Oligonucleotide therapy

original article

© The American Society of Gene & Cell Therapy

Synthetic Lethal Therapy for KRAS Mutant Non-small-cell Lung Carcinoma with Nanoparticle-mediated CDK4 siRNA Delivery Cheng-Qiong Mao1, Meng-Hua Xiong2, Yang Liu1, Song Shen1, Xiao-Jiao Du1, Xian-Zhu Yang1, Shuang Dou1, Pei-Zhuo Zhang3 and Jun Wang1 1 Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, People’s Republic of China; 2Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui, People’s Republic of China; 3Suzhou GenePharma, Suzhou, Jiangsu, People’s Republic of China

The KRAS mutation is present in ~20% of lung cancers and has not yet been effectively targeted for therapy. This mutation is associated with a poor prognosis in nonsmall-cell lung carcinomas (NSCLCs) and confers resistance to standard anticancer treatment drugs, including epidermal growth factor receptor tyrosine kinase inhibitors. In this study, we exploited a new therapeutic strategy based on the synthetic lethal interaction between cyclin-dependent kinase 4 (CDK4) downregulation and the KRAS mutation to deliver micellar nanoparticles (MNPs) containing small interfering RNA targeting CDK4 (MNPsiCDK4) for treatment in NSCLCs harboring the oncogenic KRAS mutation. Following MNPsiCDK4 administration, CDK4 expression was decreased, accompanied by inhibited cell proliferation, specifically in KRAS mutant NSCLCs. However, this intervention was harmless to normal KRAS wild-type cells, confirming the proposed mechanism of synthetic lethality. Moreover, systemic delivery of MNPsiCDK4 significantly inhibited tumor growth in an A549 NSCLC xenograft murine model, with depressed expression of CDK4 and mutational KRAS status, suggesting the therapeutic promise of MNPsiCDK4 delivery in KRAS mutant NSCLCs via a synthetic lethal interaction between KRAS and CDK4. Received 16 May 2013; accepted 21 December 2013; advance online publication 11 March 2014. doi:10.1038/mt.2014.18

INTRODUCTION

Lung cancer is the most frequent cause of cancer-related death worldwide, accounting for more than 1 million deaths per year.1,2 Non-small-cell lung carcinomas (NSCLCs), the main histological type of lung cancer with a frequency of more than 50%3,4 and with ~40% of cases diagnosed at an advanced stage of disease,5 is usually treated with a platinum-based doublet as first-line chemotherapy for advanced disease, which often leads to resistance to chemotherapy and generally a poor prognosis.6,7 Second-line treatment for recurrent or progressive disease includes treatment with chemotherapy or treatment with an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor.8–10 Thus, gefitinib and erlotinib,

the EGFR kinase inhibitors, have been shown to be effective on NSCLCs harboring an EGFR mutation in clinical trials with longer progression-free survival and have been approved by the US Food and Drug Administration for NSCLCs as a first-line therapy in patients with EGFR mutations.11–16 However, patients with mutant KRAS tumors (with a frequency of 10–30%) fail to benefit from the EGFR inhibitors.17,18 Furthermore, although KRAS mutations were identified in NSCLC tumors more than 25 years ago, this remains a challenging target for therapy.19,20 Strategies to target KRAS directly, such as downregulating its expression or disrupting its membrane localization through farnesyltransferase inhibitors, have not yet been successful in the clinic21; strategies to target KRAS indirectly via small-molecule inhibitors that target RAS effectors are still being evaluated and have shown limited therapeutic efficacy as a single inhibitor.22 Therefore, a novel treatment strategy is needed for patients with KRAS mutant NSCLCs. With a more complete understanding of the complex and extensive network of KRAS effectors and regulators, secondary dependencies on genes that are themselves not oncogenes but could lead to vulnerabilities caused by the KRAS mutation state can also be developed to provide more efficient and safe therapeutic opportunities.20,23–26 For example, Luo et al.20 screened genomewide RNA interference to identify a diverse set of proteins whose depletion selectively impaired the viability of RAS mutant cells. These genes critical for KRAS activity are considered to have “synthetic lethal” interactions with the KRAS oncogene, which occurs when alterations in a gene result in cell death only in the presence of another nonlethal genetic alteration, such as a cancer-associated mutation (as shown in Figure 1a).25 Based on the synthetic lethal interactions, many targets have been explored for the treatment of KRAS mutant cancer cells. Scholl et al.27 found a synthetic lethal interaction between STK33 suppression and a KRAS mutation using high-throughput RNA interference, which supported STK33 as a target for treatment of the broad spectrum of human cancers with mutant KRAS. Barbie et al.28 investigated another KRAS synthetic lethal gene, TBK1, by systematic RNA interference. In their study, suppression of TBK1 induced apoptosis specifically in human cancer cell lines that expressed mutant KRAS, whereas suppression of KRAS

Correspondence: Jun Wang, Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, People’s Republic of China. E-mail: [email protected]

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Figure 1 MNPsiCDK4-mediated synthetic lethal therapy for KRAS mutant non-small-cell lung carcinomas (NSCLCs). (a) A schematic view of synthetic lethality. Gene A and gene B are said to be synthetic lethal if mutation of either gene alone is compatible with viability but simultaneous mutation of both genes causes death.25 Regularly, a mutation of first gene A alone is essential to the development of cancer. In normal cells, both gene A and gene B are wild type; single mutation of gene B by stimulation has no effect on cell viability. However, in tumor cells, gene A has been mutant firstly for the tumor development. By synthetic lethal therapy, the mutation of second gene B (for example, via RNA interference or small molecule inhibiting gene B) would result in selective cytotoxicity of the tumor cells. (b) We designed an MNP delivery system self-assembled from amphiphilic block copolymers PCL29-PPEEA21 and PCL40-PEG45, which is capable of binding small interfering RNA (siRNA) and forming MNPsiCDK4 to specifically treat NSCLCs harboring mutant KRAS. CDK, cyclin-dependent kinase; MNP, micellar nanoparticle; MNPsiCDK4, MNP containing siRNA targeting CDK4; PCL29-PPEEA21, poly(ε-caprolactone)-poly(2-aminoethylethylene phosphate); PCL40-PEG45, poly(ε-caprolactone)-poly(ethylene glycol).

directly led to cell death in all cell lines, including those expressing wild-type KRAS,28 suggesting that treatments based on synthetic lethality may provide cancer-specific treatment via gene–gene interactions. Such treatments can potentially reduce the toxicity to normal cells with wild-type KRAS compared with direct downregulation of KRAS expression. The synthetic lethal interaction between Snail2 and RAS in the broad spectrum of human cancers of epithelial origin that have undergone epithelial–mesenchymal transition has also been investigated.29 Furthermore, Puyol et al.30 unveiled a synthetic lethal interaction between KRAS and cyclindependent kinase 4 (CDK4) in a mouse tumor model that closely recapitulates NSCLCs. CDK4 alleles were targeted in advanced KRAS mutant tumors, inducing apoptosis and preventing tumor progression. It is known that the RAS–RAF–ERKpathway is usually activated after the interaction between growth factors and cell surface receptors. However, once the protein KRAS is mutated, this pathway can stuck in the “active” status, which leads to sustained mitogen-activated protein kinase activation and uncontrollable translation of messenger RNA (mRNA) to proteins. Recent study has shown that the mutation of KRAS can result in an increased expression of CDK4 and cyclin D1, which facilitates cell proliferation and promotes tumorigenesis.31,32 Thus, it may be one reason why targeting CDK4 can lead to the synthetic lethality in KRAS mutant tumors, which shed new light on treatments for KRAS mutant NSCLCs. In view of this, we elected to test, by i.v. injection in a murine model, the effectiveness of the delivery of nanoparticles containing small interfering RNA (siRNA) targeting CDK4 in terms of the inhibition of KRAS mutant NSCLCs (as shown in Figure 1b). Molecular Therapy  vol. 22 no. 5 may 2014

In this work, we utilized a micellar nanoparticle (MNP) delivery system self-assembled from the amphiphilic block copolymers poly(ε-caprolactone)-block-poly(2-aminoethylethylene phosphate) (PCL29-b-PPEEA21) and poly(ε-caprolactone)-blockpoly(ethylene glycol) (PCL40-b-PEG45) at a molar ratio of 1:1.5, which was capable of binding siRNA and forming an MNP/CDK4 siRNA complex (MNPsiCDK4) to specifically treat NSCLCs harboring mutant KRAS. We studied CDK4 gene expression knockdown levels in NSCLC A549 cells (mutant KRAS), NCI-H226 cells (H226, wild-type KRAS), NCI-H661 cells (H661, wild-type KRAS), and human HL7702 hepatocytes (wild-type KRAS) following MNPsiCDK4 treatment in vitro. The investigations on cell viability and the inhibitory effect on colony formation showed a selective reduction in cell proliferation in KRAS mutant cell lines without exhibiting toxicity to wild-type KRAS cells. Additionally, MNPsiCDK4 showed antitumor activity in an A549 xenograft murine model but not in an H226 xenograft murine model, indicating that MNPsiCDK4 is promising for KRAS mutant NSCLC therapy by the synthetic lethal interaction between CDK4 downregulation and KRAS mutation.

RESULTS MNPsiCDK4 mediates CDK4 expression knock down in vitro

MNPs were self-assembled from a mixture of the block polymers PCL29-b-PPEEA21 and PCL40-b-PEG45 at a molar ratio of 1:1.5 in aqueous solution, which formed nanoparticles with locked PCL segments in the dense inner core and hydrophilic PEG and PPEEA chains in the corona shell, similar to the self-assembly 965

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of the triblock copolymer poly(ethylene glycol)-block-poly(εcaprolactone)-block-poly(2-aminoethylethylene phosphate) reported previously.33 As shown in Figure 2a, MNPs of PCL29b-PPEEA21 and PCL40-b-PEG45 exhibited particle sizes mainly between 40 and 80 nm, with an average particle diameter of 59.9 ± 2.8 nm as a function of intensity, similar to the result observed using transmission electron microscopy (Figure 2a). As we have previously reported, the cationic nature of the MNPs enables them to bind siRNA,33,34 and a gel retardation assay carried out at different atomic nitrogen to phosphorus ratios (N/P ratios) confirmed the complete complexation of siRNA by MNPs at an N/P ratio of 5 or greater (Figure 2b). In order to determine whether MNPs can deliver siCDK4 to NSCLCs for therapy, we analyzed the reduction in CDK4 mRNA and protein levels mediated by MMPsiCDK4 in A549, H226, H661, and HL7702 cells 24 and 48-hour posttransfection. CDK4 mRNA and protein levels were analyzed using real-time polymerase chain reaction and western blot analysis, respectively. As shown in Figure 2c and Supplementary Figure S1a, single delivery of siCDK4 at a concentration of 100 nmol/l by MNPsiCDK4 at an N/P ratio of 10 significantly knocked down CDK4 mRNA levels in A549, H226, H661, and HL7702 cells to a level of 56.7 ± 6.6, 60.1 ± 2.9, 62.6 ± 2.6, and 53.4 ± 3.7%, respectively. However, neither MNPs alone nor MNPsiN.C. significantly altered CDK4 mRNA levels. Additionally, a similar gene silencing efficiency was detected at an N/P ratio of 5, where MNPsiCDK4 mediated ~45% knock down of CDK4 mRNA

Downregulation of CDK4 expression induces a selective reduction in cell proliferation in KRAS mutant cells via a synthetic lethal interaction To verify the selective requirement for CDK4 in KRAS mutant cells, we analyzed the effects of suppressing CDK4 in the A549, H226, H661, and HL7702 cell lines. As seen in Figure 3a and Supplementary Figure S2a, knock down of CDK4 by MNPsiCDK4 inhibited the viability of A549 cells which harbor mutant KRAS in codon 12. When siCDK4 was delivered at a concentration of 100 nmol/l by MNPs, the cell viability of A549 cells was significantly inhibited to 75.6 ± 8.9%, and further increasing siCDK4 to a concentration of 200 nmol/l led to even lower viability of A549 cells compared to treatment with LiposiCDK4 at 50 nmol/l; viability was 45.8 ± 6.9 and 64.3 ± 3.0%, respectively. However,

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Figure 2 MNPsiCDK4 effectively transfects non-small-cell lung carcinoma (NSCLC) cell lines and human hepatocytes with small interfering RNA (siRNA). (a) Dynamic light scattering characterization of MNPs and transmission electronic microscopic images of MNPs. (b) Gel retardation assay of MNP/siRNA complexes at different N/P ratios. (c) MNPsiCDK4-mediated gene silencing in A549 cell line (mutant KRAS); H226 cell line (wild-type KRAS), and HL7702 cell line (wild-type KRAS) by real-time polymerase chain reaction at N/P = 10, siCDK4 = 100 nmol/l and N/P = 5, siCDK4 = 50, 100, and 200 nmol/l, respectively; and by western blotting at N/P = 5, siCDK4 = 100 and 200 nmol/l. The dose of siCDK4 for LiposiCDK4 was 50 nmol/l, and the dose of siN.C. depended on the maximum dose of siCDK4 at the same N/P ratio. CDK, cyclin-dependent kinase; MNP, micellar nanoparticle; MNPsiCDK4, MNP containing siRNA targeting CDK4; mRNA, messenger RNA; PBS, phosphate-buffered saline; siN.C., negative control small interfering RNA.

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the viability of the NSCLC cell lines H226 and H661 and human HL7702 hepatocytes with wild-type KRAS were almost unaffected after transfection with siCDK4, verifying the inhibition of

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Figure 3 Effects of CDK4 knock down on the viability and proliferation of multiple cell lines. (a) Cell viability in the A549, H226, and HL7702 cell lines, 72 hours after transfection with MNPsiCDK4 at different concentrations. (b) Effects of CDK4 knock down on colony formation of the A549, H661, and HL7702 cell lines. Photographs of crystal violet-stained colonies are shown. (c,d) Multicolor competition assay 72 hours after transfection with MNPsiCDK4. KRAS mutant cells expressing green fluorescent protein (GFP) (A549-GFP) and KRAS wild-type cells (H226 or HL7702 cells) were mixed and transfected with siCDK4. The mutant and wild-type cell ratio at the end of the experiment was measured using FACS . If selective toxicity to mutant cells occurred rather than to wild-type cells, a reduced percentage of mutant cells would be detected. (e) The percentage of KRAS mutant cells in the mixture transfected with different formulations was normalized against that of a phosphate-buffered saline (PBS) control. CDK, cyclin-dependent kinase; MNP, micellar nanoparticle; MNPsiCDK4, MNP containing small interfering RNA targeting CDK4; siN.C., negative control small interfering RNA.

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was also observed in the colony formation assay, which showed decreased clonogenicity only in A549 cells following treatment with MNPsiCDK4 (Figure 3b and Supplementary Figure S2b). Additionally, we performed a multicolor competition assay to confirm cell line–specific proliferation inhibition (Figure 3c and Supplementary Figure S2c). KRAS mutant cells (A549-green fluorescent protein (GFP)) and KRAS wild-type cells (H226, H661, and HL7702) were seeded at a 50:50 mixture as shown in Figure 3d. Three days after siRNA transfection, the relative ratio of KRAS mutant cells versus wild-type cells was analyzed by FACS and compared to that of the same cells transfected with a negative control siRNA (siN.C.). If there was synthetic lethality between CDK4 and KRAS, a reduction in the GFP-positive population would be seen. As shown in Figure 3e, the percentage of KRAS mutant cells in the mixture was normalized against that of phosphate-buffered saline (PBS) control. It was found that the percentage of KRAS mutant A549-GFP cells in the mixture decreased significantly after transfection with MMPsiCDK4 compared to the PBS control in the H226/A549-GFP mixture and HL7702/A549-GFP mixture, which were 25.3 versus 14.1 and 26.1 versus 15.0%, respectively, with a nearly 50% depression in the KRAS mutant population, indicating that MNPsiCDK4 could lead to preferential cytotoxicity toward KRAS mutant cells rather than KRAS wild-type cells. Thus, treatment of NSCLCs based on this synthetic lethal interaction was not only effective

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Administration of MNPsiCDK4 induced a reduction in KRAS mutant NSCLC growth by decreasing CDK4 expression in vivo We next investigated whether the anti-cell proliferation activity of MNPsiCDK4 observed in vitro would also occur in vivo following systemic administration. A tumor xenograft model was generated in female athymic (nu/nu) mice by injection with A549 cells and used to assess the tumor growth inhibition efficacy of systemic administration of MNPsiCDK4 at a dose of 2 mg/kg siCDK4 per injection, compared with the effect of MNPsiN.C. or blank MNP administration. Mice were treated every 2 days, beginning on the 12th day after xenografts were seeded. The tumor growth curve is shown in Figure 4a. Intravenous injection of MNPsiCDK4 in tumor-bearing mice showed particularly significant inhibition of tumor growth, whereas neither MNPsiN.C. at the same siRNA dose nor blank MNP at the same concentration affected tumor growth, indicating that MNPsiCDK4 displayed sequence-specific antitumor activity in vivo. In addition, as shown in Figure 4b and Supplementary Figure  S3, the weight and the volume of A549 xenograft tumors at the final time point were obviously decreased compared to treatment with MNPsiN.C. or MNP. In order to verify that antitumor activity was dependent on the KRAS status, we

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MNPsiCDK4 exhibited significantly lower levels of CDK4 mRNA (CDK4 expression level of each tumor mass: 69.6 ± 1.6, 63.1 ± 1.5, 48.1 ± 0.4, 47.6 ± 1.8, 40.6 ± 1.3, 15.7 ± 1.6, and 15.5 ± 0.9%, respectively, with an average expression level of 38.4 ± 19%) compared with tumors treated with PBS (CDK4 expression level of each tumor mass: 151.8 ± 19.3, 131.0 ± 12.8, 100.0 ± 8.9, 99.8 ± 6.4, 98.9 ± 3.5, 93.2 ± 6.0, and 91.6 ± 7.2%, respectively, with an average expression level of 102.4 ± 14.5%). In contrast, CDK4 mRNA levels remained unchanged in tumors from mice treated with either MNPsiN.C. (from 137.2 ± 4.0 to 84.8 ± 1.7%, with an average expression level of 104.0 ± 13.4%) or blank MNP (from 122.8 ± 1.2 to 83.7 ± 5.3%, with an average expression level of 94.3 ± 9.6%). Western blot analysis of total protein from each tumor mass using anti-CDK4 monoclonal antibodies showed, following 38 days of

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also established a KRAS wild-type tumor xenograft model with H226 cells. After the same dose of administration as in the A549 tumor model, we did not observe a significant difference in tumor growth or tumor volume in any of the four groups (Figure 4c,d), indicating that MNPsiCDK4 is only effective in a mutant KRAS tumor model but ineffective in a wild-type KRAS tumor model. To further evaluate whether the inhibition of tumor growth observed on treatment with MNPsiCDK4 is related to CDK4 gene silencing in tumor cells, we examined CDK4 mRNA and protein expression levels in tumors following treatment. The A549 tumor mass from each mouse was excised after 38 days of treatment, and the extracted RNA was analyzed using real-time polymerase chain reaction for CDK4 and GAPDH mRNA expression. As shown in Figure 5a, tumors from mice treated with

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Figure 5 MNPsiCDK4 mediated CDK4 expression knock down in vivo. (a) Expression level of CDK4 messenger RNA (mRNA) in A549 tumor tissue determined by quantitative real-time polymerase chain reaction at the final time point of the treatment (orange, phosphate-buffered saline (PBS); blue, MNP; pink, MNPsiN.C.; and purple, MNPsiCDK4). All the tumor samples (seven mice per group) were lysed for analysis. The histogram in upper right corner reports the average CDK4 mRNA expression level for each treatment. (b) KRAS protein expression levels and the reduction of CDK4 protein expression levels in A549 xenograft tumor tissue by MNPsiCDK4 administration at the final time point of the treatment. Two samples of each group were randomly chosen for detection. (c) Expression level of CDK4 mRNA in H226 tumor tissue at the final time point of the treatment. All the tumor samples (five mice per group) were lysed for analysis. (d) KRAS and CDK4 protein expression levels in H226 xenograft tumor tissue. Two samples of each group were randomly chosen for detection. (e) Hematoxylin and eosin (HE) (400×), CDK4 (400×), and Ki67 (400×) analyses of tumor tissues at the final time point of the treatment. Left: A549 tumor tissue, right: H226 tumor tissue. CDK, cyclin-dependent kinase; MNP, micellar nanoparticle; MNPsiCDK4, MNP containing small interfering RNA targeting CDK4; siN.C., negative control small interfering RNA.

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treatment, clear knock down of CDK4 protein expression levels in tumors from mice treated with MNPsiCDK4. Such CDK4 protein expression knock down was not seen in tumors from mice receiving control treatments (Figure 5b). We also assessed the expression of KRAS protein and found that the administration of different formulations affected neither KRAS expression nor KRAS mutant status (Supplementary Figure S4). The H226 tumor mass from each mouse was also excised and analyzed for CDK4 mRNA or protein expression as well. Although tumor suppression was not observed in the H226 tumor model, CDK4 expression was significantly downregulated both on the mRNA level (as shown in Figure 5c; the CDK4 expression levels of the four groups were PBS 98.9 ± 13.5%, MNPs 103.0 ± 37.9%, MNPsiN.C. 107.2 ± 26.2%, and MNPsiCDK4 52.5.0 ± 21.7%) and on the protein level (as shown in Figure 5d). Immunohistochemistry analyses further confirmed that this reduction in CDK4 protein levels in A549 or H226 tumor cells was due to MNPsiCDK4 administration (Figure 5e). Furthermore, in addition to CDK4 gene silencing, administration of MNPsiCDK4 in animals inoculated with A549 cells inhibited NSCLC proliferation, as indicated by Ki67 levels and a reduction in the percentage of Ki67+ tumor cells, which may lead to an inhibition of tumor growth. However, in animals inoculated with H226 cells, administration of MNPsiCDK4 did not significantly affect the percentage of Ki67+ tumor cells.

DISCUSSION

In this study, we utilized an MNP-based siRNA delivery system self-assembled from PCL29-b-PPEEA21 and PCL40-b-PEG45 for KRAS mutant NSCLC therapy. As previously reported, the amphiphilicity of PCL40-b-PEG45 and PCL29-b-PPEEA21 allows the polymers to assemble into a micellar structure in aqueous solution, forming nanoparticles with locked PCL segments in the dense inner core and hydrophilic PEG and PPEEA chains in the corona shell. The PEG corona is known to prevent particle aggregation and enhance the colloidal stability of the micelle in the presence of serum proteins, and the PPEEA corona endows MNPs with a cationic nature that enables them to bind siRNA, forming an MNP/siRNA complex33 and preventing siRNA degradation (Supplementary Figure S5). As shown in Supplementary Figure S6, at N/P = 5 or 10, the MNPsiRNA exhibited an average size of 52.2 ± 4.9 or 51.2 ± 2.6 nm and surface zeta potential of 34.5 ± 2.5 or 24.4 ± 0.6 mV, respectively, which was slightly decreased than that of MNPs due to the interaction between anionic siRNA and cationic MNPs. Delivery of MNPsiCDK4 at these two N/P ratios to NSCLCs showed effective downregulation of CDK4 expression in a dose-dependent manner, which induced a reduction in cell proliferation harboring KRAS mutation. We also performed the MTT assay in another two KRAS mutant NSCLCs (H23 and H358) to preclude the possibility of accidental sensitization to CDK4 targeting in A549 cell line. When siCDK4-1095 and siCDK4 were demonstrated to significantly downregulate CDK4 expression in A549 cells (Supplementary Figure S7), from Supplementary Figures S8 and S9, it is observed that the delivery of siCDK4 and siCDK4-1095 can effectively downregulate CDK4 expression with depressed cell viability in H23 and H358 cells, indicating that the sensitization to CDK4 was led by the KRAS status rather than their own characteristics. When coculturing A549-GFP and H23 cells 970

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as described before, the percentage of GFP-positive cells increased (Supplementary Figure S10), which was opposite to the result shown in Figure 3e. Additionally, in order to strengthen the conclusion that the inhibition was via a synthetic lethal interaction between KRAS and CDK4 rather than downregulation of CDK4 alone, we further performed the same experiments in H226, H661 (NSCLCs with wild-type KRAS), and HL7702 (human hepatocytes with wild-type KRAS) cells. If CDK4 and KRAS were synthetically lethal gene pairs, a mutation in both genes can cause death while a mutation of either gene alone is compatible with viability.25 The results consistently demonstrated that treatment with MNPsiCDK4 was specific to the mutant KRAS cell lines, as an inhibition in proliferation was not observed in wild-type KRAS cell lines by MTT, colony formation, or cell number counting (Supplementary Figure S11), indicating that MNPsiCDK4 was nontoxic to either wild-type KRAS NSCLCs or human normal cells. The knock down of CDK4 in KRAS wild-type cell lines was also confirmed, which demonstrated a synthetic lethal relationship between the two genes, as previously described.25,35 All the in vitro observations suggest that MNPsiCDK4 can decrease the proliferation of NSCLCs harboring a KRAS mutation and, furthermore, provide a therapeutic benefit via a synthetic lethal interaction which is harmless to normal cells. However, it is still necessary to improve the MNP delivery system efficiency when compared with other studies of synthetic lethal screens, which usually utilize the lentiviral short hairpin RNA vectors for gene knock down. Most of them can achieve extremely low expression of target protein for a long term with only one transfection.27,28,30 From our present result, it seems that we can obtain good transfection efficiency only at high doses of siRNA. The downregulation of CDK4 mRNA is reduced by about 25%, when the dose is decreased to 50 nmol/l, which is about 60% of LiposiCDK4 at the same dose. So our future work may partly focus on the optimization of delivery system for more effective gene downregulation. Contrary to the performance in vitro, MNP delivery system showed better potential applications than lentivirus in vivo, for example, in terms of biocompatibility and nonimmunogenicity (Supplementary Figure S12). Based on the previous study of the MNP delivery system in vivo,34,36 we generated a tumor xenograft model in female athymic mice by injection with A549 or H226 cells and systemic administration of MNPsiCDK4. Particularly significant inhibition of tumor growth was only observed in the A549 tumor model (~75% inhibition) with decreased CDK4 expression and mutant KRAS status, suggesting that MNP delivery of siCDK4 to tumor cells by i.v. injection could be effective in the treatment of KRAS mutant NSCLCs. Since KRAS mutations, thought to be a primary driver of the development of cancer, are found at high frequencies in NSCLCs and are correlated with poor prognosis and therapeutic resistance,16,37,38 many research groups have focused on the inhibition of KRAS mutations21,22,39,40; however, the quest for a KRAS inhibitor seems fraught with difficulties.20,27,28 Thus, there has been no efficient dosing regimen for KRAS mutant NSCLCs. In 2010, an interaction between KRAS and CDK4 was unveiled in a mouse tumor model, indicating a complementary strategy for KRAS mutant NSCLCs by targeting CDK4.30 In addition, therapy based on synthetic lethality can provide cancer-specific cytotoxic agents www.moleculartherapy.org  vol. 22 no. 5 may 2014

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by killing only KRAS mutant NSCLCs and sparing normal cells,25 which was validated in Figures 2 and 3 and Supplementary Figures S8 and S9. Directly targeting KRAS usually can result in cytotoxicity in wild-type KRAS cells.28 The advantages of synthetic lethality in KRAS mutant NSCLCs, including antitumor activity and cancer-specific cytotoxic effects, thus avoiding “off-target” effects in normal wild-type KRAS cells raises the possibility of curing KRAS mutant NSCLCs. Hence, this strategy can easily overcome a bottleneck in cancer drug development, i.e., identifying compounds that will kill cancer cells at concentrations that do not harm normal cells.25 In fact, before Puyol et al.30 unveiled synthetic lethality between KRAS and CDK4, several researchers have found some interactions between the two genes. For example, in 1972, Aktas et al.32 reported that Ras is required for cell cycle progression and activation of both Cdk2 and Cdk4; Groth and Willumsen41 found that the Rastransformed cells cease to proliferate and enter a quiescent-like state with low Cdk4 and Cdk2 activity. Though lots of studies indicated that CDK4 may play an important role for cell proliferation and neoplasia in KRAS activated cells, little has been realized that CDK4 can be targeted for KRAS mutant cancer therapy, especially in vivo.42,43 Zhang et al.42 tested the efficiency of a CDK4 pharmacological inhibitor CINK4 and a CDK4 targeting siRNA in KRAS mutant NSCLC cell lines, and they found that effective inhibition of CDK4 may enhance the antitumor activity of paclitaxel. However, their work also focused on the in vitro therapy. Several factors limited the CDK4 targeted synthetic lethal therapy for in vivo application, such as the “off-target” effect of CDK4 pharmacological inhibitor and the obstacles for siRNA administration in vivo. As we know, there is currently no Food and Drug Administration–approved drug that targets CDKs. Several compounds targeting CDK4 are used in clinical trials, including PD-0332991, LEE011, LY2835219, and P276-00 (www.clinicaltrials.gov).44,45 Among these, only PD-0332991 has been shown to be selective for CDK4 and CDK6 with no significant activity on other CDKs.46 It can dramatically improve progressionfree survival of women with metastatic estrogen receptor–positive breast cancer by combinational therapy with oral nonsteroidal aromatase inhibitor letrozole in phase 2 trial, suggesting the important role of CDK4 in tumorigenesis.47 However, there is no other compound that has been shown to be specific for CDK4 without activity on other CDKs. Therefore, it is not the optimal choice to select a CDK4 inhibitor for synthetic lethality-based therapy because “offtarget” side effects in wild-type KRAS cells cannot be avoided. RNA interference is an alternative approach for specific CDK4 targeting, as long as an efficient siRNA delivery system can be developed. Inspiringly, our group has explored an MNP for siRNA delivery based on poly(ethylene glycol), poly(3-caprolactone), and poly(2aminoethyl ethylene phosphate). This delivery system has been shown to be effective in breast cancer, prostate cancer, and lung cancer xenograft murine models as well as in liver administration for systemic siRNA delivery.33,34,48,49 In this work, we validated the possibility of KRAS mutant NSCLC therapy through a synthetic lethal interaction between KRAS and CDK4 via MNP-mediated delivery of siCDK4 by systemic administration in a tumor xenograft model. These investigations are promising in terms of exploiting a new therapeutic strategy that is effective and safe in KRAS mutant NSCLCs. Molecular Therapy  vol. 22 no. 5 may 2014

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MATERIALS AND METHODS

Materials. The two copolymers, monomethoxy poly(ethylene glycol)block-poly(3-caprolactone) and poly(3-caprolactone)-block-poly (2-aminoethyl ethylene phosphate) (mPEG-b-PCL and PCL-b-PPEEA), were synthesized as previously described.33 The degree of polymerization of PEG and PCL blocks was 45 and 40, and that of PCL and PPEEA blocks was 29 and 21, respectively, as calculated based on 1 HNMR analysis as previously described.33 Hereafter, the two polymers will be denoted as mPEG45-b-PCL30 and PCL29-b-PPEEA15. The Lipofectamine 2000 transfection kit (Invitrogen, Carlsbad, CA) was used as suggested by the manufacturer. siRNA targeting CDK4 mRNA (siCDK4, antisense strand, 5′-CAGAUCUCGGUGAACGAUGdTdT-3′) and siRNA with a scrambled sequence (siN.C., antisense strand, 5′-AACCACUCAACUUUUUCCCAAdTdT-3′) were obtained from GenePharma (Shanghai, China). RPMI 1640 medium was purchased from Gibco BRL (Eggenstein, Germany). MTT, crystal violet, and trypan blue were purchased from Sangon Biotech (Shanghai, China). Preparation of MNPs and MNP/siRNA complexes. Six milligrams of

mPEG45-b-PCL40 and 4 mg of PCL29-b-PPEEA15 were dissolved in 1 ml of dimethyl sulfoxide. Under moderate stirring, 5 ml of ultrapurified water (Millipore Milli-Q Synthesis, 18.2 MU) were added dropwise at 60 ml/hour. The mixture was stirred for 1 hour at ambient temperature, followed by the removal of dimethyl sulfoxide by dialysis overnight. Zeta potentials and particle size measurements were conducted using a zeta potential analyzer with dynamic light scattering capability, as previously described,33 with a Malvern Zetasizer Nano ZS90, a HeNe laser (633 nm) and 90° collecting optics. All samples were prepared in aqueous solution at a concentration of 0.2 mg ml−1. All measurements were carried out at 25 °C, and data were analyzed using Malvern Dispersion Technology Software 4.20 (Malvern Instruments, UK) for cumulants analysis according to ISO 13321:1996. The morphology of the hybrid nanoparticles was examined by JEOL-2010 transmission electron microscopy (JEOL Ltd, Japan) at an accelerating voltage of 200 kV. For siRNA loading, MNPs were diluted to different concentrations to obtain the desired N/P ratio using either water or Opti-MEM medium (Invitrogen) and added to the siRNA solution (20 mmol/l). This mixture was mixed by pipetting and allowed to stand for 15 minutes before the gel retardation assay and gene silencing experiments were performed. Gel retardation assay. Various complexes were prepared at different

N/P ratios ranging from 1 to 10, as described above. The complexes were electrophoresed on a 1% agarose gel at a constant voltage of 120 V for 5 minutes in Tris/borate/ethylenediaminetetraacetic acid buffer (89 mmol/l Tris, 89 mmol/l boric acid, 2 mmol/l ethylenediaminetetraacetic acid, pH 8.3). The siRNA bands were visualized with ethidium bromide staining under an ultraviolet transilluminator at a wavelength of 365 nm. Free siRNA was used as the control.

Cell culture. The NSCLC cell line A549, H226, and H23 from the American Type Culture Collection were maintained in RPMI 1640 and Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, respectively. The human NSCLC cell line H661, H358, and human hepatocyte cell line HL7702 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were incubated at 37 °C in a 5% CO2 atmosphere. The A549-GFP cell line, which stably expresses GFP, was obtained by transfection with a retrovirus according to a standard protocol, and clones derived from discrete colonies were isolated and amplified in medium. Proliferation assays. The reduction in cell proliferation associated with decreasing CDK4 expression was assessed by MTT viability assays and colony formation assays in A549 cells. For the MTT viability assay, cells were seeded in 96-well plates at 8,000 cells per well in 100 µl of complete RPMI 1640 supplemented with 10% fetal bovine serum and incubated at 37 °C in a 5% CO2 atmosphere for 24 hours. Then various micelleplexes were added, and cells were incubated for

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an additional 72 hours. The MTT stock solution was then added to each well to achieve a final concentration of 1 g/l, with the exception of the wells used as a blank, to which the same volume of PBS (0.01 mol/l, pH 7.4) was added. After incubation for an additional 2 hours, 125 μl of the extraction buffer (20% sodium dodecyl sulfate in 50% dimethyl formamide, pH 4.7, prepared at 37 °C) was added to the wells and incubated overnight at 37 °C. The absorbance was measured at 570 nm using a Bio-Rad 680 microplate reader (Bio-Rad, Hercules, CA). Cell viability was normalized to that of A549 cells cultured in the culture medium with PBS treatment. For cell counting, cells were seeded in 24-well plates at 50,000 cells per well in 500 μl of complete RPMI 1640 as described above. For the colony formation assay, A549 cells were treated for 72 hours with various micelleplexes, followed by trypsinization and resuspension in 3′-demethoxy-3odemethylmatairesinol medium. One thousand cells were plated in each well of six-well plates. Cells were then incubated at 37 °C with 5% CO2 for 10–14 days. After removing the medium, cells were washed twice with PBS followed by staining with 0.5% crystal violet (Sangon Biotech) in methanol for 5 minutes. Then, the cells were washed twice with distilled water and visualized.

Immunohistochemical analysis. Mice were sacrificed, and tumor tissues

Multicolor competition assay. A549-GFP and H226 cells or A549-GFP

Statistical analysis. The statistical significance of treatment outcomes was assessed using Student’s t-test; P < 0.05 was considered statistically significant in all analyses (95% confidence level).

and HL7702 cells were seeded into 24-well plates (3 × 104 per well for each cell line) and incubated for 24 hours. After 72 hours of transfection, the cells were collected, and the proportion of each cell line was detected by flow cytometry. The results were analyzed using WinMDI 2.9 software (Scripps Research Institute, La Jolla, CA). Human NSCLC xenograft tumor model and tumor suppression study.

BALB/cA-nu nude mice (6-week-old) were purchased from the Beijing HFK Bioscience (Beijing, China), and all animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals. The procedures were approved by the University of Science and Technology of China Animal Care and Use Committee. A xenograft tumor model was generated by s.c. injection of 100 μl A549 cells (3 × 106/100 μl) or H226 cells mixed in Matrigel (Becton Dickinson, Bedford, MA) (1 × 107/100 μl,) into the right flank of nude mice. When the tumor volume was around 100 mm3 at 12 days for A549 or around 50 mm3 at 40 days after cell implantation, the mice were randomly divided into four groups (seven mice per group for A549 or five mice per group for H226) and treated with PBS, MNPs, MNPsiN.C., or MNPsiCDK4 by i.v. injection every other day at a dose of 40 μg siRNA per mouse. Tumor growth was monitored by measuring the perpendicular diameter of the tumor using calipers. The estimated volume was calculated according to the formula: tumor volume (mm3) = 0.5 × length × width.2 Detection of CDK4 expression in tumor tissues. Tumor tissues were col-

lected 24 hours after the last treatment. CDK4 expression at mRNA and protein levels in the tumors was analyzed by quantitative reverse transcription PCR and western blot analyses, respectively. For CDK4 mRNA analysis, tumor tissues were lysed in RNAiso Plus (Takara, Japan), and total RNA was isolated using the RNeasy Mini kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. The procedure of reverse and real-time polymerase chain reaction was the same as that used for the in vitro analyses. For CDK4 protein and KRAS protein analysis, tumor tissues were collected and lysed in 100 µl tissue lysis buffer (50 mmol/l N-2hydroxyethylpiperazine-N′-2-ethanesulfonic acid, pH 7.5, 150 mmol/l NaCl, 1 mmol/l ethylene glycol bis(2-aminoethyl ether)tetraacetic acid, 2.5 mmol/l ethylenediaminetetraacetic acid, 10% glycerol, 0.1% Tween 20, 1 mmol/l dithiothreitol, 10 mmol/l glycerol 2-phosphate, 1 mmol/l NaF, and 0.1 mmol/l Na3VO4) freshly supplemented with Roche’s Complete Protease Inhibitor Cocktail Tablets (Roche, Indianapolis, IN). The lysates were incubated on ice for a total of 30 minutes and vortexed every 5 minutes. The lysates were centrifuged for 10 minutes at 12,000g. Proteins were then detected by western blot analyses as described above.

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were excised 24 hours after the last treatment. The tissues were fixed in 4% formaldehyde and embedded in paraffin for analysis. Paraffin-embedded 7-μm tumor sections were prepared for immunohistochemical analysis. The proliferation of tumor cells was detected using an antibody against Ki-67 (catalog: sc-15402, Santa Cruz Biotech, Santa Cruz, CA). Deparaffinized slides were boiled for 5 minutes in 0.01 mol/l sodium citrate buffer (pH 6.0) in a pressure cooker for antigen retrieval. Subsequently, slides were allowed to cool for another 5 minutes in the same buffer. After several rinses in PBS and pretreatment with blocking medium for 5 minutes, slides were incubated with the Ki-67 antibody diluted to 1:200 in antibody diluent solution for 20 minutes at room temperature and then at 4 °C overnight. After washing slides in Tris-buffered saline, a streptavidin–biotin system was used according to the manufacturer’s instructions (BioGenex, San Ramon, CA). The slides were counterstained using Aquatex (Merck, Gernsheim, Germany). The CDK4 expression of tumor cells was determined using an antibody against CDK4 and was detected as described above. All sections were examined under a Nikon TE2000 microscope (Tokyo Prefecture, Japan).

SUPPLEMENTARY MATERIAL Figure S1. CDK4 expression down-regulated by MNPsiCDK4 in H661 cells. Figure S2. Cell viability and proliferation of H661 cell lines after CDK4 knockdown. Figure S3. The weight of A549 xenograft tumors at the end time point of the treatment. Figure S4. The KRAS status in A549 xenograft tumor cells by MNPsiCDK4 administration at the end time point of the treatment. Figure S5. RNase protection assay. Figure S6. Particle size and zeta potential of MNPsiRNA at N/P = 5 or 10. Figure S7. CDK4 protein down-regulated by different siRNA targeting CDK4. Figure S8. CDK4 mRNA down-regulated by MNPsiCDK4 and MNPsiCDK4-1095 in H23 cells and H358 cells. Figure S9. Cell viability of H23 and H358 cells determined by MTT assay. Figure S10. Multicolor competition assay in the mixture of H23 cells and A549-GFP cells after transfection of MNPsiCDK4. Figure S11. Cell number counting of A549, H661, HL7702 and H226 cell lines by transfection of MNPsiCDK4. Figure S12. MNP administration does not induce the innate immune response.

ACKNOWLEDGMENTS The authors gratefully acknowledge financial support from the National Basic Research Program of China (973 Programs, 2013CB933900, 2010CB934001), the National High Technology Research and Development Program of China (863 Programs, 2012AA022501, 2014AA020708), the National Natural Science Foundation of China (51125012, 51390482, 81302724), the Specialized Research Fund for the Doctoral Program of Higher Education from the Ministry of Education of China (SRFDP 20133402110019), and the Funds by the University of Science and Technology of China Special Grant for Postgraduate Research, Innovation, and Practice. The authors declare that they have no competing financial interests.

References

1. Ferlay, J, Shin, HR, Bray, F, Forman, D, Mathers, C and Parkin, DM (2010). Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 127: 2893–2917. 2. Jemal, A, Bray, F, Center, MM, Ferlay, J, Ward, E and Forman, D (2011). Global cancer statistics. CA Cancer J Clin 61: 69–90. 3. Travis, WD, Brambilla, E, Noguchi, M, Nicholson, AG, Geisinger, KR, Yatabe, Y et al. (2011). International association for the study of lung cancer/american thoracic

www.moleculartherapy.org  vol. 22 no. 5 may 2014

© The American Society of Gene & Cell Therapy

4.

5. 6. 7. 8. 9. 10. 11.

12. 13.

14. 15. 16. 17. 18.

19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

society/european respiratory society international multidisciplinary classification of lung adenocarcinoma. J Thorac Oncol 6: 244–285. Govindan, R, Page, N, Morgensztern, D, Read, W, Tierney, R, Vlahiotis, A et al. (2006). Changing epidemiology of small-cell lung cancer in the United States over the last 30 years: analysis of the surveillance, epidemiologic, and end results database. J Clin Oncol 24: 4539–4544. Siegel, R, Naishadham, D and Jemal, A (2012). Cancer statistics, 2012. CA Cancer J Clin 62: 10–29. Kelland, L (2007). The resurgence of platinum-based cancer chemotherapy. Nat Rev Cancer 7: 573–584. Schiller, JH, Harrington, D, Belani, CP, Langer, C, Sandler, A, Krook, J et al.; Eastern Cooperative Oncology Group. (2002). Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer. N Engl J Med 346: 92–98. Shepherd, FA, Rodrigues Pereira, J, Ciuleanu, T, Tan, EH, Hirsh, V, Thongprasert, S et al.; National Cancer Institute of Canada Clinical Trials Group. (2005). Erlotinib in previously treated non-small-cell lung cancer. N Engl J Med 353: 123–132. Somerfield, MR, Ihde, DC, Pfister, DG, Olak, J, Sause, W, Smith, TJ et al. (1997). Clinical practice guidelines for the treatment of unresectable non-small-cell lung cancer. J Clin Oncol 15: 2996–3018. Paz-Ares, L (2012). Beyond first-line NSCLC therapy: chemotherapy or erlotinib? Lancet Oncol 13: 225–227. Pao, W, Miller, V, Zakowski, M, Doherty, J, Politi, K, Sarkaria, I et al. (2004). EGF receptor gene mutations are common in lung cancers from “never smokers” and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci USA 101: 13306–13311. Murphy, M and Stordal, B (2011). Erlotinib or gefitinib for the treatment of relapsed platinum pretreated non-small cell lung cancer and ovarian cancer: a systematic review. Drug Resist Updat 14: 177–190. Coudert, B, Ciuleanu, T, Park, K, Wu, YL, Giaccone, G, Brugger, W et al.; SATURN Investigators. (2012). Survival benefit with erlotinib maintenance therapy in patients with advanced non-small-cell lung cancer (NSCLC) according to response to first-line chemotherapy. Ann Oncol 23: 388–394. Cohen, MH, Johnson, JR, Chen, YF, Sridhara, R and Pazdur, R (2005). FDA drug approval summary: erlotinib (Tarceva) tablets. Oncologist 10: 461–466. Sordella, R, Bell, DW, Haber, DA and Settleman, J (2004). Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways. Science 305: 1163–1167. Gazdar, AF (2009). Activating and resistance mutations of EGFR in non-small-cell lung cancer: role in clinical response to EGFR tyrosine kinase inhibitors. Oncogene 28(suppl. 1): S24–S31. Guha, U, Chaerkady, R, Marimuthu, A, Patterson, AS, Kashyap, MK, Harsha, HC et al. (2008). Comparisons of tyrosine phosphorylated proteins in cells expressing lung cancer-specific alleles of EGFR and KRAS. Proc Natl Acad Sci USA 105: 14112–14117. Massarelli, E, Varella-Garcia, M, Tang, X, Xavier, AC, Ozburn, NC, Liu, DD et al. (2007). KRAS mutation is an important predictor of resistance to therapy with epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancer. Clin Cancer Res 13: 2890–2896. Young, A, Lyons, J, Miller, AL, Phan, VT, Alarcón, IR and McCormick, F (2009). Ras signaling and therapies. Adv Cancer Res 102: 1–17. Luo, J, Emanuele, MJ, Li, D, Creighton, CJ, Schlabach, MR, Westbrook, TF et al. (2009). A genome-wide RNAi screen identifies multiple synthetic lethal interactions with the Ras oncogene. Cell 137: 835–848. Karnoub, AE and Weinberg, RA (2008). Ras oncogenes: split personalities. Nat Rev Mol Cell Biol 9: 517–531. Engelman, JA, Chen, L, Tan, X, Crosby, K, Guimaraes, AR, Upadhyay, R et al. (2008). Effective use of PI3K and MEK inhibitors to treat mutant Kras G12D and PIK3CA H1047R murine lung cancers. Nat Med 14: 1351–1356. Solimini, NL, Luo, J and Elledge, SJ (2007). Non-oncogene addiction and the stress phenotype of cancer cells. Cell 130: 986–988. Hartwell, LH, Szankasi, P, Roberts, CJ, Murray, AW and Friend, SH (1997). Integrating genetic approaches into the discovery of anticancer drugs. Science 278: 1064–1068. Kaelin, WG Jr (2005). The concept of synthetic lethality in the context of anticancer therapy. Nat Rev Cancer 5: 689–698. Luo, B, Cheung, HW, Subramanian, A, Sharifnia, T, Okamoto, M, Yang, X et al. (2008). Highly parallel identification of essential genes in cancer cells. Proc Natl Acad Sci USA 105: 20380–20385. Scholl, C, Fröhling, S, Dunn, IF, Schinzel, AC, Barbie, DA, Kim, SY et al. (2009). Synthetic lethal interaction between oncogenic KRAS dependency and STK33 suppression in human cancer cells. Cell 137: 821–834. Barbie, DA, Tamayo, P, Boehm, JS, Kim, SY, Moody, SE, Dunn, IF et al. (2009). Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1. Nature 462: 108–112.

Molecular Therapy  vol. 22 no. 5 may 2014

Synthetic Lethal Therapy for KRAS Mutant NSCLCs

29. Wang, Y, Ngo, VN, Marani, M, Yang, Y, Wright, G, Staudt, LM et al. (2010). Critical role for transcriptional repressor Snail2 in transformation by oncogenic RAS in colorectal carcinoma cells. Oncogene 29: 4658–4670. 30. Puyol, M, Martín, A, Dubus, P, Mulero, F, Pizcueta, P, Khan, G et al. (2010). A synthetic lethal interaction between K-Ras oncogenes and Cdk4 unveils a therapeutic strategy for non-small cell lung carcinoma. Cancer Cell 18: 63–73. 31. Trobridge, P, Knoblaugh, S, Washington, MK, Munoz, NM, Tsuchiya, KD, Rojas,  A et al. (2009). TGF-beta receptor inactivation and mutant Kras induce intestinal neoplasms in mice via a beta-catenin-independent pathway. Gastroenterology 136: 1680–8.e7. 32. Aktas, H, Cai, H and Cooper, GM (1997). Ras links growth factor signaling to the cell cycle machinery via regulation of cyclin D1 and the Cdk inhibitor p27KIP1. Mol Cell Biol 17: 3850–3857. 33. Sun, TM, Du, JZ, Yan, LF, Mao, HQ and Wang, J (2008). Self-assembled biodegradable micellar nanoparticles of amphiphilic and cationic block copolymer for siRNA delivery. Biomaterials 29: 4348–4355. 34. Mao, CQ, Du, JZ, Sun, TM, Yao, YD, Zhang, PZ, Song, EW et al. (2011). A biodegradable amphiphilic and cationic triblock copolymer for the delivery of siRNA targeting the acid ceramidase gene for cancer therapy. Biomaterials 32: 3124–3133. 35. Simons, A, Dafni, N, Dotan, I, Oron, Y and Canaani, D (2001). Establishment of a chemical synthetic lethality screen in cultured human cells. Genome Res 11: 266–273. 36. Sun, TM, Du, JZ, Yao, YD, Mao, CQ, Dou, S, Huang, SY et al. (2011). Simultaneous delivery of siRNA and paclitaxel via a “two-in-one” micelleplex promotes synergistic tumor suppression. ACS Nano 5: 1483–1494. 37. Jackman, DM, Miller, VA, Cioffredi, LA, Yeap, BY, Jänne, PA, Riely, GJ et al. (2009). Impact of epidermal growth factor receptor and KRAS mutations on clinical outcomes in previously untreated non-small cell lung cancer patients: results of an online tumor registry of clinical trials. Clin Cancer Res 15: 5267–5273. 38. Raponi, M, Winkler, H and Dracopoli, NC (2008). KRAS mutations predict response to EGFR inhibitors. Curr Opin Pharmacol 8: 413–418. 39. Torrance, CJ, Agrawal, V, Vogelstein, B and Kinzler, KW (2001). Use of isogenic human cancer cells for high-throughput screening and drug discovery. Nat Biotechnol 19: 940–945. 40. Dolma, S, Lessnick, SL, Hahn, WC and Stockwell, BR (2003). Identification of genotype-selective antitumor agents using synthetic lethal chemical screening in engineered human tumor cells. Cancer Cell 3: 285–296. 41. Groth, A and Willumsen, BM (2005). High-density growth arrest in Ras-transformed cells: low Cdk kinase activities in spite of absence of p27(Kip) Cdk-complexes. Cell Signal 17: 1063–1073. 42. Zhang, XH, Cheng, Y, Shin, JY, Kim, JO, Oh, JE and Kang, JH (2013). A CDK4/6 inhibitor enhances cytotoxicity of paclitaxel in lung adenocarcinoma cells harboring mutant KRAS as well as wild-type KRAS. Cancer Biol Ther 14: 597–605. 43. Zhang, XH, Shin, JY, Kim, JO, Oh, JE, Chae, HS and Kang, JH (2012). Synergistic antitumor activity of CDK4/6 inhibitor combined with paclitaxel in NSCLC cells harboring mutant KRAS. Cancer Res 72(suppl.): 4896. 44. Roberts, PJ, Bisi, JE, Strum, JC, Combest, AJ, Darr, DB, Usary, JE et al. (2012). Multiple roles of cyclin-dependent kinase 4/6 inhibitors in cancer therapy. J Natl Cancer Inst 104: 476–487. 45. Flaherty, KT, Lorusso, PM, Demichele, A, Abramson, VG, Courtney, R, Randolph, SS et al. (2012). Phase I, dose-escalation trial of the oral cyclin-dependent kinase 4/6 inhibitor PD 0332991, administered using a 21-day schedule in patients with advanced cancer. Clin Cancer Res 18: 568–576. 46. Fry, DW, Harvey, PJ, Keller, PR, Elliott, WL, Meade, M, Trachet, E et al. (2004). Specific inhibition of cyclin-dependent kinase 4/6 by PD 0332991 and associated antitumor activity in human tumor xenografts. Mol Cancer Ther 3: 1427–1438. 47. Finn, RS, Crown, JP, Lang, I, Boer, K, Bondarenko, IM, Kulyk, SO, et al. (2012). Results of a randomized phase 2 study of PD 0332991, a cyclin-dependent kinase (CDK) 4/6 inhibitor, in combination with letrozole vs letrozole alone for first-line treatment of ER+/HER2– advanced breast cancer (BC). Cancer Res 72(suppl.): S1–S6. 48. Liu, XQ, Xiong, MH, Shu, XT, Tang, RZ and Wang, J (2012). Therapeutic delivery of siRNA silencing HIF-1 alpha with micellar nanoparticles inhibits hypoxic tumor growth. Mol Pharm 9: 2863–2874. 49. Wang, HX, Xiong, MH, Wang, YC, Zhu, J and Wang, J (2013). N-acetylgalactosamine functionalized mixed micellar nanoparticles for targeted delivery of siRNA to liver. J Control Release 166: 106–114.

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