Inhibition of PERK mediated UPR acts as a switch for reversal of residual senescence and as senolytic therapy in glioblastoma.

BACKGROUND: Glioblastoma due to recurrence is clinically challenging with 10-15months overall survival. Previously we showed therapy induced senescence (TIS) in glioblastoma reverses causing recurrence. Here, we aim to delineate TIS reversal mechanism for potential therapeutic intervention to prevent GBM recurrence. METHODS: Residual senescent (RS) and End of Residual Senescence (ERS) cells were captured from GBM patient-derived primary-cultures and cell lines mimicking clinical scenario. RNA-sequencing, transcript/protein validations, knock-down/inhibitor studies, ChIP RT-PCR, biochemical assays and IHCs were performed for mechanistics of TIS reversal. In vivo validations were conducted in GBM orthotopic mouse model. RESULTS: Transcriptome analysis showed co-expression of ER stress-UPR and senescence associated secretory phenotype (SASP) with TIS induction and reversal. Robust SASP production and secretion by RS cells could induce senescence, ROS, DNA damage and ER stress in paracrine fashion independent of radiation. Neutralization of most significantly enriched cytokine from RS-secretome IL1ß, suppressed SASP and delayed senescence reversal. Mechanistically, with SASP and massive protein accumulation in Endoplasmic reticulum, RS cells displayed stressed ER morphology, upregulated ER stress markers and PERK pathway activation via peIF2α-ATF4-CHOP which was spontaneously resolved in ERS. ChIP RT-PCR showed CHOP occupancy at CXCL8/IL8, CDKN1A/p21 and BCL2L1/BCLXL aiding survival. PERK knockdown/inhibition with GSK2606414 in combination with radiation led to sustained ER stress and senescence without SASP. PERKi in RS functioned as senolytic via apoptosis and prevented recurrence in vitro and in vivo ameliorating overall survival. CONCLUSION: We demonstrate that PERK mediated UPR regulates senescence reversal and its inhibition can be exploited as potential seno-therapeutic option in glioblastoma.

DNA-PKcs-mediated transcriptional regulation of TOP2B drives chemoresistance in acute myeloid leukemia.

Anthracyclines, topoisomerase II enzyme poisons that cause DNA damage, are the mainstay of acute myeloid leukemia (AML) treatment. However, acquired resistance to anthracyclines leads to relapse, which currently lacks effective treatment and is the cause of poor survival in individuals with AML. Therefore, the identification of the mechanisms underlying anthracycline resistance remains an unmet clinical need. Here, using patient-derived primary cultures and clinically relevant cellular models that recapitulate acquired anthracycline resistance in AML, we have found that GCN5 (also known as KAT2A) mediates transcriptional upregulation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in AML relapse, independently of the DNA-damage response. We demonstrate that anthracyclines fail to induce DNA damage in resistant cells, owing to the loss of expression of their target enzyme, TOP2B; this was caused by DNA-PKcs directly binding to its promoter upstream region as a transcriptional repressor. Importantly, DNA-PKcs kinase activity inhibition re-sensitized AML relapse primary cultures and cells resistant to mitoxantrone, and abrogated their tumorigenic potential in a xenograft mouse model. Taken together, our findings identify a GCN5-DNA-PKcs-TOP2B transcriptional regulatory axis as the mechanism underlying anthracycline resistance, and demonstrate the therapeutic potential of DNA-PKcs inhibition to re-sensitize resistant AML relapse cells to anthracycline.

Substrate stiffness regulates the recurrent glioblastoma cell morphology and aggressiveness.

Recurrent glioblastoma is highly aggressive with currently no specific treatment regime. Therefore, to identify novel therapeutic targets for recurrent GBM, we used a cellular model developed in our lab from commercially available cell line U87MG and patient-derived cultures that allows the comparison between radiation naïve (Parent) and recurrent GBM cells generated after parent cells are exposed to lethal dose of radiation. Total RNA-seq of parent and recurrent population revealed significant upregulation of cell-ECM interactions pathway in the recurrent population. These results led us to hypothesize that the physical microenvironment contributes to the aggressiveness of recurrent GBM. To verify this, we cultured parent and recurrent GBM cells on collagen-coated polyacrylamide gels mimicking the stiffness of normal brain (Young's modulus E = 0.5kPa) or tumorigenic brain (E = 10kPa) and tissue culture plastic dishes (E ∼ 1 GPa). We found that compared to parent cells, recurrent cells showed higher proliferation, invasion, migration, and resistance to EGFR inhibitor. Using orthotopic GBM mouse model and resection model, we demonstrate that recurrent cells cultured on 0.5kPa had higher in vivo tumorigenicity and recurrent disease progression than parent cells, whereas these differences were insignificant when parent and recurrent cells were cultured on plastic substrates. Furthermore, recurrent cells on 0.5kPa showed high expression of ECM proteins like Collagen, MMP2 and MMP9. These proteins were also significantly upregulated in recurrent patient biopsies. Additionally, the brain of mice injected with recurrent cells grown on 0.5kPa showed higher Young's moduli suggesting the ability of these cells to make the surrounding ECM stiffer. Total RNA-seq of parent and recurrent cells grown on plastic and 0.5kpa identified PLEKHA7 significantly upregulated specifically in recurrent cells grown on 0.5 kPa substrate. PLEKHA7 was also found to be high in recurrent GBM patient biopsies. Accordingly, PLEKHA7 knockdown reduced invasion and survival of recurrent GBM cells. Together, these data provide an in vitro model system that captures the observed in vivo and clinical behavior of recurrent GBM by mimicking mechanical microenvironment and identifies PLEKHA7 as a novel potential target for recurrent GBM.

Targeted nano-delivery of chemotherapy via intranasal route suppresses in vivo glioblastoma growth and prolongs survival in the intracranial mouse model.

Nanotechnology-based drug delivery platforms have shown great potential in overcoming the limitations of conventional therapy for glioblastoma (GBM). However, permeation across the blood-brain barrier (BBB), physiological complexity of the brain, and glioma targeting strategies cannot entirely meet the challenging requirements of distinctive therapeutic delivery stages. The objective of this research is to fabricate lipid nanoparticles (LNPs) for the co-delivery of paclitaxel (PTX) and miltefosine (HePc) a proapoptotic agent decorated with transferrin (Tf-PTX-LNPs) and investigate its anti-glioma activity both in vitro and in vivo orthotopic NOD/SCID GBM mouse model. The present study demonstrates the anti-glioma effect of the dual drug combination of PTX and proapoptotic HePc lipid-based transferrin receptor (TfR) targeted alternative delivery (direct nose to brain transportation) of the nanoparticulate system (Tf-PTX-LNPs, 364 ± 5 nm, -43 ± 9 mV) to overcome the O6-methylguanine-DNA methyltransferase induce drug-resistant for improving the effectiveness of GBM therapy. The resulting nasally targeted LNPs present good biocompatibility, stability, high BBB transcytosis through selective TfR-mediated uptake by tumor cells, and effective tumor penetration in the brain of GBM induced mice. We observed markedly enhanced anti-proliferative efficacy of the targeted LNPs in U87MG cells compared to free drug. Nasal targeted LNPs had shown significantly improved brain concentration (Cmax fivefold and AUC0-24 4.9 fold) with early tmax (0.5 h) than the free drug. In vivo intracranial GBM-bearing targeted LNPs treated mice exhibited significantly prolonged survival with improved anti-tumor efficacy accompanied by reduced toxicity compared to systemic Taxol® and nasal free drug. These findings indicate that the nasal delivery of targeted synergistic nanocarrier holds great promise as a non-invasive adjuvant chemotherapy therapy of GBM.

Multifunctional nanocarriers for delivering siRNA and miRNA in glioblastoma therapy: advances in nanobiotechnology-based cancer therapy.

Glioblastoma multiforme (GBM) is one of the most lethal cancer due to poor diagnosis and rapid resistance developed towards the drug. Genes associated to cancer-related overexpression of proteins, enzymes, and receptors can be suppressed using an RNA silencing technique. This assists in obtaining tumour targetability, resulting in less harm caused to the surrounding healthy cells. RNA interference (RNAi) has scientific basis for providing potential therapeutic applications in improving GBM treatment. However, the therapeutic application of RNAi is challenging due to its poor permeability across blood-brain barrier (BBB). Nanobiotechnology has evolved the use of nanocarriers such as liposomes, polymeric nanoparticles, gold nanoparticles, dendrimers, quantum dots and other nanostructures in encasing the RNAi entities like siRNA and miRNA. The review highlights the role of these carriers in encasing siRNA and miRNA and promising therapy in delivering them to the glioma cells.

Epigenetic Regulation Towards Acquired Drug Resistance in Cancer.

Therapy resistance remains the most challenging obstacle in cancer treatment. Substantial efforts and evidences have accumulated over decades suggesting not only genetic but non-genomic mechanisms underlying this adaptation of tumor cells. Alterations in epigenome can have a fundamental effect on cellular functions and response to stresses like anticancer therapy. This chapter discusses the principal mechanisms by which epigenetic modifications in the genome and transcriptome aid tumor cells toward acquisition of resistance to chemotherapy.

Bio-polymeric transferrin-targeted temozolomide nanoparticles in gel for synergistic post-surgical GBM therapy.

Spatiotemporal targeting of anti-glioma drugs remains a pressing issue in glioblastoma (GBM) treatment. We challenge this issue by developing a minimally invasive in situ implantable hydrogel implant comprising transferrin-targeted temozolomide-miltefosine nanovesicles in the surgically resected GBM cavity (tumour bed). Injection of the "nanovesicle in hydrogel system" in orthotopic GBM-bearing mice improved drug penetration into the peri-cavitary region (∼4.5 mm in depth) with the potential to act as a bridge therapy in the immediate postoperative period, before the initiation of adjuvant radiotherapy. The controlled and sustained release of temozolomide over a month in the surgical cavity eradicated the microscopic GBM cells present within the tumour bed, thereby augmenting the efficacy of adjuvant therapy. The drug (temozolomide and miltefosine) combination was tolerable and efficiently inhibited tumour growth, causing significant prolongation of the survival of tumour-bearing mice compared to that with the free drug. Direct implantation at the target site in the brain resulted in spatiotemporal anti-glioma activity with minimal extracranial and systemic distribution. Nanovesicle in flexible hydrogel systems can be used as potential platforms for the post-surgical management of GBM before initiating adjuvant radiation therapy.

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.

Non-invasive transferrin targeted nanovesicles sensitize resistant glioblastoma multiforme tumors and improve survival in orthotopic mouse models.

The blood-brain barrier (BBB) and tumor heterogeneity have resulted in abysmally poor clinical outcomes in glioblastoma (GBM) with the standard therapeutic regimen. Despite several anti-glioma drug delivery strategies, the lack of adequate chemotherapeutic bioavailability in gliomas has led to a suboptimal therapeutic gain in terms of improvement in survival and increased systemic toxicities. This has paved the way for designing highly specific and non-invasive drug delivery approaches for treating GBM. The intranasal (IN) route is one such delivery strategy that has the potential to reach the brain parenchyma by circumventing the BBB. We recently showed that in situ hydrogel embedded with miltefosine (HePc, proapoptotic anti-tumor agent) and temozolomide (TMZ, DNA methylating agent) loaded targeted nanovesicles prevented tumor relapses in orthotopic GBM mouse models. In this study, we specifically investigated the potential of a non-invasive IN route of TMZ delivered from lipid nanovesicles (LNs) decorated with surface transferrin (Tf) and co-encapsulated with HePc to reach the brain by circumventing the BBB in glioma bearing mice. The targeted nanovesicles (228.3 ± 10 nm, -41.7 ± 4 mV) exhibited mucoadhesiveness with 2% w/v mucin suggesting their potential to increase brain drug bioavailability after IN administration. The optimized TLNs had controlled, tunable and significantly different release kinetics in simulated cerebrospinal fluid and simulated nasal fluid demonstrating efficient release of the payload upon reaching the brain. Drug synergy (combination index, 0.7) showed a 6.4-fold enhanced cytotoxicity against resistant U87MG cells compared to free drugs. In vivo gamma scintigraphy of 99mTc labeled LNs showed 500- and 280-fold increased brain concentration post 18 h of treatment. The efficacy of the TLNs increased by 1.8-fold in terms of survival of tumor-bearing mice compared to free drugs. These findings suggested that targeted drug synergy has the potential to intranasally deliver a high therapeutic dose of the chemotherapy agent (TMZ) and could serve as a platform for future clinical application.

DUSP6 regulates radiosensitivity in glioblastoma by modulating the recruitment of phosphorylated DNAPKcs at DNA double-strand breaks.

Glioblastoma (GBM) has poor median survival due to its resistance to chemoradiotherapy, which results in tumor recurrence. Recurrent GBMs currently lack effective treatments. DUSP6 is known to be pro-tumorigenic and is upregulated in GBM. We show that DUSP6 expression is significantly higher in recurrent GBM patient biopsies compared to expression levels in primary GBM biopsies. Importantly, although it has been reported to be a cytoplasmic protein, we found nuclear localization of DUSP6 in primary and recurrent patient samples and in parent and relapse populations of GBM cell lines generated from an in vitro radiation survival model. DUSP6 inhibition using BCI resulted in decreased proliferation and clonogenic survival of parent and relapse cells. Pharmacological or genetic inhibition of DUSP6 catalytic activity radiosensitized primary and, importantly, relapse GBM cells by inhibiting the recruitment of phosphorylated DNAPKcs (also known as PRKDC), subsequently downregulating the recruitment of phosphorylated histone H2AX (γH2AX) and 53BP1 (also known as TP53BP1). This resulted in decreased cell survival and prolonged growth arrest upon irradiation in vitro and significantly increased the progression-free survival in orthotopic mouse models of GBM. Our study highlights a non-canonical function of DUSP6, emphasizing the potential application of DUSP6 inhibitors in the treatment of recurrent GBM.

14-3-3ζ negatively regulates mitochondrial biogenesis in GBM residual cells.

Glioblastoma (GBM) is the most lethal primary brain tumour with a median survival of only 15 months. We have previously demonstrated the generation of an in vitro therapy resistance model that captures the residual resistant (RR) disease cells of GBM post-radiation. We also reported the proteomic landscape of parent, residual, and relapse cells using iTRAQ based quantitative proteomics of glioma cells. The proteomics data revealed significant up-regulation (fold change >1.5) of 14-3-3ζ, specifically in GBM RR cells. This was further confirmed by western blots in residual cells generated from GBM cell lines and patient sample-derived short-term primary culture. ShRNA-mediated knockdown of 14-3-3ζ radio-sensitized GBM cells and further stimulated therapy-induced senescence (TIS) and multinucleated giant cells (MNGCs) phenotype in RR cells. Intriguingly, 14-3-3ζ knockdown residual cells also showed a significantly higher number of mitochondria and increased mtDNA content. Indeed, in vitro GST pull-down mass spectrometry analysis of GST tagged 14-3-3ζ from RR cells identified novel interacting partners of 14-3-3ζ involved in cellular metabolism. Taken together, here we identified novel interacting partners of 14-3-3ζ and proposed an unconventional function of 14-3-3ζ as a negative regulator of TIS and mitochondrial biogenesis in residual resistant cells and loss of which also radio-sensitize GBM cells.

Birth of a solid organ cancer-the cell fusion hypothesis presented with pancreatic cancer as a model: a narrative review.

OBJECTIVE: This hypothesis-driven narrative review aims to explore the evidence for the fundamental process of cell fusion between normal, but different, cell types in the genesis of a cancer cell. BACKGROUND: Finding out how a cancer is born must remain a top priority as this will allow us the opportunity to understand the disease before it acquires its largely 'untameable' heterogeneous form. The search for the cell of origin in solid organ cancers has remained elusive despite concerted attempts over many decades. There is always more than one cell type implicated in the causation of solid organ cancers. METHODS: Based on preliminary data from our laboratory and a review of the evidence in literature, we present a novel hypothesis to explain the origin of solid organ cancers using pancreatic cancer as an example. CONCLUSIONS: We hypothesize that, "Cancer is born from fusion and hybridization of normal cells from two different lineages located within the vicinity of each other that perceive a signal reminiscent of a threat to their extinction that leads to epigenetically-mediated transformations permitting them to achieve cell fusion." Addressing this hypothesis to prove, or disprove it, presents an opportunity to unravel the basis of carcinogenesis and potential re-think our strategies for treatment in terms of choice of chemotherapeutic agents, dosage of chemo- and radiation-therapy, and timing of interventions (surgery, chemotherapy and radiation therapy).

Down-regulation of metabolic pathways could offset the poor prognosis conferred by co-existent diabetes mellitus in pancreatic (head) adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) patients with diabetes mellitus (DM) have poor overall survival. Underlying mechanisms have not been fully clarified. This presents an opportunity for precision-oncology for which we systematically analysed publicly-available PDAC transcriptome data. METHODS: PDAC TCGA RNASeq data were used. Analyses were restricted to only 'high purity' and 'head' as anatomical site. Patients were characterised by: (1) Gene expression classification, and (2) Weighted gene correlation network analysis (WGCNA) to identify co-expression patterns of genes. Newly identified gene signature subclasses of pancreatic head PDAC were associated with clinical and functional characteristics of patients. RESULTS: Consensus clustering identified two patient subclasses within PDAC involving pancreatic head. WGCNA identified 11 distinct networks of gene expression patterns across two sub-classes. Class 1 patients demonstrated a significant upregulation of Module 5 and Module 6 gene expression compared to Class 2. Class 1 predominantly expressed the acinar, ductal and islet cell gene signatures. There were significantly less patients with DM in Class 1 subclass compared to Class 2 (p < 0.037). Patients with DM had significant downregulation of pathways involved in cellular metabolism, hormone secretion and paucity of islet cell markers with no reduced survival compared with non-diabetics. CONCLUSIONS: A significant proportion of patients with PDAC of pancreatic head and DM exhibit downregulation of pathways involved in cellular metabolism, hormone secretion and signalling accompanied by a paucity of islet expression. Investigating the relationship between DM and gene expression profiles in patients with PDAC presents opportunities to improve overall survival in diabetics with PDAC.

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.

Metabolic rewiring in drug resistant cells exhibit higher OXPHOS and fatty acids as preferred major source to cellular energetics.

Alteration in metabolic repertoire is associated with resistance phenotype. Although a common phenotype, not much efforts have been undertaken to design effective strategies to target the metabolic drift in cancerous cells with drug resistant properties. Here, we identified that drug resistant AML cell line HL-60/MX2 did not follow classical Warburg effect, instead these cells exhibited drastically low levels of aerobic glycolysis. Biochemical analysis confirmed reduced glucose consumption and lactic acid production by resistant population with no differences in glutamine consumption. Raman spectroscopy revealed increased lipid and cytochrome content in resistant cells which were also visualized as lipid droplets by Raman mapping, electron microscopy and lipid specific staining. Gene set enrichment analysis data from sensitive and resistant cell lines revealed significant enrichment of lipid metabolic pathways in HL-60/MX2 cells. Further, HL-60/MX2 possessed higher mitochondrial activity and increased OXPHOS suggesting the role of fatty acid metabolism as energy source which was confirmed by increased rate of fatty acid oxidation. Accordingly, OXPHOS inhibitor increased sensitivity of resistant cells to chemotherapeutic drug and fatty acid oxidation inhibitor Etomoxir reduced colony formation ability of resistant cells demonstrating the requirement of fatty acid metabolism and dependency on OXPHOS by resistant leukemic cells for survival and tumorigenicity.

Sustained inhibition of PARP-1 activity delays glioblastoma recurrence by enhancing radiation-induced senescence.

Glioblastoma (GBM) is the most common primary brain tumor and is highly aggressive with a median survival of 15 months. We have previously shown that residual cells of GBM form multinucleated giant cells (MNGCs) showing a senescent phenotype, but eventually escape from therapy induced senescence (TIS), resulting in GBM recurrence. Here we demonstrate the role of PARP-1 in TIS and its recovery. We show that genetic and pharmacological inhibition of PARP-1 has an anti-proliferative effect on GBM cell lines and primary cultures derived from patient samples. Furthermore, the PARP-1 inhibitor olaparib, in combination with radiation increased MNGCs formation and senescence as assessed by ß-galactosidase activity, and macroH2A1 levels in residual cells. Additionally, we found that reduced PARP-1 activity and not protein levels in residual cells was crucial for MNGCs formation and their maintenance in the senescent state. PARP-1 activity was restored to higher levels in recurrent cells that escaped from TIS. Importantly, olaparib + radiation treatment significantly delayed recurrence in vitro as well in vivo in orthotopic GBM mouse models with a significant increase in overall survival of mice. Overall, this study demonstrates that sustained inhibition of PARP-1 activity during radiation treatment significantly delays GBM recurrence.

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.

HER2 borderline is a negative prognostic factor for primary malignant breast cancer.

BACKGROUND: HER-(human epidermal growth factor receptor 2) gene amplification and protein overexpression are important predictive, prognosis markers, and therapeutic target for breast cancer, emphasizing the importance of categorizing patients into HER2 positive and negative. However, from immunohistochemistry scores, 2% patients are neither HER2 + nor -ve, but borderline called HER2B. To make informed treatment decisions of these patients, it is important to know how different this group is compared to HER-2 positive/negative. METHODS: We analyzed n = 104,668 breast cancer patient samples from Surveillance, Epidemiology, and End Results (SEER) database. Survival analysis was performed using open source R (Cran project R version 3.5.0) "survival" package. Hazard ratio with confidence intervals was computed using coxph function. RESULTS: Of n = 104,668, 2239 (2.13%) patients were HER2 borderline, 87,157 (83.26%) HER2-negative, and 15,272 (14.6%) HER2-positive. The breast cancer as primary malignancy was observed in 84,944 (81.16%) patients. In primary malignant breast cancer (PMBC) patients, the hazard ratio among HER2-negative patients was significantly higher than HER2-positive patient samples (HR = 0.772, 95% CI 0.715-0.833, p = < .001), whereas HER2 negative status was not significantly favorable in PMBC negative patients in HER2-positive (HR = .919, 95% 0.797-1.06, p = .248). Most importantly in PMBC patients, the HR for HER2-borderline was poor in comparison to HER2 negative (HR = 1.354, 95% CI 1.126-1.627, p = < .001). CONCLUSION: This is the first report with large cohort of patient samples and significant statistical power to demonstrate that HER2 borderline represents a negative prognostic factor for PMBC. Thus providing rationale for controlled clinical trial for HER2-targeted therapies in HER2-borderline patients.

CytoPred: 7-gene pair metric for AML cytogenetic risk prediction.

Cytogenetic-based subjective prognostication of acute myeloid leukemia (AML) patients is a cumbersome process. Top scoring pair (TSP)-based decision tree using a robust analytical algorithm with statistical rigor offers a promising alternative. We describe CytoPred as a 7-gene pair signature based on the analysis of 2547 AML patient sample gene expression data using a modified TSP algorithm to estimate cytogenetic risk. The essential modification in TSP that helped computational encumbrance includes the filtration of gene pairs above random weighted guessers as well as sampling the gene pairs from the original gene pair pool to reduce overfitting issue. The CytoPred classifies AML cohort into clinically relevant `good' and `Int_poor' prognosis groups with distinct survival differences. The 7-gene pair was derived using 1248 AML patient samples in training set and 675 samples used for internal testing of the algorithm. The finest classifier 7-gene pair was picked from an initial pool size of 6.1 × 107 gene pairs that generated 57 687 decision trees. Further, for unbiased evaluation of CytoPred performance, we did an independent validation in 624 AML patient cohort. The CytoPred well qualifies the cutoffs for diagnostic application with 98.27% sensitivity and 99.27% specificity to predictive value in Int_poor class while 97.09% sensitivity and 91.74% specificity to predictive value for good class. Furthermore, CytoPred predicts almost identical survival probabilities like cytogenetics and its performance is not much influenced by various recurrent mutations as well as individual French-American-British (FAB) subtypes. In summary, we present a robust 7-gene pair-based metric to clinically prognosticate AML patients.

Effect of Glucosamine Conjugate-Functionalized Liposomes on Glioma Cell and Healthy Brain: An Insight for Future Application in Brain Infusion.

Conjugation of D-glucosamine with lipophilic moiety can ease its application in surface modification of liposomes. Interestingly, although D-glucosamine is safe, studies have shed light on "toxic effect" of its conjugates on cancer cells and highlighted its application in targeting glioma. However, understanding the safety of such conjugates for local delivery to the brain is unavailable. Herein, after successful synthesis of D-glucosamine conjugate (GC), the toxicity of functionalized liposome was evaluated both in vitro and in vivo. The study revealed a significant effect on cytotoxicity and apoptosis in vitro as assessed on grade IV-resistant glioma cell lines, SF268, U87MG, using MTT assay and PI staining. Additionally, this effect was not observed on normal human erythrocytes in the hemolysis assay. Furthermore, we demonstrated that GC liposomes were non-toxic to the normal brain tissues of healthy Sprague-Dawley rats. Successful functionalization yielded liposome with uniform particle size, stability, and cellular uptake. With < 10% hemolysis, all the liposomal formulations demonstrated hemato-compatibility but led to high glioma cytotoxicity. The surface density of conjugate played an important role in tumor toxicity (0.5 < 1.0 ≤ 2.0% molar ratio). PI staining revealed that compared to control cell, functionalization led 26-fold increase in induction of apoptosis in glioma cells. Absence of histological and behavioral changes along with the absence of caspase-3 in brain tissue confirmed the suitability of the system for direct infusion in the brain. Thus, this study will aid the future development of clinically useful local chemotherapeutic without "add-in" side effects.