Vijaya Ramesh

Background: Neurofibromatosis type 2 (NF2)-associated meningiomas and schwannomas are vascular tumors, and while vascular endothelial growth factor (VEGF) inhibition with bevacizumab has benefited some NF2-related schwannomas, most NF2-associated meningiomas remain nonresponsive. Methods: Leveraging our transcriptomic data, we performed Gene Ontology (GO) analysis comparing NF2-deficient meningioma cells with NF2-expressing arachnoid cells (ACs). We then established a 3D in vitro angiogenesis model by co-culturing NF2-null meningioma cells with human umbilical vein endothelial cells (HUVECs). Endothelial sprouting was assessed by CD31/PECAM immunostaining. Effects of third-generation mechanistic target of rapamycin complex 1 (mTORC1)-selective inhibitor RMC-6272 as well as APLN knock-out using CRISPR-Cas9 gene editing were also examined. Results: GO analysis identified vascular development among the top significantly upregulated pathways in NF2-deficient cells. In 3D co-culture, ECs formed radially sprouting tube-like networks from the spheroid surface, and our data supports an angiogenesis phenotype driven by meningioma cells. Given these results along with hyperactivation of mTORC1 upon NF2-deficiency, we examined whether RMC-6272 disrupts meningioma-driven angiogenesis. RMC-6272 potently suppressed EC sprouting. Cross-referencing baseline transcriptomic data, we identified Apelin (APLN), the ligand for APLN receptor (APLNR), as a basally upregulated angiogenic factor in NF2-deficient meningiomas. Quantitative RT-PCR (qRT-PCR) confirmed increased APLN expression in NF2-null immortalized and patient-derived meningioma lines, with reduced expression upon mTORC1 inhibition. Apelin-13 stimulation enhanced sprouting, whereas APLN deletion reduced endothelial sprouting. Conclusions: Here we establish a 3D-tumoroid model and implicate tumor-derived Apelin as an important contributor to NF2-associated meningioma angiogenesis. Our data also suggest that APLN expression is regulated, at least in part, by mTORC1. Together, these results provide a preclinical platform for investigating angiogenic vulnerabilities beyond VEGF in NF2-deficient meningiomas.

Tuberous sclerosis complex (TSC) is an inherited multi-system neurocutaneous disorder where patients often present with neurodevelopmental manifestations such as epilepsy and TSC-associated neuropsychiatric disorder (TAND) that includes autism spectrum disorder (ASD). TSC is caused by inactivating mutations in TSC1 or TSC2 tumour suppressor genes, with encoded proteins hamartin (TSC1) and tuberin (TSC2) forming a functional complex inhibiting mechanistic target of rapamycin complex 1 (mTORC1) signalling. This has led to treatment with allosteric mTORC1 inhibitor rapamycin analogues (‘rapalogs’) for TSC tumours; however, rapalogs are ineffective for treating neurodevelopmental manifestations. mTORC1 signalling controls protein synthesis by regulating formation of the eukryotic initiation factor (eIF) 4F complex, with further modulation by MAP kinase-interacting serine/threonine protein kinases 1 and 2 (MNK1/2) via phosphorylation of the eIF4F subunit eIF4E. While both these pathways modulate translation, comparing their impact on transcriptome-wide mRNA translation, as well as effects of inhibiting these pathways in TSC has not been explored. Employing CRISPR-modified, isogenic neural progenitor cells (NPCs) derived from a female TSC2 patient, we have examined alterations in early neurodevelopmental phenotypes including proliferation and neurite outgrowth, as well as the ability of the bi-steric mTORC1-specific inhibitor RMC-6272 to rescue these phenotypes. Further, we utilized polysome profiling to examine transcriptome-wide changes in mRNA translation upon TSC2 loss and tested effects of treatment with RMC-6272 or the MNK1/2-specific inhibitor eFT-508. Our results reveal that altered early neurodevelopmental phenotypes can be rescued upon treatment with RMC-6272, but not rapamycin. We also discovered dysregulated mRNA translation in TSC2-Null NPCs, which significantly overlaps with the translatome from TSC1-Null NPCs. Interestingly, numerous non-monogenic ASD-, neurodevelopmental disorder (NDD)- and epilepsy-associated genes identified in patients harbouring putative loss-of-function mutations, were translationally suppressed in TSC2-Null NPCs. Importantly, translation of these ASD- and NDD-associated genes was reversed upon inhibition of either mTORC1 or MNK1/2 signalling using RMC-6272 or eFT-508, respectively. This study establishes the importance of mTORC1-eIF4F- and MNK-eIF4E-sensitive mRNA translation in TAND, ASD and other neurodevelopmental disorders laying the groundwork for evaluating drugs in clinical development that target these pathways as a treatment strategy for these disorders.

SUMMARY: Tuberous sclerosis complex (TSC) is an inherited neurodevelopmental disorder (NDD) with frequent manifestations of epilepsy and autism spectrum disorder (ASD). TSC is caused by inactivating mutations in TSC1 or TSC2 tumor suppressor genes, with encoded proteins hamartin (TSC1) and tuberin (TSC2) forming a functional complex inhibiting mechanistic target of rapamycin complex 1 (mTORC1) signaling. This has led to treatment with allosteric mTORC1 inhibitor rapamycin analogs (“rapalogs”) for TSC tumors; however, rapalogs are ineffective for treating neurodevelopmental manifestations. mTORC1 signaling controls protein synthesis by regulating formation of the eIF4F complex, with further modulation by MNK1/2 kinases via phosphorylation of the eIF4F subunit eIF4E. While both these pathways modulate translation, comparing their impact on transcriptome-wide mRNA translation, as well as effects of inhibiting these pathways in TSC has not been explored. Here, employing CRISPR-modified, isogenic TSC2 patient-derived neural progenitor cells (NPCs), we have examined transcriptome-wide changes in mRNA translation upon TSC2 loss. Our results reveal dysregulated translation in TSC2 -Null NPCs, which significantly overlaps with the translatome from TSC1 -Null NPCs. Interestingly, numerous non-monogenic ASD-, NDD-and epilepsy-associated genes identified in patients harboring putative loss-of-function mutations, were translationally suppressed in TSC2 -Null NPCs. Importantly, translation of these ASD- and NDD-associated genes was reversed upon inhibition of either mTORC1 or MNK1/2 signaling using RMC-6272 or eFT-508, respectively. This study establishes the importance of mTORC1-eIF4F- and MNK-eIF4E-sensitive mRNA translation in TSC, ASD and other neurodevelopmental disorders laying the groundwork for evaluating drugs in clinical development that target these pathways as a treatment strategy for these disorders.

NF2-associated meningiomas are progressive, highly morbid, and nonresponsive to chemotherapies, highlighting the need for improved treatments. We have established aberrant activation of the mechanistic target of rapamycin (mTOR) signaling in NF2-deficient tumors, leading to clinical trials with first- and second-generation mTOR inhibitors. However, results have been mixed, showing stabilized tumor growth without shrinkage offset by adverse side effects. To address these limitations, here we explored the potential of third-generation, bi-steric mTOR complex 1 (mTORC1) inhibitors using the preclinical tool compound RMC-6272. Employing human NF2-deficient meningioma lines, we compared mTOR inhibitors rapamycin (first-generation), INK128 (second-generation), and RMC-6272 (third-generation) using in vitro dose–response testing, cell-cycle analysis, and immunoblotting. Furthermore, the efficacy of RMC-6272 was assessed in NF2-null 3D-spheroid meningioma models, and its in vivo potential was evaluated in 2 orthotopic meningioma mouse models. Treatment of meningioma cells revealed that, unlike rapamycin, RMC-6272 demonstrated superior growth inhibitory effects, cell-cycle arrest, and complete inhibition of phosphorylated 4E-BP1 (mTORC1 readout). Moreover, RMC-6272 had a longer retention time than INK128 and inhibited the expression of several eIF4E-sensitive targets on the protein level. RMC-6272 treatment of NF2 spheroids showed significant shrinkage in size as well as reduced proliferation. Furthermore, in vivo studies in mice revealed effective blockage of meningioma growth by RMC-6272, compared with vehicle controls. Our study in preclinical models of NF2 supports possible future clinical evaluation of third-generation, investigational mTORC1 inhibitors, such as RMC-5552, as a potential treatment strategy for NF2.

Background: Tuberous sclerosis complex (TSC) is an inherited neurocutaneous disorder caused by mutations in the TSC1 or TSC2 genes, with patients often exhibiting neurodevelopmental (ND) manifestations termed TSC-associated neuropsychiatric disorders (TAND) including autism spectrum disorder (ASD) and intellectual disability. Hamartin (TSC1) and tuberin (TSC2) proteins form a complex inhibiting mechanistic target of rapamycin complex 1 (mTORC1) signaling. Loss of TSC1 or TSC2 activates mTORC1 that, among several targets, controls protein synthesis by inhibiting translational repressor eIF4E-binding proteins. Using TSC1 patient-derived neural progenitor cells (NPCs), we recently reported early ND phenotypic changes, including increased cell proliferation and altered neurite outgrowth in TSC1-null NPCs, which were unaffected by the mTORC1 inhibitor rapamycin. Methods: Here, we used polysome profiling, which quantifies changes in mRNA abundance and translational efficiencies at a transcriptome-wide level, to compare CRISPR-edited TSC1-null with CRISPR-corrected TSC1-WT NPCs generated from one TSC donor (one clone/genotype). To assess the relevance of identified gene expression alterations, we performed polysome profiling in postmortem brains from ASD donors and age-matched controls. We further compared effects on translation of a subset of transcripts and rescue of early ND phenotypes in NPCs following inhibition of mTORC1 using the allosteric inhibitor rapamycin versus a third-generation bi-steric, mTORC1-selective inhibitor RMC-6272. Results: Polysome profiling of NPCs revealed numerous TSC1-associated alterations in mRNA translation that were largely recapitulated in human ASD brains. Moreover, although rapamycin treatment partially reversed the TSC1-associated alterations in mRNA translation, most genes related to neural activity/synaptic regulation or ASD were rapamycin-insensitive. In contrast, treatment with RMC-6272 inhibited rapamycin-insensitive translation and reversed TSC1-associated early ND phenotypes including proliferation and neurite outgrowth that were unaffected by rapamycin. Conclusions: Our work reveals ample mRNA translation alterations in TSC1 patient-derived NPCs that recapitulate mRNA translation in ASD brain samples. Further, suppression of TSC1-associated but rapamycin-insensitive translation and ND phenotypes by RMC-6272 unveils potential implications for more efficient targeting of mTORC1 as a superior treatment strategy for TAND.

Tuberous sclerosis complex (TSC) is an inherited neurocutaneous disorder caused by mutations in TSC1 or TSC2 genes, with patients often exhibiting neurodevelopmental (ND) manifestations termed TSC-associated neuropsychiatric disorders (TAND) including autism spectrum disorder (ASD). The hamartin-tuberin (TSC1-TSC2) protein complex inactivates mechanistic target of rapamycin complex 1 (mTORC1) signaling, leading to increased protein synthesis via inactivation of translational repressor eIF4E-binding proteins (4E-BPs). In TSC1-null neural progenitor cells (NPCs), we previously reported early ND phenotypic changes, including increased proliferation/altered neurite outgrowth, which were unaffected by mTORC1-inhibitor rapamycin. Here, using polysome-profiling to quantify translational efficiencies at a transcriptome-wide level, we observed numerous TSC1-dependent alterations in NPCs, largely recapitulated in post-mortem brains from ASD donors. Although rapamycin partially reversed TSC1-associated alterations, most neural activity/synaptic- or ASD-related genes remained insensitive but were inhibited by third-generation bi-steric, mTORC1-selective inhibitor RMC-6272, which also reversed altered ND phenotypes. Together these data reveal potential implications for treatment of TAND.

Neurofibromatosis 2 (NF2) is an inherited disorder caused by bi-allelic inactivation of the NF2 tumor suppressor gene. NF2-associated tumors, including schwannoma and meningioma, are resistant to chemotherapy, often recurring despite surgery and/or radiation, and have generally shown cytostatic response to signal transduction pathway inhibitors, highlighting the need for improved cytotoxic therapies. Leveraging data from our previous high-throughput drug screening in NF2 preclinical models, we identified a class of compounds targeting the ubiquitin-proteasome pathway (UPP), and undertook studies using candidate UPP inhibitors, ixazomib/MLN9708, pevonedistat/MLN4924, and TAK-243/MLN7243. Employing human primary and immortalized meningioma (MN) cell lines, CRISPR-modified Schwann cells (SCs), and mouse Nf2 ^-/- SCs, we performed dose response testing, flow cytometry-based Annexin V and cell cycle analyses, and RNA-sequencing to identify potential underlying mechanism of apoptosis. In vivo efficacy was also assessed in orthotopic NF2-deficient meningioma and schwannoma tumor models. Testing of three UPP inhibitors demonstrated potent reduction in cell viability and induction of apoptosis for ixazomib or TAK-243, but not pevonedistat. In vitro analyses revealed that ixazomib or TAK-243 downregulates expression of c-KIT and PDGFRα, as well as the E3 ubiquitin ligase SKP2 while upregulating genes associated with endoplasmic reticulum stress-mediated activation of the unfolded protein response (UPR). In vivo treatment of mouse models revealed delayed tumor growth, suggesting a therapeutic potential. This study demonstrates the efficacy of proteasomal pathway inhibitors in meningioma and schwannoma preclinical models and lays the groundwork for use of these drugs as a promising novel treatment strategy for NF2 patients.