of K-ras protein was efficiently inhibited by K-ras siRNA in PANC-1 cells. The growth inhibition rates of the cells were significantly different to the control groups. Apoptosis rates were significantly different with that of control group. The expression of GLI1 was significantly down-regulated, E-cadherin was up-regulated, while vimentin was also down-regulated in K-ras siRNA transfected cells compared with that of control groups. Inhibiting K-ras signaling by K-ras siRNA can inhibit proliferation and induce apoptosis of pancreatic cancer cells, down-regulate GLI1's and vimentin's expression, and up-regulate E-cadherin's expression. Inhibiting K-ras signaling by K-ras siRNA may reduce epithelial to mesenchymal transition of pancreatic cancer cell PANC-1.

Effect of blocking Ras signaling pathway with K-Ras siRNA on apoptosis in esophageal squamous carcinoma cells

ObjectiveTo study the effect of RNAi silencing of the K-Ras gene on Ras signal pathway activity in EC9706 esophageal cancer cells. MethodsEC9706 cells were treated in the following six groups: blank group (no transfection), negative control group (transfection no-carrier), transfection group (transfected with pSilencer-siK-ras), taxol chemotherapy group, taxol chemotherapy plus no-carrier group, taxol chemotherapy plus transfection group. Immunocytochemistry, Reverse transcription-polymerase chain reaction and western blotting were used to analyze the expression of MAPK1 (mitogen-activated protein kinases 1) and cyclin D1 in response to siRNA (small interfering RNA) transfection and taxol treatment. ResultsK-Ras (K-Ras gene) siRNA transfection of EC9706 esophageal squamous carcinoma cells decreased the expression of K-Ras, MAPK1 and cyclin D1 at the mRNA and protein level. Reverse transcription-polymerase chain reaction indicated that the expression levels of MAPK1 and cyclin D1 mRNAs were significantly lower in the transfection group than in the blank group (P<0.05). Western blotting showed that 72 h after EC9706 cell transfection, the expression levels of MAPK1 and cyclin D1 proteins had decreased in all groups, and the expression levels in the transfection group were significantly inhibited as compared with the blank group. Apoptosis increased significantly in the transfection group or after addition of taxol as compared with the blank group and the no-carrier group. The degree of apoptosis in the taxol plus transfection group was more severe. ConclusionApoptosis increased significantly in EC9706 esophageal carcinoma cells after siRNA-mediated inhibition of Ras signaling, with the most obvious increase observed in the transfection plus taxol chemotherapy group. Ras knockdown therefore increased cellular sensitivity to the chemotherapeutic agent, taxol. Ras knockdown also down-regulated the expression of the downstream genes, MAPK1 and cyclin D1, thus inhibiting the growth, proliferation and metabolism of esophageal cancer cells.

Abstract 938: Silencing the elephant in the room: therapeutic KRAS targeting with siRNA.

Abstract Introduction: Despite being one of the first known oncogenes in human cancer, to date there are no effective therapeutic options for inhibiting KRAS. We investigated whether systemically delivered KRAS siRNA using biocompatible nanoparticles has therapeutic potential in lung cancer models. Methods: We identified two KRAS siRNA sequences with notable potency in knocking-down KRAS expression, which were combined for the in vivo experiments. Using a lung adenocarcinoma cell line (A549) with a KRAS mutation (G12S), we assessed anti-proliferative affects of KRAS silencing with MTT assays. Using luciferase-labeled A549 cells in athymic nude mice, we created an orthotopic lung cancer model in the left lower lung and performed weekly IVIS imaging beginning one week after injection. We used a nanoliposomal delivery platform, DOPC, for systemic delivery of siRNAs. Mice were randomly distributed into the following treatment groups (n=10 mice per group): 1) Control (NC) siRNA, 2) KRAS siRNA, 3) NC siRNA+cisplatin (CDDP), 4) KRAS siRNA +CDDP. One week after cell line injection, treatment began and continued for 4 weeks. At the end of the experiment mice were sacrificed, necropsied, and tumors were stained and evaluated for Ki-67 and caspase-3 by immunohistochemistry. Results: For both KRAS siRNA sequences, greater than 90% knock-down of KRAS was seen by western blot; which individually resulted in a 38% and 61% reduction in A549 cell viability (p=0.001 and p&amp;lt;0.001, respectively). Following four weeks of systemic therapy, mice in the KRAS siRNA treatment group had an 81% reduction in luminescence (p=0.008), while the addition of CDDP did not lead to significant reductions in the NC siRNA+CDDP (0% reduction, p=0.42) or KRAS siRNA+CDDP (51% reduction, p=0.22) groups. As compared with control siRNA, significant reductions in primary tumor size (KRAS siR: 50%, p=0.003; NC siR+CDDP: 6%, p=0.24; KRAS siR+CDDP: 73%, p=0.008), number of distant metastases (KRAS siR: 71%, p=0.07; NC siR+CDDP: 0%, p=0.49; KRAS siR+CDDP: 81%, p=0.02) and aggregate mass of distant metastases (KRAS siR: 77%, p=0.0002; NC siR+CDDP: 11%, p=0.85; KRAS siR+CDDP: 74%, p=0.009) was only observed in the KRAS siRNA treatment groups. KRAS siRNA significantly reduced the frequency of mediastinal metastases (NC siR: 100% versus KRAS siR: 25%, p=0.002). Compared with control siRNA, significant reductions in Ki-67 indices were seen in all treatment groups (KRAS siR: 34%, NC siR+CDDP: 52%, KRAS siR+CDDP: 36%; all p&amp;lt;0.0001), while significant increases in caspase-3 activity was only seen in the CDDP treatment groups (KRAS siR: 0%, p=0.12; NC siR+CDDP: 223%, p=0.03; KRAS siR+CDDP: 545%, p=0.0007). Discussion: Here, we demonstrate a proof-of-concept approach to therapeutic KRAS targeting using nanoparticle delivery of siRNA. This study highlights the potential translational impact of therapeutic RNA interference, which may have broad applications in oncology. Citation Format: Chad V. Pecot, Anshumaan Maharaj, Sherry Wu, Cristian Rodriguez-Aguayo, Vianey Gonzalez-Villasana, Rajesh Rupaimoole, Behrouz Zand, Thiru Arumugam, Gabriel Lopez, Anil Sood. Silencing the elephant in the room: therapeutic KRAS targeting with siRNA. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 938. doi:10.1158/1538-7445.AM2013-938

Abstract 4472: PI3K pathway dependencies in endometrioid endometrial cancer.

Abstract Background: Endometrioid endometrial cancers (EECs) frequently harbor coexisting mutations in PI3K pathway genes, including PTEN, PIK3CA, PIK3R1, and KRAS. We sought to determine the genetic determinants of PI3K pathway inhibitor response in EEC cells, and whether PTEN-mutant EEC cells rely on p110β signaling for survival. Methods: Twenty-four human EEC cell lines were characterized for their mutation profile using the Sequenom OncoCarta Panel v1.0 and Sanger sequencing for PTEN, PIK3CA, and PIK3R1 transcripts, and their protein and phospho-protein expression levels of PI3K and MAPK signaling pathway components. The cell line panel was treated with pan-class I PI3K, p110α and p110β isoform-specific, allosteric mTOR, mTOR kinase, dual PI3K/mTOR, MEK and RAF inhibitors, and the correlations between protein expression, gene mutations, and sensitivity to targeted therapeutics determined. RNA interference was employed to assess effects of KRAS silencing on EEC cells. Results: The prevalence of PI3K pathway mutations in the EEC cell line panel studied mirrored that reported for primary EECs. PI3K pathway activation as determined by levels of AKTSer473 phosphorylation was significantly associated with PTEN but not PIK3CA or KRAS mutation status. EEC cell lines harboring PIK3CA and PTEN mutations were selectively sensitive to the pan-class I PI3K inhibitor GDC-0941 and allosteric mTOR inhibitor Temsirolimus, respectively. Subsets of EEC cells with concurrent PIK3CA and/or PTEN and KRAS mutations were sensitive to PI3K pathway inhibition. Only 2/6 KRAS-mutant EEC cell lines showed response to MEK inhibition, and KRAS siRNA silencing did not induce apoptosis in KRAS-mutant EEC cells. EEC cell lines harboring PTEN mutations were resistant to the p110β inhibitors GSK2636771 and AZD6482, and only combination with the p110α selective inhibitor A66 resulted in a decrease in cell viability. Conclusions: Our findings provide evidence to suggest that selective pan-PI3K and mTOR inhibition in EEC cells may be most effective in PIK3CA- and PTEN-mutant cancers, respectively, and that these PI3K inhibitors may even be effective in a subset of EECs concurrently harboring KRAS mutations. Our results further demonstrate that p110β inhibition alone is not sufficient to sensitize PTEN-mutant EEC cells, suggesting that combination with other targeted agents may be required to increase efficacy. Citation Format: Britta Weigelt, Patricia H. Warne, Maryou B. Lambros, Jorge S. Reis-Filho, Julian Downward. PI3K pathway dependencies in endometrioid endometrial cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4472. doi:10.1158/1538-7445.AM2013-4472

Biodegradable Nanocapsules as siRNA Carriers for Mutant K‐Ras Gene Silencing of Human Pancreatic Carcinoma Cells

The application of small interfering RNA (siRNA)-based RNA interference (RNAi) for cancer gene therapy has attracted great attention. Gene therapy is a promising strategy for cancer treatment because it is relatively non-invasive and has a higher therapeutic specificity than chemotherapy. However, without the use of safe and efficient carriers, siRNAs cannot effectively penetrate the cell membranes and RNAi is impeded. In this work, cationic poly(lactic acid) (CPLA)-based degradable nanocapsules (NCs) are utilized as novel carriers of siRNA for effective gene silencing of pancreatic cancer cells. These CPLA-NCs can readily form nanoplexes with K-Ras siRNA and over 90% transfection efficiency is achieved using the nanoplexes. Cell viability studies show that the nanoparticles are highly biocompatible and non-toxic, indicating that CPLA-NC is a promising potential candidate for gene therapy in a clinical setting.

Abstract 1813: Evaluation of TBK1 as a novel cancer target in the K-Ras pathway

Abstract TBK1 (TANK-binding kinase 1) is a serine/threonine kinase best known for its role in mediating the innate immune response through modulation of the interferon and NF-kB pathways. TBK1 has also been implicated in supporting cancer cell survival and transformation as the downstream effector of the small GTPase RalB, and is overexpressed in lung, breast and colon tumors. Recently, a high-throughput RNAi screen identified TBK1 as a synthetic lethal partner of oncogenic K-Ras, suggesting that small molecule inhibition of TBK1 may provide a novel therapeutic strategy for K-Ras mutant tumors. Given the high tractability of kinases as drug targets, we sought to confirm that kinase inhibition recapitulated the synthetic lethal effects observed with shRNA silencing. We examined two small molecule inhibitors of TBK1, BX795 (6 nM) and GSK2292978A (4 nM), in a panel of lung, pancreas and colon cancer cell lines. We found only a small subset of lines that were sensitive to TBK1 inhibition and this sensitivity did not correlate with K-Ras dependence. We then used K-Ras and TBK1 siRNA in a similar panel of cell lines. As anticipated, knockdown of K-Ras using siRNA produced significant growth and signaling defects in K-Ras dependent cell lines. In contrast, knockdown of TBK1 did not result in a significant alteration of growth in any of our cell lines. Neither TBK1 siRNA nor TBK1 inhibitors revealed a sensitivity that correlated with K-Ras dependence in our lung, pancreatic, or colorectal cancer cell lines. Our data disputes the proposed synthetic lethal relationship of oncogenic K-Ras and TBK1 and does not support development of TBK1 inhibitors for treatment of K-Ras driven tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1813. doi:1538-7445.AM2012-1813

Ephrins/eph as novel synthetic lethal targets in Kras-mutant colorectal cancer.

e13578 Background: Mutations in the Kras proto-oncogene are found in 40%-45% of colorectal cancer (CRC) patients and occur mainly in codon 12 and 13. Kras mutant (MT) CRC tumours are resistant to EGFR mAb therapies. Clear understanding of the biology of KrasMT CRC is important, and identification of druggable targets uniquely required by KrasMT CRC tumours has the potential to fill a gap in the therapeutic armamentarium of advanced CRC. The aim of this study was to identify novel targets whose knock-down is synthetically lethal with Kras mutation in CRC models. Methods: Transcriptional profiling (Almac Diagnostics CRC DSA) of primary KrasMT and WT CRC tissues and paired KrasMT and WT CRC xenografts models was performed and Metacore pathway analysis was used to identify individual genes from novel Kras-dependent pathways for incorporation into a primary RNAi screen. Results: Using clinical and in vivo KrasMT/WT CRC samples and public available Kras signatures, we identified 30 signalling pathways and 160 genes associated with KrasMT CRC cancer. The primary Kras siRNA screen identified a number of novel Kras synthetic lethal target genes with a critical role in EMT and cell adhesion/migration. In particular, down-regulation of members of the EpH/Ephrin family was found to be lethal in KrasMT but not KrasWT cells. These results were confirmed in secondary and tertiary siRNA screens using a panel of KrasMT/WT CRC cell lines and multiple siRNA sequences. In addition, EpHA1, EpHA2 and EpHA4 silencing resulted in strong increase in apoptosis in KrasMT cells and this was further synergistically enhanced in the presence of chemotherapy (5-FU, CPT-11) treatment. Interestingly, EpHA1, EpHA2 and EpHA4 silencing also inhibited migration of KrasMT cells. Conclusions: This study demonstrates the utility of microarray expression data, Metacore and siRNA screens to identify novel Kras synthetic lethal targets and pathways. In addition, we found that EpHA1, EpHA2 and EpHA4 are important novel targets for KrasMT CRC tumours.