Abrogation of the G2/M checkpoint as a chemosensitization approach for alkylating agents.

BACKGROUND: The cell cycle is tightly regulated by checkpoints, which play a vital role in controlling its progression and timing. Cancer cells exploit the G2/M checkpoint, which serves as a resistance mechanism against genotoxic anticancer treatments, allowing for DNA repair prior to cell division. Manipulating cell cycle timing has emerged as a potential strategy to augment the effectiveness of DNA damage-based therapies. METHODS: In this study, we conducted a forward genome-wide CRISPR/Cas9 screening with repeated exposure to the alkylating agent temozolomide (TMZ) to investigate the mechanisms underlying tumor cell survival under genotoxic stress. RESULTS: Our findings revealed that canonical DNA repair pathways, including the Ataxia-telangiectasia mutated (ATM)/Fanconi and mismatch repair, determine cell fate under genotoxic stress. Notably, we identified the critical role of PKMYT1, in ensuring cell survival. Depletion of PKMYT1 led to overwhelming TMZ-induced cytotoxicity in cancer cells. Isobologram analysis demonstrated potent drug synergy between alkylating agents and a Myt1 kinase inhibitor, RP-6306. Mechanistically, inhibiting Myt1 forced G2/M-arrested cells into an unscheduled transition to the mitotic phase without complete resolution of DNA damage. This forced entry into mitosis, along with persistent DNA damage, resulted in severe mitotic abnormalities. Ultimately, these aberrations led to mitotic exit with substantial apoptosis. Preclinical animal studies demonstrated that the combination regimen involving TMZ and RP-6306 prolonged the overall survival of glioma-bearing mice. CONCLUSIONS: Collectively, our findings highlight the potential of targeting cell cycle timing through Myt1 inhibition as an effective strategy to enhance the efficacy of current standard cancer therapies, potentially leading to improved disease outcomes.

Effect of Strain Rate on Nano-Scale Mechanical Behavior of A-Plane (112¯0) ZnO Single Crystal by Nanoindentation.

In this study, nanoindentation tests at three different strain rates within 100 nm indentation depth were conducted on an a-plane (112¯0) ZnO single crystal to investigate the effect of strain rate on its nano-scale mechanical behavior. The load-indentation-depth curves, pop-in events, hardness and Young's moduli of an a-plane (112¯0) ZnO single crystal at different strain rates were investigated at the nano-scale level. The results indicated that, with the indentation depth increasing, the load increased gradually at each maximum indentation depth, hma, during the loading process. A distinct pop-in event occurred on each loading curve except that corresponding to the hmax of 10 nm. The applied load at the same indentation depth increased with the increasing strain rate during the nanoindentation of the a-plane (112¯0) ZnO single crystal. The higher strain rate deferred the pop-in event to a higher load and deeper indentation depth, and made the pop-in extension width larger. The hardness showed reverse indentation size effect (ISE) before the pop-in, and exhibited normal ISE after the pop-in. Both the hardness and the Young's modulus of the a-plane (112¯0) ZnO single crystal increased with the increasing strain rate, exhibiting the positive strain-rate sensitivity.

Protein Kinase B (PKB/AKT) Protects IDH-Mutated Glioma from Ferroptosis via Nrf2.

PURPOSE: Mutations of the isocitrate dehydrogenase (IDH) gene are common genetic mutations in human malignancies. Increasing evidence indicates that IDH mutations play critical roles in malignant transformation and progression. However, the therapeutic options for IDH-mutated cancers remain limited. In this study, the investigation of patient cohorts revealed that the PI3K/protein kinase B (AKT) signaling pathways were enhanced in IDH-mutated cancer cells. EXPERIMENTAL DESIGN: In this study, we investigated the gene expression profile in IDH-mutated cells using RNA sequencing after the depletion of AKT. Gene set enrichment analysis (GSEA) and pathway enrichment analysis were used to discover altered molecular pathways due to AKT depletion. We further investigated the therapeutic effect of the AKT inhibitor, ipatasertib (Ipa), combined with temozolomide (TMZ) in cell lines and preclinical animal models. RESULTS: GSEA and pathway enrichment analysis indicated that the PI3K/AKT pathway significantly correlated with Nrf2-guided gene expression and ferroptosis-related pathways. Mechanistically, AKT suppresses the activity of GSK3ß and stabilizes Nrf2. Moreover, inhibition of AKT activity with Ipa synergizes with the genotoxic agent TMZ, leading to overwhelming ferroptotic cell death in IDH-mutated cancer cells. The preclinical animal model confirmed that combining Ipa and TMZ treatment prolonged survival. CONCLUSIONS: Our findings highlighted AKT/Nrf2 pathways as a potential synthetic lethality target for IDH-mutated cancers.

Studying the Interfacial Properties of Carbon/Glass Hybrid Composites via the Nanoindentation Method.

The mechanical properties of hybrid composite interfaces are critical in determining the overall properties of composite materials. To investigate the mechanical performance of hybrid composite interfaces, an accurate and efficient method must be developed. In this work, nanoindentation is used in this work to investigate the mechanical performance of the carbon/glass interface and the influence of the distance between carbon and the glass fibers on the modulus of the thermoset matrix. The results show that the interface sizes around the carbon and glass fibers are around 1.5 and 2.0 µm, respectively. The modulus around the carbon fibers is 5-11 GPa without the fiber effect, while that around the glass fibers is 4-10 GPa. The modulus of the matrix is not affected by the two types of fibers when the distance between them is greater than 4.5 µm.

IDH mutation and cancer stem cell.

Cancer stem cells (CSCs) are a small population of cells in human malignancies that resemble the biology of human pluripotent stem cells. CSCs are closely related to the critical hallmarks in human cancers, ranging from oncogenesis to disease progression, therapeutic resistance, and overall outcome. Mutations in isocitrate dehydrogenase (IDH) were recently identified as founder mutations for human cancers. An increasing amount of evidence indicates that IDH mutations are closely related to the establishment and maintenance of CSCs. Biosynthesis of oncometabolite, metabolic reprogramming, and epigenetic shifts establish distinctive molecular signatures in IDH-mutated CSCs. Additionally, IDH mutation and IDH-related pathways could be valuable molecular targets to impact the CSC components in human cancers and to improve the disease outcome.

Identification of Isocitrate Dehydrogenase 2 (IDH2) Mutation in Carotid Body Paraganglioma.

Carotid body paragangliomas (PGLs) are rare neuroendocrine tumors that develop within the adventitia of the medial aspect of the carotid bifurcation. Carotid body PGLs comprise about 65% of head and neck paragangliomas, however, their genetic background remains elusive. In the present study, we report one case of carotid body PGL with a somatic mutation in the gene encoding isocitrate dehydrogenase 2 (IDH2). The missense mutation in IDH2 resulted in R172G amino acid substitution, which exhibits neomorphic activity and production of D-2-hydroxyglutarate.

D-2-Hydroxyglutarate in Glioma Biology.

Isocitrate dehydrogenase (IDH) mutations are common genetic abnormalities in glioma, which result in the accumulation of an "oncometabolite", D-2-hydroxyglutarate (D-2-HG). Abnormally elevated D-2-HG levels result in a distinctive pattern in cancer biology, through competitively inhibiting α-ketoglutarate (α-KG)/Fe(II)-dependent dioxgenases (α-KGDDs). Recent studies have revealed that D-2-HG affects DNA/histone methylation, hypoxia signaling, DNA repair, and redox homeostasis, which impacts the oncogenesis of IDH-mutated cancers. In this review, we will discuss the current understanding of D-2-HG in cancer biology, as well as the emerging opportunities in therapeutics in IDH-mutated glioma.

Genotoxic therapy and resistance mechanism in gliomas.

Glioma is one of the most common and lethal brain tumors. Surgical resection followed by radiotherapy plus chemotherapy is the current standard of care for patients with glioma. The existence of resistance to genotoxic therapy, as well as the nature of tumor heterogeneity greatly limits the efficacy of glioma therapy. DNA damage repair pathways play essential roles in many aspects of glioma biology such as cancer progression, therapy resistance, and tumor relapse. O6-methylguanine-DNA methyltransferase (MGMT) repairs the cytotoxic DNA lesion generated by temozolomide (TMZ), considered as the main mechanism of drug resistance. In addition, mismatch repair, base excision repair, and homologous recombination DNA repair also play pivotal roles in treatment resistance as well. Furthermore, cellular mechanisms, such as cancer stem cells, evasion from apoptosis, and metabolic reprogramming, also contribute to TMZ resistance in gliomas. Investigations over the past two decades have revealed comprehensive mechanisms of glioma therapy resistance, which has led to the development of novel therapeutic strategies and targeting molecules.

Cohesin promotes HSV-1 lytic transcription by facilitating the binding of RNA Pol II on viral genes.

BACKGROUND: Herpes Simplex Virus type I (HSV-1) is a large double-stranded DNA virus that enters productive infection in epithelial cells and reorganizes the host nucleus. Cohesin, a major constituent of interphase and mitotic chromosomes comprised of SMC1, SMC3, and SCC1 (Mcd1/Rad21), SCC3 (SA1/SA2), have diverse functions, including sister chromatid cohesion, DNA double-stranded breaks repair, and transcriptional control. Little is known about the role of cohesin in HSV-1 lytic infection. METHODS: We measured the effect on HSV-1 transcription, genome copy number, and viral titer by depleting cohesin components SMC1 or Rad21 using RNAi, followed by immunofluorescence, qPCR, and ChIP experiments to gain insight into cohesin's function in HSV-1 transcription and replication. RESULTS: Here, we report that cohesion subunits SMC1 and Rad21 are recruited to the lytic HSV-1 replication compartment. The knockdown results in decreased viral transcription, protein expression, and maturation of viral replication compartments. SMC1 and Rad21 knockdown leads to the reduced overall RNA pol II occupancy level but increased RNA pol II ser5 phosphorylation binding on viral genes. Consistent with this, the knockdown increased H3K27me3 modification on these genes. CONCLUSIONS: These results suggest that cohesin facilitates HSV-1 lytic transcription by promoting RNA Pol II transcription activity and preventing chromatin's silencing on the viral genome.

NRF2 in human neoplasm: Cancer biology and potential therapeutic target.

Nuclear factor-erythroid 2-related factor 2 (NRF2) is a master regulator of a series of cytoprotective genes, which protects cells from stress conditions such as reactive oxygen species (ROS) and electrophiles. Besides its pivotal role in cellular physiology and stress response, several recent advances revealed that NRF2-governed pathways are extensively exploited in cancer cells, and play critical roles in granting growth advantage, supporting cancer metabolism, and promoting malignant transformation. In the present review, we focus on the regulatory mechanisms of NRF2 and its roles in cancer biology. We also discuss the value of targeting NRF2-associated pathways as a means of developing future cancer therapeutics.

Isocitrate Dehydrogenase Mutations in Glioma: Genetics, Biochemistry, and Clinical Indications.

Mutations in isocitrate dehydrogenase (IDH) are commonly observed in lower-grade glioma and secondary glioblastomas. IDH mutants confer a neomorphic enzyme activity that converts α-ketoglutarate to an oncometabolite D-2-hydroxyglutarate, which impacts cellular epigenetics and metabolism. IDH mutation establishes distinctive patterns in metabolism, cancer biology, and the therapeutic sensitivity of glioma. Thus, a deeper understanding of the roles of IDH mutations is of great value to improve the therapeutic efficacy of glioma and other malignancies that share similar genetic characteristics. In this review, we focused on the genetics, biochemistry, and clinical impacts of IDH mutations in glioma.

Quassinoid analogs with enhanced efficacy for treatment of hematologic malignancies target the PI3Kγ isoform.

Development of novel PI3K inhibitors is an important strategy to overcome their resistance and poor tolerability in clinical trials. The quassinoid family member Brusatol shows specific inhibitory activity against hematologic malignancies. However, the mechanism of its anti-cancer activity is unknown. We investigated the anti-cancer activity of Brusatol on multiple hematologic malignancies derived cell lines. The results demonstrated that the PI3Kγ isoform was identified as a direct target of Brusatol, and inhibition was dramatically reduced on cells with lower PI3Kγ levels. Novel synthetic analogs were also developed and tested in vitro and in vivo. They shared comparable or superior potency in their ability to inhibit malignant hematologic cell lines, and in a xenograft transplant mouse model. One unique analog had minimal toxicity to normal human cells and in a mouse model. These new analogs have enhanced potential for development as a new class of PI3K inhibitors for treatment of hematologic malignancies.

KSHV-encoded LANA protects the cellular replication machinery from hypoxia induced degradation.

Kaposi's sarcoma associated herpesvirus (KSHV), like all herpesviruses maintains lifelong persistence with its host genome in latently infected cells with only a small fraction of cells showing signatures of productive lytic replication. Modulation of cellular signaling pathways by KSHV-encoded latent antigens, and microRNAs, as well as some level of spontaneous reactivation are important requirements for establishment of viral-associated diseases. Hypoxia, a prominent characteristic of the microenvironment of cancers, can exert specific effects on cell cycle control, and DNA replication through HIF1α-dependent pathways. Furthermore, hypoxia can induce lytic replication of KSHV. The mechanism by which KSHV-encoded RNAs and antigens regulate cellular and viral replication in the hypoxic microenvironment has yet to be fully elucidated. We investigated replication-associated events in the isogenic background of KSHV positive and negative cells grown under normoxic or hypoxic conditions and discovered an indispensable role of KSHV for sustained cellular and viral replication, through protection of critical components of the replication machinery from degradation at different stages of the process. These include proteins involved in origin recognition, pre-initiation, initiation and elongation of replicating genomes. Our results demonstrate that KSHV-encoded LANA inhibits hypoxia-mediated degradation of these proteins to sustain continued replication of both host and KSHV DNA. The present study provides a new dimension to our understanding of the role of KSHV in survival and growth of viral infected cells growing under hypoxic conditions and suggests potential new strategies for targeted treatment of KSHV-associated cancer.

EBV epitranscriptome reprogramming by METTL14 is critical for viral-associated tumorigenesis.

Epstein-Barr virus (EBV) is a ubiquitous oncogenic virus that induces many cancers. N6-Methyladenosine (m6A) modification regulates many cellular processes. We explored the role of m6A in EBV gene regulation and associated cancers. We have comprehensively defined m6A modification of EBV latent and lytic transcripts. Furthermore, m6A modification demonstrated a functional role in regulation of the stability of viral transcripts. The methyltransferase METTL14 was induced at the transcript and protein levels, and knock-down of METTL14 led to decreased expression of latent EBV transcripts. METTL14 was also significantly induced in EBV-positive tumors, promoted growth of EBV-transformed cells and tumors in Xenograft animal models. Mechanistically, the viral-encoded latent oncoprotein EBNA3C activated transcription of METTL14, and directly interacted with METTL14 to promote its stability. This demonstrated that EBV hijacks METTL14 to drive EBV-mediated tumorigenesis. METTL14 is now a new target for development of therapeutics for treatment of EBV-associated cancers.

Correction: Shugoshin 1 is dislocated by KSHV-encoded LANA inducing aneuploidy.

[This corrects the article DOI: 10.1371/journal.ppat.1007253.].

EBNA3C facilitates RASSF1A downregulation through ubiquitin-mediated degradation and promoter hypermethylation to drive B-cell proliferation.

EBV latent antigen 3C (EBNA3C) is essential for EBV-induced primary B-cell transformation. Infection by EBV induces hypermethylation of a number of tumor suppressor genes, which contributes to the development of human cancers. The Ras association domain family isoform 1A (RASSF1A) is a cellular tumor suppressor, which regulates a broad range of cellular functions, including apoptosis, cell-cycle arrest, mitotic arrest, and migration. However, the expression of RASSF1A is lost in many human cancers by epigenetic silencing. In the present study, we showed that EBNA3C promoted B-cell transformation by specifically suppressing the expression of RASSF1A. EBNA3C directly interacted with RASSF1A and induced RASSF1A degradation via the ubiquitin-proteasome-dependent pathway. SCFSkp2, an E3-ubiquitin ligase, was recruited by EBNA3C to enhance RASSF1A degradation. Moreover, EBNA3C decreased the transcriptional activity of RASSF1A promoter by enhancing its methylation through EBNA3C-mediated modulation of DNMTs expression. EBNA3C also inhibited RASSF1A-mediated cell apoptosis, disrupted RASSF1A-mediated microtubule and chromosomal stability, and promoted cell proliferation by upregulating Cyclin D1 and Cyclin E expression. Our data provides new details, which sheds light on additional mechanisms by which EBNA3C can induce B-cell transformation. This will also facilitate the development of novel therapeutic approaches through targeting of the RASSF1A pathway.

Molecular Biology of EBV in Relationship to HIV/AIDS-Associated Oncogenesis.

Herpesvirus-induced disease is one of the most lethal factors which leads to high mortality in HIV/AIDS patients. EBV, also known as human herpesvirus 4, can transform naive B cells into immortalized cells in vitro through the regulation of cell cycle, cell proliferation, and apoptosis. EBV infection is associated with several lymphoma and epithelial cancers in humans, which occurs at a much higher rate in immune deficient individuals than in healthy people, demonstrating that the immune system plays a vital role in inhibiting EBV activities. EBV latency infection proteins can mimic suppression cytokines or upregulate PD-1 on B cells to repress the cytotoxic T cells response. Many malignancies, including Hodgkin Lymphoma and non-Hodgkin's lymphomas occur at a much higher frequency in EBV positive individuals than in EBV negative people during the development of HIV infection. Importantly, understanding EBV pathogenesis at the molecular level will aid the development of novel therapies for EBV-induced diseases in HIV/AIDS patients.

Vaginal Lactobacilli Induce Differentiation of Monocytic Precursors Toward Langerhans-like Cells: in Vitro Evidence.

Lactobacilli have immunomodulatory mechanisms that affect the host cell immune system, leading to inhibition of HIV-1 transmission. Thus, lactobacilli as mucosal delivery vehicles for developing HIV-1 vaccines have attracted interest in recent years. Herein, we investigated the immunomodulatory effects of six strains of Lactobacillus naturally isolated from vaginal samples, including Lactobacillus crispatus (L. crispatus), L. fermentum, L. jensenii, L. gasseri, L. delbrueckii and L. johnsonii, on differentiation of monocytic precursors. L. crispatus, L. fermentum and L. delbrueckii could drive human monocytic cell line THP-1 cells to differentiate into dendritic-like cells according to the morphology. Moreover, L. crispatus increased costimulatory molecules including CD40, CD80 and CD86, and Langerhans cell specific C-type lectin receptors CD207, while L. fermentum decreased these molecules in THP-1 cells. Furthermore, L. crispatus promoted the differentiation of THP-1 cells with specific markers, phagocytic features, cytokine production ability and reduced the expression of receptors for HIV-1 entry of Langerhans cells. However, in the presence of L. fermentum, THP-1 cells did not show the above alterations. Moreover, similar effects of L. crispatus and L. fermentum were observed in CD14+ monocytes. These data suggested that L. crispatus facilitates the differentiation of monocytic precursors toward Langerhans-like cells in vitro. We further identified the cell wall components of Lactobacillus and found that peptidoglycans (PGNs), rather than bacteriocins, S-layer protein and lipoteichoic acid, were key contributors to the induction of CD207 expression. However, PGNs originating from Bacillus subtilis, E. coli JM109 and E. coli DH5α did not elevate CD207 expression, indicating that only PGN derived from Lactobacillus could enhance CD207 expression. Finally, the recognized receptors of L. crispatus (such as TLR2 and TLR6) and the upstream transcription factors (PU.1, TAL1, TIF1γ, and POLR2A) of CD207 were examined, and the expression of these molecules was enhanced in THP-1 cells following L. crispatus treatment. Thus, this study offers powerful evidence that vaginal lactobacilli modulate monocytic precursor differentiation into Langerhans-like cells probably via activating the TLR2/6-TFs-CD207 axis. These data provide clues for further investigation of the original occurrence, development and differentiation of Langerhans cells from monocytes.

Shugoshin 1 is dislocated by KSHV-encoded LANA inducing aneuploidy.

Shugoshin-1 (Sgo1) protects the integrity of the centromeres, and H2A phosphorylation is critical for this process. The mitotic checkpoint kinase Bub1, phosphorylates H2A and ensures fidelity of chromosome segregation and chromosome number. Oncogenic KSHV induces genetic alterations through chromosomal instability (CIN), and its essential antigen LANA regulates Bub1. We show that LANA inhibits Bub1 phosphorylation of H2A and Cdc20, important for chromosome segregation and mitotic signaling. Inhibition of H2A phosphorylation at residue T120 by LANA resulted in dislocation of Sgo1, and cohesin from the centromeres. Arrest of Cdc20 phosphorylation also rescued degradation of Securin and Cyclin B1 at mitotic exit, and interaction of H2A, and Cdc20 with Bub1 was inhibited by LANA. The N-terminal nuclear localization sequence domain of LANA was essential for LANA and Bub1 interaction, reversed LANA inhibited phosphorylation of H2A and Cdc20, and attenuated LANA-induced aneuploidy and cell proliferation. This molecular mechanism whereby KSHV-induced CIN, demonstrated that the NNLS of LANA is a promising target for development of anti-viral therapies targeting KSHV associated cancers.