Edwin Jousma

MEK inhibition exhibits efficacy in human and mouse neurofibromatosis tumors

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.

EGFR-STAT3 signaling promotes formation of malignant peripheral nerve sheath tumors

Malignant peripheral nerve sheath tumors (MPNSTs) develop sporadically or in the context of neurofibromatosis type 1. Epidermal growth factor receptor (EGFR) overexpression has been implicated in MPNST formation, but its precise role and relevant signaling pathways remain unknown. We found that EGFR overexpression promotes mouse neurofibroma transformation to aggressive MPNST (GEM-PNST). Immunohistochemistry demonstrated phosphorylated STAT3 (Tyr705) in both human MPNST and mouse GEM-PNST. A specific JAK2/STAT3 inhibitor FLLL32 delayed MPNST formation in an MPNST xenograft nude mouse model. STAT3 knockdown by shRNA prevented MPNST formation in vivo. Finally, reducing EGFR activity strongly reduced pSTAT3 in vivo. Thus, an EGFR–STAT3 pathway is necessary for MPNST transformation and establishment of MPNST xenografts growth but not for tumor maintenance. Efficacy of the FLLL32 pharmacological inhibitor in delaying MPNST growth suggests that combination therapies targeting JAK/STAT3 might be useful therapeutics.

Preclincial testing of sorafenib and RAD001 in the Nf(flox/flox) ;DhhCre mouse model of plexiform neurofibroma using magnetic resonance imaging.

Neurofibromatosis type 1 (NF1) is an inherited disease predisposing affected patients to variable numbers of benign neurofibromas. To date there are no effective chemotherapeutic drugs available for this slow growing tumor. Molecularly targeted agents that aim to slow neurofibroma growth are being tested in clinical trials. So preclinical models for testing potential therapies are urgently needed to prioritize drugs for clinical trials of neurofibromas.

Insertional mutagenesis identifies a STAT3/Arid1b/β-catenin pathway driving neurofibroma initiation

To identify genes and signaling pathways that initiate Neurofibromatosis type 1 (NF1) neurofibromas, we used unbiased insertional mutagenesis screening, mouse models, and molecular analyses. We mapped an Nf1-Stat3-Arid1b/β-catenin pathway that becomes active in the context of Nf1 loss. Genetic deletion of Stat3 in Schwann cell progenitors (SCPs) and Schwann cells (SCs) prevents neurofibroma formation, decreasing SCP self-renewal and β-catenin activity. β-catenin expression rescues effects of Stat3 loss in SCPs. Importantly, P-STAT3 and β-catenin expression correlate in human neurofibromas. Mechanistically, P-Stat3 represses Gsk3β and the SWI/SNF gene Arid1b to increase β-catenin. Knockdown of Arid1b or Gsk3β in Stat3(fl/fl);Nf1(fl/fl);DhhCre SCPs rescues neurofibroma formation after in vivo transplantation. Stat3 represses Arid1b through histone modification in a Brg1-dependent manner, indicating that epigenetic modification plays a role in early tumorigenesis. Our data map a neural tumorigenesis pathway and support testing JAK/STAT and Wnt/β-catenin pathway inhibitors in neurofibroma therapeutic trials.

Preclinical assessments of the MEK inhibitor PD-0325901 in a mouse model of neurofibromatosis type 1

Neurofibromatosis type 1 (NF1) is a genetic disorder that predisposes affected individuals to formation of benign neurofibromas, peripheral nerve tumors that can be associated with significant morbidity. Loss of the NF1 Ras–GAP protein causes increased Ras–GTP, and we previously found that inhibiting MEK signaling downstream of Ras can shrink established neurofibromas in a genetically engineered murine model.

An inflammatory gene signature distinguishes neurofibroma Schwann cells and macrophages from cells in the normal peripheral nervous system

Neurofibromas are benign peripheral nerve tumors driven by NF1 loss in Schwann cells (SCs). Macrophages are abundant in neurofibromas, and macrophage targeted interventions may have therapeutic potential in these tumors. We generated gene expression data from fluorescence-activated cell sorted (FACS) SCs and macrophages from wild-type and mutant nerve and neurofibroma to identify candidate pathways involved in SC-macrophage cross-talk. While in 1-month-old Nf1 mutant nerve neither SCs nor macrophages significantly differed from their normal counterparts, both macrophages and SCs showed significantly altered cytokine gene expression in neurofibromas. Computationally reconstructed SC-macrophage molecular networks were enriched for inflammation-associated pathways. We verified that neurofibroma SC conditioned medium contains macrophage chemo-attractants including colony stimulation factor 1 (CSF1). Network analysis confirmed previously implicated pathways and predict novel paracrine and autocrine loops involving cytokines, chemokines, and growth factors. Network analysis also predicted a central role for decreased type-I interferon signaling. We validated type-I interferon expression in neurofibroma by protein profiling, and show that treatment of neurofibroma-bearing mice with polyethylene glycolyated (PEGylated) type-I interferon-α2b reduces the expression of many cytokines overexpressed in neurofibroma. These studies reveal numerous potential targetable interactions between Nf1 mutant SCs and macrophages for further analyses.

Abstract 2937: An EGFR-STAT3 pathway promotes NF1 peripheral nerve tumorigenesis and transformation

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; Nf1 flox/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 Nf1 flox/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 Nf1 flox/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 A04: An inflammatory gene signature distinguishes neurofibroma Schwann cells and macrophages from cells in the normal peripheral nervous system

Neurofibromas are benign peripheral nerve tumors driven by NF1 loss in Schwann cells (SCs). Current evidence suggests that an inflammatory environment is critical for neurofibroma development and growth, however the roles of intercellular interactions between SCs and other cells in neurofibroma microenvironment are not clear yet. Neurofibromas contain numerous macrophages. We analyzed gene expression in FACS-sorted SCs and macrophages from wild type nerve and neurofibroma. Nerve macrophage transcriptomes differed from previously defined macrophage sub-populations. In 1-month-old Nf1 mutant nerve, neither SCs nor macrophages significantly differed from their counterparts in normal mice. In 7-month-old neurofibromas, macrophages showed significantly altered gene expression, as did neurofibroma SCs. Computational reconstruction of SC-macrophage molecular networks based on the gene expression data revealed extensive inflammatory-associated signaling and possible interplay between macrophages and Schwann cells. Among these were previously implicated pathways and novel paracrine and autocrine loops involving cytokines, chemokines, and growth factors. Specifically, the data predicted increased cytokine expression and imbalanced type-I/type-II interferon signaling, which were confirmed by protein profiling, and normalized in neurofibroma-bearing mice treated with PEGylated interferon-α2b. These studies identify Nf1 mutant SCs interactions with macrophages, which result in chronic inflammation and neurofibroma, and provide a system in which to study early changes in benign tumorigenesis. Citation Format: Kwangmin Choi, Kakajan Komurov, Jonathan S. Fletcher, Edwin Jousma, Jose A. Cancelas, Jianqiang Wu, Nancy Ratner. An inflammatory gene signature distinguishes neurofibroma Schwann cells and macrophages from cells in the normal peripheral nervous system. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A04.

Abstract A2: Using plexiform neurofibroma preclinical therapeutics to help guide clinical trials

Background: Neurofibromatosis type 1 (NF1) patients are predisposed to develop benign plexiform neurofibromas (PNs) that cause substantial morbidity. Surgery, the only standard treatment, is not feasible for most tumors. The NF1 gene product accelerates Ras-GTP hydrolysis to Ras-GDP and thus functions as a potent negative regulator of Ras. We developed a genetically engineered mouse model of neurofibroma, DhhCre;Nf1fl/fl , for use in preclinical testing; response is monitored by MRI imaging and volumetric analysis in conjunction with pharmacokinetic and pharmacodynamic readouts. The most effective neurofibroma therapy in this neurofibroma mouse model to date is drug candidates targeting inhibition of MEK1/2 (Jessen, W. et al., J. Clin. Invest. , 2013). Results: We have continued study of the allosteric MEK inhibitor PD-0325901 using the DhhCre;Nf1fl/fl model. To establish a minimum effective dose (MED) in this mouse model we administered doses of 0.5 – 10mg/kg/day for 2 months. Neurofibromas shrank at all dose levels, correlating with significant reductions in cell proliferation and tumor vasculature, despite significant differences in PD-0325901 blood levels. To test if early MEK inhibition prevents neurofibroma growth, we administered 1.5 mg/kg/day PD-0325901 before tumors formed in the mouse, for 3 months. Neurofibromas formed, but their size was reduced. There were no rebound effects off drug. Selumetinib (AZD6244; ARRY-142886), an orally bioavailable inhibitor of MEK1/2, is being tested in a Phase 1 trial in individuals with NF1 and inoperable PNs, with response monitored using MRI and volumetric analysis. Selumetinib is administered twice daily on a continuous dosing schedule (1 cycle=28 days); the maximum tolerated dose (MTD) is determined based on toxicities observed during the first 3 cycles. To date 12 subjects (median age 14 years, range, 5-18 years) have been enrolled. Six subjects each have been treated with 20 mg/m 2 /dose (50% of the dose recommended in adults with solid tumors) or 30 mg/m2/dose. The most frequent adverse events included acneiform rash, asymptomatic CPK elevation, and mild gastrointestinal toxicity. Dose limiting toxicities (DLT) in 2 of 6 patients at the 30 mg/m 2 dose level were asymptomatic and reversible CPK elevation (n=1) and decrease in left ventricular ejection fraction (n=1), so this dose exceeded the MTD. Only 1 of 6 patients enrolled at the 20 mg/m2 dose level developed DLT (cellulitis); this dose has been identified as the MTD for this patient population. Partial responses (PN volume decrease ≥20%) have been observed at both dose levels in 5/9 subjects. Tumor shrinkage occurred slowly over time in progressive and non-progressive PNs, and progressive disease has not been observed to date. Enrollment on this study is ongoing, and a Phase 2 study is being planned. Conclusions: A genetically engineered mouse model of neurofibromatosis is predictive of response to therapy in human NF1, supporting use of a drug testing platform that integrates mouse models and human trials. MEK inhibition downstream of Ras-GTP may be effective as a single agent, at low doses, to treat pediatric patients with neurofibroma and warrants further study. Supported by NIHP50-NS057531 and the Children9s Tumor Foundation Neurofibromatosis Therapeutic Consortium to NR. Citation Format: Jianqiang Wu, Edwin Jousma, Tilat Rizvi, Richard Scott Dunn, David R. Jones, Timothy P. Cripe, Mi-Ok Kim, Leigh Marcus, Eva Dombi, Andrea Baldwin, Lauren Meyerson Long, Gillespie Andrea, Patricia Whitcomb, Staci Martin Peron, Brigitte Widemann, David Janhofer, Nancy Ratner. Using plexiform neurofibroma preclinical therapeutics to help guide clinical trials. [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 A2.