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PDF file - 126K, Supplemental Figure S4. Increase in MEF2c IHC staining in AS and HSA primary tumors.

PDF file - 142K, Supplemental Figure S2. Reduction of phospho-ERK1/2 IHC staining of cutaneous-derived xenograft and cardiac-derived tumorgraft treated with CI-1040.

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. Mol Cancer Ther; 12(9); 1701–14. ©2013 AACR.

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 NH2-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 NH2-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. (Mol Cancer Res 2009;7(4):452–61)

PDF file - 146K, Supplemental Figure S3. Reduction of phospho-ERK1/2 IHC staining of cutaneous-derived xenograft and cardiac-derived tumorgraft treated with sorafenib.

Supplementary Data from Matrix Metalloproteinase–Activated Anthrax Lethal Toxin Inhibits Endothelial Invasion and Neovasculature Formation during In vitro Morphogenesis

Supplementary Figures S1-S3 from Matrix Metalloproteinase–Activated Anthrax Lethal Toxin Inhibits Endothelial Invasion and Neovasculature Formation during In vitro Morphogenesis

Supplementary Fig. S1 from 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. [Mol Cancer Ther 2008;7(5):1218–26]

Supplementary Figure 2 from Inhibition of Tumor Angiogenesis by the Matrix Metalloproteinase–Activated Anthrax Lethal Toxin in an Orthotopic Model of Anaplastic Thyroid Carcinoma

Supplementary Fig. S6 from Mitogen-activated protein kinase kinase signaling promotes growth and vascularization of fibrosarcoma

Patients with anaplastic thyroid carcinoma (ATC) typically succumb to their disease months after diagnosis despite aggressive therapy. A large percentage of ATCs have been shown to harbor the V600E B-Raf point mutation, leading to the constitutive activation of the mitogen-activated protein kinase pathway. ATC invasion, metastasis, and angiogenesis are in part dependent on the gelatinase class of matrix metalloproteinases (MMP). The explicit targeting of these two tumor markers may provide a novel therapeutic strategy for the treatment of ATC. The MMP-activated anthrax lethal toxin (LeTx), a novel recombinant protein toxin combination, shows potent mitogen-activated protein kinase pathway inhibition in gelatinase-expressing V600E B-Raf tumor cells in vitro. However, preliminary in vivo studies showed that the MMP-activated LeTx also exhibited dramatic antitumor activity against xenografts that did not show significant antiproliferative responses to the LeTx in vitro. Here, we show that the MMP-activated LeTx inhibits orthotopic ATC xenograft progression in both toxin-sensitive and toxin-resistant ATC cells via reduced endothelial cell recruitment and subsequent tumor vascularization. This in turn translates to an improved long-term survival that is comparable with that produced by the multikinase inhibitor sorafenib. Our results also indicate that therapy with the MMP-activated LeTx is extremely effective against advanced tumors with well-established vascular networks. Taken together, these results suggest that the MMP-activated LeTx-mediated endothelial cell targeting is the primary in vivo antitumor mechanism of this novel toxin. Therefore, the MMP-activated LeTx could be used not only in the clinical management of V600E B-Raf ATC but potentially in any solid tumor. Mol Cancer Ther; 9(1); 190–201

Supplementary Figure Legends from Mitogen-activated protein kinase kinase signaling promotes growth and vascularization of fibrosarcoma

Supplementary Table S1 from Mitogen-activated protein kinase kinase signaling promotes growth and vascularization of fibrosarcoma

Supplementary Figure 1 from Inhibition of Tumor Angiogenesis by the Matrix Metalloproteinase–Activated Anthrax Lethal Toxin in an Orthotopic Model of Anaplastic Thyroid Carcinoma

The mitogen-activated protein kinase (MAPK) signaling pathways play essential roles in cell proliferation and differentiation. Recent studies also show the activation of MAPK signaling pathways in tumorigenesis, metastasis, and angiogenesis of multiple human malignancies, including renal cell carcinoma (RCC). To assess the role of this pathway in regulating the proliferation and survival of RCC cells, we first examined the expression of MAPK kinase (MKK) and MAPK in clear cell RCC and confirmed the overexpression of MKK1 and extracellular signal-regulated kinase 2 (ERK2) in these tumors. We then tested the effects of pharmacologic inhibition of MKK on human RCC cell lines, both in vitro and in vivo, using anthrax lethal toxin (LeTx), which cleaves and inactivates several MKKs. Western blotting showed that the phosphorylation levels of ERK, c-Jun-NH2 kinase, and p38 MAPK decreased after 72 h of LeTx treatment. Exposure to LeTx for 72 h reduced cell proliferation by 20% without significant effects on cell cycle distribution and apoptosis. Anchorage-independent growth of RCC cells was dramatically inhibited by LeTx. In vivo studies showed that tumor growth of RCC xenografts could be suppressed by LeTx. Extensive necrosis and decreased tumor neovascularization were observed after LeTx treatment. LeTx also showed direct inhibition of proliferation of endothelial cells in vitro. Our results suggest that suppression of one or more MAPK signaling pathways may inhibit RCC growth through the disruption of tumor vasculature. [Cancer Res 2008;68(1):81–8]

Supplementary Figure 2 from Inhibition of MAPK Kinase Signaling Pathways Suppressed Renal Cell Carcinoma Growth and Angiogenesis In vivo

Supplementary Figure 3 from Inhibition of MAPK Kinase Signaling Pathways Suppressed Renal Cell Carcinoma Growth and Angiogenesis In vivo

Supplementary Legends 1-6 from Inhibition of MAPK Kinase Signaling Pathways Suppressed Renal Cell Carcinoma Growth and Angiogenesis In vivo

Supplementary Figure 1 from Inhibition of MAPK Kinase Signaling Pathways Suppressed Renal Cell Carcinoma Growth and Angiogenesis In vivo

Supplementary Figure 6 from Inhibition of MAPK Kinase Signaling Pathways Suppressed Renal Cell Carcinoma Growth and Angiogenesis In vivo

Supplementary Figure 4 from Inhibition of MAPK Kinase Signaling Pathways Suppressed Renal Cell Carcinoma Growth and Angiogenesis In vivo

Supplementary Figure 5 from Inhibition of MAPK Kinase Signaling Pathways Suppressed Renal Cell Carcinoma Growth and Angiogenesis In vivo

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.

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. © 2015 Wiley Periodicals, Inc.

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 genePTPRJin 19% of cases. NoBRAFmutations were detected, but activatingRASmutations (24% of cases) occurred in conserved hotspots in all cutaneous and acral and 13% of mucosal subtypes.MDM2amplifications (24%) andTP53mutations (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 inBRAFwild-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.AUTHOR SUMMARYMelanoma, an aggressive cancer arising from transformed melanocytes, commonly occurs in pet dogs. Unlike human melanoma, which most often occurs in sun-exposed cutaneous skin, canine melanoma typically arises in sun-shielded oral mucosa. Clinical features of canine melanoma resemble those of human melanoma, particularly the less common sun-shielded human subtypes. However, whereas the genomic basis of diverse human melanoma subtypes is well understood, canine melanoma genomics remain poorly defined. Similarly, although diverse new treatments for human melanoma based on a biologic disease understanding have recently shown dramatic improvements in outcomes for these patients, treatments for canine melanoma are limited and outcomes remain universally poor. Detailing the genomic basis of canine melanoma thus provides untapped potential for improving the lives of pet dogs while also helping to establish canine melanoma as a comparative model system for informing human melanoma biology and treatment. In order to better define the genomic landscape of canine melanoma, we performed multi-platform characterization of 37 tumors. Our integrated analysis confirms that these tumors commonly contain mutations in canine orthologs of human cancer genes such asRAS,MDM2, andTP53as well mutational patterns that share important similarities with human melanoma subtypes. We have also found a new putative cancer gene,PTPRJ, frequently mutated in canine melanoma. These data will guide additional biologic and therapeutic studies in canine melanoma while framing the utility of comparative studies of canine and human cancers more broadly.

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. Mol Cancer Ther; 12(9); 1701–14. ©2013 AACR.

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., Author summary Melanoma, a cancer arising from transformed melanocytes, commonly occurs in pet dogs. Unlike human melanoma, most often occurring in sun-exposed cutaneous skin, canine melanoma typically arises in sun-shielded oral mucosa. Canine melanoma clinically resembles human melanoma, particularly sun-shielded subtypes. However, canine melanoma genomics remain poorly defined. Similarly, although new treatments for human melanoma based on genomic understanding have shown improvements in outcomes for these patients, treatments for canine melanoma are limited and outcomes remain poor. Detailing the genomic basis of canine melanoma thus provides untapped potential for improving lives of pet dogs and helping to define canine melanoma’s role in comparative studies that also inform human melanoma understanding. To better define the genomic landscape of canine melanoma, we performed multi-platform characterization of 37 tumors. Our integrated analysis confirms that these tumors commonly contain mutations in canine orthologs of human cancer genes such as RAS, MDM2, and TP53 alongside mutational patterns sharing important similarities with human melanoma subtypes. We have also found a new putative cancer gene, PTPRJ, frequently mutated in canine melanoma. These data will guide biologic and therapeutic studies in canine melanoma while broadly framing the utility of comparative studies of canine and human cancers.

Anthrax is caused by the gram-positive bacterium Bacillus anthracis. The pathogenesis of this disease is dependent on the presence of two binary toxins, edema toxin (EdTx) and lethal toxin (LeTx). LeTx, the major virulence factor contributing to anthrax, contains the effector moiety lethal factor (LF), a zinc-dependent metalloprotease specific for targeting mitogen-activated protein kinase kinases. This review will focus on the protease-specific activity and function of LF, and will include a discussion on the implications and consequences of this activity, both in terms of anthrax disease, and how this activity can be exploited to gain insight into other pathologic conditions.

Bacillus anthracis infections are frequently associated with severe and often irreversible hypotensive shock despite appropriate antibiotics and aggressive hemodynamic and pulmonary support. Based on the observations that the anthrax secreted proteins-protective antigen (PA), lethal factor (LF), and edema factor (EF) also produce shock and mortality in animal models, we chose to characterize further the clinical chemistries and microscopic pathology of toxin treated rats. Groups of three male Sprague Dawley rats received bolus intravenous infusions of PA/LF, PA/EF, LF, or EF alone and blood samples and tissues were collected and assayed for chemistries and tissue pathology. In PA/LF and PA/EF treated animals but not other groups, chemistries showed transaminasemia and elevated lactate dehydrogenase. PA/LF treated animals alone showed elevated hemoglobin and hematocrits; PA/EF treated animals alone showed lymphopenia. Pathology was remarkable for pulmonary edema in PA/LF treated rat lungs and pulmonary hemorrhage in PA/EF treated rat lungs. These results are consistent with our and others' previous findings that the morbidity and mortality associated with anthrax are not cytokine-mediated but due to a direct effect of the toxins on the cardiovascular system along with toxin-specific alterations in blood counts. PA/LF pathology matches that seen with acute cardiac failure, and PA/EF pathology coincides with direct vascular endothelial injury. These observations provide a rational basis for drug interventions to reduce the effect of these toxins on the heart and blood vessels.

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 (

The mitogen-activated protein kinase (MAPK) signaling pathways play essential roles in cell proliferation and differentiation. Recent studies also show the activation of MAPK signaling pathways in tumorigenesis, metastasis, and angiogenesis of multiple human malignancies, including renal cell carcinoma (RCC). To assess the role of this pathway in regulating the proliferation and survival of RCC cells, we first examined the expression of MAPK kinase (MKK) and MAPK in clear cell RCC and confirmed the overexpression of MKK1 and extracellular signal-regulated kinase 2 (ERK2) in these tumors. We then tested the effects of pharmacologic inhibition of MKK on human RCC cell lines, both in vitro and in vivo, using anthrax lethal toxin (LeTx), which cleaves and inactivates several MKKs. Western blotting showed that the phosphorylation levels of ERK, c-Jun-NH2 kinase, and p38 MAPK decreased after 72 h of LeTx treatment. Exposure to LeTx for 72 h reduced cell proliferation by 20% without significant effects on cell cycle distribution and apoptosis. Anchorage-independent growth of RCC cells was dramatically inhibited by LeTx. In vivo studies showed that tumor growth of RCC xenografts could be suppressed by LeTx. Extensive necrosis and decreased tumor neovascularization were observed after LeTx treatment. LeTx also showed direct inhibition of proliferation of endothelial cells in vitro. Our results suggest that suppression of one or more MAPK signaling pathways may inhibit RCC growth through the disruption of tumor vasculature. [Cancer Res 2008;68(1):81–8]

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.

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.

Fully-grown G2-arrested Xenopus oocytes resume meiosis upon hormonal stimulation. Resumption of meiosis is characterized by germinal vesicle breakdown, chromosome condensation, and organization of a bipolar spindle. These cytological events are accompanied by activation of MPF and the p39(Mos)-MEK1-Xp42(Mpk1)-p90(Rsk) pathways. The latter cascade is activated upon p39(Mos) accumulation. Using U0126, a MEK1 inhibitor, and p39(Mos) antisense morpholino and phosphorothioate oligonucleotides, we have investigated the role of the members of the p39(Mos)-MEK1-Xp42(Mpk1)-p90(Rsk) in spindle morphogenesis. First, we have observed at a molecular level that prevention of p39(Mos) accumulation always led to MEK1 phosphorylation defects, even when meiosis was stimulated through the insulin Ras-dependent pathway. Moreover, we have observed that Raf1 phosphorylation that occurs during meiosis resumption was dependent upon the activity of MEK1 or Xp42(Mpk1) but not p90(Rsk). Second, inhibition of either p39(Mos) accumulation or MEK1 inhibition led to the formation of a cytoplasmic aster-like structure that was associated with condensed chromosomes. Spindle morphogenesis rescue experiments using constitutively active Rsk and purified murine Mos protein suggested that p39(Mos) or p90(Rsk) alone failed to promote meiotic spindle organization. Our results indicate that activation of the p39(Mos)-MEK1-Xp42(Mpk1)-p90(Rsk) pathway is required for bipolar organization of the meiotic spindle at the cortex.

Anthrax lethal factor (LF) is a Zn2+ -metalloprotease that cleaves and inactivates mitogen-activated protein kinase kinases (MEKs). We have used site-directed mutagenesis to identify a cluster of residues in domain II of LF that lie outside the active site and are required for cellular proteolytic activity toward MEKs. Alanine substituted for Leu293, Lys294, Leu514, Asn516, or Arg491 caused a 10-50-fold reduction in LF toxicity. Further, whereas pairwise substitution of alanine for Leu514 and either Leu293, Lys294, or Arg491 completely abrogated LF toxicity, pairwise mutation of Leu514 and Asn516 resulted in toxicity comparable with N516A alone. The introduction of these mutations reduced LF-mediated cleavage of MEK2 in cell-based assays but altered neither the ability of LF to bind protective antigen nor its ability to translocate across a membrane. Interestingly, direct in vitro measurement of LF activity indicated that decreased toxicity was not always accompanied by reduced proteolytic activity. However, mutations in this region significantly reduced the ability of LF to competitively inhibit B-Raf phosphorylation of MEK. These results provide evidence that elements of domain II are involved in the association of LF into productive complex with MEKs.

Anthrax lethal toxin produced by the bacterium Bacillus anthracis is the major cause of death in animals infected with anthrax. One component of this toxin, lethal factor (LF), inactivates members of the mitogen-activated protein kinase kinase or MEK family through proteolysis of their NH(2) termini. However, neither the substrate requirements for LF cleavage nor the mechanism by which proteolysis inactivates MEK have been demonstrated. By means of deletion mutant analysis and site-directed mutagenesis, we have identified an LFIR (LF interacting region) in the COOH-terminal kinase domain of MEK1 adjacent to the proline-rich region, which is essential for LF-mediated proteolysis of MEK. Point mutations in this region block proteolysis but do not alter the kinase activity of MEK. Similar mutations in MEK6 also prevent proteolysis, indicating that this region is functionally conserved among MEKs. In addition, NH(2)-terminal proteolysis of MEK1 by LF was found to reduce not only the affinity of MEK1 for its substrate mitogen-activated protein kinase but also its intrinsic kinase activity, indicating that the NH(2)-terminal end of MEK is important not only for substrate interaction but also for catalytic activity.

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.

Resumption of meiosis in oocytes of Xenopus tropicalis required translation but not transcription, and was marked by the appearance of a white spot and a dark ring, coincident with entry into metaphase I and the onset of anaphase I, respectively. Cyclin B(2)/p34(cdc2) activity increased prior to the first meiotic division, declined at the onset of anaphase I, and subsequently increased again. The capacity of egg cytoplasm to induce germinal vesicle breakdown (GVBD) was inhibited by cycloheximide, despite the fact that these oocytes contained cyclin B(2)/p34(cdc2) complexes. However, cycloheximide-treated oocytes underwent GVBD following injection of constitutively active mitogen-activated protein kinase (MAPK) kinase 2 (MEK2), p33(Ringo), or Delta 90 cyclin B. MAPK activity increased just prior to the first meiotic division and remained stable thereafter. Although injection of constitutively active MEK2 induced GVBD, treatment with the MEK inhibitors U0126 or anthrax lethal factor delayed GVBD and prevented spindle formation. Interestingly, the ability of egg cytoplasm to induce GVBD was unaffected by the inhibition of MEK activity. Our results indicate that the synthesis of a novel or short-lived protein(s) which acts in a MEK-independent fashion is required in order for egg cytoplasm to induce GVBD in X. tropicalis oocytes.

Lower limb peripheral vascular disease (PVD) results from the occlusion of arteries leading to reduced blood flow and limb ischemia. Compensatory growth of blood vessels may be sufficient to overcome limb ischemia but for some patients therapeutic intervention, even limb amputation, is required. To gain insight into the mechanisms regulating compensatory growth of blood vessels we used a mouse model of hindlimb ischemia. The ischemic lesions were confirmed and compensatory arteriogenesis was evaluated by high frequency power Doppler ultrasound. In this model, compensatory growth occurs mainly through arteriogenesis. While signaling by mitogen activated protein kinase kinases 1 and 2 (MEK 1 and 2) is required for developmental and tumor angiogenesis, it is unknown whether MEK1/2 activity drives other physiologic vascular growth such as arteriogenesis. We hypothesized that MEK 1/2 activity is necessary for hindlimb re-vascularization following femoral artery ligation. To test this we ligated the femoral artery in mice and then treated them with PD0325901, an allosteric MEK1/2 inhibitor. Following femoral artery ligation active MEK signaling was detected in spindle-shaped cells located within regions of intense angiogenesis and myocyte proliferation. MEK inhibition with PD0325901 reversibly blocked neovascularization, muscle regeneration, and caused extensive coagulative necropathy. MEK inhibition not only prevented arteriogenesis in ischemic limbs but also caused a reduction in arterial diameter. Finally, MEK inhibition prevented accumulation of CD68+ cells in ischemic tissues and skewed systemic cytokine expression towards a persistent, pro-inflammatory phenotype. Our investigation provides pharmacologic evidence demonstrating an essential role for MEK signaling in angiogenesis and arteriogenesis in response to hind limb ischemia. Further, we observed activation of MEK signaling is required for accumulation of macrophages in ischemic tissues and prevents down-regulation of systemic pro-inflammatory cytokines. Lastly, since the effects of MEK inhibition on reperfusion are reversible, we can use MEK inhibitors to create a mouse model of chronic limb ischemia that mimics aspects of PVD.

Lethal factor is a protease, one component of Bacillus anthracis exotoxin, which cleaves many of the mitogen-activated protein kinase kinases (MEKs). Given the importance of MEK signaling in tumorigenesis, we assessed the effects of anthrax lethal toxin (LeTx) on tumor cells. LeTx was very effective in inhibiting mitogen-activated protein kinase activation in V12 H-ras-transformed NIH 3T3 cells. In vitro , treatment of transformed cells with LeTx caused them to revert to a nontransformed morphology, and inhibited their abilities to form colonies in soft agar and to invade Matrigel without markedly affecting cell proliferation. In vivo , LeTx inhibited growth of ras-transformed cells implanted in athymic nude mice (in some cases causing tumor regression) at concentrations that caused no apparent animal toxicity. Unexpectedly, LeTx also greatly decreased tumor neovascularization. These results demonstrate that LeTx potently inhibits ras-mediated tumor growth and is a potential antitumor therapeutic.

A search of the National Cancer Institute's Anti-Neoplastic Drug Screen for compounds with an inhibitory profile similar to that of the mitogen-activated protein kinase kinase (MAPKK) inhibitor PD098059 yielded anthrax lethal toxin. Anthrax lethal factor was found to inhibit progesterone-induced meiotic maturation of frog oocytes by preventing the phosphorylation and activation of mitogen-activated protein kinase (MAPK). Similarly, lethal toxin prevented the activation of MAPK in serum stimulated, ras-transformed NIH3T3 cells. In vitro analyses using recombinant proteins indicated that lethal factor proteolytically modified the NH2-terminus of both MAPKK1 and 2, rendering them inactive and hence incapable of activating MAPK. The consequences of this inactivation upon meiosis and transformed cells are also discussed.

Anthrax lethal toxin, produced by the bacterium Bacillus anthracis, is the major cause of death in animals infected with anthrax. One component of this toxin, lethal factor (LF), is suspected to be a metalloprotease, but no physiological substrates have been identified. Here it is shown that LF is a protease that cleaves the amino terminus of mitogen-activated protein kinase kinases 1 and 2 (MAPKK1 and MAPKK2) and that this cleavage inactivates MAPKK1 and inhibits the MAPK signal transduction pathway. The identification of a cleavage site for LF may facilitate the development of LF inhibitors.

By monitoring (45)Ca(2+) influx and efflux from oocytes a transient increase followed by a transient decrease in the Ca(2+)-content of progesterone-treated oocytes was observed. Chelation of intracellular Ca(2+) with EGTA or BAPTA-type buffers inhibited progesterone-induced GVBD. Buffers with a mid-range Kd (∼1.5 μM) were most effective in inhibiting GVBD whereas buffers with a Kd above or below this value were less effective. These observations indicate that intracellular Ca(2+), probably in the form of a localized release, is required for progesterone-induced oocyte maturation. However, Ca(2+) alone was insufficient to induce GVBD. When the effects of nocodazole and taxol upon this Ca(2+)-requirement were tested, we observed that taxol-induced microtubule polymerization not only delayed progesterone-induced GVBD but also completely inhibited it in combination with BAPTA-AM. Conversely, nocodazole-induced microtubule depolymerization in combination with ionophore A23187 not only accelerated progesterone-induced GVBD, but also induced GVBD in the absence of progesterone. The combined treatment of oocytes with nocodazole and InsP3, or with cold treatment and ionophore A23187 also induced GVBD in the absence of progesterone. Thus, Ca(2+) and microtubule depolymerization synergistically promote GVBD. In both nocodazole- and cold-treated oocytes, the GV was displaced to the periphery of the oocyte and underwent GVBD when treated with A23187. However, when the GV was displaced to the cortex by a centrifugal force under conditions that would not cause microtubule depolymerization and the oocyte was treated with A23187, oocytes did not undergo GVBD.

Human anthrax infection caused by exposure toBacillus anthraciscannot 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 inEscherichia colicould specifically identify LF with an affinity of3.46×107 L/mol and could neutralize LeTx with an EC50of 85 μg/mL. Even after LeTx challenge at various time points, the LF8-Fab demonstrated protection of J774A.1 cellsin vitro. The results suggest that the LF8-Fab might be further characterized and potentially be used for clinical applications against anthrax infection.

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.

‘Skepticism is a healthy response to diagnosis of any tumor as angiosarcoma. ‘ (Lane, 1952) Angiosarcoma (AS) is an aggressive malignancy of vascular tissue or vessel forming cells (Requena & Sangueza 1998). AS is rare in humans, making up 1-2 % of soft-tissue sarcomas (Young et al. 2010) and having an estimated incidence of 0.2/100,000 persons per year. Although AS can present anywhere in the body, in humans they typically arise in the skin or superficial soft tissues. It is most frequently noted on the face and scalp of elderly men where their persistent growth causes ulceration and infection, as well as on breasts, and extremities (Brennan et al. 2001; Fayette et al. 2007; Glazebrook et al. 2008). Less frequently AS arises in liver, heart, and spleen (Young et al. 2010). The literature is replete with retrospective analyses and case studies on AS but the rarity of patients diagnosed with this disease makes it difficult to perform more than a superficial investigation on the biology and clinical behavior of AS. However, the occurrence of AS is not restricted to humans and there are several alternative animal models with the potential to inform human studies. We have written this review to collect, compare, and contrast diverse reports on the biology and treatment of AS in humans and alternative animal models. Our objective is to establish a comparative framework to focus further discussion and scientific enquiry.