Preclinical Characterization of E7438, a Potent, Selective Inhibitor of Protein Methyltransferase EZH2 with Robust Antitumor Activity Against EZH2 Mutated Non-Hodgkin Lymphoma Xenografts in Mice

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3712-3712 ◽  
Author(s):  
Heike Keilhack ◽  
Akira Yokoi ◽  
Sarah K Knutson ◽  
Tim Wigle ◽  
Natalie Warholic ◽  
...  
Keyword(s):  
Repeated Measures ◽  
Tumor Regression ◽  
Xenograft Model ◽  
Lymphoma Cells ◽  
Employment Equity ◽  
H3k27 Methylation ◽  
Non Hodgkin Lymphoma ◽  
Mouse Xenograft Model ◽  
Equity Ownership ◽  
Dose Dependent

Abstract Abstract 3712 The coupled enzymatic activity of wild-type and mutant EZH2 results in hyper-trimethylation of histone H3 lysine 27 (H3K27), which drives lymphomagenesis in heterozygous patients bearing the EZH2 mutations. Our group has previously reported that selective inhibition of EZH2 in cell culture results in selective killing of lymphoma cells bearing EZH2 mutations, with minimal effect on non-mutant lymphoma cells, suggesting that EZH2 enzymatic activity is a required driver of proliferation in the mutant-bearing cells [Knutson et al. (2012) Nature Chemical Biology, in press]. Through iterative medicinal chemistry we have developed a selective inhibitor of EZH2 with good pharmacological properties, E7438. E7438 binds to the enzyme in a manner competitive with S-adenosyl methionine (SAM) and a Ki for wild-type EZH2 of 2.5 ± 0.5 nM. The compound potently inhibits all known mutants of EZH2 that have been identified in non-Hodgkin lymphoma (NHL) patient samples. E7438 displays about 35-fold less activity against the closely related enzyme EZH1, and is >4500-fold selective with respect to all other protein methyltransferases tested. Lymphoma cells treated with E7438 display concentration- and time-dependent loss of H3K27 methylation with no effect on the methylation status of any other histone sites. The loss of H3K27 methylation results in selective killing of EZH2 mutant-bearing lymphoma cell lines. E7438 displays good oral bioavailability and pharmacokinetic properties. Various EZH2 mutant-bearing human lymphoma tumors were subcutaneously implanted in nude, SCID or NSG mice. Oral administration of E7438 to tumor bearing mice resulted in significant anti-tumor activity. The responses ranged from dose-dependent tumor growth inhibition to complete and sustained regressions. For example, KARPAS422 tumors in nude mice showed complete tumor elimination after 28 days of dosing, with mice remaining tumor free for up to 90 days after treatment cessation. Figure 1. E7438 causes complete and sustained tumor regression in a KARPAS422 nude mouse xenograft model of EZH2-mutated NHL. Dosing was on a BID schedule. * P< 0.05, Repeated measures ANOVA, Dunnett's post test vs. vehicle. Figure 1. E7438 causes complete and sustained tumor regression in a KARPAS422 nude mouse xenograft model of EZH2-mutated NHL. Dosing was on a BID schedule. * P< 0.05, Repeated measures ANOVA, Dunnett's post test vs. vehicle. Mice and rats tolerated E7438 administration well at doses representing high multiples of doses that show antitumor activity in mice. Activity against the EZH2 target in both species was demonstrated by dose-dependent diminution of H3K27me3 levels, assessed by ELISA, in samples of tumor, bone marrow, skin and peripheral blood mononuclear cells (PBMCs). Highly sensitive detection of existing H3K27me3 signal could also be observed with this ELISA assay in drug-naïve samples of human PBMCs. The ability to measure dose-dependent changes in H3K27me3 levels in skin and PBMCs portends the use of signal from these surrogate tissues as a non-invasive pharmacodynamics biomarker in human clinical trials. Disclosures: Keilhack: Epizyme Inc.: Employment, Equity Ownership. Yokoi:Eisai Co., Ltd.: Employment. Knutson:Epizyme Inc.: Employment, Equity Ownership. Wigle:Epizyme Inc.: Employment, Equity Ownership. Warholic:Epizyme Inc.: Employment, Equity Ownership. Kawano:Eisai Co., Ltd.: Employment. Minoshima:Eisai Co., Ltd.: Employment. Huang:Eisai Inc.: Employment. Kuznetsov:Eisai Inc.: Employment. Kumar:Eisai Inc.: Employment. Klaus:Epizyme, Inc.: Employment, Equity Ownership. Allain:Epizyme Inc.: Employment, Equity Ownership. Raimondi:Epizyme Inc.: Employment, Equity Ownership. Porter Scott:Epizyme: Employment, Equity Ownership. Chesworth:Epizyme: Employment, Equity Ownership. Moyer:Epizyme: Employment, Equity Ownership. Uenaka:Eisai Co., Ltd.: Employment. Copeland:Epizyme Inc.: Employment, Equity Ownership. Richon:Epizyme, Inc.: Employment, Equity Ownership. Pollock:Epizyme Inc.: Employment, Equity Ownership. Kuntz:Epizyme Inc.: Employment, Equity Ownership.

scholarly journals Development of Inhibitors of PIP4K2 As a Treatment for Patients with Hematologic Malignancies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 213-213
Author(s):  
David L McElligott ◽  
Edward Kesicki ◽  
Kannan Karukarichi ◽  
Hyeseok Shim ◽  
Rui Wang ◽  
...  
Keyword(s):  
Cell Survival ◽  
Human Cancer ◽  
Xenograft Model ◽  
Hematologic Malignancies ◽  
In Vivo Studies ◽  
Selective Inhibitors ◽  
Employment Equity ◽  
Mouse Xenograft Model ◽  
Equity Ownership

Abstract Phosphoinositide signaling is central to many cellular processes including the cell survival pathway known as autophagy. While there is evidence that autophagy can suppress tumorigenesis under certain circumstances, there is increasingly abundant evidence that autophagy promotes tumorigenesis and tumor cell survival in solid tumors and hematologic malignancies. Autophagy is a complex process that has evolved to promote survival of cells under a variety of stress or nutrient starvation conditions. There are a multitude of molecules and structures involved in initiating, promoting, and resolving the autophagy process. It is known that PI5P is an important component for the resolution of autophagy. PIP4K2α,β,γ are a family of lipid kinases that convert PI5P to PI(4,5)P2. These enzymes have recently been shown to be critical for the fusion of autophagosomes with lysosomes. This known activity, coupled with the observation that PIP4K2 activity is essential for the survival and leukemia initiating potential of human and mouse acute myeloid leukemia (AML) cells, suggest that the PIP4K2 family of enzymes may be a promising target for a new class of therapeutics for the treatment of hematologic malignancies. We have investigated the role of PIP4K2 enzymes in supporting the survival of cancer cells by developing potent and selective inhibitors of PIP4K2 enzymatic activity. A screen of human cancer cell lines show that potent and selective inhibitors of PIP4K2 are effective at inhibiting growth of a variety of hematologic cancers including leukemia- and lymphoma-derived lines. In vivo studies demonstrate that these inhibitors induce rapid regression of an AML tumor (MOLM-16) in a mouse xenograft model. These studies show a dose-dependent control of tumor growth with sustained regression of tumor volume with QD oral dosing of a prototype molecule. Body weights of the mice were stable over the course of the study suggesting that the molecule is well tolerated in this dosing protocol. A preliminary toxicity study in rats has not revealed any identifiable toxicity at doses up to 100 mg/Kg given QD/PO for 14 days. Additional in vitro safety studies suggest minimal safety concerns due to off-target activity. Exploration of the structure activity relationship of PIP4K2 inhibitors, using fragment and structure-based drug discovery, has led to highly potent and selective molecules with exceptional drug-like properties. A clinical development candidate has been selected and that candidate is currently in the late stages of preclinical studies preceding anticipated clinical entry in early 2019. Disclosures McElligott: Petra Pharma: Employment, Equity Ownership. Kesicki:Petra Pharma: Employment, Equity Ownership. Karukarichi:Petra Pharma: Employment, Equity Ownership. Shim:Petra Pharma: Employment, Equity Ownership. Wang:Petra Pharma: Employment, Equity Ownership. Yu:Petra Pharma: Employment, Equity Ownership. Zolfaghari:Petra Pharma: Employment, Equity Ownership. Linstrom:Sprint Bioscience: Employment, Equity Ownership. Persson:Sprint Bioscience: Employment, Equity Ownership. Hoglund:Sprint Bioscience: Employment, Equity Ownership. Ericsson:Sprint Bioscience: Employment, Equity Ownership. Trésaugues:Sprint Bioscience: Employment, Equity Ownership. Livendahl:Sprint Bioscience: Employment, Equity Ownership. Santangelo:Sprint Bioscience: Employment, Equity Ownership. Viklund:Sprint Bioscience: Employment, Equity Ownership. Pettersson:Sprint Bioscience: Employment, Equity Ownership. Wähling:Sprint Bioscience: Employment, Equity Ownership. Forsblom:Sprint Bioscience: Employment, Equity Ownership. Karlsson:Sprint Bioscience: Employment, Equity Ownership. Ginman:Sprint Bioscience: Employment, Equity Ownership. Braga:Sprint Bioscience: Employment, Equity Ownership. Henley:Sprint Bioscience: Employment, Equity Ownership. Talagas:Sprint Bioscience: Employment, Equity Ownership. Rahm:Sprint Bioscience: Employment, Equity Ownership. Johansson:Sprint Bioscience: Employment, Equity Ownership. Martinsson:Sprint Bioscience: Employment, Equity Ownership. Andersson:Sprint Bioscience: Employment, Equity Ownership. Cantley:Petra Pharma: Equity Ownership.

scholarly journals Single Cell-Level Signaling Profiling of Acute Myeloid Leukemia Following Treatment with Axl Kinase Inhibitor BGB324

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4931-4931
Author(s):  
Monica Hellesøy ◽  
Katarzyna Wnuk-Lipinska ◽  
Anna Boniecka ◽  
Eline Milde Nævdal ◽  
Hakon Reikvam ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Overall Survival ◽  
Cell Line ◽  
Research Funding ◽  
Xenograft Model ◽  
Employment Equity ◽  
In Vitro Response ◽  
Equity Ownership ◽  
Dose Dependent

Abstract Axl is a member of the Tyro3, Axl, Mer (TAM) receptor tyrosine kinase family that regulate a wide range of cellular functions, including cell survival, proliferation, migration/invasion and adhesion. Axl has been shown to play a key role in the survival and metastasis of many tumors, and has also been found to be upregulated and constitutively active in human AML. Indeed, Axl has been reported as an independent prognostic marker and a potential novel therapeutic target in AML. BGB324 is a first-in-class highly selective small molecule inhibitor of Axl. BGB324 has been shown to be safe and well tolerated in clinical safety studies in healthy volunteers at doses up to 1500 mg/day with a predictable PK profile and long plasma half-life, and is currently in phase I b clinical trials for AML and non-small cell lung cancer. In this study, we use phosphoflow cytometry to measure changes in signal transduction nodes in single AML cells treated with BGB324. We are applying this approach to monitor signaling profiles in primary AML cells harvested from patients undergoing BGB324 treatment. Results: The human AML cell line MOLM13 was treated in vitro with BGB324 (0.5 and 1µM for 1 hour) and analyzed for signal transduction changes by phosphoflow cytometry. We found a significant reduction in phosphorylation of Axl (pY779), Akt(pS473), Erk1/2(pT202/Y204) and PLCɣ1(pY783). Next we established a systemic MOLM13 preclinical AML model in NOD/SCID mice. The mice were treated with 25 or 50 mg/kg BGB324 until moribund (up to 16 days). We found a dose-dependent and significant increase in overall survival in BGB324-treated mice. We further investigated intracellular signaling in BGB324-treated cells in vivo. Mice carrying systemic AML disease (MOLM13) were treated with BGB324 at 50mg/kg for 4 days, and we monitored CD33/45-positive MOLM13 cells harvested from spleen and bone marrow by flow cytometry. BGB324-treated mice showed a significant reduction in pErk and pPLCɣ1 relative to mice in the control group. PBMCs from peripheral blood of AML patients treated with BGB324 400 mg x1 at day 1 and 2, and thereafter 100 mg daily were collected for single cell signal profiling of signal transduction changes by conventional flow cytometry (phospho-flow) and mass cytometry (CyTOF). Preliminary phopho-flow analyses show decrease of pAkt(T308) and pPLCgamma1(Y783) in one patient. Further analyses are ongoing and will be presented. Figure 1. In vitro response to 1 hour BGB324 treatment in human AML cell line MOLM13 at 0.5 and 1µM doses. Response was evaluated in pAxl, pErk1/2, pAkt and pPLCγ1. n=3, *p≤0.05, **p≤0.005. Figure 1. In vitro response to 1 hour BGB324 treatment in human AML cell line MOLM13 at 0.5 and 1µM doses. Response was evaluated in pAxl, pErk1/2, pAkt and pPLCγ1. n=3, *p≤0.05, **p≤0.005. Figure 2. Dose-dependent response in overall survival in a MOLM13 systemic xenograft model (n=10). Figure 2. Dose-dependent response in overall survival in a MOLM13 systemic xenograft model (n=10). Figure 3. Response to BGB324-treatment in pErk, pPLCγ1 and pAkt in CD33/CD45-positive cells harvested from spleens (left) and bone marrows (right) of mice with systemic MOLM13 xenografts. n=5, *p≤0.05, **p≤0.005. Figure 3. Response to BGB324-treatment in pErk, pPLCγ1 and pAkt in CD33/CD45-positive cells harvested from spleens (left) and bone marrows (right) of mice with systemic MOLM13 xenografts. n=5, *p≤0.05, **p≤0.005. Disclosures Hellesøy: BerGenBio AS: Other: Previous employee. Stock option holder. Wnuk-Lipinska:BerGenBio AS: Employment. Boniecka:BerGenBio AS: Employment. Nævdal:BerGenBio AS: Employment. Loges:BerGenBio: Honoraria, Other: travel support, Research Funding. Cortes:Teva: Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; BerGenBio AS: Research Funding; Ariad: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Ambit: Consultancy, Research Funding; Arog: Research Funding; Celator: Research Funding; Jenssen: Consultancy. Lorens:BerGenBio AS: Employment, Equity Ownership. Micklem:BerGenBio AS: Employment, Equity Ownership. Gausdal:BerGenBio AS: Employment. Gjertsen:Haukeland University Hospital: Research Funding.

Preclinical Characterization of a Potent, Selective Inhibitor of the Protein Methyltransferase DOT1L for Use in the Treatment of MLL-Rearranged Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2379-2379 ◽  
Author(s):  
Roy M Pollock ◽  
Scott R Daigle ◽  
Carly A Therkelsen ◽  
Aravind Basavapathruni ◽  
Lei Jin ◽  
...  
Keyword(s):  
Tumor Regression ◽  
Xenograft Model ◽  
Selective Inhibitor ◽  
Significant Weight Loss ◽  
Employment Equity ◽  
Nude Rat ◽  
Protein Methyltransferase ◽  
Equity Ownership ◽  
H3k79 Methylation

Abstract Abstract 2379 The enzymatic activity of the protein methyltransferase (PMT) DOT1L has been shown to be a driver of cell proliferation in MLL-rearranged leukemia. Our group has previously reported the design of potent and selective aminonucleoside inhibitors of DOT1L [Daigle et al. (2011) Cancer Cell 20: 53–65; Basavapathruni et al. (2012) Chem. Biol. Drug Design, in press]. Structure-guided design, together with robust biochemical and biological assays, was used to optimize the potency, selectivity and pharmacological features of the aminonucleosides, resulting in the compound EPZ-5676. EPZ-5676 is an S-adenosyl methionine (SAM) competitive inhibitor of DOT11L that displays a Ki value of 80 pM and a drug-target residence time of > 24 hours. The compound is highly selective for DOT1L, demonstrating > 37,000-fold selectivity against all other PMTs tested. Crystallographic studies reveal that the high affinity, durable inhibition of DOT1L by EPZ-5676 has its origin in a conformational adaptation of the protein that attends inhibitor binding, extending the compound binding pocket to include novel recognition elements beyond the SAM binding active site. Treatment of leukemia cells with EPZ-5676 results in concentration- and time-dependent reduction of H3K79 methylation without effect on the methylation status of other histone sites. The reduction of H3K79 methylation leads to inhibition of key MLL target genes and selective, apoptotic cell killing in MLL-rearranged leukemia cells, but has minimal impact on non-rearranged cells. EPZ-5676 is highly soluble in aqueous solution and can thus be formulated for intravenous administration. The effective pharmacokinetic half-life of EPZ-5676 in systemic circulation has been measured to be 0.25 and 1.5 h in rats and dogs, respectively. A nude rat subcutaneous xenograft model of MLL-rearranged leukemia has been established. Continuous intravenous infusion of EPZ-5676 for 21 days in this model leads to dose-dependent anti-tumor activity. At the highest dose, complete tumor regressions are achieved with no regrowth for up to 32 days after the cessation of treatment (Figure 1). Figure 1. EPZ-5676 causes complete and sustained tumor regression in a MV4–11 nude rat xenograft model of MLL-rearranged leukemia. No significant weight loss or obvious toxicity was observed in rats treated with EPZ-5676 during this efficacy study. EPZ-5676 is thus a potent, selective inhibitor of DOT1L that demonstrates strong efficacy in a rat xenograft model of MLL-rearranged leukemia. Details of the preclinical characterization of this compound will be presented. Figure 1. EPZ-5676 causes complete and sustained tumor regression in a MV4–11 nude rat xenograft model of MLL-rearranged leukemia. . / No significant weight loss or obvious toxicity was observed in rats treated with EPZ-5676 during this efficacy study. EPZ-5676 is thus a potent, selective inhibitor of DOT1L that demonstrates strong efficacy in a rat xenograft model of MLL-rearranged leukemia. Details of the preclinical characterization of this compound will be presented. Disclosures: Pollock: Epizyme: Employment, Equity Ownership. Daigle:Epizyme: Employment, Equity Ownership. Therkelsen:Epizyme: Employment, Equity Ownership. Basavapathruni:Epizyme: Employment, Equity Ownership. Jin:Epizyme: Employment, Equity Ownership. Allain:Epizyme: Employment, Equity Ownership. Klaus:Epizyme, Inc.: Employment, Equity Ownership. Raimondi:Epizyme: Employment, Equity Ownership. Porter Scott:Epizyme: Employment, Equity Ownership. Chesworth:Epizyme: Employment, Equity Ownership. Moyer:Epizyme: Employment, Equity Ownership. Copeland:Epizyme Inc.: Employment, Equity Ownership. Richon:Epizyme, Inc.: Employment, Equity Ownership. Olhava:Epizyme: Employment.

Transient Laboratory Findings Upon First Dosing with T-Cell Engaging BiTE® Antibody Blinatumomab in Non-Hodgkin Lymphoma Patients.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4798-4798
Author(s):  
Dirk Nagorsen ◽  
Gerhard Zugmaier ◽  
Peter Kufer ◽  
Matthias Klinger ◽  
Andreas Viardot ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Hodgkin Lymphoma ◽  
Single Chain ◽  
Employment Equity ◽  
Laboratory Findings ◽  
Non Hodgkin Lymphoma ◽  
Treatment Dose ◽  
Equity Ownership ◽  
Dose Dependent

Abstract Abstract 4798 Blinatumomab is a single-chain bispecific antibody construct with specificity for CD19 and CD3 belonging to the class of bispecific T cell engager (BiTE®). It has shown high response rates as single agent after treatment of patients with indolent and mantle cell lymphoma and B-precursor acute lymphocytic leukemia. We here report from an ongoing phase 1 trial in non-Hodgkin lymphoma patients on transient laboratory findings seen upon start of continuous i.v. infusion of blinatumomab. The vast majority of adverse events (AEs) during 4-8 weeks of infusion occurred within the first two days of treatment. Lymphopenia (up to CTCAE grade 4) was the most frequent early laboratory AE, and can be explained by rapid B cell depletion and initial redistribution of T cells. Except for B cell decline, all laboratory parameters were transient and normalized within days during continued treatment. Dose-dependent increases were seen for AST and ALT (maximally grade 1/2), CRP, and D-dimer during the first days of treatment. Dose-dependent declines during the first days of treatment were observed for platelets and hemoglobin, which normalized after a few days. Of note, no drug-related thromboembolic events were observed. Serum levels of cytokines IL-6, IL-2, IFNγ, and IL-10 showed small dose-dependent increases, which reversed to baseline within hours. No association between cytokine levels and thrombocytopenia was found. Moreover, there was no evident correlation between any laboratory abnormality and clinical adverse event observed during the first days of treatment. The laboratory AEs induced by blinatumomab appear to reflect consequences of the onset of redirected target cell lysis, T cell activation and bystander effects. In conclusion, blinatumomab causes characteristic laboratory findings during the first days of treatment, which are transient, not associated with clinically relevant AEs and do not require treatment interruption. Compared to AEs caused by chemotherapy regimens, the laboratory findings observed after start of blinatumomab treatment are considered rather mild. Disclosures: Nagorsen: Micromet: Employment, Equity Ownership. Zugmaier:Micromet: Employment, Equity Ownership. Kufer:Micromet: Employment, Equity Ownership, Patents & Royalties. Klinger:Micromet: Employment, Equity Ownership. Schmidt:Micromet: Employment, Equity Ownership. Klappers:Micromat: Employment, Equity Ownership. Wolf:Micromet: Employment, Equity Ownership. Brandl:Micromet: Employment, Equity Ownership. Baeuerle:Micromet: Employment, Equity Ownership. Bargou:Micromet: Consultancy, Patents & Royalties.

AG-221 Offers a Survival Advantage In a Primary Human IDH2 Mutant AML Xenograft Model

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 240-240 ◽  
Author(s):  
Katharine Yen ◽  
Fang Wang ◽  
Jeremy Travins ◽  
Yue Chen ◽  
Hua Yang ◽  
...  
Keyword(s):  
Cellular Differentiation ◽  
Human Study ◽  
Leukemia Cell ◽  
Xenograft Model ◽  
Clinical Activity ◽  
High Dose ◽  
Employment Equity ◽  
Blast Cells ◽  
Equity Ownership ◽  
Dose Dependent

Abstract Somatic point mutations in isocitrate dehydrogenase 1/2 (IDH1/2) confer a gain-of-function in cancer cells resulting in the accumulation and secretion of an onco-metabolite, R (-)-2-hydroxyglutarate (2HG). High levels of 2HG have been shown to inhibit aKG dependent dioxygenases including histone and DNA demethylases, which play a key role in regulating the epigenetic state of cells. Recently, ex vivo treatment with AGI-6780, a potent IDH2 R140Q inhibitor induced cellular differentiation of leukemic blast cells isolated from primary human AML patient samples harboring an IDH2 R140Q mutation. These data provided the first evidence that inhibition of mutant IDH2 can reverse the block in cellular differentiation conferred by high levels of 2HG and could provide a therapeutic benefit to patients. AG-221 is a potent and selective inhibitor of the IDH2 mutant enzyme and is currently being evaluated in a first-in-human study entitled: A Phase 1, Multicenter, Open-Label, Dose-Escalation, Safety, Pharmacokinetic, Pharmacodynamic, and Clinical Activity Study of Orally Administered AG-221 in Subjects with Advanced Hematologic Malignancies with an IDH2 Mutation. The compound has been demonstrated to reduce 2-HG levels by >90% and reverse histone and deoxyribonucleic acid (DNA) hypermethylation in vitro, and to induce differentiation in leukemia cell models. We evaluated the efficacy of AG-221 in a primary human AML xenograft model carrying the IDH2 R140Q mutation. This is an aggressive model with mortality from AML consistently occurring by day 80, following tail vein engraftment. Results show that AG-221 is able to potently reduce 2HG found in the bone marrow, plasma and urine of engrafted mice. Treatment also induced a dose dependent, statistically significant, survival benefit where all mice in the high dose treatment group survived to the end of study. We also saw a dose dependent proliferative burst of the human specific CD45+ blast cells followed by cellular differentiation as measured by the expression of CD11b, CD14 and CD15 and cell morphology. Furthermore, the onset of differentiation correlated with survival, whereas mice that died in the low dose groups failed to show signs of cellular differentiation. These data provide strong preclinical in vivo evidence that AG-221 may have clinical benefit for IDH2 mutant patients through the reduction of 2HG and the induction of blast differentiation. Disclosures: Yen: Agios Pharmaceuticals: Employment, Equity Ownership. Wang:Agios Pharmaceuticals: Employment, Equity Ownership. Travins:Agios Pharmaceuticals: Employment, Equity Ownership. Chen:agios Pharmaceuticals: Employment, Equity Ownership. Yang:Agios Pharmaceuticals: Employment, Equity Ownership. Straley:Agios Pharmaceuticals: Employment, Equity Ownership. Choe:agios Pharmaceuticals: Employment, Equity Ownership. Dorsch:agios Pharmaceuticals: Employment, Equity Ownership. Schenkein:agios Pharmaceuticals: Employment, Equity Ownership. Agresta:agios Pharmaceuticals: Employment, Equity Ownership. Biller:agios Pharmaceuticals: Employment, Equity Ownership. Su:agios Pharmaceuticals: Employment, Equity Ownership.

Dose dependent localisation of a monoclonal F(ab′)2 fragment against CEA in the mouse xenograft model

1986 ◽  
Vol 22 (6) ◽  
pp. 709-710 ◽  
Author(s):  
G.T. Rogers ◽  
J. Boden ◽  
P.J. Harwood ◽  
R.B. Pedley ◽  
G.A. Rawlins ◽  
...  
Keyword(s):  
Xenograft Model ◽  
Mouse Xenograft ◽  
Mouse Xenograft Model ◽  
Dose Dependent

Carfilzomib Exerts Anti-Neoplastic Activity in Waldenstrom Macroglobulinemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4916-4916
Author(s):  
Antonio Sacco ◽  
Aldo M. Roccaro ◽  
Monette Aujay ◽  
Hai Ngo ◽  
Feda Azab ◽  
...  
Keyword(s):  
Cell Lines ◽  
Low Grade ◽  
Dependent Manner ◽  
Caspase 9 ◽  
Employment Equity ◽  
Waldenström Macroglobulinemia ◽  
Waldenstrom Macroglobulinemia ◽  
Synergistic Cytotoxicity ◽  
Equity Ownership ◽  
Dose Dependent

Abstract Abstract 4916 Introduction Proteasome inhibition represents a valid therapeutical approach in several tumors and its use has been validated in Waldenstrom's macroglobulinemia (WM), where single-agent Bortezomib has been successfully tested in phase 2 clinical trials. Nevertheless, a significant fraction of patients relapse, or develop significant toxicity due to high toxicity in non-transformed cells. Therefore preclinical evaluation of new proteasome inhibitor with a more selective inhibition of neoplastic cells is needed in order to increase efficacy and improve patient outcome. We tested Carfilzomib, a tetrapeptide epoxyketone selective inhibitor of the chymotrypsin-like activity of the immunoproteasome and constitutive proteasome in WM. Methods WM and IgM secreting low-grade lymphoma cell lines (BCWM.1, MEC1, RL) were used. Expression of imunoproteasome and constitutive proteasome subunits (beta1, beta2, beta5; LMP2, MECL1, LMP7) were detected primary WM cells and cell lines by an ELISA-based assay. Cytotoxicity and DNA synthesis were measured by thymidine uptake and MTT, respectively. Cell signaling and apoptotic pathways were determined by Western Blot. Determination of the additive or synergistic effect of drugs combination was calculated using the CalcuSyn software based on the Chou-Talalay method. Results Primary CD19 bone-marrow derived WM cells express higher level of the immunopreoteasome as compared to the constitutive proteasome. Carfilzomib inhibited the chymotrypsin-like activity of both the immunoproteasome (LMP7) and the constitutive proteasome (beta5) and in WM cells, in a dose-dependent manner; leading to inhibition of proliferation (IC50: 5nM; 48h) and induction of cytotoxicity (IC50: 7.5nM; 48h) in WM cells. Carfilzomib mediated apoptosis in WM by increasing PARP-, caspase-9- and -3-cleavage; as well as by inducing activation of c-jun-N-terminal kinase and ER-stress in a dose-dependent manner. Moreover, combination of Carfilzomib and bortezomib induced synergistic cytotoxicity in WM cells, as shown by enhanced PARP-, caspase-9- and -3-cleavage; and synergy in inhibiting the chymotrypsin-like activity of the immunoproteasome and constitutive proteasome. Conclusion Taken together, these findings provide the pre-clinical rational for testing Carfilzomib in Waldenstrom Macroglobulinemia. Disclosures Aujay: Proteolix: Employment, Equity Ownership. Demo:Proteolix: Employment, Equity Ownership. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Survival Pathways Stabilized By Proteasome Inhibition Reveal PIM2 As a Novel Target For Potentiating The Anti-Myeloma Activity Of Carfilzomib

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4440-4440
Author(s):  
Tracey Lin ◽  
Eric Lowe ◽  
Alana Lerner ◽  
Christopher J. Kirk ◽  
Shirin Arastu-Kapur
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Proteasome Inhibition ◽  
Myeloma Cell ◽  
Protein Accumulation ◽  
Employment Equity ◽  
Survival Pathways ◽  
Equity Ownership ◽  
Dose Dependent

In recent years, new agents for multiple myeloma treatment (e.g., proteasome inhibitors) have become more efficacious, yet nearly all patients eventually relapse and develop refractory disease. Growing evidence suggests that clonal heterogeneity in multiple myeloma may constitute the basis for treatment resistance. Therefore, a multi-pronged approach with novel agents is needed to increase the efficacy of standard therapy and prevent or overcome resistance to standard treatments. We have undertaken a research effort to discover novel targets that potentiate the anti-tumor effects of proteasome inhibition in myeloma cells. We hypothesized that proteins that are stabilized in tumor cells following proteasome inhibition likely constitute components of both pro-apoptotic and pro-survival pathways. A mass spectrometry approach, referred to as UbiScan®, was employed to determine the identity and levels of cellular proteins modified with ubiquitin. MM cell lines (U266 and NCI-H929) were treated with either carfilzomib (CFZ) or bortezomib (BTZ) for 1 hour and the ubiquitome was profiled at 1 and 3 hours after culture in drug-free media. A concentration of 125 nM was chosen in order to reflect physiologically relevant drug and target inhibition levels and to induce cell death in ∼80% of cells after 48 hours. Approximately 400 proteins showed similar increases in ubiquitination with CFZ or BTZ. One of these proteins was PIM2, a serine/threonine proto-oncogene required for plasma cell proliferation that is highly expressed in multiple myeloma cell lines. We determined that ubiquitination on PIM2 was occurring at lysine 61, which is known to be associated with proteasomal degradation. Four hours after exposure to CFZ, PIM2 ubiquitination increased 34.6 and 24.9 fold in U266 and H929 cells, respectively, and similar changes were measured following BTZ treatment. Western blot analysis of CFZ-treated cells showed a dose-dependent accumulation of total PIM2 protein, confirming that the increase in ubiquitination correlated with protein accumulation. Next, we employed a siRNA-mediated knockdown approach to study the role of PIM2 in proteasome inhibitor mediated-cell death. Knockdown of PIM2 caused a 20% - 50% decrease in viability in both myeloma cell lines. When CFZ was added 48 hours after siRNA transfection, a significant and dose-dependent decrease in viability was observed, suggesting a synergistic interaction. Based on these results, we tested the combination of CFZ and (Z)-5-(4-propoxybenzylidene)thiazolidine-2,4-dione (PIM1/2 inhibitor), which is known to inhibit both PIM1 and PIM2. The PIM1/2 inhibitor decreased levels of phosphorylation on 4E-BP1, a downstream target, confirming its activity in cells. Chemical inhibition of PIM2 potentiated the effect of CFZ in both MM cell lines. These data suggest that the combination of targeting PIM2 and the proteasome will be efficacious in the treatment of multiple myeloma. Disclosures: Lin: Onyx Pharmaceuticals, Inc.: Employment. Lowe:Onyx Pharmaceuticals, Inc.: Employment, Equity Ownership. Lerner:Onyx Pharmaceuticals, Inc.: Employment, Equity Ownership. Kirk:Onyx Pharmaceuticals: Employment, Equity Ownership. Arastu-Kapur:Onyx Pharmaceuticals, Inc.: Employment, Equity Ownership.

Placebo-Controlled, Double-Blind, First-In-Human, Ascending Single Dose and Multiple Dose, Healthy Subject Study Of Intravenous-Administered SelG1, a Humanized Anti-P-Selectin Antibody In Development For Sickle Cell Disease

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 970-970 ◽  
Author(s):  
Jonathan W. Stocker ◽  
Debra Mandarino ◽  
Ziad Kawar ◽  
Richard Alvarez ◽  
David Falconer ◽  
...  
Keyword(s):  
Single Dose ◽  
Sickle Cell ◽  
Multiple Dose ◽  
Study Drug ◽  
Cell Disease ◽  
Employment Equity ◽  
Double Blind ◽  
Equity Ownership ◽  
Clinically Significant ◽  
Dose Dependent

Abstract SelG1 is a humanized anti-P-selectin monoclonal antibody being developed as a treatment for sickle cell disease (SCD). Extensive data have been published that suggest a pivotal role for P-selectin in the pathophysiology of SCD. Much of this work has been conducted in mice engineered to express human β hemoglobin S (sickle cell hemoglobin). These mice have a remarkably similar disease pathology to that observed in human SCD including vasoocclusion. Using these mice, investigators have demonstrated P-selectin interactions between the endothelium and sickled red blood cells, leukocytes and platelets. Additional studies have demonstrated direct P-selectin mediated binding of leukocytes with platelets. All of these cell-cell interactions have been implicated in SCD vasoocclusion. Further, blockade or genetic absence of P-selectin decreases or eliminates these cell-cell interactions and vasoocclusion. A Phase I clinical study was conducted to evaluate the safety, pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of SelG1. This was a single-center, double-blind, placebo-controlled, first-in-human, ascending single dose and multiple dose study of intravenous (IV)-administered SelG1 in healthy adult male and female subjects. There were 5 dosing cohorts in the study (A through E): Ascending single dose cohorts: Cohort A:0.2 mg/kg IV dose of SelG1 (n=3) or placebo (n=1); Cohort B:0.5 mg/kg IV dose of SelG1 (n=3) or placebo (n=1); Cohort C:1.0 mg/kg IV dose of SelG1 (n=3) or placebo (n=1); Cohort D:5.0 mg/kg IV dose of SelG1 (n=6) or placebo (n=2). Multi-dose cohort: Cohort E:two 8.0 mg/kg IV doses of SelG1 (n=5) or placebo (n=2); the doses were given 2 weeks apart. In Cohorts A through D, SelG1 was slowly eliminated (mean t1/2 = 75.6 to 500 hours). Mean t1/2 values increased in a dose dependent manner. The exposure to SelG1 (mean Cmax, AUC0-t, and AUC0-∞) increased in a greater than proportional manner over the dose range. In Cohort E, SelG1 was slowly eliminated (mean t1/2 = 363 hours for the second infusion). The mean Cmax and AUC0-336values were 1.6- and 1.7-fold higher, respectively, after the second infusion relative to the first infusion. The PD data demonstrate that P-selectin function was completely blocked for a minimum of 28 days in Cohort D and at least 56 days in Cohort E. Twenty-six of the 27 subjects who received study drug completed the study, with 1 placebo subject in Cohort D withdrawing himself from the study due to the required travel commitment. There were no deaths, serious adverse events, or severe AEs reported in any subject. There were no increases in the number or severity of AEs with increasing dosages or with multi-dose administration. The percentage of subjects experiencing an AE was similar between the SelG1-treated subjects and the placebo-treated subjects; in Cohorts A-D, 66.7% of SelG1-treated subjects and 60.0% of placebo subjects reported at least 1 AE, while in Cohort E, 60.0% of SelG1-treated subjects and 50.0% of placebo-treated subjects reported at least 1 AE. Only 1 AE occurred in more than 1 subject; vessel puncture site hematoma occurred in 1 subject of Cohort A, 1 subject of Cohort C, and 2 subjects of Cohort E. All other AEs occurred in only 1 subject and were mild to moderate in severity. No AEs in any subject were deemed “related” to study drug. No clinically significant findings were noted from vital sign measurements, physical examinations, or 12-lead ECGs for this study. No biochemistry, hematology, or other laboratory data were reported as clinically significant or were reported as AEs; there were no trends that indicated increases or decreases in mean or median values over time, and there were no dose-dependent increases or decreases in mean or median values. There were no demonstrable changes in coagulation parameters or increased bleeding tendencies. No specific antibody response to SelG1 occurred in any of the subjects. In summary, the administration of SelG1 was well tolerated in this group of healthy male and female subjects. A Phase II clinical study to evaluate the clinical efficacy of SelG1 in SCD patients is currently underway. Disclosures: Stocker: Selexys Pharmaceuticals: Employment, Equity Ownership. Mandarino:Selexys Pharmaceuticals: CRO Other. Kawar:Selexys Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Alvarez:Selexys Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Falconer:Selexys Pharmaceuticals: Employment, Equity Ownership. Rollins:Selexys Pharmaceuticals: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Rother:Selexys Pharmaceuticals: Employment, Equity Ownership.

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