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Immunotherapy has emerged as a powerful strategy for halting cancer progression. However, primary malignancies affecting the brain have been exempt to this success. Indeed, brain tumors continue to portend severe morbidity and remain a globally lethal disease. Extensive efforts have been directed at understanding how tumor cells survive and propagate within the unique microenvironment of the central nervous system (CNS). Cancer genetic aberrations and metabolic abnormalities provoke a state of persistent endoplasmic reticulum (ER) stress that in turn promotes tumor growth, invasion, therapeutic resistance, and the dynamic reprogramming of the infiltrating immune cells. Consequently, targeting ER stress is a potential therapeutic approach. In this work, we provide an overview of how ER stress response is advantageous to brain tumor development, discuss the significance of ER stress in governing antitumor immunity, and put forth therapeutic strategies of regulating ER stress to augment the effect of immunotherapy for primary CNS tumors.

Summary Isocitrate dehydrogenase 1–mutant (IDH1m) gliomas are recalcitrant tumors for which radiotherapy remains a standard treatment. A recent study identified ZMYND8 as a key mediator of radioresistance for IDH1m gliomas, and pharmacologic targeting of this pathway may heighten radiotherapy-induced tumor response, providing a prospect of improved clinical outcomes. See related article by Carney et al., p. 1763

Purpose:Cancer immunoediting shapes tumor progression by the selection of tumor cell variants that can evade immune recognition. Given the immune evasion and intratumor heterogeneity characteristic of gliomas, we hypothesized that CD8+ T cells mediate immunoediting in these tumors.Experimental Design:We developed retrovirus-induced PDGF+Pten−/− murine gliomas and evaluated glioma progression and tumor immunogenicity in the absence of CD8+ T cells by depleting this immune cell population. Furthermore, we characterized the genomic alterations present in gliomas that developed in the presence and absence of CD8+ T cells.Results:Upon transplantation, gliomas that developed in the absence of CD8+ T cells engrafted poorly in recipients with intact immunity but engrafted well in those with CD8+ T-cell depletion. In contrast, gliomas that developed under pressure from CD8+ T cells were able to fully engraft in both CD8+ T-cell–depleted mice and immunocompetent mice. Remarkably, gliomas developed in the absence of CD8+ T cells exhibited increased aneuploidy, MAPK pathway signaling, gene fusions, and macrophage/microglial infiltration, and showed a proinflammatory phenotype. MAPK activation correlated with macrophage/microglia recruitment in this model and in the human disease.Conclusions:Our studies indicate that, in these tumor models, CD8+ T cells influence glioma oncogenic pathways, tumor genotype, and immunogenicity. This suggests immunoediting of immunogenic tumor clones through their negative selection by CD8+ T cells during glioma formation.

Expression of upstream activators of the MAPK pathway correlates with functional activation of the ERK and p38 cascades. (A) Correlation between Braf expression and pERK in tumors from mice treated with anti-CD8+ (n=8) and isotype control (n=8) antibodies. (B) Correlation between Ptpn11 expression and pERK in tumors from mice treated with anti-CD8 (n=8) and isotype control (n=8) antibodies. (C) Correlation between Nras expression and pERK in tumors from mice treated with anti-CD8 (n=8) and isotype control (n=8) antibodies. (D) Correlation between Traf6 expression and p-p38 in tumors from mice treated with anti-CD8+ (n=8) and isotype control (n=8) antibodies. r and p values by Pearson correlation coefficient. Points represent individual mice.

Purpose:Paclitaxel (PTX) is one of the most potent and commonly used chemotherapies for breast and pancreatic cancer. Several ongoing clinical trials are investigating means of enhancing delivery of PTX across the blood–brain barrier for glioblastomas. Despite the widespread use of PTX for breast cancer, and the initiative to repurpose this drug for gliomas, there are no predictive biomarkers to inform which patients will likely benefit from this therapy.Experimental Design:To identify predictive biomarkers for susceptibility to PTX, we performed a genome-wide CRISPR knockout (KO) screen using human glioma cells. The genes whose KO was most enriched in the CRISPR screen underwent further selection based on their correlation with survival in the breast cancer patient cohorts treated with PTX and not in patients treated with other chemotherapies, a finding that was validated on a second independent patient cohort using progression-free survival.Results:Combination of CRISPR screen results with outcomes from patients with taxane-treated breast cancer led to the discovery of endoplasmic reticulum (ER) protein SSR3 as a putative predictive biomarker for PTX. SSR3 protein levels showed positive correlation with susceptibility to PTX in breast cancer cells, glioma cells, and in multiple intracranial glioma xenografts models. KO of SSR3 turned the cells resistant to PTX while its overexpression sensitized the cells to PTX. Mechanistically, SSR3 confers susceptibility to PTX through regulation of phosphorylation of ER stress sensor IRE1α.Conclusions:Our hypothesis generating study showed SSR3 as a putative biomarker for susceptibility to PTX, warranting its prospective clinical validation.

Supplementary Table from Translocon-associated Protein Subunit SSR3 Determines and Predicts Susceptibility to Paclitaxel in Breast Cancer and Glioblastoma

Supplementary Methods and Materials Figures S1: Schematic representation of LIPU generator set up. Supp Table 1: Summary of histological evaluation of CNS after one course of US-PTX Supp. Table 2: STR results of PDX cell lines. Figures S2: Cell viability in response to PTX and ABX

Supplementary Data from Translocon-associated Protein Subunit SSR3 Determines and Predicts Susceptibility to Paclitaxel in Breast Cancer and Glioblastoma

Supplementary Figure from Translocon-associated Protein Subunit SSR3 Determines and Predicts Susceptibility to Paclitaxel in Breast Cancer and Glioblastoma

Purpose:Anti-programmed cell death protein 1 (PD-1) therapy has demonstrated inconsistent therapeutic results in patients with glioblastoma (GBM) including those with profound impairments in CD8 T-cell effector responses.Experimental Design:We ablated the CD8α gene in BL6 mice and intercrossed them with Ntv-a mice to determine how CD8 T cells affect malignant progression in forming endogenous gliomas. Tumor-bearing mice were treated with PD-1 to determine the efficacy of this treatment in the absence of T cells. The tumor microenvironment of treated and control mice was analyzed by IHC and FACS.Results:We observed a survival benefit in immunocompetent mice with endogenously arising intracranial glioblastomas after intravenous administration of anti–PD-1. The therapeutic effect of PD-1 administration persisted in mice even after genetic ablation of the CD8 gene (CD8−/−). CD11b+ and Iba1+ monocytes and macrophages were enriched in the glioma microenvironment of the CD8−/− mice. The macrophages and microglia assumed a proinflammatory M1 response signature in the setting of anti–PD-1 blockade through the elimination of PD-1–expressing macrophages and microglia in the tumor microenvironment. Anti–PD-1 can inhibit the proliferation of and induce apoptosis of microglia through antibody-dependent cellular cytotoxicity, as fluorescently labeled anti–PD-1 was shown to gain direct access to the glioma microenvironment.Conclusions:Our results show that the therapeutic effect of anti–PD-1 blockade in GBM may be mediated by the innate immune system, rather than by CD8 T cells. Anti–PD-1 immunologically modulates innate immunity in the glioma microenvironment—likely a key mode of activity.

Purpose:Paclitaxel shows little benefit in the treatment of glioma due to poor penetration across the blood–brain barrier (BBB). Low-intensity pulsed ultrasound (LIPU) with microbubble injection transiently disrupts the BBB allowing for improved drug delivery to the brain. We investigated the distribution, toxicity, and efficacy of LIPU delivery of two different formulations of paclitaxel, albumin-bound paclitaxel (ABX) and paclitaxel dissolved in cremophor (CrEL-PTX), in preclinical glioma models.Experimental Design:The efficacy and biodistribution of ABX and CrEL-PTX were compared with and without LIPU delivery. Antiglioma activity was evaluated in nude mice bearing intracranial patient-derived glioma xenografts (PDX). Paclitaxel biodistribution was determined in sonicated and nonsonicated nude mice. Sonications were performed using a 1 MHz LIPU device (SonoCloud), and fluorescein was used to confirm and map BBB disruption. Toxicity of LIPU-delivered paclitaxel was assessed through clinical and histologic examination of treated mice.Results:Despite similar antiglioma activity in vitro, ABX extended survival over CrEL-PTX and untreated control mice with orthotropic PDX. Ultrasound-mediated BBB disruption enhanced paclitaxel brain concentration by 3- to 5-fold for both formulations and further augmented the therapeutic benefit of ABX. Repeated courses of LIPU-delivered CrEL-PTX and CrEL alone were lethal in 42% and 37.5% of mice, respectively, whereas similar delivery of ABX at an equivalent dose was well tolerated.Conclusions:Ultrasound delivery of paclitaxel across the BBB is a feasible and effective treatment for glioma. ABX is the preferred formulation for further investigation in the clinical setting due to its superior brain penetration and tolerability compared with CrEL-PTX.

Methods, Figures, legends and Tables

Purpose:Diffuse intrinsic pontine glioma (DIPG) is among the deadliest of pediatric brain tumors. Radiotherapy is the standard-of-care treatment for DIPG, but offers only transient relief of symptoms for patients with DIPG without providing significant survival benefit. Oncolytic virotherapy is an anticancer treatment that has been investigated for treating various types of brain tumors.Experimental Design:Here, we have explored the use of mesenchymal stem cells (MSC) for oncolytic virus (OV) delivery and evaluated treatment efficacy using preclinical models of DIPG. The survivin promoter drives the conditional replication of OV used in our studies. The efficiency of OV entry into the cells is mediated by fiber modification with seven lysine residues (CRAd.S.pK7). Patients' samples and cell lines were analyzed for the expression of viral entry proteins and survivin. The ability of MSCs to deliver OV to DIPG was studied in the context of a low dose of irradiation.Results:Our results show that DIPG cells and tumors exhibit robust expression of cell surface proteins and survivin that enable efficient OV entry and replication in DIPG cells. MSCs loaded with OV disseminate within a tumor and release OV throughout the DIPG brainstem xenografts in mice. Administration of OV-loaded MSCs with radiotherapy to mice bearing brainstem DIPG xenografts results in more prolonged survival relative to that conferred by either therapy alone (P < 0.01).Conclusions:Our study supports OV, CRAd.S.pK7, encapsulated within MSCs as a therapeutic strategy that merits further investigation and potential translation for DIPG treatment.

Activators of MAPK signaling correlate to macrophage markers in human GBM patients. (A) Correlation of mRNA expression analysis of 161 GBM patients in TCGA library between ITGAM (CD11b) and MAPK13 (n=161). (B) Correlation between AIF1 (Iba1) and RRAS (n=161). (C) Correlation between AIF1 and MAPK13 (n=161). (D) Correlation between AIF1 and MAP3K8 (n=161). r and p values by Pearson correlation coefficient. Points represent individual GBM patients.

Supplementary Legend

Transgenic murine glioma model possesses increased CD4+ and CD8+ T-cell infiltration early in tumor development. (A) Representative bioluminescence activity of tumor captured 21 and 35 days post-tumor induction (left). Scatter plot showing an example of an ex vivo flow cytometry of the brain tumors using the CD4+ and CD8+ markers (right). (B) Bioluminescence was measured at seven time points between 21 and 63 days post-tumor induction (n=8/time point). (C) Kaplan-Meier survival curve illustrating the time points of flow cytometric analysis relative to initiation of tumor-related death (n=16). (D) Bar plots showing CD4+ (left) and CD8+ (right) T-cell tumor infiltration normalized against the contralateral brain hemisphere, and bioluminescence signal at 21 and at 35 days post-tumor induction (n=16). Data in (D) is shown as mean {plus minus} SEM. p= 0.0189 and p= 0.026 by unpaired studentÂ's t-test.

Supplementary Figures 1-7

PDF - 1816K, Supplementary Figure 1: CGH probe for Cre-mediated Pten deletion provides a surrogate of tumor DNA content for mouse tumors. Supplementary Figure 2: The genomic architecture of mouse tumor gene deletions suggests independent selection pressure for genes within the same deletion in human tumors. Supplementary Figure 3: Mouse and Human Proneural GBM share a common regulatory network. Supplementary Figure 4: *PTEN/p53 tumors rapidly progress and acquire the histological features of GBM. Supplementary Figure 5: *Pten tumors acquire and express mutations in p53. Supplementary Figure 6: *p53 tumors develop the histological features of GBM, and have an intermediate survival between *PTEN and *PTEN/p53 tumors.

Proneural glioblastoma is defined by an expression pattern resembling that of oligodendrocyte progenitor cells and carries a distinctive set of genetic alterations. Whether there is a functional relationship between the proneural phenotype and the associated genetic alterations is unknown. To evaluate this possible relationship, we performed a longitudinal molecular characterization of tumor progression in a mouse model of proneural glioma. In this setting, the tumors acquired remarkably consistent genetic deletions at late stages of progression, similar to those deleted in human proneural glioblastoma. Further investigations revealed that p53 is a master regulator of the transcriptional network underlying the proneural phenotype. This p53-centric transcriptional network and its associated phenotype were observed at both the early and late stages of progression, and preceded the proneural-specific deletions. Remarkably, deletion of p53 at the time of tumor initiation obviated the acquisition of later deletions, establishing a link between the proneural transcriptional network and the subtype-specific deletions selected during glioma progression. Cancer Res; 74(5); 1440–51. ©2014 AACR.

XLSX - 1231K, Supplementary Table 1: List of genes deleted in 75 to 100% of the end-stage *PTEN tumors Supplementary Table 2: List of genes showing copy number gains in *PTEN end-stage and *PTEN/p53 mouse tumors. Supplementary Table 3: Genes that were deleted in 75-100% of *PTEN end-stage mouse tumors and at least 10% of human tumors within one of the four GBM subtypes mapped to their chromosomal location in the human genome. Supplementary Table 4: List of gene deletions that were specific for one of the four GBM subtypes. Supplementary Table 5: Correlation of proneural-specific genetic alterations and deletions identified by cross-species comparison. Supplementary Table 6: Spearman correlation for Verhaak gene sets show highest correlation for human proneural GBM subtype for 21 dpi *PTEN tumors, end-stage *PTEN tumors and *p53 end-stage tumors. Supplementary Table 7: MR identified from NB versus *PTEN 21 dpi and NB versus *PTEN end-stage mouse MARINas, and human NB versus proneural GBM (TCGA) MARINa.

PDF - 106K, Contains the legends for Supplementary Figures 1-6 and Supplementary Tables 1-7.

Purpose: Paclitaxel (PTX) is one of the most potent and commonly used chemotherapies for breast and pancreatic cancer. Several ongoing clinical trials are investigating means of enhancing delivery of PTX across the blood–brain barrier for glioblastomas. Despite the widespread use of PTX for breast cancer, and the initiative to repurpose this drug for gliomas, there are no predictive biomarkers to inform which patients will likely benefit from this therapy. Experimental Design: To identify predictive biomarkers for susceptibility to PTX, we performed a genome-wide CRISPR knockout (KO) screen using human glioma cells. The genes whose KO was most enriched in the CRISPR screen underwent further selection based on their correlation with survival in the breast cancer patient cohorts treated with PTX and not in patients treated with other chemotherapies, a finding that was validated on a second independent patient cohort using progression-free survival. Results: Combination of CRISPR screen results with outcomes from patients with taxane-treated breast cancer led to the discovery of endoplasmic reticulum (ER) protein SSR3 as a putative predictive biomarker for PTX. SSR3 protein levels showed positive correlation with susceptibility to PTX in breast cancer cells, glioma cells, and in multiple intracranial glioma xenografts models. KO of SSR3 turned the cells resistant to PTX while its overexpression sensitized the cells to PTX. Mechanistically, SSR3 confers susceptibility to PTX through regulation of phosphorylation of ER stress sensor IRE1α. Conclusions: Our hypothesis generating study showed SSR3 as a putative biomarker for susceptibility to PTX, warranting its prospective clinical validation.

Purpose: The epigenetic mechanisms involved in transcriptional regulation leading to malignant phenotype in gliomas remains poorly understood. Topoisomerase IIB (TOP2B), an enzyme that decoils and releases torsional forces in DNA, is overexpressed in a subset of gliomas. Therefore, we investigated its role in epigenetic regulation in these tumors. Experimental Design: To investigate the role of TOP2B in epigenetic regulation in gliomas, we performed paired chromatin immunoprecipitation sequencing for TOP2B and RNA-sequencing analysis of glioma cell lines with and without TOP2B inhibition and in human glioma specimens. These experiments were complemented with assay for transposase-accessible chromatin using sequencing, gene silencing, and mouse xenograft experiments to investigate the function of TOP2B and its role in glioma phenotypes. Results: We discovered that TOP2B modulates transcription of multiple oncogenes in human gliomas. TOP2B regulated transcription only at sites where it was enzymatically active, but not at all native binding sites. In particular, TOP2B activity localized in enhancers, promoters, and introns of PDGFRA and MYC, facilitating their expression. TOP2B levels and genomic localization was associated with PDGFRA and MYC expression across glioma specimens, which was not seen in nontumoral human brain tissue. In vivo, TOP2B knockdown of human glioma intracranial implants prolonged survival and downregulated PDGFRA. Conclusions: Our results indicate that TOP2B activity exerts a pleiotropic role in transcriptional regulation of oncogenes in a subset of gliomas promoting a proliferative phenotype.

Purpose: Cancer immunoediting shapes tumor progression by the selection of tumor cell variants that can evade immune recognition. Given the immune evasion and intratumor heterogeneity characteristic of gliomas, we hypothesized that CD8+ T cells mediate immunoediting in these tumors. Experimental Design: We developed retrovirus-induced PDGF+Pten−/− murine gliomas and evaluated glioma progression and tumor immunogenicity in the absence of CD8+ T cells by depleting this immune cell population. Furthermore, we characterized the genomic alterations present in gliomas that developed in the presence and absence of CD8+ T cells. Results: Upon transplantation, gliomas that developed in the absence of CD8+ T cells engrafted poorly in recipients with intact immunity but engrafted well in those with CD8+ T-cell depletion. In contrast, gliomas that developed under pressure from CD8+ T cells were able to fully engraft in both CD8+ T-cell–depleted mice and immunocompetent mice. Remarkably, gliomas developed in the absence of CD8+ T cells exhibited increased aneuploidy, MAPK pathway signaling, gene fusions, and macrophage/microglial infiltration, and showed a proinflammatory phenotype. MAPK activation correlated with macrophage/microglia recruitment in this model and in the human disease. Conclusions: Our studies indicate that, in these tumor models, CD8+ T cells influence glioma oncogenic pathways, tumor genotype, and immunogenicity. This suggests immunoediting of immunogenic tumor clones through their negative selection by CD8+ T cells during glioma formation.

As a hallmark of glioblastoma (GBM), the myeloid-rich tumor microenvironment is one of the major causes of GBM immunosuppression and therapy resistance. Therefore, tumor-associated myeloid cells (TAMCs) have been identified as a promising therapeutic target for remodeling the immunologically “cold” brain tumors and overcoming the therapy resistance of GBM. Emerging research findings have uncovered the interplay between TAMCs and radiotherapy, a key component of the standard of care for GBM. While radiotherapy is known to induce antitumor immune response, in which the functionality of the myeloid compartment, including phagocytosis of tumor and subsequent activation of effector T cells, plays a key role, irradiation also triggers immune resistance mechanisms, such as the overexpression of anti-phagocytic molecule CD47 in gliomas and immune checkpoint molecule PD-L1 in TAMCs. To tackle this, a bispecific-lipid nanoparticle (B-LNP) was designed to hijack the irradiation-induced upregulation of immunosuppressive molecules for harnessing TAMCs to elicit antitumor immune response. The B-LNP was surface functionalized with anti-CD47/PD-L1 ligands to enable a simultaneous targeting of TAMCs and glioma cells through dual ligation. The engineered B-LNP effectively bound to and blocked CD47 and PD-L1 molecules, and served as a bridge to engage TAMCs for enhanced phagocytosis of glioma cells when combined with radiotherapy. To promote the TAMC-mediated activation of adaptive antitumor immunity post-phagocytosis, diABZI, a synthetic non-nucleotidyl agonist for stimulator of interferon genes (STING), was physically encapsulated into B-LNP as a payload therapeutic. Our results indicate that B-LNP/diABZI complex enabled a TAMC-specific STING activation in preclinical murine glioma model CT-2A, which transformed the immunosuppressive TAMCs into tumor-eradicating cells in the glioma microenvironment, as evidenced by immune profiling, single-cell RNA sequencing analysis, and bulk metabolomics. As a result, the nano-engineered TAMCs dramatically promoted tumor infiltration and anti-glioma activity of T cells, which improved the therapeutic outcome of radiotherapy, eradicating tumors from about 70% of the glioma-bearing mice, and generated a long-lasting immunological memory against gliomas. The translational potential of our nano-engineering approach was further validated using a glioma model that recapitulates the genetic, histological, and immunological features of human GBM, and using the clinical tumor specimens of GBM patients. In conclusion, our work demonstrates a nanotechnology-mediated immunomodulatory approach that targets and modulates the myeloid-rich GBM microenvironment as a combinatorial treatment for improving the existing standard of care for GBM. Citation Format: Peng Zhang, Aida Rashidi, Junfei Zhao, Brandyn Castro, Abby Ellingwood, Yu Han, Aurora Lopez-Rosas, Markella Zannikou, Crismita Dmello, Rebecca Levine, Ting Xiao, Alex Cordero, Adam M Sonabend, Irina V Balyasnikova, Catalina Lee-Chang, Jason Miska, Maciej S Lesniak. Nano-engineering of immunosuppressive myeloid cells for immunostimulation in glioblastoma [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B36.

Introduction: Deconstructive immune cell profiling of central nervous system (CNS) tumors has focused on the tumor, excluding interrogation of the tumor microenvironment (TME). Integrated spatial analysis can ascertain the cell interactome and may be a key biomarker for effective anti-tumor immune responses. Methods: En bloc resections of glioma (n=10) and lung metastasis (n=10) to preserve the tissue architecture, underwent tissue segmentation and high dimension opal 7-color multiplex imaging. Bioinformatic analysis of scRNA was used to infer immune cell functionality. Results: CD3+ T cell frequency was equivalent between CNS cancer lineages. Within gliomas T cells were confined to the perivascular space and the infiltrating edge. In lung metastasis, T cells are confined to the tumor stroma. CD163+ macrophages predominate in brain metastasis throughout the TME (p Conclusion: Current therapies are targeted to cell populations and singular pathways. Immunosuppressive macrophages dominate within the TME and targeting this population may create an environment that favors T cell activation and effective immune responses. Furthermore, the immune interactome, an important event for anti-tumor immune response, is a function of cancer lineage, TME, and STAT3 expression, which will gain relevance for future therapeutics directed to modulating these interactions. Citation Format: Hinda Najem, Martina Ott, Cynthia Kassab, Arvind Rao, Ganesh Rao, Anantha Marisetty, Adam M. Sonabend, Craig Horbinski, Roel Verhaak, Anand Shakar, Santhoshi Krishnan, Frederick S. Varn, Victor A. Arietta, Pravesh Gupta, Sherise D. Ferguson, Jason Huse, Gregory N. Fuller, James Long, Dan Winskowski, Ben Freiberg, C. David James, Leonidas C. Platanias, Maciej S. Lesniak, Jared K. Burks, Amy B. Heimberger. The central nervous system immune cell interactome is a function of cancer lineage, tumor microenvironment and STAT3 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2548.

The purpose of this study is to identify novel genes that drive proliferation in glioblastoma (GBM) and likely contribute to gliomagenesis, therefore facilitating the aggressive nature of the tumor. GBM is an aggressive primary malignancy of the brain with almost a 100% recurrence rate. It is the most common malignant brain tumor in adults, with an average survival of 21 months after diagnosis, with the current standard of care. As such, new advances in therapy are desperately needed. Large scale genomic approaches have shown that GBM has a complex genetic architecture, with a great deal of intratumoral heterogeneity. However, a more comprehensive understanding of determinants of growth is required to identify new targets and provide new therapeutic strategies. CRISPR-Cas9 screening technology has enabled whole-genome screens that allow for systematic and objective identification of genes governing cell viability and proliferation. These genes may represent unique vulnerabilities that can be targeted with novel therapeutics. Here, we performed a genome-wide CRISPR knockout screen in SNB19 human GBM cells, covering 17,000 genes with 4 guides per gene. In this type of screen, we anticipate that those guides that are depleted over time are the ones that correspond to genes driving growth and proliferation of GBM cells. Initial analysis showed that there was a reduction in guides for known driver genes, as we might expect. However, in addition to known drivers, we were able to identify a list of approximately 150 new genes that showed significant depletion in our screen (p In summary, we have used a whole-genome CRISPR-Cas9 knockout screen to identify a large novel set of genes that contributes to glioma viability and proliferation. Of these genes, we have been able to further validate a limited set to show that they do in fact contribute to the survival of cells in vitro and animal survival in vivo. Finally, we have been able to show enrichment of specific pathways across these gene sets, giving us a set of genes and pathways that represent novel genetic vulnerabilities. We believe this work will contribute significantly to developing new therapies for a disease that is desperately in need of additional therapeutic options. Citation Format: Shivani Baisiwala, Cheol Park, Chidibere Awah, Jack M. Shireman, Miranda R. Saathoff, Adam Sonabend, Atique U. Ahmed. Identifying a novel network of driver genes in glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 197.

Proneural glioblastoma is defined by an expression pattern resembling that of oligodendrocyte progenitor cells and carries a distinctive set of genetic alterations. Whether there is a functional relationship between the proneural phenotype and the associated genetic alterations is unknown. To evaluate this possible relationship, we performed a longitudinal molecular characterization of tumor progression in a mouse model of proneural glioma. In this setting, the tumors acquired remarkably consistent genetic deletions at late stages of progression, similar to those deleted in human proneural glioblastoma. Further investigations revealed that p53 is a master regulator of the transcriptional network underlying the proneural phenotype. This p53-centric transcriptional network and its associated phenotype were observed at both the early and late stages of progression, and preceded the proneural-specific deletions. Remarkably, deletion of p53 at the time of tumor initiation obviated the acquisition of later deletions, establishing a link between the proneural transcriptional network and the subtype-specific deletions selected during glioma progression. Cancer Res; 74(5); 1440–51. ©2014 AACR.

Background: Recent studies of primary central nervous system lymphoma (PCNSL) have found an association between cytoreductive surgery and survival, challenging the traditional notion that surgery is not beneficial and potentially harmful. However, no studies have examined outcomes after surgery plus adjuvant treatment. We investigated treatment-related outcomes for surgery plus radiation using data from the Surveillance, Epidemiology, and End Results (SEER) Program. Methods: The SEER database was queried to collect cases of histologically confirmed non-Hodgkin's lymphoma within the CNS diagnosed between 1995-2014. Median survival times were determined by the Kaplan-Meier method and compared using the log-rank test. Predictors of overall survival and cause-specific survival were determined using the Cox proportional hazards regression model. Treatment modalities were categorized as biopsy alone, biopsy plus radiation therapy (RT), surgery alone, and surgery + RT. Biopsy alone was used as the reference category. Subgroup analysis stratified patients by extent of surgical resection and recursive partition analysis (RPA) risk group. Results: We identified 5,417 cases that met search criteria, 39% of which received surgical resection. Median survival times for biopsy alone (n=1,824, 34%), biopsy + RT (n=1,460, 27%), surgery alone (n=1,222, 23%), and surgery + RT (n=911, 17%) were 7, 8, 20, and 27 months, respectively (p Conclusion: Surgical resection of PCNSL in the general population is more common than previously thought. Radiation therapy after surgery is associated with increased survival, regardless of the extent of surgical resection or the patient's RPA risk category. Neurotoxicity, dosing, and effects of concurrent chemotherapy should be addressed in future studies. Citation Format: Connor J. Kinslow, Ali I. Rae, Alfred I. Neugut, Christopher M. Adams, Sameer A. Sheth, Guy M. McKhann, Michael B. Sisti, Jeffrey N. Bruce, Fabio M. Iwamoto, Adam M. Sonabend, Tony J. Wang. Surgery plus adjuvant radiation as a valid treatment option for primary central nervous system lymphoma (PCSNL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 664.