Versatile Nano-PROTAC-Induced Epigenetic Reader Degradation for Efficient Lung Cancer Therapy

Abstract

Recent evidence has indicated that overexpression of the epigenetic reader bromodomain-containing protein 4 (BRD4) contributes to a poor prognosis of lung cancers, and the suppression of its expression promotes cell apoptosis and leads to tumor shrinkage. Proteolysis targeting chimera (PROTAC) has recently emerged as a promising therapeutic strategy with the capability to precisely degrade targeted proteins. Herein, a novel style of versatile nano-PROTAC (CREATE (CRV-LLC membrane/DS-PLGA/dBET6)) is developed, which is constructed by using a pH/GSH (glutathione)-responsive polymer (disulfide bond-linked poly(lactic-co-glycolic acid), DS-PLGA) to load BRD4-targeted PROTAC (dBET6), followed by the camouflage with engineered lung cancer cell membranes with dual targeting capability. Notably, CREATE remarkably confers simultaneous targeting ability to lung cancer cells and tumor-associated macrophages (TAMs). The pH/GSH-responsive design improves the release of dBET6 payload from nanoparticles to induce pronounced apoptosis of both cells, which synergistically inhibits tumor growth in both subcutaneous and orthotopic tumor-bearing mouse model. Furthermore, the efficient tumor inhibition is due to the direct elimination of lung cancer cells and TAMs, which remodels the tumor microenvironment. Taken together, the results elucidate the construction of a versatile nano-PROTAC enables to eliminate both lung cancer cells and TAMs, which opens a new avenue for efficient lung cancer therapy via PROTAC.

Keywords: BRD4; PROTAC; epigenetic reader; tumor microenvironment; tumor-associated macrophages.

Scheme 1

Schematic diagram of CRV‐LLCM‐coated nanoparticles formulation and the underlying therapeutic mechanisms.

Figure 1

Characterizations of CRV‐LLCM‐based nanoparticles. A,B) TEM analysis of DPB and CREATE. C,D) DLS analysis of DPB and CREATE. E) Cumulative release of DPB in neutral (pH = 7.4) and acidic environment (pH = 5) (n = 3). F) Cumulative release of DPB in non‐GSH environment (GSH = 0 × 10⁻³ m) and GSH environment (GSH = 5 × 10⁻³ M and 10 × 10⁻³ m, respectively) (n = 3). G) Zeta potential of DPB, CRV‐LLCM, and CREATE (n = 3). H) SDS‐PAGE analysis of the protein composition of CRV‐LLCM, LDPB (LLC membrane/DS‐PLGA/dBET6), and CREATE, respectively. LDPB, LLCM/DS‐PLGA/dBET6; CREATE, CRV‐LLCM/DS‐PLGA/dBET6. Data are presented as mean ± SD. ** p < 0.01 and **** p < 0.0001.

Figure 2

Biological effect induced by different formulations. A) Dose‐dependent cellular uptake of different formulations determined by CLSM (left panel) and FACS (right panel). LLC cells were treated with various concentrations of CRV‐LLCM/DS‐PLGA/DiD (CRV‐LLCM/DS‐PLGA/DiD was equivalent to 60, 120, 240, and 480 µg mL⁻¹, respectively) for 9 h. B) Time‐dependent cellular uptake of different formulations determined by CLSM (left panel) and FACS (right panel). LLC cells were treated with CRV‐LLCM/DS‐PLGA/DiD (240 µg mL⁻¹) for 3, 6, 9, and 12 h. C) Cellular uptake of DS‐PLGA/DiD, LLCM/DS‐PLGA/DiD, and CRV‐LLCM/DS‐PLGA/DiD. LLC cells were treated with the indicated nanoparticles (DS‐PLGA/DiD equivalent to 60 µg mL⁻¹; LLCM/DS‐PLGA/DiD and CRV‐LLCM/DS‐PLGA/DiD equivalent to 240 µg mL⁻¹) for 9 h. D) Schematic diagram of LLC spheroids formulation and treatment. E) Cellular uptake evaluation of DS‐PLGA/DiD, LLCM/DS‐PLGA/DiD, and CRV‐LLCM/DS‐PLGA/DiD in LLC spheroids. The 3D tumor spheroids were treated with the indicated formulations (DS‐PLGA/DiD equivalent to 60 µg mL⁻¹; LLCM/DS‐PLGA/DiD and CRV‐LLCM/DS‐PLGA/DiD were equivalent to 240 µg mL⁻¹) for 9 h. F) Immunoblotting analysis of BRD4, c‐Myc, and apoptotic genes in LLC cells. The cells treated with different formulations (PBS, CLDP (CRV‐LLC membrane/DS‐PLGA/NC), dBET6, DPB, LDPB, or CREATE, respectively) for 9 h, and incubated with the freshly prepared medium for another 15 h. Tubulin served as a loading control.

Figure 3

Inhibitory effects induced by different formulations in vitro. A) Cell viability of LLC cells. B) Colony formation assays of LLC cells treated with different formulations. LLC cells were treated with different formulations (PBS, CLDP, dBET6, DPB, LDPB, or CREATE, respectively) for 9 h, and incubated with the freshly prepared medium for another 7 d (n = 3). C) Annexin V‐FITC/propidium iodide (PI) apoptosis assay of LLC cells. D) Quantification of JC1 monomers/aggregates of LLC cells. To evaluate the cell viability and apoptosis, LLC cells were treated with different formulations (PBS, CLDP, dBET6, DPB, LDPB, or CREATE, respectively) for 9 h, and incubated with the freshly prepared medium for another 15 h (n = 3). Data are presented as mean ± SD. ** p < 0.01 and **** p < 0.0001.

Figure 4

Inhibitory effects induced by different formulations on M2‐like macrophages in vitro. A) Schematic diagram of the induction and identification of M2‐like macrophages. B) FACS analysis of IL‐4‐induced (50 ng mL⁻¹ for 48 h) M2‐like macrophages after incubation with anti‐F4/80 and anti‐CD206 antibodies. C) Dose‐dependent cellular uptake of nanoparticles in M2‐like macrophages assessed by CLSM (left panel) and FACS (right panel). M2‐like macrophages were treated with various concentrations of CRV‐LLCM/DS‐PLGA/DiD (CRV‐LLCM/DS‐PLGA/DiD equivalent to 60, 120, 240, and 480 µg mL⁻¹, respectively) for 9 h. D) Time‐dependent cellular uptake of nanoparticles in M2‐like macrophages assessed by CLSM (left panel) and FACS (right panel). LLC cells were treated with CRV‐LLCM/DS‐PLGA/DiD (240 µg mL⁻¹) for 3, 6, 9, and 12 h. E) Cell viability of M2‐like macrophages. F) Live/Dead analysis of M2‐like macrophages. G) Annexin V‐FITC/ propidium iodide (PI) apoptosis of M2‐like macrophages. H) Quantification of apoptotic rate (%) of M2‐like macrophages. To evaluate the cell viability, Live/Dead analysis, and apoptosis, M2‐like macrophages were treated with different formulations (PBS, CLDP, dBET6, DPB, LDPB, or CREATE, respectively) for 9 h, and incubated with the freshly prepared medium for another 15 h (n = 3). Data are presented as mean ± SD. **** p < 0.0001.

Figure 5

Biological effects induced by different formulations on M2/LLC spheroids. A) Schematic diagram of M2/LLC spheroids formulation and treatment. B) Verification of M2/LLC spheroids using antibodies against TTF1 and CD206. C) Cellular uptake of DS‐PLGA/DiD, LLCM/DS‐PLGA/DiD, or CRV‐LLCM/DS‐PLGA/DiD in M2/LLC spheroids. The M2/LLC spheroids were treated with the indicated formulations (DS‐PLGA/DiD equivalent to 60 µg mL⁻¹; LLCM/DS‐PLGA/DiD, or CRV‐LLCM/DS‐PLGA/DiD equivalent to 240 µg mL⁻¹) for 9 h. The M2/LLC spheroid was conducted with Z‐stack scanning with the distance between slices of 20.35 m. D) Live/Dead assay analyzing M2/LLC spheroids treated with different formulations (PBS, CLDP, dBET6, DPB, LDPB, or CREATE, respectively) for 9 h, and incubated with the freshly prepared medium for another 15 h (n = 3). The spheroids were conducted with Z‐stack scanning with the distance between slices of 26.12 μm.

Figure 6

Biodistribution and circulation lifetime of different formulations in vivo. A) Fluorescence imaging of the biodistribution of different formulations (Saline, DiR, DS‐PLGA/DiR, LLCM/DS‐PLGA/DiR, and CRV‐LLCM/DS‐PLGA/DiR, respectively) in LLC‐allograft mice at a series of time points. B) Circulation lifetimes of different formulations (Saline, DiR, DS‐PLGA/DiR, LLCM/DS‐PLGA/DiR, and CRV‐LLCM/DS‐PLGA/DiR, respectively) at the indicated times. C) Quantitative biodistribution analysis of DiR‐labeled nanoparticles in major organs and tumors.

Figure 7

Inhibitory effects of different formulations in vivo. A) Schematic illustrating the in vivo therapeutic approach in the LLC tumor‐bearing mice. B) Gross morphology of tumors after intravenously inoculated with Saline, CLDP, dBET6, DPB, LDPB, and CREATE at the time of sacrifice (n = 5). C) Changes of relative tumor volumes detected at the indicated times. D) Changes in relative body weight detected at the indicated times. E) Representative images of HE and TUNEL staining of tumor sections treated with Saline, CLDP, dBET6, DPB, LDPB, and CREATE, respectively. Data are presented as mean ± SD. **** p < 0.0001.

Figure 8

Effects of different formulations on the tumor microenvironment. A) Representative immunofluorescence staining of CD206⁺ and TTF1⁺ cells in the tumor tissues treated with PBS, CLDP, dBET6, DPB, LDPB, and CREATE. B) Representative immunofluorescence images of CD4⁺ and CD8⁺ T cell infiltration in the tumor tissues treated with Saline, CLDP, dBET6, DPB, LDPB, and CREATE. C) Representative immunofluorescence images of α‐SMA⁺ and CD31⁺ tumor blood vessels in the tumor tissues treated as above.