Targeted Nano- Therapeutics for Drug Resistant Tumors

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Amiji, PhD University Distinguished Professor and Director of the Laboratory of Biomaterials and Advanced Nano-Delivery

1 Invited Presentation at the 2016 American Association of Pharmaceutical Scientists National Biotechnology Conference. Boston, MA. Wednesday, May 18 th, 2016 Targeted Nano- Therapeutics for Drug Resistant Tumors Mansoor M. Amiji, PhD University Distinguished Professor and Director of the Laboratory of Biomaterials and Advanced Nano-Delivery Systems (BANDS) Department of Pharmaceutical Sciences Northeastern University 140 The Fenway Building Boston, MA Website:

The Tumor Microenvironment The Hallmarks of Cancer Cells of the TME Ref: D. Hanahan and R.A. Weinberg. Cell; 144: 646-674 (2011). Ref. D. Leyva-Illades, et al.

Tumors are composite of many different cellular and non-cellular constituents that surround the malignant cancer cells harboring activating mutations in oncogenes or loss of tumor

2 The Tumor Microenvironment The Hallmarks of Cancer Cells of the TME Ref: D. Hanahan and R.A. Weinberg. Cell; 144: (2011). Ref. D. Leyva-Illades, et al., Transl Gastrointest Cancer; 1: (2012). Tumors are composite of many different cellular and non-cellular constituents that surround the malignant cancer cells harboring activating mutations in oncogenes or loss of tumor suppressors that drive tumor growth. A variety of infiltrating immune cells, cancerassociated fibroblasts, and angiogenic endothelial cells play expanding and critical functions in sustaining cell proliferation, evading growth suppressors, promoting survival, activating invasion and metastasis, and reprogramming energy metabolism.

3 Hypoxia, Glycolysis and Drug Resistance Ref. O. Trendan, et. al., J. Nat. Cancer. Inst. 99(19): (2007) Ref. M.G. Vander-Heiden, et al. Science 324 (5930): (2009)

Overcoming MDR by RNAi/Drug Therapy Circumventing antiapoptotic mechanisms (e.g., survivin and Bcl- 2 expression) Inhibiting ATPdependent drug efflux transporters (e.g., mdr-1 and mrp-1 genes) Inhibition of aerobic glycolysis (e.

4 Overcoming MDR by RNAi/Drug Therapy Circumventing antiapoptotic mechanisms (e.g., survivin and Bcl- 2 expression) Inhibiting ATPdependent drug efflux transporters (e.g., mdr-1 and mrp-1 genes) Inhibition of aerobic glycolysis (e.g., PKM-2, HK, and LDH-A genes) Affecting genes that regulate cell-cycle checkpoints (e.g., mad-2) Ref. M. Susa, et al., Pharm. Res., 28(2): (2011). Funded by NCI Alliance in Nanotechnology, Platform Partnership Grants

5 Targeting Module Combinatorial-Designed Nano-Assemblies Module Payloads Imaging Module Payload Encapsulation Pol-Lipid C 2-4 Pol-Lipid C 6-10 Pol-Lipid C Pol-Thiol Pol-PEG Peptide Fluorescence Radioactive Shanthi Ganesh Arun Iyer Amit Singh

Hyaluronic Acid Derivatives Hyaluronic acid (HA) is a natural, biocompatible, and biodegradable polymer 5 1 Long history

, for viscosupplementation therapy in arthritis) 2 Intrinsic targeting to CD44 receptors over-expressed on tumor cells

substitutions (EDC coupling or click chemical cojugation) Combinatorial library of formed nanoparticles by self-assembly

6 Hyaluronic Acid Derivatives Hyaluronic acid (HA) is a natural, biocompatible, and biodegradable polymer 5 1 Long history of safe use in clinical applications (e.g., for viscosupplementation therapy in arthritis) 2 Intrinsic targeting to CD44 receptors over-expressed on tumor cells (e.g., cancer stem cells) and macrophages 4 Modular HA nanoparticle platform synthesized using different functional substitutions (EDC coupling or click chemical cojugation) Combinatorial library of formed nanoparticles by self-assembly of the constituents at specific weight ratios of each (i.e., LEGO blocks) sirna/mirna Duplexes OR Small Molecule Drug OR Targeted Self-Assembling Nano-system HA-PEG HA-Thiol HA-PEG-Peptide 3 EGFR Peptide

$Platinum Refractory Tumor$ Duan, MGH HA shell for tumor

7 SKOV3 CD44 Expression Evaluation of Combination mdr-1 Silencing and Paclitaxel Co-Therapy in Resistant Ovarian Cancer CD44 expression in human ovarian tumor samples Platinum Refractory Tumor Sensitive Tumor 40X E Zhenfeng Duan, MGH HA shell for tumor targeting PEI for condensing mdr1 sirna PEG corona for prolonging circulation mdr-1 sirna payload CD44 Pgp CD44 Pgp β-actin SKOV-3 TR SKOV-3 Relative Expression CD44 Pgp SKOV-3TR SKOV-3 SKOV3 TR Encapsulation of mdr-1 sirna in HA-PEI/HA-PEG nanoparticles for CD44 targeted delivery in resistant SKOV3 TR human ovarian adenocarcioma model Ref. X. Yang, et al., Scientific Reports, 5(8509): 1-9 (2015).

8 R e l a t i v e M D R 1 E x p r e s s i o n Down-regulation of mdr-1 in SKOV3 TR Cells using sirna- Encapsulation in HA-PEI/HA-PEG Nanoparticles Relative mdr-1 gene silencing after 24 h by qpcr 1. 2 S K O V - 3 T R c e l l o n l y H A - P E I / H A - P E G / n o n - s p e c i f i c s i R N A % L i p o f e c t a m i n e / M D R 1 s i R N A 9 0 n M H A - P E I / H A - P E G / M D R 1 s i R N A 4 5 n M H A - P E I / H A - P E G / M D R 1 s i R N A 9 0 n M H A - P E I / H A - P E G / M D R 1 s i R N A n M 0. 0 Pgp β-actin Relative Pgp Expression Relative P-gp expression after 24 h 1: SKOV-3TR cell only 2: HA-PEI/HA-PEG/non-specific sirna 45nM 3: HA-PEI/HA-PEG/non-specific sirna 90nM 4: HA-PEI/HA-PEG/non-specific sirna 180nM 5: MDR1 sirna alone 45nM 6: MDR1 sirna alone 90nM 7: MDR1 sirna alone 180nM 8: Lipofectamine /MDR1 90nM 9: HA-PEI/HA-PEG/MDR1 sirna 45nM 10: HA-PEI/HA-PEG/MDR1 sirna 90nM 11: HA-PEI/HA-PEG/MDR1 sirna 180nM Ref. X. Yang, et al., Scientific Reports, 5(8509): 1-9 (2015).

9 Evaluation of P-gp and CD44 Expression in Tumor Tissues upon Administration of mdr-1 sirna in HA Nanoparticles CD44 Pgp CD44 Pgp 1 β-actin Relative Expression CD Pgp : saline + paclitaxel 2: MDR1 sirna + paclitaxel 3: HA-PEI/HA-PEG/none specific sirna + paclitaxel 4: HA-PEI/HA-PEG/MDR1 sirna + paclitaxel Ref. X. Yang, et al., Scientific Reports, 5(8509): 1-9 (2015).

10 R e l a t i v e T u m o r V o l u m e Ref. X. Yang, et al., Scientific Reports, 5(8509): 1-9 (2015). In Vivo Anti-tumor Efficacy of Combination mdr-1 sirna/paclitaxel Therapy in SKOV3 TR Xenograft Model Group 4: HA-PEI/HA-PEG/mdr1 sirna Group 3: HA-PEI/HA-PEG/non-specific sirna Group 2: MDR1 sirna alone Group 1: saline Group 1-4: paclitaxel (20mg/kg) Tumors collection s a l i n e + p a c l i t a x e l M D R 1 s i R N A + p a c l i t a x e l H A - P E I / H A - P E G / n o n - s p e c i f i c s i R N A + p a c l i t a x e l H A - P E I / H A - P E G / M D R 1 s i R N A + p a c l i t a x e l Saline + paclitaxel mdr-1 sirna alone + paclitaxel HA-PEI/HA-PEG/ Non-pecific sirna + paclitaxel T i m e a f t e r i n j e c t i o n ( d a y s ) HA-PEI/HA-PEG/ mdr-1 sirna + paclitaxel

PKM-2/β-actin MDR-1/β-actin Combination mdr-1/pkm-2 Gene Silencing in SKOV3 Model

6 72 h 96 h mdr-1 Gene Silencing sirna Dose: 100 nm Time: 72 and 96 hours Michael

11 PKM-2/β-actin MDR-1/β-actin Combination mdr-1/pkm-2 Gene Silencing in SKOV3 Model with HA-PEI/HA-PEG Nanoparticles h 96 h mdr-1 Gene Silencing sirna Dose: 100 nm Time: 72 and 96 hours Michael Goldberg, DFCI h 96 h PKM-2 Gene Silencing Ref. R.A. Cairns, I.S. Harris, & TW. Mak. Nat Revs Cancer, 11: (2011) Partially Funded by DFCI/NU Grant Ref. M. Talekar, et al., Mol. Cancer Therap., 14(7): (2015). Meghna Talekar

Ref. M. Talekar, et al., Mol. Cancer Therap., 14(7): 1521-1531 (2015).

Non-Targeted EGFR Targeted Whole body near-ir imaging of tumor targeted delivery with ICG-encapsulated control

12 Ref. M. Talekar, et al., Mol. Cancer Therap., 14(7): (2015). Biodistribution and Tumor-Specific Localization of sirna Upon Administration in HA-PEI/HA-PEG Nanoparticles Non-Targeted EGFR Targeted Whole body near-ir imaging of tumor targeted delivery with ICG-encapsulated control and EGFR targeted HA-PEI/HA-PEG nanoparticles and quantitative assessment of mdr-1 sirna accumulation as a function of time in plasma and various tissues.

MDR-1 and PKM-2 expression levels were assessed using PCR (A) and immunohistofluorescence (B & C) and an increase

13 Ref. M. Talekar, et al., Mol. Cancer Therap., 14(7): (2015). Establishment of Resistant SKOV3 Model by Intravenous Paclitaxel Administration in Athymic Mice In vivo PTX resistance model development. SKOV-3 wild-type tumor bearing mice were injected with 20 mg/kg PTX every alternate day for 10 days. MDR-1 and PKM-2 expression levels were assessed using PCR (A) and immunohistofluorescence (B & C) and an increase in mdr- 1/P-gp expression was observed in animals treated with PTX. However no significant difference was observed following week 1 and week 3 post last dose of PTX. We are currently investigating the efficacy of combination therapy with sirna and PTX in this model. B A C

Gene of interest/β-actin Ref. M. Talekar, et al., Mol. Cancer Therap., 14(7): 1521-1531 (2015).

14 Gene of interest/β-actin Ref. M. Talekar, et al., Mol. Cancer Therap., 14(7): (2015). Tumor volume (mm 3 ) In Vivo mdr-1 and PKM-2 Gene Silencing and Anti-Tumor Efficacy with Combination Therapy in SKOV3 Model In Vivo Silencing Efficiency simdr-1 sipkm In Vivo Anti-Tumor Response PBS PTX solution simdr-1 sipkm-2 HA-PEI (NT) HA-PEI (T) HA-PEI-MDR-1+PTX(NT) HA-PEI-MDR-1+PTX(T) HA-PEI-MDR-1/PKM-2+PTX(NT) HA-PEI-MDR-1/PKM-2+PTX(T) PBS Scrambled sirna NP sirna (NT) NP sirna (T) Time (days) qpcr analysis of in vivo gene silencing efficacy in SKOV3 tumor xenograft model in female nu/nu (athymic) mice upon intravenous administration of mdr-1 and PKM2 silencing sirna (0.5 mg/kg) and the anti-tumor therapeutic response from single and combination sirna/paclitaxel (20 mg/kg) administration

Tumor-Associated Macrophages Ref. J.W. Pollard. Nat Revs Cancer, 4, 71-78 (2004) Ref. T.L. Rogers and I. Holen.

virtually all aspects of tumor growth and progression, including stem cells, metabolism, angiogenesis, invasion,

15 Tumor-Associated Macrophages Ref. J.W. Pollard. Nat Revs Cancer, 4, (2004) Ref. T.L. Rogers and I. Holen. J Translational Med, 9:177 (2011) Tumor-associated macrophages (TAMs), which are predominantly M2 polarized, affect virtually all aspects of tumor growth and progression, including stem cells, metabolism, angiogenesis, invasion, metastasis, and therapeutic resistance. Communication between tumor cells and macrophages is critical for tumor malignancy.

TAM Repolarization from M2 to M1 Ref. S.K. Biswas & A. Montovani, Nat Immunol., 11: 889-896 (2010). Ref. R.C. Chang, et al., Cells, 3(3): 702-712 (2014).

16 TAM Repolarization from M2 to M1 Ref. S.K. Biswas & A. Montovani, Nat Immunol., 11: (2010). Ref. R.C. Chang, et al., Cells, 3(3): (2014). Reprogramming TAMs from a predominant M2 to M1 phenotype would provide an opportunity for anti-tumor response and potentially improve cancer immunotherapy. Several microrna s (e.g., mir-155 and mir-125b) have shown to change macrophage polarity from M2 to M1.

Exosome Transfer from Human Pancreatic Tumor

Cells Macrophages Luciferase Expressing Panc-1

Macrophage Polarization Markers TEM image of

17 Exosome Transfer from Human Pancreatic Tumor Cells to Macrophages using a Co-Culture System Transwell Co-Culture System Tumor Cells Macrophages 6 h After treating LPS/IFN-γ Tumor Cells Macrophages Luciferase Expressing Panc-1 Cells 48h & 72h Mei-Ju Su Microscopy and RT-PCR Analysis of Exosome-Mediated Macrophage Polarization RT-PCR Analysis of M1/M2 Macrophage Polarization Markers TEM image of Panc-1 Exosomes Ref. M.J. Su, et al., Nature - Scientific Reports, (Submitted)

MicroRNAs 155 and 125b Transfection in Panc-1 Cells with

(Panc-1) transfected with plasmid DNA expressing mir-155 and

18 MicroRNAs 155 and 125b Transfection in Panc-1 Cells with HA-PEI/HA-PEG Nanoparticles Human pancreatic cancer cells (Panc-1) transfected with plasmid DNA expressing mir-155 and mir-125b and the levels of expression in cells relative to control were measured using PCR. The plasmid dose was 20 mg per 200,000 cells. Ref. M.J. Su, et al., Nature - Scientific Reports, (Submitted)

19 Change in Macrophage Polarization from M2 to M1 with Exosome-Mediated Transfer of MicroRNAs 155 and 125b Ref. M.J. Su, et al., Nature - Scientific Reports, (Submitted)

20 MicroRNA Profiling in Cytosol and Exosomes in SK-LU1 Non-Small Cell Lung Cancer Cells Malav Trivedi TEM image of SK-LU1 exosomes Ref. M. Trivedi, et al., Oncogenesis, (Submitted) Meghna Talekar

Changes in wt-p53 and mir-125b Levels in Cytosol and Exosomes upon Transfection with HA-Based Nanoparticles A Flag p53 Transcript Levels B mir-125b Transcript Levels (A) Quantitative qrt-pcr analysis

(combi/cells) and in exosomes (combi/exo) after 18 hours of incubation.

21 Changes in wt-p53 and mir-125b Levels in Cytosol and Exosomes upon Transfection with HA-Based Nanoparticles A Flag p53 Transcript Levels B mir-125b Transcript Levels (A) Quantitative qrt-pcr analysis of expression of wt-p53 in cells (p53/cells) and in exosomes (p53/exo) when transfected with wt-p53 expressing plasmid DNA alone or in combination with mirna-125b expressing plasmid DNA in cells (combi/cells) and in exosomes (combi/exo) after 18 hours of incubation. (B) Quantitative qrt-pcr analysis of expression of mir-125b expression in cells (mir-125b/cells) and in exosomes (mir-125b /exo) when transfected with mirna-125b expressing plasmid DNA alone or in combination with wt-p53 expressing plasmid DNA in cells (combi/cells) and in exosomes (combi/exo) after 18 hours of incubation. Ref. M. Trivedi, et al., Oncogenesis, (Submitted)

Establishment of KRAS/p53 Mutant Genetically-Engineered Mouse Model of

weeks post AdeCre dosing variable viral dose Time course analysis of

weeks Large adenoma 12 weeks Adenocarcinoma 16 weeks Atypical adenomatous

22 Establishment of KRAS/p53 Mutant Genetically-Engineered Mouse Model of Non-Small Cell Lung Cancer Wild type lungs Mutant 4 wks Mutant 10 wks* 6 weeks post AdeCre dosing variable viral dose Time course analysis of stages of tumor progression in Lox-K-ras-G12D mice Papillary adenoma 6 weeks Large adenoma 12 weeks Adenocarcinoma 16 weeks Atypical adenomatous hyperplasia 2 weeks Ref. M. Talekar, et al., Molecular Therapy, 24(4): (2016).

23 R a tio F la g -p 5 3 / -a c tin In Vivo wt-p53 and mir-125b Transfection in Lung Tumor Tissues in the KRAS/p53 GEM Models Relative p53 expression wt-p53 Transcript * AdCre Kras LSL-G12D /p53 fl/fl * * * Day 1 pdna- HA NP s Day 3 pdna- HA NP s Flag p53 β-actin 2.0 Day 5 pdna- HA NP s Day 7 Cisplatin solution Therapy on week 2, 4 and 6 0 PBS CD44 T p53 NP Dual T p53 NP CD44 T combo NP Dual T combo NP 1.5 Relative mir-125b expression mir-125b Transcript * * * PBS CD44 T 125b NP Dual T 125b NP CD44 T combo NP Dual T combo NP * P B S C D 4 4 T p 5 3 N P D u a l T p 5 3 N P C D 4 4 T c o m b o N P D u a l T c o m b o N P Ref. M. Talekar, et al., Molecular Therapy, 24(4): (2016).

24 Changes in the M1/M2 Macrophage Markers and Inflammatory Cytokine Levels in Tumor Tissues Ref. M. Talekar, et al., Molecular Therapy, 24(4): (2016).

25 Changes in the Ki-67 Expression Profile in Tumor Tissues upon Transfection with wt-p53 and mir-125b Ref. M. Talekar, et al., Molecular Therapy, 24(4): (2016).

26 Summary Poor drug delivery efficiency and the various soluble and insoluble tumor microenvironmental factors are critical in the development of drug resistance. Combinatorial-designed multifunctional polymeric nano-systems offer a versatile platform for tumor-targeted delivery of RNAi and small molecule therapeutics for modulation of drug resistance. Our RNAi approaches have relies on the hallmarks of cancer focusing on down-regulation of anti-apoptotic genes (e.g., bcl-2 and survivin), ATPdependant efflux transporters (e.g., mdr-1), glycolytic enzymes (e.g., PKM-2), and cell cycle checkpoint genes (e.g., mad-2). Further evaluation of the role of tumor microenvironment, especially through regulation of tumor cell communication, epigenetics, macrophage polarity, and inflammatory pathways, will be critical to overcome resistance. In all of the above nano-therapeutic strategies, our focus continues to be on the use of safe materials and scalable manufacturing processes that allow for rapid translation of these experimental approaches in clinically-viable therapeutic options for cancer patients.

Nanotechnology applications in medical diagnosis, imaging, and therapy

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