Preclinical Solid Tumor Models Research Articles

Abstract 815: New approach for old target: W0101 antibody drug conjugate effectively inhibits tumor growth in preclinical models of IGF-1R overexpressing solid tumors

Abstract Insulin-like growth factor type 1 receptor (IGF-1R) has been recognized for decades for its role in tumorigenesis and growth1. In addition, overexpression of this receptor has been largely documented in numerous tumor tissues. However, so far, previous therapeutic approaches based on monoclonal antibodies and tyrosine kinase inhibitors failed to show clinical benefit in the overall patient population2. Here we disclose for the first time a novel IGF-1R targeted Antibody Drug Conjugate (ADC), W0101, designed to deliver highly potent cytotoxic drugs selectively to IGF-1R over-expressing tumor cells. The naked antibody for our ADC was selected on the basis of its specific binding properties to IGF-1R compared to insulin receptor (IR), and for its internalization properties. After coupling of a proprietary auristatin derivative to the naked antibody, neither the binding nor the internalization properties of W0101 evaluated by FACS analysis, were modified. Interestingly, the capacity of W0101 to induce cytotoxicity was evaluated in vitro in various cell lines and was demonstrated to be independent of effector functions. Mouse efficacy studies with various doses and schedules of administration were conducted in several models expressing different level of IGF-1Rin order to determine their sensitivity to W0101. The sensitivity of W0101 was correlated to the expression level of IGF-1R evaluated by IHC. And in a 3+ MCF-7 breast cancer model, a single injection of W0101 at 3mg/kg led to more than 90% tumor growth inhibition (TGI), associated with a modulation of the IGF-1R expression on the tumor cells (IHC study). The first in-human trial of W0101 is currently on going to address clinical safety. We believe that W0101 will bring a new therapeutic option for patients that overexpress IGF-IR. 1Pollak M. Insulin and insulin-like growth factor signaling in neoplasia. Nat Rev Cancer, 2008,8:915-928. 2Corvaia N, Beck A, Caussanel V, Goetsch L. Insulin-like growth factor receptor type I as a target for cancer therapy. Frontiers in Bioscience, Scholar, 5, 439-450, January 1, 2013 Citation Format: Barbara Akla, Noureddine Loukili, Alain Robert, Charlotte Beau-Larvor, Martine Malissard, Jean-Francois Haeuw, Alain Beck, Michel Perez, Cyrille Dreyfus, Mariya Pavlyuk, Eric Chetaille, Nathalie Corvaia. New approach for old target: W0101 antibody drug conjugate effectively inhibits tumor growth in preclinical models of IGF-1R overexpressing solid tumors [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 815.

Biochemical Properties of a Decoy Oligodeoxynucleotide Inhibitor of STAT3 Transcription Factor.

Cyclic STAT3 decoy (CS3D) is a second-generation, double-stranded oligodeoxynucleotide (ODN) that mimics a genomic response element for signal transducer and activator of transcription 3 (STAT3), an oncogenic transcription factor. CS3D competitively inhibits STAT3 binding to target gene promoters, resulting in decreased expression of proteins that promote cellular proliferation and survival. Previous studies have demonstrated antitumor activity of CS3D in preclinical models of solid tumors. However, prior to entering human clinical trials, the efficiency of generating the CS3D molecule and its stability in biological fluids should be determined. CS3D is synthesized as a single-stranded ODN and must have its free ends ligated to generate the final cyclic form. In this study, we report a ligation efficiency of nearly 95 percent. The ligated CS3D demonstrated a half-life of 7.9 h in human serum, indicating adequate stability for intravenous delivery. These results provide requisite biochemical characterization of CS3D that will inform upcoming clinical trials.

Glutathione system in animal model of solid tumors: From regulation to therapeutic target.

Glutathione (GSH) is one of the most important defenses against oxidative stress through the fine-tuned regulation of redox homeostasis. Glutathione is also involved in many metabolic processes and is important for the regulation of cell survival, proliferation, and death. Furthermore, GSH and the enzymes that are involved in its biosynthesis, catabolism, and detoxification (e.g., disulfide-oxidized glutathione, glutathione S-transferase, glutathione peroxidase, glutathione reductase, and γ-glutamyltranspetidase) play an important role in several diseases, including cancer. In cancer cells, these enzymes protect the tumor microenvironment against oxidative stress and cell death and are important for tumor growth and development. Thus, the GSH system is an important tool for investigating new pharmacological approaches for cancer treatment. Several preclinical models of solid tumors are available for this purpose. This review summarizes and discusses the regulation and dysregulation of GSH and its related enzymes in different models of solid tumors, and potential treatments that target the GSH system.

Abstract B171: PK/PD modeling of the first-in-class, potent and selective covalent CDK7 inhibitor, SY-1365, provides mechanistic basis for intermittent dosing regimens in preclinical efficacy models of hematologic and solid tumors

Abstract Introduction: CDK7 has recently emerged as an attractive gene control target in cancers driven by transcriptional dependencies, including triple-negative breast cancer (TNBC) and acute myeloid leukemia (AML). SY-1365 is a first-in-class, potent, and selective covalent CDK7 inhibitor that has shown durable and complete responses in xenograft models of AML and various solid tumors. To advance the development of this CDK7 inhibitor in patients, the current work assessed the relationship between SY-1365 pharmacokinetics (PK), pharmacodynamics (PD) measured as CDK7 target occupancy, and efficacy in mouse xenograft models of AML (HL-60) and TNBC (HCC70). Methods: SY-1365 was dosed intravenously in several xenograft experiments across a range of dose levels (5-40 mg/kg) and dose frequencies (daily to weekly). PK and changes in CDK7 occupancy were measured following single and repeat dosing, and efficacy studies measured the resultant dose-dependent changes in tumor volume over time after repeat dosing of SY-1365. To account for the temporal differences in PK, PD and tumor growth inhibition (TGI), a mathematical modeling approach was taken to integrate the resulting datasets. In addition, an assay to determine CDK7 occupancy in human PBMCs was developed to support the ongoing phase 1 study. Results: The PK in mouse was proportional with respect to dose and exhibited a terminal elimination half-life (t1/2) of 4 h. The PK/PD model was able to describe the PK, PD, and efficacy in HL-60 and HCC70 xenografts, with similar exposure and CDK7 occupancy profiles observed in these two xenografts. The model demonstrated a CDK7 turnover t1/2 of 69 h, longer than that reported for other oncogenic kinases targeted with covalent inhibitors where daily dosing is required (e.g., EGFRi, BTKi), with optimal efficacy achieved when target engagement was sustained over the dosing interval. This provides a mechanistic basis for intermittent dosing based on the dose-dependent TGI, including tumor regressions, observed with SY-1365 in in vivo models of TNBC and AML. Conclusions: The PK/PD relationship for target engagement and efficacy of the first-in-class potent and selective covalent CDK7 inhibitor, SY-1365, has been characterized in preclinical models through a mathematical modeling approach. Assays were developed to enable target occupancy quantification in xenograft tumors, and in human PBMCs as a surrogate tissue to support clinical investigation. Further investigations into tumor molecular profiles driving response are currently under way. In summary, this work supported the selection of an initial twice-weekly dosing regimen in the ongoing phase 1 trial of SY-1365 in adult patients with advanced solid tumors (NCT03134638). Citation Format: Nigel J. Waters, Shanhu Hu, Brett Matzuka, Graeme Hodgson, Yixuan Ren, Yoon Choi, Kevin Dykstra, Christopher Roberts, Kevin Sprott, Emmanuelle di Tomaso, Christian Fritz. PK/PD modeling of the first-in-class, potent and selective covalent CDK7 inhibitor, SY-1365, provides mechanistic basis for intermittent dosing regimens in preclinical efficacy models of hematologic and solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B171.

Abstract B182: The novel tyrosine kinase inhibitors UJ26 and UJ30 are active in solid tumor and hematologic cancer cell lines

Abstract Background: Tyrosine kinase (TK) inhibitors represent the class of targeted antitumoral drugs that has been so far more successful. Here, we present two novel TK inhibitors, UJ26 and UJ30, with antitumor activity in preclinical models of solid tumors and hematologic cancers. Materials and Methods: Biochemical kinase profiling was performed (DiscoverX, CA, USA). Cell lines included in the NCI60 panel and lymphoma cell lines were exposed to increasing doses of UJ26, UJ30, and, as control, to the SRC inhibitor dasatinib for 72 hours. Cell proliferation was evaluated by using MTT assay. In vitro synergy was assessed with the Chou-Talalay combination index (CI): synergism (<0.9), additive (0.9-1.1), antagonism/no benefit (> 1.1). DLBCL RI-1 cells (15x106) were s.c. inoculated in NOD-Scid (NOD.CB17-Prkdcscid/NCrHsd) mice. Tumor size was measured on regular basis and drug treatments with UJ26 and UJ30 both given i.p, at the dose of 100mg/kg started when tumors reached 5 mm in diameter (100 mm3). Results: The two novel kinase inhibitors UJ26 and UJ30 presented broad specificity comparable to that of dasatinib, able to inhibit clinically relevant kinases ABL1, EGFR, FLT3, KIT, and MET. UJ26 and UJ30 presented antitumor activity in the NCI60 cell line panel against models of hematologic malignancies and solid tumors, in particular in the K562 leukemia and KM12 colon cancer cell lines (GI50 < 50 nM). Antiproliferative activity of UJ26 and UJ30 was then assessed in 8 B-cell lymphoma cell lines derived from mantle cell lymphoma (MCL) (Jeko1, MAVER1), chronic lymphocytic leukemia (MEC-1, PCL-12), and diffuse large B cell lymphoma (DLBCL) of the activated B cell like (ABC) (RI-1, SU-DHL-2) or of the germinal center B cell (GCB) (VAL, SU-DHL-4). Compounds were active with median IC50s of 600nM (95%C.I. 300-1065nM) and 250nM (95%C.I. 100-1049nM), respectively. Their antitumor activity appeared more potent than dasatinib (median IC50=1325nM, 95%C.I. 100-16625nM), in particular in 3 of the cell lines (MAVER1, SU-DHL-2 and MEC1). UJ26 and UJ30 were evaluated in combination with the BET inhibitor OTX015, exposing the four most sensitive lymphoma cell lines to increasing concentrations of the single compound or of both compounds for 72h. Both the UJ26/OTX015 combination, as well as the UJ30/OTX015 combination, were synergistic in all the cell lines. In vivo experiments showed that UJ26 was very toxic already after the first dose with swelling of the injection area. UJ26-treated mice started to recover after 2 weeks and were not treated any more, and there was no antitumor effect. UJ30 given once every 5 days reduced tumor volume compared to control group (D5, D11, D15; P < 0.05) and was well tolerated. UJ30-treated xenografts were three times smaller than both control and UJ26 groups at the end of the experiment (D15, UJ30 median tumor volume=867, 95%C.I. 600-1267.5). Body weight loss was observed after treatment with compound UJ26 whereas UJ30 showed similar weight growth as in the control group. Experiment was terminated after 2 weeks for vehicle- and UJ26-treated groups (D15) while UJ30-treated mice were sacrificed at day 20. Conclusions: The novel TK inhibitors UJ26 and UJ30 showed preclinical antitumor activity and in particular UJ30 is worth further investigations. CN, AC, FB: co-senior authors. Citation Format: Chiara Tarantelli, Eugenio Gaudio, Andrea Unzue, Pawel Sledz, Hillarie Ekeh, Elena Bernasconi, Filippo Spriano, Cristina Nevado, Amedeo Caflisch, Francesco Bertoni. The novel tyrosine kinase inhibitors UJ26 and UJ30 are active in solid tumor and hematologic cancer cell lines [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B182.

Quantitative high-throughput phenotypic screening of pediatric cancer cell lines identifies multiple opportunities for drug repurposing.

Drug repurposing approaches have the potential advantage of facilitating rapid and cost-effective development of new therapies. Particularly, the repurposing of drugs with known safety profiles in children could bypass or streamline toxicity studies. We employed a phenotypic screening paradigm on a panel of well-characterized cell lines derived from pediatric solid tumors against a collection of ∼3,800 compounds spanning approved drugs and investigational agents. Specifically, we employed titration-based screening where compounds were tested at multiple concentrations for their effect on cell viability. Molecular and cellular target enrichment analysis indicated that numerous agents across different therapeutic categories and modes of action had an antiproliferative effect, notably antiparasitic/protozoal drugs with non-classic antineoplastic activity. Focusing on active compounds with dosing and safety information in children according to the Children's Pharmacy Collaborative database, we identified compounds with therapeutic potential through further validation using 3D tumor spheroid models. Moreover, we show that antiparasitic agents induce cell death via apoptosis induction. This study demonstrates that our screening platform enables the identification of chemical agents with cytotoxic activity in pediatric cancer cell lines of which many have known safety/toxicity profiles in children. These agents constitute attractive candidates for efficacy studies in pre-clinical models of pediatric solid tumors.

Inducible Activation of MyD88 and CD40 in CAR T Cells Results in Controllable and Potent Antitumor Activity in Preclinical Solid Tumor Models.

Adoptive immunotherapy with T cells expressing chimeric antigen receptors (CAR) has had limited success for solid tumors in early-phase clinical studies. We reasoned that introducing into CAR T cells an inducible costimulatory (iCO) molecule consisting of a chemical inducer of dimerization (CID)-binding domain and the MyD88 and CD40 signaling domains would improve and control CAR T-cell activation. In the presence of CID, T cells expressing HER2-CARζ and a MyD88/CD40-based iCO molecule (HER2ζ.iCO T cells) had superior T-cell proliferation, cytokine production, and ability to sequentially kill targets in vitro relative to HER2ζ.iCO T cells without CID and T cells expressing HER2-CAR.CD28ζ. HER2ζ.iCO T cells with CID also significantly improved survival in vivo in two xenograft models. Repeat injections of CID were able to further increase the antitumor activity of HER2ζ.iCO T cells in vivo Thus, expressing MyD88/CD40-based iCO molecules in CAR T cells has the potential to improve the efficacy of CAR T-cell therapy approaches for solid tumors.Significance: Inducible activation of MyD88 and CD40 in CAR T cells with a small-molecule drug not only enhances their effector function, resulting in potent antitumor activity in preclinical solid tumors, but also enables their remote control post infusion. Cancer Discov; 7(11); 1306-19. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1201.

Abstract 1151: SY-1365, a potent and selective CDK7 inhibitor, exhibits promising anti-tumor activity in multiple preclinical models of aggressive solid tumors

Abstract CDK7 has recently emerged as an attractive target in cancer since its inhibition decreases the transcript levels of oncogenic transcription factors, especially those driven by super-enhancers (SEs). Cancers have been hypothesized to be addicted to SE regulated genes and simultaneous suppression of multiple SE associated genes through CDK7 inhibition might represent a novel, powerful way to selectively kill cancer cells. Previously, we reported that SY-1365, a highly selective covalent CDK7 inhibitor, induces apoptosis in leukemia cells, but not in non-malignant cells, and demonstrates anti-tumor activity in in vivo models of leukemia. In this study, we extend these findings to the identification of multiple solid tumors that are susceptible to SY-1365 and compare the effects of SY-1365 on gene expression to other gene control agents. SY-1365 was screened in a panel of solid tumor cell lines, revealing activity in breast, ovarian, colorectal and lung cancer cells with low nM EC50 and rapid induction of apoptosis. In breast cancer, a subset of triple negative breast cancer (TNBC) cell lines were found to be more sensitive than luminal breast cancer cell lines, so we extended our studies to in vivo models and showed substantial tumor growth inhibition in multiple patient-derived xenograft (PDX) models of TNBC. Since other compounds have been reported to modulate the expression of SE regulated genes, we compared the transcriptional effects of SY-1365 treatment with those of a pan-CDK inhibitor (flavopiridol), a CDK9 inhibitor (NVP2) and a BRD4 inhibitor (JQ1) using microarray analysis. Applying principal component analysis, we observed a unique transcriptional response elicited by SY-1365 compared to the other inhibitors. NVP2 and flavopiridol inhibited an overlapping and much larger gene set than either JQ1 or SY-1365. Interestingly, SY-1365 treatment decreased the expression of oncogenic transcription factors, cell cycle checkpoint regulators and DNA damage response genes. The downregulation of transcripts involved in apoptosis and DNA damage response by SY-1365 suggests that CDK7 inhibition might synergize with targeted agents that affect these processes. Indeed, we observed that SY-1365 was synergistic with the PARP inhibitor niraparib and the Bcl-2 inhibitor venetoclax in triple negative breast cancer and AML cell lines, respectively. In summary, we have identified TNBC, ovarian and small cell lung cancer cell lines as highly sensitive to SY-1365 in vitro and have observed substantial tumor growth inhibition in PDX models of TNBC. SY-1365 induced a distinct transcriptional response compared with other transcriptional inhibitors, with apoptotic and DNA damage pathways being central. Finally, these mechanism of action studies support a rationale for investigating combinations of SY-1365 with inhibitors of PARP and Bcl-2. Citation Format: Shanhu Hu, Nan Ke, Yixuan Ren, Sofija Miljovska, Nisha Rajagopal, Michael McKeown, David Orlando, Kevin Sprott, Yoon J. Choi, Eric Olson, Christian C. Fritz. SY-1365, a potent and selective CDK7 inhibitor, exhibits promising anti-tumor activity in multiple preclinical models of aggressive solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1151. doi:10.1158/1538-7445.AM2017-1151

Abstract CT046: Phase I dose escalation study of the CDK4/6 inhibitor palbociclib in combination with the MEK inhibitor PD-0325901 in patients with RAS mutant solid tumors

Abstract Background: Both MEK and CDK4/6 have been investigated as therapeutic targets in preclinical models of RAS mutant solid tumors. Multiple mechanisms contribute to synergism including the development of MAP kinase-dependence in RAS mutant cells exposed to CDK4/6 inhibition, leading to increased apoptosis, as well as enhanced induction of senescence with combinatorial vs. monotherapy treatment. We conducted a Phase 1 study of combined palbociclib and PD-0325901 in patients with RAS mutant solid tumors to assess safety, tolerability, and MTD, as well as pharmacokinetic parameters, preliminary efficacy and effects on mutant RAS allelic burden in plasma. Methods: Patients with RAS mutant solid tumors were enrolled utilizing a 3 + 3 design. Palbociclib and PD-0325901 were given orally once and twice daily, respectively for three of every four weeks. The maximum planned administered doses were 125 mg palbociclib daily and 8 mg PD-025901 twice daily. Pharmacokinetic parameters were measured on cycle 1 day 21 and plasma samples to measure RAS allelic burden were serially collected. Results: Twenty-five patients (17 with KRAS mutant NSCLC) who received at least one dose of each study drug were enrolled over 5 dose levels including (palbociclib/PD-0325901) 75/2, 75/4, 100/4, 125/4 and 125/8. The maximum administered dose of 125 mg palbociclib daily and 8 mg twice daily PD-0325901 was tolerable. One DLT of pneumonitis occurred at the 100/4 dose level. The most frequent (>10%) drug-related toxicities were leukopenia (72%), anemia (72%), thrombocytopenia (72%), neutropenia (64%), acneiform rash (64%), diarrhea (52%), fatigue (44%), lower extremity edema (32%), vomiting (28%), nausea (28%), oral mucositis (24%), increased AST (20%), increased creatinine (12%), epistaxis (12%), and blurred vision (12%). The median number of cycles completed was 3 (range: 1 - 28+). Across all patients, 1 patient (4%) with KRAS mutant NSCLC achieved partial response and 18 (72%) had stable disease as the best response. Eleven patients were progression-free > 3 months, and 6 were progression-free > 6 months, including 5 with KRAS mutant NSCLC, two of whom received prior immune checkpoint blockade. Among patients with KRAS mutant NSCLC, clinical benefit was seen among those with tumors harboring KRAS mutation alone, as well as those with tumors demonstrating concomitant loss of TP53 or CDKN2AB. A dose-dependent decrease in plasma RAS allelic burden was observed across dose levels. Conclusions: Administration of combined palbociclib and PD-0325901 was tolerable and produced promising progression-free survival among patients with KRAS mutant NSCLC. Additional dose levels utilizing continuous MEK inhibition are being evaluated. Citation Format: Geoffrey I. Shapiro, John Hilton, Leena Gandi, Nicole Chau, James Cleary, Andrew Wolanski, Adrienne Anderson, Brian Beardslee, Faith Hassinger, Ketki Bhushan, Elizabeth Downey, Joseph Gibson, Solida Pruitt-Thompson, Alona Muzikansky, Suzanne Barry, Nora Feeney, Cloud Paweletz, Geoffrey Oxnard, Jeffrey Supko, Pasi Jänne, Kwok-Kin Wong, Bruce Johnson. Phase I dose escalation study of the CDK4/6 inhibitor palbociclib in combination with the MEK inhibitor PD-0325901 in patients with RAS mutant solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT046. doi:10.1158/1538-7445.AM2017-CT046

Abstract 118: Targeting cancer with a novel BET bromodomain inhibitor ODM-207

Abstract Background: Bromodomain and extra-terminal (BET) family proteins are dual bromodomain-containing epigenetic readers that bind to acetylated-lysine residues at gene promoter and enhancer elements in histones and recruit protein complexes to promote transcriptional elongation. Recent evidence demonstrates that BET bromodomain inhibition leads to anti-proliferative activity in pre-clinical models of many hematological malignancies and solid tumors. Selective inhibition of BET bromodomains by small molecule inhibitors has emerged as a promising therapeutic strategy for the treatment of cancer. In this study, we evaluated the antitumor activity of ODM-207, a novel, potent and highly selective BET bromodomain inhibitor. Methods and Results: ODM-207 is a potent and selective BET inhibitor that is structurally unrelated to the benzodiazepine like inhibitors such as JQ1, I-BET762, and OTX015. We tested the preclinical activity of ODM-207 across multiple tumor types in a 4-day growth inhibition in vitro assay. ODM-207 potently inhibits cell viability of a wide range of hematological and solid tumor cell lines. ODM-207 also shows potent antiproliferative effects in patient-derived cancer cells representing various tumor types. In VCaP prostate cancer cell lines, ODM-207 induced apoptosis consistent with increased expression of pro-apoptotic regulators, whereas potent antiproliferative effects associated with cell cycle arrest and cellular senescence were seen in e.g. LNCaP prostate cancer cell line. To gain insight on mechanisms of acquired BET inhibitor resistance, we generated a cell line with resistance to BET-inhibition by culturing LNCaP prostate cancer cells with increasing concentrations of OTX015, a potent and selective BET-inhibitor. Interestingly, ODM-207 is also able to inhibit proliferation and downregulate Myc levels in cells that have acquired resistance to BET-inhibitors OTX015 and I-BET762. In xenograft models, oral treatment with ODM-207 significantly inhibits tumor growth at a dose which is well tolerated. Conclusions: In summary, these studies demonstrate that ODM-207 is a potent inhibitor of BET proteins in models of hematologic malignancies and solid tumors in vitro and in vivo and support its clinical development for the treatment of cancer. Citation Format: Anu-Maarit Moilanen, Mari Björkman, Reetta Riikonen, Chandrasekhar Abbineni, Mahaboobi Jaleel, Sivapriya Marappan, Tarja Ikonen, Girish Daginakatte, Aravind A B, Elina Mattila, Juha Rantala, Susanta Samajdar, Murali Ramachandra, Pekka Kallio. Targeting cancer with a novel BET bromodomain inhibitor ODM-207 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 118. doi:10.1158/1538-7445.AM2017-118

Inhibiting TGF-Beta Signaling in the Tumor Micro-Environment Enhances the Cytotoxic Function of Adoptive NK Cells

Introduction: The tumor micro-environment poses a limitation to the efficacy of adoptive NK cell therapy due to several immunosuppressive cytokines. TGF-beta, produced in excess by tumor cells, regulatory T cells and stromal cells, facilitates epithelial to mesenchymal transformation thereby promoting metastasis and fibrosis. This high TGF-beta milieu in cancer patients also impairs innate natural killer (NK) cell mediated cancer immunity through various mechanisms.Adoptive transfer of healthy donor NK cells will have limited clinical efficacy when highly activated NK cells are introduced into the immunosuppressive TGF-beta rich tumor microenvironment of cancer patients. We are testing several small molecule inhibitors of the TGF-beta receptor in combination with healthy donor NK cells with a goal of enhancing adoptive NK cell therapy in various disease models.Methods: Human NK cells isolated from healthy donor peripheral blood were expanded over a 21-day period in co-culture with irradiated K562 cells genetically modified to express membrane-bound IL-21. (Somanchi et al. 2011 JoVE 48. doi: 10.3791/2540). Cell culture media was supplemented with IL-2 (50mU/mL).For in vitro assays, at the beginning of expansion week 3 the culture conditions for NK cells was continued unchanged, supplemented with TGF-beta 1 ligand (TGF-B1) at 5ng/mL or 10ng/mL either alone or in combination with EW7197 (a TGF-beta Type 1 receptor inhibitor) or with EW7197 alone. These NK cells were tested with Calcein-AM release cytotoxicity assays at various time points from addition of TGF-B1 (0h, 36h, 72h and 96h). Briefly, NK cells were co-cultured with OCI-AML cells labeled with Calcein-AM at a ratio of 5NK:1 OCI-AML. At the end of 4 hours co-incubation, cytotoxicity was measured by relative fluorescence of calcein release into the culture supernatant compared with Triton-X induced complete target cell lysis.In a murine liver metastases model using the colon cancer cell line HCT116, 105 HCT116 cells were surgically implanted into NSG mouse spleens (following hemi-splenectomy). Mice in 2 groups (3/group) subsequently received 5 x 106 NK cells each (tail vein), weekly for two weeks, starting 10 days post-op. Another control group received vehicle infusions alone while a fourth control group received the TGF-beta inhibitor LY2157299 by oral gavage twice daily at 75mg/kg for two weeks. One group of mice receiving NK cells also received twice daily LY2157299 for two weeks starting with the first NK cell infusion. Mice receiving NK cells also received IL2 (75,000U IP) three times a week for two weeks.Results: At TGF-beta levels similar to that found in AML patients (5ng/ml), NK cell killing of OCI cells was markedly impaired progressively from 36h to 96h exposure. This reduced cytotoxic activity correlated with a 3-4 fold decrease in expression of NKG2D most marked at 96h(Figure 1). CD16 expression was also significantly reduced by 2-3 fold on TGF-beta exposed NK cells after 96h. The TGF-beta inhibitor EW7197 maintained NK cell killing as well as NKG2D and CD16 expression in the presence of TGF-B1 (Figure 1).At 32 days post-splenic implantation, vehicle mice (those that received HCT116 either alone or in combination with LY2157299 alone) appeared moribund and had grossly distended abdomens with ascites. All mice were autopsied at this time point. All control mice had grossly enlarged livers with significant metastases and absence of viable liver tissue (Fig 2). In contrast, mice treated with two weekly infusions of NK cells and LY2157299 for 4 weeks had no ascites and predominantly healthy livers. All mice that received NK cells alone had some evidence of liver metastases with various degrees of disease burden. Composite H&E stains of liver sections from 3 mice/group showed <10% liver metastases microscopically in mice treated with a combination of NK cells and LY2157299. This was significantly reduced compared to 80-90% in the untreated or LY2157299 alone mice and 60-70% in the NK cell alone group (Fig 3).Discussion: Our preliminary results indicate that TGF-beta inhibition in combination with adoptive NK cell therapy can mitigate the immunosuppressive tumor microenvironment conferred by TGF-beta signaling. We are in the process of validating this approach in other preclinical models of both hematologic malignancies and solid tumors. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

RN927C, a Site-Specific Trop-2 Antibody-Drug Conjugate (ADC) with Enhanced Stability, Is Highly Efficacious in Preclinical Solid Tumor Models.

Trop-2, also known as TACSTD2, EGP-1, GA733-1, and M1S1, is frequently expressed on a variety of human carcinomas, and its expression is often associated with poor prognosis of the diseases. However, it is also present on the epithelium of several normal tissues. A comprehensively designed Trop-2-targeting antibody-drug conjugate (ADC), balancing both efficacy and toxicity, is therefore necessary to achieve clinical utility. To this end, we developed a cleavable Trop-2 ADC (RN927C) using a site-specific transglutaminase-mediated conjugation method and a proprietary microtubule inhibitor (MTI) linker-payload, PF-06380101. Robust in vitro cytotoxicity of RN927C was observed on a panel of Trop-2-expressing tumor cell lines, with IC50 generally in the subnanomolar range. As expected for an MTI-containing ADC, RN927C readily induced mitotic arrest of treated cells in vitro and in vivo, followed by subsequent cell death. The in vivo efficacy of RN927C was tested in multiple cell line and patient-derived xenograft tumor models, including pancreatic, lung, ovarian, and triple-negative breast tumor types. Single-dose administration of RN927C at 0.75 to 3 mg/kg was generally sufficient to induce sustained regression of Trop-2-expressing tumors and showed superior efficacy over standard treatment with paclitaxel or gemcitabine. Administration of RN927C in nonhuman primate toxicity studies resulted in target-mediated effects in skin and oral mucosa, consistent with Trop-2 expression in these epithelial tissues with minimal, non-dose limiting off-target toxicities. On the basis of the combined efficacy and safety results, RN927C is postulated to have a favorable therapeutic index for treatment of solid tumors. Mol Cancer Ther; 15(11); 2698-708. ©2016 AACR.

Abstract 3499: Triptolide pro-drug decreases tumor burden and halts tumor progression in a murine model of acute myeloid leukemia

Abstract Introduction Standard treatment for acute myelogenous leukemia (AML) has not changed over the past few decades and relies primarily on the traditional “7 + 3″ regimen of daunorubicin, administered over 3 days and Cytarabine, administered over 7 days. This chemo-intensive regimen is poorly tolerated by patients and is characterized by a high rate of relapse and treatment failure. Triptolide is a diterpenoid tri-epoxide compound isolated from the Chinese herb Tripterygium wilfordii. The water-soluble pro-drug of triptolide, Minnelide has been shown by our group to be highly effective in a number of pre-clinical models of solid tumors and is currently undergoing Phase I Clinical trial for gastrointestinal tumors. Interim analysis of the Phase I data has shown that the equivalent mouse dose of 0.2 mg/kg/day is well tolerated by patients with no reported dose limiting toxicity. Methods Primary AML apheresis samples from patients with acute myeloid leukemia and multiple AML cell Lines (THP1, KG1, Kasumi1, HL-60) were treated with Triptolide at doses from of 2.5 nM to 50nM and cell viability was measured using a formazan based colorimetric assay. Apoptosis was measured using Annexin V via flowcytometry and colony forming ability of AML cell lines was measured using a methylcellulose based assay. To generate an engraftment model of AML, NOD.Rag1-/-;γcnull (NRG) animals expressing human interleukin-3 (IL-3) and human GM-CSF (NRGS) were injected luciferase expressing THP1 cells after sublethal irradiation of 250 cGy. These animals were then serially evaluated using In Vivo Imaging System (IVIS) and leukemic burden was calculated the total bioluminescent signal after intra-periotneal injection of luciferin. Treatment with vehicle or Minnelide at a dose of 0.1mg/kg/day and 0.15 mg/kg/day was started on the 10th day after confirming engraftment using IVIS. Results Triptolide at a dose range of 2.5 nM to 50 nM produced a dose and time dependent killing of leukemic cells in both cell lines and primary AML patient samples. The IC-50 for THP1, KG1, Kasumi1 and HL-60 cell lines with triptolide treatment at 48 hours were 5 ± 0.8 nM, 8 ± 0.7 nM, 7.2 ± 0.6 nM, 10 nM ± 2 nM respectively. Treatment with triptolide at a dose of 2.5 nM induced cell death and apoptosis as measured by Annexin V posiitvity in primary AML apheresis samples. Colony formation by THP1 cells and KG1 cells was completely abrogated by treatment with Triptolide at 2.5 nM and 25 nM respectively. Vehicle treated mice showed a rapid progression of disease burden when compared to Minnelide treated mice. At a dose of 0.1 mg/kg/day and 0.15 mg/kg/day, these mice had no appreciable increase in leukemic burden over normal mice when assessed by IVIS. Conclusion We show that Minnelide induces cell death at therapeutically relevant concentrations in vitro and decreased leukemic burden in a murine model of AML. Minnelide may emerge as a novel therapeutic strategy in treating patients with AML. Citation Format: Bhuwan Giri, Sulagna Banerjee, John George, Shrey Modi, Vineet Kumar Gupta, Mahendra K. Singh, Vikas Dudeja, Ashok K. Saluja. Triptolide pro-drug decreases tumor burden and halts tumor progression in a murine model of acute myeloid leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3499.

Abstract 4709: Broad activity for the combination of BET and MEK inhibitors across solid and hematologic cancers

Abstract BET (bromodomain and extra-terminal) family proteins are epigenetic readers that modulate expression genes involved in cell growth and oncogenesis. Selective small molecule BET inhibitors, such as the GSK I-BETs (I-BET762, I-BET151), abrogate binding of BET proteins to acetylated chromatin and inhibit transcription of BET target genes. We and others have previously demonstrated single agent activity for BET inhibitors in a number of pre-clinical solid and hematologic tumor models. Transcriptomics and mechanistic studies from several of these tumor types indicate that BET inhibitors influence numerous signaling pathways at the transcriptional level, including RAS/RAF/MEK signaling. Here we describe the synergistic effects of combining BET and MEK inhibitors in various solid and hematologic cancer models. We observe synergistic growth inhibition and apoptosis in a subset of cell lines representing multiple tumor types, as well as patient-derived xenograft models treated with the combination ex vivo. Additionally, combination of BET and MEK inhibitors results in improved tumor growth inhibition in cell line xenograft models compared to either single agent therapy. Further exploration of the combination in sensitive tumor types highlights multiple mechanisms potentially driving synergy, and suggests possible markers associated with sensitivity to the combination. Taken together, our data highlight the potential of BET/MEK inhibitor combinations to improve upon the efficacy observed for these agents as monotherapies in a wide variety of preclinical cancer models. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed by the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed. Human biological samples were sourced ethically and their research use was in accord with the terms of the informed consents. Citation Format: Anastasia Wyce, Daniel J. Felitsky, Xi-Ping Zhang, Jeanne J. Matteo, Susan Korenchuk, Lijoy K. Mathew, Melissa Musso, Sakina Khaku, Victoria Ortiz, Kathryn Keenan, Melissa Stern, Yan Degenhardt, Ramona Plant, Charles F. McHugh, Peter J. Tummino, Christopher Carpenter, Olena Barbash. Broad activity for the combination of BET and MEK inhibitors across solid and hematologic cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4709.

Abstract B177: RXDX-107, a dodecanol alkyl ester of bendamustine, demonstrates improved pharmacokinetic properties and significant anti-tumor efficacy in preclinical models of solid tumors

Abstract Bendamustine is an alkylating agent that can form interstrand DNA crosslinks (ICLs) and cause cell death via several pathways, including intrinsic apoptosis. Bendamustine has proven to be a potent and effective treatment for patients with chronic lymphocytic leukemia (CLL) and rituximab-refractory indolent non-Hodgkin's lymphoma (NHL), with additional clinical activity seen in other B-cell malignancies and in multiple myeloma (MM). However, the activity of bendamustine in the treatment of solid tumor malignancies has been less impressive, possibly due to the pharmacokinetic and biodistribution properties of bendamustine. RXDX-107 is a dodecanol alkyl ester of bendamustine, and the clinical drug product is encapsulated in human serum albumin (HSA) to form nanoparticles. RXDX-107 was designed to improve tissue biodistribution over bendamustine, which may result in superior efficacy and tolerability in patients with solid tumors. In in vitro anti-proliferative studies, RXDX-107 displayed dose-dependent cytotoxicity against multiple solid tumor cell lines, including non-small lung cancer (NSCLC), breast and ovarian cancer. While the IC50s of RXDX-107 were comparable to those of bendamustine, RXDX-107 displayed stronger induction of pH2AX (a biomarker for DNA damage) and more complete cell killing than bendamustine in multiple cell lines. Furthermore, RXDX-107 potently inhibited subcutaneous tumor growth in cell line-derived xenograft (CDX) models of human NSCLC and breast cancer. Importantly, RXDX-107 also showed single agent anti-tumor activities, including tumor regression in multiple patient-derived xenograft (PDX) models of advanced solid tumors, such as breast, lung, and ovarian cancer. In conclusion, our preclinical data demonstrate potent and broad anti-tumor activity of RXDX-107 across a variety of solid tumor types, and support further clinical development of this novel drug candidate for the treatment of solid tumors. Citation Format: Leenus Martin, Roopal Patel, Anthony Drager, Renee Roemmele, Bradley McIntyre, Robert McKean, Piyush Patel, Peter Chua, Jerry Cao, Robert Shoemaker, Peter Brown, Pratik Multani, Robert Wild, Ralph Lin, Gang Li. RXDX-107, a dodecanol alkyl ester of bendamustine, demonstrates improved pharmacokinetic properties and significant anti-tumor efficacy in preclinical models of solid tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B177.

Abstract A187: Selective HDAC inhibition by ACY-241 enhances the activity of paclitaxel in solid tumor models

Abstract Combinations of HDAC inhibitors with chemotherapeutic agents have demonstrated evidence of clinical benefit, for example in ovarian cancer combining belinostat with carboplatin and paclitaxel (Dizon, Int J Gynecol Cancer, 2012), and disease stabilization with vorinostat and tamoxifen in hormone receptor therapy-refractory breast cancer patients (Munster, Br J Cancer, 2011). However, the toxicity profile of pan-HDAC inhibitors frequently limits their use in combination with other drugs commonly used in hematologic and solid tumors. The search for combination therapies that are better tolerated has motivated the development of more selective HDAC inhibitors with improved safety profiles. ACY-241 is a new, orally available and selective histone deacetylase (HDAC) 6 inhibitor under clinical development in combination with pomalidomide and dexamethasone in multiple myeloma (NCT02400242). ACY-241 has the potential for a substantially reduced side effect profile versus current nonselective HDAC inhibitor drug candidates due to reduced potency against Class I HDACs while retaining the potential for anticancer effectiveness. In this study the potential activity of ACY-241 in combination with paclitaxel was explored in preclinical models of solid tumors. In cell lines generated from multiple solid tumor lineages, combination treatment with ACY-241 and paclitaxel resulted in enhanced inhibition of proliferation and increased cell death relative to either single agent alone. The combination of ACY-241 plus paclitaxel also demonstrated enhanced efficacy in xenograft models of pancreatic and ovarian cancer relative to either single agent. Combination dosing was well tolerated as treated animals continued to gain weight throughout the dosing period, and similar results were obtained when ricolinostat, an alternative HDAC6 inhibitor, was tested in combination with paclitaxel. Beyond cell cycle arrest and apoptosis, treatment with paclitaxel was recently shown to induce multipolar spindle formation during mitosis, subsequently leading to aberrant cell division and death (Zasadil, Sci Transl Med, 2014). While ACY-241 treatment alone had minimal effect on mitotic spindle formation, combination treatment with ACY-241 and paclitaxel resulted in more frequent occurrence of mitotic cells with multipolar spindles. Thus, in addition to potentially blocking entry into S phase, combination treatment also leads to increased frequency of aberrant mitoses. At the molecular level, treatment with paclitaxel results in enhanced stability of microtubules, which in turn leads to increased acetylation of α-tubulin. HDAC6 directly regulates acetylation of α-tubulin, and ACY-241 treatment results in a dose-dependent increase in α-tubulin hyperacetylation. The enhanced anti-cancer efficacy resulting from combination treatment with ACY-241 and paclitaxel in cell lines was associated with further increased hyperacetylation of α-tubulin, suggesting these agents synergistically impact the regulation of tubulin biology. The enhanced efficacy of ACY-241 plus paclitaxel observed here, in addition to the anticipated superior safety profile of a selective HDAC6 inhibitor, provides a strong rationale for clinical development of this combination in patients with advanced solid tumors. Citation Format: Pengyu Huang, Ingrid Almeciga-Pinto, Morgan Jordan, Min Yang, Simon S. Jones, Steven N. Quayle. Selective HDAC inhibition by ACY-241 enhances the activity of paclitaxel in solid tumor models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A187.

Abstract C103: The combination of PI3kδ-selective inhibition and immunomodulation shows efficacy in solid tumor models

Abstract Understanding the in vivo responses to immunoregulatory agents provides a basis for building more efficacious combination regimens. Pharmacologic inhibition of the oncogenic PI3Kδ pathway has been shown to be active in patients with hematopoietic malignancies. Recently, genetic inactivation of PI3Kδ in mice was shown to delay the growth of solid tumors, through the inactivation of Treg-mediated suppression of cytotoxic CD8+ T cell responses, suggesting that it may have additional utility in this patient population. We identified a similar immunomodulatory role for the PI3Kδ-selective inhibitor INCB050465 in a preclinical model of pancreatic cancer, where an increase in the number of CD8+ T cells, a decrease in the number of suppressor cells and efficacy were seen. Therefore we explored the potential of INCB050465 in additional preclinical solid tumor models, alone and in combination with other immunotherapeutic agents. INCB050465 inhibited tumor growth in multiple established tumor models which are not dependent upon oncogenic PI3K signaling. Tumor growth inhibition was not observed in these models in immunocompromised mice, demonstrating that the anti-tumor effects of these agents require an intact immune system. To further investigate immune-mediated mechanisms, tumors were analyzed for modulation of gene expression and immune phenotype after mice received short-term treatment. INCB050465 was shown to significantly downregulate the T cell gene signature in tumors, and this was primarily due to depletion of CD4+CD25+FoxP3+ regulatory T cells. As seen previously, the number of CD8+ T cells was shown to be higher in INCB050465-treated tumors. The combination of PI3Kδ and JAK inhibition resulted in enhanced activity in a T-cell-inflamed model by reducing both Treg and M2 macrophages, which promotes re-activation of both CD4+ and CD8+ T cells. In addition, PI3Kδ inhibition and PD-L1 blockade resulted in enhanced efficacy by depleting Treg and prolonging T cell response over time. In summary, pharmacological inhibition of PI3Kδ can enhance anti-tumor immunity by depleting Treg while increasing the numbers of cytotoxic CD8+ T cells. These data support clinical evaluation of the mechanism, and further studies to understand the molecular basis of efficacy and complex cellular responses may provide rationale to identify individuals who may benefit from PI3Kδ inhibitor-based immunotherapy combinations in the clinic. Citation Format: Holly K. Koblish, Liang-Chuan Wang, Michael Hansbury, Yue Zhang, Gengjie Yang, Timothy Burn, Paul Waeltz, Mark Rupar, Eddy Yue, Brent Douty, Thomas Maduskuie, Nikoo Falahatpisheh, Yun-long Li, Andrew Combs, Gregory Hollis, Reid Huber, Peggy Scherle. The combination of PI3kδ-selective inhibition and immunomodulation shows efficacy in solid tumor models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C103.

CBIO-14TARGETING PROTEASOME ACTIVITY WITH MARIZOMIB AS A THERAPEUTIC PERSPECTIVE FOR GLIOMA PATIENTS

Inhibition of the Ubiquitin-Proteasome pathway offers promise for the treatment of gliomas, but has yet to be tested because the approved drugs in this class don't penetrate the CNS. Marizomib is a novel second-generation proteasome inhibitor, with advantages over bortezomib and carfilzomib including irreversible inhibition of all three enzymatic activities of the proteasome and superior tolerability. While there are some promising data in preclinical models of solid tumors using proteasome inhibitors, only Marizomib has proven active in intracranial GBM xenografts, prompting the initiation of a Phase I trial in GBM in combination with bevacizumab. We aim to identify potential biomarkers predictive of response to Marizomib in GBM patients. Tumor tissues obtained from glioma patients at Toronto Western Hospital and utilized for proteasome activity assay. Glioma Stem Cells (GSC) isolated from freshly resected gliomas and used for in-vitro characterization of Marizomib treatment on GSCs. DNA and RNA were isolated from tumors of the patients (including those participating in the Phase I trial) for mutational analysis and expression of a cancer gene panel. Data shows significant increase in all three proteasome activities, Chymotrypsin-Like, Trypsin-Like and Caspase-Like, in GBM tumors compared to normal brain tissues, suggesting a role for the proteasome in GBM formation. Furthermore, progression of low-grade astrocytoma to GBM is associated with enhanced Chymotrypsin-Like and Trypsin-Like activities, indicating that targeting these subunits could potentially inhibit the progression of gliomas. In vitro analysis using GSC lines indicates that the GSC cells of proneural and mesenchymal origin differ in response to Marizomib, raising the possibility that molecular signatures associated with GBM subtypes could determine the therapeutic response to Marizomib in GBM patients. In conclusion, Marizomib represents as a potential therapeutic agent for GBM. Further investigation is necessary to assess the therapeutic benefits of Marizomib as a single agent or combined with other agents.

Tasquinimod in the treatment of castrate-resistant prostate cancer – current status and future prospects

Treatment options have significantly expanded in recent years for men with metastatic castration-resistant prostate cancer (mCRPC), with the routine use of immunotherapy (sipuleucel-T) and novel hormonal agents such as enzalutamide and abiraterone acetate prior to taxane-based chemotherapy or radium-223 radiotherapy. A number of immune checkpoints limit the immune response of the host to metastatic tumor progression in prostate cancer, one of which is an immunosuppressive and pro-angiogenic cell called the myeloid-derived suppressor cell (MDSC). Tasquinimod is a small molecular oral inhibitor of S100A9, a key cell surface regulator of MDSC function, and has shown anti-angiogenic, antitumor and immune-modulatory properties in preclinical models of prostate cancer and other solid tumors. A large randomized phase II trial of tasquinimod in men with chemotherapy-naïve mCRPC demonstrated a significant prolongation in radiographic and symptomatic progression-free survival compared with placebo, which was also associated with improvements in overall survival. Tasquinimod was studied in a global phase III randomized trial in men with bone mCRPC and, while it significantly improved radiographic progression-free survival, this did not result in an overall survival benefit. However, tasquinimod is under evaluation as well as a combination therapy with other systemic agents in prostate cancer and as a single agent in other solid tumors. This review encompasses the preclinical and clinical development of tasquinimod as a therapy for men with prostate cancer.

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.