PDF file, 595K, Supplementary Figure S2. Gene Interaction Network of 339 Ras-Driven Genes Differentially Expressed in Mouse or Human Neurofibromas or MPNSTs. Supplementary Figure S3. Dot plot showing qPCR DNA-copy number results for AURKA gene locus in 37 samples analyzed. Supplementary Figure S4. Expressional down regulation or inhibition of Aurora Kinase Reduces Survival in MPNST Cells.

PDF file - 488K, Insertion into the mouse TC21 gene causes a null allele. Supplemental Table 1. Loss of TC21 partially rescues Nf1 mutant embryonic lethality. Supplemental Figure 2: Loss of TC21 decreases the survival of NPCis mice by increasing brain tumors. Supplemental Figure 3. Cell death in spheres and TGFbeta1 expression in sciatic nerves and neurofibromas. Supplemental Figure 4: Nf1-/- DRGs and spheres express high levels of phospho-AKT but not phospho-SMAD2/3. Supplemental Figure 5: Expression of TGFbeta ligands and receptors in mouse MPNST and human MPNST cell lines

Supplementary Materials and Methods; Figure S1. Western blot of tropomyosin isoforms in neuroblastoma cells used in this project; Figure S2. Effect of combining tropomyosin and microtubule inhibitors on cell growth; Figure S3. A, B, Effect of TR100 or ATM-3507 in combination with paclitaxel in CHLA- 20 neuroblastoma cells; Figure S4. Cytotoxicity of CHLA-20 cells treated with different dosing regimens of TR100 plus VCR; Figure S5. Effect of combining TR100 and vincristine on tumor growth;Figure S6. Weights of animals from Fig. 4 and S5; Figure S7. The combination of tropomyosin inhibitors plus vincristine did not impact the microtubule network of cells in interphase; Figure S8. The combination of TR100 and paclitaxel induces apoptosis in CHLA-20 neuroblastoma cells; Table S1. IC50 concentrations for TR100 and ATM-3057 in a panel of neuroblastoma cell lines; Table S2. Pharmacokinetic analysis after single dosing of ATM3507/Dexolve intravenously at 30mg/kg in non-tumor bearing immunocompetent mice (Balb/C, n=3).

PDF file - 198KB, Supplementary Figure S1. Combinatorial treatment with bortezomib and oHSV1716 induce synergistic cell killing against S462TY malignant peripheral nerve sheath tumor (MPNST) cell. Supplementary Figure S2. Induction of ER stress and unfolded protein response (UPR) in CAL27 head and neck cancer cells treated with bortezomib.

XLS file, 636K, 339 Ras-Driven Genes Differentially Expressed in Mouse or Human Neurofibromas or MPNSTs.

PDF file - 164KB, Supplementary Table S1. Median-effect doses of bortezomib and oHSV (34.5ENVE) in vitro. Supplementary Table S2. Statistical analysis of viral replication data. Data shown are the fold increase in virus replication after bortezomib treatment of the indicated cells.

Abstract Actin filaments, with their associated tropomyosin polymers, and microtubules are dynamic cytoskeletal systems regulating numerous cell functions. While antimicrotubule drugs are well-established, antiactin drugs have been more elusive. We previously targeted actin in cancer cells by inhibiting the function of a tropomyosin isoform enriched in cancer cells, Tpm3.1, using a first-in-class compound, TR100. Here, we screened over 200 other antitropomyosin analogues for anticancer and on-target activity using a series of in vitro cell-based and biochemical assays. ATM-3507 was selected as the new lead based on its ability to disable Tpm3.1-containing filaments, its cytotoxicity potency, and more favorable drug-like characteristics. We tested ATM-3507 and TR100 alone and in combination with antimicrotubule agents against neuroblastoma models in vitro and in vivo. Both ATM-3507 and TR100 showed a high degree of synergy in vitro with vinca alkaloid and taxane antimicrotubule agents. In vivo, combination-treated animals bearing human neuroblastoma xenografts treated with antitropomyosin combined with vincristine showed minimal weight loss, a significant and profound regression of tumor growth and improved survival compared with control and either drug alone. Antitropomyosin combined with vincristine resulted in G2–M phase arrest, disruption of mitotic spindle formation, and cellular apoptosis. Our data suggest that small molecules targeting the actin cytoskeleton via tropomyosin sensitize cancer cells to antimicrotubule agents and are tolerated together in vivo. This combination warrants further study. Mol Cancer Ther; 16(8); 1555–65. ©2017 AACR.

Abstract Background: Bortezomib is an FDA-approved proteasome inhibitor, and oncolytic herpes simplex virus-1 (oHSV) is a promising therapeutic approach for cancer. We tested the impact of combining bortezomib with oHSV for antitumor efficacy. Experimental Design: The synergistic interaction between oHSV and bortezomib was calculated using Chou–Talalay analysis. Viral replication was evaluated using plaque assay and immune fluorescence. Western blot assays were used to evaluate induction of estrogen receptor (ER) stress and unfolded protein response (UPR). Inhibitors targeting Hsp90 were utilized to investigate the mechanism of cell killing. Antitumor efficacy in vivo was evaluated using subcutaneous and intracranial tumor xenografts of glioma and head and neck cancer. Survival was analyzed by Kaplan–Meier curves and two-sided log-rank test. Results: Combination treatment with bortezomib and oHSV (34.5ENVE), displayed strong synergistic interaction in ovarian cancer, head and neck cancer, glioma, and malignant peripheral nerve sheath tumor (MPNST) cells. Bortezomib treatment induced ER stress, evident by strong induction of Grp78, CHOP, PERK, and IRE1α (Western blot analysis) and the UPR (induction of hsp40, 70, and 90). Bortezomib treatment of cells at both sublethal and lethal doses increased viral replication (P < 0.001), but inhibition of Hsp90 ablated this response, reducing viral replication and synergistic cell killing. The combination of bortezomib and 34.5ENVE significantly enhanced antitumor efficacy in multiple different tumor models in vivo. Conclusions: The dramatic synergy of bortezomib and 34.5ENVE is mediated by bortezomib-induced UPR and warrants future clinical testing in patients. Clin Cancer Res; 20(14); 3787–98. ©2014 AACR.

Abstract Children with high risk neuroblastoma still have a poor response rate to existing chemotherapeutics making it imperative that new classes of compounds are developed to treat this disease. The actin cytoskeleton is an ideal chemotherapeutic target due to its role in numerous biological processes essential for tumor cell growth and survival. Targeting actin however has been problematic due to unacceptable levels of toxicity associated with impacting actin containing structures essential for normal cell function. We have developed a novel class of compounds which target tropomyosin, the second core component of an actin microfilament. By targeting the cancer associated tropomyosin, Tm5NM1, we are able to discriminate between the actin filament populations in normal and transformed cells. We have demonstrated that our first in class anti-tropomyosin compound, TR100, impacts actin filament integrity leading to tumor cell death in vitro and in vivo in neuroblastoma models. In this study we elucidate the molecular mechanisms by which disruption of the actin cytoskeleton by TR100 induces tumor cell death. We also investigate the efficacy of this novel class of compound in combination with existing chemotherapeutics. Preliminary studies have demonstrated that low exposure of neuroblastoma cells to TR100 results in a G0G1 arrest. Increased exposure leads to the activation of the mitochondrial apoptotic pathway as measured by increases in both caspase activity and mitochondrial permeability. To delineate the signaling pathways involved in anti-tropomyosin compound induced apoptosis, we have conducted a kinexus phospho-array. Using this approach we have identified the activation of key stress response pathways. Treatment of the SH-EP neuroblastoma cell line with anti-tropomyosin compounds for 8h results in reduced phospho-activity of key intermediates of the MEK/ERK pathway, in particular a significant decrease in the phosphorylation of the RSK1/2 family of kinases. Preliminary data suggest that the compounds mediate their effect through the downregulation of the MEK/ERK and p38/JNK cell survival pathways. We have now extended this study to investigate the impact of the drugs in combination with other established chemotherapeutic agents. In particular compounds which target the microtubules, another key cytoskeletal structure within the cells. In CHLA20 neuroblastoma cells, TR100 showed significant synergy when used in combination with both paclitaxel (microtubule stabilizing) and vincristine (microtubule destabilizing) with a combinatorial index < 0.5. We are now extending this study to examine if this synergy is observed in a panel of neuroblastoma cell lines and determine the mechanism of action. We are also now investigating the toxicity and efficacy of the combinatorial approach in vitro and in vivo. Using this approach we hope to identify a potential molecular biomarker that would allow us to predict the response of patients to this combinatorial treatment. Delineation the molecular mechanisms and signaling pathways involved in anti-tropomyosin mediated cell death as a single agent and in combination will allow us to determine the potential use of these compounds as an adjunct therapy in the treatment of patients with neuroblastoma. Citation Format: Justine R. Stehn, Duo Chen, Mark A. Currier, David Eaves, Melissa Desouza, Matthew Moosavian, Timothy P. Cripe, Peter W. Gunning. Synergistic anti-neuroblastoma efficacy using combinatorial cytoskeletal inhibitors. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B50.

ABSTRACTThe combination of docetaxel and gemcitabine is frequently used to treat recurrent bone sarcoma. Nanoparticle albumin‐bound paclitaxel (nab‐paclitaxel) is less toxic and more active than docetaxel or paclitaxel for breast cancer patients. The combination of nab‐paclitaxel and gemcitabine has preclinical synergy and is approved to treat pancreatic cancer. We observed growth inhibition and improved survival with nab‐paclitaxel in a Ewing sarcoma xenograft, and activity was additive with gemcitabine in an osteosarcoma model. Primary Ewing sarcoma tumors expressed the transport protein SPARC, previously associated with nab‐paclitaxel activity. These findings provide rationale for further evaluation of nab‐paclitaxel with gemcitabine for bone sarcoma. Pediatr Blood Cancer 2014;61:2096–2098. © 2014 Wiley Periodicals, Inc.

Abstract Neurofibromatosis type 1 (NF1) is a very common inherited disease, affecting 1:3500 individuals worldwide. Nearly all (95%) of NF1 patients develop benign neurofibromas and malignant peripheral nerve sheath tumors(MPNSTs). Currently, their prevention is not possible, partially because the molecular mechanisms of tumorigenesis and the molecules that mark benign neurofibroma formation are poorly understood. This study is to test the relevance of EGFR expression to neurofibroma formation, and to identify possible additional pathways and genes that might contribute to neurofibroma formation. We bred the Nf1 flox/flox;DhhCre mice, 100% of which form neurofibromas (Wu et al., 2008), to CNP-hEGFR mice and to Wa2 mice, an EGFR hypomorphic allele. To test the role of EGFR in tumorigenesis, we also used sleeping beauty (SB) insertional mutagenesis to obtain quadruple transgenic mice (Rosa26-lsl-SB11;T2/Onc; Nf1flox/flox;DhhCre). To define neurofibroma initiation and progression genes, we used Pyrosequencing to identify common insertion sites (CISs) that had more SB insertions that are most likely to harbor disease-related genes. We used ingenuity pathway analysis to predict pathways and genes that might contribute to neurofibroma formation. We used a “neurofibroma sphere” culture system, a method used for detecting self-renewing stem/progenitor cells, to determine inhibitory effects of a STAT3 inhibitor (FLLL32). We immunostained human and mouse sections with anti-pSTAT3 (tyr705) to determine STAT3 activation status. We found that mouse neurofibroma number and size increased in Nf1flox/flox;DhhCre mice with hEGFR expressed in nerve Schwann cells. Diminished EGFR signaling in Nf1 flox/flox;DhhCre, Wa2/+ mice decreased neurofibroma number, not size. We used insertional mutagenesis to identify other modifiers of neurofibroma tumorigenesis. Analysis of CISs identified hubs involving GSK3B, TNF, and STAT3. STAT3 was the most significant changed pathway. Inhibition of STAT3 by shRNA or a specific STAT3 inhibitor FLLL32 blocked human neurofibroma-sphere formation. Immunohistochemistry identified STAT3(p705) in human and mouse neurofibromas and MPNSTs. FLLL32 inhibited cell proliferation and stimulated cell death as well as reduced neurofibroma growth in vivo in the Nf1flox/flox;DhhCre mouse neurofibromas. STAT3 knockdown by shRNA prevented MPNST formation in vivo. Finally, reducing EGFR activity strongly reduced pSTAT3 in vivo. Thus, an EGFR-STAT3 pathway regulates neurofibroma number and neurofibroma growth, and promotes transformation. STAT3 inhibitors may be useful in NF1 therapeutics. (*This work was supported by the National Institutes of Health (R01 NS28840 to N.R. and P50 NS057531 to N.R. and D.L.) and an Ohio State University Comprehensive Cancer Center pelotonia idea award to J.W.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2937. doi:1538-7445.AM2012-2937

Abstract Ras superfamily proteins participate in TGF-β—mediated developmental pathways that promote either tumor suppression or progression. However, the specific Ras proteins, which integrate in vivo with TGF-β signaling pathways, are unknown. As a general approach to this question, we activated all Ras proteins in vivo by genetic deletion of the RasGAP protein Nf1 and examined mice doubly deficient in a Ras protein to determine its requirement in formation of TGF-β—dependent neurofibromas that arise in Nf1-deficient mice. Animals lacking Nf1 and the Ras-related protein R-Ras2/TC21 displayed a delay in formation of neurofibromas but an acceleration in formation of brain tumors and sarcomas. Loss of R-Ras2 was associated with elevated expression of TGF-β in Nf1-deficient Schwann cell precursors, blockade of a Nf1/TGFβRII/AKT-dependent autocrine survival loop in tumor precursor cells, and decreased precursor cell numbers. Furthermore, the increase in size of sarcomas from xenografts doubly deficient in these genes was also found to be TGF-β—dependent, in this case resulting from cell nonautonomous effects on endothelial cells and myofibroblasts. Extending these findings in clinical specimens, we documented an increase in TGF-β ligands and an absence of TGF-β receptor II in malignant peripheral nerve sheath tumors, which correspond to tumors in the Nf1-deficient mouse model. Together, our findings reveal R-Ras2 as a critical regulator of TGF-β signaling in vivo. Cancer Res; 72(20); 5317–27. ©2012 AACR.

Neurofibromatosis type 1 (NF1) patients develop benign neurofibromas and malignant peripheral nerve sheath tumors (MPNST). These incurable peripheral nerve tumors result from loss of NF1 tumor suppressor gene function, causing hyperactive Ras signaling. Activated Ras controls numerous downstream effectors, but specific pathways mediating the effects of hyperactive Ras in NF1 tumors are unknown. We performed cross-species transcriptome analyses of mouse and human neurofibromas and MPNSTs and identified global negative feedback of genes that regulate Ras/Raf/MEK/ERK signaling in both species. Nonetheless, ERK activation was sustained in mouse and human neurofibromas and MPNST. We used a highly selective pharmacological inhibitor of MEK, PD0325901, to test whether sustained Ras/Raf/MEK/ERK signaling contributes to neurofibroma growth in a neurofibromatosis mouse model (Nf1(fl/fl);Dhh-Cre) or in NF1 patient MPNST cell xenografts. PD0325901 treatment reduced aberrantly proliferating cells in neurofibroma and MPNST, prolonged survival of mice implanted with human MPNST cells, and shrank neurofibromas in more than 80% of mice tested. Our data demonstrate that deregulated Ras/ERK signaling is critical for the growth of NF1 peripheral nerve tumors and provide a strong rationale for testing MEK inhibitors in NF1 clinical trials.

Ras superfamily proteins participate in TGF-b—mediated developmental pathways that promote either tumor suppression or progression. However, the specific Ras proteins, which integrate in vivo with TGF-b signaling pathways, are unknown. As a general approach to this question, we activated all Ras proteins in vivo by genetic deletion of the RasGAP protein Nf1 and examined mice doubly deficient in a Ras protein to determine its requirement in formation of TGF-b—dependent neurofibromas that arise in Nf1-deficient mice. Animals lacking Nf1 and the Ras-related protein R-Ras2/TC21 displayed a delay in formation of neurofibromas but an acceleration in formation of brain tumors and sarcomas. Loss of R-Ras2 was associated with elevated expression of TGF-b in Nf1-deficient Schwann cell precursors, blockade of a Nf1/TGFbRII/AKT-dependent autocrine survival loop in tumor precursor cells, and decreased precursor cell numbers. Furthermore, the increase in size of sarcomas from xenografts doubly deficient in these geneswas also found to be TGF-b—dependent, in this case resulting from cell nonautonomous effects on endothelial cells and myofibroblasts. Extending these findings in clinical specimens, we documented an increase in TGF-b ligands and an absence of TGF-b receptor II in malignant peripheral nerve sheath tumors, which correspond to tumors in the Nf1-deficient mouse model. Together, our findings reveal R-Ras2 as a critical regulator of TGF-b signaling in vivo. Cancer Res; 72(20); 5317–27. 2012 AACR.

The TC21/R-Ras2 protein is an oncogenic member of the Ras family. Ras proteins act as binary switches which are ‘active’ when bound to GTP and ‘inactive’ when bound to GDP. GTPase activating proteins (GAPs) accelerate the hydrolysis from Ras-GTP to Ras-GDP. This Ras-related GTPase TC21 has transforming capabilities similar to H, N and K-Ras. Activated alleles of TC21 transform epithelial and fibroblast cell lines and induce tumors in vivo. To explore the role of TC21 in cancer we used a model of Ras activation driven by loss of the NF1 tumor suppressor protein, a GAP for all Ras proteins including TC21. Thus loss of NF1 predicts increased levels of activated TC21/R-Ras2. NF1 loss in Schwann cells of the peripheral nervous system causes benign tumors known as neurofibromas. Neurofibromas can transform to sarcomas known as malignant peripheral nerve sheath tumors (MPNSTs). We found that TC21 loss delayed benign neurofibroma formation in Nf1fl/fl;DhhCre mice. Nf1 loss increased mRNA encoding the cytokine transforming growth factor-beta (TGF-β) and rendered Schwann cell progenitors insensitive to TGF-β; these phenotypes could be rescued by the Ras-related protein TC21/R-Ras2 and were mediated through TGF-β receptors. Conversely, growth of Nf1;Trp53 brain tumors and NF1−/− MPNST sarcomas were accelerated by TC21 loss. MPNST from Nf1;Trp53 mice and NF1−/− MPNST xenografts had increased levels of mRNA encoding TGF- ≤ ligands, and blocking TGF- ≤ decreased sarcoma size induced by shTC21. In benign and malignant tumors an AKT-dependent pathway regulated TGF- ≤ expression. Indicating relevance to human tumorigenesis, global gene expression analyses demonstrated increases in expression of TGF- ≤ ligands and decreases in TGF- ≤ receptors in human neurofibromas and MPNSTs. Thus, we identify critical roles for TC21 in regulation of TGF- ≤ expression. The results are important because TGF- ≤ acts as a tumor suppressor in numerous types of benign tumors and is also known for its oncogenic role in cellular transformation and tumor progression. While it has been known that Ras proteins are involved in TGF- ≤ mediated tumor suppression and oncogenesis, integration between the pathways is incompletely understood, especially in vivo. The study demonstrates that TC21 can be a major regulator of the duality of TGF- ≤ effects on tumorigenesis in vivo. This work was supported by a grant to NR (NIH P50NS057531-03). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1433. doi:1538-7445.AM2012-1433

AbstractPurpose: Patients with neurofibromatosis type 1 (NF1) develop malignant peripheral nerve sheath tumors (MPNST), which are often inoperable and do not respond well to current chemotherapies or radiation. The goal of this study was to use comprehensive gene expression analysis to identify novel therapeutic targets.Experimental Design: Nerve Schwann cells and/or their precursors are the tumorigenic cell types in MPNST because of the loss of the NF1 gene, which encodes the RasGAP protein neurofibromin. Therefore, we created a transgenic mouse model, CNP-HRas12V, expressing constitutively active HRas in Schwann cells and defined a Ras-induced gene expression signature to drive a Bayesian factor regression model analysis of differentially expressed genes in mouse and human neurofibromas and MPNSTs. We tested functional significance of Aurora kinase overexpression in MPNST in vitro and in vivo using Aurora kinase short hairpin RNAs (shRNA) and compounds that inhibit Aurora kinase.Results: We identified 2,000 genes with probability of linkage to nerve Ras signaling of which 339 were significantly differentially expressed in mouse and human NF1-related tumor samples relative to normal nerves, including Aurora kinase A (AURKA). AURKA was dramatically overexpressed and genomically amplified in MPNSTs but not neurofibromas. Aurora kinase shRNAs and Aurora kinase inhibitors blocked MPNST cell growth in vitro. Furthermore, an AURKA selective inhibitor, MLN8237, stabilized tumor volume and significantly increased survival of mice with MPNST xenografts.Conclusion: Integrative cross-species transcriptome analyses combined with preclinical testing has provided an effective method for identifying candidates for molecular-targeted therapeutics. Blocking Aurora kinases may be a viable treatment platform for MPNST. Clin Cancer Res; 18(18); 5020–30. ©2012 AACR.