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Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive Schwann cell-derived neoplasms that occur sporadically or in patients with neurofibromatosis type 1 (NF1). Preclinical research on sporadic MPNSTs has been limited as few cell lines exist. We generated and characterized a new sporadic MPNST cell line, 2XSB, which shares the molecular and genomic features of the parent tumor. These cells have a highly complex karyotype with extensive chromothripsis. 2XSB cells show robust invasive 3-dimensional and clonogenic culture capability and form solid tumors when xenografted into immunodeficient mice. High-density single nucleotide polymorphism array and whole exome sequencing analyses indicate that, unlike NF1-associated MPNSTs, 2XSB cells have intact, functional NF1 alleles with no evidence of mutations in genes encoding components of Polycomb Repressor Complex 2. However, mutations in other genes implicated in MPNST pathogenesis were identified in 2XSB cells including homozygous deletion of CDKN2A and mutations in TP53 and PTEN. We also identified mutations in genes not previously associated with MPNSTs but associated with the pathogenesis of other human cancers. These include DNMT1, NUMA1, NTRK1, PDE11A, CSMD3, LRP5 and ACTL9 . This sporadic MPNST-derived cell line provides a useful tool for investigating the biology and potential treatment regimens for sporadic MPNSTs.

Background Loss of the Ras GTPase-activating protein neurofibromin promotes nervous system tumor pathogenesis in patients with neurofibromatosis type 1 (NF1). Neurofibromin loss potentially hyperactivates classic Ras (H-Ras, N-Ras, K-Ras), M-Ras, and R-Ras (R-Ras, R-Ras2/TC21) subfamily proteins. We have shown that classic Ras proteins promote proliferation and survival, but not migration, in malignant peripheral nerve sheath tumor (MPNST) cells. However, it is unclear whether R-Ras, R-Ras2 and M-Ras are expressed and hyperactivated in MPNSTs and, if so, whether they contribute to MPNST pathogenesis. We assessed the expression and activation of these proteins in MPNST cells and inhibited them to determine the effect this had on proliferation, migration, invasion, survival and the phosphoproteome. Methods NF1-associated (ST88-14, 90-8, NMS2, NMS-PC, S462, T265-2c) and sporadic (STS-26T, YST-1) MPNST lines were used. Cells were transfected with doxycycline-inducible vectors expressing either a pan-inhibitor of the R-Ras subfamily [dominant negative (DN) R-Ras] or enhanced green fluorescent protein (eGFP). Methodologies used included immunoblotting, immunocytochemistry, PCR, Transwell migration, 3 H-thymidine incorporation, calcein cleavage assays and shRNA knockdowns. Proteins in cells with or without DN R-Ras expression were differentially labeled with SILAC and mass spectrometry was used to identify phosphoproteins and determine their relative quantities in the presence and absence of DN R-Ras. Validation of R-Ras and R-Ras2 action and R-Ras regulated networks was performed using genetic and/or pharmacologic approaches. Results R-Ras2 was uniformly expressed in MPNST cells, with R-Ras present in a major subset. Both proteins were activated in neurofibromin-null MPNST cells. Consistent with classical Ras inhibition, DN R-Ras and R-Ras2 knockdown inhibited proliferation. However, DN R-Ras inhibition impaired migration and invasion but not survival. Mass spectrometry-based phosphoproteomics identified thirteen protein networks distinctly regulated by DN R-Ras, including multiple networks regulating cellular movement and morphology. ROCK1 was a prominent mediator in these networks. DN R-Ras expression and RRAS and RRAS2 knockdown inhibited migration and ROCK1 phosphorylation; ROCK1 inhibition similarly impaired migration and invasion, altered cellular morphology and triggered the accumulation of large intracellular vesicles. Conclusions R-Ras proteins function distinctly from classic Ras proteins by regulating distinct signaling pathways that promote MPNST tumorigenesis by mediating migration and invasion. Plain English Summary Mutations of the NF1 gene potentially results in the activation of multiple Ras proteins, which are key regulators of many biologic effects. The protein encoded by the NF1 gene, neurofibromin, acts as an inhibitor of both classic Ras and R-Ras proteins; loss of neurofibromin could cause these Ras proteins to become persistently active, leading to the development of cancer. We have previously shown that three related Ras proteins (the classic Ras proteins) are highly activated in malignant peripheral nerve sheath tumor (MPNST) cells with neurofibromin loss and that they drive cancer cell proliferation and survival by activating multiple cellular signaling pathways. Here, we examined the expression, activation and action of R-Ras proteins in MPNST cells that have lost neurofibromin. Both R-Ras and R-Ras2 are expressed in MPNST cells and activated. Inhibition of R-Ras action inhibited proliferation, migration and invasion but not survival. We examined the activation of cytoplasmic signaling pathways in the presence and absence of R-Ras signaling and found that R-Ras proteins regulated 13 signaling pathways distinct from those regulated by classic Ras proteins. Closer study of an R-Ras regulated pathway containing the signaling protein ROCK1 showed that inhibition of either R-Ras, R-Ras2 or ROCK1 similarly impaired cellular migration and invasion and altered cellular morphology. Inhibition of R-Ras/R-Ras2 and ROCK1 signaling also triggered the accumulation of abnormal intracellular vesicles, indicating that these signaling molecules regulate the movement of proteins and other molecules in the cellular interior. 8c6qZW2Hind241ZLHJfeSi Video Abstract

Background We have found that erbB receptor tyrosine kinases drive Ras hyperactivation and growth in NF1 -null malignant peripheral nerve sheath tumors (MPNSTs). However, MPNSTs variably express multiple erbB receptors with distinct functional characteristics and it is not clear which of these receptors drive MPNST pathogenesis. Here, we test the hypothesis that altered erbB4 expression promotes MPNST pathogenesis by uniquely activating key cytoplasmic signaling cascades. Methods ErbB4 expression was assessed using immunohistochemistry, immunocytochemistry, immunoblotting and real-time PCR. To define erbB4 functions, we generated mice that develop MPNSTs with floxed Erbb4 alleles (P 0 -GGFβ3; Trp53 +/− ;Erbb4 flox/flox mice) and ablated Erbb4 in these tumors. MPNST cell proliferation and survival was assessed using 3 H-thymidine incorporation, MTT assays, Real-Time Glo and cell count assays. Control and Erbb4 -null MPNST cells were orthotopically xenografted in immunodeficient mice and the growth, proliferation (Ki67 labeling), apoptosis (TUNEL labeling) and angiogenesis of these grafts was analyzed. Antibody arrays querying cytoplasmic kinases were used to identify erbB4-responsive kinases. Pharmacologic or genetic inhibition was used to identify erbB4-responsive kinases that drive proliferation. Results Aberrant erbB4 expression was evident in 25/30 surgically resected human MPNSTs and in MPNSTs from genetically engineered mouse models (P 0 -GGFβ3 and P 0 -GGFβ3; Trp53 +/− mice); multiple erbB4 splice variants that differ in their ability to activate PI3 kinase and nuclear signaling were present in MPNST-derived cell lines. Erbb4 -null MPNST cells demonstrated decreased proliferation and survival and altered morphology relative to non-ablated controls. Orthotopic allografts of Erbb4 -null cells were significantly smaller than controls, with reduced proliferation, survival and vascularization. ERBB4 knockdown in human MPNST cells similarly inhibited DNA synthesis and viability. Although we have previously shown that broad-spectrum erbB inhibitors inhibit Ras activation, Erbb4 ablation did not affect Ras activation, suggesting that erbB4 drives neoplasia via non-Ras dependent pathways. An analysis of 43 candidate kinases identified multiple NRG1β-responsive and erbB4-dependent signaling cascades including the PI3K, WNK1, STAT3, STAT5 and phospholipase-Cγ pathways. Although WNK1 inhibition did not alter proliferation, inhibition of STAT3, STAT5 and phospholipase-Cγ markedly reduced proliferation. Conclusions ErbB4 promotes MPNST growth by activating key non-Ras dependent signaling cascades including the STAT3, STAT5 and phospholipase-Cγ pathways. ErbB4 and its effector pathways are thus potentially useful therapeutic targets in MPNSTs.

Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized by the occurrence of nerve sheath tumors and considerable clinical heterogeneity. Some translational studies have been limited by the lack of animal models assessing patient-specific mutations. In order to test therapeutic approaches that may restore function to the mutated gene or gene product, we developed mice harboring NF1 patient-specific mutations including a nonsense mutation (c.2041C>T; p.Arg681*) and a missense mutation (c.2542G>C; p.Gly848Arg). The latter are associated with the development of multiple plexiform neurofibromas along spinal nerve roots. We demonstrate that the human nonsense NF1Arg681* and missense NF1Gly848Arg mutations have different effects in the mouse on neurofibromin expression and each recapitulates unique aspects of the NF1 phenotype, depending upon the genetic context when assessed in the homozygous state or when paired with a conditional knockout allele. Whereas the missense Nf1Gly848Arg mutation fails to produce an overt phenotype in the mouse, animals homozygous for the nonsense Nf1Arg681* mutation are not viable. Mice with one Nf1Arg681*allele in combination with a conditional floxed Nf1 allele and the DhhCre transgene (Nf14F/Arg681*; DhhCre) display disorganized nonmyelinating axons and neurofibromas along the spinal column, which leads to compression of the spinal cord and paralysis. This model will be valuable for novel preclinical testing of nonsense suppression therapies using drugs to target in-frame point mutations that create premature termination codons in patients with NF1.

Most students in MD-PhD programs take a leave of absence from medical school to complete PhD training, which promotes a natural loss of clinical skills and knowledge and could negatively impact a student's long-term clinical knowledge. To address this concern, clinical refresher courses in the final year of PhD training have traditionally been used; however, effectiveness of such courses versus a longitudinal clinical course spanning all PhD training years is unclear. The University of Alabama at Birmingham MD-PhD Program implemented a comprehensive continuing clinical education (CCE) course spanning PhD training years that features three course components: (1) clinical skills; (2) clinical knowledge; and (3) specialty exposure activities. To evaluate course effectiveness, data from an anonymous student survey completed at the end of each semester were analyzed. Five hundred and ninety-seven surveys were completed by MD-PhD students from fall 2014 to 2022. Survey responses indicated that the majority of students found the course helpful to: maintain clinical skills and knowledge (544/597, 91% and 559/597, 94%; respectively), gain exposure to clinical specialties (568/597, 95%), and prepare them for responsibilities during clinical clerkships. During semesters following lockdowns from the COVID-19 pandemic, there were significant drops in students' perceived preparedness. Positive student survey feedback and improved preparedness to return to clinic after development of the course suggests the CCE course is a useful approach to maintain clinical knowledge during research training.

ABSTRACTChemotherapeutic agents effective against malignant peripheral nerve sheath tumors (MPNSTs) are urgently needed. We recently found that tamoxifen potently impedes xenograft growth. In vitro, tamoxifen inhibits MPNST proliferation and survival in an estrogen receptor–independent manner; these effects are phenocopied by the calmodulin inhibitor trifluoperazine. The present study was performed to establish the mechanism of action of tamoxifen in vivo and optimize its therapeutic effectiveness. To determine if tamoxifen has estrogen receptor–dependent effects in vivo, we grafted MPNST cells in castrated and ovariectomized mice; xenograft growth was unaffected by reductions in sex hormones. To establish whether tamoxifen and trifluoperazine additively or synergistically impede MPNST growth, mice xenografted with neurofibromatosis type 1–associated or sporadic MPNST cells were treated with tamoxifen, trifluoperazine, or both drugs for 30 days. Both monotherapies inhibited graft growth by 50%, whereas combinatorial treatment maximally reduced graft mass by 90% and enhanced decreases in proliferation and survival. Kinomic analyses showed that tamoxifen and trifluoperazine have both shared and distinct targets in MPNSTs. In addition, trifluoperazine prevented tamoxifen-induced increases in serum/glucocorticoid regulated kinase 1, a protein linked to tamoxifen resistance. These findings suggest that combinatorial therapy with tamoxifen and trifluoperazine is effective against MPNSTs because these agents target complementary pathways that are essential for MPNST pathogenesis.

Malignant peripheral nerve sheath tumors (MPNSTs) are Schwann cell-derived malignancies that arise from plexiform neurofibromas in patients with mutation of the neurofibromin 1 ( NF1 ) gene. We have shown that the growth factor neuregulin-1 (NRG1) also contributes to human neurofibroma and MPNST pathogenesis and that outbred C57BL/6J × SJL/J transgenic mice overexpressing NRG1 in Schwann cells (P 0 -GGFβ3 mice) recapitulate the process of neurofibroma-MPNST progression. However, it is unclear whether NRG1 acts predominantly within NF1 -regulated signaling cascades or instead activates other essential cascades that cooperate with NF1 loss to promote tumorigenesis. We now report that tumorigenesis is suppressed in inbred P 0 -GGFβ3 mice on a C57BL/6J background. To determine whether NRG1 overexpression interacts with reduced Nf1 or Trp53 gene dosage to “unmask” tumorigenesis in these animals, we followed cohorts of inbred P 0 -GGFβ3; Nf1 +/− , P 0 -GGFβ3; Trp53 + / − and control (P 0 -GGFβ3, Nf1 +/− and Trp53 +/− ) mice for 1 year. We found no reduction in survival or tumors in control and P 0 -GGFβ3 ;Nf1 + / − mice. In contrast, P 0 -GGFβ3; Trp53 + / − mice died on average at 226 days, with MPNSTs present in 95 % of these mice. MPNSTs in inbred P 0 -GGFβ3; Trp53 + / − mice arose de novo from micro-MPNSTs that uniformly develop intraganglionically. These micro-MPNSTs are of lower grade (WHO grade II–III) than the major MPNSTs (WHO grade III–IV); array comparative genomic hybridization showed that lower grade MPNSTs also had fewer genomic abnormalities. Thus, P 0 -GGFβ3; Trp53 + / − mice represent a novel model of low- to high-grade MPNST progression. We further conclude that NRG1 promotes peripheral nervous system neoplasia predominantly via its effects on the signaling cascades affected by Nf1 loss.

Glioblastoma has a poor prognosis with limited therapeutic options. To date, almost all therapeutic agents that showed promise in in vitro assays, preclinical trials, or even in early clinical trials have failed to make a substantial impact on the survival of patients with high-grade glioma. One major obstacle for small molecules, therapeutic proteins, or immune cells remains the blood brain barrier, which often prevents the efficient delivery of agents to the tumor. To address this delivery issue, we developed a cellular vector where implanted modified post-mitotic Migratory Cortical Inhibitory Interneuron Precursors (MCIPs) migrate to high-grade glioma by chemoattraction to locally secrete a therapeutic protein. The inhibitory interneurons of the cerebral cortex originate predominantly in the ventral/subcortical portion of the telencephalic neural tube. During fetal brain development, MCIP migration is guided over long distances by chemorepulsant and chemoattractant factors. Remarkably, several MCIP chemoattractant factors are also secreted by high-grade gliomas. Indeed, our in vitro and in vivo data show that MCIPs robustly migrate to the majority of glioblastoma cell lines evaluated. As a proof of principle, we modified the MCIPs to secrete bispecific T-cell engagers (BiTEs), linking the EGFR tumor antigen to CD3 on T-cells to create an adaptor molecule that induces an anti-tumor response of resident and supplied T-cells by bridging a tumor antigen and the T-cell receptor. We find that implanted BITE-secreting MCIPs significantly extended survival of mice injected with high-grade glioma. Therefore, we conclude that the use of MCIPs as a delivery vector for therapeutic agents could revolutionize the way we treat glioblastoma, as they allow for the local delivery of therapeutic agents in high concentrations, bypassing the need for these agents to cross the blood brain barrier while reducing the risk for systemic toxicities outside of the brain.

The consequences of pathogenic variants in the NF1 gene can manifest in numerous tissues as a result of loss of neurofibromin protein function(s). A known function of NF1 is negative regulation of p21ras signaling via a GTPase activating (Ras-GAP) domain. Besides modulation of Ras signaling as a tumor suppressor, other functions of this multi-domain protein are less clear. Biallelic inactivation of NF1 leads to an embryonic lethal phenotype, while neurofibromin is expressed at varying levels in most tissues beyond developmental stages. Taking advantage of the mouse genetics toolkit, we established novel tamoxifen-inducible systemic knockout Nf1 mouse models (C57BL/6) to gain a better understanding of the role of Nf1 in the adult (3-4 months) mouse. Following inactivation of floxed Nf1 alleles, adult CAGGCre-ERTM;Nf14F/4F mice lose function of Nf1 systemically. Both male and female animals do not survive beyond 11 days post-tamoxifen induction and exhibit histological changes in multiple tissues. During this acute crisis, CAGGCre-ERTM;Nf14F/4F mice are not able to maintain body temperature or body mass, and expend all adipose tissue; however, they continue to consume food and absorb calories comparable to littermate-paired controls. Targeted metabolite analyses and indirect calorimetry studies revealed altered fat metabolism, amino acid metabolism and energy expenditure, with animals undergoing metabolic crisis and torpor-like states. Thermoneutral conditions accelerated the acute, lethal phenotype coincident with lower food intake. This study reveals that systemic loss of neurofibromin in the adult mouse induces metabolic dysfunction and lethality, thus highlighting potential functions of this multi-domain protein in addition to tumor suppression. Footnotes * This version fixes two errors with abbreviations in the text that were incorporated as in-text citations.