Dewan Md Sakib Hossain

TLR9-mediated siRNA delivery for targeting of normal and malignant human hematopoietic cells in vivo

STAT3 operates in both cancer cells and tumor-associated immune cells to promote cancer progression. As a transcription factor, it is a highly desirable but difficult target for pharmacologic inhibition. We have recently shown that the TLR9 agonists CpG oligonucleotides can be used for targeted siRNA delivery to mouse immune cells. In the present study, we demonstrate that a similar strategy allows for targeted gene silencing in both normal and malignant human TLR9(+) hematopoietic cells in vivo. We have developed new human cell-specific CpG(A)-STAT3 siRNA conjugates capable of inducing TLR9-dependent gene silencing and activation of primary immune cells such as myeloid dendritic cells, plasmacytoid dendritic cells, and B cells in vitro. TLR9 is also expressed by several human hematologic malignancies, including B-cell lymphoma, multiple myeloma, and acute myeloid leukemia. We further demonstrate that oncogenic proteins such as STAT3 or BCL-X(L) are effectively knocked down by specific CpG(A)-siRNAs in TLR9(+) hematologic tumor cells in vivo. Targeting survival signaling using CpG(A)-siRNAs inhibits the growth of several xenotransplanted multiple myeloma and acute myeloid leukemia tumors. CpG(A)-STAT3 siRNA is immunostimulatory and nontoxic for normal human leukocytes in vitro. The results of the present study show the potential of using tumoricidal/immunostimulatory CpG-siRNA oligonucleotides as a novel 2-pronged therapeutic strategy for hematologic malignancies.

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.

The aptamer-siRNA conjugates: reprogramming T cells for cancer therapy

Harnessing the body’s own immune system has the potential to generate diverse, yet highly specific antitumor responses with protective long-term effects. Recent clinical advances demonstrated that new cancer immunotherapies can achieve impressive therapeutic effects, even in patients with advanced tumors of various types [1]. The rapid progress in this area was enabled by our improved understanding of the mechanisms underlying tumor immune evasion. Cancers evolved multiple strategies to block the activity of T lymphocytes and adaptive immune responses. These include upregulation and the triggering of inhibitory receptors on CD8 T cells, such as CTLA-4 or PD-1, or direct induction of effector T-cell death, which altogether shift the balance of T-cell-mediated responses from cytotoxic T cells to Tregs [2,3]. Targeting of the CTLA-4 and PD-1 immune checkpoint pathways in T cells provided the first effective tools to disengage tumor defense systems [1]. Nevertheless, the antibody-mediated neutralization of critical immunoregulatory cell populations carries a potential risk of collateral damage or autoimmune manifestations [1]. Growing evidence suggests that targeting intracellular signaling and transcriptional regulators can provide safer methods to correct T-cell dysfunctions, such as Th2 or Treg bias [4]. Transcription factors, such as STAT3, FoxP3, SMAD2/3 or HIF-1a, are commonly activated in the tumor microenvironment and promote Treg functions [5–9]. Due to their intracellular localization and lack of enzymatic activity, the targeting of transcriptional regulators using antibodies or small-molecule drugs is challenging. Oligonucleotide-based therapeutics provide alternative strategies to target the expression of transcription factors (siRNA and antisense oligonucleotides) or their binding to target DNA sequences (decoy oligonucleotides) with high specificity. However, the broad application of oligonucleotide therapeutics is limited by the difficulty in efficient and cell-specific delivery. T cells resist the delivery of formulated oligonucleotides using conventional transfection methods. The delivery of oligonucleotides using viral vectors, which has been successfully employed in some T-cell-specific gene therapies, is less suitable for cancer therapy due to potential carcinogenic effects, broad tropism and difficulty in large-scale production [10].

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.

Systemic delivery of TLR9-activating/STAT3-blocking oligonucleotides induces leukemia regression

Meeting abstracts Inhibition of transcription factors (TF) that drive tumor progression and immune evasion, such as STAT3, remains a challenge for pharmacological drugs. Blocking STAT3 binding to DNA using specific decoy oligodeoxynucleotides (dODN) provides an alternative targeted inhibitory

Abstract CT334: Pazopanib as third-line therapy for metastatic renal cell carcinoma: Clinical efficacy and temporal analysis of cytokine profile

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Pazopanib was assessed in a phase III study conducted in patients (pts) with mRCC who were either cytokine-refractory or treatment-naive, and clinical outcomes with pazopanib have been associated with a specific immunologic profile (IP). The activity of pazopanib in the third-line setting and temporal changes in molecular profile during therapy are poorly understood, however. Methods: Eligibility was limited to pts with 2 prior lines of therapy (including at least 1 VEGF-directed therapy), ECOG PS 0-2, and clear cell histology. Pts received pazopanib 800 mg/daily on a 28d cycle. A Simon MinMax 2-stage design was employed, with 80% power of declaring an encouraging overall response rate (ORR) of 23% (type I error=10%). IPs were assessed monthly on a Luminex platform using the Human Cytokine 30-plex Cytokine Immunoassay (Invitrogen). Results: 28 pts were enrolled with a median age of 63 (range, 45-86). In the pre-specified intent-to-treat analysis, 12/28 pts (43%) had a confirmed response (1 CR, 11 PR), with 1 additional unconfirmed PR. Median progression-free survival for the cohort was 17.4 mos (95% CI 14.7-NR). No grade 4 treatment-related toxicities were observed. The most common grade 3 toxicities included hypertension (46%) and proteinuria (14%). Amongst patients still on therapy at 6 months and 12 months, responders had lower levels of HGF, VEGF, IL-6, IL-8 and soluble IL-2R (P<0.05 for each). Non-responders also showed increased numbers of myeloid-derived suppressor cells (MDSCs) at both time intervals. Phenotypic and functional studies confirmed that MDSCs from these mRCC patients were granulocytic. Conclusions: The ORR observed with pazopanib in the current study is the highest to date in a third-line trial in mRCC. Differences in cytokine profile and granulocytic MDSC quantity between responders and non-responders suggest that the mechanism of pazopanib resistance is at least partly related to generation of systemic tumor immune tolerance. Citation Format: Sumanta K. Pal, Dewan Md Sakib Hossain, Qifang Zhang, Chan Gao, Jeremy O. Jones, Paul H. Frankel, Robert A. Figlin, Marcin Kortylewski. Pazopanib as third-line therapy for metastatic renal cell carcinoma: Clinical efficacy and temporal analysis of cytokine profile. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT334. doi:10.1158/1538-7445.AM2014-CT334