Low dose novel PARP-PI3K inhibition via nanoformulation improves colorectal cancer immunoradiotherapy

Abstract

Multimodal therapy is often used in oncology to overcome dosing limitations and chemoresistance. Recently, combination immunoradiotherapy has shown great promise in a select subset of patients with colorectal cancer (CRC). Furthermore, molecularly targeted agents delivered in tandem with immunotherapy regimens have been suggested to improve treatment outcomes and expand the population of responding patients. In this study, radiation-sensitizing small molecules niraparib (PARP inhibitor) and HS-173 (PI3K inhibitor) are identified as a novel combination that synergistically enhance toxicity and induce immunogenic cell death both in vitro and in vivo in a CRC model. These inhibitors were co-encapsulated in a polymer micelle to overcome solubility limitations while minimizing off-target toxicity. Mice bearing syngeneic colorectal tumors (CT26) were administered these therapeutic micelles in combination with X-ray irradiation and anti-CTLA-4 immunotherapy. This combination led to enhanced efficacy demonstrated by improved tumor control and increased tumor infiltrating lymphocytes. This report represents the first investigation of DNA damage repair inhibition combined with radiation to potentiate anti-CTLA-4 immunotherapy in a CRC model.

Keywords: Drug delivery; Immune checkpoint blockade; Nanomedicine; Poly(2-oxazoline); Radiation therapy.

Graphical abstract

Fig. 1

Evaluation of cell viability and drug synergy. (A) Chemical structures of PARP and PI3K inhibitors (B)In vitro toxicity of free drug combination treatment with () and without () radiation. Radiation only controls are shown with a dotted line. (C) Combination index (CI) values with radiation were calculated and plotted to evaluate the synergy of the combination with radiation. CI > 1 represents antagonism, CI = 1 represents additivity, and CI > 1 represents synergy. The dashed line is for reference at y = 1. The full screen combination data are in Fig. S1. The N and H combination was chosen due to data in both (B) and (C).

Fig. 2

Immunogenic cell death induction in vitro and in vivo. (A) Visualization of immunogenic cell death by surface (wheat germ albumin membrane stain) exposure of calreticulin. (B) Quantified calreticulin expression for each treatment group from images in (A). ∗ denotes significance (p < 0.05) (C) Macroscopic view of DMSO-treated tumor demonstrates dense tumor tissue (purple) with a small necrotic region (pink). (D) Microscopic view of rectangle in (C) which demonstrates the accumulation of leukocytes (purple specks) at the necrotic region (pink) periphery with minimal infiltration. (E) Macroscopic view of N-H treated tumor demonstrates less dense tumor tissue (purple) and a larger necrotic region (pink). (F) Microscopic view of (E) demonstrates a larger leukocyte (purple specks) influx to the necrotic region (pink). le represents the leading edge of immune infiltration, and nc represents the necrotic region. Arrows represent leukocytes and arrowheads represent nuclear fragments. Scale bars represent 1000 μm (black) and 100 μm (white).

Fig. 3

Particle characterization. (A) Schematic representation of formulation method showing structure of drugs, polymers, and micelles. (B) Intensity average DLS measurements for E and N-H POx. (C) Negative stain TEM image of N-H POx demonstrates a particle size of about 14 nm. Scale bar represents 100 nm. (D)In vitro toxicity of free N-H compared with E and N-H POx, with (open symbols) and without radiation (closed symbols). (E) Drug release profile for N-H POx under sink conditions at pH 7.4 PBS as determined by HPLC.

Fig. 4

In vitro radiation and N-H combination damage. (A) Representative γH2AX images demonstrate the difference in number of foci and overall signal intensity for various treatments with and without radiation. Scale bar represents 100 μm. (B) Quantification of γH2AX puncti per cell from images in (A). (C) Histograms of treated samples analyzed by flow cytometry show shift in γH2AX intensity. (D) Clonogenic assay demonstrates the radiosensitizing potential of N-H POx over free N-H or E POx at various radiation doses. Curves are fit using the linear quadratic equation. No colonies formed in the N-H POx treatment group at 6 gray.

Fig. 5

In vivo efficacy and survival studies.(A) Dosing schedule for both the efficacy and survival studies. (B) Relative tumor volume curves for mice in efficacy study treated with various therapeutic agents after treatment regimen in (A). (C) Polymer carrier (E POx) and N-H POx–treated tumors from day 14 stained with H&E, proliferation (Ki67 in brown), and apoptosis (CC3 in brown). Scale bar (100 μm) is representative for all images. (D) Relative tumor volume for α-CTLA-4 treatment groups from survival study. Curve ends when first mouse in treatment group reaches the endpoint. (E) Kaplan-Meier survival curve for α-CTLA-4 treatment groups with differences in survival calculated in accordance with the logrank test.

Fig. 6

Alternative α-CTLA-4 dosing study.(A) Treatment scheme similar to Fig. 5A with altered α-CTLA-4 dosing time. (B) Relative tumor volume curves for uncured mice responding to treatment (n = 3), ∗ denotes p < 0.016. (C) Individual growth curves for all (cured and uncured) E POx + IR + α-CTLA-4 treated mice. (D) Individual growth curves for all (cured and uncured) N-H POx + IR + α-CTLA-4–treated mice. The dotted curve represents a non-responding mouse. Responding, uncured mice in (D) show delayed growth versus responding, uncured mice in (C).

Fig. 7

In vivo safety.(A) CBC results from tumored mice treated after therapeutic regimen in 5A. Counts in K/μL except red blood cells (RBC). (B) Clinical chemistry evaluation (BUN, total protein (TP), ALT, AST) demonstrates the absence of organ-based toxicity for all treatment groups. ∗ denotes significance (p < 0.05) compared with the E POx control. (C) Histologic examination of H&E stained tissues (heart, lungs, liver, kidneys, and spleen) harvested from mice after therapeutic regimen in 5A further confirm the absence of treatment-based toxicity. Scale bar (200 μm) is representative for all images. Arrows indicate areas of hepatic vacuolization. CBC, complete blood count; ALT, alanine transaminase; AST, aspartate aminotransferase; BUN. blood urea nitrogen.