Efficacy of intermittent combined RAF and MEK inhibition in a patient with concurrent BRAF- and NRAS-mutant malignancies.

Vemurafenib, a RAF inhibitor, extends survival in patients with BRAF(V600)-mutant melanoma but activates extracellular signal-regulated kinase (ERK) signaling in RAS-mutant cells. In a patient with a BRAF(V600K)-mutant melanoma responding to vemurafenib, we observed accelerated progression of a previously unrecognized NRAS-mutant leukemia. We hypothesized that combining vemurafenib with a MAP-ERK kinase (MEK) inhibitor would inhibit ERK activation in the melanoma and prevent ERK activation by vemurafenib in the leukemia, and thus suppress both malignancies. We demonstrate that intermittent administration of vemurafenib led to a near-complete remission of the melanoma, and the addition of the MEK inhibitor cobimetinib (GDC-0973) caused suppression of vemurafenib-induced leukemic proliferation and ERK activation. Antimelanoma and antileukemia responses have been maintained for nearly 20 months, as documented by serial measurements of tumor-derived DNA in plasma in addition to conventional radiographic and clinical assessments of response. These data support testing of intermittent ERK pathway inhibition in the therapy for both RAS-mutant leukemia and BRAF-mutant melanoma.

Multi-tier regulation of the streptomycete morphogenetic peptide SapB.

Streptomyces coelicolor is a morphologically complex bacterium requiring the secretion of surface-active proteins to progress through its life cycle. SapB represents an important class of these biosurfactants, as illustrated by its ability to restore aerial hyphae formation when applied exogenously to developmental mutants. However, such aerial hyphae fail to sporulate, exemplifying the need to co-ordinate the timing of SapB production with other developmental events. SapB has an unusual lantibiotic structure. Its structural gene, ramS, is only 38 nucleotides downstream of the gene encoding its putative modification enzyme, RamC. Transient, co-ordinated expression of the operon was thought to be controlled by the response regulator RamR. However, we show that ramS is transcribed throughout the cell cycle with a dual expression profile dissimilar to the tightly controlled ramC expression. Surprisingly, post-translational modification relies on prior membrane localization of the precursor peptide, RamS, as demonstrated by the absence of RamS modification in S. coelicolor hyphae treated with the Bacillus subtilis lipoprotein surfactin. Our results demonstrate that interspecies interaction can also be mediated by interference of post-translational events. Further, temporal and spatial regulation of irreversible post-translational modification of a surface-active morphogenetic peptide suggests a new model for the control of key developmental events.

Signal perception by FNR: the role of the iron-sulfur cluster.

The metabolic flexibility of bacteria is key to their ability to survive and thrive in a wide range of environments. Optimal switching from one metabolic pathway to another is a key requirement for this flexibility. Respiration is a good example: many bacteria utilize O(2) as the terminal electron acceptor, but can switch to a range of other acceptors, such as nitrate, when O(2) becomes limiting. Sensing environmental levels of O(2) is the key step in switching from aerobic to anaerobic respiration. In Escherichia coli, the fumarate and nitrate reduction transcriptional regulator (FNR) controls this switch. Under O(2)-limiting conditions, FNR binds a [4Fe-4S](2+) cluster, generating a transcriptionally active dimeric form. Exposure to O(2) results in conversion of the cluster into a [2Fe-2S](2+) form, leading to dissociation of the protein into inactive monomers. The mechanism of cluster conversion, together with the nature of the reaction products, is of considerable current interest, and a near-complete description of the process has now emerged. The [4Fe-4S](2+) into [2Fe-2S](2+) cluster conversion proceeds via a two-step mechanism. In step 1, a one-electron oxidation of the cluster takes place, resulting in the release of a Fe(2+) ion, the formation of an intermediate [3Fe-4S](1+) cluster, together with the generation of a superoxide anion. In step 2, the intermediate [3Fe-4S](1+) cluster rearranges spontaneously to form the [2Fe-2S](2+) cluster, releasing two sulfide ions and an Fe(3+) ion in the process. The one-electron activation of the cluster, coupled to catalytic recycling of the superoxide anion back to oxygen via superoxide dismutase and catalase, provides a novel means of amplifying the sensitivity of [4Fe-4S](2+) FNR to its signal molecule.

Influence of the environment on the [4Fe-4S]2+ to [2Fe-2S]2+ cluster switch in the transcriptional regulator FNR.

In Escherichia coli, the switch between aerobic and anaerobic metabolism is primarily controlled by the fumarate and nitrate reduction transcriptional regulator FNR. In the absence of O2, FNR binds a [4Fe-4S]2+ cluster, generating a transcriptionally active dimeric form. Exposure to O2 results in the conversion of the cluster to a [2Fe-2S]2+ form, leading to dissociation of the protein into transcriptionally inactive monomers. The [4Fe-4S]2+ to [2Fe-2S]2+ cluster conversion proceeds in two steps. Step 1 involves the one-electron oxidation of the cluster, resulting in the release of Fe2+, generating a [3Fe-4S]1+ cluster intermediate, and a superoxide ion. In step 2, the cluster intermediate spontaneously rearranges to form the [2Fe-2S]2+ cluster, with the release of a Fe3+ ion and two sulfide ions. Here, we demonstrate that, in both native and reconstituted [4Fe-4S] FNR, the reaction environment and, in particular, the presence of Fe2+ and/or Fe3+ chelators can influence significantly the cluster conversion reaction. We demonstrate that while the rate of step 1 is largely insensitive to chelators, that of step 2 is significantly enhanced by both Fe2+ and Fe3+ chelators. We show that, for reactions in Fe3+-coordinating phosphate buffer, step 2 is enhanced to the extent that step 1 becomes the rate determining step and the [3Fe-4S]1+ intermediate is no longer detectable. Furthermore, Fe3+ released during this step is susceptible to reduction in the presence of Fe2+ chelators. This work, which may have significance for the in vivo FNR cluster conversion reaction in the cell cytoplasm, provides an explanation for apparently contradictory results reported from different laboratories.

RsmA is an anti-sigma factor that modulates its activity through a [2Fe-2S] cluster cofactor.

The rsmA gene of Streptomyces coelicolor lies directly upstream of the gene encoding the group 3 sigma factor sigma(M). The RsmA protein is a putative member of the HATPase_c family of anti-sigma factors but is unusual in that it contains seven cysteine residues. Bacterial two-hybrid studies demonstrate that it interacts specifically with sigma(M), and in vitro studies of the purified proteins by native PAGE and transcription assays confirmed that they form a complex. Characterization of RsmA revealed that it binds ATP and that, as isolated, it contains significant quantities of iron and inorganic sulfide, in equal proportion, with spectroscopic properties characteristic of a [2Fe-2S] cluster-containing protein. Importantly, the interaction between RsmA and sigma(M) is dependent on the presence of the iron-sulfur cluster. We propose a model in which RsmA regulates the activity of sigma(M). Loss of the cluster, in response to an as yet unidentified signal, activates sigma(M) by abolishing its interaction with the anti-sigma factor. This represents a major extension of the functional diversity of iron-sulfur cluster proteins.