Eun Joo Chung

Phase I and Pharmacokinetic Study of MS-275, a Histone Deacetylase Inhibitor, in Patients With Advanced and Refractory Solid Tumors or Lymphoma

PURPOSE The objective of this study was to define the maximum-tolerated dose (MTD), the recommended phase II dose, the dose-limiting toxicity, and determine the pharmacokinetic (PK) and pharmacodynamic profiles of MS-275. PATIENTS AND METHODS Patients with advanced solid tumors or lymphoma were treated with MS-275 orally initially on a once daily x 28 every 6 weeks (daily) and later on once every-14-days (q14-day) schedules. The starting dose was 2 mg/m2 and the dose was escalated in three- to six-patient cohorts based on toxicity assessments. RESULTS With the daily schedule, the MTD was exceeded at the first dose level. Preliminary PK analysis suggested the half-life of MS-275 in humans was 39 to 80 hours, substantially longer than predicted by preclinical studies. With the q14-day schedule, 28 patients were treated. The MTD was 10 mg/m2 and dose-limiting toxicities were nausea, vomiting, anorexia, and fatigue. Exposure to MS-275 was dose dependent, suggesting linear PK. Increased histone H3 acetylation in peripheral-blood mononuclear-cells was apparent at all dose levels by immunofluorescence analysis. Ten of 29 patients remained on treatment for > or = 3 months. CONCLUSION The MS-275 oral formulation on the daily schedule was intolerable at a dose and schedule explored. The q14-day schedule is reasonably well tolerated. Histone deacetylase inhibition was observed in peripheral-blood mononuclear-cells. Based on PK data from the q14-day schedule, a more frequent dosing schedule, weekly x 4, repeated every 6 weeks is presently being evaluated.

Phase I Trial of MS-275, a Histone Deacetylase Inhibitor, Administered Weekly in Refractory Solid Tumors and Lymphoid Malignancies

Purpose: MS-275 is a histone deacetylase inhibitor that has shown potent and unique anticancer activity in preclinical models. The aims of this phase I trial were to determine the dose-limiting toxicities and maximum tolerated dose of oral MS-275 in humans administered with food on a once weekly schedule and to study the pharmacokinetics of oral MS-275. Experimental Design: Patients with refractory solid tumors and lymphoid malignancies were treated with oral MS-275 on a once weekly schedule for 4 weeks of a 6-week cycle. Samples for pharmacokinetic and pharmacodynamic analyses were collected during cycle 1. Protein acetylation in subpopulations of peripheral blood mononuclear cells was measured using a multivariable flow cytometry assay. Results: A total of 22 patients were enrolled, and 19 were considered evaluable for toxicity. The maximum tolerated dose was 6 mg/m2. No National Cancer Institute Common Toxicity Criteria grade 4 toxicities were observed. Dose-limiting grade 3 toxicities were reversible and consisted of hypophosphatemia, hyponatremia, and hypoalbuminemia. Non–dose-limiting grade 3 myelosuppression was also observed. The mean terminal half-life of MS-275 was 33.9 ± 26.2 and the Tmax ranged from 0.5 to 24 h. Although there was considerable interpatient variability in pharmacokinetics, the area under the plasma concentration versus time curve increased linearly with dose. Conclusions: MS-275 is well tolerated at a dose of 6 mg/m2 administered weekly with food for 4 weeks every 6 weeks. Drug exposure increases linearly with dose, and protein acetylation increased in all the subpopulations of peripheral blood mononuclear cells following MS-275 administration.

A phase II study of perifosine in androgen independent prostate cancer

OBJECTIVES: Perifosine is an alkylphospholipid that has exhibited broad antineoplastic activity in preclinical studies. The primary objective of this study was to determine the clinical efficacy of this agent in the treatment of androgen-independent prostate cancer (AIPC) using PSA and clinical criteria. PATIENTS AND METHODS: Nineteen patients with progressive, metastatic AIPC were treated with oral perifosine. Cycles were 28 days in length. A loading dose of 900 mg was given on day 1 of cycle 1 followed by a maintenance dose of 150 mg daily for the next 20 days. A loading dose of 600 mg was administred on the first day of subsequent cycles by the maintenance dose of 150 mg daily for the next 20 days. Pharmacokinetic measurements were made throughout the course of the study. Circulating epithelial cells were collected via leukapheresis on day 0, 3, and 28. RESULTS: Median patient age was 67 years and median PSA was 180 ng/mL (range: 19-904 ng/ml). Grade 1-2 fatigue and gastrointestinal toxicities were common. Pharmacokinetic studies showed an average minimum concentration at steady-state of approximately 4059 ng/ml which correlated well with previous studies. Median time to progression was 4 weeks. There were no radiographic responses or PSA declines of 50% or greater related to perifosine. CONCLUSIONS: Treatment with perifosine was complicated by fatigue and gastrointestinal toxicity. No significant clinical activity against prostate cancer was observed. This agent does not merit further study in the setting of monotherapy in this population.

In vitro and In vivo Radiosensitization with AZD6244 (ARRY-142886), an Inhibitor of Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase 1/2 Kinase

Purpose: The mitogen-activated protein (MAP) kinase pathway is important for cell proliferation, survival, and differentiation, and is frequently up-regulated in cancers. The MAP kinase pathway is also activated after exposure to ionizing radiation. We investigated the effects of AZD6244 (ARRY-142886), an inhibitor of MAP kinase/extracellular signal-regulated kinase 1/2, on radiation response. Experimental Design: The effects of AZD6244 on the in vitro radiosensitivity of human cancer cell lines (A549, MiaPaCa2, and DU145) were evaluated using clonogenic assays. DNA damage repair was evaluated using γH2AX, and mitotic catastrophe was measured using nuclear fragmentation. Cell cycle effects were measured with flow cytometry. Growth delay was used to evaluate the effects of AZD6244 on in vivo tumor radiosensitivity. Results: Exposure of each cell line to AZD6244 before irradiation resulted in an increase in radiosensitivity with dose enhancement factors at a surviving fraction of 0.1, ranging from 1.16 to 2.0. No effects of AZD6244 on radiation-induced apoptosis or persistence of γH2AX foci after irradiation were detected. Cells treated with AZD6244 had an increased mitotic index and decreased Chk1 phosphorylation at 1 and 2 hours after irradiation. Mitotic catastrophe was increased in cells receiving AZD6244 and irradiation compared with the single treatments. In vivo studies revealed that AZD6244 administration to mice bearing A549 tumor xenografts resulted in a greater than additive increase in radiation-induced tumor growth delay (dose enhancement factor of 3.38). Conclusions: These results indicate that AZD6244 can enhance tumor cell radiosensitivity in vitro and in vivo and suggest that this effect involves an increase in mitotic catastrophe.

Role of Type II Pneumocyte Senescence in Radiation-Induced Lung Fibrosis

BACKGROUND Radiation is a commonly delivered therapeutic modality for cancer. The causes underlying the chronic, progressive nature of radiation injury in the lung are poorly understood. METHODS C57Bl/6NCr mice were exposed to thoracic irradiation (n = 3 per dose and time point for tissue collection). Microarray analysis of gene expression from irradiated murine lung was performed using one-way analysis of variance with post hoc Scheffe analysis. Senescence and type II airway epithelial cell (AECII) count were assayed in irradiated murine lung tissue (n = 3 per condition). Irradiated mice were treated with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase (NOX), and fibrosis was assessed by collagen assays. All statistical tests were two-tailed. RESULTS Gene expression in lung tissue from mice irradiated to 17.5 Gy clustered with that of aged unirradiated mice. Only fibrogenic exposures led to AECII senescence (0 Gy: 0.66% ± 0.67%; 5 Gy: 4.5% ± 1.19%; 17.5 Gy: 18.7% ± 3.05; P = .007) and depletion (0 Gy: 2.89 per alveolus ± 0.26; 5 Gy: 2.41 ± 0.19; 17.5 Gy: 1.6 ± 0.14; P < .001) at 30 weeks. Treatment of irradiated mice with DPI for 16 weeks markedly reduced collagen accumulation (5×6 Gy: 57.26 μg/lung ± 9.91; 5×6 Gy ± DPI: 36.54μg/lung ± 4.39; P = .03) and AECII senescence (5×6 Gy: 37.61% ± 4.82%; 5×6 Gy ± DPI: 12.38% ± 2.78; P < .001). CONCLUSIONS These studies identify senescence as an important process in AECII in vivo and indicate that NOX is a critical mediator of radiation-induced AECII senescence and pulmonary fibrosis.

Mesenchymal stem cells inhibit cutaneous radiation‐induced fibrosis by suppressing chronic inflammation

Exposure to ionizing radiation (IR) can result in the development of cutaneous fibrosis, for which few therapeutic options exist. We tested the hypothesis that bone marrow‐derived mesenchymal stem cells (BMSC) would favorably alter the progression of IR‐induced fibrosis. We found that a systemic infusion of BMSC from syngeneic or allogeneic donors reduced skin contracture, thickening, and collagen deposition in a murine model. Transcriptional profiling with a fibrosis‐targeted assay demonstrated increased expression of interleukin‐10 (IL‐10) and decreased expression of IL‐1β in the irradiated skin of mice 14 days after receiving BMSC. Similarly, immunoassay studies demonstrated durable alteration of these and several additional inflammatory mediators. Immunohistochemical studies revealed a reduction in infiltration of proinflammatory classically activated CD80+ macrophages and increased numbers of anti‐inflammatory regulatory CD163+ macrophages in irradiated skin of BMSC‐treated mice. In vitro coculture experiments confirmed that BMSC induce expression of IL‐10 by activated macrophages, suggesting polarization toward a regulatory phenotype. Furthermore, we demonstrated that tumor necrosis factor‐receptor 2 (TNF‐R2) mediates IL‐10 production and transition toward a regulatory phenotype during coculture with BMSC. Taken together, these data demonstrate that systemic infusion of BMSC can durably alter the progression of radiation‐induced fibrosis by altering macrophage phenotype and suppressing local inflammation in a TNF‐R2‐dependent fashion. Stem Cells 2013;31:2231–2241

Inhibition of Tumor Cell Motility by the Interferon-inducible GTPase MxA

To identify pathways controlling prostate cancer metastasis we performed differential display analysis of the human prostate carcinoma cell line PC-3 and its highly metastatic derivative PC-3M. This revealed that a 78-kDa interferon-inducible GTPase, MxA, was expressed in PC-3 but not in PC-3M cells. The gene encoding MxA, MX1, is located in the region of chromosome 21 deleted as a consequence of fusion of TMPRSS2 and ERG, which has been associated with aggressive, invasive prostate cancer. Stable exogenous MxA expression inhibited in vitro motility and invasiveness of PC-3M cells. In vivo exogenous MxA expression decreased the number of hepatic metastases following intrasplenic injection. Exogenous MxA also reduced motility and invasiveness of highly metastatic LOX melanoma cells. A mutation in MxA that inactivated its GTPase reversed inhibition of motility and invasion in both tumor cell lines. Co-immunoprecipitation studies demonstrated that MxA associated with tubulin, but the GTPase-inactivating mutation blocked this association. Because MxA is a highly inducible gene, an MxA-targeted drug discovery screen was initiated by placing the MxA promoter upstream of a luciferase reporter. Examination of the NCI diversity set of small molecules revealed three hits that activated the promoter. In PC-3M cells, these drugs induced MxA protein and inhibited motility. These data demonstrate that MxA inhibits tumor cell motility and invasion, and that MxA expression can be induced by small molecules, potentially offering a new approach to the prevention and treatment of metastasis.

FLT3 regulates β-catenin tyrosine phosphorylation, nuclear localization, and transcriptional activity in acute myeloid leukemia cells

Deregulated accumulation of nuclear β-catenin enhances transcription of β-catenin target genes and promotes malignant transformation. Recently, acute myeloid leukemia (AML) cells with activating mutations of FMS-like tyrosine kinase-3 (FLT3) were reported to display elevated β-catenin-dependent nuclear signaling. Tyrosine phosphorylation of β-catenin has been shown to promote its nuclear localization. Here, we examined the causal relationship between FLT3 activity and β-catenin nuclear localization. Compared to cells with wild-type FLT3 (FLT3-WT), cells with the FLT3 internal tandem duplication (FLT3-ITD) and tyrosine kinase domain mutation (FLT3-TKD) had elevated levels of tyrosine-phosphorylated β-catenin. Although β-catenin was localized mainly in the cytoplasm in FLT3-WT cells, it was primarily nuclear in FLT3-ITD cells. Treatment with FLT3 kinase inhibitors or FLT3 silencing with RNAi decreased β-catenin tyrosine phosphorylation and nuclear localization. Conversely, treatment of FLT3-WT cells with FLT3 ligand increased tyrosine phosphorylation and nuclear accumulation of β-catenin. Endogenous β-catenin co-immunoprecipitated with endogenous activated FLT3, and recombinant activated FLT3 directly phosphorylated recombinant β-catenin. Finally, FLT3 inhibitor decreased tyrosine phosphorylation of β-catenin in leukemia cells obtained from FLT3-ITD-positive AML patients. These data demonstrate that FLT3 activation induces β-catenin tyrosine phosphorylation and nuclear localization, and thus suggest a mechanism for the association of FLT3 activation and β-catenin oncogeneic signaling in AML.

Alteration of Signal-Transducing Molecules and Phenotypical Characteristics in Peripheral Blood Lymphocytes from Gastric Carcinoma Patients

The mechanisms underlying the impaired immune response frequently observed in cancer patients are not fully understood. Alteration of T-cell-associated signal transduction molecules has recently been implicated in immune suppression in tumor-bearing hosts. Furthermore, T cells from tumor-bearing host, irrespective of the presence of the ζ-chain, showed a lack of proliferative activity and cytotoxic function. In the present study, we investigated the expression of the ζ-chain molecule and the p56lck and p59fyn protein tyrosine kinase (PTK) levels in peripheral blood T lymphocytes (T-PBL) from patients with advanced gastric carcinomas; for this, flow cytometric analysis and immunoblotting, respectively, were used. We also compared the results of flow cytometric analysis of PBL between stomach cancer patients and normal healthy volunteers. In T-PBL from 22 tumor-bearing hosts, significantly reduced ζ-chain expression (16/22, 73%) was observed. Moreover, the expression level of p56lck in T-PBL from patients was significantly lower than that of p59fyn. Flow cytometric analysis of T-PBL indicated a markedly increased CD8+28– cell population in T-PBL from 19 tumor-bearing hosts.

Quercetin inhibits radiation-induced skin fibrosis.

Radiation induced fibrosis of the skin is a late toxicity that may result in loss of function due to reduced range of motion and pain. The current study sought to determine if oral delivery of quercetin mitigates radiation-induced cutaneous injury. Female C3H/HeN mice were fed control chow or quercetin-formulated chow (1% by weight). The right hind leg was exposed to 35 Gy of X rays and the mice were followed serially to assess acute toxicity and hind leg extension. Tissue samples were collected for assessment of soluble collagen and tissue cytokines. Human and murine fibroblasts were subjected to clonogenic assays to determine the effects of quercetin on radiation response. Contractility of fibroblasts was assessed with a collagen contraction assay in the presence or absence of quercetin and transforming growth factor-β (TGF-β). Western blotting of proteins involved in fibroblast contractility and TGF-β signaling were performed. Quercetin treatment significantly reduced hind limb contracture, collagen accumulation and expression of TGF-β in irradiated skin. Quercetin had no effect on the radioresponse of fibroblasts or murine tumors, but was capable of reducing the contractility of fibroblasts in response to TGF-β, an effect that correlated with partial stabilization of phosphorylated cofilin. Quercetin is capable of mitigating radiation induced skin fibrosis and should be further explored as a therapy for radiation fibrosis.