Marta Dzaman

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.

Gene Therapy for the Treatment of Neurological Disorders: Central Nervous System Neoplasms.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults with a median survival of 16.2-21.2 months post diagnosis (Stupp et al., N Engl J Med 352(10): 987-996, 2005). Because of its location, complete surgical resection is impossible; additionally because GBM is also resistant to chemotherapeutic and radiotherapy approaches, development of novel therapies is urgently needed. In this chapter we describe the development of preclinical animal models and a conditionally cytotoxic and immune-stimulatory gene therapy strategy that successfully causes tumor regression in several rodent GBM models.

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 457: Depletion of glioma infiltrating myeloid derived suppressor cells promotes anti-tumor T cell responses

MDSCs represent a population of immature myeloid cells at various stages of differentiation that have the potential to inhibit anti-tumor T cell immunity. We demonstrate the accumulation of MDSCs in GL26 and M7-induced glioma (GBM) bearing mice. Absolute numbers of Ly-6G+ (Gr-1high) MDSCs showed a 200 fold increase within the tumor microenvironment (TME) 28 days post-tumor implantation. The numbers of Ly-6C+ (Gr-1low) MDSCs also showed a similar trend within the TME. While this massive influx of MDSCs was noted within intracranial tumors, MDSC levels did not increase in the dLNs, spleen or bone marrow (BM) of intracranial tumor bearing mice. MDSC numbers were significantly elevated in the blood of GL26 and M7 intracranial tumor bearing mice at 28 days. While both Gr-1high and Gr-1low MDSCs isolated from the TME of GL26 intracranial tumor bearing mice inhibited antigen-specific T cell proliferation, Gr-1low MDSC were found to be more efficient. Gr-1high or Gr-1low MDSCs from the bone marrow of intracranial tumor bearing mice failed to suppress antigen-specific T cell proliferation suggesting that TME derived factors may activate MDSCs to exert their immune-suppressive properties. In vivo, depletion of Gr-1+ cells enhanced the median survival of GBM bearing mice. Furthermore, when combined with Ad-TK + Ad-FLT3L immune-gene therapy, Gr-1+ depletion significantly enhanced the frequency of tumor-specific T cells within the TME and spleen and increased IFN-γ production by splenic T cells. Our data therefore indicates that inhibiting the accumulation of MDSCs with in the GBM TME promotes the generation of robust anti-tumor immunity. Preliminary experiments to determine the mechanism of MDSC trafficking to the TME point towards the receptor CXCR2 and its ligand CXCL1. Microarray analysis of glioma cell lines showed elevated levels of CXCL1 mRNA. Additionally a culture of primary mixed glial cells also produced CXCL1 when stimulated with GBM cell lysates. SB225002, a CXCR2 inhibitor suppressed the migration of MDSCs towards GBM cells in an in vitro migration assay. Overall, our data suggests that strategies that inhibit MDSC recruitment to the GBM TME and/or block their activity could enhance the T cell mediated tumor clearance and provide survival benefit. Work supported by grants from NIH-NINDS Citation Format: Neha Kamran, Youping Li, Mariela Moreno-Ayala, Hikmat Assi, Marianela Candolfi, Marta Dzaman, Pedro Lowenstein, Maria Castro. Depletion of glioma infiltrating myeloid derived suppressor cells promotes anti-tumor T cell responses. [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 457. doi:10.1158/1538-7445.AM2015-457

Abstract 995: Loss of ATRX decreases survival and improves response to DNA damaging agents in a novel mouse model of glioblastoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Pediatric glioblastoma (GBM) remains one of the most difficult childhood tumors to treat, and most patients will die within the first two years of receiving this diagnosis. ATRX is a histone chaperone protein that is mutated primarily in adolescent GBMs. No previous animal model has demonstrated the effect of ATRX loss on GBM formation. In this study, we determined the contribution of ATRX knockdown to GBM formation and treatment response in a novel mouse model of GBM. Using the second-generation shRNA-mir library, we cloned an ATRX knockdown sequence into a plasmid with flanking sequences recognized by the Sleeping Beauty (SB) transposase for insertion into host genomic DNA. Glioblastomas were induced in mice using the SB transposase system injecting plasmids encoding luciferase, shp53 and NRAS, with or without shATRX, into the ventricle of neonatal mice. Uptake of plasmid DNA as well as development of intracranial tumors was monitored by bioluminescence. When animals showed symptoms of tumor burden they were euthanized and brains were processed for histological evaluation or placed in culture with neural stem cell media (with EGF and FGF supplementation). Tumors in both groups (with or without shATRX) showed histological hallmarks of human grade IV glioblastoma. The loss of ATRX was confirmed by IHC, and was specifically localized within tumors generated with the shATRX plasmid and not in the tumors generated with shp53 and NRAS alone, nor in the adjacent normal cortex. Notably, loss of ATRX reduced median survival of mice by 43% (p=0.012). Tissue was analyzed by FISH telomere probe as ATRX loss in human tumors is associated with alternative lengthening of telomeres (ALT). ATRX-deficient tumors were significantly more likely to show chromosomal aneuploidy (p=0.015) by telomere FISH. Cell lines generated from ATRX-deficient tumors were confirmed to have reduction of ATRX expression. Tumor cell lines (with and without ATRX loss) were plated, treated at 24 hours with intervention or control, and analyzed for viability at 72 hours. ATRX-deficient tumor cells were significantly (p≤0.005) more sensitive to DNA damaging agents, including: (1) 5-FU, (2) doxorubicin, (3) UV irradiation, and (4) adenoviral thymidine kinase with ganciclovir; with the notable exception of temozolomide (p=0.86), which is the standard of care for treatment of pediatric GBM. Loss of ATRX in a mouse model hastens glioblastoma formation and decreases survival. In addition, loss of ATRX leads to aneuploidy and improved response to DNA damaging agents, providing possible targeted therapies for tumors with this mutation. This mouse model prospectively validates ATRX as a tumor suppressor in pediatric GBM for the first time in an animal model; and provides a platform for analysis of relevant pathways and development of potential novel therapies. Supported by NIH/NINDS grants to MGC and PRL. Citation Format: Carl Koschmann, Alexandra Calinescu, Marta Dzaman, Rosie Lemons, Daniel Thomas, Maria G. Castro, Pedro R. Lowenstein. Loss of ATRX decreases survival and improves response to DNA damaging agents in a novel mouse model of glioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 995. doi:10.1158/1538-7445.AM2014-995