E2F Pathway Research Articles

Genomic and immunologic profiles of concurrent RB1 and CDKN1A/p21(WAF1) truncating mutations (RW+) in bladder cancer.

4571 Background: p53 target and cell cycle inhibitor CDKN1A/p21(WAF1) was initially not found to be mutated in cancer. TCGA analysis identified CDKN1A mutations are present but rare with frequencies of < 1%, but enrich in bladder cancer (̃8%). Truncating WAF1 mutations are associated with sensitivity to cisplatin and are associated with truncating Rb mutations in bladder cancer (RW+). We hypothesized RW+ bladder cancers may represent a unique subgroup with sensitivity to therapeutics. Methods: A total of 1104 urothelial tumors underwent molecular profiling at Caris Life Sciences (Phoenix, AZ) utilizing NGS of DNA (592 Gene Panel, NextSeq, or WES, NovaSeq) and RNA (NovaSeq, WTS). Wilcoxon, Fisher’s exact were used for statistical significance (p value without and q value with multi comparison correction). Immune cell fraction (QuanTIseq) and pathway analysis (ssGSEA) were assessed by mRNA analysis. Immune epitope prediction was performed using the NetMHCpan v4.0 method in the Immune Epitope Database. Results: Concurrent truncating mutation (frameshift, nonsense) for RB1 and WAF1 were detected in 47 tumors (RW+, 4.25%) and tumors with wild-type status for both RB1 and WAF1 genes were classified as RW- group (54.08%). Tumors harboring only one RB1 or WAF1 mutation were excluded for further analysis. When compared to RW- group, RW+ tumors showed lower mutation rate of TP53 (54.5% vs 80.9%, q < 0.05), ARID1A (23.5% vs 38.3%, p < 0.05), and PIK3CA (18.4% vs 31.9%, p < 0.05). Interestingly, RW+ was mutually exclusive with FGFR3 mutation (18.0% vs 0%, p < 0.05). We further evaluated RNA expression of DNA repair and checkpoint arrest pathways. Notably, E2F pathway (Normalized Enrichment Scores, NES: 0.89 vs 0.86, q < 0.01) and DNA G2M checkpoint (NES: 0.89 vs 0.86, q < 0.01) were found to be the most enriched in RW+ with respect to RW- group. In addition, mRNA levels of FANCC/A, CHEK1, WEE1, CDC25A/C, PALB2 and BRCA1/2 were found to be overexpressed in RW+ group (q < 0.05). RW+ tumors also displayed a distinct immunological profile: They were associated with higher PD-L1 status (63.8% vs 37.3%, q < 0.01), higher median TMB (11 mut/Mb vs 8 mut/Mb, q < 0.01) and with less frequent loss of heterozygosity for HLA-DPA1 (51.1% vs 66.7%, p < 0.05), with more high-binding-affinity neoantigen load (4.78 vs 3.89, p < 0.05) to MHC proteins, consistent with the significantly more myeloid dendritic cells in in RW+ group (0.3 vs 0.04, q < 0.001). Conclusions: Concurrent truncating mutation in RB1 and WAF1 (RW+) bladder carcinomas have fewer p53, ARID1A, and PIK3CA mutations but are enriched for E2F targets, G2/M checkpoint genes, FANCC/A, CHEK1, WEE1, CDC25A/C, PALB2 and BRCA1/2 and have a distinct immunological profile. The findings suggest therapeutic strategies for RW+ bladder cancers including Chk1/Wee1, PARP inhibitors, -/+ immunotherapy that may impact on clinical outcomes.

A novel pyroptosis risk model composed of NLRP6 effectively predicts the prognosis of hepatocellular carcinoma patients.

Pyroptosis is a unique inflammatory-related cell death process, and inflammation is considered to be a key factor in hepatocellular carcinoma (HCC). However, the pyroptosis landscape in HCC has not been thoroughly studied. Clinical data and RNA sequencing data of HCC patients were collected from The Cancer Genome Atlas database, and differentially expressed genes (DEGs) associated with pyroptosis were discovered. The relationship between DEGs and prognosis was studied. Using The Cancer Genome Atlas cohort, a least absolute shrinkage and selection operator regression model was built on the basis of pyroptosis-related DEGs, which was then verified by the Gene Expression Omnibus (GEO) cohort. Functional enrichment analysis and immunological states were also studied between distinct risk subgroups. Finally, the potential tumor suppressive function of NLRP6 in HCC was analyzed. In total, 26 pyroptosis-related DEGs were identified. Consensus clustering results showed patients with high levels of pyroptosis were associated with higher tumor stage, grade, and poor prognosis. The least absolute shrinkage and selection operator risk model was built using six genes linked with prognosis (GSDMC, GSDME, NOD2, NLRP6, CASP8, and SCAF11). Risk score was an independent risk factor that suggested shortened overall survival in both the training cohort (HR: 3.52, 95% CI: 1.351-9.193) and validation cohort (HR: 3.31, 95% CI: 1.435-7.617). Meanwhile, the low-risk population had higher immunological activity. We also found a novel potential tumor suppressor gene NLRP6, which may negatively regulate the E2F and MYC pathways and be associated with higher immune cell infiltration levels, which lead to better prognosis. This study revealed the pyroptosis landscape of HCC and provided a promising clinical prognosis evaluation model. Additionally, some new targets related to prognosis such as NLRP6 are proposed for further study.

Clonal evolution after treatment pressure in multiple myeloma: heterogenous genomic aberrations and transcriptomic convergence

We investigated genomic and transcriptomic changes in paired tumor samples of 29 in-house multiple myeloma (MM) patients and 28 patients from the MMRF CoMMpass study before and after treatment. A change in clonal composition was found in 46/57 (82%) of patients, and single-nucleotide variants (SNVs) increased from median 67 to 86. The highest increase in prevalence of genetic aberrations was found in RAS genes (60% to 72%), amp1q21 (18% to 35%), and TP53 (9% to 18%). The SBS-MM1 mutation signature was detected both in patients receiving high and low dose melphalan. A total of 2589 genes were differentially expressed between early and late samples (FDR < 0.05). Gene set enrichment analysis (GSEA) showed increased expression of E2F, MYC, and glycolysis pathways and a decreased expression in TNF-NFkB and TGFbeta pathways in late compared to early stage. Single sample GSEA (ssGSEA) scores of differentially expressed pathways revealed that these changes were most evident in end-stage disease. Increased expression of several potentially targetable genes was found at late disease stages, including cancer-testis antigens, XPO1 and ABC transporters. Our study demonstrates a transcriptomic convergence of pathways supporting increased proliferation and metabolism during disease progression in MM.

A Genome-Wide CRISPR Activation Screen Identifies PRRX2 as a Regulator of Enzalutamide Resistance in Prostate Cancer.

PRRX2 mediates enzalutamide resistance via activation of the E2F and BCL2 pathways, which can be targeted with CDK4/6 and BCL2 inhibitors to reverse resistance.

Long noncoding RNA Gm31629 protects against mucosal damage in experimental colitis via YB-1/E2F pathway

Mucosal healing is a key treatment goal for inflammatory bowel disease, and adequate epithelial regeneration is required for an intact gut epithelium. However, the underlying mechanism for mucosal healing is unclear. Long noncoding RNAs (lncRNAs) have been reported to be involved in the development of inflammatory bowel disease. Here, we report that a lncRNA named Gm31629 decreased in intestinal epithelial cells in response to inflammatory stimulation. Gm31629 deficiency led to exacerbated intestinal inflammation and delayed epithelial regeneration in dextran sulfate sodium–induced (DSS-induced) colitis model. Mechanistically, Gm31629 promoted E2F pathways and cell proliferation by stabilizing Y-box protein 1 (YB-1), thus facilitating epithelial regeneration. Genetic overexpression of Gm31629 protected against DSS-induced colitis in vivo. Theaflavin 3-gallate, a natural compound mimicking Gm31629, alleviated DSS-induced epithelial inflammation and mucosal damage. These results demonstrate an essential role of lncRNA Gm31629 in linking intestinal inflammation and epithelial cell proliferation, providing a potential therapeutic approach to inflammatory bowel disease.

Elevated SNRPA1, as a Promising Predictor Reflecting Severe Clinical Outcome via Effecting Tumor Immunity for ccRCC, Is Related to Cell Invasion, Metastasis, and Sunitinib Sensitivity.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal carcinoma and is associated with poor prognosis and notorious for its immune dysfunction characteristic. SNRPA1 is a spliceosome component responsible for processing pre-mRNA into mRNA, while the biological effect of SNRPA1 in ccRCC remains elusive. The aim of this study was to decipher the effect of SNRPA1 on clinical effect and tumor immunity for ccRCC patients. Multi-databases were collected to evaluate the different expression, prognostic value, DNA methylation, tumor immune microenvironment, and drug sensitivity of SNRPA1 on ccRCC. IHC was utilized to validate the expression and prognostic value of SNRPA1 in ccRCC patients from the SMMU cohort. The knockout expression of SNRPA by sgRNA plasmid inhibited the cell proliferation, migration, and metastasis ability and significantly increased the sensitivity of sunitinib treatment. In addition, we explored the role of SNRPA1 in pan-cancer level. The results indicated that SNRPA1 was differentially expressed in most cancer types. SNRPA1 may significantly influence the prognosis of multiple cancer types, especially in ccRCC patients. Notably, SNRPA1 was significantly correlated with immune cell infiltration and immune checkpoint inhibitory genes. In addition, the aggressive and immune inhibitory effects shown in SNRPA1 overexpression and the effect of SNRPA1 on ccRCC cell line invasion, metastasis, and drug sensitivity in vitro were observed. Moreover, SNRPA1 was related to Myc, MTORC, G2M, E2F, and DNA repair pathways in various cancer types. In all, SNRPA1 may prove to be a new biomarker for prognostic prediction, effect tumor immunity, and drug susceptibility in ccRCC.

DDX10 promotes the proliferation and metastasis of colorectal cancer cells via splicing RPL35

BackgroundColorectal cancer (CRC) has become the second deadliest cancer in the world and severely threatens human health. An increasing number of studies have focused on the role of the RNA helicase DEAD-box (DDX) family in CRC. However, the mechanism of DDX10 in CRC has not been elucidated.MethodsIn our study, we analysed the expression data of CRC samples from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. Subsequently, we performed cytological experiments and animal experiments to explore the role of DDX10 in CRC cells. Furthermore, we performed Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and protein–protein interaction (PPI) network analyses. Finally, we predicted the interacting protein of DDX10 by LC–MS/MS and verified it by coimmunoprecipitation (Co-IP) and qPCR.ResultsIn the present study, we identified that DDX10 mRNA was extremely highly expressed in CRC tissues compared with normal colon tissues in the TCGA and GEO databases. The protein expression of DDX10 was measured by immunochemistry (IHC) in 17 CRC patients. The biological roles of DDX10 were explored via cell and molecular biology experiments in vitro and in vivo and cell cycle assays. We found that DDX10 knockdown markedly reduced CRC cell proliferation, migration and invasion. Then, we constructed a PPI network with the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING). GO and KEGG enrichment analysis and gene set enrichment analysis (GSEA) showed that DDX10 was closely related to RNA splicing and E2F targets. Using LC–MS/MS and Co-IP assays, we discovered that RPL35 is the interacting protein of DDX10. In addition, we hypothesize that RPL35 is related to the E2F pathway and the immune response in CRC.ConclusionsIn conclusion, provides a better understanding of the molecular mechanisms of DDX10 in CRC and provides a potential biomarker for the diagnosis and treatment of CRC.

Molecular basis of longevity sustaining characteristics of Chinese medicine herbs

The balancing between tumor suppression and cell viability is the key to balance aging and tumorigenesis, and reach tumor-free longevity, or healthy aging. We speculate that the balance of tumor suppression signaling and the cell growth sustaining mechanisms in molecular genetics might reflect the balance between Yin and Yang in Chinese medicine. Some Chinese medicine herbs are known to sustain longevity, and help to prevent aging related diseases, such as Panax Ginseng, Ganoderma Lucidum etc. Based on Chinese medicine databases and the molecular signature database, we try to apply in silico systems pharmacology to analyze the function of these herbs in modulating signaling pathways. We found that most of these herbs could enhance cellular mitochondrial function: oxidative phosphorylation. Surprisingly, the lipids metabolism pathways were found in all the herbs analyzed. Interestingly, the cell proliferation pathways, such as telomere maintenance pathway, Myc pathway, E2F pathway, as well as the cell quality control pathway (apoptosis pathway) were also found to be enhanced in most herbs, which added evidences to our balancing hypothesis. Thus, our data revealed the sustainable energy production, the sustainable cell proliferation, and the quality control build the life triangle for the mechanism of action of Chinese medicinal herbs with longevity sustaining property. This methodology might help to predict the molecular mechanism of Chinese medicine herbs, and further understand the formulation of Chinese medicine in preventing aging-related diseases. The limitations of this kind of analysis are also discussed.

Molecular Signature Predictive of Long-Term Liver Fibrosis Progression to Inform Antifibrotic Drug Development

There is a major unmet need to assess the prognostic impact of antifibrotics in clinical trials because of the slow rate of liver fibrosis progression. We aimed to develop a surrogate biomarker to predict future fibrosis progression. A fibrosis progression signature (FPS) was defined to predict fibrosis progression within 5 years in patients with hepatitis C virus and nonalcoholic fatty liver disease (NAFLD) with no to minimal fibrosis at baseline (n= 421) and was validated in an independent NAFLD cohort (n= 78). The FPS was used to assess response to 13 candidate antifibrotics in organotypic exvivo cultures of clinical fibrotic liver tissues (n= 78) and cenicriviroc in patients with nonalcoholic steatohepatitis enrolled in a clinical trial (n= 19, NCT02217475). A serum protein-based surrogate FPS was developed and tested in a cohort of compensated cirrhosis patients (n= 122). A 20-gene FPS was defined and validated in an independent NAFLD cohort (adjusted odds ratio, 10.93; area under the receiver operating characteristic curve, 0.86). Among computationally inferred fibrosis-driving FPS genes, BCL2 was confirmed as a potential pharmacologic target using clinical liver tissues. Systematic exvivo evaluation of 13 candidate antifibrotics identified rational combination therapies based on epigallocatechin gallate, which were validated for enhanced antifibrotic effect in exvivo culture of clinical liver tissues. In patients with nonalcoholic steatohepatitis treated with cenicriviroc, FPS modulation was associated with 1-year fibrosis improvement accompanied by suppression of the E2F pathway. Induction of the PPARα pathway was absent in patients without fibrosis improvement, suggesting a benefit of combining PPARα agonism to improve the antifibrotic efficacy of cenicriviroc. A 7-protein serum protein-based surrogate FPS was associated with the development of decompensation in cirrhosis patients. The FPS predicts long-term fibrosis progression in an etiology-agnostic manner, which can inform antifibrotic drug development.

Delineation of gastric tumors with activated ERK/MAPK signaling cascades for the development of targeted therapeutics

The ERK/MAPK signaling pathway is activated in various cancers including gastric cancer. Targeting the ERK/MAPK/MEK pathway has been considered as a promising strategy for cancer therapy. However, MEK inhibition leads to a series of resistance mechanisms due to mutations in MEK, elevated expression of RAS or RAF proteins and activation of the associated signaling pathways. In the present study, ERK/MAPK pathway specific gene signatures were identified to be highly activated in intestinal subtype gastric tumors. Inhibition of ERK/MAPK pathway with the inhibitor PD98059 in gastric cancer cell lines by in vitro signaling pathway and genome-wide expression profiling revealed the associated signaling pathways. Functional genomic investigation of the ERK/MAPK regulated genes reveals the association of ERK/MAPK pathway with E2F, Myc, SOX-2, TGF-β, OCT4 and Notch pathways in gastric cancer cells. Of these, E2F, Myc and SOX-2 pathways are activated in intestinal subtype gastric tumors and TGF-β, OCT4, Notch pathways are activated in diffuse subtype gastric tumors. Further, the mutational load of gastric tumors was found to have association and correlation with the activation pattern of ERK/MAPK pathways across gastric tumors. ERK/MAPK activation was also found to represent the EBV and MSI activated subtypes of gastric tumors. Identification of potent drug candidates inhibiting the ERK/MAPK and associated pathways would pave a way for developing the targeted therapeutics for a subset of gastric tumors with activated ERK/MAPK signaling cascade.

Restoration of aged hematopoietic cells by their young counterparts through instructive microvesicles release.

This study addresses the potential to reverse age-associated morbidity by establishing methods to restore the aged hematopoietic system. Parabiotic animal models indicated that young secretome could restore aged tissues, leading us to establish a heterochronic transwell system with aged mobilized peripheral blood (MPB), co-cultured with young MPB or umbilical cord blood (UCB) cells. Functional studies and omics approaches indicate that the miRNA cargo of microvesicles (MVs) restores the aged hematopoietic system. The in vitro findings were validated in immune deficient (NSG) mice carrying an aged hematopoietic system, improving aged hallmarks such as increased lymphoid:myeloid ratio, decreased inflammation and cellular senescence. Elevated MYC and E2F pathways, and decreased p53 were key to hematopoietic restoration. These processes require four restorative miRs that target the genes for transcription/differentiation, namely PAX and phosphatase PPMIF. These miRs when introduced in aged cells were sufficient to restore the aged hematopoietic system in NSG mice. The aged MPBs were the drivers of their own restoration, as evidenced by the changes from distinct baseline miR profiles in MPBs and UCB to comparable expressions after exposure to aged MPBs. Restorative natural killer cells eliminated dormant breast cancer cells in vivo, indicating the broad relevance of this cellular paradigm - preventing and reversing age-associated disorders such as clearance of early malignancies and enhanced responses to vaccine and infection.

Transcriptional Deregulation Mediated By ID2-TCF3 Axis Supports MM Cell Growth and Proliferation in the Context of the Bone Marrow Milieu

Multiple myeloma (MM) is a complex and heterogenous disease which is dependent on the surrounding microenvironment for growth and survival. In MM, dysregulation of transcriptional control is a major driver of tumor transformation and progression. To evaluate transcriptional programs activated in MM cells in the context of the bone marrow milieu, we have performed extensive transcriptomic analysis by RNA-seq and ATAC-seq using bone marrow stromal cells (BMSC) derived from MM patients and stromal cell line (HS5) in co-culture with various MM cell lines. We observed that both cell-cell interactions and soluble factors secreted by BMSC or HS5 cells significantly downregulated expression of Inhibitor of DNA Binding 2 (ID2), while footprint analysis of the open chromatin regions in MM cells upon interaction with BMSC revealed enrichment for binding motifs of the TCF family of transcription factors (E proteins). Inhibitors of DNA binding (ID) proteins control crucial transcriptional programs in B cell maturation via their heterodimerization with E proteins which are members of the basic helix-loop-helix (bHLH) class I family of transcription factors, repressing their DNA binding and therefore transcriptional activity.We found that ID2 expression is significantly lower in primary CD138+ MM cells from patients compared to normal plasma cells (NPCs). Moreover, we have previously implicated the B-cell factor TCF3 as a novel MM dependency. Using MM cell lines and primary samples, we observed elevated enhancer activity at TCF3 locus in primary malignant plasma cells compared to NPCs, which resulted in significant upregulation of TCF3 expression in MM patients. We also showed that TCF3 is regulated by a large proximal enhancer that is bound by MYC and is highly sensitive to chemical perturbation of enhancer co-activators such as BRD4. Genetic perturbation of TCF3 confirmed its critical role on MM cell growth and viability especially in IgH MYC translocated MM cell lines.We here further explored the role of ID2-TCF3 axis and the hypothesis that lower expression of ID2 drives higher TCF3 activity in MM cells, which is further enhanced in presence of the bone marrow microenvironment.Genetic modulation of ID2 significantly affected MM cell viability, with MM cells ectopically expressing ID2 displaying a cell growth arrest even in the presence of the supportive BM milieu. To define the mechanism of the observed oncosuppressive role of ID2 in MM, which is in line with preliminary observations in other hematological malignancy but in contrast with the pro-tumoral role described in solid tumors, we first performed immunoprecipitation of ID2 followed by mass spectrometry in 3 MM cell lines, and identified a very consistent and specific interaction with E proteins TCF3 and TCF12. Next, to explore the transcriptional programs dependent on ID2 we performed RNA-seq of 2 MM cell lines after ID2 overexpression. In line with our in vitro data, gene ontology and gene set enrichment analysis showed a significant downregulation of genes involved in E2F pathway, cell cycle progression and regulation of gene transcription. Interestingly, among the known TCF3 targets in B cells, only XBP-1 was significantly downregulated in MM cells after ID2 overexpression, suggesting the existence of a cell-specific TCF3 dependent transcriptional program in MM. Indeed, ATAC-seq experiments revealed ID2 overexpression led to a significant decrease of TCF3 binding motifs in open chromatin regions, confirming the relevance of ID2 in regulating TCF3 transcriptional activity in MM.In conclusion, while both E and ID proteins have been implicated in malignant transformation, their role in supporting MM transcriptional deregulation and tumor growth in the context of the microenvironment is being defined. Here, we have identified ID2 as a major regulator of the TCF3 dependent transcriptional program in MM, whose downregulation is essential to maintain MM proliferation and to mediate the benefits induced by MM-stroma interaction. DisclosuresAnderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Munshi: Janssen: Consultancy; Amgen: Consultancy; Takeda: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Celgene: Consultancy; Karyopharm: Consultancy; Adaptive Biotechnology: Consultancy; Novartis: Consultancy; Legend: Consultancy; Pfizer: Consultancy; Bristol-Myers Squibb: Consultancy.

Anti-Tumor Activity of AZD4547 Against NTRK1 Fusion Positive Cancer Cells Through Inhibition of NTRKs.

Inhibitors of tropomyosin-related kinases (TRKs) display remarkable outcomes in the regression of cancers harboring the Neurotrophin Receptors Tyrosine Kinase (NTRK) fusion gene. As a result, TRKs have become attractive targets in anti-cancer drug discovery programs. Here, we demonstrate that AZD4547, a highly potent and selective inhibitor of fibroblast growth factor receptor (FGFR), displays anti-tumor activity against KM12(Luc) harboring the TPM3-NTRK1 fusion gene associated with its direct inhibition of TRKs. The results of profiling, using a 64-member in-house cancer cell panel, show that AZD4547 displays anti-proliferation activity against KM12(Luc) with a GI50 of 100 nM. In vitro biochemical assays reveal that AZD4547 has IC50 values of 18.7, 22.6 and 2.9 nM against TRKA, B and C, respectively. In a cellular context, AZD4547 blocks auto-phosphorylation of TRKs and phosphorylation of its downstream molecules including PLC-gamma and AKT in a dose dependent manner. Also, AZD4547 at 0.1 μM concentration downregulates expression of MAPK target genes (DUSP6, CCND1 and ETV1) as well as the E2F pathway. Furthermore, AZD4547 induces G0/G1 arrest and apoptosis, and suppresses anchorage independent growth of KM12(Luc). Oral administration of 40 mpk AZD4547 dramatically delays tumor growth in a KM12(Luc) implemented xenograft model, without promoting body weight changes. The capability of AZD4547 to inhibit TRKA, TRKB and clinically relevant mutants (TRKA G595R, G667S, G667C and G667A) was also evaluated using Ba/F3 cells harboring the ETV6-NTRKs fusion gene. The combined observations demonstrate the potential application of AZD4547 for treatment of NTRK fusion driven cancers.

Cellular and molecular properties of neural progenitors in the developing mammalian hypothalamus

The neuroendocrine hypothalamus is the central regulator of vital physiological homeostasis and behavior. However, the cellular and molecular properties of hypothalamic neural progenitors remain unexplored. Here, hypothalamic radial glial (hRG) and hypothalamic mantle zone radial glial (hmRG) cells are found to be neural progenitors in the developing mammalian hypothalamus. The hmRG cells originate from hRG cells and produce neurons. During the early development of hypothalamus, neurogenesis occurs in radial columns and is initiated from hRG cells. The radial glial fibers are oriented toward the locations of hypothalamic subregions which act as a scaffold for neuronal migration. Furthermore, we use single-cell RNA sequencing to reveal progenitor subtypes in human developing hypothalamus and characterize specific progenitor genes, such as TTYH1, HMGA2, and FAM107A. We also demonstrate that HMGA2 is involved in E2F1 pathway, regulating the proliferation of progenitor cells by targeting on the downstream MYBL2. Different neuronal subtypes start to differentiate and express specific genes of hypothalamic nucleus at gestational week 10. Finally, we reveal the developmental conservation of nuclear structures and marker genes in mouse and human hypothalamus. Our identification of cellular and molecular properties of neural progenitors provides a basic understanding of neurogenesis and regional formation of the non-laminated hypothalamus. The hypothalamus performs a wide range of vital physiological functions, including growth and reproductive behaviors, and circadian rhythms. The authors identify and characterize hypothalamic radial glial and hypothalamic mantle zone radial glial cells as the neural progenitors in the hypothalamus.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

NAT10 as a potential prognostic biomarker and therapeutic target for HNSCC

BackgroundIncreasing evidence has demonstrated the critical roles of mRNA modification regulators on multiple types of cancers. However, it is still poorly known about the prognostic and therapeutic value of mRNA modification regulators in HNSCC.MethodsThe gene expression profile of 36 mRNA modification regulators and their corresponding clinical data were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). Stepwise regression in R with both directions was used to construct a model for the prognosis of HNSCC. Univariate Cox regression survival analysis was performed to identify the most significant risk gene. Gene set enrichment analysis (GSEA) was applied to determine the cancer-associated pathways with NAT10. Immunohistochemistry (IHC) staining was performed to evaluate the expression of NAT10 in formalin fixed paraffin-embedded (FFPE) samples of HNSCC. Univariate and multivariate Cox regression survival analysis performed to identify the independent risk factors associated with the OS of patients with HNSCC. HNSCC cell lines (Cal-27, FaDu, and Detroit-562) were transfected with short interfering RNA (siRNA) targeting NAT10 or treated with Remodelin, a small-molecule inhibitor of NAT10. Knockdown efficiency of siRNA was assessed by quantitative real-time PCR (qRT-PCR) and western blotting. In addition, CCK-8 assay, scratch assay and transwell assay were used to examine the proliferation, migration, and invasion abilities of the three HNSCC cell lines after NAT10 was inhibited genetically and pharmaceutically. Cell cycle and cell apoptosis assays were performed by flow cytometry. Finally, the therapeutic value of Remodelin in HNSCC was evaluated via a patient-derived xenograft (PDX) model. The statistical analysis was performed with SPSS 23.0.ResultsA risk prediction model containing 10 mRNA modification regulators was constructed and showed prognostic value in HNSCC. NAT10 was further identified as a key risk gene and independent prognostic factor in TCGA HNSCC dataset. The GSEA analysis suggested that high NAT10 expression was associated with MYC, E2F, G2M checkpoint, mTORC1, DNA repair and oxidative phosphorylation pathways. NAT10 protein expression was significantly up-regulated in tumour cells compared to normal epithelial cells in FFPE samples and increased NAT10 protein expression was correlated with poor overall survival of 267 HNSCC patients. Genetic depletion of NAT10 using siRNA or chemical inhibition of NAT10 using Remodelin resulted in reduced cell proliferation, migration and invasion abilities in Cal-27, FaDu and Detroit-562 cells. Knockdown of NAT10 using siRNA significantly increased cell cycle arrest in S/G2-phase. Remodelin significantly inhibited tumour growth and tumour cell proliferation in the PDX model of HNSCC.ConclusionsNAT10 could be a potential prognostic marker and a therapeutic target for HNSCC.

Effects of the p16/cyclin D1/CDK4/Rb/E2F1 pathway on aberrant lung fibroblast proliferation in neonatal rats exposed to hyperoxia.

p16INK4a (p16) inhibits the vital G1 to S phase transition during cell cycle progression through the p16/cyclin D1/CDK4/retinoblastoma(Rb)/E2F1 pathway. Hyperoxia can suppress the G1/S checkpoint and induce more lung fibroblasts (LFs) to transition from the G1 phase to the S phase and undergo cell proliferation. The present study investigated the rate of p16 gene promoter methylation and the protein expression levels of p16, cyclin D1, CDK4, Rb and E2F1 in LFs from the lungs of rats exposed to hyperoxia and normoxia on postnatal days 3, 7 and 14. In the hyperoxia-exposed group, the methylation rate was 50 and 80% on days 7 and 14, respectively. Cyclin D1 and CDK4 overexpression was associated with p16 loss and Rb inactivation by phosphorylation. Rb phosphorylation induced E2F1 release in the G1 phase, which promoted cell proliferation. No methylation was observed in the normoxia-exposed group. These observations suggested that p16 loss may stimulate aberrant LF proliferation via the p16/cyclin D1/CDK4/Rb/E2F1 pathway.

AP-2α-Mediated Activation of E2F and EZH2 Drives Melanoma Metastasis.

In melanoma metastasis, the role of the AP-2α transcription factor, which is encoded by TFAP2A, is controversial as some findings have suggested tumor suppressor activity while other studies have shown high TFAP2A expression in node-positive melanoma associated with poor prognosis. Here we demonstrate that AP-2α facilitates melanoma metastasis through transcriptional activation of genes within the E2F pathway including EZH2. A BioID screen found that AP-2α interacts with members of the nucleosome remodeling and deacetylase (NuRD) complex. Loss of AP-2α removed activating chromatin marks in the promoters of EZH2 and other E2F target genes through activation of the NuRD repression complex. In melanoma cells, treatment with tazemetostat, an FDA-approved and highly specific EZH2 inhibitor, substantially reduced anchorage-independent colony formation and demonstrated heritable antimetastatic effects, which were dependent on AP-2α. Single-cell RNA sequencing analysis of a metastatic melanoma mouse model revealed hyperexpansion of Tfap2a High/E2F-activated cell populations in transformed melanoma relative to progenitor melanocyte stem cells. These findings demonstrate that melanoma metastasis is driven by the AP-2α/EZH2 pathway and suggest that AP-2α expression can be used as a biomarker to predict responsiveness to EZH2 inhibitors for the treatment of advanced melanomas. SIGNIFICANCE: AP-2α drives melanoma metastasis by upregulating E2F pathway genes including EZH2 through inhibition of the NuRD repression complex, serving as a biomarker to predict responsiveness to EZH2 inhibitors.

Abstract 404: A novel 4-gene score predict patient survival as well as pathologically complete (R0) resection in pancreatic cancer

Abstract BACKGROUND: Pancreatic cancer is one of the most aggressive cancers with a 5-year survival of less than 10%. Surgery is the only cure for pancreatic cancer but the majority of patients present with metastatic disease at the time of diagnosis. Systemic chemotherapy remains the primary treatment option. Given the insidious clinical presentation and poor treatment response, there is an urgent need to discover a predictive biomarker for appropriate patient selection. We hypothesized that the 4-gene score, which reflects tumor cell proliferation, is both a prognostic and predictive biomarker for pancreatic cancer. METHODS: The 4-gene score is derived from tumor expression of DOK4, HCCS, PGF, and SHCBP1 genes, which were identified based on differential mRNA expression analysis of a human breast cell line, its metastatic variant cells, and clinical outcome data of breast cancer patient cohorts, as previously reported. A total of 954 pancreatic cancer patients were analyzed for both discovery and validation of the 4-gene score from publicly available datasets to investigate the relationship between the score with clinical features such as metastasis, cancer aggressiveness, immune cell infiltration, patient survival, and resectability. RESULTS: We found that the 4-gene score correlation in pancreatic cancer was higher than in breast cancer cohorts, specifically in clinically aggressive parameters such as pathological grade and MKI67 expression. Also, the score in metastatic tumor cohorts was higher than in primary cancer cohorts (p &amp;lt; 0.001). These findings were mirrored in gene set analysis, in which the score high tumors enriched cell proliferation-related gene sets such as E2F targets, G2M checkpoint, MYC targets v1 and v2, mitotic spindle, and metastasis-related gene sets in three cohorts (TCGA, GSE62452, and GSE57495). A high score was associated with increased mutation rates in KRAS and CDKN2A genes (p = 0.006 and 0.044) in the TCGA cohort, as well as enhancement of KRAS, CDKN2A, p53, TGF-β, and E2F pathways in three cohorts. Furthermore, a high score tumor had less infiltration of CD8+ T cells but was associated with high cytolytic activity and IFN-γ scores, which reflect overall cancer immunity. The high score was correlated with the level of drug sensitivity area under the curve of Sorafenib (r = 0.648) in primary cancer, Paclitaxel (r = 0.624) and Doxorubicin (r = 0.575) in metastatic cancer. Finally, a high 4-gene score was significantly associated with worse clinical outcomes in three cohorts (p = 0.044, 0.048, and 0.022), a low rate of R0 resection (p = 0.013), and worse survival after R1/2 resection (p = 0.027). CONCLUSION: The 4-gene score identified poor survival in pancreatic cancer and has potential as a predictive biomarker for R0 resection and treatment response in metastatic pancreatic cancer. Evaluating the degree of the 4-gene score could be a valuable potential prognostic tool. Citation Format: Masanori Oshi, Lan Le, Yoshihisa Tokumaru, Ankit Patel, Ryusei Matsuyama, Itaru Endo, Li Yan, Matthew H.G. Katz, Kazuaki Takabe. A novel 4-gene score predict patient survival as well as pathologically complete (R0) resection in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 404.

Abstract 1966: Cell proliferation-related pathway scores (G2M Checkpoint, E2F Targets, MYC Targets V1 and V2, Mitotic Spindle, p53 pathway) as predictive and prognostic biomarkers in pancreatic cancer

Abstract Background: Given the poor survival and severe side effects of treatments for pancreatic cancer, we need prognostic and predictive biomarkers to guide management. We hypothesized that the cell proliferation-related pathway is associated with drug response and survival in pancreatic cancer. Methods: We studied six hallmark cell proliferation-related gene sets (G2M Checkpoint, E2F Targets, MYC Targets V1 and V2, Mitotic Spindle, p53 pathway) defined by MSigDB in gene set variant analysis (GSVA) was evaluated in 3 independent cohorts, TCGA-PDAC (n = 176), GSE57495 (n = 63), and GSE62452 (n = 69). Results: We found that although G2M and E2F correlated strongly with each other (spearman's r = 0.976), Mitotic and p53 pathway scores correlated highly with the other cell function gene sets. All pathways were significantly associated with high expression of MKI67 and with proliferation score (all p &amp;gt; 0.001), but none with pathologically complete resection (R0). Mitotic Spindle pathway alone associated with high cytolytic activity (p = 0.020). All pathways were significantly associated with high expression of KRAS and TP53 genes and with high rate of KRAS gene alteration except for MYC v1. The G2M, E2F, and p53 pathway were significantly associated with a high rate of TP53 gene alteration (p = 0.026, 0.011, and 0.07, respectively). Interestingly, different pathway correlated with AUC of different agents, such as Gemcitabine (Mitotic: r=0.706 [p = 0.01]), Paclitaxel (MYC v2: r = -0.636 [p &amp;lt; 0.05]), Apatinib (Mitotic: r = -0.556 [p = 0.03]), Palbociclib (E2F: r =0.675 [p &amp;lt; 0.01]), and Sorafenib (G2M: r = -0.593 [p = 0.03]), for pancreatic cancer. Among all six pathways, only G2M was significantly associated with worse patient survival consistently in all 3 cohorts. Conclusion: Each proliferation-related pathway was predictive of unique agent, and G2M score predicts survival of pancreatic cancer. Citation Format: Masanori Oshi, Lan Le, Yoshihisa Tokumaru, Li Yan, Ryusei Matsuyama, Itaru Endo, Kazuaki Takabe. Cell proliferation-related pathway scores (G2M Checkpoint, E2F Targets, MYC Targets V1 and V2, Mitotic Spindle, p53 pathway) as predictive and prognostic biomarkers in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1966.