NF1 Knockdown Research Articles

Abstract 705: Combinatorial therapeutic targeting of BMP2 and MEK in NF1-null malignant peripheral nerve sheath tumors

Abstract Our lab has identified and validated bone morphogenetic protein 2 (BMP2) signaling pathways as a therapeutic target in malignant peripheral nerve sheath tumors (MPNSTs), independent of the NRAS and MEK1/2 regulation. BMP2/SMAD signaling is associated with enhanced motility and invasion, a hallmark feature of the biological aggressiveness of MPNSTs. We have shown that inhibition of BMP2 leads to decreased motility and invasiveness in MPNST cell lines. Consistent with our data, gene expression studies from NF1 patient tissue samples demonstrated that increased expression of BMP2 is associated with malignancy in NF1 patients. Ras-GAP related domain (GRD) is the most widely studied functional target of Nf1 implicated in tumorigenesis, however, therapeutic interventions targeting Ras activity have met with limited success. Our objective is to target the BMP2/SMAD pathway, involved in migration and invasion, in combination with MEK inhibitors to develop a novel therapeutic approach for treatment of MPNSTs. The mechanism by which NF1-deficiency leads to activation of BMP2 signaling pathways, and consequently increased cellular motility and invasion is unknown. Using NF1(+/-) and NF1 knockdown MPNST cell lines, we are investigating the transcriptional regulation of BMP2 by NF1 to identify signaling mechanisms downstream of NF1 mediating increase in BMP2 levels. We will present data showing that BMP2 is regulated at the level of transcript stability upon NF1 deficiency. We will identify and design a rational approach of targeting of the molecular components involved in stability of the BMP2 3′ UTR in MPNSTs. Furthermore, we will present a combinatorial approach that targets the growth and proliferative potential of MPNSTs by RAS-MEK inhibitors, along with motility and invasive capability by inhibition of the BMP2-Smad axis for better targeting of these tumors. The overall goal of our research is to use the regulation of BMP2 transcriptional control to define mechanism-based novel druggable targets that can be exploited in combinatorial therapies with RAS-MEK inhibitors to treat NF1-related tumors. Citation Format: Sidra Ahsan, Daochun Sun, Michael A. Tainsky. Combinatorial therapeutic targeting of BMP2 and MEK in NF1-null malignant peripheral nerve sheath tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 705. doi:10.1158/1538-7445.AM2015-705

Abstract 2325: The mechanistic basis of BMP2 activation in NF1-deficient malignant peripheral nerve sheath tumors

Abstract Neurofibromatosis type I (NF1)-deficient malignant peripheral nerve sheath tumor (MPNST) is an aggressive tumor for which the only treatment option is surgical removal with wide margins, often leaving behind cancer cells needing chemotherapy. Ras-GAP related domain (GRD) is the most widely studied functional target of Nf1 implicated in tumorigenesis, however, therapeutic interventions targeting Ras activity have met with limited success. Using gene expression profiling, we identified bone morphogenetic protein 2 (BMP2) signaling pathways as a therapeutic target in MPNSTs, independent of the NRAS and MEK1/2 regulation. This novel finding addresses an understudied aspect of the signaling cascades downstream of neurofibromin deficiency, independent of the RAS-GRD domain. Consistent with our data, gene expression studies from NF1 patient tissue samples demonstrated that increased expression of BMP2 was associated with malignancy in NF1 patients [Miller SJ et al., EMBO Mol Med. 1:236-48. 2009]. Furthermore, we have shown that inhibition of the BMP2 receptor by LDN-193189 decreases invasiveness and cellular migration in MPNST cell lines [Sun D et al., MCR, 11: 616-27, 2013]. The mechanism by which NF1-deficiency leads to hyperactivation of BMP2 signaling pathways, and consequently increased cellular motility and invasion is unknown. Using NF1-wt and NF1-kd sporadic MPNST cell lines, we are investigating the transcriptional regulation of BMP2 upon NF1 deficiency to identify signaling mechanisms downstream of NF1 mediating increase in BMP2 levels. The goal of this work is to determine whether NF1 knockdown regulates BMP2 transcription through initiation and rate of transcription or post-transcriptionally. This will be assessed using promoter-reporter assays and mRNA half-life studies. We will present BMP2 reporter activity analysis using various constructs of the BMP2 promoter, and show BMP2 transcript stability upon NF1 kd. Using these data, we will identify and functionally validate signaling pathways mediating BMP2 activation as the readout using small molecular inhibitors. Clinical trials have demonstrated the limited efficacy of inhibition of the RAS-MEK, and PI3K-AKT-mTOR axis in NF1 patients. The BMP2, Smad pathway is involved in cellular motility and invasiveness, characteristic of the aggressive MPNSTs. Therefore, we will present a combinatorial approach that targets the growth and proliferative potential of MPNSTs by RAS-MEK and PI3K inhibitors, along with motility and invasive capability by inhibition of the BMP2-Smad axis for better targeting of these tumors. The overall goal of our research is to use the regulation of BMP2 transcriptional control to define mechanism-based novel druggable targets that can be exploited in combinatorial therapies with RAS-MEK and PI3K inhibitors to treat NF1-related tumors. Citation Format: Sidra Ahsan, Daochun Sun, Michael A. Tainsky. The mechanistic basis of BMP2 activation in NF1-deficient malignant peripheral nerve sheath tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2325. doi:10.1158/1538-7445.AM2014-2325

Down-regulated Six2 by knockdown of neurofibromin results in apoptosis of metanephric mesenchyme cells in vitro

Embryonic Six2-positive nephron progenitor cells adjacent to ureteric bud tips ultimately give rise to nephron structures, including proximal and distal tubules, podocytes, Bowman's capsules, and the glomeruli. This process requires an internal balance between self-renew and differentiation of the nephron progenitor cells, which is mediated by numerous molecules. Recent studies have shown that the neurofibromin (Nf1) null mutant mouse embryos have an 18- to 24-h developmental delay in metanephros manifesting retardation in its cephalad repositioning and reduction number of glomeruli. However, the underlying inter-/intracellular signaling mechanisms responsible for reducing number of glomeruli during nephrogenesis remain to be fully elucidated. Here, we originally detected the Nf1 expression in developing kidney and metanephric mesenchyme cells. Surprisingly, Nf1 knockdown by small interfering RNAs in the metanephric mesenchyme cells (mK3) resulted in a decreased expression of Six2, the key marker of renal progenitor cells, while the ratio of apoptotic cells was significantly increased. Furthermore, overexpression of Six2 in mk3 cells partially rescued apoptosis phenotype. Collectively, these results implied that knockdown of Nf1 resulted in apoptosis of mK3 cells in vitro probably through down-regulation of Six2 expression. Collectively, we demonstrated that down-regulated Six2 by knockdown of Nf1 resulted in apoptosis of mK3 cells in vitro. These results implied that inhibition of Nf1 may delay metanephros development via down-regulation of Six2.

Abstract LB-398: Identification of novel mechanisms of resistance to EGFR tyrosine kinase inhibitors in non-small cell lung cancer

Abstract Most lung adenocarcinoma patients with activating mutations in the Epidermal Growth Factor Receptor (EGFR) gene initially respond to EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib and gefitinib. However, most patients eventually acquire resistance to this class of drugs. In approximately half of all resistant tumors this is caused by a secondary EGFR mutation (T790M) and/or by amplification of the MET tyrosine kinase receptor, yet the cause of resistance in the remainder of cases is largely unknown. In this study, we aim to identify novel determinants of resistance to EGFR TKIs in order to find new drug targets that may increase the effectiveness of EGFR targeted therapy. We performed a genome-wide siRNA screen examining cell viability in the presence and absence of erlotinib. We used a lung adenocarcinoma cell line (PC9) with activated EGFR that is highly sensitive to erlotinib. We selected hits as genes that upon knockdown showed increased cell viability in drug treated versus untreated conditions. Within the list of candidate genes is the tumor suppressor Neurofibromin (NF1). Using multiple shRNA constructs, we confirmed the enhanced survival in the presence of erlotinib upon NF1 knockdown in a number of EGFR mutant lung adenocarcinoma cell lines using different cell viability and cell competition assays. NF1 is a known negative regulator of RAS activity. We observed increased RAS activity and MAPK signaling in cells where NF1 was knocked down using RNA interference and found that this was resistant to inhibition by erlotinib. Moreover, treatment with low doses of MEK inhibitors resensitized the NF1 knockdown cells to erlotinib. To assess the relevance of this resistance mechanism in vivo, we made use of a tetracycline-inducible mouse model of EGFR-dependent lung cancer. Prolonged erlotinib treatment of lung tumor-bearing mice leads to the outgrowth of resistant tumors. Evaluation of the expression levels of Nf1 in erlotinib-resistant tumors in this preclinical model showed low Nf1 mRNA levels in about half of all erlotinib resistant tumors when compared to the levels in normal adjacent lung. Interestingly, tumors bearing the EGFR-T790M mutation do not show decreased Nf1 expression. These observations suggest that decreased NF1 might be a novel resistance mechanism in EGFR-driven lung tumors. Together, our results show that reduced NF1 expression leads to resistance to erlotinib in EGFR mutant lung adenocarcinomas via enhanced RAS and MAPK pathway activity. Since NF1 is known to be mutated in a subset of lung adenocarcinomas, it is possible that NF1 mutations or loss of expression in EGFR mutant lung tumors could result in de novo or acquired resistance to EGFR inhibitors and that these patients may benefit from treatment with a MEK inhibitor in combination with continued EGFR tyrosine kinase inhibitor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-398. doi:10.1158/1538-7445.AM2011-LB-398

Oligodendrocyte progenitor cell numbers and migration are regulated by the zebrafish orthologs of the NF1 tumor suppressor gene

Neurofibromatosis type 1 is the most commonly inherited human cancer predisposition syndrome. Neurofibromin (NF1) gene mutations lead to increased risk of neurofibromas, schwannomas, low grade, pilocytic optic pathway gliomas, as well as malignant peripheral nerve sheath tumors and glioblastomas. Despite the evidence for NF1 tumor suppressor function in glial cell tumors, the mechanisms underlying transformation remain poorly understood. In this report, we used morpholinos to knockdown the two nf1 orthologs in zebrafish and show that oligodendrocyte progenitor cell (OPC) numbers are increased in the developing spinal cord, whereas neurons are unaffected. The increased OPC numbers in nf1 morphants resulted from increased proliferation, as detected by increased BrdU labeling, whereas TUNEL staining for apoptotic cells was unaffected. This phenotype could be rescued by the forced expression of the GTPase-activating protein (GAP)-related domain of human NF1. In addition, the in vivo analysis of OPC migration following nf1 loss using time-lapse microscopy demonstrated that olig2-EGFP(+) OPCs exhibit enhanced cell migration within the developing spinal cord. OPCs pause intermittently as they migrate, and in nf1 knockdown animals, they covered greater distances due to a decrease in average pause duration, rather than an increase in velocity while in motion. Interestingly, nf1 knockdown also leads to an increase in ERK signaling, principally in the neurons of the spinal cord. Together, these results show that negative regulation of the Ras pathway through the GAP activity of NF1 limits OPC proliferation and motility during development, providing insight into the oncogenic mechanisms through which NF1 loss contributes to human glial tumors.

Abstract A30: NF1 is a tumor suppressor in neuroblastoma that determines retinoic acid response and disease outcome

Abstract Neuroblastoma is a malignancy of early childhood that derives from the developing neural crest and accounts for 15% of all paediatric oncology deaths. The vitamin A derivative retinoic acid (RA) controls neural development and promotes differentiation, cell cycle arrest and apoptosis of neuronal and neuroblastoma cells. These diverse effects of RA are exerted primarily through the ability to differentially regulate gene expression mediated by the retinoic acid receptors (RARs). RA is used in the clinic to treat high-risk neuroblastoma patients, however with variable success. This variability in clinical response to RA is enigmatic as no mutations in components of the RA signaling cascade have been found. By using large-scale RNAi-based genetic screens, we aimed to identify predictive biomarkers for RA sensitivity in neuroblastoma. We found an unexpected crosstalk between the tumor suppressor neurofibromin (NF1) and RA signaling. NF1 is a negative regulator of RAS signaling that promotes conversion of the active GTP bound form into the inactive GDP bound form of RAS. Activation of RAS-MEK signaling by loss of NF1 repressed ZNF423, a critical transcriptional co-activator of the retinoic acid receptors, and blocked RA induced differentiation and target gene activation. Restoration of ZNF423 levels resensitized NF1 knockdown cells to the inhibitory effect of RA. Moreover, ZNF423 and NF1 expression predicted sensitivity to RA in a large panel of neuroblastoma cell lines. Neuroblastomas with low levels of both NF1 and ZNF423 have extremely poor outcome. We found NF1 mutations in neuroblastoma cell lines and also in primary tumors. Inhibition of MEK signaling downstream of NF1 restored responsiveness to RA, suggesting a therapeutic strategy to overcome RA resistance in NF1 deficient neuroblastomas. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A30