Identification of alsterpaullone as a novel small molecule inhibitor to target group 3 medulloblastoma.

Advances in the molecular biology of medulloblastoma revealed four genetically and clinically distinct subgroups. Group 3 medulloblastomas are characterized by frequent amplifications of the oncogene MYC, a high incidence of metastasis, and poor prognosis despite aggressive therapy. We investigated several potential small molecule inhibitors to target Group 3 medulloblastomas based on gene expression data using an in silico drug screen. The Connectivity Map (C-MAP) analysis identified piperlongumine as the top candidate drug for non-WNT medulloblastomas and the cyclin-dependent kinase (CDK) inhibitor alsterpaullone as the compound predicted to have specific antitumor activity against Group 3 medulloblastomas. To validate our findings we used these inhibitors against established Group 3 medulloblastoma cell lines. The C-MAP predicted drugs reduced cell proliferation in vitro and increased survival in Group 3 medulloblastoma xenografts. Alsterpaullone had the highest efficacy in Group 3 medulloblastoma cells. Genomic profiling of Group 3 medulloblastoma cells treated with alsterpaullone confirmed inhibition of cell cycle-related genes, and down-regulation of MYC. Our results demonstrate the preclinical efficacy of using a targeted therapy approach for Group 3 medulloblastomas. Specifically, we provide rationale for advancing alsterpaullone as a targeted therapy in Group 3 medulloblastoma.

Mechanism of action and therapeutic efficacy of Aurora kinase B inhibition in MYC overexpressing medulloblastoma.

Medulloblastoma comprises four molecular subgroups of which Group 3 medulloblastoma is characterized by MYC amplification and MYC overexpression. Lymphoma cells expressing high levels of MYC are susceptible to apoptosis following treatment with inhibitors of mitosis. One of the key regulatory kinases involved in multiple stages of mitosis is Aurora kinase B. We hypothesized that medulloblastoma cells that overexpress MYC would be uniquely sensitized to the apoptotic effects of Aurora B inhibition. The specific inhibition of Aurora kinase B was achieved in MYC- overexpressing medulloblastoma cells with AZD1152-HQPA. MYC overexpression sensitized medulloblastoma cells to cell death upon Aurora B inhibition. This process was found to be independent of endoreplication. Using both flank and intracranial cerebellar xenografts we demonstrate that tumors formed from MYC-overexpressing medulloblastoma cells show a response to Aurora B inhibition including growth impairment and apoptosis induction. Lastly, we show the distribution of AZD1152-HQPA within the mouse brain and the ability to inhibit intracranial tumor growth and prolong survival in mice bearing tumors formed from MYC-overexpressing medulloblastoma cells. Our results suggest the potential for therapeutic application of Aurora kinase B inhibitors in the treatment of Group 3 medulloblastoma.

Disruption of fibroblast growth factor receptor signaling in nonmyelinating Schwann cells causes sensory axonal neuropathy and impairment of thermal pain sensitivity.

Axon-glial interactions are critical for normal functioning of peripheral nerves, and their disruption leads to peripheral neuropathies. Fibroblast growth factors (FGFs) are key players in peripheral nerve regeneration after injury. We investigated the role of FGF receptor (Fgfr) signaling in Schwann cells and the consequent regulation of normal Schwann cell-axon interactions. Fgfr1 and Fgfr2 were conditionally inactivated, either singly or in combination, in myelinating and nonmyelinating Schwann cells (NMSCs) of transgenic mice. The double mutant mice displayed significant loss of thermal sensitivity accompanied by marked neuropathy of unmyelinated nociceptive sensory axons terminating in the dorsal horn of spinal cords, the primary site for integrating pain and temperature inputs. Neuropathy, although to a lesser extent, was also observed in the nociceptive C-fibers in the Remak bundles of sciatic nerves; however, there was no loss of NMSCs that ensheathe these axons. Furthermore, axons wrapped by myelinating Schwann cells and associated myelin sheaths appeared to be unaffected. Relative to the double mutants, axonal neuropathy developed much later in the Fgfr1 but not Fgfr2 single mutant, indicating a difference in signaling potential of the two receptors, with Fgfr1 being more robust than Fgfr2. These findings emphasize the importance of Fgfr1 and Fgfr2 signaling as potential mediators of axon-glial interaction in the peripheral sensory pain pathway primarily via influencing NMSC function, which in turn modulates the structure and function of unmyelinated sensory axons. This study provides a novel molecular mechanism for nociception with possible implications for pain sensitivity in peripheral sensory neuropathies.

S-phase entry of oligodendrocyte lineage cells is associated with increased levels of p21Cip1.

The mechanisms regulating the number of myelinating cells in the central nervous system are crucial for both normal development and repair in pathological conditions. Among relevant growth factors involved in this process, fibroblast growth factor-2 (FGF2) induces oligodendrocyte progenitors (OLPs) to proliferate and stimulates mature oligodendrocytes (OLs) to reenter the S-phase of the cell cycle. S-phase entry is modulated by the formation of complexes between cyclins and cyclin-dependent kinases (CDKs), on one hand, and by their interactions with cell cycle inhibitors (e.g., p18INK, p27Kip1, p21Cip1), on the other. Although the roles of cyclin E/CDK2 complexes and the inhibitor p27Kip1 have been extensively investigated relative to proliferation and differentiation in the OL lineage, less is known about the regulation of the formation of cyclin D1/CDK4 complexes and the role of p21Cip1 in these events. In this study, we show that the FGF2-mediated increase in bromodeoxyuridine (BrdU) incorporation into OL progenitors and mature OLs occurs concomitantly with increase in the levels of p21Cip1 and the formation of p21Cip1/cyclin D1/CDK4 ternary complexes. These complexes are functionally active is indicated by the ensuing FGF2-dependent hyperphosphorylation of the downstream target Rb. In untreated mature OLs that do not incorporate BrdU, the levels of p21Cip1 are low, and the level of the inhibitor p18INK is high. Furthermore, p18INK sequesters CDK2 into binary complexes, precluding the formation of p21Cip1/cyclin D1/CDK4 ternary complexes in these cells. Therefore, we propose that p21Cip1 is acting as a positive regulator, rather than an inhibitor, of cell cycle entry by favoring the assembly of active cyclin D1/CDK4 complexes.

Human immunodeficiency virus-related primary central nervous system lymphoma: factors influencing survival in 111 patients.

BACKGROUND: The current study evaluated factors influencing survival in patients diagnosed with human immunodeficiency virus (HIV)-related primary central nervous system lymphoma (PCNSL), with a focus on the effects of therapeutic radiotherapy (RT) and highly active antiretroviral therapy (HAART). METHODS: A retrospective chart review of patients with a diagnosis of HIV-related PCNSL at one of five university hospitals between 1987 and 1998 was performed. Clinical details including antiretroviral agent use, brain imaging scan results, RT use, and survival outcomes were recorded. RESULTS: One hundred eleven patients with HIV-related PCNSL were identified. The annual incidence decreased significantly between 1992 and 1995 and between 1996 and 1998 (P = 0.04). The median survival period was 50 days (mean, 109 days; range, 4-991 days), with improved survival for patients diagnosed after 1993. Patients treated with two or more antiretroviral agents had improved survival (P = 0.01), as did patients who received RT (P < 0.0001). For patients who received RT, completion of the prescribed course and treatment to > or = 30 Gray (Gy) independently predicted a more favorable outcome. RT used in conjunction with antiretroviral therapy involving two or more agents had an additive positive effect on survival. For patients who did not receive RT, poor performance status and encephalopathy predicted a shorter survival duration. CONCLUSIONS: The results of the current study suggest that HAART and treatment with RT to > or = 30 Gy improve survival for patients with HIV-related PCNSL. This combination of therapies may provide a standard of care as the basis for further trials of chemotherapy, novel adjunctive treatment, and quality of life assessment.

Insulin impairs endothelium-dependent vasodilation independent of insulin sensitivity or lipid profile.

Insulin resistance is a risk factor for atherosclerosis and is associated with hyperinsulinemia, abnormal lipid profile, and hypertension. Whether hyperinsulinemia affects vascular function independent of insulin resistance or other metabolic risk factors is unknown. This investigation aimed to assess the effects of hyperinsulinemia on endothelial function in subjects with a spectrum of insulin sensitivity and lipid profile. Endothelium-dependent (flow-mediated dilation, FMD) and -independent (nitroglycerin) responses of the brachial artery were studied by high-resolution ultrasound before and during hyperinsulinemia (euglycemic clamp) in 25 normoglycemic, normotensive subjects. Participants were divided into an insulin-sensitive and an insulin-resistant subgroup based on their sensitivity index values, with a cutoff of 8, and into a normal-cholesterol and a high-cholesterol subgroup based on their total cholesterol levels, with a cutoff of 5.2 mmol/l (200 mg/dl). In the whole population, FMD was lower during hyperinsulinemia compared with baseline (2.3 +/- 0.6% vs. 6 +/- 0.6%; P < 0.001). Resting FMD was lower in the insulin-resistant subgroup compared with the insulin-sensitive subgroup (4.2 +/- 0.9% vs. 7.4 +/- 0.8%; P = 0.014) and in the high-cholesterol subjects compared with the normal-cholesterol subjects (4.4 +/- 0.7% vs. 8 +/- 0.7%; P = 0.002). Hyperinsulinemia decreased FMD in both the insulin-sensitive (from 7.4 +/- 0.8% to 3.6 +/- 0.4%; P < 0.001) and insulin-resistant (from 4.2% to 1.22%; P = 0.012) subgroups and in both the normal-cholesterol (from 8 +/- 0.7% to 3.9 +/- 0.4%; P < 0.001) and high-cholesterol (from 4.4 +/- 0.7% to 1.1 +/- 0.8%; P = 0.01) participants. Acute hyperinsulinemia impairs conduit vessel endothelial function independent of insulin sensitivity and lipid profile. Insulin may trigger endothelial dysfunction and promote atherosclerosis.

Increased activity of endogenous endothelin in patients with type II diabetes mellitus.

BACKGROUND: Endothelial dysfunction may contribute to the risk of premature atherosclerosis in patients with diabetes. Endothelin (ET-1) may be involved in this process by activating smooth muscle cell mitogenesis and leukocyte adhesion. We sought to assess the activity of endogenous ET-1 in a group of patients with type II diabetes mellitus with the use of antagonists of ET-1 receptors. METHODS AND RESULTS: Forearm blood flow (FBF) responses (strain gauge plethysmography) to intraarterial infusion of a selective blocker of ET(A) receptors (BQ-123) and, on a different occasion, to ET-1, were measured in 15 patients with diabetes and 12 healthy controls. In addition, 5 patients with diabetes received coinfusion of BQ-123 and BQ-788 (a selective blocker of ET(B) receptors). In normal subjects, BQ-123 did not significantly modify FBF from baseline (P=0.16). In contrast, BQ-123 administration resulted in a significant vasodilator response in patients with diabetes (P<0.001). Infusion of exogenous ET-1 resulted in lower vasoconstrictor responses in patients with diabetes than in controls (P=0.001), whereas the vasoconstrictor response to norepinephrine was similar in the 2 groups (P=0.78). In patients with diabetes, the vasodilator response to selective ET(A) blockade was not significantly modified by nonselective blockade of ET-1 receptors obtained by coinfusion of BQ-123 and BQ-788. CONCLUSIONS: The activity of endogenous ET-1 on ET(A) receptors is enhanced in the resistance vessels of patients with diabetes, whereas their sensitivity to exogenous ET-1 is blunted. This abnormality may participate in the pathophysiology of vascular complications associated with diabetes.

Reduced endothelium-dependent and -independent dilation of conductance arteries in African Americans.

OBJECTIVES: The goal of this study was to determine whether racial differences exist in the functional behavior of conduit vessels. BACKGROUND: Compared with Caucasians, African Americans have a higher prevalence of cardiovascular disease and its complications, which may be related to reduced nitric oxide (NO)-dependent and -independent vasodilation of the microvasculature. However, whether a similar impairment is also present at the level of the conductance arteries is unknown. METHODS: To this end, we studied endothelium-dependent (posthyperemia flow-mediated dilation) and -independent (nitroglycerin) vascular responses of the brachial artery by high-resolution ultrasound imaging. There were 46 black subjects (23 men and 23 women; age 37 +/- 8 years and 38 +/- 9 years, respectively) and 46 white subjects (23 men and 23 women; age 38 +/- 11 years and 36 +/- 9 years, respectively) in this study. RESULTS: Baseline diameter was similar in blacks and in whites (4.4 +/- 0.9 mm and 4.1 +/- 0.7 mm, respectively). Mean reactive hyperemia after cuff deflation was similar in the two groups (793 +/- 653% in black and 852 +/- 734% in white subjects, respectively; p = 0.5). Flow-mediated dilation was significantly lower in black compared with white individuals (4.79 +/- 3.5% vs. 8.87 +/- 4.5%, respectively; p < 0.0001). Nitroglycerin-mediated dilation was also significantly lower in black individuals compared with white individuals (10.99 +/- 4.6% vs. 14.98 +/- 5.4%, respectively; p = 0.0002). CONCLUSIONS: African Americans show reduced responsiveness of conductance vessels to both endogenous and exogenous NO compared with Caucasian Americans. These findings expand our understanding of racial differences in vascular function and suggest a mechanistic explanation for the increased incidence and severity of cardiovascular disease observed in African Americans.

Role of cyclooxygenase products in the regulation of vascular tone and in the endothelial vasodilator function of normal, hypertensive, and hypercholesterolemic humans.

A defective vascular activity of endothelial vasoactive substances is observed in essential hypertension and hypercholesterolemia, and is believed to participate in the vascular abnormalities characteristic of these conditions. The present study aimed to determine the role of cyclooxygenase (COX) products in the maintenance of vascular tone and in the endothelium-mediated vasodilation of healthy subjects, and to investigate their contribution to the endothelial dysfunction of essential hypertensive and hypercholesterolemic patients. The effects of intra-arterial aspirin (acetylsalicylic acid [ASA], 1, 3, and 10 mg/min) were assessed on basal forearm blood flow (strain gauge plethysmography) and on responses to acetylcholine (7.5, 15 and 30 microg/min) and sodium nitroprusside (0.8, 1.6 and 3.2 microg/min) in 24 normal, 23 hypertensive, and 24 hypercholesterolemic subjects. Basal forearm blood flow was not different among the 3 groups (p = 0.95). ASA resulted in a significant reduction of forearm blood flow from baseline in normal (p = 0.003), hypertensive (p = 0.001), and hypercholesterolemic subjects (p = 0.001), without any difference among the 3 groups (p = 0.90). ASA significantly improved the effect of acetylcholine in normal (p = 0.008), hypertensive (p = 0.008), and hypercholesterolemic subjects (p = 0.022), without significant difference among the 3 groups (p = 0.46). ASA did not significantly modify the vasodilator effect of sodium nitroprusside in any of the 3 groups. These findings suggest that in humans, vasodilator prostanoids actively contribute to the maintenance of basal vascular tone, whereas vasoconstrictor products of COX activity limit endothelium-dependent vasodilation. COX products do not appear to play a major role in the endothelial dysfunction of hypertensive or hypercholesterolemic patients.

Improved endothelium-dependent vasodilation after blockade of endothelin receptors in patients with essential hypertension.

BACKGROUND: Hypertensive patients have both impaired endothelium-dependent vasodilation and increased activity of the endothelin (ET-1) system, which participate in their increased vascular tone and may predispose them to atherosclerosis. This study investigated the contribution of increased ET-1 activity to the impaired endothelium-dependent vasodilator function of hypertensive patients. METHODS AND RESULTS: Forearm blood flow (FBF) responses to intraarterial infusion of acetylcholine (ACh; 7.5, 15, and 30 microg/min) and sodium nitroprusside (SNP; 0.8,1.6, and 3.2 microg/min) were assessed by strain-gauge plethysmography before and after nonselective blockade of ET(A) and ET(B) receptors by combined infusion of BQ-123 (ET(A) blocker; 100 nmol/min) and BQ-788 (ET(B) blocker; 50 nmol/min). During saline administration, the vasodilator response to ACh was significantly blunted in hypertensive patients compared with controls (P<0.001), whereas the vasodilator effect of SNP was not different between groups (P=0.74). Blockade of ET-1 receptors resulted in a significant increase in FBF from baseline in hypertensive patients (P<0.008) but not in controls (P=0.15). In hypertensive patients, a combined ET(A/B) blockade resulted in a significant potentiation of the vasodilator response to ACh compared with saline (P=0.01), whereas the response to SNP was unchanged (P=0.44). In contrast, the response to ACh was not significantly modified by ET-1 receptor antagonism in healthy subjects (P=0.14 compared with saline). CONCLUSIONS: These findings indicate that blockade of ET-1 receptors improves endothelium-dependent vasodilator function in hypertensive patients, thereby suggesting that an increased ET-1 activity may play a role in the pathophysiology of this abnormality.