Activation Of Stimulator Of Interferon Genes Pathway Research Articles

Abstract LB-118: SR8541A is a potent inhibitor of ENPP1 and exhibits dendritic cell mediated antitumor activity

Abstract Purpose: In recent years, programs targeting the innate immune pathways alone and alongside the adaptive immune pathways have become increasingly popular. A main area of interest in innate immunity is the STING (STimulator of INterferon Genes) pathway. STING plays an integral role by activating type 1 interferons in response to detection of cytosolic nucleic acid by cGAS (Cyclic GMP-AMP Synthase). cGAS detects cytosolic DNA and converts it into 2′3′ cGAMP, which is the direct ligand of STING. Targeting the direct activation of this pathway using synthetic, non-hydrolyzable forms of this ligand has shown to be effective in preclinical efficacy models, but has failed to impress in phase 1 clinical trials. Another method of activating this pathway is to inhibit the direct negative regulator, ENPP1 (Ectonucleotide pyrophosphatase/phosphodiesterase 1). ENPP1 constitutively hydrolyzes 2′3′-cGAMP, suppressing the pathway both in the cancer cells and in the surrounding tumor microenvironment. Previously, we reported that SR-8314, a highly selective and potent ENPP1 inhibitor, induces the activation of the STING pathway via type I interferon response in both in vitro and in vivo models. In this study, we continue this research and present our nominated clinical candidate, SR-8541A, which shows improved physiochemical characteristics and increased immune response. Methods: Direct binding of SR-8541A to ENPP1 was evaluated using a thermal shift assay. Inhibition of ENPP1 enzymatic activity was shown using TMP or ATP as substrates in either cell free or cell based assays. RT-PCR, western blots, immune infiltration using peripheral blood mononuclear cells (PBMCs), and MesoScale Disovery (MSD) ELISA assays were performed to evaluate the effect of SR-8541A on the STING pathway. ENPP1 CRISPR knockout cells were generated to demonstrate on-target activity of SR-8541A. Pharmacokinetics, stability, and selectivity were completed with outside vendors. Results: SR-8541A exhibits strong binding to ENPP1 and shows inhibitory activity at an IC50 of 1.4 nM. STING pathway activation was confirmed by a significant increase in gene expression of IFNβ, ISG15 and CXCL10 in SR-8541A treated cells. An increase in secreted type 1 interferon and other cytokines was also observed in these treated cells. Using immune infiltration assays, we show that SR-8541A stimulates the migration and infiltration of immune cells (PBMC) into cancer spheroids. Importantly, depletion of dendritic cells results in loss of infiltration of remaining PBMCs into cancer spheroids. Additionally, natural killer (NK) cells do not infiltrate into SR-8541A treated cancer spheroids unless co-cultured with dendritic cells. ENPP1 CRISPR knockout cell models confirmed that the drug effect was dependent on the presence of ENPP1. SR-8541A was stable for >60 mins in liver microsomes and S9 fractions from various species. Rodent pharmacokinetics showed a strong oral bioavailability of ~50% and tumor efficacy studies are ongoing. Conclusions: In summary, we show that SR-8541A is a potent and selective inhibitor of ENPP1 that displayed strong immune response in 3D spheroid models. The results from these studies along with physiochemical properties and tumor model data will be presented. The promising physiochemical properties make it a strong candidate for clinical studies. Citation Format: ALEXIS S. WESTON, Trason G. Thode, Ryan Rodriguez del Villar, Srikanta Dana, Srinivas Kasibhatla, Mohan R. Kaadige, Sunil Sharma. SR8541A is a potent inhibitor of ENPP1 and exhibits dendritic cell mediated antitumor activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-118.

Abstract 3317: Discovery of novel STING agonists with robust anti-tumor activity

Abstract Introduction: Stimulator of interferon genes (STING) is an endoplasmic reticulum (ER) membrane signaling protein, which plays a key role in the sensing of cytoplasmic DNA arising from bacterial and viral infection, as well as self-DNA. Several studies have shown that STING signaling is essential for antitumor immunity by inducing the production of type I IFN and activating both innate and adaptive immunity. Therefore, activation of STING pathway in the tumor microenvironment could be a potential effective approach for cancer immunotherapy. Here, we present the characterization of novel STING agonists in a variety of in vitro cell lines and in vivo murine models. Methods: THP-1 monocyte cells and HEK 293-derived cells transfected with STING variants were used for cellular potency evaluation. Multiple murine tumor models were used for evaluation of in vivo efficacy. Tumor cells were inoculated into C57BL/6 mice and compounds were injected intratumorally three times at every three days when tumors reached a mean volume of approximately 100 mm3. Compounds-treated mice free from tumor for 30 days were re-challenged with MC38 tumor cells in MC38 murine model. Results: Several lead compounds CS-1018, CS-1020 and CS-1010 activate mouse and human STING variants in vitro with much better potency than natural ligand cGAMP and reference compound X. CS-1018 demonstrated dose dependent robust antitumor activity in B16F10 and MC38 mouse models. 3 out of 8 mice achieve complete responses at the dose of 100 ug in B16F10, 25% or 75% of mice were tumor free when treated with 5 ug or 25 ug of compound respectively in MC38. The survival mice from 5 ug and 25 ug groups remained tumor-free after re-challenged with another inoculation of MC38 cancer cells. The antitumor activity of compound CS-1020 and CS-1010 was also demonstrated in MC38 mouse model. When CS-1020 was dosed at 5 ug or 25 ug, complete responses were observed in 63% or 88% of mice respectively. In the case of CS-1010, the dose of 5 ug provides complete responses in 100% of treated mice. Similarly, both compounds induced immune-mediated tumor rejection when re-challenged with the same cancer cells. Conclusions: We have identified several STING agonists, which demonstrated better cellular potency to mouse and human STING alleles compared to natural ligand cGAMP and reference compound. All compounds showed dose dependent antitumor activity in MC38 or B16F10 syngeneic models, in addition, tumor-free mice after treatment of compounds developed tumor specific immunologic memory response in MC38 murine model. Combination studies of identified STING agonists with other IO agents are currently ongoing. The results will be discussed. Citation Format: Anrong Li, Yuntao Song, Chen Dong, Xiaoqi Chen, Junbao Yang. Discovery of novel STING agonists with robust anti-tumor activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3317.

Clinical outcomes of prexasertib monotherapy in recurrent BRCA wild-type high-grade serous ovarian cancer involve innate and adaptive immune responses

BackgroundPreclinical data suggest cell cycle checkpoint blockade may induce an immunostimulatory tumor microenvironment. However, it remains elusive whether immunomodulation occurs in the clinical setting. To test this, we used blood...

Abstract B78: Improving chemotherapy response of immunologically cold high-grade serous ovarian cancer with loss of PTEN using STING agonist

Abstract High-grade serous ovarian carcinoma (HGSC) is the most lethal gynecologic malignancy, with high rates of chemotherapy resistance and poor outcomes. We previously showed that tumors from chemotherapy-resistant patients show an immunologically cold phenotype, exhibiting lower density of CD8+ T cells and low expression of interferon genes. Subsequently, using the ID8-Trp53−/− murine model of HGSC, we further demonstrated the potential of Stimulator of Interferon Genes (STING) pathway activation in enhancing response of HGSC to carboplatin chemotherapy. Currently, we aim to better characterize the effect of cancer cell genotype on response to treatment in HGSC. The evolution of the tumor immune microenvironment (TIME) as immunologically hot or cold can be to some extent dictated by cancer cell-specific genetic alterations. While loss of the tumor suppressor TP53 is a universal mutation (>96% of cases), previous reports in HGSC have shown that patients with BRCA1 mutations (~25% of cases) have higher CD8+ T-cell infiltration and higher chemosensitivity. In contrast, loss of PTEN (seen in ~20% of cases) is associated with poor outcomes and chemoresistance. We hypothesized that HGSC tumors with loss of PTEN expression can benefit via immunomodulatory treatment approaches that activate the STING pathway following chemotherapy. C57/BL6 mice were implanted with either ID8-Trp53−/−; Brca1−/− cells or ID8-Trp53−/−; Pten−/− cells and subjected to one of three treatment groups: vehicle, carboplatin, or carboplatin + STING agonist. Characterization of the TIME generated from ID8-Trp53−/−; Brca1−/− cells, and those from ID8-Trp53−/−; Pten−/− cells, through local and systemic immune profiling, showed significant immunologic differences between different genotypes. Addition of STING agonist significantly increased chemosensitivity and improved overall response in ID8-Trp53−/−; Pten−/− injected mice compared to those treated with carboplatin alone. This study helps to determine the potential of STING pathway activation in inducing an activated tumor immune state in genotypes of HGSC that result in a “cold” TIME, such as loss of PTEN, to augment responses to chemotherapy and prolong survival. Citation Format: Noor Shakfa, Elizabeth Lightbody, Afrakoma Afriyie-Asante, Vinicius Kannen, Madhuri Koti. Improving chemotherapy response of immunologically cold high-grade serous ovarian cancer with loss of PTEN using STING agonist [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B78.

Combination of dual immune checkpoint inhibition (ICI) with stereotactic radiation (SBRT) in metastatic renal cell carcinoma (mRCC) (RADVAX RCC).

614 Background: Dual ICI with nivolumab/ipilimumab (N/I) has become a standard of care (SOC) for patients (pts) with mRCC. Since the failure to respond to ICI may be due to a lack of immune recognition, novel vaccination approaches and stimulation of the innate immune system (e.g. stimulator of interferon genes (STING) agonists) are being pursued in clinical trials. Because preclinical studies using SBRT have demonstrated the a.) release of tumor antigens, b.) STING pathway activation and c.) synergy with ICI, we have conducted a study to explore the safety and efficacy of this approach in mRCC pts. Methods: Pts with clear cell mRCC were screened and enrolled at two sites (UT Southwestern and Johns Hopkins). Prior treatment with tyrosine kinase inhibitors (TKI) and IL2 were allowed. Enrolled pts received standard of care dosing with Nivolumab (3 mg/kg) and Ipilimumab (1mg/kg) IV q3weeks followed by nivolumab monotherapy. SBRT was administered to 1-2 disease sites with a dose of 50 Gy in 5 fractions between the first and the second dose of N/I. The primary goals of this exploratory study were to determine the safety and tolerability as well as the objective response rate (ORR) by RECIST 1.1 of non-irradiated lesions. Results: A total of 29 pts were screened and 25 pts were enrolled. 11 (44%) of pts received at least one prior systemic therapy: 4 (16%) pts received IL2 and 7 (28%) TKI therapy. The cohort was primarily intermediate and poor risk pts (favorable risk n = 2 (8%), intermediate risk n = 20 (80%), poor risk n = 3 (12%)). 10 (40%) pts required immune suppressive therapy with prednisone for classic immune related adverse events as seen with dual ICI. Radiation pneumonitis limited to the radiation field (grade 2) was seen in 2 pts and responded promptly to oral steroids. At the time of analysis, partial responses (PR) were observed in 14/25 patients with an ORR of 56%. Additional analyses including duration of response, progression free survival and overall survival will be presented. Conclusions: Dual ICI with SBRT showed an acceptable safety and encouraging antitumor activity in mRCC warranting further investigations. Clinical trial information: NCT03065179.

STING Pathway Expression Identifies NSCLC With an Immune-Responsive Phenotype.

Although the combination of anti-programmed cell death-1 or anti-programmed cell death ligand-1 (PD-L1) with platinum chemotherapy is a standard of care for NSCLC, clinical responses vary. Even though predictive biomarkers (which include PD-L1 expression, tumor mutational burden, and inflamed immune microenvironment) are validated for immunotherapy, their relevance to chemoimmunotherapy combinations is less clear. We have recently reported that activation of the stimulator of interferon genes (STING) innate immune pathway enhances immunotherapy response in SCLC. Here, we hypothesize that STING pathway activation may predict and underlie predictive correlates of antitumor immunity in NSCLC. We analyzed transcriptomic and proteomic profiles in two NSCLC cohorts from our institution (treatment-naive patients in the Profiling of Resistance Patterns and Oncogenic Signaling Pathways in Evaluation of Cancers of the Thorax study and relapsed patients in the Biomarker-Integrated Approaches of Targeted Therapy for Lung CancerElimination study) and The Cancer Genome Atlas (N= 1320). Tumors were stratified by STING activation on the basis of protein or mRNA expression of cyclic GMP-AMPsynthase, phospho-STING, and STING-mediated chemokines (chemokine ligand 5 [CCL5] and C-X-C motif chemokine 10 [CXCL10]). STING activation in patient tumors and in platinum-treated preclinical NSCLC models was correlated with biomarkers of immunotherapy response. STING activation is associated with higher levels ofintrinsic DNA damage, targetable immune checkpoints, and chemokines in treatment-naive and relapsed lung adenocarcinoma. We observed that tumors with lower STING and immune gene expression show higher frequency of serine-threonine kinase 11 (STK11) mutations; however, we identified a subset of these tumors that are TP53 comutated and display high immune- and STING-related gene expression. Treatment with cisplatin increases STING pathway activation and PD-L1 expression in multiple NSCLC preclinical models, including adeno- and squamous cell carcinoma. STING pathway activation in NSCLC predicts features of immunotherapy response and is enhanced by cisplatin treatment. This suggests a possible predictive biomarker and mechanism for improved response to chemoimmunotherapy combinations.

Abstract 4983: Discovery and characterization of next-generation small molecule direct STING agonists

Abstract Accumulating evidence highlights an important role of type I interferon response in the immune surveillance mechanisms. IFNβ release by antigen-presenting cells promotes spontaneous anti-tumor CD8+ T cell priming being largely dependent on activation of Stimulator of Interferon Genes (STING). STING agonists promote regression of established tumors and generation of long-term immunologic memory in preclinical animal models. Herein we report the discovery of potent and selective, first-in-class non-nucleotide, non-macrocyclic, small molecule direct STING agonists with molecular weight below 500, structurally unrelated to known cyclic dinucleotide chemotypes with potential for systemic administration. Activation of STING pathway was monitored in THP-1 Dual reporter monocytic cell line as well as peripheral blood mononuclear cells (PBMC) or antigen presenting cells from human and mouse origin. Surface expression of the antigen-presenting cell maturation markers i.e. CD80, CD86, CD83 and HLA-DR was assessed by flow cytometry. Binding affinity was confirmed by three independent assays. RNA sequencing was performed on total RNA isolated from THP-1 cells and PBMC isolated from 2 healthy human donors. Direct binding to both mouse and human STING protein of Selvita agonists have been confirmed in biophysical binding assays (FTS, MST and FP) and by crystallography studies. The compounds have fine-tunable ADME properties with good solubility, permeability and human plasma stability. They selectively activates STING-dependent signaling in both THP-1 reporter assays and in primary cells of human and mouse origin. In addition, RNA sequencing data confirmed selectivity of the Selvita compounds. In vitro functional assays demonstrated their ability to induce cytokine responses (IFNβ, TNFα) in a panel of human peripheral blood mononuclear cell (PBMC), human monocyte derived macrophage (HMDM) and human dendritic cells samples with various STING haplotypes including refractory alleles. Additionally, the compounds efficiently induced cytokine release in mouse bone marrow-derived macrophages and dendritic cells. Pro-inflammatory cytokine profile was accompanied by up-regulation of the maturation markers, i.e. CD80, CD86, CD83 and HLA-DR, on the surface of human antigen presenting cells. These data demonstrate potent, novel, next-generation small molecule STING agonists activating STING-dependent signaling in both mouse and human immune cells to promote potential antitumor immunity. The compounds show good selectivity and in vitro ADME properties enabling further development for systemic administration as a single agent or in combinatory immunotherapies for cancer treatment. Citation Format: Monika Dobrzańska, Stefan Chmielewski, Magdalena Zawadzka, Jolanta Mazurek, Karolina Gluza, Katarzyna Wójcik-Jaszczyńska, Maciej Kujawa, Grzegorz Topolnicki, Grzegorz Ćwiertnia, Aleksandra Poczkaj, Izabela Dolata, Magdalena Mroczkowska, Agnieszka Gibas, Marcin Leś, Sylwia Sudoł, Adam Radzimierski, Kinga Michalik, Magdalena Sieprawska-Lupa, Katarzyna Banaszak, Katarzyna Wiklik, Federico Malusa, Michał Combik, Karolina Wiatrowska, Agnieszka Adamus, Lukasz Dudek, Jose Alvarez, Charles Fabritius, Anna Rajda, Maciej Rogacki, Faustyna Gajdosz, Peter Littlewood, Luigi Stasi, Krzysztof Brzózka. Discovery and characterization of next-generation small molecule direct STING agonists [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4983.

Covering the Cover

Covering the Cover

Abstract 1932: Discovery of ENPP1 inhibitors as agonists of STING pathway

Abstract The discovery of STING (Stimulator of interferon genes) pathway and the second messenger 2′3′-cGAMP that activate it opened up several new possibilities for the development of anti-cancer and anti-pathogen therapeutics. STING is a pattern-recognition receptor anchored on endoplasmic reticulum and has pivotal roles in surveillance and innate immune response. Binding of 2′3′-cGAMP with STING induces conformational changes, which subsequently leads to perinuclear migration of STING and recruitment of TANK-binding kinase 1 (TBK1) and IFN regulatory factor 3 (IRF3). TBK1 phosphorylates IRF3, which then translocates to the nucleus and drives the transcription of innate immune genes, including IFN-β. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is a dominant hydrolyzing enzyme of 2′3′-cGAMP. In ENPP-/- cells, 2′3′-cGAMP is more active in stimulating STING activation, and this is predicted to be due to increased half-life. Similarly, hydrolysis resistant 2′3′-cGAMP analogs are reported to be potent STING agonists. We hypothesized that inhibition of ENPP1 activity with an orally available small molecule would be a novel approach to activate STING pathway when compared with the cyclic dinucleotides (CDNs) due to their parenteral route of administration. We have utilized computational methods successfully discovered small molecule candidates that showed a strong binding affinity towards ENPP1. With the help of in vitro enzymatic inhibition and direct binding assays we have identified few lead candidates with an IC50 of <100 nM. Cellular assays further showed target specific inhibition of ENPP1 activity and subsequent activation of STING pathway. Additionally, our lead series of compounds showed no effect on other members of the ENPP family, a kinome panel, and several epigenetic regulators, suggesting the specificity towards ENPP1. In summary, we have developed small molecule inhibitors of ENPP1 and one of our lead candidate's is currently under investigation for ADME-Tox, PK and efficacy. The results from these studies will be presented. Citation Format: Sunil Sharma, Alexis Weston, Trason Thode, Eric Gomez, Mohan Kaadige, Hariprasad Vankayalapti. Discovery of ENPP1 inhibitors as agonists of STING pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1932.

Differences in Innate immune cell production of IFNα and IFNβ to cell activation in the Autoinflammatory Diseases CANDLE (Chronic Atypical Neutrophilic Dermatosis with Lipodystrophy and Elevated temperature) and SAVI (STING Associated Vasculopathy with Onset in Infancy)

Abstract The Type I IFN mediated autoinflammatory diseases SAVI and CANDLE are caused by gain-of-function mutations in Stimulator of Interferon Genes (STING) and loss-of-function mutations in proteasome components. We evaluated the innate immune cellular origin of IFN Type I in SAVI and CANDLE. We examined expression of IFN genes from FACS sorted T, B, NK and monocytes in SAVI (n=5) and CANDLE (n=3) normalized to healthy controls (HC). The Interquartile Range of Type I IFN expression was 78(4–151) and 481-fold(56-38,242) of IFNB1 and 60(3–116) and 0.4-fold(0.1–39) of IFNA7/IFNA17 in CANDLE and SAVI monocytes, respectively. Monocyte depletion reduced IFN message by 90% and FACS analysis confirmed that monocytes and not dendritic cells are the main source of IFNs. The overall mean ratio of IFNβ to IFNα was 989:1 in SAVI and 1.3:1 in CANDLE monocytes. Mean IFNα plasma levels measured by pan IFNα ELISA with 31pg/ml ±58 SEM in CANDLE and 230pg/ml ±112 SEM in SAVI. IP10 serum levels were 2215pg/ml ±702 SEM and 3632pg/ml ±860 SEM in CANDLE and SAVI, respectively. STING pathway activation with cGAMP in the presence of cycloheximide (CHX), a blocker of translational elongation, led to IFNB1 but not IFNA1, 2, 7, 17, 21 while CHX + epoxomicin, a proteasome inhibitor led to transcription of IFNAs and IFNB1 suggesting secondary upregulation of IFNAs in SAVI. Compared to healthy controls, CANDLE and SAVI had increased left-shifted immature CD13lo CD16+ neutrophils (9 and 4-fold, respectively), as well as higher expression of the activation marker CD36+ (6.9 and 6.9-fold, respectively) in total granulocytes. In conclusion, Type I IFN is mainly produced in monocytes and analyses of the IFN signaling in CANDLE and SAVI will allow us to develop improved targeted therapies.

Abstract A10: Activation of the STING pathway enhances immunity and improves survival in a murine myeloid leukemia model.

Abstract Type I interferon (IFN) production by innate immune cells is critical to prime spontaneous T cell responses against solid tumors. Until recently, the tumor-derived signals which stimulate host type I IFN production have remained elusive. However, emerging data suggest that tumor-derived DNA is sufficient to induce IFN-β production in responding cells through activation of a cytosolic DNA sensing receptor called STING (Stimulator of Interferon Genes). STING-mediated IFN-β production, in turn, leads to functional priming of antigen-specific T cells which mediate tumor rejection in pre-clinical solid tumor models. However, because of their disseminated growth pattern, hematological cancers, such as acute leukemia, may not release sufficient quantities of DNA upon apoptosis to activate the STING pathway in innate immune cells, such as dendritic cells (DCs). The failure of acute leukemias to induce a type I IFN response may contribute to their ability to generate T cell tolerance in the host. Thus, we hypothesized that activating the STING pathway would promote a type I IFN response sufficient to facilitate DC-mediated priming of leukemia-specific T cells and to prolong survival of mice with acute myeloid leukemia (AML). Murine C1498 AML cells engineered to express a model peptide antigen called SIY (C1498.SIY) were inoculated intravenously into syngeneic C57BL/6 mice which were then treated with a selective murine STING agonist, DMXAA (5,6-dimethylxanthenone-4-acetic acid), or vehicle control. Preliminary experiments confirmed that DMXAA administration resulted in robust IFN-β production by C57BL/6 spleen cells (90-fold induction over vehicle control-treated spleen cells). STING pathway activation via DMXAA treatment of C1498.SIY-bearing animals resulted in a massive expansion (10-fold over vehicle control-treated animals) of endogenous SIY antigen-specific T cells as determined by SIY/Kb pentamer analysis. Intracellular cytokine staining of SIY-peptide restimulated spleen cells from DMXAA-treated, leukemia-bearing animals, revealed that the expanded SIY-specific CD8+ T cells were functionally active, and produced high-levels of IFN-γ. To determine if this enhanced immune response translated to a benefit in survival, C1498.SIY AML cell-challenged mice were treated with a single dose of DMXAA or vehicle control five days after tumor inoculation. Among mice treated with DMXAA, 80% of mice rejected the leukemia and survived long-term, while control-treated mice succumbed to AML within approximately 3 weeks, as expected. DMXAA-treated mice surviving a primary C1498.SIY cell challenge also rejected a secondary challenge with parental C1498 cells (SIY-negative), suggesting the DMXAA treatment led to potent immunological memory against native C1498-expressed antigens. DMXAA treatment of mice following a systemic challenge with parental C1498 cells also led to enhanced survival compared to control-treated animals. This effect was T and or B cell-dependent, as DMXAA treatment of RAG-/- mice had no effect on survival. Collectively, these data suggest that DMXAA-mediated activation of the STING pathway upstream of type I IFN potently enhances adaptive immunity to AML which culminates in prolonged survival or even disease cure in treated animals. It will be important to determine whether the STING pathway may be successfully manipulated in other murine leukemia models. If so, then STING may be a promising therapeutic target to enhance endogenous immunity in AML patients. Citation Format: Emily K. Curran, Xiufen Chen, Leticia Corrales, Justin Kline. Activation of the STING pathway enhances immunity and improves survival in a murine myeloid leukemia model. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A10.

Abstract 4272: Potent in situ cancer immunotherapy with synthetic human STING-activating cyclic dinucleotides

Abstract Spontaneous tumor-initiated T cell priming is dependent on IFN-β production by tumor-resident dendritic cells. Based on recent observations indicating that IFN-β expression was dependent upon activation of the host STING (Stimulator of Interferon Genes) pathway, we hypothesized that direct engagement of STING through intratumoral administration of specific agonists would result in effective anti-tumor therapy. To this end, we generated novel synthetic cyclic dinucleotide (CDN) derivatives with superior STING-activating and anti-tumor properties. The most potent molecule contains a 2′-5′ and 3′-5′ phosphate bridge that increases its affinity for STING at least 10-fold. Additionally, sulfur atoms at the non-bridging oxygens of the internucleotide phosphate bridge enable both superior STING binding affinity, as well as resistance to host cell phosphodiesterases. Importantly, the lead molecule activates PBMCs from a panel of human donors representing all known STING alleles, including homozygotes for the most refractory allele. Intratumoral injection of the lead CDN molecule induced profound regression of established B16 melanoma tumors, generated substantial systemic immune responses capable of rejecting distant metastases and provided long-lived immunologic memory. Cures in entire cohorts of mice bearing well-established 4T1 mammary and CT26 colon carcinoma models correlated with induction of cytokine-mediated innate and adaptive antigen-specific T cell immunity, both systemically and in the tumor microenvironment. Immune activation and anti-tumor efficacy was lost in mice encoding non-functional STING. In vivo activation of STING with the lead CDN administered by multiple routes demonstrated a high safety and tolerability margin in preclinical toxicology studies. In addition, pharmacokinetic analysis indicates that the lead CDN compound was rapidly cleared from systemic circulation, consistent with pulsatile STING activation and transient systemic cytokine and hematological changes. These results demonstrate that CDNs have high translation potential for treatment strategies to induce activation of the tumor microenvironment, leading to effective tumor-initiated T cell priming and anti-tumor immunity. Citation Format: Laura Hix Glickman, David B. Kanne, Kelsey E. Gauthier, George E. Katibah, Justin J. Leong, Ken Metchette, Thomas W. Dubensky, Sarah M. McWhirter. Potent in situ cancer immunotherapy with synthetic human STING-activating cyclic dinucleotides. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4272. doi:10.1158/1538-7445.AM2015-4272

Comparative studies connecting IFN dysregulation with pathway dysregulation in 2 autoinflammatory diseases, SAVI and CANDLE (HUM1P.274)

Abstract Type I interferon dysregulation is associated with some autoinflammatory diseases. Gain-of-function mutations in TMEM173 encoding Stimulator of Interferon Genes (STING) lead to constitutive production of Type I IFN and the clinical syndrome, STING associated vasculopathy with onset in infancy (SAVI). In another IFN mediated disease, Chronic Atypical Neutrophilic Dermatosis with Lipodystrophy and Elevated Temperature (CANDLE), disease causing mutations cause global proteasome dysfunction and increase in IFN message. The cellular source of the IFNs in blood and the dominant isoforms remain unclear. We examined the expression of 5 type I IFN genes among FACS sorted PBMCs: T-, B-, NK cells and monocytes in SAVI (n=2) and total PBMCs from CANDLE patients (n=2) by qRTPCR. We found in SAVI that monocytes expressed the highest level of type I IFN message. Among the IFN genes tested and normalized to healthy monocytes, patients’ IFNβ is over 250-fold higher than IFNα; following the notion that STING pathway activation leads to IFNβ transcription. Depleting monocytes from SAVI PBMCs reduced 90% of the IFN message, confirming that monocytes not dendritic cells are the main source of IFNs. In contrast, CANDLE patients’ IFN alpha and beta genes are comparably upregulated. Although IFN dysregulation is identified in blood, determining the cellular origin of the IFN production and the pathways that drive IFN dysregulation will allow us to develop better targets for therapy.

Activation of the Sting Pathway Enhances Immunity and Improves Survival in a Murine Myeloid Leukemia Model

Type I interferon (IFN) production by innate immune cells is critical to prime spontaneous T cell responses against solid tumors. Emerging data suggests that tumor-derived DNA induces IFN-β production through activation of a cytosolic DNA sensing receptor called STING (Stimulator of Interferon Genes). While this leads to functional priming of antigen-specific T cells and tumor rejection in solid tumor models, the disseminated growth pattern of hematological malignancies may result in insufficient quantities of DNA being released upon apoptosis to activate the STING pathway and may contribute to T cell tolerance. Thus, we hypothesized that activating the STING pathway would promote a type I IFN response sufficient to facilitate priming of leukemia-specific T cells and prolong survival of mice with acute myeloid leukemia (AML). Murine C1498 AML cells engineered to express a model peptide antigen called SIY (C1498.SIY) were inoculated intravenously into syngeneic C57BL/6 mice and then treated with a selective murine STING agonist, DMXAA (5,6-dimethylxanthenone-4-acetic acid), or vehicle control. Preliminary experiments confirmed that DMXAA administration resulted in robust IFN-β production by C57BL/6 spleen cells (90-fold induction over vehicle control). STING pathway activation via DMXAA treatment of C1498.SIY-bearing animals resulted in a massive expansion (10-fold over vehicle control-treated animals) of endogenous SIY antigen-specific T cells as analyzed by SIY/Kb pentamer analysis. Intracellular cytokine staining of SIY-peptide restimulated spleen cells from these animals revealed that the expanded SIY-specific CD8+ T cells were functionally active, and produced high-levels of IFN-gamma. This enhanced immune response also translated to a benefit in survival. After C1498.SIY AML cell-challenge, the majority (80%) of mice treated with DMXAA rejected the leukemia and survived long-term, while control-treated mice succumbed to AML within approximately 3 weeks, as expected. DMXAA-treated mice surviving a primary C1498.SIY cell challenge also rejected a secondary challenge with parental C1498 cells (SIY-negative), suggesting DMXAA treatment led to potent immunological memory against native C1498-expressed antigens. DMXAA treatment of mice following a systemic challenge with parental C1498 cells also led to enhanced survival compared to control-treated animals. This effect was T and or B cell-dependent, as DMXAA treatment of RAG-/- mice had no effect on survival. IFNAR-/- mice unable to sense type I IFNs succumbed more rapidly following AML induction compared to C57BL/6 mice, but demonstrated prolonged survival following DMXAA treatment compared to vehicle-control-treated IFNAR-/- hosts, suggesting that STING activation may stimulate other pathways important for generating anti-leukemia immunity in addition to type I IFN or may directly affect the viability of AML cells directly as was observed following in vitro cultures of AML cells with DMXAA. Collectively, these data suggest that DMXAA-mediated activation of the STING pathway potently enhances adaptive immunity to AML, culminating in prolonged survival and even disease cure. If successfully manipulated in other murine leukemia models, the STING pathway may be a promising therapeutic target to enhance endogenous immunity in AML patients. DisclosuresNo relevant conflicts of interest to declare.

Activation of the STING pathway enhances immunity and improves survival in a murine myeloid leukemia model

Type I interferon (IFN) production by innate immune cells is critical to prime spontaneous T cell responses against solid tumors. Emerging pre-clinical data suggests that tumor-derived DNA induces potent IFN-β production by activating a cytosolic DNA sensing receptor called STING (Stimulator of Interferon Genes), ultimately resulting in tumor antigen-specific T cell priming and in some cases, tumor rejection. However, because of their disseminated growth pattern, hematological malignancies, such as acute myeloid leukemia (AML), may not release sufficient quantities of DNA during cell death to activate the STING pathway in innate immune cells, which contributes to the T cell tolerance observed in these hosts. Thus, we hypothesized that STING pathway activation would promote a host type I IFN response sufficient to facilitate leukemia-specific T cell priming and immune-mediated control of AML progression. Murine C1498 AML cells expressing the model SIY antigen (C1498.SIY) were inoculated intravenously into syngeneic C57BL/6 mice which then received a single dose of the murine STING agonist, DMXAA (5,6-dimethylxanthenone-4-acetic acid), or vehicle control 5 days later. DMXAA induced potent IFN-β production by C57BL/6 spleen cells (90-fold induction over vehicle control). STING pathway activation via DMXAA treatment of C1498.SIY-bearing animals resulted in a massive expansion (10-fold over vehicle control) of endogenous SIY antigen-specific T cells as analyzed by SIY/Kb pentamer staining and flow cytometry. Intracellular cytokine staining of SIY-peptide restimulated spleen cells from DMXAA-treated animals revealed that the expanded SIY-specific CD8+ T cells produced high-levels of IFN-γ. This enhanced immune response also translated to a benefit in survival. After C1498.SIY AML cell-challenge, 80% of DMXAA-treated mice survived long-term, while controls succumbed to AML within approximately 3-4 weeks, as expected. DMXAA-treated mice surviving a primary C1498.SIY cell challenge also rejected a secondary challenge with parental C1498 cells (SIY-negative), suggesting DMXAA-induced STING activation led to potent immunological memory against native C1498-expressed antigens. DMXAA treatment of mice following a systemic challenge with parental C1498 cells also led to significantly enhanced survival compared to control-treated animals. This effect was T and/or B cell-dependent, as DMXAA treatment of leukemia-bearing RAG-/- mice had no effect on survival. Collectively, these data suggest that DMXAA-mediated activation of the STING pathway enhances adaptive immunity to AML, culminating in prolonged survival and even disease cure. Human STING agonists are currently in development and, if STING pathway activation demonstrates efficacy in additional pre-clinical leukemia models, it may be of interest to target this pathway in AML patients.

Anticancer Flavonoids Are Mouse-Selective STING Agonists

The flavonoids FAA and DMXAA showed impressive activity against solid tumors in mice but failed clinical trials. They act on a previously unknown molecular target(s) to trigger cytokine release from leukocytes, which causes tumor-specific vascular damage and other antitumor effects. We show that DMXAA is a competitive agonist ligand for mouse STING (stimulator of interferon genes), a receptor for the bacterial PAMP cyclic-di-GMP (c-di-GMP) and an endogenous second messenger cyclic-GMP-AMP. In our structure-activity relationship studies, STING binding affinity and pathway activation activity of four flavonoids correlated with activity in a mouse tumor model measured previously. We propose that STING agonist activity accounts for the antitumor effects of FAA and DMXAA in mice. Importantly, DMXAA does not bind to human STING, which may account for its lack of efficacy or mechanism-related toxicity in man. We propose that STING is a druggable target for a novel innate immune activation mechanism of chemotherapy.