BACKGROUND Bruton's tyrosine kinase (BTK) is a mediator of the B-cell–receptor signaling pathway implicated in the pathogenesis of B-cell cancers. In a phase 1 study, ibrutinib, a BTK inhibitor, showed antitumor activity in several types of non-Hodgkin's lymphoma, including mantle-cell lymphoma. METHODS In this phase 2 study, we investigated oral ibrutinib, at a daily dose of 560 mg, in 111 patients with relapsed or refractory mantle-cell lymphoma. Patients were enrolled into two groups: those who had previously received at least 2 cycles of bortezomib therapy and those who had received less than 2 complete cycles of bortezomib or had received no prior bortezomib therapy. The primary end point was the overall response rate. Secondary end points were duration of response, progression-free survival, overall survival, and safety. RESULTS The median age was 68 years, and 86% of patients had intermediate-risk or high-risk mantle-cell lymphoma according to clinical prognostic factors. Patients had received a median of three prior therapies. The most common treatment-related adverse events were mild or moderate diarrhea, fatigue, and nausea. Grade 3 or higher hematologic events were infrequent and included neutropenia (in 16% of patients), thrombocytopenia (in 11%), and anemia (in 10%). A response rate of 68% (75 patients) was observed, with a complete response rate of 21% and a partial response rate of 47%; prior treatment with bortezomib had no effect on the response rate. With an estimated median follow-up of 15.3 months, the estimated median response duration was 17.5 months (95% confidence interval [CI], 15.8 to not reached), the estimated median progression-free survival was 13.9 months (95% CI, 7.0 to not reached), and the median overall survival was not reached. The estimated rate of overall survival was 58% at 18 months. CONCLUSIONS Ibrutinib shows durable single-agent efficacy in relapsed or refractory mantle-cell lymphoma. (Funded by Pharmacyclics and others; ClinicalTrials.gov number, NCT01236391.)

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Transcription regulators of the MerR family respond to myriad stress signals to activate sigma70/sigmaA-targeted genes, which contain suboptimal 19-bp spacers between their -35 and -10 promoter elements. The crystal structure of a BmrR-TPP(+)-DNA complex provided initial insight into the transcription activation mechanism of the MerR family, which involves base pair distortion, DNA undertwisting and shortening of the spacer, and realignment of the -35 and -10 boxes. Here, we describe the crystal structure of MerR family member MtaN bound to the mta promoter. Although the global DNA binding modes of MtaN and BmrR differ somewhat, homologous protein-DNA interactions are maintained. Moreover, despite their different sequences, the mta promoter conformation is essentially identical to that of the BmrR-TPP(+)-bound bmr promoter, indicating that this DNA distortion mechanism is common to the entire MerR family. Interestingly, DNA binding experiments reveal that the identity of the two central bases of the mta and bmr promoters, which are conserved as either a thymidine or an adenine in nearly all MerR promoters, is not important for DNA affinity. Comparison of the free and DNA-bound MtaN structures reveals that a conformational hinge, centered at residues N-terminal to the ubiquitous coiled coil, is key for mta promoter binding. Analysis of the structures of BmrR, CueR, and ZntR indicates that this hinge may be common to all MerR family members.

Despite significant improvements in the signs and symptoms of myelofibrosis (MF), and possible prolongation of patients' survival, some have disease that is refractory to ruxolitinib and many lose their response over time. Furthermore, patients with ≥3 mutations are less likely to respond to ruxolitinib. Here we describe outcomes after ruxolitinib discontinuation in MF patients enrolled in a phase 1/2 study at our center. After a median follow-up of 79 months, 86 patients had discontinued ruxolitinib (30 of whom died while on therapy). The median follow-up after ruxolitinib discontinuation for the remaining 56 patients was 32 months, with median survival after discontinuation of 14 months. Platelets <260 × 10/L at the start of therapy or <100 × 10/L at the time of discontinuation were associated with shorter survival after discontinuation. Of 62 patients with molecular data at baseline and follow-up, 22 (35%) acquired a new mutation while receiving ruxolitinib (14 [61%] in ). Patients showing clonal evolution had significantly shorter survival after discontinuation (6 vs 16 months). Transfusion dependency was the only clinical variable associated with clonal evolution. These findings underscore the need for novel therapies and suggest that clonal evolution or decreasing platelet counts while on ruxolitinib therapy may be markers of poor prognosis.

The 2.15-Å resolution cocrystal structure of EcoRV endonuclease mutant T93A complexed with DNA and Ca 2؉ ions reveals two divalent metals bound in one of the active sites. One of these metals is ligated through an innersphere water molecule to the phosphate group located 3 to the scissile phosphate. A second inner-sphere water on this metal is positioned approximately in-line for attack on the scissile phosphate. This structure corroborates the observation that the pro-S P phosphoryl oxygen on the adjacent 3 phosphate cannot be modified without severe loss of catalytic efficiency. The structural equivalence of key groups, conserved in the active sites of EcoRV, EcoRI, PvuII, and BamHI endonucleases, suggests that ligation of a catalytic divalent metal ion to this phosphate may occur in many type II restriction enzymes. Together with previous cocrystal structures, these data allow construction of a detailed model for the pretransition state configuration in EcoRV. This model features three divalent metal ions per active site and invokes assistance in the bond-making step by a conserved lysine, which stabilizes the attacking hydroxide ion nucleophile.Recently determined crystal structures of type II restriction endonucleases have produced a wealth of information on the basis for target site sequence selectivity (1-6). However, these structures do not resolve the detailed structural mechanism of catalysis. Although acidic and basic groups in the active sites can be identified, and in some cases divalent-metal binding sites delineated, a convincing picture clarifying the way in which the attacking hydroxide ion is generated, and the leaving group stabilized, has not been elucidated for any of the enzymes.EcoRV endonuclease is a homodimer of 244 aa per monomer. It cleaves the duplex sequence 5Ј-GATATC at the central TA step in a blunt-ended fashion (7). As in all type II enzymes, phosphoryl transfer proceeds via attack of a hydroxide ion nucleophile on the scissile phosphorus, generating products containing a 5Ј phosphate group. This reaction occurs by in-line displacement through a pentacovalent transition state, with inversion of stereochemistry at phosphorus and an absolute requirement for divalent metal cations (8, 9). Highresolution crystallographic analyses of EcoRV show that the scissile phosphates of the DNA are located adjacent to the carboxylates of Asp-90 and Asp-74, and that a divalent metal ion bridges the enzyme and DNA at this position (2, 3). Although this metal could stabilize the additional negative charge that develops in the transition state, it is not correctly positioned to generate the attacking hydroxide ion. Thus, speculative mechanisms in which the nucleophile arises from dissociation of a metal-ligated water have invoked significant rearrangements of the DNA from its observed conformation in the crystal structures (2, 10).An alternative mechanism involving substrate-assisted catalysis by the adjacent 3Ј-phosphate of the DNA also has been proposed (11,12). In support of this mechanis...