Experimental models of undifferentiated pleomorphic sarcoma and malignant peripheral nerve sheath tumor.

Undifferentiated pleomorphic sarcoma (UPS) and malignant peripheral nerve sheath tumor (MPNST) are aggressive soft tissue sarcomas that do not respond well to current treatment modalities. The limited availability of UPS and MPNST cell lines makes it challenging to identify potential therapeutic targets in a laboratory setting. Understanding the urgent need for improved treatments for these tumors and the limited cellular models available, we generated additional cell lines to study these rare cancers. Patient-derived tumors were used to establish 4 new UPS models, including one radiation-associated UPS-UPS271.1, UPS511, UPS0103, and RIS620, one unclassified spindle cell sarcoma-USC060.1, and 3 new models of MPNST-MPNST007, MPNST3813E, and MPNST4970. This study examined the utility of the new cell lines as sarcoma models by assessing their tumorigenic potential and mutation status for known sarcoma-related genes. All the cell lines formed colonies and migrated in vitro. The in vivo tumorigenic potential of the cell lines and corresponding xenografts was determined by subcutaneous injection or xenograft re-passaging into immunocompromised mice. USC060.1 and UPS511 cells formed tumors in mice upon subcutaneous injection. UPS0103 and RIS620 tumor implants formed tumors in vivo, as did MPNST007 and MPNST3813E tumor implants. Targeted sequencing analysis of a panel of genes frequently mutated in sarcomas identified TP53, RB1, and ATRX mutations in a subset of the cell lines. These new cellular models provide the scientific community with powerful tools for detailed studies of tumorigenesis and for investigating novel therapies for UPS and MPNST.

Enhancer reprogramming in PRC2-deficient malignant peripheral nerve sheath tumors induces a targetable de-differentiated state.

Malignant peripheral nerve sheath tumors (MPNSTs) are soft tissue sarcomas that frequently harbor genetic alterations in polycomb repressor complex 2 (PRC2) components-SUZ12 and EED. Here, we show that PRC2 loss confers a dedifferentiated early neural-crest phenotype which is exclusive to PRC2-mutant MPNSTs and not a feature of neurofibromas. Neural crest phenotype in PRC2 mutant MPNSTs was validated via cross-species comparative analysis using spontaneous and transgenic MPNST models. Systematic chromatin state profiling of the MPNST cells showed extensive epigenomic reprogramming or chromatin states associated with PRC2 loss and identified gains of active enhancer states/super-enhancers on early neural crest regulators in PRC2-mutant conditions around genomic loci that harbored repressed/poised states in PRC2-WT MPNST cells. Consistently, inverse correlation between H3K27me3 loss and H3K27Ac gain was noted in MPNSTs. Epigenetic editing experiments established functional roles for enhancer gains on DLX5-a key regulator of neural crest phenotype. Consistently, blockade of enhancer activity by bromodomain inhibitors specifically suppressed this neural crest phenotype and tumor burden in PRC2-mutant PDXs. Together, these findings reveal accumulation of dedifferentiated neural crest like state in PRC2-mutant MPNSTs that can be targeted by enhancer blockade.

AXL Inhibition Enhances MEK Inhibitor Sensitivity in Malignant Peripheral Nerve Sheath Tumors.

Dysregulation of the receptor tyrosine kinase AXL is known to promote cancer cell growth and survival in many sarcomas, including the rare subtype, malignant peripheral nerve sheath tumors (MPNST). MPNSTs are largely chemoresistant and carry a poor prognosis. AXL is an attractive potential therapeutic target, as it is aberrantly expressed, and its activation may be an early event in MPNST. However, the effect of AXL inhibition on MPNST development and progression is not known. Here, we investigated the role of AXL in MPNST development and the effects of AXL and MEK1/2 co-inhibition on MPNSTs. We used western blotting to examine AXL expression and activation in MPNST cell lines. We analyzed the effects of exogenous growth arrest-specific 6 (GAS6) expression on downstream signaling and the proliferation, migration, and invasion of MPNST cells. The effect of AXL knockdown with or without mitogen-activated protein kinase (MAPK) inhibition on downstream signal transduction and tumorigenesis was also examined in vivo and in vitro. We found that AXL knockdown increased MAPK pathway signaling. This compensation, in turn, abrogated the antitumorigenic effects linked to AXL knockdown in vivo. AXL knockdown, combined with pharmacological MEK inhibition, reduced the proliferation and increased the apoptosis of MPNST cells both in vitro and in vivo. The pharmacological co-inhibition of AXL and MEK1/2 reduced MPNST volumes. Together these findings suggest that AXL inhibition enhances the sensitivity of MPNST to other small molecule inhibitors. We conclude that combination therapy with AXL inhibitor may be a therapeutic option for MPNST.

Co-targeting PI3K, mTOR, and IGF1R with small molecule inhibitors for treating undifferentiated pleomorphic sarcoma.

Undifferentiated pleomorphic sarcomas (UPSs) are aggressive mesenchymal malignancies with no definitive cell of origin or specific recurrent genetic hallmarks. These tumors are largely chemoresistant; thus, identification of potential therapeutic targets is necessary to improve patient outcome. Previous studies demonstrated that high expression of activated protein kinase B (AKT) in patients with UPS corresponds to poor disease-specific survival. Here, we demonstrate that inhibiting phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR) signaling using a small molecule inhibitor reduced UPS cell proliferation and motility and xenograft growth; however, increased phosphorylation of insulin-like growth factor 1 receptor (IGF1R) indicated the potential for adaptive resistance following treatment through compensatory receptor activation. Co-treatment with a dual PI3K/mTOR inhibitor and an anti-IGF1R kinase inhibitor reduced in vivo tumor growth rates despite a lack of antiproliferative effects in vitro. Moreover, this combination treatment significantly decreased UPS cell migration and invasion, which is linked to changes in p27 subcellular localization. Our results demonstrate that targeted inhibition of multiple components of the IGF1R/PI3K/mTOR pathway was more efficacious than single-agent therapy and suggest that co-targeting this pathway could be a beneficial therapeutic strategy for patients with UPS.

Patterns of recurrence and survival in sporadic, neurofibromatosis Type 1-associated, and radiation-associated malignant peripheral nerve sheath tumors.

OBJECTIVE Malignant peripheral nerve sheath tumors (MPNSTs) are an aggressive group of soft tissue sarcomas that can arise sporadically, in the context of neurofibromatosis Type 1 (NF1) or at a site of prior irradiation. Large series profiling the features and outcomes of sporadic, NF1-associated, and radiation-associated MPNSTs are limited. The goal of this study was to elucidate differences between MPNST etiologies in a large single-institution retrospective study. METHODS Patients (n = 317) were identified through the tumor registry of The University of Texas MD Anderson Cancer Center. Clinicopathological features were retrospectively collected. Features were compared among MPNST subtypes for patients who had sufficient clinical history (n = 289), and clinicopathological features were used to identify adverse predictors of recurrence and survival outcomes. RESULTS Five-year local recurrence-free survival (LRFS), distant recurrence-free survival (DRFS), and disease-specific survival (DSS) estimates were 56.6%, 49.6%, and 53.6%, respectively, for the high-grade MPNST cohort. Five-year DSS was lower in NF1-associated and radiation-associated MPNST than in sporadic MPNST (52%, 47%, and 67%, respectively, p = 0.140). Patients with radiation-associated MPNST had worse 5-year LRFS than those with the sporadic and NF1-associated subtypes (RT-associated vs sporadic, p = 0.010; RT-associated vs NF1-associated, p = 0.232). Truncally located tumors, positive surgical margins, local recurrence, and metastasis were predictors of adverse DSS in multivariate analysis. CONCLUSIONS Radiation-associated MPNSTs are associated with poorer local recurrence-free and disease-specific survival than sporadic and NF1-associated tumors. NF1-associated MPNSTs may have worse survival outcomes owing to large tumor size, compromising truncal location, and lower rate of negative resection margins compared with sporadic tumors.

Poly (ADP) ribose polymerase inhibition: A potential treatment of malignant peripheral nerve sheath tumor.

Poly (ADP) ribose polymerase (PARP) inhibitors, first evaluated nearly a decade ago, are primarily used in malignancies with known defects in DNA repair genes, such as alterations in breast cancer, early onset 1/2 (BRCA1/2). While no specific mutations in BRCA1/2 have been reported in malignant peripheral nerve sheath tumors (MPNSTs), MPNST cells could be effectively targeted with a PARP inhibitor to drive cells to synthetic lethality due to their complex karyotype and high level of inherent genomic instability. In this study, we assessed the expression levels of PARP1 and PARP2 in MPNST patient tumor samples and correlated these findings with overall survival. We also determined the level of PARP activity in MPNST cell lines. In addition, we evaluated the efficacy of the PARP inhibitor AZD2281 (Olaparib) in MPNST cell lines. We observed decreased MPNST cell proliferation and enhanced apoptosis in vitro at doses similar to, or less than, the doses used in cell lines with established defective DNA repair genes. Furthermore, AZD2281 significantly reduced local growth of MPNST xenografts, decreased the development of macroscopic lung metastases, and increased survival of mice with metastatic disease. Our results suggest that AZD2281 could be an effective therapeutic option in MPNST and should be further investigated for its potential clinical use in this malignancy.

AXL is a potential therapeutic target in dedifferentiated and pleomorphic liposarcomas.

BACKGROUND: AXL is a well-characterized, protumorigenic receptor tyrosine kinase that is highly expressed and activated in numerous human carcinomas and sarcomas, including aggressive subtypes of liposarcoma. However, the role of AXL in the pathogenesis of well-differentiated (WDLPS), dedifferentiated (DDLPS), and pleomorphic liposarcoma (PLS) has not yet been determined. METHODS: Immunohistochemical analysis of AXL expression was conducted on two tissue microarrays containing patient WDLPS, DDLPS, and PLS samples. A panel of DDLPS and PLS cell lines were interrogated via western blot for AXL expression and activity and by ELISA for growth arrest-specific 6 (GAS6) production. AXL knockdown was achieved by siRNA or shRNA. The effects of AXL knockdown on cell proliferation, migration, and invasion were measured in vitro. In addition, AXL shRNA-containing DDLPS cells were assessed for their tumor-forming capacity in vivo. RESULTS: In this study, we determined that AXL is expressed in a subset of WDLPS, DDLPS, and PLS patient tumor samples. In addition, AXL and its ligand GAS6 are expressed in a panel of DDLPS and PLS cell lines. We show that the in vitro activation of AXL via stimulation with exogenous GAS6 resulted in a significant increase in cell proliferation, migration, and invasion in DDLPS and PLS cell lines. Transient knockdown of AXL resulted in attenuation of these protumorigenic phenotypes in vitro. Stable AXL knockdown not only decreased migratory and invasive characteristics of DDLPS and PLS cells in vitro but also significantly diminished tumorigenicity of two dedifferentiated liposarcoma xenograft models in vivo. CONCLUSIONS: Our results suggest that AXL signaling contributes to the aggressiveness of DDLPS and PLS, and that AXL is therefore a potential therapeutic target for treatment of these rare, yet devastating tumors.

She3p possesses a novel activity required for ASH1 mRNA localization in Saccharomyces cerevisiae.

Intracellular and intercellular polarity requires that specific proteins be sorted to discreet locations within and between cells. One mechanism for sorting proteins is through RNA localization. In Saccharomyces cerevisiae, ASH1 mRNA localizes to the distal tip of the bud, resulting in the asymmetric sorting of the transcriptional repressor Ash1p. ASH1 mRNA localization requires four cis-acting localization elements and the trans-acting factors Myo4p, She3p, and She2p. Myo4p is a type V myosin motor that functions to directly transport ASH1 mRNA to the bud. She2p is an RNA-binding protein that directly interacts with the ASH1 mRNA cis-acting elements. Currently, the role for She3p in ASH1 mRNA localization is as an adaptor protein, since it can simultaneously associate with Myo4p and She2p. Here, we present data for two novel mutants of She3p, S348E and the double mutant S343E S361E, that are defective for ASH1 mRNA localization, and yet both of these mutants retain the ability to associate with Myo4p and She2p. These observations suggest that She3p possesses a novel activity required for ASH1 mRNA localization, and our data imply that this function is related to the ability of She3p to associate with ASH1 mRNA. Interestingly, we determined that She3p is phosphorylated, and global mass spectrometry approaches have determined that Ser 343, 348, and 361 are sites of phosphorylation, suggesting that the novel function for She3p could be negatively regulated by phosphorylation. The present study reveals that the current accepted model for ASH1 mRNA localization does not fully account for the function of She3p in ASH1 mRNA localization.

Nonsense-mediated decay of ash1 nonsense transcripts in Saccharomyces cerevisiae.

Nonsense-mediated mRNA decay (NMD) performs two functions in eukaryotes, one in controlling the expression level of a substantial subset of genes and the other in RNA surveillance. In the vast majority of genes, nonsense mutations render the corresponding transcripts prone to surveillance and subject to rapid degradation by NMD. To examine whether some classes of nonsense transcripts escape surveillance, we asked whether NMD acts on mRNAs that undergo subcellular localization prior to translation. In Saccharomyces cerevisiae, wild-type ASH1 mRNA is one of several dozen transcripts that are exported from the mother-cell nucleus during mitotic anaphase, transported to the bud tip on actin cables, anchored at the bud tip, and translated. Although repressed during transport, translation is a prerequisite for NMD. We found that ash1 nonsense mutations affect transport and/or anchoring independently of NMD. The nonsense transcripts respond to NMD in a manner dependent on the position of the mutation. Maximal sensitivity to NMD occurs when transport and translational repression are simultaneously impaired. Overall, our results suggest a model in which ash1 mRNAs are insensitive to NMD while translation is repressed during transport but become sensitive once repression is relieved.