Raffi Manoukian

Macroscopic cartilage formation with embryonic stem-cell-derived mesodermal progenitor cells.

The totipotent embryonic stem cell generates various mesodermal cells when stimulated with BMP4. Among the resulting cells, those expressing flk-1 and/or PDGFRα displayed chondrogenic activity in the presence of TGFβ3 and expressed cartilage-specific genes in 7 to 16 day pellet cultures. Depositions of cartilage matrix and type II collagen were detected by day 14. TGFβ-stimulated chondrogenesis was synergistically enhanced by PDGF-BB, resulting in a larger cartilage particle filled with a cartilaginous area containing type II collagen, with a surface cell layer expressing type I collagen. In contrast, noggin inhibited both the TGFβ- and TGFβ+PDGF-stimulated cartilage formation, suggesting that a BMP-dependent pathway is involved. In fact, replacement of TGFβ3 with BMP4 on days 10 to 12 markedly elevated the cartilage matrix deposition during the following 7 to 8 days. Moreover, culture with TGFβ3 and PDGF-BB, followed by the incubation with BMP4 alone, resulted in a cartilage particle lacking type I collagen in the matrix and the surface layer, which suggests hyaline cartilage formation. Furthermore, such hyaline cartilage particles were mineralized. These studies indicate that the PDGFRα+ and/or flk-1+ cells derived from embryonic stem cells possess the full developmental potential toward chondrocytes, in common with embryonic mesenchymal cells.

Polyglutamine repeat length-dependent proteolysis of huntingtin.

Amino-terminal fragments of huntingtin, which contain the expanded polyglutamine repeat, have been proposed to contribute to the pathology of Huntingtons disease (HD). Data supporting this claim have been generated from patients with HD in which truncated amino-terminal fragments forming intranuclear inclusions have been observed, and from animal and cell-based models of HD where it has been demonstrated that truncated polyglutamine-containing fragments of htt are more toxic than full-length huntingtin. We report here the identification of a region within huntingtin, spanning from amino acids 63 to 111, that is cleaved in cultured cells to generate a fragment of similar size to those observed in patients with HD. Importantly, proteolytic cleavage within this region appears dependent upon the length of the polyglutamine repeat within huntingtin, with pathological polyglutamine repeat-containing huntingtin being more efficiently cleaved than huntingtin containing polyglutamine repeats of nonpathological size.

An antibody to IP-10 is a potent antagonist of cell migration in vitro and in vivo and does not affect disease in several animal models of inflammation

IP-10 secretion is induced by pro-inflammatory cytokines and mediates the migration of CXCR3+ cells. Its elevation in clinical samples has been associated with multiple inflammatory diseases and its antagonism has been reported to be effective in several animal models of inflammatory disease. We generated a mouse anti-mouse IP-10 monoclonal antibody (mAb; Clone 20A9) that specifically bound murine IP-10 with high affinity and inhibited in vitro IP-10 induced BaF3/mCXCR3 cell migration with an IC50 of ∼4 nM. The 20A9 mAb was completely absorbed in vivo and had dose proportional pharmacokinetic exposure with a serum half life of 2.4–6 days. The 20A9 mAb inhibited IP-10 mediated T-cell recruitment to the airways, indicating that it is effective in vivo. However, administration of the 20A9 mAb had no significant effect on disease in mouse models of delayed type hypersensitivity, collagen induced arthritis, cardiac allograft transplantation tolerance, EAE or CD4+ CD45RBHi T-cell transfer-induced IBD. These data suggest that the 20A9 mAb can antagonize IP-10 mediated chemotaxis in vitro and in vivo and that this is insufficient to cause a therapeutic benefit in multiple mouse models of inflammatory disease.

Motesanib inhibits Kit mutations associated with gastrointestinal stromal tumors

BackgroundActivating mutations in Kit receptor tyrosine kinase or the related platelet-derived growth factor receptor (PDGFR) play an important role in the pathogenesis of gastrointestinal stromal tumors (GIST).MethodsThis study investigated the activity of motesanib, an inhibitor of vascular endothelial growth factor receptors (VEGFR) 1, 2, and 3; PDGFR; and Kit, against primary activating Kit mutants and mutants associated with secondary resistance to imatinib. Single- and double-mutant isoforms of Kit were evaluated for their sensitivity to motesanib or imatinib in autophosphorylation assays and in Ba/F3 cell proliferation assays.ResultsMotesanib inhibited Kit autophosphorylation in CHO cell lines expressing primary activating mutations in exon 9 (AYins503-504, IC50 = 18 nM) and exon 11 (V560 D, IC50 = 5 nM; Δ552-559, IC50 = 1 nM). Motesanib also demonstrated activity against kinase domain mutations conferring imatinib resistance (V560D/V654A, IC50 = 77 nM; V560D/T670I, IC50 = 277 nM; Y823 D, IC50 = 64 nM) but failed to inhibit the imatinib-resistant D816V mutant (IC50 > 3000 nM). Motesanib suppressed the proliferation of Ba/F3 cells expressing Kit mutants with IC50 values in good agreement with those observed in the autophosphorylation assays.ConclusionsIn conclusion, our data suggest that motesanib possesses inhibitory activity against primary Kit mutations and some imatinib-resistant secondary mutations.

AMG 900, a potent inhibitor of aurora kinases causes pharmacodynamic changes in p-Histone H3 immunoreactivity in human tumor xenografts and proliferating mouse tissues

BackgroundThe Aurora family of serine-threonine kinases are essential regulators of cell division in mammalian cells. Aurora-A and -B expression and kinase activity is elevated in a variety of human cancers and is associated with high proliferation rates and poor prognosis. AMG 900 is a highly potent and selective pan-aurora kinase inhibitor that has entered clinical evaluation in adult patients with advanced cancers. In mice, oral administration of AMG 900 blocks the phosphorylation of histone H3 on serine-10 (p-Histone H3), a proximal substrate of aurora-B and inhibits the growth of multiple human tumor xenografts, including multidrug-resistant models.MethodsIn order to establish a preclinical pharmacokinetic-pharmacodynamic (PK-PD) relationship for AMG 900 that could be translated to the clinic, we used flow cytometry and laser scanning cytometry detection platforms to assess the effects on p-Histone H3 inhibition in terms of sensitivity, precision, and specificity, in human tumor xenografts in conjunction with mouse skin and bone marrow tissues. Mice with established COLO 205 tumors were administered AMG 900 at 3.75, 7.5, and 15 mg/kg and assessed after 3 hours.ResultsSignificant suppression of p-Histone H3 in mouse skin was only observed at 15 mg/kg (p <0.0001), whereas in mouse bone marrow and in tumor a dose-dependent inhibition was achieved at all three doses (p ≤0.00015). These studies demonstrate that AMG 900 inhibits p-Histone H3 in tumors and surrogate tissues (although tissues such as skin may be less sensitive for assessing PD effects). To further extend our work, we evaluated the feasibility of measuring p-Histone H3 using fine-needle aspirate (FNA) tumor xenograft biopsies. Treatment with AMG 900 significantly inhibited p-Histone H3 (>99% inhibition, p <0.0001) in COLO 205 tumors. Lastly, we illustrate this LSC-based approach can detect p-Histone H3 positive cells using mock FNAs from primary human breast tumor tissues.ConclusionPhosphorylation of histone H3 is a useful biomarker to determine the pharmacodynamics (PD) activity of AMG 900. FNA biopsies may be a viable approach for assessing AMG 900 PD effects in the clinic.

Antagonism of Ang-Tie2 and Dll4-Notch signaling has opposing effects on tumor endothelial cell proliferation, evidenced by a new flow cytometry method

Sustained angiogenesis is essential for tumor growth as it provides the tumor with a network of blood vessels that supply both oxygen and essential nutrients. Limiting tumor-associated angiogenesis is a proven strategy for the treatment of human cancer. To date, the rapid detection and quantitation of tumor-associated endothelial cell (TAEC) proliferation has been challenging, largely due to the low frequency of endothelial cells (ECs) within the tumor microenvironment. In this report, we address this problem using a new multiparametric flow cytometry method capable of rapid and precise quantitation of proliferation by measuring bromodeoxyuridine (BrdUrd) uptake in mouse TAECs from established human tumor xenografts. We determined the basal proliferation labeling index of TAECs in two human tumor xenografts representing two distinct histologies, COLO 205 (colorectal cancer) and U-87 (glioblastoma). We then investigated the effects of two large-molecule antiangiogenic agents targeting different biochemical pathways. Blocking angiopoietin-Tie2 signaling with the peptide-Fc fusion protein, trebananib (AMG 386), inhibited proliferation of TAECs, whereas blocking Dll4-Notch signaling with an anti-Dll4-specific antibody induced hyperproliferation of TAECs. These pharmacodynamic studies highlight the sensitivity and utility of this flow cytometry-based method and demonstrate the value of this assay to rapidly assess the in vivo proliferative effects of angiogenesis-targeted agents on both the tumor and the associated vasculature.

Abstract 3600: Preclinical pharmacokinetic and biomarker analysis of AMG 900, an orally available small molecule inhibitor of aurora kinases, in human xenograft tumor and surrogate tissues

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: The aurora family of serine-threonine kinases (Aurora-A, -B, -C) regulate cell-cycle progression in mammalian cells. Aurora kinases A and B are essential for proper chromosome congression, segregation, and cytokinesis during mitosis, whereas aurora kinase C function appears restricted to male meiosis. Aurora kinase B is responsible for the direct phosphorylation of histone H3 on serine-10 (p-histone H3). Aurora kinase A and B expression is elevated in a variety of human cancers and is associated with advanced clinical staging and poor prognosis. With the emergence of aurora kinases as key mitotic regulators and their potential role in tumorigenesis, we developed AMG 900, a novel ATP competitive small molecule inhibitor, that is highly potent and selective for aurora kinases A, B, and C. In vivo, AMG 900 inhibits the growth of multiple human tumor xenografts, including multidrug-resistant models. Objective: The aim of this study was to establish a pharmacokinetic-pharmacodynamic (PK-PD) relationship for AMG 900 in human tumor xenografts and surrogate tissues. Results: In nude mice, oral administration of AMG 900 inhibited p-histone H3 in tumors and bone marrow in a concentration- and dose-dependent manner. To further refine the AMG 900 PK-PD relationship, p-histone H3 IC50 values for bone marrow and dissociated COLO 205 tumors from mice were associated with plasma concentrations of 294 and 273 ng/mL, respectively. The duration of target coverage required for anti-tumor efficacy was determined to be > 6 hours per day above the in vivo p-histone H3 IC50 using different dosing schedules. To explore whether skin could be a suitable surrogate tissue for measuring p-histone H3 inhibition, tumor-bearing mice were administered AMG 900 at 3.75, 7.5, and 15 mg/kg for 3 hours. Significant suppression of p-histone H3 in skin was only observed at 15 mg/kg (p 99%, p < 0.0001) in COLO 205 tumors, suggesting that FNA biopsies may be a viable approach for assessing AMG 900 PD effects in the clinic. Conclusion: These studies demonstrate that AMG 900 inhibits p-histone H3 in tumors and surrogate tissues, although tissues such as skin may be less sensitive to assess PD effects. The use of p-histone H3 as a biomarker to determine plasma levels of AMG 900 required to inhibit aurora B activity may be useful in clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3600.