Coordinate regulation of residual bone marrow function by paracrine trafficking of AML exosomes.

We recently demonstrated that acute myeloid leukemia (AML) cell lines and patient-derived blasts release exosomes that carry RNA and protein; following an in vitro transfer, AML exosomes produce proangiogenic changes in bystander cells. We reasoned that paracrine exosome trafficking may have a broader role in shaping the leukemic niche. In a series of in vitro studies and murine xenografts, we demonstrate that AML exosomes downregulate critical retention factors (Scf, Cxcl12) in stromal cells, leading to hematopoietic stem and progenitor cell (HSPC) mobilization from the bone marrow. Exosome trafficking also regulates HSPC directly, and we demonstrate declining clonogenicity, loss of CXCR4 and c-Kit expression, and the consistent repression of several hematopoietic transcription factors, including c-Myb, Cebp-ß and Hoxa-9. Additional experiments using a model of extramedullary AML or direct intrafemoral injection of purified exosomes reveal that the erosion of HSPC function can occur independent of direct cell-cell contact with leukemia cells. Finally, using a novel multiplex proteomics technique, we identified candidate pathways involved in the direct exosome-mediated modulation of HSPC function. In aggregate, this work suggests that AML exosomes participate in the suppression of residual hematopoietic function that precedes widespread leukemic invasion of the bone marrow directly and indirectly via stromal components.

Stent-grafts for de novo TIPS: technique and early results.

PURPOSE: To evaluate the potential benefits of placing a polytetrafluoroethylene (PTFE)-covered stent-graft during initial creation of a transjugular intrahepatic portosystemic shunt (TIPS) in clinical practice. MATERIALS AND METHODS: De novo TIPS were created with a PTFE stent-graft in four male and four female patients with symptomatic portal hypertension awaiting liver transplant. Their ages ranged from 35 to 62 (mean, 47) years. Patients were followed with TIPS ultrasound (US) and/or venography until liver transplantation or death; one remains under active study. Six recovered specimens underwent gross and microscopic evaluation. RESULTS: All TIPS placements were successful. Six shunts were primarily patent, with a mean patency of 289 days, through completion of the study. Five were found to be patent at transplant and one was found to be patent at autopsy. Explant evaluation revealed a smooth, thin layer of neointima and exclusion of biliary secretions. Three patients developed a total of four stenoses (one tandem lesion) during follow-up, leading to revision in two patients. Mean primary and total patency in these patients was achieved after 279 and 463 days, respectively. A previously occult moderate stenosis was detected after explant in another patient. Only one (nonsignificant) stenosis clearly developed in an area covered by PTFE. CONCLUSION: Placement of a de novo PTFE stent-graft during TIPS creation is feasible and may extend primary shunt patency. Appropriate positioning of the stent-graft is critical.

Modulatory function of CREB.CREM alpha heterodimers depends upon CREM alpha phosphorylation.

The cAMP-responsive element (CRE) modulator protein CREM alpha has been proposed to be a negative regulator of the CRE-binding protein (CREB). Precisely how CREM alpha inhibits CREB function is unclear, however. CREM alpha and CREB have highly related structures, and both proteins bind to consensus CRE sequences with similar affinities. Furthermore, both proteins can be phosphorylated by cAMP-dependent protein kinase A (PKA). Two models have been proposed to explain how CREM alpha could prevent the activation of genes by PKA-phosphorylated CREB: inhibitory CREM alpha homodimers could prevent occupancy of the CRE by CREB, or CREM alpha could block gene activation by forming non-functional CREB.CREM alpha heterodimers. To determine whether CREB-CREM alpha heterodimers are indeed non-functional, we engineered the leucine zipper regions of the two proteins to direct the pattern of dimerization. We then tested the biological activities of the phosphorylated and nonphosphorylated complexes in in vivo transcription assays. Our results indicate that CREM alpha can contribute to PKA-mediated gene activation when selectively heterodimerized with CREB. Furthermore, this transcriptional activity depends upon the ability of the complexes to be phosphorylated by PKA.

Engineered leucine zippers show that hemiphosphorylated CREB complexes are transcriptionally active.

The ability of basic/leucine zipper transcription factors to form homo- and heterodimers potentially increases the diversity of signaling pathways that can impinge upon a single genetic element. The capacity of these proteins to dimerize in various combinations complicates the analysis of their functional properties, however. To simplify the functional analysis of CREB dimers, we mutated selected residues within the leucine zipper region to generate proteins that could only heterodimerize. These mutants allowed us to determine whether phosphorylation of both CREB subunits was necessary for transcriptional activation. Our results reveal that hemiphosphorylated CREB dimers are half as active as fully phosphorylated dimers. It is possible, therefore, that the degree of phosphorylation of CREB complexes could modulate the transcriptional responses of specific genes to cAMP.

The cAMP-regulated enhancer-binding protein ATF-1 activates transcription in response to cAMP-dependent protein kinase A.

Many promoters respond transcriptionally to elevated levels of cAMP through the cAMP-responsive enhancer (CRE). Several proteins have been characterized which bind to the CRE and presumably modulate CRE-dependent transcription. Of these CRE-binding proteins, only CREB has been shown to be activated by cAMP-dependent protein kinase A (PKA), and as such, CREB represents the only basis for our understanding of cAMP-regulated transcriptional activity. In this report, we describe the complete cDNA sequence of another CRE-binding protein, ATF-1. This protein contains a consensus phosphorylation site for PKA and shares extensive homology with CREB in the region surrounding and carboxyl-terminal to the PKA site. ATF-1 does not contain sequences homologous to the glutamine-rich amino-terminal domain found in CREB, however. ATF-1, like CREB, is expressed in a wide variety of cell types, and ATF-1 is capable of dimerizing with CREB. Both ATF-1 homodimers and ATF-1/CREB heterodimers bind to the CRE but not to the related phorbol ester response element. ATF-1 is as active as CREB in its ability to mediate the transcriptional effects of PKA, and, because ATF-1 has a smaller effect on basal expression, it is actually more responsive than CREB to cAMP. These findings indicate that CREB is not unique in its ability to mediate cAMP-dependent transcriptional regulation.