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1.
Clin Cancer Res ; 23(18): 5339-5348, 2017 Sep 15.
Article in English | MEDLINE | ID: mdl-28611198

ABSTRACT

Purpose: Encorafenib, a selective BRAF inhibitor (BRAFi), has a pharmacologic profile that is distinct from that of other clinically active BRAFis. We evaluated encorafenib in a phase I study in patients with BRAFi treatment-naïve and pretreated BRAF-mutant melanoma.Experimental Design: The pharmacologic activity of encorafenib was first characterized preclinically. Encorafenib monotherapy was then tested across a range of once-daily (50-700 mg) or twice-daily (75-150 mg) regimens in a phase I, open-label, dose-escalation and -expansion study in adult patients with histologically confirmed advanced/metastatic BRAF-mutant melanoma. Study objectives were to determine the maximum tolerated dose (MTD) and/or recommended phase II dose (RP2D), characterize the safety and tolerability and pharmacokinetic profile, and assess the preliminary antitumor activity of encorafenib.Results: Preclinical data demonstrated that encorafenib inhibited BRAF V600E kinase activity with a prolonged off-rate and suppressed proliferation and tumor growth of BRAF V600E-mutant melanoma models. In the dose-escalation phase, 54 patients (29 BRAFi-pretreated and 25 BRAFi-naïve) were enrolled. Seven patients in the dose-determining set experienced dose-limiting toxicities. Encorafenib at a dose of 300 mg once daily was declared the RP2D. In the expansion phase, the most common all-cause adverse events were nausea (66%), myalgia (63%), and palmar-plantar erythrodysesthesia (54%). In BRAFi-naïve patients, the overall response rate (ORR) and median progression-free survival (mPFS) were 60% and 12.4 months [95% confidence interval (CI), 7.4-not reached (NR)]. In BRAFi-pretreated patients, the ORR and mPFS were 22% and 1.9 months (95% CI, 0.9-3.7).Conclusions: Once-daily dosing of single-agent encorafenib had a distinct tolerability profile and showed varying antitumor activity across BRAFi-pretreated and BRAFi-naïve patients with advanced/metastatic melanoma. Clin Cancer Res; 23(18); 5339-48. ©2017 AACR.


Subject(s)
Antineoplastic Agents/therapeutic use , Carbamates/therapeutic use , Melanoma/drug therapy , Melanoma/genetics , Mutation , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/genetics , Sulfonamides/therapeutic use , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/adverse effects , Antineoplastic Agents/pharmacokinetics , Carbamates/administration & dosage , Carbamates/adverse effects , Carbamates/pharmacokinetics , Disease Models, Animal , Drug Evaluation, Preclinical , Drug Monitoring , Female , Humans , Kaplan-Meier Estimate , Male , Maximum Tolerated Dose , Melanoma/mortality , Melanoma/pathology , Mice , Molecular Targeted Therapy , Neoplasm Staging , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/adverse effects , Protein Kinase Inhibitors/pharmacokinetics , Sulfonamides/administration & dosage , Sulfonamides/adverse effects , Sulfonamides/pharmacokinetics , Treatment Outcome , Xenograft Model Antitumor Assays
2.
Cancer Discov ; 7(6): 610-619, 2017 06.
Article in English | MEDLINE | ID: mdl-28363909

ABSTRACT

Preclinical evidence suggests that concomitant BRAF and EGFR inhibition leads to sustained suppression of MAPK signaling and suppressed tumor growth in BRAFV600E colorectal cancer models. Patients with refractory BRAFV600-mutant metastatic CRC (mCRC) were treated with a selective RAF kinase inhibitor (encorafenib) plus a monoclonal antibody targeting EGFR (cetuximab), with (n = 28) or without (n = 26) a PI3Kα inhibitor (alpelisib). The primary objective was to determine the maximum tolerated dose (MTD) or a recommended phase II dose. Dose-limiting toxicities were reported in 3 patients receiving dual treatment and 2 patients receiving triple treatment. The MTD was not reached for either group and the phase II doses were selected as 200 mg encorafenib (both groups) and 300 mg alpelisib. Combinations of cetuximab and encorafenib showed promising clinical activity and tolerability in patients with BRAF-mutant mCRC; confirmed overall response rates of 19% and 18% were observed and median progression-free survival was 3.7 and 4.2 months for the dual- and triple-therapy groups, respectively.Significance: Herein, we demonstrate that dual- (encorafenib plus cetuximab) and triple- (encorafenib plus cetuximab and alpelisib) combination treatments are tolerable and provide promising clinical activity in the difficult-to-treat patient population with BRAF-mutant mCRC. Cancer Discov; 7(6); 610-9. ©2017 AACR.See related commentary by Sundar et al., p. 558This article is highlighted in the In This Issue feature, p. 539.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carbamates/therapeutic use , Cetuximab/therapeutic use , Colorectal Neoplasms/drug therapy , Protein Kinase Inhibitors/therapeutic use , Sulfonamides/therapeutic use , Thiazoles/therapeutic use , Adult , Aged , Aged, 80 and over , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/adverse effects , Antineoplastic Agents/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Carbamates/administration & dosage , Carbamates/adverse effects , Cetuximab/administration & dosage , Cetuximab/adverse effects , Colorectal Neoplasms/genetics , Disease-Free Survival , Female , Humans , Male , Maximum Tolerated Dose , Middle Aged , Mutation , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/adverse effects , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/genetics , Sulfonamides/administration & dosage , Sulfonamides/adverse effects , Thiazoles/administration & dosage , Thiazoles/adverse effects
3.
J Clin Oncol ; 34(36): 4371-4380, 2016 12 20.
Article in English | MEDLINE | ID: mdl-27601554

ABSTRACT

Purpose AZD1775 is a WEE1 kinase inhibitor targeting G2 checkpoint control, preferentially sensitizing TP53-deficient tumor cells to DNA damage. This phase I study evaluated safety, tolerability, pharmacokinetics, and pharmacodynamics of oral AZD1775 as monotherapy or in combination with chemotherapy in patients with refractory solid tumors. Patients and Methods In part 1, patients received a single dose of AZD1775 followed by 14 days of observation. In part 2, patients received AZD1775 as a single dose (part 2A) or as five twice per day doses or two once per day doses (part 2B) in combination with one of the following chemotherapy agents: gemcitabine (1,000 mg/m2), cisplatin (75 mg/m2), or carboplatin (area under the curve, 5 mg/mL⋅min). Skin biopsies were collected for pharmacodynamic assessments. TP53 status was determined retrospectively in archival tumor tissue. Results Two hundred two patients were enrolled onto the study, including nine patients in part 1, 43 in part 2A (including eight rollover patients from part 1), and 158 in part 2B. AZD1775 monotherapy given as single dose was well tolerated, and the maximum-tolerated dose was not reached. In the combination regimens, the most common adverse events consisted of fatigue, nausea and vomiting, diarrhea, and hematologic toxicity. The maximum-tolerated doses and biologically effective doses were established for each combination. Target engagement, as a predefined 50% pCDK1 reduction in surrogate tissue, was observed in combination with cisplatin and carboplatin. Of 176 patients evaluable for efficacy, 94 (53%) had stable disease as best response, and 17 (10%) achieved a partial response. The response rate in TP53-mutated patients (n = 19) was 21% compared with 12% in TP53 wild-type patients (n = 33). Conclusion AZD1775 was safe and tolerable as a single agent and in combination with chemotherapy at doses associated with target engagement.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Neoplasms/drug therapy , Neoplasms/mortality , Pyrazoles/administration & dosage , Pyrimidines/administration & dosage , Adult , Aged , Analysis of Variance , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Carboplatin/administration & dosage , Carboplatin/adverse effects , Cisplatin/administration & dosage , Cisplatin/adverse effects , Deoxycytidine/administration & dosage , Deoxycytidine/adverse effects , Deoxycytidine/analogs & derivatives , Disease-Free Survival , Dose-Response Relationship, Drug , Drug Administration Schedule , Drug-Related Side Effects and Adverse Reactions/epidemiology , Female , Humans , Male , Maximum Tolerated Dose , Middle Aged , Neoplasms/pathology , Prognosis , Pyrazoles/adverse effects , Pyrimidines/adverse effects , Pyrimidinones , Risk Assessment , Survival Analysis , Treatment Outcome , Gemcitabine
4.
Br J Haematol ; 161(5): 688-694, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23573950

ABSTRACT

Givinostat, a histone-deacetylase inhibitor (HDACi), inhibits proliferation of cells bearing the JAK2 V617F mutation and has shown significant activity with good tolerability in patients with chronic myeloproliferative neoplasms (MPN). In this multicentre, open-label, phase II study, 44 patients with polycythaemia vera (PV), unresponsive to the maximum tolerated doses (MTD) of hydroxycarbamide (HC), were treated with Givinostat (50 or 100 mg/d) in combination with MTD of HC. The European LeukaemiaNet response criteria were used to assess the primary endpoint after 12 weeks of treatment. Complete or partial response was observed in 55% and 50% of patients receiving 50 or 100 mg of Givinostat, respectively. Control of pruritus was observed in 64% and 67% of patients in the 50 and 100 mg groups, respectively. The combination of Givinostat and HC was well tolerated: eight patients (18%) discontinued, four in each treatment arm; grade 3 adverse events were reported in one patient (4·5%) in each treatment arm. The combined use of Givinostat and HC was safe and clinically effective in HC-unresponsive PV patients.


Subject(s)
Carbamates/therapeutic use , Histone Deacetylase Inhibitors/therapeutic use , Hydroxyurea/therapeutic use , Nucleic Acid Synthesis Inhibitors/therapeutic use , Polycythemia Vera/drug therapy , Adult , Aged , Aged, 80 and over , Carbamates/administration & dosage , Carbamates/adverse effects , Dose-Response Relationship, Drug , Drug Administration Schedule , Drug Therapy, Combination , Female , Histone Deacetylase Inhibitors/administration & dosage , Histone Deacetylase Inhibitors/adverse effects , Humans , Hydroxyurea/administration & dosage , Hydroxyurea/adverse effects , Male , Middle Aged , Nucleic Acid Synthesis Inhibitors/administration & dosage , Nucleic Acid Synthesis Inhibitors/adverse effects , Treatment Failure , Treatment Outcome
5.
Neurosci Lett ; 534: 316-21, 2013 Feb 08.
Article in English | MEDLINE | ID: mdl-23262078

ABSTRACT

Molecular targeted therapy can potentially provide more effective treatment for patients with high-grade gliomas. Notch and Akt are notable target molecules as they play important roles in a variety of cellular processes, such as regeneration, differentiation, proliferation, migration, and invasion. Here, we assessed the therapeutic possibility of inhibiting Notch and Akt in gliomas using the clinically available, selective small molecule inhibitors MRK003 and MK-2206. We evaluated their efficacy individually and as a combination therapy in U251 and U87 glioma cell lines. We confirmed that MK-2206 effectively inhibits Akt phosphorylation in a dose-dependent manner, whereas MRK003 inhibits Notch signaling and Akt phosphorylation. Both MRK003 and MK-2206 significantly inhibited cell growth, migration, and invasion in a dose-dependent manner. Akt dephosphorylation was enhanced by combination therapy with MRK003 and MK-2206. However, the effect of combination treatment did not exceed that of MK-2206 monotherapy in proliferation assay. Inhibition of invasion, further enhanced by combination therapy, correlated with increased Akt inactivation. In summary, combination therapy with MRK003 and MK-2206 may be effective for inhibiting invasion but not proliferation.


Subject(s)
Antineoplastic Agents/pharmacology , Brain Neoplasms/pathology , Glioma/pathology , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Receptors, Notch/antagonists & inhibitors , Brain Neoplasms/drug therapy , Brain Neoplasms/metabolism , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cyclic S-Oxides/pharmacology , Drug Interactions , Glioma/drug therapy , Glioma/metabolism , Heterocyclic Compounds, 3-Ring/pharmacology , Humans , Molecular Targeted Therapy , Neoplasm Invasiveness , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects , Thiadiazoles/pharmacology
6.
J Clin Oncol ; 30(19): 2307-13, 2012 Jul 01.
Article in English | MEDLINE | ID: mdl-22547604

ABSTRACT

PURPOSE: Aberrant Notch signaling has been implicated in the pathogenesis of many human cancers. MK-0752 is a potent, oral inhibitor of γ-secretase, an enzyme required for Notch pathway activation. Safety, maximum-tolerated dose, pharmacokinetics (PKs), pharmacodynamics, and preliminary antitumor efficacy were assessed in a phase I study of MK-0752. PATIENTS AND METHODS: MK-0752 was administered in three different schedules to patients with advanced solid tumors. Hair follicles were collected at higher dose levels to assess a gene signature of Notch inhibition. RESULTS: Of 103 patients who received MK-0752, 21 patients received a continuous once-daily dosing at 450 and 600 mg; 17 were dosed on an intermittent schedule of 3 of 7 days at 450 and 600 mg; and 65 were dosed once per week at 600, 900, 1,200, 1,500, 1,800, 2,400, 3,200, and 4,200 mg. The most common drug-related toxicities were diarrhea, nausea, vomiting, and fatigue. PKs (area under the concentration-time curve and maximum measured plasma concentration) increased in a less than dose proportional manner, with a half-life of approximately 15 hours. Significant inhibition of Notch signaling was observed with the 1,800- to 4,200-mg weekly dose levels, confirming target engagement at those doses. One objective complete response and an additional 10 patients with stable disease longer than 4 months were observed among patients with high-grade gliomas. CONCLUSION: MK-0752 toxicity was schedule dependent. Weekly dosing was generally well tolerated and resulted in strong modulation of a Notch gene signature. Clinical benefit was observed, and rational combination trials are currently ongoing to maximize clinical benefit with this novel agent.


Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Neoplasms/drug therapy , Neoplasms/metabolism , Propionates/pharmacology , Propionates/pharmacokinetics , Sulfones/pharmacology , Sulfones/pharmacokinetics , Administration, Oral , Adult , Aged , Aged, 80 and over , Benzene Derivatives , Cohort Studies , Dose-Response Relationship, Drug , Drug Administration Schedule , Female , Humans , Male , Middle Aged , Neoplasms/pathology , Propionates/adverse effects , Sulfones/adverse effects , Treatment Outcome
7.
J Exp Med ; 209(3): 437-44, 2012 Mar 12.
Article in English | MEDLINE | ID: mdl-22351932

ABSTRACT

Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease that is refractory to medical intervention. Notch pathway antagonism has been shown to prevent pancreatic preneoplasia progression in mouse models, but potential benefits in the setting of an established PDA tumor have not been established. We demonstrate that the gamma secretase inhibitor MRK003 effectively inhibits intratumoral Notch signaling in the KPC mouse model of advanced PDA. Although MRK003 monotherapy fails to extend the lifespan of KPC mice, the combination of MRK003 with the chemotherapeutic gemcitabine prolongs survival. Combination treatment kills tumor endothelial cells and synergistically promotes widespread hypoxic necrosis. These results indicate that the paucivascular nature of PDA can be exploited as a therapeutic vulnerability, and the dual targeting of the tumor endothelium and neoplastic cells by gamma secretase inhibition constitutes a rationale for clinical translation.


Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Carcinoma, Pancreatic Ductal/drug therapy , Cyclic S-Oxides/pharmacology , Pancreatic Neoplasms/drug therapy , Thiadiazoles/pharmacology , Animals , Carcinoma, Pancreatic Ductal/blood supply , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclic S-Oxides/administration & dosage , Deoxycytidine/administration & dosage , Deoxycytidine/analogs & derivatives , Drug Synergism , Drug Therapy, Combination , Endothelial Cells/drug effects , Endothelial Cells/pathology , Humans , Hypoxia/chemically induced , Mice , Mice, Mutant Strains , Necrosis , Pancreatic Neoplasms/blood supply , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Protease Inhibitors/administration & dosage , Protease Inhibitors/pharmacology , Receptors, Notch/metabolism , Signal Transduction/drug effects , Thiadiazoles/administration & dosage , Translational Research, Biomedical , Gemcitabine
8.
Blood ; 119(12): 2863-72, 2012 Mar 22.
Article in English | MEDLINE | ID: mdl-22267604

ABSTRACT

To identify rational therapeutic combinations with cytarabine (Ara-C), we developed a high-throughput, small-interference RNA (siRNA) platform for myeloid leukemia cells. Of 572 kinases individually silenced in combination with Ara-C, silencing of 10 (1.7%) and 8 (1.4%) kinases strongly increased Ara-C activity in TF-1 and THP-1 cells, respectively. The strongest molecular concepts emerged around kinases involved in cell-cycle checkpoints and DNA-damage repair. In confirmatory siRNA assays, inhibition of WEE1 resulted in more potent and universal sensitization across myeloid cell lines than siRNA inhibition of PKMYT1, CHEK1, or ATR. Treatment of 8 acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic myeloid leukemia (CML) cell lines with commercial and the first-in-class clinical WEE1 kinase inhibitor MK1775 confirmed sensitization to Ara-C up to 97-fold. Ex vivo, adding MK1775 substantially reduced viability in 13 of 14 AML, CML, and myelodysplastic syndrome patient samples compared with Ara-C alone. Maximum sensitization occurred at lower to moderate concentrations of both drugs. Induction of apoptosis was increased using a combination of Ara-C and MK1775 compared with using either drug alone. WEE1 is expressed in primary AML, ALL, and CML specimens. Data from this first siRNA-kinome sensitizer screen suggests that inhibiting WEE1 in combination with Ara-C is a rational combination for the treatment of myeloid and lymphoid leukemias.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Cell Cycle Proteins/metabolism , Cytarabine/pharmacology , Leukemia, Myeloid/enzymology , Nuclear Proteins/metabolism , Phosphotransferases/analysis , Protein-Tyrosine Kinases/metabolism , Blotting, Western , Cell Line, Tumor , Drug Screening Assays, Antitumor , High-Throughput Screening Assays , Humans , Phosphotransferases/metabolism , RNA, Small Interfering , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction
9.
Mol Cancer Ther ; 10(12): 2405-14, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21992793

ABSTRACT

The purpose of this study was to determine the capacity of MK-1775, a potent Wee-1 inhibitor, to abrogate the radiation-induced G(2) checkpoint arrest and modulate radiosensitivity in glioblastoma cell models and normal human astrocytes. The radiation-induced checkpoint response of established glioblastoma cell lines, glioblastoma neural stem (GNS) cells, and astrocytes were determined in vitro by flow cytometry and in vivo by mitosis-specific staining using immunohistochemistry. Mechanisms underlying MK-1775 radiosensitization were determined by mitotic catastrophe and γH2AX expression. Radiosensitivity was determined in vitro by the clonogenic assay and in vivo by tumor growth delay. MK-1775 abrogated the radiation-induced G(2) checkpoint and enhanced radiosensitivity in established glioblastoma cell lines in vitro and in vivo, without modulating radiation response in normal human astrocytes. MK-1775 appeared to attenuate the early-phase of the G(2) checkpoint arrest in GNS cell lines, although the arrest was not sustained and did not lead to increased radiosensitivity. These results show that MK-1775 can selectively enhance radiosensitivity in established glioblastoma cell lines. Further work is required to determine the role Wee-1 plays in checkpoint activation of GNS cells.


Subject(s)
Brain Neoplasms/drug therapy , G2 Phase Cell Cycle Checkpoints/drug effects , G2 Phase Cell Cycle Checkpoints/radiation effects , Glioblastoma/drug therapy , Pyrazoles/therapeutic use , Pyrimidines/therapeutic use , Animals , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Cycle Proteins/antagonists & inhibitors , Cell Line, Tumor , Glioblastoma/metabolism , Glioblastoma/pathology , Humans , Mice , Mice, Nude , Molecular Targeted Therapy , Nuclear Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrazoles/pharmacokinetics , Pyrazoles/pharmacology , Pyrimidines/pharmacokinetics , Pyrimidines/pharmacology , Pyrimidinones , Radiation Tolerance/drug effects , Radiation-Sensitizing Agents/pharmacokinetics , Radiation-Sensitizing Agents/pharmacology , Up-Regulation/drug effects , Up-Regulation/radiation effects , Xenograft Model Antitumor Assays
10.
J Clin Oncol ; 29(26): 3529-34, 2011 Sep 10.
Article in English | MEDLINE | ID: mdl-21825264

ABSTRACT

PURPOSE: To estimate the maximum-tolerated dose (MTD), describe dose-limiting toxicities (DLTs), and characterize pharmacokinetic properties of MK-0752, a gamma secretase inhibitor, in children with refractory or recurrent CNS malignancies. PATIENTS AND METHODS: MK-0752 was administered once daily for 3 consecutive days of every 7 days at escalating dosages starting at 200 mg/m(2). The modified continual reassessment method was used to estimate the MTD. A course was 28 days in duration. Pharmacokinetic analysis was performed during the first course. Expression of NOTCH and hairy enhancer of split (HES) proteins was assessed in peripheral-blood mononuclear cells (PBMCs) before and following treatment with MK-0752. RESULTS: Twenty-three eligible patients were enrolled: 10 males (median age, 8.1 years; range, 2.6 to 17.7 years) with diagnoses of brainstem glioma (n = 6), ependymoma (n = 8), medulloblastoma/primitive neuroectodermal tumor (n = 4), glioblastoma multiforme (n = 2), atypical teratoid/rhabdoid tumor (n = 1), malignant glioma (n = 1), and choroid plexus carcinoma, (n = 1). Seventeen patients were fully evaluable for toxicity. No DLTs occurred in the three patients enrolled at 200 mg/m(2)/dose. At 260 mg/m(2)/dose, DLTs occurred in two of six patients, both of whom experienced grade 3 ALT and AST. There were no grade 4 toxicities; non-dose-limiting grade 3 toxicities included hypokalemia and lymphopenia. Population pharmacokinetic values (% coefficient of variation) for MK-0752 were apparent oral clearance, 0.444 (38%) L/h/m(2); apparent volume of distribution, 7.36 (24%) L/m(2); and k(a), 0.358 (99%) hr(-1). CONCLUSION: MK-0752 is well-tolerated in children with recurrent CNS malignancies. The recommended phase II dose using the 3 days on followed by 4 days off schedule is 260 mg/m(2)/dose once daily.


Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Antineoplastic Agents/therapeutic use , Central Nervous System Neoplasms/drug therapy , Drug-Related Side Effects and Adverse Reactions , Propionates/therapeutic use , Sulfones/therapeutic use , Adolescent , Antineoplastic Agents/adverse effects , Antineoplastic Agents/pharmacokinetics , Basic Helix-Loop-Helix Transcription Factors/drug effects , Basic Helix-Loop-Helix Transcription Factors/metabolism , Benzene Derivatives , Child , Child, Preschool , Female , Homeodomain Proteins/drug effects , Homeodomain Proteins/metabolism , Humans , Male , Maximum Tolerated Dose , Propionates/adverse effects , Propionates/pharmacokinetics , Receptor, Notch1/drug effects , Receptor, Notch1/metabolism , Recurrence , Repressor Proteins/drug effects , Repressor Proteins/metabolism , Sulfones/adverse effects , Sulfones/pharmacokinetics , Transcription Factor HES-1
11.
Clin Cancer Res ; 17(9): 2799-806, 2011 May 01.
Article in English | MEDLINE | ID: mdl-21389100

ABSTRACT

PURPOSE: Investigate the efficacy and pharmacodynamic effects of MK-1775, a potent Wee1 inhibitor, in both monotherapy and in combination with gemcitabine (GEM) using a panel of p53-deficient and p53 wild-type human pancreatic cancer xenografts. EXPERIMENTAL DESIGN: Nine individual patient-derived pancreatic cancer xenografts (6 with p53-deficient and 3 with p53 wild-type status) from the PancXenoBank collection at Johns Hopkins were treated with MK-1775, GEM, or GEM followed 24 hour later by MK-1775, for 4 weeks. Tumor growth rate/regressions were calculated on day 28. Target modulation was assessed by Western blotting and immunohistochemistry. RESULTS: MK-1775 treatment led to the inhibition of Wee1 kinase and reduced inhibitory phosphorylation of its substrate Cdc2. MK-1775, when dosed with GEM, abrogated the checkpoint arrest to promote mitotic entry and facilitated tumor cell death as compared to control and GEM-treated tumors. MK-1775 monotherapy did not induce tumor regressions. However, the combination of GEM with MK-1775 produced robust antitumor activity and remarkably enhanced tumor regression response (4.01-fold) compared to GEM treatment in p53-deficient tumors. Tumor regrowth curves plotted after the drug treatment period suggest that the effect of the combination therapy is longer-lasting than that of GEM. None of the agents produced tumor regressions in p53 wild-type xenografts. CONCLUSIONS: These results indicate that MK-1775 selectively synergizes with GEM to achieve tumor regressions, selectively in p53-deficient pancreatic cancer xenografts.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carcinoma, Pancreatic Ductal/drug therapy , Deoxycytidine/analogs & derivatives , Pancreatic Neoplasms/drug therapy , Pyrazoles/administration & dosage , Pyrimidines/administration & dosage , Animals , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/pathology , Cell Cycle Proteins/antagonists & inhibitors , Cell Line, Tumor , Deoxycytidine/administration & dosage , Disease Progression , Drug Synergism , Female , Genes, p53 , Humans , Mice , Mice, Nude , Mutation/physiology , Neoplasms/drug therapy , Neoplasms/pathology , Nuclear Proteins/antagonists & inhibitors , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Pyrimidinones , Tumor Burden/drug effects , Xenograft Model Antitumor Assays , Gemcitabine
12.
Int J Cancer ; 126(5): 1155-65, 2010 Mar 01.
Article in English | MEDLINE | ID: mdl-19728339

ABSTRACT

To reveal molecular drivers of glioma invasion, two distinct glioblastoma (GBM) cell phenotypes (invading cells and tumor core cells) were collected from 19 GBM specimens using laser capture microdissection. Isolated RNA underwent whole human genome expression profiling to identify differentially expressed genes. Pathway enrichment analysis highlighted the bidirectional receptor/ligand tyrosine kinase system, EphB/ephrin-B, as the most tightly linked system to the invading cell phenotype. Clinical relevance of ephrin-B genes was confirmed in a clinically annotated expression data set of 195 brain biopsy specimens. Levels of ephrin-B1 and -B2 mRNA were significantly higher in GBM (n = 82) than in normal brain (n = 24). Kaplan-Meier analysis demonstrated ephrin-B2, but not ephrin-B1, expression levels were significantly associated with short term survival in malignant astrocytomas (n = 97, p = 0.016). In human brain tumor specimens, the production and phosphorylation of ephrin-B2 were high in GBM. Immunohistochemistry demonstrated ephrin-B2 localization primarily in GBM cells but not in normal brain. A highly invasive glioma cell line, U87, expressed high levels of ephrin-B2 compared with relatively less invasive cell lines. Treatment with EphB2/Fc chimera further enhanced migration and invasion of U87 cells, whereas treatment with an ephrin-B2 blocking antibody significantly slowed migration and invasion. Forced expression of ephrin-B2 in the U251 cell line stimulated migration and invasion in vitro and ex vivo, concomitant with tyrosine phosphorylation of ephrin-B2. These results demonstrate that high expression of ephrin-B2 is a strong predictor of short-term survival and that ephrin-B2 plays a critical role in glioma invasion rendering this signaling pathway as a potential therapeutic target.


Subject(s)
Brain Neoplasms/metabolism , Ephrin-B2/metabolism , Glioma/metabolism , Signal Transduction/physiology , Blotting, Western , Brain Neoplasms/genetics , Brain Neoplasms/mortality , Cell Movement , Cells, Cultured , Ephrin-B1/genetics , Ephrin-B1/metabolism , Ephrin-B2/genetics , Gene Expression , Gene Expression Profiling , Glioma/genetics , Glioma/mortality , Humans , Immunohistochemistry , Immunoprecipitation , Kaplan-Meier Estimate , Lasers , Ligands , Microdissection , Neoplasm Invasiveness , Oligonucleotide Array Sequence Analysis , Phosphorylation , Prognosis , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Transfection
13.
Cancer Res ; 69(23): 8949-57, 2009 Dec 01.
Article in English | MEDLINE | ID: mdl-19903844

ABSTRACT

Notch pathway signaling plays a fundamental role in normal biological processes and is frequently deregulated in many cancers. Although several hypotheses regarding cancer subpopulations most likely to respond to therapies targeting the Notch pathway have been proposed, clinical utility of these predictive markers has not been shown. To understand the molecular basis of gamma-secretase inhibitor (GSI) sensitivity in breast cancer, we undertook an unbiased, de novo responder identification study using a novel genetically engineered in vivo breast cancer model. We show that tumors arising from this model are heterogeneous on the levels of gene expression, histopathology, growth rate, expression of Notch pathway markers, and response to GSI treatment. In addition, GSI treatment of this model was associated with inhibition of Hes1 and proliferation markers, indicating that GSI treatment inhibits Notch signaling. We then identified a pretreatment gene expression signature comprising 768 genes that is significantly associated with in vivo GSI efficacy across 99 tumor lines. Pathway analysis showed that the GSI responder signature is enriched for Notch pathway components and inflammation/immune-related genes. These data show the power of this novel in vivo model system for the discovery of biomarkers predictive of response to targeted therapies, and provide a basis for the identification of human breast cancers most likely to be sensitive to GSI treatment.


Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Cyclic S-Oxides/administration & dosage , Enzyme Inhibitors/administration & dosage , Mammary Neoplasms, Experimental/drug therapy , Mammary Neoplasms, Experimental/enzymology , Thiadiazoles/administration & dosage , Animals , Cell Growth Processes/drug effects , Drug Administration Schedule , Gene Regulatory Networks , Humans , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Inbred C57BL , Mice, Transgenic
14.
BMC Genomics ; 9: 54, 2008 Jan 29.
Article in English | MEDLINE | ID: mdl-18230158

ABSTRACT

BACKGROUND: Glioblastoma multiforme (GBM) is the most common primary intracranial tumor and despite recent advances in treatment regimens, prognosis for affected patients remains poor. Active cell migration and invasion of GBM cells ultimately lead to ubiquitous tumor recurrence and patient death. To further understand the genetic mechanisms underlying the ability of glioma cells to migrate, we compared the matched transcriptional profiles of migratory and stationary populations of human glioma cells. Using a monolayer radial migration assay, motile and stationary cell populations from seven human long term glioma cell lines and three primary GBM cultures were isolated and prepared for expression analysis. RESULTS: Gene expression signatures of stationary and migratory populations across all cell lines were identified using a pattern recognition approach that integrates a priori knowledge with expression data. Principal component analysis (PCA) revealed two discriminating patterns between migrating and stationary glioma cells: i) global down-regulation and ii) global up-regulation profiles that were used in a proband-based rule function implemented in GABRIEL to find subsets of genes having similar expression patterns. Genes with up-regulation pattern in migrating glioma cells were found to be overexpressed in 75% of human GBM biopsy specimens compared to normal brain. A 22 gene signature capable of classifying glioma cultures based on their migration rate was developed. Fidelity of this discovery algorithm was assessed by validation of the invasion candidate gene, connective tissue growth factor (CTGF). siRNA mediated knockdown yielded reduced in vitro migration and ex vivo invasion; immunohistochemistry on glioma invasion tissue microarray confirmed up-regulation of CTGF in invasive glioma cells. CONCLUSION: Gene expression profiling of migratory glioma cells induced to disperse in vitro affords discovery of genomic signatures; selected candidates were validated clinically at the transcriptional and translational levels as well as through functional assays thereby underscoring the fidelity of the discovery algorithm.


Subject(s)
Brain Neoplasms/genetics , Brain Neoplasms/pathology , Cell Movement/genetics , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Glioma/genetics , Glioma/pathology , Cell Line, Tumor , Humans , Immunohistochemistry , Models, Biological , Oligonucleotide Array Sequence Analysis , Principal Component Analysis , Reproducibility of Results , Survival Rate
15.
J Neurooncol ; 86(3): 297-309, 2008 Feb.
Article in English | MEDLINE | ID: mdl-17928955

ABSTRACT

Glioblastoma multiforme (GBM) is inherently invasive, and it is from the invasive cell population that the tumor recurs. The GBM invasion transcriptome reveals over-expression of various autocrine factors that could act as motility drivers, such as autotaxin (ATX). Some of these factors could also have paracrine roles, modulating the behavior of cells in the peri-tumoral brain parenchyma. ATX generates lysophosphatidic acid (LPA), which signals through LPA receptors expressed by GBM as well as in astrocytes, oligodendrocytes (ODC) and microglia; their activation manifest cell specific effects. ATX stimulates invasion of GBM cells in vitro and ex vivo invasion assays. ATX activity enhances GBM adhesion in cells expressing the LPA1 receptor, as well as stimulating rac activation. GBM secreted ATX can also have paracrine effects: ATX activity results in reduced ODC adhesion. ODC monolayer invasion showed that U87 and U251 GBM cells expressing ATX invaded through an ODC monolayer significantly more than cells depleted of ATX or cells expressing inactive ATX, suggesting that GBM cells secreting ATX find ODCs less of a barrier than cells that do not express ATX. Secreted factors that drive GBM invasion can have autocrine and paracrine roles; one stimulates GBM motility and the other results in ODC dis-adhesion.


Subject(s)
Brain Neoplasms/metabolism , Gene Expression Regulation, Neoplastic/physiology , Glioblastoma/metabolism , Glucose-6-Phosphate Isomerase/metabolism , Multienzyme Complexes/metabolism , Phosphodiesterase I/metabolism , Pyrophosphatases/metabolism , Animals , Brain/physiopathology , Cell Adhesion/drug effects , Cell Adhesion/physiology , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/physiology , Gene Expression Regulation, Neoplastic/drug effects , Humans , In Vitro Techniques , Lipopolysaccharides/pharmacology , Multienzyme Complexes/genetics , Neoplasm Invasiveness , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Neoplasms, Experimental/secondary , Phosphodiesterase I/genetics , Phosphoric Diester Hydrolases , Pyrophosphatases/genetics , RNA, Small Interfering/pharmacology , Rats , Time Factors , Transplants , rac1 GTP-Binding Protein/metabolism
16.
J Natl Cancer Inst ; 99(21): 1583-93, 2007 Nov 07.
Article in English | MEDLINE | ID: mdl-17971532

ABSTRACT

Invasion is a defining hallmark of glioblastoma multiforme, just as metastasis characterizes other high-grade tumors. Glial tumors invariably recur due to the regrowth of invasive cells, which are unaffected by standard treatment modalities. Drivers of glioma invasion include autocrine signals propagated by secreted factors that signal through receptors on the tumor. These secreted factors are able to diffuse through the peritumoral stroma, thereby influencing parenchymal cells that surround the tumor mass. Here we describe various autocrine motility factors that are expressed by invasive glioma cells and explore the effects that they may have on normal cells present in the path of invasion. Conversely, normal brain parenchymal cells secrete ligands that can stimulate receptors on invasive glioma cells and potentially facilitate glioma invasion or create a permissive microenvironment for malignant progression. Parallel observations have been made for solid tumors of epithelial origin, in which parenchymal and stromal cells either support or suppress tumor invasion. Most autocrine and paracrine interactions involved in glioma invasion constitute known signaling systems in stages of central nervous system development that involve the migration of precursor cells that populate the developing brain. Key paracrine interactions between glioma cells and the brain microenvironment can influence glioma pathobiology and therefore contribute to its poor prognosis. Current therapies for glioma that could have an impact on paracrine communication between tumors and normal cells are discussed. We suggest that cells in the normal brain parenchyma be considered as potential targets for adjuvant therapies to control glioma growth because such cells are less likely to develop resistance than glioma cells.


Subject(s)
Autocrine Communication , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Glioma/metabolism , Glioma/pathology , Paracrine Communication , Animals , Antineoplastic Agents/pharmacology , Astrocytes/metabolism , Astrocytes/pathology , Astrocytoma/metabolism , Astrocytoma/pathology , Autocrine Communication/drug effects , Axons/metabolism , Axons/pathology , Brain Neoplasms/blood supply , Brain Neoplasms/drug therapy , Glioblastoma/metabolism , Glioblastoma/pathology , Glioma/blood supply , Glioma/drug therapy , Glucose-6-Phosphate Isomerase/metabolism , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Neoplasm Invasiveness , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Neurons/metabolism , Neurons/pathology , Oligodendroglia/metabolism , Oligodendroglia/pathology , Paracrine Communication/drug effects
17.
Appl Opt ; 46(22): 5110-8, 2007 Aug 01.
Article in English | MEDLINE | ID: mdl-17676121

ABSTRACT

To gain insight into brain tumor invasion, experiments are conducted on multicellular tumor spheroids grown in collagen gel. Typically, a radius of invasion is reported, which is obtained by human measurement. We present a simple, heuristic algorithm for automated invasive radii estimation (AIRE) that uses local fluctuations of the image intensity. We then derive an analytical expression relating the image graininess to the cell density for a model imaging system. The result agrees with the experiment up to a multiplicative constant and thus describes a novel method for estimating the cell density from bright-field images.


Subject(s)
Brain Neoplasms/pathology , Glioblastoma/pathology , Optics and Photonics , Spheroids, Cellular/pathology , Tumor Cells, Cultured/pathology , Algorithms , Biopsy , Cell Count , Humans , Image Processing, Computer-Assisted , Microscopy, Confocal , Models, Statistical , Neoplasm Invasiveness
18.
Mol Cancer Ther ; 6(4): 1212-22, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17406030

ABSTRACT

Although astrocytic brain tumors do not metastasize systemically, during tumorigenesis glioma cells adopt an invasive phenotype that is poorly targeted by conventional therapies; hence, glioma patients die of recurrence from the locally invasive tumor population. Our work is aimed at identifying and validating novel therapeutic targets and biomarkers in invasive human gliomas. Transcriptomes of invasive glioma cells relative to stationary cognates were produced from a three-dimensional spheroid in vitro invasion assay by laser capture microdissection and whole human genome expression microarrays. Qualitative differential expression of candidate invasion genes was confirmed by quantitative reverse transcription-PCR, clinically by immunohistochemistry on tissue microarray, by immunoblotting on surgical specimens, and on two independent gene expression data sets of glial tumors. Cell-based assays and ex vivo brain slice invasion studies were used for functional validation. We identify mitogen-activated protein kinase (MAPK) kinase 3 (MKK3) as a key activator of p38 MAPK in glioma; MKK3 activation is strongly correlated with p38 activation in vitro and in vivo. We further report that these members of the MAPK family are strong promoters of tumor invasion, progression, and poor patient survival. Inhibition of either candidate leads to significantly reduced glioma invasiveness in vitro. Consistent with the concept of synthetic lethality, we show that inhibition of invasion by interference with these genes greatly sensitizes arrested glioma cells to cytotoxic therapies. Our findings therefore argue that interference with MKK3 signaling through a novel treatment combination of p38 inhibitor plus temozolomide heightens the vulnerability of glioma to chemotherapy.


Subject(s)
Glioma/enzymology , Glioma/pathology , MAP Kinase Kinase 3/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Apoptosis/drug effects , Astrocytoma/enzymology , Astrocytoma/pathology , Biomarkers/metabolism , Cell Line, Tumor , Collagen Type I/metabolism , Disease Progression , Enzyme Activation/drug effects , Gene Expression Profiling , Glioma/diagnosis , Glioma/genetics , Humans , MAP Kinase Kinase 3/antagonists & inhibitors , MAP Kinase Kinase 3/genetics , Male , Neoplasm Invasiveness , Phosphorylation/drug effects , Prognosis , Protein Kinase Inhibitors/pharmacology , Rats , Rats, Wistar , Survival Analysis , Up-Regulation/drug effects , Up-Regulation/genetics , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/genetics
19.
Biophys J ; 92(1): 356-65, 2007 Jan 01.
Article in English | MEDLINE | ID: mdl-17040992

ABSTRACT

Glioblastoma, the most malignant form of brain cancer, is responsible for 23% of primary brain tumors and has extremely poor outcome. Confounding the clinical management of glioblastomas is the extreme local invasiveness of these cancer cells. The mechanisms that govern invasion are poorly understood. To gain insight into glioblastoma invasion, we conducted experiments on the patterns of growth and dispersion of U87 glioblastoma tumor spheroids in a three-dimensional collagen gel. We studied two different cell lines, one with a mutation to the EGFR (U87DeltaEGFR) that is associated with increased malignancy, and one with an endogenous (wild-type) receptor (U87WT). We developed a continuum mathematical model of the dispersion behaviors with the aim of identifying and characterizing discrete cellular mechanisms underlying invasive cell motility. The mathematical model quantitatively reproduces the experimental data, and indicates that the U87WT invasive cells have a stronger directional motility bias away from the spheroid center as well as a faster rate of cell shedding compared to the U87DeltaEGFR cells. The model suggests that differences in tumor cell dispersion may be due to differences in the chemical factors produced by cells, differences in how the two cell lines remodel the gel, or different cell-cell adhesion characteristics.


Subject(s)
Brain Neoplasms/pathology , Glioblastoma/pathology , Neoplasm Invasiveness , Spheroids, Cellular/metabolism , Brain Neoplasms/metabolism , Cell Adhesion , Cell Line, Tumor , Collagen/chemistry , ErbB Receptors/chemistry , Glioblastoma/metabolism , Humans , Models, Statistical , Models, Theoretical , Mutation , Time Factors , Tumor Cells, Cultured
20.
Acta Biotheor ; 53(3): 181-90, 2005.
Article in English | MEDLINE | ID: mdl-16329007

ABSTRACT

Highly malignant neuroepithelial tumors are known for their extensive tissue invasion. Investigating the relationship between their spatial behavior and temporal patterns by employing detrended fluctuation analysis (DFA), we report here that faster glioma cell motility is accompanied by both greater predictability of the cells' migration velocity and concomitantly, more directionality in the cells' migration paths. Implications of this finding for both experimental and clinical cancer research are discussed.


Subject(s)
Brain Neoplasms/pathology , Cell Movement/physiology , Glioma/pathology , Neoplasm Invasiveness/pathology , Tumor Cells, Cultured/physiology , Acceleration , Cell Line, Tumor , Humans , Linear Models , Orientation/physiology
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