STAT3 Inhibition Combined with CpG Immunostimulation Activates Antitumor Immunity to Eradicate Genetically Distinct Castration-Resistant Prostate Cancers

Abstract

Purpose: Prostate cancers show remarkable resistance to emerging immunotherapies, partly due to tolerogenic STAT3 signaling in tumor-associated myeloid cells. Here, we describe a novel strategy combining STAT3 inhibition with Toll-like Receptor-9 (TLR9) stimulation to unleash immune response against prostate cancers regardless of the genetic background. Experimental Design: We developed and validated a conjugate of the STAT3 antisense oligonucleotide (ASO) tethered to immunostimulatory TLR9 agonist (CpG oligonucleotide) to improve targeting of human and mouse prostate cancer and myeloid immune cells, such as myeloid-derived suppressor cells (MDSCs). Results: CpG-STAT3ASO conjugates showed improved biodistribution and potency of STAT3 knockdown in target cells in vitro and in vivo. Systemic administration of CpG-STAT3ASO (5mg/kg) eradicated bone-localized, Ras-/Myc-driven and Ptenpc−/−Smad4pc−/−Trp53c−/− prostate tumors in the majority of treated mice. These antitumor effects were primarily immune-mediated and correlated with an increased ratio of CD8+ to regulatory T-cells and reduced pSTAT3+/PD-L1+ MDSCs. Both innate and adaptive immunity contributed to systemic antitumor responses as verified by the depletion of Gr1+ myeloid cells and CD8+, CD4+ T-cells, respectively. Importantly, only the bi-functional CpG-STAT3ASO, but not control CpG oligonucleotides, STAT3ASO alone nor the co-injection of both oligonucleotides, succeeded in recruiting neutrophils and CD8+ T-cells into tumors. Thus, the concurrence of TLR9 activation with STAT3 inhibition in the same cellular compartment is indispensable for overcoming tumor immune tolerance and effective antitumor immunity against prostate cancer. Conclusions: The bi-functional, immunostimulatory and tolerance-breaking design of CpG-STAT3ASO offers a blueprint for the development of effective and safer oligonucleotide strategies for treatment of immunologically “cold” human cancers.

Structure and Function of the Nuclear Receptor Superfamily and Current Targeted Therapies of Prostate Cancer

“…However, Targeting of STAT3 has been challenging due to the essential role of STAT3 in normal physiological function [61] . Activation of STAT3 requires phosphorylation at Y705 and Y727 by Src kinase and MAP kinase, respectively [62] .…”

“…An innovative approach to STAT3 targeting is the use of CpG conjugated-STAT3 ASO, which is a bi-functional molecule that utilizes two processes to attack tumors [61] .…”

“…This molecule is able to activate the immune system and inactivate STAT3 expression [61] . The CpG motifs represent a synthetic modification of cysteine triphosphate and guanine triphosphate bound by a phosphodiester bond and is recognized by the pattern recognition receptor toll-like receptor 9 (TLR-9) expressed on myeloid-derived suppressor cells (MDSC) [61] . The STAT3-ASO component binds to complementary STAT3 mRNA strands, preventing translation of STAT3 [63,64] .…”

“…The use of CpG conjugated STAT3-ASO in the treatment of androgen-independent prostate cancer would provide a treatment option for a stage of prostate cancer that currently has no treatment. For the first time, CpG-STAT3 ASO has shown promise in reducing the tumor size of localized bone metastasis using in vivo models [61] .…”

“…Furthermore, the combination of STAT3 inhibitors with a selective target that allows only cells expressing TLR-9 (i.e. MDSC) to be affected, reduces off-target effects that would arise from STAT3 inhibition in other tissues [61] .…”