Dayson Moreira

Leukemia cell–targeted STAT3 silencing and TLR9 triggering generate systemic antitumor immunity

Signal transducer and activator of transcription 3 (STAT3) is an oncogene and immune checkpoint commonly activated in cancer cells and in tumor-associated immune cells. We previously developed an immunostimulatory strategy based on targeted Stat3 silencing in Toll-like receptor 9 (TLR9)-positive hematopoietic cells using CpG-small interfering RNA (siRNA) conjugates. Here, we assessed the therapeutic effect of systemic STAT3 blocking/TLR9 triggering in disseminated acute myeloid leukemia (AML). We used mouse Cbfb-MYH11/Mpl-induced leukemia model, which mimics human inv(16) AML. Our results demonstrate that intravenously delivered CpG-Stat3 siRNA, but not control oligonucleotides, can eradicate established AML and impair leukemia-initiating potential. These antitumor effects require hosts effector T cells but not TLR9-positive antigen-presenting cells. Instead, CpG-Stat3 siRNA has direct immunogenic effect on AML cells in vivo upregulating major histocompatibility complex class-II, costimulatory and proinflammatory mediators, such as interleukin-12, while downregulating coinhibitory PD-L1 molecule. Systemic injections of CpG-Stat3 siRNA generate potent tumor antigen-specific immune responses, increase the ratio of tumor-infiltrating CD8(+) T cells to regulatory T cells in various organs, and result in CD8(+) T-cell-dependent regression of leukemia. Our findings underscore the potential of using targeted STAT3 inhibition/TLR9 triggering to break tumor tolerance and induce immunity against AML and potentially other TLR9-positive blood cancers.

Myeloid cells as a target for oligonucleotide therapeutics: turning obstacles into opportunities

Immunotherapies emerged as an alternative for cancer treatment, yet their clinical efficacies are still limited, especially in case of solid tumors. Myeloid immune cells, such as macrophages and myeloid-derived suppressor cells (MDSCs), are often hijacked by tumors and become pivotal inhibitors of antitumor immunity. Immunosuppressive functions of tumor-associated myeloid cells result from the activity of Signal Transducer and Activator of Transcription 3 (STAT3), a transcription factor with well-defined tumorigenic and tolerogenic roles in human cancers. To overcome challenges in the development of pharmacological STAT3 inhibitors, we recently developed oligonucleotide-based strategies for cell-selective, in vivo STAT3 targeting. Conjugation of a STAT3siRNA or decoy STAT3 inhibitors to synthetic Toll-like Receptor 9 (TLR9) agonists, CpG oligonucleotides, allowed for selective delivery into TLR9-positive cells. Cellular target for CpG-STAT3 inhibitors include non-malignant, tumor-associated myeloid cells, such as polymorphonuclear MDSCs, as well as cancer cells in acute myeloid leukemia, B cell lymphoma and in certain solid tumors. The chemically modified CpG-STAT3 inhibitors resist serum nucleases and thus can be administered intravenously. Their potency relies on the intracellular gain-of-function effect: release of the central immune checkpoint regulator (STAT3) to unleash proinflammatory signaling (CpG/TLR9) in the same antigen-presenting cell. At the cellular level, CpG-STAT3 inhibitors exert two-pronged effect by rescuing T cells from the immune checkpoint control while decreasing survival of cancer cells. In this article, we review the preclinical data on CpG-STAT3 inhibitors and discuss perspectives of using TLR9-targeted delivery of oligonucleotide therapeutics for the generation of novel, more effective and safer cancer immunotherapies.

TLR9 expression and secretion of LIF by prostate cancer cells stimulates accumulation and activity of polymorphonuclear MDSCs

Proinflammatory signals promote prostate tumorigenesis and progression, but their origins and downstream effects remain unclear. We recently demonstrated that the expression of an innate immune receptor, TLR9, by prostate cancer cells is critical for their tumor‐propagating potential. We investigated whether cancer cell–intrinsic TLR9 signaling alters composition of the prostate tumor microenvironment. We generated Ras/Myc (RM9) and Myc‐driven (Myc‐CaP) prostate cancer cells expressing the tetracycline‐inducible gene Tlr9 (Tlr9ON) or the control LacZ (LacZON). When engrafted into mice and treated with tetracycline, Tlr9ON, but not LacZON, tumors showed accelerated growth kinetics compared with tumors in PBS‐treated mice. Tlr9 upregulation in cancer cells triggered the selective accumulation of CD11b+Ly6GHILy6CLO myeloid cells, phenotypically similar to PMN‐MDSCs. The PMN‐MDSCs from tetracycline‐treated RM9‐Tlr9ON tumors increased the immunosuppressive activity of the STAT3 transcription factor, thereby more potently inhibiting T cell proliferation. We identified LIF, an IL‐6‐type cytokine and STAT3 activator, as a potential mediator of crosstalk between TLR9‐expressing prostate cancer cells and PMN‐MDSCs. Antibody‐mediated LIF neutralization reduced the percentage of tumor‐infiltrating PMN‐MDSCs and inhibited tumor growth in mice. The clinical relevance of LIF is confirmed by the correlation between TLR9 and LIF expression in prostate cancer specimens. Furthermore, blood samples from patients with prostate cancer showed elevated levels of LIF and high LIFR expression on circulating PMN‐MDSCs. Our results suggest that TLR9+ prostate cancers promote immune evasion via LIF‐mediated expansion and activation of PMN‐MDSCs. Finally, targeting TLR9/LIF/STAT3 signaling using oligonucleotide‐based inhibitors, such as CpG‐STAT3dODN, can offer new opportunities for prostate cancer immunotherapy.

B-cell lymphoma immunotherapy using TLR9-targeted oligonucleotide STAT3 inhibitors

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.

261. Gain-of-Function Effect Augments Therapeutic Efficacy of CpG-STAT3 Anti-Sense Oligonucleotide Against Castration-Resistant Prostate Cancers

The treatment for castration-resistant prostate cancers (CRPC) remains a clinical challenge. STAT3 expression level correlates with poor prognosis in prostate cancer patients and resistance to hormone therapy. In addition to oncogenic role of STAT3 in cancer cells, it is also commonly activated in the prostate tumor microenvironment. Therefore, STAT3 is a promising target targeting STAT3 is expected to generate dual effects; anti-tumor activity and anti-immunosuppression in CRPC. Targeting transcriptions factors for cancer therapy has not been successful due to the lack of enzymatic activity. Recent advances in oligonucleotide-based therapy using anti-sense oligonucleotide (ASO) targeting STAT3 had shown promising results in phase I clinical trials in various human cancers, including liver metastasis. However, the limitation fo these reagents remained targeting We previously demonstrated that Toll-like Receptor 9 (TLR9) is up-regulated in prostate cancer stem-like cells in the late-stage prostate cancer. STAT3 signaling is activated in TLR9+ prostate cancer cells and tumor associated immune cells. Here, we describe a new molecule that enables specific delivery of STAT3 ASO to TLR9+ tumor and immune by linking STAT3 ASO to CpG ODN, TLR9 ligand. CpG-STAT3 ASO was quickly and efficiently internalized by human and mouse prostate cancer cell lines showing cytoplasmic localization after 15 minutes after incubation in vitro. Human and mouse TLR9+ immune cells; DCs, Macrophages and B cells were able to efficiently uptake CpG-STAT3 ASO in vitro. In a biodistribution experiment, CpG-STAT3 ASOCy3 was effectively internalized by TLR9+ myeloid cells in various organs including lymph node and bone marrow. Treatment with CpG-STAT3 ASO conjugates in vitro decreased the expression of STAT3 in TLR9+ human (DU145 and LNCaP-TLR9) and mouse (RM1 and RM9) prostate cancer cells more efficiently than unconjugated STAT3 ASO. Notably, CpG-STAT3 ASO showed significantly augmented cytotoxicity in prostate cancer cells compared to STAT3 ASO. In a preliminary anti-tumor efficacy study using syngeneic mouse model, repeated intra-tumoral injections of CpG-STAT3 ASO reduced the tumor growth of RM9. In contrast, the inhibitory effect of STAT3 ASO was transient leading to tumor relapse after the initial treatments. Noteworthy, CpG-STAT3 ASO reduced tumor growth and STAT3 expression in the untreated tumors in the contra-lateral site. Moreover, CpG-STAT3 ASO, but not the unconjugated STAT3 ASO, reduced activation of STAT3 in infiltrated myeloid-derived suppressor cells (MDSC) (CD11b+Gr1+) in both tumors and the percentage of T reg (CD4+FoxP3+) cells in the tumor draining lymph nodes. These results implicate that only when combined with concomitant TLR9 stimulation STAT3 inhibition can systemically overcome tumor immune tolerance. Overall, these results suggest that CpG-STAT3 ASO conjugate holds a great promise for an anti-cancer therapy of CRPC by simultaneously targeting STAT3 signaling in TLR9+ prostate cancer cells and tumor-associated immune cells.

698. Eliminating TLR9+ Prostate Cancer Stem Cells In Vivo Using NF-kB/RELA- or STAT3-Targeting CpG-siRNA Conjugates

Treatment of hormone-refractory and frequently reoccurring prostate cancers is a major clinical challenge. Here, we demonstrate that Toll-like Receptor 9 (TLR9) is commonly upregulated in late-stage prostate cancers and provides a potential therapeutic target. As a sensor of immunogenic cell death, TLR9 bridges intra-prostatic inflammation to cancer stem cell phenotype. Our limited dilution/serial transplantation experiments in vivo demonstrate that TLR9 is essential for prostate cancer cells’ potential to propagate and self-renew. In addition, low expression or silencing of TLR9 limits the clonogenic potential and mesenchymal stem cell-like properties of prostate cancer cells. Genome-wide transcriptional analysis of cancer cells isolated from xenotransplanted prostate tumors revealed a unique TLR9-dependent gene expression signature. Further analysis of the TLR9 downstream signaling indicated that tumorigenic transcription factors NF-kB/RELA and STAT3 cooperate to orchestrate expression of stem cell-related genes. Both RELA and STAT3 bound and co-regulated promoters of NKX3.1 and KLF4 prostate cancer stem cell-related genes. We further demonstrated the feasibility of targeting prostate cancer-propagating potential in vivo by TLR9-targeted siRNA delivery using CpG-siRNA conjugates. Local administration of CpG-RELAsiRNA or CpG-STAT3siRNA but not control conjugates, inhibited tumor growth and cancer cell clonogenic potential in two xenotransplanted prostate cancer models. Selective elimination of tumor-propagating cells using TLR9-targeted blockade of NF-?B/RELA and STAT3 signaling has potential for clinical translation to benefit patients with late-stage prostate cancers.

216. TLR9-Targeted STAT3 Silencing Abrogates Immunosuppressive Activity of Myeloid-Derived Suppressor Cells from Prostate Cancer Patients

Recent advances in immunotherapy of advanced human cancers underscored the need to eliminate tumor-induced immunosuppression. The myeloid-derived suppressor cells (MDSCs) are important negative regulators of T cell responses in advanced solid tumors, such as prostate cancers. Targeting MDSCs proved challenging due to heterogeneity and phenotypic similarities between myeloid cell lineages. Here, we identify a population of Lin–CD15HICD33LO granulocytic MDSCs that accumulate in patients’ blood during prostate cancer progression from localized to metastatic disease. The prostate cancer-associated MDSCs potently inhibit autologous CD8+ T cell proliferation, IFNg and Granzyme-B production. The circulating MDSCs have high levels of activated STAT3, a central immune checkpoint regulator. As shown by immunofluorescent microscopy, the CD15+/pSTAT3+ cells are also important component of immune infiltrate in primary tumor and tumor-draining lymph node tissues in these patients. We previously generated an original strategy to silence genes specifically in TLR9+ cells using naked CpG-siRNA conjugates. Here, we demonstrate that the CpG-STAT3siRNA is quickly internalized by human MDSCs effectively reducing STAT3 expression. The TLR9-dependent STAT3 blocking abrogates immunosuppressive effects of patients-derived MDSCs on CD8+ T cells. Our further studies demonstrated that these effects resulted from reduced expression and enzymatic activity of Arginase-1, a downstream STAT3 target gene and T cell inhibitor. Overall, we demonstrate the feasibility of using targeted STAT3 blockade to alleviate MDSC-mediated immunosuppression without depletion of myeloid cells in prostate cancer patients. We anticipate that further development of this oligonucleotide strategy will generate safer and more effective immunotherapy for advanced prostate cancers and potentially other solid tumors.