Data published in the journal Cancer Cell presented possible new treatment options and elaborated on the contributions of key cancer-associated genes, phosphosites, and signaling pathways in human papillomavirus (HPV)negative head and neck squamous cell carcinomas (HNSCC).1

The data systematically recorded information regarding the disease, with multi-omic analysis determining 3 distinct subtypes with high potential for treatment with respective available therapeutics.

This study extends our biological understanding of HPV[-negative] HNSCC and generates therapeutic hypotheses that may serve as the basis for future preclinical studies and clinical trials toward molecularly guided precision treatment of this aggressive cancer type, wrote the investigators.2

The first subtype, called CIN for chromosome instability, was determined to have the worst prognosis. It was associated with the larynx, a history of smoking, and increased instability of chromosomes. The research team suggested that this cancer type would respond best to CDK4/6 inhibitor treatment given its relation to aberrations of the CCND1 and CDKN2A genes as well as a high activity of the CDK4 and CDK6 enzymes.

The investigators analyzed a number of protein elevations of basal factors in the second subtype discovered, which was in turn called Basal. These represent the most basic proteins necessary for gene transcription activation. The subtype had both high activity in the EGFR signaling pathway and high expression of the AREG and TNFA molecules. This led the investigators to suggest that treatment with monoclonal antibodies targeting EGFR would best treat this subtype.

Immune, the final subtype, was discovered among patients who did not smoke and had high expression of multiple immune checkpoint proteins. The data suggest patients with this subtype would respond best to immune checkpoint inhibitors.

The overall data found high potential for treatment response in 32% of patients with the CIN subtype, 62% of those with the basal subtype, and 83% with the immune subtype.

This study extends our biological understanding of HPV-negative HNSCCs and generates therapeutic hypotheses that may serve as the basis for future studies and clinical trials toward molecularly guided precision medicine treatment of this aggressive cancer type, Daniel Chan, PhD, principal investigator on the trial and director of the Center for Biomarker Discovery and Translation at the Johns Hopkins University School of Medicine, said in a press release.

The team also determined that there were 2 modes of activation of EGFR. This determination suggests a potentially new way to stratify this cancer type based on the number of molecules bound to EGFR. Moreover, the investigators concluded that the loss of the ability to produce immune responses is credited to the widespread deletion of immune modulatory genes.

Investigators from both the United States and Poland analyzed 110 treatment-nave primary HNSCC tumors and matched blood samples. A total of 66 tumors matched normal adjacent tissues.

We have made the primary and processed datasets available in publicly accessible data repositories and portals, which will allow full investigation of this extensively characterized cohort by both the HNSCC and broader scientific communities. We also expect wide application of the demonstrated proteogenomics framework to future studies of HNSCC and other cancer types, the investigators concluded

References:

1. Huang C, Chen L, Savage SR, et al. Proteogenomic insights into the biology and treatment of HPV-negative head and neck squamous cell carcinoma. Cancer Cell. January 5, 2021. doi: 10.1016/j.ccell.2020.12.007

2. Researchers create comprehensive database of head and neck cancers. News release. Hopkins Medicine. January 7, 2021. Accessed January 25, 2021. https://www.hopkinsmedicine.org/news/newsroom/news-releases/researchers-create-comprehensive-database-of-head-and-neck-cancers

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Distinct Subtypes and Potential Treatment Options Found in Analysis of Head and Neck Cancers - Cancer Network

Introduction

Ovarian cancer (OC) is the seventh most common cancer among women in the world and the second leading cause of gynecological cancer death.1 Most OC is often diagnosed at the advanced stage.2 In the past few decades, many treatment methods, such as active surgery, targeted therapy, intraperitoneal hyperthermia chemotherapy, small molecular inhibitors, neoadjuvant chemotherapy, and intraperitoneal chemotherapy, were hired to cure OC.3,4 However, most patients with ovarian cancer will suffer from tumor recurrence after first-line therapy. Although OC is sensitive to chemotherapy with platinum and taxane following debulking surgery, resistance to chemotherapy will eventually develop in almost all patients. Once the disease recurs, the interval between subsequent treatments steadily decreases due to rapid development and chemoresistance.5 Therefore, it is urgent to explore more effective reagents for the treatment of OC.

Natural plant products have been widely used in the treatment of various diseases. Sinomenine (7,8-didehydro-4-hydroxy-3,7-dimethoxy-17-methylmorphinan-6-one), an alkaloid monomer extracted from Sinomenium acutum plants, contains four rings, A, B, C and D.6 Many studies have proved that sinomenine has anti-inflammatory, anti-rheumatic, anti-oxidant, analgesic, immunosuppressive, and anti-angiogenic effects.711 Although the shorter biological half-life of sinomenine and the side-effects, such as increasing histamine release, restrained its clinical applications,6,12 sinomenine has also been developed into Zhengqingfengtongning (ZQFTN), a Chinese proprietary medicine approved by the Chinese government for treating RA and other autoimmune diseases in China. The anti-tumor effect of sinomenine has been preliminarily addressed in many kinds of tumors, such as liver cancer,13 breast cancer,14 lung cancer,15 renal cell carcinoma,16 and glioblastoma.17 Recently, the inhibitory effect of sinomenine on growth and metastasis of ovarian cancer cells has drawn considerable attention.18,19 Li et al showed that sinomenine inhibits ovarian cancer cell growth and metastasis by inhibiting the Wnt/-catenin pathway via targeting MCM2.18 Xu et al identified that sinomenine exerted the antitumor effect in ovarian cancer cells by hindering the expression of long non-coding RNA HOST2.19 However, these studies and findings are basic and preliminary for the effect of sinomenine on ovarian cancer. The mechanism of sinomenine inhibiting ovarian cancer remains to be further elucidated.

High-throughput sequencing, especially RNA sequencing (RNA-seq) has become an effective way to explore functional genes and mechanisms in anti-tumor research because of its low cost and ultra-high data output.20 Recently, RNA-seq has been successfully applied in ovarian cancer research for earlier detection, identification of pathological origin, defining the aberrant genes and dysregulated molecular pathways across patient groups, and identification of novel genes and molecular pathways in the development of multidrug resistance.21 Here, we hired high-throughput RNA-seq to explore the potential mechanism of the sinomenine mediated growth inhibition of ovarian cancer HeyA8 cells. Then, the results of the high-throughput mRNA sequence were validated by real-time PCR. Furthermore, we also preliminarily clarified that sinomenine inhibited the growth of ovarian cancer cells through the suppression of mitosis by down-regulating the expression and the activity of CDK1. This study was expected to lay a theoretical foundation for the future application of sinomenine in the treatment of ovarian cancers.

The human ovarian cancer cell line HeyA8-MDR was obtained from Stem Cell Bank, Chinese Academy of Sciences and was maintained in RPMI-1640 (Hyclone) supplemented with 10% fetal bovine serum (Gibcol) and 1% penicillin/streptomycin (Gibcol) at 37 C in a humidified atmosphere with 5% CO2.

For cell survival rate assay, HeyA8-MDR cells were seeded at a density of 5000 cells per well into 96-well plates. After incubation for 24 hours, sinomenine (0, 0.25, 0.5, 1, 2, 4 and 8 mM) was added to each well. The control cells (0 M) were only treated with an equivalent volume of DMSO. Each concentration of sinomenine was tested with six replicates. After incubation for 48 hours, the culture medium was supplemented with 10 ul CCK8 solution for 2 hours at 37 C. Then the absorbance was measured at 450 nm. Cell survival rates were shown as a percentage of the absorbance reading of the control cells. IC50 was calculated with the IC50 Calculator (https://www.aatbio.com/tools/ic50-calculator).

Cell proliferation assay was detected by CCK8 as described in our previous study.22 Briefly, 1 103 HeyA8-MDR cells per well were plated on 96-well plates. After being incubated for 24 hours, the cells were treated with 1.56 mM sinomenine and DMSO separately. Then, 10 L CCK8 reagent was added to each well and incubated for 2 hours at 37 C. The optical density value was measured at 450 nm every day for 7 days.

HeyA8-MDR cells were seeded at a density of 200 cells per well into 6-well plates. HeyA8-MDR cells, treated with 1.56 mM sinomenine and DMSO separately, were incubated for 10 days in a cell incubator at 37 C. Then, the cells were fixed with 4% PFA and stained with crystal violet staining solution (#Y1232, Yuxiu Biotech, China). The number of colonies, containing more than 50 cells observed under a microscope, were counted.

RNA extraction, library construction and sequencing were performed at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China). Total RNA was extracted using TRIzol Reagent (Thermo Scientific) and genomic DNA was removed by digestion with DNase I (Takara). Then RNA quality was determined by 2100 Bioanalyser (Agilent) and quantified using the ND-2000 (NanoDrop Technologies). Only high-quality RNA samples (OD260/280 = 1.8~2.2, OD260/2302.0, RIN6.5, 28S:18S1.0, >2 g) were used to construct the sequencing library.

RNA-seq transcriptome library was constructed with the TruSeqTM RNA sample preparation Kit from Illumina (San Diego, CA) using 1 g of total RNA. Shortly, messenger RNA was purified using oligo (dT) magnetic beads and then fragmented by fragmentation buffer. Double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen) with random hexamer primers (Illumina). Then the synthesized cDNA was subjected to end-repair, phosphorylation and A base addition according to Illuminas library construction protocol. Libraries were size selected for cDNA target fragments of 200300 bp on 2% Low Range Ultra Agarose followed by PCR amplified using Phusion DNA polymerase (NEB) for 15 PCR cycles. After being quantified by TBS380, the paired-end RNA-seq sequencing library was sequenced with the Illumina HiSeq 4000 (2150 bp read length). The RNA-seq data were deposited at NCBI (BioProject id: PRJNA641485).

The raw paired end reads were trimmed and quality controlled by SeqPrep (https://github.com/jstjohn/SeqPrep) and Sickle (https://github.com/najoshi/sickle) with default parameters. Then clean reads were separately aligned to reference the genome with orientation mode using TopHat (http://tophat.cbcb.umd.edu/, version2.1.1) software.23 The mapping criteria of bowtie were as follows: sequencing reads should be uniquely matched to the genome allowing up to 2 mismatches, without insertions or deletions. Then the region of the gene was expanded following depths of sites and the operon was obtained. In addition, the whole genome was split into multiple 15 kb windows that share 5 kb. New transcribed regions were defined as more than 2 consecutive windows without an overlapped region of gene, where at least 2 reads were mapped per window in the same orientation.

The expression level of each transcript was calculated according to the Transcripts Per Kilobase of exon model per Million mapped reads (TPM) using RSEM (http://deweylab.biostat.wisc.edu/rsem/).24 The differentially expressed genes (DEGs) (fold changes 2 and corrected P-value 0.05) between control and sinomenine-treated HeyA8 cell line were identified by R statistical package software EdgeR (Empirical analysis of Digital Gene Expression in R, http://www.bioconductor.org/packages/2.12/bioc/html/edgeR.html).25 GO functional enrichment and KEGG pathway analysis were carried out by Goatools (https://github.com/tanghaibao/Goatools) and KOBAS 2.1.1 (http://kobas.cbi.pku.edu.cn/download.php).26 The GO terms and the KEGG pathways were considered statistically significant when Bonferroni-corrected P-value 0.05.

The PPI for DEGs (fold changes4 and corrected P-value 0.05) were calculated on the web of the Retrieval of Interacting Genes (STRING) database (https://string-db.org/)27 and the cut-off criterion of the combined score was set as >0.4. Then, PPI networks for DEGs were visualized by Cytoscape (Version: 3.8.0).28

Total RNAs were reverse transcribed by the MMLV Reverse Transcriptase (Promega) according to the manufactures protocol. Real-time PCR analysis was performed on the LightCycler 96 System (Roche) with ChamQ SYBR qPCR Master Mix (Q321-02, Vazyme, China). All samples were examined in triplicate. The fold changes of each target gene were calculated using the 2Ct method relative to GAPDH.

Cells, which were treated with sinomenine and DMSO separately, were harvested and washed twice with PBS. Then the cells were fixed with 4% paraformaldehyde for 10 min at 4 C, and permeabilized for 10 minutes with 0.3% Triton X-100. The cells were incubated with anti-P-Histone H3 (53348, CST) antibodies for 30 minutes. After staining, the cells were washed twice and then incubated with FITC-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (SA00003-2, Proteintech) for 30 minutes. After being washed twice, the stained cells were stained with 50 ug/mL propidium iodide (PI) and analyzed with Beckman Coulter CytoFLEX flow cytometry system.

Western blots were performed as described previously.29 The primary antibodies were listed as follows: Geminin (52508), CDT1 (8064), Thymidine Kinase 1 (28755), P-Histone H3 (53348), Cyclin A2 (91500), Cyclin B1 (12231), Cyclin E1 (20808) and P-cdc2 (Tyr15) (4539) from Cell Signaling Technology, P-Cdk1/2 (Thr14) (DF2944) and P-CDK1 (Thr161) (AF8001) from Affinity, CDK1 (67575-1-Ig) from Protein Tech Group.

The data were shown as means standard deviation (SD). The difference between means was analyzed using SPSS 16.0 software (SPSS Inc., Chicago, IL, USA). The methods of statistical analysis were indicated in figure legends. p < 0.05 was considered as statistically significant.

To explore the inhibitory effect of sinomenine on the cell growth of ovarian cancer HeyA8 cells, the cell viability of HeyA8 were measured by CCK8 assay. As shown in Figure 1A, the inhibitory effect of sinomenine on HeyA8 cells was dose-dependent. The IC50 of sinomenine was 1.56 mM in HeyA8 cells (Figure 1B). The result of the CCK8 assay showed that sinomenine significantly suppressed the proliferation of HeyA8 at the concentration of 1.56 mM (Figure 1C). In addition, the result of the clone formation assay also indicated that sinomenine significantly inhibit the ability of the clone formation of HeyA8 cells (Figure 1D and E). Taken together, these results indicated that sinomenine inhibited the growth of ovarian cancer HeyA8 cells, which is consistent with results in previous reports.18,19

Figure 1 Sinomenine inhibits proliferation of ovarian cancer cell line HeyA8. (A) The survival rate of HeyA8 cells treated with different concentrations of sinomenine (0, 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 mM) for 48 hours were measured by CCK8 assay. (B) The IC50 of sinomenine was 1.56 mM in HeyA8 cells, which was calculated with the IC50 Calculator on the website (https://www.aatbio.com/tools/ic50-calculator). (C) The growth curves of HeyA8 treated with DMSO as control and sinomenine (1.56 mM) were measured by CCK8 assay. (D) HeyA8 treated with sinomenine formed fewer and smaller colonies than those treated with DMSO. (E) The number of colonies of HeyA8 cells treated with sinomenine and DMSO separately in (D), the value represents the mean SD for triplicate samples, **P<0.01, Students t-test.

To investigate the DEGs caused by the treatment of sinomenine, the gene expression of HeyA8 cells, which were treated with 1.56 mM sinomenine and an equal volume DMSO for 48 hours separately, were analyzed by high-throughput RNA sequencing. After gene mapping and the expression level of each transcript was converted to the value of TPM, the comparison at corrected P-value p 0.05 and log2FC fold change 1 (for up-regulation) or 1 (for down-regulation) was made to identify the DEGs for two groups (Figure 2A). The list of DEGs, along with their TPM and annotations, are presented in Supplementary Table S1. A total of 2679 genes were identified as DEGs, including 1323 down-regulated genes and 1356 up-regulated genes (Figure 2B), and were displayed by cluster heatmaps (Figure 2C).

Figure 2 Differentially expressed genes (DEGs) between different treatment groups. (A) Volcano map of DEGs. The horizontal axis represents expression changes (log) of the genes in sinomenine and DMSO treated groups, while the vertical axis showed the statistical significance of the changes in gene expression. The discrepancy was more significant with smaller p values and bigger log10 (corrected p-value). Each dot in the image represents one gene, the grey dots represent genes with no significant discrepancy, red dots were significantly up-regulated genes and green dots were significantly down-regulated genes. (B) The numbers of DEGs. There were 1323 down-regulated genes and 1356 up-regulated genes. (C) Heatmap of DEGs. Red represents high relative gene expression level and blue represents low relative gene expression level. Con: groups of HeyA8 cells treated with DMSO, SIN: groups of HeyA8 cells treated with sinomenine.

To study the characteristics of the 2679 DEGs, gene ontology (GO) enrichment analysis was performed. The top 20 ranked GO terms of DEGs were shown in Figure 3A. Kinetochore organization occupied the strongest enrichment degree as it possessed the highest rich factor (0.65), followed by strand displacement, meiotic chromosome segregation, attachment of spindle microtubules to kinetochore and DNA strand elongation. These enriched GO terms were obviously involved in the regulation of cell cycle, especially involved in the DNA replication in S phase, Kinetochore organization in G2 phase and chromosome segregation in M phase. These indicated that sinomenine inhibits the HeyA8 cell growth by regulating the process of the cell cycle.

Figure 3 GO and KEGG enrichment analysis of DEGs. (A) Bubble diagram of top 20 ranked GO terms of DEGs. The vertical axis indicates GO terms and the horizontal axis represents the rich factor. The enrichment degree was stronger with a bigger rich factor. The size of dots indicates the number of genes in the GO term. (B) KEGG pathway enrichment analysis of DEGs. The vertical axis represents KEGG pathways. The upper horizontal axis represents the number of genes enriched in the indicated pathway (dots on the yellow line). The lower horizontal axis represents the significant level of enrichment (green bar).

To further investigate the biological functions of DEGs, KEGG pathway enrichment analysis was performed. The results also showed the top 20 statistically significant pathways (Figure 3B). The top 5 significant pathways are DNA replication, cell cycle, homologous recombination, Fanconi anemia pathway, and microRNAs in cancer. In addition to the above pathways, these pathways including purine metabolism, pyrimidine metabolism, mismatch repair and nucleotide excision repair are also closely related to the cell cycle. Taken together, these results of GO and KEGG pathway enrichment could provide essential information on the investigation of sinomenine in ovarian cancer HeyA8 cells.

In order to systemically analyze the functions of DEGs in sinomenine-treated ovarian cancer HeyA8 cells, a total of 856 DEGs (foldchange 4, corrected P-value 0.05) were mapped to the PPI database to obtain the PPI networks. As shown in Figure 4A, a total of 600 relationships between 104 genes (nodes) were identified. Table 1 shows the node genes with the top 18 ranked degree (number of interactions). The DEGs of CDK1 (degree = 62), PLK1 (degree = 50), BUB1 (degree = 48), NDC80 (degree = 48) and BUB1B (degree = 44) formed networks with high degrees. The top 3 ranked degree genes (CDK1, PLK1, and BUB1) play important roles in the progression of G2/M phase in the cell cycle.30,31 Furthermore, real-time PCR analysis was used to verify the results of transcriptome sequencing and the results indicated that the expression trends of the top 18 ranked degree genes were consistent with those obtained by RNA-seq, suggesting that the RNA-seq data reliably reflected the gene expression alterations (Figure 4B). According to the RNA-seq and real-time PCR results, the expressions of cell cycle regulated genes, such as CDK1, PLK1, and BUB1, were significantly decreased after sinomenine treatment. These results were also in accord with the cell phenotype experiments.

Figure 4 The network of protein-protein interactions (PPI) of DEGs. (A) The network of PPI. The size of the circles (nodes) represents the degrees of the gene in the PPI network. A greater size indicates a greater degree. (B) Real-time PCR validation of the expressions of top18 degree DEGs. Yellow lines were from the results of the transcriptome data (TPM Value); Black lines were from the results of real-time PCR (Relative Expression). Con: HeyA8 cells treated with DMSO; SIN: HeyA8 cells treated with sinomenine for 48 hours. Three replicates were carried out in the real-time PCR analysis. Bars represent means SD (n = 3). (C) Histogram of top 20 ranked GO terms of the real-time PCR validated 18 genes in the PPI network.

Table 1 Node Genes with Top 18 Ranked Degree in the PPI Network

In addition, GO enrichment analysis indicated that the validated 18 genes were enriched in regulation of sister chromatid segregation, chromosome, condensed chromosome, microtubule cytoskeleton organization involved in mitosis, condensed nuclear chromosome, mitotic cell cycle process, and mitotic nuclear division, etc. (Figure 4C). These results suggested that sinomenine may affect the proliferation of ovarian cancer HeyA8 cells by regulating the progression of M phase in cell cycle through these target genes and their classical pathways.

The results of RNA-seq and real-time PCR indicated that sinomenine inhibited the mRNA expression level of CDK1 in ovarian cancer HeyA8 cells. While, the cyclin B1/Cdk1 complex was reported to play an important role in regulating the entry into mitosis.32,33 In order to clarify the effect of sinomenine on cell cycle distribution, firstly, we analyzed the cell cycle distribution of HeyA8 cells by flow cytometry assay and the results showed the population of G2/M were decreased from 55.6% 2.85% to 44.08% 3.22% in HeyA8 cells treated with sinomenine for 48 hours (Figure 5A and B). Meanwhile, the percentage of M phase was decreased from 14.46 1.10% to 6.35% 0.61% (Figure 5C and D). Then, the expression levels of some cell cycle related proteins including cyclin E1, cyclin A2, cyclin B1, Geminin, CDT1, Thymidine kinase 1 and P-Histone H3 (Ser10), which presented in cells at various phases of the cell cycle, were analyzed by Western blot (Figure 5E). The results showed that the expression level of P-Histone H3 (Ser10), which is present only in the M phase, was significantly decreased in sinomenine treated HeyA8 cells when compared with DMSO treated negative control HeyA8 cells and blank control HeyA8 cells. However, there were no significant changes in the expression levels of other cell cycle related proteins (Figure 5F). These results indicated that sinomenine decreased the number of M-phase cells, suggesting the suppression of mitosis in ovarian cancer HeyA8 cells.

Figure 5 Sinomenine suppressed G2/M transition in HeyA8 cells by inhibiting the expression and the activity of CDK1. (A and B) The cell cycle transition of HeyA8 cells was examined by flow cytometry assay (A). The results showed that the percentage of cells in the G2/M phase was decreased significantly after treatment with sinomenine for 48 hours (B). Each value represents the mean SD for triplicate samples. ***P<0.001, Students t-test. (C and D) The percentage of M phase cells were analyzed by flow cytometry assay staining with P-Histone H3 (Ser10) and PI (C). The results showed that the percentage of M phase in HeyA8 cells treated with sinomenine for 48 hours were obviously decreased (D). Each value represents the mean SD for triplicate samples. ***P<0.001, Students t-test. (E) The expression of specific proteins in different cell cycle phase were determined by Western blot. (F) The expression level of P-Histone H3 (ser10), which only expressed in the M phase, were clearly decreased in sinomenine treated HeyA8 cells. Each value represents the mean SD for triplicate samples. ***P<0.001, one-way ANOVA analysis. (G) The expression of CDK1 and the phosphorylation of CDK1 were detected by Western blot. (H) The expression level CDK1 and P-CDK1 (Thr161) were clearly decreased in sinomenine treated HeyA8 cells. Each value represents the mean SD for triplicate samples. **P<0.01, ***P<0.001, one-way ANOVA analysis.

Meanwhile, the activity of CDK1 depends on its phosphorylation state.34 Phosphorylation of the Thr161 residue of Cdk1 can stabilize its interaction with cyclins and lead to further activation of the kinase.35 On the other hand, the cyclin B1/Cdk1 complex can be inactivated via inhibitory phosphorylation of Thr14 and Tyr15 residues of the CDK1.36 Therefore, we detected the protein expression level and phosphorylation levels of CDK1 by Western blot (Figure 5G). The results showed that the expression level of CDK1 and the phosphorylation level of CDK1 on Thr161 in sinomenine treated HeyA8 cells were significantly lower than those in DMSO treated HeyA8 cells and control HeyA8 cells. While, the phosphorylation levels of CDK1 on Thr14 and Tyr15 did not change significantly (Figure 5H). These results indicated that sinomenine suppressed the expression of CDK1 and the activity of CDK1 by inhibiting the phosphorylation of CDK1 on Thr161.

OC is the most lethal gynecologic malignancy and no significant treatment progress has been made in the past 30 years. It is easy to relapse and form drug resistance after the first comprehensive treatment. In order to improve the survival rate and prolong the survival period of patients, it is very important to find new effective chemotherapy drugs for the treatment of OC. Many studies showed that a large number of natural plant products possess the ability of anti-tumor. Up to now, many natural anti-tumor products, such as vinblastine, vincristine, podophyllotoxin, paclitaxel (Taxol) and camptothecin, have been tested clinically.37 In recent years, sinomenine, an alkaloid extracted from Sinomenium acutum plants, have been proved to have the ability of anti-tumor. However, the study of its anti-ovarian cancer effect is still rare and its mechanism is unclear. Here, we studied the anti-tumor effect of sinomenine on ovarian cancer cells and found that sinomenine inhibits the capacities of proliferation and colony formation of ovarian cancer HeyA8 cells, which was consistent with previous reports.18,19 Furthermore, we firstly preliminarily explored its mechanism by High-throughput RNA-seq. The results of GO and KEGG pathway enrichment and the ProteinProtein Interaction Analysis indicated that the DEGs were mainly involved in the cell cycle.

Cell survival and proliferation mainly depend on the progression of the cell cycle, which is a series of tightly-regulated molecular events controlling DNA replication and mitosis.38,39 The entry into mitosis is regulated by the activation of Cdc2/Cdk1 kinase, which was controlled by several steps including cyclin B1 nuclear accumulation and binding, dephosphorylation of Cdc2/Cdk1 at Thr14 and Tyr15, and phosphorylation of Cdc2/Cdk1 at Thr161.40,41 In our experiment, sinomenine decreased the expression level of CDK1 and especially suppressed the activated phosphorylation Thr161 on CDK1. At the same time, sinomenine also decreased the phosphorylation of Histone H3 at Ser10, which is tightly correlated with chromosome condensation during mitosis.42 These results suggested that sinomenine inhibited the proliferation of ovarian cancer HeyA8 cells through suppressing the mitosis by down-regulating the expression and the activity of CDK1.

While the process of the M phase is composed of many biological events, such as chromatin condenses, Spindle formation, chromosome separation, mitotic nuclear division, and mitotic DNA damage checkpoint.43 The results of GO enrichment assay indicated that many of the top 18 ranked DEGs in PPI were involved in multiple biological events in phase M. For instance, PLK1, serine/threonine-protein kinase 1, performs several important functions throughout M phase of the cell cycle, including the regulation of centrosome maturation and spindle assembly, the removal of cohesions from chromosome arms, the inactivation of anaphase-promoting complex/cyclosome (APC/C) inhibitors, and the regulation of mitotic exit and cytokinesis.44,45 Mitotic checkpoint serine/threonine-protein kinase BUB1 performs two crucial functions, including spindle-assembly checkpoint signaling and correct chromosome alignment, during mitosis.46 NDC80, as a component of the essential kinetochore-associated NDC80 complex, is required for chromosome segregation and spindle checkpoint activity.47 However, how sinomenine affects the process of M phase still needs to be further explored.

In summary, our results indicated that sinomenine plays anti-tumor functions by inhibiting the growth of the ovarian cancer HeyA8 cells through the suppression of mitosis by down-regulating the expression and the activity of CDK1. These results may provide a preliminary research basis for the application of sinomenine in anti-ovarian cancer.

This work was supported by the National Key R&D Program of China (2018YFA0107500), the Key Specialty Construction Project of the Shanghai Municipal Commission of Health and Family Planning (No. ZK2015A33, 201740117), National Natural Science Foundation of China (31771511) and Foundation strengthening program in technical field of China (2019-JCJQ-JJ-068).

The authors declare no conflicts of interest.

1. Bray F, Ferlay J, Soerjomataram I, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68:394424.

2. Tew WP. Ovarian cancer in the older woman. J Geriatr Oncol. 2016;7:354361.

3. Pujade-Lauraine E. New treatments in ovarian cancer. Ann Oncol. 2017;28:viii57viii60.

4. Cortez AJ, Tudrej P, Kujawa KA, et al. Advances in ovarian cancer therapy. Cancer Chemother Pharmacol. 2018;81:1738.

5. Leung CS, Yeung TL, Yip KP, et al. Calcium-dependent FAK/CREB/TNNC1 signalling mediates the effect of stromal MFAP5 on ovarian cancer metastatic potential. Nat Commun. 2014;5:5092.

6. Zhang YS, Han JY, Iqbal O, et al. Research advances and prospects on mechanism of sinomenin on histamine release and the binding to histamine receptors. Int J Mol Sci. 2018;20:70.

7. Zhou H, Liu JX, Luo JF, et al. Suppressing mPGES-1 expression by sinomenine ameliorates inflammation and arthritis. Biochem Pharmacol. 2017;142:133144.

8. Wu Y, Lin Z, Yan Z, et al. Sinomenine contributes to the inhibition of the inflammatory response and the improvement of osteoarthritis in mouse-cartilage cells by acting on the Nrf2/HO-1 and NF-B signaling pathways. Int Immunopharmacol. 2019;75:105715.

9. Liu S, Chen Q, Liu J, et al. Sinomenine protects against E.coli-induced acute lung injury in mice through Nrf2-NF-B pathway. Biomed Pharmacother. 2018;107:696702.

10. Zhu Q, Sun Y, Zhu J, et al. Antinociceptive effects of sinomenine in a rat model of neuropathic pain. Sci Rep. 2014;4:7270.

11. Feng ZT, Yang T, Hou XQ, et al. Sinomenine mitigates collagen-induced arthritis mice by inhibiting angiogenesis. Biomed Pharmacother. 2019;13:108759.

12. Tang J, Raza A, Chen J, et al. A systematic review on the sinomenine derivatives. Mini Rev Med Chem. 2018;18::906917.

13. Hong Y, Yang J, Shen X, et al. Sinomenine hydrochloride enhancement of the inhibitory effects of anti-transferrin receptor antibody-dependent on the COX-2 pathway in human hepatoma cells. Cancer Immunol Immunother. 2013;62:447454.

14. Li X, Wang K, Ren Y, et al. MAPK signaling mediates sinomenine hydrochloride-induced human breast cancer cell death via both reactive oxygen species-dependent and -independent pathways: an in vitro and in vivo study. Cell Death Dis. 2014;5:e1356.

15. Shen KH, Hung JH, Liao YC, et al. Sinomenine inhibits migration and invasion of human lung cancer cell through downregulating expression of miR-21 and MMPs. Int J Mol Sci. 2020;21:3080.

16. Zhao B, Liu L, Mao J, et al. Sinomenine hydrochloride attenuates the proliferation, migration, invasiveness, angiogenesis and epithelial-mesenchymal transition of clear-cell renal cell carcinoma cells via targeting Smad in vitro. Biomed Pharmacother. 2017;96:10361044.

17. Jiang Y, Jiao Y, Liu Y, et al. Sinomenine hydrochloride inhibits the metastasis of human glioblastoma cells by suppressing the expression of matrix metalloproteinase-2/-9 and reversing the endogenous and exogenous epithelial-mesenchymal transition. Int J Mol Sci. 2018;19:844.

18. Li H, Lin Z, Bai Y, et al. Sinomenine inhibits ovarian cancer cell growth and metastasis by mediating the Wnt/-catenin pathway via targeting MCM2. RSC Adv. 2017;7:5001750026.

19. Xu Y, Jiang T, Wang C, et al. Sinomenine hydrochloride exerts antitumor outcome in ovarian cancer cells by inhibition of long non-coding RNA HOST2 expression. Artif Cells Nanomed Biotechnol. 2019;47:41314138.

20. Tang F, Barbacioru C, Wang Y, et al. mRNA-seq whole-transcriptome analysis of a single cell. Nat Methods. 2009;6:377382.

21. Wang J, Dean DC, Hornicek FJ, et al. RNA sequencing (RNA-Seq) and its application in ovarian cancer. Gynecol Oncol. 2019;152:194201.

22. Yu B, Li H, Chen J, et al. Extensively expanded murine-induced hepatic stem cells maintain high-efficient hepatic differentiation potential for repopulation of injured livers. Liver Int. 2020. doi:10.1111/liv.14509

23. Trapnell C, Pachter L, Salzberg SL. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics. 2009;25:11051111.

24. Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinform. 2011;12:323.

25. Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010;26:139140.

26. Xie C, Mao X, Huang J, et al. KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases. Nucleic Acids Res. 2011;39:W316W322.

27. Szklarczyk D, Morris JH, Cook H, et al. The STRING database in 2017: quality-controlled protein-protein association networks, made broadly accessible. Nucleic Acids Res. 2017;45:D362D368.

28. Shannon P, Markiel A, Ozier O, et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;13:24982504.

29. Yang LH, Wang Y, Qiao S, et al. Liver-enriched activator protein 1 as an isoform of ccaat/enhancer-binding protein beta suppresses stem cell features of hepatocellular carcinoma. Cancer Manag Res. 2018;10:873885.

30. Liao H, Ji F, Ying S. CDK1: beyond cell cycle regulation. Aging. 2017;9:24652466.

31. Qi W, Tang Z, Yu H. Phosphorylation- and polo-box-dependent binding of Plk1 to bub1 is required for the kinetochore localization of Plk1. Mol Biol Cell. 2006;17:37053716.

32. Hunt T. Under arrest in the cell cycle. Nature. 1989;342:483484.

33. Nurse P. Universal control mechanism regulating onset of M-phase. Nature. 1990;344:503508.

34. Woodbury EL, Morgan DO. Cdk and APC activities limit the spindle-stabilizing function of Fin1 to anaphase. Nat Cell Biol. 2007;9:106112.

35. Ducommun B, Brambilla P, Flix MA, et al. cdc2 phosphorylation is required for its interaction with cyclin. EMBO J. 1991;10:33113319.

36. Atherton-Fessler S, Liu F, Gabrielli B, et al. Cell cycle regulation of the p34cdc2 inhibitory kinases. Mol Biol Cell. 1994;5:9891001.

37. Gezici S, ekerolu N. Current perspectives in the application of medicinal plants against cancer: novel therapeutic agents. Anticancer Agents Med Chem. 2019;19:101111.

38. Hall PA. Cell proliferation. J Pathol. 1991;165:349354.

39. Matson JP, Cook JG. Cell cycle proliferation decisions: the impact of single cell analyses. FEBS J. 2017;284:362375.

40. Mochida S, Rata S, Hino H, et al. Two bistable switches govern M phase entry. Curr Biol. 2016;26:33613367.

41. Norbury C, Blow J, Nurse P. Regulatory phosphorylation of the p34cdc2 protein kinase in vertebrates. EMBO J. 1991;10:33213329.

42. Hendzel MJ, Wei Y, Mancini MA, et al. Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma. 1997;106:348360.

43. McIntosh JR. Mitosis. Cold Spring Harb Perspect Biol. 2016;8:a023218.

44. Petronczki M, Lnrt P, Peters JM. Polo on the rise-from mitotic entry to cytokinesis with Plk1. Dev Cell. 2008;14:646659.

45. Kishi K, van Vugt MA, Okamoto K, et al. Functional dynamics of Polo-like kinase 1 at the centrosome. Mol Cell Biol. 2009;29:31343150.

46. Raaijmakers JA, van Heesbeen R, Blomen VA, et al. BUB1 is essential for the viability of human cells in which the spindle assembly checkpoint is compromised. Cell Rep. 2018;22:14241438.

47. Amin MA, McKenney RJ, Varma D. Antagonism between the dynein and Ndc80 complexes at kinetochores controls the stability of kinetochore-microtubule attachments during mitosis. J Biol Chem. 2018;293:57555765.

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[Full text] Sinomenine Inhibits the Growth of Ovarian Cancer Cells Through the Sup | OTT - Dove Medical Press

Reactivating neural crest pluripotency

Cranial neural crest cells (CNCCs) are a transient cell group with an extraordinary differentiation potential that extends beyond its ectodermal lineage to form the majority of facial mesenchyme. Zalc et al. identified a neuroepithelial precursor population that transiently reactivates pluripotency factors to generate CNCCs. The pluripotency factor Oct4 is required for the expansion of CNCC developmental potential to form facial mesenchyme. Analysis of the chromatin landscape of Oct4+ CNCC precursors showed that these cells resemble those of epiblast stem cells, with additional features suggestive of future priming for neural crest programs. Thus, to expand their cellular potency, CNCC precursors undergo a natural in vivo reprogramming event.

Science, this issue p. eabb4776

Cell differentiation is classically described as a unidirectional process that progresses through a series of lineage restriction events, with cellular potential being increasingly reduced as the embryo develops, a concept famously illustrated by Conrad Waddington in his epigenetic landscape. However, the vertebrate-specific transient cell population called cranial neural crest cells (CNCCs) challenges this paradigm. Although they originate in the ectoderm and are capable of differentiating into cell types typical of this germ layer, CNCCs can also give rise to mesenchymal cell types canonically associated with the mesoderm lineage, such as bone, cartilage, and smooth muscle. How CNCCs expand their differentiation potential beyond their germ layer of origin remains unresolved.

We hypothesized that unbiased analysis of transcriptional heterogeneity during the early stages of mammalian CNCC development may identify a precursor population and provide clues as to how these specialized cells gain their extraordinary differentiation potential. To test this, we combined single-cell RNA-sequencing analysis of murine CNCCs from staged mouse embryos with follow-up lineage-tracing, loss-of-function, and epigenomic-profiling experiments.

We found that premigratory CNCCs are heterogeneous and carry positional information reflective of their origin in the neuroepithelium, but this early positional information is subsequently erased, with delaminating CNCCs showing a relatively uniform transcriptional signature that later rediversifies as CNCCs undergo first commitment events. We identify an early precursor population that expresses canonical pluripotency transcription factors and gives rise to CNCCs and craniofacial structures. Rather than being maintained from the epiblast, pluripotency factor Oct4 is transiently reactivated in the prospective CNCCs after head-fold formation, and its expression shifts from the most anterior to the more posterior part of the cranial domain as development progresses. Oct4 is not required for the induction of CNCCs in the neuroepithelium, but instead is important for the specification and survival of facial mesenchyme, thus directly linking this pluripotency factor with the expansion of CNCC cellular potential. Open chromatin landscapes of Oct4+ CNCC precursors are consistent with their neuroepithelial origin while also broadly resembling those of pluripotent epiblast stem cells. In addition, we saw priming of distal regulatory regions at a subset of loci associated with future neural crest migration and mesenchyme formation.

Our results show that premigratory CNCCs first form as a heterogeneous population that rapidly changes its transcriptional identity during delamination, resulting in the formation of a transcriptionally (and likely also functionally) equivalent cell group capable of adapting to future locations during and after migration. Such functional equivalency and plasticity of CNCCs is consistent with previous embryological studies. Furthermore, the demonstration that CNCC precursors transiently reactivate pluripotency factors suggests that these cells undergo a natural in vivo reprogramming event that allows them to climb uphill on Waddingtons epigenetic landscape. Indeed, our results show that at least one of the pluripotency factors, Oct4, is required for the expansion of CNCC developmental potential to include formation of facial mesenchyme. Whether this mechanism is specific to CNCCs and if such expansion of cellular plasticity could be harnessed for regenerative medicine purposes remain interesting questions for future investigations.

(A) Single-cell RNA (scRNA) sequencing of genetically labeled murine CNCCs over 14 hours of development revealed rapid transcriptional changes and identified a precursor population expressing pluripotency factors. (B) Uphill on Waddingtons epigenetic landscape, reactivation of Oct4 endows CNCC precursors with the ability to form derivatives typical of mesoderm, such as mesenchyme.

During development, cells progress from a pluripotent state to a more restricted fate within a particular germ layer. However, cranial neural crest cells (CNCCs), a transient cell population that generates most of the craniofacial skeleton, have much broader differentiation potential than their ectodermal lineage of origin. Here, we identify a neuroepithelial precursor population characterized by expression of canonical pluripotency transcription factors that gives rise to CNCCs and is essential for craniofacial development. Pluripotency factor Oct4 is transiently reactivated in CNCCs and is required for the subsequent formation of ectomesenchyme. Furthermore, open chromatin landscapes of Oct4+ CNCC precursors resemble those of epiblast stem cells, with additional features suggestive of priming for mesenchymal programs. We propose that CNCCs expand their developmental potential through a transient reacquisition of molecular signatures of pluripotency.

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Reactivation of the pluripotency program precedes formation of the cranial neural crest - Science Magazine

For some cancer patients, a "poop transplant" could boost the positive effects of immunotherapy, a treatment designed to rally the immune system against cancer cells.

Not all cancer patients respond to immunotherapy drugs. For example, only about 40% of patients with advanced melanoma, a type of skin cancer, reap long-term benefit from the drugs, according to recent estimates. In trying to pinpoint the differences between patients who respond well to immunotherapy and those who don't, scientists have zeroed in on a likely suspect: the microorganisms living in their guts.

Now, a new study, published Feb. 4 in the journal Science, adds to the growing evidence that having the right gut bugs can improve a patient's response to immunotherapy, helping to stop disease progression or even shrink tumors.

In the study, scientists collected stool from melanoma patients who responded well to immunotherapy and then transplanted their feces (and microbes) into the guts of 15 patients who had never previously responded to the drugs. After the transplant, six of the 15 patients responded to immunotherapy for the first time, showing either tumor reduction or disease stabilization that lasted more than a year.

Related: 7 odd things that raise your risk of cancer (and 1 that doesn't)

"The microbes really appear to drive the immunological changes we see in patients," said study author Dr. Hassane Zarour, a cancer immunologist, co-leader of the Cancer Immunology and Immunotherapy Program at University of Pittsburgh Medical Center Hillman and a professor of medicine at the University of Pittsburgh. The team linked the changes in gut bugs to changes in both tumor growth and the immune system; for instance, some of the participants showed an increase in specific immune cells and antibodies that appeared in their blood.

Despite the positive changes seen in some patients, fecal transplants likely won't help all patients whose cancer resists immunotherapy, Zarour said. In the new study, for instance, nine of the 15 patients did not benefit from the treatment. As part of their research, the team began to sift through the differences between those who improved after the transplant and those who didn't.

The idea for combining fecal transplants with immunotherapy first came from studies in mice with tumors, in which the rodents responded differently to the drugs depending on which gut microbes they carried, according to Science Magazine. By tweaking the mice's gut microbiomes the collection of bacteria, viruses and other microbes in their digestive tracts scientists found that they could improve this response, but they weren't sure which microbes made the difference.

That said, mice's responses to immunotherapy improved after they were given fecal matter from human cancer patients whose tumors had shrunk under immunotherapy. "When they took non-responding mice and gave them the right bugs they could convert non-responding mice into responding mice," Zarour said.

Other research showed that when human patients took antibiotics, which alter the gut microbiome, they were less likely to respond to immunotherapy, providing more evidence that gut bugs make a big difference in people, too.

Having seen the positive effects of fecal transplants in mice, scientists began testing the treatment in humans, starting with a few small clinical trials.

In two such trials, led by researchers at Sheba Medical Center in Ramat Gan, Israel, patients received both fecal transplants and oral pills containing dried stool. The patients then took immunotherapy drugs called "checkpoint blockades," which essentially rip the brakes off of immune cells and help amplify their activity against tumors. A subset of these patients, who had previously not responded to the drugs, suddenly began responding.

The new study by Zarour and his colleagues echoes these positive results, but it also starts to address a crucial question: How do gut bugs boost the effects of immunotherapy?

To answer this question, the team closely analyzed the microbes present in the donor stool samples and the recipients, before and after fecal transplants. The team also collected blood and tumor cell samples to assess the patients' immune responses over time, and computed tomography (CT) scans, to track tumor growth. They then used artificial intelligence to find connections between all these data points.

Out of the 15 patients, nine still didn't respond to immunotherapy after their transplant. But of the six who did respond, one showed a complete response to checkpoint blockade drugs, meaning their tumors shrunk so much they were no longer detectable; two others showed a partial response, meaning their tumors shrunk but did not disappear, and three have shown no disease progression for over a year. In all six of these patients, the microbes from the donor's stool quickly colonized their guts, and several of the newcomer bugs that were previously linked to positive immunotherapy outcomes increased in number.

Related: 11 surprising facts about your immune system

This change in gut bacteria triggered an immune response in the six patients, as their bodies began building antibodies that recognized the new bugs; these antibodies showed up in their blood. While the link between bacteria-specific antibodies and cancer is not well understood, it's thought that some of these antibodies can help prime the immune system to hunt down tumor cells, Zarour said.

"The bugs that increased in the responders were really correlated with positive immunological changes," he said. These patients also built up a larger arsenal of activated T cells immune cells that can target and kill cancer cells while substances that suppress the immune system decreased. For example, a protein called interleukin-8 (IL-8) can summon immunosuppressive cells to tumor sites and therefore blunt the effects of immunotherapy; but IL-8 decreased in the six responsive patients.

By comparison, cells that secrete IL-8 increased in the nine patients who didn't respond to the fecal transplant. Based on this new data, "IL-8 seems to really play a critical role in regulating patients' responses" to the two-part treatment, Zarour said.

Compared with the six responsive patients, the nine others also showed less pronounced immune responses to the transplant and lower levels of the noted beneficial bacteria; some even had dissimilar gut microbiomes to their fecal donors, suggesting the bacteria didn't take over their guts as seen in responsive patients.

In general, "the gut microbiome may be just one of the many reasons we don't respond to a specific treatment," Zarour said, so fecal transplants wouldn't be expected to work for everyone. That said, the immune changes seen in the six responders, including the decline in IL-8, provide hints as to why it works for some people.

In the future, these results will need to be validated in larger groups of melanoma patients, as well as other cancer patients whose disease resists immunotherapy, Zarour said.

Though small, the new trial provides "firm evidence that manipulating the microbiome can yield benefit when added to immunotherapy for cancer," said Dr. Jeffrey Weber, a medical oncologist and co-director of the Melanoma Research Program at New York University Langone Health, who was not involved in the research. Assuming these results hold up in other patients, though, fecal transplants may not be the best way to deliver helpful microbes into the gut, Weber said in an email.

The future may lie in ingesting the bacteria orally, after they've been freeze-dried, Weber said. This approach could include something similar to the oral pills used in other trials, for example. Either that, or scientists could isolate specific metabolites produced by the helpful bacteria and use those as drugs, Weber said. "The big question is, what metabolites from the 'favorable' bacterial species are actually responsible for benefit," he said.

Originally published on Live Science.

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Cancer patients weren't responding to therapy. Then they got a poop transplant. - Livescience.com

UKONIQ is approved for adult patients with relapsed or refractory marginal zone lymphoma after at least one prior anti-CD20 based regimen

UKONIQ is approved for adult patients with relapsed or refractory follicular lymphoma after at least three prior lines of systemic therapy

UKONIQ is the first and only inhibitor of PI3K-delta and CK1-epsilon for relapsed/refractory MZL and FL

U.S. Commercial launch now underway

Company to host conference call on Monday, February 8, 2021 at 8:30 AM ET

NEW YORK, Feb. 05, 2021 (GLOBE NEWSWIRE) -- TG Therapeutics, Inc.(NASDAQ: TGTX), today announced theU.S. Food and Drug Administration(FDA) has approved UKONIQ (umbralisib), for the treatment of adult patients with relapsed or refractory marginal zone lymphoma (MZL) who have received at least one prior anti-CD20 based regimen and adult patients with relapsed or refractory follicular lymphoma (FL) who have received at least three prior lines of systemic therapy.

UKONIQ is the first and only, oral, once daily, inhibitor of phosphoinositide 3 kinase (PI3K) delta and casein kinase 1(CK1) epsilon. Accelerated approval was granted for these indications based on overall response rate (ORR) data from the Phase 2 UNITY-NHL Trial (NCT02793583). Continued approval for these indications may be contingent upon verification and description of clinical benefit in a confirmatory trial.This application was granted priority review for the MZL indication. In addition, UKONIQ was granted Breakthrough Therapy Designation (BTD) for the treatment of MZL and orphan drug designation (ODD) for the treatment of MZL and FL.

Michael S. Weiss, Executive Chairman and Chief Executive Officer of TG Therapeutics stated, Todays approval of UKONIQ marks a historic day for our Company with this being our first approval and we are extremely pleased to be able to bring our novel inhibitor of PI3K-delta and CK1-epsilon to patients with relapsed/refractory MZL and FL. We have built a commercial team with significant experience who will immediately start to engage our customers to educate them on UKONIQ and how to access the product for patients in needand expect to make UKONIQ available to US distributors in the next few days. Mr. Weiss continued, We want to thank the patients, physicians, nurses and clinical coordinators for their support and participation in our clinical trials, and the FDA for their collaboration throughout this process. We remain dedicated to patients with B-cell diseases and our mission of developing treatment options for those in need.

Despite treatment advances, MZL and FL remain incurable diseases with limited treatment options for patients who relapse after prior therapy and no defined standard of care. With the approval ofumbralisib we now have a targeted, oral, once-daily option, offering a needed treatment alternative for patients, stated Dr. Nathan Fowler, Professor of Medicine at The University of Texas MD Anderson Cancer Center and the Study Chair of the UNITY-NHL MZL &FL cohorts.

The approval ofumbralisib for the treatment of relapsed/refractory marginal zone lymphoma and follicular lymphoma offers patients a new treatment option, and new hope in the fight against these diseases, stated Meghan Gutierrez, Chief Executive Officer of the Lymphoma Research Foundation.

EFFICACY & SAFETY DATA IN RELAPSED/REFRACTORY MZL AND FLThe efficacy of UKONIQ monotherapy was evaluated in two single-arm cohorts, within the Phase 2 UNITY-NHL clinical trial, in 69 patients with MZL who received at least 1 prior therapy, including an anti-CD20 regimen, and in 117 patients with FL who received at least 2 prior systemic therapies, including an anti-CD20 monoclonal antibody and an alkylating agent. The UNITY-NHL Phase 2 trial is an open-label, multi-center, multi-cohort study with patients receiving UKONIQ 800 mg once daily. The primary endpoint was independent review committee (IRC) assessed overall response rate (ORR) according to the Revised International Working Group Criteria.

CI, confidence interval; NR, not reached; NE, not evaluable +Denotes censored observation

The safety of UKONIQ monotherapy was based on a pooled population from the 221 adults with MZL and FL in three single arm, open label trials and one open label extension trial. Patients received UKONIQ 800 mg orally once daily. Serious adverse reactions occurred in 18% of patients who received UKONIQ. Serious adverse reactions that occurred in 2% of patients were diarrhea-colitis (4%), pneumonia (3%), sepsis (2%), and urinary tract infection (2%). The most common adverse reactions (>15%), including laboratory abnormalities, were increased creatinine (79%), diarrhea-colitis (58%, 2%), fatigue (41%), nausea (38%), neutropenia (33%), ALT increase (33%), AST increase (32%), musculoskeletal pain (27%), anemia (27%), thrombocytopenia (26%), upper respiratory tract infection (21%), vomiting (21%), abdominal pain (19%), decreased appetite (19%), and rash (18%).

ABOUT UKONIQ(umbralisib) 200 MG TABLETSUKONIQ is the first and onlyoral inhibitor of phosphoinositide 3 kinase (PI3K) delta and casein kinase 1(CK1) epsilon. PI3K-deltais known to play an important role in supporting cell proliferation and survival, cell differentiation, intercellular trafficking and immunity and is expressed in both normal and malignant B-cells. CK1-epsilonis a regulator of oncoprotein translation and has been implicated in the pathogenesis of cancer cells, including lymphoid malignancies.

UKONIQ is indicated for the treatment of adult patients with relapsed or refractory marginal zone lymphoma (MZL) who have received at least one prior anti-CD20-based regimen and for the treatment of adult patients with relapsed or refractory follicular lymphoma (FL) who have received at least three prior lines of systemic therapy.

These indications are approved under accelerated approval based on overall response rate. Continued approval for this indication may be contingent upon verification and description of clinical benefit in a confirmatory trial.

IMPORTANT SAFETY INFORMATIONInfections: Serious, including fatal, infections occurred in patients treated with UKONIQ. Grade 3 or higher infections occurred in 10%of 335 patients, with fatal infections occurring in <1%. The most frequent Grade 3 infections included pneumonia, sepsis, and urinary tract infection. Provide prophylaxis for Pneumocystis jirovecii pneumonia (PJP) and consider prophylactic antivirals during treatment with UKONIQ to prevent CMV infection, including CMV reactivation. Monitor for any new or worsening signs and symptoms of infection, including suspected PJP or CMV, during treatment with UKONIQ. For Grade 3 or 4 infection, withhold UKONIQ until infection has resolved. Resume UKONIQ at the same or a reduced dose. Withhold UKONIQ in patients with suspected PJP of any grade and permanently discontinue in patients with confirmed PJP. For clinical CMV infection or viremia, withhold UKONIQ until infection or viremia resolves. If UKONIQ is resumed, administer the same or reduced dose and monitor patients for CMV reactivation by PCR or antigen test at least monthly.

Neutropenia: Serious neutropenia occurred in patients treated with UKONIQ. Grade 3 neutropenia developed in 9% of 335 patients and Grade 4 neutropenia developed in 9%. Monitor neutrophil counts at least every 2 weeks for the first 2 months of UKONIQ and at least weekly in patients with neutrophil count <1 x 109/L (Grade 3-4) neutropenia during treatment with UKONIQ. Consider supportive care as appropriate. Withhold, reduce dose, or discontinue UKONIQ depending on the severity and persistence of neutropenia.

Diarrhea or Non-Infectious Colitis: Serious diarrhea or non-infectious colitis occurred in patients treated with UKONIQ. Any grade diarrhea or colitis occurred in 53% of 335 patients and Grade 3 occurred in 9%.For patients with severe diarrhea (Grade 3, i.e., > 6 stools per day over baseline) or abdominal pain, stool with mucus or blood, change in bowel habits, or peritoneal signs, withhold UKONIQ until resolved and provide supportive care with antidiarrheals or enteric acting steroids as appropriate. Upon resolution, resume UKONIQ at a reduced dose. For recurrent Grade 3 diarrhea or recurrent colitis of any grade, discontinue UKONIQ. Discontinue UKONIQ for life-threatening diarrhea or colitis.

Hepatotoxicity: Serious hepatotoxicityoccurred in patients treated with UKONIQ. Grade 3 and 4 transaminase elevations (ALT and/or AST) occurred in 8% and <1%, respectively, in 335 patients. Monitor hepatic function at baseline and during treatment with UKONIQ. For ALT/AST greater than 5 to less than 20 times ULN, withhold UKONIQ until return to less than 3 times ULN, then resume at a reduced dose. For ALT/AST elevation greater than 20 times ULN, discontinue UKONIQ.

Severe Cutaneous Reactions: Severe cutaneous reactions, including a fatal case of exfoliative dermatitis, occurred in patients treated with UKONIQ. Grade 3 cutaneous reactions occurred in 2% of 335 patients and included exfoliative dermatitis, erythema, and rash (primarily maculo-papular). Monitor patients for new or worsening cutaneous reactions. Review all concomitant medications and discontinue any potentially contributing medications. Withhold UKONIQ for severe (Grade 3) cutaneous reactions until resolution. Monitor at least weekly until resolved. Upon resolution, resume UKONIQ at a reduced dose. Discontinue UKONIQ if severe cutaneous reaction does not improve, worsens, or recurs. Discontinue UKONIQ for life-threatening cutaneous reactions or SJS, TEN, or DRESS of any grade. Provide supportive care as appropriate.

Allergic Reactions Due to Inactive Ingredient FD&C Yellow No. 5: UKONIQ contains FD&C Yellow No. 5 (tartrazine), which may cause allergic-type reactions (including bronchial asthma) in certain susceptible persons, frequently in patients who also have aspirin hypersensitivity.

Embryo-fetal Toxicity: Based on findings in animals and its mechanism of action, UKONIQ can cause fetal harm when administered to a pregnant woman. Advise pregnant women of the potential risk to a fetus. Advise females and males with female partners of reproductive potential to use effective contraception during treatment and for at least one month after the last dose.

Serious adverse reactions occurred in 18% of 221 patients who received UKONIQ. Serious adverse reactions that occurred in 2% of patients were diarrhea-colitis (4%), pneumonia (3%), sepsis (2%), and urinary tract infection (2%). Permanent discontinuation of UKONIQ due to an adverse reaction occurred in 14% of patients. Dose reductions of UKONIQ due to an adverse reaction occurred in 11% of patients. Dosage interruptions of UKONIQ due to an adverse reaction occurred in 43% of patients.

The most common adverse reactions (>15%), including laboratory abnormalities, in 221 patients who received UKONIQ were increased creatinine (79%), diarrhea-colitis (58%, 2%), fatigue (41%), nausea (38%), neutropenia (33%), ALT increase (33%), AST increase (32%), musculoskeletal pain (27%), anemia (27%), thrombocytopenia (26%), upper respiratory tract infection (21%), vomiting (21%), abdominal pain (19%), decreased appetite (19%), and rash (18%).

Lactation: Because of the potential for serious adverse reactions from umbralisib in the breastfed child, advise women not to breastfeed during treatment with UKONIQ and for at least one month after the last dose.

Please visit http://www.tgtherapeutics.com/prescribing-information/uspi-ukon for full Prescribing Information and Medication Guide.

Physicians, pharmacists, or other healthcare professionals with questions about UKONIQ should visitwww.UKONIQ.com.

ABOUT TG PATIENT SUPPORTThe TG Patient Support is a comprehensive program designed by TG Therapeutics to support patients through their treatment journey and the reimbursement process. More information about the TG Patient Support program is accessible by phone at 1-877-TGTXPSP (1-877-848-9777); by fax at 1-877-778-1329 or at http://www.UKONIQ.com/patient/patientsupport.

ABOUT MARGINAL ZONE LYMPHOMAMarginal zone lymphoma (MZL) comprises a group of indolent (slow growing) mature B-cell non-Hodgkin lymphomas (NHLs). MZL is generally considered a chronic and incurable disease. With an annual incidence of approximately 8,200 newly diagnosed patients in the United States1,2, MZL is the third most common B-cell NHL, accounting for approximately ten percent of all NHL cases. MZL consists of three different subtypes: extranodal MZL of the mucosal-associated lymphoid tissue (MALT), nodal marginal zone lymphoma (NMZL), and splenic marginal zone lymphoma (SMZL)3.

ABOUT FOLLICULAR LYMPHOMAFollicular lymphoma (FL) is typically an indolent form of non-Hodgkin lymphoma (NHL) that arises from B-lymphocytes. It is the second most common form of NHL. FL is generally not curable and is considered a chronic disease, as patients can live for many years with this form of lymphoma.With an annual incidence inthe United Statesof approximately 13,200 newly diagnosed patients1,2, FL is the most common indolent lymphoma accounting for approximately 17 percent of all NHL cases4.

CONFERENCE CALL INFORMATIONThe Company will host a conference call on Monday, February 8, 2021 at 8:30 AM ET to discuss the UKONIQ approval. In order to participate in the conference call, please call 1-877-407-8029 (U.S.), 1-201-689-8029 (outside theU.S.), Conference Title: TG Therapeutics. A live webcast will be available on the Events page, located within the Investors & Media section, of the Company's website atwww.tgtherapeutics.com. An audio recording of the conference call will also be available for replay atwww.tgtherapeutics.com, for a period of 30 days after the call.

ABOUT TG THERAPEUTICS, INC.TG Therapeuticsis a fully-integrated, commercial stage biopharmaceutical company focused on the acquisition, development and commercialization of novel treatments for B-cell malignancies and autoimmune diseases. In addition to an active research pipeline including five investigational medicines across these therapeutic areas, TG has received accelerated approval from the U.S. FDA for UKONIQTM (umbralisib), for the treatment of adult patients with relapsed/refractory marginal zone lymphoma who have received at least one prior anti-CD20-based regimen and relapsed/refractory follicular lymphoma who have received at least three prior lines of systemic therapies. Currently, the Company has two programs in Phase 3 development for the treatment of patients with relapsing forms of multiple sclerosis (RMS) and patients with chronic lymphocytic leukemia (CLL) and several investigational medicines in Phase 1 clinical development.For more information, visit http://www.tgtherapeutics.com, and follow us on Twitter @TGTherapeutics and Linkedin.UKONIQTM is a registered trademark of TG Therapeutics, Inc.

__________________________________________________

1National Cancer Institute. SEER Cancer Statistics Review 2008-2017: Non-Hodgkin Lymphoma. Table 19.26. https://seer.cancer.gov/csr/1975_2017/results_single/sect_19_table.26_2pgs.pdf. Accessed January 19, 2021.2National Cancer Institute. SEER Cancer Stat Facts: Non-Hodgkin Lymphoma. https://seer.cancer.gov/statfacts/html/nhl.html. Accessed January 19, 2021.3Lymphoma Research Foundation: Marginal Zone Lymphomahttps://lymphoma.org/aboutlymphoma/nhl/mzl/4Lymphoma Research FoundationFollicular Lymphoma

Cautionary StatementThis press release contains forward-looking statements that involve a number of risks and uncertainties. For those statements, we claim the protection of the safe harbor for forward-looking statements contained in the Private Securities Litigation Reform Act of 1995.

Such forward looking statements include but are not limited to statements regarding expectations for the timing and commercial launch and availability of UKONIQ (umbralisib) for relapsed or refractory (R/R) marginal zone lymphoma (MZL) and follicular lymphoma (FL); clinical trials, including the confirmatory trial for UKONIQ in R/R MZL and FL; and anticipated healthcare professional and patient acceptance and use of UKONIQ for the FDA-approved indications.

In addition to the risk factors identified from time to time in our reports filed with theSecurities and Exchange Commission, factors that could cause our actual results to differ materially include the following: the Companys ability to establish and maintain a commercial infrastructure, and to successfully launch, market and sell UKONIQ or future products, if approved; failure to obtain and maintain requisite regulatory approvals, including the risk that the Company fails to satisfy post-approval regulatory requirements, such as the submission of sufficient data from a confirmatory clinical study; the potential for variation from the Companys projections and estimates about the potential market for UKONIQ or the Companys product candidates due to a number of factors, including for example, limitations that regulators may impose on the required labeling for the proposed treatment population for UKONIQ or our other product candidates; the Companys ability to meet post-approval compliance obligations (on topics including but not limited to product quality, product distribution and supply chain, pharmacovigilance, and sales and marketing); potential regulatory challenges to the Companys plans to seek expanded or additional indications for UKONIQ in the U.S. or plans to seek marketing approval for the product in additional geographies, outside of the U.S.; the Companys reliance on third parties for manufacturing, distribution and supply, and a range of other support functions for its clinical and commercial products, including UKONIQ; the uncertainties inherent in research and development; and the risk that the ongoing COVID-19 pandemic and associated government control measures have an adverse impact on our research and development plans or commercialization efforts. Further discussion about these and other risks and uncertainties can be found in our Annual Report on Form 10-K for the fiscal year endedDecember 31, 2019and in our other filings with theU.S. Securities and Exchange Commission.

Any forward-looking statements set forth in this press release speak only as of the date of this press release. We do not undertake to update any of these forward-looking statements to reflect events or circumstances that occur after the date hereof. This press release and prior releases are available atwww.tgtherapeutics.com. The information found on our website is not incorporated by reference into this press release and is included for reference purposes only.

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TG Therapeutics Announces FDA Accelerated Approval of UKONIQ (umbralisib) - GlobeNewswire

DetailsCategory: AntibodiesPublished on Friday, 05 February 2021 10:41Hits: 447

LONDON, UK I February 05, 2021 I The KESTREL Phase III trial for AstraZenecas Imfinzi (durvalumab) did not meet the primary endpoint of improving overall survival (OS) versus the EXTREME treatment regimen (chemotherapy plus cetuximab), a standard of care, in the 1st-line treatment of patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) whose tumours expressed high levels of PD-L1. Also, the combination of Imfinzi plus tremelimumab did not indicate an OS benefit in all-comer patients, a secondary endpoint.

Dave Fredrickson, Executive Vice President, Oncology Business Unit, said: Metastatic head and neck cancer is a complex and challenging disease with a poor prognosis. While we are disappointed by these results, insights from the KESTREL Phase III trial will advance our understanding and application of immunotherapy across our clinical development programme. We will continue to build on the established benefits of Imfinzi in early lung cancer and small cell lung cancer, to bring immunotherapy treatment options to all patients who may benefit.

The safety and tolerability profiles forImfinzias a monotherapy and in combination with tremelimumab were consistent with previous trials. The data will be shared in due course.

HNSCCNearly 750,000 patients were diagnosed with head and neck cancer around the world in 2020.1 Two thirds of these patients are diagnosed in advanced stages, and more than half of those treated eventually relapse.2,3 Median survival for a patient with an uncurable or metastatic relapse remains under one year.3 More than 90% of all head and neck cancers start in the squamous cells that line the mouth, nose and throat and are called head and neck squamous cell carcinomas.4

KESTRELThe KESTREL Phase III trial was a randomised, open-label, multi-centre, global trial in the 1st-line treatment of recurrent or metastatic HNSCC. The trial tested Imfinzi or Imfinzi plus a second immunotherapy, tremelimumab, versus the EXTREME treatment regimen (cetuximab with cisplatinor carboplatin plus 5-fluorouracil), a standard of care treatment. High PD-L1 was defined as either 50% or more tumour cells or 25% or more tumour-infiltrating immune cells expressing PD-L1.

The trial was conducted in more than 200 centres across 23 countries, including centres in the US, Europe, South America and Asia. The primary endpoint was OS in patients with high PD-L1 expression in the Imfinzi monotherapy arm. OS in all-comer patients treated with the combination of Imfinzi plus tremelimumab was being tested as a key secondary endpoint.

ImfinziImfinzi (durvalumab) is a human monoclonal antibody that binds to PD-L1 and blocks the interaction of PD-L1 with PD-1 and CD80, countering the tumour's immune-evading tactics and releasing the inhibition of immune responses.

Imfinziis approved in the curative-intent setting of unresectable, Stage III non-small cell lung cancer (NSCLC) after chemoradiation therapy in the EU, US, Japan, China and many other countries, based on the PACIFIC Phase III trial. Additionally, it is approved in the EU, US, Japan and many other countries for the treatment of extensive-stage small cell lung cancer (SCLC) based on the CASPIAN Phase III trial. Imfinziis also approved for previously treated patients with advanced bladder cancer in the US and several other countries.

As part of a broad development programme, Imfinzi is being tested as a monotherapy and in combination with other anti-cancer treatments for patients with NSCLC, SCLC, bladder cancer, hepatocellular carcinoma (HCC), biliary tract cancer, oesophageal cancer, gastric and gastroesophageal cancer, cervical cancer, ovarian cancer, endometrial cancer and other solid tumours.

TremelimumabTremelimumab is a human monoclonal antibody and potential new medicine that targets the activity of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Tremelimumab blocks the activity of CTLA-4, contributing to T cell activation, priming the immune response to cancer and fostering cancer cell death. Tremelimumab is being tested in a clinical trial programme in combination with Imfinzi in NSCLC, SCLC, bladder cancer and HCC.

AstraZeneca in immunotherapyImmunotherapy is a therapeutic approach designed to stimulate the bodys immune system to attack tumours. The Companys Immuno-Oncology (IO) portfolio is anchored by immunotherapies that have been designed to overcome anti-tumour immune suppression. AstraZeneca is invested in using IO approaches that deliver long-term survival for new groups of patients across tumour types.

The Company is pursuing a comprehensive clinical trial programme that includes Imfinzi as a monotherapy and in combination with tremelimumab in multiple tumour types, stages of disease, and lines of therapy, and where relevant using the PD-L1 biomarker as a decision-making tool to define the best potential treatment path for a patient. In addition, the ability to combine the IO portfolio with radiation, chemotherapy, and small, targeted molecules from across AstraZenecas oncology pipeline, and from research partners, may provide new treatment options across a broad range of tumours.

In head and neck cancer, the Company is also testing monalizumab, a first-in-class humanised anti-NKG2A antibody, in combination with cetuximab in the INTERLINK-1 Phase III trial in patients with recurrent or metastatic HNSCC previously treated with IO and chemotherapy. AstraZeneca obtained full oncology rights to monalizumab from Innate Pharma in October 2018 through a co-development and commercialisation agreement initiated in 2015.

AstraZeneca in oncologyAstraZeneca has a deep-rooted heritage in oncology and offers a quickly growing portfolio of new medicines that has the potential to transform patients' lives and the Company's future. With seven new medicines launched between 2014 and 2020, and a broad pipeline of small molecules and biologics in development, the Company is committed to advance oncology as a key growth driver for AstraZeneca focused on lung, ovarian, breast and blood cancers.

By harnessing the power of six scientific platforms - Immuno-Oncology, Tumour Drivers and Resistance, DNA Damage Response, Antibody Drug Conjugates, Epigenetics, and Cell Therapies - and by championing the development of personalised combinations, AstraZeneca has the vision to redefine cancer treatment and one day eliminate cancer as a cause of death.

AstraZenecaAstraZeneca (LSE/STO/Nasdaq: AZN) is a global, science-led biopharmaceutical company that focuses on the discovery, development and commercialisation of prescription medicines, primarily for the treatment of diseases in three therapy areas - Oncology, Cardiovascular, Renal & Metabolism, and Respiratory & Immunology. Based in Cambridge, UK, AstraZeneca operates in over 100 countries and its innovative medicines are used by millions of patients worldwide. Please visitastrazeneca.comand follow the Company on Twitter@AstraZeneca.

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References

1. World Health Organization. World GLOBOCAN 2020. Available at https://gco.iarc.fr/today/home. Accessed January 2021.

2. Heriou A, et al. Multiple Cancers of the Head and Neck. MAEDICA a Journal of Clinical Medicine 2013;8(1):80-852.

3. Rothschild U, et al. Immunotherapy in head and neck cancer scientific rationale, current treatment options and future directions. Swiss Med Wkly. 2018;148:w14625.

4. Palka K, et al. Update in Molecular Diagnostic Tests in Head and Neck Cancer. Semin Oncol. 2008 June;35(3):198-210.

SOURCE: AstraZeneca

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Update on KESTREL Phase III trial of Imfinzi with or without tremelimumab in the 1st-line treatment of recurrent or metastatic head and neck cancer |...