Pro bone morphogenetic protein-4 (BMP-4) is initially cleaved at a consensus furin motif adjacent to the mature ligand domain (the S1 site), and this allows for subsequent cleavage at an upstream motif (the S2 site). Previous studies have shown that S2 cleavage regulates the activity and signaling range of mature BMP-4, but the mechanism by which this occurs is unknown. Here, we show that the pro-and mature domains of BMP-4 remain noncovalently associated after S1 cleavage, generating a complex that is targeted for rapid degradation. Degradation requires lysosomal and proteosomal function and is enhanced by interaction with heparin sulfate proteoglycans. Subsequent cleavage at the S2 site liberates mature BMP-4 from the prodomain, thereby stabilizing the protein. We also show that cleavage at the S2, but not the S1 site, is enhanced at reduced pH, consistent with the possibility that the two cleavages occur in distinct subcellular compartments. Based on these results, we propose a model for how cleavage at the upstream site regulates the activity and signaling range of mature BMP-4 after it has been released from the prodomain. INTRODUCTIONBone morphogenetic protein-4 (BMP-4) is a signaling molecule that acts as a morphogen to influence cell fate in a concentration-dependent manner. BMP-4 was originally identified as a protein that is capable of inducing ectopic bone formation, but more recent studies have shown that it plays many different roles during embryonic development and in adults (Hogan, 1996).BMP-4 function is essential for normal embryogenesis as illustrated by the fact that mice homozygous for a null allele of BMP-4 form little or no mesoderm and die near the time of gastrulation (Winnier et al., 1995). BMP-4 heterozygous mutant mice are viable but display a variety of birth defects, including reduced numbers of primordial germ cells, polydactyly, and kidney, eye, and craniofacial abnormalities (Dunn et al., 1997;Lawson et al., 1999;Miyazaki et al., 2000;Chang et al., 2001). These data indicate that control of BMP-4 gene dosage is essential for normal embryonic patterning.Excess BMP-4 activity also leads to birth defects. Mice mutant for the BMP antagonists gremlin, noggin, and/or chordin show early lethality and/or defects in the spinal cord, forebrain, somites, skeleton, and kidney (Brunet et al., 1998;McMahon et al., 1998;Gong et al., 1999;Khokha et al., 2003). In humans, mutations in the noggin gene are responsible for multiple synostoses syndrome, a genetic disease characterized by fusion of the joints (Gong et al., 1999), and abnormally high levels of BMP-4 protein are a key feature of fibrodysplasia ossificans progressiva, a crippling hereditary disorder in which ectopic bone forms throughout the body (Kaplan and Shore, 1998).The requirement for strict regulation of BMP-4 dosage is met by controlling BMP-4 activity at multiple levels. At the extracellular level, BMP-4 is regulated by binding proteins, such as chordin and noggin, that block activation of cell surface receptors, and by the prote...

PURPOSE Colony-stimulating factor-3 receptor ( CSF3R)-T618I is a recurrent activating mutation in chronic neutrophilic leukemia (CNL) and to a lesser extent in atypical chronic myeloid leukemia (aCML) resulting in constitutive JAK-STAT signaling. We sought to evaluate safety and efficacy of the JAK1/2 inhibitor ruxolitinib in patients with CNL and aCML, irrespective of CSF3R mutation status. METHODS We conducted a phase II study of ruxolitinib in 44 patients (21 CNL and 23 aCML). The primary end point was overall hematologic response rate (ORR) by the end of 6 continuous 28-day cycles for the first 25 patients enrolled. We considered a response as either partial (PR) or complete response (CR). We expanded accrual to 44 patients to increase our ability to evaluate secondary end points, including grade ≥ 3 adverse events, spleen volume, symptom assessment, genetic correlates of response, and 2-year survival. RESULTS ORR was 32% for the first 25 enrolled patients (8 PR [7 CNL and 1 aCML]). In the larger cohort of 44 patients, 35% had a response (11 PR [9 CNL and 2 aCML] and 4 CR [CNL]), and 50% had oncogenic CSF3R mutations. The mean absolute allele burden reduction of CSF3R-T618I after 6 cycles was greatest in the CR group, compared with the PR and no response groups. The most common cause of death is due to disease progression. Grade ≥ 3 anemia and thrombocytopenia were observed in 34% and 14% of patients, respectively. No serious adverse events attributed to ruxolitinib were observed. CONCLUSION Ruxolitinib was well tolerated and demonstrated an estimated response rate of 32%. Patients with a diagnosis of CNL and/or harboring CSF3R-T618I were most likely to respond.

Patient-subjects reported that they can, at least partially, control the benefits they receive from participating in an early-phase oncology trial. This sense of control may underlie unrealistic optimism about benefiting personally from trial participation. Effective interventions to counteract unrealistic optimism may need to address the psychological factors that give rise to distorted risk/benefit processing.

Transfer of human chromosome 11, which contains the myoD locus, from primary fibroblasts into 10T1/2 cells results in activation of myoD. In contrast, hybrids that retain human chromosome 11 and additional human chromosomes fail to activate myoD. We show that human chromosome 4 inhibits myoD activation. myoD enhancer/promoter reporter constructs show that repression is at the transcriptional level. Chromosome fragment-containing hybrids localize the repressing activity to the region of 4p that contains the homeobox gene MSX1. MSX1 is expressed in primary human fibroblasts and in 10T1/2 cells containing human chromosome 4, while parental 10T1/2 cells do not express Msx1. Forced expression of Msx1 represses myoD enhancer activity. Msx1 protein binds to the myoD enhancer and likely represses myoD transcription directly. Antisense MSX1 relieves repression mediated by chromosome 4. We conclude that MSX1 inhibits transcription of myoD and that myoD is a target for homeobox gene regulation.

The human cytomegalovirus (CMV) virion glycoprotein gpUL4 (gp48) gene expresses a transcript that contains three AUG codons upstream from the one used to initiate synthesis of the gp48 protein. Previously we reported that the second of these AUG codons, AUG2, was necessary but insufficient for inhibition of downstream translation (M. Schleiss, C. R. Degnin, and A. P. Geballe, J. Virol. 65:6782-6789, 1991). We now demonstrate that the coding information of the upstream open reading frame initiated by AUG2 (uORF2) is critical for the inhibitory signal. Several missense mutations, particularly those involving the carboxy-terminal codons of uORF2, inactivate the inhibitory signal, while mutations that preserve the coding content of uORF2 uniformly retain the inhibitory signal. The uORF2 termination codon is essential for inhibition, but leader sequences further downstream are not critical. Conservation of uORF2 among clinical strains of CMV suggests that uORF2 provides an important function in the CMV infectious cycle. Although these results indicate that the peptide product of uORF2 mediates the inhibitory effect, we demonstrate that the uORF2 signal acts only in cis, and we propose a model of inhibition by the gp48 uORF2 signal.