Increase In Annexin V-positive Cells Research Articles

An increase in ER stress and unfolded protein response in iPSCs-derived neuronal cells from neuronopathic Gaucher disease patients

Gaucher disease (GD) is a lysosomal storage disorder caused by a mutation in the GBA1 gene, responsible for encoding the enzyme Glucocerebrosidase (GCase). Although neuronal death and neuroinflammation have been observed in the brains of individuals with neuronopathic Gaucher disease (nGD), the exact mechanism underlying neurodegeneration in nGD remains unclear. In this study, we used two induced pluripotent stem cells (iPSCs)-derived neuronal cell lines acquired from two type-3 GD patients (GD3-1 and GD3-2) to investigate the mechanisms underlying nGD by biochemical analyses. These iPSCs-derived neuronal cells from GD3-1 and GD3-2 exhibit an impairment in endoplasmic reticulum (ER) calcium homeostasis and an increase in unfolded protein response markers (BiP and CHOP), indicating the presence of ER stress in nGD. A significant increase in the BAX/BCL-2 ratio and an increase in Annexin V-positive cells demonstrate a notable increase in apoptotic cell death in GD iPSCs-derived neurons, suggesting downstream signaling after an increase in the unfolded protein response. Our study involves the establishment of iPSCs-derived neuronal models for GD and proposes a possible mechanism underlying nGD. This mechanism involves the activation of ER stress and the unfolded protein response, ultimately leading to apoptotic cell death in neurons.

Epidemic dropsy toxin, sanguinarine chloride, stimulates sucrose-sensitive hemolysis and breakdown of membrane phospholipid asymmetry in human erythrocytes

Sanguinarine (SGN) is a benzophenathridine alkaloid extracted from Sanguinaria canadensis plant. SGN is incriminated in epidemic dropsy (ED) characterized by multiple-organ failure and anemia. Nevertheless, how SGN leads to anemia of ED remains poorly understood. This study was thus initiated to investigate the interaction of SGN with human red blood cells (RBCs) and to delineate associated molecular mechanisms. Heparin- and EDTA-anticoagulated blood was collected from healthy participants and whole blood was analyzed for a complete blood count, while isolated RBCs were examined for hemolytic and eryptotic markers following exposure to 1–100 μM SGN for 24 h at 37 °C. Calcium was measured by Fluo4/AM, hemolysis by hemoglobin leakage, membrane scrambling by Annexin V-FITC, cell size by forward scatter (FSC), cell granularity by side scatter (SSC), and oxidative stress by H2DCFDA. SGN led to increased Fluo4 fluorescence and dose-dependent hemolysis which was not ameliorated by exclusion of extracellular Ca2+ but was nevertheless sensitive to hyperosmotic conditions and to the presence of aspirin. SGN also caused significant increase in Annexin V-positive cells, decreased FSC and SSC values, and elevated DCF fluorescence. Moreover, significantly reduced lymphocyte and basophil percentages along with selective toxicity to platelets was noted. Collectively, SGN possesses sucrose- and cyclooxygenase-sensitive hemolytic potential and elicits eryptosis characterized by Ca2+ accumulation, phosphatidylserine externalization, morphological alterations including cell shrinkage and loss of granularity, and oxidative stress. In conclusion, this report reveals a novel activity of SGN against human RBCs and informs prospective policies in ED prevention and management.

Cytotoxicity of Gymnopilus purpureosquamulosus extracts on hematologic malignant cells through activation of the SAPK/JNK signaling pathway.

Treatment of hematologic malignancies is a formidable challenge for hematologists and there is an urgent need to identify safe and efficacious agents either via synthesis in the laboratory or isolation from natural products. Here, we report the cytotoxicity of extracts from mushroom Gymnopilus purpureosquamulosus Høil (G. pps) and describe its molecular mechanisms. Using leukemia, lymphoma and multiple myeloma cell lines, 28–35 ppm G. pps extract inhibited cell proliferation by ~46–79%, which correlates with activation of apoptosis as indicated by increase in annexin V-positive cells (~5–8-fold), production of reactive oxygen species (~2–3-fold), cells in sub G0/G1 phase (~3–13-fold), caspase 3 enzymatic activity (~1.6–2.9-fold), DNA fragmentation, PARP1 cleavage and down-regulation of prosurvival proteins. Mitochondrial membrane potential decreased and leakage of pro-apoptotic factors to cytoplasm was observed, consistent with the activation of intrinsic apoptosis. Western blot analysis showed activation of the ASK1-MEK-SAPK/JNK and ASK1-P38 MAPK pathways possibly due to changes in the cellular redox status as suggested by decreased protein levels of peroxiredoxin, thioredoxin and thioredoxin reductase. Moreover, antioxidant N-acetylcysteine alleviated the cytotoxicity of G. pps. Pharmacological inhibition of SAPK/JNK and P38 alleviated the G. pps-mediated cytotoxicity. The extract activated apoptosis in leukemia and lymphoma patient cell samples but not in mononuclear cells from healthy donors further supporting the therapeutic values of G. pps for hematologic malignancies.

Preclinical Investigation of the p53-MDM2 Antagonist Idasanutlin (RG7388) Demonstrates Significant Activity in High Risk Adult Acute Lymphoblastic Leukemia

Outcomes for adult patients with acute lymphoblastic leukemia (ALL) are dismal and have not kept pace with their pediatric counterparts, with five-year survival rates of less than 45%. TP53 mutations are infrequent in ALL, but activity of the oncoprotein MDM2 may otherwise phenotypically disrupt and circumvent normal p53 function, positing the p53-MDM2 signaling axis as a potential therapeutic target for the engagement of intrinsic cell death programs. Given the clinical safety and responses to p53-MDM2 antagonist idasanutlin (RG7388) in other hematological cancers and solid tumors, we aimed to evaluate the therapeutic potential of idasanutlin in ALL. Single-agent activity of idasanutlin was investigated in 17 high-risk ALL patient and patient-derived xenograft samples (aged 4 to 51 years), both B-ALL (n=15) and T-ALL (n=2), including KMT2A-rearranged, TCF3-rearranged, and Philadelphia-positive ALL. An ex vivo coculture of ALL blasts and hTERT-immortalized mesenchymal stem cells (MSC) was employed to support growth of the ALL blasts during short-term culture, complemented by a fluorescent image-based microscopy platform which identifies and discriminates the two cellular compartments using random forest machine learning algorithms based on cellular nuclear staining. Idasanutlin demonstrated sub-micromolar, dose-dependent anti-leukemic activity against 15 of 17 samples tested, with half maximal effective concentrations (EC50) in the range of 10 to 220 nM (mean EC50 = 45.1nM); the two exceptions were later determined to harbor homozygous inactivating TP53 mutations; p.Y220C and p.S241P within the p53 DNA binding domain. The idasanutlin EC50 concentrations determined are clinically achievable, well below the peak plasma concentrations reported in patients for other disease indications. Furthermore, idasanutlin concentrations below 10µM had no impact upon MSC survival. Conforming to p53-MDM2 auto-regulatory feedback mechanisms, we demonstrated that idasanutlin efficiently stabilized and activated p53 at the protein level within 6 hours when treated with their respective idasanutlin EC50, to a level greater than 4-fold increased relative to their respective vehicle-only controls (p=0.001, n=7). Further, p53-regulated transcriptional target gene products, MDM2 and p21, were increased by 5-fold and 2-fold respectively, validating engagement of the p53 pathway by idasanutlin (p=0.036 and 0.125, respectively). By contrast, idasanutlin did not elicit increased expression of either p53-regulated transcriptional target gene product in the identified TP53-mutant patient samples (n=2). On-target specificity of idasanutlin was further confirmed in a NALM6 isogenic cell line model, whereby the TP53 wildtype line was sensitive to idasanutlin (EC50 = 74nM) and effectively activated p53 signaling whereas the TP53 homozygous null line was highly resistant (EC50 = ~10µM). To determine whether the decreased cell numbers and engagement of p53 signaling observed were accompanied by cell death, the capacity of idasanutlin to induce apoptosis in the ALL samples was next investigated. Patient-derived ALL samples (n=6) were treated with vehicle or idasanutlin at their respective EC50s for 24 and 48 hours, and then analyzed by flow cytometry. There was an increase in annexin-V positive cells within 24 hours compared to the vehicle-only treated cells (mean±SD 14.3±6.6% vs 27.0±21.2% respectively (p=0.125). By 48 hours significant apoptosis was attained, with a mean±SD of 59.0±23.8% annexin-V positive cells compared to the mean of vehicle-treated cells at 29.1±11.6% (p=0.004). Cleaved poly(ADP-ribose) polymerase (PARP) levels were also increased greater than 3-fold compared to vehicle-only control cells as assessed by immunoblotting (p=0.045, n=3), corroborating these findings. These data emphasize the potential of pharmacologically targeting the p53-MDM2 axis in ALL, demonstrating potent, on-target, cytotoxic activity in a range of high-risk ALL cytogenetic subgroups. Taken together, these findings support further preclinical investigations into idasanutlin and other p53-MDM2 antagonists and potential combinations to improve the treatment of adult ALL. Disclosures Irving: F. Hoffmann-La Roche: Research Funding.

Abstract 2960: Anti-cancer activity of NCTD: promotion of pro-apoptosis and anti-invasion in human oral cancer cells

Abstract Norcantharidin (NCTD), a demethylated analog of cantharidin isolated from blister beetles, has been used as a promising anticancer agent; however, the anti-tumor potentials of NCTD against human oral cancer has not been fully understood. Here, this study was aimed to investigate the pro-apoptotic and anti-invasive effect and its molecular targets of NCTD in human oral cancer cell lines. Trypan blue exclusion assay, live/dead assay, western blotting, 4-6-Diamidino-2-Phenylindole staining, flow cytometric analysis, Terminal Deoxynucleotidyl Transferase dUTP Nick end Labeling (TUNEL) assay, immunohistochemistry and tumor xenograft mice models were performed to investigate the pro-apoptotic effect of NCTD. Clonogenic, wound healing, invasion, zymography, western blotting and immunocytochemistry assays were performed to investigate the anti-invasive effect of NCTD. NCTD significantly inhibited cell growth and increased the number of dead cells in oral cancer cell lines. It induced the following apoptotic phenomena: (1) the cleavages of poly (ADP-ribose) polymerase and casepase-3; (2) increase in apoptotic morphological changes (nuclear condensation and fragmentation); (3) increase in annexin V-positive cells or sub-G1 population of cells. NCTD significantly activated the p38 mitogen-activated protein kinase (MAPK) pathway. A p38 MAPK inhibitor (SB203580) partially attenuated NCTD-induced apoptosis in oral cancer cell lines. NCTD strongly suppressed tumor growth in the tumor xenograft bearing HSC-3 cells, and the number of TUNEL-positive cells increased in NCTD-treated tumor tissues. Also, NCTD markedly suppressed the colony formation, migration, and invasion of YD-15 cells as well as the activities of MMP-2 and MMP-9. It disrupted F-actin reorganization through suppressing the FAK/Paxillin axis. Moreover, NCTD exhibited a powerful anti-invasive effect compared with that of PF-562271. Collectively, these results suggest that NCTD has a potential pro-apoptotic and anti-invasive activity against oral cancer cell lines. Therefore, these results suggest that NCTD could be a potential anticancer drug candidate for the treatment of human oral cancer. This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, and future Planning [2017R1D1A1B03029317, 2018R1D1A1B07043080, and 2019R1A2C1085896]. Citation Format: Chi-Hyun Ahn, Kyoung-Ok Hong, Ji-Ae Shin, Seong Doo Hong, Sung-Dae Cho. Anti-cancer activity of NCTD: promotion of pro-apoptosis and anti-invasion in human oral cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2960.

Novel 3,4-diarylpyrazole as prospective anti-cancerous agents

Cancer is a leading cause of death globally. Despite therapeutic advancements the mortality rate of cancer is continuously increasing. Thus, it is important to identify and design potential therapeutic agents which can specifically bind with most common targets of cancer and inhibit tumor progression. The present work discloses the potential therapeutic application of the novel 3,4-diaryl 1H-pyrazoles as prospective anti-cancerous agent. The in silico molecular docking studies performed with 3,4-disubstituted pyrazoles as ligand with targets including DNA, BCL-2 and F1-ATP Synthase revealed strong binding affinity with DNA (-7.5 kcal/mol), BCL-2 (-8.1 kcal/mol) and F1-ATP Synthase (-7.2 kcal/mol). Furthermore, the in silico finding was validated with the in vitro cytotoxicity assay with human breast cancer cell line (MDA-MB-231). MDA-MB-231 cells treated with 3,4-diarylpyrazole resulted in an increase in annexin-V positive cells, production of reactive oxygen species (ROS), dissipation of the mitochondrial membrane potential and activation of caspase-3. Taken together, this study demonstrate that a novel synthesized 3,4-diarylpyrazoles, showed strong binding affinity against DNA, anti-proliferative activity and executed apoptosis through ROS-dependent caspase-3-mediated mitochondrial intrinsic apoptotic pathway against MDA-MB-231 cells. These findings increase our understanding of the molecular mechanism (s) by which 3,4-diarylpyrazoles can exert their anticancer activity and may contribute towards development of novel therapeutic agent against breast cancer.

Molecular mechanisms of apoptosis induced by a novel synthetic quinolinone derivative in HL-60 human leukemia cells

The mortality rates for acute myeloid leukemia are very high, necessitating the search for novel chemotherapeutic candidates. Herein, we investigated the anticancer potential of a new synthetic compound, 2-ethyl-3-methyliden-1-tosyl-2,3-dihydroquinolin-4-(1H)-one (AJ-374) against myeloid leukemia HL-60 cell line. This analog was selected from the small library of synthetic dihydroquinolinones on the basis of its strong antiproliferative activity against HL-60 cells and 30-fold lower cytotoxicity towards healthy HUVEC cells. AJ-374 promoted the arrest of the cells in the subG0/G1 phase of the cell cycle in the first 24 h. Treatment of HL-60 cells with AJ-374 caused an increase in annexin-V positive cells, activation of caspase-8, -9 and -3, dissipation of the mitochondrial membrane potential and enhancement of FAS protein level. Apoptosis induction triggered by this quinolinone was blocked by the pre-treatment of the cells with caspase-8, -9 and -3 inhibitors. The obtained results indicated that AJ-374-induced apoptosis was executed by both, the extrinsic and intrinsic pathways. The cytotoxic activity of AJ-374 was also associated with down-regulation of the mitogen-activated protein kinase (MAPK) pathway and was independent of reactive oxygen species generation. Taken together, these results suggest that AJ-374 exerts a potent anticancer effect on leukemia cells, with a wide safety margin, which makes this analog an attractive drug candidate for further testing.

Isoflavones Isolated from the Seeds of Millettia ferruginea Induced Apoptotic Cell Death in Human Ovarian Cancer Cells.

The seeds of Millettia ferruginea are used in fishing, pesticides, and folk medicine in Ethiopia. Here, the anti-cancer effects of isoflavones isolated from M. ferruginea were evaluated in human ovarian cancer cells. We found that isoflavone ferrugone and 6,7-dimethoxy-3’,4’-methylenedioxy-8-(3,3-dimethylallyl)isoflavone (DMI) had potent cytotoxic effects on human ovarian cancer cell A2780 and SKOV3. Ferrugone and DMI treatment increased the sub-G1 cell population in a dose-dependent manner in A2780 cells. The cytotoxic activity of ferrugone and DMI was associated with the induction of apoptosis, as shown by an increase in annexin V-positive cells. Z-VAD-fmk, a broad-spectrum caspase inhibitor, and z-DEVD-fmk, a caspase-3 inhibitor, significantly reversed both the ferrugone and DMI-induced apoptosis, suggesting that cell death stimulated by the isoflavones is mediated by caspase-3-dependent apoptosis. Additionally, ferrugone-induced apoptosis was found to be caspase-8-dependent, while DMI-induced apoptosis was caspase-9-dependent. Notably, DMI, but not ferrugone, increased the intracellular levels of reactive oxygen species (ROS), and antioxidant N-acetyl-L-cysteine (NAC) attenuated the pro-apoptotic activity of DMI. These data suggest that DMI induced apoptotic cell death through the intrinsic pathway via ROS production, while ferrugone stimulated the extrinsic pathway in human ovarian cancer cells.

Contribution of p38 MAPK Pathway to Norcantharidin-Induced Programmed Cell Death in Human Oral Squamous Cell Carcinoma.

Norcantharidin (NCTD), a demethylated analog of cantharidin isolated from blister beetles, has been used as a promising anticancer agent; however, the underlying function of NCTD against human oral squamous cell carcinoma (OSCC) has not been fully understood. Here, this study was aimed to investigate the apoptotic effect and molecular targets of NCTD in human OSCC in vitro and in vivo. The anticancer effects of NCTD and its related molecular mechanisms were evaluated by trypan blue exclusion assay, live/dead assay, western blotting, 4-6-Diamidino-2-Phenylindole (DAPI) staining, flow cytometric analysis, Terminal Deoxynucleotidyl Transferase dUTP Nick end Labeling (TUNEL) assay, and immunohistochemistry. NCTD significantly inhibited cell growth and increased the number of dead cells in HSC-3 and HN22 cell lines. It induced the following apoptotic phenomena: (1) the cleavages of poly (ADP-ribose) polymerase and casepase-3; (2) increase in apoptotic morphological changes (nuclear condensation and fragmentation); (3) increase in annexin V-positive cells or sub-G1 population of cells. NCTD significantly activated the p38 mitogen-activated protein kinase (MAPK) pathway but inactivated the signal transducer and activator of transcription (STAT)3 pathway. A p38 MAPK inhibitor (SB203580) partially attenuated NCTD-induced programmed cell death (apoptosis) in both cell lines, whereas ectopic overexpression of STAT3 did not affect it. NCTD strongly suppressed tumor growth in the tumor xenograft bearing HSC-3 cells, and the number of TUNEL-positive cells increased in NCTD-treated tumor tissues. In addition, NCTD did not cause any histopathological changes in the liver nor the kidney. NCTD induced programmed cell death via the activation of p38 MAPK in OSCC. Therefore, these results suggest that NCTD could be a potential anticancer drug candidate for the treatment of OSCC.

Abstract P2-03-06: PTEN expression at the nexus of oncogenic signals in TNBC: Testing combination of p110beta-isoform-specific inhibitor with five PARP inhibitors

Abstract Background: TNBC is the most aggressive form of BRCA-associated BC subtype. The loss of PTEN is a common “first event” associated with basal-like subtype (Martins et al., 2012) and this mode of PI3K-pathway activation (deletion/mutation/loss of PTEN) occurs more frequently (35%) than PIK3CA (5-10%) mutations in TNBC (Ellis & Perou, 2013). Several reports suggest upregulation of PI3K/AKT mediated by PTEN loss depends on the PI3Kbeta-isoform. PARP1 is identified as a target of BRCA-defined cancers. Inhibitors of PARP1 have been recently approved by the FDA as targeted agents in BRCA-defined breast cancers. Failure to repair damaged DNA upon PARP inhibition causes accumulation of DNA double-strand breaks and leads to apoptosis of cancer cells. Also, nuclear PTEN controls DNA repair (Bassi et al., 2013). Here we hypothesized that a combination of PARP inhibitor with p110beta-isoform-specific inhibitor would sensitize the effect of PARP inhibitor(s) in PTEN-deprived TNBC model. Methods: We tested five PARP inhibitors, Talazoparib (BMN673, B), Niraparib (N), Olaparib (O), Rucaparib (R) and Veliparib (V) in combination with p110beta specific inhibitor, AZD6482. Four PTEN-null TNBC cell lines (BRCA WT/null), MDA-MB468, HCC70 (p.F90fs*9), BT549 (p.V275fs*1) and SUM149 cell lines were used for the study. Proliferative, apoptotic and PARylation signals following drug combinations were demonstrated by WB in a dose and time-dependent manner. Pro-apoptotic and anti-proliferative effects were verified using complementary 3D ON-TOP assay, real-time proliferation (Incucyte), AnnexinV and cl-caspase3 analyses. As an internal control, we also compared anti-proliferative signals of GDC-0032 (p110 beta sparing inhibitor) and GDC-0941 (pan PI3K inhibitor) with that of AZD6482 at 3/6 hours in MDA-MB468 and SUM149 cells. Mode of apoptosis was tested using triple fluorescence staining in live cells. Results: A dose of 500 nM BMN673 alone was effective in slowing cell proliferation and induced apoptosis. AZD6482 (5-10 uM) did not have anti-proliferative or pro-apoptotic effects at 48 and 72 hours. When compared, combinations of different PARPi (100nM of B, 1uM of N, 10uM of O, 10uM of R and 10uM of V) with AZD6482 abrogated 3D growth in BT549 and MDA-MB468 TNBC cells in a time-dependent manner. The effect of PARP inhibitor was tested by PAR signals which were abrogated either alone or in combination with carboplatin in both BRCA1/2 WT and mutated cells. The most pronounced anti-tumor effect was observed with the combination of B and AZD6482 which was mechanistically explained by the robust increase of AnnexinV positive cells following a single 500nM dose of B at both 48 and 72 hours. Pan PI3K inhibitor, GDC-0941, and AZD6482 blocked the activation of PI3K and its downstream effectors in contrast to p110 alpha-specific inhibitor, GDC-0032 in MDA-MB468 and SUM149 cells. Summary: We demonstrated a remarkable sensitivity of tumor cells to PARP inhibition in PTEN-defined TNBC models and identified PTEN-nullness as a potential predictive biomarker for a possible co-targeting of the PI3K pathway to further sensitize TNBC to PARP inhibitors. Citation Format: Dey N, Carlson JH, De P, Leyland-Jones B. PTEN expression at the nexus of oncogenic signals in TNBC: Testing combination of p110beta-isoform-specific inhibitor with five PARP inhibitors [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-03-06.

Thymoquinone attenuates phosphorylation of AKT to inhibit kidney cancer cell proliferation.

Thymoquinone (Tq) is an active compound from Nigella sativa which is used in traditional medicine. The effect of Tq on kidney cancer and the pathway of action remain unproven. Herein, we report the anticancer properties of Tq on kidney cancer cells. Tq demonstrated anti-proliferative effects in A498 cells with a GI50 value of 40.07µM and Caki-1 cells with a GI50 of 51.04µM by the MTT assay. Tq exhibited nuclear fragmentation and inhibited trans-endothelial migration of A498 and Caki-1 cells in a dose-responsive manner. Time-dependent increase in Annexin V-positive cells and sub-G0 /G1 cell population was observed post-Tq treatment. The compound increased Bax protein levels and reduced Bcl-2 protein levels dose dependently in cells, thereby favoring apoptosis. Tq decreased the phosphorylation of Akt in both kidney cell types. The results suggest effective anticancer activity of Tq on kidney cancer cells which may be mediated by the Akt pathway. PRACTICAL APPLICATIONS: Results from the current investigation will through more light on the mechanistic pathway of Tq activity on the inhibition of kidney cancer cell proliferation. The output of this preclinical in vitro study may be translated into better chemotherapeutics of Tq and its analogs to treat kidney cancer. However, a detailed investigation on in vivo models is recommended.

PP-22 promotes autophagy and apoptosis in the nasopharyngeal carcinoma cell line CNE-2 by inducing endoplasmic reticulum stress, downregulating STAT3 signaling, and modulating the MAPK pathway.

Paris polyphylla var. yunnanensis, named Chong Lou, is considered an antitumor substance. In this study, we investigated the effect of PP-22, a monomer purified from P. polyphylla var. yunnanensis, on the nasopharyngeal carcinoma cell line CNE-2 in vitro. The results showed that PP-22 could inhibit the proliferation of CNE-2 cells via the induction of apoptosis, with evidence of the characteristic morphological changes in the apoptosis in the nucleus and an increase in Annexin V-positive cells. In addition, we found that PP-22 could activate the p38 mitogen-activated protein kinase (MAPK) pathway and that this activation was reversed by SB203580, a specific inhibitor of the p38 MAPK pathway. In contrast, PP-22 promoted apoptosis via an intrinsic pathway, including the endoplasmic reticulum stress pathway, in a caspase-dependent manner. A further study showed that PP-22 also induced apoptosis by downregulating the signal transducers and activators of transcription 3 (STAT3) pathway, and the inhibitory effect was also confirmed by STAT3 small interfering RNA. In addition, PP-22 could promote autophagy by inhibiting the extracellular regulated protein kinases (ERK) pathway. And autophagy plays a protective role against apoptosis. Together, these data show that PP-22 promotes autophagy and apoptosis in the nasopharyngeal carcinoma CNE-2 cell line.

Cis-[RuCl(BzCN)(bipy)(dppe)]PF6 induces anti-angiogenesis and apoptosis by a mechanism of caspase-dependent involving DNA damage, PARP activation, and Tp53 induction in Ehrlich tumor cells

Antimetastatic activities, low toxicity to normal cells and high selectivity for tumor cells make of the ruthenium complexes promising candidates in the search for develop new chemotherapeutic agents for the treatment of cancer. This study aimed to determine the cytotoxic, genotoxic and to elucidate the signaling pathway involved in the death cell process induced by cis-[RuCl(BzCN)(bipy)(dppb)]PF6(1) and cis-[RuCl(BzCN)(bipy)(dppe)]PF6(2) in Ehrlich ascites carcinoma (EAC) in vitro. Moreover, we report for the first time the anti-angiogenic potential on chick embryo chorioallantoic membrane (CAM) model. Peripheral blood mononuclear cells (PBMC) were isolated from healthy controls with an age range of 20–30 years and used to calculate the selectivity index (SI). The complex 2 (IC50 = 8.5 ± 0.4/SI = 6.3) showed high cytotoxic and selectivity index against EAC cells than complex 1 (IC50 = 14.9 ± 0.2/SI = 0.2) using the MTT assay. Complex 2 induced DNA damage on Ehrlich tumor cells at concentrations and time periods evalueted. In consequence, it was observed an increase of Tp53 gene expression, G0/G1-arrest cells, and increased levels of cleaved PARP protein. Beside that, the treatment of EAC with complex 2 led to an increase in Annexin V-positive cells and apoptosis induction by Caspase-7. Additionally, the complex 2 inhibited the angiogenesis caused by Ehrlich tumor cells in CAM model. This complex is active and selective for Ehrlich tumor cells, inducing DNA damage, cell cycle arrest and cell death by caspase-dependent apoptosis involving PARP activation (PARP1), and Tp53 induction.

Abstract 1532: Mithramycin induces the antiproliferative activity of chemotherapeutic agents in Ewing sarcoma cells

Abstract Ewing sarcoma (ES) is one of the most frequent primary bone and soft tissue tumors that occur in pediatric and adolescent population. ES is often diagnosed after the disease has already metastasized. With available treatment options, the prognosis of metastatic ES patients is poor and the 5-year survival rate is less than 20%. Therefore, identifying precise targeted therapies to induce therapeutic response in ES patients is urgently needed. An oncogenic fusion protein and transcription factor EWS-FLI1 is associated with more than 85% of ES tumors. Mithramycin has been identified as an effective agent to target EWS-FLI1 and is currently in clinical trials. In this study, we investigated the efficacy of Mithramycin to induce the anti-proliferative activity of chemotherapeutic agents commonly used for the treatment of this malignancy using human ES cell lines. TC71, TC32, CHLA32, CHLA10 and TC205 cells were treated with increasing concentrations of Mithramycin or Vincristine or Doxorubicin or Etoposide. Cell growth inhibition was evaluated at 24 and 48 h post-treatment using CellTiter Glo kit. Results showed a dose/time-dependent anti-proliferative effect for all agents. To confirm the effect of Mithramycin on selected cell lines, mRNA expression of downstream targets of EWS-FLI1 (ID2, LDB2, NROB1 and RCOR1 genes) was determined by quantitative PCR following treatment with Mithramycin. Mithramycin significantly down-regulated all tested genes and these results are in agreement with published work. In order to determine the combination response, cell growth inhibition of the chemotherapeutic drugs was assessed in the presence of added Mithramycin. The combination Index was evaluated by Chou-Talalay method. Further studies were performed for the combination of Mithramycin and Vincristine to investigate its effect on apoptosis and cell cycle arrest using CHLA10 and TC205 cells. Apoptotic markers such as the expression of cleaved Poly (ADP-ribose) polymerase (c-PARP) and survivin were determined by Western blot analysis. The apoptotic cell population was measured by Annexin-V staining using flow cytometry. When compared to individual agents, the combination of Mithramycin and Vincristine showed greater effect on apoptosis in both cell lines as evinced by an increase in Annexin-V positive cells, decrease in survivin expression and up-regulation of c-PARP. Overall, these results indicate the potential clinical advantage of combination treatment involving Mithramycin along with other chemotherapeutic agents for inhibiting the growth of ES cells. Molecular profiling analysis is underway to elucidate the candidate pathways associated with beneficial effect of this combination treatment. Note: This abstract was not presented at the meeting. Citation Format: Anish Ray, Bhavani Nagarajan, Umesh T. Sankpal, Sagar Shelake, W. Paul Bowman, Andras Lacko, Riyaz M. Basha. Mithramycin induces the antiproliferative activity of chemotherapeutic agents in Ewing sarcoma cells [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 1532. doi:10.1158/1538-7445.AM2017-1532

Gilteritinib (ASP2215), a Novel FLT3/AXL Inhibitor: Preclinical Evaluation in Combination with Azacitidine in Acute Myeloid Leukemia

Background FMS-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes in acute myeloid leukemia (AML). Activating mutations in FLT3 such as internal tandem duplications (ITD) at the juxtamembrane domain are present in approximately 25-30% of newly diagnosed AML cases. Patients with AML harboring the FLT3-ITD mutation have poorer prognosis following the current induction chemotherapy treatment of cytarabine (AraC) and an anthracycline (daunorubicin [DNR] or idarubicin [IDR]). Azacitidine (Aza) is a treatment option for AML patients who are not eligible for intensive chemotherapy. Gilteritinib, a novel, small molecule inhibitor of the tyrosine kinases FLT3/AXL, is in Phase 3 development for treatment of FLT3 mutation-positive AML, including patients with FLT3-ITD mutations. In previous preclinical studies, gilteritinib has demonstrated superior antitumor effects when given in combination with AraC and either DNR or IDR compared with combination chemotherapy. Here, we report preclinical antileukemic activity of gilteritinib in combination with Aza against FLT3-ITD mutation-positive AML.Methods: The antitumor effects of gilteritinib alone and in combination with Aza were investigated in MV4-11 cells harboring the FLT3-ITD mutation. Apoptosis was detected using annexin V staining via flow cytometry; PARP cleavage was evaluated via Western blot analysis. Anti-apoptotic protein expression was evaluated by Western blot. Furthermore, the combination antitumor effects were evaluated in MV4-11-xenografted nude mice administered once-daily, oral gilteritinib at 3 mg/kg for 21 days alone and concomitantly with once-daily intravenous Aza at 3 mg/kg for 5 days. Pharmacokinetic parameters were also investigated in MV4-11 xenografted nude mice. Statistical differences in antitumor effects of combination therapy versus gilteritinib alone and combination therapy versus Aza alone were assessed on Day 21 using the Student's t-test.Results: Gilteritinib treatment for 48h resulted in an induction of apoptosis in MV4-11 cells as determined by an increase in annexin V-positive cells. When used in combination, gilteritinib augmented the Aza-induced increase in annexin V-positive cells. Gilteritinib also decreased the expression of anti-apoptotic proteins such as MCL-1, BCL2L10, and survivin, which are reported to be important in chemotherapy sensitivity, following 24h treatment. In combination with annexin results, an increase in PARP cleavage was also observed in MV4-11 cells following gilteritinib treatment; cells co-treated with gilteritinib and Aza showed a further increase in PARP cleavage. In mice xenografted with MV4-11 cells, oral gilteritinib (3 mg/kg/day) inhibited the tumor growth by 71%; Aza alone did not suppress tumor growth. The tumor volume in the doublet combination group (94% growth inhibition) was significantly smaller than in each single treatment group on Day 21 (Figure). No obvious influences on body weight, behavior, or diarrhea were noted in the combination group. Plasma concentrations for either gilteritinib or Aza were not considerably increased by their use in combination therapy when compared with the concentrations observed with single treatment.Conclusions: Gilteritinib treatment augmented Aza-induced apoptosis in part by reductions of anti-apoptotic protein expressions in vitro. In the non-clinical models used, gilteritinib, in combination with Aza, showed superior antitumor efficacy compared with each single agent alone. These findings support the development of gilteritinib in combination with Aza as a potential treatment of FLT3-ITD mutation-positive AML. [Display omitted] DisclosuresUeno:Astellas Pharma Inc: Employment. Mori:Astellas Pharma Inc: Employment. Kamiyama:Astellas Pharma Inc: Employment. Kaneko:Astellas Pharma Inc: Employment. Isshiki:Astellas Pharma Inc: Employment. Takeuchi:Astellas Pharma Inc: Employment.

CM363, a novel naphthoquinone derivative which acts as multikinase modulator and overcomes imatinib resistance in chronic myelogenous leukemia

Human Chronic Myelogenous Leukemia (CML) is a hematological stem cell disorder which is associated with activation of Bcr-Abl-Stat5 oncogenic pathway. Direct Bcr-Abl inhibitors are initially successful for the treatment of CML but over time many patients develop drug resistance. In the present study, the effects of CM363, a novel naphthoquinone (NPQ) derivative, were evaluated on human CML-derived K562 cells. CM363 revealed an effective cell growth inhibition (IC50 = 0.7 ± 0.5 μM) by inducing cancer cells to undergo cell cycle arrest and apoptosis. CM363 caused a dose- and time-dependent reduction of cells in G0/G1 and G2/M phases. This cell cycle arrest was associated with increased levels of cyclin E, pChk1 and pChk2 whereas CM363 downregulated cyclin B, cyclin D3, p27, pRB, Wee1, and BUBR1. CM363 increased the double-strand DNA break marker γH2AX. CM363 caused a time-dependent increase of annexin V-positive cells, DNA fragmentation and increased number of apoptotic nuclei. CM363 triggered the mitochondrial apoptotic pathway as reflected by a release of cytochrome C from mitochondria and induction of the cleavage of caspase-3 and -9, and PARP. CM363 showed multikinase modulatory effects through an early increased JNK phosphorylation followed by inhibition of pY-Bcrl-Abl and pY-Stat5. CM363 worked synergistically with imatinib to inhibit cell viability and maintained its activity in imatinib-resistant cells. Finally, CM363 (10 mg/Kg) suppressed the growth of K562 xenograft tumors in athymic mice. In summary, CM363 is a novel multikinase modulator that offers advantages to circumvent imanitib resistance and might be therapeutically effective in Bcrl-Abl-Stat5 related malignancies.

Abstract B12: Co-targeting PTEN-defined TNBC with p110beta-isoform specific inhibitor and PARP inhibitor BMN 673: A predictive context to sensitize PARP inhibitors in TNBC

Abstract Background: TNBC is a highly aggressive form of BRCA-associated BC subtype for which chemotherapy still remains a major form of treatment to which patients initially respond yet a majority of them inevitably relapse. PARP1 has been identified as a target in BRCA-defined cancers and inhibitors of PARP1 has been recently approved by FDA as targeted agents in BRCA-defined cancers. The loss of PTEN is the most common “first event” associated with basal-like subtype (Martins et al., 2012) and this PI3K-pathway activating event (deletion/mutation/loss of PTEN) occurs more frequently (35%) than PIK3CA mutations in this subtype of BC (Ellis & Perou, 2013). In the cytoplasm, PTEN-loss mediated upregulation of PI3K/AKT signal is known to depend on the PI3Kbeta-isoform. In contrast nuclear PTEN controls DNA repair (Bassi et al., 2013). A failure to repair the damaged DNA upon inhibition of PARP causes accumulation of DNA double-strand breaks and leads to apoptosis of cancer cells. Thus we hypothesized that a combination of p110beta-isoform specific inhibitor and PARP inhibitor will sensitize PARP inhibitors in PTEN-null TNBC model. Methods: Five PARP inhibitors, Talazoparib (BMN673, B), Niraparib (N), Olaparib (O), Rucaparib (R) and Veliparib (V) were tested in four PTEN-null TNBC cell lines, MDA-MB468, HCC70 (p.F90fs*9), BT549 (p.V275fs*1) and SUM149 cell lines in combination with p110beta specific inhibitor, AZD6482. Proliferative, apoptotic and PARylation signals following drug combinations were demonstrated by WB in a dose and time dependent manner. Pro-apoptotic and anti-proliferative effects were verified using complementary 3D ON-TOP assay, real time proliferation (Incucyte), AnnexinV and cl-caspase3 analyses. In order to understand the initial effects of isoform-specific inhibitors of PI3K we also compared anti-proliferative signals of GDC-0032 (p110 beta sparing inhibitor) and GDC-0941 (pan PI3K inhibitor) with that of AZD6482 at 3/6 hours in MDA-MB468 and SUM149 cells. Results: In PTEN null TNBC cells 500 nM BMN673 alone was effective in slowing cell proliferation and inducing apoptosis while AZD6482 (both 5-10 uM) did not have anti-proliferative or pro-apoptotic effects at 48 and 72 hours. Combinations of different PARPi (100nM of B, 1uM of N, 10uM of O, 10uM of R and 10uM of V) with AZD6482 blocked 3D growth of PTEN null BT549 and MDA-MB468 TNBT cells in a time dependent manner (96 hours & 7 days). PARP inhibitors abrogated PAR signals either alone or in combination with carboplatin in both BRCA1/2 WT and mutated cells. The most pronounced anti-tumor effect was observed with the combination of B and AZD6482 which was mechanistically explained by the robust increase of AnnexinV positive cells following a single 500nM dose of B at both 48 and 72 hours. In both MDA-MB468 and SUM149 cells although panPI3K inhibitor, GDC-0941 downregulated pAKT (S473, T308), pP70S6K (T389), pS6RP and p4EBP1 (T37/46) levels, p110 alpha-specific inhibitor GDC-0032 failed to perturb these signals while AZD6482 blocked them all. In summary, we demonstrate a remarkable sensitivity of tumor cells to PARP inhibition in PTEN-defined TNBC models and identify that PTEN-nullness as a potential predictive context for a possible co-targeting of PI3K pathway to further sensitize TNBC to PARP inhibitors. Significance: Our findings squarely place PTEN expression at the nexus of oncogenic signals in TNBC and indicate that TNBC patients with no PTEN activity in their tumors might benefit from combined p110beta-isoform specific inhibitor and PARP inhibitor. Citation Format: Nandini Dey, Jennifer H. Carlson, Pradip De, Brian Leyland-Jones. Co-targeting PTEN-defined TNBC with p110beta-isoform specific inhibitor and PARP inhibitor BMN 673: A predictive context to sensitize PARP inhibitors in TNBC. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B12.

Proteolytic Targeting Chimeric Molecules (PROTACs) Specific for Bromodomain-Containing Protein (BRD) 4 Are Active Against Pre-Clinical Models of Multiple Myeloma

Background:BRD4, a bromodomain and extraterminal domain (BET) family member, has an important role in modulating the expression of essential oncogenes such as c-MYC, and is emerged as a promising therapeutic target in diverse cancer types. Pharmacologic BET inhibitors in development such as JQ1 and OTX015 display preclinical anti-myeloma activity, and induce preferential loss of BRD4 bound to super-enhancers leading to transcriptional repression of c-MYC. Another approach to target this pathway is through the use of bi-functional molecules, which incorporate a small molecule BRD4 binding moiety with an E3 ubiquitin ligase recognition motif, such as ARV-825 and dBET1 (Lu et al. Chem Biol. 22:755, 2015, Winter et al. Science 348:1376, 2015). These agents induce Cereblon (CRBN)-dependent BRD4 ubiquitination and then proteasome-mediated degradation, thereby also reducing downstream c-MYC protein levels.Methods:We performed pre-clinical studies in myeloma cell lines and primary samples using ARV-825 and ARV-763, which are PROTACs that target BRD4 to either the CRBN or the Von Hippel-Lindau (VHL) E3 ligases, respectively. Downstream effects were studied using viability and apoptosis assays, cell cycle profiling, and Western blotting, among others.Results:Tetrazolium assays showed that both PROTACs were able to reduce the viability of a panel of myeloma cell lines, including MM1.S, U266, RPMI 8226, ANBL-6, KAS-6/1, and OPM-2 cells, and this occurred with greater potency than was the case for the BRD4 inhibitors JQ1 or OTX015. Median inhibitory concentrations were 5.66-91.98 nM for ARV-825, and 13.22-1522 nM for ARV-763, respectively. This reduction in viability was both time- and concentration-dependent, and was associated with a reduction of cells in the S phase, and an increase in G0/G1 cells, as well as cells with sub-G0/G1 DNA content, suggesting the onset of apoptosis. Programmed cell death was indeed found to be induced based on the appearance of an increase in Annexin V-positive cells by flow cytometry, and in cleaved caspase 8, caspase 9, caspase 3, and poly-ADP-ribose polymerase by Western blotting. The latter was associated with a specific reduction in the expression levels of both BRD4 and c-MYC that did not influence the abundance of other cellular proteins that were not BRD4 targets, and in a reduction in BRD4 and c-MYC mRNA. In contrast, JQ1 and OTX015 exposure resulted in a slight increase in BRD4 protein expression and a lesser decrease of c-MYC protein. Studies of drug combinations showed that, as expected, lenalidomide and pomalidomide were antagonistic to the effects of the CRBN-targeted ARV-825 PROTAC, but these immunomodulatory drugs showed additive or synergistic effects in combination with the VHL-targeted agent ARV-763. Also as expected, bortezomib and carfilzomib reduced the ability of both ARV-825 and ARV-763 to induce BRD4 degradation, but enhanced anti-proliferative and pro-apoptotic effects were seen in a manner that was influenced by the sequence of drug addition. In studies of drug-resistant cell lines, both PROTACs were able to overcome dexamethasone, melphalan, lenalidomide, and bortezomib resistance, but cross-resistance was seen in RPMI 8226/Dox40 cells, suggesting that these compounds are substrates for P-glycoprotein, which is over-expressed in these cells. Finally, we tested BRD4 PROTACs in primary cells isolated from patients with multiple myeloma, and observed rapid loss of viability of these plasma cells.Conclusions:Taken together, our data demonstrate that BRD4 degraders have promising activity against pre-clinical models of multiple myeloma, and support their translation to the clinic for patients with relapsed/refractory disease. Additional combination and mechanistic studies, as well as data from ongoing in vivo studies, will be presented at the meeting. DisclosuresLu:Arvinas, LLC: Employment, Equity Ownership. Qian:Arvinas, LLC: Employment, Equity Ownership. Orlowski:Acetylon: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Research Funding; Forma Therapeutics: Consultancy; Celgene: Consultancy, Research Funding; Millennium Pharmaceuticals: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding; Onyx Pharmaceuticals: Consultancy, Research Funding; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy; BioTheryX, Inc.: Membership on an entity's Board of Directors or advisory committees; Spectrum Pharmaceuticals: Research Funding.

Targeting Heme Oxygenase-1 Improves Poor Therapeutical Outcomes of BCR-ABL-Positive Adult B-Allwithikaros Isoform 6

Genomic profiling studies have demonstrated that the dominant-negative Ikaros isoform 6 (IK6) is overexpressed in BCR-ABL1-positive B-lineage acute lymphoblastic leukemia (BCR-ABL1-positive B-ALL), and it is strongly associated with a poor outcome. In addition, IK6 expression leads to BCR-ABL-positive B-ALL patients insensitive to tyrosine kinase inhibitors (TKIs). Moreover, a study has revealed that Heme oxygenase-1 (HO-1), an essential survival factor, could be a potential target in treating ALL. Therefore, we were wondering whether targeting HO-1 could improve poor therapeutical outcomes of BCR-ABL-positive adult B-ALL with IK6. Firstly, we detected that IK6 existed in 20 of 42 (47.6%) adult BCR-ABL1-positive B-ALL by using reverse transcribed polymerase chain reaction (PCR) and nucleotide sequencing. We also found that early clinical response was poor in BCR-ABL1-positive B-ALL patients with IK6, and they were usually high WBC counts at diagnosis and a high relapse rate. Compared to healthy donors' CD34+ cells, the levels of HO-1 expression were higher in primary CD34+ cells derived from adult BCR-ABL1-positive B-ALL patients with IK6 by qRT-PCR and western blot studies. And there was a strong correlation between the expression of IK6 and HO-1. In primary CD34+ leukemic cells derived from our IK6-positive patients' pool, silencing HO-1 resulted in the reduce of proliferation and a substantial proapoptotic effect, including a significantly enriched subG1 population, an increase of Annexin V-positive cells and of caspase3 cleavage. Furthermore, silencing HO-1 also increased their sensitivity to TKIs. This phenomenon was also supported by the observation that ZnPP IX substantially augmented the growth-inhibitory effects of imatinib on primary leukemic cells. Finally, we found that IK6 led to STAT5 activiation, and HO-1 was one of the downstream target genes of STAT5. In conclusion, the existence of extra IK6 seems to exacerbate poor prognosis and risks of relapse in adult BCR-ABL1-positive B-ALL. HO-1 plays an important role as a survival factor in BCR-ABL1-positive B-ALL with IK6, and targeting HO-1 can probably improve the clinical outcomes. DisclosuresNo relevant conflicts of interest to declare.

The radiosensitizing activity of the SMAC-mimetic, Debio 1143, is TNFα-mediated in head and neck squamous cell carcinoma

Background and purposeSecond mitochondria-derived activator of caspase (SMAC)-mimetics are a new class of targeted drugs that specifically induce apoptotic cancer cell death and block pro-survival signaling by antagonizing selected members of the inhibitor of apoptosis protein (IAP) family. Material and methodsThe present study was designed to investigate the radiosensitizing effect and optimal sequence of administration of the novel SMAC-mimetic Debio 1143 in vitro and in vivo. Apoptosis, alteration of DNA damage repair (DDR), and tumor necrosis factor-alpha (TNF-α) signaling were examined. ResultsIn vitro, Debio 1143 displayed anti-proliferative activity and enhanced intrinsic radiation sensitivity in 5/6 head and neck squamous cell carcinoma (HNSCC) cell lines in a synergistic manner. In vivo, Debio 1143 dose-dependently radio-sensitized FaDu and SQ20B xenografts, resulting in complete tumor regression in 8/10 FaDu-xenografted mice at the high dose level. At the molecular level, Debio 1143 combined with radiotherapy (RT) induced enhancement of caspase-3 activity, increase in Annexin V-positive cells and karyopyknosis, and increase in TNF-α mRNA levels. Finally, in a neutralization experiment using a TNF-α-blocking antibody and a caspase inhibitor, it was shown that the radiosensitizing effect of Debio 1143 is mediated by caspases and TNF-α. ConclusionsThese results demonstrate that the novel SMAC-mimetic Debio 1143 is a radiosensitizing agent that is worthy of further investigation in clinical trials in combination with radiotherapy.