Malignant Peripheral Nerve Sheath Tumors Cell Lines Research Articles

Decorin suppresses stemness and migration potential of malignant peripheral nerve sheath tumor through inhibiting epidermal growth factor receptor signaling

Cancer stem cells (CSCs) play pivotal roles in the growth, invasion, metastasis, chemo-resistance in malignant peripheral nerve sheath tumor (MPNST). The current characterization of CSCs in MPNST is not complete. Decorin is a critical regulator of microenvironment, but its expression and function in CSCs of MPNST has not been studied. In the current study, Decorin levels and its relationship with lung and liver metastasis were determined in clinical specimens. Decorin expression in CD133-positive or CD44-positive CSCs was analyzed by RT-qPCR on cytospun MPNST cells after flow cytometry-based cell sorting. Decorin-positive cells were separated from Decorin-negative cells in transfected MPNST cell lines using a designed plasmid expressing red fluorescent protein (RFP) under a Decorin promoter. Tumor sphere formation, tumor growth, cell invasion, cell migration, and the resistance to chemotherapy-induced apoptosis were determined on Decorin-positive versus Decorin-negative MPNST cells. In vivo tumor growth was analyzed in mice receiving subcutaneous transplantation of Decorin-positive versus Decorin-negative MPNSTs. We found that Decorin levels were significantly downregulated in MPNST specimens, compared to non-tumorous adjacent tissue. Significantly lower Decorin levels were detected in MPNSTs with lung or liver metastasis compared to those without. Poorer patient survival was detected in Decorin-low MPNST, compared to Decorin-high subjects. More Decorin-negative cells were detected in CD133-positive MPNST cells than CD133-negative MPNST cells, and in CD44-positive MPNST cells than in CD44-negative MPNST cells. Compared to Decorin-positive MPNST cells, Decorin-negative MPNST cells generated significantly more tumor spheres in culture, were more invasive and migratory, and were more resistant to chemotherapy-induced apoptosis, likely due to the inhibition of epidermal growth factor receptor signaling by Decorin. Decorin-negative MPNST cells grew significantly larger tumor in vivo. Thus, depletion of Decorin may occur in CSCs in MPNSTs, serving possibly as a new therapeutic target.

Abstract 1675: Investigating the role of SRC-family kinases in MPNST tumorigenesis

Abstract Purpose: Malignant peripheral nerve sheath tumor (MPNST), an aggressive soft-tissue sarcoma, is the leading cause of mortality in patients with neurofibromatosis type 1. Over the past two decades, significant advancement has been made in understanding the pathogenesis of MPNST. Still, this progress has not improved overall survival of patients. Therefore, ongoing efforts to identify alterations contributing to the transformation, progression, and metastasis of MPNST are essential for pinpointing targetable oncogenic signaling pathways for this cancer. Previous studies have revealed an increase in YAP/TAZ activity through both LATS1/2-dependent and LATS1/2-independent mechanisms, suggesting that YAP/TAZ hyperactivity may act as a convergence point for MPNST pathogenesis. YES and SRC, two members of the SRC-family kinases (SFKs), were shown to phosphorylate LATS1/2 and YAP/TAZ, and stimulate the transcriptional activity of YAP/TAZ by promoting their nuclear accumulation in hepatocellular carcinoma and colorectal cancer cells. Thus, we have investigated whether YES and SRC are involved in the pathogenesis of MPNSTs. Experimental Design: To assess whether the SFK-YAP/TAZ signaling axis is implicated in the development of MPNST, we have used genetic and pharmacological approaches to investigate the roles of YES and SRC in the proliferation of a subset of human MPNST cell lines, and in the growth of xenografted MPNST tumors in ectopic and orthotopic mouse models. We also performed a phosphoproteome and a transcriptome analysis of YES and SRC-depleted MPNST cells to assess their oncogenic signaling network. Lastly, we evaluated the clinical significance of phospho-SFK and YAP in human MPNST TMAs. Results: Our findings reveal that YES and SRC activity play an essential and redundant role in sustaining the proliferation of human MPNST cell lines. Further, the simultaneous depletion of both kinases impedes tumor growth in xenograft MPNST mouse models. Transcriptomic analyses identified YAP and TAZ as potential effectors of SFKs, as well as other family of transcription factors. Finally, TMA data suggested a correlation between the expressions of YAP, YAP/TAZ, and phospho-SFK and the progression of MPNST. Citation Format: Elamine Zereg, Laure Voisin, Karl Grenier, Mathieu Courcelles, Sungmi Jung, Pierre Thibault, Sylvain Meloche. Investigating the role of SRC-family kinases in MPNST tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1675.

Abstract 1972: Preclinical activity of the type II RAF inhibitor tovorafenib in tumor models harboring either a BRAF fusion or a NF1-LOF mutation

Abstract Genomic alterations and dysregulation of the MAPK pathway have been described in many different types of cancers. BRAF V600 point mutations and BRAF fusions, which are found in both pediatric and adult cancers, are oncogenic drivers that drive constitutive activation of the RAF pathway (Jones 2009, Yaeger 2019). Neurofibromatosis 1 (NF1) loss-of-function (LOF) mutations, which occur in many cancer types, result in decreased neurofibromin GAP function, thus activating RAS (Basu 1992). Tovorafenib is an investigational, selective, CNS-penetrant, small molecule type II RAF inhibitor which inhibits both RAF monomers and dimers and has been shown preclinically to impact the KIAA1549::BRAF fusion (Sun 2017). In this study, the impact of tovorafenib alone or in combination with MEK inhibitor, pimasertib, was explored in adult or pediatric tumor models harboring BRAF fusions or NF1-LOF mutations. At clinically relevant doses, tovorafenib treatment resulted in tumor regression in an AGK::BRAF fusion melanoma patient-derived xenograft (PDX) model in vivo. In contrast, little anti-tumor activity was observed in response to tovorafenib in vivo in two tumor models harboring NF1-LOF mutations. Ongoing PK-PD studies in both the AGK::BRAF fusion model and the NF1-LOF models will assess the extent and duration of target inhibition. In vitro, anti-proliferative activity of tovorafenib was also evaluated in NF1-LOF mutant tumor cell lines or PDX models of different tumor types, including melanoma, lung and malignant peripheral nerve sheath tumor (MPNST). While tovorafenib was potent in the BRAF V600E tumor cell line, little activity was observed in the NF1-LOF tumor cell lines. Modulation of phosphorylated-ERK (pERK) was also assessed. At very low concentrations, modest induction of pERK was observed in the NF1-LOF tumor cell lines, with inhibition at higher concentrations. In vitro combination studies were conducted using pimasertib and tovorafenib, or a tool compound. Synergy was observed in a NF1-LOF embryonal rhabdomyosarcoma PDX model ex vivo and a NF1-LOF MPNST cell line in vitro, suggesting that vertical pathway inhibition is impactful in the NF1-LOF mutant setting. Tovorafenib is currently being evaluated as a monotherapy in relapsed or progressive pediatric low-grade glioma (pLGG) harboring RAF alterations (NCT04775485) and in combination with pimasertib in patients ≥12 years of age with recurrent, progressive, or refractory solid tumors harboring MAPK pathway alterations (NCT04985604). Ongoing preclinical translational work will continue to explore potential biomarker-defined tumor indications for these agents. Citation Format: Shubhra Rastogi, Sam Perino, Madhu Lal-Nag, Yujin Wang, Samuel C. Blackman, Eleni Venetsanakos. Preclinical activity of the type II RAF inhibitor tovorafenib in tumor models harboring either a BRAF fusion or a NF1-LOF mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1972.

Spatial Gene-Expression Profiling Unveils Immuno-oncogenic Programs of NF1-Associated Peripheral Nerve Sheath Tumor Progression.

Plexiform neurofibromas (PNF) are benign peripheral nerve sheath tumors (PNST) associated with neurofibromatosis type 1 (NF1). Despite similar histologic appearance, these neoplasms exhibit diverse evolutionary trajectories, with a subset progressing to malignant peripheral nerve sheath tumor (MPNST), the leading cause of premature death in individuals with NF1. Malignant transformation of PNF often occurs through the development of atypical neurofibroma (ANF) precursor lesions characterized by distinct histopathologic features and CDKN2A copy-number loss. Although genomic studies have uncovered key driver events promoting tumor progression, the transcriptional changes preceding malignant transformation remain poorly defined. Here we resolve gene-expression profiles in PNST across the neurofibroma-to-MPNST continuum in NF1 patients and mouse models, revealing early molecular features associated with neurofibroma evolution and transformation. Our findings demonstrate that ANF exhibit enhanced signatures of antigen presentation and immune response, which are suppressed as malignant transformation ensues. MPNST further displayed deregulated survival and mitotic fidelity pathways, and targeting key mediators of these pathways, CENPF and BIRC5, disrupted the growth and viability of human MPNST cell lines and primary murine Nf1-Cdkn2a-mutant Schwann cell precursors. Finally, neurofibromas contiguous with MPNST manifested distinct alterations in core oncogenic and immune surveillance programs, suggesting that early molecular events driving disease progression may precede histopathologic evidence of malignancy. If validated prospectively in future studies, these signatures may serve as molecular diagnostic tools to augment conventional histopathologic diagnosis by identifying neurofibromas at high risk of undergoing malignant transformation, facilitating risk-adapted care.

Abstract A061: The LSD1 inhibitor iadademstat shows preclinical efficacy in malignant peripheral nerve sheath tumor cells and synergistic effects in combination

Abstract Background: Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive and rare malignancies accounting for 10% of all tissue sarcomas. Around 50% develop in patients with neurofibromatosis type 1 carrying mutations in the NF1 gene and the other 50% occur sporadically. NF1 is a tumor suppressor gene and a negative regulator of RAS signaling pathway. However, mutation of the NF1 gene is not sufficient to drive MPNST and a series of further mutational events must accumulate in genes other than NF1, such as in CDKN2A and PRC2 complex components. Mutations in p53 or PTEN and/or mutations or copy number variations in oncogenes, such as EGFR or c-Met can also be detected in MPNST. Herein we explore the effects of LSD1 inhibition with iadademstat as a single agent in a panel of MPNST cell lines, both NF1-related and sporadic, and in combination with inhibitors of the MEK-ERK and PI3K/AKT signaling pathways. Materials and Methods: The cell viability in 12 MPNST cell lines, both commercial and derived from patients’ primary tumors, was assessed after 6 days of treatment with iadademstat as a single agent. Synergisms between iadademstat and the MEK inhibitor selumetinib or the PI3K inhibitor copanlisib were also evaluated after 6 days of treatment in three selected responsive cell lines and analyzed with Calcusyn and Combenefit software. Additionally, the effects of iadademstat in RAS pathways (pAKT and pERK levels) were also evaluated in these cell lines. Results: Iadademstat displayed sub-nanomolar activity as a single agent in 8 out of 12 cell lines tested, both NF1-related and sporadic, with different degrees of viability reduction. This was further confirmed with two additional LSD1 inhibitors, with iadademstat being the most potent inhibitor tested. Highly-responsive cell lines showed a decrease in pAKT levels while medium- and low-responsive cell lines displayed no significant differences. pERK levels were not affected. Moreover, the combination of iadademstat and selumetinib or copanlisib displayed strong synergy in cell viability assays in both commercial and patient-derived iadademstat-sensitive cell lines. Conclusions: The LSD1 inhibitor iadademstat is efficacious as single agent in nearly 70% of the MPNST cell lines tested, both NF1-related and sporadic and this efficacy increases synergistically in combination with selumetinib or copanlisib. Therefore, LSD1 inhibitors alone or in combination emerge as a potential treatment for MPNST, an indication with high unmet needs with no approved targeted therapies available. Citation Format: Natalia Sacilotto, Cristina Mascaró, Edgar Creus, Juana Fernández, Conxi Lázaro, Ana Limón, Robert Soliva, Jordi Xaus. The LSD1 inhibitor iadademstat shows preclinical efficacy in malignant peripheral nerve sheath tumor cells and synergistic effects in combination [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A061.

CSIG-02. SHH PATHWAY ACTIVATION DRIVES NEURAL CREST CELL SIGNATURE IN A SUBGROUP OF MPNSTS

Abstract INTRODUCTION Neurofibromatosis Type 1 is hereditary tumor predisposition syndrome that results in the development of innumerable nerve sheath tumors that fall within the spectrum of benign, premalignant and malignant lesions. Our previous work has demonstrated that SHH pathway activation drives malignant transformation in a subset of malignant peripheral nerve sheath tumors (MPNSTs). Here, we demonstrate that SHH pathway activation induces a neural crest cell-like state in a subset of MPNSTs. METHODS We performed single nuclear RNA sequencing on 6 tumors (5 MPNSTS and 1 atypical neurofibroma). We performed in vitro experiments to assess the correlation between SHH pathway activation and neural crest signatures. We inhibited SHH pathway with sonidegib, a SMO inhibitor, in MPNST cell lines and performed RT-PCR to assess markers of neural crest cell signatures. In addition, we assessed cellular viability with treatment. RESULTS We observed that cells that express SMO at high levels, with SHH pathway activation, express neural crest cell makers. Single cell trajectory analysis further demonstrates a pseudotemporal continuum from atypical neurofibromas (Schwann cells), MPNSTs (Schwann cell precursor cells) and other MPNSTs (neural crest cells), which supports that these tumors fall along the developmental trajectory of neural crest cell lineage. SHH pathway activity was high in S462TY MPNST cell line and low in STS-26T MPNST cell line. S462TY also demonstrated prominent elevation in expression of neural crest cell markers TWIST1, SOX9, SNAI2, OTX2, PAX4 and PAX6 . We found that treatment with sonidegib treatment attenuates the expression of neural crest cell signatures. Finally, inhibiting SHH pathway activation reduced cellular viability in MPNST cell lines. CONCLUSION SHH pathway activation drives dedifferentiation into a neural crest cell-like state in a subset of MPNSTs. We confirm that inhibiting SHH pathway activity in a subset of MPNSTS prevent growth and malignant progression, providing a rational for future clinical trials.

CDK4/6-MEK Inhibition in MPNSTs Causes Plasma Cell Infiltration, Sensitization to PD-L1 Blockade, and Tumor Regression.

Malignant peripheral nerve sheath tumors (MPNST) are lethal, Ras-driven sarcomas that lack effective therapies. We investigated effects of targeting cyclin-dependent kinases 4 and 6 (CDK4/6), MEK, and/or programmed death-ligand 1 (PD-L1) in preclinical MPNST models. Patient-matched MPNSTs and precursor lesions were examined by FISH, RNA sequencing, IHC, and Connectivity-Map analyses. Antitumor activity of CDK4/6 and MEK inhibitors was measured in MPNST cell lines, patient-derived xenografts (PDX), and de novo mouse MPNSTs, with the latter used to determine anti-PD-L1 response. Patient tumor analyses identified CDK4/6 and MEK as actionable targets for MPNST therapy. Low-dose combinations of CDK4/6 and MEK inhibitors synergistically reactivated the retinoblastoma (RB1) tumor suppressor, induced cell death, and decreased clonogenic survival of MPNST cells. In immune-deficient mice, dual CDK4/6-MEK inhibition slowed tumor growth in 4 of 5 MPNST PDXs. In immunocompetent mice, combination therapy of de novo MPNSTs caused tumor regression, delayed resistant tumor outgrowth, and improved survival relative to monotherapies. Drug-sensitive tumors that regressed contained plasma cells and increased cytotoxic T cells, whereas drug-resistant tumors adopted an immunosuppressive microenvironment with elevated MHC II-low macrophages and increased tumor cell PD-L1 expression. Excitingly, CDK4/6-MEK inhibition sensitized MPNSTs to anti-PD-L1 immune checkpoint blockade (ICB) with some mice showing complete tumor regression. CDK4/6-MEK inhibition induces a novel plasma cell-associated immune response and extended antitumor activity in MPNSTs, which dramatically enhances anti-PD-L1 therapy. These preclinical findings provide strong rationale for clinical translation of CDK4/6-MEK-ICB targeted therapies in MPNST as they may yield sustained antitumor responses and improved patient outcomes.

Inhibition of Erb-B2 Receptor Tyrosine Kinase 3 and Associated Regulatory Pathways Potently Impairs Malignant Peripheral Nerve Sheath Tumor Proliferation and Survival

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive, currently untreatable Schwann cell-derived neoplasms with hyperactive mitogen-activated protein kinase and mammalian target of rapamycin signaling pathways. To identify potential therapeutic targets, previous studies used genome-scale shRNA screens that implicated the neuregulin-1 receptor erb-B2 receptor tyrosine kinase 3 (erbB3) in MPNST proliferation and/or survival. The current study shows that erbB3 is commonly expressed in MPNSTs and MPNST cell lines and that erbB3 knockdown inhibits MPNST proliferation and survival. Kinomic and microarray analyses of Schwann and MPNST cells implicate Src- and erbB3-mediated calmodulin-regulated signaling as key pathways. Consistent with this, inhibition of upstream (canertinib, sapitinib, saracatinib, and calmodulin) and parallel (AZD1208) signaling pathways involving mitogen-activated protein kinase and mammalian target of rapamycin reduced MPNST proliferation and survival. ErbB inhibitors (canertinib and sapitinib) or erbB3 knockdown in combination with Src (saracatinib), calmodulin [trifluoperazine (TFP)], or proviral integration site of Moloney murine leukemia kinase (AZD1208) inhibition even more effectively reduces proliferation and survival. Drug inhibition enhances an unstudied calmodulin-dependent protein kinase IIα phosphorylation site in an Src-dependent manner. The Src family kinase inhibitor saracatinib reduces both basal and TFP-induced erbB3 and calmodulin-dependent protein kinase IIα phosphorylation. Src inhibition (saracatinib), like erbB3 knockdown, prevents these phosphorylation events; and when combined with TFP, it even more effectively reduces proliferation and survival compared with monotherapy. These findings implicate erbB3, calmodulin, proviral integration site of Moloney murine leukemia kinases, and Src family members as important therapeutic targets in MPNSTs and demonstrate that combinatorial therapies targeting critical MPNST signaling pathways are more effective.

Integrating analysis of proteome profile and drug screening identifies therapeutic potential of MET pathway for the treatment of malignant peripheral nerve sheath tumor

ABSTRACT Background Malignant peripheral nerve sheath tumor (MPNST) is an aggressive sarcoma with a poor prognosis that requires novel therapeutic agents. Proteome information is useful for identifying new therapeutic candidates because it directly reflects the biological phenotype. Additionally, in vitro drug screening is an effective tool to identify candidate drugs for common cancers. Hence, we attempted to identify novel therapeutic candidates for MPNST by integrating proteomic analysis and drug screening. Methods We performed comprehensive proteomic analysis on 23 MPNST tumor samples using liquid chromatography – tandem mass spectrometry to identify therapeutic targets. We also conducted drug screening of six MPNST cell lines using 214 drugs. Results Proteomic analysis revealed that the MET and IGF pathways were significantly enriched in the local recurrence/distant metastasis group of MPNST, whereas drug screening revealed that 24 drugs showed remarkable antitumor effects on the MPNST cell lines. By integrating the results of these two approaches, MET inhibitors, crizotinib and foretinib, were identified as novel therapeutic candidates for the treatment of MPNST. Conclusions We successfully identified novel therapeutic candidates for the treatment of MPNST, namely crizotinib and foretinib, which target the MET pathway. We hope that these candidate drugs will contribute to the treatment of MPNST.

Abstract 4471: Reprogramming “cold” NF1 malignancies into “hot” tumors for immunotherapy

Abstract Introduction: Neurofibromatosis Type 1 (NF1) results from mutations in the NF1 gene that inactivate the tumor suppressor, neurofibromin. This leads to hyperactive RAS signaling that predisposes NF1 patients to tumor development. Over time, some of these tumors can develop into malignant peripheral nerve sheath tumors (MPNST) which are currently incurable.Immune checkpoint blocking (ICB) programs a patient’s immune system to enhance tumor destruction. However, MPNSTs are cold tumors characterized by low immune cell presence in the tumor microenvironment. Interestingly, MPNSTs show checkpoint protein PD-L1 expression suggesting ICB as potential therapy for MPNST, given the ability to increase immune cell density in the tumor microenvironment. Activation of the intracellular receptor stimulator of interferon genes (STING) enhances antitumor immunity through the induction of pro-inflammatory cytokines and chemokines, including type I interferons. Cyclic GMP-AMP synthase (cGAS) is an enzyme that once bound to cytosolic DNA, synthesizes cyclic GMP-AMP (cGAMP), which activates STING to induce inflammatory cytokines and other immune mediators. Preclinical studies using mouse tumor models have assessed the efficacy of STING agonists that trigger the cGAS-STING-IFN axis, ultimately leading to augmented innate immunity and a T cell-rich tumor environment. Additionally, combining STING agonists with ICB has a synergistic effect in treating tumors. Therefore, we hypothesize that treatment with STING agonists would turn “cold” MPNSTs into “hot” tumors making them susceptible to targeting with ICB. Methods: We treated MPNST cell lines with synthetic STING agonist ADU-S100 for varying durations of 8, 18, 24, and 48 hours. At the end of the treatment, cells were harvested for qRT-PCR and immunoblot analysis. Results: We measured STING-IFN pathway activation through immunoblotting and observed that phosphorylation of STING effector proteins - a readout of STING activation – was increased followingby 8 hours after treatment. qRT-PCR analysis showed that STING target cytokine/chemokine gene expression was also upregulated by 8 hours after ADU-S100 treatment. Conclusions: Theise data demonstrates that ADU-S100 was able to activate the STING pathway in MPNST cell lines leading to proinflammatory cytokine/chemokine production. We will next test STING agonist in vivo use to determine whether ADU-S100 treatment inin mouse MPNST modelss of MPNST to is able to reprogram the tumor microenvironment into an immune inflamed one amenable to immunotherapy. Citation Format: Laasya Madana, Lu Q. Le, Nipunika Somatilaka, Renee McKay. Reprogramming “cold” NF1 malignancies into “hot” tumors for immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4471.

MEK Inhibition Synergizes with TYK2 Inhibitors in NF1-Associated Malignant Peripheral Nerve Sheath Tumors.

Malignant peripheral nerve sheath tumors (MPNST) are aggressive sarcomas with limited treatment options and poor survival rates. About half of MPNST cases are associated with the neurofibromatosis type 1 (NF1) cancer predisposition syndrome. Overexpression of TYK2 occurs in the majority of MPNST, implicating TYK2 as a therapeutic target. The effects of pharmacologic TYK2 inhibition on MPNST cell proliferation and survival were examined using IncuCyte live cell assays in vitro, and downstream actions were analyzed using RNA-sequencing (RNA-seq), qPCR arrays, and validation of protein changes with the WES automated Western system. Inhibition of TYK2 alone and in combination with MEK inhibition was evaluated in vivo using both murine and human MPNST cell lines, as well as MPNST PDX. Pharmacologic inhibition of TYK2 dose-dependently decreased proliferation and induced apoptosis over time. RNA-seq pathway analysis on TYK2 inhibitor-treated MPNST demonstrated decreased expression of cell cycle, mitotic, and glycolysis pathways. TYK2 inhibition resulted in upregulation of the MEK/ERK pathway gene expression, by both RNA-seq and qPCR array, as well as increased pERK1/2 levels by the WES Western system. The compensatory response was tested with dual treatment with TYK2 and MEK inhibitors, which synergistically decreased proliferation and increased apoptosis in vitro. Finally, combination therapy was shown to inhibit growth of MPNST in multiple in vivo models. These data provide the preclinical rationale for the development of a phase I clinical trial of deucravacitinib and mirdametinib in NF1-assosciated MPNST.

Deep genomic analysis of malignant peripheral nerve sheath tumor cell lines challenges current malignant peripheral nerve sheath tumor diagnosis

Malignant peripheral nerve sheath tumors (MPNSTs) are soft-tissue sarcomas of the peripheral nervous system that develop either sporadically or in the context of neurofibromatosis type 1 (NF1). MPNST diagnosis can be challenging and treatment outcomes are poor. We present here a resource consisting of the genomic characterization of 9 widely used human MPNST cell lines for their use in translational research. NF1-related cell lines recapitulated primary MPNST copy number profiles, exhibited NF1, CDKN2A, and SUZ12/EED tumor suppressor gene (TSG) inactivation, and presented no gain-of-function mutations. In contrast, sporadic cell lines collectively displayed different TSG inactivation patterns and presented kinase-activating mutations, fusion genes, altered mutational frequencies and COSMIC signatures, and different methylome-based classifications. Cell lines re-classified as melanomas and other sarcomas exhibited a different drug-treatment response. Deep genomic analysis, methylome-based classification, and cell-identity marker expression, challenged the identity of common MPNST cell lines, opening an opportunity to revise MPNST differential diagnosis.

Abstract B011: Single cell CRISPR screen of malignant peripheral nerve sheath tumors identifies SWI/SNF genes of regulatory importance

Abstract Background: Malignant peripheral nerve sheath tumors (MPNST) are rare and aggressive soft tissue sarcomas of the peripheral nervous system which are associated with a poor prognosis. Recurrent genetic mutations of the polycomb repressive complex 2 (PRC2) have been identified in up to 75% of MPNST cases. PRC2 is involved in an intricate epigenetic regulatory network in mammalian cells, where it is known to function antagonistically to the SWI/SNF protein complexes which are commonly associated with active transcription. The regulatory relationship between these complexes is critical to normal cell development and maintenance of cellular identity. However, little is known about how PRC2 aberrations affect this regulatory balance in MPNST. Therefore, this study aims to investigate the role of SWI/SNF in MPNST. Methods: A CRISPR knock-out (KO) screen combined with single cell RNA sequencing (RNAseq) was used to target 44 components of SWI/SNF complexes. The transcriptional regulatory effects of resulting genes of interest were further investigated via bulk CRISPR RNAseq experiments, while phenotypic effects were studied using loss-of-function assays coupled with colony formation assays. The dynamic complex formation was further deciphered using glycerol gradient sedimentation and co-immunoprecipitation (co-IP) assays. Results: Double PHD Fingers 1 gene, DPF1, was identified as a unique transcriptional regulator of MPNST via the single cell CRISPR KO screen. These findings were confirmed using a bulk RNAseq screen, where gene set enrichment analysis suggested DPF1 KO has an inhibitory effect on MPNST cell cycle and proliferation through enrichment of genes involved in the P53 pathway (FDR<0.0001) and the apoptotic pathway (FDR<0.0001). This hypothesis was validated using siRNA and CRISPR KO studies in MPNST cell lines, where DPF1 KO led to reduced proliferation and viability of these cells in both 2D and 3D cell culture assays. Soft agar assays were then used to highlight a role of DPF1 to in anchorage independent cell growth of MPNST cells. We then used glycerol gradient sedimentation assays to confirm DPF1 function as a member of the SWI/SNF complexes in MPNST, where DPF1 was observed to co-migrate with SMARCA4, a core ATPase commonly associated with the BAF complex of the SWI/SNF family. This assay was also used to eliminate DPF1 as a member of the GBAF and PBAF SWI/SNF complexes in MPNST, as we identified distinct sedimentation of DPF1 and proteins unique to these complexes. These findings were further validated using co-immunoprecipitation assays, where SMARCA4 had the ability to pull down DPF1, while components of the GBAF and PBAF complexes did not. Conclusions: DPF1 has been identified as a unique regulator of MPNST cells and acts as a member of the BAF SWI/SNF complex. Citation Format: Bega Murray, Xiyuan Zhang, Shahroze Abbas, Haiyan Lei, Hilda Jafarah, Michael C. Kelly, Jack F. Shern. Single cell CRISPR screen of malignant peripheral nerve sheath tumors identifies SWI/SNF genes of regulatory importance. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B011.

LPAR1 and aberrantly expressed LPAR3 differentially promote the migration and proliferation of malignant peripheral nerve sheath tumor cells.

Schwann cell-derived neoplasms known as malignant peripheral nerve sheath tumors (MPNSTs) are the most common malignancy and the leading cause of death in individuals with neurofibromatosis Type 1. Using genome-scale shRNA screens, we have previously found evidence suggesting that lysophosphatidic acid receptors (LPARs) are essential for MPNST proliferation and/or survival. Here, we examine the expression and mutational status of all six LPA receptors in MPNSTs, assess the role that individual LPA receptors play in MPNST physiology and examine their ability to activate key neurofibromin-regulated signaling cascades. We found that human Schwann cells express LPAR1 and LPAR6, while MPNST cells express predominantly LPAR1 and LPAR3. Whole exome sequencing of 16 MPNST cell lines showed no evidence of mutations in any LPAR genes or ENPP2, a gene encoding a major LPA biosynthetic enzyme. Oleoyl-LPA, an LPA variant with an unsaturated side chain, promoted MPNST cell proliferation and migration. LPAR1 knockdown ablated the promigratory effect of LPA, while LPAR3 knockdown decreased proliferation. Inhibition of R-Ras signaling with a doxycycline-inducible dominant negative (DN) R-Ras mutant, which inhibits both R-Ras and R-Ras2, blocked LPA's promigratory effect. In contrast, DN R-Ras did not affect migration induced by neuregulin-1β (NRG1β), suggesting that LPA and NRG1β promote MPNST migration via distinct pathways. LPA-induced migration was also inhibited by Y27632, an inhibitor of the ROCK1/2 kinases that mediate R-Ras effects in MPNSTs. Thus, LPAR1 and aberrantly expressed LPAR3 mediate distinct effects in MPNSTs. These receptors and the signaling pathways that they regulate are potentially useful therapeutic targets in MPNSTs.

Photonic hyperthermia of malignant peripheral nerve sheath tumors at the third near-infrared biowindow.

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas that typically carry a dismal prognosis. Given the insensitivity of these tumors to traditional chemotherapy and the absence of effective targeted drugs, new therapeutic strategies are urgently needed. Photothermal therapy (PTT) including near-infrared laser at the third biowindow (NIR-III) has demonstrated significant potential in cancer theranostics due to its minimally invasive nature and excellent therapeutic outcomes. However, the passive utilization of photothermal agents (PTAs) with poor target specificity and biocompatibility substantially hinders the clinical translation and application of this method. We evaluated the efficiency, safety, and underlying mechanisms of NIR-III without PTAs in the treatment of MPNSTs. The photothermal performance and tissue penetration capability of the NIR-III laser were evaluated in human MPNST cell lines using CCK-8, Calcein-AM and propidium iodide (PI) staining, and Annexin V-FITC/PI assays. The tumor xenografted mice model was used for evaluating the efficacy and biosafety of NIR-III photothermal ablation. Finally, the underlying mechanisms of NIR-III treatment, explored by whole-transcriptome sequencing, are further verified by RT-qPCR. We found that although the NIR-III photothermal treatment efficiency varied among individuals, which was possibly influenced by different endoplasmic reticulum stress responses, the expected antineoplastic effect was ultimately achieved after adjustment of the power density and radiation duration. The present study provides an intriguing noninvasive therapy for MPNSTs that accelerates the clinical translation of PTT while avoiding the biocompatibility issues arising from PTAs. This work was supported by grants from National Natural Science Foundation of China (82102344; 82172228); Shanghai Rising Star Program supported by Science and Technology Commission of Shanghai Municipality (20QA1405600); Natural Science Foundation of Shanghai (22ZR1422300); Science and Technology Commission of Shanghai Municipality (19JC1413) ; "Chenguang Program" supported by Shanghai Education Development Foundation (SHEDF) (19CG18); Shanghai Municipal Key Clinical Specialty (shslczdzk00901); Innovative research team of high-level local universities in Shanghai (SSMU-ZDCX20180700).

VEGF and VEGFR family members are expressed by neoplastic cells of NF1-associated tumors and may play an oncogenic role in malignant peripheral nerve sheath tumor growth through an autocrine loop

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder. The role of angiogenesis and VEGF pathway in the pathogenesis of neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs) remains poorly understood.We assessed the expression of VEGF and VEGFR family members in cohorts of plexiform neurofibromas (pNF), MPNSTs and MPNST cell lines at transcript [pNF, n = 49; MPNST, n = 34] and protein levels [pNF, n = 21; MPNST, n = 9].VEGF and VEGFR members were variably expressed in cell lines. VEGFA (p = 3.10−5), VEGFR1 (p = 0.08), and VEGFR2 (p = 2.10−4) mRNAs were overexpressed in MPNSTs in comparison with pNFs. Both VEGFA and VEGFR1 proteins were expressed by spindle tumor cells of pNFs and MPNSTs. VEGFA was expressed more in MPNSTs than in pNFs (p = 9.10−6) and a trend for VEGFR1 overexpression was observed (p = 0.06). VEGFR2 was not found at the protein level. The microvascular density was significantly reduced in MPNSTs as compared to pNFs (p = 0.0025), with no differences regarding the expression of the activated phosphorylated forms of ERK (P-ERK [p = 0.63]) and AKT (P-AKT [p = 0.41]) in endothelial cells, suggesting that VEGF-dependant angiogenesis may not be critical for MPNST oncogenesis.Altogether, these results indicate that the VEGF-VEGFR pathway may play a role in the development of pNFs and MPNSTs, independently of angiogenesis. Whether or not it drives an oncogenic autocrine/paracrine loop in neoplastic cells, participating in an increased activation of signaling pathways downstream of tyrosine kinase receptors, including VEGFRs, is a tempting hypothesis. Nevertheless, the specific targeting of angiogenesis in MPNSTs may not be sufficient to slow down tumor growth.

Abstract LB060: Single-cell CRISPR screen of malignant peripheral nerve sheath tumors (MPNST)

Abstract Background: Malignant peripheral nerve sheath tumors (MPNST) are aggressive sarcomas of the peripheral nervous system. Loss of function of the polycomb repressive complex 2 (PRC2) have been identified in up to 80% of MPNST cases, via recurrent genetic mutations in the core components, EED and/or SUZ12. However, the transcriptional mis-regulation incurred by the loss of transcriptional repression in MPNST, through loss of H3K27me3, has yet to be fully deciphered. We hypothesize that PRC2 loss leads to transcriptional regulatory imbalance between key epigenetic regulating complexes in this malignancy, which may contribute to the oncogenesis of this disease. Therefore, this project aims to elucidate the role of the SWI/SNF chromatin remodeling complexes in MPNST, epigenetic regulators known to act antagonistically to PRC2. Method: This project uses a CRISPR knock out screen combined with a single cell RNA sequencing readout to target 44 known components of the SWI/SNF complexes. This technology enables large scale phenotypic assays, which we leverage to decipher the complicated epigenetic regulatory network within these lesions. Resulting genes of interest were further studied via loss-of-function assays coupled with colony formation assay and chromatin immunoprecipitation DNA sequencing (ChIP-seq) experiments. Results: This CRISPR screen highlighted core SWI/SNF components, SMARCA4 and SMARCE1, as genes of interest in MPNST. Knockdown of these genes in MPNST cell lines reduced proliferation, indicating a role for these genes in MPNST growth and viability. Further, investigations via soft agar assay found both SMARCA4 and SMARCE1 knock out to decrease the tumorigenic potential of MPNST cells. The potential dependency of SMARCA4 function in MPNST cells on PRC2 mutation was investigated using a doxycycline-inducible PRC2-restoration MPNST cell line model. ChIP-seq results suggest that loss of PRC2 does not substantially affect the binding of SMARCA4-containing SWI/SNF complexes across the genome. DPF1, a member of the neuronal-specific SWI/SNF chromatin remodeling complex, was further highlighted as a unique regulator of MPNST transcription via the CRISPR single-cell screen. CRISPR knockdown of this gene in MPNST cell lines has been found to decrease proliferation, while soft agar assay experiments highlighted that DPF1 loss reduces cell tumorigenicity. The unique dependency of this gene observed in MPNST but not in any other cancer subtypes highlights the potential of DPF1 to be a novel and unique therapeutic target in MPNST. Conclusion: Thus far, our studies have shown that members of the SWI/SNF complex play key roles in the MPNST cell proliferation and tumorigenicity. The function of these complexes in MPNST, however, may be independent of the transcriptional imbalance caused by PRC2 mutation. Citation Format: Béga Murray, Xiyuan Zhang, Shahroze Abbas, Hilda Jafarah, Jack F. Shern. Single-cell CRISPR screen of malignant peripheral nerve sheath tumors (MPNST) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB060.

Abstract 1614: Creation of malignant peripheral nerve sheath tumor models deficient for polycomb repressive complex 2 and identification of therapeutic vulnerabilities

Abstract We constructed models of nerve sheath tumors that arise in Neurofibromatosis Type 1 (NF1) patients and conducted therapeutics discovery using synthetic lethal pharmacogenomic screens. Given both plexiform neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs) arise within the Schwann cell lineage, we developed a drug discovery pipeline to identify targeted therapeutics for treating NF1-related neoplasia. Using CRISPR/Cas9, immortalized human Schwann cell lines were created lacking the NF1 gene or NF1 and components of Polycomb Repressive Complex 2 (PRC2), such as SUZ12. ~80% of all MPNST harbor loss of function mutations in PRC2 genes, which is highly suggestive that perturbation of epigenetic homeostasis plays a role in malignant transformation of neurofibromas. Our models mimic the genetics of MPNSTs and allowed us to identify selectively lethal drugs which exploit vulnerabilities specific to these genetic drivers. We describe the identification of these novel drugs, extensive in vivo testing in models of MPNST, and mechanistic studies to explain why they act specifically on MPNSTs. We identified compounds showing selective lethality towards NF1/SUZ12 double mutant cells. These include drugs affecting epigenetic homeostasis, such as HDAC inhibitors (HDACi). Moreover, many of these drugs showed strong synergy when tested in combination with a MEK inhibitor (MEKi) against NF1/SUZ12 deficient human Schwann and MPNST cell lines. We investigated the mechanism of this synergy using genetics, transcriptome, and proteome analysis. Interestingly, these mechanistic studies revealed possible emerging avenues of resistance the cells are utilizing to compensate for and survive therapeutic intervention. For instance, proteomics analysis of PRC2 deficient MPNST cell lines treated with combination therapy of MEKi and HDACi indicates activation of specific survival and stress response pathways. Targeting these pathways in conjunction with the therapeutics we have identified could prevent emergence of resistance and tumor escape. Clinically interesting drug candidates were advanced and tested as single agents and in combination in multiple in vivo models of MPNST (including patient derived and cell line xenografts). The FDA approved drugs vorinostat (HDACi) and selumetinib (MEKi) have shown dramatic efficacy, with combination therapy exhibiting strong synergy in vivo. This includes durable responses in survival studies and the ability to dramatically shrink established tumors, with treated tumors showing markers of apoptosis. The discovery of novel agents effective against several models of MPNST is exciting as there are currently no approved targeted therapies for MPNSTs. Our results implicate targeting of epigenetic homeostasis, in combination with MEKi, as a major vulnerability of MPNSTs deficient for PRC2 activity. Citation Format: Kyle B. Williams, Alex Larsson, Justin Tibbits, Christopher L. Moertel, David A. Largaespada. Creation of malignant peripheral nerve sheath tumor models deficient for polycomb repressive complex 2 and identification of therapeutic vulnerabilities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1614.

Abstract 701: Loss of PRC2 enforces a mesenchymal neural crest stem cell phenotype in NF1-deficient cancer through activation of core transcription factors

Abstract Objective: Recurrent mutations in the polycomb repressive complex 2 (PRC2) occur in ~80% of malignant peripheral nerve sheath tumors (MPNST), which originate from NF1-deficient Schwann cells. In MPNST, PRC2 loss results in a diverse set of transcriptomic and phenotypic consequences, including gain of acetylated H3K27 (H3K27ac) accompanying the loss of trimethylated H3K27 (H3K27me3), hyperactivated Ras signaling, and tumor escape from immune surveillance. Mechanistic understanding of how PRC2 loss results in these diverse consequences remains unresolved. Methods: To delineate the oncogenic mechanisms mediated by PRC2 loss, we engineered MPNST cell lines to dynamically reassemble a functional PRC2 and evaluated the transcriptomic and epigenetic consequences of PRC2 restoration. We further extended these finding using single cell RNA sequencing (scRNAseq) from human MPNST. Results: Through integrative analysis of RNAseq and H3K27me3 ChIP-seq in PRC2-deficient MPNST cells, we identified 6134 H3K27me3 peaks gained when a functional PRC2 was restored. Of the 876 significantly altered genes, 699 genes were downregulated, and 177 genes were upregulated when a restored PRC2 caused genome-wide redistribution of H3K27me3 peaks. Detailed mechanistic dissection of these PRC2-regulated genes revealed a two-pronged oncogenic process mediated by PRC2 loss and they cooperatively contribute to the tumorigenesis of MPNST. First, PRC2 loss leads to the upregulation of a transcriptional circuit that remodels the enhancer landscape of MPNST cells and establishes an enhancer-driven transcription factor, FOXC1, as a master regulator and thus a core vulnerability of the cell. Second, PRC2 loss reduces type I interferon (IFN) signaling and antigen presentation as a downstream consequence of the upregulated Ras signaling. These data were further integrated with scRNAseq of human MPNST. Importantly, we discovered that the PRC2-deficient tumor cells have a corrupted transcriptional program characteristic of a mesenchymal precursor cell found during the trajectory of normal Schwann cell development. Interestingly, malignant cells expressing high levels of IFN and antigen presentation genes were missing in the metastatic MPNST, potentially allowing the tumor cell to escape the immune surveillance. The transcriptional circuit established by PRC2-regulated FOXC1 and its downstream targets sustains the malignant program in both primary and metastatic MPNST. Conclusion: In summary, we discovered the activation of a lineage specific oncogenic transcription program in PRC2-deficient MPNST, which is characteristic of a neural crest-derived mesenchymal stem cell. Our findings provide mechanistic understanding of the inherent metastatic potential, chemotherapy and radiotherapy resistance, and immune escape that are clinically characteristic of these tumors. Citation Format: Xiyuan Zhang, Hannah E. Lou, Vishaka Gopalan, Zhihui Liu, Hilda Jafarah, Haiyan Lei, Paige Jones, Carly M. Sayers, Marielle E. Yohe, Prashant Chittiboina, Brigitte C. Widemann, Carol J. Thiele, Michael C. Kelly, Sridhar Hannenhalli, Jack F. Shern. Loss of PRC2 enforces a mesenchymal neural crest stem cell phenotype in NF1-deficient cancer through activation of core transcription factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 701.

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

AbstractUndifferentiated 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.