Tumor-Targeted Synergistic Blockade of MAPK and PI3K from a Layer-By-Layer Nanoparticle Erik C
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Published OnlineFirst June 1, 2015; DOI: 10.1158/1078-0432.CCR-15-0013 Cancer Therapy: Preclinical Clinical Cancer Research Tumor-Targeted Synergistic Blockade of MAPK and PI3K from a Layer-by-Layer Nanoparticle Erik C. Dreaden1,2, Yi Wen Kong1,3, Stephen W. Morton1,2, Santiago Correa1,4, Ki Young Choi1,2, Kevin E. Shopsowitz1,2, Kasper Renggli1,2,4, Ronny Drapkin5,6,7, Michael B. Yaffe1,3,4,8, and Paula T. Hammond1,2,9 Abstract Purpose: Cross-talk and feedback between the RAS/RAF/ bioluminescence imaging, Western blotting, serum cytokine MEK/ERK and PI3K/AKT/mTOR cell signaling pathways is analysis, and immunohistochemistry. critical for tumor initiation, maintenance, and adaptive Results: Combined MAPK and PI3K axis blockade from the resistance to targeted therapy in a variety of solid tumors. nanoscale formulations (160 Æ 20 nm, À40 Æ 1 mV) was Combined blockade of these pathways—horizontal block- synergistically toxic toward triple-negative breast (MDA-MB- ade—is a promising therapeutic strategy; however, com- 231) and RAS-mutant lung tumor cells (KP7B) in vitro, effects pounded dose-limiting toxicity of free small molecule inhib- that were further enhanced upon encapsulation. In vivo, system- itor combinations is a significant barrier to its clinical ically administered LbL nanoparticles preferentially targeted application. subcutaneous MDA-MB-231 tumor xenografts, simultaneously Experimental Design: AZD6244 (selumetinib), an alloste- blocked tumor-specific phosphorylation of the terminal kinases ric inhibitor of Mek1/2, and PX-866, a covalent inhibitor of Erk and Akt, and elicited significant disease stabilization in PI3K, were co-encapsulated in a tumor-targeting nanoscale the absence of dose-limiting hepatotoxic effects observed from drug formulation—layer-by-layer (LbL) nanoparticles. Struc- the free drug combination. Mice receiving untargeted, but dual ture, size, and surface charge of the nanoscale formulations drug–loaded nanoparticles exhibited progressive disease. were characterized, in addition to in vitro cell entry, synergistic Conclusions: Tumor-targeting nanoscale drug formulations cell killing, and combined signal blockade. In vivo tumor could provide a more safe and effective means to synergisti- targeting and therapy was investigated in breast tumor xeno- cally block MAPK and PI3K in the clinic. Clin Cancer Res; 21(19); graft–bearing NCR nude mice by live animal fluorescence/ 4410–9. Ó2015 AACR. Introduction 1Koch Institute for Integrative Cancer Research, Massachusetts Insti- tute of Technology, Cambridge, Massachusetts. 2Department of Combination chemotherapy has been a mainstay of clinical Chemical Engineering, Massachusetts Institute of Technology, Cam- oncology since the mid 1960s (1, 2). While initially proposed to 3 bridge, Massachusetts. Department of Biology, Massachusetts Insti- exploit nonoverlapping toxicity profiles of cytotoxic chemothera- tute of Technology, Cambridge, Massachusetts. 4Department of Bio- logical Engineering, Massachusetts Institute of Technology, Cam- peutics, recent advances in cancer cell signaling and the discovery of bridge, Massachusetts. 5Penn Ovarian Cancer Research Center, Basser potent and selective small-molecule inhibitors have led to the Research Center for BRCA, University of Pennsylvania, Philadelphia, development of rational combination approaches to cancer chemo- Pennsylvania. 6Perelman Center for Advanced Medicine, Abramson Cancer Center, University of Pennsylvania, Philadelphia, Pennsylvania. therapy. These strategies can prime cancer cells for apoptosis (3, 4), 7Department of Obstetrics and Gynecology, University of Pennsylva- abrogate mechanisms for resistance (5–9), block cancer cell-cycle nia, Philadelphia, Pennsylvania. 8Division of Acute Care Surgery,Trau- progression (10), or dynamically rewire DNA damage response ma, and Critical Care, Department of Surgery, Beth Israel Deaconess — Medical Center, Harvard Medical School, Boston, Massachusetts. pathways (11, 12) all leading to enhanced tumor cell killing. 9Institute for Soldier Nanotechnologies, Massachusetts Institute of Although combination therapies can be curative in a subset of Technology, Cambridge, Massachusetts. malignancies (13), inefficient delivery limits the safety and effi- Note: Supplementary data for this article are available at Clinical Cancer cacy of cooperative drug combinations that require cellular colo- Research Online (http://clincancerres.aacrjournals.org/). calization for effective cell killing. This can be difficult to achieve Corresponding Authors: Paula T. Hammond, Massachusetts Institute of Tech- with traditional pharmaceutical excipients as synergistic com- nology, 77 Massachusetts Avenue, Cambridge, MA 02139. Phone: 1-617-2587577; pounds often exhibit vastly different physiochemical properties Fax: 1-617-2588992; E-mail: [email protected]; and Michael B. Yaffe, Massa- (e.g., size, charge, lipophilicity, and stability) that necessitate chusetts Institute of Technology, Broad Institute, Beth Israel Deaconess Medical separate drug carriers. Colocalized delivery of cooperative drug Center and Harvard Medical School, 77 Massachusetts Avenue, Cambridge, MA combinations from single, multicomponent delivery vehicles can 02139. Phone: 1-617-4522442; E-mail: [email protected] improve both therapeutic index (5) and treatment outcomes doi: 10.1158/1078-0432.CCR-15-0013 (6, 9, 12, 14) compared with combinations of individual carriers; Ó2015 American Association for Cancer Research. however, tissue-specific delivery remains a significant challenge. 4410 Clin Cancer Res; 21(19) October 1, 2015 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst June 1, 2015; DOI: 10.1158/1078-0432.CCR-15-0013 Nanoparticle-Targeted Synergistic Blockade of MAPK and PI3K Horizontal blockade of MAPK and PI3K has been proposed as a Translational Relevance means to achieve synergistic cell killing and to abrogate adaptive The MAPK and PI3K cell signaling pathways encompass resistance to single-axis targeted therapies. Engelman and collea- numerous clinically approved drug targets, and recent evidence gues found that combined MEK and PI3K/mTOR inhibition was suggests that cross-talk/feedback between these pathways is synergistic against genetically engineered mouse models of KRAS- critical for initiation and maintenance of various solid tumors, mutant non–small cell lung cancer (33). Similarly, Posch and as well as resistance to targeted therapies. Simultaneous block- colleagues reported that that dual inhibition of MEK and PI3K/ ade of these two signaling axes is a promising therapeutic stra- mTOR was both synergistic and required for durable treatment tegy; however, rational combinations approaches with small- response in NRAS-mutant melanoma in vitro and in vivo (44). A molecule inhibitors are poorly tolerated in patients. In this recent phase I clinical trial investigating simultaneous (i.e., hor- work, we develop a tumor-targeting nanoscale drug formula- izontal) blockade of MAPK and PI3K in patients with refractory tion based on layer-by-layer (LbL) self-assembly that horizon- solid tumors found that dual inhibition may exhibit favorable tally blocks MAPK and PI3K signaling in vitro and in vivo, efficacy compared with single-axis therapy, albeit with com- selectively inhibiting disease progression in tumor-bearing pounded and often dose-limiting toxicity (45). Here, we address mice. These self-assembled drug carriers co-delivered synergis- this unmet clinical need by engineering nanoscale drug carriers tic drug compounds with vastly differing physiochemical pro- that improve the therapeutic index of this novel combination perties, in a manner that pre-empted adaptive resistance and therapy through multimodal targeted delivery to solid tumors. diminished toxic effects of the free drug combination. This These modular, dual drug–containing delivery vehicles horizon- approach could provide a more safe and effective means to tally block MAPK and PI3K signaling to diminish tumor growth in synergistically block MAPK and PI3K in the clinic. vivo. Nanotechnology-enabled horizontal blockade of MAPK and PI3K is a promising strategy for the treatment of several solid tumor types and is demonstrated here in triple-negative breast cancer tumor models. Nanoscale drug carriers that deliver cooperative drug combina- tions in a tumor-targeted fashion are urgently needed in the clinic. Materials and Methods Layer-by-layer (LbL) nanoparticles (15–24) are a new class of self-assembled polymer drug carriers that addresses challenges in Materials the delivery of combination therapeutics. These structures consist Poly(L-lysine) HBr (PLL, 15–30 kDa; Sigma-Aldrich), hyalur- of a functional nanoparticle core, a polyelectrolyte multilayer onan (HA, 200 kDa; Lifecore), and dextran sulfate sodium salt shell, and an exterior tumor-targeting stealth layer. Previously, we (DXS, 20 kDa; Sigma-Aldrich) were used as-received without developed an LbL nanoparticle architecture that targets solid further modification. Antibodies included total Akt (CST tumors through 3 independent mechanisms: (i) size-dependent 4691), cleaved caspase-3 (CST 9661), total Erk (CST 4370), pAkt passive tumor targeting, (ii) active, ligand-directed targeting of (CST 4060; S473), pAkt-Alexa Fluor 488 (Millipore CS203310; cell surface CD44 receptor, and (iii) hypoxic tumor pH-responsive S473), pErk1/2 (CST 4370; T202/204, T185/187), and pErk1/ cellular delivery (24). Here, we apply multimodal