Lay Summary

Neurofibromatosis type 1 (NF1) is common, occurring in 1/3000 live births, and results in skin pigmentation and the growth of tumors along nerves in the skin, brain, and other parts of the body. It arises from a mutated and non-functional tumor suppressor bearing its name.  Tumors of the peripheral nerves, called neurofibromas, and of the main eye nerve, called  optic glioma, are debilitating and deadly.  Neurofibromas contain cancerous NF1-deficient Schwann cells as well as NF1^+/- mast cells, endothelial cells and fibroblasts that contribute to neurofibroma and growth.  Optic glioma is composed of NF1-deficient neoplastic glial cell types (astrocytes, glioma stem cells) coupled with NF1^+/- astrocytes, neurons and microglia. Although there are several human clinical trials underway, there is no known cure or treatment of NF1.   The main objective of this proposal is to explore a novel treatment strategy for the NF1. 

          My lab discovered steps leading to the loss of a subset of cells in the pediatric nervous system called the Schwann cells that become tumors.  In this proposal I am putting forth an entirely novel approach by which the growth of these Schwann cells that form tumors can be arrested, and the same cells can be made to function as normal Schwann cells by a novel compound called Ocimum Santum hydrophilic fraction 1 (OSHP-1) which interferes with the proteins called histones that sit on the DNA and remodels them in such a manner so as to stop the bad cancer genes from expressing. Obviously, before trying this new investigational drug on humans, it must be tested on the animal model of the human disease.  So, we are using a mouse model of the NF1 malignant peripheral nerves sheath tumor to test the hypothesis that OSHP-1 can treat NF1 by shrinking the tumor and revert the tumorigenic Schwann Cells to normal Schwann Cells.  

The ultimate applicability of this research is arriving at a cure for NF.  OSHP-1 will treat NF1 MPNST rather than treat the symptoms. 

1. The outcome of this study will help pediatric patients that develop benign growths called Lisch nodules which appear in the colored part of the eye (the iris). Affected individuals may develop tumors that grow along the nerve leading from the eye to the brain (the optic nerve). These tumors, which are called optic gliomas, may lead to reduced vision or total vision loss.
2. Secondly, the types of patients that the discoveries from this work will help are malignant peripheral nerve sheath tumors (MPNSTs) and glioma NF1 patients who are at risk of developing cancerous tumors that grow along nerves. These tumors, which usually develop in adolescence or adulthood, are called malignant peripheral nerve sheath tumors (MPNST). People with NF1 also have an increased risk of developing other cancers, including brain tumors and leukemia (cancer of blood-forming tissue).
3. Potential clinical applications of the use of HPLC purified compound of OSHP-1 are to treat the cancerous tumors that grow along the nerves as well as optic glioma in NF patients.

Projected time to achieve patient-related outcome: The anticipated short term outcome will identify the efficacy of OSHP-1 in treating tumors in mouse model of NF1-MPNST.  The anticipated long term gain (5-10 years) is treatment of NF1 associated MPNST in human population using chemically synthesized active compound in OSHP-1 on a peptide amphiphile nanofiber (PAN) platform.

Likely contributions to advancing the field of NF research and/or patient care: My lab’s research focus is to understand what causes the Schwann cells to become tumorigenic neurofibroma and what causes the neurofibromas to become malignant and how to stop that from happening.  We discovered that OSHP-1 can block the Schwann cells from becoming neurofibroma and neurofibroma from becoming malignant (MPNST).   

Executive Summary

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder with an incidence of 1: 3000 [1]. NF1 is characterized by diverse, progressive cutaneous, neurologic, skeletal, and neoplastic manifestations with limited therapeutic options. The leading cause of death in NF1 patients is the malignant peripheral nerve sheath tumor (MPNST), a highly aggressive soft tissue sarcoma [2]. Half of all MPNST develop in individuals with NF1, with a 5-year survival of about 20% to 50%, and the outcome is especially dismal in those with unresectable or metastatic disease [2, 3].  About 65–88% NF1 MPNST arise from plexiform neurofibromas (PN) [4].  Surgical resection is the only known therapy for MPNST [4].  In NF1, MPNSTs commonly arise within a preexisting plexiform neurofibroma [5], the remainder arises sporadically. 

Epigenetic mechanisms have been shown to play a very important role in the neurofibroma progression to a malignant phenotype. Along with neurofibromin, loss of polycomb repressive complex 2 (PRC2) has been implicated in the tumorigenesis of Schwann cells (SCs).  NF1 MPNSTs with PRC2 loss showed complete loss of trimethylation at lysine 27 of histone H3 (H3K27me3) and aberrant transcriptional activation of multiple PRC2-repressed homeobox master regulators and their regulated developmental pathways [6]. NF1 MPNSTs have poor prognoses with 5-year survival rates as low as 35% and recurrence rate range from 40-65%.  Despite advances in the field of MPNST and several clinical trials underway, this pediatric malignancy remains an incurable chronic disease. It is necessary to define effective therapeutic targets, preferably based on the understanding of the genetic and epigenetic mechanisms that affect the tumor development and progression of this disease. 

We have recently identified and purified a novel naturally occurring multi-target cocktail in the methanolic extracts from the hydrophilic fraction 1 of Ocimum Sanctum (OS) leaves (OSHP-1) as potential treatment option for NF1 MPNSTs.  OS is granted “Generally Recognized as Safe” (GRAS) status by the FDA in USA. Our preliminary results show that the OSHP-1 (i) prevented the proliferation of NF1 MPNST cells in cultures by increasing the expression of HDAC2 (which removes the acetyl group from H3K27Ac) and decreasing the expression of lysine acetyl transferase 7 (KAT7); (ii) reversed the expression of certain actively transcribed genes PAX2, FOXN4, IGF2 and TLX1 in NF1MPNST cells; (iii) increased the expression of a Schwann cell differentiation.  Individual HPLC purified fractions of OSHP-1 show anti-tumor properties addressing different aspects of tumorigenesis. The purified peak 1 showed epigenome/chromatin remodeling properties; peak 2 has the property to downregulate SOX2, responsible for stem cell proliferation; peak 3 constituent can re-differentiate the de-differentiated MPNST cell line ST88-14 by increasing NCAM, p75^NTR expression and decreasing folate receptor alpha (FRα) levels in the nucleus.

With a strong in vitro data, we are set to test our hypothesis in animal model of NF1 MPNST.  We will test the hypothesis that reprograming of the epigenome in the animal model of induced NF1 MPNSTs, is a potential treatment option for its treatment. The overall goal of this project is to treat NF1 by reprograming the epigenome to stop the aberrant expression of tumorigenic genes in plexiform neurofibroma that leads to tumor metastasis. To test this hypothesis, we propose one aim to be completed in one year.  In this aim we will determine the anti-tumor effects of OSHP-1 on the mouse model of induced NF1 MPNST.  We will use the Periostin-Cre; Nf1^flox/flox mice mouse generated by Dr David Gutmann, Washington University.  To induce MPNSTs, pTomo-shNf1; shp53 lentivirus or vehicle will be injected into the right sciatic nerves of 6-8-week-old Periostin-Cre; Nf1^flox/flox mice, resulting in GFP expression following Cre-mediated recombination.  Mice will be monitored weekly for tumor induction along sciatic nerve.  The induced MPNSTs will exhibit reduced S100β-staining, increased Ki67 labeling, increased mast cell infiltration (tryptase staining), and collagen-4A (Col4A) basement membrane immunoreactivity. The ones that show limping gait, will be divided into two categories: -(i) euthanized and (ii) treated with OSHP-1 (oral gavage twice daily-dose divided in two-2.5, 5.0, 7.5 mg/kg body weight) till the gait is normalized. The sciatic nerve will be dissected and immune-stained for NCAM, p75^NTR, S100β, Ki67, HDAC2, Tryptase and Col4A. It is expected that the OSHP-1 treatment will result in an increase in the expression of NCAM, p75, S100β and HDAC2, and decreased Ki67, trytase and Col4A levels.  Whole exome sequencing of the lentivirus infected sciatic nerve will be done to evaluate the gene expression profiles and reversal thereof with OSHP-1. The expectation is that the animals receiving treatment with OSHP-1 will show reversal of tumor markers.  Significance will be tested by the Wald test, and P-values will be adjusted by Bonferroni’s correction to address the problem of multiple comparisons due to multiple testing.

Description of Research Proposal

Budget

Collaborations and Conflicts of Interest