Somatic inactivating PTPRJ mutations and dysregulated pathways identified in canine malignant melanoma by integrated comparative genomic analysis.

Canine malignant melanoma, a significant cause of mortality in domestic dogs, is a powerful comparative model for human melanoma, but little is known about its genetic etiology. We mapped the genomic landscape of canine melanoma through multi-platform analysis of 37 tumors (31 mucosal, 3 acral, 2 cutaneous, and 1 uveal) and 17 matching constitutional samples including long- and short-insert whole genome sequencing, RNA sequencing, array comparative genomic hybridization, single nucleotide polymorphism array, and targeted Sanger sequencing analyses. We identified novel predominantly truncating mutations in the putative tumor suppressor gene PTPRJ in 19% of cases. No BRAF mutations were detected, but activating RAS mutations (24% of cases) occurred in conserved hotspots in all cutaneous and acral and 13% of mucosal subtypes. MDM2 amplifications (24%) and TP53 mutations (19%) were mutually exclusive. Additional low-frequency recurrent alterations were observed amidst low point mutation rates, an absence of ultraviolet light mutational signatures, and an abundance of copy number and structural alterations. Mutations that modulate cell proliferation and cell cycle control were common and highlight therapeutic axes such as MEK and MDM2 inhibition. This mutational landscape resembles that seen in BRAF wild-type and sun-shielded human melanoma subtypes. Overall, these data inform biological comparisons between canine and human melanoma while suggesting actionable targets in both species.

Oculoectodermal syndrome is a mosaic RASopathy associated with KRAS alterations.

Oculoectodermal syndrome (OES) is a rare disease characterized by a combination of congenital scalp lesions and ocular dermoids, with additional manifestations including non-ossifying fibromas and giant cell granulomas of the jaw occurring during the first decade of life. To identify the genetic etiology of OES, we conducted whole-genome sequencing of several tissues in an affected individual. Comparison of DNA from a non-ossifying fibroma to blood-derived DNA allowed identification of a somatic missense alteration in KRAS NM_033360.3(KRAS):c.38G>A, resulting in p.Gly13Asp. This alteration was also observed in the patient's other affected tissues including the skin and muscle. Targeted sequencing in a second, unrelated OES patient identified an NM_033360.3(KRAS):c.57G>C, p.Leu19Phe alteration. Allelic frequencies fell below 40% in all tissues examined in both patients, suggesting that OES is a mosaic RAS-related disorder, or RASopathy. The characteristic findings in OES, including scalp lesions, ocular dermoids, and benign tumors, are found in other mosaic and germline RASopathies. This discovery also broadens our understanding of the spectrum of phenotypes resulting from KRAS alterations. Future research into disease progression with regard to malignancy risk and investigation of RAS-targeted therapies in OES is warranted. KRAS sequencing is clinically available and may also now improve OES diagnostic criteria.

A human/murine chimeric fab antibody neutralizes anthrax lethal toxin in vitro.

Human anthrax infection caused by exposure to Bacillus anthracis cannot always be treated by antibiotics. This is mostly because of the effect of the remaining anthrax toxin in the body. Lethal factor (LF) is a component of lethal toxin (LeTx), which is the major virulence of anthrax toxin. A murine IgG monoclonal antibody (mAb) against LF with blocking activity (coded LF8) was produced in a previous study. In this report, a human/murine chimeric Fab mAb (coded LF8-Fab) was developed from LF8 by inserting murine variable regions into human constant regions using antibody engineering to reduce the incompatibility of the murine antibody for human use. The LF8-Fab expressed in Escherichia coli could specifically identify LF with an affinity of 3.46 × 10(7) L/mol and could neutralize LeTx with an EC50 of 85 µ g/mL. Even after LeTx challenge at various time points, the LF8-Fab demonstrated protection of J774A.1 cells in vitro. The results suggest that the LF8-Fab might be further characterized and potentially be used for clinical applications against anthrax infection.

Matrix metalloproteinase-activated anthrax lethal toxin inhibits endothelial invasion and neovasculature formation during in vitro morphogenesis.

Solid tumor growth is dependent on angiogenesis, the formation of neovasculature from existing vessels. Endothelial activation of the extracellular signal-regulated kinase 1/2, c-jun NH(2)-terminal kinase, and p38 mitogen-activated protein kinase pathways is central to this process, and thus presents an attractive target for the development of angiogenesis inhibitors. Anthrax lethal toxin (LeTx) has potent catalytic mitogen-activated protein kinase inhibition activity. Preclinical studies showed that LeTx induced potent tumor growth inhibition via the inhibition of xenograft vascularization. However, LeTx receptors and the essential furin-like activating proteases are expressed in many normal tissues, potentially limiting the specificity of LeTx as an antitumor agent. To circumvent nonspecific LeTx activation and simultaneously enhance tumor vascular targeting, a substrate preferably cleaved by the gelatinases class of matrix metalloproteinases (MMP) was substituted for the furin LeTx activation site. In vivo efficacy studies showed that this MMP-activated LeTx inhibited tumor xenografts growth via the reduced migration of endothelial cells into the tumor parenchyma. Here we have expanded on these initial findings by showing that this MMP-activated LeTx reduces endothelial proangiogenic MMP expression, thus causing a diminished proteolytic capacity for extracellular matrix remodeling and endothelial differentiation into capillary networks. Additionally, our data suggest that inhibition of the c-jun NH(2)-terminal kinase and p38, but not extracellular signal-regulated kinase-1/2, pathways is significant in the antiangiogenic activity of the MMP-activated LeTx. Collectively, these results support the clinical development of the MMP-activated LeTx for the treatment of solid tumors.

LRP5 and LRP6 are not required for protective antigen-mediated internalization or lethality of anthrax lethal toxin.

Anthrax toxin (AnTx) plays a key role in the pathogenesis of anthrax. AnTx is composed of three proteins: protective antigen (PA), edema factor, and lethal factor (LF). PA is not toxic but serves to bind cells and translocate the toxic edema factor or LF moieties to the cytosol. Recently, the low-density lipoprotein receptor-related protein LRP6 has been reported to mediate internalization and lethality of AnTx. Based on its similarity to LRP6, we hypothesized that LRP5 may also play a role in cellular uptake of AnTx. We assayed PA-dependent uptake of anthrax LF or a cytotoxic LF fusion protein (FP59) in cells and mice harboring targeted deletions of Lrp5 or Lrp6. Unexpectedly, we observed that uptake was unaltered in the presence or absence of either Lrp5 or Lrp6 expression. Moreover, we observed efficient PA-mediated uptake into anthrax toxin receptor (ANTXR)-deficient Chinese hamster ovary cells (PR230) that had been stably engineered to express either human ANTXR1 or human ANTXR2 in the presence or absence of siRNA specific for LRP5 or LRP6. Our results demonstrate that neither LRP5 nor LRP6 is necessary for PA-mediated internalization or lethality of anthrax lethal toxin.

Mitogen-activated protein kinase kinase signaling promotes growth and vascularization of fibrosarcoma.

We hypothesized that signaling through multiple mitogen-activated protein kinase (MAPK) kinase (MKK) pathways is essential for the growth and vascularization of soft-tissue sarcomas, which are malignant tumors derived from mesenchymal tissues. We tested this using HT-1080, NCI, and Shac fibrosarcoma-derived cell lines and anthrax lethal toxin (LeTx), a bacterial toxin that inactivates MKKs. Western blots confirmed that LeTx treatment reduced the levels of phosphorylated extracellular signal-regulated kinase and p38 MAPK in vitro. Although short treatments with LeTx only modestly affected cell proliferation, sustained treatment markedly reduced cell numbers. LeTx also substantially inhibited the extracellular release of angioproliferative factors including vascular endothelial growth factor, interleukin-8, and basic fibroblast growth factor. Similar results were obtained with cell lines derived from malignant fibrous histiocytomas, leiomyosarcomas, and liposarcomas. In vivo, LeTx decreased MAPK activity and blocked fibrosarcoma growth. Growth inhibition correlated with decreased cellular proliferation and extensive necrosis, and it was accompanied by a decrease in tumor mean vessel density as well as a reduction in serum expression of angioproliferative cytokines. Vital imaging using high-resolution ultrasound enhanced with contrast microbubbles revealed that the effects of LeTx on tumor perfusion were remarkably rapid (<24 h) and resulted in a marked reduction of perfusion within the tumor but not in nontumor tissues. These results are consistent with our initial hypothesis and lead us to propose that MKK inhibition by LeTx is a broadly effective strategy for targeting neovascularization in fibrosarcomas and other similar proliferative lesions.

Cytotoxicity of the matrix metalloproteinase-activated anthrax lethal toxin is dependent on gelatinase expression and B-RAF status in human melanoma cells.

Anthrax lethal toxin (LeTx) shows potent mitogen-activated protein kinase pathway inhibition and apoptosis in melanoma cells that harbor the activating V600E B-RAF mutation. LeTx is composed of two proteins, protective antigen and lethal factor. Uptake of the toxin into cells is dependent on proteolytic activation of protective antigen by the ubiquitously expressed furin or furin-like proteases. To circumvent nonspecific LeTx activation, a substrate preferably cleaved by gelatinases was substituted for the furin LeTx activation site. Here, we have shown that the toxicity of this matrix metalloproteinase (MMP)-activated LeTx is dependent on host cell surface MMP-2 and MMP-9 activity as well as the presence of the activating V600E B-RAF mutation, making this toxin dual specific. This additional layer of tumor cell specificity would potentially decrease systemic toxicity from the reduction of nonspecific toxin activation while retaining antitumor efficacy in patients with V600E B-RAF melanomas. Moreover, our results indicate that cell surface-associated gelatinase expression can be used to predict sensitivity among V600E B-RAF melanomas. This finding will aid in the better selection of patients that will potentially respond to MMP-activated LeTx therapy.

Biological and biochemical characterization of anthrax lethal factor, a proteolytic inhibitor of MEK signaling pathways.

The secretion of factors that block critical intracellular signaling pathways is a common strategy used by pathogenic bacteria for disabling host defenses and causing disease. Anthrax lethal toxin (LeTx) has been shown to cleave and inactivate mitogen-activated protein kinase (MAPK) kinases (MKKs or MEKs) and to inhibit MKK signaling. Cleavage of MKKs by LeTx prevents activation of their downstream substrates, the MAPKs. Because MAPK pathways regulate a variety of crucial cellular functions including proliferation, survival, differentiation, adhesion, and motility, LeTx has become a focus of study as an investigative tool as well as for the treatment and prevention of diseases due to malfunctions in MAPK signaling. This chapter describes methods for expressing and purifying the components of LeTx and focuses on techniques available for assessing its activity.

The systemic administration of lethal toxin achieves a growth delay of human melanoma and neuroblastoma xenografts: assessment of receptor contribution.

Two of the three components of anthrax toxin, protective antigen (PA) and lethal factor (LF), together known as lethal toxin (LeTx), reportedly show anti-tumor activity in melanoma in vitro and in vivo. The growth inhibitory activity of LeTx in culture was determined in nine human cancer cell lines, including melanoma, neuroblastoma and adenocarcinoma cells, as well as in human umbilical vein endothelial cells (HUVEC). The contribution of the two known PA receptor proteins, ANTXR1/TEM8 and ANTXR2/CMG2, to the sensitivity of the cells was assessed. The efficacy of LeTx was evaluated in vivo in the SK-N-AS neuroblastoma and SK-MEL-28 melanoma tumor xenograft models. Sensitivity to LeTx in vitro was observed in the neuroblastoma and colorectal adenocarcinoma cells and HUVEC, as well as melanoma cells. ANTXR1/TEM8 and ANTXR2/CMG2 protein expression studies suggested that a certain threshold of the PA receptor protein level must be met for sensitivity to LeTx to be observed. However, although the SK-N-AS neuroblastoma cells expressed the highest levels of receptor proteins and achieved the lowest IC50 in vitro (0.1 ng/ml), we observed no correlation between either the ANTXR1/TEM8 or ANTXR2/CMG2 protein levels and sensitivity to LeTx in vitro. In vivo, LeTx was an active anti-tumor agent when administered intravenously to mice bearing the human SK-N-AS or SK-MEL-28 tumor xenografts. The tumor growth delays were 6-8 days with a lower dose regimen and 14-16 days with a higher dose regimen for the two tumor models. These in vitro data suggest that LeTx may have broad therapeutic indications in cancer and the in vivo studies demonstrate that LeTx has systemic efficacy in neuroblastoma as well as melanoma. The therapeutic potential of LeTx needs to be further investigated in non-melanoma tumor models expressing the ANTXR1/TEM8 and/or ANTXR2/CMG2 protein.

Anthrax lethal toxin inhibits growth of and vascular endothelial growth factor release from endothelial cells expressing the human herpes virus 8 viral G protein coupled receptor.

PURPOSE: In this study, we tested the hypothesis that inhibition of mitogen-activated protein kinase kinases (MKK) inhibits tumor growth by acting on angiogenic signaling and by extension may form the basis of an effective strategy for treatment of Kaposi's sarcoma. EXPERIMENTAL DESIGN: Murine endothelial cells expressing the human herpes virus 8 G protein-coupled receptor (vGPCR-SVEC) were treated with anthrax lethal toxin (LeTx). LeTx is a binary toxin ordinarily secreted by Bacillus anthracis and is composed of two proteins: protective antigen (the binding moiety) and lethal factor (the active moiety). Lethal factor is a protease that cleaves and inactivates MKKs. RESULTS: In vitro, treatment of vGPCR-SVEC with LeTx inhibited MKK signaling, moderately inhibited cell proliferation, and blocked the ability of these cells to form colonies in soft agar. Treatment with LeTx also blocked the ability of these cells to release several angioproliferative cytokines, notably vascular endothelial growth factor (VEGF). In contrast, inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 with U0126 caused a substantial inhibition of proliferation but only modestly inhibited VEGF release. In xenograft models, i.v. injection of LeTx caused reduced tumor growth characterized immunohistochemically by inhibition of MKK signaling, decreased rates of proliferation, and reduced levels of VEGF and VEGF receptor 2, with a corresponding decrease in vascular density. CONCLUSIONS: These data support a role for MKK signaling in tumor growth and vascularization and are consistent with the hypothesis that inhibition of MKK signaling by LeTx or a similar agent may be an effective strategy for the treatment of Kaposi's sarcoma as well as other vascular tumors.

Systemic anthrax lethal toxin therapy produces regressions of subcutaneous human melanoma tumors in athymic nude mice.

PURPOSE: Anthrax Lethal Toxin (LeTx), composed of protective antigen and lethal factor, catalytically cleaves mitogen-activated protein kinase (MAPK) kinases and inhibits the MAPK signaling pathways. The majority of metastatic melanomas possess the V599E BRAF mutation, which constitutively activates MAPK1/2 signaling. LeTx is cytotoxic to BRAF mutant melanoma cell lines in vitro, whereas most normal cells are resistant to this toxin. In this study, we determine the in vivo potency and safety of systemically administered LeTx. EXPERIMENTAL DESIGN: A s.c. xenograft melanoma model in athymic nude mice was treated with different i.p. doses of LeTx. RESULTS: In this study, we show that in vivo systemic LeTx treatment of s.c. xenograft melanoma tumors in athymic nude mice yields partial and complete tumor regressions with minor toxicity to mice. When animal toxicity was observed, we did not find any histologic evidence of tissue damage. CONCLUSIONS: LeTx is one of the rare targeted agents to produce complete remissions of human melanomas in an animal model and thus warrants further preclinical development.

Xenopus tropicalis oocytes: more than just a beautiful genome.

For more than 30 yr, Xenopus laevis has been the animal of choice for studying the biochemical regulation of the meiotic and early mitotic vertebrate cell cycles. Attracted by its diploid genome, several laboratories have begun using the similar, although evolutionarily distinct, frog Xenopus tropicalis for studies of vertebrate development. Comparisons between the two species indicate that their development is similar in most respects. Both frogs share many advantages, including their amenability to manipulation and their ability to produce large numbers of high-quality oocytes and eggs year round. In addition, X. tropicalis possesses several advantages that, when combined with its potential for genetic studies, makes it an attractive, complementary model for vertebrate developmental biology. In this chapter, we note some of these advantages and describe in detail techniques we have adapted for the study of meiosis in X. tropicalis.

BRAF status and mitogen-activated protein/extracellular signal-regulated kinase kinase 1/2 activity indicate sensitivity of melanoma cells to anthrax lethal toxin.

Anthrax lethal toxin, composed of protective antigen and lethal factor, was tested for cytotoxicity to human melanoma cell lines and normal human cells. Eleven of 18 melanoma cell lines were sensitive to anthrax lethal toxin (IC(50) < 400 pmol/L) and 10 of these 11 sensitive cell lines carried the V599E BRAF mutation. Most normal cell types (10 of 15) were not sensitive to anthrax lethal toxin and only 5 of 15 normal human cell types were sensitive to anthrax lethal toxin (IC(50) < 400 pmol/L). These cells included monocytes and a subset of endothelial cells. In both melanoma cell lines and normal cells, anthrax toxin receptor expression levels did not correlate with anthrax lethal toxin cytotoxicity. Furthermore, an anthrax toxin receptor-deficient cell line (PR230) did not show any enhanced sensitivity to anthrax lethal toxin when transfected with anthrax toxin receptor. Anthrax lethal toxin toxicity correlated with elevated phosphorylation levels of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) 1/2 in both melanoma cell lines and normal cells. Anthrax lethal toxin-sensitive melanoma cell lines and normal cells had higher phospho-MEK1/2 levels than anthrax lethal toxin-resistant melanoma cell lines and normal tissue types. U0126, a specific MEK1/2 inhibitor, was not toxic to anthrax lethal toxin-resistant melanoma cell lines but was toxic to 8 of 11 anthrax lethal toxin-sensitive cell lines. These results show that anthrax lethal toxin toxicity correlates with elevated levels of active MEK1/2 pathway but not with anthrax toxin receptor expression levels in both normal and malignant tissues. Anthrax lethal toxin may be a useful therapeutic for melanoma patients, especially those carrying the V599E BRAF mutation with constitutive activation of the mitogen-activated protein kinase pathway.

Combined inhibition of MEK and mTOR has a synergic effect on angiosarcoma tumorgrafts.

Angiosarcoma (AS) is a rare neoplasm of endothelial origin that has limited treatment options and poor five-year survival. Using tumorgraft models, we previously showed that AS is sensitive to small-molecule inhibitors that target mitogen-activated/extracellular-signal-regulated protein kinase kinases 1 and 2 (MEK). The objective of this study was to identify drugs that combine with MEK inhibitors to more effectively inhibit AS growth. We examined the in vitro synergy between the MEK inhibitor PD0325901 and inhibitors of eleven common cancer pathways in melanoma cell lines and canine angiosarcoma cell isolates. Combination indices were calculated using the Chou-Talalay method. Optimized combination therapies were evaluated in vivo for toxicity and efficacy using canine angiosarcoma tumorgrafts. Among the drugs we tested, rapamycin stood out because it showed strong synergy with PD0325901 at nanomolar concentrations. We observed that angiosarcomas are insensitive to mTOR inhibition. However, treatment with nanomolar levels of mTOR inhibitor renders these cells as sensitive to MEK inhibition as a melanoma cell line with mutant BRAF. Similar results were observed in B-Raf wild-type melanoma cells as well as in vivo, where treatment of canine AS tumorgrafts with MEK and mTOR inhibitors was more effective than monotherapy. Our data show that a low dose of an mTOR inhibitor can dramatically enhance angiosarcoma and melanoma response to MEK inhibition, potentially widening the field of applications for MEK-targeted therapy.

Melanoma patient derived xenografts acquire distinct Vemurafenib resistance mechanisms.

Variable clinical responses, tumor heterogeneity, and drug resistance reduce long-term survival outcomes for metastatic melanoma patients. To guide and accelerate drug development, we characterized tumor responses for five melanoma patient derived xenograft models treated with Vemurafenib. Three BRAF(V600E) models showed acquired drug resistance, one BRAF(V600E) model had a complete and durable response, and a BRAF(V600V) model was expectedly unresponsive. In progressing tumors, a variety of resistance mechanisms to BRAF inhibition were uncovered, including mutant BRAF alternative splicing, NRAS mutation, COT (MAP3K8) overexpression, and increased mutant BRAF gene amplification and copy number. The resistance mechanisms among the patient derived xenograft models were similar to the resistance pathways identified in clinical specimens from patients progressing on BRAF inhibitor therapy. In addition, there was both inter- and intra-patient heterogeneity in resistance mechanisms, accompanied by heterogeneous pERK expression immunostaining profiles. MEK monotherapy of Vemurafenib-resistant tumors caused toxicity and acquired drug resistance. However, tumors were eradicated when Vemurafenib was combined the MEK inhibitor. The diversity of drug responses among the xenograft models; the distinct mechanisms of resistance; and the ability to overcome resistance by the addition of a MEK inhibitor provide a scheduling rationale for clinical trials of next-generation drug combinations.

Novel protein targeted therapy of metastatic melanoma.

Metastatic melanoma patients have a dismal prognosis with poor responsiveness to chemotherapy, radiation therapy and current immunotherapy regimens and a median survival of less than six months. Novel therapies directed at melanoma-selective molecular targets are urgently needed. Based on the frequent constitutive activation of the mitogen-activated protein kinase (MAPK) signaling pathway in malignant melanomas and the selective inhibition of MAPK signaling by anthrax lethal factor which proteolytically cleaves MAPK kinases, anthrax lethal toxin may be a useful agent for patients with metastatic melanoma. Anthrax lethal toxin consists of two proteins--protective antigen and lethal factor. These two proteins have been separately produced in good yields and in high purity. The three-dimensional structures of these proteins have been solved, and their molecular mechanisms of cell binding and action determined. Preclinical studies with anthrax lethal toxin show sensitivity of malignant melanoma cell lines in tissue culture and anti-tumor efficacy in melanoma xenograft models. Additional studies to define the maximal tolerated doses and dose-limiting toxicity of anthrax lethal toxin in rodent and primate models should pave the way for phase I studies testing the efficacy of the anthrax lethal toxin in patients with metastatic melanoma.

Identification of VEGF-independent cytokines in proliferative diabetic retinopathy vitreous.

PURPOSE: To identify inflammatory cytokines significantly elevated and independent of VEGF levels in the vitreous of proliferative diabetic retinopathy (PDR) patients that may serve as novel diagnostic factors or therapeutic targets. METHODS: Thirty-nine cytokines and chemokines were measured from the vitreous of 72 patients undergoing vitrectomy (29 controls and 43 PDR) via a magnetic bead-based immunoassay. Patient information, including sex, age, history of smoking, cancer diagnosis and treatment, and presence of diabetes and hypertension were also collected. Univariate and multivariate logistic regression analyses were performed to assess the association of cytokine concentrations and patient demographics with disease. RESULTS: Nineteen cytokines were significantly elevated in the vitreous of PDR patients compared with controls, including five novel cytokines that have not previously been associated with PDR: sCD40L, GM-CSF, IFNα2, IL-12p40, and MCP-3. Sixteen cytokines were found to be statistically independent of VEGF. Of these, 14 show a statistically significant interaction with VEGF, while two do not. With regards to patient demographics, age and hypertension were statistically significant risk factors with the odds of disease decreasing with increasing age and increasing 3-fold for hypertensive patients. CONCLUSIONS: This is the first report of a comprehensive multiplex analysis to identify novel VEGF-independent cytokines associated with PDR. Of the 39 inflammatory cytokines tested, 16 are predictive of disease risk, independent of VEGF levels. These PDR-associated cytokines represent potential targets in the treatment of PDR, both in conjunction with anti-VEGF therapy, as well as for patients that are nonresponders to such therapy.

Pharmacologic inhibition of MEK signaling prevents growth of canine hemangiosarcoma.

Angiosarcoma is a rare neoplasm of endothelial origin that has limited treatment options and poor five-year survival. As a model for human angiosarcoma, we studied primary cells and tumorgrafts derived from canine hemangiosarcoma (HSA), which is also an endothelial malignancy with similar presentation and histology. Primary cells isolated from HSA showed constitutive extracellular signal-regulated kinase (ERK) activation. The mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor CI-1040 reduced ERK activation and the viability of primary cells derived from visceral, cutaneous, and cardiac HSA in vitro. HSA-derived primary cells were also sensitive to sorafenib, an inhibitor of B-Raf and multireceptor tyrosine kinases. In vivo, CI-1040 or PD0325901 decreased the growth of cutaneous cell-derived xenografts and cardiac-derived tumorgrafts. Sorafenib decreased tumor size in both in vivo models, although cardiac tumorgrafts were more sensitive. In human angiosarcoma, we noted that 50% of tumors stained positively for phosphorylated ERK1/2 and that the expression of several MEK-responsive transcription factors was upregulated. Our data showed that MEK signaling is essential for the growth of HSA in vitro and in vivo and provided evidence that the same pathways are activated in human angiosarcoma. This indicates that MEK inhibitors may form part of an effective therapeutic strategy for the treatment of canine HSA or human angiosarcoma, and it highlights the use of spontaneous canine cancers as a model of human disease.

MEK genomics in development and disease.

The mitogen-activated protein kinase kinases (the MAPK/ERK kinases; MKKs or MEKs) and their downstream substrates, the extracellular-regulated kinases have been intensively studied for their roles in development and disease. Until recently, it had been assumed any mutation affecting their function would have lethal consequences. However, the identification of MEK1 and MEK2 mutations in developmental syndromes as well as chemotherapy-resistant tumors, and the discovery of genomic variants in MEK1 and MEK2 have led to the realization the extent of genomic variation associated with MEKs is much greater than had been appreciated. In this review, we will discuss these recent advances, relating them to what is currently understood about the structure and function of MEKs, and describe how they change our understanding of the role of MEKs in development and disease.