Abstract BACKGROUND Comprehensive molecular characterization of pediatric brain tumors has led to a more refined diagnosis. However, the feasibility of performing multi-omic and functional precision medicine approaches using ex-vivo drug screening in the clinical setting is unknown. METHODS Patients with newly diagnosed or recurrent central nervous system tumors were enrolled in a feasibility study of multi-omic analysis including whole genome trio germline sequencing, tumor whole exome/RNA sequencing, RNA-based DiSCoVER analysis, methylation profiling, immunogenic potential analysis, and ex-vivo drug screening utilizing a customized panel of 175 FDA approved/investigational drugs. Findings were presented at a multidisciplinary molecular neuro-oncology tumor board. RESULTS Eighteen patients (9 female; average age 9.4 years; 12 newly diagnosed, 6 with recurrent disease; 5 high grade gliomas, 4 ependymomas, 4 embryonal tumors, 3 low grade gliomas, 2 others) were enrolled between 2020-2022. Whole genome germline testing (N=18) was normal in half of patients (pathogenic germline mutations in 3 patients, variants of unknown significance in 6 patients). Ex-vivo drug screening results (N=14) varied among patients, however sub-micromolar efficacy was commonly observed for proteosome inhibitors, HDAC inhibitors, and topoisomerase II inhibitors. Average time from surgery to receipt of finalized results varied across platforms (drug screening 5.7 days, whole exome/RNA sequencing 14.8 days, whole genome germline 10.6 days, methylation 21 days). Fifteen patients had a modification in diagnosis and 4 patients had a change in tumor classification. Multi-omic and functional precision medicine results alone or in combination led to changes in management in 50% of patients (Tumor Molecular Sequencing 6/18, Ex-Vivo Drug Screening 4/14, RNA DiSCoVER Analysis 2/15, Methylation 1/14). CONCLUSION Our studies demonstrate the feasibility of timely ex-vivo drug screening and multi-omic analysis and show that these data may lead to changes in patient diagnosis/management. These findings are the basis of an ongoing trial for recurrent medulloblastoma (NCT05057702).

Figure S7 shows effects of CDK4/6 inhibitors on viability of mouse CPC cells and drug synergy between Triptolide and Dinaciclib.

Table S1 - Primers and Probes Table S2 - Antibodies Table S3 - Cell line information Table S4 - SB-induced medulloblastoma and CNS-PNET characteristics Table S5 - gene lists in mouse and human medulloblastoma Table S6 - gene lists in mouse and human CNS-PNET Table S7 - CIS genes for medulloblastoma and CNS-PNET Table S8 - CIS comparison between this and previous SB studies in medulloblastoma Table S9 - CIS comparison in this SB screen and a previous screen in osteosarcoma

Identifying the cells from which cancers arise is critical for understanding the molecular underpinnings of tumor evolution. To determine whether stem/progenitor cells can serve as cells of origin, we created a Msi2-CreERT2 knock-in mouse. When crossed to CAG-LSL-MycT58A mice, Msi2-CreERT2 mice developed multiple pancreatic cancer subtypes: ductal, acinar, adenosquamous, and rare anaplastic tumors. Combining single-cell genomics with computational analysis of developmental states and lineage trajectories, we demonstrate that MYC preferentially triggers transformation of the most immature MSI2+ pancreas cells into multi-lineage pre-cancer cells. These pre-cancer cells subsequently diverge to establish pancreatic cancer subtypes by activating distinct transcriptional programs and large-scale genomic changes, and enforced expression of specific signals like Ras can redirect subtype specification. This study shows that multiple pancreatic cancer subtypes can arise from a common pool of MSI2+ cells and provides a powerful model to understand and control the programs that shape divergent fates in pancreatic cancer.

Accumulating evidence has shown that H6 Family Homeobox 3 (HMX3) plays a crucial role in nervous system regulation. However, the regulatory mechanism of HMX3 in colorectal cancer (CRC) has seldom been studied. Herein, HMX3 was significantly downregulated in CRC, as demonstrated by qRT-PCR and WB analysis on clinical samples and a panel of cell lines. Besides, it was found that the expression of HMX3 was negatively correlated with survival of CRC patients. The functional analyses (EdU staining, CCK-8, colony formation, Transwell, and wound scratch assays) showed that CRC cell proliferation, migration, and invasion were significantly suppressed by HMX3 overexpression, while enhanced by HMX3 knockdown. Moreover, in vivo experiment revealed HMX3 overexpression could also suppress tumor growth. Combining bioinformatics and WB analysis, we preliminarily uncovered that HMX3 was involved in apoptosis and KRAS signaling pathways. Mechanistically, Ubiquitin-specific protease 38 (USP38) was identified as a novel post-translational regulator of HMX3, which could directly interact with HMX3 to stabilize its protein expression via deubiquitination. Furthermore, the role of USP38 silencing in promoting cell proliferation, migration, and invasion of CRC cells was blocked by HMX3 overexpression. In conclusion, our findings suggested that USP38/HMX3 axis is a novel promising therapeutic candidate for CRC.

Figure S4. Shows ARHGAP36 expression in mouse and human neural tissue. Figure S5. Shows characterization of ARHGAP36 and Megf10 in C17.2 cells Figures S6-S7 show mechanistic analysis of FOXR2 in C17.2 and HSC1L cells

Characterization of SB-induced medulloblastomas and CNS-PNETs.

Multiciliated cells (MCCs) in the brain reside in the ependyma and the choroid plexus (CP) epithelia. The CP secretes cerebrospinal fluid that circulates within the ventricular system, driven by ependymal cilia movement. Tumors of the CP are rare primary brain neoplasms mostly found in children. CP tumors exist in three forms: CP papilloma (CPP), atypical CPP, and CP carcinoma (CPC). Though CPP and atypical CPP are generally benign and can be resolved by surgery, CPC is a particularly aggressive and little understood cancer with a poor survival rate and a tendency for recurrence and metastasis. In contrast to MCCs in the CP epithelia, CPCs in humans are characterized by solitary cilia, frequent TP53 mutations, and disturbances to multiciliogenesis program directed by the GMNC-MCIDAS transcriptional network. GMNC and MCIDAS are early transcriptional regulators of MCC fate differentiation in diverse tissues. Consistently, components of the GMNC-MCIDAS transcriptional program are expressed during CP development and required for multiciliation in the CP, while CPC driven by deletion of Trp53 and Rb1 in mice exhibits multiciliation defects consequent to deficiencies in the GMNC-MCIDAS program. Previous studies revealed that abnormal NOTCH pathway activation leads to CPP. Here we show that combined defects in NOTCH and Sonic Hedgehog signaling in mice generates tumors that are similar to CPC in humans. NOTCH-driven CP tumors are monociliated, and disruption of the NOTCH complex restores multiciliation and decreases tumor growth. NOTCH suppresses multiciliation in tumor cells by inhibiting the expression of GMNC and MCIDAS, while Gmnc-Mcidas overexpression rescues multiciliation defects and suppresses tumor cell proliferation. Taken together, these findings indicate that reactivation of the GMNC-MCIDAS multiciliogenesis program is critical for inhibiting tumorigenesis in the CP, and it may have therapeutic implications for the treatment of CPC.

Identifying the cells from which cancers arise, and the paths they take towards malignancy, is critical for understanding the molecular basis of tumor initiation and progression. To understand if stem and progenitor cells can serve as cells of origin for cancer, we created a model in which the CreER T2 recombinase was knocked into the endogenous locus of the stem cell determinant Msi2. When crossed to a conditional CAG-LSL-Myc T58A model, Msi2-Cre ERT2 mice developed a diversity of tumors across tissues, including multiple pancreatic cancer subtypes: pancreatic ductal adenocarcinoma (PDAC), adenosquamous carcinoma of the pancreas (ASCP), acinar cell carcinoma (ACC), and rare anaplastic tumors. Using single cell analysis, we traced the temporal changes that occurred as normal Msi2+ cells evolved through a pre-cancerous stage to PDAC, ASCP or ACC. These results revealed that Msi2+ cells were present predominantly in pancreatic ducts and exhibited heterogeneous differentiation states in the normal pancreas. At initiation, Myc triggered oncogenic transformation in the most undifferentiated subset leading to the rise of pre-cancer cells with multi-lineage properties. Subsequently, these pre-cancer cells were driven along different fates by epigenetically activated transcriptional programs with genomic changes amplifying the progression into distinct pancreatic cancer subtypes. Finally, integrating transcriptomic and functional genomic approaches in this new model allowed us to define Ifne , Ifitm3 , Atf3 , Hmmr , and Raet1e as novel functional dependencies of ASCP, giving us unique insight into the most lethal of pancreatic malignancies. These data show that multiple pancreatic cancer subtypes can arise from a common pool of pre-malignant cells and provide a powerful molecular framework to understand the programs that shape divergent fates in pancreas cancer and develop approaches for early detection and interception.

Multiciliated cells (MCCs) in the brain include the ependymal cells and choroid plexus (CP) epithelial cells. The CP secretes cerebrospinal fluid that circulates within the ventricular system, driven by ependymal cilia movement. However, the mechanisms and functional significance of multiciliogenesis in the CP remain unknown. Deregulated oncogenic signals cause CP carcinoma (CPC), a rare but aggressive pediatric brain cancer. Here we show that aberrant NOTCH and Sonic Hedgehog signaling in mice drive tumors that resemble CPC in humans. NOTCH-driven CP tumors were monociliated, whereas disruption of the NOTCH complex restored multiciliation and decreased tumor growth. NOTCH suppressed multiciliation in tumor cells by inhibiting the expression of GEMC1 and MCIDAS, early regulators of multiciliogenesis. Consistently, GEMC1-MCIDAS function is essential for multiciliation in the CP, and is critical for correcting multiciliation defect in tumor cells by a NOTCH inhibitor. Disturbances to the GEMC1 program are commonly observed in human CPCs characterized by solitary cilia. Consistently, CPC driven by deletion of Trp53 and Rb1 in mice exhibits a cilia deficit consequent to loss of Gemc1-Mcidas expression. Taken together, these findings reveal a GEMC1-MCIDAS multiciliation program in the CP critical for inhibiting tumorigenesis, and it may have therapeutic implications for the treatment of CPC.

Choroid plexus carcinoma (CPC) is a rare brain tumor that occurs most commonly in very young children and has a dismal prognosis despite intensive therapy. Improved outcomes for patients with CPC depend on a deeper understanding of the mechanisms underlying the disease. Here we developed transgenic models of CPCs by activating the Myc oncogene and deleting the Trp53 tumor suppressor gene in murine neural stem cells or progenitors. Murine CPC resembled their human counterparts at a histologic level, and like the hypodiploid subset of human CPC, exhibited multiple whole-chromosome losses, particularly of chromosomes 8, 12, and 19. Analysis of murine and human CPC gene expression profiles and copy number changes revealed altered expression of genes involved in cell cycle, DNA damage response, and cilium function. High-throughput drug screening identified small molecule inhibitors that decreased the viability of CPC. These models will be valuable tools for understanding the biology of choroid plexus tumors and for testing novel approaches to therapy. Significance: This study describes new mouse models of choroid plexus carcinoma and uses them to investigate the biology and therapeutic responsiveness of this highly malignant pediatric brain tumor.

N-6-methyladenosine (m(6)A) constitutes one of the most abundant internal RNA modifications and is critical for RNA metabolism and function. It has been previously reported that viral RNA contains internal m(6)A modifications; however, only recently the function of m(6)A modification in viral RNAs has been elucidated during infections of HIV, hepatitis C virus and Zika virus. In the present study, we found that enterovirus 71 (EV71) RNA undergoes m(6)A modification during viral infection, which alters the expression and localization of the methyltransferase and demethylase of m(6)A, and its binding proteins. Moreover, knockdown of m(6)A methyltransferase resulted in decreased EV71 replication, whereas knockdown of the demethylase had the opposite effect. Further study showed that the m(6)A binding proteins also participate in the regulation of viral replication. In particular, two m(6)A modification sites were identified in the viral genome, of which mutations resulted in decreased virus replication, suggesting that m(6)A modification plays an important role in EV71 replication. Notably, we found that METTL3 interacted with viral RNA-dependent RNA polymerase 3D and induced enhanced sumoylation and ubiquitination of the 3D polymerase that boosted viral replication. Taken together, our findings demonstrated that the host m(6)A modification complex interacts with viral proteins to modulate EV71 replication.

Abstract Medulloblastoma and central nervous system primitive neuroectodermal tumors (CNS-PNET) are aggressive, poorly differentiated brain tumors with limited effective therapies. Using Sleeping Beauty (SB) transposon mutagenesis, we identified novel genetic drivers of medulloblastoma and CNS-PNET. Cross-species gene expression analyses classified SB-driven tumors into distinct medulloblastoma and CNS-PNET subgroups, indicating they resemble human Sonic hedgehog and group 3 and 4 medulloblastoma and CNS neuroblastoma with FOXR2 activation. This represents the first genetically induced mouse model of CNS-PNET and a rare model of group 3 and 4 medulloblastoma. We identified several putative proto-oncogenes including Arhgap36, Megf10, and Foxr2. Genetic manipulation of these genes demonstrated a robust impact on tumorigenesis in vitro and in vivo. We also determined that FOXR2 interacts with N-MYC, increases C-MYC protein stability, and activates FAK/SRC signaling. Altogether, our study identified several promising therapeutic targets in medulloblastoma and CNS-PNET. Significance: A transposon-induced mouse model identifies several novel genetic drivers and potential therapeutic targets in medulloblastoma and CNS-PNET.

Current research has identified several potential biomarkers for lung cancer diagnosis or prognosis. However, most of these biomarkers are derived from a relatively small number of samples using algorithms at the gene level. Hence, gene expression signatures discovered in these studies have little overlaps. In this study, we proposed a new strategy to identify biomarkers from multiple datasets at the pathway level. We integrated the genome-wide expression data of lung cancer tissues from 13 published studies and applied our strategy to identify lung cancer diagnostic and prognostic biomarkers. We identified a 32-gene signature that differentiates lung adenocarcinomas from other lung cancer subtypes. We also discovered a 43-gene signature that can predict the outcome of human lung cancers. We tested their performance in several independent cohorts, which confirmed their robust prognostic and diagnostic power. Furthermore, we showed that the proposed gene expression signatures were independent of several traditional clinical indicators in lung cancer management. Our results suggest that the pathway-based strategy is useful to identify transcriptomic biomarkers from large-scale gene expression datasets that were collected from multiple sources.

In cancer, recurrent somatic single-nucleotide variants—which are rare in most paediatric cancers—are confined largely to protein-coding genes1–3. Here we report highly recurrent hotspot mutations (r.3A>G) of U1 spliceosomal small nuclear RNAs (snRNAs) in about 50% of Sonic hedgehog (SHH) medulloblastomas. These mutations were not present across other subgroups of medulloblastoma, and we identified these hotspot mutations in U1 snRNA in only <0.1% of 2,442 cancers, across 36 other tumour types. The mutations occur in 97% of adults (subtype SHHδ) and 25% of adolescents (subtype SHHα) with SHH medulloblastoma, but are largely absent from SHH medulloblastoma in infants. The U1 snRNA mutations occur in the 5′ splice-site binding region, and snRNA-mutant tumours have significantly disrupted RNA splicing and an excess of 5′ cryptic splicing events. Alternative splicing mediated by mutant U1 snRNA inactivates tumour-suppressor genes (PTCH1) and activates oncogenes (GLI2 and CCND2), and represents a target for therapy. These U1 snRNA mutations provide an example of highly recurrent and tissue-specific mutations of a non-protein-coding gene in cancer. Highly recurrent hotspot r.3A>G mutations are identified in U1 splicesomal small nuclear RNAs in about 50% of Sonic hedgehog medulloblastomas, which result in disrupted RNA splicing and the activation of oncogenes.

In the version of this article initially published online, there were errors in URLs for www.southernbiotech.com, appearing in Methods sections "m6A dot-blot" and "Western blot analysis." The first two URLs should be https://www.southernbiotech.com/?catno=4030-05&type=Polyclonal#&panel1-1 and the third should be https://www.southernbiotech.com/?catno=6170-05&type=Polyclonal. In addition, some Methods URLs for bioz.com, www.abcam.com and www.sysy.com were printed correctly but not properly linked. The errors have been corrected in the PDF and HTML versions of this article.

Brain tumors are the leading cause of cancer-related death in children, and medulloblastoma (MB) is the most common malignant pediatric brain tumor. Advances in surgery, radiation, and chemotherapy have improved the survival of MB patients. But despite these advances, 25-30% of patients still die from the disease, and survivors suffer severe long-term side effects from the aggressive therapies they receive. Although MB is often considered a single disease, molecular profiling has revealed a significant degree of heterogeneity, and there is a growing consensus that MB consists of multiple subgroups with distinct driver mutations, cells of origin, and prognosis. Here, we review recent progress in MB research, with a focus on the genes and pathways that drive tumorigenesis, the animal models that have been developed to study tumor biology, and the advances in conventional and targeted therapy.

Abstract The mTOR inhibitors, including the second generation of mTOR kinase inhibitors, for cancer treatment have been evaluated in clinical trials, but their overall activity as monotherapy is limited. The cap-dependent translation repressor, 4E-BP1, is an important effector of the mTOR kinase in controlling cell proliferation and tumor growth. Loss of 4E-BP1 expression in cancer has been linked to malignant progression and poor prognosis. However, the molecular mechanism(s) by which 4E-BP1 is downregulated in cancer remain largely unknown. Here, we identify Snail as a novel repressor of 4E-BP1 expression that can be exploited to alter mTOR-targeted therapies. We find that 4E-BP1 expression inversely correlates with Snail level in cancer cell lines and clinical specimens. Mechanistically, we show that Snail binds to three E-boxes present in the human 4E-BP1 promoter to repress transcription of 4E-BP1. Ectopic expression of Snail in cancer cell lines lacking Snail profoundly inhibits 4E-BP1 expression, and promotes cap-dependent translation and resistance to the anti-proliferative effect of mTOR kinase inhibitors. Conversely, genetic depletion of Snail restores 4E-BP1 expression and sensitizes cancer cells to mTOR inhibition by enhancing the translation-repressive function of 4E-BP1 on cell proliferation and tumor growth. Thus, our findings reveal a critical role for Snail in determining the therapeutic response to mTOR kinase inhibitors, and suggest that Snail may be a potential marker for predicting the efficacy of mTOR-target therapies in the clinic. Moreover, our work highlights the importance of combining Snail inhibition with mTOR kinase inhibitors for the treatment of tumors in which Snail overexpression occurs with reduced 4E-BP1 expression. Citation Format: Jun Wang, Qing Ye, Yubin Guo, Yanan Cao, Xiuping Huang, Wenting Mi, Side Liu, Chi Wang, B. Mark Evers, Qing-Bai She. Snail determines the efficacy of mTOR-targeted therapies by transcriptional repression of 4E-BP1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-125. doi:10.1158/1538-7445.AM2017-LB-125

Loss of the tumor suppressors RB1 and TP53 and MYC amplification are frequent oncogenic events in small cell lung cancer (SCLC). We show that Myc expression cooperates with Rb1 and Trp53 loss in the mouse lung to promote aggressive, highly metastatic tumors, that are initially sensitive to chemotherapy followed by relapse, similar to human SCLC. Importantly, MYC drives a neuroendocrine-low “variant” subset of SCLC with high NEUROD1 expression corresponding to transcriptional profiles of human SCLC. Targeted drug screening reveals that SCLC with high MYC expression is vulnerable to Aurora kinase inhibition, which, combined with chemotherapy, strongly suppresses tumor progression and increases survival. These data identify molecular features for patient stratification and uncover a potential targeted treatment approach for MYC-driven SCLC.

We previously showed that different pathologic subtypes were associated with different prognostic values in patients with stage IA lung adenocarcinoma (AC). We hypothesize that differential gene expression profiles of different subtypes may be valuable factors for prognosis in stage IA lung adenocarcinoma. We performed microarray gene expression profiling on tumor tissues micro-dissected from patients with acinar and solid predominant subtypes of stage IA lung adenocarcinoma. These patients had undergone a lobectomy and mediastinal lymph node dissection at the Shanghai Chest Hospital, Shanghai, China in 2012. No patient had preoperative treatment. We performed the Gene Set Enrichment Analysis (GSEA) analysis to look for gene expression signatures associated with tumor subtypes. The histologic subtypes of all patients were classified according to the 2015 WHO lung Adenocarcinoma classification. We found that patients with the solid predominant subtype are enriched for genes involved in RNA polymerase activity as well as inactivation of the p53 pathway. Further, we identified a list of genes that may serve as prognostic markers for stage IA lung adenocarcinoma. Validation in the TCGA database shows that these genes are correlated with survival, suggesting that they are novel prognostic factors for stage IA lung adenocarcinoma. In conclusion, we have uncovered novel prognostic factors for stage IA lung adenocarcinoma using gene expression profiling in combination with histopathology subtyping.