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PDF file, 1661K, Supplemental Figure S1- Chemical Structures. Supplemental Figure S2-Oxygen Consumption in MCF-7 Cells treated with or without betalapchone and dicoumarol. Supplemental Figure S2 legend. Supplemental Figure S3-Survival assay for normal mammary epithelial cells treated with betalapachone with or without dicoumarol. Supplemental Figure S3 legend. Supplemental Figure S4-Survival assay for MDA-MB231 cells treated with or without quinones and ROS scavengers. Supplemental Figure S4 legend. Supplemental Figure S5-Westernblot of MCF-7 cells treated with betalapachone with or without catalase. Supplemental Figure S5 legend. Supplemental Figure S6-Proteolysis of PARP in MCF-7 cells treated with betalapachone or staurosporine. Supplemental Figure S6 legend. Supplemental Figure S7- NQO1 mediated futile cycle of betalapachone. Supplemental Figure S7 legend. Supplemental Figure S8- Western blot and analysis of NQO1 to Catalase ratios in Breast Cancer Cell lines. Supplemental Table 1- NQO1 enzymatic activity in breast cancer cell lines. Supplemental Table 1 legend.

Supplementary Methods, Figures, and Tables

Purpose:Identification of novel strategies to expand the use of PARP inhibitors beyond BRCA deficiency is of great interest in personalized medicine. Here, we investigated the unannotated role of Kub5-HeraRPRD1B (K-H) in homologous recombination (HR) repair and its potential clinical significance in targeted cancer therapy.Experimental Design:Functional characterization of K-H alterations on HR repair of double-strand breaks (DSB) were assessed by targeted gene silencing, plasmid reporter assays, immunofluorescence, and Western blots. Cell survival with PARP inhibitors was evaluated through colony-forming assays and statistically analyzed for correlation with K-H expression in various BRCA1/2 nonmutated breast cancers. Gene expression microarray/qPCR analyses, chromatin immunoprecipitation, and rescue experiments were used to investigate molecular mechanisms of action.Results:K-H expression loss correlates with rucaparib LD50 values in a panel of BRCA1/2 nonmutated breast cancers. Mechanistically, K-H depletion promotes BRCAness, where extensive upregulation of PARP1 activity was required for the survival of breast cancer cells. PARP inhibition in these cells led to synthetic lethality that was rescued by wild-type K-H reexpression, but not by a mutant K-H (p.R106A) that weakly binds RNAPII. K-H mediates HR by facilitating recruitment of RNAPII to the promoter region of a critical DNA damage response and repair effector, cyclin-dependent kinase 1 (CDK1).Conclusions:Cancer cells with low K-H expression may have exploitable BRCAness properties that greatly expand the use of PARP inhibitors beyond BRCA mutations. Our results suggest that aberrant K-H alterations may have vital translational implications in cellular responses/survival to DNA damage, carcinogenesis, and personalized medicine.

Loss of K-H genetic alterations

Studies supporting a strong association between tau deposition and neuronal loss, neurodegeneration, and cognitive decline have heightened the allure of tau and tau-related mechanisms as therapeutic targets. In February 2020, leading tau experts from around the world convened for the first-ever Tau2020 Global Conference in Washington, DC, co-organized and cosponsored by the Rainwater Charitable Foundation, the Alzheimer's Association, and CurePSP. Representing academia, industry, government, and the philanthropic sector, presenters and attendees discussed recent advances and current directions in tau research. The meeting provided a unique opportunity to move tau research forward by fostering global partnerships among academia, industry, and other stakeholders and by providing support for new drug discovery programs, groundbreaking research, and emerging tau researchers. The meeting also provided an opportunity for experts to present critical research-advancing tools and insights that are now rapidly accelerating the pace of tau research., info:eu-repo/semantics/published

The circadian clock imposes daily rhythms in cell proliferation, metabolism, inflammation and DNA damage response1, 2. Perturbations of these processes are hallmarks of cancer3 and chronic circadian rhythm disruption predisposes to tumor development1, 4. This raises the hypothesis that pharmacological modulation of the circadian machinery may be an effective therapeutic strategy for combatting cancer. The nuclear hormone receptors REV-ERBα and REV-ERBβ (REV-ERBs) are essential components of the circadian clock5, 6. Here we show that SR9009 and SR9011, two different agonists of REV-ERBs are specifically lethal to cancer cells and oncogene-induced senescent (OIS) cells, including melanocytic naevi, while having no effect on viability of normal cells or tissues. Anticancer activity of SR9009 and SR9011 affects a number of oncogenic drivers (such as H-RAS, BRAF, PIK3CA, and others), and persists in the absence of p53 and under hypoxic conditions. The regulation of autophagy and de novo lipogenesis by SR9009 and SR9011 plays a critical role in evoking an apoptotic response in malignant cells. Importantly, the selective anticancer properties of these REV-ERB agonists impair glioblastoma growth in vivo and improve survival without causing any overt toxicity in mice. These results indicate that pharmacological modulation of circadian regulators is an effective novel antitumor strategy, identifying the existence of a previously unknown class of anticancer agents with a wide therapeutic window. We propose that REV-ERB agonists are novel autophagy and de novo lipogenesis inhibitors with selective activity towards malignant and benign neoplasms.

The multikinase inhibitor and FDA-approved drug dovitinib (Dov) crosses the blood-brain barrier and was recently used as single drug application in clinical trials for GB patients with recurrent disease. The Dov-mediated molecular mechanisms in GB cells are unknown. We used GB patient cells and cell lines to show that Dov downregulated the stem cell protein Lin28 and its target high-mobility group protein A2 (HMGA2). The Dov-induced reduction in pSTAT3Tyr705 phosphorylation demonstrated that Dov negatively affects the STAT3/LIN28/Let-7/HMGA2 regulatory axis in GB cells. Consistent with the known function of LIN28 and HMGA2 in GB self-renewal, Dov reduced GB tumor sphere formation. Dov treatment also caused the downregulation of key base excision repair factors and O6 -methylguanine-DNA-methyltransferase (MGMT), which are known to have important roles in the repair of temozolomide (TMZ)-induced alkylating DNA damage. Combined Dov/TMZ treatment enhanced TMZ-induced DNA damage as quantified by nuclear γH2AX foci and comet assays, and increased GB cell apoptosis. Pretreatment of GB cells with Dov ('Dov priming') prior to TMZ treatment reduced GB cell viability independent of p53 status. Sequential treatment involving 'Dov priming' and alternating treatment cycles with TMZ and Dov substantially reduced long-term GB cell survival in MGMT+ patient GB cells. Our results may have immediate clinical implications to improve TMZ response in patients with LIN28+ /HMGA2+ GB, independent of their MGMT methylation status.

Purpose: Identification of novel strategies to expand the use of PARP inhibitors beyond BRCA deficiency is of great interest in personalized medicine. Here, we investigated the unannotated role of Kub5-HeraRPRD1B (K-H) in homologous recombination (HR) repair and its potential clinical significance in targeted cancer therapy. Experimental Design: Functional characterization of K-H alterations on HR repair of double-strand breaks (DSB) were assessed by targeted gene silencing, plasmid reporter assays, immunofluorescence, and Western blots. Cell survival with PARP inhibitors was evaluated through colony-forming assays and statistically analyzed for correlation with K-H expression in various BRCA1/2 nonmutated breast cancers. Gene expression microarray/qPCR analyses, chromatin immunoprecipitation, and rescue experiments were used to investigate molecular mechanisms of action. Results: K-H expression loss correlates with rucaparib LD50 values in a panel of BRCA1/2 nonmutated breast cancers. Mechanistically, K-H depletion promotes BRCAness, where extensive upregulation of PARP1 activity was required for the survival of breast cancer cells. PARP inhibition in these cells led to synthetic lethality that was rescued by wild-type K-H reexpression, but not by a mutant K-H (p.R106A) that weakly binds RNAPII. K-H mediates HR by facilitating recruitment of RNAPII to the promoter region of a critical DNA damage response and repair effector, cyclin-dependent kinase 1 (CDK1). Conclusions: Cancer cells with low K-H expression may have exploitable BRCAness properties that greatly expand the use of PARP inhibitors beyond BRCA mutations. Our results suggest that aberrant K-H alterations may have vital translational implications in cellular responses/survival to DNA damage, carcinogenesis, and personalized medicine.

Functions of Kub5-Hera (In Greek Mythology Hera controlled Artemis) (K-H), the human homolog of the yeast transcription termination factor Rtt103, remain undefined. Here, we show that K-H has functions in both transcription termination and DNA double-strand break (DSB) repair. K-H forms distinct protein complexes with factors that repair DSBs (e.g. Ku70, Ku86, Artemis) and terminate transcription (e.g. RNA polymerase II). K-H loss resulted in increased basal R-loop levels, DSBs, activated DNA-damage responses and enhanced genomic instability. Significantly lowered Artemis protein levels were detected in K-H knockdown cells, which were restored with specific K-H cDNA re-expression. K-H deficient cells were hypersensitive to cytotoxic agents that induce DSBs, unable to reseal complex DSB ends, and showed significantly delayed γ-H2AX and 53BP1 repair-related foci regression. Artemis re-expression in K-H-deficient cells restored DNA-repair function and resistance to DSB-inducing agents. However, R loops persisted consistent with dual roles of K-H in transcription termination and DSB repair.

The circadian clock drives daily rhythms in cell proliferation, metabolism, inflammation and DNA damage response. Perturbations of these processes are hallmarks of cancer, and circadian rhythm disruption predisposes to tumor development in animal models and humans. This raises the hypothesis that pharmacologic modulation of the circadian machinery may be an effective therapeutic strategy for combating cancer. The nuclear hormone receptors REV-ERBα and REV-ERBβ (REV-ERBs) are essential components of the circadian clock. Here we show that SR9009 and SR9011, two different agonists of REV-ERBs, are specifically lethal to cancer cells and oncogene-induced senescent (OIS) cells, including melanocytic naevi, while having no effect on viability of normal cells or tissues. Anticancer activity of SR9009 and SR9011 affects several oncogenic drivers (such as H-RAS, BRAF, PIK3CA, and others), and persists in the absence of p53 and under hypoxic conditions. The regulation of autophagy and de novo lipogenesis by SR9009 and SR9011 plays a critical role in evoking an apoptotic response in malignant cells. Importantly, the selective anticancer properties of these REV-ERB agonists impair glioblastoma growth in vivo and improve survival without causing any overt toxicity in mice. These results indicate that pharmacologic modulation of circadian regulators is an effective novel antitumor strategy, identifying the existence of a previously unknown class of anticancer agents with a wide therapeutic window. We propose that REV-ERB agonists are novel autophagy and de novo lipogenesis inhibitors with selective activity towards malignant and benign neoplasms. Citation Format: Gabriele Sulli, Amy Rommel, Xiaojie Wang, Matthew J. Kolar, Francesca Puca, Alan Saghatelian, Maksim V. Plikus, Inder M. Verma, Satchidananda Panda. Pharmacologic activation of REV-ERBs is lethal in cancer and oncogene-induced senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5464.

Glioblastoma multiforme (GBM) is the most malignant brain tumor and highly resistant to intensive combination therapies. GBM is one of the most vascularized tumors and vascular endothelial growth factor (VEGF) produced by tumor cells is a major factor regulating angiogenesis. Successful results of preclinical studies of anti-angiogenic therapies using xenograft mouse models of human GBM cell lines encouraged clinical studies of anti-angiogenic drugs, such as bevacizumab (Avastin), an anti-VEGF antibody. However, these clinical studies have shown that most patients become resistant to anti-VEGF therapy after an initial response. Recent studies have revealed some resistance mechanisms against anti-VEGF therapies involved in several types of cancer. In this review, we address mechanisms of angiogenesis, including unique features in GBMs, and resistance to anti-VEGF therapies frequently observed in GBM. Enhanced invasiveness is one such resistance mechanism and recent works report the contribution of activated MET signaling induced by inhibition of VEGF signaling. On the other hand, tumor cell-originated neovascularization including tumor-derived endothelial cell-induced angiogenesis and vasculogenic mimicry has been suggested to be involved in the resistance to anti-VEGF therapy. Therefore, these mechanisms should be targeted in addition to anti-angiogenic therapies to achieve better results for patients with GBM.

Recent cellular and molecular biological examination of DNA repair, cell cycle checkpoints, apoptosis, differentiation, and stress-induced premature senescence (SIPS) in response to genotoxic stress is largely responsible for the current understanding of the interplay between cell stress responses and human diseases, such as cancer. Human pathologies associated with older individuals show homeostatic alterations of several cell stress factors, including Bax, p53, Hdm2, and secretory clusterin (sCLU) that can influence survival, senescence, and ultimately the risk of cancer. Understanding cell stress responses in the context of genetic predispositions is vital to the understanding of cancer risk from environmental stress, such as exposure to low doses of ionizing radiation (IR). Increased expression of sCLU appears to be a sensitive marker of exposure. Its interplay and regulation with stress-induced alterations in Bax, Ku70, Hdm2, p53, and their roles in regulation of survival and carcinogenesis are discussed. Understanding the mechanisms of induction and repression of the cellular responses to genotoxic agents is essential for understanding downstream cancer initiation and progression.

Aberrant CDK1 regulation induces impulsive proliferation as well as genomic and chromosomal instability in cancer cells. However, the mechanism of how CDK1 is regulated at the transcriptional level, especially under pathological conditions (e.g. cancer), remains elusive. Here, we show that Kub5-Hera (K-H), a known transcription termination factor, plays a novel role in promoting CDK1 transcription to mediate DNA repair and cellular response to PARP1 inhibition in various cancer cell lines. At the molecular level, we demonstrate via luciferase reporter assays and mutagenesis studies that K-H binds the phospho-Serine 2-containing C-terminal domain (CTD) of RNA Pol II, which is then recruited to the cell-cycle homology region (CHR) of the CDK1 promoter to efficiently stimulate CDK1 transcription. Mechanistically, the loss of K-H concomitantly reduces CDK1 mRNA/protein expression levels, which consequently compromises homologous recombination (HR)-mediated DNA repair due to the lack of BRCA1 phosphorylation for efficient function. Intriguingly, loss of K-H stimulates PARP1 activity needed for cancer cell survival. Genetic and pharmacological ablation of PARP1 activity in BRCA-proficient cells with aberrant K-H reduction leads to synthetic lethality, which is reversed by re-expression of wild-type K-H but not the mutant with attenuated RNAPII binding. The mechanistic insights gained from this study should lay foundation toward promising new tumor biology-based targets (i.e., K-H as a novel therapeutic target) and innovative strategies to reduce normal tissue toxicity, increase tumor control, and expand the use of PARP1 inhibitors (e.g., Rucaparib) against BRCA-proficient cancers with aberrant K-H loss. Citation Format: Edward A. Motea, Farjana Fattah, Ling Xiao, Luc Girard, Amy Rommel, Julio Morales, Praveen Patidar, Andrew Porter, John Minna, David Boothman. Kub5-Hera/RPRD1B controls CDK1 regulation and synthetic lethality to PARP1 inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-071. doi:10.1158/1538-7445.AM2015-LB-071