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Figure S1 related to Figure 1 shows that MLN4924 reduces oligo-ubiquitylation of Lats2 and that Merlin-mutant schwannoma cells have a lower on-target sensitivity to MLN4924 than a genetically matched Merlin-positive Schwann line. Also shows that canonical YAP target genes are not expressed highly in FC-1801 cells.

Figure S4 related to Figure 4 shows that GDC-0980 reduces mTORC1 and PI3K signaling in Merlin-mutant Meso-33 cells and the MSK-LX19 PDX model, p27 levels are dramatically increased by combined MLN4914 and GDC-0980 therapy, and that the treatments in Figure 4 were well-tolerated.

Figure S3 related to Figure 3 shows that animals maintained weight during the treatments administered in Figure 3.

Supplementary Materials and Methods, Supplementary Figure Legends, Supplementary Table 1, Supplementary References

Inactivation of NF2/Merlin causes the autosomal-dominant cancer predisposition syndrome familial neurofibromatosis type 2 (NF2) and contributes to the development of malignant pleural mesothelioma (MPM). To develop a targeted therapy for NF2-mutant tumors, we have exploited the recent realization that Merlin loss drives tumorigenesis by activating the E3 ubiquitin ligase CRL4DCAF1, thereby inhibiting the Hippo pathway component Lats. Here, we show that MLN4924, a NEDD8-activating enzyme (NAE) inhibitor, suppresses CRL4DCAF1 and attenuates activation of YAP in NF2-mutant tumor cells. In addition, MLN4924 sensitizes MPM to traditional chemotherapy, presumably as a result of collateral inhibition of cullin-RING ubiquitin ligases (CRL) involved in DNA repair. However, even in combination with chemotherapy, MLN4924 does not exhibit significant preclinical activity. Further analysis revealed that depletion of DCAF1 or treatment with MLN4924 does not affect mTOR hyperactivation in NF2-mutant tumor cells, suggesting that loss of Merlin activates mTOR independently of CRL4DCAF1. Intriguingly, combining MLN4924 with the mTOR/PI3K inhibitor GDC-0980 suppresses the growth of NF2-mutant tumor cells in vitro as well as in mouse and patient-derived xenografts. These results provide preclinical rationale for the use of NAE inhibitors in combination with mTOR/PI3K inhibitors in NF2-mutant tumors.

Figure S2 related to Figure 2 shows that DCAF1 knockdown suppresses the growth of Merlin-mutant mesothelioma cells.

Deficiency of the tumor suppressor Merlin causes development of schwannoma, meningioma, and ependymoma tumors, which can occur spontaneously or in the hereditary disease neurofibromatosis type 2 (NF2). Merlin mutations are also relevant in a variety of other tumors. Surgery and radiotherapy are current first-line treatments; however, tumors frequently recur with limited treatment options. Here, we use human Merlin-negative schwannoma and meningioma primary cells to investigate the involvement of the endogenous retrovirus HERV-K in tumor development. HERV-K proteins previously implicated in tumorigenesis were overexpressed in schwannoma and all meningioma grades, and disease-associated CRL4DCAF1 and YAP/TEAD pathways were implicated in this overexpression. In normal Schwann cells, ectopic overexpression of HERV-K Env increased proliferation and upregulated expression of c-Jun and pERK1/2, which are key components of known tumorigenic pathways in schwannoma, JNK/c-Jun, and RAS/RAF/MEK/ERK. Furthermore, FDA-approved retroviral protease inhibitors ritonavir, atazanavir, and lopinavir reduced proliferation of schwannoma and grade I meningioma cells. These results identify HERV-K as a critical regulator of progression in Merlin-deficient tumors and offer potential strategies for therapeutic intervention.Significance:The endogenous retrovirus HERV-K activates oncogenic signaling pathways and promotes proliferation of Merlin-deficient schwannomas and meningiomas, which can be targeted with antiretroviral drugs and TEAD inhibitors.

Supplementary Table 1Table 1 and legend

Figure S1Figure S1 and figure legend

Supplementary Table 2Table 2 and legend

Figure S6Figure S6 and figure legend

Figure S3Figure S3 and figure legend

Figure S7Figure S7 and figure legend

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder characterized by the development of multiple tumors in the central nervous system, most notably schwannomas, and meningiomas. Mutational inactivation of the NF2 gene encoding the protein Merlin is found in most sporadic and inherited schwannomas, but the molecular mechanisms underlying neoplastic changes in schwannoma cells remain unclear. We report here that Nf2-deficient cells display elevated expression levels of key enzymes involved in lipogenesis and that this upregulation is caused by increased activity of Torc1. Inhibition or knockdown of fatty acid synthase (FASN), the enzyme that catalyzes the formation of palmitic acid from malonyl-CoA, drove NF2-deficient cells into apoptosis. Treatment of NF2-mutant cells with agents that inhibit the production of malonyl-CoA reduced their sensitivity to FASN inhibitors. Collectively, these results suggest that the altered lipid metabolism found in NF2-mutant cells renders them sensitive to elevated levels of malonyl-CoA, as occurs following blockade of FASN, suggesting new targeted strategies in the treatment of NF2-deficient tumors. Cancer Res; 77(18); 5026–38. ©2017 AACR.

Figure S5Figure S5 and legend

Supplemental files describing metabolic alterations in NF2-deficient cells, supplementary materials and methods, and supportive data regarding the effects of FASN inhibitors or knockdown on various metabolic processes in WT and NF2-deficient cells.

Supplementary Table 3Table 3 and legend

Figure S4Figure S4 and figure legend

Schwannomas are tumors of the nervous system that occur sporadically and in patients with the cancer predisposition syndrome neurofibromatosis type 2 (NF2). Schwannomas and all NF2-related tumors are caused by loss of the tumor suppressor merlin. Using our human in vitro model for schwannoma, we analyzed extracellular signal-regulated kinase 1/2 (ERK1/2) and AKT signaling pathways, their upstream growth factor receptors, and their role in schwannoma cell proliferation and adhesion to find new systemic therapies for these tumors that, to date, are very difficult to treat. We show here that human primary schwannoma cells show an enhanced basal Raf/mitogen-activated protein/ERK kinase/ERK1/2 pathway activity compared with healthy Schwann cells. Due to a strong and prolonged activation of platelet-derived growth factor receptor β (PDGFRβ), which is highly overexpressed, ERK1/2 and AKT activation was further increased in schwannoma, leading to increased proliferation. Using specific inhibitors, we discovered that ERK1/2 activation involves the integrin/focal adhesion kinase/Src/Ras signaling cascades and PDGFRβ-mediated ERK1/2 activation is triggered through the phosphatidylinositol 3-kinase/protein kinase C/Src/c-Raf pathway. Due to the complexity of signals leading to schwannoma cell proliferation, potential new therapeutic agents should target several signaling pathways. The PDGFR and c-Raf inhibitor sorafenib (BAY 43-9006; Bayer Pharmaceuticals), currently approved for treatment of advanced renal cell cancer, inhibits both basal and PDGFRβ-mediated ERK1/2 and AKT activity and decreases cell proliferation in human schwannoma cells, suggesting that this drug constitutes a promising tool to treat schwannomas. We conclude that our schwannoma in vitro model can be used to screen for new therapeutic targets in general and that sorafenib is possible candidate for future clinical trials. [Cancer Res 2008;68(13):5236–45]

Supplementary Figure 1 from Dissecting and Targeting the Growth Factor–Dependent and Growth Factor–Independent Extracellular Signal-Regulated Kinase Pathway in Human Schwannoma

Supplementary Figure 3 from Dissecting and Targeting the Growth Factor–Dependent and Growth Factor–Independent Extracellular Signal-Regulated Kinase Pathway in Human Schwannoma

Supplementary Figure Legends 1-3 from Dissecting and Targeting the Growth Factor–Dependent and Growth Factor–Independent Extracellular Signal-Regulated Kinase Pathway in Human Schwannoma

Supplementary Figure 2 from Dissecting and Targeting the Growth Factor–Dependent and Growth Factor–Independent Extracellular Signal-Regulated Kinase Pathway in Human Schwannoma

Deficiency of the tumor suppressor Merlin causes development of schwannoma, meningioma, and ependymoma tumors, which can occur spontaneously or in the hereditary disease neurofibromatosis type 2 (NF2). Merlin mutations are also relevant in a variety of other tumors. Surgery and radiotherapy are current first-line treatments; however, tumors frequently recur with limited treatment options. Here, we use human Merlin-negative schwannoma and meningioma primary cells to investigate the involvement of the endogenous retrovirus HERV-K in tumor development. HERV-K proteins previously implicated in tumorigenesis were overexpressed in schwannoma and all meningioma grades, and disease-associated CRL4DCAF1 and YAP/TEAD pathways were implicated in this overexpression. In normal Schwann cells, ectopic overexpression of HERV-K Env increased proliferation and upregulated expression of c-Jun and pERK1/2, which are key components of known tumorigenic pathways in schwannoma, JNK/c-Jun, and RAS/RAF/MEK/ERK. Furthermore, FDA-approved retroviral protease inhibitors ritonavir, atazanavir, and lopinavir reduced proliferation of schwannoma and grade I meningioma cells. These results identify HERV-K as a critical regulator of progression in Merlin-deficient tumors and offer potential strategies for therapeutic intervention. Significance: The endogenous retrovirus HERV-K activates oncogenic signaling pathways and promotes proliferation of Merlin-deficient schwannomas and meningiomas, which can be targeted with antiretroviral drugs and TEAD inhibitors.

Background: Mutations in the NF2 gene, which codes for the tumor suppressor Merlin, is responsible for the development of all Neurofibromatosis Type 2 (NF2)-related tumors including schwannomas and meningiomas. These tumors can also occur spontaneously in non-NF2 patients. The only available treatments for this group of tumors are surgery and radiosurgery/radiotherapy. Therefore, in a search of effective drug-based treatments for NF2-mutated schwannomas and meningiomas, we are investigating TYRO3, AXL and MERTK (TAM) receptors as new therapeutic targets. Methods: The expression and activation of TAM receptors were investigated using human Merlin-deficient meningioma and schwannoma tissues compared to control by Western blotting. Infiltration of tumor associated macrophages and their correlation to TAM family receptors were investigated using immunofluorescence double staining and flow cytometry. To understand the role of TAM receptors in the pathogenesis of schwannomas and meningiomas, in vitro shRNA knock-down and pharmacological inhibition analysis were conducted using patient-derived schwannoma and meningioma cells. Results: The results revealed overexpression and hyper-activation of AXL, MERTK, TYRO3 in human Merlin-deficient meningiomas and schwannomas. Meningiomas and schwannomas show a varied level of infiltration by M2 macrophages (CD163+) and TAM receptors are expressed on these CD163+ macrophages in addition to the tumor cells. The expression of MERTK correlates to the level of infiltrated CD163+ macrophages. MERTK forms a complex with TYRO3 in both meningioma and schwannoma tissues, and its expression is mandatory to maintain AXL and TYRO3 levels in both cell types. MERTK and AXL contribute to the increased proliferation and survival of schwannoma and meningioma primary cells in vitro. Pathological proliferation and survival of both cell types were successfully reversed by AXL inhibitor BGB324 (BerGenBio, FDA approved for AML) and MERTK inhibitor UNC2025 (preclinical) in vitro, with UNC2025 being more effective. TYRO3 did not affect the proliferation of either schwannoma or meningioma primary cells. Conclusion: Our findings suggest that TAM receptors are aberrantly expressed and activated in human Merlin-deficient meningiomas and schwannomas. Infiltration of M2 macrophages may contribute to an overall increase in the expression of TAM receptors in these tumors. AXL and MERTK are important in schwannoma and meningioma pathogenesis and are potentially good therapeutic targets. MERTK inhibitor UNC2025 has the potential to be used as a common candidate for the treatment of NF2-related tumors. Citation Format: Foram Dave, Sylwia Ammoun, C Oliver Hanemann. TAM receptors as potential therapeutic targets in NF2-related schwannomas and meningiomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 941.

Deficiency of the tumor suppressor Merlin causes the development of schwannoma, meningioma and ependymoma tumors. These can occur spontaneously or in the hereditary disease Neurofibromatosis type 2 (NF2). Merlin mutations are also relevant in a variety of other tumors. Currently available treatments are surgery or radiosurgery which are only partially effective, with tumors frequently reoccurring and in case of meningioma often as a higher grade. There is an urgent need for effective drug treatments.In this study, we investigated the role of Human Endogenous Retrovirus Type K (HERV-K) and its targetability in Merlin negative schwannoma and meningioma tumors by using tissues and primary cells isolated form patients employing immunohistochemistry, immunocytochemistry, western blotting and proliferation assays. We demonstrate that HERV-K proteins are overexpressed in Merlin negative schwannoma and all meningioma grades. Furthermore, we implicate CRL4DCAF1 and YAPT/EAD Hippo pathway in this overexpression and suggest that exosome transport of the HERV-K Envelope (Env) protein might contribute to schwannoma development. We also show that: (i) Ectopic overexpression of HERV-K Env in normal Schwann cells increased proliferation and upregulated the transcription factor c-Jun. Env overexpression also increased activity of the extracellular signal-regulated kinase 1/2 (pERK1/2), a known mitogenic pathway in schwannoma. (ii) An anti-Env monoclonal antibody decreased pERK1/2 and reduced proliferation of schwannoma cells. iii) FDA-approved retroviral protease inhibitors Ritonavir, Atazanavir and Lopinavir decreased pERK1/2 and cyclin D1 and reduced proliferation of schwannoma and grade I meningioma cells. We provide evidence for HERV-K Env contributing to the development of NF2-associated schwannomas and meningiomas. Considering the urgent need of drugs to treat NF2, we suggest the trialling of antiretroviral protease inhibitors, and consideration of anti-HERV-K Env immunotherapy, as well as TEAD-specific inhibitors in these patients. The above treatments would also be potentially beneficial for patients with other tumors caused by Merlin deficiency. Citation Format: Sylwia Ammoun, Emmanuel A. Maze, Bora Agit, Robert Belshaw, C Oliver Hanemann. Human endogenous retrovirus type K promotes proliferation of Merlin negative schwannoma and meningioma which can be inhibited by anti-retroviral and anti-TEAD drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1164.

Inactivation of NF2/Merlin causes the autosomal-dominant cancer predisposition syndrome familial neurofibromatosis type 2 (NF2) and contributes to the development of malignant pleural mesothelioma (MPM). To develop a targeted therapy for NF2-mutant tumors, we have exploited the recent realization that Merlin loss drives tumorigenesis by activating the E3 ubiquitin ligase CRL4DCAF1, thereby inhibiting the Hippo pathway component Lats. Here, we show that MLN4924, a NEDD8-activating enzyme (NAE) inhibitor, suppresses CRL4DCAF1 and attenuates activation of YAP in NF2-mutant tumor cells. In addition, MLN4924 sensitizes MPM to traditional chemotherapy, presumably as a result of collateral inhibition of cullin-RING ubiquitin ligases (CRL) involved in DNA repair. However, even in combination with chemotherapy, MLN4924 does not exhibit significant preclinical activity. Further analysis revealed that depletion of DCAF1 or treatment with MLN4924 does not affect mTOR hyperactivation in NF2-mutant tumor cells, suggesting that loss of Merlin activates mTOR independently of CRL4DCAF1. Intriguingly, combining MLN4924 with the mTOR/PI3K inhibitor GDC-0980 suppresses the growth of NF2-mutant tumor cells in vitro as well as in mouse and patient-derived xenografts. These results provide preclinical rationale for the use of NAE inhibitors in combination with mTOR/PI3K inhibitors in NF2-mutant tumors.

In the original version of [this article][1] ([1][2]), the CYR61 Western blot on the left-hand side of Fig. 2C was accidentally duplicated to the right-hand portion of the figure panel. The error has been corrected in the latest online HTML and PDF versions of the article. The authors regret this

Merlin-deficient tumors of the nervous system are common. The most frequent ones are schwannomas and meningiomas. Classical chemotherapeutic treatments are not effective and surgery/radiosurgery leave patients with considerable morbidity. Therefore there is a need for new effective and systemic treatments. Thus, our goal is to analyze schwannomas pathobiology to define new therapeutic targets in order to develop effective treatments for those tumors. We have previously observed overexpression/activation of platelet-derived growth factor receptor beta (PDGFRβ) in human primary schwannoma cells leading to strong activation of Raf/MEK1/2/ERK1/2 and AKT pathways, and enhanced cell proliferation. These effects were completely inhibited by commercially available Raf/PDGFR inhibitor sorafenib (Bay 43-9006; Bayer Pharmaceuticals). Although sorafenib could be a promising drug for the treatment of schwannomas, the benign character of those tumors would require long-term therapy which may result in the development of side effects in some patients. Thus, alternative drugs inhibiting PDGFR signaling may be necessary. Moreover, in addition to PDGFR, aberrant activation of other receptor tyrosine kinases (RTKs) such as EGFR and ErbB2, have also been reported in schwannoma. Thus, PDGFR, EGFR and ErbB2 and their downstream signaling pathways ERK1/2 and AKT may represent attractive therapeutic targets in schwannoma. Although several novel agents targeting RTKs and their downstream signaling pathways are under clinical investigation, those drugs have not been evaluated for schwannoma. Thus, using our human in vitro schwannoma model, we have tested additional inhibitors of PDGFR imatinib and nilotinib (Novartis), EGFR/ErbB2 inhibitor lapatinib (GlaxoSmithKline) and MEK1/2 inhibitor AZD6244 (ARRY-142886; Astra Zeneca). Using western blotting and proliferation assays we have shown that PDGFR inhibitors imatinib and nilotinib efficiently inhibited PDGF-DD mediated ERK1/2 and AKT activation/phosphorylation and cell proliferation. Similarly, EGFR/ErbB2 inhibitor lapatinib efficiently inhibited heregulin mediated activation/phosphorylation of ErbB2, ERK1/2, AKT and S6 ribosomal protein and cell proliferation. The expression of anti-apoptotic protein survivin was also decreased. MEK1/2 inhibitor AZD6244, was also very effective abolishing PDGFRβ mediated ERK1/2 activation and cell proliferation. In summary, imatinib, nilotinib, lapatinib and AZD6244 effectively inhibited signaling pathways involved in enhanced schwannoma proliferation and survival, suggesting that those agents used separately or in combination, could be promising drug candidates for schwannoma treatment. Therefore, next approach will be the translation of our in vitro studies into a clinical setting. Those drugs are under investigation and have been successfully evaluated in both Phase I and II studies, so the re-profiling of these drugs should be straightforward. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C164.