Genome-scale CRISPR-Cas9 knockout screening in hepatocellular carcinoma with lenvatinib resistance

Lenvatinib is the first target drug approved for advanced hepatocellular carcinoma (HCC). However, the development of drug resistance is common, and the mechanisms of lenvatinib resistance and resistant targets in HCC are poorly understood. By using CRISPR/Cas9 library screening, we screened out two key resistance genes, neurofibromin 1(NF1), and dual specificity phosphatase 9 (DUSP9), as critical drivers for lenvatinib resistance in HCC. With RNAi knockdown and CRISPR/Cas9 knockout models, we further clarified the mechanisms by which NF1 loss reactivates the PI3K/AKT and MAPK/ERK signaling pathways, while DUSP9 loss activates the MAPK/ERK signaling pathways, thereby inactivating FOXO3, followed by degradation of FOXO3, finally induced lenvatinib resistance. We also screened out trametinib, a small molecule pathway inhibitor for MEK, that can be used to reverse resistance induced by NF1 and DUSP9 loss in HCC cells. Trametinib was still able to halt HCC growth even when NF1 was knocked out in mice. Collectively, the findings indicate that NF1 and DUSP9 takes critical role in lenvatinib resistance and may be novel specific targets and predictive markers for lenvatinib resistance in HCC.

Novel Variants and Clinical Characteristics of 16 Patients from Southeast Asia with Genetic Variants in Neurofibromin-1

Neurofibromatosis type 1 (NF1) is one of the most common inherited disorders. It is caused by mutations in the neurofibromin-1 gene (NF1) and affects the formation and growth of nerve tissues. More than 3,600 pathogenic variants in the NF1 gene have been identified from patients with most of the germline variants are from the Western populations. We found 16 patients (15 Chinese and 1 Asian Indian) who had heterozygous variants in NF1 through targeted next-generation sequencing. There were 15 different variants: 4 frameshift, 4 nonsense, 5 missense, and 2 splice variants. One nonsense variant and three frameshift variants had never been reported in any population or patient database. Twelve of the 16 patients met the NF1 diagnostic criteria, and each was found to have a pathogenic or likely pathogenic variant. Three different missense variants of unknown significance were discovered in the other four patients who did not meet NF1 diagnostic criteria. Our findings add four novel variants to the list of genetic mutations linked to NF1's various clinical manifestations.

Neurofibromin 1 in mushroom body neurons mediates circadian wake drive through activating cAMP–PKA signaling

Various behavioral and cognitive states exhibit circadian variations in animals across phyla including Drosophila melanogaster, in which only ~0.1% of the brain’s neurons contain circadian clocks. Clock neurons transmit the timing information to a plethora of non-clock neurons via poorly understood mechanisms. Here, we address the molecular underpinning of this phenomenon by profiling circadian gene expression in non-clock neurons that constitute the mushroom body, the center of associative learning and sleep regulation. We show that circadian clocks drive rhythmic expression of hundreds of genes in mushroom body neurons, including the Neurofibromin 1 (Nf1) tumor suppressor gene and Pka-C1. Circadian clocks also drive calcium rhythms in mushroom body neurons via NF1-cAMP/PKA-C1 signaling, eliciting higher mushroom body activity during the day than at night, thereby promoting daytime wakefulness. These findings reveal the pervasive, non-cell-autonomous circadian regulation of gene expression in the brain and its role in sleep.

Neurofibromin 1 controls metabolic balance and Notch-dependent quiescence of juvenile myogenic progenitors

Patients affected by neurofibromatosis type 1 (NF1) frequently show muscle weakness with unknown etiology. Here we show that Neurofibromin-1 (Nf1) is not required in muscle fibers, but specifically in early postnatal myogenic progenitors (MPs), where Nf1 loss led to cell cycle exit and differentiation blockade, depleting the MP pool resulting in reduced myonuclear accrual as well as reduced muscle stem cell numbers. This was caused by precocious induction of stem cell quiescence coupled to metabolic reprogramming of MPs impinging on glycolytic shutdown, which was conserved in muscle fibers. We show that a Mek/Erk/NOS pathway hypersensitizes Nf1-deficient MPs to Notch signaling, consequently, early postnatal Notch pathway inhibition ameliorated premature quiescence, metabolic reprogramming and muscle growth. This reveals an unexpected role of Ras/Mek/Erk signaling supporting postnatal MP quiescence in concert with Notch signaling, which is controlled by Nf1 safeguarding coordinated muscle growth and muscle stem cell pool establishment. Furthermore, our data suggest transmission of metabolic reprogramming across cellular differentiation, affecting fiber metabolism and function in NF1.

HGG-39. ALTERNATIVE SPLICING OF NEUROFIBROMIN 1 IS ASSOCIATED WITH ELEVATED MAPK ACTIVITY AND POOR PROGNOSIS IN HIGH-GRADE GLIOMA

Despite a good understanding of the coding mutations underlying high-grade gliomas (HGG), their prognosis remains poor. We sought to characterize their transcriptional alterations and how this contributes to pathogenesis. We analyzed a large cohort of pediatric HGG (pHGG) by DNA sequencing (n=79) and RNA-Seq (n=63 plus normal brain, n=20), finding spliceosome mutations that are associated with increased splicing burden. High levels of alternative splicing were found in known cancer driver genes, with enrichment for chromatin regulators (including the SWI/SNF and NuRD complexes) and the RAS/MAPK pathway, in particular neurofibromin 1 (NF1). Both pediatric and adult HGG preferentially expressed NF1-II, a less active RAS GTPase, resulting in increased RAS/MAPK activity resulting from inclusion of exon23a into the GAP-related domain of NF1. In IDH wild-type, adult HGG, NF1-II was associated with reduced survival independently from RAS/MAPK pathway mutations. NF1 exon23a splicing was regulated by REST-mediated suppression of splicing factors controlling its inclusion. Together, our results identify a novel mechanism by which HGG can activate RAS/MAPK signaling and other oncogenic pathways to promote tumorigenesis independently from direct mutations.

Rhythmic expression of Neurofibromin 1 in mushroom body neurons mediates circadian wake drive through activating cAMP−PKA signaling

SUMMARYVarious behavioral and cognitive states exhibit circadian variations in animals across phyla including Drosophila, in which only ∼0.1% of the entire brain neurons contain circadian clocks. This suggests that clock neurons communicate with a plethora of non-clock neurons to transmit the timing information to gate various behavioral outputs in Drosophila. Here, we address the molecular underpinning of this phenomenon by performing circadian RNA-seq analysis of non-clock neurons that constitute the mushroom body (MB), the center of information processing and sleep regulation. We identify hundreds of genes rhythmically expressed in the MB, including the Drosophila ortholog of Neurofibromin 1 (Nf1), the gene responsible for neurofibromatosis type 1 (NF1). Rhythmic expression of Nf1 promotes daytime wakefulness by activating cAMP−PKA signaling and increasing excitability of the MB. These findings reveal the pervasive, non-cell-autonomous circadian regulation of gene expression in the brain and its role in sleep, with implications in the pathology of NF1.

Identification of a Novel NF1 Frameshift Variant in a Chinese Family with Neurofibromatosis Type 1

Neurofibromatosis type 1 (NF1) is a progressive neurocutaneous disorder in humans, mainly characterized by café-au-lait macules (CALMs) and neurofibromas. NF1 is caused by variants of the neurofibromin 1 gene (NF1), which encodes a Ras-GTPase-activating protein called neurofibromin. NF1 variants may result in loss of neurofibromin function and elevation of cell proliferation and tumor formation. In this study, a Chinese NF1 family with an autosomal dominant inheritance pattern was recruited. Exome sequencing and Sanger sequencing were performed to discover the causative variant responsible for the family, followed by molecular analysis of effect of the mutated NF1 protein on Ras activity. A novel frameshift variant c.541dupC (p.(Gln181Profs∗20)) in the NF1 gene was identified in all three affected family members. The variant cosegregated with the disease phenotypes in the pedigree and was absent in 100 healthy controls. Bioinformatic analysis showed that the variant c.541dupC (p.(Gln181Profs∗20)) was pathogenic. The further molecular analysis verified the cells expressing NF1 variant p.(Gln181Profs∗20) partially enhanced Ras activity and elevated cell proliferation and tumor formation due to loss of neurofibromin function caused by the variant. Taken together, the data strongly advocate the c.541dupC (p.(Gln181Profs∗20)) variant as the underlying genetic cause of the Chinese family with NF1. Moreover, our findings broaden the spectrum of NF1 variants and provide molecular insights into the pathogenesis of NF1.

Conjunctival Melanoma: Genetic and Epigenetic Insights of a Distinct Type of Melanoma

Conjunctival melanoma (CjM) is a rare, primary cancer of the ocular region. Genetic and epigenetic characteristics of conjunctival melanoma have not been completely elucidated yet. Conjunctival melanoma presents similarities with cutaneous melanoma, with substantial differences in the biological behavior. We reviewed the genetic and epigenetic insights of CjM involved in invasion and metastatic spread. CjM is commonly characterized by mutations of v-raf murine sarcoma viral oncogene homolog B1 (BRAF), neurofibromin 1 (NF1) and telomerase reverse transcriptase (TERT), high expression of mammalian target of rapamycin (mTOR) and heat shock protein 90 (HSP90), frequent phosphatase and tensin homolog (PTEN) loss and upregulation of specific miRNAs. These features should identify CjM as a distinct subset of melanoma with its own profile, which is more similar to cutaneous melanoma than mucosal melanoma and remarkably different from uveal melanoma.