Tim Demuth

Molecular targets of glioma invasion

Abstract.Glioblastoma multiforme is the most common and lethal primary malignant brain tumor. Although considerable progress has been made in technical proficiencies of surgical and radiation treatment for brain tumor patients, the impact of these advances on clinical outcome has been disappointing, with median survival time not exceeding 15 months. Over the last 30 years, no significant increase in survival of patients suffering from this disease has been achieved. A fundamental source of the management challenge presented in glioma patients is the insidious propensity of tumor invasion into distant brain tissue. Invasive tumor cells escape surgical removal and geographically dodge lethal radiation exposure and chemotherapy. Recent improved understanding of biochemical and molecular determinants of glioma cell invasion provide valuable insight into the underlying biological features of the disease, as well as illuminating possible new therapeutic targets. These findings are moving forward to translational research and clinical trials as novel antiglioma therapies.

Phase I Pharmacologic and Pharmacodynamic Study of the Gamma Secretase (Notch) Inhibitor MK-0752 in Adult Patients With Advanced Solid Tumors

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.

Migrating glioma cells activate the PI3-K pathway and display decreased susceptibility to apoptosis

Glioma cells that migrate out of the main tumor mass into normal brain tissue contribute to the failure of most gliomas to respond to treatment. Treatments that target migratory glioma cells may enhance the therapeutic response. Multiple lines of evidence suggest that suppression of apoptosis accompanies activation of the migratory phenotype. Here, we determine whether migration and apoptosis are consistently linked in glioma cells and whether manipulation of migration influences cytotoxic therapy-induced apoptosis. Camptothecin and Trail-induced apoptosis were decreased 2-5-fold in actively migrating glioma cells relative to migration-restricted cells. Consistent with a mechanistic link between migration and apoptosis, the dose-response for stimulation of migration on laminin was inversely proportional to apoptosis induction. Treatment of glioma cells with migration inhibitors alone had little effect on basal rates of apoptosis and had little effect on Trail-induced or camptothecin-induced apoptosis in migration-restricted cells. By contrast, migration inhibitors increased camptothecin and Trail-induced apoptosis in actively migrating glioma cells. Migrating glioma cells have increased amounts of phosphorylated Akt and its downstream substrate glycogen synthase kinase-3 relative to migration restricted cells. Treatment of migrating cells with a specific inhibitor of phosphoinositide 3-kinase (PI3-K), LY294002, blocked the phosphorylation of Akt and increased the sensitivity to apoptosis. LY294002 had no effect on the migration of restricted cells. This suggests that migrating glioma cells activate the PI3-K survival pathway, protecting migrating cells from apoptosis. Taken together, these data provide support for a link between migration and apoptosis in glioma cells. In addition, evidence indicates that treatment with migration inhibitors, while not affecting apoptosis-induction in migration-restricted cells, can sensitize migrating glioma cells to cytotoxic agents.

MK-1775, a potent Wee1 inhibitor, synergizes with gemcitabine to achieve tumor regressions, selectively in p53-deficient pancreatic cancer xenografts

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. Clin Cancer Res; 17(9); 2799–806. ©2011 AACR.

Phase I Trial of MK-0752 in Children With Refractory CNS Malignancies: A Pediatric Brain Tumor Consortium Study

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.

MAP-ing glioma invasion: Mitogen-activated protein kinase kinase 3 and p38 drive glioma invasion and progression and predict patient survival

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. [Mol Cancer Ther 2007;6(4):1212–22]

Gamma secretase inhibition promotes hypoxic necrosis in mouse pancreatic ductal adenocarcinoma

Blocking Notch signaling in pancreatic cancer promotes hypoxia and cell death.

Targeting radiation-induced G(2) checkpoint activation with the Wee-1 inhibitor MK-1775 in glioblastoma cell lines.

The purpose of this study was to determine the capacity of MK-1775, a potent Wee-1 inhibitor, to abrogate the radiation-induced G2 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 G2 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 G2 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. Mol Cancer Ther; 10(12); 2405–14. ©2011 AACR.

The Phosphorylation of Ephrin-B2 Ligand Promotes Glioma Cell Migration and Invasion

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.

RNAi screening of the kinome with cytarabine in leukemias

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.