The oscillation of mitotic kinase governs cell cycle latches in mammalian cells.

The mammalian cell cycle alternates between two phases - S-G2-M with high levels of A- and B-type cyclins (CycA and CycB, respectively) bound to cyclin-dependent kinases (CDKs), and G1 with persistent degradation of CycA and CycB by an activated anaphase promoting complex/cyclosome (APC/C) bound to Cdh1 (also known as FZR1 in mammals; denoted APC/C:Cdh1). Because CDKs phosphorylate and inactivate Cdh1, these two phases are mutually exclusive. This 'toggle switch' is flipped from G1 to S by cyclin-E bound to a CDK (CycE:CDK), which is not degraded by APC/C:Cdh1, and from M to G1 by Cdc20-bound APC/C (APC/C:Cdc20), which is not inactivated by CycA:CDK or CycB:CDK. After flipping the switch, cyclin E is degraded and APC/C:Cdc20 is inactivated. Combining mathematical modelling with single-cell timelapse imaging, we show that dysregulation of CycB:CDK disrupts strict alternation of the G1-S and M-G1 switches. Inhibition of CycB:CDK results in Cdc20-independent Cdh1 'endocycles', and sustained activity of CycB:CDK drives Cdh1-independent Cdc20 endocycles. Our model provides a mechanistic explanation for how whole-genome doubling can arise, a common event in tumorigenesis that can drive tumour evolution.

MITOL-dependent ubiquitylation negatively regulates the entry of PolγA into mitochondria.

Mutations in mitochondrial replicative polymerase PolγA lead to progressive external ophthalmoplegia (PEO). While PolγA is the known central player in mitochondrial DNA (mtDNA) replication, it is unknown whether a regulatory process exists on the mitochondrial outer membrane which controlled its entry into the mitochondria. We now demonstrate that PolγA is ubiquitylated by mitochondrial E3 ligase, MITOL (or MARCH5, RNF153). Ubiquitylation in wild-type (WT) PolγA occurs at Lysine 1060 residue via K6 linkage. Ubiquitylation of PolγA negatively regulates its binding to Tom20 and thereby its mitochondrial entry. While screening different PEO patients for mitochondrial entry, we found that a subset of the PolγA mutants is hyperubiquitylated by MITOL and interact less with Tom20. These PolγA variants cannot enter into mitochondria, instead becomes enriched in the insoluble fraction and undergo enhanced degradation. Hence, mtDNA replication, as observed via BrdU incorporation into the mtDNA, was compromised in these PEO mutants. However, by manipulating their ubiquitylation status by 2 independent techniques, these PEO mutants were reactivated, which allowed the incorporation of BrdU into mtDNA. Thus, regulated entry of non-ubiquitylated PolγA may have beneficial consequences for certain PEO patients.

Nuclear localization of p65 reverses therapy-induced senescence.

Senescence is the arrest of cell proliferation and is a tumor suppressor phenomenon. In a previous study, we have shown that therapy-induced senescence of glioblastoma multiforme (GBM) cells can prevent relapse of GBM tumors. Here, we demonstrate that ciprofloxacin-induced senescence in glioma-derived cell lines and primary glioma cultures is defined by SA-ß-gal positivity, a senescence-associated secretory phenotype (SASP), a giant cell (GC) phenotype, increased levels of reactive oxygen species (ROS), γ-H2AX and a senescence-associated gene expression signature, and has three stages of senescence -initiation, pseudo-senescence and permanent senescence. Ciprofloxacin withdrawal during initiation and pseudo-senescence reinitiated proliferation in vitro and tumor formation in vivo Importantly, prolonged treatment with ciprofloxacin induced permanent senescence that failed to reverse following ciprofloxacin withdrawal. RNA-seq revealed downregulation of the p65 (RELA) transcription network, as well as incremental expression of SMAD pathway genes from initiation to permanent senescence. Ciprofloxacin withdrawal during initiation and pseudo-senescence, but not permanent senescence, increased the nuclear localization of p65 and escape from ciprofloxacin-induced senescence. By contrast, permanently senescent cells showed loss of nuclear p65 and increased apoptosis. Pharmacological inhibition or genetic knockdown of p65 upheld senescence in vitro and inhibited tumor formation in vivo Our study demonstrates that levels of nuclear p65 define the window of reversibility of therapy-induced senescence and that permanent senescence can be induced in GBM cells when the use of senotherapeutics is coupled with p65 inhibitors.

CDX2 inducible microRNAs sustain colon cancer by targeting multiple DNA damage response pathway factors.

Meta-analysis of transcripts in colon adenocarcinoma patient tissues led to the identification of a DNA damage responsive miR signature called DNA damage sensitive miRs (DDSMs). DDSMs were experimentally validated in the cancerous colon tissues obtained from an independent cohort of colon cancer patients and in multiple cellular systems with high levels of endogenous DNA damage. All the tested DDSMs were transcriptionally upregulated by a common intestine-specific transcription factor, CDX2. Reciprocally, DDSMs were repressed via the recruitment of HDAC1/2-containing complexes onto the CDX2 promoter. These miRs downregulated multiple key targets in the DNA damage response (DDR) pathway, namely BRCA1, ATM, Chk1 (also known as CHEK1) and RNF8. CDX2 directly regulated the DDSMs, which led to increased tumor volume and metastasis in multiple preclinical models. In colon cancer patient tissues, the DDSMs negatively correlated with BRCA1 levels, were associated with decreased probability of survival and thereby could be used as a prognostic biomarker. This article has an associated First Person interview with the first author of the paper.

De novo transcriptome analysis provides insights into formation of in vitro adventitious root from leaf explants of Arnebia euchroma.

BACKGROUND: Adventitious root formation is considered a major developmental step during the propagation of difficult to root plants, especially in horticultural crops. Recently, adventitious roots induced through plant tissue culture methods have also been used for production of phytochemicals such as flavonoids, anthocyanins and anthraquinones. It is rather well understood which horticultural species will easily form adventitious roots, but the factors affecting this process at molecular level or regulating the induction process in in vitro conditions are far less known. The present study was conducted to identify transcripts involved in in vitro induction and formation of adventitious roots using Arnebia euchroma leaves at different time points (intact leaf (control), 3 h, 12 h, 24 h, 3 d, 7 d, 10 d and 15 d). A. euchroma is an endangered medicinal Himalayan herb whose root contains red naphthoquinone pigments. These phytoconstituents are widely used as an herbal ingredient in Asian traditional medicine as well as natural colouring agent in food and cosmetics. RESULTS: A total of 137.93 to 293.76 million raw reads were generated and assembled to 54,587 transcripts with average length of 1512.27 bps and N50 of 2193 bps, respectively. In addition, 50,107 differentially expressed genes were identified and found to be involved in plant hormone signal transduction, cell wall modification and wound induced mitogen activated protein kinase signalling. The data exhibited dominance of auxin responsive (AUXIN RESPONSE FACTOR8, IAA13, GRETCHEN HAGEN3.1) and sucrose translocation (BETA-31 FRUCTOFURANOSIDASE and MONOSACCHARIDE-SENSING protein1) genes during induction phase. In the initiation phase, the expression of LATERAL ORGAN BOUNDARIES DOMAIN16, EXPANSIN-B15, ENDOGLUCANASE25 and LEUCINE-rich repeat EXTENSION-like proteins was increased. During the expression phase, the same transcripts, with exception of LATERAL ORGAN BOUNDARIES DOMAIN16 were identified. Overall, the transcriptomic analysis revealed a similar patterns of genes, however, their expression level varied in subsequent phases of in vitro adventitious root formation in A. euchroma. CONCLUSION: The results presented here will be helpful in understanding key regulators of in vitro adventitious root development in Arnebia species, which may be deployed in the future for phytochemical production at a commercial scale.

Abrogation of FBW7α-dependent p53 degradation enhances p53's function as a tumor suppressor.

The gene encoding the tumor suppressor p53 is mutated in most cancers. p53 expression is known to be tightly controlled by several E3 ligases. Here, we show that F-box and WD repeat domain-containing 7α (FBW7α), the substrate-recognition component of the SCFFBW7 multiprotein E3 ligase complex, targets both WT and tumor-derived mutants of p53 for proteasomal degradation in multiple human cancer cell lines (HCT116 and U2OS). We found that lack of FBW7α stabilizes p53 levels, thereby increasing its half-life. p53 ubiquitylation and subsequent degradation require the F-box and the C-terminal WD40 repeats in FBW7α. The polyubiquitylation of p53 occurred via Lys-48 linkage and involved phosphorylation on p53 at Ser-33 and Ser-37 by glycogen synthase kinase 3ß (GSK3ß) and DNA-dependent protein kinase (DNA-PK), respectively. These phosphorylation events created a phosphodegron that enhanced p53 binding to FBW7α, allowing for the attachment of polyubiquitin moieties at Lys-132 in p53. FBW7α-dependent p53 polyubiquitylation apparently occurred during and immediately after DNA double-strand breaks induced by either doxorubicin or ionizing radiation. Accordingly, in cells lacking FBW7α, p53 induction was enhanced after DNA damage. Phosphodegron-mediated polyubiquitylation of p53 on Lys-132 had functional consequences, with cells in which FBW7α-mediated p53 degradation was abrogated exhibiting enhancement of their tumorigenic potential. We conclude that p53, which previously has been reported to transactivate FBW7, is also targeted by the same E3 ligase for degradation, suggesting the presence of a regulatory feedback loop that controls p53 levels and functions during DNA damage.

Novel analogs of sulfasalazine as system xc- antiporter inhibitors: Insights from the molecular modeling studies.

System xc- (Sxc- ), a cystine-glutamate antiporter, is established as an interesting target for the treatment of several pathologies including epileptic seizures, glioma, neurodegenerative diseases, and multiple sclerosis. Erastin, sorafenib, and sulfasalazine (SSZ) are a few of the established inhibitors of Sxc- . However, its pharmacological inhibition with novel and potent agents is still very much required due to potential issues, for example, potency, bioavailability, and blood-brain barrier (BBB) permeability, with the current lead molecules such as SSZ. Therefore, in this study, we report the synthesis and structure-activity relationships (SAR) of SSZ derivatives along with molecular docking and dynamics simulations using the developed homology model of xCT chain of Sxc- antiporter. The generated homology model attempted to address the limitations of previously reported comparative protein models, thereby increasing the confidence in the computational modeling studies. The main objective of the present study was to derive a suitable lead structure from SSZ eliminating its potential issues for the treatment of glioblastoma multiforme (GBM), a deadly and malignant grade IV astrocytoma. The designed compounds with favorable Sxc- inhibitory activity following in vitro Sxc- inhibition studies, showed moderately potent cytotoxicity in patient-derived human glioblastoma cells, thereby generating potential interest in these compounds. The xCT-ligand model can be further optimized in search of potent lead molecules for novel drug discovery and development studies.

Inhibition of novel GCN5-ATM axis restricts the onset of acquired drug resistance in leukemia.

Leukemia is majorly treated by topoisomerase inhibitors that induce DNA double strand breaks (DSB) resulting in cell death. Consequently, modulation of DSB repair pathway renders leukemic cells resistant to therapy. As we do not fully understand the regulation of DSB repair acquired by resistant cells, targeting these cells has been a challenge. Here we investigated the regulation of DSB repair pathway in early drug resistant population (EDRP) and late drug resistant population (LDRP). We found that doxorubicin induced equal DSBs in parent and EDRP cells; however, cell death is induced only in the parent cells. Further analysis revealed that EDRP cells acquire relaxed chromatin via upregulation of lysine acetyl transferase KAT2A (GCN5). Drug treatment induces GCN5 interaction with ATM facilitating its recruitment to DSB sites. Hyperactivated ATM maximize H2AX, NBS1, BRCA1, Chk2, and Mcl-1 activation, accelerating DNA repair and survival of EDRP cells. Consequently, inhibition of GCN5 significantly reduces ATM activation and survival of EDRP cells. Contrary to EDRP, doxorubicin failed to induce DSBs in LDRP because of reduced drug uptake and downregulation of TOP2ß. Accordingly, ATM inhibition prior to doxorubicin treatment completely eliminated EDRP but not LDRP. Furthermore, baseline AML samples (n = 44) showed significantly higher GCN5 at mRNA and protein levels in MRD positive compared to MRD negative samples. Additionally, meta-analysis (n = 221) showed high GCN5 expression correlates with poor overall survival. Together, these results provide important insights into the molecular mechanism specific to EDRP and will have implications for the development of novel therapeutics for AML.

Radiation-induced homotypic cell fusions of innately resistant glioblastoma cells mediate their sustained survival and recurrence.

Understanding of molecular events underlying resistance and relapse in glioblastoma (GBM) is hampered due to lack of accessibility to resistant cells from patients undergone therapy. Therefore, we mimicked clinical scenario in an in vitro cellular model developed from five GBM grade IV primary patient samples and two cell lines. We show that upon exposure to lethal dose of radiation, a subpopulation of GBM cells, innately resistant to radiation, survive and transiently arrest in G2/M phase via inhibitory pCdk1(Y15). Although arrested, these cells show multinucleated and giant cell phenotype (MNGC). Significantly, we demonstrate that these MNGCs are not pre-existing giant cells from parent population but formed via radiation-induced homotypic cell fusions among resistant cells. Furthermore, cell fusions induce senescence, high expression of senescence-associated secretory proteins (SASPs) and activation of pro-survival signals (pAKT, BIRC3 and Bcl-xL) in MNGCs. Importantly, following transient non-proliferation, MNGCs escape senescence and despite having multiple spindle poles during mitosis, they overcome mitotic catastrophe to undergo normal cytokinesis forming mononucleated relapse population. This is the first report showing radiation-induced homotypic cell fusions as novel non-genetic mechanism in radiation-resistant cells to sustain survival. These data also underscore the importance of non-proliferative phase in resistant glioma cells. Accordingly, we show that pushing resistant cells into premature mitosis by Wee1 kinase inhibitor prevents pCdk1(Y15)-mediated cell cycle arrest and relapse. Taken together, our data provide novel molecular insights into a multistep process of radiation survival and relapse in GBM that can be exploited for therapeutic interventions.

Clinical implications of MTA proteins in human cancer.

Metastasis-associated gene or metastasis tumor antigen 1 (MTA1) is a new member of cancer progression-related gene family. It was first identified in rat mammary adenocarcinoma and later recognized as an important constituent of nucleosomal remodeling complex (NuRD), displaying dual regulatory functions as a co-repressor and co-activator for a large number of genes. Chromatin remodelers are ATP-dependent multi-protein chromatin modifying machines. These complexes alter the nucleosome positioning regulating the accessibility of genomic DNA to various transcription factors and thus modulate eukaryotic gene transcription. Since its identification two decades ago, MTA1 has been reported to be overexpressed in many cancers. Moreover, its overexpression has also been correlated with transformation and tumor progression. Furthermore, MTA1 has been shown to modulate the response of several tumor suppressor genes like p53 and oncogenes like c-myc. Taken together, current literature suggests that MTA proteins, especially MTA1, act as a master co-regulatory molecule involved in the carcinogenesis and progression of various malignant tumors. The primary focus of this review is to provide an overview of the MTA proteins with special emphasis on its role in cancer and use as a marker for cancer progression and potential target for therapy.

Small molecules that disrupt RAD54-BLM interaction hamper tumor proliferation in colon cancer chemoresistance models.

RAD54 and BLM helicase play pivotal roles during homologous recombination repair (HRR) to ensure genome maintenance. BLM amino acids (aa 181-212) interact with RAD54 and enhance its chromatin remodeling activity. Functionally, this interaction heightens HRR, leading to a decrease in residual DNA damage in colon cancer cells. This contributes to chemoresistance in colon cancer cells against cisplatin, camptothecin, and oxaliplatin, eventually promoting tumorigenesis in preclinical colon cancer mouse models. ChIP-Seq analysis and validation revealed increased BLM and RAD54 corecruitment on the MRP2 promoter in camptothecin-resistant colon cancer cells, leading to BLM-dependent enhancement of RAD54-mediated chromatin remodeling. We screened the Prestwick small-molecule library, with the intent to revert camptothecin- and oxaliplatin-induced chemoresistance by disrupting the RAD54-BLM interaction. Three FDA/European Medicines Agency-approved candidates were identified that could disrupt this interaction. These drugs bound to RAD54, altered its conformation, and abrogated RAD54-BLM-dependent chromatin remodeling on G5E4 and MRP2 arrays. Notably, the small molecules also reduced HRR efficiency in resistant lines, diminished anchorage-independent growth, and hampered the proliferation of tumors generated using camptothecin- and oxaliplatin-resistant colon cancer cells in both xenograft and syngeneic mouse models in BLM-dependent manner. Therefore, the 3 identified small molecules can serve as possible viable candidates for adjunct therapy in colon cancer treatment.

Systematic analyses with genomic and metabolomic insights reveal a new species, Ophiocordyceps indica sp. nov. from treeline area of Indian Western Himalayan region.

Ophiocordyceps is a species-rich genus in the order Hypocreales (Sordariomycetes, Ascomycota) depicting a fascinating relationship between microbes and insects. In the present study, a new species, Ophiocordyceps indica sp. nov., is discovered infecting lepidopteran larvae from tree line locations (2,202-2,653 m AMSL) of the Kullu District, Himachal Pradesh, Indian Western Himalayan region, using combinations of morphological and molecular phylogenetic analyses. A phylogeny for Ophiocordyceps based on a combined multigene (nrSSU, nrLSU, tef-1α, and RPB1) dataset is provided, and its taxonomic status within Ophiocordycipitaceae is briefly discussed. Its genome size (~59 Mb) revealed 94% genetic similarity with O. sinensis; however, it differs from other extant Ophiocordyceps species based on morphological characteristics, molecular phylogenetic relationships, and genetic distance. O. indica is identified as the second homothallic species in the family Ophiocordycipitaceae, after O. sinensis. The presence of targeted marker components, viz. nucleosides (2,303.25 µg/g), amino acids (6.15%), mannitol (10.13%), and biological activity data, suggests it to be a new potential source of nutraceutical importance. Data generated around this economically important species will expand our understanding regarding the diversity of Ophiocordyceps-like taxa from new locations, thus providing new research avenues.

Glioblastoma recurrent cells switch between ATM and ATR pathway as an alternative strategy to survive radiation stress.

Primary treatment modality for glioblastoma (GBM) post-surgery is radiation therapy. Due to increased DNA damage repair capacity of resistant residual GBM cells, recurrence is inevitable in glioblastoma and unfortunately the recurrent tumours are resistant to the conventional therapy. Here we used our previously described in vitro radiation survival model generated from primary GBM patient samples and cell lines, which recapitulates the clinical scenario of therapy resistance and relapse. Using the parent and recurrent GBM cells from these models, we show that similar to parent GBM, the recurrent GBM cells also elicit a competent DNA damage response (DDR) post irradiation. However, the use of apical DNA damage repair sensory kinase (ATM and/or ATR) is different in the recurrent cells compared to parent cells. Consistently, we demonstrate that there is a differential clonogenic response of parent and recurrent GBM cells to the ATM and ATR kinase inhibitors with recurrent samples switching between these sensory kinases for survival emphasizing on the underlying heterogeneity within and across GBM samples. Taken together, here we report that recurrent tumours utilize an alternate DDR kinase to overcome radiation induced DNA damage. Since there is no effective treatment specifically for recurred GBM patients, these findings provide a rationale for developing newer treatment option to sensitize recurrent GBM samples by detecting in clinics the ability of cells to activate a DNA damage repair kinase different from their parent counterparts.

Comparative transcriptome and tissue-specific expression analysis of genes reveal tissue-cultured plants as an alternative source for phenylethanoids and phenylpropanoids in Rhodiola imbricata (Edgew.).

Rhodiola imbricata (Crassulaceae) is a traditional trans-Himalayan endangered medicinal herb with immense therapeutic applications. Over the years, over-exploitation, un-managed harvesting, and lack of captive cultivation procedures persuaded threat to its wild habitat. Plant tissue culture and RNA-Seq-based molecular bioprospection of key regulatory genes aid the understanding of molecular dynamics involved in specialized metabolites (phenylethanoids and phenylpropanoids) biosynthesis and its sustainable production. Hence, comparative transcriptomic analysis was performed using leaf and root tissues from the wild and tissue-cultured plants, revealing tissue-specific production of salidroside and rosavin. The transcriptome profiling resulted in 345 million high-quality reads yielding 92,380 unique transcripts with an N50 of 1260 bp. Tissue-specific gene expression analysis revealed that both phenylethanoids and phenylpropanoids biosynthesis are predominantly associated with the shikimate pathway. In addition to RNA-Seq data, the downstream biosynthesis pathways genes viz., phospho-2-dehydro-3-deoxyheptonate aldolase (DAHPS), 3-dehydroquinate synthase (DHQS), shikimate kinase (SK), chorismate mutase (CM), arogenate dehydrogenase (TYRAAT), aromatic-L-amino-acid decarboxylase (TDC), phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4-CL), cinnamoyl-CoA reductase (CCR), and cinnamyl alcohol dehydrogenase (CAD) showed higher expression pattern in wild plant tissues compared to tissue-cultured plants. The transcript fold expression determined by RT-qPCR results followed similar patterns as those observed in RNA-seq and targeted metabolite profiling data. Salidroside and rosavin content in wild plants exhibited 2.40 fold and 1.77 fold increase accumulation compared to the tissue-cultured plant. The present investigation explained the tissue and condition-specific significant differences between the expression of proposed biosynthetic pathway genes and salidroside and rosavin content. Additionally, NAC, bHLH, and ARF were the most abundant transcription factor families found in the transcriptomic analysis of R. imbricata. The generated transcriptome dataset provides a valuable gene(s)/transcription factors hub that can be used for the sustainable production of salidroside and rosavin in R. imbricata under tissue culture conditions.

Functions of BLM Helicase in Cells: Is It Acting Like a Double-Edged Sword?

DNA damage repair response is an important biological process involved in maintaining the fidelity of the genome in eukaryotes and prokaryotes. Several proteins that play a key role in this process have been identified. Alterations in these key proteins have been linked to different diseases including cancer. BLM is a 3'-5' ATP-dependent RecQ DNA helicase that is one of the most essential genome stabilizers involved in the regulation of DNA replication, recombination, and both homologous and non-homologous pathways of double-strand break repair. BLM structure and functions are known to be conserved across many species like yeast, Drosophila, mouse, and human. Genetic mutations in the BLM gene cause a rare, autosomal recessive disorder, Bloom syndrome (BS). BS is a monogenic disease characterized by genomic instability, premature aging, predisposition to cancer, immunodeficiency, and pulmonary diseases. Hence, these characteristics point toward BLM being a tumor suppressor. However, in addition to mutations, BLM gene undergoes various types of alterations including increase in the copy number, transcript, and protein levels in multiple types of cancers. These results, along with the fact that the lack of wild-type BLM in these cancers has been associated with increased sensitivity to chemotherapeutic drugs, indicate that BLM also has a pro-oncogenic function. While a plethora of studies have reported the effect of BLM gene mutations in various model organisms, there is a dearth in the studies undertaken to investigate the effect of its oncogenic alterations. We propose to rationalize and integrate the dual functions of BLM both as a tumor suppressor and maybe as a proto-oncogene, and enlist the plausible mechanisms of its deregulation in cancers.

Inhibition of SETMAR-H3K36me2-NHEJ repair axis in residual disease cells prevents glioblastoma recurrence.

BACKGROUND: Residual disease of glioblastoma (GBM) causes recurrence. However, targeting residual cells has failed, due to their inaccessibility and our lack of understanding of their survival mechanisms to radiation therapy. Here we deciphered a residual cell-specific survival mechanism essential for GBM relapse. METHODS: Therapy resistant residual (RR) cells were captured from primary patient samples and cell line models mimicking clinical scenario of radiation resistance. Molecular signaling of resistance in RR cells was identified using RNA sequencing, genetic and pharmacological perturbations, overexpression systems, and molecular and biochemical assays. Findings were validated in patient samples and an orthotopic mouse model. RESULTS: RR cells form more aggressive tumors than the parental cells in an orthotopic mouse model. Upon radiation-induced damage, RR cells preferentially activated a nonhomologous end joining (NHEJ) repair pathway, upregulating Ku80 and Artemis while downregulating meiotic recombination 11 (Mre11) at protein but not RNA levels. Mechanistically, RR cells upregulate the Su(var)3-9/enhancer-of-zeste/trithorax (SET) domain and mariner transposase fusion gene (SETMAR), mediating high levels of H3K36me2 and global euchromatization. High H3K36me2 leads to efficiently recruiting NHEJ proteins. Conditional knockdown of SETMAR in RR cells induced irreversible senescence partly mediated by reduced H3K36me2. RR cells expressing mutant H3K36A could not retain Ku80 at double-strand breaks, thus compromising NHEJ repair, leading to apoptosis and abrogation of tumorigenicity in vitro and in vivo. Pharmacological inhibition of the NHEJ pathway phenocopied H3K36 mutation effect, confirming dependency of RR cells on the NHEJ pathway for their survival. CONCLUSIONS: We demonstrate that the SETMAR-NHEJ regulatory axis is essential for the survival of clinically relevant radiation RR cells, abrogation of which prevents recurrence in GBM.

Molecular features unique to glioblastoma radiation resistant residual cells may affect patient outcome - a short report.

PURPOSE: Previously we have shown, using a primary glioblastoma (GBM) cell model, that a subpopulation of innately radiation resistant (RR) GBM cells survive radiotherapy and form multinucleated and giant cells (MNGCs) by homotypic fusions. We also showed that MNGCs may cause relapse. Here, we set out to explore whether molecular characteristics of RR cells captured from patient-derived primary GBM cultures bear clinical relevance. METHODS: Primary cultures were derived from 19 naive GBM tumor samples. RR cells generated from these cultures were characterized using various cell biological assays. We also collected clinicopathological data of the 19 patients and assessed associations with RR variables using Spearman's correlation test and with patient survival using Kaplan-Meier analysis. Significance was determined using a log-rank test. RESULTS: We found that SF2 (surviving fraction 2) values (p = 0.029), days of RR cell formation (p = 0.019) and percentage of giant cells (p = 0.034) in the RR population independently correlated with a poor patient survival. We also found that low ATM (Ataxia-telangiectasia mutated) expression levels in RR cells showed a significant (p = 0.002) negative correlation with SF2 values. A low ATM expression level in RR cells along with a high tumor volume was also found to negatively correlate with patient survival (p = 0.011). Finally, we found that the ATM expression levels in RR cells independently correlated with a poor patient survival (p = 0.014). CONCLUSIONS: Our data indicate that molecular features of innately radiation resistant GBM cells independently correlate with clinical outcome. Our study also highlights the relevance of using patient-derived primary GBM cultures for the characterization of RR cells that are otherwise inaccessible for analysis.

BLM Potentiates c-Jun Degradation and Alters Its Function as an Oncogenic Transcription Factor.

Mutations in BLM helicase predispose Bloom syndrome (BS) patients to a wide spectrum of cancers. We demonstrate that MIB1-ubiquitylated BLM in G1 phase functions as an adaptor protein by enhancing the binding of transcription factor c-Jun and its E3 ligase, Fbw7α. BLM enhances the K48/K63-linked ubiquitylation on c-Jun, thereby enhancing the rate of its subsequent degradation. Functionally defective Fbw7α mutants prevalent in multiple human cancers are reactivated by BLM. However, BS patient-derived BLM mutants cannot potentiate Fbw7α-dependent c-Jun degradation. The decrease in the levels of c-Jun in cells expressing BLM prevents effective c-Jun binding to 2,584 gene promoters. This causes decreases in the transcript and protein levels of c-Jun targets in BLM-expressing cells, resulting in attenuated c-Jun-dependent effects during neoplastic transformation. Thus, BLM carries out its function as a tumor suppressor by enhancing c-Jun turnover and thereby preventing its activity as a proto-oncogene.

Enhanced proteasomal activity is essential for long term survival and recurrence of innately radiation resistant residual glioblastoma cells.

Therapy resistance and recurrence in Glioblastoma is due to the presence of residual radiation resistant cells. However, because of their inaccessibility from patient biopsies, the molecular mechanisms driving their survival remain unexplored. Residual Radiation Resistant (RR) and Relapse (R) cells were captured using cellular radiation resistant model generated from patient derived primary cultures and cell lines. iTRAQ based quantitative proteomics was performed to identify pathways unique to RR cells followed by in vitro and in vivo experiments showing their role in radio-resistance. 2720 proteins were identified across Parent (P), RR and R population with 824 and 874 differential proteins in RR and R cells. Unsupervised clustering showed proteasome pathway as the most significantly deregulated pathway in RR cells. Concordantly, the RR cells displayed enhanced expression and activity of proteasome subunits, which triggered NFkB signalling. Pharmacological inhibition of proteasome activity led to impeded NFkB transcriptional activity, radio-sensitization of RR cells in vitro, and significantly reduced capacity to form orthotopic tumours in vivo. We demonstrate that combination of proteasome inhibitor with radio-therapy abolish the inaccessible residual resistant cells thereby preventing GBM recurrence. Furthermore, we identified first proteomic signature of RR cells that can be exploited for GBM therapeutics.

Unique spectral markers discern recurrent Glioblastoma cells from heterogeneous parent population.

An inability to discern resistant cells from bulk tumour cell population contributes to poor prognosis in Glioblastoma. Here, we compared parent and recurrent cells generated from patient derived primary cultures and cell lines to identify their unique molecular hallmarks. Although morphologically similar, parent and recurrent cells from different samples showed variable biological properties like proliferation and radiation resistance. However, total RNA-sequencing revealed transcriptional landscape unique to parent and recurrent populations. These data suggest that global molecular differences but not individual biological phenotype could differentiate parent and recurrent cells. We demonstrate that Raman Spectroscopy a label-free, non-invasive technique, yields global information about biochemical milieu of recurrent and parent cells thus, classifying them into distinct clusters based on Principal-Component-Analysis and Principal-Component-Linear-Discriminant-Analysis. Additionally, higher lipid related spectral peaks were observed in recurrent population. Importantly, Raman spectroscopic analysis could further classify an independent set of naïve primary glioblastoma tumour tissues into non-responder and responder groups. Interestingly, spectral features from the non-responder patient samples show a considerable overlap with the in-vitro generated recurrent cells suggesting their similar biological behaviour. This feasibility study necessitates analysis of a larger cohort of naïve primary glioblastoma samples to fully envisage clinical utility of Raman spectroscopy in predicting therapeutic response.