Felipe J. Nunez

ATRX loss promotes tumor growth and impairs nonhomologous end joining DNA repair in glioma

The loss of ATRX impairs DNA repair, promoting glioma growth but enhancing sensitivity to DNA-damaging therapies. Aggressive gliomas’ Achilles’ heel ATRX is a protein that is often mutated in glioma, a lethal and relatively common brain tumor. Koschmann et al. developed a mouse model of ATRX-deficient glioma and discovered that these tumors grow more aggressively than their counterparts with wild-type ATRX. The reason this happens is that the loss of ATRX impairs DNA repair, resulting in genetically unstable tumors that can accumulate oncogenic mutations more quickly. However, because of their DNA repair defect, these tumors also proved to be more sensitive to treatments that damage the DNA, such as radiation and some types of chemotherapy. Consistent with these findings, the presence of ATRX mutation correlated with better outcomes in patients, because these tumors were more susceptible to treatment. Recent work in human glioblastoma (GBM) has documented recurrent mutations in the histone chaperone protein ATRX. We developed an animal model of ATRX-deficient GBM and showed that loss of ATRX reduces median survival and increases genetic instability. Further, analysis of genome-wide data for human gliomas showed that ATRX mutation is associated with increased mutation rate at the single-nucleotide variant (SNV) level. In mouse tumors, ATRX deficiency impairs nonhomologous end joining and increases sensitivity to DNA-damaging agents that induce double-stranded DNA breaks. We propose that ATRX loss results in a genetically unstable tumor, which is more aggressive when left untreated but is more responsive to double-stranded DNA-damaging agents, resulting in improved overall survival.

Recent advances and future of immunotherapy for glioblastoma

ABSTRACT Introduction: Outcome for glioma (GBM) remains dismal despite advances in therapeutic interventions including chemotherapy, radiotherapy and surgical resection. The overall survival benefit observed with immunotherapies in cancers such as melanoma and prostate cancer has fuelled research into evaluating immunotherapies for GBM. Areas covered: Preclinical studies have brought a wealth of information for improving the prognosis of GBM and multiple clinical studies are evaluating a wide array of immunotherapies for GBM patients. This review highlights advances in the development of immunotherapeutic approaches. We discuss the strategies and outcomes of active and passive immunotherapies for GBM including vaccination strategies, gene therapy, check point blockade and adoptive T cell therapies. We also focus on immunoediting and tumor neoantigens that can impact the efficacy of immunotherapies. Expert opinion: Encouraging results have been observed with immunotherapeutic strategies; some clinical trials are reaching phase III. Significant progress has been made in unraveling the molecular and genetic heterogeneity of GBM and its implications to disease prognosis. There is now consensus related to the critical need to incorporate tumor heterogeneity into the design of therapeutic approaches. Recent data also indicates that an efficacious treatment strategy will need to be combinatorial and personalized to the tumor genetic signature.

Mutated Chromatin Regulatory Factors as Tumor Drivers in Cancer

Genes encoding proteins that regulate chromatin structure and DNA modifications [i.e., chromatin regulatory factors (CRF)] and genes encoding histone proteins harbor recurrent mutations in most human cancers. These mutations lead to modifications in tumor chromatin and DNA structure and an altered epigenetic state that contribute to tumorigenesis. Mutated CRFs have now been identified in most types of cancer and are increasingly regarded as novel therapeutic targets. In this review, we discuss DNA alterations in CRFs and how these influence tumor chromatin structure and function, which in turn leads to tumorigenesis. We also discuss the clinical implications and review concepts of targeted treatments for these mutations. Continued research on CRF mutations will be critical for our future understanding of cancer biology and the development and implementation of novel cancer therapies. Cancer Res; 77(2); 227-33. ©2017 AACR.

Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary brain tumor in adults with dismal prognosis. New potential therapies are rapidly emerging from the bench and require systematic testing in experimental models which closely reproduce the salient features of the human disease. Herein we describe in detail a method to induce new models of GBM with transposon-mediated integration of plasmid DNA into cells of the subventricular zone of neonatal mice. We present a simple way to clone new transposons amenable for genomic integration using the Sleeping Beauty transposon system and illustrate how to monitor plasmid uptake and disease progression using bioluminescence, histology and immuno-histochemistry. We also describe a method to create new primary GBM cell lines. Ideally, this report will allow further dissemination of the Sleeping Beauty transposon system among brain tumor researchers, leading to an in depth understanding of GBM pathogenesis and progression and to the timely design and testing of effective therapies for patients.

Current state and future prospects of immunotherapy for glioma

There is a large unmet need for effective therapeutic approaches for glioma, the most malignant brain tumor. Clinical and preclinical studies have enormously expanded our knowledge about the molecular aspects of this deadly disease and its interaction with the host immune system. In this review we highlight the wide array of immunotherapeutic interventions that are currently being tested in glioma patients. Given the molecular heterogeneity, tumor immunoediting and the profound immunosuppression that characterize glioma, it has become clear that combinatorial approaches targeting multiple pathways tailored to the genetic signature of the tumor will be required in order to achieve optimal therapeutic efficacy.

IDH1R132H acts as a tumor suppressor in glioma via epigenetic upregulation of the DNA damage response

One sentence summary Mutant IDH1 acts as a tumor suppressor when co-expressed together with TP53 and ATRX inactivating mutations in glioma, inducing genomic stability, DNA repair and resistance to genotoxic therapies. Abstract Glioma patients whose tumors carry a mutation in the Isocitrate Dehydrogenase 1 (IDH1R132H) gene are younger at the time of diagnosis and survive longer. The molecular glioma subtype which we modelled, harbors IDH1R132H, tumor protein 53 (TP53) and alpha-thalassemia/mental retardation syndrome X-linked (ATRX) loss. The impact of IDH1R132H on genomic stability, DNA damage response (DDR) and DNA repair in this molecular glioma subtype is unknown. We discovered that IDH1R132H expression in the genetic context of ATRX and TP53 inactivation: (i) increases median survival (MS), (ii) enhances DDR activity via epigenetic upregulation of Ataxia-telangiectasia mutated (ATM) signaling, and (iii) elicits tumor radioresistance. Pharmacological inhibition of ATM or checkpoint kinase 1 and 2 (CHK1/2), two essential kinases in the DDR pathways, restored tumors’ radiosensitivity. Translation of these findings for mlDH1 glioma patients could significantly improve the therapeutic efficacy of radiotherapy, and thus have a major impact on patient survival.

Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory regions of oncogenes and tumor suppressors can lead to changes in gene expression and play an important role in the pathogenesis of brain tumors. Native chromatin immunoprecipitation (ChIP) is a popular technique that allows the detection of modifications or other proteins tightly bound to DNA. In contrast to cross-linked ChIP, in native ChIP, cells are not treated with formaldehyde to covalently link protein to DNA. This is advantageous because sometimes crosslinking may fix proteins that only transiently interact with DNA and do not have functional significance in gene regulation. In addition, antibodies are generally raised against unfixed peptides. Therefore, antibody specificity is increased in native ChIP. However, it is important to keep in mind that native ChIP is only applicable to study histones or other proteins that bind tightly to DNA. This protocol describes the native chromatin immunoprecipitation on murine brain tumor neurospheres.

Mutant ATRX: uncovering a new therapeutic target for glioma

ABSTRACT Introduction: ATRX is a chromatin remodeling protein whose main function is the deposition of the histone variant H3.3. ATRX mutations are widely distributed in glioma, and correlate with alternative lengthening of telomeres (ALT) development, but they also affect other cellular functions related to epigenetic regulation. Areas covered: We discuss the main molecular characteristics of ATRX, from its various functions in normal development to the effects of its loss in ATRX syndrome patients and animal models. We focus on the salient consequences of ATRX mutations in cancer, from a clinical to a molecular point of view, focusing on both adult and pediatric glioma. Finally, we will discuss the therapeutic opportunities future research perspectives. Expert opinion: ATRX is a major component of various essential cellular pathways, exceeding its functions as a histone chaperone (e.g. DNA replication and repair, chromatin higher-order structure regulation, gene transcriptional regulation, etc.). However, it is unclear how the loss of these functions in ATRX-null cancer cells affects cancer development and progression. We anticipate new treatments and clinical approaches will emerge for glioma and other cancer types as mechanistic and molecular studies on ATRX are only just beginning to reveal the many critical functions of this protein in cancer.

Abstract 3009: ATRX validated as tumor suppressor in a novel mouse model of pediatric and young adult GBM

Pediatric Glioblastoma (GBM) remains one of the most difficult childhood tumors to treat, and most children with this diagnosis will not survive longer than two years. ATRX is a histone chaperone protein that is mutated primarily in pediatric patients with GBM and younger adults with secondary GBM. No previous animal model has demonstrated the effect of ATRX loss on GBM formation. We cloned an ATRX knockdown sequence into a Sleeping Beauty (SB) transposase-responsive plasmid (shATRX) for insertion into host genomic DNA. Glioblastomas were induced in neonatal mice by injecting plasmids encoding SB transposase/ luciferase, shp53 and NRAS, with or without shATRX, into the ventricle of neonatal mice. Tumors in both groups (with or without shATRX) showed histological hallmarks of human glioblastoma. The loss of ATRX was specifically localized only within tumors generated with the shATRX plasmid and not in the adjacent cortex. Notably, loss of ATRX reduced median survival of mice by 43% (p = 0.012). ATRX-deficient tumors displayed evidence of telomeric lengthening using telomeric FISH assay for alternative lengthening of telomeres (ALT). ATRX-deficient tumors were significantly more likely to develop microsatellite instability (p = 0.014), a hallmark of impaired DNA-damage repair. Analysis of three human GBM sequencing datasets confirmed increased number of somatic nucleotide mutations in ATRX-deficient tumors. Treatment of primary cell cultures generated from mouse GBMs showed that ATRX-deficient tumor cells are significantly more sensitive to certain DNA damaging agents, with greater evidence of double-stranded DNA breakage, by gH2A.X. In addition, mice with ATRX-deficient GBM treated with whole brain irradiation showed reduced tumor growth by luminescence, with some long-term survivors. In summary, this mouse model prospectively validates ATRX as a tumor suppressor in human GBM for the first time in an animal model. In addition, loss of ATRX leads to increased genetic instability and response to DNA-damaging therapy. Based on these results, we have generated the hypothesis that ATRX loss leads to a genetically unstable tumor; which is more aggressive when untreated, but more responsive to DNA-damaging therapy, ultimately resulting in equivalent or improved overall survival. Supported by St. Baldrick9s Fellowship and Alex9s Lemonade Stand /Northwest Mutual Young Investigator Award to CK and NIH/NINDS grants to MGC and PRL. Citation Format: Carl Koschmann, Alexandra Calinescu, Daniel Thomas, Felipe J. Nunez, Marta Dzaman, Johnny Krasinkiewicz, Rosie Lemons, Neha Kamran, Flor Mendez, Soyeon Roh, David Ferguson, Pedro R. Lowenstein, Maria G. Castro. ATRX validated as tumor suppressor in a novel mouse model of pediatric and young adult GBM. [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 3009. doi:10.1158/1538-7445.AM2015-3009

Abstract 3033: Generation of a mouse model of young adult glioblatoma: In vivo expression of mutated IDH1-R132H gene using the sleeping beauty transposase system

Background: Glioblastoma Multiforme (GBM) is a malignant primary brain tumor. Genomic analysis of GBMs revealed somatic mutations associated with particular subtypes, which correlate with age at the time of diagnosis. Distinctive features of young adult GBMs include a mutation in Isocitrate Dehydrogenase 1 gene (IDH1-R132H) and mutations in epigenetic regulators, ATRX and H3.3G34, which define an epigenetic and biological subgroup. IDH1-R132H results in gain of an alternative enzymatic function producing an excess of the metabolite 2-hydroxglutarate (2HG) and alterations in histone methylation patterns. Animal models are critical to study and understand disease progression and pathogenesis; therefore we aimed to develop an in vivo model of young adult GBM. Objectives: Generate an animal model of young adult GBM through expression of IDH1-R132H, H3.G34 and ATRX knockdown in neonatal mouse brain using the Sleeping Beauty Transposase system. In this model we will study the effects of these genetic lesions in tumor development and DNA repair mechanisms. Experimental approach: To induce tumors we injected NRAS and shP53 plasmids in the lateral ventricle of C57BL/6 neonatal mice with or without pKT/IDH1-R132H, Pkt/H3.G34 or PT2/shATRX using the sleeping beauty transpose system. The delivery of the plasmid was confirmed by luciferase expression and tumor development was monitored by imaging. The tumors were also used to generate GBM cell for genetic analysis and moribund animals were perfused and analyzed using immunocytochemistry. Results: Pkt-IDH1-R132H expression increases the levels of H3K27me3. Currently we have five groups of animals to asses tumor progression: 1) NRAS/shP53 (n = 7) (median survival (MS) = 88 days post injection (DPI); 2) NRAS/shP53/shATRX (n = 7) (MS = 50 DPI); 3) NRAS/shP53/shATRX/IDH1-R132H (n = 18) (MS = 90 DPI); 4) NRAS/shP53/IDH1-R132H (n = 16) (MS = 100 DPI) and 5) NRAS/shP53/shATRX/H3.G34 (n = 5). Conclusion: Using the Sleeping Beauty System (SB) we expressed mutated IDH1-R132H or mutated H3.G34 together with ATRX knockdown in mice. Genetically engineering mice with IDH1-R132H have increased survival. Mice developed brain tumors harboring key features of young adult GBMs. We aim to determine the impact of these genetic lesions in median survival, tumor progression and DNA repair “in vivo”. Note: This abstract was not presented at the meeting. Citation Format: Felipe J. Nunez, Flor M. Mendez, Carl Koschmann, Alexandra Calinescu, Pedro R. Lowenstein, Maria G. Castro. Generation of a mouse model of young adult glioblatoma: In vivo expression of mutated IDH1-R132H gene using the sleeping beauty transposase system. [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 3033. doi:10.1158/1538-7445.AM2015-3033