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.

The promising results of clinical trials using immune checkpoint inhibitors revived interests in cancer immunotherapy. However, it also became apparent that efficacy of immune checkpoint blockade can benefit from combining it with immunostimulatory strategies. Here, we review prior and re-emerging approaches using Toll-like Receptor 9 (TLR9) agonists, CpG oligodeoxynucleotides (ODNs), focused on the generation of antitumor immune responses in cancer patients. While numerous early clinical trials using TLR9 ligands in monotherapies provided evidence of CpG ODNs tolerability and safety, they failed to demonstrate sufficient antitumor efficacy. Recent studies unraveled multiple levels of negative regulation of immunostimulatory TLR9 signaling in immune cells by the tumor microenvironment that can stifle immune activity in cancer patients. Therefore, CpG ODNs-based strategies can greatly benefit from combination with strategies targeting immune checkpoint regulation. The most recent clinical trials of CpG ODNs together with immune checkpoint inhibitors have a chance to generate novel, more effective and safer cancer immunotherapies.

Rhabdomyosarcoma (RMS) is a mesenchymal tumor of soft tissue in children that originates from a myogenic differentiation defect. Expression of SNAIL transcription factor is elevated in the alveolar subtype of RMS (ARMS), characterized by a low myogenic differentiation status and high aggressiveness. In RMS patients SNAIL level increases with higher stage. Moreover, SNAIL level negatively correlates with MYF5 expression. The differentiation of human ARMS cells diminishes SNAIL level. SNAIL silencing in ARMS cells inhibits proliferation and induces differentiation in vitro, and thereby completely abolishes the growth of human ARMS xenotransplants in vivo. SNAIL silencing induces myogenic differentiation by upregulation of myogenic factors and muscle-specific microRNAs, such as miR-206. SNAIL binds to the MYF5 promoter suppressing its expression. SNAIL displaces MYOD from E-box sequences (CANNTG) that are associated with genes expressed during differentiation and G/C rich in their central dinucleotides. SNAIL silencing allows the re-expression of MYF5 and canonical MYOD binding, promoting ARMS cell myogenic differentiation. In differentiating ARMS cells SNAIL forms repressive complex with histone deacetylates 1 and 2 (HDAC1/2) and regulates their expression. Accordingly, in human myoblasts SNAIL silencing induces differentiation by upregulation of myogenic factors. Our data clearly point to SNAIL as a key regulator of myogenic differentiation and a new promising target for future ARMS therapies.

NF-κB is a key regulator of inflammation and cancer progression, with an important role in leukemogenesis. Despite its therapeutic potential, targeting NF-κB using pharmacologic inhibitors has proven challenging. Here, we describe a myeloid cell–selective NF-κB inhibitor using an miR-146a mimic oligonucleotide conjugated to a scavenger receptor/Toll-like receptor 9 agonist (C-miR146a). Unlike an unconjugated miR146a, C-miR146a was rapidly internalized and delivered to the cytoplasm of target myeloid cells and leukemic cells. C-miR146a reduced expression of classic miR-146a targets (IRAK1 and TRAF6), thereby blocking activation of NF-κB in target cells. IV injections of C-miR146a mimic to miR-146a–deficient mice prevented excessive NF-κB activation in myeloid cells, and thus alleviated myeloproliferation and mice hypersensitivity to bacterial challenge. Importantly, C-miR146a showed efficacy in dampening severe inflammation in clinically relevant models of chimeric antigen receptor (CAR) T-cell–induced cytokine release syndrome. Systemic administration of C-miR146a oligonucleotide alleviated human monocyte-dependent release of IL-1 and IL-6 in a xenotransplanted B-cell lymphoma model without affecting CD19-specific CAR T-cell antitumor activity. Beyond anti-inflammatory functions, miR-146a is a known tumor suppressor commonly deleted or expressed at reduced levels in human myeloid leukemia. Using The Cancer Genome Atlas acute myeloid leukemia data set, we found an inverse correlation of miR-146a levels with NF-κB–related genes and with patient survival. Correspondingly, C-miR146a induced cytotoxic effects in human MDSL, HL-60, and MV4-11 leukemia cells in vitro. The repeated IV administration of C-miR146a inhibited expression of NF-κB target genes and thereby thwarted progression of disseminated HL-60 leukemia. Our results show the potential of using myeloid cell–targeted miR-146a mimics for the treatment of inflammatory and myeloproliferative disorders.

Rhabdomyosarcoma (RMS) is a soft tissue sarcoma, which may originate from impaired differentiation of mesenchymal stem cells (MSC). Expression of MET receptor is elevated in alveolar RMS subtype (ARMS) which is associated with worse prognosis, compared to embryonal RMS (ERMS). Forced differentiation of ARMS cells diminishes MET level and, as shown previously, MET silencing induces differentiation of ARMS. In ERMS cells introduction of TPR-MET oncogene leads to an uncontrolled overstimulation of the MET receptor downstream signaling pathways. In vivo, tumors formed by those cells in NOD-SCID mice display inhibited differentiation, enhanced proliferation, diminished apoptosis and increased infiltration of neutrophils. Consequently, tumors grow significantly faster and they display enhanced ability to metastasize to lungs and to vascularize due to elevated VEGF, MMP9 and miR-378 expression. In vitro, TPR-MET ERMS cells display enhanced migration, chemotaxis and invasion toward HGF and SDF-1. Introduction of TPR-MET into MSC increases survival and may induce expression of early myogenic factors depending on the genetic background, and it blocks terminal differentiation of skeletal myoblasts. To conclude, our results suggest that activation of MET signaling may cause defects in myogenic differentiation leading to rhabdomyosarcoma development and progression.

Tropism of neural stem cells (NSCs) to hypoxic tumor areas provides an opportunity for the drug delivery. Here, we demonstrate that NSCs effectively transport antisense oligonucleotides (ASOs) targeting oncogenic and tolerogenic signal transducer and activator of transcription 3 (STAT3) protein into glioma microenvironment. To enable spontaneous, scavenger receptor-mediated endocytosis by NSCs, we used previously described CpG-STAT3ASO conjugates. Following uptake and endosomal escape, CpG-STAT3ASO colocalized with CD63 + vesicles and later with CD63 + CD81 + exosomes. Over 3 days, NSCs secreted exosomes loaded up to 80% with CpG-STAT3ASO. Compared to native NSC exosomes, the CpG-STAT3ASO-loaded exosomes potently stimulated immune activity of human dendritic cells or mouse macrophages, inducing nuclear factor kB (NF-kB) signaling and interleukin-12 (IL-12) production. Using orthotopic GL261 tumors, we confirmed that NSC-mediated delivery improved oligonucleotide transfer from a distant injection site into the glioma microenvironment versus naked oligonucleotides. Correspondingly, the NSC-delivered CpG-STAT3ASO enhanced activation of glioma-associated microglia. Finally, we demonstrated that NSC-mediated CpG-STAT3ASO delivery resulted in enhanced antitumor effects against GL261 glioma in mice. Peritumoral injections of 5 Â 10 5 NSCs loaded ex vivo with CpG-STAT3ASO inhibited subcutaneous tumor growth more effectively than the equivalent amount of oligonucleotide alone. Based on these results, we anticipate that NSCs and NSC-derived exosomes will provide a clinically relevant strategy to improve delivery and safety of oligonucleotide therapeutics for glioma treatment.

Growing evidence links the aggressiveness of non-Hodgkin's lymphoma, especially the activated B cell-like type diffuse large B cell lymphomas (ABC-DLBCLs) to Toll-like receptor 9 (TLR9)/MyD88 and STAT3 transcription factor signaling. Here, we describe a dual-function molecule consisting of a clinically relevant TLR9 agonist (CpG7909) and a STAT3 inhibitor in the form of a high-affinity decoy oligodeoxynucleotide (dODN). The CpG-STAT3dODN blocked STAT3 DNA binding and activity, thus reducing expression of downstream target genes, such as MYC and BCL2L1, in human and mouse lymphoma cells. We further demonstrated that injections (i.v.) of CpG-STAT3dODN inhibited growth of human OCI-Ly3 lymphoma in immunodeficient mice. Moreover, systemic CpG-STAT3dODN administration induced complete regression of the syngeneic A20 lymphoma, resulting in long-term survival of immunocompetent mice. Both TLR9 stimulation and concurrent STAT3 inhibition were critical for immune-mediated therapeutic effects, since neither CpG7909 alone nor CpG7909 co-injected with unconjugated STAT3dODN extended mouse survival. The CpG-STAT3dODN induced expression of genes critical to antigen-processing/presentation and Th1 cell activation while suppressing survival signaling. These effects resulted in the generation of lymphoma cell-specific CD8/CD4-dependent T cell immunity protecting mice from tumor rechallenge. Our results suggest that CpG-STAT3dODN as a systemic/local monotherapy or in combination with PD1 blockade can provide an opportunity for treating patients with B cell NHL.

Both the extracellular matrix (ECM) and DNA epigenetic regulation are critical for maintaining stem cell phenotype and cancer progression. Whether and how ECM regulates epigenetic alterations to influence cancer stem cells (CSCs) remain to be explored. Here we report that ECM through laminin-integrin α6 upregulates ten-eleven translocation enzyme 3 (TET3) dioxygenase. TET3 in turn mediates DNA cytosine 5'-hydroxymethylation (5hmC) and upregulates genes critical for maintenance of glioma stem cells (GSCs). Activating integrin α6-FAK pathway increases STAT3 activity, TET3 expression and 5hmC levels in GSCs. Moreover, targeting STAT3 disrupts integrin α6-FAK signaling and inhibits TET3 + GSC maturation in vivo. STAT3 directly regulates TET3 expression and the two proteins are co-localized with 5hmC in GSC clusters. 5hmC is upregulated by STAT3 at the promoters of several tumorigenic genes, including c-Myc, known to be critical for GSCs. In vivo silencing of TET3 in GSC-enriched tumors reduces 5hmC accumulation and expression of the GSC critical genes, leading to tumor growth inhibition. TET3 Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: