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.

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Summary The mammalian Ajuba LIM proteins (Ajuba, LIMD1, WTIP) are cytosolic adapter proteins that exhibit the potential to communicate cell adhesive events with nuclear responses to remodel epithelia [1] [2]. Determining their role(s) in vivo, however, has been challenging due to overlapping tissue expression and functional redundancy. Thus, we turned to Drosophila where a single gene, CG11063 or djub, exists. Drosophila containing djub mutant loss-of-function alleles or depleted of dJub by RNAi identify djub as an essential gene for development and novel regulator of epithelial organ size as a component of the conserved Hippo pathway, which has been implicated in both tissue size control and cancer development [3-5] [6-9]. djub-deficient tissues were small, had decreased cell numbers as a result of increased apoptosis and decreased proliferation, due to downregulation of DIAP1 and cyclin E. This phenocopies tissues deficient for Yorkie (Yki), the downstream target of the Hippo pathway. djub genetically interacts with the Hippo pathway, and epistasis suggests that djub lies downstream of hpo. In mammalian and Drosophila cells, Ajuba LIM proteins/dJub interact with LATS/Wts and WW45/Sav to inhibit phosphorylation of YAP/Yki. This work describes a novel role for the Ajuba LIM proteins as negative regulators of the Hippo signaling pathway.

SummaryThe HERBY trial was a phase II open-label, randomized, multicenter trial evaluating bevacizumab (BEV) in addition to temozolomide/radiotherapy in patients with newly diagnosed non-brainstem high-grade glioma (HGG) between the ages of 3 and 18 years. We carried out comprehensive molecular analysis integrated with pathology, radiology, and immune profiling. In post-hoc subgroup analysis, hypermutator tumors (mismatch repair deficiency and somatic POLE/POLD1 mutations) and those biologically resembling pleomorphic xanthoastrocytoma ([PXA]-like, driven by BRAF_V600E or NF1 mutation) had significantly more CD8+ tumor-infiltrating lymphocytes, and longer survival with the addition of BEV. Histone H3 subgroups (hemispheric G34R/V and midline K27M) had a worse outcome and were immune cold. Future clinical trials will need to take into account the diversity represented by the term “HGG” in the pediatric population.

Many patients with BRAF inhibitor resistance can develop disease at new sites, suggesting that drug-induced selection pressure drives metastasis. Here we used mass spectrometry-based phosphoproteomic screening to uncover ligand-independent EphA2 signaling as an adaptation to BRAF inhibitor therapy that led to the adoption of a metastatic phenotype. The EphA2-mediated invasion was AKT-dependent and readily reversible upon removal of drug as well as through PI3K and AKT inhibition. In xenograft models, BRAF inhibition led to the development of EphA2 positive metastases. A retrospective analysis of melanoma patients on BRAF inhibitor therapy showed that 68% of those failing therapy develop metastases at new disease sites, compared to 35% in patients on dacarbazine. Further IHC staining of melanoma specimens taken from patients on BRAF inhibitor therapy as well as metastatic samples taken from patients failing therapy showed increased EphA2 staining. We suggest that inhibition of ligand-independent EphA2 signaling may limit metastases associated with BRAF inhibitor therapy.

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Sphingosine 1-phosphate (S1P) is a potent chemokinetic agent for endothelial cells that is released by activated platelets. We previously developed Arg-Gly-Asp (RGD)-containing polyethylene glycol biomaterials for the controlled delivery of S1P to promote endothelialization. Here, we studied the effects of cell adhesion strength on S1P-stimulated endothelial cell migration in the presence of arterial levels of fluid shear stress, since an upward shift in optimal cell adhesion strengths may be beneficial for promoting long-term cell adhesion to materials. Two RGD peptides with different integrin-binding specificities were added to the polyethylene glycol hydrogels. A linear RGD bound primarily to beta(3) integrins, whereas a cyclic RGD bound through both beta(1) and beta(3) integrins. We observed increased focal adhesion formation and better long-term adhesion in flow with endothelial cells on linear RGD peptide, versus cyclic RGD, even though initial adhesion strengths were higher for cells on cyclic RGD. Addition of 100 nM S1P increased cell speed and random motility coefficients on both RGD peptides, with the largest increases found on cyclic RGD. For both peptides, much of the increase in cell migration speed was found for smaller cells (<1522 microm(2) projected area), although the large increases on cyclic RGD were also due to medium-sized cells (2288-3519 microm(2)). Overall, a compromise between high cell migration rates and long-term adhesion will be important in the design of materials that endothelialize after implantation.

The RAS-RAF-MEK-ERK pathway is a key driver of proliferation and survival signals in tumor cells and has been the focus of intense drug development efforts over the past 20 years. The recent regulatory approval of RAF inhibitors and a MAP-ERK kinase (MEK) inhibitor for metastatic melanoma provides clinical validation of tumor dependency on this pathway. Unfortunately, the therapeutic benefit of these agents is often short lived and resistance develops within a matter of months. Preclinical models of resistance to vemurafenib have provided critical insights into predicting, validating, and characterizing potential mechanisms. A key observation has been that vemurafenib-resistant tumor cells suffer a fitness deficit in the absence of drug treatment and this led to the predication that modulating the selective pressure of drug treatment through intermittent dosing could delay or prevent the emergence of resistant tumors. Most importantly, the preclinical data are supported by observations in vemurafenib-treated patients with melanoma providing a strong rationale for clinical testing of alternative dosing regimens. Cancer Res; 73(20); 6106-10. Ó2013 AACR.