Malignant Peripheral Nerve Sheath Tumors Cell Research Articles

BIOM-37. EXPLORING L-TYPE AMINO ACID TRANSPORTER 1 (LAT1) AS A DIAGNOSTIC MARKER AND THERAPEUTIC TARGET IN MALIGNANT PERIPHERAL NERVE SHEATH TUMORS (MPNST)

Abstract OBJECTIVES Malignant peripheral nerve sheath tumors (MPNST) are aggressive soft tissue sarcomas that frequently arise from benign plexiform neurofibromas (PN). Approximately half of MPNST are associated with Neurofibromatosis type 1 (NF1). Despite partial elucidation of the molecular mechanisms underlying MPNST, diagnosis remains challenging, and prognosis is poor. Previously, we found that MPNST cells have elevated βIII-spectrin, a cytoskeletal protein involved in cell morphology and regulation of transporters, including L-type Amino Acid Transporter 1 (LAT1). LAT1 facilitates transport of large neutral amino acids and is upregulated in other cancer types. Our objective is to investigate LAT1 as a diagnostic marker and therapeutic target for MPNST. METHODS Gene and protein expression were determined by qPCR and WES protein analysis, respectively. The effect of shRNA knockdown of LAT1 in MPNST cells was assessed using IncuCyte proliferation assays as well as multiple mouse models. Tumor uptake of [18F] FDOPA, primarily transported by LAT1, in MPNST PDX tumors was assessed using PET/CT imaging. RESULTS LAT1 was expressed in MPNST (n=14) vs. benign PN tissues (n=6). Additionally, in human and murine MPNST cells, shRNA-mediated knockdown of LAT1 suppressed proliferation of MPNST cells in vitro and tumor growth in vivo. In a subcutaneous tumor growth model, intratumor injection of shLAT1 lentivirus reduced tumor volume to approximately 50% of control. Furthermore, uptake of [18F] FDOPA by LAT1 in MPNST PDX tumors was detected by PET/CT imaging. WU-356 PDX tissues exhibited higher LAT1 expression as well as increased uptake of [18F] FDOPA in tumors. Conversely, PET imaging showed significantly lower uptake of [18F] FDOPA in LAT1 knockdown tumors (p<0.05). CONCLUSIONS These results suggest that LAT1 promotes the growth and survival of MPNST cells. In addition, these findings demonstrate the potential use of LAT1 and the radiotracer [18F] FDOPA as a diagnostic biomarker in the management of MPNST.

RBIO-07. IMMUNOMODULATORY PATHWAYS MEDIATE RADIOTHERAPY RESPONSE IN MALIGNANT PERIPHERAL NERVE SHEATH TUMORS

Abstract PURPOSE Malignant peripheral nerve sheath tumors (MPNSTs) are the most common cause of death in neurofibromatosis type 1 (NF-1) and are resistant to radiation therapy (RT), yet the mechanisms underlying RT response are poorly understood. Here, we elucidate mechanisms of RT response in NF-1 associated peripheral nerve sheath tumors through genome-wide CRISPRi screens, genomic analysis of mouse models, and molecular analysis of patient-derived tumor specimens. METHODS Patient derived NF1 mutant plexiform neurofibroma (pNF) cells (NF9511b, NF95.6), and MPNST cells (ST88-14; JH2-002) were used to measure RT responses by cell counts and flow cytometry. Genome-wide CRISPRi screens were used to identify functional modifiers of RT response in ST88-14 and JH2-002 MPNST cells. Nf1-/-; Tp53-/- mouse MPNSTs were subcutaneously implanted in immunocompetent C57/B6 mice, irradiated (2Gyx5), and dissociated for single-cell RNA sequencing (scRNA-seq) using 10x Genomics. Human MPNST tumor specimens (n=45) were analyzed with targeted DNA mutation sequencing and methylation arrays for cell type deconvolution. RESULTS Radiation single-dose response curves revealed MPNST cells (IC50 6.85Gy) were radioresistant compared to pNF cells (IC50 3.13Gy). Genome wide CRISPRi screens in ST88-14 and JH2-002 MPNST cells converged on cell cycle, DNA repair, and immunomodulatory gene sets mediating RT sensitivity. Irradiation of subcutaneous mouse Nf1-/-; Tp53-/- MPNSTs significantly decreased tumor growth (p=0.01, t-test). scRNA-seq of 32,763 cells from 4 irradiated and 3 unirradiated mouse Nf1-/-; Tp53-/- MPNSTs identified 8 tumor clusters and 7 non-tumor clusters. Irradiation demonstrated a pro-inflammatory effect with increased T-cell presence (5.8% versus 3.3%, p=0.04, t-test) and greater activation of immunostimulatory genes (Cxcl10, Stat1, Irf7) in T cells. In human MPNSTs, CDKN2A/B deletion (p=0.04, log-rank test) and decreased immune cell composition (p=0.03, log-rank test) were associated with significantly worse overall survival in response to RT. CONCLUSION MPNST RT responses may depend on immunomodulatory mechanisms that could be leveraged to improve clinical RT response.

CNSC-13. CROSSTALK BETWEEN SENSORY NEURONAL ACTIVITY AND TUMOR CELLS PROMOTES MALIGNANCY AND CANCER PAIN

Abstract BACKGROUND Nerve infiltration of solid tumors is associated with poor prognosis in multiple cancer types. Understanding the role of neuronal activity in cancer progression offers innovative approaches for treating malignancies and cancer-associated neurological issues such as pain. Pain perception is mediated by the activity of peripheral sensory neurons. Many cancer types induce pain, and sensory innervation could support tumor growth, raising the interesting hypothesis that cancer pain-associated neuronal activity promotes malignancies. Our study aims to test the hypothesis using malignant peripheral nerve sheath tumor (MPNST) as the experimental platform. METHODS in vivo allograft models were established by implanting mouse MPNST cells into the sciatic nerves of male and female mice, followed by performing pain behavior assays (von Frey, Hargreaves, and conditioned place preference tests) and tumor growth measurements. Primary mouse dorsal root ganglion (DRG) sensory neuron cultures were prepared for calcium imaging and neuron-tumor co-culture experiments. RESULTS MPNST tumor-bearing mice exhibited hypersensitivity to noxious and non-noxious stimuli (evoked pain) and ongoing pain, compared to the sham controls. MPNST-conditioned media increased intracellular calcium concentration and ATF3 (indicator for nerve injury/stress) expression in primary sensory neuron culture, suggesting that MPNST tumor cell secretory factors may induce pain by triggering sensory neuron injury/stress responses that increase sensory neuron activity/sensitivity. Reciprocally, we found that stimulation of sensory neuron activity accelerates MPNST tumor growth. The conditioned media from stimulated sensory neurons increased tumor proliferation (EdU assay) in vitro, suggesting that sensory neuron activity-induced paracrine factors increase MPNST cell growth. CONCLUSIONS MPNST is a devastating cancer that grows within peripheral nerves which induce intractable pain. Our findings demonstrate a feedforward cycle where the cancer cells induce sensory neuron hypersensitivity/hyperactivity (pain) which in turn further accelerates tumor growth. Future studies will identify the molecular mechanisms underlying this crosstalk between MPNST and sensory neuron activity.

EXTH-62. FOCAL ADHESION KINASE (FAK) IN CHROMOSOME 8 GAIN-ASSOCIATED MALIGNANT PERIPHERAL NERVE SHEATH TUMORS (MPNST)

Abstract OBJECTIVES Malignant peripheral nerve sheath tumors (MPNST) are aggressive sarcomas often associated with Neurofibromatosis type 1 (NF1), a genetic syndrome driven by overactivation of the RAS pathway. MEK inhibitors (MEKi) target downstream components of the RAS pathway and have been approved for NF1 patients with plexiform neurofibromas (PN). However, MPNST often develop resistance to MEKi. Our lab recently discovered the critical role of chromosome 8 (Chr8) genes in MPNST. The Chr8 gene, protein tyrosine kinase 2 (PTK2), also known as focal adhesion kinase (FAK) is overexpressed in multiple cancers, suggesting its importance in tumor progression. In this study, we aim to evaluate FAK as a therapeutic target for MPNST and investigate the FAKi and RAF/MEKi combination. METHODS A CRISPR gRNA screen was used to identify Chr8 genes involved in MPNST survival. The effect of FAK shRNA knockdown and a FAK inhibitor (FAKi), alone or combined with a MEKi, in MPNST cells was assessed using IncuCyte proliferation assays and multiple murine models. RESULTS The CRISPR gRNA screen identified 57 genes on Chr8 important for MPNST cell survival, including PTK2/FAK. Knockdown of FAK by shRNA in MPNST inhibited cell proliferation in vitro and reduced tumorigenesis in vivo. Consistently, the FAKi defactinib significantly inhibited the proliferation of MPNST (IC50-values: 1.72-4.65μM). Moreover, the defactinib/avutometinib combination was synergistic in vitro and the combination of FAKi with RAF/MEKi reduced tumor growth more that either drug alone in Chr8-gain MPNST PDX models. Avutometinib induced compensatory activation of FAK, while defactinib/avutometinib combination decreased phosphorylation of FAK, ERK1/2 and STAT3, and augmented cleavage of Caspase-3 and PARP-1, shown using the WES protein simple system. CONCLUSION These results demonstrate the potential therapeutic efficacy of FAK inhibitors, both alone and in combination with RAF/MEKi, for the treatment of MPNST.

Multiomic Analyses Reveal New Targets of Polycomb Repressor Complex 2 in Schwann Lineage Cells and Malignant Peripheral Nerve Sheath Tumors

Abstract Background Malignant peripheral nerve sheath tumors (MPNST) can arise from atypical neurofibromas (ANF). Loss of the polycomb repressor complex 2 (PRC2) is a common event. Previous studies on PRC2-regulated genes in MPNST used genetic addback experiments in highly aneuploid MPNST cell lines which may miss PRC2-regulated genes in NF1-mutant ANF-like precursor cells. A set of PRC2-regulated genes in human Schwann cells has not been defined. We hypothesized PRC2 loss has direct and indirect effects on gene expression resulting in MPNST, so we sought to identify PRC2-regulated genes in immortalized human Schwann cells (iHSCs). Methods We engineered NF1-deficient iHSCs with loss of function SUZ12 or EED mutations. RNA sequencing revealed 1,327 differentially expressed genes to define PRC2-regulated genes. To investigate MPNST pathogenesis we compared genes in iHSCs to consistent gene expression differences between ANF and MPNSTs. Chromatin immunoprecipitation sequencing was used to further define targets. Methylome and proteomic analyses were performed to further identify enriched pathways. Results We identified potential PRC2-regulated drivers of MPNST progression. Pathway analysis indicates many upregulated cancer-related pathways. We found transcriptional evidence for activated Notch and Sonic Hedgehog signaling in PRC2-deficient iHSCs. Functional studies confirm Notch signaling is active in MPNST cell lines, patient-derived xenografts, and transient cell models of PRC2 deficiency. A combination of MEK and γ-secretase inhibition shows synergy in MPNST cell lines. Conclusions We identified PRC2-regulated genes and potential drivers of MPNSTs. Our findings support the Notch pathway as a druggable target in MPNSTs. Our identification of PRC2-regulated genes and pathways could result in more novel therapeutic approaches.

TSPO deficiency promotes the progression of malignant peripheral sheath tumors by regulating the G2/M phase of the cell cycle via CDK1

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive Schwann cell-derived sarcomas that are sporadic or associated with Neurofibromatosis 1 (NF1) gene mutations. Traditional therapies are usually ineffective for treating MPNSTs, so new targets need to be identified for the treatment of MPNSTs. In the present study, the role of the mitochondrial translocator protein (TSPO) in the regulation of cell proliferation and the cell cycle in MPNSTs was investigated. TSPO expression was lower in MPNSTs than in NFs. Loss-of-function experiments revealed that TSPO deficiency promoted MPNST cell growth, migration, and invasion and influenced the cell cycle in vitro and in vivo. In addition, TSPO depletion suppressed cell apoptosis by downregulating the expression of caspase-3, caspase-8, HSP60, p27, p53, and BCL-2 and suppressed the cell cycle by upregulating CDK1, CDK2, CCNB1 and CCNA2. Furthermore, CDK1 was determined to be an upstream target of TSPO-mediated regulation via RNA-seq, qPCR, and Western blotting. Specifically, depletion of CDK1 weakened the effect of TSPO deficiency on cell proliferation and migration. More importantly, CDK1 knockdown induced significant cell cycle arrest in the G2/M phase. In summary, TSPO deficiency regulates the cell cycle in MPNSTs by targeting CDK1, which may be an effective molecular target for prognosis evaluation and treatment.

Targeting the Galectin-1/Ras Interaction for Treating Malignant Peripheral Nerve Sheath Tumors.

Background: Neurofibromatosis type 1 (NF1) is a common inherited neurological disorder that can lead to the development of malignant peripheral nerve sheath tumors (MPNSTs), a highly aggressive form of sarcoma. Current treatment options for MPNSTs are limited, with poor prognosis and high recurrence rates. This study aims to explore the potential of targeting the Galectin-1 (Gal-1) and Ras interaction as a novel therapeutic strategy for MPNSTs. Methods: Molecular docking simulations were conducted to identify specific residues involved in the Gal-1 and H-Ras(G12V) interaction. LLS30, a compound designed to target the Ras binding pocket on Gal-1, was developed and tested. The efficacy of LLS30 was evaluated through in vitro assays, including cell viability, apoptosis, and co-immunoprecipitation studies, as well as in vivo assays using orthotopic MPNST xenograft and experimental lung metastasis models. Transcriptome sequencing was performed to analyze the impact of LLS30 on gene expression and signaling pathways. Results: Molecular docking revealed key residues involved in the Gal-1/Ras interaction, and LLS30 was shown to bind to these residues, disrupting the interaction. LLS30 treatment resulted in Ras delocalization from the plasma membrane and suppression of the Ras/Erk signaling pathway. In vitro, LLS30 significantly reduced MPNST cell proliferation and induced apoptosis. In vivo, LLS30 demonstrated potent anti-tumor activity, reducing tumor burden and metastasis while improving survival in animal models. Transcriptome analysis showed that LLS30 downregulates critical pathways, including KRAS signaling and epithelial-mesenchymal transition (EMT). Conclusions: Interference with the Gal-1/Ras interaction could lead to suppression of the Ras signaling pathway. LLS30 effectively disrupts the Gal-1/Ras interaction, resulting in significant anti-tumor and anti-metastatic effects in MPNST models. These findings indicated that targeting Gal-1 with LLS30 offers a promising therapeutic approach for treating MPNSTs and may also be applicable to other malignancies where Gal-1 and Ras are key oncogenic drivers.

Interferon-Induced Transmembrane Protein 1 (IFITM1) Is Downregulated in Neurofibromatosis Type 1-Associated Malignant Peripheral Nerve Sheath Tumors.

Neurofibromatosis type 1 (NF1), an autosomal dominant genetic disorder, is caused by mutations in the NF1 gene, which encodes the GTPase-activating protein neurofibromin. The pathogenesis of the tumor progression of benign plexiform neurofibromas (PNs) and malignant peripheral nerve sheath tumors (MPNSTs) remain unclear. Here, we found that interferon-induced transmembrane protein 1 (IFITM1) was downregulated in MPNST tissues compared to those in PN tissues from patients with NF1. Overexpression of IFITM1 in NF1-associated MPNST cells resulted in a significant decrease in Ras activation (GTP-Ras) and downstream extracellular regulatory kinase 1/2 (ERK1/2) phosphorylation, whereas downregulation of IFITM1 via treatment with small interfering RNA in normal Schwann cells had the opposite result, indicating that expression levels of IFITM1 are closely associated with tumor progression in NF1. Treatment of MPNST cells with interferon-gamma (IFN-γ) significantly augmented the expression of IFITM1, thereby leading to a decrease in Ras and ERK1/2 activation. Despite the small number of patient samples, these findings may potentially provide a new target for chemotherapy in patients with NF1-associated MPNSTs. In xenograft mice injected with MPNST cells, IFN-γ treatment successfully suppressed tumor progression with increased IFITM1 expression and decreased Ras and ERK1/2 activation in tumor tissues. Collectively, these results suggest that IFITM1 is closely involved in MPNST pathogenesis and that IFN-γ is a good candidate for the therapeutic treatment of MPNSTs in NF1.

The CoREST complex is a therapeutic vulnerability in malignant peripheral nerve sheath tumors.

Malignant peripheral nerve sheath tumor (MPNST) is a highly aggressive sarcoma that may be seen in patients with neurofibromatosis type 1 (NF1) or occur sporadically. While surgery is the primary treatment for localized MPNST with a 61.9% overall survival rate, metastatic disease is often fatal due to resistance to systemic therapies which underscores the urgent need for effective treatments. MPNSTs frequently harbor inactivating driver mutations in the PRC2 epigenetic repressor complex suggesting epigenetic therapies may represent a specific vulnerability in these tumors. Here, we investigate the role of the LSD1-HDAC1-CoREST (LHC) repressor complex in mediating MPNST tumor growth and progression. Our findings demonstrate that the LHC small molecule inhibitor, corin, induces apoptosis and significantly inhibits proliferation in MPNST cells. Transcriptomic analysis of corin-treated MPNST cells demonstrates specific increases in genes associated with axonogenesis and neuronal differentiation as well as altered extracellular matrix; additionally, corin treatment is shown to inhibit MPNST invasion in vitro. These results underscore the critical role of the LHC complex in facilitating MPNST growth and progression and suggest that targeting the LHC complex represents a promising therapeutic approach for this aggressive malignancy.

The Use of Hexokinase 2-Displacing Peptides as an Anti-Neoplastic Approach for Malignant Peripheral Nerve Sheath Tumors.

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are aggressive sarcomas that can arise both sporadically and in patients with the genetic syndrome Neurofibromatosis type 1 (NF1). Prognosis is dismal, as large dimensions, risk of relapse, and anatomical localization make surgery poorly effective, and no therapy is known. Hence, the identification of MPNST molecular features that could be hit in an efficient and selective way is mandatory to envision treatment options. Here, we find that MPNSTs express high levels of the glycolytic enzyme Hexokinase 2 (HK2), which is known to shield cancer cells from noxious stimuli when it localizes at MAMs (mitochondria-associated membranes), contact sites between mitochondria and endoplasmic reticulum. A HK2-targeting peptide that dislodges HK2 from MAMs rapidly induces a massive death of MPNST cells. After identifying different matrix metalloproteases (MMPs) expressed in the MPNST microenvironment, we have designed HK2-targeting peptide variants that harbor cleavage sites for these MMPs, making such peptides activatable in the proximity of cancer cells. We find that the peptide carrying the MMP2/9 cleavage site is the most effective, both in inhibiting the in vitro tumorigenicity of MPNST cells and in hampering their growth in mice. Our data indicate that detaching HK2 from MAMs could pave the way for a novel anti-MPNST therapeutic strategy, which could be flexibly adapted to the protease expression features of the tumor microenvironment.

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 5649: PRRX1-TOP2A interaction is a malignancy-promoting factor in human malignant peripheral nerve sheath tumors

Abstract Background: Paired related-homeobox 1 (PRRX1) is a transcription factor in the regulation of developmental morphogenetic processes. There is growing evidence that PRRX1 is highly expressed in certain cancers and is critically involved in human survival prognosis. However, the molecular mechanism of PRRX1 in cancer malignancy remains to be elucidated. Methods: PRRX1 expression in human Malignant peripheral nerve sheath tumors (MPNSTs) samples was detected immunohistochemically to evaluate survival prognosis. MPNST models with PRRX1 gene knockdown or overexpression were constructed in vitro, and the phenotype of MPNST cells was evaluated. Bioinformatics analysis combined with co-immunoprecipitation, mass spectrometry, RNA seq, and structural prediction were used to identify proteins interacting with PRRX1. Results: High expression of PRRX1 was associated with a poor prognosis for MPNST. PRRX1 knockdown suppressed the tumorigenic potential. PRRX1 overexpressed in MPNSTs directly interacts with topoisomerase 2A (TOP2A) to cooperatively promote epithelial-mesenchymal transition and increase expression of tumor malignancy-related gene sets including mTORC1, KRAS, and SRC signaling pathways. Etoposide, a TOP2A inhibitor used in the treatment of MPNST, may exhibit one of its anticancer effects by inhibiting the PRRX1-TOP2A interaction. Conclusion: Targeting the PRRX1-TOP2A interaction in malignant tumors with high PRRX1 expression might provide a novel tumor-selective therapeutic strategy. Significance: MPNST is a malignant tumor with a poor prognosis and still lacks definitive treatment. Our results could hold promise as an alternative therapeutic strategy to inhibit the interaction between PRRX1 and TOP2A, and may improve the prognosis for malignant tumor patients with high expression of PRRX1 and TOP2A such as MPNSTs. Citation Format: Shota Takihira, Eiji Nakata, Daisuke Yamada, Tatsunori Osone, Tomoka Takao, Takeshi Takarada, Akira Hirasawa, Takuto Itano, Tomohiro Fujiwara, Toshiyuki Kunisada, Toshifumi Ozaki. PRRX1-TOP2A interaction is a malignancy-promoting factor in human malignant peripheral nerve sheath tumors [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 5649.

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.

PRRX1-TOP2A interaction is a malignancy-promoting factor in human malignant peripheral nerve sheath tumours

BackgroundPaired related-homeobox 1 (PRRX1) is a transcription factor in the regulation of developmental morphogenetic processes. There is growing evidence that PRRX1 is highly expressed in certain cancers and is critically involved in human survival prognosis. However, the molecular mechanism of PRRX1 in cancer malignancy remains to be elucidated.MethodsPRRX1 expression in human Malignant peripheral nerve sheath tumours (MPNSTs) samples was detected immunohistochemically to evaluate survival prognosis. MPNST models with PRRX1 gene knockdown or overexpression were constructed in vitro and the phenotype of MPNST cells was evaluated. Bioinformatics analysis combined with co-immunoprecipitation, mass spectrometry, RNA-seq and structural prediction were used to identify proteins interacting with PRRX1.ResultsHigh expression of PRRX1 was associated with a poor prognosis for MPNST. PRRX1 knockdown suppressed the tumorigenic potential. PRRX1 overexpressed in MPNSTs directly interacts with topoisomerase 2 A (TOP2A) to cooperatively promote epithelial-mesenchymal transition and increase expression of tumour malignancy-related gene sets including mTORC1, KRAS and SRC signalling pathways. Etoposide, a TOP2A inhibitor used in the treatment of MPNST, may exhibit one of its anticancer effects by inhibiting the PRRX1–TOP2A interaction.ConclusionTargeting the PRRX1–TOP2A interaction in malignant tumours with high PRRX1 expression might provide a novel tumour-selective therapeutic strategy.

Role of catecholamine synthases in the maintenance of cancer stem-like cells in malignant peripheral nerve sheath tumors.

Malignant peripheral nerve sheath tumors (MPNSTs) are malignant tumors that are derived from Schwann cell lineage around peripheral nerves. As in many other cancer types, cancer stem cells (CSCs) have been identified in MPNSTs, and they are considered the cause of treatment resistance, recurrence, and metastasis. As an element defining the cancer stemness of MPNSTs, we previously reported a molecular mechanism by which exogenous adrenaline activates a core cancer stemness factor, YAP/TAZ, through β2 adrenoceptor (ADRB2). In this study, we found that MPNST cells express catecholamine synthases and that these enzymes are essential for maintaining cancer stemness, such as the ability to self-renew and maintain an undifferentiated state. Through gene knockdown and inhibition of these enzymes, we confirmed that catecholamines are indeed synthesized in MPNST cells. The results confirmed that catecholamine synthase knockdown in MPNST cells reduces the activity of YAP/TAZ. These data suggest that a mechanism of YAP/TAZ activation by de novo synthesized adrenaline, as well as exogenous adrenaline, may exist in the maintenance of cancer stemness of MPNST cells. This mechanism not only helps to understand the pathology of MPNST, but could also contribute to the development of therapeutic strategies for MPNST.

FT895 Impairs Mitochondrial Function in Malignant Peripheral Nerve Sheath Tumor Cells.

Neurofibromatosis type 1 (NF1) stands as a prevalent neurocutaneous disorder. Approximately a quarter of NF1 patients experience the development of plexiform neurofibromas, potentially progressing into malignant peripheral nerve sheath tumors (MPNST). FT895, an HDAC11 inhibitor, exhibits potent anti-tumor effects on MPNST cells and enhances the cytotoxicity of cordycepin against MPNST. The study aims to investigate the molecular mechanism underlying FT895's efficacy against MPNST cells. Initially, our study unveiled that FT895 disrupts mitochondrial biogenesis and function. Post-FT895 treatment, reactive oxygen species (ROS) in MPNST notably increased, while mitochondrial DNA copy numbers decreased significantly. Seahorse analysis indicated a considerable decrease in basal, maximal, and ATP-production-coupled respiration following FT895 treatment. Immunostaining highlighted FT895's role in promoting mitochondrial aggregation without triggering mitophagy, possibly due to reduced levels of XBP1, Parkin, and PINK1 proteins. Moreover, the study using CHIP-qPCR analysis revealed a significant reduction in the copy numbers of promoters of the MPV17L2, POLG, TFAM, PINK1, and Parkin genes. The RNA-seq analysis underscored the prominent role of the HIF-1α signaling pathway post-FT895 treatment, aligning with the observed impairment in mitochondrial respiration. In summary, the study pioneers the revelation that FT895 induces mitochondrial respiratory damage in MPNST cells.

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.