Malignant Cell Death Research Articles

126 - Selectively targeting the malignant clone through the cellular redox state

Antioxidant compounds that can mimic the activity of endogenous enzymes via redox cycling offer a novel method of targeted cancer treatment. We investigated the therapeutic effect of a mitochondrial- targeted, MnSOD mimetic, Mn(III) mesotetrakis (N-n-butoxyethylpyridinium-2yl) porphyrin, (MnTnBuOE) within myeloid lineage hematopoietic malignancies and utilize the hematopoietic disorder myelodysplastic syndrome (MDS) for the presentation of mechanistic insights to MnTnBuOE efficacy. Consistent with our previous report, MnTnBuOE treatment offers protection of normal HSCs demonstrated by an increase in both CD34+CD38- and CD34+CD38+ stem/progenitor cell viability. However, similar treatment shows significant cytotoxicity mediated by MnTnBuOE induced hydrogen peroxide within acute myeloid leukemia (AML) and MDS models. Transcriptional array analysis indicates that MnTnBuOE treatment initiates activator protein 1 (AP-1) activity. DNA mediated pull down and immunoprecipitation identifies JunB as a transcriptional mediator of MnTnBuOE induced cytotoxicity. Stable knock down of JunB provides protection against MnTnBuOE induced cytotoxicity confirming JunB’s critical role in MnTnBuOE induced hematopoietic malignant cell death. Precipitation with biotin-1,3-cyclopentadione combined with LC-MS/MS analysis of JunB identified Cys285 as the critical cysteine within the DNA binding region of JunB that is susceptible to oxidation by hydrogen peroxide. This, in concert with co-immunoprecipitation of JunB placed Ref-1 as a protein specifically involved in the redox-modification of JunB. Lentiviral mediated knock down of Ref-1 indicates that the interaction between Ref-1 and JunB is critical for MnTnBuOE induced cytotoxicity. The AP-1 ability to bind consensus promoter sequence regardless of methylation status, indicates MnTnBuOE mediated activation of JunB as a potential alternative therapy for aggressive or chemoresistant malignancies in a p53 independent manner. The finding that MnTnBuOE can exert selective cytotoxicity in malignant cells via redox-dependent differential activation of molecular pathways controlling cell fate will significantly aid further development of safe, effective redox-active therapeutics to prevent normal tissue injury and enhance chemotherapeutic efficacy.

An investigation of metronomic photodynamic therapies for local prostate cancer: supplementary fluorescent photography and proteomic studies, as measurable parameters of this treatment.

Aim: To investigate the clinical effects of metronomic Photodynamic therapy in prostate cancer patients, using different chlorophyll based photosensitisers. Methods and materials: The patients who enrolled in this ethics approved Phase 1 prostate cancer trial were treated with a variable combination of targeted photo-dynamic therapies and coupled ultrasound energies, mediated through orally/sub-lingually administered chlorophyll based photosensitisers. The metronomic technique involves the repeated administration of both the photosensitiser(s) and energies over set time intervals. As scientific assessment of the prostate cancer effects, intra-treatment fluorescent photography of the prostate gland /prostatic fossa, together with proteomic based urine specimen studies were performed. Results: The clinical data involved 666 treatment episodes, 333 photosensitiser dosages and includes 3-5 years patient follow-up. The effects on the prostate symptoms and cancer are documented, prostate size reduction being a serendipitous finding. Real time fluorescent photography has documented qualitative reproducible data. The quantitative urinary proteomic studies demonstrated statistically significant results, as documentation of the known pathological effects, attributable to this treatment. Conclusion: This study has been shown to be safe and to meet the criteria required of a TGA Phase 1 trial and reports the clinical and investigational prostate cancer responses to the metronomic therapy, involving the three different photosensitisers. The scientific bases for this therapy have been validated with the real-time fluorescent photographic and proteomic analyses data, of particular interest and importance are the immune system results. The new word metapoptosis is suggested as the description for PDT induced malignant cell death in association with immune stimulation, as described in this study.

Abstract 2618: The thymidine kinase 1 (TK1) protein concentration in sera from Hodgkin lymphoma patients: A marker for cell death and disruption of malignant cells

Abstract TK activity measurements have been used for many years to monitor cancer disease activity. However, what the blood concentrations of TK1 protein reflect is still uncertain. A TK1 immunoassay, the AroCell TK 210 ELISA, has been developed based on specific monoclonal antibodies against the C-terminal region of TK1. We utilized this assay to monitor the blood concentrations of TK1 in a cohort of patients with Hodgkin lymphoma (HL) before and after conventional treatment. TK1 concentrations in serum or plasma were measured using the AroCell TK 210 ELISA. For comparison, a large number of other biomarkers and cells were measured in blood such as C-reactive protein (CRP), Lactate dehydrogenase (LDH), hemoglobin, WBC, platelet counts, erythrocyte sedimentation rates (ESR), albumin, liver enzymes, creatinine. Fifty-eight patients with HL were included before start of treatment. Seventy three percent had the nodular sclerosis subtype and 52% stages I-II and 48% stages III-IV disease. Blood was sampled before treatment, during treatment and twice after completion of treatment. The upper normal TK1 concentration of healthy subjects (n=250) was 0.45 μg/L with a median of 0.25 μg/L (range 0.15-0.66 μg/L) as compared to HL, which was 0.26 μg/L (range 0.03-17 μg/L). The differences in ranges were significant (p<0.0001, F-statistics). Nodular sclerosis patients had higher TK1 levels as compared to the other subtypes (p=0.03). TK1 increased after treatment (p=0.001) in patients with normal concentrations from start i.e. <0.45 μg/L, whereas the concentrations decreased in 5/6 patients with TK1 >0.45 μg/L. TK1 concentrations before start of treatment was correlated to high CRP (p=0.01), low Hemoglobin (p=0.03), high WBC (p<0.001) and high platelet counts (p<0.01), but not to albumin or ESR or any other of the measured biomarkers. In a multivariate regression analysis, TK1 was correlated to LDH as the only independent variable (p<0.0001). In Hodgkin lymphoma patients, serum or plasma concentrations of TK1 may reflect malignant cell death and cell disruption rather than cell proliferation during treatment. Our results and hypothesis may help to interpret TK1 protein measurements as clinical tools in cancer management. Citation Format: Jagarlamudi Kiran Kumar, Staffan Eriksson, Johan Mattsson Ulfstedt, Per Venge, Daniel Molin. The thymidine kinase 1 (TK1) protein concentration in sera from Hodgkin lymphoma patients: A marker for cell death and disruption of malignant cells [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 2618.

Ginkgolic acids induce HepG2 cell death via a combination of apoptosis, autophagy and the mitochondrial pathway.

Ginkgolic acids may induce malignant cell death via the B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 apoptosis pathway. Concurrently, apoptosis, autophagy and mitochondrial dysfunction may also be involved in bringing about this endpoint. The anticancer effect of Ginkgolic acids (GAs) was investigated using the HepG2 cell line. The median lethal dose of the GAs of the HepG2 was measured via an MTT assay, the dose-response curves were evaluated and changes in cell morphology were monitored by microscopy. Autophagy in HepG2 cells was down regulated using 3-methyladenine (3-MA) or Beclin-1-specific small interfering RNA (siRNA) and the expression of apoptosis associated proteins caspase-3, Bax/Bcl-2, and the autophagy-associated protein 5 and microtubule-associated protein 1A/1B-light chain 3 in the GA-treated HepG2 cells were all measured by western blot analysis. The level of apoptosis in the GA-treated cells was also assessed using terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling (TUNEL) assay, and the mitochondrial membrane potential (Δψm) was detected by immunofluorescence. The results of the MTT and TUNEL assays indicated that the proliferation of HepG2 cells treated with GAs was significantly reduced compared with the control group, and the rate of the inhibition was dose-dependent. Western blot analysis indicated that treatment with the Gas induced apoptosis and autophagy in the HepG2 cells. The Δψm of the GA-treated HepG2 cells was decreased compared with the control, as monitored by immunofluorescence. However, upon the administration of 3-MA or Beclin-1-specific siRNAs (inhibitors of the autophagy), the expression levels of the apoptosis- and autophagy-associated proteins were decreased. In conclusion, the results of the present study indicated that GAs are potent anticancer agents that function through a combination of the apoptosis, autophagy and mitochondrial pathways.

Monanchoxymycalin C with anticancer properties, new analogue of crambescidin 800 from the marine sponge Monanchora pulchra

A new pentacyclic guanidine alkaloid, monanchoxymycalin C (1) was isolated from a new collection of marine sponge Monanchora pulchra along with the known monanchoxymycalin A (2). The structure of 1 was elucidated on the basis of spectroscopic data. Monanchoxymycalin C exhibits cytotoxic activity against human cancer HeLa cells at low micromolar concentrations, induces apoptosis-related death of malignant cells and inhibits cancer cell colony formation. In addition, synergistic and additive effects have been observed in combination with cisplatin.

Structure–Activity Relationships within a Series of σ1 and σ2 Receptor Ligands: Identification of a σ2 Receptor Agonist (BS148) with Selective Toxicity against Metastatic Melanoma

A new series of spirocyclic σ receptor (σR) ligands were prepared and studied. Most were found to have a high affinity and selectivity for σ1 R; three compounds were shown to be σ1 R agonists, while another proved to be the only σ1 R antagonist. Only one of the σ1 R agonists (BS148) also exhibited σ2 R selectivity and was able to inhibit the growth of metastatic malignant melanoma cell lines without affecting normal human melanocytes. The antiproliferative activity of this compound suggested an σ2 R agonist profile. Further, preliminary investigations indicated that the mechanism of metastatic malignant melanoma cell death induced by BS148 is due, at least in part, to apoptosis.

The renaissance of anti-neoplastic immunity from tumor cell demise.

Cancer therapies can temporarily reduce tumor burdens by inducing malignant cell death. However, cancer cure is still far from realization because tumors often gain resistance to current treatment and eventually relapse. Accumulating evidence suggests that successful cancer interventions require anti-tumor immunity. Therapy-induced cell stress responses ultimately result in one or more cell death modalities, including apoptosis, autophagy, necroptosis, and pyroptosis. These irreversible dying processes are accompanied by active or passive release of cell death-associated molecular patterns (CDAMPs), which can be sensed by corresponding pattern recognition receptors (PRR) on tumor-infiltrating immune cells. This crosstalk with the immune system can reawaken immune surveillance in the tumor microenvironment (TME). This review focuses on immune-modulatory properties of anti-cancer regimens and CDAMP-mediated communications between cell stress responses and the immune contexture of TME. In addition, we describe how immunogenic cell death can elicit strong and durable anti-tumor immune responses.

ID:3005 Combination of vaccine-strain measles and mumps viruses enhances oncolytic activity against human solid malignancies: special focus on prostate cancer

Oncolytic viruses (OLVs) including measles and mumps viruses (MeV and MuV) have a potential to serve as a therapeutic option for cancers. We have previously shown that the combination of MeV and MuV synergistically kills various human haematological cancer cells. This study aims to investigate the anti-tumor activity of MeV, MuV and MeV-MuV combination (MM) against human solid malignancies in vitro and in vivo. The results showed that MeV, MuV and MM combination targeted and effectively killed various cancer cell lines of human solid malignancies but not normal cells. Notably, MM combination demonstrated a greater anti-tumor effect and prolonged survival in a human prostate cancer (PC3) xenograft tumour model compared to MeV and MuV. MeV, MuV and MM combination significantly induced the expression of immunogenic cell death (ICD) markers and enhanced spleen-infiltrating immune cells such as macrophages, natural killer and dendritic cells. Our study demonstrated that MM combination is a promising option for treatment of human solid malignancies and suggested that MM could induce immunogenic cell death of malignant cells and activate immunity against cancers.

Abstract 4296: A real-time annexin V method for monitoring programmed cell death

Abstract Efficacious and durable anti-cancer responses are driven by both the selective death of malignant cells and the induction of immunostimulatory activities. Limited but promising clinical evidence suggests that provocation of an unknown balance of inflammatory- and non-inflammatory cell death may be key for orchestrating these positive outcomes. Therefore, identifying and characterizing new clinically useful small molecules and biologic inducers (or combinations thereof) which promote a spectrum of programmed cell death in in vitro screening environments remains critically important. Unfortunately, current screening methods are either insufficiently robust, cost- or resource-prohibitive, or provide no means for initial characterization of the kinetics of programmed cell death. To address this unmet need, we developed a real-time, live cell assay method that utilizes a fully homogeneous, bioluminescent annexin V reagent. The method does not require laborious washing and sample preparation steps associated with traditional annexin methods and is fully compatible with plate-based multimodal signal detection systems. The system contains two annexin proteins which have been engineered to contain separate and distinct complementing domains of a binary luciferase. Additionally, the system contains a novel time-released luciferase substrate and a cell impermeable, fluorogenic DNA dye for monitoring necrosis. Because the annexin-luciferase fusion pairs have only modest affinity for each other, luminescence remains low until phosphatidylserine exposure, a hallmark of the programmed cell death phenotype, brings annexin monomers into close proximity facilitating complementation of the luciferase sensor. The assay reagent can be applied at dosing for real-time measurement of the dose-dependency and magnitude of programmed cell death progression. This work describes our efforts to characterize and validate the performance of the bioluminescent annexin assay using relevant cell death induction models. First, the assay was shown to be functionally concordant with a flow cytometry annexin method in a dose-response model with bortezomib at exposure periods known to produce both early and late-apoptosis (with necrosis) phenotypes. Next, we assessed the performance of the assay using a limited training set of small molecule and biologic inducers of programmed cell death that utilize different mechanisms of action (i.e., apoptosis and necroptosis) and by using a number of diverse but representative cancer cell lines. We conclude that the bioluminescent annexin method provides a new kinetic approach to efficient and effective detection of programmed cell death mechanisms in real time in a convenient homogeneous format. Citation Format: Kevin Kupcho, Andrew Niles, John Shultz, Jamison Grailer, Wenhui Zhou, Robin Hurst, Jim Hartnett, Terry Riss, Dan Lazar, James Cali. A real-time annexin V method for monitoring programmed cell death [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 4296. doi:10.1158/1538-7445.AM2017-4296

AKT Axis, miR-21, and RECK Play Pivotal Roles in Dihydroartemisinin Killing Malignant Glioma Cells.

Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, is known to play important roles in inhibiting proliferation rate, inducing apoptosis, as well as hindering the metastasis and invasion of glioma cells, but the underlying mechanisms are still unclear so far. In this study, methyl thiazolyl tetrazolium (MTT), colony-forming, wound healing, invasion, and apoptosis assays were performed to investigate the effect of DHA on malignant glioma cells. Results showed that DHA induced apoptosis of malignant glioma cells through Protein Kinase B (AKT) axis, induced death of malignant glioma cells by downregulating miR-21, and inhibited the invasion of malignant glioma cells corresponding with up-regulation of the reversion-inducing-cysteine-rich protein with kazal motifs (RECK). These results revealed that AKT axis, miR-21, and RECK play pivotal roles in DHA killing malignant glioma cells, suggesting that DHA is a potential agent for treating glioma.

All-Atom Molecular Dynamics Reveals Mechanism of Zinc Complexation with Therapeutic F10.

Advancing the use of therapeutic nucleic acids requires understanding the chemical and structural properties that allow these polymers to promote the death of malignant cells. Here we explore Zn2+ complexation by the fluoropyrimidine polymer F10, which has strong activities in multiple preclinical models of cancer. Delivery of fluoropyrimidine FdUMP in the 10-residue polymer F10 rather than the nucleobase (5-fluorouracil) allows consideration of metal ion binding effects on drug delivery. The differences in metal ion interactions with fluoropyrimidine compared to normal DNA results in conformation changes that affect protein binding, cell uptake, and codelivery of metals such as zinc, and the cytoxicity thereof. Microsecond-time-scale, all-atom simulations of F10 predict that zinc selectively stabilizes the polymer via interactions with backbone phosphate groups and suggest a mechanism of complexation for the zinc-base interactions shown in previous experimental work. The positive zinc ions are attracted to the negatively charged phosphate groups. Once the Zn2+ ions are near F10, they cause the base's N3 nitrogen to deprotonate. Subsequently, magnesium atoms displace zinc from their interactions with phosphate, freeing the zinc ions to interact with the FdU bases by forming weak interactions with the O4 oxygen and the fluorine attached to C5. These interactions of magnesium with phosphate groups and zinc with nucleobases agree with previous experimental results and are seen in MD simulations only when magnesium is introduced after N3 deprotonation, indicating a specific order of metal binding events. Additionally, we predict interactions between zinc and F10's O2 atoms, which were not previously observed. By comparison to 10mers of polyU and polydT, we also predict that the presence of fluorine increases the binding affinity of zinc to F10 relative to analogous strands of RNA and DNA consisting of only native nucleotides.

Abstract 3556: CXCR4 controls BCL-2 expression and function by regulating miR-15a/16-1 expression in tumor cells

Abstract Background: CXCR4 is overexpressed in the majority of tumor cells and its degree of expression often correlates with disease severity. Binding of CXCL12 to the CXCR4 receptor activates signaling pathways which are crucial for the interaction of hematopoietic cells with the microenvironment and cell survival. Signaling activated through CXCR4 was shown to be detrimental by increasing survival of tumor cells and promoting resistance to therapy in many types of cancer. CXCR4-antagonists, such as BL-8040, currently in phase II trials, were shown to selectively inhibit tumor cell growth and to induce apoptosis in vitro and in vivo. However, the molecular mechanism by which CXCR4 overexpression triggers tumor cell survival and its inhibition leads to cell death is not fully understood. Objective: To study the mechanism by which the CXCR4 pathway controls malignant cell survival and death through regulating miR-15a/16-1 expression. Method: We assessed the effect of CXCR4 overexpression, its activation and inhibition, on the expression of miR-15a/16-1 and their target genes, BCL-2, MCL-1 and cyclin D1, in a variety of tumor cells in vitro and in vivo. Results: We found that overexpression of CXCR4 in tumor cells or stimulation of cells with its ligand, CXCL12, lead to up-regulation of miR-15a/16-1, resulting in down-regulation of their target genes BCL-2, MCL-1 and cyclin D1. Furthermore, overexpression of CXCR4 in these cells increases tumorgenesis and shifts their oncogenic dependency from the BCL-2 to the CXCR4/ERK signaling pathway. Antagonists of CXCR4 such as BL-8040 were shown to induce apoptotic cell death of malignant cells. BL-8040 was found to increase the expression of miR-15a/16-1 and reduce the expression of BCL2, MCL1 and cyclin D1. Importantly, CXCR4 inhibition using BL-8040 induced apoptosis in vitro and in vivo in AML and neuroblastoma tumors. This was mediated by inhibition of survival signals by ERK and down-regulation of BCL-2 expression. In support of these results overexpression of miR-15a/16-1 in AML and neuroblastoma cells was shown to induce their apoptosis. Conclusions: Our results demonstrate, for the first time to the best of our knowledge, that CXCR4 signaling regulates the expression of miR-15a/16-1 and their target genes. Our results suggest that overexpression of CXCR4 may override the survival dependency of tumor cells on BCL-2, MCL-1, and cyclin D1 leading to resistance of tumor cells to inhibition of these pathways. Furthermore, these results indicate that ligands of CXCR4 may tip the balance toward cell death by down- regulating survival signals through miR-15a/16-1 suppression of BCL2, MCL1 and cyclin D1 expression. Citation Format: Shiri Klein Silberman, Michal Abraham, Baruch Bulvik, Hanna Wald, Orly Eizenberg, Dvorah Olam, Lola Weiss, Katia Beider, Ori Wald, Shlomo Bulvik, Abraham Avigdor, Ohad Benjamini, Eithan Galun, Arnon Nagler, Yaron Pereg, Amnon Peled. CXCR4 controls BCL-2 expression and function by regulating miR-15a/16-1 expression in tumor cells. [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 3556.

Abstract 2326: LTX-315, an oncolytic peptide, increases anticancer immunity mediated by CTLA4 blockade in an interleukin-2 receptor beta-chain-dependent manner

Abstract Intratumoral immunotherapies aim at reducing local immunosuppression as well as reinstating and enhancing systemic anticancer T cell functions without inducing side effects. LTX-315 is a first-in-class oncolytic peptide -based local immunotherapy that meets these criteria by inducing a type of malignant cell death that elicits anticancer immune responses. Here, we show that LTX-315 rapidly reprograms the tumour microenvironment by decreasing the local abundance of immunosuppressive Tregs and myeloid derived-suppressor cells and by increasing the frequency of polyfunctional TH1/TC1 cells with a concomitant raise in their expression of CTLA4 and a drop in PD-1 expression. Logically, in tumours that were resistant to intratumoural or systemic CTLA4 blockade, subsequent local inoculation of LTX-315 cured the animals or caused tumour regressions with abscopal effects(meaning that even malignant lesions that were established in the opposite flank and were not treated by LTX-315 responded to the therapy). This synergistic interaction between CTLA4 blockade and LTX-315 was reduced upon blockade of the β-chain of the interleukin-2 receptor (CD122) which is required for signalling in response to IL-2 and IL-15. This preclinical study provides a strong rationale for administering the oncolytic peptide LTX-315 to patients who are receiving treatment with the CTLA4 blocking antibody ipilimumab. Citation Format: Takahiro Yamazaki, Marie Vetizou, Camila Flores, Aurelien Marabelle, Baldur Sveinbjørnsson, Øystein Rekdal, Guido Kroemer, Laurence Zitvogel. LTX-315, an oncolytic peptide, increases anticancer immunity mediated by CTLA4 blockade in an interleukin-2 receptor beta-chain-dependent manner. [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 2326.

A novel manganese complex selectively induces malignant glioma cell death by targeting mitochondria.

Despite advances in treatment, malignant glioma commonly exhibits recurrence, subsequently leading to a poor prognosis. As manganese (Mn) compounds can be transported by the transferrin-transferrin receptor system, the present study synthesized and examined the potential use of Adpa-Mn as a novel antitumor agent. Adpa-Mn time and dose-dependently inhibited U251 and C6 cell proliferation; however, it had little effect on normal astrocytes. Apoptosis was significantly elevated following treatment with Adpa-Mn, as detected by chromatin condensation, Annexin V/propidium iodide staining, cytochrome c release from mitochondria to the cytoplasm, and the activation of caspases-9, -7 and -3 and poly (ADP-ribose) polymerase. In addition, Adpa-Mn enhanced fluorescence intensity of monodansylcadaverine and elevated the expression levels of the autophagy-related protein microtubule-associated protein 1 light chain 3. Pretreatment with the autophagy inhibitors 3-methyladenine and chloroquine enhanced Adpa-Mn-induced cell inhibition, thus indicating that autophagy has an essential role in this process. Furthermore, evidence of mitochondrial dysfunction was detected in the Adpa-Mn-treated group, including disrupted membrane potential, elevated levels of reactive oxygen species (ROS) and depleted adenosine triphosphate. Conversely, treatment with the mitochondrial permeability transition inhibitor cyclosporin A reversed Adpa-Mn-induced ROS production, mitochondrial damage and cell apoptosis, thus suggesting that Adpa-Mn may target the mitochondria. Taken together, these data suggested that Adpa-Mn may be considered for use as a novel anti-glioma therapeutic option.

4EBP1/c-MYC/PUMA and NF-κB/EGR1/BIM pathways underlie cytotoxicity of mTOR dual inhibitors in malignant lymphoid cells

AbstractThe mammalian target of rapamycin (mTOR), a kinase that regulates proliferation and apoptosis, has been extensively evaluated as a therapeutic target in multiple malignancies. Rapamycin analogs, which partially inhibit mTOR complex 1 (mTORC1), exhibit immunosuppressive and limited antitumor activity, but sometimes activate survival pathways through feedback mechanisms involving mTORC2. Thus, attention has turned to agents targeting both mTOR complexes by binding the mTOR active site. Here we show that disruption of either mTOR-containing complex is toxic to acute lymphocytic leukemia (ALL) cells and identify 2 previously unrecognized pathways leading to this cell death. Inhibition of mTORC1-mediated 4EBP1 phosphorylation leads to decreased expression of c-MYC and subsequent upregulation of the proapoptotic BCL2 family member PUMA, whereas inhibition of mTORC2 results in nuclear factor-κB–mediated expression of the Early Growth Response 1 (EGR1) gene, which encodes a transcription factor that binds and transactivates the proapoptotic BCL2L11 locus encoding BIM. Importantly, 1 or both pathways contribute to death of malignant lymphoid cells after treatment with dual mTORC1/mTORC2 inhibitors. Collectively, these observations not only provide new insight into the survival roles of mTOR in lymphoid malignancies, but also identify alterations that potentially modulate the action of mTOR dual inhibitors in ALL.

MB-67DUAL TARGETING OF PI3K AND mTOR SIGNALING IN MEDULLOBLASTOMA

MB-67. DUAL TARGETING OF PI3K AND mTOR SIGNALING IN MEDULLOBLASTOMA Jessica Clymer1,2, Frank Eckerdt2, Jonathan Bell2, Rishi Lulla1,2, Stewart Goldman1,2, and Leonidas Platanias2,3; Division of Hematology/ Oncology/Stem Cell Transplantation, Department of Pediatrics, Ann & Robert H. Lurie Children’s Hospital, Chicago, IL, USA; Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA; Division of Hematology/Oncology, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA BACKGROUND: Aberrant activation of the PI3K pathway has been shown to play a role in medulloblastoma cell proliferation, while mTOR activation contributes to therapy resistance. Here we sought to evaluate the effects of combined targeting of the PI3K and mTOR pathways in medulloblastoma. METHODS: Medulloblastoma cell lines Daoy and D556 were grown in 2-D cultures to investigate the effects of pharmacologic inhibition of PI3K and mTORon kinase signaling, medulloblastomacell proliferation, colony formation, and apoptosis. After knockdown of p110 isoforms, cells were also grown under stem cell conditions as 3-D neurospheres and subjected to extreme limiting dilution analysis (ELDA) for assessment of stem-like cancer cell growth. RESULTS: Of all class I PI3K isoforms (p110a, p110b, p110d, and p110g), only p110a was essential for medulloblastoma cell proliferation, colony formation and neurosphere growth. Accordingly, pharmacologic targeting of the p110a isoform of PI3K with BYL-719 synergizedwith the mTOR inhibitor OSI-027, as it resulted in inhibition of effector pathways, blocked cell proliferation and colony formation, and increased malignant cell death by apoptosis. Finally,BYL-719and OSI-027 greatly impairedgrowthof stem-likecancer cells as 3-D neurospheres. CONCLUSIONS: The p110a isoform of PI3K exerts crucial roles in medulloblastoma. Inhibition of p110a and mTOR exerts potent antineoplastic effects on medulloblastoma cells and also blocks neurosphere growth. Dual inhibition of p110a and mTOR might be promising for targeting both malignant medulloblastoma cells and the therapy-resistant cancer stem cell population. Neuro-Oncology 18:iii97–iii122, 2016. doi:10.1093/neuonc/now076.63 #The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The oncolytic peptide LTX-315 overcomes resistance of cancers to immunotherapy with CTLA4 checkpoint blockade.

Intratumoral immunotherapies aim at reducing local immunosuppression, as well as reinstating and enhancing systemic anticancer T-cell functions, without inducing side effects. LTX-315 is a first-in-class oncolytic peptide-based local immunotherapy that meets these criteria by inducing a type of malignant cell death that elicits anticancer immune responses. Here, we show that LTX-315 rapidly reprograms the tumor microenvironment by decreasing the local abundance of immunosuppressive Tregs and myeloid-derived suppressor cells and by increasing the frequency of polyfunctional T helper type 1/type 1 cytotoxic T cells with a concomitant increase in cytotoxic T-lymphocyte antigen-4 (CTLA4) and drop in PD-1 expression levels. Logically, in tumors that were resistant to intratumoral or systemic CTLA4 blockade, subsequent local inoculation of LTX-315 cured the animals or caused tumor regressions with abscopal effects. This synergistic interaction between CTLA4 blockade and LTX-315 was reduced upon blockade of the β-chain of the interleukin-2 receptor (CD122). This preclinical study provides a strong rationale for administering the oncolytic peptide LTX-315 to patients who are receiving treatment with the CTLA4 blocking antibody ipilimumab.

Transduction of Recombinant M3-p53-R12 Protein Enhances Human Leukemia Cell Apoptosis.

Tumor suppressor protein p53 plays important roles in initiating cell cycle arrest and promoting tumor cell apoptosis. Previous studies have shown that p53 is either mutated or defective in approximately 50% of human cancers; therefore restoring normal p53 activity in cancer cells might be an effective anticancer therapeutic approach. Herein, we designed a chimeric p53 protein flanked with the MyoD N-terminal transcriptional activation domain (amino acids 1-62, called M3) and a poly-arginine (R12) cell penetrating signal in its N-and C-termini respectively. This chimeric protein, M3-p53-R12, can be expressed in E. coli and purified using immobilized metal ion chromatography followed by serial refolding dialysis. The purified M3-p53-R12 protein retains DNA-binding activity and gains of cell penetrating ability. Using MTT assay, we demonstrated that M3-p53-R12 inhibited the growth of K562, Jurkat as well as HL-60 leukemia cells carrying mutant p53 genes. Results from FACS analysis also demonstrated that transduction of M3-p53-R12 protein induced cell cycle arrest of these leukemia cells. Of special note, M3-p53-R12 has no apoptotic effect on normal mesenchymal stem cells (MSC) and leukocytes, highlighting its differential effects on normal and tumor cells. To sum up, our results reveal that purified recombinant M3-p53-R12 protein has functions of suppressing the leukemia cell lines' proliferation and launching cell apoptosis, suggesting the feasibility of using M3-p53-R12 protein as an anticancer drug. In the future we will test whether this chimeric protein can preferentially trigger the death of malignant cancer cells without affecting normal cells in animals carrying endogenous or xenographic tumors.

Cathepsin-Cleavable BIM BH3 Peptide Amphiphiles Are Potent Inducers of Cellular Apoptosis

The BCL-2 family of proteins forms a complex interaction network that regulates cellular life and death decisions and contributes to cancer development, maintenance, and chemoresistance. BH3 only member proteins (e.g. BIM) serve as cellular stress sentinels and, when triggered, signal irreversible activation of apoptosis through their α-helical BH3 death domains. These pro-apoptotic signals are normally held in check by the multidomain anti-apoptotic proteins (e.g. BCL-XL, MCL-1) but when they are unable to do so the multidomain pro-apoptotic proteins BAX and BAK induce cell death through pore formation in the mitochondrial outer membrane. Therapeutic manipulation of the BCL-2 family with BH3 mimetics (including small molecules and synthetic peptides) is an emerging paradigm in cancer treatment and immune modulation. The design of next-generation therapeutics based on the BIM BH3 helix offers the unique advantage of recapitulating BIM's natural capacity to directly target the full complement of anti- and pro-apoptotic BCL-2 proteins. Here, we utilize the highly active BH3 domain of BIM as part of a peptide amphiphile nanostructure designed to overcome malignant cell death blockade. Peptide amphiphiles consist of bioactive peptides linked to hydrophobic lipid-like tail groups. In aqueous solutions, amphiphiles spontaneously assemble into micelles. Micelle-based peptide delivery provides several advantages: single micelles deliver high concentration of peptides into cells, they stabilize peptide secondary structure(s), and they have the potential for combinatorial synthesis using multiple bioactive moieties targeting non-redundant cell death escape pathways. While the exact mechanism behind cellular uptake of peptide amphiphiles remains controversial, recent work has shown that peptide amphiphiles intracellularly traffic through lysosomes and endosomes. In order to prevent the bioactive peptides from being sequestered within these structures, a system of escape is needed. Lysosomes contain many well-characterized proteases, and cathepsin B has previously been utilized to release chemotherapeutics in the context of targetable antibody-based treatments. Here, we generate peptide amphiphiles with BIM BH3 peptides and show that these nanostructures are able to specifically bind recombinant BCL-2 proteins, are stable at physiologic temperatures and pH, quickly enter into cells, and induce dose-responsive apoptosis in malignant hematologic cancers as measured by viability and caspase 3/7 activation. We further demonstrate that incorporating a cathepsin B-cleavable linker between the BIM BH3 peptide and the hydrophobic tail within individual amphiphiles results in increased binding to recombinant BCL-2 proteins while also allowing for increased cellular uptake and mitochondrial localization leading to faster and more potent dose-dependent cytotoxicity and caspase activation in malignant cells. Thus, we have developed a modular and potentially targetable nanostructure that represents a new promising strategy for BCL-2 family modulation and apoptosis induction in cancer. DisclosuresNo relevant conflicts of interest to declare.

Abstract 683: DEBIO0932, an Hsp90 inhibitor downregulates key signaling pathways and sensitizes glioma cells to temozolomide

Abstract Heat shock protein 90 (Hsp90), a molecular chaperone that regulates proper folding, stabilization and degradation of key regulatory client proteins, has also been implicated in maintenance of high levels of aberrantly expressed proteins such as kinases and transcription factors in several malignancies. Hence Hsp90 inhibition has emerged as a novel strategy for therapeutic targeting of cancers. Several Hsp90 inhibitors (Hsp90i) have demonstrated efficacy as anti-neoplastic agents in pre-clinical studies by inducing downregulation of key oncogenic proteins such as Akt, EGFR, ErKs, STAT3 and VEGFR resulting in differentiation or death of malignant cells. DEBIO0932 is an orally bioavailable second generation HSP90i which can cross the blood brain barrier (BBB) and has shown preclinical efficacy both as a single agent and in combination therapy against several malignancies. However, its possible efficacy in gliomas has not been fully characterized. We examined the effects of DEBIO0932 as a single agent and in combination with temozolomide (TMZ) using glioma cell lines (U373, LN229, U251HF) and glioma stem-like cells (GSC11, GSC23). DEBIO0932-treated glioma cells and GSC showed decreased proliferation in a WST-1 assay and induction of apoptosis by flow cytometry. Western blot analysis of DEBIO0932 treated cells showed activation of apoptotic pathways and downregulation of EGFR, Akt and MAPK which are key regulators of survival pathways and drivers of malignancy in glioblastoma. Wound healing and migration assay showed reduced Glioma cell motility, decreased migration and invasion after DEBIO0932 treatment. Analysis of effects of DEBIO0932 on key signaling molecules using a kinase array showed downregulation of several key kinases relevant to glioma biology; of particular interest, treatment with DEBIO0932 caused downregulation of β-catenin which has been implicated in maintenance of tumor stem cell state. Corresponding to this, we also observed downregulation of CD133, a marker enriched in glioma stem cells. Additionally, given the downregulation of several proteins implicated in cell survival and treatment resistance, we tested the ability of DEBIO0932 to increase the cytotoxic effect of temozolomide (TMZ) in glioma cells. Combination studies showed a synergistic effect of DEBIO0932 and TMZ against gliomas cells and GSC indicating the potential for Hsp90i in overcoming tumor cell resistance to cytotoxic signals. Our results strongly support the potential for Hsp90 inhibition as a therapeutic strategy against gliomas particularly in combination with cytotoxic agents such as TMZ. The oral bioavailability and ability to cross the BBB make DEBIO0932 a promising agent for therapeutic targeting of glioblastoma. Citation Format: Alessandro Canella, Jihong Xu, W. Hans Meisen, Balveen Kaur, Lara Rizzotto, Divya Kesanakurti, Prabakaran Nagarajan, Vinay K. Puduvalli. DEBIO0932, an Hsp90 inhibitor downregulates key signaling pathways and sensitizes glioma cells to temozolomide. [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 683. doi:10.1158/1538-7445.AM2015-683