Alexandra Calinescu

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.

Dural Cells Release Factors Which Promote Cancer Cell Malignancy and Induce Immunosuppressive Markers in Bone Marrow Myeloid Cells

BACKGROUND Thirty per cent of cancer patients develop spine metastases with a substantial number leading to spinal cord compression and neurological deficits. Many demonstrate a propensity toward metastasis to the posterior third of the vertebral body. The dura, the outer layer of the meninges, lies in intimate contact with the posterior border of the vertebral body and has been shown to influence adjacent bone. The effects of the dura on bone marrow and cancer cells have not been examined. Understanding the biology of spinal metastasis will provide insights into mechanisms of cancer growth and allow for new treatment strategies. OBJECTIVE To examine the extent to which dura influences bone marrow/tumor cell metastatic characteristics. METHODS Dura conditioned media (DCM) from primary dura was examined for the ability to stimulate tumor cell proliferation/invasion and to alter bone marrow cell populations. RNA sequencing of dural fibroblasts was performed to examine expression of cytokines and growth factors. RESULTS DCM induced a significant increase in invasion and proliferation of multiple tumor cell lines, and of patient-derived primary spinal metastatic cells. DCM also increased the proliferation of bone marrow myeloid cells, inducing expression of immunosuppressive markers. RNA sequencing of dural fibroblasts demonstrated abundant expression of cytokines and growth factors involved in cancer/immune pathways. CONCLUSION Factors released by primary dural cells induce proliferation of tumor cells and alter bone marrow to create a fertile environment for tumor growth. The dura therefore may play an important role in the increased incidence of metastases to adjacent bone.

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

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

Abstract 187: Absence of S100A9 confers survival advantage in an aggressive de novo mouse model of glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common (60%) primary brain tumor in adults and constitutes a major challenge for both patients and clinicians, with a median survival of only 15-21 months when treated with surgery, radiation therapy and temozolomide. To improve patient outcome, a promising and safe avenue of adjuvant treatment is immune therapy with dendritic cell vaccines or adoptive T cell transfer. These treatments are however hindered by the immune suppressive environment induced by GBMs. Understanding the mechanisms by which GBM suppress the anti-tumor immune response is paramount in order to develop successful immune therapies. The Sleeping Beauty transposase system was used to modify the genetic makeup of stem cells in the sub-ventricular zone of neonatal mice and thus induce de novo glioblastomas with several combinations of genes. The most aggressive tumors where induced when combining NRAS and SV-40 Large T antigen (NLgT) generating invasive tumors which show the histological hallmarks human GBM (WHO grade IV) with pseudo-pallisading necrosis, neovascularization and hemorrhages, rendering the mice moribund with a median survival of 30 days. Myeloid derived suppressive cells, a heterogeneous population of immature bone marrow derived cells, are induced by cancers and other consumptive diseases and strongly inhibit adaptive and innate immune responses. We show that MDSCs isolated from de novo GBMs, inhibit antigen-specific and antigen non-specific T cell proliferation. Tumor infiltrating MDSCs highly express the pro-inflammatory secreted calcium binding protein S100A9 and its cognate receptors RAGE and TLR4. In bone marrow cultures, conditioned media from primary cell lines derived from NLgT tumors induce a marked expansion (60-70%) of myeloid derived suppressor cells (MDSCs) and enhance the expression of S100A9. Mice deficient for S100A9 with de novo NLgT tumors show an increased survival compared to wild-type animals (median survival= 47days, a 56% improvement). Analysis of tumor-infiltrating mononuclear cells in moribund NLgT wild-type and S100A9KO mice shows no significant difference in percent infiltrating MDSCs, macrophages and dendritic cells (% of CD45+), however the percent of CD8+ cytotoxic T lymphocytes is increased in S100A9 KO (16% vs. 6% p=0.0228). In addition, expression of MHCII is increased in macrophages and dendritic cells from S100A9 KO mice when compared to wild type mice. Taken together these data suggest that the absence of S100A9 confers a survival advantage by allowing a stronger anti-tumor immune response with:(1) increased number of cytotoxic T lymphocytes and (2) increased maturation of antigen presenting cells. Experiments are currently underway to identify the detailed cellular and molecular events, which give rise to this phenotype. This study was supported by NIH/RO1 NS057711 and NS074387. Citation Format: Alexandra Calinescu, Hikmat Assi, Bradley Kolb, Carl Koschmann, Pedro R. Lowenstein, John Ohlfest, Maria G. Castro. Absence of S100A9 confers survival advantage in an aggressive de novo mouse model of glioblastoma multiforme. [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 187. doi:10.1158/1538-7445.AM2014-187