Mutational activation of BRAF is the most prevalent genetic alteration in human melanoma, with ≥ 50% of tumours expressing the BRAF(V600E) oncoprotein1,2. Moreover, the marked tumour regression and improved survival of late-stage BRAF-mutated melanoma patients in response to treatment with vemurafenib demonstrates the essential role of oncogenic BRAF in melanoma maintenance3,4. However, as most patients relapse with lethal drug-resistant disease, understanding and preventing mechanism(s) of resistance is critical to providing improved therapy5. Here we investigate the cause and consequences of vemurafenib resistance using two independently derived primary human melanoma xeno-graft models in which drugresistanceisselected by continuous vemurafenib administration. In one of these models, resistant tumours show continued dependency on BRAF(V600E) → MEK → ERK signalling owing to elevated BRAF(V600E) expression. Most importantly, we demonstrate that vemurafenib-resistant melanomas become drug dependent for their continued proliferation, such that cessation of drug administration leads to regression of established drug-resistant tumours. We further demonstrate that a discontinuous dosing strategy, which exploits the fitness disadvantage displayed by drug-resistant cells in the absence of the drug, forestalls the onset of lethal drug-resistant disease. These data highlight the concept that drug-resistant cells may also display drug dependency, such that altered dosing may prevent the emergence of lethal drug resistance. Such observations may contribute to sustaining the durability of the vemurafenib response with the ultimate goal of curative therapy for the subset of melanoma patients with BRAF mutations.

Mutation of the RB-1 and p53 tumor suppressors is associated with the development of human osteosarcoma. With the goal of generating a mouse model of this disease, we used conditional and transgenic mouse strains to inactivate Rb and/or p53 specifically in osteoblast precursors. The resulting Rb;p53 double mutant (DKO) animals are viable but develop early onset osteosarcomas with complete penetrance. These tumors display many of the characteristics of human osteosarcomas, including being highly metastatic. We established cell lines from the DKO osteosarcomas to further investigate their properties. These immortalized cell lines are highly proliferative and they retain their tumorigenic potential, as judged by their ability to form metastatic tumors in immunocompromised mice. Moreover, they can be induced to differentiate and, depending on the inductive signal, will adopt either the osteogenic or adipogenic fate. Consistent with this multipotency, a significant portion of these tumor cells express Sca-1, a marker that is typically associated with stem cells/uncommitted progenitors. By assaying sorted cells in transplant assays, we demonstrate that the tumorigenicity of the osteosarcoma cell lines correlates with the presence of the Sca-1 marker. Finally, we show that loss of Rb and p53 in Sca-1-positive mesenchymal stem/progenitor cells is sufficient to yield transformed cells that can initiate osteosarcoma formation in vivo.osx-cre ͉ Sca-1 ͉ hibernoma mouse model O steosarcomas account for Ϸ30% of malignant bone tumors and 3-4% of all childhood malignancies (1, 2). They arise primarily around the knee joint, lower femur and upper tibia, which are all regions of active bone growth and repair. These tumors are predominantly osteoblastic in nature, although there is a correlation between loss of differentiation and poor prognosis. The generation of new therapeutic treatments for osteosarcoma has improved the 5-year survival rate of affected individuals. However, like other mesenchymal neoplasms, osteosarcomas are predisposed to metastasize via the hematogenous route, and thus, pulmonary metastasis is a major cause of death. Analyses of both sporadic and hereditary tumors show that inactivation of the p53 and RB-1 tumor suppressors plays a key role in the development of this tumor type (1, 2). Li-Fraumeni patients, who often carry germ-line mutations in p53, are predisposed to a variety of tumors, 12% of which are bone sarcomas (3, 4). p53 mutations are also observed in 20-60% of sporadic osteosarcomas (5-7). Similarly, patients carrying germline mutations in RB-1 have an Ϸ500-fold higher incidence of osteosarcoma than the general population (8). Moreover, RB-1 mutations are detected in 70% of all adolescent osteosarcomas (9). Finally, human osteosarcomas can carry mutations in both p53 and RB-1 (10).Mouse models have provided considerable insight into the role of p53 in bone development and tumorigenesis. Experiments from three different settings suggest that p53 plays an important role in bone development by modul...

In Saccharomyces cerevisiae (budding yeast), commitment to cell division in late G 1 is promoted by the G 1 cyclin Cln3 and its associated cyclin-dependent kinase, Cdc28. We show here that all known aspects of the function of Cln3 in G 1 phase, including control of cell size, pheromone sensitivity, cell cycle progress, and transcription, require the protein Swi6. Swi6 is a component of two related transcription factors, SBF and MBF, which are known to regulate many genes at the G 1 -S transition. The Cln3-Cdc28 complex somehow activates SBF and MBF, but there was no evidence for direct phosphorylation of SBF/MBF by Cln3-Cdc28 or for a stable complex between SBF/MBF and Cln3-Cdc28. The activation also does not depend on the ability of Cln3 to activate transcription when artificially recruited directly to a promoter. The amino terminus and the leucine zipper of Swi6 are important for the ability of Swi6 to respond to Cln3 but are not essential for the basal transcriptional activity of Swi6. Cln3-Cdc28 may activate SBF and MBF indirectly, perhaps by phosphorylating some intermediary protein.

In wild-type diploid cells, heteroallelic recombination between his4A and his4C alleles leads mostly to His 1 gene conversions that have a parental configuration of flanking markers, but $22% of recombinants have associated reciprocal crossovers. In rad52 strains, gene conversion is reduced 75-fold and the majority of His 1 recombinants are crossover associated, with the largest class being half-crossovers in which the other participating chromatid is lost. We report that UV irradiating rad52 cells results in an increase in overall recombination frequency, comparable to increases induced in wild-type (WT) cells, and surprisingly results in a pattern of recombination products quite similar to RAD52 cells: gene conversion without exchange is favored, and the number of 2n À 1 events is markedly reduced. Both spontaneous and UV-induced RAD52-independent recombination depends strongly on Rad50, whereas rad50 has no effect in cells restored to RAD52. The high level of noncrossover gene conversion outcomes in UV-induced rad52 cells depends on Rad51, but not on Rad59. Those outcomes also rely on the UV-inducible kinase Dun1 and Dun1's target, the repressor Crt1, whereas gene conversion events arising spontaneously depend on Rad59 and Crt1. Thus, there are at least two Rad52-independent recombination pathways in budding yeast.