We recently reported that chronic lympho-cytic leukemia (CLL) cells synthesize and release vascular endothelial growth factor (VEGF) under normoxic and hypoxic conditions. CLL B cells also express VEGF membrane receptors (VEGF-R1 and VEGF-R2), suggesting that they use VEGF as a survival factor. To assess the mechanism of apoptosis resistance related to VEGF, we determined the impact of VEGF on CLL B cells, and we studied the impact of epigallocatechin-3-gallate (EGCG), a known receptor tyrosine kinase (RTK) inhibitor, on VEGF receptor status and viability of CLL B cells. VEGF 165 significantly increased apoptotic resistance of CLL B cells, and immunoblotting revealed that VEGF-R1 and VEGF-R2 are spontaneously phosphorylated on CLL B cells. EGCG significantly increased apo-ptosis/cell death in 8 of 10 CLL samples measured by annexin V/propidium iodide (PI) staining. The increase in annexin V/PI staining was accompanied by caspase-3 activation and poly-adenosine diphos-phate ribose polymerase (PARP) cleav-age at low concentrations of EGCG (3 g/mL). Moreover, EGCG suppressed the proteins B-cell leukemia/lymphoma-2 protein (Bcl-2), X-linked inhibitor of apopto-sis protein (XIAP), and myeloid cell leuke-mia-1 (Mcl-1) in CLL B cells. Finally, EGCG (3-25 g/mL) suppressed VEGF-R1 and VEGF-R2 phosphorylation, albeit incompletely. Thus, these results suggest that VEGF signaling regulates survival signals in CLL cells and that interruption of this autocrine pathway results in caspase activation and subsequent leukemic cell death. (Blood. 2004;104:788-794)

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The prevalence of germline pathogenic mutations in a comprehensive panel of cancer predisposition genes is not well defined for patients with pancreatic ductal adenocarcinoma (PDAC). To estimate the frequency of mutations in a panel of 22 cancer predisposition genes, 96 patients unselected for a family history of cancer who were recruited to the Mayo Clinic Pancreatic Cancer patient registry over a 12 month period were screened by next-generation sequencing. Fourteen pathogenic mutations in 13 patients (13.5%) were identified in eight genes: four in ATM, two in BRCA2, CHEK2, and MSH6, and one in BARD1, BRCA1, FANCM, and NBN. These included nine mutations (9.4%) in established pancreatic cancer genes. Three mutations were found in patients with a first degree relative with PDAC, and 10 mutations were found in patients with first or second-degree relatives with breast, pancreas, colorectal, ovarian, or endometrial cancer. These results suggest that a substantial proportion of patients with PDAC carry germline mutations in predisposition genes associated with other cancers, and that a better understanding of pancreatic cancer risk will depend on evaluation of families with broad constellations of tumors. These findings highlight the need for recommendations governing germline gene-panel testing of pancreatic cancer patients.

The molecular mechanism of autocrine regulation of vascular endothelial growth factor (VEGF) in chronic lymphocytic leukemia (CLL) B cells is unknown. Here, we report that CLL B cells express constitutive levels of HIF-1␣ under normoxia. We have examined the status of the von Hippel-Lindau gene product (pVHL) that is responsible for HIF-1␣ degradation and found it to be at a notably low level in CLL B cells compared with normal B cells. We demonstrate that the microRNA, miR-92-1, overexpressed in CLL B cells, can target the VHL transcript to repress its expression. We found that the stabilized HIF-1␣ can form an active complex with the transcriptional coactivator p300 and phosphorylated-STAT3 at the VEGF promoter and recruit RNA polymerase II. This is initial evidence that pVHL, without any genetic alteration, can be regulated by microRNA and explains the aberrant autocrine VEGF secretion in CLL. (Blood. 2009; 113:5568-5574) Introduction B-cell chronic lymphocytic leukemia (CLL) continues to be a more common leukemia with no obvious curative approaches. 1,2 Elevated plasma vascular endothelial growth factor (VEGF) levels in CLL are associated with advancing disease, even in early-stage disease. 3 CLL B cells have high endogenous levels of mRNA for VEGF and are able to spontaneously secrete VEGF. 4,5 The tissue neovascularization has been shown to be elevated in the marrow 6 and lymph nodes 5 of patients with CLL, possibly related to the increased VEGF levels. Furthermore, the ability of VEGF to alter CLL B-cell apoptosis resistance is linked to the expression of VEGF receptors VEGF-R1, -R2, and neuropilin receptor-1, found on CLL B cells. 7,8 Therefore, the advantage of understanding these pathways is that interference with critical components of these receptor-ligand/receptor-mediated pathways should induce CLL B-cell death or apoptosis. 9 We believe the VEGF-based autocrine system that leads to increased apoptosis resistance in CLL B cells 7 is an important pathway, as it lends itself to therapeutic exploitation given the increasing repertoire of anti-VEGF agents 10 ; however, to date, the molecular mechanism of autocrine regulation of VEGF in CLL B cells is unknown.In this study, we describe a plausible mechanism for activation of the VEGF-based autocrine pathway in CLL B cells. We found that HIF-1␣, a known key transcription factor for VEGF, 11,12 is overexpressed in leukemic CLL B cells under normoxia primarily because of notably reduced levels of von Hippel-Lindau (pVHL) protein without having any genetic alterations of the VHL gene. Importantly, HIF-1␣ then is able to form an active complex with the transcriptional coactivator p300 and phosphorylated-STAT3 as the VEGF promoter, and recruit RNA polymerase II. Finally, we present initial evidence that the microRNA, miR-92-1 (formerly known as miR-92a) known to be elevated in CLL B cells, 13 is able to target the VHL transcript and repress translation. We believe this novel activation/regulation of the HIF-1␣/VEGF axis in CLL B cells explains spontaneous VEG...

A novel bone biopsy technique was used to generate a robust stromal cell system to study how stroma modulates CLL B-cell apoptosis and how the leukemic cell-stromal interaction influences secretion of vascular factors. Marrow stromal elements (MSE) rescued CLL B-cells from both spontaneous and drug induced apoptosis, partly due to soluble factors. When CLL B-cells were added to the MSE cultures, a dramatic increase in the secretion of basic fibroblast growth factor and decrease in the secretion of thrombospondin was observed. These results indicate the interaction between CLL Bcells and marrow stromal elements regulates angiogenic switching and may be linked to disease progression.

Purpose: Flavopiridol, a cyclin-dependent kinase inhibitor, transcription inhibitor, and DNA-interacting agent, was combined with cisplatin or carboplatin to establish toxicities, evaluate pharmacokinetics, and examine its effects on patient cancers and levels of selected polypeptides in patient peripheral blood mononuclear cells (PBMC). Experimental Design: Therapy was given every 3 weeks. Stage I: cisplatin was fixed at 30 mg/m 2 with escalating flavopiridol. Stage II: flavopiridol was fixed at the stage I maximum tolerated dose (MTD) with escalation of cisplatin. Stage III: flavopiridol was fixed at the stage I MTD with escalation of carboplatin. Results: Thirty-nine patients were treated with 136 cycles of chemotherapy. Neutropenia was seen in only 11% of patients. Grade 3 flavopiridol/CDDP toxicities were nausea (30%), vomiting (19%), diarrhea (15%), dehydration (15%), and neutropenia (10%). Flavopiridol combined with carboplatin resulted in unexpectedly high toxicities and one treatment-related death. Stable disease (>3 months) was seen in 34% of treated patients, but there were no objective responses. The stage II MTD was 60 mg/m 2 cisplatin and 100 mg/m 2 /24 hours flavopiridol. As given, CDDP did not alter flavopiridol pharmacokinetics. Flavopiridol induced increased p53 and pSTAT3 levels in patient PBMCs but had no effects on cyclin D1, phosphoRNA polymerase II, or Mcl-1. Conclusions: Flavopiridol and cisplatin can be safely combined in the treatment of cancer patients. Unexpected toxicity in flavopiridol/carboplatin-treated patients attenuates enthusiasm for this alternative combination. Analysis of polypeptide levels in patient PBMCs suggests that flavopiridol may be affecting some, but not all, of its known in vitro molecular targets in vivo.

Boesenbergia rotunda is a herb from the Boesenbergia genera under the Zingiberaceae family. B. rotunda is widely found in Asian countries where it is commonly used as a food ingredient and in ethnomedicinal preparations. The popularity of its ethnomedicinal usage has drawn the attention of scientists worldwide to further investigate its medicinal properties. Advancement in drug design and discovery research has led to the development of synthetic drugs from B. rotunda metabolites via bioinformatics and medicinal chemistry studies. Furthermore, with the advent of genomics, transcriptomics, proteomics, and metabolomics, new insights on the biosynthetic pathways of B. rotunda metabolites can be elucidated, enabling researchers to predict the potential bioactive compounds responsible for the medicinal properties of the plant. The vast biological activities exhibited by the compounds obtained from B. rotunda warrant further investigation through studies such as drug discovery, polypharmacology, and drug delivery using nanotechnology.

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