Hematological Cancer Cell Research Articles

Abstract LB-007: Interaction between integrin α4β1 and IGF1 and its role in IGF signaling in hematologic malignancies

Abstract Integrins are transmembrane αβ heterodimers that function as cell adhesion receptors. Integrins are aberrantly overexpressed in cancer cells and also in tumor-associated cells. They are involved in cancer cell proliferation, invasion and survival. It has been proposed that the interaction between integrin α4β1, a major integrin in hematologic cancer, and VCAM-1 and fibronectin is responsible for drug resistance in these malignances, since antagonists to this integrin reduce drug resistance. However these results are based on the assumption that VCAM-1 and fibronectin are the only ligands for α4β1. Insulin-like growth factor 1 (IGF1) signaling plays a critical role in drug resistance and radio resistance hematologic cancer. IGF1 is a polypeptide hormone that binds to tyrosine kinase receptor IGF1R and activates the PI3K/Akt signaling pathway. This pathway is involved in cell proliferation, survival, and inhibition of apoptosis. In various types of cancer IGF1 is over expressed and the PI3K/Akt pathway is activated at a much higher rate than in normal cells. This results in the growth of cancer cells and resistance to chemotherapy. IGF1 signaling is implicated in drug resistance, and is a good therapeutic target in hematologic cancers. It is unclear if α4β1 and IGF signaling work independently in drug resistance. Recently we established that IGF1 binds to integrins αvβ3 and α6β4, and subsequent integrin-IGF1-IGF1R ternary complex formation is a critical step in IGF signaling in non-hematologic cancer. We developed the R36E/R37E IGF1 mutant that is defective in integrin binding and a dominant-negative antagonist of IGF signaling. Here we show that IGF1 is a ligand for integrin α4β1 and induces a ternary complex with IGF1R and α4β1. Antagonists to integrin α4β1 and the R36E/R37E IGF1 mutant reduced intracellular signaling in Chinese hamster ovary cells expressing human integrin α4 (α4-CHO cells) and also in different hematologic cancer cells such as Reh, Molt-4, U937 and JY cells. These results suggest that integrin α4β1 is involved in IGF signaling through direct binding to IGF1 in hematologic cancer. The α4β1-IGF1 interaction is a novel therapeutic target for drug resistance in hematologic cancer. Citation Format: Dora M. Cedano Prieto, Jessica Yu, Yoko Takada, Yoshikazu Takada. Interaction between integrin α4β1 and IGF1 and its role in IGF signaling in hematologic malignancies. [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 LB-007.

Abstract 3726: Predicting cellular response to small molecule Mcl-1 antagonists

Abstract Myeloid cell leukemia -1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 protein family which many cancers overexpress and depend on for survival. Mcl-1 inhibits apoptosis by binding to Bax and Bak, inhibiting the effectors of apoptosis from oligomerizing at the outer membrane of the mitochondria to promote cell death. Due to its role in cancer and resistance development to conventional chemotherapies, efforts to potently and specifically target MCL-1 have been ongoing. We have discovered novel small molecule inhibitors that selectively disrupt Mcl-1-Bim binding in the sub-nanomolar range. Anti-proliferative activities of our leading Mcl-1 inhibitors were assessed in a panel of hematological and solid cancer cell lines. We found that our compound promoted apoptosis in a subset of cell lines tested. As there was a variable response, we used BH3 profiling, which is a functional assay that rapidly measures dependence on any of the anti-apoptotic Bcl-2 family proteins for an individual sample to predict in vitro sensitivity to our Mcl-1 antagonists. Using the MS1 peptide, a known potent and selective Mcl-1 binder as a positive control in the BH3 profiling assay, we found a correlation between the BH3 profiling measurement of anti-apoptotic dependence to Mcl-1 and increased cytotoxicity caused by our compounds. These studies demonstrate that our compounds promote apoptosis through a selective inhibition of Mcl-1 mechanism of action and supports further investigation of our compounds in in vivo models of cancer. Citation Format: Leah Hogdal, John Sensintaffar, Allison Arnold, Craig Goodwin, Zhiguo Bian, Subrata Shaw, Chris Tarr, Taekyu Lee, Edward Olejniczak, Stephen Fesik. Predicting cellular response to small molecule Mcl-1 antagonists. [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 3726.

Abstract 3572: AZ5576, a novel potent and selective CDK9 inhibitor, induces rapid cell death and achieves efficacy in multiple preclinical hematological models

Abstract Cyclin-dependent kinase 9 (Cdk9) is a serine/threonine kinase that regulates elongation of transcription through phosphorylation of RNA polymerase II at serine 2 (pSer2-RNAPII). Mcl1, an anti-apoptotic protein that has been linked to increased survival and chemotherapy resistance in various cancers, can be indirectly modulated through transient inhibition of Cdk9 due to it having a short-lived transcript and being a labile protein. Transient inhibition of Cdk9, therefore, represents a potential therapeutic opportunity in tumors dependent on Mcl1 for survival, including various hematological malignancies. AZ5576 is a potent, highly selective, and orally bioavailable inhibitor of Cdk9 that inhibits Cdk9 enzyme activity with an IC50 <5nM and decreases phosphorlyation of Ser2-RNAPII in cells with an IC50 of 96nM. In the MLL-fusion containing acute myeloid leukemia (AML) line, MV411, short-term treatment with AZ5576 led to a rapid dose-dependent decrease in pSer2-RNAPII with concomitant loss of Mcl1 mRNA and protein, resulting in the cleavage and activation of caspase 3 and loss of cell viability. However, protein expression of other Bcl2 family members (e.g. Bcl2, BclxL, Bim) remained unchanged. Further, AZ5576 induced rapid caspase activation and loss of viability in a diverse set of hematological cancer cell lines, including those from acute myeloid leukemia (16/20), multiple myeloma (10/19), and diffuse large B-cell lymphoma (8/13). In vivo efficacy with intermittent dosing of AZ5576 was also demonstrated in multiple xenograft models from each hematological tumor type. Finally, AZ5576 was still able to induce loss of viability in various multiple myeloma lines cultured in the presence of cytokines or bone marrow stromal cells that represent potential protective effects of the tumor microenvironment. Together, these results highlight the therapeutic potential for selective CDK9 inhibition in the treatment of hematological malignancies. Citation Format: Justin Cidado, Minhui Shen, Michael Grondine, Scott Boiko, Haiyun Wang, Alexandra Borodovsky, Anne Marie Mazzola, Alan Wu, Deborah Lawson, Douglas Ferguson, Beirong Gao, Andy Cui, Celina D’Cruz, Lisa Drew. AZ5576, a novel potent and selective CDK9 inhibitor, induces rapid cell death and achieves efficacy in multiple preclinical hematological models. [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 3572.

402. Bioengineering of Peripheral Blood Derived Gamma Delta T Cells in a Serum-Free Expansion Medium

To date the main source of effector cells in preclinical and clinical immunotherapy trials has been αβ T cells. Although cellular products utilizing these cells have shown some promise, there is a need for better control of antitumor immunogenicity, especially in the context of chimeric antigen receptor technologies. Therefore, as an alternative, we are investigating the use of a T cell subset, γΔ T cells. These are a potentially attractive cellular therapeutic as they can be expanded to clinically useful levels and have potent MHC independent intrinsic anti-tumorigenicity driven by their recognition of stress antigens on tumor cells. Our efforts have focused on development of a clinically reliable expansion and manufacturing process of genetically engineered γΔ T cells. In the current study we describe the optimization of culture conditions in serum free media (SFM) that can support robust expansion followed by genetic modification of the expanded cells. Peripheral blood mononuclear cells (PBMC) isolated from healthy donors were cultured in several SFMs including: OpTmizer, X-Vivo, SCGM, and AIM V as well as commonly used media containing serum, RPMI1640 supplemented with either 10% FBS or human serum. Complete growth media also included 2 mM L-glutamine, zoledronic acid (5µM added only at the start of culture), and IL-2 (either 100 or 1000 lU/ml added twice weekly). Of the SFM cultures, robust γΔ T cell expansion was only observed in OpTmizer supplemented with high-dose IL-2, which resulted in expansion and γΔ T cell percentages to levels sufficient for clinical use. Specifically, in OpTmizer, the percentage of γΔ T cells increased from a mean of 2.4% (0.8%-3.6%) in the starting product to 66.4% (35%-94.5%, 6 of 9 cultures were >70% γΔ T cell), resulting in 80-fold expansion at 14 days. The SFM-expanded γΔ T cells were evaluated for their anti-tumor cytotoxicity and shown to be equally effective at killing both hematologic and solid cancer cell lines as γΔ T cells expanded in serum containing media. Transduction efficiency was tested using GFP encoding self-inactivating lentiviral vectors (LV). These studies demonstrated that SIV and HIV transduce expanded γΔ T cells equally, and that compared to EF1α, cassettes employing an MSCV promoter provide greater transgene expression. The timing of LV addition was optimized with respect to i) γΔ T cell expansion, ii) LDL Receptor (LDL-R) expression, and iii) LV dose (3-4 consecutive transductions over 48 hours). The greatest transduction efficiencies were achieved in cultures with the greatest expansion. As expected a range of transduction efficiencies was observed for various donors with a mean of approximately 20%, which is similar to LV transduction levels observed for αβ T cell transductions with similar multiplicity of infection and rates of expansion. Therefore, an optimized strategy to expand and transduce γΔ T cells with demonstrated anti-cancer properties was developed. The utilization of SFM to expand these cells to clinically useful doses and redirection toward tumor cells via bioengineering can result in a clinically safe, widely applicable, and potentially more efficacious cellular immunotherapy.

TRAIL-coated lipid-nanoparticles overcome resistance to soluble recombinant TRAIL in non-small cell lung cancer cells

Purpose. Non-small cell lung cancer (NSCLC) is one the types of cancer with higher prevalence and mortality. Apo2-Ligand/TRAIL is a TNF family member able to induce apoptosis in tumor cells but not in normal cells. It has been tested in clinical trials against different types of human cancer including NSCLC. However, results of clinical trials have shown a limited efficacy of TRAIL-based therapies. Recently we have demonstrated that artificial lipid nanoparticles coated with bioactive Apo2L/TRAIL (LUV-TRAIL) greatly improved TRAIL cytotoxic ability being capable of killing chemoresistant hematological cancer cells. In the present work we have extended the study to NSCLC. Methods/patients. LUV-TRAIL-induced cytotoxicity was assessed on different NSCLC cell lines with different sensitivity to soluble TRAIL and on primary human tumor cells from three patients suffering from NSCLC cancer. We also tested LUV-TRAIL-cytotoxic ability in combination with several anti-tumor agents. Results. LUV-TRAIL exhibited a greater cytotoxic effect compared to soluble TRAIL both in A549 cells and primary human NSCLC cells. LUV-TRAIL-induced cell death was dependent on caspase-8 and caspase-3 activation. Moreover, combination of LUV-TRAIL with other anti-tumor agents such as flavopiridol, and SNS-032 clearly enhanced LUV-TRAIL-induced cytotoxicity against NSCLC cancer cells. Conclusion. The novel formulation of TRAIL based on displaying it on the surface of lipid nanoparticles greatly increases its anti-tumor activity and has clinical potential in cancer treatment.

Traf2- and Nck-interacting kinase (TNIK) is involved in the anti-cancer mechanism of dovitinib in human multiple myeloma IM-9 cells.

Traf2- and Nck-interacting kinase (TNIK) is a member of the germinal center kinase family. TNIK was first identified as a kinase that is involved in regulating cytoskeletal organization in many types of cells, and it was recently proposed as a novel therapeutic target in several types of human cancers. Although previous studies suggest that TNIK plays a pivotal role in cancer cell survival and prognosis, its function in hematological cancer cell survival has not been investigated. Here we investigated the relationship between TNIK function and cell viability in multiple myeloma IM-9 cells using TNIK small interfering RNA (siRNA) transfection and dovitinib treatment. Treatment of IM-9 cells with TNIK siRNA and dovitinib treatment reduced cell proliferation. The ATP competing kinase assay and western blot analysis showed that dovitinib strongly inhibited both the interaction of TNIK with ATP (K i, 13nM) and the activation of Wnt signaling effectors such as β-catenin and TCF4. Dovitinib also induced caspase-dependent apoptosis in IM-9 cells without significant cytotoxicity in PBMCs. Our results provide new evidence that TNIK may be involved in the proliferation of multiple myeloma IM-9 cells and in the anti-cancer activity of dovitinib via inhibition of the endogenous Wnt signaling pathway.

Inhibition of CXCR4 by LY2624587, a Fully Humanized Anti-CXCR4 Antibody Induces Apoptosis of Hematologic Malignancies.

SDF-1 and CXCR4 are a chemokine and chemokine receptor pair playing critical roles in tumorigenesis. Overexpression of CXCR4 is a hallmark of many hematological malignancies including acute myeloid leukemia, chronic lymphocytic leukemia and non-Hodgkin’s lymphoma, and generally correlates with a poor prognosis. In this study, we developed a humanized anti-CXCR4 monoclonal antibody, LY2624587 as a potent CXCR4 antagonist that was advanced into clinical study for cancer. LY2624587 blocked SDF-1 binding to CXCR4 with an IC50 of 0.26 nM, and inhibited SDF-1-induced GTP binding with a Kb of 0.66 nM. In human lymphoma U937 and leukemia CCRF-CEM cells expressing endogenous CXCR4, LY2624587 inhibited SDF-1-induced cell migration with IC50 values of 3.7 and 0.26 nM, respectively. This antibody also inhibited CXCR4 and SDF-1 mediated cell signaling including activation of MAPK and AKT in tumor cells expressing CXCR4. Bifocal microscopic and flow cytometry analyses revealed that LY2624587 mediated receptor internalization and caused CXCR4 down-regulation on the cell surface. In human hematologic cancer cells, LY2624587 caused dose dependent apoptosis in vitro and in vivo. In mouse xenograft models developed with human leukemia and lymphoma cells expressing high levels of CXCR4, LY2624587 exhibited dose-dependent tumor growth inhibition and provided significant survival benefit in a disseminated lymphoma model. Collectively, we have demonstrated that CXCR4 inhibition by LY2624587 has the potential for the treatment of human hematological malignancies.

Dissecting Signaling Network Responses to PAK4 Allosteric Modulators (PAMs) in Cell Subsets within Primary Human Acute Myeloid Leukemia Samples

Background: Acute myeloid leukemia (AML) is the most common acute leukemia in adults and has a poor outcome with limited treatment options in patients with relapsed or resistant disease. Therapy resistance in AML is likely related to the inadequacy of therapy within leukemia cell subsets, including leukemia stem cells (LSCs). The p21-activated kinase (PAK) family of proteins was shown to be overexpressed in cancer cells and to play a key role in proliferation, survival, and maintenance of cellular structure. The series of orally bioavailable PAK4 allosteric modulators (PAM) have previously been shown to have activity in hematological cancer cell lines, including those derived from acute myeloid leukemia (AML) (Senapedis et al. Blood124, 2208-2208). Understanding how therapies target cellular subsets within primary patient samples could aid drug development by revealing any subset specific drug effects. In this project, we studied the effects of p21-activated kinase 4 (PAK4) modulation in AML samples. PAK4 modulation has been shown to have significant effects on many intracellular signaling pathways, including PI3K/AKT, MAPK/ERK and WNT/β-catenin pathways (Senapedis et al. Blood124, 2208-2208). It is unknown whether PAMs will have similar activity in primary leukemia cells. Likewise, it is currently unclear to what extent PAMs will differentially impact primary cell subsets including leukemia stem cells and non-malignant cell subsets that may be critical to recovery of bone marrow functions. We have previously shown that the single cell biology platform of flow cytometry is well-suited for dissecting clinically relevant signaling network mechanisms in primary human AML (Irish et al. Cell, 118(2):217-28).Methods: Flow cytometry was used to dissect the impact of an orally bioavailable PAM in AML cell lines and primary patient tissue. Cell lines chosen for this study included NRAS mutant KG-1 and Kasumi-1, which carry t(8;21) and express the AML1:ETO fusion protein. Primary AML biopsies were acquired from bone marrow or blood prior to any treatment and patients were identified and consented for this study according to a local Institutional Review Board-approved protocol. AML tissue samples were viably cryopreserved and then assayed ex vivo. Established protocols were used for phospho-specific flow cytometry, fluorescent cell barcoding, and data analysis in Cytobank (Irish et al. Cell, 118(2):217-28, Doxie and Irish, Curr Top Microbiol Immunol. 377:1-21).Results: Differential effects of PAK4 inhibition were observed between cell lines and among cell subsets from AML patient bone marrow. In leukemia cell lines and patient samples, p-ERK and p-S6 showed marked inhibition via PAM, though degree of inhibition varied. In AML patient samples, PAMs blocked signaling responses in p-ERK specifically in AML blasts, but spared normal CD45hi mononuclear cells (0.88 vs. 0.29-fold reduction (arcsinh scale) in p-ERK at 10 nM). Within the AML blast population, CD34+ CD38- and CD34+ CD38+ AML subsets showed similar PAM dose response via p-ERK.Conclusions: Single cell analysis effectively distinguishes effects of PAK4 inhibition via a series of allosteric modulators of PAK4 (PAMs) on leukemia and non-leukemia subsets in the same sample. PAM reduced immediate p-ERK and p-S6 levels in primary leukemia and cell lines. Notably, inhibition in various subsets within human AML was successfully measured by phospho-flow cytometry. Signaling changes in p-ERK were minimal within non-leukemic mature CD45+ mononuclear cells found in primary patient biopsies. Analysis of CD34+ CD38- cells indicates that PAMs could have activity within leukemia stem cells, and, at least, effect the AML progenitors. These findings support further investigation into the mechanism of action and treatment potential of PAMs in AML. DisclosuresSenapedis:Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties. Baloglu:Karyopharm Therapeutics Inc.: Employment, Equity Ownership. Landesman:Karyopharm: Employment. Irish:Novartis: Honoraria; Cytobank, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Research Funding; InCyte: Research Funding. Savona:Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

The Combination of Entospletinib and Vincristine Demonstrates Synergistic Activity in a Broad Panel of Hematological Cancer Cell Lines and Anti-Tumor Efficacy in a DLBCL Xenograft Model

BackgroundEntospletinib (GS-9973) selectively inhibits spleen tyrosine kinase (SYK), a critical signaling component of the BCR pathway that is expressed primarily in cells of hematopoietic lineage including normal and malignant B-lymphocytes. Entospletinib is currently in phase II clinical trials, where it has demonstrated both a high degree of safety as well as efficacy against chronic lymphocytic leukemia (Sharman, J., et al. Blood, 2015) and other B cell malignancies. Despite these successes, new therapeutic options, including combinations with standard of care agents, are needed in order to achieve the goal of curing disease through finite treatment. We show here that the combination of entospletinib and vincristine causes synergistic apoptosis in vitro in a broad panel of cell lines derived from hematological cancers including diffuse large B cell lymphoma (DLBCL), acute lymphocytic leukemia, follicular lymphom), multiple myeloma, and acute myelogenous leukemia. We also evaluated and compared the in vivo efficacy of entospletinib and vincristine as singe agents and in combination in a DLBCL tumor xenograft model using the SU-DHL-10 cell line.MethodsIn vitro growth inhibition of a panel of malignant hematological cell lines was assessed using CellTiter-Glo™ Assay (Promega) after 72h incubation with entospletinib or vincristine alone or in combination. Synergy was evaluated using the Bliss model of independence (Meletiadis, J., et al., Med Mycol, 2005). In vivo, SU-DHL-10 cells (5 x 106 cells) were implanted subcutaneously in the axilla in male SCID beige mice. All mice were sorted into study groups on Day 16 such that each group's mean tumor volume fell within 10% of the overall mean (197mm3). Dosing was initiated on Day 16 and animals were dosed for 17 days. Plasma concentrations of entospletinib and vincristine were assessed on Day 19, and the entospletinib 75 mg/kg dose was lowered on Day 22 to 50 mg/kg to approximate the human achievable SYK target coverage of EC80. Efficacy and tolerability were evaluated by tumor measurements and body weight monitored three times weekly. Tumor burden data were analyzed by the application of a two-way analysis of variance (ANOVA), with post-hoc analysis.ResultsIn vitro combinations of entospletinib with low concentrations of vincristine resulted in marked inhibition of cell proliferation and induction of apoptosis in a broad panel of 19 tumor cell lines representing major B cell malignancies including DLBCL. The combination of entospletinib with vincristine had a profound inhibitory effect on proliferation in all subtypes of DLBCL. Entospletinib was evaluated at a concentration equivalent to the Cminof the clinical dose and vincristine was used at concentrations (≤ 10 nM) that had little to no significant single agent effect in these cell lines.In vivo in a SU-DHL-10 xenograft model, entospletinib dosed alone at 25 or 75/50 mg/kg significantly inhibited tumor growth, causing 39% and 20% tumor growth inhibition (TGI), respectively, compared to the vehicle-treated control group. Vincristine administered at either 0.15 and 0.5 mg/kg Q7D x 3 also resulted in significant TGI (42% and 85% TGI, respectively). The addition of entospletinib (75/50 mg/kg) to 0.5 mg/kg or 0.15 mg/kg vincristine resulted in a significant increase in TGI from 85% to 96% (p= 0.001) and 42% to 71% (p< 0.0001), respectively. The addition of entospletinib (25 mg/kg) to vincristine did not significantly increase the tumor growth inhibition. While the groups receiving either entospletinib or vincristine as single agents had no complete or partial tumor regression, 50% of the mice receiving the combination of 75/50 mg/kg entospletinib with 0.5 mg/kg vincristine had partial responses, 8% had complete regression and 8% were tumor free at the end of study (Figure 1).ConclusionEntospletinib and vincristine demonstrated efficacy and tolerability both alone and in combination in the SU-DHL-10 DLBCL cell line xenograft model in SCID beige mice. Vincristine combinations with entospletinib showed significantly greater efficacy than vincristine alone. These data support the further clinical development of entospletinib in combination with vincristine for the treatment of DLBCL.a ENTO: PO: Q12H x 2 (Day 16-32)b VCR: IV: Q7D x 3 (Days 18, 25, 32) [Display omitted] DisclosuresAxelrod:Gilead Sciences: Employment, Equity Ownership. Fowles:Gilead Sciences: Employment, Equity Ownership. Silverman:Gilead Sciences: Employment, Equity Ownership. Clarke:Gilead Sciences: Employment, Equity Ownership. Tang:Gilead Sciences: Employment, Equity Ownership. Rousseau:Gilead Sciences: Employment, Equity Ownership. Webb:Gilead Sciences: Employment, Equity Ownership. Di Paolo:Gilead Sciences: Employment, Equity Ownership.

Astarabine, a Cytarabine Pro-Drug, Is Safe and Efficacious in the Treatment of Leukemia - Results of Animal Studies

The chemotherapy drug cytarabine is a standard of care in the treatment of Acute Myeloid Leukemia (AML) and relapsed Acute Lymphoblastic Leukemia (ALL), and is the backbone of induction and consolidation chemotherapy for AML. However, cytarabine is highly toxic, with severe side effects such as cerebellar toxicity and bone marrow suppression. Moreover, doses are significantly attenuated for older patients and patients with hepatic or renal dysfunction. Hence, the toxicity of cytarabine considerably limits its use.Astarabine is a construct of cytarabine covalently bound to asparagine. Unlike cytarabine, Astarabine's toxicity was shown to be mostly specific to leukemia cells. Leukemia cells lack asparagine synthetase and are dependent on external source for asparagine, hence asparagine was shown to be selectively taken up by leukemia cells by active transport. Astarabine is taken up by leukemia cells, where it is metabolized into the cytotoxic cytarabine molecules, resulting in leukemia cell death.The effect of Astarabine on 14 different cell lines was investigated. The studies demonstrated that most of the hematological cancer cell lines were sensitive to both Astarabine and cytarabine, while solid tumors cell lines were significantly less sensitive. Astarabine metabolite assay in human leukemia cell lines detected both Astarabine and free Cytarabine metabolites, indicating that Astarabine is a pro-drug of cytarabine.Animal pharmacokinetics studies were performed in mice and pigs. Pharmacokinetics analysis in mice was preformed by Astarabine single intraperitoneal (IP) injection. Astarabine half-life t1/2 of distribution and elimination in mice plasma ranged between 0.645-1.105 hours. Pharmacokinetics analysis in pigs preformed by Astarabine single intraveneous (IV) injection indicated that 50% (t1/2) of Astarabine is cleared from the pigs blood within 17.5-22.8 hours, a significantly higher t1/2 compared to the reported Cytarabine half-life.In vivo tolerability/toxicology studies determinedAstarabine IP repeated maximal tolerated dose (MTD) in IRC mice as 781 mg/kg/day, administrated for 7 consecutive days. IP repeated MTD of Astarabine in SCID mice was determined as 1275-1462 mg/kg/day, administrated for 7 consecutive days. IV repeated MTD in SCID mice was determined as 400 mg/kg/day, administrated for 14 days. Astarabine IV repeated MTD in young pigs was determinedto be 28 mg/kg/day, administrated for 6 consecutive days. Hence, it is concluded Astarabine improves drug tolerability and reduce toxicity in animals by 10-20 fold compared to the reported toxicity of cytarabine.In vivo efficacy studies were performed in a leukemia mouse model (L1210). The L1210 mouse leukemia creates a semi-solid tumor in the abdomen of the mice. Leukemia transplanted mice (12 mice per group) received 14 consecutive daily IP doses of 312 mg/kg/day or 1250 mg/kg/day of Astarabine, or saline control. The control mice died or sacrificed on day 17 due to cancer growth. The abdominal diameter of mice in the 312 mg/kg/day dose Astarabine and the control groups increased during the study (by 41% and 57.8%, respectively), while the abdominal diameter of the 1250 mg/kg/day dose Astarabine group decreased by 8.8%. The abdominal diameter results correlated with the rate of tumor growth: tumors were found in necropsies of mice of both control and 312 mg/kg/day dose Astarabine, however, no tumors were found in necropsies of mice treated with the 1250 mg/kg/day dose Astarabine during recovery period and on termination day. The results present a dose response of Astarabine in leukemia models.In conclusion, Astarabine may serve as a safer, non-toxic alternative to cytarabine for treatment of AML and ALL. Furthermore, Astarabine may offer effective treatment to older patients where current available treatment results in poor outcome. Studies in humans are ongoing. DisclosuresGengrinovitch:BioSight Ltd.: Employment, Equity Ownership, Patents & Royalties. Ben Yakar:BioSight Ltd.: Employment, Equity Ownership. Rowe:Amgen: Consultancy; BioSight Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx Ltd.: Consultancy.

Abstract C176: TAS4464, a novel, highly potent, and selective inhibitor of NEDD8 activating enzyme demonstrates sustained target inhibition and antitumor activity in a preclinical model

Abstract BACKGROUND: NEDD8 activating enzyme (NAE) is an essential E1 enzyme in the NEDD8 conjugation (neddylation) pathway, which controls cancer cell growth and survival through activation of cullin-RING ligase complexes (CRLs). Therefore, NAE is considered to be a promising target for cancer therapy. Here we describe the profile of a novel, highly potent, and selective NAE inhibitor, TAS4464, which inactivates CRLs in tumors and has strong antitumor efficacy in a preclinical model. MATERIALS AND METHODS: The selectivity of TAS4464 to E1 enzymes were assessed by various ubiquitin-like protein conjugation assays. The effects of TAS4464 on NEDD8 conjugation and the amounts of CRL substrates were evaluated by Western blot analysis. Cell viability was measured with a Cell Titer-Glo assay. The antitumor activity of TAS4464 was evaluated in an acute lymphoblastic leukemia (CCRF-CEM) xenograft model. RESULTS: We demonstrated that TAS4464 was a mechanism-based NAE inhibitor. TAS4464 formed an NEDD8-TAS4464 adduct that could achieve nanomolar inhibition of NAE. TAS4464 selectively inhibited NAE enzyme activity relative to the other E1s, ubiquitin activating enzyme (UAE) and SUMO activating enzyme (SAE). TAS4464 treatment inhibited cullin neddylation and induced accumulation of key CRL substrate proteins such as CDT1, p27, and phosphorylated IκB in human cancer cell lines. Enzyme inhibitory and cellular NAE inhibitory activities of TAS4464 were higher than those of a known NAE inhibitor, MLN4924. Cytotoxicity profiling of TAS4464 revealed widespread antiproliferative activity against a panel of cancer cell lines. Almost all cell lines derived from hematological malignancies were highly sensitive to TAS4464. Whereas carbonic anhydrase inhibition by MLN4924 resulted in accumulation in red blood cells, the blood-to-plasma ratio of TAS4464 was close to equivalent because of higher selectivity. In addition, TAS4464 did not inhibit major cytochrome p450 enzymes and showed good metabolic stability in isolated hepatocytes in vitro. The pharmacodynamic action of TAS4464 in tumors assessed by measuring the amount of cullin-NEDD8 conjugation in CCRF-CEM xenografts revealed strong cullin neddylation inhibitory activity of TAS4464; the inhibitory activity was sustained longer than that of MLN4924. In the CCRF-CEM xenograft model, weekly administration of TAS4464 at a dose of 100 mg/kg was more efficacious than twice-weekly administration of MLN4924 at a dose of 120 mg/kg: TAS4464 led to complete tumor regression without marked weight loss. CONCLUSION: TAS4464 is the most potent and selective NAE inhibitor reported to date, with good pharmaceutical properties and superior efficacy. Hematological cancer cell lines are highly sensitive to TAS4464. Based on its promising preclinical activity, a phase 1 clinical trial of TAS4464 is being planned. Citation Format: Chihoko Yoshimura, Hiromi Muraoka, Hiroaki Ochiiwa, Shingo Tsuji, Akihiro Hashimoto, Toru Takenaka, Hiromi Kazuno, Keiji Ishida, Takashi Mizutani, Shuichi Ohkubo, Kenichi Matsuo, Teruhiro Utsugi, Yoshikazu Iwasawa. TAS4464, a novel, highly potent, and selective inhibitor of NEDD8 activating enzyme demonstrates sustained target inhibition and antitumor activity in a preclinical model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C176.

2,5-Dimethyl-celecoxib inhibits cell cycle progression and induces apoptosis in human leukemia cells.

Cyclooxygenase-2 (COX-2) is an essential regulator of cancer promotion and progression. Extensive efforts to target this enzyme have been developed to reduce growth of cancer cells for chemopreventive and therapeutic reasons. In this context, cyclooxygenase-2 inhibitors present interesting antitumor effects. However, inhibition of COX-2 by anti-COX-2 compounds such as celecoxib was recently associated with detrimental cardiovascular side effects limiting their clinical use. As many anticancer effects of celecoxib are COX-2 independent, analogs such as 2,5-dimethyl-celecoxib (DMC), which lacks COX-2-inhibitory activity, represent a promising alternative strategy. In this study, we investigated the effect of this molecule on growth of hematologic cancer cell lines (U937, Jurkat, Hel, Raji, and K562). We found that this molecule is able to reduce the growth and induces apoptosis more efficiently than celecoxib in all the leukemic cell lines tested. Cell death was associated with downregulation of Mcl-1 protein expression. We also found that DMC induces endoplasmic reticulum stress, which is associated with a decreased of GRP78 protein expression and an alteration of cell cycle progression at the G1/S transition in U937 cells. Accordingly, typical downregulation of c-Myc and cyclin D1 and an upregulation of p27 were observed. Interestingly, for shorter time points, an alteration of mitotic progression, associated with the downregulation of survivin protein expression was observed. Altogether, our data provide new evidence about the mode of action of this compound on hematologic malignancies.

Lipid raft-mediated Fas/CD95 apoptotic signaling in leukemic cells and normal leukocytes and therapeutic implications.

Plasma membrane is now recognized to contain tightly packed cholesterol/sphingolipid-rich domains, known as lipid or membrane rafts, which are more ordered than the surrounding lipid bilayer. Lipid rafts are crucial for the compartmentalization of signaling processes in the membrane, mostly involved in cell survival and immune response. However, in the last 15 years, a large body of evidence has also identified raft platforms as scaffolds for the recruitment and clustering of death receptor Fas/CD95 and downstream signaling molecules, leading to the concept of death-promoting lipid rafts. This raft-Fas/CD95 coclustering was first described at the early 2000s as the underlying mechanism for the proapoptotic action of the alkylphospholipid analog edelfosine in leukemic cells, hence facilitating protein-protein interactions and conveying apoptotic signals independently of Fas/CD95 ligand. Edelfosine induces apoptosis in hematologic cancer cells and activated T-lymphocytes. Fas/CD95 raft coclustering is also promoted by Fas/CD95 ligand, agonistic Fas/CD95 antibodies, and additional antitumor drugs. Thus, death receptor recruitment in rafts is a physiologic process leading to cell demise that can be pharmacologically modulated. This redistribution and local accumulation of apoptotic molecules in membrane rafts, which are usually accompanied by displacement of survival signaling molecules, highlight how alterations in the apoptosis/survival signaling balance in specialized membrane regions modulate cell fate. Membrane rafts might also modulate apoptotic and nonapoptotic death receptor signaling. Here, we discuss the role of lipid rafts in Fas/CD95-mediated apoptotic cell signaling in hematologic cancer cells and normal leukocytes, with a special emphasis on their involvement as putative therapeutic targets in cancer and autoimmune diseases.

Abstract 5472: Selinexor, a selective inhibitor of nuclear export (SINE), acts through NF-κB deactivation and combines with proteasome inhibitors to synergistically induce tumor cell death

Abstract Background: The primary nuclear export protein, Exportin 1 (XPO1/CRM1) is overexpressed in most cancers, which frequently correlates with poor prognosis. SINE compounds are a family of small-molecule bioavailable drugs that bind covalently to XPO1 leading to nuclear retention of major tumor suppressor proteins such as p53 and IκB, resulting in selective tumor cell death. Selinexor is the most advanced SINE with &amp;gt;500 hematological and solid tumor cancer patients treated to date in Phase I/II clinical trials. Here we investigated the role of NF-κB transcriptional deactivation in SINE induced anti tumor activity by nuclear retention of IκB. We report the results of combining selinexor or related SINE compounds with proteasome inhibitors, for which inhibition of NF-κB has also been shown to be an important mechanism for cancer cell killing. Methods: Whole protein cell lysates from solid and hematological cancer cell lines treated with selinexor with or without proteasome inhibitors were analyzed by immunoblots. IκB localization was evaluated by immunofluorescence. Cytotoxic effects of SINE compounds were evaluated in the presence or absence of proteasome inhibitors. NF-κB transcriptional activity was analyzed using an ELISA assay. H929 multiple myeloma xenografts in NOD-SCID mice were treated with selinexor alone or in combination with carfilzomib to determine effects on tumor growth. Results: NF-κB transcriptional activity was upregulated in SINE resistant sarcoma cell lines HT1080R (IC50 = 2.4 μM versus HT1080 parental IC50 = 0.03 μM) and ASPS-KY (IC50&amp;gt;10μM). In those resistant lines, combination of the proteasome inhibitors bortezomib or carfilzomib with SINE compound showed dramatic synergistic cytotoxic potency of more than 10 folds. Also, the combination of bortezomib with selinexor led to synergistic nuclear retention of IκB. Furthermore, IκB silencing by siRNA led to reduced selinexor cytotoxic potency, increasing IC50 from 58 nM to 830 nM (9-fold) in IM-9 multiple myeloma cells and from 27 nM to 1.9 μM (66-fold) in U2OS osteosarcoma cells. Finally, the combination of selinexor with carfilzomib was synergistic in reducing tumor growth in the H929 multiple myeloma xenograft model. Conclusion: Inhibition of NF-κB transcriptional activity through forced nuclear retention of IκB appears to be an important mechanism in the selective tumor cell cytotoxicity of selinexor and related SINE compounds. Furthermore, the combination of selinexor and proteasome inhibitors, which are also known to act at least in part through inhibition of NF-κB, leads to synergistic activity in vitro and in vivo, suggesting that such combinations may provide clinically more effective than the single agents. A Phase 1 trial to study the safety and efficacy of selinexor in combination with carfilzomib in multiple myeloma is ongoing (NCT02199665). Citation Format: Yosef Landesman, Trinayan Kashyap, Marsha Crochiere, Boris Klebanov, Sharon Friedlander, William Senapedis, Robert Carlson, Michael Kauffman, Sharon Shacham. Selinexor, a selective inhibitor of nuclear export (SINE), acts through NF-κB deactivation and combines with proteasome inhibitors to synergistically induce tumor cell death. [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 5472. doi:10.1158/1538-7445.AM2015-5472

Anticancer activity of Ophiobolin A, isolated from the endophytic fungus Bipolaris setariae

The present work describes the anticancer activity of Ophiobolin A isolated from the endophytic fungus Bipolaris setariae. Ophiobolin A was isolated using preparative HPLC and its structure was confirmed by HRMS, 1H NMR, 13C NMR, COSY, DEPT, HSQC and HMBC. It inhibited solid and haematological cancer cell proliferation with IC50 of 0.4–4.3 μM. In comparison, IC50 against normal cells was 20.9 μM. It was found to inhibit the phosphorylation of S6 (IC50 = 1.9 ± 0.2 μM), ERK (IC50 = 0.28 ± 0.02 μM) and RB (IC50 = 1.42 ± 0.1 μM), the effector proteins of PI3K/mTOR, Ras/Raf/ERK and CDK/RB pathways, respectively. It induced apoptosis and inhibited cell cycle progression in MDA-MB-231 cancer cells with concomitant inhibition of signalling proteins. Thus, this study reveals that anticancer activity of Ophiobolin A is associated with simultaneous inhibition of multiple oncogenic signalling pathways namely PI3K/mTOR, Ras/Raf/ERK and CDK/RB.

Mangrove dolabrane-type of diterpenes tagalsins suppresses tumor growth via ROS-mediated apoptosis and ATM/ATR-Chk1/Chk2-regulated cell cycle arrest.

Natural compounds are an important source for drug development. With an increasing cancer rate worldwide there is an urgent quest for new anti‐cancer drugs. In this study, we show that a group of dolabrane‐type of diterpenes, collectively named tagalsins, isolated from the Chinese mangrove genus Ceriops has potent cytotoxicity on a panel of hematologic cancer cells. Investigation of the molecular mechanisms by which tagalsins kill malignant cells revealed that it induces a ROS‐mediated damage of DNA. This event leads to apoptosis induction and blockage of cell cycle progression at S‐G2 phase via activation of the ATM/ATR—Chk1/Chk2 check point pathway. We further show that tagalsins suppress growth of human T‐cell leukemia xenografts in vivo. Tagalsins show only minor toxicity on healthy cells and are well tolerated by mice. Our study shows a therapeutic potential of tagalsins for the treatment of hematologic malignancies and a new source of anticancer drugs.

Phage-AgNPs complex as SERS probe for U937 cell identification.

The early diagnosis of malignancy is the most critical factor for patient survival and the treatment of cancer. In particular, leukemic cells are highly heterogeneous, and there is a need to develop new rapid and accurate detection systems for early diagnosis and monitoring of minimal residual disease. This study reports the utilization of molecular networks consisting of entire bacteriophage structure, displaying specific peptides, directly assembled with silver nanoparticles as a new Surface Enhanced Raman Spectroscopy (SERS) probe for U937 cells identification in vitro. A 9-mer pVIII M13 phage display library is screened against U937 to identify peptides that selectively recognize these cells. Then, phage clone is assembled with silver nanoparticles and the resulting network is used to obtain a SERS signal on cell-type specific molecular targets. The proposed strategy could be a very sensitive tool for the design of biosensors for highly specific and selective identification of hematological cancer cells and for detection of minimal residual disease in a significant proportion of human blood malignancy.

CC-223, a Potent and Selective Inhibitor of mTOR Kinase: In Vitro and In Vivo Characterization.

mTOR is a serine/threonine kinase that regulates cell growth, metabolism, proliferation, and survival. mTOR complex-1 (mTORC1) and mTOR complex-2 (mTORC2) are critical mediators of the PI3K-AKT pathway, which is frequently mutated in many cancers, leading to hyperactivation of mTOR signaling. Although rapamycin analogues, allosteric inhibitors that target only the mTORC1 complex, have shown some clinical activity, it is hypothesized that mTOR kinase inhibitors, blocking both mTORC1 and mTORC2 signaling, will have expanded therapeutic potential. Here, we describe the preclinical characterization of CC-223. CC-223 is a potent, selective, and orally bioavailable inhibitor of mTOR kinase, demonstrating inhibition of mTORC1 (pS6RP and p4EBP1) and mTORC2 [pAKT(S473)] in cellular systems. Growth inhibitory activity was demonstrated in hematologic and solid tumor cell lines. mTOR kinase inhibition in cells, by CC-223, resulted in more complete inhibition of the mTOR pathway biomarkers and improved antiproliferative activity as compared with rapamycin. Growth inhibitory activity and apoptosis was demonstrated in a panel of hematologic cancer cell lines. Correlative analysis revealed that IRF4 expression level associates with resistance, whereas mTOR pathway activation seems to associate with sensitivity. Treatment with CC-223 afforded in vivo tumor biomarker inhibition in tumor-bearing mice, after a single oral dose. CC-223 exhibited dose-dependent tumor growth inhibition in multiple solid tumor xenografts. Significant inhibition of mTOR pathway markers pS6RP and pAKT in CC-223-treated tumors suggests that the observed antitumor activity of CC-223 was mediated through inhibition of both mTORC1 and mTORC2. CC-223 is currently in phase I clinical trials.

Synthesis and antiproliferative activity of α-branched α,β-unsaturated ketones in human hematological and solid cancer cell lines

A series of α-branched α,β-unsaturated ketones were prepared via boron trifluoride etherate mediated reaction between arylalkynes and carboxaldehydes. The evaluation of the antiproliferative activity over hematological (NB4) and solid cancer (A549, MCF-7) cell lines provided a structure-activity relationship. 5-Parameter QSAR equations were built which were able to explain 80%–92% of the variance in activity. The resulting selective lead compound showed IC50 value 0.6 μM against the hematological cell line and did not cause apoptosis, but blocked cell cycle in G0/G1. Moreover, it was demonstrated that this compound enhances and accelerates retinoic acid induced granulocytic differentiation.

Human NK cells activated by EBV+ lymphoblastoid cells overcome anti-apoptotic mechanisms of drug resistance in haematological cancer cells

Natural killer (NK) cells recognize and eliminate transformed or infected cells that have downregulated MHC class-I and express specific activating ligands. Recent evidence indicates that allogeneic NK cells are useful to eliminate haematological cancer cells independently of MHC-I expression. However, it is unclear if transformed cells expressing mutations that confer anti-apoptotic properties and chemoresistance will be susceptible to NK cells. Allogeneic primary human NK cells were activated using different protocols and prospectively tested for their ability to eliminate diverse mutant haematological and apoptotic-resistant cancer cell lines as well as patient-derived B-cell chronic lymphocytic leukemia cells with chemotherapy multiresistance. Here, we show that human NK cells from healthy donors activated in vitro with Epstein Barr virus positive (EBV+)-lymphoblastoid cells display an enhanced cytotoxic and proliferative potential in comparison to other protocols of activation such a K562 cells plus interleukin (IL)2. This enhancement enables them to kill more efficiently a variety of haematological cancer cell lines, including a panel of transfectants that mimic natural mutations leading to oncogenic transformation and chemoresistance (e.g., overexpression of Bcl-2, Bcl-XL and Mcl-1 or downregulation of p53, Bak/Bax or caspase activity). The effect was also observed against blasts from B-cell chronic lymphocytic leukemia patients showing multi-resistance to chemotherapy. Our findings demonstrate that particular in vitro activated NK cells may overcome anti-apoptotic mechanisms and oncogenic alterations frequently occurring in transformed cells, pointing toward the use of EBV+-lymphoblastoid cells as a desirable strategy to activate NK cells in vitro for the purpose of treating haematological neoplasia with poor prognosis.