Publications by authors named "Sandeep Burma"

63 Publications

Comprehensive targeting of resistance to inhibition of RTK signaling pathways by using glucocorticoids.

Nat Commun 2021 12 1;12(1):7014. Epub 2021 Dec 1.

Department of Neurology, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.

Inhibition of RTK pathways in cancer triggers an adaptive response that promotes therapeutic resistance. Because the adaptive response is multifaceted, the optimal approach to blunting it remains undetermined. TNF upregulation is a biologically significant response to EGFR inhibition in NSCLC. Here, we compared a specific TNF inhibitor (etanercept) to thalidomide and prednisone, two drugs that block TNF and also other inflammatory pathways. Prednisone is significantly more effective in suppressing EGFR inhibition-induced inflammatory signals. Remarkably, prednisone induces a shutdown of bypass RTK signaling and inhibits key resistance signals such as STAT3, YAP and TNF-NF-κB. Combined with EGFR inhibition, prednisone is significantly superior to etanercept or thalidomide in durably suppressing tumor growth in multiple mouse models, indicating that a broad suppression of adaptive signals is more effective than blocking a single component. We identify prednisone as a drug that can effectively inhibit adaptive resistance with acceptable toxicity in NSCLC and other cancers.
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http://dx.doi.org/10.1038/s41467-021-27276-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8636603PMC
December 2021

Elimination of Radiation-Induced Senescence in the Brain Tumor Microenvironment Attenuates Glioblastoma Recurrence.

Cancer Res 2021 12 27;81(23):5935-5947. Epub 2021 Sep 27.

Department of Neurosurgery, University of Texas Health, San Antonio, Texas.

Glioblastomas (GBM) are routinely treated with ionizing radiation (IR) but inevitably recur and develop therapy resistance. During treatment, the tissue surrounding tumors is also irradiated. IR potently induces senescence, and senescent stromal cells can promote the growth of neighboring tumor cells by secreting factors that create a senescence-associated secretory phenotype (SASP). Here, we carried out transcriptomic and tumorigenicity analyses in irradiated mouse brains to elucidate how radiotherapy-induced senescence of non-neoplastic brain cells promotes tumor growth. Following cranial irradiation, widespread senescence in the brain occurred, with the astrocytic population being particularly susceptible. Irradiated brains showed an altered transcriptomic profile characterized by upregulation of CDKN1A (p21), a key enforcer of senescence, and several SASP factors, including HGF, the ligand of the receptor tyrosine kinase (RTK) Met. Preirradiation of mouse brains increased Met-driven growth and invasiveness of orthotopically implanted glioma cells. Importantly, irradiated p21 mouse brains did not exhibit senescence and consequently failed to promote tumor growth. Senescent astrocytes secreted HGF to activate Met in glioma cells and to promote their migration and invasion , which could be blocked by HGF-neutralizing antibodies or the Met inhibitor crizotinib. Crizotinib also slowed the growth of glioma cells implanted in preirradiated brains. Treatment with the senolytic drug ABT-263 (navitoclax) selectively killed senescent astrocytes , significantly attenuating growth of glioma cells implanted in preirradiated brains. These results indicate that SASP factors in the irradiated tumor microenvironment drive GBM growth via RTK activation, underscoring the potential utility of adjuvant senolytic therapy for preventing GBM recurrence after radiotherapy. SIGNIFICANCE: This study uncovers mechanisms by which radiotherapy can promote GBM recurrence by inducing senescence in non-neoplastic brain cells, suggesting that senolytic therapy can blunt recurrent GBM growth and aggressiveness.
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http://dx.doi.org/10.1158/0008-5472.CAN-21-0752DOI Listing
December 2021

Therapy-Induced Senescence: Opportunities to Improve Anticancer Therapy.

J Natl Cancer Inst 2021 Oct;113(10):1285-1298

Departments of Neurosurgery and Biochemistry & Structural Biology, University of Texas Health Science Center, San Antonio, TX, USA.

Cellular senescence is an essential tumor suppressive mechanism that prevents the propagation of oncogenically activated, genetically unstable, and/or damaged cells. Induction of tumor cell senescence is also one of the underlying mechanisms by which cancer therapies exert antitumor activity. However, an increasing body of evidence from preclinical studies demonstrates that radiation and chemotherapy cause accumulation of senescent cells (SnCs) both in tumor and normal tissue. SnCs in tumors can, paradoxically, promote tumor relapse, metastasis, and resistance to therapy, in part, through expression of the senescence-associated secretory phenotype. In addition, SnCs in normal tissue can contribute to certain radiation- and chemotherapy-induced side effects. Because of its multiple roles, cellular senescence could serve as an important target in the fight against cancer. This commentary provides a summary of the discussion at the National Cancer Institute Workshop on Radiation, Senescence, and Cancer (August 10-11, 2020, National Cancer Institute, Bethesda, MD) regarding the current status of senescence research, heterogeneity of therapy-induced senescence, current status of senotherapeutics and molecular biomarkers, a concept of "one-two punch" cancer therapy (consisting of therapeutics to induce tumor cell senescence followed by selective clearance of SnCs), and its integration with personalized adaptive tumor therapy. It also identifies key knowledge gaps and outlines future directions in this emerging field to improve treatment outcomes for cancer patients.
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http://dx.doi.org/10.1093/jnci/djab064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8486333PMC
October 2021

EGFR inhibition triggers an adaptive response by co-opting antiviral signaling pathways in lung cancer.

Nat Cancer 2020 Apr 6;1(4):394-409. Epub 2020 Apr 6.

Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center, Dallas TX 75390.

EGFR inhibition is an effective treatment in the minority of non-small cell lung cancer (NSCLC) cases harboring EGFR-activating mutations, but not in EGFR wild type (EGFRwt) tumors. Here, we demonstrate that EGFR inhibition triggers an antiviral defense pathway in NSCLC. Inhibiting mutant EGFR triggers Type I IFN-I upregulation via a RIG-I-TBK1-IRF3 pathway. The ubiquitin ligase TRIM32 associates with TBK1 upon EGFR inhibition, and is required for K63-linked ubiquitination and TBK1 activation. Inhibiting EGFRwt upregulates interferons via an NF-κB-dependent pathway. Inhibition of IFN signaling enhances EGFR-TKI sensitivity in EGFR mutant NSCLC and renders EGFRwt/KRAS mutant NSCLC sensitive to EGFR inhibition in xenograft and immunocompetent mouse models. Furthermore, NSCLC tumors with decreased IFN-I expression are more responsive to EGFR TKI treatment. We propose that IFN-I signaling is a major determinant of EGFR-TKI sensitivity in NSCLC and that a combination of EGFR TKI plus IFN-neutralizing antibody could be useful in most NSCLC patients.
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http://dx.doi.org/10.1038/s43018-020-0048-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7706867PMC
April 2020

Inhibition of the de novo pyrimidine biosynthesis pathway limits ribosomal RNA transcription causing nucleolar stress in glioblastoma cells.

PLoS Genet 2020 11 17;16(11):e1009117. Epub 2020 Nov 17.

Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.

Glioblastoma is the most common and aggressive type of cancer in the brain; its poor prognosis is often marked by reoccurrence due to resistance to the chemotherapeutic agent temozolomide, which is triggered by an increase in the expression of DNA repair enzymes such as MGMT. The poor prognosis and limited therapeutic options led to studies targeted at understanding specific vulnerabilities of glioblastoma cells. Metabolic adaptations leading to increased synthesis of nucleotides by de novo biosynthesis pathways are emerging as key alterations driving glioblastoma growth. In this study, we show that enzymes necessary for the de novo biosynthesis of pyrimidines, DHODH and UMPS, are elevated in high grade gliomas and in glioblastoma cell lines. We demonstrate that DHODH's activity is necessary to maintain ribosomal DNA transcription (rDNA). Pharmacological inhibition of DHODH with the specific inhibitors brequinar or ML390 effectively depleted the pool of pyrimidines in glioblastoma cells grown in vitro and in vivo and impaired rDNA transcription, leading to nucleolar stress. Nucleolar stress was visualized by the aberrant redistribution of the transcription factor UBF and the nucleolar organizer nucleophosmin 1 (NPM1), as well as the stabilization of the transcription factor p53. Moreover, DHODH inhibition decreased the proliferation of glioblastoma cells, including temozolomide-resistant cells. Importantly, the addition of exogenous uridine, which reconstitutes the cellular pool of pyrimidine by the salvage pathway, to the culture media recovered the impaired rDNA transcription, nucleolar morphology, p53 levels, and proliferation of glioblastoma cells caused by the DHODH inhibitors. Our in vivo data indicate that while inhibition of DHODH caused a dramatic reduction in pyrimidines in tumor cells, it did not affect the overall pyrimidine levels in normal brain and liver tissues, suggesting that pyrimidine production by the salvage pathway may play an important role in maintaining these nucleotides in normal cells. Our study demonstrates that glioblastoma cells heavily rely on the de novo pyrimidine biosynthesis pathway to generate ribosomal RNA (rRNA) and thus, we identified an approach to inhibit ribosome production and consequently the proliferation of glioblastoma cells through the specific inhibition of the de novo pyrimidine biosynthesis pathway.
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http://dx.doi.org/10.1371/journal.pgen.1009117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7707548PMC
November 2020

Specificity of end resection pathways for double-strand break regions containing ribonucleotides and base lesions.

Nat Commun 2020 06 18;11(1):3088. Epub 2020 Jun 18.

Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT, 06510, USA.

DNA double-strand break repair by homologous recombination begins with nucleolytic resection of the 5' DNA strand at the break ends. Long-range resection is catalyzed by EXO1 and BLM-DNA2, which likely have to navigate through ribonucleotides and damaged bases. Here, we show that a short stretch of ribonucleotides at the 5' terminus stimulates resection by EXO1. Ribonucleotides within a 5' flap are resistant to cleavage by DNA2, and extended RNA:DNA hybrids inhibit both strand separation by BLM and resection by EXO1. Moreover, 8-oxo-guanine impedes EXO1 but enhances resection by BLM-DNA2, and an apurinic/apyrimidinic site stimulates resection by BLM-DNA2 and DNA strand unwinding by BLM. Accordingly, depletion of OGG1 or APE1 leads to greater dependence of DNA resection on DNA2. Importantly, RNase H2A deficiency impairs resection overall, which we attribute to the accumulation of long RNA:DNA hybrids at DNA ends. Our results help explain why eukaryotic cells possess multiple resection nucleases.
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http://dx.doi.org/10.1038/s41467-020-16903-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7303207PMC
June 2020

MiR223-3p promotes synthetic lethality in BRCA1-deficient cancers.

Proc Natl Acad Sci U S A 2019 08 8;116(35):17438-17443. Epub 2019 Aug 8.

Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229;

Defects in DNA repair give rise to genomic instability, leading to neoplasia. Cancer cells defective in one DNA repair pathway can become reliant on remaining repair pathways for survival and proliferation. This attribute of cancer cells can be exploited therapeutically, by inhibiting the remaining repair pathway, a process termed synthetic lethality. This process underlies the mechanism of the Poly-ADP ribose polymerase-1 (PARP1) inhibitors in clinical use, which target BRCA1 deficient cancers, which is indispensable for homologous recombination (HR) DNA repair. HR is the major repair pathway for stressed replication forks, but when BRCA1 is deficient, stressed forks are repaired by back-up pathways such as alternative nonhomologous end-joining (aNHEJ). Unlike HR, aNHEJ is nonconservative, and can mediate chromosomal translocations. In this study we have found that miR223-3p decreases expression of PARP1, CtIP, and Pso4, each of which are aNHEJ components. In most cells, high levels of microRNA (miR) 223-3p repress aNHEJ, decreasing the risk of chromosomal translocations. Deletion of the miR223 locus in mice increases PARP1 levels in hematopoietic cells and enhances their risk of unprovoked chromosomal translocations. We also discovered that cancer cells deficient in BRCA1 or its obligate partner BRCA1-Associated Protein-1 (BAP1) routinely repress miR223-3p to permit repair of stressed replication forks via aNHEJ. Reconstituting the expression of miR223-3p in BRCA1- and BAP1-deficient cancer cells results in reduced repair of stressed replication forks and synthetic lethality. Thus, miR223-3p is a negative regulator of the aNHEJ DNA repair and represents a therapeutic pathway for BRCA1- or BAP1-deficient cancers.
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http://dx.doi.org/10.1073/pnas.1903150116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6717293PMC
August 2019

Efficacy of EGFR plus TNF inhibition in a preclinical model of temozolomide-resistant glioblastoma.

Neuro Oncol 2019 12;21(12):1529-1539

Department of Neurology and Neurotherapeutics, Division of Hematology-Oncology, Dallas, Texas.

Background: Glioblastoma (GBM) is the most common primary malignant adult brain tumor. Temozolomide (TMZ) is the standard of care and is most effective in GBMs that lack the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). Moreover, even initially responsive tumors develop a secondary resistance to TMZ and become untreatable. Since aberrant epidermal growth factor receptor (EGFR) signaling is widespread in GBM, EGFR inhibition has been tried in multiple clinical trials without success. We recently reported that inhibiting EGFR leads to increased secretion of tumor necrosis factor (TNF) and activation of a survival pathway in GBM. Here, we compare the efficacy of TMZ versus EGFR plus TNF inhibition in an orthotopic mouse model of GBM.

Methods: We use an orthotopic model to examine the efficacy of TMZ versus EGFR plus TNF inhibition in multiple subsets of GBMs, including MGMT methylated and unmethylated primary GBMs, recurrent GBMs, and GBMs rendered experimentally resistant to TMZ.

Results: The efficacy of the 2 treatments was similar in MGMT methylated GBMs. However, in MGMT unmethylated GBMs, a combination of EGFR plus TNF inhibition was more effective. We demonstrate that the 2 treatment approaches target distinct and non-overlapping pathways. Thus, importantly, EGFR plus TNF inhibition remains effective in TMZ-resistant recurrent GBMs and in GBMs rendered experimentally resistant to TMZ.

Conclusion: EGFR inhibition combined with a blunting of the accompanying TNF-driven adaptive response could be a viable therapeutic approach in MGMT unmethylated and recurrent EGFR-expressing GBMs.
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http://dx.doi.org/10.1093/neuonc/noz127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6917414PMC
December 2019

Radiation-Induced DNA Damage Cooperates with Heterozygosity of TP53 and PTEN to Generate High-Grade Gliomas.

Cancer Res 2019 07 14;79(14):3749-3761. Epub 2019 May 14.

Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas.

Glioblastomas are lethal brain tumors that are treated with conventional radiation (X-rays and gamma rays) or particle radiation (protons and carbon ions). Paradoxically, radiation is also a risk factor for GBM development, raising the possibility that radiotherapy of brain tumors could promote tumor recurrence or trigger secondary gliomas. In this study, we determined whether tumor suppressor losses commonly displayed by patients with GBM confer susceptibility to radiation-induced glioma. Mice with Nestin-Cre-driven deletions of and alleles were intracranially irradiated with X-rays or charged particles of increasing atomic number and linear energy transfer (LET). Mice with loss of one allele each of and did not develop spontaneous gliomas, but were highly susceptible to radiation-induced gliomagenesis. Tumor development frequency after exposure to high-LET particle radiation was significantly higher compared with X-rays, in accordance with the irreparability of DNA double-strand breaks (DSB) induced by high-LET radiation. All resultant gliomas, regardless of radiation quality, presented histopathologic features of grade IV lesions and harbored populations of cancer stem-like cells with tumor-propagating properties. Furthermore, all tumors displayed concomitant loss of heterozygosity of and along with frequent amplification of the receptor tyrosine kinase, which conferred a stem cell phenotype to tumor cells. Our results demonstrate that radiation-induced DSBs cooperate with preexisting tumor suppressor losses to generate high-grade gliomas. Moreover, our mouse model can be used for studies on radiation-induced development of GBM and therapeutic strategies. SIGNIFICANCE: This study uncovers mechanisms by which ionizing radiation, especially particle radiation, promote GBM development or recurrence.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-0680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635038PMC
July 2019

TNF-driven adaptive response mediates resistance to EGFR inhibition in lung cancer.

J Clin Invest 2018 06 7;128(6):2500-2518. Epub 2018 May 7.

Department of Neurology and Neurotherapeutics.

Although aberrant EGFR signaling is widespread in cancer, EGFR inhibition is effective only in a subset of non-small cell lung cancer (NSCLC) with EGFR activating mutations. A majority of NSCLCs express EGFR wild type (EGFRwt) and do not respond to EGFR inhibition. TNF is a major mediator of inflammation-induced cancer. We find that a rapid increase in TNF level is a universal adaptive response to EGFR inhibition in NSCLC, regardless of EGFR status. EGFR signaling actively suppresses TNF mRNA levels by inducing expression of miR-21, resulting in decreased TNF mRNA stability. Conversely, EGFR inhibition results in loss of miR-21 and increased TNF mRNA stability. In addition, TNF-induced NF-κB activation leads to increased TNF transcription in a feed-forward loop. Inhibition of TNF signaling renders EGFRwt-expressing NSCLC cell lines and an EGFRwt patient-derived xenograft (PDX) model highly sensitive to EGFR inhibition. In EGFR-mutant oncogene-addicted cells, blocking TNF enhances the effectiveness of EGFR inhibition. EGFR plus TNF inhibition is also effective in NSCLC with acquired resistance to EGFR inhibition. We suggest concomitant EGFR and TNF inhibition as a potentially new treatment approach that could be beneficial for a majority of lung cancer patients.
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http://dx.doi.org/10.1172/JCI96148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983340PMC
June 2018

BRD4 Promotes DNA Repair and Mediates the Formation of TMPRSS2-ERG Gene Rearrangements in Prostate Cancer.

Cell Rep 2018 01;22(3):796-808

Department of Pathology, UT Southwestern Medical Center, Dallas, TX 75390, USA; Department of Urology, UT Southwestern Medical Center, Dallas, TX 75390, USA; Harold C. Simmons Comprehensive Cancer Center, UT Southwestern Medical Center, Dallas, TX 75390, USA. Electronic address:

BRD4 belongs to the bromodomain and extraterminal (BET) family of chromatin reader proteins that bind acetylated histones and regulate gene expression. Pharmacological inhibition of BRD4 by BET inhibitors (BETi) has indicated antitumor activity against multiple cancer types. We show that BRD4 is essential for the repair of DNA double-strand breaks (DSBs) and mediates the formation of oncogenic gene rearrangements by engaging the non-homologous end joining (NHEJ) pathway. Mechanistically, genome-wide DNA breaks are associated with enhanced acetylation of histone H4, leading to BRD4 recruitment, and stable establishment of the DNA repair complex. In support of this, we also show that, in clinical tumor samples, BRD4 protein levels are negatively associated with outcome after prostate cancer (PCa) radiation therapy. Thus, in addition to regulating gene expression, BRD4 is also a central player in the repair of DNA DSBs, with significant implications for cancer therapy.
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http://dx.doi.org/10.1016/j.celrep.2017.12.078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5843368PMC
January 2018

EGFR Mutations Compromise Hypoxia-Associated Radiation Resistance through Impaired Replication Fork-Associated DNA Damage Repair.

Mol Cancer Res 2017 11 11;15(11):1503-1516. Epub 2017 Aug 11.

Department of Oncologic Sciences, University of South Alabama Mitchell Cancer Institute, Mobile, Alabama.

EGFR signaling has been implicated in hypoxia-associated resistance to radiation or chemotherapy. Non-small cell lung carcinomas (NSCLC) with activating L858R or ΔE746-E750 EGFR mutations exhibit elevated EGFR activity and downstream signaling. Here, relative to wild-type (WT) EGFR, mutant (MT) EGFR expression significantly increases radiosensitivity in hypoxic cells. Gene expression profiling in human bronchial epithelial cells (HBEC) revealed that MT-EGFR expression elevated transcripts related to cell cycle and replication in aerobic and hypoxic conditions and downregulated RAD50, a critical component of nonhomologous end joining and homologous recombination DNA repair pathways. NSCLCs and HBEC with MT-EGFR revealed elevated basal and hypoxia-induced γ-H2AX-associated DNA lesions that were coincident with replication protein A in the S-phase nuclei. DNA fiber analysis showed that, relative to WT-EGFR, MT-EGFR NSCLCs harbored significantly higher levels of stalled replication forks and decreased fork velocities in aerobic and hypoxic conditions. EGFR blockade by cetuximab significantly increased radiosensitivity in hypoxic cells, recapitulating MT-EGFR expression and closely resembling synthetic lethality of PARP inhibition. This study demonstrates that within an altered DNA damage response of hypoxic NSCLC cells, mutant EGFR expression, or EGFR blockade by cetuximab exerts a synthetic lethality effect and significantly compromises radiation resistance in hypoxic tumor cells. .
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http://dx.doi.org/10.1158/1541-7786.MCR-17-0136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5668182PMC
November 2017

Androgen Receptor Variants Mediate DNA Repair after Prostate Cancer Irradiation.

Cancer Res 2017 09 28;77(18):4745-4754. Epub 2017 Jul 28.

Department of Urology, University of Texas Southwestern Medical Center, Dallas, Texas.

In prostate cancer, androgen deprivation therapy (ADT) enhances the cytotoxic effects of radiotherapy. This effect is associated with weakening of the DNA damage response (DDR) normally supported by the androgen receptor. As a significant number of patients will fail combined ADT and radiotherapy, we hypothesized that DDR may be driven by androgen receptor splice variants (ARV) induced by ADT. Investigating this hypothesis, we found that ARVs increase the clonogenic survival of prostate cancer cells after irradiation in an ADT-independent manner. Notably, prostate cancer cell irradiation triggers binding of ARV to the catalytic subunit of the critical DNA repair kinase DNA-PK. Pharmacologic inhibition of DNA-PKc blocked this interaction, increased DNA damage, and elevated prostate cancer cell death after irradiation. Our findings provide a mechanistic rationale for therapeutic targeting of DNA-PK in the context of combined ADT and radiotherapy as a strategy to radiosensitize clinically localized prostate cancer. .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-0164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5600864PMC
September 2017

A TNF-JNK-Axl-ERK signaling axis mediates primary resistance to EGFR inhibition in glioblastoma.

Nat Neurosci 2017 Aug 12;20(8):1074-1084. Epub 2017 Jun 12.

Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

Aberrant epidermal growth factor receptor (EGFR) signaling is widespread in cancer, making the EGFR an important target for therapy. EGFR gene amplification and mutation are common in glioblastoma (GBM), but EGFR inhibition has not been effective in treating this tumor. Here we propose that primary resistance to EGFR inhibition in glioma cells results from a rapid compensatory response to EGFR inhibition that mediates cell survival. We show that in glioma cells expressing either EGFR wild type or the mutant EGFRvIII, EGFR inhibition triggers a rapid adaptive response driven by increased tumor necrosis factor (TNF) secretion, which leads to activation in turn of c-Jun N-terminal kinase (JNK), the Axl receptor tyrosine kinase and extracellular signal-regulated kinases (ERK). Inhibition of this adaptive axis at multiple nodes rendered glioma cells with primary resistance sensitive to EGFR inhibition. Our findings provide a possible explanation for the failures of anti-EGFR therapy in GBM and suggest a new approach to the treatment of EGFR-expressing GBM using a combination of EGFR and TNF inhibition.
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http://dx.doi.org/10.1038/nn.4584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5529219PMC
August 2017

DNA-damage-induced degradation of EXO1 exonuclease limits DNA end resection to ensure accurate DNA repair.

J Biol Chem 2017 06 17;292(26):10779-10790. Epub 2017 May 17.

From the Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas 75390,

End resection of DNA double-strand breaks (DSBs) to generate 3'-single-stranded DNA facilitates DSB repair via error-free homologous recombination (HR) while stymieing repair by the error-prone non-homologous end joining (NHEJ) pathway. Activation of DNA end resection involves phosphorylation of the 5' to 3' exonuclease EXO1 by the phosphoinositide 3-kinase-like kinases ATM (ataxia telangiectasia-mutated) and ATR (ATM and Rad3-related) and by the cyclin-dependent kinases 1 and 2. After activation, EXO1 must also be restrained to prevent over-resection that is known to hamper optimal HR and trigger global genomic instability. However, mechanisms by which EXO1 is restrained are still unclear. Here, we report that EXO1 is rapidly degraded by the ubiquitin-proteasome system soon after DSB induction in human cells. ATR inhibition attenuated DNA-damage-induced EXO1 degradation, indicating that ATR-mediated phosphorylation of EXO1 targets it for degradation. In accord with these results, EXO1 became resistant to degradation when its SQ motifs required for ATR-mediated phosphorylation were mutated. We show that upon the induction of DNA damage, EXO1 is ubiquitinated by a member of the Skp1-Cullin1-F-box (SCF) family of ubiquitin ligases in a phosphorylation-dependent manner. Importantly, expression of degradation-resistant EXO1 resulted in hyper-resection, which attenuated both NHEJ and HR and severely compromised DSB repair resulting in chromosomal instability. These findings indicate that the coupling of EXO1 activation with its eventual degradation is a timing mechanism that limits the extent of DNA end resection for accurate DNA repair.
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http://dx.doi.org/10.1074/jbc.M116.772475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5491765PMC
June 2017

MET signaling promotes DNA repair and radiation resistance in glioblastoma stem-like cells.

Ann Transl Med 2017 Feb;5(3):61

Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX, USA.

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http://dx.doi.org/10.21037/atm.2017.01.67DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5326644PMC
February 2017

Enhanced dependency of KRAS-mutant colorectal cancer cells on RAD51-dependent homologous recombination repair identified from genetic interactions in Saccharomyces cerevisiae.

Mol Oncol 2017 05 27;11(5):470-490. Epub 2017 Mar 27.

Signal Transduction Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Australia.

Activating KRAS mutations drive colorectal cancer tumorigenesis and influence response to anti-EGFR-targeted therapy. Despite recent advances in understanding Ras signaling biology and the revolution in therapies for melanoma using BRAF inhibitors, no targeted agents have been effective in KRAS-mutant cancers, mainly due to activation of compensatory pathways. Here, by leveraging the largest synthetic lethal genetic interactome in yeast, we identify that KRAS-mutated colorectal cancer cells have augmented homologous recombination repair (HRR) signaling. We found that KRAS mutation resulted in slowing and stalling of the replication fork and accumulation of DNA damage. Moreover, we found that KRAS-mutant HCT116 cells have an increase in MYC-mediated RAD51 expression with a corresponding increase in RAD51 recruitment to irradiation-induced DNA double-strand breaks (DSBs) compared to genetically complemented isogenic cells. MYC depletion using RNA interference significantly reduced IR-induced RAD51 foci formation and HRR. On the contrary, overexpression of either HA-tagged wild-type (WT) MYC or phospho-mutant S62A increased RAD51 protein levels and hence IR-induced RAD51 foci. Likewise, depletion of RAD51 selectively induced apoptosis in HCT116-mutant cells by increasing DSBs. Pharmacological inhibition targeting HRR signaling combined with PARP inhibition selectivity killed KRAS-mutant cells. Interestingly, these differences were not seen in a second isogenic pair of KRAS WT and mutant cells (DLD-1), likely due to their nondependency on the KRAS mutation for survival. Our data thus highlight a possible mechanism by which KRAS-mutant-dependent cells drive HRR in vitro by upregulating MYC-RAD51 expression. These data may offer a promising therapeutic vulnerability in colorectal cancer cells harboring otherwise nondruggable KRAS mutations, which warrants further investigation in vivo.
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http://dx.doi.org/10.1002/1878-0261.12040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5527460PMC
May 2017

Endonuclease EEPD1 Is a Gatekeeper for Repair of Stressed Replication Forks.

J Biol Chem 2017 02 3;292(7):2795-2804. Epub 2017 Jan 3.

the Department of Medicine and the Cancer Center, University of Florida Health, Gainesville, Florida 32610, and

Replication is not as continuous as once thought, with DNA damage frequently stalling replication forks. Aberrant repair of stressed replication forks can result in cell death or genome instability and resulting transformation to malignancy. Stressed replication forks are most commonly repaired via homologous recombination (HR), which begins with 5' end resection, mediated by exonuclease complexes, one of which contains Exo1. However, Exo1 requires free 5'-DNA ends upon which to act, and these are not commonly present in non-reversed stalled replication forks. To generate a free 5' end, stalled replication forks must therefore be cleaved. Although several candidate endonucleases have been implicated in cleavage of stalled replication forks to permit end resection, the identity of such an endonuclease remains elusive. Here we show that the 5'-endonuclease EEPD1 cleaves replication forks at the junction between the lagging parental strand and the unreplicated DNA parental double strands. This cleavage creates the structure that Exo1 requires for 5' end resection and HR initiation. We observed that EEPD1 and Exo1 interact constitutively, and Exo1 repairs stalled replication forks poorly without EEPD1. Thus, EEPD1 performs a gatekeeper function for replication fork repair by mediating the fork cleavage that permits initiation of HR-mediated repair and restart of stressed forks.
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http://dx.doi.org/10.1074/jbc.M116.758235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5314175PMC
February 2017

INT6/EIF3E Controls the RNF8-Dependent Ubiquitylation Pathway and Facilitates DNA Double-Strand Break Repair in Human Cells.

Cancer Res 2016 10 22;76(20):6054-6065. Epub 2016 Aug 22.

Laboratory of Biology and Modelling of the Cell, CNRS UMR 5239, INSERM U1210, ENS de Lyon, University of Lyon, Lyon, France.

Unrepaired DNA double-strand breaks (DSB) are the most destructive chromosomal lesions driving genomic instability, a core hallmark of cancer. Here, we identify the antioncogenic breast cancer factor INT6/EIF3E as an essential regulator of DSB repair that promotes homologous recombination (HR)-mediated repair and, to a lesser extent, nonhomologous end-joining repair. INT6 silencing impaired the accrual of the ubiquitin ligase RNF8 at DSBs and the formation of ubiquitin conjugates at DSB sites, especially Lys63-linked polyubiquitin chains, resulting in impaired recruitment of BRCA1, BRCA2, and RAD51, which are all involved in HR repair. In contrast, INT6 deficiency did not affect the accumulation of RNF168, 53BP1, or RPA at DSBs. In INT6-silenced cells, there was also an alteration in DNA damage-induced localization of MDC1, a key target for ATM phosphorylation, which is a prerequisite for RNF8 recruitment. The attenuated DNA damage localization of RNF8 resulting from INT6 depletion could be attributed to the defective retention of ATM previously reported by us. Our findings deepen insights into how INT6 protects against breast cancer by showing how it functions in DSB repair, with potential clinical implications for cancer therapy. Cancer Res; 76(20); 6054-65. ©2016 AACR.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5065779PMC
http://dx.doi.org/10.1158/0008-5472.CAN-16-0723DOI Listing
October 2016

Augmented HR Repair Mediates Acquired Temozolomide Resistance in Glioblastoma.

Mol Cancer Res 2016 10 29;14(10):928-940. Epub 2016 Jun 29.

Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults and is universally fatal. The DNA alkylating agent temozolomide is part of the standard-of-care for GBM. However, these tumors eventually develop therapy-driven resistance and inevitably recur. While loss of mismatch repair (MMR) and re-expression of MGMT have been shown to underlie chemoresistance in a fraction of GBMs, resistance mechanisms operating in the remaining GBMs are not well understood. To better understand the molecular basis for therapy-driven temozolomide resistance, mice bearing orthotopic GBM xenografts were subjected to protracted temozolomide treatment, and cell lines were generated from the primary (untreated) and recurrent (temozolomide-treated) tumors. As expected, the cells derived from primary tumors were sensitive to temozolomide, whereas the cells from the recurrent tumors were significantly resistant to the drug. Importantly, the acquired resistance to temozolomide in the recurrent lines was not driven by re-expression of MGMT or loss of MMR but was due to accelerated repair of temozolomide-induced DNA double-strand breaks (DSB). Temozolomide induces DNA replication-associated DSBs that are primarily repaired by the homologous recombination (HR) pathway. Augmented HR appears to underpin temozolomide resistance in the recurrent lines, as these cells were cross-resistant to other agents that induced replication-associated DSBs, exhibited faster resolution of damage-induced Rad51 foci, and displayed higher levels of sister chromatid exchanges (SCE). Furthermore, in light of recent studies demonstrating that CDK1 and CDK2 promote HR, it was found that CDK1/2 inhibitors countered the heightened HR in recurrent tumors and sensitized these therapy-resistant tumor cells to temozolomide.

Implications: Augmented HR repair is a novel mechanism underlying acquired temozolomide resistance in GBM, and this raises the possibility of improving the therapeutic response to temozolomide by targeting HR with small-molecule inhibitors of CDK1/2. Mol Cancer Res; 14(10); 928-40. ©2016 AACR.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5065752PMC
http://dx.doi.org/10.1158/1541-7786.MCR-16-0125DOI Listing
October 2016

Mouse models of radiation-induced glioblastoma.

Oncoscience 2015 28;2(12):934-5. Epub 2015 Dec 28.

Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX, USA.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4741397PMC
http://dx.doi.org/10.18632/oncoscience.278DOI Listing
February 2016

EEPD1 Rescues Stressed Replication Forks and Maintains Genome Stability by Promoting End Resection and Homologous Recombination Repair.

PLoS Genet 2015 Dec 18;11(12):e1005675. Epub 2015 Dec 18.

Department of Medicine and the Cancer Center, University of Florida Health, Gainesville, Florida, United States of America.

Replication fork stalling and collapse is a major source of genome instability leading to neoplastic transformation or cell death. Such stressed replication forks can be conservatively repaired and restarted using homologous recombination (HR) or non-conservatively repaired using micro-homology mediated end joining (MMEJ). HR repair of stressed forks is initiated by 5' end resection near the fork junction, which permits 3' single strand invasion of a homologous template for fork restart. This 5' end resection also prevents classical non-homologous end-joining (cNHEJ), a competing pathway for DNA double-strand break (DSB) repair. Unopposed NHEJ can cause genome instability during replication stress by abnormally fusing free double strand ends that occur as unstable replication fork repair intermediates. We show here that the previously uncharacterized Exonuclease/Endonuclease/Phosphatase Domain-1 (EEPD1) protein is required for initiating repair and restart of stalled forks. EEPD1 is recruited to stalled forks, enhances 5' DNA end resection, and promotes restart of stalled forks. Interestingly, EEPD1 directs DSB repair away from cNHEJ, and also away from MMEJ, which requires limited end resection for initiation. EEPD1 is also required for proper ATR and CHK1 phosphorylation, and formation of gamma-H2AX, RAD51 and phospho-RPA32 foci. Consistent with a direct role in stalled replication fork cleavage, EEPD1 is a 5' overhang nuclease in an obligate complex with the end resection nuclease Exo1 and BLM. EEPD1 depletion causes nuclear and cytogenetic defects, which are made worse by replication stress. Depleting 53BP1, which slows cNHEJ, fully rescues the nuclear and cytogenetic abnormalities seen with EEPD1 depletion. These data demonstrate that genome stability during replication stress is maintained by EEPD1, which initiates HR and inhibits cNHEJ and MMEJ.
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http://dx.doi.org/10.1371/journal.pgen.1005675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4684289PMC
December 2015

Tumor-selective use of DNA base excision repair inhibition in pancreatic cancer using the NQO1 bioactivatable drug, β-lapachone.

Sci Rep 2015 Nov 25;5:17066. Epub 2015 Nov 25.

Departments of Pharmacology, Dallas, TX 75390-8807.

Unlabelled: Base excision repair (BER) is an essential pathway for pancreatic ductal adenocarcinoma (PDA) survival. Attempts to target this repair pathway have failed due to lack of tumor-selectivity and very limited efficacy. The

Nad(p)h: Quinone Oxidoreductase 1 (NQO1) bioactivatable drug, ß-lapachone (ARQ761 in clinical form), can provide tumor-selective and enhanced synergy with BER inhibition. ß-Lapachone undergoes NQO1-dependent futile redox cycling, generating massive intracellular hydrogen peroxide levels and oxidative DNA lesions that stimulate poly(ADP-ribose) polymerase 1 (PARP1) hyperactivation. Rapid NAD(+)/ATP depletion and programmed necrosis results. To identify BER modulators essential for repair of ß-lapachone-induced DNA base damage, a focused synthetic lethal RNAi screen demonstrated that silencing the BER scaffolding protein, XRCC1, sensitized PDA cells. In contrast, depleting OGG1 N-glycosylase spared cells from ß-lap-induced lethality and blunted PARP1 hyperactivation. Combining ß-lapachone with XRCC1 knockdown or methoxyamine (MeOX), an apyrimidinic/apurinic (AP)-modifying agent, led to NQO1-dependent synergistic killing in PDA, NSCLC, breast and head and neck cancers. OGG1 knockdown, dicoumarol-treatment or NQO1- cancer cells were spared. MeOX + ß-lapachone exposure resulted in elevated DNA double-strand breaks, PARP1 hyperactivation and TUNEL+ programmed necrosis. Combination treatment caused dramatic antitumor activity, enhanced PARP1-hyperactivation in tumor tissue, and improved survival of mice bearing MiaPaca2-derived xenografts, with 33% apparent cures.

Significance: Targeting base excision repair (BER) alone has limited therapeutic potential for pancreatic or other cancers due to a general lack of tumor-selectivity. Here, we present a treatment strategy that makes BER inhibition tumor-selective and NQO1-dependent for therapy of most solid neoplasms, particularly for pancreatic cancer.
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http://dx.doi.org/10.1038/srep17066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4658501PMC
November 2015

Targeting glutamine metabolism sensitizes pancreatic cancer to PARP-driven metabolic catastrophe induced by ß-lapachone.

Cancer Metab 2015 12;3:12. Epub 2015 Oct 12.

Department of Pharmacology, University of Texas Southwestern Medical Center, 6001 Forest Park Drive, Dallas, 75390-8807 TX USA ; Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX USA.

Background: Pancreatic ductal adenocarcinomas (PDA) activate a glutamine-dependent pathway of cytosolic nicotinamide adenine dinucleotide phosphate (NADPH) production to maintain redox homeostasis and support proliferation. Enzymes involved in this pathway (GLS1 (mitochondrial glutaminase 1), GOT1 (cytoplasmic glutamate oxaloacetate transaminase 1), and GOT2 (mitochondrial glutamate oxaloacetate transaminase 2)) are highly upregulated in PDA, and among these, inhibitors of GLS1 were recently deployed in clinical trials to target anabolic glutamine metabolism. However, single-agent inhibition of this pathway is cytostatic and unlikely to provide durable benefit in controlling advanced disease.

Results: Here, we report that reducing NADPH pools by genetically or pharmacologically (bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) or CB-839) inhibiting glutamine metabolism in mutant Kirsten rat sarcoma viral oncogene homolog (KRAS) PDA sensitizes cell lines and tumors to ß-lapachone (ß-lap, clinical form ARQ761). ß-Lap is an NADPH:quinone oxidoreductase (NQO1)-bioactivatable drug that leads to NADPH depletion through high levels of reactive oxygen species (ROS) from the futile redox cycling of the drug and subsequently nicotinamide adenine dinucleotide (NAD)+ depletion through poly(ADP ribose) polymerase (PARP) hyperactivation. NQO1 expression is highly activated by mutant KRAS signaling. As such, ß-lap treatment concurrent with inhibition of glutamine metabolism in mutant KRAS, NQO1 overexpressing PDA leads to massive redox imbalance, extensive DNA damage, rapid PARP-mediated NAD+ consumption, and PDA cell death-features not observed in NQO1-low, wild-type KRAS expressing cells.

Conclusions: This treatment strategy illustrates proof of principle that simultaneously decreasing glutamine metabolism-dependent tumor anti-oxidant defenses and inducing supra-physiological ROS formation are tumoricidal and that this rationally designed combination strategy lowers the required doses of both agents in vitro and in vivo. The non-overlapping specificities of GLS1 inhibitors and ß-lap for PDA tumors afford high tumor selectivity, while sparing normal tissue.
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http://dx.doi.org/10.1186/s40170-015-0137-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4601138PMC
October 2015

Concepts and challenges in cancer risk prediction for the space radiation environment.

Life Sci Space Res (Amst) 2015 Jul 17;6:92-103. Epub 2015 Jul 17.

Colorado State University, Ft. Collins, CO, USA.

Cancer is an important long-term risk for astronauts exposed to protons and high-energy charged particles during travel and residence on asteroids, the moon, and other planets. NASA's Biomedical Critical Path Roadmap defines the carcinogenic risks of radiation exposure as one of four type I risks. A type I risk represents a demonstrated, serious problem with no countermeasure concepts, and may be a potential "show-stopper" for long duration spaceflight. Estimating the carcinogenic risks for humans who will be exposed to heavy ions during deep space exploration has very large uncertainties at present. There are no human data that address risk from extended exposure to complex radiation fields. The overarching goal in this area to improve risk modeling is to provide biological insight and mechanistic analysis of radiation quality effects on carcinogenesis. Understanding mechanisms will provide routes to modeling and predicting risk and designing countermeasures. This white paper reviews broad issues related to experimental models and concepts in space radiation carcinogenesis as well as the current state of the field to place into context recent findings and concepts derived from the NASA Space Radiation Program.
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http://dx.doi.org/10.1016/j.lssr.2015.07.006DOI Listing
July 2015

Immunofluorescence-based methods to monitor DNA end resection.

Methods Mol Biol 2015 ;1292:67-75

Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, 2201 Inwood Road, NC7.208, Dallas, TX, 75390, USA.

Double-strand breaks (DSBs) are the most deleterious among all types of DNA damage that can occur in the cell. These breaks arise from both endogenous (e.g., DNA replication stress) and exogenous insults (e.g., ionizing radiation). DSBs are principally repaired by one of two major pathways: nonhomologous end joining (NHEJ) or homologous recombination (HR). NHEJ is an error-prone process that can occur in all phases of the cell cycle, while HR is limited to the S and G2 phases of the cell cycle when a sister chromatid is available as a template for error-free repair. The first step in HR is "DNA end resection," a process during which the broken DNA end is converted into a long stretch of 3'-ended single-stranded DNA (ssDNA). In recent years, DNA end resection has been identified as a pivotal step that controls "repair pathway choice," i.e., the appropriate choice between NHEJ and HR for DSB repair. Therefore, methods to quantitatively or semiquantitatively assess DNA end resection have gained importance in laboratories working on DNA repair. In this chapter, we describe two simple immunofluorescence-based techniques to monitor DNA end resection in mammalian cells. The first technique involves immuno-detection of replication protein A (RPA), an ssDNA-binding protein that binds to resected DNA. The second technique involves labeling of genomic DNA with 5-bromo-2'-deoxyuridine (BrdU) that can be detected by anti-BrdU antibody only after the DNA becomes single stranded due to resection. These methods are not complicated, do not involve sophisticated instrumentation or reporter constructs, and can be applied to most mammalian cell lines and, therefore, should be of broad utility as simple ways of monitoring DNA end resection in vivo.
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http://dx.doi.org/10.1007/978-1-4939-2522-3_5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5482411PMC
December 2015

Phosphorylation of EXO1 by CDKs 1 and 2 regulates DNA end resection and repair pathway choice.

Nat Commun 2014 Apr 7;5:3561. Epub 2014 Apr 7.

Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, 2201 Inwood Road, NC7, 214E, Dallas, Texas 75390, USA.

Resection of DNA double-strand breaks (DSBs) is a pivotal step during which the choice between NHEJ and HR DNA repair pathways is made. Although CDKs are known to control initiation of resection, their role in regulating long-range resection remains elusive. Here we show that CDKs 1/2 phosphorylate the long-range resection nuclease EXO1 at four C-terminal S/TP sites during S/G2 phases of the cell cycle. Impairment of EXO1 phosphorylation attenuates resection, chromosomal integrity, cell survival and HR, but augments NHEJ upon DNA damage. In contrast, cells expressing phospho-mimic EXO1 are proficient in resection even after CDK inhibition and favour HR over NHEJ. Mutation of cyclin-binding sites on EXO1 attenuates CDK binding and EXO1 phosphorylation, causing a resection defect that can be rescued by phospho-mimic mutations. Mechanistically, phosphorylation of EXO1 augments its recruitment to DNA breaks possibly via interactions with BRCA1. In summary, phosphorylation of EXO1 by CDKs is a novel mechanism regulating repair pathway choice.
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http://dx.doi.org/10.1038/ncomms4561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4041212PMC
April 2014

Inhibition of DNA double-strand break repair by the dual PI3K/mTOR inhibitor NVP-BEZ235 as a strategy for radiosensitization of glioblastoma.

Clin Cancer Res 2014 Mar 23;20(5):1235-48. Epub 2013 Dec 23.

Authors' Affiliations: Departments of Radiation Oncology, Clinical Sciences, and Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center; and VA North Texas Health Care System, Dallas, Texas.

Purpose: Inhibitors of the DNA damage response (DDR) have great potential for radiosensitization of numerous cancers, including glioblastomas, which are extremely radio- and chemoresistant brain tumors. Currently, there are no DNA double-strand break (DSB) repair inhibitors that have been successful in treating glioblastoma. Our laboratory previously demonstrated that the dual phosphoinositide 3-kinase/mTOR inhibitor NVP-BEZ235 can potently inhibit the two central DDR kinases, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia-telangiectasia mutated (ATM), in vitro. Here, we tested whether NVP-BEZ235 could also inhibit ATM and DNA-PKcs in tumors in vivo and assessed its potential as a radio- and chemosensitizer in preclinical mouse glioblastoma models.

Experimental Design: The radiosensitizing effect of NVP-BEZ235 was tested by following tumor growth in subcutaneous and orthotopic glioblastoma models. Tumors were generated using the radioresistant U87-vIII glioma cell line and GBM9 neurospheres in nude mice. These tumors were then treated with ionizing radiation and/or NVP-BEZ235 and analyzed for DNA-PKcs and ATM activation, DSB repair inhibition, and attenuation of growth.

Results: NVP-BEZ235 potently inhibited both DNA-PKcs and ATM kinases and attenuated the repair of ionizing radiation-induced DNA damage in tumors. This resulted in striking tumor radiosensitization, which extended the survival of brain tumor-bearing mice. Notably, tumors displayed a higher DSB-load when compared with normal brain tissue. NVP-BEZ235 also sensitized a subset of subcutaneous tumors to temozolomide, a drug routinely used concurrently with ionizing radiation for the treatment of glioblastoma.

Conclusions: These results demonstrate that it may be possible to significantly improve glioblastoma therapy by combining ionizing radiation with potent and bioavailable DNA repair inhibitors such as NVP-BEZ235.
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http://dx.doi.org/10.1158/1078-0432.CCR-13-1607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3947495PMC
March 2014

Opposing effect of EGFRWT on EGFRvIII-mediated NF-κB activation with RIP1 as a cell death switch.

Cell Rep 2013 Aug 22;4(4):764-75. Epub 2013 Aug 22.

Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

RIP1 is a central mediator of cell death in response to cell stress but can also mediate cell survival by activating NF-κB. Here, we show that RIP1 acts as a switch in EGFR signaling. EGFRvIII is an oncogenic mutant that does not bind ligand and is coexpressed with EGFRWT in glioblastoma multiforme (GBM). EGFRvIII recruits ubiquitin ligases to RIP1, resulting in K63-linked ubiquitination of RIP1. RIP1 binds to TAK1 and NEMO, forming an EGFRvIII-RIP1 signalosome that activates NF-κB. RIP1 is essential for EGFRvIII-mediated oncogenicity and correlates with NF-κB activation in GBM. Surprisingly, activation of EGFRWT with EGF results in a negative regulation of EGFRvIII, with dissociation of the EGFRvIII-RIP1 signalosome, loss of RIP1 ubiquitination and NF-κB activation, and association of RIP1 with FADD and caspase-8. If EGFRWT is overexpressed with EGFRvIII, the addition of EGF leads to a RIP1 kinase-dependent cell death. The EGFRWT-EGFRvIII-RIP1 interplay may regulate oncogenicity and vulnerability to targeted treatment in GBM.
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http://dx.doi.org/10.1016/j.celrep.2013.07.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3780989PMC
August 2013

Cytoplasmic TRADD confers a worse prognosis in glioblastoma.

Neoplasia 2013 Aug;15(8):888-97

Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center, Dallas, TX, USA.

Tumor necrosis factor receptor 1 (TNFR1)-associated death domain protein (TRADD) is an important adaptor in TNFR1 signaling and has an essential role in nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation and survival signaling. Increased expression of TRADD is sufficient to activate NF-κB. Recent studies have highlighted the importance of NF-κB activation as a key pathogenic mechanism in glioblastoma multiforme (GBM), the most common primary malignant brain tumor in adults.We examined the expression of TRADD by immunohistochemistry (IHC) and find that TRADD is commonly expressed at high levels in GBM and is detected in both cytoplasmic and nuclear distribution. Cytoplasmic IHC TRADD scoring is significantly associated with worse progression-free survival (PFS) both in univariate and multivariate analysis but is not associated with overall survival (n = 43 GBMs). PFS is a marker for responsiveness to treatment. We propose that TRADD-mediated NF-κB activation confers chemoresistance and thus a worse PFS in GBM. Consistent with the effect on PFS, silencing TRADD in glioma cells results in decreased NF-κB activity, decreased proliferation of cells, and increased sensitivity to temozolomide. TRADD expression is common in glioma-initiating cells. Importantly, silencing TRADD in GBM-initiating stem cell cultures results in decreased viability of stem cells, suggesting that TRADD may be required for maintenance of GBM stem cell populations. Thus, our study suggests that increased expression of cytoplasmic TRADD is both an important biomarker and a key driver of NF-κB activation in GBM and supports an oncogenic role for TRADD in GBM.
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http://dx.doi.org/10.1593/neo.13608DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3730041PMC
August 2013
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