Abstract: Redox homeostasis in an inflammatory environment is crucial to ensure a balanced immune response. Several inflammatory disorders are associated with impairment of redox homeostasis, leading to DNA damage and subsequent immune dysregulation. The transcription factor STAT3 has anti-inflammatory activity in myeloid cells, mediated by inhibition of Ubc13, a key Toll-like receptor (TLR) signaling component. STAT3 anti-inflammatory activity is crucial in protection from systemic inflammation, maintenance of lineage-balanced hematopoiesis, and inhibition of DNA damage accumulation in hematopoietic progenitors. The molecular cues by which STAT3 mitigates DNA damage in an inflammatory environment, however, are poorly understood. Recently, we found that STAT3-deficient bone marrow derived-macrophages (BMDMs) exhibited increased oxidative stress upon TLR4 stimulation, including reduced antioxidant amounts and elevated production of reactive oxygen species (ROS). These responses were accompanied by increased gH2AX foci formation, suggesting accumulation of DNA damage. By contrast, BMDMs lacking Ubc13 exhibited increased expression of antioxidants and reduced DNA damage. RNA-sequencing analysis identified transcriptional targets of STAT3 and Ubc13 in steady state and TLR-stimulated BMDMs. Collectively, our findings suggest STAT3-mediated control of Ubc13 is important in regulating inflammation-induced oxidative stress and DNA damage by modulating antioxidant signaling and DNA damage repair pathways. Thus, STAT3 plays a crucial role in metabolic reprogramming of macrophages during inflammation to preserve redox homeostasis.

Abstract: INTRODUCTION: Tumor-released exosomes have pleiotropic functions in promoting autocrine signaling to distant cells. Elucidating the mechanistic modulation of the immune system by these exosomes provides insights into potential biomarkers for detection, recurrence and response and identifies potential new therapeutic targets. METHODS: Exosomes were isolated from human glioblastoma stem cells (GSCs) and fibroblasts (control) using differential centrifugation and characterized by nanosight technology, electron microscopy, and western blotting. Fluorescent-labeled exosomes were co-cultured with human immune cells. Confocal microscopy was used to determine the preferential uptake in various immune populations and to evaluate the intracellular trafficking. The cell-secreted exosome content was characterized by mass spectrometry and nanostring technology. The phenotypic and functional skewing of the monocyte lineage was analyzed given its propensity to take up exosomes. RESULTS: GSC-secreted exosomes were homogenous in morphology, ranged from 50-120 nm in size, and expressed CD63 and CD9 surface molecules. The GSCs-produced exosomes were preferentially absorbed by CD14+ monocytes (precursors to macrophages) and Gr-1+ derived myeloid cells isolated from healthy volunteers and/or glioblastoma patients. When activated, CD4+ and CD8+ T cells, but not NK cells could also uptake exosomes. Longitudinal kinetic studies established that the highest uptake of PKH67-labeled GCS-secreted exosomes occurred at 48 hours after exposure. Confocal microscopy revealed that monocytes could only internalize GSC-released exosomes but not fibroblast-secreted exosomes. The exposure to GSC-secreted exosomes induced a phenotypic change in monocytes and prevented them from undergoing apoptosis. Studies of M1/M2 macrophage markers by flow cytometry revealed that GSCs-secreted exosomes, but not the fibroblast-secreted exosomes, increased expression of CD80, CD163, CD206 and decreased expression of MHC class II. This profile was similar to myeloid suppressor cells and macrophages that were obtained directly ex vivo from glioblastomas (n = 17). The GSC-secreted exosomes were preferentially enriched relative to fibroblast-secreted exosomes in transcriptional regulators that induced the M2 phenotype. CONCLUSIONS: Monocytes demonstrate preferential uptake of GSC-secreted exosomes which then induces a glioma-supportive M2 phenotype - similar to the phenotype observed in myeloid cells and macrophages isolated from human glioblastomas. This data indicates that the GSC-secreted exosomes can be a contributing factor in the M2 skewing within the tumor microenvironment. Citation Format: Konrad Gabrusiewicz, Yuuri Hashimoto, Jun Wei, Maiti Sourindra, John Yu, Shinji Yamashita, Anna Zal, Tomasz Zal, Laurence Cooper, Amy B. Heimberger. Glioblastoma stem cell-secreted exosomes can induce a tumor supportive M2 response. [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 5088. doi:10.1158/1538-7445.AM2015-5088

Abstract: Regulatory T cells (Tregs) expressing the forkhead transcription factor Foxp3 play a central role in the dominant control of immunological tolerance. Compelling evidence obtained from both animal and clinical studies have now linked the expansion and accumulation of Foxp3+ Tregs at the tumor site to the failure of immune-mediated tumor rejection. However, little is known regarding tumor-associated Foxp3+ Tregs within tumor tissues. Using both subcutaneous (s.c.) tumor and lung metastasis models of MCA-205 fibrosarcoma, we have found that tumor-associated Foxp3+ Tregs are highly activated and undergo vigorous cellular proliferation. Tumor-associated Foxp3+ Tregs were also found to be more potent functionally by in-vitro suppression assays and express higher levels of membrane-bound TGF-b1. To visualize endogenous Foxp3+ Tregs, we have generated Foxp3-GFP transgenic reporter mice by inserting GFP into a bacterial artificial chromosome (BAC) encompassing the Foxp3 locus. Using multi-photon microscopy, we observed a rapid recruitment and accumulation of Foxp3+ Tregs to the nascent lung tumor metastasis as early as 3 days after tumor inoculation. Intravital imaging of established s.c. tumors revealed a distinctive behavior of Foxp3+Tregs: the suppressor cells form a well-defined periperipheral layer encasing the tumor mass, moving rapidly within the superficial plain but not perpendicularly in and out of the tumor. Together, these data highlight that tumor-associated Foxp3+ Tregs are highly dynamic in function, behavior, and origin, perhaps contributing to overall poor immunogenicty of tumors.

Abstract: Annamycin (Ann) is an anti-tumoral anthracycline whose anti-leukemia activity is relatively unaffected by P-glycoprotein-related multidrug resistance. Unlike for the related doxorubicin (DOX), Ann accumulates in multidrug resistant cell lines, which is accompanied by DNA damage and apoptosis. In preclinical toxicology studies, in contrast to DOX, free Ann displayed a greatly reduced cardiotoxicity, while L-Ann appeared to be non-cardiotoxic. A liposomal formulation of Ann, termed L-Annamycin (L-Ann), is currently evaluated in patients with acute myeloid leukemia (AML). Anti-leukemia activity of Ann was demonstrated in several leukemia models as judged by circulating blast cytoreduction and extension of overall survival. However, the efficacy of L-Ann in the microenvironment of the bone marrow and other organ tissues remains unclear. In the current study, we assessed the anti-AML efficacy of Ann in a novel AML model that allows visualizing the dynamics of individual AML cells in vivo by two-photon microscopy. In this model, mouse AML cells bearing the MLL/ENL-FLT3/ITD[p53-/-] mutations co-express high levels of the cyan fluorescent protein mTurquoise2. Upon intravenous infusion of several tens of thousands cells into syngeneic immunocompetent C57BL6 mice, lethal AML disease reliably develops within 2 weeks. Using host mice expressing appropriate fluorescence reporter genes, the bright cyan fluorescence enables sensitive intravital imaging of individual AML cells in the context of organ architecture. Using this model in Thy1-RFP reporter mice expressing red fluorescence in all organ tissues with the blood flow marked by BSA-AF647 fluorescence, we evaluated AML cellularity reduction in the bone marrow and other organs after a single dose of L-Ann as well as in response to chronic treatment. In addition, we assessed the localization of the surviving AML cells at a high spatial resolution. We evaluated the in vivo organ biodistribution of intravenously infused L-Ann in C57BL6 mice by flow cytometry and two-photon microscopy based on the intrinsic fluorescence of the drug. In addition, we visualized the intracellular compartmentalization of L-Ann using confocal microscopy. Consistent with in vitro findings, we observed a rapid and deep reduction of AML blasts in the peripheral blood after a single dose of L-Ann in a dose-dependent manner (1-4 mg/kg). This reduction was strongly correlated with prolongation of animal survival from 14 days (vehicle) to 37 days (L-Ann 4 mg/kg once weekly started on day 10). In vitro and intravital microscopy revealed a distinct pattern of L-Ann distribution in organ tissues, which correlated in part with the local index of AML cellularity reduction and residual disease localization. Interestingly, in addition to the expected uptake of Ann in the cell's nucleus, Ann was also accumulated in the cytosol of the cells. This bi-compartmental intracellular distribution pattern contrasted with the nuclear-only localization of DOX. Administration of L-Ann early in the course of AML resulted in occasional complete responses some of which associated with resistance to AML re-challenge, suggesting capacity for anti-AML immune memory induction. This study confirms the efficacy of the drug in the model setting of syngeneic, immune-competent AML. Besides reinforcing the rationale for further development of Annamycin in AML, this study demonstrates a highly advantageous AML mouse model that is highly informative in studies of AML pharmacology, minimum residual disease (MRD), microenvironment and immunology. Disclosures Fokt: Moleculin Biotech, Inc.: Equity Ownership, Research Funding. Andreeff:Oncoceutics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AstaZeneca: Consultancy; 6 Dimensions Capital: Consultancy; Reata: Equity Ownership; Aptose: Equity Ownership; Eutropics: Equity Ownership; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees. Priebe:Moleculin Biotech, Inc.: Consultancy, Equity Ownership, Research Funding. Zal:VueBio.com: Equity Ownership; BioLineRx: Research Funding; Daiichi-Sankyo: Research Funding; Moleculin Biotech, Inc.: Research Funding; NIH-CTEP: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding.

Abstract: We and others have previously reported that leukemia progression is associated with vast expansion of the hypoxic niches and stabilization of hypoxia-inducible factor 1 alpha (HIF-1α) in leukemic cells (Frolova et al. Cancer Biol Ther. 2012, 10:858; Benito et al. PLoS One 2011, 6(8); e23108:1). Interactions of leukemia and the bone marrow (BM) microenvironment are known to play a key role in the survival and growth of leukemic cells, and we have shown that HIF-1α stabilization in stromal cells of the microenvironment facilitates leukemia homing and progression (Chen et al. Blood 2012, 119:4971). In this study, we aimed to characterize the time-dependent progression of BM hypoxia involving both leukemia cells and components of the BM niche, using the multiphoton intravital microscopy (MP-IVM) technique. We first generated a transplantable, imageable leukemia model by retrovirally transducing C57Bl6-Ai14 murine BM cells that express red fluorescing tdTomato with the p190-Bcr/Abl oncogene. The resulting p190-Bcr/Abl tdTomato cells caused rapid development of acute lymphocytic leukemia (ALL) in un-irradiated C57Bl6 immunocompetent mice, manifested by infiltration of the spleen, liver, BM within long bones, skull, and central nervous system followed by death within 28 days. Leukemia cells collected from the BM (LBC) of these animals were transplantable into secondary recipients and triggered accelerated ALL development (14-16 days). Time-course analysis of skull and femur bones in the secondary recipients by MP-IVM demonstrated LBC lodging on day 1 after ALL cell injection, followed by rapid accumulation of leukemia cells localized predominantly within the sinusoidal spaces, which were visualized by injecting the vascular fluorescent dye BSA-647 (Fig. 1a). To detect in vivo hypoxia development, we utilized HS680 (HypoxiSense 680), a carbonic anhydrase IX (CAIX)–targeted fluorescent agent that can be used to image overexpression of CAIX, a direct HIF-1α target, in tumors in response to regional hypoxia. C57Bl6 mice were engrafted with 2 x 105 LBC , and HS680 was injected intravenously at serial intervals followed by MP-IVM. In two separate experiments, increased HS680 fluorescence was detected in bone-lining cells in the BM niches of mice harboring ALL on days 8 and 13, but not in their healthy littermates (Fig 1b). To obtain an independent confirmation of hypoxia, additional mice (n=3) at the same stage (day 14) of leukemia development were sacrificed 3 hr after injection of chemical hypoxia probe pimonidazole (Pimo), and hypoxic BM cells that bound the hypoxia probe were detected by immunohistochemistry. Pimo staining demonstrated vastly spread areas of hypoxia that enclosed both leukemia cells and BM niche cells (Fig 1c), consistent with our previously published observations in different leukemia models. In summary, these findings demonstrate rapid development of intra-BM hypoxia that parallels leukemia progression and involves not only leukemia cells, but also BM niche cells. The HS680 probe can detect hypoxia in vivo within niche cells but not in leukemia cells, likely because of differential expression of CAIX. Our ongoing studies will characterize the cellular origin of hypoxic niche cells by utilizing immunohistochemical techniques and Col2.3-GFPemd transgenic mice to visualize osteoblasts. We postulate that the tumor microenvironment altered with hypoxic niche cells will influence leukemia development or responses to therapy. To this end, we have generated mice with conditionally deleted HIF-1α within BM stromal cells and are investigating the differences in leukemia homing, progression, and chemoresistance between these mice and mice whose BM stromal cells express HIF-1a. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Abstract: Background: Presence at diagnosis or acquisition of activating RAS pathway mutations is a pervasive mechanism of resistance to therapy in AML. Efforts to directly target mutant RAS have been unsuccessful and the efficacy of BRAF and MEK inhibitors has been limited due to compensatory reactivation of MAPK signaling. ERK1/2 (ERK) is a key downstream component in the MAPK pathway and therefore represents an attractive target for inhibiting MAPK signaling. Compound 27 (1) is a dual-mechanism inhibitor of ERK that inhibits both the catalytic activity of ERK and its phosphorylation by MEK. It is a close analog of ASTX029, a dual-mechanism ERK inhibitor currently under clinical investigation in solid tumors (NCT03520075). Objectives: We analysed the preclinical activity of Compound 27 in AML, investigated its mechanism of action and ability to overcome resistance. Results: Using a panel of 9 AML cell lines, the IC50 value for single agent Compound 27 was in the low to intermediate nanomolar range (1.89-388 nM). Decreased ERK phosphorylation was confirmed by Western blot analysis. To better characterize the biological effects of Compound 27, we performed mass cytometry (CyTOF) analysis of NRAS-mutated OCI-AML3 cells. This experiment showed approximately 75% downregulation of CyclinB1 and cMyc in 250 nM drug-treated cells versus untreated cells (Figure 1a). The expression of anti-apoptotic proteins, including MCL1, BclXL and Bcl2, were also decreased. Western blot analysis confirmed increased cleaved PARP, and reduced cMyc and cell cycle-related proteins CyclinB1, CyclinD1 and CDK4 with Compound 27 treatment. In isogenic cells, p53 knock-down had no effect on the efficacy of Compound 27. We next investigated the efficacy of simultaneous inhibition of ERK and Bcl-2 in AML cells. Compound 27 sensitized OCI-AML3 cells, which are intrinsically resistant to ABT-199 (a Bcl-2 inhibitor), to treatment with ABT-199 and shifted the cytostatic effect of the single agents to a cytotoxic effect with a combination index (CI) of 0.008 (cell death 91% for combination versus 20% with ABT-199 alone). This suggests strong synergistic effects of combination treatment (Figure 1b). In OCI-AML2 cells with acquired resistance to ABT-199, the combination increased apoptosis to 80% as compared to 20% with ABT-199 alone. Compound 27 sensitized bulk CD45+ as well as CD34+CD38-leukemia progenitor cells to ABT-199. Compound 27 also sensitized FLT3-ITD mutant human AML cell lines MOLM13, MOLM14, MV-4-11 and murine Ba/F3-ITD cells to the FLT3 inhibitor AC220 (CI in MOLM13=0.3). Synergy of Compound 27 and 5-azacitidine was also observed (p=0.009). Leukemia microenvironment-mediated resistance to therapy is partly mediated by MAPK activation. We co-cultured OCI-AML3 and MOLM13 cells with normal bone marrow-derived mesenchymal stromal cells (NMSCs) to mimic the bone marrow microenvironment and analysed the effect of Compound 27 in combination with either ABT-199 or AC220. Combination drug treatment were more effective in terms of cytoreduction and apoptosis induction in coculture. However, neither combination was able to completely overcome stroma-mediated resistance (Figure 1b). Analysis of other stroma-relevant molecules in coculture showed that CXCR4 was increased while CD44 was decreased in response to ERK inhibition. Effective reactive oxygen species (ROS) mitigation and hyper-active mitochondrial fission is important for maintaining "stemness" of AML cells (2). ERK phosphorylates DRP1, which is necessary for mitochondrial fission. Treatment of OCI-AML3 cells with Compound 27 led to increased mitochondrial ROS, decreased levels of pDRP1(Ser616) and increased mitochondrial length, suggesting impaired fission and reduced "stemness" of AML cells (Figure 1c). Conclusion: ERK inhibition by Compound 27 synergizes with 5-azacitidine, ABT-199 and AC220 and can overcome primary or acquired resistance. The impact on mitochondrial dynamics suggests a potential impact on leukemia stem cells. Additional mechanistic confirmatory work is in progress. References: 1. Heightman TD, Berdini V, Braithwaite H, et al. Fragment-based discovery of a potent, orally bioavailable inhibitor that modulates the phosphorylation and catalytic activity of ERK1/2. J Med Chem. 2018;61(11):4978-4992. 2. Schimmer AD. Mitochondrial Shapeshifting Impacts AML Stemness and Differentiation. Cell Stem Cell. 2018;23(1):3-4. Figure 1 Figure 1. Disclosures Hindley: Astex Pharmaceuticals: Current Employment. Dao: Astex Pharmaceuticals, Inc.: Current Employment. Sims: Astex Pharmaceuticals: Current Employment. Andreeff: Medicxi: Consultancy; Syndax: Consultancy; Aptose: Consultancy; ONO Pharmaceuticals: Research Funding; AstraZeneca: Research Funding; Amgen: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Breast Cancer Research Foundation: Research Funding; Karyopharm: Research Funding; Glycomimetics: Consultancy; Senti-Bio: Consultancy; Oxford Biomedica UK: Research Funding; Daiichi-Sankyo: Consultancy, Research Funding; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees. Borthakur: University of Texas MD Anderson Cancer Center: Current Employment; Takeda: Membership on an entity's Board of Directors or advisory committees; Astex: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ryvu: Research Funding; ArgenX: Membership on an entity's Board of Directors or advisory committees; Protagonist: Consultancy; GSK: Consultancy.