NF1 Tumor Suppressor Research Articles

Abstract 2823: Low merlin level as a biomarker for sensitivity of ovarian cancer cell lines to FAK inhibition

Abstract Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that undergoes gene amplification in >20% of serous ovarian cancers. In addition to increased tumor growth and spread, ovarian cancer patients with FAK amplification have a poorer prognosis with decreased overall survival. Merlin, a product of the NF2 tumor suppressor gene, is being evaluated as a biomarker for sensitivity to FAK inhibition in human clinical trials for malignant mesothelioma. As it remains undetermined whether linkages exist between merlin and FAK in other tumor types, we evaluated nine human and two murine ovarian cancer cell lines with sub-micromolar levels of FAK inhibitor (PF-271) for effects on anchorage-independent cell growth. Cells were identified as sensitive (OVCAR3, OVCAR8, HEY, ID8-IP), intermediate (A2780, IGROV1), or resistant (OVCAR10, IGROV1-IP, SKOV3, SKOV3-IP, 5009) to FAK inhibition. Notably, merlin protein levels were high in all resistant cell lines, moderate in intermediate cell lines, and low or undetectable in sensitive cell lines. Oral FAK inhibitor administration reduced orthotopic tumor growth of sensitive (ID8-IP, low merlin) but not resistant (5009, high merlin) murine ovarian carcinoma cells. However, stable knockdown of merlin expression in resistant SKOV3-IP and OVCAR10 human ovarian carcinoma cells did not induce sensitivity to FAK inhibition in vitro. These results support the notion that high merlin expression is correlated with a FAK-inhibitor resistant phenotype, likely through an indirect mechanistic linkage. Nevertheless, merlin protein levels may serve as a biomarker to predict ovarian cancer sensitivity or resistance to FAK inhibitor treatment. Supported by NIH CA102310 Citation Format: Nina R. Shah, Isabelle Tancioni, Kristy K. Ward, Christine Lawson, Xiao Lei Chen, Nichol L.G. Miller, Florian J. Sulzmaier, Sean Uryu, David D. Schlaepfer. Low merlin level as a biomarker for sensitivity of ovarian cancer cell lines to FAK inhibition. [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 2823. doi:10.1158/1538-7445.AM2014-2823

Primary culture of human vestibular schwannomas.

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

Primary Culture of Human Vestibular Schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

P75NTR is highly expressed in vestibular schwannomas and promotes cell survival by activating nuclear transcription factor κB

Vestibular schwannomas (VSs) arise from Schwann cells (SCs) and result from the loss of function of merlin, the protein product of the NF2 tumor suppressor gene. In contrast to non‐neoplastic SCs, VS cells survive long‐term in the absence of axons. We find that p75NTR is overexpressed in VSs compared with normal nerves, both at the transcript and protein level, similar to the response of non‐neoplastic SCs following axotomy. Despite elevated p75NTR expression, VS cells are resistant to apoptosis due to treatment with proNGF, a high affinity ligand for p75NTR. Furthermore, treatment with proNGF protects VS cells from apoptosis due to c‐Jun N‐terminal kinase (JNK) inhibition indicating that p75NTR promotes VS cell survival. Treatment of VS cells with proNGF activated NF‐κB while inhibition of JNK with SP600125 or siRNA‐mediated knockdown reduced NF‐κB activity. Significantly, proNGF also activated NF‐κB in cultures treated with JNK inhibitors. Thus, JNK activity appears to be required for basal levels of NF‐κB activity but not for proNGF‐induced NF‐κB activity. To confirm that the increase in NF‐κB activity contributes to the prosurvival effect of proNGF, we infected VS cultures with Ad.IκB.SerS32/36A virus, which inhibits NF‐κB activation. Compared with control virus, Ad.IκB.SerS32/36A significantly increased apoptosis including in VS cells treated with proNGF. Thus, in contrast to non‐neoplastic SCs, p75NTR signaling provides a prosurvival response in VS cells by activating NF‐κB independent of JNK. Such differences may contribute to the ability of VS cells to survive long‐term in the absence of axons. GLIA 2014;62:1699–1712

Induction of mitotic catastrophe by PKC inhibition in Nf1-deficient cells

Mutations of tumor suppressor Nf1 gene deregulate Ras-mediated signaling, which confers the predisposition for developing benign or malignant tumors. Inhibition of protein kinase C (PKC) was shown to be in synergy with aberrant Ras for the induction of apoptosis in various types of cancer cells. However, it has not been investigated whether loss of PKC is lethal for Nf1-deficient cells. In this study, using HMG (3-hydroxy-3-methylgutaryl, a PKC inhibitor), we demonstrate that the inhibition of PKC by HMG treatment triggered a persistently mitotic arrest, resulting in the occurrence of mitotic catastrophe in Nf1-deficient ST8814 cells. However, the introduction of the Nf1 effective domain gene into ST8814 cells abolished this mitotic crisis. In addition, HMG injection significantly attenuated the growth of the xenografted ST8814 tumors. Moreover, Chk1 was phosphorylated, accompanied with the persistent increase of cyclin B1 expression in HMG-treated ST8814 cells. The knockdown of Chk1 by the siRNA prevented the Nf1-deficient cells from undergoing HMG-mediated mitotic arrest as well as mitotic catastrophe. Thus, our data suggested that the suppression of PKC activates the Chk1-mediated mitotic exit checkpoint in Nf1-deficient cells, leading to the induction of apoptosis via mitotic catastrophe. Collectively, the study indicates that targeting PKC may be a potential option for developing new strategies to treat Nf1-deficiency-related diseases.

Paths of resistance to EGFR inhibitors: is NF enough?

Although the majority of patients with EGFR-mutant lung cancer respond well to EGF receptor (EGFR) tyrosine kinase inhibitors (TKI), all patients eventually develop resistance. The mechanism of acquired resistance is still unknown for a considerable subset of cases. This study reveals the NF1 tumor suppressor gene as a new mediator of resistance to EGFR TKIs and provides a mechanistic rationale for developing combination therapies.

Molecular insights into NF2/Merlin tumor suppressor function.

The FERM domain protein Merlin, encoded by the NF2 tumor suppressor gene, regulates cell proliferation in response to adhesive signaling. The growth inhibitory function of Merlin is induced by intercellular adhesion and inactivated by joint integrin/receptor tyrosine kinase signaling. Merlin contributes to the formation of cell junctions in polarized tissues, activates anti-mitogenic signaling at tight-junctions, and inhibits oncogenic gene expression. Thus, inactivation of Merlin causes uncontrolled mitogenic signaling and tumorigenesis. Merlin's predominant tumor suppressive functions are attributable to its control of oncogenic gene expression through regulation of Hippo signaling. Notably, Merlin translocates to the nucleus where it directly inhibits the CRL4(DCAF1) E3 ubiquitin ligase, thereby suppressing inhibition of the Lats kinases. A dichotomy in NF2 function has emerged whereby Merlin acts at the cell cortex to organize cell junctions and propagate anti-mitogenic signaling, whereas it inhibits oncogenic gene expression through the inhibition of CRL4(DCAF1) and activation of Hippo signaling. The biochemical events underlying Merlin's normal function and tumor suppressive activity will be discussed in this Review, with emphasis on recent discoveries that have greatly influenced our understanding of Merlin biology.

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

Tumor Suppressor NF2/Merlin Is a Microtubule Stabilizer

Cancer-associated mutations in oncogene products and tumor suppressors contributing to tumor progression manifest themselves, at least in part, by deregulating microtubule-dependent cellular processes that play important roles in many cell biological pathways, including intracellular transport, cell architecture, and primary cilium and mitotic spindle organization. An essential characteristic of microtubules in the performance of these varied cell processes is their ability to continuously remodel, a phenomenon known as dynamic instability. It is therefore conceivable that part of the normal function of certain cancer-causing genes is to regulate microtubule dynamic instability. Here, we report the results of a high-resolution live-cell image-based RNA interference screen targeting a collection of 70 human tumor suppressor genes to uncover cancer genes affecting microtubule dynamic instability. Extraction and computational analysis of microtubule dynamics from EB3-GFP time-lapse image sequences identified the products of the tumor suppressor genes NF1 and NF2 as potent microtubule-stabilizing proteins. Further in-depth characterization of NF2 revealed that it binds to and stabilizes microtubules through attenuation of tubulin turnover by lowering both rates of microtubule polymerization and depolymerization as well as by reducing the frequency of microtubule catastrophes. The latter function appears to be mediated, in part, by inhibition of hydrolysis of tubulin-bound GTP on the growing microtubule plus end.

Abstract C262: Malignant mesothelioma lacking merlin shows enhanced sensitivity to the FAK inhibitor defactinib (VS-6063): Elucidation of the merlin-FAK relationship.

Abstract Malignant pleural mesothelioma (MPM) is an aggressive tumor in the lining of the lung often resulting from prior exposure to asbestos. Median overall survival with the standard of care chemotherapy is only 12 months from diagnosis. Thus, new therapeutic modalities are urgently needed to improve the prognosis of MPM patients. ∼50% of MPM patients exhibit biallelic inactivation of the NF2 tumor suppressor gene by mutation and/or deletion resulting in lack of expression of functional merlin protein. Merlin inactivation is a critical step in MPM tumorigenesis and is related, at least in part, to up-regulation of focal adhesion kinase (FAK) activity. FAK has also been demonstrated to play a role in maintenance of cancer stem cells (CSCs), a subpopulation of tumor cells associated with drug resistance and tumor recurrence. Therefore, therapeutic targeting of FAK using defactinib (VS-6063), a potent, orally active FAK kinase inhibitor, may benefit cancer patients through targeting of both CSCs and bulk tumor cells. Among a panel of MPM cell lines in 3-dimensional Matrigel culture, MPM lines lacking expression of merlin were found to be especially sensitive to defactinib. In contrast, MPM cell lines with wild-type merlin expression were less sensitive with average EC50 values of 5.1 μM. Oral dosing of defactinib induced significant tumor growth inhibition in a merlin-negative MPM model pre-implanted in the lungs of mice. Using Aldefluor activity as a marker for CSCs in MPM, we find that chemotherapeutic agents enriched for Aldefluor-positive CSCs, while the FAK inhibitor defactinib reduced the proportion of CSCs. When used in combination with pemetrexed or cisplatin, defactinib blocked the enrichment of CSCs cells by these chemotherapeutic agents. Disruption of cell-cell or cell-extracellular matrix (ECM) contacts with blocking antibodies allowed us to propose a molecular model to account for the observed merlin-FAK inhibitor synthetic lethal relationship. We find that weak cell-cell adhesions in merlin-negative MPM cells lead to a greater dependence of tumor cell survival and proliferation on cell-ECM-induced FAK signaling rendering merlin-negative cells especially vulnerable to FAK inhibitor treatment. Taken together, these data support the clinical development of defactinib in an ongoing Phase 2 registration-directed clinical trial evaluating the activity of defactinib in MPM patients (stratified by merlin status) with a confirmed response (PR or SD) following first line platinum/pemetrexed therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C262. Citation Format: Irina M. Shapiro, Vihren N. Kolev, Yuwaraj Kadariya, Christian M. Vidal, Quentin G. Wright, Jennifer E. Ring, Craig Menges, Joseph R. Testa, Qunli Xu, Jonathan A. Pachter. Malignant mesothelioma lacking merlin shows enhanced sensitivity to the FAK inhibitor defactinib (VS-6063): Elucidation of the merlin-FAK relationship. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C262.

Abstract IA13: Glioma stem cells and cancer

Abstract Malignant GBMs are incurable tumors that progress within a year's time. Genome wide studies have pinpointed prevalent cancer associated mutions in GBM and among these, the tumor suppressors p53, Pten and NF1 are among the most frequently found. We have developed mouse models of GBM via mutation of p53, Pten and NF1 in the brain. Among the many key insights gleaned from studies with our mouse models have been: 1) The first demonstration that stem cells are the major source of these tumors. 2) The demonstration that all tumor cells are not equivalent and that the Cancer Stem Cells, a subpopulation of cells, are the root of tumor progression. 3) The discovery of a distinct form of GBM that arises in a different cell population and that has unique properties. This novel form of GBM has until now been grouped together with the predominant form of GBM. Our work will lead to a separation of prognosis and treatment for the different forms. In addition, using high throughput screens, we have small compounds in development that specifically target GBM cells but not normally dividing cells. I will discuss these advances and the implications for glioma, solid tumors and cancer in general. Citation Format: Luis F. Parada. Glioma stem cells and cancer. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr IA13.

Reduced Brain Neurofibromatosis Type 1 (Nf1) Tumor Suppressor Gene Expression Confers Differential Sensitivity to Ionizing Radiation In Vivo

Reduced Brain Neurofibromatosis Type 1 (Nf1) Tumor Suppressor Gene Expression Confers Differential Sensitivity to Ionizing Radiation In Vivo

Merlin Expression in Spiral Ganglion Neurons Is Decreased in Deafened Mice

Objectives: 1) Evaluate the expression and phosphorylation status of merlin, the protein product of the Nf2 tumor suppressor gene, in auditory neural structures before and after deafening. 2) Determine the extent to which merlin influences neurite growth in spiral ganglion neurons (SGNs). 3) Determine whether merlin expression in SGNs or spiral ganglion Schwann cells (SGSCs) inhibits neurite growth. Methods: Immunostaining of cochlear sections before and after deafening and organotypic explants with anti-merlin antibodies were used to assess merlin expression in SGNs and SGSCs. A (Tx)-inducible Cre-mediated recombination system was used to knock out merlin in neonatal SGNs. Cultures were maintained with (+) or without (-) Tx for 24h to induce Cre expression. To evaluate loss of merlin expression in SGNs versus SGSCs, we plated (+)Tx and (-)Tx neurons onto a bed of (+)Tx and (-)Tx SGSCs. Neurite length and branching were determined for each condition. Results: Merlin expression in SGNs raises the possibility that merlin regulates SGN neurite growth. Merlin expression in SGNs decreased in cochlear sections taken from P30 versus P60 control rats and in P30 and P60 deafened versus P30 and P60 control rats respectively. Reduced merlin expression in cultured neurons but not SCs significantly increases both the number and length of SGN neurites in SGNs. Conclusions: The pattern of merlin expression and phosphorylation following deafening in SGNs and SGSCs taken together with results indicating that neuronal expression of merlin inhibits neurite growth raises the possibility that merlin plays a role in auditory neural maintenance, regeneration, or both.

Tumor suppressor Nf2 limits expansion of the neural progenitor pool by inhibiting Yap/Taz transcriptional coactivators

Brain development requires a precise balance between expansion of the neural progenitor pool and the production of postmitotic neurons and glia. Disruption of this equilibrium results in a myriad of structural abnormalities and disorders of the nervous system. The molecular mechanism that restricts neural progenitor expansion is poorly understood. Here we show that the tumor suppressor neurofibromatosis 2 (Nf2; merlin) limits the expansion of neural progenitor cells (NPCs) in the mammalian dorsal telencephalon. Nf2 is localized at the apical region of NPCs. In the absence of Nf2, NPCs of the cortical hem, hippocampal primordium and neocortical primordium overexpand, while production of Cajal-Retzius cells and hippocampal neurons decreases, resulting in severe malformation of the hippocampus in adult mice. We further show that Nf2 functions by inhibiting the Yap/Taz transcriptional coactivators, probably through a mechanism that is distinct from the canonical Hippo pathway. Overexpressing human YAP in NPCs causes a hippocampal malformation phenotype that closely resembles that of Nf2 mutants and, importantly, deleting Yap in the Nf2 mutant background largely restores hippocampal development. Our studies uncover Nf2 as an important inhibitor of neural progenitor expansion and establish Yap/Taz as key downstream effectors of Nf2 during brain development.

Identifying the Ubiquitin Ligase Complex That Regulates the NF1 Tumor Suppressor and Ras

The NF1 tumor suppressor protein neurofibromin is a negative regulator of Ras. Neurofibromin is dynamically regulated by the proteasome, and its degradation and reexpression are essential for maintaining appropriate levels of Ras-GTP. Like p53, NF1/neurofibromin can be inactivated in cancer by both mutations and excessive proteasomal destruction; however, little is known about the mechanisms that underlie this latter process. Here, we show that a Cullin 3 (Cul3)/kelch repeat and BTB domain-containing 7 complex controls both the regulated proteasomal degradation of neurofibromin and the pathogenic destabilization of neurofibromin in glioblastomas. Importantly, RNAi-mediated Cul3 ablation and a dominant-negative Cul3 directly stabilize neurofibromin, suppress Ras and extracellular signal-regulated kinase, and inhibit proliferation in an NF1-dependent manner. Moreover, in glioblastomas where neurofibromin is chronically destabilized, Cul3 inhibition restabilizes the protein and suppresses tumor development. Collectively, these studies show a previously unrecognized role for Cul3 in regulating Ras and provide a molecular framework that can be exploited to develop potential cancer therapies.

Tumor suppressor Nf2 limits expansion of the neural progenitor pool by inhibiting Yap/Taz transcriptional coactivators

Brain development requires a precise balance between expansion of the neural progenitor pool and the production of postmitotic neurons and glia. Disruption of this equilibrium results in a myriad of structural abnormalities and disorders of the nervous system. The molecular mechanism that restricts neural progenitor expansion is poorly understood. Here we show that the tumor suppressor neurofibromatosis 2 (Nf2; merlin) limits the expansion of neural progenitor cells (NPCs) in the mammalian dorsal telencephalon. Nf2 is localized at the apical region of NPCs. In the absence of Nf2, NPCs of the cortical hem, hippocampal primordium and neocortical primordium overexpand, while production of Cajal-Retzius cells and hippocampal neurons decreases, resulting in severe malformation of the hippocampus in adult mice. We further show that Nf2 functions by inhibiting the Yap/Taz transcriptional coactivators, probably through a mechanism that is distinct from the canonical Hippo pathway. Overexpressing human YAP in NPCs causes a hippocampal malformation phenotype that closely resembles that of Nf2 mutants and, importantly, deleting Yap in the Nf2 mutant background largely restores hippocampal development. Our studies uncover Nf2 as an important inhibitor of neural progenitor expansion and establish Yap/Taz as key downstream effectors of Nf2 during brain development.

Abstract PR15: SUZ12: A novel tumor suppressor and potential biomarker for efficacy of BRD4 inhibition

Abstract Patients with the familial cancer syndrome neurofibromatosis type I possess mutations in the NF1 tumor suppressor gene, which encodes a RasGAP. However a subset of patients carry a larger deletion that encompasses NF1 and the surrounding 13 genes. These patients develop hundreds of benign nervous system tumors, are at much higher risk for developing malignancies, and exhibit specific developmental defects. Therefore we hypothesized that a cooperating tumor suppressor must lie within this region. Notably, this entire region is frequently deleted in in a number of different cancers, including glioblastoma (GBM). To identify a potential cooperating tumor suppressor we analyzed a large number of NF1-associated malignancies by array CGH and detected homozygous loss of SUZ12, a member of the PRC2 repressor complex. Subsequent mutation analysis showed that SUZ12 was homozygously inactivated in a large subset of MPNSTs. Importantly, reintroduction of SUZ12 in SUZ12 deficient Malignant Peripheral Nerve Sheath tumors (MPNST) cell lines restored the function of the PRC2 complex and induced cell death. Conversely, RNAi mediated inactivation of SUZ12 significantly enhanced the tumorigenic properties of NF1-deficient GBMs. Additionally, we found that mice carrying compound heterozygous mutations in Nf1 and Suz12 developed a variety of tumor types and had a marked decrease in overall survival. As expected, knockdown of SUZ12 dramatically decreased the global H3K27 tri-methylation (repressive mark) and increased the global H3K27 acetylation (activation mark). This suggests that the pathogenic effect of SUZ12-loss is, at least in part, caused by an aberrant activation of genes due to an increase in H3K27 acetylation. Interestingly this H3K27 acetylation mark is read by BRD4, suggesting that inhibition of BRD4 might decrease the expression of critical targets. In vitro and in vivo studies indeed confirmed a hypersensitivity to BRD4 inhibition of cell lines and primary tumors lacking SUZ12. Additionally our preliminary data show an inverse correlation between the H3K27 tri-methylation level and the response to treatment with the BRD4 inhibitor JQ1, suggesting that SUZ12 levels, and thus H3K27 tri-methylation status, could be used as a biomarker. In conclusion, we showed that SUZ12 is a tumor suppressor in a number of different cancers, including MPNSTs and gliomas. These tumors are exquisitely sensitive to inhibition of BRD4. Loss of SUZ12 leads to complete ablation of the H3K27 tri-methylation mark, which could serve as a biomarker for treatment with the BRD4 inhibitor JQ1. This abstract is also presented as Poster B51. Citation Format: Thomas De Raedt, Eline Beert, Eric Pasmant, James E. Bradner, Pierre Wolkenstein, Eric Legius, Karen Cichowski. SUZ12: A novel tumor suppressor and potential biomarker for efficacy of BRD4 inhibition. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr PR15.

Clinical next generation sequencing (NGS) to reveal high frequency of alterations to guide targeted therapy in lung cancer patients.

8020 Background: Cancer genomic profiling via NGS in a clinical setting can reveal additional actionable genomic alterations (GA) in patients with lung cancer (LC) previously tested only by hotspot analysis and leading to unanticipated avenues of targeted treatment. Methods: We performed an NGS-based diagnostic test (FoundationOne) to characterize all classes of GA across 3,320 exons of 182 cancer-related genes and 37 introns of 14 genes frequently rearranged in cancer on 386 LC FFPE specimens in a CLIA-certified lab (Foundation Medicine). Specimens included fine needle aspirates, core needle biopsies, and malignant effusions. 95% of cases were NSCLC (367/386). Actionable GAs are defined as those linked to targeted anti-cancer therapies approved or being evaluated in clinical trials. NGS confirmed known hotspot results for EGFR, KRAS and EML4:ALK in 100% of cases. Results: Genomic profiles were generated from 364/386 (94%) of lung cancer cases, identifying 1205 GA, averaging 3.31 alterations per tumor (range 0 to 10). 85% of tumors (310) harbored at least one actionable GA, with a mean of 1.79 GA per tumor (range 0 to 6). In 68% of tumors (248), at least one GA was detected that would be missed by current ‘hotspot assays’. ERBB2 harbored base substitutions or indels in 1.3% of cases. BRAF and C-Kit were altered at frequencies of 2% and 1% respectively. The mTOR/PI3K pathway is likely to be activated via alterations in tumor suppressors STK11 (11%), NF1 (6%) and PTEN (4%), as well as by alterations of PIK3CA (10%) and in AKT1/2/3 (4%), suggesting possible benefit from mTOR/PI3K inhibitors. The Hedgehog pathway (PTCH1/SMO/SUFU) was altered in 2% of cases. ALK and RET were rearranged in 4% and 2% of cases, respectively, with several cases initially diagnosed negative by FISH testing. Conclusions: Profiling the tumor genomes of 364 LC patients led to the identification of a series of GA not detectable by hotspot testing that could significantly inform targeted treatment decisions. Moreover, actionable GA appeared in unexpected tumor type, i.e. an EGFR mutation in a SCLC, reinforcing the likely utility of clinical cancer genomic profiling for the personalized treatment of LC patients.

Sensitivity of malignant mesothelioma lacking Merlin to the FAK inhibitor VS-6063: Evaluation of merlin/NF2 status in clinical samples.

e18541 Background: Malignant pleural mesothelioma (MPM) is an aggressive tumor in the pleural lining of the lung often resulting from prior exposure to asbestos. MPM patients are usually diagnosed at an advanced stage of disease and the prognosis is poor. Median survival after diagnosis is 9 to 12 months and standard-of-care agents such as cisplatin and pemetrexed have only a modest impact on median survival time for MPM patients. New therapeutic modalities are urgently needed to improve the prognosis of MPM patients. 40-50% of MPM patients exhibit homozygous disruption of the NF2 tumor suppressor gene by mutation and/or deletion resulting in lack of expression of functional merlin protein. Methods: Proliferation of drug-treated mesothelioma cell lines in 3-dimensional (3D) Matrigel culture was assessed by MTS, and MPM xenograft growth was measured in vehicle- vs. FAK inhibitor-treated SCID mice. Since absence of merlin expression can theoretically result from several mechanisms including NF2 mutation and chromosome 22 abnormalities, we assessed NF2 gene deletion by FISH and merlin protein levels by IHC in the same human mesothelioma tumor samples. Results: Among a panel of mesothelioma cell lines in 3D culture, MPM lines lacking expression of merlin protein were found to be especially sensitive to the selective FAK inhibitor VS-6063. In contrast, MPM cell lines with wildtype merlin were less sensitive with EC50 values greater than 1 μM. Accordingly, oral dosing with a FAK inhibitor induced significant tumor growth inhibition in a merlin-negative mesothelioma model in mice. To enable the planned stratification of MPM patients by merlin status, an immunohistochemistry (IHC) assay has been optimized to quantify merlin protein levels. A merlin IHC H-score below the defined cutoff was associated with loss of at least one copy of chromosome 22, indicating that chromosomal deletion is an important mechanism of merlin loss in mesothelioma patients. Conclusions: These data support the clinical development of VS-6063 for treatment of malignant pleural mesothelioma patients stratified by merlin/NF2 status. A potentially pivotal mesothelioma trial is set to initiate in 2013.

Abstract 355: Generation of a murine model of Neurofibromatosis Type 2 that accurately recapitulates the spontaneous development of vestibular and spinal schwannomas found in NF2 patients.

Abstract Neurofibromatosis type 2 (NF2) is a genetic disease resulting from germline loss of the NF2 tumor suppressor gene. The hallmark of NF2 is hearing and vestibular loss of function in young adults caused by the development of vestibular schwannomas, which occur bilaterally in nearly all patients and are associated with homozygous loss of NF2. Individuals affected with NF2 also acquire schwannomas of other cranial, spinal and peripheral nerves. Importantly, non-familial schwannomas, the most common tumor of the peripheral nervous system, are also caused by bi-allelic loss of NF2 gene function. A major limitation in the identification of genetic and pharmacologic targets for schwannomas has been the lack of a robust preclinical model that accurately recapitulates human NF2 disease. In order to develop a more robust model, we intercrossed a mouse expressing Cre Recombinase under the control of the 3.9PeriCre promoter with an Nf2 conditional knockout mouse previously generated by Dr. Marco Giovannini to conditionally ablate Nf2 in neural crest-derived cell populations. Histological analysis of the dorsal root ganglion (DRG) and proximal spinal nerves of 3.9PeriCre+;Nf2flox/flox mice revealed that intercrossed mice spontaneously develop tumors between 8 and 10 months at every single spinal level. Microscopic analysis implicated these tumors as schwannomas, as they display a dense pattern of cellularity consistent with the established criteria of GEM schwannoma, including S100β1 positivity. Quantitation of the DRG volume indicated that 3.9PeriCre+ mice have a statistically significant increase when compared to 3.9PeriCre- littermates. FDG-PET studies corroborated this finding, as comparative studies showed an increase in the metabolic activity of paraspinal tumors found in 3.9PeriCre+ mice. Auditory Brainstem Response testing has revealed that 3.9PeriCre+ mice acquire an age-dependent increase in hearing threshold by 8 months when compared to age-matched 3.9PeriCre- controls. Histological analysis of Cranial Nerve VIII confirmed that the hearing loss was directly associated with the development of vestibular schwannomas. To test for concomitant vestibular loss of function, three different behavioral studies (contact righting, trunk curl, and swim) were performed to isolate and test the integrity of the vestibular sense in 3.9PeriCre;Nf2flox/flox mice. These studies indicated that 3.9PeriCre+ mice develop vestibular deficits, as all three studies identified highly significant differences between the two genotypes. Collectively, these genetically engineered mice acquire a physiologic phenotype that closely recapitulates important features of human NF2 disease and provides opportunities for testing putative therapeutic targets using genetic intercrosses or novel small molecule inhibitors. Citation Format: Jeff R. Gehlhausen, Su Jung Park, Matthew Shew, Wade Clapp, Charles Yates. Generation of a murine model of Neurofibromatosis Type 2 that accurately recapitulates the spontaneous development of vestibular and spinal schwannomas found in NF2 patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 355. doi:10.1158/1538-7445.AM2013-355