RNA‐Peptide nanoplexes drug DNA damage pathways in high‐grade serous ovarian tumors

Abstract DNA damaging chemotherapy is a cornerstone of current front‐line treatments for advanced ovarian cancer (OC). Despite the fact that a majority of these patients initially respond to therapy, most will relapse with chemo‐resistant disease; therefore, adjuvant treatments that synergize with DNA‐damaging chemotherapy could improve treatment outcomes and survival in patients with this deadly disease. Here, we report the development of a nanoscale peptide‐nucleic acid complex that facilitates tumor‐specific RNA interference therapy to chemosensitize advanced ovarian tumors to frontline platinum/taxane therapy. We found that the nanoplex‐mediated silencing of the protein kinase, MK2, profoundly sensitized mouse models of high‐grade serous OC to cytotoxic chemotherapy by blocking p38/MK2‐dependent cell cycle checkpoint maintenance. Combined RNAi therapy improved overall survival by 37% compared with platinum/taxane chemotherapy alone and decreased metastatic spread to the lungs without observable toxic side effects. These findings suggest (a) that peptide nanoplexes can serve as safe and effective delivery vectors for siRNA and (b) that combined inhibition of MK2 could improve treatment outcomes in patients currently receiving frontline chemotherapy for advanced OC.


| I N TR ODU C TI ON
RNA interference (RNAi) therapy is a powerful treatment strategy that can be used to transiently deplete disease-causing proteins. 1 While promising, systemic administration of RNAi therapeutics such as small interfering RNA (siRNA) in patients is challenging due to rapid urinary clearance, nuclease degradation, and inefficient tissue, cellular, or cytosolic delivery. Formulation strategies for siRNA based on phospholipid 2,3 and lipid-like 4,5 carriers have conferred therapeutic benefits in animal disease models and in clinical trials 6,7 ; however, efficient tissuespecific delivery beyond the liver, and avoidance of dose-limiting side effects remains a key challenge for the field. In contrast to antisense oligonucleotide therapies that have been FDA approved since 1998, no siRNA-based therapies have been approved to-date.
Polymer-based drug carriers [8][9][10][11][12][13][14] represent a versatile and potentially safe alternative to lipidic oligonucleotide drug carriers, and related architectures have been utilized in a range of clinically approved drug formulations for more than three decades. Due to their modular nature, polymers can be engineered to package, protect, and deliver siRNAs to tumors, and can afford therapeutic nucleic acids access to the cytosol where they can inhibit the expression of oncogenic and pro-survival genes, among others.
Recently, we demonstrated that a family of polypeptides synthesized by N-carboxyanhydride (NCA) ring-opening polymerization [15][16][17] can serve as modular scaffolds for the delivery of both small molecule drugs 16,18,19 and nucleic acids. 20 Previous strategies to formulate siRNA using synthetic polypeptides have focused largely on the formation of electrostatic complexes with cationic block copolymers. 21 The resulting systems facilitate efficient complexation of nucleic acids; however, highly charged polycationic blocks can be challenging to engineer such that siRNAs are efficiently released from complexes entrapped within endosomal compartments. Design strategies for nucleic acid carriers thus require a balance between high valency of charge (complexation) and intracellular release or cytotoxicity. In place of a more traditional diblock copolymer with a charged or neutral water-soluble block, we have engineered a family of NCA polymers derived from short oligopeptides that spatially isolate varying delivery functions within the final nanoparticle ( Figure 1a). By lowering the charge density of the positively charged component and introducing other intermolecular interactions, here we can employ a more dynamic polyelectrolyte complex system than the typical ionically crosslinked matrix. Supramolecular NCA architectures in which siRNA is electrostatically complexed within a dynamically complexed core could thus provide new opportunities for tumor-targeted siRNA delivery in vivo.
Despite many encouraging advances in addressing other common cancers such as breast and prostate cancer, there has been little to no improvement in the survival rate of ovarian cancer (OC) patients over the past 30 years. 22 The 5-year survival rates for the disease have hovered at approximately 30-40% in the past several decades, and OC is now the fifth most lethal cancer for women. 23 Today, nearly all patients diagnosed with epithelial OC will be treated with DNA-damaging platinum-based chemotherapy. 24 Particularly for advanced serous OC, surgical debulking followed by platinum therapy is the treatment of choice. Of these patients, approximately 20-30% will not respond to the initial therapy, and of the remaining patients, approximately 70% eventually relapse with a platinum-resistant form of the disease. 25 Methods to sensitize aggressive or recurrent ovarian tumors to platinum chemotherapy could thus improve response rates and overall survival following diagnosis, which most often occurs in advanced stages. Recently, we identified a p38MAPK/MK2-dependent DNA damage response pathway that is selectively required for survival of cells deficient in the tumor suppressor, p53. [26][27][28][29] We found 28 that loss of the protein kinase, MK2, profoundly sensitized non-small cell lung cancers to DNA damaging chemotherapy in vivo, resulting from corresponding loss of cell cycle checkpoint maintenance and subsequent mitotic catastrophe. 27 Importantly, we observed that loss of this response pathway is inconsequential in normal (i.e., p53proficient) cells, thus limiting chemosensitization to tumor cells harboring genetic mutations in the p53 tumor suppressor protein. [26][27][28][29] Given that TP53 mutation and/or loss is a hallmark of high-grade serous OC and is observed in >96% of patients, 30 we seek here to develop this therapeutic approach for the treatment of OC.
Although MK2 is a promising drug target due to its role in both cancer and inflammation, commercial development of small molecule inhibitors against the protein have experienced little clinical success due in part to the kinase's shallow binding pocket and sequence homology with other physiologically important kinases (e.g., MK3, MK5, MNK1, MNK2).
Furthermore, MK2 plays an important role in the immune response. 31 Thus, highly selective and tissue-specific silencing of this "undruggable"   Photon correlation spectroscopy and laser Doppler electrophoresis measurements were carried out in molecular biology grade deionized water using a Malvern Zetasizer Nano ZS90 particle analyser (k 5 633 nm, material/dispersant RI 1.590/1.330). Critical aggregation concentration was determined as described previously 32 using a fourparameter logistic fit (k ex 5 335 nm, k em,I1 5 373 nm, k em,I3 5 384 nm; n 5 3 replicates).

| Retrovirus transduction
For VSVG-pseudotyped virus production, 293T cells were transfected using the calcium phosphate method (Clontech) using pMLP along with packaging and structural vectors VSVG and GAG/POL. Supernatants containing virus were then used to transduce target cells in the presence of 8 lg/ml polybrene for three rounds of infection. Successfully transduced cells were selected either with puromycin (3 lg/ml) for pMLP infected cells, or were sorted for GFP expression by flow cytometry for GFP-Luciferase used to infect OVCAR8 cells for bioluminescent imaging and was a gift from B. Huang (MIT).

| qRT-PCR
For qRT-PCR analysis, RNA was extracted from mouse tumor or cells using TRIzol reagent (Ambion) according to the manufacturer's instructions and 1 lg of total RNA was used for reverse transcription using the Superscript III First-Strand Synthesis kit (Invitrogen) as per the manufacturer's instructions. For qPCR, data cDNA was amplified using SYBR green PCR mastermix (Applied Biosystems) according to the manufacturer's cycling conditions for 40 cycles on a Bio-Rad C1000 Thermal Cycler. Data were analyzed using the delta-delta Ct method as described previously and plotted as fold change versus control. 34

| Western blot
Cells were lysed in RIPA lysis buffer containing protease and phosphatase inhibitor (Roche). Protein concentration was measured using BCA (Pierce) and cell extracts containing the same amount of protein were mixed with 63 reducing sample buffer, boiled at 958C for 5 min, and subject to electrophoresis using standard SDS-PAGE methods. For LICOR-based blotting, proteins were transferred to nitrocellulose membranes (Biorad) and blocked with Odyssey blocking buffer for 1 hr. Primary antibodies were incubated overnight at 48C followed by secondary antibodies conjugated with LICOR fluorophores, then scanned with a LICOR/Odyssey infrared imaging system (LICOR Biosciences). Band densitometries were quantified using ImageStudio. For ECL-based blotting, proteins were transferred to methanol-activated PVDF membrane (Biorad) and blocked with 5% nonfat dried milk for 1h. Primary antibodies were incubated overnight at 48C followed by secondary antibodies conjugated with HRP for developing with ECL (Perkin Elmer).   Figure S1c). This morphology is presented in contrast to a more traditional micellar or lamellar core-shell structure in which siRNA complexation is expected to be limited to the outer surface or leaflet bilayer. This less organized but uniform morphology suggests strong interactions between siRNA and the charged and hydrophobic blocks to yield a blended core nanoparticle which we hypothesize enables more facile intracellular release of siRNA. The critical aggregation concentration of these peptide-siRNA suprastructures was 18 6 7 mM as measured by pyrene fluorescence assay, approximately 2.3-fold greater than the estimated minimum initial blood concentration following systemic (e.g., intravenous) administration at 1 mg/kg siRNA in mice (Figure 1f). The resulting nanoparticle system was designed for intraperitoneal administration in which nanoparticles would be more directly exposed to tumor sites; therefore, in these initial studies, a specific tumor cell targeting ligand was not incorporated.

| In vivo studies
In vitro, nanoplex-transfected OC cells exhibited diffuse, cytoplasmic fluorescence, as well as puncta colocalized with acidic organelles

| Loss of MK2 chemosensitizes ovarian tumor cells
Having established a suitable platform for in vivo RNAi, we next sought a therapeutic target that was not currently druggable using small molecules, and whose functional silencing could improve treatment outcomes in patients currently receiving frontline chemotherapy for OC.
We previously showed that pairwise loss of p53 and MK2 was synthetically lethal in chemotherapy-treated, non-small cell lung tumors; 28 however, at present it was unclear to what extent these findings extend to other tumor types. We hypothesized that an MK2-dependent DNA damage response would likewise be required for survival in chemotherapy treated ovarian tumors that are genotypically characterized by lossof-function p53 mutations. Indeed, we found that activation of MK2's upstream signal effector, p38, was significantly enriched in serous ovarian tumors relative to matched normal tissues (Supporting Information Figure S2) and that clonogenicity of p53-mutant ovarian tumor cells (TOV112D) was dramatically diminished following shRNA-mediated MK2 depletion and platinum or taxane treatment in vitro (Figure 2a-c, Supporting Information Figure S3). Together, these findings provide strong support that loss of MK2 can chemosensitize p53-mutant ovarian tumors to frontline therapeutic interventions for the disease (i.e., platinum/taxane doublet therapy). In vitro, we also observed nominally improved MK2 silencing efficiency from RNA-peptide nanoplexes com- thatin contrast to lines often employedmore closely resembles the copy-number changes, mutations, and mRNA expression profiles presented by high-grade serous ovarian tumors. We 39 and others 40,41 later identified cell lines among these whose phenotypic and histopathological features, in vivo, further aligned with that of the human disease. In these studies, we selected the OVCAR8 cell line due to its relatively aggressive growth rate, strongly mesenchymal phenotype,

| Neoadjuvant siMK2 therapy improves OC treatment outcomes
After demonstrating efficient in vivo delivery of siRNA to orthotopic, high-grade serous ovarian tumor models, we next investigated treatment outcomes in OVCAR8 tumor-bearing mice. In prior work, 44 we Having shown that nanoplex-mediated RNAi of MK2 chemosensitized ovarian tumors to frontline chemotherapy in vivo, we next examined differential pharmacodynamics that may have predicated improved treatment outcomes in mice receiving combined siRNA and chemotherapy. In prior work (unpublished), we found that repeated intraperitoneal dosing of nanoplex-siMK2 (1 mg/kg siRNA, BIW) did not significantly affect serum biochemical markers of liver and kidney damage. 44 At the study endpoints, we observed no significant differences in serum biochemistry from mice receiving chemotherapy alone or that with those receiving concurrent RNAi therapy (Figure 4i-k). Renal and hepatic function was impaired in both groups; however, we could not attribute differential survival to varying morbidity as measured from sera. We also examined lung tissues obtained from mice that reached study endpoints. Immunohistochemical staining of these tissues using an ovarian lineage-specific marker (PAX8) found no observable pulmonary metastases in mice receiving combined siMK2 and chemotherapy, while multiple tumor lesions were observed in all other groups (Figure 4l). Moreover, we found that ascitic fluids from mice obtained at study endpoints were significantly enriched in monocyte chemoattractant protein 1 (MCP-1, CCL2) following treatment with combined RNAi/chemotherapy versus chemotherapy alone (p 5 .0384; Figure 4m). Interestingly, ascitic MCP-1 elevations were absent in mice treated with peptide nanoplexes, alone. Additional, independent studies are currently underway to better understand the molecular mechanisms by which loss of MK2 modulates tumor invasiveness and overall survival in OVCAR8 tumor-bearing mice that receive platinum/taxane chemotherapy.

| C ONC LUSI ON S
Peptide nanoplexes are a promising, modular drug delivery platform that can enable enhanced tumor accumulation of a wide range of therapeutics including small molecules, siRNA, plasmid DNA, and mRNA. 5,15,46,47 Here, we show that a novel polymer blend architecture consisting of three NCA peptide homologs can self-assemble to form stable siRNA nanoplexes that silence a key DNA-damage response pathway in p53-deficient advanced ovarian tumors. Nanoplexmediated silencing of MK2 profoundly sensitized high-grade serous ovarian tumors to concurrent platinum/taxane chemotherapy in vivo, improved overall survival by 37%, and decreased metastatic spread to the lungs without observable toxic side effects.
Given their peptide backbone and FDA GRAS (generally regarded as safe) designation of their amino acid metabolites, these structures could serve as uniquely biocompatible alternatives to conventional, non-natural siRNA delivery vectors. Kataoka and coworkers, for example, have shown that chemotherapy-appended NCA polypeptides are well-tolerated in patients both in the United States 48 and in Japan, [49][50][51] and protein-mimetic NCA polypeptides such as glatiramer acetate have been approved for clinical use in the United States since 1997. In contrast to peptides synthesized by solid-phase techniques or those produced through recombinant expression, the RNAi delivery technologies described here could be manufactured both in large-scale and with diverse (i.e., non-natural) chemical functionality, potentially enabling more facile commercial development.
Therapeutic inhibition of MK2 also holds promise for a range of other disease indications beyond that demonstrated here in ovarian tumors. Duvall and coworkers, for example, have shown that polymermediated delivery of peptide inhibitors of MK2 can block intimal hyperplasia and failure of autologous vein graft transplants in rabbits, a procedure commonly used for coronary and peripheral artery bypass in patients. 52 There, inhibition of the p38/MK2 pathway-mediated stress response induced following graft harvest/transplantation greatly decreased profibrotic response and corresponding graft failure. Inhibition of MK2 using small molecule tool compounds has also been shown to synergize with blockade of other cell cycle checkpoint kinases in Ras-driven solid tumors, augmenting tumor cell killing in a p53dependent manner. 53 The discovery of safe and specific small molecule inhibitors of MK2 for rheumatoid arthritis 54 therapy is also an active area of industrial research. RNAi-mediated silencing of MK2 may obviate the need for such development as small molecules targeting MK2 have historically exhibited off-target toxic effects and limited selectivity. Given the promising results shown here, we anticipate that peptide nanoplex-mediated RNAi may serve as a safe, specific, and effective neoadjuvant therapy that can sensitize advanced ovarianand other solidtumors to frontline cytotoxic chemotherapy. Center for technical support, specifically the FACS, Peterson nanotechnology, high-throughput screening, microscopy, ATWAI, and Tang Histology facilities. We also wish to thank members of the Hammond, Yaffe and Hemann labs for helpful discussions.