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Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel—the MYC pathway and the cyclin D–cyclin-dependent kinase (CDK)–retinoblastoma protein (RB) pathway1,2. Both MYC and the cyclin D–CDK–RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1–cyclin D pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis. AMBRA1-mediated degradation of cyclin D through CRL4–DDB1 regulates cell proliferation and prevents replication stress in neurodevelopment and cancer.

Glioblastoma cancer-stem like cells (GSCs) display marked resistance to ionizing radiation (IR), a standard of care for glioblastoma patients. Mechanisms underpinning radio-resistance of GSCs remain largely unknown. Chromatin state and the accessibility of DNA lesions to DNA repair machineries are crucial for the maintenance of genomic stability. Understanding the functional impact of chromatin remodeling on DNA repair in GSCs may lay the foundation for advancing the efficacy of radio-sensitizing therapies. Here, we present the results of a high-content siRNA microscopy screen, revealing the transcriptional elongation factor SPT6 to be critical for the genomic stability and self-renewal of GSCs. Mechanistically, SPT6 transcriptionally up-regulates BRCA1 and thereby drives an error-free DNA repair in GSCs. SPT6 loss impairs the self-renewal, genomic stability and tumor initiating capacity of GSCs. Collectively, our results provide mechanistic insights into how SPT6 regulates DNA repair and identify SPT6 as a putative therapeutic target in glioblastoma., Cancer stem cells can evade treatment. Here, the authors perform an in vitro screen to identify proteins that are involved in protecting glioma cancer stem cells from therapy and find that SPT6 increases BRCA1 expression and drives error-free DNA repair, thereby ensuring the survival of the cells.

Glioblastoma (GBM) ranks among the most lethal cancers, with current therapies offering only palliation. Inter- and intrapatient heterogeneity is a hallmark of GBM, with epigenetically distinct cancer stem-like cells (CSCs) at the apex. Targeting GSCs remains a challenging task because of their unique biology, resemblance to normal neural stem/progenitor cells, and resistance to standard cytotoxic therapy. Here, we find that the chromatin regulator, JmjC domain histone H3K36me2/me1 demethylase KDM2B, is highly expressed in glioblastoma surgical specimens compared to normal brain. Targeting KDM2B function genetically or pharmacologically impaired the survival of patient-derived primary glioblastoma cells through the induction of DNA damage and apoptosis, sensitizing them to chemotherapy. KDM2B loss decreased the GSC pool, which was potentiated by coadministration of chemotherapy. Collectively, our results demonstrate KDM2B is crucial for glioblastoma maintenance, with inhibition causing loss of GSC survival, genomic stability, and chemoresistance.

Glioblastoma (GBM, WHO grade IV astrocytoma) is among the deadliest of solid tumors with median survival rates of only approximately 15 months. In spite of concerted efforts, molecular mechanisms/genes causing high recurrence and treatment resistance are poorly understood. The tumor suppressor BRCA1 plays a critical role in maintaining genomic stability, in general, and replication stress (RS), in particular. Numerous reports have demonstrated that wild type BRCA1 loss impairs the growth of several cancers (breast, ovarian, lung, prostate and colon). Our data show considerably higher level of RS in malignant gliomas compared to other solid tumors (breast, prostate ovarian) and identify a novel role for BRCA1, where high BRCA1 expression negatively associates with glioma patient survival. BRCA1 plays an unexpected tumor-promoting role in GBM, safeguarding a protective response to supraphysiological RS levels via transcriptional regulation of RRM2 expression. shRNA-mediated knockdown of BRCA1 or chemical inhibition of RRM2 both induce GBM cell death due to accumulation of RS-induced DNA damage in vitro and extend survival of tumor bearing mice in vivo, highlighting the clinical relevance of our findings. Acknowledgements: This work was supported by Danish Cancer Society Foundation.

Oncogene-evoked replication stress (RS) fuels genomic instability in diverse cancer types. Here we report that BRCA1, traditionally regarded a tumour suppressor, plays an unexpected tumour-promoting role in glioblastoma (GBM), safeguarding a protective response to supraphysiological RS levels. Higher BRCA1 positivity is associated with shorter survival of glioma patients and the abrogation of BRCA1 function in GBM enhances RS, DNA damage (DD) accumulation and impairs tumour growth. Mechanistically, we identify a novel role of BRCA1 as a transcriptional co-activator of RRM2 (catalytic subunit of ribonucleotide reductase), whereby BRCA1-mediated RRM2 expression protects GBM cells from endogenous RS, DD and apoptosis. Notably, we show that treatment with a RRM2 inhibitor triapine reproduces the BRCA1-depletion GBM-repressive phenotypes and sensitizes GBM cells to PARP inhibition. We propose that GBM cells are addicted to the RS-protective role of the BRCA1-RRM2 axis, targeting of which may represent a novel paradigm for therapeutic intervention in GBM.

Glioblastoma (GBM) ranks among the deadliest solid cancers worldwide and its prognosis has remained dismal, despite the use of aggressive chemo-irradiation treatment regimens. Limited drug delivery into the brain parenchyma and frequent resistance to currently available therapies are problems that call for a prompt development of novel therapeutic strategies. While only displaying modest efficacies as mono-therapy in pre-clinical settings, histone deacetylase inhibitors (HDACi) have shown promising sensitizing effects to a number of cytotoxic agents. Here, we sought to investigate the sensitizing effect of the HDACi trichostatin A (TSA) to the alkylating agent lomustine (CCNU), which is used in the clinic for the treatment of GBM. Twelve primary GBM cell cultures grown as neurospheres were used in this study, as well as one established GBM-derived cell line (U87 MG). Histone deacetylase (HDAC) expression levels were determined using quantitative real-time PCR and Western blotting. The efficacy of either CCNU alone or its combination with TSA was assessed using various assays, i.e., cell viability assays (MTT), cell cycle assays (flow cytometry, FACS), double-strand DNA break (DSB) quantification assays (microscopy/immunofluorescence) and expression profiling assays of proteins involved in apoptosis and cell stress (Western blotting and protein array). We found that the HDAC1, 3 and 6 expression levels were significantly increased in GBM samples compared to non-neoplastic brain control samples. Additionally, we found that pre-treatment of GBM cells with TSA resulted in an enhancement of their sensitivity to CCNU, possibly via the accumulation of DSBs, decreased cell proliferation and viability rates, and an increased apoptotic rate. From our data we conclude that the combined administration of TSA and CCNU eradicates GBM cells with a higher efficacy than either drug alone, thereby opening a novel avenue for the treatment of GBM.

Glioblastoma (GBM) is the most advanced and lethal tumor with epigenetically distinct cancer stem-like cells (CSCs) resistant to chemo- and radio-therapy. Spt6 is a histone chaperon involved in nucleosome assembly and regulates RNA polymerase II-mediated transcription. Our preliminary data identified Spt6 to be crucial for glioblastoma stem cells (GSCs) genomic stability and survival. Spt6 depletion in GSCs decreased global transcription rates and cell cycle entry. Cycloheximide and proteasome inhibitor (MG132) experiments confirmed that Spt6 stabilizes RNA polymerase II and so regulates GSC maintenance at a transcriptional level. Spt6 loss impaired GSC survival in vitro and tumor initiating capacity in vivo, thereby making it an attractive target for glioblastoma therapy.

Glioblastoma multiforme (GBM) is among the most lethal of solid cancers in adults. It is associated with a median survival of only approximately 15 months despite aggressive radio- and chemotherapies and recurrence is inevitable. Despite recent advances in our understanding of this deadly disease, the molecular mechanism and/or genes that cause high recurrence rates and treatment resistance in GBM are poorly understood. Histone chaperones affect the structure of the chromatin and expression of genes through interaction with histones and RNA polymerase II (PolII). Spt6 is a highly conserved transcription elongation factor and histone chaperone that has been found to counteract tri- methylation of the histone variant H3, at various lysines, an otherwise epigenetic mark which is associated with repression of transcription. We have identified a novel role of Spt6 in glioblastoma cells. Our data show that siRNA-mediated knockdown of Spt6 in GBM cells results in decreased proliferation rates and progression through the cell cycle, increased levels of DNA damage (measured by γH2AX) as well as delayed capacity for DNA repair after exposure to ionizing radiation. Altogether, our data imply an important role of Spt6 in glioblastoma maintenance and therapeutic resistance.

Glioblastoma-initiating cells (GICs) are self-renewing tumorigenic sub-populations, contributing to therapeutic resistance via decreased sensitivity to ionizing radiation (IR). GIC survival following IR is attributed to an augmented response to genotoxic stress. We now report that GICs are primed to handle additional stress due to basal activation of single-strand break repair (SSBR), the main DNA damage response pathway activated by reactive oxygen species (ROS), compared with non-GICs. ROS levels were higher in GICs and likely contributed to the oxidative base damage and single-strand DNA breaks found elevated in GICs. To tolerate constitutive DNA damage, GICs exhibited a reliance on the key SSBR mediator, poly-ADP-ribose polymerase (PARP), with decreased viability seen upon small molecule inhibition to PARP. PARP inhibition (PARPi) sensitized GICs to radiation and inhibited growth, self-renewal, and DNA damage repair. In vivo treatment with PARPi and radiotherapy attenuated radiation-induced enrichment of GICs and inhibited the central cancer stem cell phenotype of tumor initiation. These results indicate that elevated PARP activation within GICs permits exploitation of this dependence, potently augmenting therapeutic efficacy of IR against GICs. In addition, our results support further development of clinical trials with PARPi and radiation in glioblastoma.

BACKGROUND: Glioblastoma multiforme (GBM) remains one of the most lethal tumors and is associated with a median survival of only approximately 15 months, despite aggressive combination radio-chemo-therapy. Regardless of recent advances in our understanding of this deadly disease, the molecular mechanisms/genes that cause high recurrence rates and treatment resistance in GBM are poorly understood. It is commonly known that cancer cells including GBM utilize glucose as their main energy source, a phenomenon known as the Warburg effect. Phosphofructosekinase-1 (PFK-1) is one of two rate-limiting enzymes in glycolysis and presents a potential critical control point for these glucose ‘addictive’ cells. In this study we investigated the role of PFK-1 and its effect on cell proliferation and maintenance of GBM. METHODS: To elucidate the role of PFK-1 in GBM, we abrogated its function using shRNA-mediated knockdown or chemical inhibition. Next, we evaluated the cellular responses utilizing a broad range of in vitro assays (cell viability, apoptosis, cell cycle, DNA damage checkpoint activation) under unperturbed and/or genotoxic stress conditions. RESULTS: Inhibition of PFK-1 led to decreased viability, cell cycle de-regulation and increased rates of apoptosis. Furthermore, impaired function of PFK-1 affected cytoskeleton assembly and IR-induced checkpoint activation. CONCLUSION: The novel role of PFK-1 identified in our studies, together with successful therapeutic targeting in vitro and in vivo, furthers our understanding of the role that glycolysis plays in GBM maintenance and therapeutic resistance.