Citations

Abstract Introduction:The histone deacetylase inhibitor (HDACi) Panobinostat (LBH-589, Novartis Pharmaceutical Corp) belongs to a new class of chemotherapeutics with demonstrated anticancer activities in laboratory studies and within the clinic. Using pre-clinical models of leukemia and lymphoma we have demonstrated that HDACi mediate their therapeutic effects via the intrinsic apoptosis pathway. Here we investigated the ability of Panobinostat to induce apoptosis in human multiple myeloma (MM) cell lines, alone and in combination with agents that target pro-survival Bcl-2 proteins (ABT-737) and the extrinsic apoptosis pathway (rhTRAIL). Moreover, we have used the Vk*myc murine model of MM to demonstrate the therapeutic efficacy of Panobinostat and its ability to induce apoptosis in MM cells in vivo. Methods: JJN3, OPM-2, RPMI-8226 and U266 MM cell lines were incubated with Panobinostat, ABT-737 or rhTRAIL alone or in combination. Cell viability and cell cycle were assessed using flow cytometry. Histone acetylation was assessed by western blot. C57BL/6 mice (n = 32) bearing transplanted Vk*myc MM (2 months post-transplant) were treated with Panobinostat (25mg/kg, 4 days; 15mg/kg 5 days/week, 3 weeks) or vehicle (D5W). Serum paraprotein levels were assessed weekly in all mice. Mice were sacrificed at various time points to measure apoptotic and therapeutic effects. Results: Treatment of MM cell lines with Panobinostat resulted in time- and dose-dependent induction of apoptosis associated with G1 phase arrest and histone H3/H4 acetylation. Combinations of Panobinostat with ABT-737 or rhTRAIL synergised to mediate robust and rapid apoptosis in the human MM cells. Serum paraprotein in mice bearing Vk*myc MM cells was significantly reduced after 4 days of high dose (25mg/ml) Panobinostat compared with vehicle treated mice (p<0.01) and remained at this reduced level for the treatment period (≤60% reduction compared with vehicle treated mice). This was associated with a significant induction of apoptosis and a reduction in CD-138+ plasma cells within the bone marrow. Removal of treatment was associated with increased paraprotein levels, progression of extramedullary disease and death endpoints. Overall, Panobinostat induced a significant survival advantage compared with vehicle (median=36.5 vs. 16 days, p<0.01). Conclusions: These results suggest that the HDACi Panobinostat has single agent activity against MM cell lines in vitro and provides a significant therapeutic benefit to mice bearing syngeneic MM tumors. Moreover, combination therapies consisting of HDAC inhibitors with agents that target pro-survival Bcl-2 proteins or the TRAIL apoptosis pathway may provide a rational approach to the treatment of MM. Future studies will investigate the molecular events underpinning the effects mediated by Panobinostat, ABT-737 and TRAIL in the Vk*myc MM model. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B176.

Abstract It is now widely accepted that a spectrum of somatic alterations occur in human cancer and that particular alterations are associated with response or lack of response to anti-cancer therapies. Here, we undertook a systematic correlation analysis of somatic mutations, copy number aberrations and drug response across 300+ human cancer cell lines. We studied the RAF and VEGFR inhibitor, sorafenib, evaluating associations between sorafenib response and somatic mutations in 35 common cancer genes and high-level amplifications and homozygous deletions in more than 100 minimal common regions of gain and loss, which include many known oncogenes and tumor suppressor genes. Notably, we found that NRAS mutations, among other alterations, were significantly associated with sensitivity to sorafenib (odds ratio = 7, p-value = 0.003), whereas activating BRAF mutations were strongly associated with a lack of response (odds ratio = 4.9, p-value = 0.012). Specifically, 64% (7 of 11) of NRAS mutant cell lines were sensitive to sorafenib, while only 20% (34 of 170) of NRAS wild-type lines were sensitive. On the contrary, just 6.7% (2 of 30) of BRAF mutant lines were sensitive to sorafenib. Because NRAS mutations occur frequently in leukemia and no leukemia cell lines were included in the original analysis, we selected 5 NRAS mutant and 5 NRAS wild-type leukemia cell lines for further evaluation. Consistent with our previous observation, the four most sensitive leukemia cell lines were all NRAS mutant, each line exhibiting greater than 50% growth inhibition after 72 hours of exposure to 4uM sorafenib. These results confirm the original association and suggest that across both solid tumors and hematologic malignancies, NRAS mutations may identify a subset of cancers that are likely to respond sorafenib. In addition, this work demonstrates that systematic analysis of somatic alterations and drug response across a large collection of cancer cell lines is capable of discovering novel predictive biomarkers. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A47.

Abstract Background: Ingested nitrate can form N-nitroso compounds endogenously, which may lead to DNA adducts and activating mutations in oncogenes. However, previous studies have reported null or inverse associations between dietary nitrate intake and cancer risk, while nitrate intake from drinking water has shown null or positive associations with cancer risk. These associations may be confounded by protective effects of dietary folate, which provides methyl groups for nucleotide synthesis, and thus plays a vital role in the DNA repair system. In a small case-control study, an increased risk of breast cancer was observed among individuals with high dietary nitrate relative to folate intake. We therefore evaluated interaction of total folate with nitrate intake from diet and drinking water on breast cancer risk in the Iowa Women's Health Study (IWHS). Methods: The IWHS is a prospective cohort study of cancer among 41,836 women in Iowa, initiated in 1986. Dietary intake was assessed at study baseline using a food frequency questionnaire (FFQ). Nitrate intake from foods and total folate intake from foods and dietary supplements were computed using consumption frequencies collected by the FFQ. Nitrate intake from drinking water was assessed using a historical analytical database on Iowa municipal water supplies in 15,151 women using municipal water supply. We evaluated the interaction of nitrate and total folate intake with breast cancer risk using Cox proportional hazard regression modeling, adjusting for potential confounders (age, body mass index, waist-hip-ratio, education, smoking, family history of breast cancer, age at menopause, age at first live birth, estrogen use, total energy intake, and intake of vitamin C and E and flavonoids). Results: After baseline exclusions (history of cancer at baseline, previous mastectomy or partial breast resection, implausible energy intake, or ≥30 missing responses on the FFQ), there were 34,388 postmenopausal women in the analytic cohort. Through 2008, 2,875 incident breast cancer cases were identified. Nitrate intake from diet or drinking water was not associated with breast cancer risk. After stratification by total folate intake, high dietary nitrate intake (>106.1 mg/day) was associated with a marginally decreased risk of breast cancer compared to those with low nitrate intake (≤106.1 mg/day) (HR=0.90, 95% confidence interval=0.81–1.00) only among women with folate intake <400 μg/day (p for interaction = 0.65). There was no interaction of nitrate intake from drinking water and total folate intake on breast cancer risk. Conclusions: Our findings do not support the previously reported increased risk of breast cancer risk among individuals with high dietary nitrate and low folate intake. The inverse association between nitrate intake and breast cancer risk observed among women with low folate intake in the present study may be attributed to residual confounding due to high correlation among intakes of nitrate, folate and antioxidants. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A86.

AbstractCellular and interpatient heterogeneity and the involvement of different stem and progenitor compartments in leukemogenesis are challenges for the identification of common pathways contributing to the initiation and maintenance of acute myeloid leukemia (AML). Here we used a strategy of parallel transcriptional analysis of phenotypic long-term hematopoietic stem cells (HSCs), short-term HSCs, and granulocyte-monocyte progenitors from individuals with high-risk (−7/7q−) AML and compared them with the corresponding cell populations from healthy controls. This analysis revealed dysregulated expression of 11 genes, including IL-1 receptor accessory protein (IL1RAP), in all leukemic stem and progenitor cell compartments. IL1RAP protein was found to be overexpressed on the surface of HSCs of AML patients, and marked cells with the −7/7q− anomaly. IL1RAP was also overexpressed on HSCs of patients with normal karyotype AML and high-risk myelodysplastic syndrome, suggesting a pervasive role in different disease subtypes. High IL1RAP expression was independently associated with poor overall survival in 3 independent cohorts of AML patients (P = 2.2 × 10−7). Knockdown of IL1RAP decreased clonogenicity and increased cell death of AML cells. Our study identified genes dysregulated in stem and progenitor cells in −7/7q− AML, and suggests that IL1RAP may be a promising therapeutic and prognostic target in AML and high-risk myelodysplastic syndrome.

OBJECTIVE To describe trends of primary efficacy and safety outcomes of islet transplantation in type 1 diabetes recipients with severe hypoglycemia from the Collaborative Islet Transplant Registry (CITR) from 1999 to 2010. RESEARCH DESIGN AND METHODS A total of 677 islet transplant-alone or islet-after-kidney recipients with type 1 diabetes in the CITR were analyzed for five primary efficacy outcomes and overall safety to identify any differences by early (1999–2002), mid (2003–2006), or recent (2007–2010) transplant era based on annual follow-up to 5 years. RESULTS Insulin independence at 3 years after transplant improved from 27% in the early era (1999–2002, n = 214) to 37% in the mid (2003–2006, n = 255) and to 44% in the most recent era (2007–2010, n = 208; P = 0.006 for years-by-era; P = 0.01 for era alone). C-peptide ≥0.3 ng/mL, indicative of islet graft function, was retained longer in the most recent era (P < 0.001). Reduction of HbA1c and resolution of severe hypoglycemia exhibited enduring long-term effects. Fasting blood glucose stabilization also showed improvements in the most recent era. There were also modest reductions in the occurrence of adverse events. The islet reinfusion rate was lower: 48% by 1 year in 2007–2010 vs. 60–65% in 1999–2006 (P < 0.01). Recipients that ever achieved insulin-independence experienced longer duration of islet graft function (P < 0.001). CONCLUSIONS The CITR shows improvement in primary efficacy and safety outcomes of islet transplantation in recipients who received transplants in 2007–2010 compared with those in 1999–2006, with fewer islet infusions and adverse events per recipient.

Abstract We performed a meta-analysis of 3 genome-wide association studies to identify additional common variants influencing chronic lymphocytic leukemia (CLL) risk. The discovery phase was composed of genome-wide association study data from 1121 cases and 3745 controls. Replication analysis was performed in 861 cases and 2033 controls. We identified a novel CLL risk locus at 6p21.33 (rs210142; intronic to the BAK1 gene, BCL2 antagonist killer 1; P = 9.47 × 10−16). A strong relationship between risk genotype and reduced BAK1 expression was shown in lymphoblastoid cell lines. This finding provides additional support for polygenic inheritance to CLL and provides further insight into the biologic basis of disease development.

Oncolytic reovirus is carried by cells to tumors and protected from neutralizing antibodies in the circulation.

AbstractWe tested the effects of overexpression of the endothelial nitric oxide synthase (eNOS) gene in the normal arterial wall by adenoviral-mediated gene transfer. Rabbit carotid arteries were surgically isolated and exposed to adenoviral vectors encoding eNOS (AdeNOS) or β-galactosidase (AdβGal) on the contralateral side. Vector solutions at a concentration of 1×1010plaque forming units/mL were instilled for 20 minutes before restoration of flow. Arteries were harvested 4 days later for immunostaining, measurement of cGMP, and vasomotor studies. Endothelium-specific gene transfer was confirmed by staining for β-galactosidase in the AdβGal arteries. Immunostaining of en face endothelial cell imprints from AdeNOS-transduced arteries with a monoclonal antibody to eNOS showed increased immunoreactivity. Basal cGMP levels were significantly greater in the AdeNOS-transduced arteries (18.4±4.6 versus 4.2±0.5 pmol/mg protein;P<.05). Contractions to phenylephrine were significantly reduced in the AdeNOS-transduced arteries (area under curve, 106±5 versus 119±7;P<.05), but in the presence of the eNOS inhibitor,NG-monomethyl-l-arginine (L-NMMA, 3×10−4mol/L), there was no difference between the two (area under curve, 148±5 versus 153±6;P=NS). Relaxations to acetylcholine obtained during submaximal contractions to phenylephrine were significantly enhanced in the AdeNOS-transduced arteries (EC50, 7.45±0.05 versus 7.23±0.03;P<.05). We conclude that overexpression of eNOS in the endothelium results in diminished contractile responses, as well as enhanced endothelium-dependent relaxations. These findings imply a possible role for vascular eNOS gene transfer in the treatment of vasospasm and endothelial dysfunction.

Abstract—A defining property of ATP-sensitive K+(KATP) channels is inhibition by sulfonylurea drugs, yet the response of cardiac KATPchannels toward sulfonylureas during myocardial ischemia is not consistent. Altered channel sensitivity toward sulfonylureas has, in part, been ascribed to antagonism by cytosolic nucleotide diphosphates, although the mechanism of interaction remains unclear. Herein, in inside-out patches excised from cardiomyocytes, we observed a dual response of KATPchannels toward the sulfonylurea drug, glyburide, in the presence of cytosolic UDP. Specifically, glyburide failed to inhibit spontaneous KATPchannel activity in the presence of UDP but inhibited UDP-induced channel activity after rundown of spontaneous channel openings. Such behavior of KATPchannels cannot be explained by differences in the level of channel activity or by UDP-induced displacement of glyburide. Rather, the dual response toward the sulfonylurea could be attributed to a property of KATPchannels to switch between operative conditions (spontaneous versus UDP-induced) each associated with a distinct responsiveness toward ligands. Conversion of post-rundown KATPchannels to the spontaneously operative channel condition, by Mg-ATP, restored the ability of UDP to antagonize the inhibitory action of glyburide lost after rundown, suggesting that the response of the channel to glyburide is phosphorylation dependent. The existence of distinct operative conditions of cardiac KATPchannels could be the basis for the inconsistent response of the channel toward sulfonylurea drugs and should be considered when sulfonylureas are used to implicate the opening of KATPchannels in the myocardium.

Isolated cat lungs perfused at constant flow (75 ml/min per kg total body weight) were ventilated with gases of varying CO 2 content (0 to 10%), 20% O 2 , and the balance N 2 . Tidal volume was constant, and airway pressure did not change. Left atrial pressure was held constant, and changes in pulmonary artery pressure (PPA) were taken to reflect changes in pulmonary vascular resistance. Progressive hypercapnia (Pco 2 = 0 to 60 mm Hg) resulted in an increase in PPA as pH decreased. Equivalent degrees of acidosis, produced by the infusion of 0.3 N lactic acid, resulted in a higher PPA. Changes in PPA produced by lactic acid were reversible with 0.89 M sodium bicarbonate. With Pco 2 constant, and pH changed by lactic acid or sodium bicarbonate infusion, PPA was higher in lungs ventilated with room air (Pco 2 = 0) than in those ventilated with 5 or 10% CO 2 (Pco 2 = 30 or 60 mm Hg) at the same hydrogen ion concentration of the perfusate. These findings can be explained by two opposing actions of CO 2 on pulmonary vessels: a dilator action due to the direct effect of CO 2 and a constrictor action caused by the increase in hydrogen ion concentration.