Although clinical trials have shown that RAF inhibitors prolong the survival of patients with BRAF-mutant melanoma, resistance inevitably develops; resistance is shown here to be frequently mediated by the expression of splicing variants of mutant BRAF. Although recent clinical trials have shown the efficacy of B-RAF inhibitors in the treatment of melanomas with activating B-RAF mutations, the patients inevitably develop resistance. David Solit and colleagues now identify a mechanism of acquired resistance conferred by a structural change in B-RAF itself. The expression of a 61-kilodalton splice variant of mutant B-RAF leads to enhanced B-RAF dimerization, rendering it resistant to kinase inhibitors. This variant was found to be expressed in 6 of 19 patients who had developed resistance to the B-RAF inhibitor PLX4032. Activated RAS promotes dimerization of members of the RAF kinase family1,2,3. ATP-competitive RAF inhibitors activate ERK signalling4,5,6,7 by transactivating RAF dimers4. In melanomas with mutant BRAF(V600E), levels of RAS activation are low and these drugs bind to BRAF(V600E) monomers and inhibit their activity. This tumour-specific inhibition of ERK signalling results in a broad therapeutic index and RAF inhibitors have remarkable clinical activity in patients with melanomas that harbour mutant BRAF(V600E)8. However, resistance invariably develops. Here, we identify a new resistance mechanism. We find that a subset of cells resistant to vemurafenib (PLX4032, RG7204) express a 61-kDa variant form of BRAF(V600E), p61BRAF(V600E), which lacks exons 4–8, a region that encompasses the RAS-binding domain. p61BRAF(V600E) shows enhanced dimerization in cells with low levels of RAS activation, as compared to full-length BRAF(V600E). In cells in which p61BRAF(V600E) is expressed endogenously or ectopically, ERK signalling is resistant to the RAF inhibitor. Moreover, a mutation that abolishes the dimerization of p61BRAF(V600E) restores its sensitivity to vemurafenib. Finally, we identified BRAF(V600E) splicing variants lacking the RAS-binding domain in the tumours of six of nineteen patients with acquired resistance to vemurafenib. These data support the model that inhibition of ERK signalling by RAF inhibitors is dependent on levels of RAS–GTP too low to support RAF dimerization and identify a novel mechanism of acquired resistance in patients: expression of splicing isoforms of BRAF(V600E) that dimerize in a RAS-independent manner.

Purpose: PD-L1 is the main ligand for the immune inhibitory receptor PD-1. This ligand is frequently expressed by melanoma cells. In this study, we investigated whether PD-L1 expression is controlled by melanoma driver mutations and modified by oncogenic signaling inhibition. Experimental Design: Expression of PD-L1 was investigated in a panel of 51 melanoma cell lines containing different oncogenic mutations, including cell lines with innate and acquired resistance to BRAF inhibitors (BRAFi). The effects of targeted therapy drugs on expression of PD-L1 by melanoma cells were investigated. Results: No association was found between the level of PD-L1 expression and mutations in BRAF, NRAS, PTEN, or amplification of AKT. Resistance to vemurafenib due to the activation of alternative signaling pathways was accompanied with the induction of PD-L1 expression, whereas the resistance due to the reactivation of the MAPK pathway had no effect on PD-L1 expression. In melanoma cell lines, the effects of BRAF, MEK, and PI3K inhibitors on expression of PD-L1 were variable from reduction to induction, particularly in the presence of INFγ. In PD-L1–exposed lymphocytes, vemurafenib paradoxically restored activity of the MAPK pathway and increased the secretion of cytokines. Conclusions: In melanoma cell lines, including BRAFi-resistant cells, PD-L1 expression is variably regulated by oncogenic signaling pathways. PD-L1–exposed lymphocytes decrease MAPK signaling, which is corrected by exposure to vemurafenib, providing potential benefits of combining this drug with immunotherapies. Clin Cancer Res; 20(13); 3446–57. ©2014 AACR.

Significance: MITF, a master regulator of melanocytes and a major melanoma oncogene amplified in 30-40% of melanomas, determines proliferative or invasive phenotypes. Previously unrecognized as a driver of lysosomal biogenesis, we found that MITF expression correlates with many lysosomal genes and generates late endosomes that are not functional in proteolysis. This accumulation of incomplete organelles expands the late endosomal compartment, enhancing Wnt signaling by entrapping the Wnt machinery in multivesicular bodies. Wnt signaling can stabilize many proteins besides β-catenin. Our study identifies MITF as an oncogenic protein stabilized by Wnt, and describes three novel glycogen synthase kinase 3-regulated phosphorylation sites in this oncogene. This study deepens our knowledge on proliferative stages of melanoma: MITF, multivesicular bodies, and Wnt may form a feedback loop that drives proliferation.

Herpesviruses constitute a family of large DNA viruses widely spread in vertebrates and causing a variety of different diseases. They possess dsDNA genomes ranging from 120 to 240 kbp encoding between 70 to 170 open reading frames. We previously reported the protein interaction networks of two herpesviruses, varicella-zoster virus (VZV) and Kaposi's sarcoma-associated herpesvirus (KSHV). In this study, we systematically tested three additional herpesvirus species, herpes simplex virus 1 (HSV-1), murine cytomegalovirus and Epstein-Barr virus, for protein interactions in order to be able to perform a comparative analysis of all three herpesvirus subfamilies. We identified 735 interactions by genome-wide yeast-two-hybrid screens (Y2H), and, together with the interactomes of VZV and KSHV, included a total of 1,007 intraviral protein interactions in the analysis. Whereas a large number of interactions have not been reported previously, we were able to identify a core set of highly conserved protein interactions, like the interaction between HSV-1 UL33 with the nuclear egress proteins UL31/UL34. Interactions were conserved between orthologous proteins despite generally low sequence similarity, suggesting that function may be more conserved than sequence. By combining interactomes of different species we were able to systematically address the low coverage of the Y2H system and to extract biologically relevant interactions which were not evident from single species. Herpesvirus proteins interact with each other in a complex manner throughout the infectious cycle. This is probably best exemplified in the process where a large number of viral proteins come together to form new viral particles which are subsequently released from the infected cell. A more detailed understanding of how viral proteins interact with each other might assist the development of drugs which may inhibit these interactions and consequently block viral replication. Here we present three genome-wide studies of protein-protein interactions in the herpesviruses herpes simplex virus I, murine cytomegalovirus and Epstein-Barr virus. Altogether we identified 735 interactions in the three viruses, most of which have not previously been reported. By combining these studies with our previously published studies for Kaposi's sarcoma-associated herpesvirus and varicella-zoster virus we were able to perform a comparative analysis of interactions in five related viral species. We observed that a high proportion of interactions were conserved between the different species, despite a low degree of sequence conservation. This implies that by comparing interaction data, we were able to increase the coverage of our viral networks and thus obtain a better and more complete picture of interactions between herpesviral proteins.

This study shows that, despite malignant transformation, autoimmune checkpoints are still functional in B-cell leukaemia, with targeted activation of these checkpoints effectively killing patient-derived B-cell leukaemia in a transplant model; the results represent a novel strategy to overcome drug resistance in leukaemia patients. Markus Müschen and colleagues reason that in certain B cell malignancies with constitutive B cell receptor (BCR) signalling — acute lymphoblastic leukaemias carrying BCR–ABL translocations — it may be possible to modify the normal selection process that favours B cells with an intermediate level of BCR signalling to instead drive BCR signalling over a threshold at which malignant B cells fail to survive. They show that this can be achieved by hyperactivation of the kinase SYK, for example, in a mouse model where pharmacological activation of a SYK pathway reduces the growth of patient-derived tumour xenografts. This concept is distinct from approaches to B lymphoma therapies that seek to block BCR signalling, and may be worth exploring in the clinic. B cells are selected for an intermediate level of B-cell antigen receptor (BCR) signalling strength: attenuation below minimum (for example, non-functional BCR)1 or hyperactivation above maximum (for example, self-reactive BCR)2,3 thresholds of signalling strength causes negative selection. In ∼25% of cases, acute lymphoblastic leukaemia (ALL) cells carry the oncogenic BCR-ABL1 tyrosine kinase (Philadelphia chromosome positive), which mimics constitutively active pre-BCR signalling4,5. Current therapeutic approaches are largely focused on the development of more potent tyrosine kinase inhibitors to suppress oncogenic signalling below a minimum threshold for survival6. We tested the hypothesis that targeted hyperactivation—above a maximum threshold—will engage a deletional checkpoint for removal of self-reactive B cells and selectively kill ALL cells. Here we find, by testing various components of proximal pre-BCR signalling in mouse BCR–ABL1 cells, that an incremental increase of Syk tyrosine kinase activity was required and sufficient to induce cell death. Hyperactive Syk was functionally equivalent to acute activation of a self-reactive BCR on ALL cells. Despite oncogenic transformation, this basic mechanism of negative selection was still functional in ALL cells. Unlike normal pre-B cells, patient-derived ALL cells express the inhibitory receptors PECAM1, CD300A and LAIR1 at high levels. Genetic studies revealed that Pecam1, Cd300a and Lair1 are critical to calibrate oncogenic signalling strength through recruitment of the inhibitory phosphatases Ptpn6 (ref. 7) and Inpp5d (ref. 8). Using a novel small-molecule inhibitor of INPP5D (also known as SHIP1)9, we demonstrated that pharmacological hyperactivation of SYK and engagement of negative B-cell selection represents a promising new strategy to overcome drug resistance in human ALL.